Principles of Gender-Specific Medicine: Gender in the Genomic Era [3 ed.] 9780128035061

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PRINCIPLES OF GENDER-SPECIFIC MEDICINE

PRINCIPLES OF GENDER-SPECIFIC MEDICINE GENDER IN THE GENOMIC ERA THIRD EDITION Edited by

Marianne J. Legato, M.D., Ph.D. (hon. c.), F.A.C.P. Emerita Professor of Clinical Medicine, Columbia University Adjunct Professor of Medicine, Johns Hopkins

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2017 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-803506-1 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

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Dedication

For my brother, Gerard, who explained it all.

Praise for Principles of Gender-Specific Medicine, 3rd edition As much as the first edition of Principles of GenderSpecific Medicine defined the field of sex and gender health a decade ago, this third edition redefines it, taking into account the evolution and complexity of genomic, hormonal, and immunological science of sex differences. Marianne Legato uses an innovative approach to organizing cutting edge topics outside of the typical format of medical textbooks, which aids the reader in viewing the science in novel ways. Even for those working in the field of sex- and gender-based medicine, there is so much to learn from this rich collection of experts and content. Thank you once again Marianne Legato! Alyson J. McGregor, MD, MA, FACEP Director, Division of Sex and Gender in Emergency Medicine (SGEM) Director, Sex and Gender in Emergency Medicine (SGEM) Fellowship Associate Professor of Emergency Medicine Warren Alpert Medical School of Brown University The name Marianne Legato has become almost synonymous with Gender and Sex-specific Medicine. She was founder and Editor-in Chief of the first scientific journal dedicated to this new science (and has recently founded a new journal—“Gender and the Genome”). She was and is Editor-in-Chief of the first textbook on Gender Medicine, which appears now in the present third edition and she has been a scout in promoting Gender Medicine throughout the world. Her books have been eye-openers for many of us and this new edition of Principles of Gender-Specific Medicine is again a breakthrough. It includes carefully chosen contributions from leading scholars, researchers, and clinicians from all

over the globe, who present the state-of-the-art in their respective fields. What “The Harrison” is for Internal Medicine, what “The Williams” is for Obstetrics, and what the “The DSM-5” is for Psychiatry, “The Legato” is for Gender Medicine—an absolutely essential Must Have and Must Read for any professional who deals with the differences in physiology and pathophysiology between males and females. Marek Glezerman, MD Professor Emeritus of Obstetrics and Gynecology Chairman of the Ethics Committee Sackler School of Medicine Tel Aviv University The new Principles of Gender-Specific Medicine, edited by Dr. Marianne Legato from Columbia University, is a compilation of the most up-to-date topics in sex and gender difference research. The chapters cover the major genomic, cellular, molecular, and whole body “hot” topics in the field. In addition, there are innovative pieces such as “Robots and Gender” by Tatsuya Nomura. Get this book and keep it on your shelf as a ready reference for state-of-the-art research in sex and gender differences that will inspire current and future scientists for years to come. Jane F. Reckelhoff, PhD Billy S. Guyton Distinguished Professor Chair, Department of Biochemistry Director, Women’s Health Research Center Director, Research Development, Office of Research and Sponsored Programs University of Mississippi Medical Center Jackson, MS

List of Contributors

Dov Feldberg  Tel Aviv University, Tel Aviv, Israel

Catherine E.M. Aiken  University of Cambridge, Cambridge, United Kingdom

Betiel Fesseha  Johns Hopkins University School of Medicine, Baltimore, MD, United States

Nicholas B. Allen  The University of Melbourne, Melbourne, VIC, Australia; University of Oregon, Eugene, OR, United States

Roger B. Fillingim  University of Florida, Gainesville, FL, United States

Shambhu Aryal  Duke University, Durham, NC, United States

Karen H. Frith  The University of Alabama in Huntsville, Huntsville, AL, United States

Hrayr Attarian  Northwestern University, Chicago, IL, United States

Andreas M. Fritzen  University of Copenhagen, Copenhagen, Denmark

Uma Mahesh R. Avula  Columbia University Medical Center, New York, NY, United States

Robert C. Froemke  New York University School of Medicine, New York, NY, United States

Davide Bolignano  CNR-Istituto di Fisiologia Clinica (IFC), Reggio Calabria, Italy

Juri Fujimura  Nippon Medical School, Tokyo, Japan

Neil A. Bradbury  Rosalind Franklin University of Medicine and Science, North Chicago, IL, United States Jan Burns  Canterbury Christ Church University, Canterbury, England Larry Cahill  University of California, Irvine, Irvine, CA, United States Ioana Carcea  New York University, New York, NY, United States Yvonne Y. Chan  University of California, Davis, Sacramento, CA, United States Howard Chang  Stanford University, Stanford, CA, United States Nisha Charkoudian  University of Bristol, Bristol, United Kingdom Dorte M. Christiansen  University of Aarhus, Aarhus, Denmark; University of Southern Denmark, Odense, Denmark Sharon Saunderson Coffey  The University of Alabama in Huntsville, Huntsville, AL, United States Eli Coleman  University of Minnesota, Minneapolis, MN, United States Marsh Cuttino  Emergency Medicine Physician, Richmond, VA, United States; Orbital Medicine, Inc., Richmond, VA, United States Adrian Dobs  Johns Hopkins University School of Medicine, Baltimore, MD, United States Mehmet Tevfik Dorak  Liverpool Hope University, Liverpool, United Kingdom Jack Drescher  New York University, New York, NY, United States Ines Ana Ederer  Medical University Vienna, Vienna, Austria Alireza Fazeli  University of Sheffield, Sheffield, United Kingdom

Dan Gazit  Hebrew University of Jerusalem, Jerusalem, Israel; Cedars-Sinai Medical Center, Los Angeles, CA, United States Marek Glezerman  Tel Aviv University, Tel Aviv, Israel; Rabin Medical Center, Petah Tikva, Israel Thomas Goodwin  Advanced Pattern Analysis & Countermeasures Group, Research Innovation Center, Colorado State University, Fort Collins, CO, United States; NASA, Johnson Space Center (Retired), Houston, TX, United States Tiffany Guess  Middle Tennessee State University, Murfreesboro, TN, United States Florian Hackl  Tufts School of Medicine, Brighton, MA, United States Kristie Hadley  Ben Gurion University of the Negev, Beer-Sheva, Israel Susanne B. Haga  Duke University School of Medicine, Durham, NC, United States Emma C. Hart  University Hospitals Bristol NHS Foundation Trust, Bristol, United Kingdom Olaf Hiort  University of Lübeck, Lübeck, Germany Oliver Hobert  Columbia University, New York, NY, United States William V. Holt  University of Sheffield, Sheffield, United Kingdom B.M.A. Huisstede  Leiden University Medical Center, Leiden, The Netherlands; University Medical Center Utrecht, Utrecht, The Netherlands Amy Hunter  The University of Alabama in Huntsville, Huntsville, AL, United States Hiko Hyakusoku  Nippon Medical School, Tokyo, Japan Emil Jovanov  The University of Alabama in Huntsville, Huntsville, AL, United States

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Natsuko Kakudo  Kansai Medical University, Moriguchi, Japan Bente Kiens  University of Copenhagen, Copenhagen, Denmark Jin Kyung Kim  The University of California Irvine Medical Center, Orange, CA, United States Laura M. Kok  Leiden University Medical Center, Leiden, The Netherlands

Jose M. Ordovas  JM-USDA-Human Nutrition Research Center on Aging at Tufts University, Boston, MA, United States Meital Oren-Suissa  Columbia University, New York, NY, United States Reinhard Pauzenberger  Medical University Vienna, Vienna, Austria

Stephan M. Korn  Medical University of Vienna, Vienna, Austria

Gadi Pelled  Hebrew University of Jerusalem, Jerusalem, Israel; Cedars-Sinai Medical Center, Los Angeles, CA, United States

Kenji Kusumoto  Kansai Medical University, Moriguchi, Japan

Anna Pisano  CNR-Istituto di Fisiologia Clinica (IFC), Reggio Calabria, Italy

Congxing Lin  Washington University School of Medicine, St. Louis, MO, United States

Vera Regitz-Zagrosek  Charité Universitätsmedizin, Berlin, Germany; DZHK partner site Berlin, Berlin, Germany

Ana Lleo  Humanitas Clinical and Research Center, Milan, Italy

Ibis Sánchez-Serrano  The Core Model Corporation, S.A., Panamá City, Panamá

Michelle R. Longmire  Stanford University, Stanford, CA, United States

Michael A. Schmidt  Advanced Pattern Analysis & Countermeasures Group, Research Innovation Center, Colorado State University, Fort Collins, CO, United States; Sovaris Aerospace, LLC, Boulder, CO, United States

Anne-Marie Lundsgaard  University of Copenhagen, Copenhagen, Denmark Liang Ma  Washington University School of Medicine, St. Louis, MO, United States Priyanka Madhushri  The University of Alabama in Huntsville, Huntsville, AL, United States Leanna W. Mah  University of California, Davis, Sacramento, CA, United States Lesley Marson  University of North Carolina, Chapel Hill, NC, United States Margaret M. McCarthy  University of Maryland School of Medicine, Baltimore, MD, United States

Fidaa Shaib  Baylor College of Medicine, Houston, TX, United States Galina Shapiro  Hebrew University of Jerusalem, Jerusalem, Israel Shahrokh F. Shariat  Medical University of Vienna, Vienna, Austria; Karl-Landsteiner-Institute of Urology and Andrology, Vienna, Austria; University of Texas Southwestern Medical Center, Dallas, TX, United States; Weill Cornell Medical College, New York, NY, United States

Erin E. McClelland  Middle Tennessee State University, Murfreesboro, TN, United States

Eyal Sheiner  Ben Gurion University of the Negev, Beer-Sheva, Israel; Soroka University Medical Center, Beer-Sheva, Israel

Aleksandar Milenkovic  The University of Alabama in Huntsville, Huntsville, AL, United States

Julian G. Simmons  The University of Melbourne, Melbourne, VIC, Australia

Hiroshi Mizuno  Juntendo University School of Medicine, Tokyo, Japan

Veena Taneja  Mayo Clinic College of Medicine, Rochester, MN, United States

Shaun K. Morris  The Hospital for Sick Children, Toronto, ON, Canada

Lisa M. Thurston  University of London, London, United Kingdom

Alexandra Müller  University of Cape Town, Cape Town, South Africa

Siobhan Tierney  Canterbury Christ Church University, Canterbury, England

Rob G.H.H. Nelissen  Leiden University Medical Center, Leiden, The Netherlands

Morikuni Tobita  Juntendo University School of Medicine, Tokyo, Japan

Santosh K. Nepal  Carilion Roanoke Memorial Hospital, Roanoke, VA, United States

Elaine Wan  Columbia University Medical Center, New York, NY, United States

Claire K. Nguyen  The Hospital for Sick Children, Toronto, ON, Canada

Kevin Warwick  Coventry University, Coventry, United Kingdom

Tatsuya Nomura  Ryukoku University, Otsu, Japan

Ursula Wesselmann  University of Alabama at Birmingham, Birmingham, AL, United States

Meghana Noonavath  Columbia University Medical Center, New York, NY, United States Sabine Oertelt-Prigione  Charité – Universitätsmedizin, Berlin, Germany Rei Ogawa  Nippon Medical School, Tokyo, Japan Shimpei Ono  Nippon Medical School, Tokyo, Japan Hakan Orbay  University of California, Davis, CA, United States; University of California, Sacramento, CA, United States

Sarah Whittle  The University of Melbourne, Melbourne, VIC, Australia Swaytha Yalamanchi  Johns Hopkins University School of Medicine, Baltimore, MD, United States Jennifer H. Yang  University of California, Davis, Sacramento, CA, United States

Foreword

During the past 25 years, the scientific and medical communities have intensified efforts to define the broader context of women’s health beyond traditional concepts that had been primarily limited to the female reproductive system, reflecting what Doctor Marianne Legato referred to as “the bikini view of women’s health.” Now, expanded concepts inclusive of conditions that affect both women and men across the lifespan bring into focus the importance of determining sex and gender influences on the human phenotype. From early advocacy demands to include women in clinical research protocols, to the current status of identifying the effects of sex and gender factors ranging from molecular and genomic science to the translational aspects of health and disease, women’s health and health care have evolved into the discipline of gender-specific medicine, bringing a broad and comprehensive approach to medical care. This evolution was significantly accelerated, expanded, and defined by the activities of the Office of Research on Women’s Health (ORWH) at the National Institutes of Health (NIH). It was a coalition of women’s health advocates, scientists, and legislators who expressed concern that clinical research receiving federal funding by the NIH did not consistently include women, and if included, not in significant numbers to determine if gender differences existed in the response to the intervention being studied. In response the NIH in 1990 established the ORWH to ensure that women would be included in biomedical and behavioral clinical research studies, the first office within the US Department of Health and Human Services to have women’s health as its primary mission. Over the years since its inception, however, the ORWH has achieved two important things: it validated research in women’s health as a legitimate scientific pursuit rather than a political imperative and it helped establish the concept that learning the unique characteristics of female physiology offered an opportunity to compare and contrast them to those of men. This was an essential step in developing the concept of gender-specific medicine which is a broader concept than women’s health; it investigates the impact of biological sex on human physiology and the experience of disease. Before a robust discipline of gender-specific medicine could be achieved, however, the enormous gaps in our information about women’s biology and their experience of disease had to be filled. To achieve this, the Office

mounted a series of symposia throughout the 1990s to develop a strategic plan for achieving its aims. The first initiative was developed in 1991 and the second resulted in the 1999 report, Agenda for Research on Woment’s Health for the 21st Century. This report not only provided an important roadmap for biological investigators but also called for interdisciplinary research and career development.1 An additional strategic plan, based on five regional workshops and public hearings, provided essential input for the generation of the 2010 report, Moving Into the Future With New Dimensions and Strategies: A Vision for 2020 for Women’s Health Research, and yielded more than 400 specific recommendations for women’s health research and career priorities. This planning effort provided a collaboratively and purposefully developed foundation for future research priorities and programmatic initiatives.2 Perhaps because of the early focus on clinical research, which is usually based on an hypothesis first tested by basic research, it required further effort to underscore the importance of determining sex differences in basic or preclinical research. The landmark 2001 Institute of Medicine report, Exploring the Biological Contributions to Human Health: Does Sex Matter? documented known sex differences and strongly urged exploration of sex differences in basic cellular, molecular, and biochemical investigations, not just clinical research.3 It also emphasized advances in molecular biology that have revealed genetic and molecular bases for differences in health and human diseases, some of which result from the sexual genotype and therefore sex differences in health. It has been surprising that even today, there are investigators who are using cell lines in their research but had never considered if the cells are derived from males or females, or how the sex of the cells might affect their results! Fortunately, the ORWH and NIH announced a newly implemented policy in 2014 to require applicants to include in their research design their plans for the balance of male and female cells and animals in future grant applications for preclinical studies.4,5 While principles of women’s health were becoming part of the new norm of scientific design, another scientific area of discovery was taking place—that of defining the human genome. The goal of identifying and mapping the genes of the human genome was formally launched in 1990 and its completion was announced in 2003. While the “human genome” is considered a

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mosaic, the “genome” of each individual is considered unique. Although attention had been given to the genetic determination of health and disease and the role in health and disease of women, the revelations of genomic science, including ever-expanding studies of the sex chromosomes, have brought about a composite of modern concepts of biomedical research. That is, not just behavioral or biological factors must be considered in defining women’s health and gender-specific medical practices, but the variations of the individual’s genes, environment, and lifestyle must be taken into account for disease prevention and treatment. The Precision Medicine Initiative, implemented in 2015, is an emerging approach for recommendations about prevention and treatment based on the individual’s genes, environments, and lifestyles. The long-standing approach to gender-based studies and our understanding of the importance of factors, including sex, that can influence the penetrance of genetic predisposition for diseases seems to reflect what this new initiative is advancing. Attention to sex and gender differences in etiology, presentation, diagnosis, treatment, or responses to therapy has become a norm of scientific design of research and evidence-based medicine. However, it has been disappointing that the definition of precision medicine does not specifically cite the consideration of the effects of biological sex on gene expression and, therefore, it is essential that this be integrated into recommendations for disease prevention or therapy. It is valuable that such principles are well defined in this textbook. Advances in our knowledge about contributions of each individual’s genetic and chromosomal make-up have furthered our concept of how research must yield data that will be of great import for the clinical care of patients. The ethical and social implications of geneticbased medicine are challenges that are being addressed. We have learned that it is necessary to examine variables of sex and gender across the spectrum of research, from basic molecular and cellular studies to clinical investigation, and ultimately to clinical application—providing personalized, gender-appropriate health care to the individual patient through Gender-Specific Medicine. The first two editions of the Principles of GenderSpecific Medicine6,7 summarized available data about the differences in men and women’s physiology and in their unique experience of the same diseases in a traditional format, i.e., experts in specific clinical areas edited each of the books’ sections and produced an overview of the information we had accumulated in our investigation of gender-specific medicine. The present edition is a novel departure from the traditional format, in that Dr. Legato has structured it around the cutting edge science that

has developed since the description of the structure of the human genome. The 21st century has witnessed a remarkable expansion in our ability to manipulate the genetic information that underlies the phenotype of life forms at all levels, including that of single cells, and has produced information about the molecular mechanisms of normal function and disease. Scientists are now also expanding the marriage of technology to living species including humans, and are implementing research into allied topics like the development of advanced robots. There is increasing interest in the assessment of the ability of men and women to navigate space and eventually colonize extraterrestrial locations. All these topics, which are significantly affected by biological sex, are included in this edition of the book. Fifty authorities have contributed their unique expertise to this edition. Thus, this third edition departs from the conventional textbook approach to diseases, providing a vision of how the expansion of knowledge about sexual dimorphism and sex-based biology in this genomic era characterizes the future for improved health care. All who are scholars, educators, scientists, or are involved in the delivery of, or beneficiaries of, health care will benefit from the clear and comprehensive presentation of the role of gender in the genomic era but also of the challenges and tremendous benefits of the future of gender-specific medicine that this textbook clearly describes.

References 1. National Institutes of Health. Agenda for Research on Women’s Health for the 21st Century, Bethesda, MD: [NIH Publication No. 99-4385, 137 p.], 1999. 2. National Institutes of Health. Moving Into the Future With New Dimensions and Strategies: A Vision for 2020 for Women’s Health, Vol. II, Bethesda, MD: [NIH Publication No. 10-7606-C 415 p.], 2010. 3. Wizemann TM, Pardue M-L, eds. Exploring the Biological Contributions to Human Health: Does Sex Matter?. Washington, DC: Institute of Medicine, National Academy Press; 2001. 276 p. 4. National Institutes of Health. Internet. Consideration of sex as a biological variable in NIH-funded research. 2015. Available from: https:// grants.nih.gov/grants/guide/notice-files/NOT-OD-15-102.html. 5. National Institutes of Health. Internet. Consideration of sex as a biological variable in NIH-funded research. 2015. Available from: https:// grants.nih.gov/grants/guide/notice-files/NOT-OD-15-102.html. 6. Legato MD MJ, ed. Principles of Gender-Specific Medicine. New York, NY: Academy Press/Elsevier; 2004. 1396 p. 7. Legato MD MJ, ed. Principles of Gender-Specific Medicine. 2nd ed. New York, NY: Academy Press/Elsevier; 2010. 800 p.

Vivian W. Pinn Former Associate Director for Research on Women’s Health, NIH (Retired), Founding Director (Retired), NIH Office of Research on Women’s Health

Preface

Every century has produced profoundly important scientific achievements, discoveries, and insights that not only changed the quality of our lives but expanded our understanding of the nature of the universe and the laws that govern it. The last two centuries are no exception: Darwin’s articulation of the process of evolution (1859) explained how we came to be human. The Manhattan Project of the 1940s produced an entirely new energy force that could be harnessed for enormous good, or colossal destruction. This century’s spectacular contribution began with the announcement that we had deciphered the structure of the human genome. The implications of this new knowledge opened the door to powers we had never had before. Understanding the genome meant we could manipulate it and even create entirely new forms of life, generating the new discipline of synthetic biology. Craig Venter explained that if we could describe a chromosome, we could create one. George Church has proposed creating an entire genome from scratch—The Human Genome Writing Project— which he thinks could be a reality by 2020. Thus, the biology of this new century is quintessentially different from the biology of those that have gone before, and we wanted to shape this edition accordingly. The genesis of the science of gender-specific medicine, the detailed study of the differences in male and female physiology and of the unique experience of same diseases that biological sex confers, began about 20 years ago; it became evident as the science progressed that men and women were significantly different in every tissue of the body. A whole cornucopia of discoveries quickly accumulated and we went from the centuriesold observations in medicine that some diseases were more likely to occur in one sex than the other to an everdeepening exploration and delineation of why those differences existed. Many of these new insights were summarized for clinicians in the first two editions of this book. Those editions followed the organizational tradition of soliciting experts in the various subspecialties of medicine to invite their colleagues to summarize the new gender-specific features of human physiology and the experience of disease in their particular disciplines. We planned this new edition as a complete departure from the traditional organization of a clinical text. Instead of recruiting a coterie of experts well versed in the gender-specific aspects of clinical medicine, we

surveyed the literature to invite the most original and innovative thinkers employing cutting edge methodology and insights to address the exciting new frontiers that characterize the age of genomic medicine. The collection of 50 chapters we assembled is not meant to be a comprehensive summary of the latest advances in gender-specific medicine. Rather we have touched on a wide variety of topics that report novel genderspecific information and/or that reflected cutting edge techniques and insights into the molecular basis for differences in males and females. The book begins with nine chapters that deal with variations in the nature of biological sex. We have long maintained that scholars interested in gender-specific science must consider the spectrum of variation in sex. Current new information sets out important principles for the adequate care of individuals who do not fit into the binary classification of humans as male or female, but exist on an extensive spectrum of sexual identity. The next six chapters set out recent insights into brain biology and development. These are followed by a series of discussion by five expert groups about the intricacies of sex-specific intrauterine development. Specific aspects of human physiology and disease states follow: three chapters discuss gender-specific aspects of immunity. The next three set out the genderspecific molecular basis for cardiovascular physiology and pathophysiology. New insights into the mechanisms of chronic pulmonary disease, cancer, infection, and some of the causes for gender-specific mortality in children are the next four topics covered. Two groups discuss the biology of stem cell therapy followed by a series of three chapters on the gender-specific nature of pain and the response to trauma. The latest insights in gender-specific exercise physiology and repetitive strain disorders in musicians are included in the next segments of the book. The sex-specific aspects of nutrition and the gender-specific impact of the microbiome on gastrointestinal function are followed by a chapter on sleep disorders and on some of the gender-specific changes of aging in the renal, musculoskeletal, and circulatory systems. We also wanted to touch on the increasing implementation of technology and engineering achievements on human life, and thus have included discussions of robots, cyborgs, and the gender-specific aspects of preparing

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humans for space travel and ultimately for prolonged stays in extraterrestrial sites. Finally, two chapters end the volume with a discussion of challenges in personalized medicine and of how the pharmaceutical industry has (and in some ways, has not) addressed issues in gender-specific drug development. In summary, this is not a compendium of everything we’ve learned about gender-specific medicine since the

second edition. Rather it is an attempt to open an exciting window on some of the most interesting new developments in 21st century science. Many might wonder about the selection of specific topics and our not touching on others, but this is ultimately a personal, albeit inevitably incomplete, assessment of some of the most interesting features of this unparalleled age of achievement in biomedical and technological investigation. Marianne J. Legato

C H A P T E R

1 Normal and Variant Sex Development Olaf Hiort University of Lübeck, Lübeck, Germany

O U T L I N E 1.2.4 Disorders of Hormone Synthesis and Actions 6 1.2.5 Unclassified Conditions 8

1.1 Physiology of Biological Sex Development 1 1.1.1 Background 1 1.1.2 Chromosomal Sex 2 1.1.3 Gonadal Sex 2 1.1.4 Development of Internal Genital Structures 3 1.1.5 Development of External Genital Structures 3 1.1.6 Extragenital Sexual Dimorphism and Brain Sex 3 1.2 Pathophysiology 3 1.2.1 Disorders or Differences of Sex Development—Nomenclature and Classification 3 1.2.2 Sex Chromosome Abnormalities— Turner Syndrome and Klinefelter Syndrome 5 1.2.3 Abnormalities of Gonadal Development 5

8 8 9 9

1.4 Management of DSD 1.4.1 Centers of Competence and the Interdisciplinary Team Approach 1.4.2 Sex Assignment

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1.5 Therapy 1.5.1 Endocrine Therapy 1.5.2 Surgical Therapy

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1.6 Conclusion and Outlook

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for hormone synthesis, which in turn allows the shaping of the individual phenotype into either male or female. These processes occur in a precise stepwise and timedependent manner and any disruption at sensitive time intervals will allow deviation from the usual pathways. These conditions are described as Disorders or Differences of Sex Development (DSD).1 DSD encompasses mostly rare to very rare genetically determined variants of a discrepancy between chromosomal, gonadal, and phenotypic sex. It is unclear at this time if also external associated, nongenetic factors may disrupt the endocrine pathways and thus lead to DSD. Sex development is not only related to development of genital structures, but also includes sexually-dimorphic development of

1.1  PHYSIOLOGY OF BIOLOGICAL SEX DEVELOPMENT 1.1.1 Background The biological sex of the human being will be defined by the genetic determination of the differentiation of the gonads to develop either into a testis or an ovary. The genetic pathway usually starts with the initial setting of either a 46,XX or a 46,XY karyotype. Subsequently, a tightly regulated cascade of both sex chromosome and autosome derived genetic expression pathways is initiated. From the bipotent gonadal anlagen then ovarian or testicular cells develop, which in due course set the stage Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00014-0

1.3 Diagnostic Procedures 1.3.1 Clinical Approach 1.3.2 Genetic and Laboratory Analysis 1.3.3 Imaging

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1.  Normal and Variant Sex Development

brain structures. Therefore, DSD will affect not only the appearance of primary genital structures and subsequent sexual maturation at puberty, but also gender identity. Furthermore, especially the initial genetic steps of gonadal development may also be involved in the differentiation and function of other organs, such as the adrenal, the skeletal system, or even the neurological system. Therefore, patients with DSD conditions may have associated endocrine illnesses and other system disorders, requiring expert medical attention.

1.1.2  Chromosomal Sex The chromosomal sex of an individual is determined with the fusion of egg and sperm at the time of conception. Usually, one X chromosome is inherited from the mother, while the sperm from the father may provide either an X or a Y chromosome to account for the 46,XX or 46,XY karyotype of the embryo. This genetic layout is seen as a starting point of sex development in the principally “unsexed” child. The chromosomes themselves may harbor sex-specific and sex-unspecific, pseudo-autosomal genes. Sex chromosome division may be altered either prior to conception or during the first cell divisions of the blastocyst, leading to numerical aberrations. Sex chromosome trisomies as 47,XXY or monosomies as 45,X are well recognized, but also higher numbers of sex chromosomes have been described in humans. Mosaicism like 45,X/46,XY are the result of the loss of a sex chromosome during the first cell cycles. Also chimeric alterations like 46,XX/46,XY may occur. It is believed that the presence of Y-chromosomal material in any individual may lead at least to partial testicular development of the gonadal structures and, hence, may also lead to differences in phenotypic development of that individual.

1.1.3  Gonadal Sex For more than 25 years it has been known that testicular determination from the bipotent gonadal anlagen is initiated with the expression of the SRY-gene from the Y-chromosome.2 SRY stands for “Sex Determining Region of the Y-Chromosome.” Subsequently, the consecutive genetic cascade has been elucidated, where SRY in turn leads to upregulation of SOX9 (SRY-related HMG-box gene 9) in the pre-Sertoli cells of the developing testes.3 The physiologic role of SRY and SOX9 has been shown in elaborate animal studies, which demonstrated that in the absence of SRY, mice were able to develop ovaries despite a typical male karyotype, while mice with a typical female karyotype were able to develop testes if SOX9 was overexpressed. In humans, duplication of SOX9 in 46,XX individuals is associated with the development of testes, while deleterious mutations in SOX9 lead to

a failure of gonadal development in 46,XY individuals and also produces associated bone malformations called campomelic dysplasia. Following expression of SRY and SOX9, a battery of other genes is expressed with specific functions (for review see Ref. 4). One example is DHH, called Desert Hedge Hog, which is expressed in the Sertoli cells, secreted, and acts as an initiator of the differentiation of the Leydig cells.5 Other examples include NR5A1, which codes for a protein called SF-1 (Steroidogenic Factor 1), involved both in testicular differentiation as well as the initiation of steroidogenesis in the Leydig cells.6 It also regulates the secretion of Anti-Mullerian Hormone from the Leydig cells and positively regulates SOX9. GATA4 in turn regulates not only several of the genes involved in steroidogenesis, but is also involved in the development of the heart7–9; therefore patients have been identified with DSD and severe heart defects like atrioventricular septal defects. If testicular development is not initiated because of SRY not being expressed in the 46,XX karyotype, ovarian development begins at about the 10th week of gestation. WNT4 (Wingless type MMTV integration side family, member 4) will foster ovarian determination by suppressing Leydig cells. Homozygous mutations in WNT4 may lead to testicular development in genetically female mice, while in contrast humans with 46,XY karyotype and gene duplication of WNT4 develop 46,XY DSD.10 Several further genes have been described that actively pursue ovarian development in an expression levelrelated and therefore dose-dependent manner. A major focus of ongoing research is identifying more genes whose expression is directly involved in gonadal development and to elucidate the interplay between these factors in sex development. It seems that in developmental biology, the sex determining genes are involved in a “battle of the sexes,” in which the sexspecific factors defend their own sex-developing program, while they downregulate the contra-sex side. At this time, testicular development is seen as the more determined part, with ovarian development being more labile11 due to the leading role of SRY in gonadal determination and the decisive activity of androgens. 1.1.3.1  Sex Differentiation and Somatic Sex Sex differentiation refers to the development of the phenotypic features of sex, namely the development of the internal and external male or female structures. This is mostly a hormone driven process, and depends on the presence or absence of either the Anti-Mullerian Hormone or androgenic sex steroids. This finally results in the male or female somatic sex.12 While female somatic sex does not depend on endocrine activity, male sex differentiation has to be seen as the active, hormone-dependent, and irreversible

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modification of the primarily bipotent tissue structures of the internal and external genital anlagen.13 Androgenic steroids such as testosterone will lead to irreversible downstream expression patterns of genes corresponding to induction of the male phenotype in cells of the genital tubercle.14 Thus, these androgenic steroids program the transcriptome and epigenome of the cells in a so-called “male programming window” in the developing embryo.12 Clinically, the effects are seen as the anogenital distance, the development of the scrotum, and the penile differentiation and length.15 However, these effects most likely are also seen in reproductive issues such as sperm count and long-term testosterone levels in the adult male. We have to presume that the androgenization effects are also major players of body composition like muscle–fat ratio, bone mineral density, etc.16

1.1.4  Development of Internal Genital Structures In both sexes the internal genital structures arise again from the bipotent anlagen, the Wolff ducts and the Mullerian ducts. Under the influence of AMH secreted from the Sertoli cells of the testes during the critical period, the Mullerian ducts regress. So in typical male development, the differentiation and growth of the uterus, upper third of the vagina, and fallopian tubes are actively suppressed. In contrast, the Wolff ducts develop under the influence of testosterone, giving rise to the epidydimis, the seminal vesicles, and some parts of the prostate. Again, the male development is dominant and hormone dependent. In some types of DSD with high amounts of testosterone formed in the adrenal, such as 21-hydroxylase deficiency leading to congenital adrenal hyperplasia (CAH), there are residues of prostate tissue even in 46,XX individuals.17

1.1.5  Development of External Genital Structures The external genital anlagen are in a bipotent stage around the 7th week of gestation. If no sex steroids are acting on the tissue, female external structures will arise with a clitoris, labia minora and majora. Under the influence of androgens, namely dihydrotestosterone converted peripherally from testosterone, the phallic structure arising from the genital tubercle will form into a penis with elongation of the urethra to the tip of the phallus. Likewise, the urogenital folds will fuse and form the labia majora, while the labia minora are the equivalent to parts of the shaft of the phallus in the male. This differentiation will be finished by the end of the 12th week of gestation with the end of embryonic

development. After this, high levels of dihydrotestosterone are needed for the growth of the phallus and of the prostate. Interestingly, these differentiation processes can only take place during very defined and strict time intervals, which represent the above described “male programming window.”18 Elaborate animal model experiments demonstrate that the lack of androgens will lead to a female phenotype regardless of karyotype. Androgenization leads to a male phenotype regardless of karyotype.19

1.1.6  Extragenital Sexual Dimorphism and Brain Sex Sex development is not restricted to the genitals. Functional studies in mouse models predict that up to 75% of all transcribed genes are expressed in a sexually dimorphic pattern.20 The brain plays a special role as it is the basis for “psychic sex,” which encompasses gender identity, gender role behavior, and sexual orientation. These very important aspects of gender are modified by biologic factors such as genes and hormones, but undergo modulation through psychological, social, and cultural factors. It has been known for more than 30 years that prenatal androgen exposure produces the sexspecific development of certain brain areas.21 In DSD, we have been able to demonstrate the importance of prenatal androgen exposure on play behavior of boys and girls.22,23

1.2 PATHOPHYSIOLOGY 1.2.1  Disorders or Differences of Sex Development—Nomenclature and Classification Historically, the individual was reared according to the external phenotype. By the end of the 19th century, as a result of increasing knowledge of the developmental aspects and the importance of gonadal differentiation, people with external female genitalia, but testicular tissues such as in complete androgen insensitivity, could be considered male.24 In the middle of the last century, more elaborate views were reflected, especially by Money and coworkers, emphasizing gender development rather than the chromosomal aspects of sexual identity.25 He also emphasized the importance of potential fertility in sex assignment. In his publication, he suggested seven different categories of conditions. Very thorough explanations of the development of these conditions were offered, based on the assumption that gender was undefined at the time of birth, but would develop during childhood. Later, Money and coworkers were condemned for their advice of early corrective surgery and secrecy about the condition to both the

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affected person and the family. In the 1980s, more and more evidence was presented that prenatal hormone actions play a role not only in genital development, but also in the determination of gender identity.26 Both professional and societal awareness lead to increasing dissatisfaction with the then current modes of communication and care. This led to a consensus conference of the main (pediatric) endocrine societies as well as other experts and representatives of support groups in 2005.27 First, the then used nomenclature was abolished and changed.1 The terms used previously such as “hermaphroditism” and “sex reversal” as well as “intersex” were seen as nonmedical terms, which were not helpful for any diagnosis or prognosis for a given individual. A new nomenclature was proposed, based purely on a biomedical assessment. It was believed that this was helpful to keep discrimination at bay and to be useful for the outlining of diagnostic pathways and guidelines for future management. With this aim, the term “Disorders of Sex Development,” abbreviated to DSD, was proposed and it found its way rapidly into the professional community and text books.28 Further examples for choices for words were “androgen insensitivity” instead of “testicular feminization,” as the latter term may be seen as pejorative. Hence, the classification that was proposed was also purely based on biomedical pathways: Three different

categories of DSD were subdivided: Sex Chromosome abnormalities, 46,XX, and 46,XY DSD. In the latter two categories, subgroups of “disorders of gonadal development,” “disorders of hormone synthesis and action,” and “unclassified” were formed (see Table 1.1). This classification still proves very useful, because it allows all known conditions to be placed into one of the subgroups. Furthermore, it has the potential to include newly identified genetically determined DSD conditions, possibly moving then from “unclassified” to one of the other subgroups. However, the term “Disorder” in DSD is subject to a fierce discussion, as it is understandably offensive to some affected people.29 The term is seen as pejorative and discriminating. Therefore, nowadays, DSD is translated often as “Differences in Sex Development.” Many people even speak of “Variations of Sex Development.” In the professional societies, the acronym DSD seems to be firmly entrenched. For specific support groups and affected people, the term “intersex” has been revived as the identification with a specific gender that is not congruent with chromosomal or genital sex. In modern practice it is therefore mandatory to distinguish between medical management and its nomenclature and classification for purposes of diagnosis and treatment, and the sociocultural nomenclature which might differ enormously from group to group and between societies.

TABLE 1.1  Biological Classification of DSD as Agreed Upon in the 2005 Consensus Conference Biological classification of DSD 46,XX

46,XY

Chromosomal DSD

Disorders of Gonadal Development ● Ovotesticular DSD ● Testicular DSD ● syndromic forms

Disorders of Gonadal Development ● ovotesticular DSD ● complete or partial gonadal dysgenesis, monogenic (SRY, NR5A1, WT-1 and others) ● syndromic

45,X and variants ● Turner syndrome ● 45,X/46,XY ● mixed gonadal dysgenesis ● 47,XXY and variants ● Klinefelter syndrome Other forms of complex chromosomal rearrangement

Disorders of Androgen excess ● congenital adrenal hyperplasia ● aromatase deficiency ● luteoma ● iatrogenic

Disorders of androgen synthesis ● syndromic (e.g., Smith-Lemli-Opitz-syndrome) ● associated with CAH (early defects of androgen synthesis) (StAR, P450 scc, 3β-hydroxysteroid-dehydrogenase-Type-2, 17α-hydroxylase/17,20-lyase,P450-oxidoreduktase) ● solely affecting androgen synthesis (17β-hydroxysteroiddehydrogenase-Type-3, 5α-reduktase-Type-2) Disorders of androgen action ● complete and partial androgen insensitivity ● endocrine disruptors

Unclassified disorders ● Mayer-Rokitansky-Küster-Hauser Syndrome ● Complex syndromic disorders

Unclassified disorders ● Hypospadias of unknown genetic origin ● Epispadias ● Complex syndromic disorders

Adapted from Hughes IA, Houk C, Ahmed SF, et al. LWPES consensus group; ESPE consensus group. Consensus statement on management of intersex disorders. Arch Dis Child 2006;91:554–563.

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The new classification is based on karyotype, being either 46,XY, 46,XX, or other. It is mandatory to keep in mind that the karyotype does not in any way reflect sex of rearing or gender identity as stated in the previous paragraphs. For medical diagnostic decision making, the karyotype is of utmost importance to understand the underlying pathophysiology of the specific condition.

1.2.2  Sex Chromosome Abnormalities—Turner Syndrome and Klinefelter Syndrome Sex chromosome monosomies or trisomies are common. Klinefelter syndrome with 47,XXY karyotype has been estimated to occur in 1:500 males,30 while the incidence of 45,X and variant karyotypes occurs in 1:2000–1:5.000 females.31 Apparently, the prenatal incidence may be even higher, but abortive loss may be as high as 90% during the first trimester. Due to increasing use of prenatal diagnostics, many fetuses with chromosomal aberrations may be deliberately aborted, this rate may be falling due to expert counseling during pregnancy. Since some prenatally diagnosed cases cannot be confirmed postnatally, the clinical symptoms have been described as variable and sometimes mild, and a genotype–phenotype correlation does not really exist, any prenatal consultations should be balanced and very comprehensive.32 In 40–60% of the cases, an early loss of the paternal sex chromosome occurs, leading to an unambiguously 45,X karyotype. If the loss occurs later, a chromosomal mosaicism occurs with different cell lines, leading to mixed gonadal dysgenesis. The presence of dosage-dependent functional sex chromosome material will determine the fate of the gonadal development, with 46,XX cell lines leading to ovarian development, while 46,XY cell lines will favor testicular development. Therefore, the phenotypic spectrum of Turner syndrome is very broad from complete loss of gonadal function to ovarian differentiation even with fertility in patients with mosaicism, and complete male phenotype with seemingly unimpaired testicular function. Further associated features of Turner syndrome include renal and cardiac abnormalities, phenotypic features like pterygium colli, lateralized mamillae, cubita valga, and others.32,33 Furthermore, patients with Turner syndrome have an increased risk for autoimmune disease.34 A main feature is short stature, due to haploinsufficiency of the “short-stature homeobox-containing gene on the X-chromosome” or SHOX-gene, located in the pseudo-autosomal region of the X-Chromosome. In Klinefelter syndrome, the aneuploidy occurs due to nondisjunction within in maternal oogenesis or in one third of the cases in paternal spermatogenesis. The testes of patients with Klinefelter syndrome contain primary germ cells, but these undergo rapid degeneration,

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leading to a complete or almost complete loss of germ cells at the time of puberty.30 Therefore patients with Klinefelter syndrome have primarily normal appearing testes, but almost no tubuli with spermatogenesis. The Leydig cells are hyperplastic, but are not able to produce testosterone in the usual amounts and this will lead to progressive testosterone deficiency. A clinical hallmark of Klinefelter syndrome is the hypergonadotropic hypogonadism and small, firm testes in adolescents and adulthood.30 Additional endocrine related features are tall stature due to overdosage of SHOX, gynecomastia due to lack of testosterone, and variable features of underandrogenization. However, although Klinefelter syndrome is the form of DSD with the highest incidence, the diagnosis is missed in many cases due to clinical variability. Based on registries in Denmark, only 25% of all patients with Klinefelter syndrome are diagnosed throughout their lifetime. However, diagnosis is very important, not only for treatment of infertility and underandrogenization, but also because the patients have a highly increased morbidity, with varicosis, thrombosis, type 2 diabetes mellitus, increased fracture risk, and neurologic and psychiatric disorders to be considered. In childhood, the learning disabilities and speech problems often lead to an odyssey of inaccurate diagnoses before the correct diagnosis is made, since the genital features are not apparent prepubertally.

1.2.3  Abnormalities of Gonadal Development In women with 46,XX karyotype, lack of gonadal development will not lead to any obvious clinical features. Both internal and external genital structures have developed female. Most patients come to attention because of lack of pubertal development due to gonadal failure in adolescence and at this time medical investigation will demonstrate hypergonadotropic hypogonadism. Associations with a variety of genes have been made and an exact diagnosis should be sought.35,36 In some very rare cases, testicular development may be triggered in humans with 46,XX karyotype.37,38 This is mainly caused by translocation of the SRY gene to another chromosome, but other downstream genes may also be abnormally regulated to start testicular development. In these cases, often normal appearing internal and external male genitalia are present and testicular endocrine function is normal. However, 46,XX males are usually infertile, as germ cells have degenerated. In the DSD classification, this is called 46,XX testicular DSD. In some cases with 46,XX karyotype, testicular development is partial, leading to an ovotesticular gonadal differentiation.39 In these cases, either one gonad is differentiated as a testis and the other as an ovary, but mostly one side contains both testicular and ovarian tissue, while the other side will be an ovary. Depending

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on the amount of testicular tissue and the endocrine activity, the children may have variable uterine development, often appearing as a hemi-uterus on the ovarian side. The external genitalia may be partially virilized depending on the testosterone levels and activity. This form of DSD is called ovotesticular DSD, and it may present both with 46,XX and 46,XY karyotype, although the latter is much rarer.40 More often, 46,XY gonadal dysgenesis may occur.41 In these patients a broad clinical spectrum has been described, usually named as partial or complete gonadal dysgenesis. In 46,XY complete gonadal dysgenesis, the phenotype is completely female and cannot be distinguished from 46,XX gonadal dysgenesis. The women have a normally formed uterus and the genital anatomy is normal female. Therefore, these patients will come to attention again at adolescence because of lack of pubertal development and hypergonadotropic hypogonadism will be diagnosed. It is important to detect the karyotype only for the possibility of tumor development from the gonadal remnants, and to detect possible associated clinical features. The genetic causes of 46,XY gonadal dysgenesis are manifold, and often not elucidated yet. One reason may be a deletion or deleterious mutation of the SRY-gene. Also SOX9 mutations and mutations in other genes of the testicular development cascade have been described.42 Of importance is the detection of mutations in Wilms-tumor 1 gene, because this gene is also involved in kidney development.43 Patients with 46,XY complete gonadal dysgenesis, also called Frasier syndrome, will develop progressive renal failure and usually require dialysis before the age of 20 years. Also, they have a high risk of developing gonadal tumors, mainly dysgerminomas, at an early age. But also other genetic causes of 46,XY gonadal dysgenesis may have associated diseases. In patients with SOX9 mutations, a bone manifestation called campomelic dysplasia has been described. Desert Hedge Hog mutations are associated with a sensory polyneuropathy etc.44 It is therefore important for the patient to elucidate the genetic basis of 46,XY gonadal dysgenesis. Partial 46,XY gonadal dysgenesis is a main differential diagnosis in DSD.40,41 In these children, partial androgenization of the external genitalia with high variability is apparent. Furthermore, the evaluation of the internal genitalia might reveal a variable development of uterus and fallopian tubes. This is due to the diminished secretion of AMH from the partially dysgenetic testes. However, within the broad spectrum of patients with partial gonadal dysgenesis, some patients have Mullerian structures which are not apparent macroscopically, but can be seen on histological evaluation. The phenotypic spectrum is so variable that some children are raised unequivocally as females, while others,

even with mutations in the same gene, may be assigned to a male sex. Others may be frankly ambiguous. The genetic causes are as wide spread as in complete gonadal dysgenesis. More recently, quite a number of patients have been described with mutations in NR5A1 leading to SF-1 deficiency.45 A genotype-phenotype correlation does not exist. Rarely, additional adrenal insufficiency is described.46 Other genes are associated with gonadal dysgenesis and are also involved in the genesis of other organs.

1.2.4  Disorders of Hormone Synthesis and Actions In people with a normal structural and numerical normal chromosomal set, the second large group of DSD are those with disorders of hormonal synthesis and action.47 In 46,XX DSD, this leads to hyperandrogenism, mostly as a consequence of bypassing enzymatic steps involved in the adrenal gluco- and mineralocorticoid synthesis.48 These conditions are called CAH. The most common form of CAH is 21-hydroxylase deficiency due to mutations in the CYP21A2-gene, which leads to diminished synthesis of cortisol and in a subset  also of aldosterone and consequently to increased adrenal testosterone synthesis via dehydroepiandosterone.49 Both 46,XX and 46,XY children can be affected and 21-hydroxylase deficiency is—as most disorders of hormone synthesis— inherited in an autosomal recessive fashion48 (Fig. 1.1). Children with 21-hydroxylase deficiency may present with acute adrenal insufficiency and salt loosing crisis (Addison’s disease) shortly after birth, a potentially lifethreatening condition. Therefore, in several Western countries, 21-hydroxylase deficiency has been included in the newborn screening. This has provided us with very good information about the overall incidence, which is usually around 1:10.000.50 The effects of the prenatal and postnatal hyperandrogenism are different between 46,XX and 46,XY children with this condition. While 46,XX children may have obvious signs of virilization at birth, ranging from slight clitoromegaly to an unequivocally male appearing external genitalia, the 46,XY children are usually not clinically apparent at birth, except perhaps for darkening of the genital skin. However, if 21-hydroxylase deficiency remains undetected because Addison’s crisis does not occur, all affected children will have acceleration of maturation with early puberty and subsequent short stature. By definition, only 46,XX children with 21-hydroxylase deficiency are included in the DSD classification because of the effects on the external genitalia.27 Within the cascade of adrenal steroid synthesis a variety of enzymatic defects other than 21-hydroxylase deficiency have been described leading to CAH.51–53 Depending on the position of the enzyme and the effects on testosterone synthesis through the gonadal and/or

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FIGURE 1.1  Graphic design of steps of steroidogenesis. The gray boxes depict different enzymes, while the yellow circles stand for coenzymes and blue boxes represent transport proteins. Reproduced with permission from Steroid Biochemistry: Kamrath C, Wudy SA, Krone N. In: Hiort O, Ahmed SF, eds. Understanding Differences and Disorders of Sex Development (DSD). Endocr Dev. Basel: Karger 2014; 27: 41–52 (DOI:10.1159/ 000363612).

adrenal pathways, either 46,XX or 46,XY children may have a DSD condition. Examples for 46,XY DSD due to CAH are the P450scc and StAR deficiencies.54,55 P450 side chain cleavage (scc) enzyme regulates the first step of steroid synthesis from cholesterol to pregnenolone. Hence, children with a blockade of this enzyme will not be able to synthesize any steroid hormones, including sex steroids. Affected 46,XY children will therefore not be able to synthesize testosterone and therefore the external genitalia will not undergo male differentiation but depending on the severity of enzyme deficiency will have either female or ambiguous genitalia. StAR stands for Steroidogenic Acute Regulatory Protein and describes a transporter involved in the shuttling of cholesterol from the outer to the inner mitochondrial membrane. Patients with StAR deficiency will accumulate cholesterol in their mitochondria in the adrenals and will have a lipoid adrenal hyperplasia leading to an enlarged

organ.55 The clinical appearance and spectrum is similar to P450scc deficiency. Further enzymatic disorders have been described to lead to CAH in selected cases, and most of them are not detected in the newborn screening. In 46,XY children, the spectrum of DSD due to disorders of hormone synthesis and action is much broader than in 46,XX children. While defects of early steroid hormone synthesis are usually associated with one of the above described rare CAH conditions, late defects of steroid hormone synthesis may also occur and affect only the testosterone or dihydrotestosterone synthesis. The 17-beta-hydroxysteroid dehydrogenase type 3 is located mainly in the Leydig cells of the testes, and mutations affecting this enzyme have been described in several patient cohorts.56 Affected 46,XY children may have almost female appearing or ambiguous genitalia at birth. The same holds true for 46,XY children with 5 alpha-reductase type 2 deficiency, where the peripheral

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dihydrotestosterone synthesis is insufficient.57 Both conditions are also inherited in an autosomal-recessive fashion and therefore the incidence is higher in countries where consanguinity is prevalent. Interestingly, in both conditions, affected 46,XX people have no clinical symptoms and are fertile.58 Androgen action is facilitated through a single androgen receptor. The responsible gene is located on the X-chromosome.59 Therefore, this is a single-copy gene in 46,XY individuals and mutations inherited in a hemizygous fashion from unaffected, fertile 46,XX mothers will have a deleterious effect. The 46,XY androgen insensitivity due to mutation of the androgen receptor is one of the most often described forms of DSD.60 The phenotypic spectrum is very broad, ranging from a completely female phenotype to males with in- or subfertility. The androgen receptor acts as a transcription factor facilitating androgen action in every cell of the body.61,62 Therefore, affected 46,XY people will have secondary effects like lack of sexual hair and very little acne at the time of puberty. Since testosterone will be aromatized to estrogens, persons with androgen insensitivity will develop gynecomastia at puberty.63 This explains why in the past this condition was called testicular feminization, because in contrast to the hormone synthesis disorders, 46,XY females with androgen insensitivity have a female pubertal development. Recently, it was demonstrated that the hormone levels are very peculiar in androgen insensitivity, mostly with elevated LH and testosterone compared to females, but estradiol levels are more in the male reference range. This demonstrates that it may be the lack of testosterone action that promotes breast development rather than the level of estrogens.63 Mutations in the androgen receptor gene have been associated with other conditions, namely a neurologic disorder called spinal and bulbar muscular atrophy (Kennedy’s disease), a late-onset neurodegenerative disorder affecting only men.64 Also, prostate cancers may be associated with mutations in the androgen receptor,65 as well as certain breast cancers.66 It is of note that 46,XX patients with 21 hydroxylase deficiency will have a normally formed uterus. In contrast, in the 46,XY hormone synthesis disorders as well as in androgen insensitivity, a uterus is not found due to the uninhibited secretion of AMH from the testes.67 However, there is a group of 46,XY patients with normal male external genitalia, but internal formation of Mullerian structures. These children are usually detected because of undescended testes. It is then found that they have a uterus and the gonads are usually associated with the fallopian tubes. The cause maybe either an inhibited AMH secretion, usually due to mutation of the AMH gene itself, or the AMH type II receptor may not be acting appropriately.68 This can also be proved by molecular genetic analysis and detection of deleterious mutations.

1.2.5  Unclassified Conditions The pathophysiology and underlying genetic basis of many clinical phenotypes cannot be explained.68 Examples are the bladder exstrophies, conditions with abdominal wall defects leading to severe malformations of the genital area. Usually, the gonads are intact and functioning, so that an endocrine condition can be excluded.27 Furthermore, malformations of the uterus and vagina, often in association with renal abnormalities, have been described in 46,XX women with normal ovarian function and have been named as Mayer-Rokitansky-KüsterHauser syndrome.27 The most often occurring conditions are hypospadias and undescended testes in males with a very broad clinical spectrum. Maldescended testis occurs in about 3% of all boys, with even higher frequency in premature children. Simple hypospadias as mild to moderate defects of urethra formation affect about 1 in 300 boys. It is debatable if these conditions fulfill the definition of DSD, but if complex conditions, for instance severe hypospadias with undescended testes, are present, a complete DSD diagnostic analysis is warranted.69

1.3  DIAGNOSTIC PROCEDURES Due to the broad phenotypic variability as well as the variety of underlying conditions, patients with DSD may come to medical attention at very different times.70 Ambiguous genitalia may be obvious at birth and warrant immediate investigations, but otherwise children may be seen for other issues (for instance because of inguinal hernia in which testicular tissue is detected) in a child so far raised as a female. Or males come to attention due to premature pubertal development and CAH is detected. Klinefelter syndrome, Turner syndrome, but also many forms of gonadal dysgenesis or even disorders of androgen biosynthesis and action are not detected until adolescence, when adverse or lack of pubertal development leads to medical attention. Because DSD is mainly a genetic condition, a very thorough family history, optimally including at least three generations, should be taken.71 Inquiries should be made not only about genital peculiarities but also about infertility or complex malformations. Moreover, the clinician should ask about ingestion of medicines or toxic substances during pregnancy with the propositus. It is known that “small for gestational” age children have a higher risk for genital malformations. Also very small premature girls may have clitoromegaly, but the cause is mostly unknown. In any case, a DSD should be considered.

1.3.1  Clinical Approach Any clinical investigation of a patient with suspected DSD must be complete and very thorough; in particular,

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it should take into account associated malformations.72 The genital inspection needs an understanding and empathetic approach. Children and adolescents need their privacy and their consent to any examination must be sought. Every painful investigation should be avoided and anesthesia should be considered if necessary. Also the number of investigators must be limited to a minimum. On the other hand, documentation needs to be discussed and is currently under debate, because genital photography is usually banned.69 In the description of the genital findings, objective data should be obtained. This includes phallic length and circumference, measurement of the anogenital distance, which has proven to be a good standard to assess androgenization of the genitalia.73,74 The rugation of the labioscrotal folds needs to be assessed, as well as the location of the introitus vaginae in the female genital status. Also the pubertal stage needs to be described in adolescence. This includes secondary hair distribution as well as breast development, which should include the exact size of the breast tissue as well as the Tanner stage of development. It becomes clear that this investigation needs an experienced professional.

1.3.2  Genetic and Laboratory Analysis Any analysis of a DSD will depend on the history and the clinical findings. However, in most instances it is useful to perform a chromosomal analysis to define the resulting optimal diagnostic pathway as well as to detect any numerical or structural chromosomal abnormality.27,70,71 A hormone analysis should be targeted and have a defined diagnostic hypothesis.75 In children with ambiguous genitalia at birth in whom a uterus is seen on ultrasound imaging, the most important differential diagnosis will include CAH due to 21-hydroxylase deficiency, and therefore determination of the 17-hydroxy progesterone is mandatory. In general, depending on the clinical findings, both adrenal and gonadal status should be assessed. In 46,XY children with DSD, this includes both analysis of the sex steroid pathway as well as Sertoli cell markers. The latter are AMH and Inhibin B, which have been found useful in discriminating between conditions of gonadal determination and those affecting hormone synthesis and action. The Sertoli cell markers are usually low in 46,XY patients with gonadal dysgenesis and normal to elevated in conditions of hormone synthesis and action.68 The only untargeted investigation is the determination of the urinary steroid excretion profile, which can be helpful in assessing the whole adrenal steroid spectrum for detection of almost all forms of CAH.76 Of course, the approach in diagnostics also depends on the age of the propositus. In the infant, basal hormone determinations are usually useful, because these children experience a “minipuberty” with endogenous

9

stimulations of the hormone synthesis pathways. In the toddler and prepubertal child, the basal hormone measurements are usually undiscriminatory.77 In this age group, stimulation tests for both adrenal and gonadal pathways must be considered. During adolescence or in the adult, basal determinations may suffice. For many of the hormone synthesis disorders, ratios of the analytes before and after the enzymatic block have been described as diagnostic, however, these cannot be taken as real reference values and need genetic confirmation.52,70 Nowadays, primary molecular genetic analysis as a diagnostic feature has also been established as a first step before gene analysis is commenced.78,79 Also, as in many other rare conditions, genetic analysis might reveal novel mutations and/or novel genes, therefore a proof of principle has to be established employing sophisticated cell or even animal models. Thus a hormone analysis with a suspicious finding may lead to a targeted gene analysis with a verification process.70 Furthermore, this approach may be also much faster and cost-effective. In the near future, modern next-generation sequencing procedures may be available; however, the principles of proof and verification still need to be followed.80 Therefore, Fig. 1.2 outlines a method of linking clinical, biochemical, and genetic diagnostic procedures in a rational way. All final decisions about diagnostic steps should be made in a center for DSD that will also use the diagnosis for management suggestions.

1.3.3 Imaging The diagnostic workup has to include an analysis of the gonads and internal genital structures.81 Modern imaging techniques can be quite useful. Ultrasound is usually used as the first-line modality in all age groups, because it is noninvasive, widely available, and quick. It can answer readily in most cases if a uterus is present and usually the presence, size, and location of intraabdominal gonads. If these cannot be visualized, magnetic resonance imaging is the next often used procedure, especially to visualize sex duct anatomy and details of the prostate and pelvic muscle anatomy. While the overall resolution is better, the use of MRI in childhood is debatable, because children often need anesthesia and images are hampered because of movement and breathing artefacts. Finally, laparoscopy may be the ultimate strategy to describe the internal anatomy that adds information about gonadal anatomy and as it is an invasive surgical procedure, it can also be used to obtain a gonadal histology.81,82 This may be needed in cases for primary diagnostic purposes, but also for the evaluation of possible tumor formation in the dysgenetic gonad. Often laparoscopy is accompanied by a cystoscopy to get an exact measurement of vaginal– urethral confluence in order to counsel the patient about the possibility of future surgical correction.

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1.  Normal and Variant Sex Development

Initial investigation (History, family history, clinical assessment, ultrasound of internal structures)

Karyotype

Inner genitalia

46,XY

Müller structures

Müller structures

Yes

Clinical diagnosis

45,X/46,XY

46,XX

No

No

Yes

Presumed CAH

Testicular DSD

Gonadal dysgenesis

Presumed defect of androgen synthesis or action

Laboratory

17-OHP, adrenal steroidprofile

LH, FSH, inhibin B, androgens

LH, FSH, Inhibin B, Androgens

LH, FSH, A, T, DHT hCG-stimulation and steroidprofiling

Genetics

Targeted gene analysis

FISH for SRY

CGH array

Targeted gene analysis

Diagnostic verification

Mixed gonadal dysgenesis

Targeted next generation sequencing (Panel, MLPA etc.) Broad next generation sequencing on scientific basis (WES, WGS), proof of concept

FIGURE 1.2  Evaluation of DSD by clinical, laboratory, and genetic investigations. Diagnostic procedures should be done in a stepwise fashion. Reproduced from Hiort O, Birnbaum W, Marshall L, et al. Management of disorders of sex development. Nat Endocr Rev 2014;10(9):520–529, permission granted by authorship.

1.4  MANAGEMENT OF DSD The management of people with DSD and their families is very challenging due to diagnostic dilemmas and the ethical questions that arise in establishing a precise prognosis for the affected individual.27,70,71 This dilemma is especially difficult in childhood, because children are not able to consent to any procedures and parental anxieties play a major role. This has to be taken into account in any decision-making process and also in the communication with the families.

1.4.1  Centers of Competence and the Interdisciplinary Team Approach Recently concepts about offering different treatment choices and options to patients have changed.83,84 There is increasing emphasis on children’s rights and parents’ responsibilities. In DSDs, every approach has to consider that there are both biological and social aspects of the condition.29 Since the end of the 20th century, the

public discussions on the dissatisfaction with treatment of people with intersex conditions has at first led to the new consensus on nomenclature and classification, but more and more also to a novel concept of management in centers of competence offering an array of specialties to address both the medical and psychological needs of patients with DSD.85 A very special management element is peer counseling, which has been proposed recently to add to the professional team in centers of competence. A peer in this regard is an affected person, who has achieved a view of his/her own personal history formed as a result of professional counseling he/she and his/her family have received. This is different from usual peer support, which is outside of the medical team.86 The difficulty in counseling parents of a child with DSD is to have them accept that the condition is not a primarily genital anomaly which can be “fixed” surgically, but that the life of the child may eventually differ considerably from the parents’ expectations.87 This requires time, information, and understanding. Even the German Ethical Committee and the German Chamber

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1.5 Therapy

of Physicians recently published statements that people with DSD need to be evaluated and managed in centers of competence, because in the past medical management was seen as devastating to many.88,89 However, the actual optimal structure of the centers of competence remains unclear at this time. Moreover, neither referral mechanisms nor reimbursement procedures have been defined yet.70 In Europe, special efforts are made to create national plans for centers of excellence for rare diseases and to include DSD centers among them. Very recently, European Reference Networks (ERN) for Rare Diseases have been called for and DSD specialists will be included in the ERN for rare endocrinopathies.90 The visibility of such centers might improve DSD care in the future and lead to better reflection on how to improve the overall management.

1.4.2  Sex Assignment Sex assignment in children with DSD has been a very controversial issue both in the past and at the present time.88,91–93 Female sex assignment is usually based on the existence of external female genitals in people with complete gonadal dysgenesis and complete androgen insensitivity, as it is generally felt that these 46,XY individuals mainly have a female gender identity. However, for some specific genetically determined conditions, recommendations can be made on the basis of the diagnosis rather than genital phenotype, reflecting the understanding how an individual with this diagnosis will develop at the time of puberty, but also based on possibilities of future fertility.94 This holds true for 46,XX androgenized individuals with 21-hydroxylase deficiency, where the internal genitalia are female and ovaries are functional.48 These children are most often raised as females, although gender role behavior may be altered. Only children with 46,XX CAH who are completely virilized have been reported to stay in their initially assigned male sex and to opt for removal of ovaries and uterus later in life.48 In contrast, 46,XY children with 5-alpha-reductase type 2 deficiency 57 often have female appearing genitalia with only slight virilization postnatally. However, at the time of puberty, marked androgenization occurs and a higher proportion of affected individuals have changed gender at this time to live as males. In rare instances, fertility may be reported. Therefore, nowadays often a male sex assignment is favored even for those children with 5-alpha-reductase type 2 deficiency, who have more female appearing external genital status.70 At this time, no straightforward recommendation exists for sex assignment in neonates with DSD. In some countries like Germany, a new personal status law was instituted, which allows for these children not to have a sex assigned.88,89 While this should relieve parents and

professionals from making premature decisions about the child and wait for the child’s own development for sexual behavior and gender role behavior, most parents opt to assign a social sex to their child in order to protect and maintain the integrity of the family. However, these recommendations are constantly challenged currently and might change in the near future.

1.5 THERAPY Generally, both medical and surgical therapeutic regimens have to be considered in DSD. Medications might encompass treatment of adrenal insufficiency with glucocorticoids and mineralocorticoids in CAH and therapy with sex steroids for induction and maintenance of puberty in forms of DSD with gonadal insufficiency. Surgery is mostly done for reconstruction of the genitalia, but more and more surgical disciplines have a role in guiding preventive aspects like gonadal tumor prevention in 46,XY DSD or in diagnostic procedures for description of exact anatomy employing laparoscopy and cystoscopy.

1.5.1  Endocrine Therapy In CAH, generally therapy with glucocorticoids is started immediately after diagnosis. In the newborn, severe Addison’s crisis may otherwise occur.95 Often, also mineralocorticoid therapy is necessary. The dosage and also the substitution regimen and medication itself may be variable. In classic 21-hydroxylase deficiency, hydrocortisone is given in a dose of 15–20  mg/m², while in other forms of CAH the substitution dose is lower.95 This is due to the fact that in 21-hydroxylase deficiency not only adrenal hormone replacement is necessary but also the suppression of excess androgenic adrenal steroids has to be achieved. For small children, dosage is very difficult, because first the very low dose preparations are not on the regular market and second they are usually not licensed for children under the age of 6 years. Furthermore, the dosage is very difficult to achieve in a manner to reflect the daytime-dependent secretion of the endogenous cortisol. One way to circumvent these practical difficulties would be to use a device like an insulin-pump to deliver the hydrocortisone continuously. Or, special capsules could be used that release the hydrocortisone in a very specific manner. Administration of the mineralocorticoid fludrocortisone is usually not that difficult, but the market availability of this drug was recently challenged, giving rise to insecurity in the patient cohort. A very special type of therapy is the concept of administering potent glucocorticoids to a pregnant mother with a child with suspected severe 21-hydroxylase

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1.  Normal and Variant Sex Development

deficiency to prevent unwanted androgenization of female fetuses.96 While case reports have described the effectiveness of such a therapy, it is regarded as highly experimental, because of the possible side-effects on both the mothers as well as the children. Mothers may experience hypertension, massive weight gain, and secondary effects like striae distensae. Only one out of eight children of two heterozygous mutation carriers will be an affected female. Therefore, seven out of eight children will be treated unnecessarily. This therapy is only warranted in couples with confirmed molecular genetic defects in the CYP21A2 gene. Early confirmation of mutation status in the child can be achieved through chorionic villous biopsy sample analysis. Nowadays screening for the chromosomal sex of the child can even be done earlier by investigating maternal blood DNA.97 Children have been reported to suffer from neurologic deficits after maternal glucocorticoid treatment. Sex hormone therapy is mostly warranted for induction of puberty in patients with gonadal insufficiency and DSD. Treatment in infancy, e.g., to increase phallic size, however, is debatable, because it is regarded as an irreversible procedure as is surgery, and in line with the current discussion, such procedures should be done only with the consent of the patient (which cannot be obtained from a small child). Hormone therapy at the time of puberty is usually adapted from regimen used for hormone replacement in other conditions with gonadal insufficiency. The medication should be dosed in a manner to allow a normal time frame of puberty, to establish and maintain secondary sexual characteristics in adulthood and to achieve adult height and body proportions, as well as to optimize bone health and to allow a satisfactory sexual life and well-being.98 It has to be noted that at this time no clinical randomized trials on sex steroid replacement or therapy have been reported in patients with DSD. Only one trial is registered, which is a crossover, double-dummy trial in 46,XY women with complete androgen insensitivity using testosterone or estradiol.99 In order to follow the full-consent policy, every child starting with hormone therapy should be fully informed about the nature of the underlying condition and the effects of the medication.70 As the adolescents are then confronted with the nature of their condition, they should be offered psychological support during the disclosure and the intervention. A reassessment of the sex assignment and gender identity may be necessary. A special situation arises if the adolescent is confronted with an opposite-sex pubertal development, e.g., androgenization in 46,XY 17-beta-hydroxysteroid dehydrogenase deficiency raised as female. In such cases an additional therapy with GnRH-analogs may be warranted to suppress endogenous pubertal development.98 For induction of male puberty, usually either topic testosterone gels or intramuscular application of testosterone

derivates is used.98 The gel preparations can have advantages due to following the physiological pattern of natural testosterone values. The intramuscular injection of testosterone enanthate often leads to highly supraphysiological levels after administration which slowly taper off during the following days. Injectable testosterone undecanoate may induce more stable serum levels of testosterone and mimic the physiology, but its use for pubertal induction has not been investigated yet. Oral preparations of testosterone do not reach the hormone serum levels needed for a full replacement. Dihydrotestosteron (DHT) is also available as a gel preparation, which can be used topically especially in male patients where aromatization to estrogens is unwanted. DHT could potentially be useful for male patients with androgen insensitivity. However, our own experience demonstrates that DHT gels cannot prevent the gynecomastia in these boys, which may be marked and persistent. Estrogen formulations are used for induction of female puberty. The primary estrogen is 17-beta-estradiol, which stems from ovarian testosterone precursors, while estrone is the weaker estrogen, which may have estradiol or androstendione as a source. Both oral and topic estrogens are usually used for both starting puberty as well as maintenance therapy.98,100 We rarely use ethinylestradiol, a compound often contained in contraceptive pills, for treatment of hypogonadism. All medications should be used in low doses in order to avoid rapid bone maturation and resulting stunted stature. Gestagens are added in those patients who have a uterus in order to induce regular menses. In those people without uterus, often continuous estrogen therapy is recommended. Side effects in patients with DSD are rare and might include the general risks of increased cardiovascular events, breast cancer, and thromboembolic disease. This has to be weighed against the positive effects on body contour, bone health, and general and sexual health related quality of life.101 Recently, some advocacy groups support hormone replacement therapy that is congruent with chromosomal sex. However, with knowledge of the different DSD conditions it becomes clear that any testosterone therapy in those assigned female will have virilization effects while estrogen therapy will have feminizing effects on males. The only exceptions are 46,XY women with CAIS, who endogenously have high testosterone levels without any effects due to mutation of the androgen receptor.63 A trial has been planned (see Ref. 99) to investigate whether women with CAIS will profit more from high dose testosterone replacement than the usually recommended estrogen therapy.

1.5.2  Surgical Therapy Surgical procedures in people with DSD have been condemned in recent years. While the reports of now

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1.6  Conclusion and Outlook

adult patients who underwent surgery are really devastating in many instances,102–104 one has to take into account that the approach toward DSD has changed since then. Not only the means of communication and consent have changed, but also the technical approaches needed in surgery as well as the understanding of the needs of patients have changed.105 Moreover the scope of surgery has been reformed, in that more diagnostic procedures as well as reconstruction for physiology rather than anatomic sex adherence are performed.81 Overall, it is generally agreed that all nonemergency surgery should be done at a time that the understanding and consent of the child can be obtained. In feminizing genitoplasty a rethinking of criteria has taken place.106 The operation may include separation of the urogenital sinus, creation of a functionally wide enough vagina, the remodeling of the labioscrotal folds, and in some instances also reduction clitoroplasty. These measures are most often encountered in androgenized patients with CAH.107 Any evidence regarding the choice of early or late surgery is lacking today, so it would be most important to create prospective studies on the success as rated by the patients themselves to any procedure. Also, whether one- or two-stage procedures are better remains to be assessed. However, it is most evident that adolescents with DSD may require a reconstructive surgery in order to have vaginal intercourse and also to prepare for possible vaginal delivery in cases of androgenized 46,XX CAH. Any decision on timing of surgery has to be made individually at this time. This holds true even more for 46,XY DSD patients with female sex of rearing. Late operations are favored so that the gender identity of the patient is very clear at surgery.106 In patients staying with male sex assignment, most often procedures like orchidopexy and hypospadias repair are performed.105 A major problem can be a micropenis and the wish of the patient to regain adequate penile length. Phalloplasty may be a possibility to achieve a satisfactory genital appearance and sexual function.108 The procedure has gained acceptance in transgender surgery. However, any assessment of patients’ expectations and experiences is so far lacking. Also, the repair of severe hypospadias poses a problem, as many surgeons still favor an early approach between 6–18 months for such a procedure. Again, postponement of the intervention until the age when informed consent can be obtained must be discussed. Any surgical procedure in a child with a possibility of a DSD should be discussed thoroughly by a multidisciplinary team in a center of competence for DSD.85 This will meet expectations to discuss necessity and timing of surgery from different points of view, and it will also allow for the necessary diagnostic workup prior to any procedures. One major task of surgeons in DSD is the assessment of the gonads.109 Especially in 46,XY DSD, an increased risk of germ cell cancers exists. Since there are no reliable

13

screening tools for these tumors, management of germ cell cancer risk requires a constant discussion on if and when to perform a biopsy of the gonad or even prophylactic gonadectomy. The risk for germ cell cancers is elevated only in patients with Y-chromosomal material in the gonads. Gonadoblastoma has been reported even in very young children with gonadal dysgenesis and either a 46,XY karyotype or a 45,X/46,XY mosaicism (mixed gonadal dysgenesis).85 Since in the past, early gonadectomy was favored for many forms of DSD, data on gonadal tumor risk in adults are mostly lacking. Therefore, for each patient an individual risk assessment should be done based on genetic diagnosis, clinical findings, especially with respect to cryptorchidism, and general tumor risks in the family.109

1.6  CONCLUSION AND OUTLOOK Since the beginning of the 21st century, both the clinical and the scientific approach to DSD have changed considerably. Sex development is seen in a more holistic manner than just the appearance of the genital organs. A better understanding of the genetics and the physiologic pathways has given new insights into the biology. Nevertheless, patients with DSD have taught us to accept that treatment does not consist merely of medical procedures, but that we must make sure that those interventions produce a significant gain in the quality of their lives. DSD belong to the rare conditions, a spectrum most likely encompassing more than 8000 different entities. The rare diseases have gained considerable attention recently on both sides of the Atlantic. In Europe, the establishment of national plans for the creation of Centers of Excellence with certain structural elements like coordinator positions, case conferences, etc. will also help management of DSD in the near future.110 With the formation of ERN for Rare Diseases, international collaboration will be enhanced and the standards of care will hopefully find a uniform standard. This in turn will promote research which further advances the understanding of rare conditions like DSD. For DSD, several networking activities have already been established.110 These include national and international surveys of past treatments,111 which have led to changes in the perception of patients’ views. Networking activities of basic scientists with ethicists are fostering improved world-wide understanding of DSD and variant sex development.112 An international database to include clinical and genetic data was formed with highest standards of data protection and this I-DSD registry is among the few examples of successful international collaborative efforts.113 All these activities provide the basis for important additional studies which can be the catalyst for progress in the future for better care and support for families with DSD.

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References 1. Vilain E, Achermann JC, Eugster EA, et al. We used to call them hermaphrodites. Genet Med. 2007;9:65–66. 2. Sinclair AH, Berta P, Palmer MS, et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature. 1990;346:240–244. 3. Foster JW, Dominguez-Steglich MA, Guioli S, et al. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature. 1994;372:525–530. 4. Lucas-Herald AK, Bashamboo A. Gonadal development. Endocr Dev. 2014;27:1–16. 5. Clark AM, Garland KK, Russell LD. Desert hedgehog (Dhh) gene is required in the mouse testis for formation of adult-type Leydig cells and normal development of peritubular cells and seminiferous tubules. Biol Reprod. 2000;63:1825–1838. 6. El-Khairi R, Achermann JC. Steroidogenic factor-1 and human disease. Semin Reprod Med. 2012;30:374–381. 7. Bouma GJ, Washburn LL, Albrecht KH, Eicher EM. Correct dosage of Fog2 and Gata4 transcription factors is critical for fetal testis development in mice. Proc Natl Acad Sci U S A. 2007;104:14994–14999. 8. Lourenço D, Brauner R, Rybczynska M, Nihoul- Fékété C, McElreavey K, Bashamboo A. Loss-of-function mutation in GATA4 causes anomalies of human testicular development. Proc Natl Acad Sci U S A. 2011;108:1597–1602. 9. Miyamoto Y, Taniguchi H, Hamel F, Silversides DW, Viger RS. A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation. BMC Mol Biol. 2008;9:44. 10. Vainio S, Heikkilä M, Kispert A, et  al. Female development in mammals is regulated by Wnt-4 signalling. Nature. 1999;397:405–409. 11. Svingen T, Koopman P. Building the mammalian testis: origins, differentiation, and assembly of the component cell populations. Genes Dev. 2013;27:2409–2426. 12. Welsh M, Suzuki H, Yamada G. The masculinization programming window. Endocr Dev. 2014;27:17–27. 13. Holterhus PM, Deppe U, Werner R, et  al. Intrinsic androgendependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome. BMC Genomics. 2007;8:376. 14. Hiort O. The differential role of androgens in early human sex development. BMC Med. 2013;11:152. http://dx.doi.org/ 10.1186/1741-7015-11-152. 15. Thankamony A, Lek N, Carroll D, et  al. Anogenital distance and penile length in infants with hypospadias or cryptorchidism: comparison with normative data. Environ Health Perspect. 2014;122:207–211. 16. Hiort O, Reinecke S, Thyen U, et  al. Aspects of puberty in disorders of somatosexual differentiation. J PediatrEndocMet. 2003;16:297–306. 17. Flück CE, Meyer-Böni M, Pandey AV, et  al. Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation. Am J Hum Genet. 2011;89(2):201–218. . Epub 2011 Jul 28. Erratum in: Am J Hum Genet. 2011 Aug 12;89(2):347. 18 Ammerpohl O, Bens S, Appari M, et  al. Androgen receptor function links human sexual dimorphism to DNA methylation. PLoS One. 2013;8:e73288. 19. Miyagawa S, Satoh Y, Haraguchi R, et al. Genetic interactions of the androgen and Wnt/beta-catenin pathways for the masculinization of external genitalia. Mol Endocrinol. 2009;23:871–880.

20. Dewing P, Shi T, Horvath S, et  al. Sexually dimorphic gene expression in mouse brain precedes gonadal differentiation. Brain Res Mol Brain Res. 2003;118:82–90. 21. Döhler KD, Coquelin A, Davis F, et  al. Differentiation of the sexually dimorphic nucleus in the preoptic area of the rat brain is determined by the perinatal hormone environment. Neurosci Lett. 1982;33:295–298. 22. Hines M. Sex steroids and human behavior: prenatal androgen exposure and sex-typical play behavior in children. Ann N Y Acad Sci. 2003;1007:272–282. 23. Jürgensen M, Hiort O, Holterhus PM, Thyen U. Gender role behavior in children with XY karyotype and disorders of sex development. Horm Behav. 2007;51:443–453. 24. Klebs E. Handbuch der Pathologischen Anatomie. Berlin: August Hirschwald; 1876. 1. Band, Zweite Abteilung, p. 718. 25. Money J, Hampson JG, Hampson JL. Hermaphroditism: recommendations concerning assignment of sex, change of sex, and psychologic management. Bull Johns Hopkins Hospit. 1955;97:284–300. 26. Diamond M, Beh HG. Changes in the management of children with intersex conditions. Nat Clin Pract Endocrinol Metab. 2008;4(1):4–5. 27. Hughes IA, Houk C, Ahmed SF, et al. LWPES consensus group; ESPE consensus group. Consensus statement on management of intersex disorders. Arch Dis Child. 2006;91:554–563. 28. Pasterski V, Prentice P, Hughes IA. Consequences of the Chicago consensus on disorders of sex development (DSD): current practices in Europe. Arch Dis Child. 2010;95:618–623. 29. Wiesemann C, Ude-Koeller S, Sinnecker GH, Thyen U. Ethical principles and recommendations for the medical management of differences of sex development (DSD)/intersex in children and adolescents. Eur J Pediatr. 2010;169(6):671–679. 30. Nieschlag E. Klinefelter-Syndrom. Dtsch Arztebl. 2013;110(20): 347–353. 31. Gravholt CH. Epidemiological, endocrine and metabolic features in Turner syndrome. Eur J Endocrinol. 2004;151(6):657–687. 32. Gravholt CH. Clinical practice in Turner syndrome. Nat Clin Pract Endocrinol Metab. 2005;1(1):41–52. 33. Bondy CA, Turner Syndrome Study Group. Care of girls and women with Turner syndrome: a guideline of the Turner Syndrome Study Group. J Clin Endocrinol Metab. 2007;92(1): 10–25. 34. Freriks K, Timmermans J, Beerendonk CCM, et  al. Significant previously undiagnosed morbidity in adult women with Turner Syndrome. J ClinEndocrinolMetab. 2011;96:E1517–E1526. 35. Huang H, Wang CQ, Tian QJ. Clinical features and management of 33 patients with 46,XX pure gonadal dysgenesis. Gynecol Endocrinol. 2016:1–4. [Epub ahead of print]. 36. Weinberg-Shukron A, Renbaum P, Kalifa R, et al. A mutation in the nucleoporin-107 gene causes XX gonadal dysgenesis. J Clin Invest. 2015;125(11):4295–4304. 37. Li TF, Wu QY, Zhang C, et al. 46, XX testicular disorder of sexual development with SRY-negative caused by some unidentified mechanisms: a case report and review of the literature. BMC Urol. 2014;14:104. 38. Huang B, Wang S, Ning Y, et  al. Autosomal XX sex reversal caused by duplication of SOX9. Am J Med Genet. 1999;87:349–353. 39. Ledig S, Hiort O, Wünsch L, Wieacker P. Partial deletion of DMRT1 causes 46,XYovotesticular disorder of sexual development. Eur J Endocrinol. 2012;167(1):119–124. 40. Bashamboo A, McElreavey K. Human sex-determination and disorders of sex-development (DSD). Semin Cell Dev Biol. 2015;45:77–83. 41. Bashamboo A, McElreavey K. Gene mutations associated with anomalies of human gonad formation. Sex Dev. 2013;7:126–146.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

42. Schafer AJ, Foster JW, Kwok C, et  al. Campomelic dysplasia with XY sex reversal: diverse phenotypes resulting from mutations in a single gene. Ann N Y Acad Sci. 1996;785:137–149. 43. Pelletier J, Bruening W, Kashtan CE, et al. Germline mutations in the Wilms' tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome. Cell. 1991;67:437–447. 44. Werner R, Merz H, Birnbaum W, et al. 46,XY Gonadal Dysgenesis due to a Homozygous Mutation in Desert Hedgehog (DHH) Identified by Exome Sequencing. J Clin Endocrinol Metab. 2015;100:E1022–E1029. 45. Werner R, Mönig I, August J, et  al. Novel Insights into 46,XY Disorders of Sex Development due to NR5A1 Gene Mutation. Sex Dev. 2015;9(5):260–268. 46. Achermann JC, Ito M, Ito M, et al. A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans. Nat Genet. 1999;22:125–126. 47. Werner R, Grötsch H, Hiort O. 46,XY disorders of sex development--the undermasculinised male with disorders of androgen action. Best Pract Res Clin Endocrinol Metab. 2010;24(2):263–277. 48. Joint LWPES/ESPE CAH Working Group. Consensus statement on 21-hydroxylase deficiency from the Lawson Wilkins Pediatric Endocrine Society and the European Society for Paediatric Endocrinology. J Clin Endocrinol Metab. 2002;87(9): 4048–4053. 49. Kulle AE, Welzel M, Holterhus PM, Riepe FG. Principles and clinical applications of liquid chromatography - tandem mass spectrometry for the determination of adrenal and gonadal steroid hormones. J Endocrinol Invest. 2011;34(9):702–708. 50. Parsa AA, New MI. Steroid 21-hydroxylase deficiency in congenital adrenal hyperplasia. J Steroid Biochem Mol Biol. 2016;pii: S0960-0760(16):30198–30204. 51. Kulle AE, Welzel M, Holterhus PM, Riepe FG. Implementation of a liquid chromatography tandem mass spectrometry assay for eight adrenal C-21 steroids and pediatric reference data. Horm Res Paediatr. 2013;79(1):22–31. 52. Krone N, Hughes BA, Lavery GG, Stewart PM, Arlt W, Shackleton CH. Gas chromatography/mass spectrometry (GC/ MS) remains a pre-eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry (LC/MS/MS). J Steroid Biochem Mol Biol. 2010;121:496–504. 53. Krone N, Reisch N, Idkowiak J, et al. Genotype-phenotype analysis in congenital adrenal hyperplasia due to P450 oxidoreductase deficiency. J Clin Endocrinol Metab. 2012;97(2):E257–E267. 54. Hauffa B, Hiort O. P450 side-chain cleavage deficiency-a rare cause of congenital adrenal hyperplasia. Endocr Dev. 2011;20:54–62. 55. Bens S, Mohn A, Yüksel B, et al. Congenital lipoid adrenal hyperplasia: functional characterization of three novel mutations in the STAR gene. J Clin Endocrinol Metab. 2010;95:1301–1308. 56. Bertelloni S, Balsamo A, Giordani L, et  al. 17beta-Hydroxysteroid dehydrogenase-3 deficiency: from pregnancy to adolescence. J Endocrinol Invest. 2009;32(8):666–670. 57. Hochberg Z, Chayen R, Reis N, et al. Clinical and genetic findings in male and female patients with 5α-reductase deficiency. J Clin Endocrinol Metab. 1996;81:2821–2827. 58. Kang HJ, Imperato-McGinley J, Zhu YS, Rosenwaks Z. The effect of 5α-reductase-2 deficiency on human fertility. Fertil Steril. 2014;101(2):310–316. 59. Hughes IA, Werner R, Bunch T, Hiort O. Androgen insensitivity syndrome. Semin Reprod Med. 2012;30(5):432–442. 60. Gottlieb B, Beitel LK, Nadarajah A, et  al. The androgen receptor gene mutations database: 2012 update. Hum Mutat. 2012;33:887–894.

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61. Holterhus PM, Brüggenwirth HT, Hiort O, et al. Mosaicism due to a somatic mutation of the androgen receptor gene determines phenotype in androgen insensitivity syndrome. J Clin Endocrinol Metab. 1997;82(11):3584–3589. 62. Quigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS. Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev. 1995;16(3):271–321. 63. Doehnert U, Bertelloni S, Werner R, Dati E, Hiort O. Characteristic features of reproductive hormone profiles in late adolescent and adult females with complete androgen insensitivity syndrome. Sex Dev. 2015;9(2):69–74. 64. Grunseich C, Fischbeck KH. Spinal and bulbar muscular atrophy. Neurol Clin. 2015;33(4):847–854. 65. Schweizer MT, Yu EY. Persistent androgen receptor addiction in castration-resistant prostate cancer. J Hematol Oncol. 2015;8:128. http://dx.doi.org/10.1186/s13045-015-0225-2. 66. Mao Q, Qiu M, Dong G, et  al. CAG repeat polymorphisms in the androgen receptor and breast cancer risk in women: a metaanalysis of 17 studies. Onco Targets Ther. 2015;8:2111–2120. 67. Rey RA, Belville C, Nihoul-Fékété C, et  al. Evaluation of gonadal function in 107 intersex patients by means of serum antimüllerian hormone measurement. J Clin Endocrinol Metab. 1999;84(2):627–631. 68. Josso N, Rey RA, Picard JY. Anti-müllerian hormone: a valuable addition to the toolbox of the pediatric endocrinologist. Int J Endocrinol. 2013;2013:674105. 69. Wünsch L. Checklist for the structural description of the deep phenotype in disorders of sexual development. Int J Endocrinol. 2012;2012:816365. 70. Hiort O, Birnbaum W, Marshall L, et al. Management of disorders of sex development. Nat Endocr Rev. 2014;10(9):520–529. 71. Ahmed SF, Achermann JC, Arlt W, et  al. UK guidance on the initial evaluation of an infant or an adolescent with a suspected disorder of sex development. Clin Endocrinol (Oxf). 2011;75:12–26. 72. Ahmed SF, Khwaja O, Hughes IA. Clinical features and gender assignment in cases of male undermasculinisation: the role for a masculinisation score. BJU Int. 2000;85:120–124. 73. Dean A, Sharpe RM. Anogenital distance or digit length ratio as measures of fetal androgen exposure: relationship to male reproductive development and its disorders. J Clin Endocrinol Metab. 2013;98:2230–2238. 74. Thankamony A, Lek N, Carroll D, et  al. Anogenital distance and penile length in infants with hypospadias or cryptorchidism: comparison with normative data. Environ Health Perspect. 2014;122:207–211. 75. Courant F, Aksglaede L, Antignac JP, et  al. Assessment of circulating sex steroid levels in prepubertal and pubertal boys and girls by a novel ultrasensitive gas chromatography-tandem mass spectrometry method. J Clin Endocrinol Metab. 2010;95(1): 82–92. 76. Wudy SA, Hartmann M, Homoki J. Hormonal diagnosis of 21-hydroxylase deficiency in plasma and urine of neonates using benchtop gas chromatography-mass spectrometry. J Endocrinol. 2000;165:679–683. 77. Koal T, Schmiederer D, Pham-Tuan H, Röhring C, Rauh M. Standardized LC-MS/MS based steroid hormone profile-analysis. J Steroid Biochem Mol Biol. 2012;129(3–5):129–138. 78. Baxter RM, Vilain E. Translational genetics for diagnosis of human disorders of sex development. Annu Rev Genomics Hum Genet. 2013;14:371–392. 79. Arboleda VA, Lee H, Sánchez FJ, et  al. Targeted massively parallel sequencing provides comprehensive genetic diagnosis for patients with disorders of sex development. Clin Genet. 2013;83(1):35–43.

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1.  Normal and Variant Sex Development

80. Koboldt DC, Steinberg KM, Larson DE, Wilson RK, Mardis ER. The next-generation sequencing revolution and its impact on genomics. Cell. 2013;155:27–38. 81. Wünsch L, Buchholz M. Imaging, endoscopy and diagnostic surgery. Endocr Dev. 2014;27:76–86. 82. Wünsch L, Holterhus PM, Wessel L, Hiort O. Patients with disorders of sex development (DSD) at risk of gonadal tumour development: management based on laparoscopic biopsy and molecular diagnosis. BJU Int. 2012. http://dx.doi. org/10.1111/j.1464-410X.2012.11181. 83. Thyen U, Richter-Appelt H, Wiesemann C, Holterhus PM, Hiort O. Deciding on gender in children with intersex-conditions: considerations and controversies. Treat Endocrinol. 2005;4:1–8. 84. Diamond M. Developmental, sexual and reproductive neuroendocrinology: historical, clinical and ethical considerations. Front Neuroendocrinol. 2011;32(2):255–263. 85. Moran ME, Karkazis K. Developing a multidisciplinary team for disorders of sex development: planning, implementation, and operation tools for care providers. Adv Urol. 2012 Article ID 604135. 86. Baratz AB, Sharp MK, Sandberg DE. Disorders of sex development peer support. Endocr Dev. 2014;27:99–112. 87. Nordenström A, Thyen U. Improving the communication of healthcare professionals with affected children and adolescence. Endocr Dev. 2014;27:113–127. 88. http://www.ethikrat.org/dateien/pdf/stellungnahme-intersexualitaet.pdf. 89. http://www.bundesaerztekammer.de/fileadmin/user_ upload/downloads/BAeK-Stn_DSD.pdf. 90. http://ec.europa.eu/health/rare_diseases/european_ reference_networks/erf/index_en.htm. 91. Diamond M, Sigmundson HK. Sex reassignment at birth. Longterm review and clinical implications. Arch Pediatr Adolesc Med. 1997;151(3):298–304. 92. Diamond M, Sigmundson HK. Sexual identity and sexual orientation in children with traumatised or ambiguous genitalia. J Sex Res. 1997;34:199–211. 93. Gillam LH, Hewitt JK, Warne GL. Ethical principles: an essential part of the process in disorders of sex development care. Horm Res Paediatr. 2011;76(5):367–368. 94. Hiort O, Thyen U, Holterhus PM. The Basis of Gender Assignment in Disorders of Somatosexual Differentiation. Horm Res. 2005;64(Suppl 2):18–22. 95. Blankenstein O. Hydrocortisone replacement in disorders of sex development. Endocr Dev. 2014;27:160–171. 96. Yau M, Khattab A, New MI. Prenatal Diagnosis of Congenital Adrenal Hyperplasia. Endocrinol Metab Clin North Am. 2016;45(2):267–281. 97. Wright CF, Wei Y, Higgins JP, Sagoo GS. Non-invasive prenatal diagnostic test accuracy for fetal sex using cell-free DNA a review and meta-analysis. BMC Res Notes. 2012;5:476. http:// dx.doi.org/10.1186/1756-0500-5-476.

98. Birnbaum W, Bertelloni S. Sex hormone replacement in disorders of sex development. Endocr Dev. 2014;27:149–159. 99. https://idw-online.de/de/news593873. 100. Kiess W, Conway G, Ritzen M, et al. Induction of puberty in the hypogonadal girl. Practices and attitudes of pediatric endocrinologists in Europe. Horm Res. 2002;57:66–71. 101. Han TS, Goswami D, Trikudanathan S, Creighton SM, Conway GS. Comparison of bone mineral density and body proportions between women with complete androgen insensitivity syndrome and women with gonadal dysgenesis. Eur J Endocrinol. 2008;159(2):179–185. 102. Köhler B, Kleinemeier E, Lux A, Hiort O, Grüters A, Thyen U, The DSD Network Working Group. Satisfaction with Genital Surgery and Sexual Life of Adults with XY Disorders of Sex Development: Results from the German Clinical Evaluation Study. J ClinEndocrinolMetab. 2012;97(2):577–588. 103. Jürgensen M, Kleinemeier E, Lux A, The DSD Network Working Group. Psychosexual Development in Adolescents and Adults with Disorders of Sex Development-Results from the German Clinical Evaluation Study. J Sex Med. 2012. http://dx.doi. org/10.1111/j.1743-6109.2012.02751.x. 104. Thyen U, Lux A, Jürgensen M, Hiort O, Köhler B. Utilization of health care services and satisfaction with care in adults affected by disorders of sex development (DSD). J Gen Intern Med. 2014(Suppl 3):S752–S759. 105. Steven L, Cherian A, Yankovic F, Mathur A, Kulkarni M, Cuckow P. Current practice in paediatric hypospadias surgery; a specialist survey. J Pediatr Urol. 2013;9(6 Pt B):1126–1130. 106. Wolffenbuttel KP, Crouch NS. Timing of feminizing surgery in disorders of sex development. Endocr Dev. 2014;27:210–221. 107. Yankovic F, Cherian A, Steven L, Mathur A, Cuckow P. Current practice in feminizing surgery for congenital adrenal hyperplasia; a specialist survey. J Pediatr Urol. 2013;9(6 Pt B):1103–1107. 108. Callens N, Hoebeke P. Phalloplasty: a panacea for 46,XY disorders of sex development conditions with penile deficiency. Endocr Dev. 2014;27:222–233. 109. Cools M, Looijenga LHJ, Wolffenbuttel KP, T’Soen G. Managing the risk of germ cell tumourigenesis in disorders of sex development. Endocr Dev. 2014;27:185–196. 110. Ahmed SF, Bryce J, Hiort O. International Networks For Supporting Research & Clinical Care In The Field Of DSD. Endocr Dev. 2014;27:284–292. 111. Cox K, Bryce J, Jiang J, et  al. Novel Associations in Disorders of Sex Development: Findings From the I-DSD Registry. J Clin Endocrinol Metab. 2014;99(2):E348–E355. 112. Hiort O, Wünsch L, Cools M, Looijenga L, Cuckow P. Requirements for a multicentric multidisciplinary registry on patients with disorders of sex development. J Pediatr Urol. 2012;8(6):624–628. 113. Ahmed SF, Rodie M, Jiang J, Sinnott RO. The European disorder of sex development registry: a virtual research environment. Sex Dev. 2010;4(4–5):192–198.

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C H A P T E R

2 Gender Diagnoses Jack Drescher New York University, New York, NY, United States

O U T L I N E 2.3.1 Child Gender Diagnoses 2.3.2 The Treatment of Prepubescent Children 2.3.3 Diagnostic Placement: Stigma Versus Access to Care

21 21

2.1 Early History

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2.2 History of Gender Diagnoses in DSM and ICD 2.2.1 Gender Diagnoses in the DSM 2.2.2 Gender Diagnoses in the ICD

19 19 19

2.4 Conclusion

2.3 Controversies Surrounding Gender Diagnoses

20

References 24

24

wear men’s clothes. A short time ago she seriously asked a relative who was in the police department to obtain permission for her to go about in male attire. She felt quite happy in her abnormal sexual condition, and did not recognize it as pathological. She could not comprehend that her sexual instinct differed from that of other women. (pp. 353–354).

2.1  EARLY HISTORY The scientific study of transgender presentations began in the 19th century and, continuing into the middle of the 20th century, usually classified them as forms of psychopathology. An early exception was Carl Westphal’s1 account of Contrary Sexual Feeling, the case of a young woman who cross-dressed as a boy and who was attracted to women with whom she formed sexual relationships. Richard von Krafft-Ebing,2 on the other hand, was a more influential proponent of the psychopathological view. His most famous work, Psychopathia Sexualis, could be read as an early psychiatric diagnostic manual. In it, he documents several cases of individuals who desired to live as members of the other gender as well as those who had been born to one gender and were living as members of the other. In a chapter entitled “General Pathology” he presented the case of what he called Viraginity (#163) in a natal woman:

From today’s perspective, the 19th century’s early theories conflated what are now referred to as transgender presentations with same sex attractions. Karl Ulrichs3 proposed a third sex theory in which men’s spirits trapped in women’s bodies (urningen) and women’s spirits trapped in male bodies (urnings) “caused” what is today called homosexuality, years before the latter term was coined in 1869.4 Yet female spirits in men’s bodies resonate with some 20th century narratives of transsexual individuals. Even Sigmund Freud, who disagreed with Krafft-Ebing’s degeneracy theory about the “causes” of homosexuality, reported a case of it in a woman in which he claimed its origins derived from the fact that “she foreswore her womanhood” (p. 157).5 In a similar vein, Freud called Leonardo da Vinci “a man who loved like a woman,” and claimed the latter’s identifying with his mother was the cause of the artist’s homosexuality.6 From a narrative perspective, a

For several years [Miss O.] attracted much attention by her bold, mannish behavior, and by wearing her hair and attire in male fashion. She did not seek to conceal her passionate fondness for persons of her own sex. She felt as a man toward women; though she looked like a man, and would much rather Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00001-2

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© 2017 Elsevier Inc. All rights reserved.

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2.  Gender Diagnoses

man’s identification with his mother is not altogether unlike a woman’s spirit trapped in a man’s body. In both cases, homosexuality is explained by a binary belief system in which there are only two genders and purports that some quality of one gender has found its way into the other. In contrast, German psychiatrist Magnus Hirschfeld7 made distinctions between what are today understood to be the desires of homosexuality (to have partners of the same-sex) from the desires of transsexualism (to live as the other sex). Further, it may come as a surprise to some that physicians in Europe were experimenting and performing gender reassignment surgery (GRS, historically referred to as sex reassignment surgery) in the 1920s. One dramatic case was that of Lily Elbe, whose life was portrayed in a 2016 Academy Award nominated film based on the novel, The Danish Girl.8 Hirschfeld collaborated with the physicians who performed Elbe’s surgeries. It would take several decades before the psychiatric and medical communities more broadly understood and accepted Hirschfeld’s early distinctions between sexual orientation and gender identity. Wider recognition of the phenomenon of transsexualism and GRS occurred in 1952 after George Jorgensen went to Denmark a natal man and returned to the United States in 1952 as a transwoman with a new body and a new name: Christine.9 Referring to her condition as transvestism, Hamburger et  al., the Danish physicians who performed Jorgensen’s surgery, published a report of their medical and surgical treatment in the Journal of the American Medical Association.10 While relatively few ever read the Hamburger paper in JAMA, the publicity surrounding Jorgensen’s transition was a significant factor leading to greater popular, medical, and psychiatric awareness of the concepts of transition, transsexual, gender reassignment, gender identity, and in later years, terms like expressed gender and experienced gender. So did the public appearances of an increasing number of people who were more openly able to express the wish to “cross over” from their birth-assigned sex to another. Within medical and mental health communities, increased research and changes in attitude toward transition were facilitated by the work of pioneering figures such as Harry Benjamin,11 John Money,12 Robert Stoller,13 and Richard Green.14 Increased public discussions of gender reassignment and gender identity would provide those who would eventually come to identify as transsexual or transgender with a category and a name for their feelings and desires.15 Eventually, what was once considered exceedingly rare gradually became more publicly visible. Consequently, in recent years an increasing number of nations, provinces, and municipalities have enacted laws establishing “gender identity” as a protected group along the line of categories like race, ethnicity, age, sex, and sexual orientation.16 On the other hand, and of great concern, increased visibility has also

led to the recent passing of laws in several US states denying transgender people access to the bathrooms that conform to their gender identity.17 However, changes in general societal awareness and attitudes, not to mention changes in the medical and psychiatric communities, did not occur overnight. For several decades following Jorgensen’s 1950s GRS, many mental health practitioners were critical of and strongly opposed gender reassignment as a treatment for gender dysphoria (GD).18–21 In part this was due to the fact that psychiatric theorizing of the time continued to conflate sexual orientation and gender identity. Many physicians and psychiatrists criticized using surgery and hormones to irreversibly treat people suffering from what they perceived to be either a severe neurotic or psychotic, delusional condition in need of psychotherapy and “reality testing.” This mainstream view of the time was captured in Richard Green’s 1960s survey of 400 physicians that included psychiatrists, urologists, gynecologists, and general medical practitioners who were asked to give a professional opinion about what to do in the following case of an individual seeking GRS:22 Since early childhood, this 30-year-old biological male has been very effeminate in his mannerisms, interests, and daydreams. His sexual desires have always been directed toward other males. He would like to be able to dress exclusively in woman’s clothes. This person feels inwardly and insists to the world that he is a female trapped in a male body. He is convinced that he can only be happy if he is operated on to make his body look like that of a woman. Specifically, he requests the removal of both testes, his penis, and the creation of an artificial vagina (all of which can, in fact, be done surgically). He also requests that his breasts be made to appear like a woman’s, either surgically or by the use of hormones (this, too, is medically possible). (p. 236).

Green22 summarized the survey’s findings as follows: Eight percent [8%] of the respondents considered the transsexual “severely neurotic” and fifteen percent [15%] considered the person “psychotic.” The majority of the responding physicians were opposed to the transsexual’s request for sex reassignment even when the patient was judged nonpsychotic by a psychiatrist, had undergone two years of psychotherapy, had convinced the treating psychiatrist of the indications for surgery, and would probably commit suicide if denied sex reassignment. Physicians were opposed to the procedure because of legal, professional, and moral and/or religious reasons. In contrast to the conservatism with which granting of sexreassignment procedures was viewed, there was a paradoxical liberalism in the approach to these patients should they already have been successful in obtaining their surgery elsewhere. Among the respondents, three quarters [75%] were willing to allow the postoperative patient to change legal papers such as a birth certificate and to marry in the new gender, and one-half [50%] would allow the person to adopt a child as a parent in the new gender. (pp. 241–242).

While these were once psychiatry’s and medicine’s prevailing views, they are no longer part of the mainstream

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2.2  History of Gender Diagnoses in DSM and ICD

of psychiatric or general medical thought and practice. In the 21st century, international expert guidelines support transition in carefully evaluated individuals,23 although the health care systems in only a minority of countries around the world now cover needed medical services for gender reassignment.16,24 Today, organizations like the American Psychiatric Association (APA) now support access to medical and surgical treatment for transgender individuals.25 Further, the US Department of Health and Human Services changed its guidelines in 2014 allowing Medicare reimbursement for gender transition services.26

2.2  HISTORY OF GENDER DIAGNOSES IN DSM AND ICD The placement of gender identity and gender role diagnoses has changed over time within both the APA Diagnostic and Statistical Manual of Mental Disorders (DSM) and the World Health Organization’s (WHO) International Statistical Classification of Diseases and Related Health Problems (ICD). Given the shifting names and categories, these categories are collectively referred to here as gender diagnoses.

2.2.1  Gender Diagnoses in the DSM Neither DSM-I27 or DSM-II28 make reference to gender diagnoses. However, beginning with 1980s DSMIII,29 gender diagnoses were introduced followed later by category migration and renaming in subsequent editions of DSM. It is worth underscoring that this migration illustrates the field’s shifting views about what to call the diagnosis, what it means, and where to place it over time. From a broader perspective, DSM-III abandoned the psychodynamic theorizing of the earlier two editions and adopted a neo-Kraepelian, descriptive, symptom-based framework drawing upon contemporary research findings of that time. In 2005, Zucker and Spitzer30 described the thinking that led to the decision to include the gender diagnoses in DSM: During the 1960s, North American psychiatry had begun to take a look at the phenomenon of transsexualism in adults (see, for example, Green & Money 1969;31 Stoller 196832). It became apparent that psychiatrists and other mental-health professionals had become increasingly aware of the phenomenon, that is, of adult patients reporting substantial distress about their gender identity and seeking treatment for it, typically hormonal and surgical sex-reassignment. Indeed, there were enough observed cases that it was possible in the 1960s to establish the first university- and hospital-based gender identity clinics for adults. Many clinicians and researchers were writing about transsexualism, and by 1980, there was a large enough database to support its uniqueness as a clinical entity and a great deal of empirical research that examined its phenomenology, natural history, psychologic and biologic correlates. Thus, by the time

19

DSM-III was in its planning phase in the mid-1970s, there were sufficient clinical data available to describe the phenomenon and to propose diagnostic criteria. (p. 37).

Zucker and Spitzer30 go on to summarize the vicissitudes of the current gender diagnoses from DSM-III through DSM-IV-TR: In the third edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-III),29 there appeared for the first time two psychiatric diagnoses pertaining to gender dysphoria in children, adolescents, and adults: gender identity disorder of childhood (GIDC) and transsexualism (the latter was to be used for adolescents and adults). In the DSM-III-R,33 a third diagnosis was added: gender identity disorder of adolescence and adulthood, nontranssexual type. In DSM-IV34 and DSM-IV-TR,35 this last diagnosis was eliminated (“sunsetted”), and the diagnoses of GIDC and transsexualism were collapsed into one overarching diagnosis, gender identity disorder (GID), with different criteria sets for children versus adolescents and adults. (p. 32).

It addition to name changes, the diagnostic category also migrated into different DSM chapters. In DSM-III,29 both GIDC and transsexualism are listed among the psychosexual disorders. In DSM-III-R,33 both are moved to a category called disorders usually first evident in infancy, childhood, or adolescence. In DSM-IV34 and DSM-IV-TR,35 they are moved again to a new parent category, sexual and gender identity disorders, and transsexualism is renamed gender identity disorder (GID) in adolescents or adults. The gender diagnoses, adult and child, were then clustered with the paraphilias and sexual dysfunctions. DSM-5 was published in 201336 and the gender diagnoses were renamed with one overarching diagnosis, GD, that includes separate, developmentally appropriate criteria sets for children (gender dysphoria in children) and another for adolescents and adults (gender dsyphoria in adolescents and adults). In addition, the gender diagnoses were moved into a separate chapter that separated them from the sexual dysfunctions and paraphilia diagnoses.37 Table 2.1 summarizes the placements of gender diagnoses in the DSM:

2.2.2  Gender Diagnoses in the ICD Prior to ICD-6 and the founding of the WHO, ICD was exclusively a mortality classification. Mental disorders in general and sexual disorders in particular were not considered to be causes of mortality, so they were not included in these classifications. ICD-638 was the first version of ICD published by WHO, the first to include a classification of morbidity, and the first version that included a classification of mental disorders.39 ICD-638 had no gender diagnoses, nor did they appear in ICD-7.40 Parenthetically, and, as previously noted, sexual orientation and gender identity were often conflated at that time, so a diagnosis of homosexuality did appear in both ICD-6 and ICD-7. Presumably reflecting changing

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2.  Gender Diagnoses

TABLE 2.1  Gender Diagnoses in the DSM

TABLE 2.2  Gender Diagnoses in the ICD

Year

DSM

Parent category

Diagnosis name

Year

ICD

Parent category

Diagnosis name

1952

DSM-I

N/A

N/A

1948

ICD-6

N/A

N/A

1968

DSM-II

Sexual deviations

Transvestitism

1955

ICD-7

N/A

N/A

1980

DSM-III

Psychosexual disorders

Transsexualism

1965

ICD-8

Sexual deviations

Transvestitism

Gender identity disorder of childhood

1975

ICD-9

Sexual deviations

Transvestism

Disorders usually first evident in infancy, childhood or adolescence

Transsexualism

1987

DSM-III-R

Transsexualism 1900

ICD-10

Gender identity disorder of childhood

Gender identity disorders

Gender identity disorder of adolescence and adulthood, nontranssexual type 1994

DSM-IV

Sexual and gender identity disorders

Gender identity disorder in adolescents or adults Gender identity disorder in children

2000

DSM-IV-TR

Sexual and gender identity disorders

Transsexualism Dual-role transvestism Gender identity disorder of childhood Other gender identity disorders Gender identity disorder, unspecified

2018

ICD-11

Gender identity disorder in adolescents or adults

Conditions related to sexual health

Gender incongruence of adolescents and adults Gender incongruence of children (Proposed)

Gender identity disorder in children 2013

DSM-5

Gender dysphoria

Gender dysphoria in adolescents or adults Gender dysphoria in children

clinical and theoretical views, ICD-8 separated sexual deviations from personality disorders.41 The sexual deviations included the new diagnosis of transvestitism for the first time. Given that definitions of diagnostic categories were not provided in ICD-8, its meaning is not entirely clear, although an alternative spelling, transvestism, was an early synonym for what later came to be known as transsexualism (as mentioned above, Hamburger and his collaborators10 use the term transvestism as Christine Jorgensen’s diagnosis). ICD-942 replaced transvestitism with transvestism, defined as a “Sexual deviation in which sexual pleasure is derived from dressing in clothes of the opposite sex. There is no consistent attempt to take on the identity or behavior of the opposite sex.” Although still categorized as a sexual deviation, it was now both a separate and exclusionary category from the newly added diagnosis of trans-sexualism [sic]. ICD-1043 offered a significant reorganization of the classification system and some new gender diagnoses. Listed under disorders of adult behavior and personality appears a new category of GID (F64) which includes five

diagnoses: transsexualism, dual-role transvestism, gender identity disorder of childhood (GIDC), other gender identity disorders, and GID, unspecified. At present, proposals have been made for the ICD11 revision scheduled to be published in 2018. WHO’s Working Group on the Classification of Sexual Disorders and Sexual Health (WGSDSH) has been charged with evaluating clinical and research data to inform revision of diagnostic categories related to sexuality and gender identity currently in the Mental and Behavioural Disorders chapter of ICD-10, and making recommendations regarding whether and how these categories should be represented in ICD-11. The proposals include a name change of the diagnosis to gender incongruence and a recommendation that the entire diagnostic category be moved outside the mental disorders section of ICD.39,44 Those recommendations have been accepted and the gender incongruence diagnoses will appear in a new ICD chapter called Conditions Related to Sexual Health. Table 2.2 summarizes the placements of gender diagnoses in the ICD:

2.3  CONTROVERSIES SURROUNDING GENDER DIAGNOSES Arguments have been made that it is wrong, perhaps even unethical, for psychiatrists and other mental health

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

2.3  Controversies Surrounding Gender Diagnoses

professionals to label any variations of gender expression as symptoms of a mental disorder.23 Consequently, the mere existence of DSM and ICD gender identity diagnoses have generated controversy in recent years.44–47 Some of those controversies are reviewed below.

2.3.1  Child Gender Diagnoses Criticisms of child gender diagnoses have existed for decades. Some activists and academics in the field of queer theory48,49 assert that the child diagnosis’ first appearance in DSM-III29 was an attempt by psychiatrists to prevent homosexuality in adults. Zucker and Spitzer30 refuted that interpretation of historical events for three reasons: (1) there was no need for a veiled backdoor diagnosis to prevent homosexuality because DSM-III still contained a diagnosis of ego-dystonic homosexuality (EDH); (2) that EDH was itself eventually removed from the DSM-III-R in 1987 because of a lack of any empirical basis to support the diagnosis; and (3) “several clinicians and scientists who argued in favor of delisting homosexuality from the DSM-II were members of the DSM-III subcommittee on psychosexual disorders that recommended the inclusion of the GIDC diagnosis in DSM-III” (p. 35). In other words, those responsible for removing homosexuality from DSM had no motive to find a “backdoor” way to put it back in. Nevertheless, in the last two decades there have been repeated calls for removal of the child gender diagnoses from psychiatric diagnostic manuals altogether.50–54 Most of these authors call it unscientific, unethical, and misguided to use childhood gender diagnoses as a way to justify clinical efforts aimed at getting children to reject their expressed gender identity and to accept the sex (and gender) they were assigned at birth. In doing so, they frequently compare clinical efforts to treat gender variant children with clinical efforts to change homosexuality.45 Bartlett et  al.,50 in recommending removal of the GIDC diagnosis from DSM, argued “children who experience a sense of inappropriateness in the culturally prescribed gender role of their sex but do not experience discomfort with their biological sex should not be considered to have GID. Because of flaws in the DSM-IV definition of mental disorder, and limitations of the current research base, there is insufficient evidence to make any conclusive statement regarding children who experience discomfort with their biological sex” (p. 753). Hill et  al.51 offer similar criticisms, “Overall, there is deepening discomfort with pathologizing children and youth for extreme gender variance. Since this is a highly contentious diagnosis—with little established reliability and validity, and problematic assessment and treatment approaches—researchers and clinicians need to establish that GID is validly diagnosed with nonbiased

21

assessments and treated effectively in accordance with current standards” (p. 57). Isay52 claims the GIDC diagnosis “implicitly labels homosexual boys as mentally disordered” (p. 9), since research indicates that a certain degree of gender atypical behavior was common in many of the adult gay men he treated. In a similar vein, Richardson54 warns of the slippery slope between GIDC’s pathologizing “extreme” gender atypical behavior and the “normal” childhood gender atypical behavior of many “proto-gay” men and women who do not meet diagnostic criteria for GIDC and never come to clinical attention. Arguments for retention of the diagnosis44,55–57 include: (1) the need of children with GD or incongruence to access care that is often complex and involves treatment of both the family and social environment; (2) increased efforts to narrow clinical criteria to exclude gender atypical behavior unrelated to GD; and (3) the need to make it clear to clinicians that the gender diagnoses of childhood do not progress directly into the gender diagnoses of adolescence and adulthood. In fact, the current research shows that most prepubescent children given a gender diagnosis grow up to be gay and cisgender (nontransgender).36,58–60 It should be further underscored that clinicians working from a variety of perspectives (see below) are unable to differentiate between children whose GD persists into adolescence and adulthood (persisters) and those who will desist or grow out of it (desisters).59–61

2.3.2  The Treatment of Prepubescent Children Little is actually known about the origins of any gender identity, be it cisgender or transgender, or about the long-term outcomes of the various treatments currently being offered to children. As noted in a 2012 APA Task Force report,58 “Opinions vary widely among experts, and are influenced by theoretical orientation, as well as assumptions and beliefs (including religious) regarding the origins, meanings, and perceived fixity or malleability of gender identity. Primary caregivers may, therefore, seek out providers for their children who mirror their own world views, believing that goals consistent with their views are in the best interest of their children” (pp. 762–763). The Task Force58 further noted, “The overarching goal of psychotherapeutic treatment for childhood GID is to optimize the psychological adjustment and well-being of the child. What is viewed as essential for promoting the well-being of the child, however, differs, as does the selection and prioritization of goals of treatment. In particular, opinions differ regarding the questions of whether or not minimization of gender atypical behaviors and prevention of adult transsexualism are acceptable goals of therapy” (p. 763). The Task Force58 outlined

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

22

2.  Gender Diagnoses

three general approaches to child treatments in the professional literature: In the first approach,62 clinicians work with the child and caregivers to lessen GD and to decrease cross-gender behaviors and identification. The assumption is that this approach decreases the likelihood of GD persisting into adolescence and culminating in adult transsexualism. For various reasons (e.g., social stigma, likelihood of hormonal and surgical procedures with their associated risks and costs), persistence is considered to be an undesirable outcome by some but not all clinicians who work in this area of practice. Critics of this approach have likened it to “reparative therapy,”53 a term more commonly used to describe efforts to change homosexuality in gay adults. In 2015, after passage of a law banning “conversion therapy for minors,” and following complaints from the local transgender community, the Toronto center advocating this approach was closed down by its parent hospital and its long-time director dismissed.63 A second treatment modality,64 often referred to as “the Dutch approach” because it was developed in the Netherlands, makes no direct effort to lessen GD or gender atypical behaviors. This method is premised on evidence that GD or incongruence diagnosed in childhood usually does not persist into adolescence and beyond, and on the lack of reliable markers to predict in whom it will or will not persist. One aspect of this approach is to have no therapeutic target with respect to adult gender identity outcome. Instead, the goal is to allow the developmental trajectory of gender identity to unfold naturally without pursuing or encouraging a specific outcome. This approach entails combined child, parent, and community-based interventions to support the child in navigating the potential social risks. A third treatment modality outlined by the APA Task Force is sometimes referred to as “gender affirmative.”65,66 Here, clinicians and family members “affirm” a child’s cross-gender identification. This means the child is supported in transitioning to a cross-gendered role, with the option of endocrine treatment to suspend puberty in order to suppress the development of unwanted secondary sex characteristics if the cross-gendered identification persists into puberty. The rationale for supporting transition before puberty is the belief that a transgender

outcome is to be expected in some children, and that these children can be identified so that primary caregivers and clinicians may opt to support early social transition. A supporting argument is that children who transition this way can revert to their originally assigned gender if necessary since the transition is done solely at a social level and without medical intervention. Critics of this approach believe supporting gender transition in childhood increases the likelihood of persistence and a lifetime of medical treatment and retrospective studies suggest that early social transition may be a factor in persistence.60 Table 2.3 compares the three approaches outlined in the APA’s Task Force report:

2.3.3  Diagnostic Placement: Stigma Versus Access to Care How a psychiatric diagnosis can promote stigma is exemplified most dramatically by the history of homosexuality’s 1973 removal from DSM-II.67 In the aftermath of that APA decision, psychiatry and medicine abandoned their historical participation in stigmatizing homosexuality. Those who accepted scientific authority on such matters gradually came to accept the APA’s position and a new cultural perspective emerged: (1) if homosexuality is not an illness, (2) if one does not literally accept biblical prohibitions against homosexuality, (3) if contemporary, secular democracy separates church and state, and (4) if openly gay people are able and prepared to function as productive citizens, then what is wrong with being gay? And if there is nothing wrong with being gay, then what moral and legal principles should the larger society endorse in helping gay people openly live their lives? The aftermath of that decision led to an historically unprecedented social acceptance of gay men and women across much of the world which, in many countries and cultures, has culminated in the contemporary social and policy debates about gay civil rights and marriage equality.68 Although the gay rights movement can attribute much of its advances to those events, transgender rights have progressed despite the continued existence of gender diagnoses in both DSM and ICD.45,69 The movement for transgender civil rights began more slowly but

TABLE 2.3  Comparing Clinical Approaches to Treating Gender Dysphoric/Gender Variant Children Clinic Toronto62 64

Amsterdam

66

San Francisco

Cross gender interests and play

Social transition before puberty

Puberty suppression

Try to prevent homosexuality

Try to prevent transsexualism

Discouraged

Discouraged

Yes

No

Yes

Permitted

Discouraged

Yes

No

No

Permitted

Permitted

Yes

No

No

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

2.3  Controversies Surrounding Gender Diagnoses

followed in the wake of the larger gay rights movement. By the late 1990s, trans inclusion had increasingly become a focus of LGBT rights groups and support by these groups for transgender rights continues to this day. Nevertheless, given how removing homosexuality from DSM was a watershed in the gay civil rights movement, some seek a similar goal for transgender people as well. For example, as the ICD revision process unfolded, many advocates, several countries, the Council of Europe Commissioner for Human Rights70 and the European Parliament71 took strong positions that issues related to transgender identity should not be classified as mental disorders in ICD-11. The European Parliament resolution71 “roundly condemns the fact that homosexuality, bisexuality and transsexuality are still regarded as mental illnesses by some countries, some of which are members of the EU, and calls on states to combat this. In particular, the resolution calls for the depsychiatrisation of the transsexual, transgender journey, for free choice of care providers, for changing identity to be simplified, and for costs to be met by social security schemes.” The document goes on and “calls on the Commission and the WHO to withdraw GID from the list of mental and behavioural disorders, and to ensure a nonpathologising reclassification in the negotiations on the 11th version of the International Classification of Diseases (ICD-11).” Similar concerns about diagnostic retention were raised in the DSM revision process. For example, the World Professional Association for Transgender Health (WPATH) called for depathologization and removal of transgender diagnoses from the DSM-5.72 In a survey by the DSM-5 GID subworkgroup of 201 organizations concerned with the welfare of transgender people from North America, Europe, Africa, Asia, Oceania, and Latin America, a majority of 55.8% believed the diagnosis should be removed from the DSM. The primary reason for wanting to keep a gender diagnosis in DSM was health care reimbursement. Regardless of whether groups were for or against the removal of the diagnosis from the mental disorders classification, the survey revealed a broad consensus that if the diagnosis remained in DSM, an overhaul of the name, criteria, and language to minimize stigmatization of transgender individuals was needed.73 Yet while many transgender advocacy groups call for removal of the gender diagnoses from DSM and ICD, other advocacy groups have raised their concerns about transgender individuals’ ability to access medical and surgical treatment. Removing the adolescent and adult gender diagnoses would undoubtedly lead to loss of private and public insurance coverage for necessary medical and surgical treatment since all of them require a diagnostic code. In addition, at least in the United States, removing gender diagnoses from the diagnostic manual would lead to the loss of potent and increasingly

23

successful arguments of “medical necessity” in legal cases challenging denial of medical and surgical treatment to transgender individuals. For example, US courts are increasingly recognizing a “medically necessity” for transgender inmates to have access to transition services while incarcerated.74 Recognizing the important role medical necessity plays in such matters, in both the DSM-5 and ICD-11 revision processes, the professionals involved have argued for finding a balance between the competing issues of stigma versus access to care.39,44,45 In 2010, France tried to resolve this stigma versus access to care problem by removing the diagnosis of “transsexualism” from the mental disorder section of its diagnostic manual and placing it in a category known as maladie rare.75 Others argue from the position of a growing number of studies that posit physical rather than mental causes of transgender presentations76–82 in support of efforts to classify gender diagnoses as purely medical conditions. Another alternative might be placing these diagnoses in either the endocrinological or genitourinary sections. The latter approach solves the problem of the diagnoses being stigmatized as a mental disorder while still allowing access to care. On the other hand, much of the health care accessed by this patient population is not directly related to endocrinology, although the case could be made that other health and mental health services required are indirectly related in many cases. A genitourinary placement is also problematic since many people who might be diagnosed do not seek or require such surgery. Some transgender advocacy groups have suggested using DSM’s V-codes and ICD’s Z-codes as a way to maintain access to care.83 These codes are used to indicate conditions or clinical situations that might come to the attention of a mental health professional but are not in and of themselves considered to be mental disorders. Such a change in the diagnostic systems would serve the purpose of depathologizing and destigmatizing the category; it would be neither a mental disorder nor a physical one (physical disorders are also stigmatized, although not often as much as mental ones). While such a move would reduce stigma, it is likely to improve access to care as third party payers rarely reimburse V- and Z-codes. For that reason, neither APA nor WHO chose to exercise that option.39,45 In reviewing the history and placement options, WGSDSH concluded that the historical classification of gender diagnoses as mental disorders was serendipitous. If, in the mid-20th century, the narrative had been that transsexualism was related to a “hormone imbalance” rather than being a “sexual deviation,” the category could very well have been placed in the ICD-10 chapter on “Endocrine, nutritional, and metabolic diseases.” In fact, the etiology of the phenomenon was unknown

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

24

2.  Gender Diagnoses

when placement decisions were made in the past and remains unknown today. Further, there are no scientifically based criteria to differentiate “normal” and pathological gender identity and the manner in which any gender identity develops remains unknown and to this day a matter of theoretical speculation. The extant scientific database cannot empirically answer the question of whether this diagnosis is purely a “mental disorder” or a disorder with another physical cause. As mentioned above, this led to the Working Group’s recommendation that the entire diagnostic category be moved outside the mental disorders section of ICD.39 Those recommendations have been accepted and the gender incongruence diagnoses will appear in a new ICD chapter called Conditions Related to Sexual Health.44

2.4 CONCLUSION Psychiatric diagnoses have a long history of generating controversy, hearkening back to an era when deciding what should and should not be a mental disorder was done by medical practitioners. However, one egregious example of medicine’s history of diagnostic excess was drapetomania, a 19th century “disorder of slaves who have a tendency to run away from their owner due to an inborn propensity for wanderlust.”84 Similarly, the unfortunate history of diagnosing homosexuality and gender variance as mental disorders supports longstanding arguments that they are not like pure medical diagnoses, such as renal or cardiac failure, and that psychiatric formulations, are completely arbitrary, subjective, culture bound, and little more than disguised societal efforts to control the behavior of its citizens.85 Further, with changing times and changing cultural attitudes, beliefs about what constitutes a mental disorder have also changed.86 Forty years after APA’s decision, marriage equality is now legal in many countries.68 Consequently, in the not too distant future, individuals seeking gender transitions might be treated by medical specialists who, like obstetricians, avail themselves of interventions to facilitate what society will consider to be normal life events. Consider that the ICD diagnoses of Normal Spontaneous Delivery (JA80) and Normal Menopause (HD00.3) were long ago “medicalized” as a way to provide access to care, despite the fact that both are natural life events and not “pathological” in any strict sense. The issues “gender” raises are complex and many do not lend themselves to easy solutions. In recent years, the author’s efforts to see and grasp the “big picture” of gender repeatedly brought to mind the tale of six blind men (inadequately) trying to describe an elephant as each of them separately touches one of its body parts. Is an elephant like a wall (its side), a spear (its tusk), a

snake (its trunk), a tree (its leg), a fan (its ear), or a rope (its tail)? The answer is obviously “none of the above” as the big picture entirety of the elephant is greater than the sum of all the analogies referring to its individual parts.45 This author cannot claim to have access to a “bigger picture” vision of gender’s meaning beyond others who have theorized or written about the subject yet hopes that future research and clinical work will increase everyone’s understanding.

References 1. Westphal C. Die KonträreSexualempfindung: Symptom einesneuropathologischen (psychopathischen) Zustandes. Arch Psychiat Nerven. 1870;2:73–108. 2. Krafft-Ebing R.. In: Wedeck H, ed. Psychopathiasexualis. New York: Putnam; 1886:1965. 3. Ulrichs K.. In: Lombardi-Nash M, ed. The Riddle of “man-manly” Love. Buffalo, NY Buffalo, NY: Prometheus Books; 1864 1994.. In: Lombardi-Nash M, ed. The Riddle of “man-manly” Love. Buffalo, NY Buffalo, NY: Prometheus Books; 1864 1994. 4. Bullough V. Homosexuality: A History. New York: Meridian; 1979. 5. Freud S. The Psychogenesis of a Case of Homosexuality in a Woman. Standard ed., 18. London: Hogarth Press; 1920:145–172 [1955]. 6. Freud S. Leonardo da Vinci and a memory of his childhood. In: Strachey J, ed. The Standard Edition of the Complete Psychological Works of Sigmund Freud, Vol. 11. London: Hogarth Press; 1910: 59–138 [1957]. 7. Hirschfeld M. Die intersexuellekonstitution. Jahrb Sex Zwischenstufen. 1923;23:3–27. 8. Ebershoff D. The Danish Girl. New York: Viking; 2000. 9. Jorgensen C. Christine Jorgensen: A Personal Autobiography. New York: Paul S. Ericksson, Inc; 1967. 10. Hamburger C, Stürup GK, Dahl-Iversen E. Transvestism: hormonal, psychiatric, and surgical treatment. J Am Med Assoc. 1953;12(6):391–396. 11. Benjamin H. The Transsexual Phenomenon: A Scientific Report on Transsexualism and Sex Conversion in the Human Male and Female. New York: Julian Press; 1966. 12. Money J. The concept of gender identity disorder in childhood and adolescence after 39 years. J Sex Marital Ther. 1994;20:163–177. 13. Stoller RJ. A contribution to the study of gender identity. Int J Psycho-anal. 1964;45:220–226. 14. Green R. Sexual Identity Conflict in Children and Adults. New York: Basic Books; 1974. 15. Denny D. A selective bibliography of transsexualism. J Gay & Lesbian Psychother. 2002;6(2):35–66. 16. Yogyakarta Principles. Principles on the application of international human rights law in relation to sexual orientation and gender identity. ; (2007). Accessed 07.06.09. 17. Bruni F. Tempest in a toilet. The New York Times, April 24, 2016. p. SR3. 18. Socarides CW. The desire for sexual transformation: a psychiatric evaluation of transsexualism. Am J Psychiatry. 1969;125(10):1419–1425. 19. Hertoft P, Sørensen T. Transsexuality: some remarks based on clinical experience. Ciba Found Symp. 1978;62:165–181. 20. Meyer JK, Reter D. Sex reassignment: follow-up. Arch Gen Psychiatry. 1979;36(9):1010–1015. 21. McHugh PR. Psychiatric misadventures. Am Scholar. 1992;61(4):497–510. 22. Green R. Attitudes toward transsexualism and sex-reassignment procedures. In: Green R, Money J, eds. Transsexualism and Sex

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

23.

24.

25.

26.

27.

28.

29.

30.

31. 32. 33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

Reassignment. Baltimore, MD: The Johns Hopkins University Press; 1969:235–251. World Professional Association for Transgender Health (WPATH). Standards of care for the health of transsexual, transgender and gender non-conforming people, 7th version. ; 2011 Accessed 01.08.12. Kreukels BP, Haraldsen IR, De Cuypere G, Richter-Appelt H, Gijs L, Cohen-Kettenis PT. A European network for the investigation of gender incongruence: the ENIGI initiative. Eur Psychiatry. 2012;27(6):445–450. American Psychiatric Association. Position statement on access to care for transgender and gender variant individuals. ; 2012 Accessed 01.03.16. U.S. Department of Health and Human Services. NCD 140.3, Transsexual surgery: decision. < http://www.hhs.gov/dab/ decisions/dabdecisions/dab2576.pdf > ; May 30, 2014 Accessed 22.03.15. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. Washington, DC: American Psychiatric Association; 1952. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. 2nd ed. Washington, DC: American Psychiatric Press; 1968. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. 3rd ed. Washington, DC: American Psychiatric Press; 1980. Zucker KJ, Spitzer RL. Was the gender identity disorder of childhood diagnosis introduced into DSM-III as a backdoor maneuver to replace homosexuality? a historical note. J Sex & Marital Ther. 2005;31:31–42. Green R, Money J, eds. Transsexualism and Sex Reassignment. Baltimore, MD: The Johns Hopkins University Press; 1969. Stoller RJ. Sex and Gender. New York: Science House; 1968. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, 3rd ed.—Revised. Washington, DC: American Psychiatric Press; 1987. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. 4th ed. Washington, DC: American Psychiatric Press; 1994. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, 4th ed.—Text Revision. Washington, DC: American Psychiatric Press; 2000. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. 5th ed. Washington, DC: American Psychiatric Press; 2013. Zucker KJ, Cohen-Kettenis PT, Drescher J, Meyer-Bahlburg HFL, Pfäfflin F, Womack WM. Memo outlining evidence for change forgender identity disorder in the DSM-5. Arch Sex Behav. 2013;42:901–914. World Health Organization. International Classification of Diseases and Related Health Problems, 6th Revision. Geneva, Switzerland: World Health Organization; 1948. Drescher J, Cohen-Kettenis P, Winter S. Minding the body: situating gender diagnoses in the ICD-11. Int Rev Psychiatry. 2012;24(6):568–577. World Health Organization. International Classification of Diseases and Related Health Problems, 7th Revision. Geneva, Switzerland: World Health Organization; 1955. World Health Organization. International Classification of Diseases and Related Health Problems, 8th Revision. Geneva, Switzerland: World Health Organization; 1965. World Health Organization. International Classification of Diseases and Related Health Problems, 9th Revision. Geneva, Switzerland: World Health Organization; 1975.

25

43. World Health Organization. International Statistical Classification of Diseases and Related Health Problems, 10th Revision. Geneva: World Health Organization; 1992. 44. Drescher J, Cohen-Kettenis PT, Reed GM. Gender incongruence of childhood in the ICD-11: controversies, proposal, and rationale. Lancet Psychiatry. 2016;3(3):297–304. 45. Drescher J. Queer diagnoses: parallels and contrasts in the history of homosexuality, gender variance, and the diagnostic and statistical manual (DSM). Arch Sex Behav. 2010;39:427–460. 46. Drescher J. Controversies in gender diagnoses. JLGBT Health. 2014;1(1):9–15. 47. Drescher J, Pula J. Ethical issues raised by the treatment of gender variant prepubescent children. Hastings Cent Rep. 2014;44(Suppl 4):S17–S22. 48. Mass L. “Sissyness” as metaphor: a conversation with Richard Green. In: Homosexuality and Sexuality: Dialogues of the Sexual Revolution, Vol. 1. New York: Harrington Park Press; 1990:213–222 49. Sedgwick E. How to bring your kids up gaySedgwick E, editor. Tendencies, 1993. Durham, NC: Duke University Press; 1991:154–166. 50. Bartlett NH, Vasey PL, Bukowski WM. Is gender identity disorder in children a mental disorder? Sex Roles. 2000;43:753–785. 51. Hill DB, Rozanski C, Carfagnini J, Willoughby B. Gender identity disorders in childhood and adolescence: a critical inquiry. Int J Sex Health. 2007;19(1):57–74. 52. Isay RA. Remove gender identity disorder in DSM. Psychiatric News. 1997, November 21;32(22):9, 13. 53. Pickstone-Taylor SD. Children with gender nonconformity [Letter to the editor]. J Am Acad Child Adolesc Psychiatry. 2003;42:266. 54. Richardson J. Setting limits on gender health. Harvard Rev Psychiat. 1996;4:49–53. 55. Meyer-Bahlburg HF. From mental disorder to iatrogenic hypogonadism: dilemmas in conceptualizing gender identity variants as psychiatric conditions. Arch Sex Behav. 2010;39:461–476. 56. Zucker KJ. Children with gender identity disorder: Is there a best practice? Neuropsychiatr Enfance Adolesc. 2008;56:358–364. 57. Zucker KJ. The DSM diagnostic criteria for gender identity disorder in children. Arch Sex Behav. 2010;39(2):477–498. 58. Byne W, Bradley SJ, Coleman E, et  al. Report of the American psychiatric association task force on treatment of gender identity disorder. Arch Sex Behav. 2012;41(4):759–796. 59. Drescher J, Byne W. Treating Transgender Children and Adolescents: An Interdisciplinary Discussion. New York: Routledge; 2013. 60. Steensma TD, McGuire JK, Kreukels BP, Beekman AJ, CohenKettenis PT. Factors associated with desistence and persistence of childhood gender dysphoria: a quantitative follow-up study. J Am Acad Child & Adolesc Psychiatry. 2013;52(6):582–590. 61. Wallien MSC, Cohen-Kettenis PT. Psychosexual outcome of gender-dysphoric children. J Am Acad Child & Adolesc Psychiatry. 2008;47(12):1413–1423. 62. Zucker KJ, Wood H, Singh D, Bradley SJ. A developmental, biopsychosocial model for the treatment of children with gender identity disorder. J Homosexual. 2012;59(3):369–397. 63. Singal, J. How the fight over transgender kids got a leading sex researcher fired. New York Magazine, February 7, 2016. ; Accessed 01.04.16. 64. de Vries AL, Cohen-Kettenis PT. Clinical management of gender dysphoria in children and adolescents: the Dutch approach. J Homosexual. 2012;59(3):301–320. 65. Brill S, Pepper R. The Transgendered Child: A Handbook for Families and Professionals. San Francisco, CA: Cleis Press; 2008. 66. Ehrensaft D. From gender identity disorder to gender identity creativity: True gender self child therapy. J Homosexual. 2012;59(3):337–356. 67. Bayer R. Homosexuality and American Psychiatry: The Politics of Diagnosis. Princeton, NJ: Princeton University Press; 1987.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

26

2.  Gender Diagnoses

68. Drescher J. The removal of homosexuality from the DSM: its impact on today’s marriage equality debate. J Gay & Lesbian Men Health. 2012;16(2):124–135. 69. Grant JM, Mottet LA, Tanis, J. National transgender discrimination survey report on health and health care: findings of a study by the national center for transgender equality and the national gay and lesbian task force. ; 2010 Accessed 30.04.13. 70. Council of Europe. Commissioner for Human Rights. Issue paper: human rights and gender identity. ; 2009 Accessed 1.08.12. 71. European Parliament. 28 September 2011 on human rights, sexual orientation and gender identity at the United Nations. ; 2011 Accessed 01.08.12. 72. Knudson GA, DeCuypere G, Bockting W. Recommendations for revision of the DSM diagnoses of gender identity disorders: consensus statement of the world professional association for transgender health. Int J Transgenderism. 2010;12(2):115–118. 73. Vance SR, Cohen-Kettenis PT, Drescher J, Meyer-Bahlburg HFL, Pfäfflin F, Zucker KJ. Opinions about the DSM gender identity disorder diagnosis: results from an international survey administered to organizations concerned with the welfare of transgender people. Int J Transgenderism. 2010;12(1):1–24. 74. Associated Press. Chelsea Manning’s hormone treatment ok’d, but not long hair. ; February 13, 2015 Accessed 29.03.15. 75. Brunet, M. Le transsexualismen’est plus unemaladiementale. Le Figaro, February 12. ; 2010 Accessed 22.11.12.

76. Berglund H, Lindström P, Dhejne-Helmy C, Savic I. Male-to-female transsexuals show sex-atypical hypothalamus activation when smelling odorous steroids. Cereb Cortex. 2008;18(8):1900–1908. 77. Garcia-Falgueras A, Swaab DF. A sex difference in the hypothalamic uncinate nucleus: relationship to gender identity. Brain. 2008;131:3132–3146. 78. Herbert J. Who do we think we are? the brain and gender identity. Brain. 2008;131:3115–3117. 79. Kruijver FPM, Zhou J-N, Pool CW, Hofman MA, Gooren LJG, Swaab DF. Male-to-female transsexuals have female neuron numbers in a limbic nucleus. J Clin Endocrinol Metab. 2000;85(5):2034–2041. 80. Rametti G, Carrillo B, Gómez-Gil E, et al. The microstructure of white matter in male to female transsexuals before cross-sex hormonal treatment: a DTI study. J Psychiat Res. 2011;45(7):949–954. 81. Schöning S, Engelien A, Bauer C, et al. Male-to-female transsexuals before and during hormone therapy. J Sex Med. 2010;7(5):1858–1867. 82. Zhou J-N, Hofman MA, Gooren LJG, Swaab DF. A sex difference in the human brain and its relation to transsexuality. Nature. 1995;378:68–70. Nov 2. 83. GATE (Global Action for Trans∗ Equality). It’s time forreform. Trans global action for trans equality health issues in the international classification of diseases. Report on the GATE experts meeting. November 16–18, 2011; The Hague. ; 2012 Accessed 30.08.14. 84. Schwartz S. The role of values in the nature/nurture debate about psychiatric disorders. Soc Psych Psych Epid. 1998;33:356–362. 85. Szasz TS. The myth of mental illness. Am Psychol. 1960;15:113–118. 86. Greco M. What is the DSM? Diagnostic manual, cultural icon, political battleground: an overview with suggestions for a critical research agenda. Psychol Sex. 2015:6. http://dx.doi.org/10.1080/ 19419899.2015.1024470.

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C H A P T E R

3 Gender Identity in Disorders of Sex Development Leanna W. Mah, Yvonne Y. Chan and Jennifer H. Yang University of California, Davis, Sacramento, CA, United States

O U T L I N E 3.1 Introduction

27

3.2 History of Disorders of Sex Development

27

3.3 Psychosexual Development

29

3.4 Disorders of Sex Development 3.4.1 46, XX Disorders of Sex Development 3.4.2 46, XY Disorders of Sex Development 3.4.3 Ovotesticular DSD

30 30 34 36

3.4.4 Sex Chromosomal DSD 37 3.4.5 Other: Micropenis, Aphallia, Cloacal Exstrophy 38 39

3.6 Conclusions

40

References 40

3.1 INTRODUCTION

and the need for comprehensive and multidisciplinary approaches toward management. Hereafter, we will review the history of DSD, our current understanding of psychosexual development, and the management of gender identity, gender assignment, and surgical interventions in patients with DSD as well as other congenital anomalies that commonly present with ambiguous genitalia.

Disorders of sex development (DSD) include a variety of medical conditions that involve the reproductive systems and refer to atypical, congenital presentations of anatomic, gonadal, or chromosomal sex. As a group, they are rare and often create challenging circumstances for patients, their families, and health care providers. The scarcity of these disorders has resulted in slow progress in our efforts to understand the underlying pathophysiology and to develop comprehensive approaches toward caring for patients with these conditions. Nevertheless, there have been great advances over the last century. Our conception of gender identity has evolved from our initial belief in the predominant influence of upbringing and social influences on gender identity development to the appreciation of a more complex psychosexual developmental process involving androgen imprinting. Furthermore, there have been increasing data illustrating the complexity of gender identity Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00012-7

3.5 Future Directions

3.2  HISTORY OF DISORDERS OF SEX DEVELOPMENT Hermaphroditism and gender identities have been documented since ancient times. In Plato’s Banquet from 400BC, Aristophanes addresses gender identities by categorizing people into one of three genders: male, female, and androgyne—a gender identity with simultaneous feminine and masculine characteristics. In Greek mythology, various legends exist of the god

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© 2017 Elsevier Inc. All rights reserved.

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3.  Gender Identity in Disorders of Sex Development

Hermaphroditus and his gender identity, with the common theme describing Hermaphroditus as a union of a female body with the male sexual organ.1 Whereas Hermaphroditus was seen as a symbol of beauty, persons of “intersex” were not always considered so. In the early 16th century United States, “hermaphrodites” were categorized as “monstrous births,” and their births were often considered acts of Satan.2 In the late 19th and early 20th century Britain and France, the so-called “hermaphrodites” were people of “doubtful” or “mistaken” sex. During this period which Alice Dreger refers to as the “Age of the Gonads,” “true” sex was narrowly defined by the presence of ovarian or testicular tissue. The belief at the time was that “the possession of a [single] sex is a necessity of our social order, for hermaphrodites as well as normal subjects.”3 Fortunately, we have moved beyond these simplistic and often demeaning views as we began to question the environmental and biological influences underlying these conditions. In the modern era, discussions and theories of hermaphroditism have been addressed under two frameworks: the “Money Era” and the “Chicago Consensus.”4 The “Money Era,” which extended from 1955–90, purported that gender identity was based upon upbringing and environmental influences regardless of chromosomal, gonadal, or hormonal sex.5 The key example reported by John Money, a psychologist, sexologist, and expert on gender identity at the time, was the allegedly successful sex reassignment of a male to female after a botched circumcision.5 Titled “The Twins Case,” the report described the female gender assignment of a 7-month-old male infant. The infant reportedly led a happy, well-adjusted life with a female identity while his male twin, who served as a genetic control, led a healthy life with a male identity. This case exemplified Money’s theory that nurture overpowers nature. Money believed that there was gender neutrality at birth. Therefore, surgical alterations up to the age of 18 months, prior to children being aware of their sex, coupled with a congruent environment, would determine gender identity. This idea was later refuted by reports that the patient requested to return to his initial sex at birth when he reached adolescence.6 This revelation suggested that factors other than sex of rearing were influential in gender identity development. Follow-up reports revealed that the patient eventually committed suicide and highlighted the importance of psychological care in these patients.7 While our understanding of hermaphroditism, now known as DSD, is still evolving, we have made significant advancements since the “Money Era” that eventually led to the “Chicago Consensus.” The “Chicago Consensus,” developed at The International Conference of Management of Intersex in 2005, introduced a multidisciplinary approach by incorporating genetics, molecular biology, and hormonal

influences into our understanding of psychosexual development.8 Further appraisal of data has shaped how DSD is currently managed, including medical, psychological, and surgical treatments. Under this framework, it is recommended that patient evaluation take place at multidisciplinary centers with multiple subspecialists, including pediatric urologists, pediatric endocrinologists, psychologists/psychiatrists, geneticists, and social workers. In addition, emphasis has been placed on shared decision-making. The Chicago Consensus marked a new generation of attitudes toward the management and treatment of DSD. The Consensus recognized the complexity of DSD and the many areas for improvement. For example, a need to refine the controversial terminology surrounding these conditions led to the development of new terms that are now more commonly accepted (Table 3.1). The Consensus also noted the need for long-term outcomes studies in quality of life that address patients’ and families’ perceptions of DSD, genital and sexual satisfaction, and overall happiness. Furthermore, it acknowledged that there was still a significant need for further collaboration between centers of expertise amongst institutions. In 2016, Lee et  al. published an update to the 2006 Consensus that addressed changes in clinical management, gender assignments, timing of surgical interventions, biochemical and genetic assessments, and risk of germ cell tumor (GCT) development. The update emphasized the importance of shared decision-making with the patient, preserving fertility potential, decreasing physical and psychosocial risks in management decisions, disclosing full medical knowledge to families, and providing adequate psychosocial support.9

TABLE 3.1  Nomenclature Proposed by the 2006 Chicago Consensus.8 Proposed 2006 nomenclature

Prior nomenclature

Disorders of sex development (DSD)

Intersex

46, XX DSD

Female pseudohermaphrodite Overvirilization of an XX female Masculinization of an XX female

46, XY DSD

Male pseudohermaphrodite Undervirilization of an XY male Undermasculinization of an XY male

Ovotesticular DSD

True hermaphrodite

46, XX testicular disorder

XX male or XX sex reversal

46, XY complete gonadal dysgenesis

XY sex reversal

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3.3 Psychosexual Development

Additionally, the update addressed criticisms of the 2006 Consensus, such as concerns over the new nomenclature. In a survey of 589 patients with congenital adrenal hyperplasia (CAH) published in 2015, a majority of the participants disliked the term “disorder” and felt it to be “stigmatizing” and “misleading.”10 Furthermore, some have noted that the term “sex” may be deceptive and imply “sexual behavior.”9 Despite the limitations of the nomenclature, an established terminology helps facilitate communication across disciplines which is necessary in the continued study of DSD. Perhaps the most notable contribution of the update was its frank acknowledgment that our understanding of the psychosocial and psychosexual well-being of patients with DSD remains limited. Moreover, it is still impossible to predict gender development with certainty. Therefore, continued emphasis on patient and family involvement, improved understanding of outcomes, psychosexual development, and individualized management remain imperative.

3.3  PSYCHOSEXUAL DEVELOPMENT While the Money Era strongly emphasized psychological neutrality at birth, recent data have suggested a stronger role for nature in psychosexual development. In addition, studies of DSD at the genetic and hormonal levels in the last few decades have yielded a better understanding of psychosexual development. Psychosexual development is a complex process involving genetic, hormonal, and environmental factors and can be further defined by one’s gender identity, gender role, and sexual orientation.11 Gender identity refers to a person’s self-representation as male or female, with a caveat that some individuals may not identify with either. Gender role describes psychological characteristics, behavioral patterns, and attitudes that are sexually dimorphic within the general population, such as toy preferences and physical aggression. These qualities can vary from culture to culture. Sexual orientation describes one’s erotic interest (e.g., heterosexual, bisexual, homosexual) and includes behavior, fantasies, and attractions. In DSD, where chromosomal, gonadal, and phenotypic sex are misaligned, the three components of psychosexual development may also not be congruent. Therefore, a principal goal of DSD management is to understand factors that influence the development of gender identity so that appropriate gender assignments may be proposed to decrease the risk of gender dysphoria. Androgen imprinting has been proposed to be a critical factor in gender identity development. Androgens influence sexual differentiation of the mammalian brain and behavior.12 In mammals, feminization of the brain is the default pathway, and the presence of androgens during the perinatal sensitive period results in male

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differentiation.13 Androgen effects on neurodevelopment are best studied in the rodent model. The role of androgens in sexual differentiation of the brain was first suggested in 1982 by Döhler et  al., who demonstrated that the sexually dimorphic nucleus in the preoptic area of the rat brain was larger in males than in females. Exposure of genetically female rats to perinatal androgen increased the size of the nucleus to that of male rats.14 The effects of perinatal androgen on neuronal development are long-lasting, and recently scientists have been able to better elucidate the underlying mechanisms. Nugent et al. demonstrated that gonadal steroids reduced DNA methyltransferase enzymes in the sexually dimorphic preoptic area, which decreased DNA methylation and induced epigenetic repression of masculinizing genes. This finding suggested that brain feminization is the result of the active suppression of masculinizing genes.13 These effects at the biological level have direct behavioral effects. For example, female XX rats injected with androgen on the day of birth exhibit increased roughand-tumble play which is typically observed in males.15 Currently, more animal studies are being conducted to further identify and understand androgen effects on epigenetic and genetic changes in sexual differentiation of the brain and their effects on behavior. Androgens also have similar effects in nonhuman primates. Previous studies concluded that prenatal androgens influence sexually differentiated behavior and genital anatomy in rhesus monkeys. During gestation, rhesus testes differentiate between days 38 and 40 and become active, peaking at days 40–75. Fetal ovaries produce no steroids, and the fetal hypothalamicpituitary-gonadal axis is quiescent. Female rhesus monkeys exposed to testosterone at day 40 developed male external genitalia and both female and male internal reproductive structures. They showed behaviorally masculinized traits, such as rough-and-tumble play and mounting of other females. In a separate study, Goy and colleagues manipulated prenatal testosterone at two different gestational time points in females: gestational day 40 and gestational day 115. Early androgen delivery resulted in male-like genitalia without rough-and-tough play. Late androgen delivery resulted in normal female genitalia with increased rough play compared to control females and females with early androgen delivery. The study concluded that behavioral and physical sexual differentiation occur independently, suggesting that timing of androgen exposure affects both phenotypical and psychosexual gender development.16 While androgen effects on psychosexual development are most pronounced during the gestational period, recent studies have shown that they also affect sexual behavior in the adolescent period. Schultz et al. reported that sex steroid-dependent organization in Syrian hamsters also occurs in adolescence. Male hamsters that

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underwent gonadectomy after the perinatal period, but prior to onset of puberty, were compared to hamsters that underwent gonadectomy after puberty. Sexual behavior and aggressive behavior were reduced in those castrated prior to puberty suggesting that testicular hormones play a role in psychosexual development during puberty. In addition, studies have also demonstrated that social experience may modulate the steroid-dependent adolescent brain and behavioral development. Male hamsters that were castrated prior to puberty but exposed to female hamsters restored some reproductive behaviors, including genital grooming and ejaculation, compared to male hamsters with no social sexual exposure.17 Though limited to animal models, these findings can help us further understand the complex role of androgens during the perinatal and postnatal periods. While findings in animal studies are not directly applicable to humans, the effects of prenatal androgen exposure in human psychosexual development have been seen in observational studies. Genetically 46, XX females with CAH who have been exposed to high levels of androgen prenatally exhibit masculinized behavior and prefer male playmates. Genetically 46, XY males with complete androgen insensitivity exhibit female typical behavior.18–20 Therefore, the hormonal milieu during the prenatal and postnatal period may have profound effects on the individual’s predisposition to a more masculine or more feminine gender identity. As the human “critical period” during which androgen affects psychosexual development is not welldefined, there has also been increasing interest in postnatal androgen exposure. After birth, there is a transient rise in testosterone levels that peak at 1–3 months of age in boys; this period is commonly referred to as the “minipuberty.” Lamminmäki et  al. assessed urine testosterone levels in 48 full term infants (22 boys and 26 girls) from age 7 days to 6 months and correlated the area under the curve (AUC) for testosterone to subsequent behavior. They found that play with toy trains correlated positively with testosterone AUC in girls and play with dolls correlated negatively with testosterone AUC in boys.21 Correlation between postnatal androgen levels and subsequent gender-related behaviors has also been demonstrated in a more recent study by Pasterski et al.22 More studies are needed to better delineate hormonal effects on psychosexual development in humans. In comparison to biological influences on psychosexual development, the effects of environmental factors are more difficult to elucidate. In 2000, Rust et al. demonstrated that the sex of the older siblings influenced gender role development. In particular, having an older brother resulted in more masculine and less feminine behavior in both male and female younger siblings.23 Observational studies have been used to comment on the role of “nurture” on gender identity in people with

DSD. A recent report on three siblings with partial androgen insensitivity syndrome (AIS) demonstrated that gender identity was concordant with sex of rearing.24 It is important to note that these remain case studies and results are not representative of other individuals with DSD, making evaluation of environmental effects on psychosexual development challenging.

3.4  DISORDERS OF SEX DEVELOPMENT The following sections will discuss the epidemiology, clinical presentation, past and current literature on gender identity and assignment, and associated surgical interventions of DSD and congenital anomalies that commonly present with ambiguous genitalia (Table 3.2). Ambiguous genitalia have been historically referred to as intersex, hermaphroditism, transsexualism, sex reversal, and other gender-based diagnostic labels. In 2006, the Chicago Consensus renamed labels that were often considered controversial, unclear, and pejorative by patients. The term “DSD” was proposed and defined as congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical.8 “Male pseudohermaphrodite” was previously used to describe a patient with 46, XY karyotype with undervirilized or undermasculinized external genitalia. This has been relabeled as “46, XY DSD.” “Female pseudohermaphrodite,” which previously described 46, XX karyotype patients with overvirilization or masculinization, has been replaced by “46, XX DSD.” “True hermaphrodites” with both testicular and ovarian tissue have been renamed “ovotesticular DSD (OTDSD).” The revision of nomenclature hoped to incorporate both genetic and phenotypic aspects of sex development. Though there have been critiques of the new nomenclature, these terms have generally been accepted by the worldwide community.25

3.4.1  46, XX Disorders of Sex Development Overvirilization or masculinization of the female fetus derives from high levels of androgen from the fetus or an exogenous source. Exposure to androgens between weeks 8 and 14 of gestation results in posterior fusion of the labioscrotal folds and exposure after weeks 12 and 14 results in clitoromegaly. Despite the presence of ambiguous genitalia, normal ovaries and Müllerian derivatives are present. 3.4.1.1  Congenital Adrenal Hyperplasia CAH is a group of autosomal recessive disorders of cortisol synthesis (Fig. 3.1). The enzymatic defect results in a decrease in cortisol, which increases adrenocorticotrophic hormone (ACTH) via negative feedback. ACTH

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3.4  Disorders of Sex Development

TABLE 3.2 Recommendations for Gender Assignment and Projected Gender Identity by DSD Diagnosis. Sex assignment

Projected gender identity

Level of evidence

CAH

Female

Female

II-230,32,33 III19,34-37

CAH with Prader 4 or 5

Female or male Female or male

Diagnosis 46, XX DSD

46, XY DSD Androgen insensitivity syndrome Complete

Female

Female

III20,56

Partial

Male or female

Male or female

III51,56,59

5α-Reductase deficiency

Male

Male

II-267 III71, 72

17β-HSD3 deficiency

Male

Male

III74,75

Ovotesticular DSD

Male or female

Male or female

III81,82,84,85

Mixed gonadal dysgenesis

Male or female

Male or female

III88,91,93

46, XY, WITH SEVERE GENITALANOMALY Aphallia

Male

Male

III96,101,102

Micropenis

Male

Male

II-2109,110III56,109

Cloacal extrophy

Male or female

Male or female

III113

Level of evidence according to evidence-based medicine system of stratifying quality of research design developed by the US Preventative Services Task Force, defined as follows: I: Properly powered and conducted randomized controlled trial (RCT); wellconducted systematic review or meta-analysis of homogenous RCTs. II-1: Well-designed controlled trial without randomization. II-2: Well-designed cohort or case-control analytic study. II-3: Multiple time series with or without the intervention; dramatic results from uncontrolled experiments. III: Opinions of respective authorities, based on clinical experience; descriptive studies or case reports; reports of expert committees.

induces adrenal hyperplasia. As the pathway to production of adrenal androgens is normal, this causes virilization of the fetus. CAH accounts for approximately 95% of 46, XX DSD and approximately 50% of all cases of ambiguous genitalia.26 3.4.1.1.1  Clinical Presentation 21-hydroxylase deficiency occurs in 1/10,000–20,000 individuals.27 Presentation of 21-hydroxylase deficiency varies depending on the severity of the mutation, but there are three phenotypes: (1) salt wasting with severe virilization; (2) simple virilization; or (3) late onset of

31

virilization. Salt wasting with severe virilization can present with symptoms of electrolyte and fluid losses due to the lack of aldosterone and cortisol, including hyponatremia, hyperkalemia, acidosis, dehydration, and vascular collapse, requiring emergent attention. Milder cases of 21-hydroxylase deficiency may be detected on newborn screening. Milder forms of virilization include clitoromegaly or enlarged phallus. Late-onset symptoms include excessive or premature acne and/or sexual hair in childhood or adolescence. Whereas newborn males present with macrogenitalia, females typically present with ambiguous genitalia (Fig. 3.2A). Other less common mutations that cause CAH include 11β-hydroxylase deficiency and 3β-hydroxysteroid dehydrogenase type 2 deficiency. More recently, glucocorticoid receptor gene mutations and P450 oxidoreductase mutations have been found to also cause CAH and can also present with ambiguous genitalia.28,29 As most studies are retrospective and the prevalence of CAH is low, literature reviews on the topic generally include all etiologies of CAH as opposed to examining specific mutations. The studies address outcomes based on the severity of virilization. A grading score often used in studies of CAH is the Prader scale. The scale is a six-point score system that starts from zero, which describes normal female external genitalia, and extends to five, which describes normal male external genitalia (Fig. 3.3).30 Our understanding of the effects of prenatal androgen exposure indicates that a greater androgen excess leads to more severe virilization.31 3.4.1.1.2  Gender Assignment and Identity The Chicago Consensus initially recommended assigning all patients with 46, XX due to CAH, including those with severely virilized genitalia, to the female gender.8 Historically, correctly diagnosed females with CAH were assigned to the female gender due to fertility potential with ovaries, uterus, vagina, and available medical management with glucocorticoid replacement therapy. In cases of missed or delayed diagnoses, gender reassignments from male to female were supported for those less than 2 years of age, as it was previously believed that stable gender identity was not yet established. In a literature review of 46, XX CAH assigned males and females from 1950–2005 by Dessens et al., 95% (n = 250) of 46, XX CAH raised as females later identified with female identity. In comparison, 12% of 46, XX females that were raised as males later regarded themselves as females or had gender dysphoria.19 Berenbaum and Bailey administered a 9 point gender identity interview to 43 girls with CAH due to 21-hydroxylase deficiency, 7 tomboys, and 29 control girls. Girls with CAH had more male-typical scores than controls but less than that of tomboys. Only 5 of the 43 girls with CAH scored outside the range scored by the controls. Girls with CAH

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3.  Gender Identity in Disorders of Sex Development

FIGURE 3.1  Adrenal steroid biosynthesis pathway. Enzymatic defects that commonly lead to ambiguous genitalia are indicated by a star. Reprinted and adapted from Li J, Papadopoulos V, Vihma V. Steroid Biosynthesis in Adipose Tissue. Steroids. 103:89–104, Copyright (2015), with permission from Elsevier.

FIGURE 3.2  Examples of ambiguous genitalia of various DSD. (A) 46, XX DSD—Female infant with CAH, demonstrating markedly virilized external genitalia. (B) 46, XY DSD—Initially identified as female but found to have 46, XY with hypospadias, bilateral undescended testes; workup suggestive of PAIS. (C) 46, XY DSD—Infant with mixed gonadal dysgenesis. (D) 46, XY DSD—Infant with severe hypospadias, bilateral undescended testes, and retained Müllerian structures. Reprinted and adapted from Yang JH, Baskin LS, DiSandro M, Gender identity in disorders of sex development: review article. Urology. 75:153–159, Copyright (2010), with permission from Elsevier.

were similar to controls in regard to individual items of gender identity such as feelings of discomfort as a girl or wish to be a boy. The girls with CAH with male typical scores did not necessarily have greater genital virilization or age at genital reconstructive surgery.32 Similar results were reported by Meyer-Bahlburg et  al. who compared 15 girls with CAH, 30 control girls, and 15 control boys and found no differences in gender identity between control girls and girls with CAH.33 Studies such as these suggest that gender identity may not correlate to degree of virilization, and girls with CAH should be raised females. With new outcome studies on severely virilized 46, XX CAH raised as males, the updated Consensus recommended consideration of male gender assignment in fully masculinized individuals with Prader 4 or 5.8,9 Meyer-Bahlburg et  al. compared the three subtypes of CAH with controls and found that degree of CAH severity correlated with degree of masculinization, and severity of masculinization has been shown to correlate with gender identity scores.30,31 Hines et al. reported that 31% of females with CAH over the age of 18 (n = 16) reported recent behavior as bisexual or homosexual and endorsed gender dysphoria.34 Reiner provided personal interviews of 84 patients at a pediatric psychodevelopment clinic of various DSD. Eleven of the eighty-four

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3.4  Disorders of Sex Development

33

FIGURE 3.3  Prader scale. Female external genitalia viewed from lithotomy (top) and in cross-section (bottom). Reprinted from Turcu AF, Auchus RJ, Adrenal steroidogenesis and congenital adrenal hyperplasia. Endocrinol Metab Clin North Am. 2015;44(2):275–96. Copyright (2015), with permission from Elsevier.

patients had CAH. Of the 11, 6 identified with female identity, while 5 declared male identity. Those who identified as male scored a Prader 5, supporting the assertion that increased prenatal androgens influence gender identity and promote masculine traits.35 In a response to the Chicago Consensus, Lee and Houk reported a case series of twelve 46, XX CAH individuals born with Prader 4 or 5 genitalia who were assigned male gender at birth prior to diagnosis. Follow-up was obtained at ages 35–69 years old, and these individuals had male gender identity, heterosexual orientation to females, normal libido, and appropriate social interactions. Gender satisfaction correlated with good counseling and family support.36 These findings have led Houk and Lee to recommend male gender assignment in highly virilized 46, XX CAH individuals. In their review, arguments supporting this recommendation include the high-risk of gender dysphoria in 46, XX CAH patients raised as females, reports of male gender reassignment in femaleraised individuals, and no reports of gender change in those raised as males. Furthermore, male gender assignment in severely virilized patients result in no loss of genital tissue, and therefore provide more options for adult gender reassignment if desired.37 These outcomes studies are limited by small study populations, but the results do suggest that male assignment is feasible and may have improved outcomes, particularly when there is good social support.

performed prepubertally.38 If surgery is pursued, there is inconclusive evidence on the timing of surgery. Factors favoring early surgical repair include technical ease, opportunity for patients to live with their stated gender, and decreased anxiety for parents and patients regarding anticipated gender assignments and possible surgical interventions.39 Advocates for delayed surgery argue for allowing gender identity to develop before commitment due to the potential harm of gender dysphoria or gender reassignment surgery.40 In a survey of pediatric urologists, 78% reported that early surgery before 2 years of age was preferred.40 However, in 2614 individuals with CAH in the United States, only 18% proceeded with surgery within the first 4 years.41 Good surgical outcome optimizes urinary and sexual function, including genital appearance, normal menstrual outflow, and satisfactory intercourse in adult life with painless penetration.42 Outcome studies have yielded variable reports. Creighton et  al. reported in a single institution review that 41% of patients perceived poor cosmetics; 98% needed further surgery for cosmetic results, tampon use, or intercourse; and 89% required second-stage surgeries after planned one-stage procedures.43 In comparison, Lean et  al. revealed that 72% of patients had good cosmetic results and 22% were satisfied.44 Despite the variable reports, it is generally thought that surgical interventions should be performed at high volume centers for improved outcomes.45

3.4.1.1.3  Surgical Interventions For those with CAH and severe virilization (classified as Prader 3–5), the 2006 and 2016 guidelines state that surgical repair, including clitoroplasty, labioplasty, vaginoplasty, perineoplasty, and/or urogenital sinus mobilization, can be considered.8,9 Vaginal dilation should only occur in adolescents as it can cause significant psychological distress if

3.4.1.1.4  Quality of Life Conflicting data have been presented on the quality of life and satisfaction of individuals with CAH. Berenbaum et  al. showed no differences between females with CAH and their unaffected female relatives on measures of well-being, social closeness, stress reaction, and alienation. These measures were also not

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3.  Gender Identity in Disorders of Sex Development

significantly associated with genital virilization or age at genital surgery.46 In contrast, Gilban et al. evaluated the health-related quality of life of children and adolescents with CAH due to 21-hydroxlase and found decreased overall satisfaction in physical and psychosocial dimensions in comparison to controls; there was also no difference in regards to degree of virilization.47 Strandqvist et al. assessed how individuals adapted to life and community by measuring outcomes such as education, employment, marriage, and fertility. Individuals with CAH registered in the Sweden National Registry were analyzed and compared to other females. Those with CAH received less primary education, took more sick leave, were more likely to be single, and were less likely to have biological children.48 Despite the variability in outcome studies and results in comparison to controls, the majority of those affected have positive results, suggesting that while there is room for improvement, current management has shown some success. These data again emphasize the need for standardized, long-term follow-up by multidisciplinary DSD teams examining outcomes from surgical and functional standpoints, as well as tracking psychosexual well-being.

3.4.2  46, XY Disorders of Sex Development 3.4.2.1  Androgen Insensitivity Syndrome AIS characterizes clinical disorders due to dysfunction of the androgen receptor (AR) leading to hormone resistance despite age-appropriate production. Severity may range from complete androgen insensitivity syndrome (CAIS), partial insensitivity (PAIS), to mild insensitivity (MAIS).49,50 The AR is encoded by an 8 exon gene on the X chromosome, and to date, more than 800 mutations have been found to cause androgen insensitivity.50 A nationwide survey in the Netherlands estimated the prevalence of AIS to be 1 in 99,000 genetic males based on the presence of mutations in the AR gene.51 Other reports estimate the prevalence of AIS to be 1 in 90,000– 100,000 individuals. Prevalence of complete androgen insensitivity is 1 in 20,000–64,000 newborn males; the prevalence of MAIS and PAIS is unknown.52 3.4.2.1.1  Clinical Presentation Patients with CAIS have the female phenotype with normal breast development due to aromatization of androgens to estrogens.53 Patients have blind-ending vaginas with absent uteri due to regression of the Müllerian ducts.50,52,53 Diagnoses are typically made during puberty when individuals with CAIS present with primary amenorrhea; more infrequently, they may be diagnosed during infertility workup.52 Diagnoses may also be made during infancy when testicles are found during inguinal hernia repairs in female-appearing infants.53 A prospective study conducted by Sarpel

et  al. found 1.1% of prepubescent girls with hernias to have CAIS.54 In contrast, there is great variability in PAIS phenotypes, which is dependent on residual AR function (Fig. 3.2B). As such, the true incidence of PAIS is more difficult to ascertain. AR gene mutations can only be found in approximately 28% of patients diagnosed with PAIS.55 Patients may present with micropenis, severe hypospadias, and bifid scrotum. In some cases, they may present with female-appearing external genitalia and clitoramegaly.50 Given the variability in phenotype, PAIS may sometimes be a diagnosis of exclusion, making outcomes studies challenging. MAIS is the mildest form of AIS. Patients may present without overt genital anomalies, but may have evidence of oligospermia.50 Some may present with gynecomastia or decreased virilization.52,55 3.4.2.1.2  Gender Assignmentand Identity The degree of AIS (complete, partial, or mild) and age at diagnosis influence management.50 Given the female genitalia, patients with CAIS are generally raised as females with few reports of gender dysphoria. In 2000, Wisniewski et  al. studied 14 subjects with CAIS. They reported a high degree of femininity throughout their development; 13 of the 14 individuals endorsed female heterosexual orientation. All 14 individuals were raised as women and were satisfied with their sex of rearing.20 Mazur examined 156 case reports of patients with CAIS raised as females, and none initiated gender reassignment, though gender dysphoria was not mentioned in most cases.56 In 2010, T’Sjoen et  al. reported the first case of persistent male gender identity in a patient with CAIS.57 Although female sex of rearing for patients with CAIS remains the standard recommendation, it is important to note that case by case variations remain, and management should ultimately be tailored to each individual patient.9 Gender assignment is difficult in patients with PAIS given the wide phenotypic range.50 In 2000, Ahmed et al. proposed the external masculinization score to assist in objectively describing the degree of masculinization of patients with ambiguous genitalia; the score takes into account the degree of scrotal fusion, the presence of a micropenis, the location of the urethral meatus, and the locations of the gonads.58 It is suggested that the external masculine score in infancy may help predict the degree of virilization at puberty, which may influence the decision for sex of rearing.50 As opposed to individuals with CAIS who are often raised as females, patients with PAIS may be raised as males or females. Kolesinska et  al. examined 118 cases of PAIS and noted an increase in the male to female gender assignment ratio. The male to female gender assignment ratio was 1.4 in the pre-1990 cohort, 2.3 in the 1990–99 cohort, and 6.3 in the post-1999 cohort.59

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Mazur evaluated 99 cases of PAIS of which a majority of the individuals was satisfied with their gender assignments. Nine underwent gender reassignment based on the patients’ wishes; three individuals reassigned from female to male gender, and six reassigned from male to female gender.56 It is interesting to note that neither male nor female sex of rearing correlated with increased need for psychological counseling for depression and substance abuse in patients with PAIS.60 3.4.2.1.3  Surgical Intervention Timing of surgical intervention remains highly controversial. Similar to cases of CAH, some argue that early intervention can create an appearance congruent to the sex of rearing and relieve familial distress regarding need for future intervention. Arguments have also been made for delayed intervention to involve the patient in the decision-making process.61 For patients with CAIS, vaginal dilation has been shown to be an effective treatment. Ismail-Pratt et  al. showed in patients with CAIS and Mayer-RokintanskyKuster-Hauser syndrome (MRKH) that vaginal dilation increased vaginal length from 4.0 to 8.5 cm with improved patient perception of vaginal length.62 At times, vaginal surgery may be indicated, and options include open or laparoscopic vaginal reconstruction/ substitution.63,64 CAIS patients are often raised as females, and gonadectomy is therefore recommended to reduce the risk of gonadal tumors. Recent studies estimate malignancy risk to be at 0.8–2.0% while others report rates as high as 5%.52,65 Risk of malignancy is low in prepubescent patients, so ideal timing of gonadectomy remains a question. Delayed gonadectomy has the benefit of allowing for spontaneous puberty whereas prepubescent gonadectomy would require hormone replacement. If gonadectomy does not occur in childhood, it is recommended in early adulthood and may be pursued laparoscopically.50 According to the recent update to the 2006 Consensus, there remains no agreement amongst surgeons regarding surgical timing. However, there is increasing emphasis on patient input, and this may argue for delayed gonadectomy.9 Surgical management is variable with PAIS given the wide phenotypic range. Infants who are assigned to the female gender will require genitoplasty and gonadectomy prior to puberty to reduce risk of virilization.50 Patients raised as males will require hypospadias repair and orchiopexy at an early age. As adolescents, they may require reduction mammoplasty should gynecomastia occur.50,52 3.4.2.2 5α-Reductase Type 2 Deficiency 5α-reductase type 2 deficiency is a rare autosomal recessive disorder caused by mutations in the SRD5A2

35

gene. In normal male development, 5α-reductase converts testosterone to dihydrotestosterone (DHT), which is necessary for male genital development. SRD5A2 gene mutations result in DHT deficiency and disrupt virilization. This condition has been reported in the Dominican Republic, Lebanon, and Papua New Guinea. Given the rarity of the disease, its true prevalence is unknown.66 3.4.2.2.1  Clinical Presentation Individuals with 5α-reductase type 2 deficiency have reduced or absent function of the steroid-5α-reductase and often present with pseudovaginal perineoscrotal hypospadias, microphallus, and undescended testicles.67 Enzymatic function varies with the underlying mutation, resulting in phenotypic variations at birth.68 In 2010, Maimoun et al. studied a cohort of 55 patients with SRD5A2 mutations and found that the most frequent phenotypes were clitoromegaly, which occurred in 49.1% of the patients, and microphallus with hypospadias, which was found in 32.7% of patients. Female genitalia and isolated microphallus were rare presentations and accounted for 7.3% and 3.6% of the study group, respectively.67 This condition is characterized by normal or elevated testosterone levels and low DHT levels. A testosterone/ DHT ratio greater than 10 after hCG stimulation generally indicates 5α-reductase deficiency. However, there are certain mutations that do not present with increased plasma testosterone/DHT ratios, making diagnosis challenging.66,67 Patients with 5α-reductase type 2 deficiency undergo phenotypical changes during puberty. At the onset of puberty, the testes secrete testosterone and increase circulating testosterone levels. It is hypothesized that the rising testosterone levels during puberty are able to produce virilization through AR binding of testosterone at lower affinity. In addition, there may be generation of sufficient levels of DHT either by the action of 5α-reductase type I (active in nongenital skin and some brain areas) or through the expression of low levels of 5α-reductase type III by unknown mechanisms.69 Patients undergo male-like pubertal changes with characteristics such as phallic growth, testicular descent, development of pubic hair, increased muscle mass, deepening of the voice, and a growth spurt.70 3.4.2.2.2  Gender Assignment and Identity Gender assignment is dependent on the phenotype at diagnosis. In cases of severe defects of the external genitalia, some individuals are raised as females.67 Those who were raised as males live as males.71 However, approximately two-thirds of patients raised as females convert to male gender after virilization at puberty. In areas such as Dominican Republic, New Guinea, and Turkey where 5α-reductase type 2 deficiency is more prevalent, local

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3.  Gender Identity in Disorders of Sex Development

beliefs, rituals, and cultures influence the process of gender assignment. Nevertheless, many eventually identify with the male gender.72 As such, male gender assignment is recommended for these individuals.9 3.4.2.2.3  Surgical Intervention If male gender is intended, surgical intervention is recommended to correct hypospadias, chordee, and undescended testicles. Androgen administration may be necessary to increase phallic length prior to repair. If female gender assignment is made, gonadal tissue should be removed prior to virilization at puberty, and clitoral reduction and vaginoplasty may be considered in cases of severe virilization.66 3.4.2.3 17β-Hydroxysteroid Dehydrogenase Type 3 Deficiency 17β-hydroxysteroid dehydrogenase 3(17β-HSD3) deficiency is a rare autosomal recessive disorder. The 17β-HSD isoenzyme converts androstenedione into testosterone in the male testis, which is vital in development of the male genitalia. Mutations in the HSD17ß3 gene result in normal development of Wolffian duct derivatives with undervirilization of the external genitalia. True incidence of the disease is unknown, but it has been reported in people of Arab descent in Gaza and in the Dutch population with an estimated prevalence of 1 in 150,000.73 3.4.2.3.1  Clinical Presentation Patients with 17β-HSD3 deficiency often appear female, with evidence of clitoromegaly, blind-ending vagina, and labial fusion. They are raised as females, and virilization tends to occur at puberty due to peripheral conversion of androstenedione to testosterone by other 17ß-HSD isoenzymes. These patients have elevated serum androstenedione concentrations with testosterone/androstenedione ratios of 3 weeks after resolution of infection/inflammation) localized mechanical allodynia and hyperinnervation of peptidergic nociceptor and sympathetic fibers.229,230 Around 40% of the infected animals (Candida + fluconazole) were allodynic versus 5.5% of the fluconazole controls (saline + fluconazole). The increased nerve innervation of the

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4.8  Future Directions

vagina was measured by comparing protein gene product 9.5, calcitonin gene-related peptide, and vesicular monoamine transporter 2 immunoreactivity in infected and noninfected mice. Long-lasting behavioral allodynia in a subset of mice was also observed after a single, extended Candida infection, as well as after repeated vulvar inflammation induced with zymosan, a mixture of fungal antigens. This model resembles provoked localized vulvodynia in women, the most common form of vulvodynia, an idiopathic pain disorder associated with a history of recurrent candidiasis, which is characterized by vulvar allodynia and hyper innervation.229,231 A reduction in sexual motivation in female mice was also reported after induction of inflammatory pain.31

4.6.3  Pelvic Visceral/Muscle Pain Uterine inflammation in female rodents can be initiated by injection of mustard oil into one uterine horn. This induces behaviors that mimic pelvic pain and referred muscle hyperalgesia in women with inflammatory conditions of their reproductive area. After 2–4 days, the majority of rats show spontaneous pain behavior (major episodes of movements/postures indicative of pelvic pain) and referred hyperalgesia (measured by vocalization) in the ipsilateral flank muscles, in response to stimulation.232,233 The areas of referred muscle hyperalgesia are also the site of neurogenic plasma extravasation in the skin, which is the first experimental evidence of trophic changes in sites of referred pain from viscera, a well-known phenomenon in the clinical setting.234 Ovariectomy has been used in mice and rats to produce a condition of visceral pain/hypersensitivity of the pelvic area, which has been proposed as a model of a hormonally dependent hyperalgesia resembling functional pain in women.235–238 Ovariectomy also increases depression in rodents.236–238 Ovariectomized mice and rats present a hyperalgesic state (a robust mechanical and thermal hyperalgesia in the abdominal and pelvic regions) of slow onset (4 weeks) and long duration, as well as visceral hypersensitivity. Hormone replacement with 17β-estradiol prevents and reverses the development of hyperalgesia but does not stop the involution of the internal reproductive organs.239 Experiments in mice have shown that spinal ERK 1/2 is involved in the estrogen-dependent chronic visceral hyperalgesia.240 Ovariectomized mice show a significant increase in the activation of ERK 1/2 (the extracellular signal-regulated kinases 1 and 2, members of the MAPK (mitogen-activated protein kinases) family) in the lumbosacral spinal cord which followed the time course of the hyperalgesia. Estrogen replacement reversed both the development of the hyperalgesia and the enhanced activation of ERK 1/2, while intrathecal injections of the ERK 1/2 inhibitor,

U0126, significantly attenuated the abdominal hyperalgesia (up to 24 h after the injection) and reversed the enhanced expression of ERK 1/2.240

4.7 ENDOMETRIOSIS Endometriosis can result in pain, related to secondary dysmenorrhea or a more generalized chronic pelvic pain syndrome; in addition, vaginal hyperalgesia can occur that results in sexual dysfunction.241 In women, the intensity of painful symptoms is not related to the size or location of the lesions.242 A number of nonhuman and rodent models of endometriosis have been developed to study subfertility; more recently, these techniques are being used to study pain.243 For example, endometriosis can be induced in female rats by grafting pieces of autologous endometrium (from one uterine horn) in different locations of the abdominal cavity: on alternate cascade mesenteric arteries that supply the caudal small intestine, at the level of the ovary and inner surface of abdominal muscles. Two to three weeks later, fluidfilled cysts develop at the implantation sites. Estradiol is required for cyst maintenance, and the severity of vaginal hyperalgesia varies with the estrus cycle. Interestingly, the cysts develop their own sensory and sympathetic nerve supply, which may be useful for future vascular studies. Evidence has been provided for a role of local cannabinoids (CB1) in the expression of the hyperalgesia associated with the endometrial cysts.242 Mouse studies of this endometrial model have been developed244 allowing future studies to utilize the availability of the many transgenic mouse strains. When combined with an artificial ureteral calculosis, endometriosis in rats produces a notable enhancement of the poststone spontaneous pain behavior (increase in both “ureteral” and “uterine” typical behavior, monitored over several days at video tape recordings, and increase in pelvic muscle hyperalgesia).220 This combination model closely resembles the clinical condition of “viscero-visceral hyperalgesia” (i.e., increased spontaneous pelvic and urinary pain, as well as in referred pelvic muscle hyperalgesia) extensively documented in women with endometriosis plus ureteral calculosis, and therefore is particularly suitable for studies understanding the pathophysiological mechanisms of this condition in women.221,245

4.8  FUTURE DIRECTIONS Studies on gonadal steroid hormones have been conducted, but there remains a lack of studies in females examining other hormones, such as thyroid and stress hormones. Animal models for pathological and disease

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4.  Female Sexual Function: The Role of Animal Models in Assessing Female Sexual Dysfunction

states are lacking, such as cancer, stroke, cardiovascular dysfunction, diabetes, and aging. The pelvic and urogenital pain models should also be used to explore the impact of pain on sexual dysfunction; effective pharmacologic treatments (classic and newer) for the pain should also be tested with respect to their effectiveness on the sexual dysfunction parameters. The study of pain comorbidities (e.g., bladder pain syndrome, irritable bowel syndrome, fibromyalgia, and myofascial pain syndromes) related to pelvic and urogenital pain that may lead to sexual dysfunction in females has yet to be fully explored. These research approaches may mimic the clinical condition of extensive co-occurrence of several pain conditions in the urogenital area and sexual dysfunction observed in patients.245–248

Acknowledgments Ursula Wesselmann’s work has been supported by NIH grant HD39699 (NICHD) and the Office of Research on Women’s Health (ORWH). Lesley Marson’s work was supported by NIH grants NS0039166 and NS029420 (NINDS) and HD30149 (NICHD). Permission granted for use of figures and portion of text from Marson L, Giamberardino MA, Costantini R, Czakanski P, Wesselmann U. Animal models for the study of female sexual dysfunction. Sex Med Rev. 2013;1(2):108–122.

References 1. Giraldi A, Marson L, Nappi R, et al. Physiology of female sexual function: animal models. J Sex Med. 2004;1(3):237–253. 2. Giraldi A, Rellini AH, Pfaus J, Laan E. Female sexual arousal disorders. J Sex Med. 2012;10(1):58–73. 3. Basson R, Althof S, Davis S, et  al. Summary of the recommendations on sexual dysfunctions in women. J Sex Med. 2004;1(1):24–34. 4. Bachmann GA, Rosen R, Pinn VW, et  al. Vulvodynia: a stateof-the-art consensus on definitions, diagnosis and management. J Reprod Med. 2006;51(6):447–456. 5. Kingsberg S, Althof SE. Evaluation and treatment of female sexual disorders. Int Urogynecol J Pelvic Floor Dysfunct. 2009;20(Suppl 1):S33–S43. 6. Nguyen RH, Veasley C, Smolenski D. Latent class analysis of comorbidity patterns among women with generalized and localized vulvodynia: preliminary findings. J Pain Res. 2013;6:303–309. 7. Nguyen RH, Ecklund AM, Maclehose RF, Veasley C, Harlow BL. Co-morbid pain conditions and feelings of invalidation and isolation among women with vulvodynia. Psychol Health Med. 2012;17(5):589–598. 8. Levin RJ. Sex and the human female reproductive tract-what really happens during and after coitus. Int J Impot Res. 1998;10(Suppl 1):S14–S21. 9. Masters WH, Johnson VE. In Human Sexual Response. Boston, MA: Little, Brown & Co; 1966. 10. Marson L, Giamberardino MA, Costantini R, Czakanski P, Wesselmann U. Animal models for the study of female sexual dysfunction. Sex Med Rev. 2013;1(2):108–122. 11. Tan O, Bradshaw K, Carr BR. Management of vulvovaginal atrophy-related sexual dysfunction in postmenopausal women: an up-to-date review. Menopause. 2012;19(1):109–117.

12. Brotto LA, Bitzer J, Laan E, Leiblum S, Luria M. Women’s sexual desire and arousal disorders. J Sex Med. 2010;7(1 Pt 2):586–614. 13. Laan E, Rellini AH, Barnes T. Standard operating procedures for female orgasmic disorder: consensus of the international society for sexual medicine. J Sex Med. 2012;10(1):74–82. 14. Wallen K, Hassett JM. Sexual differentiation of behaviour in monkeys: role of prenatal hormones. J Neuroendocrinol. 2009;21(4):421–426. 15. Borrow AP, Cameron NM. The role of oxytocin in mating and pregnancy. Horm Behav. 2012;61(3):266–276. 16. Wen S, Gotze IN, Mai O, Schauer C, Leinders-Zufall T, Boehm U. Genetic identification of GnRH receptor neurons: a new model for studying neural circuits underlying reproductive physiology in the mouse brain. Endocrinology. 2011;152(4):1515–1526. 17. Spiteri T, Ogawa S, Musatov S, Pfaff DW, Agmo A. The role of the estrogen receptor alpha in the medial preoptic area in sexual incentive motivation, proceptivity and receptivity, anxiety, and wheel running in female rats. Behav Brain Res. 2012;230(1):11–20. 18. Spiteri T, Musatov S, Ogawa S, Ribeiro A, Pfaff DW, Agmo A. Estrogen-induced sexual incentive motivation, proceptivity and receptivity depend on a functional estrogen receptor alpha in the ventromedial nucleus of the hypothalamus but not in the amygdala. Neuroendocrinology. 2010;91(2):142–154. 19. Klingerman CM, Patel A, Hedges VL, Meisel RL, Schneider JE. Food restriction dissociates sexual motivation, sexual performance, and the rewarding consequences of copulation in female Syrian hamsters. Behav Brain Res. 2011;223(2):356–370. 20. Martinez LA, Albers HE, Petrulis A. Blocking oxytocin receptors inhibits vaginal marking to male odors in female Syrian hamsters. Physiol Behav. 2010;101(5):685–692. 21. Hedges VL, Chakravarty S, Nestler EJ, Meisel RL. Delta FosB overexpression in the nucleus accumbens enhances sexual reward in female Syrian hamsters. Genes Brain Behav. 2009;8(4):442–449. 22. Wood RI. Thinking about networks in the control of male hamster sexual behavior. Horm Behav. 1997;32(1):40–45. 23. Adkins-Regan E, Leung CH. Hormonal and social modulation of cloacal muscle activity in female Japanese quail. Physiol Behav. 2006;87(1):82–87. 24. Ball GF, Balthazart J. Hormonal regulation of brain circuits mediating male sexual behavior in birds. Physiol Behav. 2004;83(2):329–346. 25. Simmons DA, Yahr P. Distribution of catecholaminergic and peptidergic cells in the gerbil medial amygdala, caudal preoptic area and caudal bed nuclei of the stria terminalis with a focus on areas activated at ejaculation. J Chem Neuroanat. 2011;41(1):13–19. 26. Ross HE, Freeman SM, Spiegel LL, Ren X, Terwilliger EF, Young LJ. Variation in oxytocin receptor density in the nucleus accumbens has differential effects on affiliative behaviors in monogamous and polygamous voles. J Neurosci. 2009;29(5):1312–1318. 27. Yamamoto Y, Carter CS, Cushing BS. Neonatal manipulation of oxytocin affects expression of estrogen receptor alpha. Neuroscience. 2006;137(1):157–164. 28. Hammock EA, Young LJ. Oxytocin, vasopressin and pair bonding: implications for autism. Philos Trans R Soc Lond B Biol Sci. 2006;361(1476):2187–2198. 29. Zipse LR, Brandling-Bennett EM, Clark AS. Paced mating behavior in the naturally cycling and the hormone-treated female rat. Physiol Behav. 2000;70(1-2):205–209. 30. Beach FA. Sexual attractivity, proceptivity, and receptivity in female mammals. Horm Behav. 1976;7(1):105–138. 31. Farmer MA, Leja A, Foxen-Craft E, et  al. Pain reduces sexual motivation in female but not male mice. J Neurosci. 2014;34(17):5747–5753.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

32. Pfaus JG. Neurobiology of sexual behavior. Curr opin Neurobiol. 1999;9(6):751–758. 33. Pfaus JG. Revisiting the concept of sexual motivation. Annu Rev Sex Res. 1999;10:120–156. 34. McClintock MK, Anisko JJ. Group mating among Norway rats: I. sex differences in the pattern and neuroendocrine consequences of copulation. Animal Behav. 1982;30:398–409. 35. Ismail N, Gelez H, Lachapelle I, Pfaus JG. Pacing conditions contribute to the conditioned ejaculatory preference for a familiar female in the male rat. Physiol Behav. 2009;96(2):201–208. 36. Johansen JA, Clemens LG, Nunez AA. Characterization of copulatory behavior in female mice: evidence for paced mating. Physiol Behav. 2008;95(3):425–429. 37. Jenkins WJ, Becker JB. Female rats develop conditioned place preferences for sex at their preferred interval. Horm Behav. 2003;43(4):503–507. 38. Erskine MS. Solicitation behavior in the estrous female rat: a review. Horm Behav. 1989;23(4):473–502. 39. Jenkins WJ, Becker JB. Dynamic increases in dopamine during paced copulation in the female rat. Eur J Neurosci. 2003;18(7):1997–2001. 40. Snoeren EM, Chan JS, de Jong TR, Waldinger MD, Olivier B, Oosting RS. A new female rat animal model for hypoactive sexual desire disorder; behavioral and pharmacological evidence. J Sex Med. 2011;8(1):44–56. 41. Wallen K. Hormonal influences on sexually differentiated behavior in nonhuman primates. Front Neuroendocrinol. 2005;26(1):7–26. 42. Wallen K. Desire and ability: hormones and the regulation of female sexual behavior. Neurosci Biobehav Rev. 1990;14(2):233–241. 43. Tarin JJ, Gomez-Piquer V. Do women have a hidden heat period? Hum Reprod. 2002;17(9):2243–2248. 44. Wallen K, Zehr JL. Hormones and history: the evolution and development of primate female sexuality. J Sex Res. 2004;41(1):101–112. 45. Thorp Jr. J, Palacios S, Symons J, Simon J, Barbour K. Improving prospects for treating hypoactive sexual desire disorder (HSDD): development status of flibanserin. BJOG. 2014;121(11):1328–1331. 46. Simon JA, Kingsberg SA, Shumel B, Hanes V, Garcia Jr. M, Sand M. Efficacy and safety of flibanserin in postmenopausal women with hypoactive sexual desire disorder: results of the SNOWDROP trial. Menopause. 2014;21(6):633–640. 47. Derogatis LR, Komer L, Katz M, et al. Treatment of hypoactive sexual desire disorder in premenopausal women: efficacy of flibanserin in the VIOLET Study. J Sex Med. 2012;9(4):1074–1085. 48. Stahl SM, Sommer B, Allers KA. Multifunctional pharmacology of flibanserin: possible mechanism of therapeutic action in hypoactive sexual desire disorder. J Sex Med. 2011;8(1):15–27. 49. Kennedy S. Flibanserin: initial evidence of efficacy on sexual dysfunction, in patients with major depressive disorder. J Sex Med. 2010;7(10):3449–3459. 50. Aubert Y, Gustison ML, Gardner LA, et  al. Flibanserin and 8-OH-DPAT implicate serotonin in association between female marmoset monkey sexual behavior and changes in pair-bond quality. J Sex Med. 2012;9(3):694–707. 51. Gelez H, Greggain-Mohr J, Pfaus JG, Allers KA, Giuliano F. Flibanserin treatment increases appetitive sexual motivation in the female rat. J Sex Med. 2013;10(5):1231–1239. 52. Allers KA, Dremencov E, Ceci A, et  al. Acute and repeated flibanserin administration in female rats modulates monoamines differentially across brain areas: a microdialysis study. J Sex Med. 2010;7(5):1757–1767. 53. Miryala CS, Hiegel C. Uphouse L. Sprague-Dawley and Fischer female rats differ in acute effects of fluoxetine on sexual behavior. J Sex Med. 2013;10(2):350–361.

55

54. Rosen RC, Lane RM, Menza M. Effects of SSRIs on sexual function: a critical review. J Clin Psychopharmacol. 1999;19(1):67–85. 55. Rowland D, McMahon CG, Abdo C, et al. Disorders of orgasm and ejaculation in men. J Sex Med. 2010;7(4 Pt 2):1668–1686. 56. Corona G, Ricca V, Bandini E, et  al. Selective serotonin reuptake inhibitor-induced sexual dysfunction. J Sex Med. 2009;6(5):1259–1269. 57. Seidman S. Ejaculatory dysfunction and depression: pharmacological and psychobiological interactions. Int J Impot Res. 2006;18(Suppl 1):S33–S38. 58. McMahon CG, Abdo C, Incrocci L, et  al. Disorders of orgasm and ejaculation in men. J Sex Med. 2004;1(1):58–65. 59. Clayton AH. Female sexual dysfunction related to depression and antidepressant medications. Curr Womens Health Rep. 2002;2(3):182–187. 60. Uphouse L. Pharmacology of serotonin and female sexual behavior. Pharmacol Biochem Behav. 2014;121:31–42. 61. Adams S, Heckard D, Hassell J, Uphouse L. Factors influencing fluoxetine-induced sexual dysfunction in female rats. Behav Brain Res. 2012;235(1):73–81. 62. Guptarak J, Sarkar J, Hiegel C, Uphouse L. Role of 5-HT(1A) receptors in fluoxetine-induced lordosis inhibition. Horm Behav. 2010;58(2):290–296. 63. Sinclair-Worley L, Uphouse L. Effect of estrogen on the lordosisinhibiting action of ketanserin and SB 206553. Behav Brain Res. 2004;152(1):129–135. 64. Snoeren EM, Veening JG, Olivier B, Oosting RS. Serotonin 1A receptors and sexual behavior in female rats: a review. Pharmacol Biochem Behav. 2014;121:43–52. 65. Snoeren E, Chan J, Bovens A, et  al. Serotonin transporter null mutation and sexual behavior in female rats: 5-HT1A receptor desensitization. J Sex Med. 2010;7(7):2424–2434. 66. Olivier JD, Van Der Hart MG, Van Swelm RP, et  al. A study in male and female 5-HT transporter knockout rats: an animal model for anxiety and depression disorders. Neuroscience. 2008;152(3):573–584. 67. Meston CM, Frohlich PF. The neurobiology of sexual function. Arch Gen Psychiatry. 2000;57(11):1012–1030. 68. Blaicher W, Gruber D, Bieglmayer C, Blaicher AM, Knogler W, Huber JC. The role of oxytocin in relation to female sexual arousal. Gynecol Obstet Invest. 1999;47(2):125–126. 69. Carmichael MS, Warburton VL, Dixen J, Davidson JM. Relationships among cardiovascular, muscular, and oxytocin responses during human sexual activity. Arch Sex Behav. 1994;23(1):59–79. 70. Rubio-Casillas A, Rodriguez-Quintero CM, Rodriguez-Manzo G, Fernandez-Guasti A. Unraveling the modulatory actions of serotonin on male rat sexual responses. Neurosci Biobehav Rev. 2015;55:234–246. 71. Veening JG, de Jong TR, Waldinger MD, Korte SM, Olivier B. The role of oxytocin in male and female reproductive behavior. Eur J Pharmacol. 2015;753:209–228. 72. Argiolas A, Melis MR. Neuropeptides and central control of sexual behaviour from the past to the present: a review. Prog Neurobiol. 2013;108:80–107. 73. Baskerville TA, Douglas AJ. Dopamine and oxytocin interactions underlying behaviors: potential contributions to behavioral disorders. CNS Neurosci Ther. 2010;16(3):e92–123. 74. Donaldson ZR, Young LJ. Oxytocin, vasopressin, and the neurogenetics of sociality. Science. 2008;322(5903):900–904. 75. Boccia ML, Goursaud AP, Bachevalier J, Anderson KD, Pedersen CA. Peripherally administered non-peptide oxytocin antagonist, L368,899((R)), accumulates in limbic brain areas: A new pharmacological tool for the study of social motivation in nonhuman primates. Horm Behav. 2007;52(3):344–351.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

56

4.  Female Sexual Function: The Role of Animal Models in Assessing Female Sexual Dysfunction

76. Pedersen CA, Vadlamudi SV, Boccia ML, Amico JA. Maternal behavior deficits in nulliparous oxytocin knockout mice. Genes Brain Behav. 2006;5(3):274–281. 77. Pedersen CA, Boccia ML. Oxytocin antagonism alters rat dams’ oral grooming and upright posturing over pups. Physiol Behav. 2003;80(2-3):233–241. 78. Pedersen CA, Boccia ML. Oxytocin links mothering received, mothering bestowed and adult stress responses. Stress. 2002;5(4):259–267. 79. Pedersen CA, Boccia ML. Oxytocin maintains as well as initiates female sexual behavior: effects of a highly selective oxytocin antagonist. Horm Behav. 2002;41(2):170–177. 80. Komisaruk BR, Sansone G. Neural pathways mediating vaginal function: the vagus nerves and spinal cord oxytocin. Scand J Psychol. 2003;44(3):241–250. 81. Gelez H, Poirier S, Facchinetti P, et  al. Neuroanatomical evidence for a role of central melanocortin-4 receptors and oxytocin in the efferent control of the rodent clitoris and vagina. J Sex Med. 2010;7(6):2056–2067. 82. Diamond LE, Earle DC, Heiman JR, Rosen RC, Perelman MA, Harning R. An effect on the subjective sexual response in premenopausal women with sexual arousal disorder by bremelanotide (PT-141), a melanocortin receptor agonist. J Sex Med. 2006;3(4):628–638. 83. Uckert S, Bannowsky A, Albrecht K, Kuczyk MA. Melanocortin receptor agonists in the treatment of male and female sexual dysfunctions: results from basic research and clinical studies. Expert opin Investig Drugs. 2014;23(11):1477–1483. 84. Rossler AS, Pfaus JG, Kia HK, Bernabe J, Alexandre L, Giuliano F. The melanocortin agonist, melanotan II, enhances proceptive sexual behaviors in the female rat. Pharmacol Biochem Behav. 2006;85(3):514–521. 85. Pfaus JG, Shadiack A, Van Soest T, Tse M, Molinoff P. Selective facilitation of sexual solicitation in the female rat by a melanocortin receptor agonist. Proc Natl Acad Sci USA. 2004;101(27):10201–10204. 86. Modi ME, Inoue K, Barrett CE, et  al. Melanocortin receptor agonists facilitate oxytocin-dependent partner preference formation in the prairie vole. Neuropsychopharmacology. 2015;40(8):1856–1865. 87. Allahdadi KJ, Hannan JL, Tostes RC, Webb RC. Endothelin-1 induces contraction of female rat internal pudendal and clitoral arteries through ET(A) receptor and rho-kinase activation. J Sex Med. 2010;7(6):2096–2103. 88. Traish AM, Botchevar E, Kim NN. Biochemical factors modulating female genital sexual arousal physiology. J Sex Med. 2010;7(9):2925–2946. 89. Min K, O’Connell L, Munarriz R, et al. Experimental models for the investigation of female sexual function and dysfunction. Int J Impot Res. 2001;13(3):151–156. 90. Musicki B, Liu T, Lagoda GA, Bivalacqua TJ, Strong TD, Burnett AL. Endothelial nitric oxide synthase regulation in female genital tract structures. J Sex Med. 2009;6(Suppl 3):247–253. 91. Cushman TT, Kim N, Hoyt R, Traish AM. Estradiol ameliorates diabetes-induced changes in vaginal structure of db/db mouse model. J Sex Med. 2009;6(9):2467–2479. 92. Pfaus JG. Pathways of sexual desire. J Sex Med. 2009;6(6):1506–1533. 93. van Rooij K, Poels S, Worst P, et al. Efficacy of testosterone combined with a PDE5 inhibitor and testosterone combined with a serotonin (1A) receptor agonist in women with SSRI-induced sexual dysfunction. A preliminary study. Eur J Pharmacol. 2015;753:246–251. 94. van Rooij K, de Leede L, Frijlink HW, et  al. Pharmacokinetics of a prototype formulation of sublingual testosterone and a buspirone tablet, versus an advanced combination tablet of

testosterone and buspirone in healthy premenopausal women. Drugs R D. 2014;14(2):125–132. 95. Poels S, Bloemers J, van Rooij K, Koppeschaar H, Olivier B, Tuiten A. Two novel combined drug treatments for women with hypoactive sexual desire disorder. Pharmacol Biochem Behav. 2014;121:71–79. 96. Bloemers J, van Rooij K, de Leede L, et  al. Single dose sublingual testosterone and oral sildenafil versus a dual-route/ dual-release fixed-dose combination tablet: a pharmacokinetic comparison. BJ Clin Pharm. 2016;81(6):1091–1102. 97. Carp SB, Rothwell ES, Bourdon A, Freeman SM, Ferrer E, Bales KL. Development of a partner preference test that differentiates between established pair bonds and other relationships in socially monogamous titi monkeys (Callicebus cupreus). Am J Primatol. 2016;78(3):326–339. 98. Snowdon CT, Tannenbaum PL, Schultz-Darken NJ, Ziegler TE, Ferris CF. Conditioned sexual arousal in a nonhuman primate. Horm Behav. 2011;59(5):696–701. 99. Ferris CF, Snowdon CT, King JA, et al. Activation of neural pathways associated with sexual arousal in non-human primates. J Magn Reson Imaging. 2004;19(2):168–175. 100. Cummings JA, Becker JB. Quantitative assessment of female sexual motivation in the rat: Hormonal control of motivation. J Neurosci Methods. 2012;204(2):227–233. 101. Uphouse L, Pinkston J, Baade D, Solano C, Onaiwu B. Use of an operant paradigm for the study of antidepressant-induced sexual dysfunction. Behav Pharmacol. 2015;26(7 Spec No): 697–705. 102. Meerts SH, Boisvert EM, Spjut KA, Clark AS. Paced mating behavior persists in rats with vaginocervical Lidocaine. Physiol Behav. 2010;99(1):139–141. 103. Meerts SH, Clark AS. Artificial vaginocervical stimulation induces a conditioned place preference in female rats. Horm Behav. 2009;55(1):128–132. 104. Lehmann ML, Erskine MS. Induction of pseudopregnancy using artificial VCS: importance of lordosis intensity and prestimulus estrous cycle length. Horm Behav. 2004;45(2):75–83. 105. Cibrian-Llanderal T, Tecamachaltzi-Silvaran M, Triana-Del Rio R, Pfaus JG, Manzo J, Coria-Avila GA. Clitoral stimulation modulates appetitive sexual behavior and facilitates reproduction in rats. Physiol Behav. 2010;100(2):148–153. 106. Parada M, Abdul-Ahad F, Censi S, Sparks L, Pfaus JG. Context alters the ability of clitoral stimulation to induce a sexually-conditioned partner preference in the rat. Horm Behav. 2011;59(4):520–527. 107. Parada M, Chamas L, Censi S, Coria-Avila G, Pfaus JG. Clitoral stimulation induces conditioned place preference and Fos activation in the rat. Horm Behav. 2010;57(2):112–118. 108. Paredes-Ramos P, Pfaus JG, Miquel M, Manzo J, Coria-Avila GA. Sexual reward induces Fos in the cerebellum of female rats. Physiol Behav. 2011;102(2):143–148. 109. Coria-Avila GA, Gavrila AM, Boulard B, Charron N, Stanley G, Pfaus JG. Neurochemical basis of conditioned partner preference in the female rat: II. Disruption by flupenthixol. Behav Neurosci. 2008;122(2):396–406. 110. Coria-Avila GA, Pfaus JG. Neuronal activation by stimuli that predict sexual reward in female rats. Neuroscience. 2007;148(3):623–632. 111. Georgiadis JR, Kringelbach ML. The human sexual response cycle: brain imaging evidence linking sex to other pleasures. Prog Neurobiol. 2012;98(1):49–81. 112. Georgiadis JR, Reinders AA, Paans AM, Renken R, Kortekaas R. Men versus women on sexual brain function: prominent differences during tactile genital stimulation, but not during orgasm. Hum Brain Mapp. 2009;30(10):3089–3101.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

113. Georgiadis JR, Kortekaas R, Kuipers R, et al. Regional cerebral blood flow changes associated with clitorally induced orgasm in healthy women. Eur J Neurosci. 2006;24(11):3305–3316. 114. Borg C, de Jong PJ, Georgiadis JR. Subcortical BOLD responses during visual sexual stimulation vary as a function of implicit porn associations in women. Soc Cogn Affect Neurosci. 2014;9(2):158–166. 115. Komisaruk BR, Wise N, Frangos E, Liu WC, Allen K, Brody S. Women’s clitoris, vagina, and cervix mapped on the sensory cortex: fMRI evidence. J Sex Med. 2011;8(10):2822–2830. 116. Komisaruk BR, Whipple B. Functional MRI of the brain during orgasm in women. Annu Rev Sex Res. 2005;16:62–86. 117. Pfaus JG, Kippin TE, Coria-Avila GA, et al. Who, what, where, when (and maybe even why)? How the experience of sexual reward connects sexual desire, preference, and performance. Arch Sex Behav. 2012;41(1):31–62. 118. Lorenz TK, Harte CB, Meston CM. Changes in autonomic nervous system activity are associated with changes in sexual function in women with a history of childhood sexual abuse. J Sex Med. 2015;12(7):1545–1554. 119. Nugent AC, Bain EE, Thayer JF, Sollers JJ, Drevets WC. Sex differences in the neural correlates of autonomic arousal: a pilot PET study. Int J Psychophysiol. 2011;80(3):182–191. 120. Salonia A, Giraldi A, Chivers ML, et al. Physiology of women’s sexual function: basic knowledge and new findings. J Sex Med. 2010;7(8):2637–2660. 121. Maravilla KR, Yang CC. Magnetic resonance imaging and the female sexual response: overview of techniques, results, and future directions. J Sex Med. 2008;5(7):1559–1571. 122. Agmo A. On the intricate relationship between sexual motivation and arousal. Horm Behav. 2011;59(5):681–688. 123. Weil ZM, Zhang Q, Hornung A, Blizard D, Pfaff DW. Impact of generalized brain arousal on sexual behavior. Proc Natl Acad Sci USA. 2010;107(5):2265–2270. 124. Dampney RA. Central mechanisms regulating coordinated cardiovascular and respiratory function during stress and arousal. Am J Physiol Regul Integr Comp Physiol. 2015;309(5): R429–R443. 125. Pfaff DW, Martin EM, Faber D. Origins of arousal: roles for medullary reticular neurons. Trends Neuroscis. 2012;35(8):468–476. 126. Schober J, Weil Z, Pfaff D. How generalized CNS arousal strengthens sexual arousal (and vice versa). Horm Behav. 2011;59(5):689–695. 127. Frohmader KS, Wiskerke J, Wise RA, Lehman MN, Coolen LM. Methamphetamine acts on subpopulations of neurons regulating sexual behavior in male rats. Neuroscience. 2010;166(3):771–784. 128. Frohmader KS, Bateman KL, Lehman MN, Coolen LM. Effects of methamphetamine on sexual performance and compulsive sex behavior in male rats. Psychopharmacology (Berl). 2010;212(1):93–104. 129. Afonso VM, Mueller D, Stewart J, Pfaus JG. Amphetamine pretreatment facilitates appetitive sexual behaviors in the female rat. Psychopharmacology (Berl). 2009;205(1):35–43. 130. Pfaus JG, Wilkins MF, Dipietro N, et  al. Inhibitory and disinhibitory effects of psychomotor stimulants and depressants on the sexual behavior of male and female rats. Horm Behav. 2010;58(1):163–176. 131. Holder MK, Mong JA. Methamphetamine enhances paced mating behaviors and neuroplasticity in the medial amygdala of female rats. Horm Behav. 2010;58(3):519–525. 132. Guarraci FA, Clark AS. Amphetamine modulation of paced mating behavior. Pharmacol Biochem Behav. 2003;76(3-4):505–515. 133. Winland C, Haycox C, Bolton JL, et  al. Methamphetamine enhances sexual behavior in female rats. Pharmacol Biochem Behav. 2011;98(4):575–582.

57

134. Holder MK, Hadjimarkou MM, Zup SL, et al. Methamphetamine facilitates female sexual behavior and enhances neuronal activation in the medial amygdala and ventromedial nucleus of the hypothalamus. Psychoneuroendocrinology. 2010;35(2):197–208. 135. Holder MK, Veichweg SS, Mong JA. Methamphetamineenhanced female sexual motivation is dependent on dopamine and progesterone signaling in the medial amygdala. Horm Behav. 2015;67:1–11. 136. Puppo V. Persistent genital arousal in women with pelvic and genital pain. J Obstet Gynaecol Can. 2014;36(9):765–766. 137. Facelle TM, Sadeghi-Nejad H, Goldmeier D. Persistent genital arousal disorder: characterization, etiology, and management. J Sex Med. 2013;10(2):439–450. 138. Goetsch MF, Lim JY, Caughey AB. A practical solution for dyspareunia in breast cancer survivors: a randomized controlled trial. J Clin Oncol. 2015;33(30):3394–3400. 139. Bultrini A, Carosa E, Colpi EM, et  al. Possible correlation between type 1 diabetes mellitus and female sexual dysfunction: case report and literature review. J Sex Med. 2004;1(3):337–340. 140. Mezones-Holguin E, Blumel JE, Huezo M, et al. Impact of diabetes mellitus on the sexuality of Peruvian postmenopausal. Gynecol Endocrinol. 2008;24(8):470–474. 141. Levin RJ. The physiology of sexual arousal in the human female: a recreational and procreational synthesis. Arch Sex Behav. 2002;31(5):405–411. 142. Ottesen B, Wagner G, Fahrenkrug J. Peptidergic Innervation of the Sexual Organs. Amsterdam: Elsevier Science Publishers; 1988. 143. Rahardjo HE, Brauer A, Magert HJ, et al. Endogenous vasoactive peptides and the human vagina--a molecular biology and functional study. J Sex Med. 2011;8(1):35–43. 144. Pei L, Jiang J, Jiang R, et al. Expression of aquaporin proteins in vagina of diabetes mellitus rats. J Sex Med. 2013;10(2):342–349. 145. Zhu J, Xia J, Jiang J, Jiang R, He Y, Lin H. Effects of estrogen deprivation on expression of aquaporins in rat vagina. Menopause (New York, NY). 2015;22(8):893–898. 146. Baldassarre M, Alvisi S, Berra M, et  al. Changes in vaginal physiology of menopausal women with type 2 diabetes. J Sex Med. 2015;12(6):1346–1355. 147. Lee HS, Li Z, Kim SO, Ahn K, Kim NN, Park K. Effect of hyperglycemia on expression of aquaporins in the rat vagina. Urology. 2012;80(3) 737.e7-12. 148. Kim SO, Oh KJ, Lee HS, Ahn K, Kim SW, Park K. Expression of aquaporin water channels in the vagina in premenopausal women. J Sex Med. 2011;8(7):1925–1930. 149. Wayman CP, Baxter D, Turner L, Van Der Graaf PH, Naylor AM. UK-414,495, a selective inhibitor of neutral endopeptidase, potentiates pelvic nerve-stimulated increases in female genital blood flow in the anaesthetized rabbit. Br J Pharmacol. 2010;160(1):51–59. 150. Kim NN, Stankovic M, Cushman TT, Goldstein I, Munarriz R, Traish AM. Streptozotocin-induced diabetes in the rat is associated with changes in vaginal hemodynamics, morphology and biochemical markers. BMC Physiol. 2006;6:4. 151. Cai RS, Alexander MS, Marson L. Activation of somatosensory afferents elicit changes in vaginal blood flow and the urethrogenital reflex via autonomic efferents. J Urol. 2008;180(3):1167–1172. 152. Meston CM. Sympathetic nervous system activity and female sexual arousal. Am J Cardiol. 2000;86(2A):30F–34FF. 153. Meston CM, Gorzalka BB. Differential effects of sympathetic activation on sexual arousal in sexually dysfunctional and functional women. J Abnorm Psychol. 1996;105(4):582–591. 154. Meston CM, Heiman JR. Ephedrine-activated physiological sexual arousal in women. Arch Gen Psychiatry. 1998;55(7):652–656. 155. Sun Q, Huang J, Yang DL, Cao XN, Zhou WL. Activation of beta-adrenergic receptors during sexual arousal facilitates

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

58

156.

157.

158.

159. 160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

170.

171.

172.

173.

174.

4.  Female Sexual Function: The Role of Animal Models in Assessing Female Sexual Dysfunction

vaginal lubrication by regulating vaginal epithelial Cl(-) secretion. J Sex Med. 2014;11(8):1936–1948. Allers KA, Richards N, Sultana S, et  al. I. Slow oscillations in vaginal blood flow: alterations during sexual arousal in rodents and humans. J Sex Med. 2010;7(3):1074–1087. Allers KA, Richards N, Scott L, et  al. II. Slow oscillations in vaginal blood flow: regulation of vaginal blood flow patterns in rat by central and autonomic mechanisms. J Sex Med. 2010;7(3):1088–1103. Aughton KL, Hamilton-Smith K, Gupta J, Morton JS, Wayman CP, Jackson VM. Pharmacological profiling of neuropeptides on rabbit vaginal wall and vaginal artery smooth muscle in vitro. Br J Pharmacol. 2008;155(2):236–243. Wesselmann U, Burnett AL, Heinberg LJ. The urogenital and rectal pain syndromes. Pain. 1997;73(3):269–294. Giuliano F, Pfaus J, Srilatha B, et  al. Experimental models for the study of female and male sexual function. J Sex Med. 2010;7(9):2970–2995. Marson L, Murphy AZ. Identification of neural circuits involved in female genital responses in the rat: a dual virus and anterograde tracing study. Am J Physiol Regul Integr Comp Physiol. 2006;291(2):R419–R428. Wiedey J, Alexander MS, Marson L. Spinal neurons activated in response to pudendal or pelvic nerve stimulation in female rats. Brain Res. 2008;1197:106–114. Marson L, Cai R, Makhanova N. Identification of spinal neurons involved in the urethrogenital reflex in the female rat. J Comp Neurol. 2003;462(4):355–370. Marson L, Foley KA. Identification of neural pathways involved in genital reflexes in the female: a combined anterograde and retrograde tracing study. Neuroscience. 2004;127(3):723–736. Gill BC, Swartz MA, Firoozi F, et  al. Improved sexual and urinary function in women with sacral nerve stimulation. Neuromodulation. 2011;14(5):436–443. discussion 43. Fariello JY, Whitmore K. Sacral neuromodulation stimulation for IC/PBS, chronic pelvic pain, and sexual dysfunction. Int Urogynecol J. 2010;21(12):1553–1558. Giuliano F, Allard J, Compagnie S, Alexandre L, Droupy S, Bernabe J. Vaginal physiological changes in a model of sexual arousal in anesthetized rats. Am J Physiol Regul Integr Comp Physiol. 2001;281(1):R140–R149. Kim NN, Min K, Huang YH, Goldstein I, Traish AM. Biochemical and functional characterization of alpha-adrenergic receptors in the rabbit vagina. Life Sci. 2002;71(24):2909–2920. Traish AM, Kim SW, Stankovic M, Goldstein I, Kim NN. Testosterone increases blood flow and expression of androgen and estrogen receptors in the rat vagina. J Sex Med. 2007;4(3):609–619. Waxman SE, Pukall CF. Laser Doppler imaging of genital blood flow: a direct measure of female sexual arousal. J Sex Med. 2009;6(8):2278–2285. Kimura Y. [Studies of ejaculation. 1. On peripheral nerves controlling ejaculation--with special reference to the posterior urethrogram]. Nippon Hinyokika Gakkai Zasshi. 1970;61(3):284–295. Vachon P, Simmerman N, Zahran AR, Carrier S. Increases in clitoral and vaginal blood flow following clitoral and pelvic plexus nerve stimulations in the female rat. Int J Impot Res. 2000;12(1):53–57. Damaser MS, Broxton-King C, Ferguson C, Kim FJ, Kerns JM. Functional and neuroanatomical effects of vaginal distention and pudendal nerve crush in the female rat. J Urol. 2003;170(3):1027–1031. Tovar A, Lara-Garcia M, Cruz Y, Pacheco P. Dorsal root activity evoked by stimulation of vagina-cervix-uterus junction in the rat. Brain Res. 2012.

175. Marson L, McKenna KE. Stimulation of the hypothalamus initiates the urethrogenital reflex in male rats. Brain Res. 1994;638(1-2):103–108. 176. Alexander MS, Kozyrev N, Bosma RL, Figley CR, Richards JS, Stroman PW. fMRI localization of spinal cord processing underlying female sexual arousal. J Sex Marital Ther. 2015:1–12. 177. Vaccaro CM. The use of magnetic resonance imaging for studying female sexual function: A review. Clin Anat. 2015;28(3):324–330. 178. Wehrum S, Klucken T, Kagerer S, et al. Gender commonalities and differences in the neural processing of visual sexual stimuli. J Sex Med. 2013;10(5):1328–1342. 179. Musicki B, Liu T, Strong TD, Lagoda GA, Bivalacqua TJ, Burnett AL. Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina. J Sex Med. 2010;7(5):1768–1777. 180. Tajkarimi K, Burnett AL. The role of genital nerve afferents in the physiology of the sexual response and pelvic floor function. J Sex Med. 2011;8(5):1299–1312. 181. Oliver JL, Kavoussi PK, Smith RP, et al. The role of regulatory proteins and S-nitrosylation of endothelial nitric oxide synthase in the human clitoris: implications for female sexual function. J Sex Med. 2014;11(8):1927–1935. 182. Leddy LS, Yang CC, Stuckey BG, et  al. Influence of sildenafil on genital engorgement in women with female sexual arousal disorder. J Sex Med. 2012;9(10):2693–2697. 183. Alexander MS, Rosen RC, Steinberg S, Symonds T, Haughie S, Hultling C. Sildenafil in women with sexual arousal disorder following spinal cord injury. Spinal Cord. 2011;49(2):273–279. 184. Chivers ML, Seto MC, Lalumiere ML, Laan E, Grimbos T. Agreement of self-reported and genital measures of sexual arousal in men and women: a meta-analysis. Arch Sex Behav. 2010;39(1):5–56. 185. Uckert S, Ehlers V, Nuser V, et al. In vitro functional responses of isolated human vaginal tissue to selective phosphodiesterase inhibitors. World J Urol. 2005;23(6):398–404. 186. Giraldi A, Alm P, Werkstrom V, Myllymaki L, Wagner G, Andersson KE. Morphological and functional characterization of a rat vaginal smooth muscle sphincter. Int J Impot Res. 2002;14(4):271–282. 187. Ghatei MA, Gu J, Mulderry PK, et  al. Calcitonin gene-related peptide (CGRP) in the female rat urogenital tract. Peptides. 1985;6(5):809–815. 188. Kim NN, Stankovic M, Armagan A, Cushman TT, Goldstein I, Traish AM. Effects of tamoxifen on vaginal blood flow and epithelial morphology in the rat. BMC Womens Health. 2006;6:14. 189. De Bernardis F, Lucciarini R, Boccanera M, et  al. Phenotypic and functional characterization of vaginal dendritic cells in a rat model of Candida albicans vaginitis. Infect Immun. 2006;74(7):4282–4294. 190. Maswood N, Caldarola-Pastuszka M, Uphouse L. Functional integration among 5-hydroxytryptamine receptor families in the control of female rat sexual behavior. Brain Res. 1998;802(1–2):98–103. 191. Ahlenius S, Larsson K, Arvidsson LE. Effects of stereoselective 5-HT1A agonists on male rat sexual behavior. Pharmacol Biochem Behav. 1989;33(3):691–695. 192. Schijven D, Sousa VC, Roelofs J, Olivier B, Olivier JD. Serotonin 1A receptors and sexual behavior in a genetic model of depression. Pharmacol Biochem Behav. 2014;121:82–87. 193. Felton TM, Kang TB, Hjorth S, Auerbach SB. Effects of selective serotonin and serotonin/noradrenaline reuptake inhibitors on extracellular serotonin in rat diencephalon and frontal cortex. Naunyn-Schmiedebergs Arch Pharmacol. 2003;367(3):297–305. 194. Frohlich PF, Meston CM. Evidence that serotonin affects female sexual functioning via peripheral mechanisms. Physiol Behav. 2000;71(3-4):383–393.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

59

REFERENCES

195. Kishitake M, Yamanouchi K. Effects of 5-HT1A-receptor agonist, 8-OH-DPAT, and GABAB-receptor agonist, baclofen, on lordosis in female rats with lesions in either the dorsal raphe nucleus or septum. J Pharmacol Sci. 2005;98(4):419–424. 196. Kishitake M, Yamanouchi K. Facilitatory effects of WAY-100635, a 5-HT1A receptor antagonist, on lordosis in female rats. Neurosci Lett. 2004;371(2–3):147–151. 197. Uphouse L, Guptarak J, Hiegel C. Progesterone reduces the inhibitory effect of a serotonin 1B receptor agonist on lordosis behavior. Pharmacol Biochem Behav. 2010;97(2):317–324. 198. Uphouse L, Andrade M, Caldarola-Pastuszka M, Jackson A. 5-HT1A receptor antagonists and lordosis behavior. Neuropharmacology. 1996;35(4):489–495. 199. Mendelson SD. A review and reevaluation of the role of serotonin in the modulation of lordosis behavior in the female rat. Neurosci Biobehav Rev. 1992;16(3):309–350. 200. Wolf A, Caldarola-Pastuszka M, Uphouse L. Facilitation of female rat lordosis behavior by hypothalamic infusion of 5-HT(2A/2C) receptor agonists. Brain Res. 1998;779(1–2):84–95. 201. Meerts SH, Clark AS. Conditioned place preference for mating is preserved in rats with pelvic nerve transection. Behav Neurosci. 2009;123(3):539–546. 202. Shafik A, Shafik IA, El Sibai O, Shafik AA. An electrophysiologic study of female ejaculation. J Sex Marital Ther. 2009;35(5):337–346. 203. Rowland DL. Neurobiology of sexual response in men and women. CNS Spectr. 2006;11(8 Suppl 9):6–12. 204. Sipski ML, Alexander CJ, Rosen R. Sexual arousal and orgasm in women: effects of spinal cord injury. Ann Neurol. 2001;49(1):35–44. 205. Exton MS, Bindert A, Kruger T, Scheller F, Hartmann U, Schedlowski M. Cardiovascular and endocrine alterations after masturbation-induced orgasm in women. Psychosom Med. 1999;61(3):280–289. 206. Bohlen JG, Held JP, Sanderson MO, Ahlgren A. The female orgasm: pelvic contractions. Arch Sex Behav. 1982;11(5):367–386. 207. McKenna KE, Chung SK, McVary KT. A model for the study of sexual function in anesthetized male and female rats. Am J Physiol. 1991;261(5 Pt 2):R1276–R1285. 208. McKenna KE, Knight KC, Mayers R. Modulation by peripheral serotonin of the threshold for sexual reflexes in female rats. Pharmacol Biochem Behav. 1991;40(1):151–156. 209. Marson L, McKenna KE. Serotonergic neurotoxic lesions facilitate male sexual reflexes. Pharmacol Biochem Behav. 1994;47(4):883–888. 210. Marson L, McKenna KE. The identification of a brainstem site controlling spinal sexual reflexes in male rats. Brain Res. 1990;515(1–2):303–308. 211. Tanahashi M, Karicheti V, Thor KB, Marson L. Characterization of bulbospongiosus muscle reflexes activated by urethral distension in male rats. Am J Physiol Regul Integr Comp Physiol. 2012;303(7):R737–R747. 212. Marson L TM, Thor KB, Karicheti V. Peripheral nerves mediating the urethrogenital reflex (UGR) in urethane-anesthetized, acutely spinalized male rats evoked by penile clamping or retraction. Soc Neurosci. 2011;391:09. 213. Ishigami T, Yoshioka K, Karicheti V, Marson L. A role for peripheral 5-HT2 receptors in serotonin-induced facilitation of the expulsion phase of ejaculation in male rats. J Sex Med. 2013;10(11):2688–2702. 214. Pfaus JG, Manitt C, Coopersmith CB. Effects of pelvic, pudendal, or hypogastric nerve cuts on Fos induction in the rat brain following vaginocervical stimulation. Physiol Behav. 2006;89(5):627–636. 215. Pfaus JG, Marcangione C, Smith WJ, Manitt C, Abillamaa H. Differential induction of Fos in the female rat brain following

216.

217. 218. 219. 220.

221.

222.

223. 224.

225.

226.

227.

228.

229.

230.

231.

232.

233.

234.

235. 236.

different amounts of vaginocervical stimulation: modulation by steroid hormones. Brain Res. 1996;741(1–2):314–330. Kim GW, Jeong GW. A comparative study of brain activation patterns associated with sexual arousal between males and females using 3.0-T functional magnetic resonance imaging. Sex Health. 2014;11(1):11–16. Janszky J, Szucs A, Halasz P, et  al. Orgasmic aura originates from the right hemisphere. Neurology. 2002;58(2):302–304. Wesselmann U, Czakanski PP. Pelvic pain: a chronic visceral pain syndrome. Curr Pain Headache Rep. 2001;5(1):13–19. Wesselmann U, Bonham A, Foster D. Vulvodynia: Vulvodynia Current state of the biological science. Pain. 2014;155(9):1696–1701. Giamberardino MA, Berkley KJ, Affaitati G, et  al. Influence of endometriosis on pain behaviors and muscle hyperalgesia induced by a ureteral calculosis in female rats. Pain. 2002;95(3):247–257. Giamberardino MA, De Laurentis S, Affaitati G, Lerza R, Lapenna D, Vecchiet L. Modulation of pain and hyperalgesia from the urinary tract by algogenic conditions of the reproductive organs in women. Neurosci Lett. 2001;304(1–2):61–64. Bradshaw HB, Temple JL, Wood E, Berkley KJ. Estrous variations in behavioral responses to vaginal and uterine distention in the rat. Pain. 1999;82(2):187–197. Berkley KJ, Wood E, Scofield SL, Little M. Behavioral responses to uterine or vaginal distension in the rat. Pain. 1995;61(1):121–131. Shin SW, Sandner-Kiesling A, Eisenach JC. Systemic, but not intrathecal ketorolac is antinociceptive to uterine cervical distension in rats. Pain. 2003;105(1–2):109–114. Sandner-Kiesling A, Pan HL, Chen SR, et al. Effect of kappa opioid agonists on visceral nociception induced by uterine cervical distension in rats. Pain. 2002;96(1–2):13–22. Du D, Eisenach JC, Ririe DG, Tong C. The antinociceptive effects of spinal cyclooxygenase inhibitors on uterine cervical distension. Brain Res. 2004;1024(1–2):130–136. Boscan P, Monnet E, Mama K, et  al. A dog model to study ovary, ovarian ligament and visceral pain. Vet Anaesth Analg. 2011;38(3):260–266. Taneja A, Di Iorio VL, Danhof M, Della Pasqua O. Translation of drug effects from experimental models of neuropathic pain and analgesia to humans. Drug Discov Today. 2012;17(15–16):837–849. Farmer MA, Taylor AM, Bailey AL, et al. Repeated vulvovaginal fungal infections cause persistent pain in a mouse model of vulvodynia. Sci Transl Med. 2011;3(101):101ra91. Farmer MA, Taylor A, MacIntyre LC, et al. Of Mice and Women: A Mouse Model of Vestibulodynia Following Recurrent Vulvovaginal Candidiasis. Glasgow: International Association for the Study of Pain (IASP) meeting; 2008. Moyal-Barracco M, Lynch PJ. 2003 ISSVD terminology and classification of vulvodynia: a historical perspective. J Reprod Med. 2004;49(10):772–777. Giamberardino MA, Berkley KJ, Iezzi S, de Bigontina P, Vecchiet L. Pain threshold variations in somatic wall tissues as a function of menstrual cycle, segmental site and tissue depth in nondysmenorrheic women, dysmenorrheic women and men. Pain. 1997;71(2):187–197. Wesselmann U, Czakanski PP, Affaitati G, Giamberardino MA. Uterine inflammation as a noxious visceral stimulus: behavioral characterization in the rat. Neurosci Lett. 1998;246(2):73–76. Wesselmann U, Lai J. Mechanisms of referred visceral pain: uterine inflammation in the adult virgin rat results in neurogenic plasma extravasation in the skin. Pain. 1997;73(3):309–317. Sanoja R, Cervero F. Estrogen-dependent changes in visceral afferent sensitivity. Auton Neurosci. 2010;153(1-2):84–89. Walf AA, Paris JJ, Frye CA. Chronic estradiol replacement to aged female rats reduces anxiety-like and depression-like behavior

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237.

238.

239.

240.

241.

4.  Female Sexual Function: The Role of Animal Models in Assessing Female Sexual Dysfunction

and enhances cognitive performance. Psychoneuroendocrinology. 2009;34(6):909–916. Frye CA, Wawrzycki J. Effect of prenatal stress and gonadal hormone condition on depressive behaviors of female and male rats. Horm Behav. 2003;44(4):319–326. Bekku N, Yoshimura H. Animal model of menopausal depressive-like state in female mice: prolongation of immobility time in the forced swimming test following ovariectomy. Psychopharmacology (Berl). 2005;183(3):300–307. Snoeren EM, Refsgaard LK, Waldinger MD, Olivier B, Oosting RS. Chronic paroxetine treatment does not affect sexual behavior in hormonally sub-primed female rats despite 5-HT(1)(A) receptor desensitization. J Sex Med. 2011;8(4):976–988. Klinger MB, Sacks S, Cervero F. A role for extracellular signal-regulated kinases 1 and 2 in the maintenance of persistent mechanical hyperalgesia in ovariectomized mice. Neuroscience. 2011;172:483–493. Burney RO, Giudice LC. Pathogenesis and pathophysiology of endometriosis. FertilSteril. 2012;98(3):511–519.

242. Dmitrieva N, Nagabukuro H, Resuehr D, et al. Endocannabinoid involvement in endometriosis. Pain. 2010;151(3):703–710. 243. Grummer R. Animal models in endometriosis research. Hum Reprod Update. 2006;12(5):641–649. 244. Pelch KE, Sharpe-Timms KL, Nagel SC. Mouse model of surgically-induced endometriosis by auto-transplantation of uterine tissue. J Vis Exp. 2012;59:e3396. 245. Giamberardino MA, Costantini R, Affaitati G, et  al. Viscerovisceral hyperalgesia: characterization in different clinical models. Pain. 2010;151(2):307–322. 246. Giamberardino MA, Affaitati G, Fabrizio A, Costantini R. Myofascial pain syndromes and their evaluation. Best Pract Res Clin Rheumatol. 2011;25(2):185–198. 247. Rodriguez MA, Afari N, Buchwald DS. Evidence for overlap between urological and nonurological unexplained clinical conditions. J Urol. 2009;182(5):2123–2131. 248. Smith KA, Pearson CB, Hachey AM, Xia DL, Wachtman LM. Alternative activation of macrophages in rhesus macaques (Macaca mulatta) with endometriosis. Comp Med. 2012;62(4):303–310.

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C H A P T E R

5 Fertility Treatment and Preservation in Transgender Men and Women Dov Feldberg Tel Aviv University, Tel Aviv, Israel

O U T L I N E 5.1 Introduction

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Reproductive Wish in Transgender Men

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Fertility and Achievement of Pregnancy in Transgender Men

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5.5 Reproductive Options for Trans People 5.5.1 Reproductive Options for Trans Women 5.5.2 Reproductive Options for Trans Men

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shows all over America. In one of the shows he stated: “I feel it is not a male or a female desire to want to have a child; it is a human desire. I am a person, and I have the right to have my own biological child.” Prior to this case, other trans men had children, but they were not reported in the media and were neither legally recognized as males, nor as married individuals.1 Thomas Beatie’s story created an enormous focus on the problem of transgenderism and helped promote this phenomenon, which during the last decades became an integral part of many societies all over the world. In 2012 a Swedish team of gynecologists reported on the first successful delivery after uterine transplantation.2 Also in 2012 a Canadian research team proposed “The Montreal Criteria of the Ethical Feasibility of Uterine Transplantation.”3 All these extensive medical

One of the most famous individuals in the history of transgenderism was Tracy LaGondino from Oregon, Arizona, who in 1990 decided to become a male. This change reflected his character and gender identity. This special event in medical history happened in his conservative home town in the United States. Using his new name, Thomas Beatie, he was legally recognized as a man. He married Nancy, a beautiful woman who was unfortunately infertile. Thomas stopped his testosterone treatment and conceived twice, after insemination with donor sperm. He underwent two pregnancies without any complications and delivered two healthy children. Thomas and his wife gave full publicity to these events and became television stars, appearing in many talk

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and scientific achievements were developed for biological females. The possibility of uterine transplantation for trans individuals, became a reality, placing gestation by males on the scientific agenda. This may allow male-tofemale transgender persons to carry a pregnancy, and to become gestational mothers. The tremendous technological and medical progress in reproductive medicine, with a radical change in the ethical and the legal approaches toward transgender men and women during the last years, positions the phenomenon of transgenderism as an integral part of our societies and demands very serious ethical, legal, and social considerations.

5.2  DEFINITION OF TRANSGENDER PEOPLE Trans people is a term for individuals who were born with a tremendous conflict between their chromosomal sex at birth (and/or their genital anatomy) and their sense of their own gender. In our advanced liberal modern world, with novel medical and reproductive technologies, the change of gender has become achievable. Individuals that were born as females, but whose selfimage and identity are male (trans men), can be treated with testosterone preparations to grow facial hair and to develop their body musculature and a deeper voice. Some of these individuals choose to have surgery to remove their breasts, and less commonly, to undergo total abdominal hysterectomy and bilateral salpingooophorectomy (TAH + BSO) and penis construction surgery.4 Individuals who were born as males (trans women) usually undergo androgen suppression and treatment with products of estrogen, in order to develop breasts and to establish a feminine contour. About 60% of trans women chose to have augmentation mammoplasty. In most cases, the hormonal suppression provokes oligo- or azoospermia; and some individuals cryopreserve their sperm before beginning these treatments. Some trans women undergo orchiectomy and surgical creation of a vagina.5 In many countries, the major obstacles for transgender individuals are the reaction of the society and achieving their changed social and legal status. One of the most important issues for the transgender community is the legal possibility of having their own biological children and establishing a normative family.6 For this reason, some transgender individuals choose to cryopreserve their gametes, before the radical transition.

5.3  STANDARDS OF CARE FOR THE HEALTH OF TRANSSEXUAL, TRANSGENDER, AND GENDERNONCONFORMING PEOPLE Prior to the publication of the first standards of care (SOC) for transgender people, there was absolutely no consensus on psychiatric, medical, and surgical needs of this cohort of individuals. Until the late 1960s transitioning was considered a criminal procedure, and transgenderism a psychiatric disease with personality disturbances. It was Harry Benjamin who established the World Professional Association for Transgender Health (WPATH).7 This organization published clinical guidelines in order to ensure “lasting personal comfort with the gendered self in order to maximize overall psychological well-being and self-fulfillment.” These SOC are still the most widely accepted guidelines, although additional SOCs have been published in the United States and in Europe. The most recent, the 7th version of WPATH-SOC was published in 2012.8 These guidelines deal with such issues as the diagnostics of gender nonconformity versus gender dysphoria, epidemiology, and special approaches in the treatment of children, adolescents, and adults. These guidelines also summarize the attitude toward hormone replacement therapy, reproductive health, sex reassignment therapy, preventive and primary care. In 2013, the Royal College of Psychiatrists in the United Kingdom released new guidelines for the diagnosis and treatment of adult gender dysphoria.9 These guidelines were composed by a multidisciplinary team that included 13 disciplines, including psychiatrists, endocrinologists, gynecologists, urologists, and other professionals. This outstanding team created a series of six recommendations for the optimal care of transgender people, covering all aspects of their primary care needs, and additionally encouraging the development and funding of research in transgender medicine.9 In 2009, the Journal of Clinical Endocrinology and Metabolism published the Clinical Practice Guidelines of the Endocrine Treatment of Transgender People. These guidelines were released with the participation of the Endocrine Society Counsel, European Society of Endocrinology, European Society of Pediatric Endocrinology, and many others.10 It should be noted that this composition was based on data confirmed by evidence-based medicine. These guidelines contain the following sections: 1. Diagnostic procedures. 2. Treatment of adolescents.

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5.5  Reproductive Options for Trans People

3. Hormonal therapy for transsexual adults. 4. Adverse outcome prevention and long-term care. 5. Surgery for sex reassignment. In a 2015 issue of the American Psychologist journal,11 the Psychological Association released their Guidelines for Transgender and Gender-Nonconforming People. These 16 paragraphs of guidelines summarized the understanding, involvement, attitudes, and experiences of psychologists in treatment and prevention measures resulting from their work with transgender individuals of different ages, couples, families, and groups.

5.4  EPIDEMIOLOGY OF GENDER IDENTITY DISORDER No recent studies on the epidemiology of transgenderism have been performed. Thus the incidence of transgender men and women remains unknown. Some publications and estimations present numbers of adult patients who applied to clinics and hospitals in order to be treated by contra-set hormonal treatment or to undergo transformative surgery.12 In the general population, the incidence of the trans-phenomenon is more frequent in girls; however, boys are more often referred to clinics that treat transgender patients.13 In general, statistical observations show that the prevalence of gender identity disturbances is higher in males than in females. Nonetheless, such observations may not reflect the special character of such disturbances in biological females. Reports from transgender clinics show an increase in referrals to these clinics during recent years. However, it is unclear whether this reflects a true increase in prevalence or simply an improved capacity and quality of medical services of transgender clinics.13 During the last decade, we have seen a more open, empathic, and understanding approach, toward this cohort of men and women, in medicine, law, the media, and in public acceptance of them. The current assumption is that transgenderism has a very broad and variable spectrum in different individuals in terms of perception of cross-gender identity. Corroboration of this requires undertaking large-scale evidence-based epidemiological studies. Until definitive data are published, we can only speculate on the prevalence of this phenomenon, as compared to the traditional male–female binary.14–15

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5.5  REPRODUCTIVE OPTIONS FOR TRANS PEOPLE The tremendous advances and novel technologies in reproductive medicine, together with legalization of transgenderism in many countries all over the globe, brought new hopes and options for trans people in terms of reproduction and building a family.16 Transgender people usually establish heterosexual or homosexual relationships, following sex-transition. There are no medical or legal reasons to prevent them from getting pregnant themselves or via surrogacy, or to reproduce together and raise their own biological children. Unfortunately, very little research has been published on the reproductive problems of trans people, or on the options that are available for them. These issues should be present on the medical and public agenda, similar to the treatment offered to cases of patients suffering from malignancies who need fertility preservation and treatment.17

5.5.1  Reproductive Options for Trans Women While planning the hormonal treatment in trans women, and before performing the surgical transition, it is important to discuss fertility problems with these males and to ask for their written informed consent. Males should be offered sperm cryopreservation after the performance of serological tests for HIV, hepatitis B and C, in accordance with the sperm bank regulations. If the sperm quality is good, it can be used in the future for intrauterine inseminations (IUI). If the decision to store the sperm is made during the hormonal therapy for trans women, the therapy should be stopped for 2–3 months, in order to produce a new cohort of sperm for cryopreservation. If the patient refuses to stop the treatment and the sperm quality is compromised, he should be informed about the possibilities of in vitro fertilization (IVF) and IUI treatment. Even sperm of a very low quality should be frozen, for future use by IVF procedure and/or by ICSI (Intracytoplasmic Sperm Injection)-assisted fertilization. In the case of azoospermia, surgical sperm extraction technique by testicular sperm aspiration (TESA) or testicular sperm extraction (TESE), should be offered in the future.18 To date, no studies on infertility in prepubertal trans girls have been published. Testicular biopsy and freezing for fertility preservation have been reported in prepubertal boys with cancer in Belgium and other countries.19–20 The quality of prepubertal sperm extracted by testicular biopsy is absolutely different from that of postpubertal

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one. This is the reason that experiments have been performed to cause in vitro maturation (IVM) of such premature sperm cells, or spermatogonial stem cells.21 These interventions have not yet been introduced into clinical practice. The remote possibility of trans women getting pregnant, carrying a pregnancy, and undergoing delivery is still far from becoming reality. However, successful uterine transplantations, with healthy children delivered, can open new horizons and give hope to trans women who dream of becoming gestational carriers.22

5.5.2  Reproductive Options for Trans Men One of the most effective modes infertility treatment and preservation in trans men, is to obtain pieces of ovarian tissue during reassignment surgery, and to cryopreserve them for future use.23 This technology is currently applied in women with malignant diseases in order to preserve their fertility after the oncological treatments. At least 80 children have already been born worldwide via ovarian tissue transplantation.24 Even under hormonal treatment with androgens, the oocytic quality, fertilization, and implantation potential is usually not compromised. IVM process is an additional new technology that can be used in these cases.25 The best fertility preservation option in trans men is to perform an IVF procedure with ovarian stimulation, prior to reassignment therapy or surgery. Cryopreservation of vitrified oocytes or embryos created by partners, or of donor sperm, will provide the best opportunities for their future reproduction.

5.6  THE ETHICAL/LEGAL STATUS OF MEDICALLY-ASSISTED REPRODUCTION IN TRANSSEXUAL PEOPLE Only a decade ago transgenderism was considered a psychiatric disease and was classified as “gender identity disorder.” That term has been recently replaced by “gender dysphoria” and no longer appears in the Diagnostic and Statistical Manual of Mental Disorders (DMS-5) as a mental illness.26 In 1948, the UN Assembly released the declaration of human rights which stated that “every human being on the globe has the right to found a family.”27 Despite this declaration, in many countries in the world this right is denied to people in nonstandard relationships that differ from the traditional “nuclear family.” For example, in many parts of the word, access is denied to assisted reproductive technology (ART) treatment and reproductive technologies for trans individuals. The argument for this approach is based mainly on alleged future consequences for the medical health of trans people and

presumed harm to children and to society.28 The only potential objection against ART or fertility treatment for trans people is to demonstrate “high risk of serious harm” to the future children. Such an outcome has to be proven by the standard evaluation of a professional team, just as is done for regular patients and couples. The evaluation is composed of investigation of the family history of a transgender parent including psychiatric and genetic diseases. A full investigation and report should be conducted by a psychiatrist, psychologist, or social worker and should be taken into consideration. The ability of such a couple to raise children should be thoroughly discussed. A recent French study29 reports a 12-year prospective follow-up of 42 children born into families with transgender men and their wives. This extensive study could not demonstrate any harm to the children mentally and physically. They were healthy and well-adjusted to their parents. No evidence of any gender variant behavior was presented. The aim of the United Nations declaration is to avoid a double standard for the general population, on the one hand, and nonconforming people on the other. The decision that there is no risk to the offspring of the transgender parent should be made by a multidisciplinary team of gynecologists, psychologists, social workers, and psychiatrists.30 The main goal is to shorten the transition period, which is very stressful for these trans individuals, until they are in a stable enough condition to start the ART treatment.31 In 2014, the European Society of Human Reproduction and Embryology (ESHRE) Task Force for Ethics and Law published the guidelines for ethical and legal aspects of ART treatment in transgender people.32 Their recommendations deal with prevention of discrimination in ART treatments, prevention of double standards for regular and nonconforming people, referral of trans patients to other clinics by professionals who refuse to perform the therapy, and the use of objective criteria for welfare of future children. The criteria should not be based on the gold standard of the traditional heterosexual nuclear family but by the psychosocial fitness of the transgender couple to manage a stable family life and to raise their offsprings together. The fertility treatment should be planned before the reassignment treatment. These guidelines stress the need for more research in this specific complicated issue of transgender patients. In November 2015, The American Society for Reproductive Medicine (ASRM) published the Ethics Committee Opinion on access to fertility services by transgender people.33 The committee indicated that transgenderism itself is not a condition for automatic refusal to receive the ART treatment and reproduce. There is no ethical basis to reject or prevent these individuals from receiving any form of fertility treatment. Every case should be investigated individually and the decisions should be made by a multidisciplinary team

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5.9  Fertility and Achievement of Pregnancy in Transgender Men

that includes endocrinologists, infertility specialists, and mental health professionals.

5.7  ART TREATMENT FOR TRANSGENDER MEN AND WOMEN Until recently, most of the literature on reproduction in trans people focused on the ethical and legal status of this specific group of individuals and their rights to become parents and to have access to the ART treatment. Some publications claimed that they are mentally unfit to be offered ART services.34 Other publications claimed that although these people have a mental disorder, it is a discrimination to withhold from them the possibility of parenthood, when there is no scientific proof that the well-being of their children will be compromised.35 WPATH strongly supports this approach and recommends that trans individuals interested in infertility treatment should be counseled by a multidisciplinary team concerning preservation and treatment of their fertility prior to the initiation of the medical–surgical transition. One of the recent publications that investigated the desire of trans people for parenthood indicates that most of them do expect to become parents and many have already become parents.36 A recent study performed in the province of Ontario in Canada,37 investigated the positive and negative experiences of trans patients with ART services and clinics in their area. The researchers also studied the strategies of counteracting transphobia and cissexism. The results of the study very strongly indicate the negative experience and emotions that trans people encounter when applying to ART centers. Many of them finally chose not to interact with these clinics and their personnel and further not to complain to the authorities, because of the fear being rejected by all other institutions. This pioneering study draws attention to the need for special reproductive services for transgender individuals provided by experienced personnel of reproductive specialists in collaboration with social workers and psychotherapists, in order to establish a friendly environment for these special patients. Trans people and their partners should be equally welcomed and assisted as are the couples of “nuclear families.”

5.8  REPRODUCTIVE WISH IN TRANSGENDER MEN With the development of ART, promising opportunities have appeared allowing trans men to preserve their fertility, in order to have genetically-related children. One of the rare studies that exists,38 recently examined

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50 trans men undergoing hormonal treatment and sex reassignment surgery. Among the participants, 22% reported already having children, 64% were involved in a relationship, 54% had a great desire to have children in the future, and 37.5% cryopreserved by vitrification their oocytes for future use. This unique study indicated that the majority of trans men plan to have geneticallyrelated children. Thus this issue should be discussed with them very thoroughly prior to hormonal and surgical transition.

5.9  FERTILITY AND ACHIEVEMENT OF PREGNANCY IN TRANSGENDER MEN One of the few studies related to trans men shows that the majority avoid extensive surgical operations during their transitional period and prefer hormonal therapy only.39 These individuals, born with female reproductive organs, at any stage of their transgender life are capable of carrying a pregnancy, and undergoing delivery. In the scarce amount of literature on the topic we encounter the great concern of the influence of testosterone on fertility, pregnancy, and neonatal outcome.40–41 Testosterone preparations for the process of female– male transition treatment are given in excessive doses. There is no evidence-based data concerning the impact of the steady state of elevated testosterone levels and their influence on oocytic quality, embryonic development, and the aspect of virilization in the female fetus. Furthermore, there are no data on the tentative influence of testosterone on the fetal female brain and CNS system. Even in cases of male fetus, such excessive testosterone levels might cause some changes including an impact on gender identity that we still cannot comprehend or predict. Nonetheless, many testosterone users successfully conceived and delivered healthy babies, without a negative effect on fetal development. In general, it is recommended that trans men stop androgenic treatment during pregnancy. In some case reports, trans men with amenorrhea had ovulated sporadically and were unaware of the pregnancy. Although they had continued with testosterone therapy for several months, they successfully gave birth to healthy children.42 Finally, in cases of extensive reassignment surgery, including hysterectomy and oophorectomy, prior to surgery oocytic cryopreservation can be used for future pregnancy via surrogated motherhood.43 Concerning the mode of delivery, one of the studies reported in a cohort of transgender patients who had cesarian section delivery, 36% were testosterone users, only 19% did not use testosterone. Among the testosterone users, 33% had cesarean section at their own request.44 Some publications reported a reduced birth weight in testosterone users,45 while other studies

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contradict this observation, reporting an absolutely normal pregnancy outcome with healthy neonates.46

5.10  REPRODUCTIVE WISH IN TRANSGENDER WOMEN It is well known from the literature that some men would like to gestate a child.47 How widespread this phenomenon is, is unknown. Usually such a wish is expressed in trans couples, but some single trans women might also desire to gestate their children.48 There are many ethical considerations against gestation in maleto-female transgender people. Foremost, males do not need to gestate in order to have a genetically-related child. Despite the progress in the development of uterine transplantation technology, its application in trans women currently seems a very remote perspective, and lacks moral and legal grounds for recognition as a human right for reproduction.

5.11  FERTILITY AND ACHIEVEMENT OF PREGNANCY IN TRANSGENDER WOMEN Gestation in male-to-female transgender people, is a subject of constant debate and extensive discussions in the literature. Moreover, uterine transplantation technology, having proved its efficiency in women, after the delivery of healthy children, still causes constant moral and legal debates.21 The debates are based on the cost-effectiveness of such a very complicated surgical procedure. The success and complication rates in terms of pregnancy and delivery are also considered. There are many ethical considerations, beyond the technical and reproductive issues of uterine transplantation in trans women that should receive special consideration. Among them is the acceptance of the unlimited right of human beings to gestate and reproduce.49 Meanwhile, the option of cryopreservation of trans women’s sperm, before the hormonal and surgical therapy, should be offered and thoroughly discussed with this population.

5.12 CONCLUSION Transgender individuals who wish to transition to their desired gender often have to undergo hormonal and surgical procedures that may lead to irreversible loss of their reproductive potential. The goal of this chapter is to state, and furthermore, to emphasize that trans men and women should be offered various options of ART treatment, to an extent which is equal to those offered to cancer patients prior to radio- or chemotherapy.

Specifically, transgender women (male-to-female transsexual patients), prior to hormonal therapy, should be offered sperm cryopreservation. In adolescents, testicular biopsy (experimental procedure) with testicular tissue cryopreservation should be offered for future use: for transplantation, or for the use of spermatogonial stem cells for IVM into spermatozoa. Trans men (female-to-male transsexual patients) should be offered cryopreservation of oocytes, or of ovarian tissue, before and during the androgenic therapy or oophorectomy. Novel technologies of ART provide trans individuals with the possibilities of gamete banking and storage, before hormonal and surgical reassignment. It is especially important for those people who are diagnosed and treated at a younger age. The scientific and medical world, as well as society at large, will need more time to accept and internalize the concept of transsexual reproduction, in order to help turn the plans of transgender people to procreate into reality. Data accumulated on gay and lesbian couples suggest that with the new technologies and treatment options, transgender people too can benefit from equal opportunities, as other gender-nonconforming people.

References 1. Califia-Rice P. Family values: two dads with a difference—neither of us was born male. Village Voice. 2000;27:46–48. 2. Hansen A. Swedish surgeons report world’s first uterus transplantations from mother to daughter. BMJ. 2012;345:6357. 3. Lefkowitz A. The Montreal Criteria for the Ethical Feasibility of Uterine Transplantation. Transpl Int. 2012;25:439–447. 4. Haraldsen IR, Dahl AA. Symptom profiles of gender dysphoric patients of transsexual type compared to patients with personality disorders and healthy adults. Acta Psychiatrica Scandinavica. 2000;102:276–281. 5. Nuttbrock L, Hwahng S, Bockting W, et  al. Psychiatric impact of gender-related abuse across the life course of male-to-female transgender persons. J Sex Res. 2010;47:12–23. 6. Richards C, Seal L. Trans people’s reproductive options and outcomes. J Fam Plann Reprod Health Care. 2014;40:245–247. 7. De Sutter P. Reproductive options for transpeople: recommendations for revision of the WPATH’s standards of care. Int J Transgend. 2009;11:183–185. 8. Coleman E, Bockting W, Botzer M, et al. Standards of care for the health of transsexual, transgender, and gender-nonconforming people, version 7. Int J Transgend. 2012;13:165–232. 9. Royal College of Psychiatrists. Good Practice Guidelines for the Assessment and Treatment of Adults with gender dysphoria. Endorsed by 13 organizations. (College Report CR181). RCPsych. ; 2013. 10. Hembree WC, Cohen-Kettenis P, Delemarre-van de Waal HA, et al. Endocrine treatment of transsexual persons: An Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2009;94:3132–3154. 11. American Psychological Association Guidelines for psychological practice with transgender and gender nonconforming people. Am Psychol. 2015;70:832. 12. Zucker KJ, Lawrence AA. Epidemiology of gender identity disorder: recommendations for the standards of care of The World

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

13. 14. 15. 16. 17.

18.

19.

20.

21.

22. 23.

24.

25.

26. 27.

28. 29.

30. 31. 32.

Professional Association for Transgender Health. Int J Transgend. 2009;11:8–18. Zucker KJ. Gender identity disorder in children and adolescents. Annu Rev Clin Psycho. 2005;1:467–492. Luders E, Sánchez FJ, Tosun D, et al. Increased cortical thickness in male-to-female transsexualism. J Behav Brain Sci. 2012;2:357. Herbert J. Who do we think we are? The brain and gender identity. Brain. 2008;131:3115–3117. Robertson JA. Cancer and fertility: ethical and legal challenges. J Natl Cancer Inst Monogr. 2005;34:104–106. Ethics Committee of the American Society for Reproductive Medicine Fertility preservation and reproduction in patients facing gonadotoxic therapies: a committee opinion. Fertil Steril. 2013;100:1224–1231. De Sutter P. Gender reassignment and assisted reproduction present and future reproductive options for transsexual people. Hum Reprod. 2001;16:612–614. Tournaye H, Goossens E, Verheyen G, et al. Preserving the reproductive potential of men and boys with cancer: current concepts and future prospects. Human Reprod Update. 2004;10:525–532. Picton HM, Wyns C, Anderson RA, et  al. A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys. Human Reprod. 2015;30:2463–2475. Goossens E, Tournaye H. Préservation de la fertilité chez le garçonprépubère: transplantation de cellules souchesspermatogoniales et greffetesticulaire. Gynécol Obstét Fertil. 2013;41:529–531. Akar ME, Ozkan O, Aydinuraz B, et al. Clinical pregnancy after uterus transplantation. Fertil Steril. 2013;100:1358–1363. Wallace SA, Blough KL, Kondapalli LA. Fertility preservation in the transgender patient: expanding on cofertility care beyond cancer. Gynecol Endocrinol. 2014;30:868–871. Demeestere I, Simon P, Dedeken L, et al. Live birth after autograft of ovarian tissue cryopreserved during childhood. Hum Reprod. 2015;30:2107–2109. Van den Broecke R, Van der Elst J, Liu J, Hovatta O, Dhont M. The female-to-male transsexual patient: A source of human ovarian cortical tissue for experimental use. Hum Reprod. 2001;16:145–147. Safer J, Tangpricha V. Out of the shadows: it is time to mainstream treatment for transgender patients. Endocr Pract. 2008;14:248–250. The Universal Declaration of Human Rights (UDHR). General Assembly resolution 217(III) A. ; 10 December 1948. Murphy TF. The ethics of fertility preservation in transgender body modifications. J Bioeth Inq. 2012;9:311–316. Chiland C, Clouet AM, Golse B, Guinot M, Wolf JP. A new type of family: transmen as fathers thanks to donor sperm insemination. A 12-year follow-up exploratory study of their children. Neuropsychiatr Enfance Adolesc. 2013;61:365–370. Murphy TF. The ethics of helping transgender men and women have children. Perspect Biol Med. 2010;53:46–60. National Center for Transgender Equality. . De Wert G, Dondorp W, Shenfield F, et al. ESHRE Task Force on Ethics and Law 23: medically assisted reproduction in singles,

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lesbian and gay couples, and transsexual people. Hum Reprod. 2014;29:1859–1865. 33. Ethics Committee of the American Society for Reproductive Medicine Access to fertility services by transgender persons: An Ethics Committee opinion. Fertil Steril. 2015;104:1111–1115. 34. Newfield E, Hart S, Dibble S, Kohler L. Female-to-male transgender quality of life. Qual Life Res. 2006;15(9):1447–1457. 35. Holman CW, Goldberg JM. Social and medical transgender case advocacy. Intl J Transgend. 2006;9:197–217. 36. Lyons T, Shannon K, Pierre L, et al. A qualitative study of transgender individuals’ experiences in residential addiction treatment settings: stigma and inclusivity. Subst Abuse Treat Prev Policy. 2015;10:1. 37. Bauer GR, Scheim AI, Deutsch MB, Massarella C. Reported emergency department avoidance, use, and experiences of transgender persons in Ontario, Canada: results from a respondentdriven sampling survey. Ann Emerg Med. 2014;63:713–720. 38. Wierckx K, Van Caenegem E, Pennings G, et  al. Reproductive wish in transsexual men. Hum Reprod. 2012;27:483–487. 39. Obedin-Maliver J, Makadon HJ. Transgender men and pregnancy. Obstet Med. 2016;9:4–8. 40. Ross LE, Tarasoff LA, Anderson S, Epstein R, Stu M. Sexual and gender minority peoples’ recommendations for assisted human reproduction services. J Obstet Gynaecol Can. 2014;36:146–153. 41. James-Abra S, Tarasoff LA, Green D, et al. Trans people’s experiences with assisted reproduction services: a qualitative study. Hum Reprod. 2015;30:1365–1374. 42. Light AD, Obedin-Maliver J, Sevelius JM, Kerns JL. Transgender men who experienced pregnancy after female-to-male gender transitioning. Obstet Gynecol. 2014;124:1120–1127. 43. Practice Committees of American Society for Reproductive Medicine; Society for Assisted Reproductive Technology Mature oocyte cryopreservation: a guideline. Fertil Steril. 2013;99:37–43. 44. Caanen MR, Soleman RS, Kuijper EA, et al. Antimüllerian hormone levels decrease in female-to-male transsexuals using testosterone as cross-sex therapy. Fertil Steril. 2015;103:1340–1345. 45. Noble F. ‘Male pregnancy’ figures reveal how 54 MEN gave birth in Australia in the past 12 months. Daily Mail. 2014. 46. The American College of Nurse-Midwives (ACNM) position statement: transgender/transsexual/gender variant health care. Task Force on Gender Bias; Clinical Standards and Documents Section DOSP. ; 2012. 47. Murphy TF. Assisted gestation and transgender women. Bioethics. 2015;29:389–397. 48. Practice Committee of the American Society for Reproductive Medicine Practice Committee of the Society for Assisted Reproductive Technology. Recommendations for practices utilizing gestational carriers: a committee opinion. Fertil Steril. 2015;103:e1–e8. 49. Brännström M, Johannesson L, Bokström H, et al. Livebirth after uterus transplantation. Lancet. 2015;385:607–616.

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6 Standards of Care for the Health of Transsexual, Transgender, and GenderNonconforming People: An Introduction Eli Coleman* University of Minnesota, Minneapolis, MN, United States

O U T L I N E 6.1 Introduction to the SOC

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6.10 Criteria or Hormone and Surgery

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6.2 Basic Principles

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6.11 Therapy Before Surgery

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6.3 The Issue of Medical Necessity

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6.12 Surgery Criteria for Adolescents

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6.4 Clinical Effectiveness

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6.5 The Standards of Care are Flexible

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6.13 A Note About Social Transitions for Children and Adolescents

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6.6 Beyond the Binary

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6.14 Risks of Withholding or Delaying Medical Treatment

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6.7 Competency for Adult Assessment

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6.15 Important Role of Advocacy

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6.16 Conclusion

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6.8 Competency for Childhood and Adolescent Assessment 72 6.9 Surgical Competency

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Clinicians working with transsexual, transgender, and gender-nonconforming people should be well aware of and adhere to the World Professional Association for Transgender Health’s (WPATH) Standards of Care (SOC).1 The overall goal of the SOC is to guide health professionals in providing safe and effective pathways to transsexuals, transgender, and gender nonconforming individuals in order to maximize their overall health, psychological well-being, and self-fulfillment.

The WPATH, formerly the Harry Benjamin International Gender Dysphoria Association2 is an international, multidisciplinary, professional association whose mission is to promote evidence-based care, education, research, advocacy, public policy, and respect in transsexual and transgender health. The vision of WPATH is a world wherein transsexual, transgender, and gender-nonconforming people benefit from access to evidence-based health care, social services, justice,

*

 Eli Coleman is Professor and Director of the Program in Human Sexuality, Department of Family Medicine and Community Health, University of Minnesota Medical School. He is one of the Past Presidents of the World Professional Association for Transgender Health (WPATH). He is the Chair of the Standards of Care Committee for WPATH and was responsible for Version 7 and currently Version 8. Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00058-9

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and equality. Clinicians should also consider becoming a member of this organization in order to remain current with the state of the science and practice. WPATH offers biennial international congresses and now has divided itself into regions offering conferences on the off years in the various regions of the world. In addition, WPATH has begun a Global Education Initiative to provide a pathway to certification and continuing education opportunity. Another way of keeping up with developments is to subscribe and read WPATH’s official journal— the International Journal of Transgenderism. Textbooks are available that are useful for in depth discussion of clinical issues (e.g., Ref. [2]). Clinical competence in this area is clearly dependent upon sound specialized training and continuing education. Clinical care goes beyond provision of hormone treatment or surgical interventions. Clinical care includes primary care, gynecologic and urologic care, reproductive health care, voice and communication therapy, and mental health services (e.g., assessment, counseling, psychotherapy).

6.1  INTRODUCTION TO THE SOC The SOC were initially developed in 1979. The SOC have gone through six revisions since then (1980, 1981, 1990, 1998, 2001, and 2012). The most recent version (SOC 7) constituted a complete overhaul of the previous versions and has been translated into 12 languages. The current SOC and the translations are available for free download on the WPATH website or through purchase of a hard copy. The SOC are also available in a mobile app. For a more complete history of the SOC and a description of the process and background papers which were commissioned to help write the new standards, see Refs. [3–6]. The SOC contains many useful sections which review the epidemiology, the role of the mental health clinician, components of assessment of gender dysphoria and other comorbid conditions, components of an assessment and letter of recommendations for hormone therapy or surgical procedures, the value and limits of E-therapy, online or distance, counseling, reproductive health issues, the role of voice and communication therapy, assessment and treatment of gender dysphoria with intersexed individuals, applicability of the SOC to people living in institutional environments, and lifelong preventative and primary care considerations. It also contains appendices, including a glossary, an overview of medical risks of hormone therapy, a summary of criteria for hormone therapy and surgery, and evidence for clinical outcomes of therapeutic approaches. Finally, the evidence for supporting these standards is clearly articulated and referenced. WPATH has begun the process of revising the SOC and it is expected that SOC 8 will be published in 2018. The

SOC are updated regularly so it is important to consult the WPATH to make sure clinicians use the latest version. A useful companion document to the SOC are the Endocrine Society Guidelines.7 They are mostly aligned with the WPATH SOC and are also under revision and expected to be published in early 2017. Efforts are being made to align the Endocrine Guidelines to SOC 7 and to be consistent with SOC 8. The endocrine guidelines are a useful companion document, especially for the provision of hormone therapy.

6.2  BASIC PRINCIPLES There are basic principles which underlie the SOC.1 These are useful principles that can address the applicability of the SOC in areas which are not clearly defined or in applying these standards to different cultures and resource-poor settings. These principles include: Exhibit respect for patients with nonconforming gender identities (do not pathologize differences in gender identity or expression). ● Provide care (or refer to knowledgeable colleagues) that affirms patients’ gender identities and reduces the distress of gender dysphoria, when present. ● Become knowledgeable about the health care needs of transsexual, transgender, and gendernonconforming people, including the benefits and risks of treatment options for gender dysphoria. ● Match the treatment approach to the specific needs of patients, particularly their goals for gender expression and need for relief from gender dysphoria. ● Facilitate access to appropriate care. ● Seek patients’ informed consent before providing treatment. ● Offer continuity of care and be prepared to support and advocate for patients within their families and communities (schools, workplaces, and other settings). ●

Probably the most significant aspect of Version 7 of the SOC is the tone of the document. It emphasizes what professionals need to do rather than placing emphasis on what the client needs to do. It also emphasizes that health care for trans individuals is more than just providing hormones and surgery—it is about promoting overall health and well-being.

6.3  THE ISSUE OF MEDICAL NECESSITY One of the greatest challenges today is getting full recognition of the medical necessity of these procedures and having them covered by national health and insurance policies and services. The SOC has always been, and is currently being utilized to advocate for these services to be covered. The SOC

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6.7  Competency for Adult Assessment

clearly outlines and documents the fact that hormone and surgical treatments for this population are, in most cases, medically necessary in order to resolve gender dysphoria. There are other procedures that might enhance a sense of masculinity or femininity or “good looks,” that individuals might be interested in pursuing. However, clinicians must be cognizant of the difference between medically necessary gender affirming procedures and those that would be cosmetic and not necessarily medically necessary. WPATH has clearly stated in the SOC and in a subsequent clarifying document that procedures such as hysterectomy, bilateral mastectomy, chest reconstruction or augmentation, genital reconstruction, scrotoplasty, facial hair removal, and facial reconstruction (facial feminization) are medically necessary procedures for many individuals.8 This is not to say that all procedures are necessary for each patient. It is important for the clinician to evaluate which procedures will relieve their gender dysphoria.

6.4  CLINICAL EFFECTIVENESS Behind the question of medical necessity is the question of clinical effectiveness of these procedures. In medicine one of the most important clinical principles is “Do no harm.” So, the question is: why alter the natural body which is not in any way deemed to be diseased or malignant? Yet, providing hormone and surgical procedures has been shown to be the most effective way to alleviate gender dysphoria. The SOC provide the review of the evidence and the expert consensus on the validity and effectiveness of these procedures. There is clear evidence that they can no longer be called “experimental.” They are highly effective procedures that have been shown to reduce or eliminate gender dysphoria. They have also been shown to be effective in improving comorbid conditions (e.g., anxiety, depression, and substance abuse). These procedures reduce the risk of suicide. Patients seldom regret going through these procedures. All of this evidence is reviewed in the text of the SOC and in Appendix D.

6.5  THE STANDARDS OF CARE ARE FLEXIBLE While the SOC does set standards that are designed to provide optimal care based upon the best available science, they are designed to be flexible. It allows clinicians to make decisions that are well thought through and are well-documented regarding the reasons they may deviate from the accepted SOC. Clinical departures from the SOC may arise because of a patient's unique anatomic, social, or psychological situation, an experienced health professional’s evolving method of handling a common situation, a research protocol, or the need for harm reduction strategies. These departures should be recognized as such, explained

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to the patient, and documented through informed consent for quality patient care and legal protection. Flexibility is especially important when the SOC needs to be modified in culturally appropriate ways without sacrificing essential principles and to adjust to resource limitations, especially in underdeveloped parts of the world.

6.6  BEYOND THE BINARY As there is greater public visibility and awareness of gender diversity, this has expanded the options for people with gender dysphoria to actualize an identity and find a gender role and expression that are comfortable for them. In many other cultures where the gender binary is not so rigid and other gender identities are acknowledged, there are also many options other than the binary (e.g., Refs. [9–11]). As more and more people learn about these cultures, they find them a source of inspiration for people living beyond the binary. Thus, clinicians are seeing more and more individuals coming for treatment who want to live completely in another role than their assigned gender. Some individuals describe themselves not as merely gender nonconforming but as unambiguously cross-sexed (i.e., as a member of the other sex). Others consider themselves neither male or female. Instead, they may describe their gender identity as trans, trans*, transmasculine, transfeminine, transgender, bigender, or genderqueer. Terminology is rapidly evolving to describe various ways that individuals define themselves that transcend a male/female binary concept. In evaluating someone for hormone or surgical interventions, the clinician would use the same criteria for adults or adolescents. In all likelihood, these individuals might be helped with hormone therapy alone (and low doses can sometimes be as effective as higher doses). Others will require hysterectomy, bilateral mastectomy, or chest reconstruction or augmentation. Again it is important that the clinician help the individual consider the risks and benefits and avoid unnecessary surgeries that one might regret later on. There will be a much larger discussion of treatment strategies with this population in SOC Version 8.

6.7  COMPETENCY FOR ADULT ASSESSMENT A common issue arises when considering the competence of the health care professional in making an assessment for providing health care for transsexual, transgender, or other gender nonconforming individuals. Probably the most important characteristic that the health care professional should possess is cultural competence. This means interacting with the community and being involved in or at least knowledgeable about

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the sociopolitical issues that the community and their families are facing. This is why trans-identified health care workers are especially valued. However, cis-gendered professionals can develop this cultural competency through interacting with people in the community. Cultural competency can also be gained through more formal and continuing education opportunities. Beyond this essential characteristic, there are minimal requirements that are stated in SOC 7: 1. A master's degree or its equivalent in a clinical behavioral science field. This degree, or a more advanced one, should be granted by an institution accredited by the appropriate national or regional accrediting board. The mental health professional should have documented credentials from a relevant licensing board or equivalent for that country. 2. Competence in using the Diagnostic and Statistical Manual of Mental Disorders and/or the International Classification of Diseases for diagnostic purposes. 3. Ability to recognize and diagnose coexisting mental health concerns and to distinguish these from gender dysphoria. 4. Documented supervised training and competence in psychotherapy or counseling. 5. Knowledgeable about gender nonconforming identities and expressions, and the assessment and treatment of gender dysphoria. 6. Continuing education in the assessment and treatment of gender dysphoria. This may include attending relevant professional meetings, workshops, or seminars; obtaining supervision from a mental health professional with relevant experience; or participating in research related to gender nonconformity and gender dysphoria.

6.8  COMPETENCY FOR CHILDHOOD AND ADOLESCENT ASSESSMENT Working with children and adolescents is particularly challenging. Helping individuals and families make decisions regarding social transitions, administering puberty-blocking hormones or cross sex hormones, or considering any kind of surgical intervention can be quite difficult. These are weighty decisions that need to be made with the individual who has evolving abilities to consent for various treatments. Family and peer pressure can complicate these decision-making processes. In addition to the competency requirements for adult assessments, there are a number of other critical competencies that the health care professional should possess. These are articulated in SOC 7. 1. Meet the competency requirements for mental health professionals working with adults.

2. Trained in childhood and adolescent developmental psychopathology. 3. Competence in diagnosing and treating the ordinary problems of children and adolescents. An important consideration for health care professionals beginning work in this area is that they should work under the supervision of a mental health professional with established competence in the assessment and treatment of gender dysphoria and have general competence in working with comorbid conditions as well.

6.9  SURGICAL COMPETENCY Successful outcome from surgical interventions is dependent upon surgeons with the competence in their surgical field but also have the specialized knowledge of gender affirming surgeries. A positive physical outcome, i.e., avoidance of infection or other medical complication, as well as a satisfactory aesthetic outcome are important. All of this takes additional training. Not only is a good physical outcome important for medical reasons, but this result can be critical in psychosocial adjustment postsurgery. Poor outcomes can lead to regret or difficulty in adjusting to a new physical gender expression. The SOC outlines minimal criteria for surgeons to establish competency in performing these procedures. 1. Physicians who perform surgical treatments for gender dsyphoria should be board certified urologists, gynecologists, plastic surgeons, or general surgeons. 2. Surgeons should have specialized competence in genital reconstructive techniques as indicated by documented supervised training with a more experienced surgeon. 3. Even experienced surgeons must be willing to have their surgical skills reviewed by their peers. 4. Surgeons should regularly attend professional meetings where new techniques are presented. 5. Surgeons should be knowledgeable about more than one surgical technique for genital reconstruction so that they, in consultation with patients, can choose the ideal technique for each individual. Alternatively, if a surgeon is skilled in a single technique and this procedure is either not suitable for or desired by a patient, the surgeon should inform the patient about other procedures and offer referral to another appropriately skilled surgeon.

6.10  CRITERIA OR HORMONE AND SURGERY Certainly one of the most important things for health care professionals to understand are the criteria for hormonal

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6.10  Criteria or Hormone and Surgery

treatment and surgical procedures. A psychosocial assessment must be conducted by a health care professional with the competence to conduct these types of assessment. This includes the ability to diagnose gender dysphoria and comorbid conditions. This is often done by a mental health professional but can also be conducted by a health care

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professional with competence in conducting psychosocial evaluations. Different procedures require different levels of assessments and in particular, a determination about whether one assessment or two are required. Below are two useful tables summarizing the requirements for evaluations for different hormone and surgical procedures.

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There are more specific requirements for hormonal or surgical interventions with children and adolescents and they are determined by the level of intervention: reversible, partially reversible, and irreversible interventions. It is important to carefully consult the SOC to understand these requirements.

6.11  THERAPY BEFORE SURGERY There is quite a bit of misunderstanding regarding whether therapy is required before any surgical intervention. It is clear that this would be required for ado­ lescents. However, for adults, the SOC is clear that there is no requirement for therapy before surgery unless this is recommended based upon evaluation of the individual. There is simply no evidence that it is a necessary requirement for all individuals. The SOC clearly outlines the advantages of psychotherapy and the therapeutic issues that might be addressed through psychotherapy. These can be recommended, but are not required. In cases of comorbid serious psychopathology, therapy is clearly recommended as comorbid conditions are expected to be “reasonably managed” prior to surgery. The assumption here is that serious psychopathology should be addressed appropriately so that the patient can better understand the risks and advantages of hormone treatment or surgical procedures and be better prepared to deal with the adjustment issues related to undergoing these procedures and the psychosocial adjustment of transitioning. It is a balancing act for the clinician because if the individual is psychologically unstable, the effectiveness of the interventions can be compromised and lead to worsening of their conditions and/or regret about the procedures. At the same time, we know that hormone treatments and surgical procedures can improve a person’s psychological functioning and alleviate not only gender dysphoria but comorbid conditions. This involves careful assessment and psychotherapy to decide when the individual is ready for gender-affirming interventions.

6.12  SURGERY CRITERIA FOR ADOLESCENTS As these involve irreversible interventions, decisions regarding surgery during adolescence before the age of majority or age of consent for medical decision making must be done with great care and consideration. There is no question that in carefully evaluated situations psychotherapy, such as when considering or undergoing bilateral mastectomy, can be very useful for some individuals. Long-standing gender dysphoria, the likelihood

of successful living in the preferred gender role, and the extent of family and social support are all some of the things that the clinician must establish. For example, chest surgery in transmasculine individuals could be considered after ample time of living in the desired gender role and after 1 year of testosterone treatment. The intent of this suggested sequence is to give adolescents sufficient opportunity to experience and socially adjust to a more masculine gender role prior to undergoing surgery. Genital surgery, being much more irreversible, is a very difficult decision. However, we have evidence that in some cases that these procedures could be beneficial before the age of 18.12 The SOC, under the proviso of making well-documented exceptions, would condone genital surgery before 18. SOC 8 will certainly be addressing this issue in more detail in order to provide more guidance in this challenging area.

6.13  A NOTE ABOUT SOCIAL TRANSITIONS FOR CHILDREN AND ADOLESCENTS A very difficult decision in working with children and adolescents is whether to support social transitions at an early age. The SOC cautions about the encouragement of social transitions during childhood without careful assessment and working with the system to accommodate the child’s gender expression. Mental health professionals are encouraged not to impose a binary view of gender and to challenge children and adolescents. Their parents should be encouraged to avoid this as well. They should give ample room for children and adolescents to explore different options for gender expression. Early social transitions might be appropriate for some, but must be carefully considered. In these cases, a very important caveat to keep in mind is that many children do not persist in their gender dysphoria into adolescence so it is very important that they are well aware that there are pathways back to the gender assigned at birth.

6.14  RISKS OF WITHHOLDING OR DELAYING MEDICAL TREATMENT While there are clear criteria for making decisions regarding hormone and surgical interventions, a very important consideration is often neglected: refusing timely medical interventions might prolong gender dysphoria and contribute to poor psychosocial adjustment and development or exacerbation of mental health issues. The barriers for proper assessment, therapy, access to timely health care, and undue restrictions on eligibility for medical procedures are not benign or a neutral

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REFERENCES

option. These barriers and delays can have negative consequences. The SOC clearly warns of these problems and advocates for timely interventions. Again, it is a balancing act of having enough time to conduct adequate evaluations, complete any recommended therapies prior to hormonal or surgical interventions, and avoiding unnecessary delays of gender affirming treatment.

6.15  IMPORTANT ROLE OF ADVOCACY WPATH recognizes that health is dependent upon not only good clinical care but also social and political climates that provide and ensure social tolerance, equality, and the full rights of citizenship. There are clearly social determinants of health. You cannot have health without rights. WPATH encourages health care professionals to become involved in advocating for public policies and legal reforms that promote tolerance and equity for gender and sexual diversity and that eliminate prejudice, discrimination, and stigma.

6.16 CONCLUSION Health care professionals working with transsexual, transgender, and gender nonconforming individuals should be well acquainted with the WPATH SOC and adhere to those standards. The SOC are evolving and will be revised periodically, and it is important that clinicians stay current with revisions of the SOC and the developments in the field between revisions. The field is advancing at a rapid rate as more research is being conducted and as there is a large shift in the sociopolitical environment for trans individuals. For beginners working in this area, supervised training is essential. For more advanced clinicians, continuing education is mandatory. As WPATH develops its certification process, clinicians are encouraged to ensure their competency by becoming certified. Hopefully we will have more systematic and scientifically based guidance on providing the best possible care to these individuals. An area needing particular attention are guidelines for the treatment of children and adolescents.

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Health professionals can use the SOC to help patients consider the full range of health services open to them, in accordance with their clinical needs and goals for gender expression. And, as the SOC emphasizes, health care for trans individuals is more than just providing hormones and surgery—it is about promoting overall health and well-being.

References 1. Coleman E, Bockting W, Botzer M, et  al. Standards of care for the health of transsexual, transgender, and gender nonconforming people, 7th version. Int J Transgenderism. 2012;13(4):165–232. http://dx.doi.org/10.1080/15532739.2011.700873. 2. Ettner R, Monstrey S, Coleman E. Principles of Transgender Medicine and Surgery. 2nd Ed New York: Routledge; 2016. 3. Coleman E. Toward Version 7 of the World Professional Association for Transgender Health's Standards of Care: medical and therapeutic approaches to treatment. Int J Transgenderism. 2009;11(4):215– 219. http://dx.doi.org/10.1080/15532730903439450. 4. Coleman E. Toward Version 7 of the World Professional Association for Transgender Health's Standards of Care: hormonal and surgical approaches to treatment. Int J Transgenderism. 2009;11(3):141– 145. http://dx.doi.org/10.1080/15532730903383740. 5. Coleman E. Toward Version 7 of the World Professional Association for Transgender Health's Standards of Care: psychological assessment and approaches to treatment. Int J Transgenderism. 2009;11(2):69–73. http://dx.doi.org/10.1080/15532730903008008. 6. Coleman E. Toward Version 7 of the World Professional Association for Transgender Health's Standards of Care. Int J Transgenderism. 2009;11(1):1–7. http://dx.doi.org/10.1080/15532730902799912. 7. Hembree WC, Cohen-Kettenis P, Delemarre-van de Waal HA, et al. Endocrine treatment of transsexual persons: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2009;94(9):3132–3154. http://dx.doi.org/10.1210/jc.2009-0345. 8. World Professional Association for Transgender Health. WPATH clarification on medical necessity of treatment, sex reassignment, and insurance coverage for transgender and transsexual people worldwide. Retrieved from ; 2008, 16.10.16. 9. Coleman E, Colgan P, Gooren L. Male cross-gender behavior in Myanmar (Burma): a description of the acault. Arch Sex Behav. 1992;21(3):313–321. 10. Nanda S. Neither Man nor Woman, The Hijras of India. 2nd Ed Belmont CA, USA: Wadsworth Publishing Company; 1998. 11. Peletz MG. Transgenderism and gender pluralism in southeast Asia since early modern times. Curr Anthropol. 2006;47(2):309–340. 12. Milrod C. How young is too young: ethical concerns in genital surgery of the transgender MTF adolescent. J Sex Med. 2014;11(2):338–346. http://dx.doi.org/10.1111/jsm.12387.

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7 Sex-Dependent and -Independent Mechanisms in External Genitalia Development Congxing Lin and Liang Ma Washington University School of Medicine, St. Louis, MO, United States

O U T L I N E 7.1 Overview 7.1.1 Background and Clinical Relevance 7.1.2 Clocal Development 7.1.3 The Use of Mouse Models in Studying Urogenital Development

7.2.5 Genes Involved in Maintaining Epithelial Structure 84 7.2.6 Other Genes with Relevance to GT Development 85

77 77 78 80

7.3 Hormone-Dependent Sexual Differentiation 86 7.3.1 The Window of Hormone-Dependent Sexual Differentiation 86 7.3.2 Endocrine Disruptors and Congenital Penile Anomaly 87 7.3.3 Discordant Brain and Genital Sexualization 87

7.2 The Androgen-Independent Phase of Genital Tubercle Development 80 7.2.1 Overview 80 7.2.2 Canonical WNT Signaling Pathway 81 7.2.3 Shh Signaling Pathway 83 7.2.4 Fibroblast Growth Factor Signaling Pathway 83

References 88

7.1 OVERVIEW

In addition to its significance in evolution, the development of the external genitalia is also of particular interest to pediatricians because of the high incidence of congenital malformations. External genital malformation is one of the most common inborn errors in humans. Hypospadias, a condition characteristic of mispositioning of the urethral meatus at the penile shaft, scrotum, or perineum, affects every 1 in 200 boys and the incidence has been widely reported to be increased recently.1–3 Epispadias is a less common but more severe genital defect. Patients with epispadias often has a bifurcated penis and a dorsal urethral opening accompanied by other malformation in the lower urogenital tract, especially that of the bladder. Although most of

7.1.1  Background and Clinical Relevance The external genitalia, i.e., penis in males and clitoris in females, are the most posterior part of the genitourinary tract. All therian mammals possess an external genital organ at the posterior end of the urogenital sinus, which is completely separated from the rectum. External genitalia, in particular the penis, are of obvious significance for internal fertilization. Acquiring this organ is an indispensable component in the transition from aquatic life to terrestrial life, and therefore represents a key step in the mammalian evolution.

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the hypospadias/epispadias can be corrected by surgical procedures, some of the most severe cases often need multiple operations and may result in compromised reproductive health. Ejaculation problems and negative genital appraisal have been widely reported. Nonetheless, even with successful surgical correction, patients might develop psychological problem later in their adolescence. Despite the great health concern posed by external genital abnormalities, the genetic basis for genital development and the etiology of hypospadias/ epispadias is not well understood. Both endocrine disruptors and genetic factors have all been suggested to play a role in the pathogenesis of hypospadias. The development of external genitalia, i.e., penis in males and clitoris in females, undergoes two distinct phases: an early androgen-independent genital tubercle (GT) growth and a late androgen-dependent sexual differentiation. The early phase of genital growth is achieved by the coordinated growth and patterning of the cloaca-endoderm-derived urethra and mesodermally derived genital mesenchyme within a capsule of ventral skin. Up to 12 weeks of gestation in human and 16 days in mice, male and female GT are morphologically identical; this phase of development is controlled by the same genetic program in both sexes.4 After that, the prepuce in males undergoes growth under the action of androgen, and eventually results in fusion of the opposing urethral folds and formation of definitive tubular urethra. In females, the growth of prepuce is less prominent, and urethral epithelium remains as an epithelial cord (Fig. 7.1). The process of urethral fusion has been compared to that of palatal fusion. It involves growth of genital

FIGURE 7.1  Embryonic development of external genitalia in mice. The embryonic anlage of the external genitalia, the GT, develops on the ventral side of the cloacal membrane. During androgen-independent development, GT undergoes continuous outgrowth from E11  to  E15. Afterwards, the GT in males masculinize to form a penis with an internalized urethra. While in females, the clitoris develops in the absence of androgen.

mesenchyme, fusion of the opposing epithelium, and subsequent removal of the residual epithelial seam by apoptosis.2,4 Disruption in either androgen-independent or androgen-dependent developmental processes may cause hypospadias, epispadias, or other genital malformations. Hypospadias has traditionally been considered the result of feminization of the external genitalia. Indeed, reduced androgen responsiveness due to mutation in either the androgen receptor (AR) or androgen synthetic enzymes in humans, and/or androgen antagonist flutamide treatment in rodents both cause hypospadias. It’s noteworthy that although androgen insufficiency is believed to be the leading cause of the hypospadias, it only explains less than 5% of the cases.

7.1.2  Clocal Development It’s important to note that many severe hypospadias or micropenis cases are also accompanied by malformations in other part of the urogenital and anorectal tracts. This is predictable because the rectum, urethra, vagina in females, and the external genitalia all derive from a common embryonic structure, the cloaca. The morphogenesis of these caudal organs is highly coordinated and likely regulated by a shared genetic program. Therefore, the development of external genitalia has to be studied in the context of cloacal patterning. In placental mammals, the cloaca is a transient embryonic structure that later becomes part of the urinary, genital, and digestive tracts. It forms at the caudal end of the endodermderived hindgut, and subsequently gives rise to the urogenital sinus ventrally and anorectal sinus dorsally through a process termed cloaca septation. Following that, the urogenital sinus develops into the external and internal urethra, bladder, and prostate, and in females, the posterior vagina and urethra. The anorectal sinus gives rise to the rectum and anal channel (Fig. 7.2 control panels). Along with cloacal division, the GT emerges around the cloacal membrane. The ventral cloacal endoderm extends distally and forms the urethral epithelium, whereas the paracloacal mesenchyme (PCM) constitutes the genital mesenchyme. The GT outgrowth depends on a signaling center located at the distal-most part of the cloacal endoderm (later urethral epithelium), termed the distal urethral epithelium (dUE). Abnormal development of cloaca-related structures causes a spectrum of inborn defects from isolated hypospadias to complex urogenital-anorectal malformations such as OEIS (omphalocele, exstrophy, imperforate anus, and spinal defects). Patients with ARM often present ectopic connections between rectum and genital-urinary tract with an absence of normal anal opening, suggesting a defective cloacal septation (β-catenin-cKOs are shown as an example for persistent cloaca in Fig. 7.2). Only

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FIGURE 7.2  Normal and abnormal cloacal development in mice. 3D reconstruction of the cloacal field in E12.0 WT (A, C, E, and G) and

β-Catenin-cKO (B, D, F, and H). Different tissue lineages were indicated by different colors. (A–B) Reconstructed 3D structures of embryos with cloaca indicated with magenta (light gray in print version). Note the vertical growth of urethral epithelium within the GT in the control, and lack of GT and urethral epithelium in the cKO. (C–D) Reconstructed embryos with cloaca-lineage (magenta (light gray in print version)) together with wolffian ducts/ureter (white) and neural tube (blue  (black in print version)). Note the cloacal membrane is disintegrated and cloaca is exposed (arrow and inset). (E–F) Reconstructed cloaca showing overall smaller volume, abnormal shape, and ectopic anterior connection between hindgut and the urogenital tract in the cKO mutants. (G–H) Reconstructed cloaca-derivatives and wolffian ducts/ureter showing normal connection between wolffian ducts and urogenital sinus in the control and disconnection in the mutant. cl, cloaca; urs, urogenital sinus; ur, ureter; wd, wolffian duct; hg, hindgut; ue, urethral epithelium; tg, tail bud; bl, bladder.

in rare cases, the rectum ends blindly without forming a fistula. The ectopic connection can appear anywhere from the bladder neck to the perineum. In most severely affected females, the rectum, vagina, and urethra join together and form a common channel, which is termed persistent cloaca.5,6 The etiology of these defects is largely unknown. However, 50% of the patients with imperforate anus also present anomalies in other organ systems.7 The embryology for cloacal morphogenesis is still a matter of speculation. The prevailing view is that the invasion of urorectal septum (URS), either from the anterior side8 or lateral side of the cloaca,9,10 is the driving force in this process,8–11 whereas the cloacal endoderm plays a rather passive role. Therefore, it is commonly perceived that septation defects are due to underdevelopment of the URS, in which case the rectum and the urogenital tract remain joined at the most posterior position, resembling the morphology of the embryonic cloaca, with normal anal opening maintained. This theory, however, could not explain the abnormal cloacal development where a fistula forms at an anterior position such as the bladder neck. An alternative theory is that the formation of URS is the result rather than the cause of normal cloacal patterning.5,12,13 According to

this model, underdevelopment of the dorsal cloaca can directly cause defective septation in abnormal cloacal development. However, direct evidence supporting this model is still lacking. Studies in mouse genetics models have provided valuable insights into the molecular mechanisms underlying cloacal development. The Sonic Hedgehog (Shh) signaling pathway plays a critical role in cloacal patterning and subsequent development of the urogenital and anorectal organs. Knocking out Shh14–17 or Hh signaling mediator Gli2/Gli318 both result in failed cloacal septation, with underdeveloped GT in mice. In a recent study, we demonstrated that the target tissue for endodermally expressed Shh is the neighboring PCM, as conditional deletion of Shh signaling mediator Smoothened (Smo) gene in the PCM led to defective cloacal septation, while abolishing Smo in the cloacal endoderm did not affect this process.14 We also found that loss of Hh signaling led to downregulation of regulatory genes in the PCM, including Hoxa13 and Hoxd13.14 Intriguingly, mice deficient in both Hoxa13 and Hoxd1319 also developed persistent cloaca and GT agenesis. Other than Shh signaling, Six1/Eya1,20 Wnt5a,21 Fgf10,22 EphrinB2/ EphB2,23 and Bmp724,25 have also been implicated in cloacal development.

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7.1.3  The Use of Mouse Models in Studying Urogenital Development The development of the human genitals is largely similar to that in rodents. This makes rodent models, both transgenic and drug-induced, useful tools to investigate the etiology of human congenital genital malformations. Indeed, most current knowledge about external genital development has originated from studies using rodent models. With the help of the state-of-art knockout/transgenic mouse technology, especially the newly developed Cre/LoxP technology, researchers have revealed that disrupting signaling pathways governing early androgen-independent development can also lead to hypospadias and other external genital abnormalities. Shh is strongly expressed in the urethra throughout the GT development, and Shh-/- mice have complete agenesis of external genitalia.15,26 Consistently, recent work showed that ablating Hh receptor Smoothened in genital mesenchyme14 also causes genital malformation. Notably, dysregulated Shh signaling, as a consequence of reduced cholesterol level, is implicated in Smith-Lemli-Opitz syndrome (SLOS) in humans. SLOS patients also present with a high incidence of hypospadias. Hoxa13 mutant mouse27 develop failure of urethral closure due to dysregulated Bmp7 and Fgf8 expression. Mutation in the same gene causes Hand-Foot-Genital syndrome (HFGS) in humans. Moreover, recent work also implicated disruption of WNT signaling in the pathogenesis of mouse hypospadias and agenesis of genitalia, although its relevance to the human condition is still not clear. Mice homozygous of Fgf10 null alleles or Fgfr2IIIb null alleles developed severe posterior hypospadias,28–32 and it is believed that the defective urethral maturation was the main cause of the phenotype. This work is further supported by a recent study describing the defective external genital development in the tissue-specific FGF-receptor knockout mice.31,32 It’s also noteworthy that many studies have suggested that deficiencies in cell adhesion may also cause hypospadias-like phenotype. Mice lacking basement membrane protein laminin α-5 (LAMA5) exhibit a severe proximal hypospadias.33 A similar phenotype has also been observed in the ectodermal-specific knockout of β-Catenin mice.34 In addition, transgenic mice with Six1/Eya1,20 Wnt5a,21,35,36 EphrinB2/EphB2,23 Alx4,37 Mafb,38–40 Sp8,41 Fgf8,28,34,36,41,42 BMPR,35 Bmp7,24,25 and/or AR mutations40,43 have been used to investigate the development of the external genitalia. Rodents have also been used to characterize hormone (androgen) regulated sexual differentiation. Several recent studies43,44 have used mouse models to establish the time window during which the GT is responsive to either estrogenic and/or androgenic signals.

7.2  THE ANDROGEN-INDEPENDENT PHASE OF GENITAL TUBERCLE DEVELOPMENT 7.2.1 Overview The external genitalia of both sexes are developed from the same embryonic anlage, the GT. In mice, development of the GT starts at around E10.5. First, two paired swellings appear on either side of the cloacal membrane. These two swellings are later joined by a third swelling on the dorsal side of the cloacal membrane, and form a single tubercle. The ventral-distal part of the cloacal epithelium can be seen across the distal tip of the GT. Following that, the GT undergoes an outgrowth phase to form a cone-like structure at around E12.5. During this process, the dorsal GT grows much faster. As a result, the urethral epithelium, which is derived from the cloacal epithelium, is pushed to the ventral side of the GT. Thereafter, GT outgrowth continues along the proximal–distal axis (Fig. 7.3). At the same time, the prepuce starts to develop on either side of the GT. Up until E15, the GT in males and females are morphologically identical to each other and there has not been any report of gene expression difference between the sexes. Therefore, we consider this phase of GT development to be hormone-independent. The most important and prominent event in early GT development is its outgrowth. The GT include cells from all three germ layers. The GT mesenchymal cells are mesodermal in origin, the urethral epithelium is derived from the endodermal cloacal epithelium, while the whole structure is under the cover of an ectodermal surface epithelium. The early GT development can be described as coordinated growth of the GT mesenchyme and urethra within an ectodermal capsule. Despite its apparent difference in structure and functionality, the outgrowth of the GT shares a lot in common with another type of body appendages, the limb bud.45,46 The outgrowth of both organs depends heavily on epithelial–mesenchymal interactions. First, an epithelial signaling center is formed at the distal end of both structures; the apical ectodermal ridge (AER) and the dUE in the developing limb bud and GT, respectively (Fig. 7.3). These signaling centers secrete instructive signals, and establish an independent proximodistal axis away from the main body axis. These signals are maintained through reciprocal interactions between the mesenchyme and epithelium, and provide continuous guidance to regulate the shape of future structures during the whole process. The GT signaling center dUE was first described in 2000 by Dr. Yamada’s group.28 They demonstrated that a strip of endodermal urethral cells around the cloaca region starts to express Fgf8 at around E10 in mouse embryos. These cells are the endodermal part of the cloacal membrane

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FIGURE 7.3  Distal signaling epithelia in the outgrowth of body appendages. (A) The outgrowth of the limb is instructed by a specialized ectodermal epithelium, the AER (in red (dark gray in print version)), positioned at the distal edge of the limb bud expanding across the anteroposterior axis. Inset shows a cross-section through the plain indicated by the arrow. (B) The Fgf8-expressing GT signaling center dUE (in red (dark gray in print version)) is positioned at the distal most part of the cloacal endoderm right below the ventral ectoderm (inset, coronal section through the plain indicated by the arrow). (C, D) The dUE remains at the distal end of the urethral epithelium as the GT and the urethra undergo continuous proximodistal outgrowth. Endodermal cloaca was illustrated in yellow (light gray in print version) in B–D. Fgf8 expression marks the AER (E) and dUE (F-H). cl, cloaca; hg, hindgut; ue, urethral epithelium; tg, tail bud; ls, lateral swellings; ds, dorsal swelling; ugs, urogenital sinus; bl, bladder.

that is positioned right below the ectodermal epithelial cells. As the GT grows out of the body wall, the more prominent dorsal growth pushes the cloacal membrane to the ventral side of the GT, which becomes the distal end of the urethral epithelium. At the same time, the lower part of the cloacal endoderm extends into the GT to form the more proximal part of the urethral epithelium. During the whole process, Fgf8 expression is maintained in the endodermal part of the dUE. These Fgf8-expressing cells are termed dUE cells. Organ culture experiments further supported that these cells play a key role in promoting GT outgrowth.28 Follow-up studies demonstrated that the expression of genes in distal GT mesenchyme are tightly regulated by dUE signals.4,28,34,41 This signal transduction echos that between AER and the underlying limb mesenchyme during limb development, a prevalent model used to study organogenesis. Major signaling pathways such as WNT, Hh, BMP, and FGF pathways, along with transcription factors such as Hox and Tbx genes, have all been extensively studied in context of limb development. The early GT development appears to involve a very similar set of players, as mounting evidence indicate that all these aforementioned genes also participate in regulating GT outgrowth. Despite the similarities, it’s also noteworthy that GT development has some unique features. The key difference is that in addition to the mesenchyme, GT also involves two types of epithelia: the ectodermal surface epithelium and the urethral epithelium within. In a recent study, it was clearly demonstrated that the contact between

ectodermal epithelium and endodermal cloacal epithelium is obligatory for the initiation of GT development.36 However, the molecular basis for this requirement remains to be determined. In addition, the outgrowth of the GT is coupled with the tubulogenesis of the urethra, which is derived from the caudal end of the cloacal endoderm. Therefore, GT development also has to be put in context of the whole cloaca patterning. These differences indicate a more complicated epithelial–mesenchymal interaction in the GT. In the following sections, we will review the current knowledge on each major signaling pathway in GT development.

7.2.2  Canonical WNT Signaling Pathway In the absence of the canonical WNT signaling, β-catenin is bound to a complex involving Axin, APC, and GSK3β which normally leads to its degradation. The canonical WNT signaling pathway is initiated by the binding of WNT ligand to its surface receptor frizzled proteins. This binding will trigger a downstream signaling cascade, which frees β-catenin from a degradation complex in the cytoplasm. As a result, β-catenin accumulates and enters the nucleus. In the nucleus, β-catenin activates TCF-dependent downstream gene expression. The canonical WNT signaling has been shown to play a dynamic role in regulating GT development.34,44 Multiple WNT ligands, receptors, and downstream signaling mediators are expressed in the GT.34 The activity of the canonical WNT pathway, evidenced by a

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WNT reporter TOPGAL transgenic expression, can be observed in the urethral epithelium throughout early GT outgrowth.34 Urethral epithelium-specific knockout of β-catenin before GT formation results in a complete abolishment of the GT accompanied by a cloacal septation defect.34,47 Additionally, ablation of β-catenin during GT outgrowth causes developmental arrest, resulting in an underdeveloped GT with proximal hypospadias in both sexes. Notably, early deletion leads to a more severe phenotype, suggesting a continuous requirement of canonical WNT activity during GT outgrowth. β-catenin knockout embryos also demonstrate a defective urethral epithelium.34 On the other hand, induced expression of a gain-of-function β-catenin results in an overdeveloped GT with urethral hyperplasia.34 Taken together, these results indicate an obligatory role for the canonical WNT signaling pathway in the initiation and outgrowth of the GT. Further gene expression analyses and transcription profiling on these mutant embryos revealed many downstream genes that are regulated by WNT signaling, including members of the Hh and BMP pathways. But the most prominent target is Fgf8.34,41 Fgf8 is only expressed in the dUE, the presumptive signaling center of the GT. Fgf8 also plays key role in mediating limb development, and was proposed by many to be the key growth factor in GT development as well. Forced expression of Fgf8 using an inducible Rosa-Fgf8 allele largely rescued the GT development in the urethral epitheliumspecific β-catenin-cKO41 (Fig. 7.4). The regulation of Fgf8 by the canonical WNT signaling pathway is later demonstrated to be mediated at least in part by transcription factor SP8.41 Sp8 is regulated by the canonical WNT signaling pathway. Sp8 knockout embryos also demonstrate a defective GT development as observed in the β-catenin KOs. Moreover, in a sensitized genetic background with β-catenin GOF mutations, SP8 regulates Fgf8 in a dose-dependent manner.41 Intriguingly, this genetic cassette appears to be conserved between the limb and the GT, as the limb phenotype of AER-specific β-catenin, Sp8, and Fgf8 mutant embryos parallel the GT phenotype of urethra epithelium-specific knockouts of the same mutations.34,41,48,49 In the limb, the activation of β-catenin is mediated by the ectodermally expressed canonical WNT ligand WNT3.48 In other words, the ectodermal epithelium is both the source, and the target of WNT signals. However in the GT, the source of WNT ligands is yet to be determined. Both WNT3 and WNT7a can be detected in the developing GT.34 However, their expression is only detected in the surface ectoderm overlying the dUE, but not in the dUE itself. It’s plausible that the ectodermally expressed WNT ligands induce canonical WNT signaling in the neighboring dUE cells. This hypothesis remains to be tested by compound genetic knockout

experiments. If true, it would represent a major difference in WNT transduction between the limb and the GT. Canonical WNT signaling may also play a role in regulating the proliferation of the GT mesenchyme. The mesenchymal specific deletion of β-catenin by Dermo1-Cre

FIGURE 7.4  The canonical WNT signaling regulates GT outgrowth through Fgf8. (A–C) SEM analyses revealed an absence of GT in UE-βCat-LOFs (B), and a distinct tubercle structure in the LOF mutant carrying R26Fgf8 allele (C). (D–F) Histological and in situ analyses on E12.0 β-Cat-LOF; R26Fgf8 GT showing a normal patterning (D) and Hox genes expression (E and F). (G-L) Skeleton preparation on E18.5 embryos showing an absence of autopod and radius, truncation of ulna, underdeveloped humerus in the forelimb (I), and a complete absence of all stylopod, zeugopod and autopod elements in the hindlimb of AER-bCat-LOF mutant (J); and a presence of autopod rudiments (arrow and inset in K) and radius with proper humerus and ulna in the forelimb (K), and a fully developed femur in the hindlimb of R26Fgf8-rescued b-Cat-LOF mutant (L).

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resulted in an underdeveloped and malformed GT, which is accompanied by an obvious reduction in proliferation.34 Notably, the GT mesenchyme expresses Lef1, a cofactor of β-catenin and mediator of the canonical WNT signaling, and cyclin D1, the downstream target of the canonical WNT. However, it is also noteworthy that the TOPGAL reporter analysis did not reveal strong WNT activity in the GT mesenchyme.34 Given the fact that β-catenin may also participate in regulating cell adhesion (this will be discussed in detail in the following section), whether and to what extent the mesenchymal phenotype can be attributed to loss of canonical WNT activity remains to be further determined.

7.2.3  Shh Signaling Pathway Shh plays a dynamic role in the development of cloacal derivatives. Strong Shh expression can be detected throughout the endodermally derived cloacal epithelium before GT initiation, and its expression is later maintained in the urogenital sinus epithelium and the urethral epithelium. Shh signaling is essential for GT development. Global Shh knockout embryos demonstrate agenesis of the GT, along with a persistent cloaca.15,26 This phenotype is accompanied by a significant reduction or in some cases loss of genital-specific gene expression from both the urethral epithelium and the neighboring genital mesenchyme.15,26 Moreover, the requirement of Shh signaling extends beyond GT initiation. Using an inducible conditional knockout model, both Lin et al. and Seifert et al. independently showed that removal of Shh at later stages of GT outgrowth leads to moderate to mild GT underdevelopment with an open urethra (hypospadias).14,16 Shh exerts its growth promoting role, at least in part, through maintaining the GT signaling center dUE. In both straight and conditional Shh mutant embryos, expression of Fgf8, the best characterized marker for dUE, was diminished. More importantly, restoration of Fgf8 genetically by forced expression of a gain-offunction β-catenin allele in the urethral epithelium partially rescues the GT initiation defect and a subset of GT-specific gene expression.14,42 How exactly Shh maintains the dUE signaling center remains elusive. Existing evidence indicates that Shh does not act cell-autonomously on the urethral cells, as conditional deletion of its signal effector Smoothened in the urethral epithelium does not affect normal GT development.14,16 On the contrary, a series of tissue-specific knockout studies showed that the urethral epithelial-expressed Shh acts on the surrounding genital mesenchyme and the overlying genital ectoderm.14,16 Conditional ablation of Smoothened in the genital mesenchyme leads to a severe GT underdevelopment similar to that observed in global Shh knockouts.14 In a separate report, conditional knockout

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of Smoothened in the genital ectoderm was shown to be associated with a defect in urethral tube closure.16 However, this urethral phenotype does not seem to be as severe as in other Shh mutants. Taken together, it is more likely that Shh maintains dUE signaling through reciprocal interactions with the neighboring mesenchymal cells. BMP signaling might be an important target in this feedback loop. A simultaneous upregulation of Bmp4 expression, and a downregulation of its antagonist Noggin expression, was observed in the conditional Shh knockout embryos.14 Whether this alteration in Bmp pathway activity directly leads to the failure of dUE maintenance remains to be tested. On the other hand, Shh also regulates GT and cloaca development independent of dUE signaling. Shh plays an essential role in patterning the cloacal field and this function cannot be duplicated by either WNT or Fgf.14 Even with Fgf signaling restored, the urethra remains trapped within the body in the absence of Shh, while the cloaca remains unseparated.14 The GT outgrowth in these embryos is stalled at around E12.5, indicating that a properly formed cloaca is a prerequisite for continued GT development. The failure of cloaca septation is also observed in the conditional Smoothened knockout animals, which further supports an obligatory requirement for Shh signaling in the PCM. Shh exerts its function in the PCM and GT mesenchyme by regulating the length of the cell cycle.17 In the absence of Shh signaling, the cell cycle extends from 8 h to 14.5 h, which leads to a stunning 75% reduction in growth. In addition, Shh also maintains the expression of Hoxa13 and Hoxd13 genes in the developing GT. The expression of these genes cannot be detected in the Shh knockout embryos even with Fgf signaling restored.14 It’s obvious that Shh plays a key role in regulating growth and patterning of both the limb and the GT. However, the expression pattern of Shh also marks the intriguing difference between the two types of appendages. Shh expression was only detected in the cloacal epithelium and later in the urethral epithelium of the GT, while in the limb bud, its expression was detected in the posterior limb mesenchyme.15,26,50 The function of Shh in the GT appears to be more complicated. This is because GT development and cloacal patterning are intrinsically coupled, and both processes are an integral part of caudal embryonic development. Experimental evidence so far suggests that Shh is a key factor that coordinates the whole process, as Shh expression can be detected days before GT development starts.

7.2.4  Fibroblast Growth Factor Signaling Pathway Given the apparent similarity between limb and GT development, and its significance in limb development,

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Fgf signaling in the GT has been extensively studied. Fgf family genes are dynamically expressed in the developing GT. Several Fgf ligands, including Fgf8, Fgf9, Fgf3, and Fgf4 are detected in the GT signaling center dUE,28,36,41,42 while Fgf10 is expressed in the distal GT mesenchyme.28 Among FGF receptors, Fgfr1 and two isoforms of Fgfr2, the epithelial Fgfr2b and the mesenchymal Fgf2c, are expressed in the GT.29–32 Most attention has been concentrated on Fgf8 early on as it marks both AER and dUE. Earlier studies demonstrated that application of FGF8-soaked beads stimulated GT growth and activated GT-specific gene expression in organ culture.28 Downregulation of Fgf8 expression in the dUE has also been reported in a variety of mutants with GT underdevelopment, including Shh,14,15,26,42 β-catenin,34 and Fgfr2IIIb30 knockout animals. This downregulation has been proposed to be a potential mechanism leading to the abnormal GT development in these models. However, two independent reports in 2009 showed that GT development is not affected in the absence of Fgf8.36,42 Both studies described a normal GT formation in urethral specific Fgf8 knockout, and one study reported that Fgf4 and Fgf8 double knockout also have a normal GT.42 These findings apparently argue against a functional role for Fgf8 in GT outgrowth. The study by Cohn’s group also showed evidence that although Fgf8 transcripts were made in the dUE, the protein might not be translated. The authors went on to suggest that Fgf8 is only a marker for dUE, but does not function in GT development.36 However, a study by Ma’s group using an inducible Fgf8 overexpressor model seems to argue against this view. In a PLOS Genetics paper published in 2013, Lin et al. showed that Fgf8 transcripts expressed from the Rosa26 locus can definitely be translated into protein.41 The authors went on to show that a slight upregulation of Fgf8 level triggered a negative feedback loop on the endogenous Fgf8 expression level. More importantly, this brief period of Fgf8 upregulation led to GT overdevelopment in these conditional Fgf8 gain-of-function embryos. In addition, the authors also demonstrated that forced expression of Fgf8, albeit at a level much lower than the endogenous Fgf8 expression, could successfully rescue the GT outgrowth defect in the urethral epithelium-specific β-catenin conditional knockout animals.41 Taken together, these data argue against Fgf8 as being just a dUE marker but rather characterize it as an important functional player in GT outgrowth. This apparent discrepancy might be explained by the well documented genetic redundancy among different Fgf ligands. The study by Yamada’s group reported an ectopic upregulation of Fgf4 in the absence of Fgf8, and an ectopic upregulation of Fgf3 when both Fgf4 and Fgf8 are conditionally abolished.42 In addition to Fgf3, 4, and 8, Fgf9 expression was also observed in the dUE. It’s plausible that multiple Fgf ligands are compensating for each other in the developing GT. Although which ligand is the

most responsible for GT development remains elusive, the growth promoting role of Fgf signaling is very clear as supported by findings from conditional Fgf receptor knockout mice. It has been demonstrated that the removal of both Fgfr1 and Fgfr2 from the genital mesenchyme results in severe genital underdevelopment at a very early stage; this is consistent with the notion that FGF signaling is obligatory for appendage outgrowth.41 In addition to its role in promoting outgrowth, Fgf signaling is also required in maintaining epithelial structure.31 This function will be discussed in the following section.

7.2.5  Genes Involved in Maintaining Epithelial Structure Genes involved in regulating epithelial structure are also important in the development of the GT and the urethra. One key structure during early GT development is the double layered cloacal membrane, which is composed of endodermally derived cloacal epithelium and an overlying ectodermal surface epithelium. This structure is of particular importance not only because it later develops into the signaling center urethral epithelium, but also because it provides structural support for GT outgrowth. The rupture of this membrane will inevitably result in an open urethra and hypospadias. If this occurs early, the abnormal structure would misplace the source of growth signals and cause profound malformation of the whole GT. The cloacal membrane is one of the rare places where two types of epithelia make contact to form a junction. Therefore, disruption in epithelial cell adhesion and differentiation will significantly affect its structure. This notion is first supported by Lin et al.’s study published in 2008.34 The authors demonstrated that conditional ablation of β-catenin in the surface ectoderm unexpectedly led to a severe GT malformation although WNT activity is not detected in these cells. Further investigations revealed that in the absence of β-catenin, the surface epithelium is much thinner and does not express correct keratin markers. Moreover, the junction between two types of epithelia is disrupted in the cloaca membrane. On the other hand, all regulatory genes examined showed a normal expression level and pattern. It is also noteworthy that the disruption of the double layered cloaca membrane precedes the open urethra phenotype.34 Similarly, a rather normal gene expression in a severely malformed GT was also observed in embryos lacking basement membrane protein LAMA5.33 LAMA5 mutants also showed an open urethra with an underdeveloped GT as early as E13.5. Again in these mutants, there is also clear evidence of premature disintegration of the cloaca membrane (Fig. 7.5). The urethral epithelium is already exposed before the hypospadias is evident. Two independent studies in 2015 demonstrated that conditional ablation of Fgfr2 in the surface ectodermal

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FIGURE 7.5  The premature disintegration of cloacal membrane in LAMA5 mutant GT. Double immunofluorescent staining on 12.5 control (A–C) and LAMA5-KO (E–G) embryos. E-Cadherin was detected in both the ectodermal and urethral epithelia (A and E), whereas KRT14 was expressed in the ventral ectodermal epithelium (B and F). Note that in the LAMA5-KO, the KRT14-expressing ectodermal surface epithelium at the ventral midline (cloacal membrane) was missing (white arrows in F). (D, H) Shh in situ hybridization on E12.5 control (D), and LAMA5-KO (H) embryos. Note that the Shh-expressing urethral cells were covered by Shh-negative ectodermal epithelium in the control (D). While in the KO GT, the outermost layer of the midline epithelium was clearly Shh-positive (arrows in H).

epithelium also leads to GT malformation.31,32 The targeted epithelia show a variety of defects in epithelial cell shape, adhesion, cytoskeletal organization, and stratification. In the ectoderm specific Fgfr2 knockout, the entire GT is malformed and a severe hypospadias is obvious. The phenotypes of all three aforementioned global or conditional knockout mice are very similar to each other, which start with an early disruption in the cloaca membrane leading to ectopic urethral opening. Notably, the prepuce development in these mutants is also affected as secondary defects to the open urethra. One of the unique mutant phenotypes is observed in the urethral epithelium-specific Fgfr2 mutant mice.31 In these animals, the GT development is largely normal, while the maturation of the urethra tube is perturbed, resulting in a mild hypospadias phenotype. These embryos show a defective urethral epithelial stratification, and a variety of abnormalities in cell shape and epithelial organization. It is noteworthy that epithelial proliferation in these mutants is also reduced.30,31 The FGF activity in the urethral epithelium is also supported by recent findings that simultaneous disruption of Sprouty1 and Sprouty2, modulators of FGF activity, leads to elevated urethral cell proliferation.51 These data indicate FGF activity is not only required for maintaining proper structure of the urethral epithelium, but also essential for urethral epithelial cell proliferation.

7.2.6  Other Genes with Relevance to GT Development Other regulatory genes, including Six1/Eya1,20 Wnt5a,35 ROR2,52 p63,53 Dlx5/Dlx6,53 Alx4,37 HoxA13/ HoxD13,19 EphrinB2/EphB2,22,23 and Bmp7,24,25 are also linked to abnormal cloaca and GT development.

Loss of function mutation in ROR2 gene is responsible for the recessive Robinow syndrome, a congenital disorder affecting multiple organs. The male patients of Robinow syndrome often display microgenitalia.52 The expression of ROR2, and its presumptive ligand WNT5a,35 are both detected in the developing GT. Both ROR2 and Wnt5a global knockout mice showed retarded GT outgrowth. Wnt5a−/− embryos also demonstrate a caudal truncation which affects clocal development in the severely affected individuals.35,36,52 Moreover, the expression of Wnt5a is tightly regulated by dUE signaling.34 P63, Dlx5, and Dlx6 mutations are linked to SplitHand/Split-Foot Malformations (SHFM). P63 is normally expressed in the urethral epithelium. Ablation of p63 results in a profound genital defect with an open urethra.53 This defect is correlated with a downregulation of Bmp7. Dlx5 is initially expressed in the urethral epithelium, and later the expression shifts to the genital mesenchyme. Dlx5/6 double knockout embryos showed a complete open urethra.53 In these embryos, Fgf8 expression was not detected. Alx4 demonstrates a unique expression pattern which is restricted to the dorsal part of the GT.37 Mice homozygous of an Alx4 mutation Alx4Lst demonstrate dorsal GT hypoplasia, which is associated with an abnormally augmented Hh signaling in the same area. Six1 and Eya1 are widely involved in organogenesis. Disruption of either one or both genes leads to a spectrum of urogenital defects, involving both cloaca and the external genitalia. These defects are correlated with an augmented Bmp signaling activity.20 Transcription factor Hoxa13 and Hoxd13 are strongly expressed in both GT mesenchyme and the urethral epithelium.19,27 Mutation of HoxA13 has been linked to human HFGS. Hoxa13 knockout embryos present

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hypospadias similar to what is observed in HFGS.27 This phenotype is believed to be caused by downregulation of Bmp7 and Fgf8 from the dUE. In addition, Hoxa13 regulates the expression of EphA6 and EphA7 in the vascular endothelia of the GT.54

7.3  HORMONE-DEPENDENT SEXUAL DIFFERENTIATION The second phase of the external genitalia development is hormone-dependent sexual differentiation. During this late phase of genitalia development, the genital mesenchyme of the penis continues to grow and pinches the opposing urethral epithelial walls together. This leads to formation of the definitive urethra tube in males. The remaining epithelial cells are removed through apoptosis.2 In the female, the growth of genital mesenchyme is less prominent. As a result, the urethra remains as a cord at the ventral side of the clitoris. This sexually dimorphic differentiation event is controlled by the balance between androgenic signaling activity and estrogenic signaling activity.

7.3.1  The Window of Hormone-Dependent Sexual Differentiation Dr. Yamada’s group first defined the window of androgen-dependent phase of genitalia development in mice.44 They injected an antiandrogen, flutamide, into pregnant mothers at different times in gestation and found that it can demasculinize male embryos starting from E15.5 but not before. Consistent with this observation, both AR and estrogen receptor (ER) α exhibit indistinguishable expression patterns regardless of embryo sexes prior to E15. After E15, AR and ERα expressions become sexually dimorphic. AR is highly expressed in male GTs but is almost undetectable in females. In contrast, ERα expression is downregulated in male GT but augmented in females. Such sexually dimorphic expression of these steroid hormone receptors is achieved by their respective ligands as treatment with estrogen and testosterone in male and female embryos, respectively, can upregulate the expression of their own receptor expression while downregulating the expression of the other receptor.43 In addition to the androgen signaling, estrogen signaling can also influence the development of the external genitalia. Ectopic exposure to estrogenic compound estradiol benzoate (EB) leads to the nuclear translocation of the ERα, resulting in augmented estrogenic signaling activity.43 At the same time, EB treatment also reduces the AR-positive cells and consequently androgenic activity. The combination of augmented ER signaling and repressed AR signaling results in the

impaired masculinization, and male-to-female sexual reversal of the external genitalia. It is important to note that different aspects of external genitalia development are responsive to different hormones in specific time windows. This is evidenced by the fact that exposure to different types of endocrine disruptive compounds (EDC) during these time windows may cause developmental defects of the penis. An elegant study conducted by Cohn’s group43 has demonstrated that genetic or pharmaceutical ablation of androgen signaling in different developmental stages cause a spectrum of penile defect. Abolishing androgen signaling before E13.5 in mice causes a phenotype resembling human complete androgen insensitive phenotype, while disrupting AR around E17.5 causes micropenis but the urethral closure is not affected. On the other hand, inhibition of androgen signaling between E12.5  and  E16.5 causes hypospadias with chordee. In comparison, animals receiving neonatal EB treatment (P0–P6) developed micropenis, while animals receiving embryonic EB treatment had a normal penile development.43 Together, this indicates the balance between andogenic and estrogenic signals are essential for the sexual differentiation of the external genitalia. The most striking difference between males and females is that the proliferation of the genital mesenchyme is much higher in males. This high proliferation rate of the penile mesenchyme is believed to be mediated by androgen–AR signaling. AR is strongly expressed in the genital mesenchyme. Tissue-specific knockout analyses show that ablation of AR in the urethral epithelium does not cause any genital malformation, whereas ablation of AR in the genital mesenchyme leads to maleto-female sex reversal of the external genitalia. This clearly indicates that the primary target tissue of androgen signaling is the genital mesenchyme. Gene profiling experiments have revealed profound changes in regulatory gene expression in flutamide-treated mouse penile tissues. These gene expression alterations reflect a general reduction of Hh, WNT, and FGF signaling activities.43 How exactly androgen relays its growth promoting signal, and which downstream target genes are directly responsible for the augmented proliferation of the penile mesenchyme, remains to be further clarified. Nonetheless, recent studies suggested Hh and WNT signaling might play an important role in this process. The conditional ablation of Shh,55 Indian Hedgehog (Ihh),43 or β-catenin44 in the genital mesenchyme both impair the masculinization of the penis. It is further demonstrated that WNT activity in the genital mesenchyme is much higher in males than in females,44 which is inversely correlated with the expression level of WNT inhibitor Dkk. Another interesting target gene is Mafb, a transcription factor that shows sexually dimorphic expression pattern in the genital mesenchyme.38,39 The expression of Mafb

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7.3 Hormone-Dependent Sexual Differentiation

depends on androgen signaling, and loss of Mafb results in feminization of the penis. Later development of the penile shaft in mice involves a series of fusion events that culminate in the formation of urethral meatus as well as joining of the male urogenital mating protuberance (MUMP) to the MUMP ridge.3 Development of these structures as well as their counterparts in human and rat is well described in a recent review by Dr. Cunha.3 Very little is known about the molecular players in these processes. However, recent studies by Dr. Cohn’s group43 suggest that Ihh is a crucial player in MUMP and MUMP ridge formation.

7.3.2  Endocrine Disruptors and Congenital Penile Anomaly The increase in hypospadias incidence along with other male dysgenesis in developed countries raised the possibility that environmental factors, such as fetal exposure to endocrine disruptors, may contribute to the development of hypospadias.56 Endocrine disruptors are chemical compounds found in the environment, including industrial and agricultural compounds, and even natural products found in plants. These compounds, also called xenobiotics, can interfere with human physiology by binding to hormone receptors and altering gene expression during development. Quite a number of xenobiotics structurally resemble estrogen and can bind to the ER and affect target gene expression. Exposure to these compounds has been linked to cancer, the steady decline of sperm counts, premature onset of puberty, and abnormal development of the reproductive tract.57 Diethylstilbestrol (DES) is the first synthetic estrogenic compound orally administered to pregnant women from 1947 to 1971 in an effort to preserve pregnancy. Despite controlled studies in the 1950s showing no protective effect of DES toward pregnancy, this drug was continually prescribed to pregnant women in the United States and elsewhere in the world for another two decades. During that time at least four million pregnant women and their fetuses were exposed to DES in the United States alone.58 DES was also introduced into the environment for its ability to accelerate cattle growth. In addition to a plethora of developmental abnormalities observed in the female reproductive tract, in utero exposure to DES in males has been associated with cryptorchidism, hypospadias, microphallus, testicular hypoplasia, and epididymal cysts.59 In humans, only a limited number of studies have examined the potential connection between xenobiotics exposure and hypospadias incidence. “DES-sons” exhibit a whopping ~20fold increase in the incidence of hypospadias although the absolute number of incidence is less impressive.60 Moreover, several reports demonstrated a higher incidence of hypospadias in DES-grandsons, suggesting a

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transgeneration effect for these endocrine disruptors.60,61 It is also noteworthy that roughly 50% of mice exposed to DES embryonically develop extensive embryonic hypospadias neonatally. However, expected hypospadias does not develop when the treated animals reach adulthood.62 In addition to DES, exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to be associated with hypospadias exemplified by the study in Seveso, Italy.63 Other studies have linked higher hypospadias incidence with heavy pesticide usage.64 Compared to estrogenic compounds, the effect of antiandrogenic compounds on genitalia development is more obvious. This is expected as patients with 5α-reductase mutations develop hypospadias with 100% penetrance.65 In utero exposure to other xenoestrogens such as p,p’-DDE (a DDT breakdown product), TCDD, and polychlorinated biphenyls (PCBs) all led to hypospadias in laboratory animals (although the latter two are female-specific).66–68 p,p’-DDE is able to interfere with the conformational change of the AR which is required to stabilize AR to bind DNA and activate downstream target genes.68 Other endocrine disrupting compounds affecting genitalia development include Vinclozolin (fungicide), Procymidone (fungicide), and dibutylphthalate (DBP).67,69 In the case of Vinclozolin, its two metabolites competitively bind to AR and interfere with AR function thereby causing hypospadias at 50 mg/kg per day.67,69 Procymidone can also interfere with AR-dependent transcriptional activation in cell lines and can cause hypospadias at a dose as low as 25 mg/kg per day.70 On the other hand, DBP can affect genital development at all doses examined and it does not function by interfering with AR activity but appears to affect fetal androgen production.71 These data suggest that in utero exposure to a variety of chemical compounds during a critical period of sexual differentiation can affect genital development and leads to hypospadias. They may do so not by causing somatic mutations, but by antagonizing AR function and subsequently alter gene expression which is essential for sexual differentiation. Thus it is conceivable that transient exposure to xenobiotics may affect genital development by altering the normal genetic program or cell fate determination and lead to hypospadias. These data are in agreement with laboratory animal studies and raise significant health concerns over the effect of xenobiotics on male genitalia development.

7.3.3  Discordant Brain and Genital Sexualization Numerous studies documented anatomical differences between male and female brains in a number of mammalian species. Early work by Pfeiffer and Pheonix demonstrated the role of testerosterone in organizing

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the pattern of pituitary secretion and male-type behavior, respectively.72,73 Following those pioneer works, more sex differences were observed in the mammalian brain, such as a larger frontal cortex, a larger area of the anteroventral periventricular nucleus in females, and conversely, a larger sexually dimorphic nucleus of the preoptic area, a higher number of dopamine neurons in rodent substantia nigra in males.74–79 The current view of brain sexualization posits that a perinatal surge of organizational sex hormones, likely testosterone from the gonad, leads to permanent irreversible changes in brain development which is followed by pubertal hormones acting to induce sex-specific behaviors. During prenatal development, only testosterone has the organizational role in masculinizing and defeminizing the brain.80 It is converted to estrogen by cytochrome P450 aromatase in the brain and acts locally to masculinize the brain.81 It is interesting to note that estradiol functions to masculinize brain and raises the questions how female embryos protect themselves from maternal estrogen. It turns out that at least in rodents, the fetus synthesizes α-fetoprotein, a high affinity estrogen binding protein, which sequesters estradiol in fetal blood and prevents them from entering the female brain.82 On the other hand, female brain development does not depend on sex hormones. During the critical period of hormone sensitivity, both genitalia and brain will respond to the gonadal hormones. In mice, there are two peaks of gonadal testosterone secretion, one around day 17 of gestation and another immediately after birth.83,84 Disruption of these secretions will affect masculinization of the brain and genitalia differently, and lead to behavioral defects in adulthood. One interesting difference between the two organs is that they respond differently to estrogen. Estrogen will masculinize the brain while feminizing the genitalia only during the neonatal period. This is a potentially very intriguing difference and could underscore a mechanism for endocrine disruption and altered sexual behaviors in transgenders. We would like to posit that if female embryos are exposed to certain estrogenic endocrine disruptors around the hormone sensitive period, they should masculinize the brain but have no effect on genitalia development, provided that they are not sequestered by the α-fetoprotein. In this case, the female will possess female external genitalia but a masculinized brain and possibly male mating behavior. Conversely, because of the different time windows of masculinization between the brain and genitalia, exposure to chemicals disrupting androgen signaling, such as flutamide and vinclozolin from E17.5 onward will not perturb genitalia development but will definitely feminize the brain in mice. These disruptions of endocrine signaling will uncouple brain and genitalia masculinization events and could provide a molecular basis for transgender development in addition to

genetic causes. These predictions, if validated in mice, may be relevant to humans, however, it is not clear at present whether estrogens are responsible for masculinizing brains in humans. Nonetheless, even though formal epidemiological studies on gender identity disorder (GID) are still lacking, at least a three fold increase in GID patients was estimated between the 1960s and 1990s.85 Although many factors may contribute to this increase, such as better awareness of this disease, exposure to endocrine disruptors during the hormone sensitive window and the discordance between brain and genital masculinization could be a major contributor to this increase. In fact, a number of EDCs including bisphenol A and phthalates have been shown to affect animal behavior providing partial support to our hypothesis.86 However, more research is needed to test the link between endocrine disruption and transgender development in animal models as well as in humans.

References 1. Baskin LS, Ebbers MB. Hypospadias: anatomy, etiology, and technique. J Pediatr Surg. 2006;41(3):463–472. 2. Baskin LS, Erol A, Jegatheesan P, Li Y, Liu W, Cunha GR. Urethral seam formation and hypospadias. Cell Tissue Res. 2001;305(3):379–387. 3. Cunha GR, Sinclair A, Risbridger G, Hutson J, Baskin LS. Current understanding of hypospadias: relevance of animal models. Nat Rev Urol. 2015;12(5):271–280. 4. Yamada G, Satoh Y, Baskin LS, Cunha GR. Cellular and molecular mechanisms of development of the external genitalia. Differentiation. 2003;71(8):445–460. 5. Kluth D, Hillen M, Lambrecht W. The principles of normal and abnormal hindgut development. J Pediatr Surg. 1995;30(8):1143–1147. 6. Levitt MA, Pena A. Anorectal malformations. Orphanet J Rare Dis. 2007;2:33. 7. Stoll C, Alembik Y, Dott B, Roth MP. Associated malformations in patients with anorectal anomalies. Eur J Med Genet. 2007;50(4):281–290. 8. Tourneux F. Sur les premiers developpements du cloaques du turbercule genital et de l’anus chez l’embryon de mouton. J Anat. 1888;24:503–517. 9. Rathke H. Abhandlungen zur bildungs- und entwicklungsgeschichte der tiere. Leipzig: F.C.W. Vogel; 1832. 10. Retterer E. Region ano-genitale des mammiferes. J Anat. 1890;26:126–216. 11. Stephens F. Congenital Malformations of the Rectum, Anus and Genito-Urinary Tracts. Edinburgh: E. & S. Livingstone, Ltd.; 1963. 12. Kluth D. Embryology of anorectal malformations. Semin Pediatr Surg. 2010;19(3):201–208. 13. van der Putte SC. Normal and abnormal development of the anorectum. J Pediatr Surg. 1986;21(5):434–440. 14. Lin C, Yin Y, Veith GM, Fisher AV, Long F, Ma L. Temporal and spatial dissection of Shh signaling in genital tubercle development. Development. 2009;136(23):3959–3967. 15. Perriton CL, Powles N, Chiang C, Maconochie MK, Cohn MJ. Sonic hedgehog signaling from the urethral epithelium controls external genital development. Dev Biol. 2002;247(1):26–46. 16. Seifert AW, Bouldin CM, Choi KS, Harfe BD, Cohn MJ. Multiphasic and tissue-specific roles of sonic hedgehog in

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

17.

18.

19.

20.

21. 22.

23.

24.

25. 26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

cloacal septation and external genitalia development. Development. 2009;136(23):3949–3957. Seifert AW, Zheng Z, Ormerod BK, Cohn MJ. Sonic hedgehog controls growth of external genitalia by regulating cell cycle kinetics. Nat Commun. 2010;1:23. Mo R, Kim JH, Zhang J, Chiang C, Hui CC, Kim PC. Anorectal malformations caused by defects in sonic hedgehog signaling. Am J Pathol. 2001;159(2):765–774. Warot X, Fromental-Ramain C, Fraulob V, Chambon P, Dolle P. Gene dosage-dependent effects of the Hoxa-13 and Hoxd13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts. Development. 1997;124(23): 4781–4791. Wang C, Gargollo P, Guo C, et  al. Six1 and Eya1 are critical regulators of peri-cloacal mesenchymal progenitors during genitourinary tract development. Dev Biol. 2011;360(1):186–194. Tai CC, Sala FG, Ford HR, et al. Wnt5a knock-out mouse as a new model of anorectal malformation. J Surg Res. 2009;156(2):278–282. Fairbanks TJ, De Langhe S, Sala FG, et al. Fibroblast growth factor 10 (Fgf10) invalidation results in anorectal malformation in mice. J Pediatr Surg. 2004;39(3):360–365. discussion -5. Dravis C, Yokoyama N, Chumley MJ, et al. Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development. Dev Biol. 2004;271(2):272–290. Wu X, Ferrara C, Shapiro E, Grishina I. Bmp7 expression and null phenotype in the urogenital system suggest a role in reorganization of the urethral epithelium. Gene Expr Patterns. 2009;9(4):224–230. Xu K, Wu X, Shapiro E, et al. Bmp7 functions via a polarity mechanism to promote cloacal septation. PloS One. 2012;7(1):e29372. Haraguchi R, Mo R, Hui C, et  al. Unique functions of Sonic hedgehog signaling during external genitalia development. Development. 2001;128(21):4241–4250. Morgan EA, Nguyen SB, Scott V, Stadler HS. Loss of Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia. Development. 2003;130(14):3095–3109. Haraguchi R, Suzuki K, Murakami R, et  al. Molecular analysis of external genitalia formation: the role of fibroblast growth factor (Fgf) genes during genital tubercle formation. Development. 2000;127(11):2471–2479. Satoh Y, Haraguchi R, Wright TJ, et  al. Regulation of external genitalia development by concerted actions of FGF ligands and FGF receptors. Anat Embryol. 2004;208(6):479–486. Petiot A, Perriton CL, Dickson C, Cohn MJ. Development of the mammalian urethra is controlled by Fgfr2-IIIb. Development. 2005;132(10):2441–2450. Gredler ML, Seifert AW, Cohn MJ. Tissue-specific roles of Fgfr2 in development of the external genitalia. Development. 2015;142(12):2203–2212. Harada M, Omori A, Nakahara C, Nakagata N, Akita K, Yamada G. Tissue-specific roles of FGF signaling in external genitalia development. Dev Dyn. 2015;244(6):759–773. Lin C, Werner R, Ma L, Miner JH. Requirement for basement membrane laminin alpha5 during urethral and external genital development. MechDev. 2016;141:62–69. Lin C, Yin Y, Long F, Ma L. Tissue-specific requirements of beta-catenin in external genitalia development. Development. 2008;135(16):2815–2825. Suzuki K, Bachiller D, Chen YP, et  al. Regulation of outgrowth and apoptosis for the terminal appendage: external genitalia development by concerted actions of BMP signaling [corrected]. Development. 2003;130(25):6209–6220. Seifert AW, Yamaguchi T, Cohn MJ. Functional and phylogenetic analysis shows that Fgf8 is a marker of genital induction in mammals but is not required for external genital development. Development. 2009;136(15):2643–2651.

89

37. Matsumaru D, Haraguchi R, Moon AM, et al. Genetic analysis of the role of Alx4 in the coordination of lower body and external genitalia formation. Eur J Hum Genet. 2014;22(3):350–357. 38. Suzuki K, Numata T, Suzuki H, et al. Sexually dimorphic expression of Mafb regulates masculinization of the embryonic urethral formation. Proc Natl Acad Sci USA. 2014;111(46):16407–16412. 39. Suzuki H, Suzuki K, Yamada G. Systematic analyses of murine masculinization processes based on genital sex differentiation parameters. Dev Growth Differ. 2015;57(9):639–647. 40. Matsushita S, Suzuki K, Ogino Y, et al. Androgen regulates Mafb expression through its 3’UTR during mouse urethral masculinization. Endocrinology. 2016;157(2):844–857. 41. Lin C, Yin Y, Bell SM, et  al. Delineating a conserved genetic cassette promoting outgrowth of body appendages. PLoS Genet. 2013;9(1):e1003231. 42. Miyagawa S, Moon A, Haraguchi R, et  al. Dosage-dependent hedgehog signals integrated with Wnt/beta-catenin signaling regulate external genitalia formation as an appendicular program. Development. 2009;136(23):3969–3978. 43. Zheng Z, Armfield BA, Cohn MJ. Timing of androgen receptor disruption and estrogen exposure underlies a spectrum of congenital penile anomalies. Proc Natl Acad Sci USA. 2015;112 (52):E7194–E7203. 44. Miyagawa S, Satoh Y, Haraguchi R, et al. Genetic interactions of the androgen and Wnt/beta-catenin pathways for the masculinization of external genitalia. Mol Endocrinol. 2009;23(6):871–880. 45. Yamada G, Suzuki K, Haraguchi R, et al. Molecular genetic cascades for external genitalia formation: an emerging organogenesis program. Dev Dyn. 2006;235(7):1738–1752. 46. Cohn MJ. Development of the external genitalia: conserved and divergent mechanisms of appendage patterning. Dev Dyn. 2011;240(5):1108–1115. 47. Miyagawa S, Harada M, Matsumaru D, et  al. Disruption of the temporally regulated cloaca endodermal beta-catenin signaling causes anorectal malformations. Cell Death Differ. 2014;21(6):990–997. 48. Barrow JR, Thomas KR, Boussadia-Zahui O, et  al. Ectodermal Wnt3/beta-catenin signaling is required for the establishment and maintenance of the apical ectodermal ridge. Genes Dev. 2003;17(3):394–409. 49. Bell SM, Schreiner CM, Waclaw RR, Campbell K, Potter SS, Scott WJ. Sp8 is crucial for limb outgrowth and neuropore closure. Proc Natl Acad Sci USA. 2003;100(21):12195–12200. 50. Chiang C, Litingtung Y, Lee E, et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature. 1996;383(6599):407–413. 51. Ching ST, Cunha GR, Baskin LS, Basson MA, Klein OD. Coordinated activity of Spry1 and Spry2 is required for normal development of the external genitalia. Dev Biol. 2014;386 (1):1–11. 52. Schwabe GC, Trepczik B, Suring K, et al. Ror2 knockout mouse as a model for the developmental pathology of autosomal recessive Robinow syndrome. Dev Dyn. 2004;229(2):400–410. 53. Suzuki K, Haraguchi R, Ogata T, et al. Abnormal urethra formation in mouse models of split-hand/split-foot malformation type 1 and type 4. Eur J Hum Genet. 2008;16(1):36–44. 54. Shaut CA, Saneyoshi C, Morgan EA, Knosp WM, Sexton DR, Stadler HS. HOXA13 directly regulates EphA6 and EphA7 expression in the genital tubercle vascular endothelia. Dev Dyn. 2007;236(4):951–960. 55. Miyagawa S, Matsumaru D, Murashima A, et  al. The role of sonic hedgehog-Gli2 pathway in the masculinization of external genitalia. Endocrinology. 2011;152(7):2894–2903. 56. Toppari J, Larsen JC, Christiansen P, et  al. Male reproductive health and environmental xenoestrogens. Environ Health Perspect. 1996;104(Suppl 4):741–803.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

90

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57. Bigsby R, Chapin RE, Daston GP, et  al. Evaluating the effects of endocrine disruptors on endocrine function during development. Environ Health Perspect. 1999;107(Suppl 4):613–618. 58. Mittendorf R. Teratogen update: carcinogenesis and teratogenesis associated with exposure to diethylstilbestrol (DES) in utero. Teratology. 1995;51(6):435–445. 59. Newbold R. Cellular and Molecular Effects of Developmental Exposure to Diethylstilbestrol: Implications for Other Environmental Estrogens. Environ Health Perspect. 1994;103(Suppl 7):83–87. 60. Klip H, Verloop J, van Gool JD, et  al. Hypospadias in sons of women exposed to diethylstilbestrol in utero: a cohort study. Lancet. 2002;359(9312):1102–1107. 61. Pons JC, Papiernik E, Billon A, Hessabi M, Duyme M. Hypospadias in sons of women exposed to diethylstilbestrol in utero. Prenat Diagn. 2005;25(5):418–419. 62. Kim KS, Torres Jr. CR, Yucel S, Raimondo K, Cunha GR, Baskin LS. Induction of hypospadias in a murine model by maternal exposure to synthetic estrogens. Environ Res. 2004;94(3):267–275. 63. Mastroiacovo P, Spagnolo A, Marni E, et  al. Birth defects in the Seveso area after TCDD contamination. JAMA. 1988;259(11):1668–1672. 64. Garry VF, Schreinemachers D, Harkins ME, Griffith J. Pesticide appliers, biocides, and birth defects in rural Minnesota. Environ Health Perspect. 1996;104(4):394–399. 65. Imperato-McGinley J, Guerrero L, Gautier T, German JL, Peterson RE. Steroid 5alpha-reductase deficiency in man. An inherited form of male pseudohermaphroditism. Birth Defects Orig Artic Ser. 1975;11(4):91–103. 66. Hurst CH, DeVito MJ, Setzer RW, Birnbaum LS. Acute administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in pregnant Long Evans rats: association of measured tissue concentrations with developmental effects. Toxicol Sci. 2000;53(2):411–420. 67. Gray Jr. LE, Wolf C, Lambright C, et  al. Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p’-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol Ind Health. 1999;15(1–2):94–118. 68. Kelce WR, Stone CR, Laws SC, et al. Metabolite p,’p’-DDE is a potent androgen receptor antagonist. Nature. 1995;375(6532):581–585. 69. Gray Jr. LE, Ostby J, Monosson E, Kelce WR. Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicol Ind Health. 1999;15(1–2):48–64. 70. Ostby J, Kelce WR, Lambright C, Wolf CJ, Mann P, Gray Jr. LE. The fungicide procymidone alters sexual differentiation in the male rat by acting as an androgen-receptor antagonist in vivo and in vitro. Toxicol Ind Health. 1999;15(1–2):80–93. 71. Gray Jr. LE. Xenoendocrine disrupters: laboratory studies on male reproductive effects. Toxicol Lett. 1998;102–103:331–335.

72. Pfeiffer CA. Sexual differences of the hypophyses and their determination by the gonads. Am J Anatomy. 1936;58:195–225. 73. Phoenix CH, Goy RW, Gerall AA, Young WC. Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology. 1959;65:369–382. 74. Bleier R, Byne W, Siggelkow I. Cytoarchitectonic sexual dimorphisms of the medial preoptic and anterior hypothalamic areas in guinea pig, rat, hamster, and mouse. J Comp Neurol. 1982;212(2):118–130. 75. Gorski RA, Gordon JH, Shryne JE, Southam AM. Evidence for a morphological sex difference within the medial preoptic area of the rat brain. Brain Res. 1978;148(2):333–346. 76. Gorski RA, Harlan RE, Jacobson CD, Shryne JE, Southam AM. Evidence for the existence of a sexually dimorphic nucleus in the preoptic area of the rat. J Comp Neurol. 1980;193(2):529–539. 77. Dewing P, Chiang CW, Sinchak K, et  al. Direct regulation of adult brain function by the male-specific factor SRY. Curr Biol. 2006;16(4):415–420. 78. McArthur S, McHale E, Gillies GE. The size and distribution of midbrain dopaminergic populations are permanently altered by perinatal glucocorticoid exposure in a sex- region- and timespecific manner. Neuropsychopharmacology. 2007;32(7):1462–1476. 79. Murray HE, Pillai AV, McArthur SR, et al. Dose- and sex-dependent effects of the neurotoxin 6-hydroxydopamine on the nigrostriatal dopaminergic pathway of adult rats: differential actions of estrogen in males and females. Neuroscience. 2003;116(1):213–222. 80. Grgurevic N, Majdic G. Sex differences in the brain-an interplay of sex steroid hormones and sex chromosomes. Clin Sci (Lond). 2016;130(17):1481–1497. 81. Naftolin F, Ryan KJ, Davies IJ, et  al. The formation of estrogens by central neuroendocrine tissues. Recent Prog Horm Res. 1975;31:295–319. 82. McEwen BS, Plapinger L, Chaptal C, Gerlach J, Wallach G. Role of fetoneonatal estrogen binding proteins in the associations of estrogen with neonatal brain cell nuclear receptors. Brain Res. 1975;96(2):400–406. 83. Motelica-Heino I, Castanier M, Corbier P, Edwards DA, Roffi J. Testosterone levels in plasma and testes of neonatal mice. J Steroid Biochem. 1988;31(3):283–286. 84. vom Saal FS, Bronson FH. Sexual characteristics of adult female mice are correlated with their blood testosterone levels during prenatal development. Science. 1980;208(4444):597–599. 85. Coleman E. Toward Version 7 of the World Professional Association for Transgender Health’s Standards of Care: Medical and Therapeutic Approaches to Treatment. Int J Transgenderism. 2009;11:215–219. 86. Palanza P, Nagel SC, Parmigiani S, Vom Saal FS. Perinatal exposure to endocrine disruptors: sex, timing and behavioral endpoints. Curr Opin Behav Sci. 2016;7:69–75.

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8 The Transsexual Adult Swaytha Yalamanchi, Betiel Fesseha and Adrian Dobs Johns Hopkins University School of Medicine, Baltimore, MD, United States

O U T L I N E 8.6.3 Criteria for Initiation of Hormone Replacement Therapy 8.6.4 Male to Female Transsexual Individuals 8.6.5 Female to Male Transsexual Individuals 8.6.6 Surgical Therapy 8.6.7 Reproductive Health 8.6.8 Lifelong Preventative and Primary Care

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individual to undergo hormonal and surgical treatment, her role as a former veteran in a time when gender roles were in flux in the post-World II era likely contributed to significant media coverage. Shortly after publishing the Transsexual Phenomenon, gender identity clinics, notably including the clinic at Johns Hopkins University, began to develop. The Harry Benjamin International Gender Dysphoria Association was then founded in 1979. Presently known as the World Professional Association of Transgender Health (WPATH), the organization is a leader in the care of transgendered, transsexual, and gender confused individuals.1,3 In recent years, there has been accelerated openness and awareness in both the lay and medical community about health concerns specific to transsexual individuals. Notably, the 2011 Institute of Medicine report on health of sexual and gender minorities specifically highlighted the need for more research in this arena.4 However, there are significant knowledge gaps in the

Descriptions of transgendered individuals date back centuries with initial medical literature describing transsexualism as a form of psychosis.1 Pioneer Magnus Hirschfeld in the early 20th century put forward a less pathologized description of transsexualism for the first time and first coined the terms “transvestites.” Importantly, he was also the first to separate sexuality from transsexualism. His advances in the field were followed by the development of synthetic testosterone and estrogen in the 1930s. In the 1950–60s, Harry Benjamin, widely considered a pioneer in the field, coined the term “transsexual” and The Transsexual Phenomenon, published in 1966, is considered a seminal publication.2 Benjamin was also the first to separate transsexualism from transvestism. Christine Jorgensen, who became a hallmark figure in the transsexual movement, was described in The Transsexual Phenomenon. Although not the first

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care of transgender, transsexual, and gender nonconforming individuals. Major limitations include lack of clinical exposure and education for many providers, limited available research on the subject matter, and associated social stigma. Data have demonstrated that only 63% of endocrinologists were willing to provide transgender care with the majority of these providers not presently managing transgender patients. Furthermore, a total of 20% of providers surveyed were “very” comfortable in discussing gender identity and/or sexual orientation with 41% rating themselves as “somewhat” or “very” competent in providing transgender care.5 These findings highlight the need for further research and educational efforts in this arena. Thus, the purpose of this chapter is to review the current body of literature with regard to available data on epidemiology, pathophysiology, diagnostic evaluation, and suggested treatment and monitoring plan of transsexual adults.

8.2 TERMINOLOGY Definitions utilized throughout this chapter are as described in both the Endocrine Society and WPATH guidelines.3,6 While sex refers to attributes felt to constitute biologic maleness or femaleness (most commonly based on primary sexual characteristics), gender identity describes an individual’s inherent sense of being male, female, both, or neither.7,8 Gender role refers to behaviors, attitudes, and personality traits defined as masculine or feminine based on societal norms. Gender dysphoria refers to discomfort or distress caused by discordant gender identity and assigned sex at birth. Gender dysphoria has replaced gender identity disorder in the most recent edition of the Diagnostic Statistical Manual of Mental Disorders, 5th Edition to be more consistent with present sexology terminology and to avoid the stigma associated with connotations of patients being “disordered.”9 The formal diagnostic criteria for gender dysphoria are as outlined below in the section on “Clinical Evaluation.” For some individuals, gender dysphoria warrants gender affirmation, which may include medical and/or surgical treatment. The term transgender describes individuals who cross culturally defined categories of gender; gender identity varies to different degrees from assigned sex at birth. Transsexual is a term often applied by the medical community to describe individuals who seek to or who have changed sex characteristics by medically or surgical intervention, typically accompanied by a permanent change in gender role. While the terms transsexual and transgender are frequently used interchangeably with some arguing that the former is an outdated term, we utilize the term transsexual throughout this chapter, as was the style in the Endocrine Society guidelines, because the term transgender also encompasses individuals whose

gender identity does not conform to gender roles and who may not seek endocrine treatment.6 The terms female to male (FTM) and transsexual man are used to describe individuals assigned a female sex at birth who have changed their body and/or gender role to a more masculine body or role, while the terms male to female (MTF) and transsexual woman refer to individuals assigned a male sex at birth who have changed their body and/or gender role to a more feminine body or role. Transition refers to the period during which transsexual individuals change their physical, social, and legal characteristics to that of the other biological sex, consistent with their identified gender. The terms also encompass ongoing physical and psychological changes and adaptation.3,6

8.3 EPIDEMIOLOGY For approximately 66% of transsexual individuals, the onset of gender dysphoria is in childhood.8,10 Although gender dysphoria in childhood often resolves, transsexualism after puberty rarely does.11–13 There is considerable variability in the ratio of MTF versus FTM individuals throughout the world. In Western society, the ratio is estimated to be 3:1.14 It is difficult to assess the true prevalence and incidence of adult transsexualism for multiple reasons. Limited epidemiologic studies have been performed and such studies are difficult to design in part because widely varying social and cultural norms worldwide confound assessment. Thus, epidemiologic studies have largely focused on transsexual individuals presenting for transition-related care at specialty clinics and, less commonly, transgender-related diagnoses.3 The majority of prevalence estimates have ranged from 1 to 10 per 100,000 individuals and have largely been based on European data.15–19 The Veterans Health Administration (VA) includes one of the largest cohorts of transgendered individuals in the United States. Blosnich et al. utilized ICD-9 codes to identify 3177 veterans from the years 2000–11 with gender identity disorder.20 Data from the study demonstrated that the prevalence of gender identity disorder in the VA population was greater than fivefold higher (22.9 per 100,000 individuals) than older estimates in the general population. An incident rate of 246 new diagnoses of gender identity disorder per 100,000 persons was also reported. Suicide risk was jointly evaluated in this study and reported to be 4000–5000 per 100,000 individuals in 2010, over 20-fold higher than veterans without gender identity disorder. There are several important limitations in interpreting this study, most notably its applicability to the general population given that this was a population of veterans. Additionally, the VA passed an important directive in 2011 to standardize treatment services

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for transgendered veterans,21 thus further contributing to what is likely an underestimate of true prevalence and incidence of transgendered and transsexual individuals prior to that time. Kauth et  al. addressed some of these limitations by their subsequent study extending the included ICD-9 codes to include “transsexualism” and by analyzing data from the cohort from 2006 to 2013.22 The authors identified 2662 individuals with diagnoses related to transgender status from 2006 to 2013 with 40% of those new cases in the 2 years status post the 2011 directive. In 2013 specifically, the prevalence of transgender-related diagnoses was 32.9 (95% confidence interval (CI): 21.6, 44.1) per 100,000 individuals and incidence was 6.7 (95% CI: 1.6, 11.8) per 100,000 individuals. Although these studies cannot be compared directly due to different time frames and different inclusion criteria, both studies did show that the prevalence and incidence of transgender-related diagnoses is higher than that of the general population. There are several theories about why this may be, including a “hypermasculinity theory” such that young men with gender identity disorder enlist in the military during critical periods in psychosocial development in an effort purge their inner gender conflict through the rigors of the military and focus on overtly masculine activities.23 It is also possible that given that the VA population is predominantly male, and in the United States natal males are more likely to have gender identity disorder as compared to females, males are overrepresented. However, veterans are also exposed to very unique stressors in the military and it is unclear how this, among other factors, may also be playing a role.22 That being said, Roblin et al. described a novel computer algorithm utilizing the Kaiser Permanente Georgia electronic medical record system (a non-VA based cohort) to identify the prevalence of transgendered individuals and their respective gender identities while addressing the aforementioned limitations with exclusive reliance on ICD-9 codes. The authors reported a similar prevalence of 4.4 per 100,000 people (95% CI: 2.6–7.4) in 2006 and 38.7 per 100,000 people (95% CI: 32.4–46.2) in 2014. Of the 185 transgendered individuals identified, 99 (54%, 95% CI: 46–61) were MTF and 84 (45%, 95% CI: 38–53%) were FTM.19 Most recently, Collin et  al. performed a systematic review and meta-analysis including 27 studies. The authors reported the following meta-prevalence estimates per 100,000: 9.2 (95% CI: 4.9–13.6) for surgical or hormonal gender affirmation therapy and 6.8 (95% CI: 4.6–9.1) for transgender-related diagnoses. After excluding a study with outlying results, the meta-prevalence estimate of self-reported transgender identity was 355 per 100,000 (95% CI: 144–566). Significant heterogeneity was noted in most studies.24 In conclusion, it is difficult to design epidemiologic studies able to capture the true prevalence and incidence

of transsexualism and existing data likely underestimate true rates. Overall, data from both European and American populations suggest an increase in transgendered-related diagnoses, possibly related to increasing societal and medical awareness. Veterans also represent a specific population in which rates of transgenderedrelated disorders and associated suicidality are higher than that of the general population. This warrants further attention.

8.4 PATHOPHYSIOLOGY As mentioned, transsexualism previously was felt to be a psychological disorder, a theory which has been debunked.25 Transsexualism is now increasingly hypothesized to be partly due to an interrelated process of prenatal hormone exposure, genetics, and variations in the structure/function of the brain.

8.4.1  Prenatal Sex Hormones Sex differences in brain structure and function have been attributed in part to sex hormone effects during prenatal development, during sexual differentiation (organizing effects of sex hormones), and later in life (activating effects of sex hormones).26,27 Male differentiation of the fetal brain occurs in the presence of androgens, while female differentiation occurs in the absence of androgens. Sexual differentiation of the brain occurs later than genital development. It is thus hypothesized that these two processes could be independently influenced, and when developing in opposite directions, may be important in transsexualism.28 While disorders of sexual development (DSD) and transsexualism are separate entities, the former may serve as a model for understanding the role of prenatal sex hormone exposure in the latter condition. For example, cross-gender identification has been examined in girls exposed to high levels of prenatal androgens in the setting of classic congenital adrenal hyperplasia. Dessens et al. reported a 1.6% rate of FTM transgenderism in 46,XX women with CAH, considerably higher than the frequency in the general population (0.003%), though still overall low.29 There are also suggestive data on the relevance of prenatal sex hormonal milieu amongst individuals without DSDs. While the ratio of the index finger and ring finger length, which is considered a sexually dimorphic feature,30 has been demonstrated to be higher in MTF transsexuals relative to controls, the ratio of FTM transsexuals is similar to that of control males suggesting that prenatal hormonal exposure may be important in MTF transsexualism.31,32 It is unclear if prenatal sex hormone exposure has a genetic basis, including altered synthesis of sex hormones or altered sensitivity of function of sex hormone receptors.27

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8.4.2 Genetics Sibling studies have also suggested a possible genetic link. Gomez Gil et al. (n = 995) demonstrated a higher risk of transsexuality among nontwin siblings, with higher risk being noted in brothers as compared to sisters, and in MTF as compared to FTM transsexuals, though the overall risk is low.33 Furthermore, twin studies have shown that a higher concordance of gender identity disorder in monozygotic, as compared to dizygotic twins.34

Data are particularly limited on FTM transsexuals and in children/adolescents with transsexualism.27 In conclusion, it is hypothesized that a combination of prenatal sex hormonal milieu, genetics, and differences in sexually dimorphic brain structures may be important in the pathophysiology of transsexualism. However, our understanding of the pathophysiologic underpinnings of transsexualism is limited by a lack of high-quality data and variability amongst available studies in methodology and in accounting for confounding factors.

8.4.3  Sexual Dimorphism of the Brain Data have also suggested that brains of transsexual individuals may be more similar to those with their selfidentified gender, rather than those of the same physical sex, albeit brain structure and function of transsexual individuals appear to be selectively, rather than entirely, masculinized or feminized.27 There are sex differences in the shape and size of certain hypothalamic nuclei with sexual differentiation reported to occur between the ages of 2–4. Sexual differentiation of the bed nuclei of the striae terminalis extends into adulthood. Swaab et al. reported that based on autopsy findings, the size and number of neurons in the bed nucleus of striata terminalis and the third interstitial nucleus of the anterior hypothalamus of MTF transsexual patients were similar in size and neuron numbers to females.35–38 Signs of masculinization/feminization have also been reported in cortical thickness and in several white matter fiber tracts.27 With treatment, MTF transsexual individuals have a decrease in cortical and subcortical gray matter volume, while FTM transsexuals have an increase in cortical subcortical gray matter after treatment. Changes are also seen in performance of cognitive tasks, such as mental rotation and visual memory tasks. Data on event-related fMRI in untreated MTF transsexuals are limited to studies measuring cerebral activation while smelling odorous steroids, viewing erotic film scenes, and in discriminating male versus female voices, but are suggestive of activation patterns more similar to female as opposed to male patterns.27,39,40 For example, Berglund et  al. demonstrated that hypothalamic blood flow in response to odorous compound was more similar among MTF transsexual individuals to control female participants than to males.39 Such data are unavailable in FTM transsexual individuals.27 However, there are several notable limitations to the available literature on the pathophysiology of transsexualism including minimal prospective data, lack of controlling for confounding factors such as sexual orientation in many studies, and lack of data on age of development of onset of transsexualism. It is further unclear if MTF and FTM transsexualism are similar, i.e., comparable entities.

8.5  CLINICAL PRESENTATION AND DIAGNOSIS The diagnosis of transsexualism is generally made by a mental health professional and it is key to identify potential psychiatric comorbidities prior to consideration of hormonal or surgical therapy. Both the Endocrine Society guidelines and WPATH Standards of Care provide excellent clinical guidance.3,6 Per the Diagnostic and Statistical Manual of Mental Disorders (DSM)-V, the diagnosis of gender dysphoria requires the experience of at least two of the following criteria for at least 6 months duration in either adolescents or adults:9 1. Marked incongruence between one’s experienced/ expressed gender and primary and/or secondary sex characteristics (or, in young adolescents, the anticipated secondary sex characteristics) 2. Strong desire to be rid of one’s primary and/or secondary sex characteristics because of a marked incongruence with one’s experienced/expressed gender (or, in young adolescents, a desire to prevent the development of the anticipated secondary sex characteristics) 3. Strong desire for the primary and/or secondary sex characteristics of the other gender 4. Strong desire to be of the other gender (or some alternative gender different from one’s assigned gender) 5. Strong desire to be treated as the other gender (or some alternative gender different from one’s assigned gender) 6. Strong conviction that one has the typical feelings and reactions of the other gender (or some alternative gender different from one’s assigned gender) ICD-10 lists three diagnostic criteria for “transsexualism”: The desire to live and be accepted as a member of the opposite sex, usually accompanied by the wish to transition ● The presence of transsexual identity persistent for at least 2 years ●

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The absence of another mental disorder or a genetic, intersex, or chromosomal abnormality.

●

There are limited data on comorbidities in transsexual individuals prior to hormonal or surgical gender affirmation therapy. A large retrospective multicenter study including sites in the United States and Europe with >2000 transsexual adults reported that at the start of hormone therapy, >20% of transsexual men and women had depression, 3% of transsexual women had HIV, and 3% of transsexual women had type 2 diabetes.41 There are mixed data regarding the prevalence of polycystic ovarian syndrome among FTM transsexual individuals.42–46 Baba et al. demonstrated in a Japanese population of FTM individuals (n = 69), a total of 58% had polycystic ovarian syndrome defined by the Rotterdam criteria.43 A total of 39.1% of FTM participants had hyperandrogenemia (n = 29/69) with 30.6% having insulin resistance as defined by HOMA-IR (n = 15/49), and 30.5% having hypoadiponectinemia (n = 18/59). Obesity was seen in 27.5% of FTM individuals with PCOS (n = 11/40) and overall in 23.2% (n = 16/69) of FTM participants. This is in contrast to estimations of obesity in at least 50–65% of women with PCOS. While hyperandrogenemia was associated with PCOS and obesity, insulin resistance was associated with obesity alone. It should be noted that exogenous androgen administration could not be ruled out in this population, potentially resulting in an artificially high prevalence of PCOS. A subsequent study by Mueller et  al. evaluated the prevalence of PCOS in 61 FTM individuals by the 1990 National Institutes of Health (NIH) criteria and 2003 Rotterdam criteria.42 The authors reported the prevalence of PCOS was 11.5% in FTM individuals and 9.6% in controls by the NIH criteria (p = .912) and 14.8% in FTM and 12.8% in controls by the Rotterdam criteria (p = .909). In both unadjusted and adjusted models (accounting for age, BMI, and calculated free testosterone), the odds ratio for the prevalence of PCOS when compared to control subjects was not significantly increased in FTM individuals using the NIH criteria (OR= 0.33, p = .142), nor the Rotterdam criteria (OR= 0.28, p = .062). There was, however, an increased prevalence of biochemical hyperandrogenism in FTM individuals, as compared to control participants (calculated free testosterone of 0.031 ± 0.019 vs 0.019 ± 0.012, p < .001). It is unclear if hyperandrogenism was due to undetected self-medication with androgens prior to enrollment in the study, though considerable overlap was noted with control subjects.

8.6 TREATMENT Both WPATH and the Endocrine Society have published guidelines on the diagnosis and treatment of

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transsexual individuals.3,6 It is presently recommended to delay puberty among transgendered children with a GnRH agonist with initiation of hormone replacement therapy at the age of 16 and consideration of surgical intervention after the age of 18. This is a complex and nuanced area of clinical care and research and outside the scope of this chapter. Protocols are outlined elsewhere.3,6

8.6.1  Overall Goals of Treatment The overall goals of initial hormone treatment are to suppress endogenous hormone production and to induce secondary characteristics of the new sex.47 However, specific goals must be individualized based on the patient’s desires, relative risks and benefits of medications, comorbidities, as well as socioeconomic factors.3 Treatment is initially hormonal and may also include surgical management as outlined below.

8.6.2 Counseling A mental health provider should make the initial diagnosis of transsexualism prior to the initiation of hormonal therapy and/or surgical intervention. Psychotherapy with the overall goal of helping individuals achieve long-term comfort in their gender identity expression is highly recommended prior to treatment. A detailed description of the role of mental health providers is published elsewhere.3,6 Prior to initiation of hormone therapy, it is important to establish realistic goals.

8.6.3  Criteria for Initiation of Hormone Replacement Therapy The 2009 Endocrine Society guidelines suggest that the patient satisfy both eligibility criteria and readiness criteria prior to initiation of hormone replacement therapy. Eligibility criteria include: (1) fulfillment of DSM-IV or ICD-10 criteria for gender identity disorder or transsexualism; (2) absence of psychiatric comorbidities that interfere with diagnostic work up or treatment; (3) knowledge and understanding of the expected outcomes of treatment, as well as medical and social risks and benefits; and (4) experience of a documented real life experience of living in the desired gender role for at least 3 months or a period of psychotherapy (duration determined by the mental health provider, but usually a minimum of 3 months). Readiness criteria include: (1) further consolidation of gender identity during real life experience or psychotherapy; (2) progress in mastering other identified problems leading to improvement or continuing stable mental health; and (3) likelihood of taking hormones in a responsible manner.6 However, more recently, the WPATH put forth the following criteria for starting hormone replacement

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therapy, which notably has eliminated the need for the real life experience component of living as the identified gender: (1) persistent, well-documented gender dysphoria; (2) capacity to make a well-informed decision; (3) being of legal age; and (4) reasonably well-controlled medical or mental health.3 Data have suggested that gender-affirming hormone therapy is associated with improvements, or at least stabilization, in depression and anxiety.48–50 Murad et  al. performed a systematic review and meta-analysis of low quality observational studies and demonstrated that hormonal therapy in individuals with gender identity disorder resulted in an improvement in gender identity dysphoria in 80%, an improvement in psychological symptoms in 78%, an improvement in quality of life in 80%, and an improvement in sexual function in 72%.51

8.6.4  Male to Female Transsexual Individuals 8.6.4.1  Gender-Affirming Hormone Therapy The goals of hormone therapy in MTF transsexuals are to induce breast formation, to cause more female fat distribution, and to reduce male pattern hair growth.8,52 Estrogen also suppresses the hypothalamic-gonadotropin axis (HPG), thus reducing testosterone levels. However, combination of estrogen with a second agent that suppresses androgen secretion or action has been demonstrated to be more effective and permits use of lower doses of estrogen. The latter category generally includes an antiandrogen (spironolactone or cyproterone acetate), or, less commonly, a progestin (medroxyprogesterone), GnRH agonist (such as goserelin acetate), 5-α-reductase inhibitor (finasteride and dutasteride), or nonsteroidal antiandrogen (flutamide, nilutamide). Many formulations of estrogens are available and can be administered via an oral, transdermal, or intramuscular route. However, oral ethinyl estradiol, has been associated with increased risk of venous thromboembolism53 and cardiovascular-related death54 and use is not recommended in the management of the transsexual patient.3,6 Oral 17β-estradiol valerate, which is not available in the United States, is preferred to ethinyl estradiol.3,6 Oral estradiol doses are often double to quadruple that needed for postmenopausal women (i.e., 1–2 mg) and higher doses may be needed for individuals with testes present (up to 4 or 8 mg/day). The goal estradiol level is in the premenopausal range with some advocating reduction in dosing when MTF transsexual individuals are the age of menopause depending on the clinical circumstances. The risk of adverse events associated with estradiol, including thrombosis risk, may be dose-dependent, most notably at supraphysiologic levels. In particular for individuals with risk factors for venous thromoboembolism, transdermal

estradiol is recommended and parental products may also be considered to avoid first-past metabolism and stimulation of hepatic clotting factors.3,6,8,55 Please refer to below section on Adverse Effects of Hormone Therapy for a more detailed discussion on the risk of venous thromboembolism. Spironolactone is a mineralocorticoid receptor antagonist, typically utilized as an antihypertensive agent. Spironolactone is also an effective antiandrogen, postulated to work via the following mechanisms: (1) antagonism of androgen receptors and inhibition of androgen biosynthesis56; (2) reduction in 17-betahydroxysteroid dehydrogenase, thus inhibiting conversion of androstenedione to testosterone57; (3) inhibition of 5-α-reductase activity thus preventing the conversion of testosterone to dihydrotestosterone (DHT); and (4) increase in sex hormone binding globulin.58–60 Higher doses are typically needed to achieve the desired effect, ranging from 100–200 mg/day and may need to be administered in divided doses for tolerability. Given its activity as a mineralocorticoid antagonist, blood pressure and electrolytes, most notably potassium, need to be monitored with the latter every 2–3 months in the first year.3,6 In the United States, spironolactone is the most commonly used and generally the most cost-effective antiandrogen.61 Cyproterone acetate is both an androgen receptor antagonist and a progestin. Its progestin-like activity results in suppression of the HPG axis as well. Although widely used in Europe, this medication is not available in the United States. Use of progestins, with the exception of cyproterone acetate, is controversial. Progestins are important in mammary development in the female sex and it has been hypothesized that these medications are necessary to enhance breast development, to decrease breast sensitivity and to decrease irritability.60,62,63 However, addition of progestins has not been shown to enhance breast growth.64 Furthermore, progestins have been associated with depression, weight gain, lipid changes. Thus, overall, the data reveals that while progestins suppress androgen production, they have no role in feminization of the body and may have adverse metabolic effects.8 Furthermore, based on evidence from the Women’s Health Initiative, the use of medroxyprogesterone may increase the risk of breast cancer and cardiovascular risk in women.65 Micronized progesterone is preferable, as it may be better tolerated and have a more neutral effect on lipids. It has been suggested that intramuscular injections (i.e., Depo Provera) may also minimize excess risk.66 As outlined below, cardio-metabolic screening is indicated, most notably for individuals treated with more lipid-adverse forms of cross-sex hormones. GnRH agonists (e.g., goserelin, buserelin, and triptorelin), are effective in suppression of the hypothalamic

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pituitary gonadal axis, but are expensive and not commonly used for this indication.61 Dittrich et al. reported 60 MTF transsexual individuals who were treated with a combination of oral estradiol 17-β valerate daily and injections of goserelin acetate monthly for 2 years. The regimen was effective in reducing testosterone levels and reported adverse reactions were low.67 Mueller et al. further described 84 MTF individuals treated with oral estradiol 17-β valerate daily and injections of goserelin acetate monthly who demonstrated an increase in lumbar spine bone mineral density without effects on femoral bone mineral density.68 There is limited clinical experience with use of 5-α-reductase inhibitors (finasteride and dutasteride), which inhibit conversion from testosterone to 5-α-dihydrotesterone, and nonsteroidal antiandrogens (flutamide, nilutamide), which increase testosterone and estrogen via increased gonadotropin secretion (only the increase in estrogen is desirable). Neither class of medications is recommended for routine clinical use due to limited clinical experience with these agents. There is considerable individual variability in response to hormone therapy. Typically, after 1–3 months of estrogen and antiandrogen therapy, a decrease in facial and body hair (although facial hair tends to respond less favorably and additional measures such as hair removal or electrolysis are often needed),52 decrease in skin oiliness,52 redistribution of fat (increase in subcutaneous fat), and decrease in lean body mass69 is seen. Although breast tissue growth may occur early, generally breast development is maximal 2 years after initiation of hormone therapy.59,70 The testes and prostate may atrophy over the course of years,6 with atrophy of the former allowing for potential retraction into the inguinal canal and potential discomfort. Hormonal therapy is unlikely to alter the effects of prior androgen exposure on bone configuration such as shape of hands, feet, jaw, and pelvis, or height. Hormonal therapy is also unlikely to change the presence of laryngeal prominence or alter the voice.6 It is recommended that MTF individuals who undergo feminization surgery also consult a voice and communication specialist to maximize surgical outcome, help protect vocal health, and learn nonpitch-related aspects of communication.3 8.6.4.2  Adverse Effects of Hormone Therapy Adverse effects associated with hormone therapy are outlined in Table 8.1. The most salient concerns with hormone therapy in MTF transsexual individuals include hyperprolactinemia, cancer risk, venous thromboembolism, coronary heart disease, and overall mortality. Estrogen therapy can result in growth of pituitary lactotroph cells, resulting in increased prolactin levels (potentially compounded by concurrent use of antipsychotic agents) with a reported prevalence of up to 21%

TABLE 8.1  Risk Associated With Feminizing and Masculinizing Hormone Therapy in Transsexual Men and Transsexual Women Respectively Feminizing hormones

Masculinizing hormones

Venous thromboembolic diseasea

Polycythemia

Gallstones

Weight gain

Elevated liver enzymes

Acne

Weight gain

Androgenic alopecia

Hypertriglyceridemia

Sleep apnea

Likely increased risk with presence of additional risk factors

Cardiovascular disease

–

Possible increased risk

Hypertension

Elevated liver enzymes

Hyperprolactinemia or prolactinoma

Hyperlipidemia

Type 2 diabetesa

Destabilization of certain psychiatric disordersb

Risk level Likely increased risk

Possible increased risk with additional risk factors (such as age)

Cardiovascular disease Hypertension Type 2 diabetes

No increased risk or inconclusive

Breast cancer

Loss of bone density Breast cancer Cervical cancer Ovarian cancer Uterine cancer

Adapted from Coleman E, Bockting W, Botzer M, et al. Standards of care for the health of transsexual, transgender, and gender-nonconforming people, version 7. Int J Transgenderism. 2012;13(4):165–232. a Risk is greater with oral estrogen administration than with transdermal estrogen administration. b Includes bipolar, schizoaffective and other disorders that may include manic or psychotic symptoms. Appears to be associated with higher or supraphysiologic doses of testosterone.

in MTF individuals on high dose estrogen therapy and incidence of 3.7–7.2% per year.71 Hyperprolactinemia is less common with lower doses of estrogen and should improve with a reduction in dosing. While most common after initiation of hormone replacement therapy, elevated prolactin levels may be seen years later.72 Case reports of prolactinomas have been reported in this population following high dose estrogen therapy.73 Some have thus recommended measurement of prolactin at baseline, annually for 2 years, and then every 2 years with a pituitary MRI if prolactin levels do not normalize with a reduction in estrogen dose.6

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There have also been case reports of breast cancer occurring in the MTF population, hypothesized to relate to high dose estrogen therapy, but this is overall rare and not reported to be higher than that of men in the general population.74–78 Specifically, Gooren et  al. reported an estimated rate of 4.1 cases/100,000 person years (95% CI: 0.8–13.0) in a Dutch cohort of 2307 MTF transsexual individuals receiving hormone therapy, in total, one case of confirmed breast cancer and one possible case of breast cancer were identified. The mean duration of follow up on hormone therapy was 21.4± 8.7 years. It is unclear if breast cancer risk increases with duration of treatment of feminizing hormones and use of progestins in this population. Cases of prostate cancer have also been reported.79,80 A retrospective Dutch study of orchidectomized MTF individuals reporting a single case, corresponding to an overall incidence of prostate cancer of 0.04% and in those >40 years, of 0.13%.81 However, these data must be interpreted cautiously in light of relatively young age at screening and the lack of rigorous screening with low likelihood of prostate specific antigen (PSA) levels being elevated in androgen deficiency. Case reports of meningiomas also exist, hypothesized to be hormonally related.82–86 Traditionally, the risk of thromboembolic disease has been reported to be high in transsexual women.87–89 As outlined above, however, the risk of venous thromoboembolism is highest among individuals using ethinyl estradiol with rates of 2–6% with the highest risk of events in the first year and in the setting of established risk factors such as immobility and perioperative period.53,88 Use of transdermal estradiol has been associated with a lower risk of venous thromboembolism.88,90,91 Some have advocated that among MTF transsexual individuals with risk factors, anticoagulation with initiation of hormonal therapy may be a feasible option. However, presently, increased risk of thromoboembolic events have not been seen with other forms of estrogen therapy including oral 17-β-estradiol.7,53 Although conflicting recommendations exist, some advocate discontinuation of cross-sex hormones for at least 2–4 weeks prior to elective surgery and resumption 3 weeks later after full mobilization.88 With regards to cardiovascular disease, Asscheman et  al. reported increased cardiovascular death in MTF versus FTM, with current use of ethinyl estradiol conferring a threefold risk.54 It was thus hypothesized that ethinyl estradiol conferred a thrombotic risk that explained cardiovascular mortality. However, subsequent studies in a Belgian population have shown increased cardiovascular morbidity in populations in which ethinyl estradiol use is low. The prevalence of myocardial infarction was similar in transsexual women compared to control men (18.7 vs 12.5 cases/1000 persons), but higher as compared to control women (0 cases/1000 persons, p = .001). The prevalence of cerebrovascular disease

(including transient ischemic attacks) was significantly higher in transsexual women compared to male controls (23.4 vs 9.4 cases/1000 persons, p = .03), but not compared to women (14.9 cases/1000 persons).92 Wiercx et  al. reported that 6% of transsexual women (n = 6) experienced cardiovascular problems, including cerebral thrombosis, TIA, peripheral arterial disease, venous ulcer, and myocardial infarction after an average of 11.3 years on hormone therapy.87 This increase may partly be due to higher prevalence of cardiometabolic risk factors. In this population, tobacco use and diabetes were higher among MTF individuals than control populations.92 Limited data have suggested that with estrogen and antiandrogen therapy, transsexual women have decreased insulin sensitivity,93 along with increased subcutaneous fat, and to a proportional and lesser extent, visceral fat.94 Although two short-term prospective studies have not shown a significant increase in blood pressure with hormone therapy,95,96 a cross-sectional European study reported that 22% of individuals on feminizing hormones for a mean of 10 years had elevated blood pressure or treated hypertension.50,87 Short-term prospective, cross-sectional and retrospective studies have consistently reported an increase in both HDL cholesterol and triglycerides with feminizing hormonal therapy.50,87,97 A meta-analysis including 16 studies demonstrated only an increase in triglycerides, but no significant changes in other lipid fractions or blood pressure among transsexual women. However, the quality of evidence was low in the study due to the fact that the included studies were largely uncontrolled and observational, had brief follow-up, and heterogeneity of treatment regimens.98 A subsequent 2-year prospective study99 that enrolled metabolically healthy transsexual women (n = 79) and transsexual men (n = 43) also demonstrated increased insulin resistance, an increase in fasting glucose (85.9 vs 89.7 mg/dL, p < .001), increased systolic (111.3 vs 129.1 mm Hg, p < .001), diastolic blood pressure (75.6 vs 78.8 mm Hg, p < .001), waist circumference (81.2 vs 85.7 cm, p < .001), increased triglycerides (77.1 vs 139.0 mg/dL, p < .01) and LDL (100.1 vs 130.1 mg/dL, p < .001), and decreased HDL (54.3 vs 42.0 mg/dL, p < .001). Glyco-insulinemic alterations were more pronounced in transsexual women, as compared to men, while the remainder of metabolic parameters were similar between the two groups. Further, with the exception of changes in lipid parameters that increased over time, the remainder of metabolic parameters had a lesser change after year 1. Overall, 13.9% (n = 13) of transsexual women developed metabolic syndrome at year 1 (n = 11) and 16.5% (n = 13) had metabolic syndrome at year 2. The study also noted that individuals treated for psychiatric problems may be more likely to develop metabolic syndrome (50.0% (n = 6) vs 10.4% (n = 7)), but it is difficult to extrapolate

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8.6 Treatment

with such a small sample size. It has also been suggested that, similar to postmenopausal women, timing of estrogen therapy with regards to effects on cardiovascular health is important, with early treatment generally consistent with a more favorable cardiovascular response. Taken together, these findings highlight the importance of aggressive control of cardiometabolic risk factors prior to initiation of hormone replacement therapy. Additionally, overall mortality is up to 51% higher in MTF individuals as compared to FTM individuals and the general population, and largely attributed to drug abuse, HIV, cardiovascular disease, and suicide.54 These findings highlight the need for long-term follow-up with a mental health professional and screening for high-risk behaviors such as unprotected intercourse. Limited and mixed data exist on bone health in transsexual women. Prior to hormone therapy, transsexual women have lower bone mass with a higher prevalence of osteoporosis (18% vs 4%) and lower muscle mass and strength when compared with biologic men in a Belgian population.100 Untreated transsexual women in this study were less likely to participate in sports and the majority were vitamin D deficient ( females)

Postimplantation fetus

Sry gene expressed in males only Androgen exposure (males > females) Morphological differences (anogenital distance, gonadal development) Growth rate (males > females)

Placental development

Gene expression and proteome dimorphism Morphological differences in overweight mothers (females > males) MicroRNA expression differences provoked by maternal obesity (females only)

Maternal physiology

Increased gestational diabetes (males > females) Risk of adverse obstetric outcomes (increased rates of cesarean section etc., males > females)

Perinatal life

Higher birth weight Risk of preterm delivery Gestational age-adjusted morbidity and mortality (all males > females)

which consist of a series of highly regulated, specialized, and unique cleavage divisions. These divisions characterize the preimplantation and early postimplantation phases of embryonic development.19 At this stage, there are no overt morphological features of the developing organism to indicate a phenotypic sex, but even at these very early developmental stages, differences between male and female embryos can be detected. At the 2-cell stage there is already a difference in successful culture rates between blastomeres biopsied from male and female embryos.20 A little later in preimplantation development, sex-specific differences in gene expression become apparent. These have been demonstrated initially in genes that derive from the sex chromosomes (e.g., at the 8-cell stage in the mouse21), and later in the autosomes (e.g., at the blastocyst stage in the mouse22). Furthermore, the preimplantation period is a key stage of development when DNA methylation is reestablished and cells are committed to various lineages. Both DNA methylation and cell-lineage specification are potential

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mechanisms for developmental programming of adult disease. The observation in cattle that newly established methylation patterns during preimplantation development may differ depending on the sex of the embryo is thus of particular interest.23,24 Moreover, it has been demonstrated in bovine embryos that the expression of key enzymes that are involved in establishing genome methylation is upregulated in male blastocysts cultured in vitro compared to their female counterparts,25 which may account in part for findings of sexually dimorphic differences in methylation amounts and patterns. However, not only methylation patterns, but also other aspects of epigenetic regulation are sexually dimorphic in the preimplantation embryo. Expression of genes involved in histone methylation (HMT1 and ILF3) is also higher in male than female bovine embryos.25 This finding is of particular interest as our understanding continues to develop of how histone modifications impact on gene expression and play a central role in later onset of developmentally programmed phenotypes.26 Experimental manipulations of culture conditions of the preimplantation mouse embryo in vitro lead to altered phenotypes in adult life in both sexes,27–29 but with subtle variations between the sexes. For example, although the addition of fetal calf serum to preimplantation culture medium increased later obesity in both male and female mice, this was only associated with elevated plasma glucose in the male, whereas female mice were hypertensive and developed liver steatosis.27 Addition of the embryokine Colony Stimulating-factor 2 (CSF2) to culture medium for bovine embryos at the morula stage has recently been shown to increase the survival of female, but not male preimplantation embryos.30 Hence karyotypic sex plays an important role in the adaptation of the blastocyst to its early environment. Preimplantation mouse embryos that carry a Y chromosome develop more quickly in vitro than do XX embryos,31 although this is influenced by the glucose concentration of the culture medium and the effect is not consistently observed across species. Sex differences in growth and cell division rates are detectable in the preimplantation embryo, considerably prior to the expression of the Sry gene in the genital ridge, which commences in the mouse between embryonic day 10.5 and 12.5, and is generally taken to herald the start of overt morphological differences between male and female embryos. The mechanism by which different growth rates between the sexes prior to the expression of the Sry gene is controlled is unknown, but may be related to a period of activation of both of the X chromosomes in the female preimplantation embryo at the blastocyst stage.32 In rodents, this period of double X chromosome activation occurs between the 8-cell and blastocyst stages,33 but the equivalent timing (if any) is uncertain in human embryos.34,35 However, the burst of transcription from

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both X chromosomes, before one of the pair is inactivated in the female embryo, results in a proteome that is distinct between in the sexes36 and involves approximately 600 differentially expressed genes.22 This early transcriptional burst distinguishes male from female embryos, and could have important effects on both limiting the growth rate of female embryos and potentially on later gene expression. The rate of growth during specific time periods when developmental programming stimuli may have long-term effects is important, because a higher rate of growth and metabolic activity during the same time window can effectively magnify the exposure. Hence male embryos that are developing faster than their female counterparts may be more affected by the same environmental modulation during these critical periods of development.37 Linked to growth rates, and requirement for cellular energy production, there may be sex-specific differences in mitochondrial activity in blastocysts.38 Aside from the genome itself, and modifications to the structure and expression patterns of DNA, the preimplantation conceptus also contains very high copy numbers of the mitochondrial genome.39 The mitochondrial DNA copy number is higher in male blastocysts than in females,25 and this increased potential for energy production may not only be instrumental in facilitating an increased growth rate, but may have implications for longer-term cellular metabolism and later life oxidative stress accumulation.40 After the activation of the Sry gene, there are morphologically detectable differences between male and female embryos, which commence in the genital ridge with the formation of the gonads. Formation of the gonad leads to differential expression of sex steroids, which in turn can influence a myriad of other aspects of fetal physiology and anatomy. The most reliable marker identified thus far of fetal androgen exposure in utero appears to be anogenital distance.41 This parameter is clearly sexually dimorphic at birth, a difference that is preserved across species and often used as a means of sex determination in newborn animals.42 Recently, it has been demonstrated that this overt morphological difference arises in human fetuses as early as 11–13 weeks gestation during the so-called “masculinization programming window,” by which stage males have a longer anogenital distance than their female counterparts.41 Anogenital distance can thus be used as a relatively sensitive marker of intrauterine androgen exposure. The physiological sensitivity of the developing fetus to endogenenous sex steroid levels means that maternal exposures altering in utero androgen levels have important developmental effects, which are differently expressed in male and female fetuses. Exposures that can alter fetal androgen levels include common exposures in human pregnancy, such as maternal smoking41 and exposure to a number of environmental toxins.43–45 The female

fetus, which is normally exposed to much lower levels of endogenous androgens than the male, is particularly sensitive to fluctuations in the sex-steroid milieu. It has long been established that exposure of the female fetus to increased androgen levels (similar to those seen in the male) can influence the normal growth trajectory, and disrupt the normal development of the female reproductive system.46 More recently, studies have shown that excess androgen exposure in the female fetus affects a multitude of physiological parameters including the pancreatic development,47 renal gluconeogenesis,48 and liver metabolism.49 If androgen levels are significantly above the normal range, then reproductive development in male fetuses is also affected.50 Much of the current evidence regarding developmental programming effects comes from animal models generated in species that, unlike humans, reproduce litters rather than single offspring. In such species there is a further complication when considering fetal exposure to sex steroids, which is that the ratio of male-to-female fetuses in the litter may affect the circulating androgen level. Hence where the ratio is skewed in favor of the male, female fetuses may be exposed to more than usual levels of androgens.51 Going a step further, it has also been suggested that the physical position of female fetuses in relation to the male fetuses within the womb could affect their androgen exposure. If sex steroid exposure is the key to sex-specific developmental programming effects then we would expect females from male-heavy litters, and possibly also those positioned between two male fetuses in utero to have later life phenotypes more akin to their male counterparts. This is seen in both increased anogenital distance,51 and in changes in the preoptic nucleus in the rat brain, which becomes more masculine in morphology in females that have developed in utero between two male siblings.52 Although steroid exposure level is perhaps the most obvious factor differing between male and female fetuses in late gestation, it should be noted that there still exist other differences at a cellular physiology level that are unrelated to steroid levels (reviewed in Ref. 53). Although the reason is not known, it is well described that male fetuses have higher rates of congenital anomalies than their female counterparts,54 and higher rates of term or near-term stillbirth.55 In considering possible mechanisms for sexually dimorphic development in late pregnancy an important consideration is the dose dependence of genes expressed from the X chromosome. Although this is partially normalized between the sexes by X inactivation much earlier in fetal development, it is estimated that approximately 15% of genes on the X chromosome can escape inactivation.56 Thus in later fetal development, there remains potential for X chromosome-dosage to impact on the differential development of the female versus the male fetus. These differences

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20.2  Sex-Specific Differences in Normal Intrauterine Development

may be further exacerbated by imprinting of genes on the X chromosome specifically inhibiting the expression of alleles derived from either parent.53 By the time of birth, it is evident both morphologically and physiologically that male and female infants respond differently to the same environmental stressors57 and have different epigenetic profiles.58 Although male infants are larger at birth than females, they have higher morbidity and mortality rates at and around the time of birth.55,59 Moreover, male infants have higher rates of premature delivery, and poorer gestational-age adjusted outcomes after birth.59,60 It has been suggested that, after birth in the early neonatal phase, organ growth and maturation remains sexually dimorphic, which may be related to the sex-steroid milieu in early postnatal life and could have important effects on outcomes in infancy.61 However, the intrauterine and neonatal development do not occur in isolation. During the later stages of intrauterine development, the fetus exists in a complex interplay involving maternal physiology and the placenta, which acts as the interface between the two. The placental karyotype reflects that of the embryo, and so has the potential to interact with both the mother and the conceptus in a sex-dependent manner. If the placentas of females are better adapting to the environmental challenges faced during development, or vice versa, then this may account for at least some of the sexually dimorphic developmental programming effects observed in humans and in animal models.62 However, there are several major difficulties in defining precisely how the placental sex and fetal sex interact during development. The first is that disentangling the relative contribution of placental and fetal sex to any given phenotype is necessarily complex. The second is that the morphology and function of the placenta varies considerably between species, making inferences from animal models less secure than is generally the case with other organ systems.63 However, given the key role of the placenta in nourishing and supporting fetal growth, any difference in placental structure or function could be critical in determining differences between the early development of male and female fetuses.62,64 Several studies have shown that gene expression in the placenta varies depending on fetal sex in both humans65–67 and rodents.68 In rodents the epigenome of the placenta shows clear sexual dimorphism (reviewed in Ref. 69), and it has been demonstrated that these sexually dimorphic gene expression patterns are modifiable in response to alterations in maternal diet.70,71 Such changes have been demonstrated to influence parameters likely to impact directly on offspring growth, including placental nutrient transport,71 establishing a putative mechanism by which placental epigenetic changes could bring about sex-specific development programming in offspring. In humans, morphological differences have been observed in term placentas

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of female fetuses in response to maternal overweight/ obesity but corresponding changes did not occur in the placentas associated with a male fetus.72 Moreover, the placentas of human female fetuses show differences in mitochondrial function and miRNA expression in response to maternal obesity that are not observed in the placentas of males.73 Intriguingly, it has recently been reported that not only maternal diet, but also paternal high-fat diet in a mouse model may impact on placental physiology.74 The placentas of female offspring showed increased placental gene methylation patterns, whereas gene expression profiles were upregulated in the placentas of their male counterparts.74 Combined with gene pathway analyses,75 such studies may cast further light on the mechanisms that underlie the sexually dimorphic response of the placenta to developmental programming and in turn contribute to the difference in later life phenotypes between the sexes. The role of maternal physiology is also of interest. It has been suggested that mothers may respond differently in their adaptations to the pregnancy environment depending on the sex of the conceptus, and that this may influence pregnancy outcome. The earliest possibility for such a mechanism occurs in the oviductal environment, which supports the free-floating embryo during the preimplantation phase of development. It is known that alterations to the maternal diet can influence the concentrations of important signaling molecules such as mTORC176 and amino acids77 within the oviductal and uterine fluid, and hence provide a molecular stimulus to developmental programming of the conceptus. However, it is yet to be determined whether there may be any sexual dimorphism in the very early maternal reproductive tract environment. Beyond the implantation stage however, it has been observed that female fetuses signal their presence to the mother via higher levels of human chorionic gonadotropin than do male fetuses,78 which may be influential in eliciting the fetal sex-specific response of the maternal physiology to the presence of the conceptus. An interesting example of maternal physiological response to fetal sex is that there is a higher likelihood of a woman developing gestational diabetes when carrying a male fetus than when carrying a female. A recent systematic review, which included data from 2,402,643 individual pregnancies, has shown that the risk of gestational diabetes is 4% higher when the fetus is male than female.79 While this difference is small enough to make the fetal sex-difference unlikely to have clinically useful ramifications for assessing the likelihood of gestational diabetes, it illustrates that the maternal adaptation to pregnancy is highly likely to involve a two-way interplay between fetal and maternal physiology. The sex of the fetus thus impacts on the overall pregnancy environment and this could be influential in producing the subtle but lasting cellular

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changes that characterize developmental programming. Other important maternal obstetric outcomes that are dependent on maternal adaptation to pregnancy and are highly influential for the later health of the newborn, may also differ with fetal sex. These include the risk of dysfunctional labor and requiring cesarean section, both of which are higher when the fetus is male.55,80 The poorer perinatal outcomes observed in male fetuses than in females could be at least in part a result of impaired maternal adaptation to pregnancy. These in turn could lead to an excess of developmentally programmed adverse phenotypes in adult and later life.

20.3  DEFINING A SEX-SPECIFIC RESPONSE IN DEVELOPMENTAL PROGRAMMING—METHODOLOGICAL ANALYSIS Given the variation in physiological intrauterine growth and development between male and female fetuses, it is unsurprising to find that altering the normal early environment could affect developing males and females differently. Numerous experiments have been performed altering the early environment in animal models, mainly via alterations in maternal diet, and such studies have frequently reported different effects of the altered environment depending on fetal sex.81 However, a note of caution should be introduced in interpreting the results of many studies in the field. In order to report an effect as “sex-specific” the basic criterion should be that the results from both sexes are reported. However, it is not sufficient to conclude that if the sexes are analyzed separately, and an effect is deemed statistically significant (usually defined as an alpha level less than 0.05) in one sex but not the other that the effect is sex-specific. Such analysis compares the results from each sex separately to the null hypothesis (that there is no influence of the treatment on the specified parameter), but makes no direct comparison between the sexes. The majority of published studies in this area do not compare male and female offspring directly to one another, and yet often conclude that results are sex-specific.82 An improvement on the method of comparing the sexes separately to the null hypothesis is to specify an interaction term between the effect of sex and the effect of treatment. This can be achieved using a two-way ANOVA, which is a relatively common means of analyzing such studies. However, this approach is often of limited value in smaller animal studies, where the n numbers are kept to the minimum possible in order to reduce costs and animal use, and hence do not have sufficient power for true sex-specificity analysis. A small study may be adequately powered for the main treatment effect, but not to detect differences in interaction terms at the usually quoted alpha

level of 0.05. Other statistical approaches more suitable for small sample sizes are possible, including considering the difference in probability distributions of the effect detected in each sex or using a Bayesian approach to test the likelihood of a biologically meaningful difference in treatment effect between male and female subjects.82 Cohorts in which sex-specific differences in transgenerational developmental programming effects are to be tested are subject to a further methodological difficulty; that of multiple comparisons. In studies that test transmission of developmental programming effects via both paternal and maternal lines, when sex differences in outcomes are taken into account, comparisons a minimum of eight different groups of second generation offspring are being considered (both male and female offspring exposed to a developmental programming stimulus via each of four possible grandparents). If any further factors are added, such as different exposures, combinations of exposures or treatment groups, then at least 16 separate but nonindependent hypotheses are being simultaneously tested. When such analyses are performed, the possibility of generating spurious results by multiple hypothesis testing should be very carefully considered in the experimental and analytic design. Corrections for nonindependent multiple comparisons including the use of Bonferroni corrections, cross-validation, permutation testing, or boot-strapping are often required. The most appropriate correction for each individual study design will vary (e.g., the Bonferroni correction is often too conservative when testing large numbers of hypotheses) and expert statistical advice should be sought at the initial design stage where such experiments are contemplated. This is particularly important given the time, effort, and expense involved in maintaining transgenerational experimental cohorts. A further important consideration when defining sexspecific effects in animal models of developmental programming is to consider the litter of origin of the male and female offspring. Most animal cohorts in developmental programming are structured such that the reported n values refer to the number of litters tested, rather than the number of offspring. Because the intervention (often, but not limited to, dietary manipulation) is performed at the level of the mother, rather than the offspring, this is a sound analytic approach. However, when male and female offspring are both tested, cohorts are often structured such that a brother and sister that are derived from the same litter are included in the analysis. This approach is not incorrect, but should be accounted for in the modeling structure, rather than treating the siblings are independent. A useful approach to analyzing cohorts with this structure is to add a random effect for litter of origin to the model, which takes account of the nonindependence between siblings, but retains much of the power related to sample size. Again, in practice, this approach is underutilized.

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20.4  Sex-Specific Implications of Adverse Environmental Stimuli in Human Early Development

The difficulties outlined with analysis of animal studies reporting sex-specific effects makes interpretation of much of the literature in this area at least somewhat problematic. The publication and adoption of ARRIVE (Animal Research: Reporting of in vivo experiments) guidelines,83 which aim to improve the standard of statistical analysis of animal cohorts such as those utilized in developmental programming studies, is likely to lead to gradual improvements in the robustness of study conclusions. Human studies that report sex-specific developmental programming effects are also subject to problematic analysis, although rarely due to the same issues that are common in animal studies. Human studies are more likely to be affected by the long generation time inherent in developmental programming effects, which by definition are programmed in utero, but persist into postnatal and usually adult life. Associated difficulties in human populations include ensuring sufficiently high follow-up rates, and the myriad influences of the hugely variable postnatal environment on later health and disease. The further away from the time of birth at which offspring outcomes are measured, the greater is the likelihood of the study being significantly impacted by one or both of these issues. Even where retention rates of participants in the study cohort are good, offspring sex differences may exist in follow-up rates, which could bias the findings considerably. Despite these methodological issues, the weight of evidence is very much in favor of sex differences existing in a number of animal models of developmental programming and in human cohorts. Key examples of such studies are reviewed in the next section.

20.4  SEX-SPECIFIC IMPLICATIONS OF ADVERSE ENVIRONMENTAL STIMULI IN HUMAN EARLY DEVELOPMENT 20.4.1  Neonatal Effects Exposures leading to developmental programming are difficult to define and control in human pregnancy. Partly because of the complexity of defining in utero exposures in humans, the natural experiments provided by stress related to various environmental disasters affecting populations at specific times are often studied.10,84–86 Such studies have revealed a number of sex-specific effects of the exposure to psychological and physical stress related to such events in infants at birth, although in many instances it is difficult to separate out which aspect of the exposure is primarily responsible for the resulting phenotype. Israeli women who were exposed to stress through unpredictable attacks during their pregnancies were more likely to deliver

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preterm if their fetus was female, but not if the fetus was male. Exposure to prenatal stress in the female newborn was furthermore associated with reduced intrauterine growth.85 Very similar outcomes in female infants were observed in a birth cohort that had been in utero during an earthquake in Chile.10 Aside from prenatal stress related to natural disasters, the effects of exposure in utero to self-reported stressful events in the mother have also been examined.87 In female infants, such exposures were associated with an increased anogenital distance. Interestingly, although the association was not statistically significant, male infants who were in utero during stressful maternal life events tended to have shorter anogenital distances. In response to maternal asthma during pregnancy the intrauterine growth of female fetuses is reduced, while male fetuses maintain their growth trajectory.88 Sex differences in fetal growth are observed in conjunction with differences in placental glucocorticoid metabolism in this human cohort, emphasizing the key role of the placenta in buffering the fetal exposure to various maternal stresses.88

20.4.2  Later Life Effects Human studies into later life effects in adulthood are limited, mainly due to long generation times. However, several studies have examined developmental programming effects into mid or late childhood. Key examples of such studies come from a cohort of women who were pregnant during severe ice storms in Quebec, which were a cause of prenatal stress (Project Ice Storm84,89). Follow-up of the babies that were in utero during the storms concluded that at the age of 5 there was a sexspecific difference in motor skills, with boys performing more poorly on testing than their female counterparts, however the timing of the exposure during gestation was interactive with the effect of sex on motor outcomes.84 In the same cohort by the age of 12, girls who had been in utero during the storms had an increased likelihood of developing asthma, but there was no such effect observed in boys.89 Boys, but not girls, whose mothers had experienced prenatal bereavement were more likely to be diagnosed with ADHD by the age of 3 years than control children,90 however a cohort that measured maternal anxiety during pregnancy found that positive associations between childhood ADHD and prenatal stress in both sexes.91 When observations were made at 5 years of age on female who had been exposed to stressful maternal life events in utero, they showed an increase in “masculinized” play-behaviors92—this is particularly interesting in view of their tendency to increased anogenital distance in infanthood and the putative effect of prenatal stress level on in utero androgen levels. Beyond childhood and into adolescence, studies have shown sexually dimorphic developmental programming

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influences on behavioral and psychological traits. For example, female adolescents whose mothers experienced more antenatal anxiety were more likely to self-report depressive symptoms than controls, but this association was not seen in male adolescents.93 Teenagers that had been exposed to prenatal stress related to an earthquake in utero were followed up at 18 years of age, and were found to have a higher likelihood of experiencing depressive symptoms. The likelihood was highest in males who had been in utero during the second trimester of pregnancy during the earthquake, which raises the interesting possibility of an interaction between sex and gestational age in determining the overall impact of the prenatal environment on later phenotype.94,95 Outcomes in adulthood are even more rarely reported in humans following exposure to well-defined developmental programming challenges. Birth weight is often used as a proxy for a suboptimal intrauterine environment, and many of the early seminal studies in the field rested on this parameter.96 However, environmental challenges that are sufficient to substantially impact on the growth of the baby are wide-ranging in nature. This means that a highly heterogeneous exposure group, in terms of both the nature and the magnitude of the adverse pregnancy environment, may be included in studies where the birth weight is used as a proxy for fetal exposure. Conversely, intrauterine challenges that are highly influential in altering gene expression or metabolic set points may not alter birth weight. While birth weight is a relatively crude measure of the “health” of the intrauterine environment, nonetheless some interesting insight can be gained into sexually dimorphic impacts on disease in adulthood from its study, because it is one of the very few parameters reliably available from older human cohorts. An interesting example of such a study utilizes Danish adults born between 1936 and 1983.97 In this cohort, low birth weight was associated with the development of type 2 diabetes in adulthood in both males and females. However high birth weight was only associated with diabetes in females, but not in males.97 Furthermore, the magnitude of the association between birth weight and diabetes was greater in females than in males.97 Several human studies have also reported an association between birth weight and hypertension in adult life, which is specific to males.98 Interestingly however, a meta-analysis of such studies found that the association between birth weight and systolic blood pressure existed in both sexes and was of roughly equal magnitude.99 Attempts to cast mechanistic light on the long-term effects of developmental programming in human cohorts have also been made. Later life outcomes in humans have also been correlated with recorded discrete exposures to maternal stress during pregnancy. Individuals who were in utero during famine (1944–45) had their methylation patterns compared

to their control same-sex siblings, and showed variations that were related to both sex and timing of famine exposure.100

20.5  SEX-SPECIFIC IMPLICATIONS OF ADVERSE ENVIRONMENTAL STIMULI IN ANIMAL MODELS The difficulties inherent in precisely phenotyping human pregnancies, and in long-term follow-up of the offspring make animal models vital in understanding the pathogenesis of adverse outcomes programmed in early life. A huge array of different exposures and outcomes has thus far been measured in a variety of species, and sexual dimorphism is a common finding. However, outcomes are observed in either males or females without a great deal of consistency regarding which sex is more or less vulnerable to developmental programming effects overall, even when the same intervention is applied. There are a number of possible explanations for this observation, including the differences in study protocols regarding the severity and the timing of the intervention, and the age of study of the offspring. One factor that is not often considered is the estrous cycle stage of adult females during which observations are made. However, many aspects of physiology and particularly behavior are known to be hormone-dependent, and the implications of estrous cycle stage for measuring dynamic outcomes in adult female offspring merits closer consideration.101 An interesting example of the importance of estrous cycle staging has been observed in rats that were subjected to an early postnatal handling stress. In adulthood their prolactin response to an applied stress was measured. In males and females in diestrous the rats exposed to the prenatal stress had a blunted prolactin response, but when the females were tested in estrous their prolactin responses to stress were identical to those of controls.102 This illustrates the importance of careful and consistent experimental design when dynamic parameters are being assessed, and is particularly important given that sex steroids may play a key role in inducing and influencing developmental programming phenotypes. Sexually dimorphic changes in gene expression patterns have been observed in virtually all organ systems studied in response to developmental programming. Sexdependent gene expression in rodent models has been observed in key organ systems closely linked to common developmental programming outcomes, including the brain,103,104 liver,105 placenta,106 and adipose tissue.107 Such sex-specific differences in gene expression are also observed in baboons108 and other experimental mammals.109,110 In this section, important illustrative examples of studies that shed light on the differences between

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20.6  Developmental Programming of the Reproductive Tract: A Special Case

male and female offspring outcomes following exposure to adverse early life environments are considered. One of the important mechanisms that potentially accounts for the adult phenotypes arising from adverse early life environments is structural alterations in organs that cannot be compensated by later growth. Sex differences in adult tissue morphology have been observed in a range of organ systems. A key example is nephron number in the kidney, which is an important parameter in determining renal function in later life, as well as contributing to blood pressure regulation. Adult female rats exposed to a low-protein maternal diet were resistant to the hypertension that affected their male counterparts,111 and maintained their glomerular numbers despite the in utero challenge.112 In the neonatal period male pups who were repeatedly separated from their mothers, causing separation stress, also showed significant structural changes in the kidney notably damage to the interstitial arteries.113 Their female counterparts subjected to the same neonatal stress were robust to the effect and maintained kidney structure equivalent to that seen in control females.113 Aside from the kidney, the brain of programmed animals also shows sexually dimorphic structural changes in response to a suboptimal early life environment. Female mouse offspring that have been subjected to prenatal stress have a reduced glial cell number in the hippocampus, which is accompanied by a phenotype consistent with depressive symptoms, but no such changes are apparent in their male counterparts.114 By contrast, brain structural changes confined to male offspring have been observed in response to prenatal stress in the rat. In this model, the dendritic spine density in the prefrontal cortex was reduced in male offspring only.115 One of the most sensitive homeostasis mechanisms affected by developmental programming is the renin–angiotensin system. In rodent models of low protein diet, sex specific alterations in renin–angiotensin function have been observed in the pancreas116 (where female offspring only are affected) and in the kidney itself (where male offspring are specifically affected; reviewed in Ref. 117). Excess accumulation of oxidative stress is commonly observed in tissues of animals exposed to a suboptimal early life environment.118 The early generation and accumulation of free radical species can cause long-term damage to the intracellular environment, including key macromolecules such as DNA and complex proteins. Several studies have observed sex-specific differences in oxidative stress levels following developmental programming exposures, e.g., in rats exposed to a maternal diabetes during pregnancy the male offspring, but not their female counterparts, developed hypertension in adulthood associated with accumulation of oxidative stress-markers.119 Evidence for sex-specific responses to oxidative stress accumulation also comes from study of

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adult rats exposed to placental insufficiency in utero.120 Again, only the male offspring were hypertensive in later life, and this was observed in conjunction with upregulated oxidative stress-related proteins. Antioxidant defense mechanisms were assayed in both the male and female offspring in this model, and were found to be higher in the female tissues than in the male.120 This finding suggests that both sexes are potentially vulnerable to programming by oxidative stress, but that females are relatively well protected by compensatory mechanisms. Accelerated aging has frequently been implicated as the final common pathway in many developmental programming models, although the subtleties of this interpretation of the observed phenotypes are yet to fully emerge.121 It is commonly observed that the phenotype of programmed offspring diverges further from the control with increased age.122,123 The sexually dimorphic exacerbation of programmed phenotypes with age has been reported in several animal models, e.g., a rat model of placental insufficiency.123,124 The male offspring exposed to placental insufficiency are hypertensive from young adulthood onwards,124 whereas by contrast the female offspring are normotensive in young adulthood but develop both hypertension and obesity with elevated leptin levels by 1 year old.123 In male rat offspring, but not females, prenatal nicotine exposure also gives rise to hypertension that is not apparent in early adulthood but becomes overt with the added physiological challenge of aging.125 Interesting observations on rats that have been subjected to a prenatal restraint stress suggest that with aging male rats have increasing problems with hippocampus-dependent memory-related tasks, whereas the hippocampus of their female counterparts remains relatively plastic through adulthood.126 Many of the phenotypic changes in adulthood observed in animals that have been exposed to a suboptimal early life environment may ultimately be linked to behavioral alterations, e.g., eating behaviors and stress responses. Sexual dimorphism in such phenotypes is very common, with female offspring tending to show more anxiety-related traits,127,128 whereas males tend to show more deficits in cognition and memory.115,129 In response to a prenatal calorie-restricted diet in the rat, expression of hypothalamic neuropeptide Y mRNA (central to appetite regulation and feeding behavior) was reduced in female but not male offspring.130

20.6  DEVELOPMENTAL PROGRAMMING OF THE REPRODUCTIVE TRACT: A SPECIAL CASE When considering the developmental influences on the adult function of most organ systems, e.g., the cardiovascular or renal systems, it is straightforward to

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FIGURE 20.1  Sex differences in developmental programming of the (A) female and (B) male adult reproductive tract.

compare the adult male and adult female offspring. However, programming of the reproductive tract is necessarily sex-specific, due to the fundamental differences in the role of the adult male and adult female in reproduction (these effects are summarized in Fig. 20.1). Programming of the adult reproductive tract is of key importance in understanding the implications of developmental programming for future generations. In both males and female adults the reproductive tract nurtures and protects the germline cells that form the next generation, and in which any mutation or adverse stimuli can have profound consequences for the health of the future second generation offspring. In females, however, there is also the added complication of developmental programming effects on the somatic reproductive tract, in which the conceptus must develop. It has been proposed that programming effects on the oviduct and uterus may be sufficient to program adverse phenotypes in a second generation of offspring.6 In the male, it is known that prenatal exposure to a suboptimal environment can negatively influence reproductive capability. In a rat model combining prenatal exposure to the antiandrogenic d-dibutyl phthalate (DBP) and dexamethasone, the male offspring had significant morphological anomalies in the external genitalia and deranged steroid hormone production.131,132 Administration of DBP to the mother during the masculinization window in the rat pregnancy led to reduced testes weight and anogenital distance, but the relationship between antiandrogen exposure and Sertoli cell number was less well defined.133 Aside from the direct effect of an antiandrogen administered during early life, development of the somatic male reproductive system may also be altered by more conventional developmental programming exposures, e.g., maternal stress exposure during gestation (via restraint) leads to reduced testes development and alters the reproductive hormone profile in the male rat.134 Interestingly the effect of prenatal

stress in this context may still be dependent on androgen exposure level, as it has been demonstrated in the guinea pig that at least some effects of prenatal stress on behavioral parameters can be “rescued” in male offspring via postnatal testosterone administration.135 Aside from structural influences on the somatic reproductive system, it has also been observed that male puberty timing can be influenced by adverse early life exposures, e.g., uterine artery ligation in a rodent model.136 Developmental programming effects on the male gametes themselves however have been examined in a wide range of models, including paternal obesity (reviewed in Ref. 137), maternal undernutrition,138 and postnatal xenostrogen exposure.139 The findings in these models center mainly on (1) damage to the structure and sequence of the DNA itself and (2) alterations in the epigenome in response to the developmental programming intervention.137 It has furthermore been demonstrated that such changes are sufficient to induce programmed phenotypes in subsequent generations,138 and that transgenerational effects can be transmitted by alteration of sperm microRNAs.140 Impairment of male fertility by maternal protein restriction has been demonstrated across the male reproductive lifespan.141 The female somatic reproductive tract is also highly sensitive during development to adverse environmental influence (reviewed in Refs. 5, 142). In a rat low-protein maternal diet model, the oviducts and somatic ovarian tissue of the adult offspring display increased levels of oxidative stress and reduced telomere length, in keeping with a phenotype of accelerated cellular aging.118 This is of particular interest given that the female somatic reproductive tract undergoes senescence earlier than senescence of other organ systems, hence the occurrence of menopause (humans) and estropause (rodents) prior to the onset of old age. In a mouse model of high-fat maternal diet, germ cell number is reduced in young adult females and this appears to be linked closely with

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20.7  Sex-Specific Transgenerational Developmental Programming

a phenotype of oxidative stress and increased lipid peroxidation in the somatic ovary, which supports and promotes germ cell survival throughout adult life.40 The steroidogenic response and gonadotropin receptor levels in the somatic ovary are also influenced by maternal protein restriction.143 Moreover, it is of interest that these changes are recapitulated in a second generation of offspring, albeit at an older age (6 months of age rather than 3 months in the rat maternal low-protein diet model).122 The germline cells themselves, despite being depleted in number,118,122,144 seem to be relatively protected from the adverse consequences of exposure to a suboptimal early life environment.118 Although many effects of developmental programing on the adult female reproductive tract are manifested via germ-cell numbers and somatic tissue aging, there are several other important aspects of female reproductive function that are influenced by the early life environment. These include estrous cycling asynchronicity,145,146 oviductal microenvironment alterations,147 and adaptations of the uterine vasculature.148–150 An interesting recent study has demonstrated in a mouse model of postnatal anxiety that miRNA expression in the sperm can be altered, and subsequently that transfer of these purified miRNAs to the ooplasm could recapitulate some of the metabolic effects in a second generation of offspring.151 This experiment suggested miRNA expression in the ooplasm as a novel mechanism for transmitting developmental programming phenotypes across generations.

20.7 SEX-SPECIFIC TRANSGENERATIONAL DEVELOPMENTAL PROGRAMMING 20.7.1  Sexual Dimorphism in Offspring Beyond the F1 Generation Developmental programming can be understood as an adaptive environmental response to align offspring physiology with the maternal environment, where pathology can arise if the pre- and postnatal environmental conditions vary substantially.16 This is more likely to be the case when developmental programming effects are considered beyond the immediate F1 offspring of an effected pregnancy. Many studies have now observed that developmental programming effects can persist into an F2 or even F3 generation (reviewed in Ref. 6). The question thus arises of whether these transgenerational effects are also likely to be sexually dimorphic. Several studies in rodent models suggest that sexually dimorphic developmental programming can be observed across generations. In the F2 generation, grand-maternal protein restriction in a rat model causes alterations in glucose and insulin metabolism

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that depend not only on offspring sex, but also the time period of the dietary exposure in the F0 generation.152 Grand-maternal protein restriction also alters behavioral parameters in F2 female but not male offspring when transmitted via the paternal F1 line.153 In humans, it has been observed that grandparental diets affect all-cause mortality within a defined lifeperiod in their grandchildren, with an intriguing sexual dimorphism.154 In this study the mortality rate of males was linked to the food available to the paternal grandfather, whereas the same parameter in females was unconnected with the paternal grandfather’s food availability, but was influenced by the food available to the paternal grandmother.154 Subsequent studies have confirmed similar findings and strengthened the conclusions by adding adjustment for the early life environment faced by the grandchildren.155

20.7.2  Transmission of Programming Effects Beyond the F1 Generation via Paternal Versus Maternal Lines An important sex-specific difference occurs in how developmentally programmed F1 generation adult males and females can influence the programming of their own offspring. In humans, the paradigmatic example of such uniparental transmission of developmental programming effects comes from study of the grandchildren of adults exposed to the Dutch Hunger Winter of 1944–45, in whom first generation developmental programming effects were first described.156 Grandchildren whose paternal grandfather had been exposed to famine were significantly more like to be obese in adulthood than those whose grandparental famine exposure occurred via the maternal line.157 The mechanism by which transgenerational developmental programming in humans may occur through the paternal line, but not the maternal line has not yet been elucidated, but this is an important area for further study. Several animal studies have demonstrated that transgenerational developmental programming is possible via both the paternal137 and the maternal F1 lines.122 Such complex transgenerational experiments have been performed mainly, but not exclusively, in rodents.152,158,159 Particularly interesting evidence from a mouse grandmaternal undernutrition model suggests that the same F0 generation exposure can produce different F2 offspring effects depending on whether transmission occurs via the maternal or paternal line. F2 offspring who were exposed to developmental programming via their maternal grandmother showed a phenotype of obesity, whereas those exposed via their paternal grandmother showed low birth weight.160 Exposure via either grandmother gave rise to impaired glucose tolerance in the second-generation offspring.160 The complexity of these

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results demonstrates the lack of certainty that currently exists about the mechanisms of sex-specific transgenerational programming. In keeping with these results, observations on the second generation offspring of a rat maternal low-protein model have shown increased body weight only in the males, whereas impaired glucose tolerance was again seen in both sexes.158 Although studies of transgenerational developmental programming effects demonstrate transmission via both parental lineages, in many models a combination of transmission routes are clearly possible. It has been shown that sperm methylation variations in the F1 generation are a likely candidate mechanism for transmitting effects via the paternal lineage,138 although the potential mechanism for maternal transmission are more complex due to the wider role of the female in producing the F2 generation offspring (reviewed in Ref. 6).

20.8 CONCLUSIONS Many developmental programming challenges have fundamentally different effects on the later-life health of male and female offspring.37,86,128 Often these effects are similar in both sexes, but of differing magnitudes. However, there are cases in which a developmental challenge causes opposite effects in offspring of each sex.129 Effects that differ in magnitude between the sexes, but in which the overall direction of change is the same should have a test of contrasts applied to determine whether the observed effect is clearly sexually dimorphic. More careful study is required to define clear trends in which exposures consistently lead to clearly sexually dimorphic outcomes, not only in effect-magnitude but also in the direction of the effect. While the precise early developmental differences between males and females are useful to consider in attempting clarifying the mechanisms by which sexually dimorphic developmental programming effects might arise, the broader biological question of why developmental programming effects should be sexually dimorphic at all is also of interest. Developmental programming is often viewed as a negative developmental influence, adversely affecting otherwise normal metabolic development.16 However, programming during early development can also be viewed as an optimization strategy for fetal physiology to give offspring a competitive advantage in harsh environments.1,16 The competitive advantage obtained may differ for male and female offspring, depending on their reproductive roles in later life. To understand why the development of male and female concepti should be influenced differently by the same early life environment, the Trivers-Willard hypothesis has been invoked.37,161 The Trivers-Willard hypothesis suggests that in order to

maximize the chances of perpetuating transmission of their alleles during times of poor resources, mammalian parents should preferentially allocate resources to producing female rather than male offspring.162,163 The rationale for this in mammalian species is that females are the less reproductively variable sex and therefore have a greater overall chance of reproductive success. This reflects the inherently different role of the female as the provider of the protoplasm of the oocyte and the gestational carrier of the next generation, compared to the male as the provider of complementary gametes in competition with other equivalent males. The original framing of the Trivers-Willard hypothesis suggests that the sex ratio in litters produced by mammals in times of nutritional or other environmental hardship should swing in favor of females.161 There is some evidence deriving from calorie-restricted164 and low-fat165 animal models of developmental programming that such a shift can be observed. Although controversial, such effects are also seen in large studies of human populations.10,166,167 However, survival of the conceptus is an extreme outcome measure, and it is plausible that many of the sexually dimorphic phenotypes observed in response to early life environmental manipulations represent less severe examples of the same effect. The observation that the male is the more severely affected sex in many developmental programming studies10 fits well with the basic premise of the Trivers-Willard hypothesis, that fewer resources are allocated to male concepti in times of environmental hardship. A great deal of work remains to be done in fully understanding how an adverse early life environment impacts on the long-term health of both immediate offspring and of further generations.6 Current understanding of how offspring sex interacts with early life environmental influences mainly arises from animal models, but is increasingly supplemented with data from human cohorts. The observed effects remain far from consistent or predictable, although some patterns can be discerned.81 It is clear however from the body of literature amassed that male and female offspring do differ in their developmental response to the same environmental challenge, and that it is likely that these basic differences rest on the impact of interventions during windows of development when growth patterns, growth rates, and gene expression profiles differ between male and female concepti.

References 1. Barker DJ. The origins of the developmental origins theory. J Intern Med. 2007;261(5):412–417. 2. Dearden L, Ozanne SE. Early life origins of metabolic disease: developmental programming of hypothalamic pathways controlling energy homeostasis. Front Neuroendocrinol. 2015;39:3–16. 3. Blackmore HL, Ozanne SE. Programming of cardiovascular disease across the life-course. J Mol Cell Cardiol. 2015;83:122–130.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

4. Veru F, Laplante DP, Luheshi G, et  al. Prenatal maternal stress exposure and immune function in the offspring. Stress. 2014;17(2):133–148. 5. Zambrano E, Guzman C, Rodriguez-Gonzalez GL, et  al. Fetal programming of sexual development and reproductive function. Mol Cell Endocrinol. 2014;382(1):538–549. 6. Aiken CE, Ozanne SE. Transgenerational developmental programming. Hum Reprod Update. 2014;20(1):63–75. 7. Stein AD, Ravelli AC, Famine Lumey LH. Third-trimester pregnancy weight gain, and intrauterine growth: the Dutch Famine Birth Cohort Study. Hum Biol. 1995;67(1):135–150. 8. Fuller SC. The effect of prenatal natural disaster exposure on school outcomes. Demography. 2014;51(4):1501–1525. 9. Cao-Lei L, Dancause KN, Elgbeili G, et  al. DNA methylation mediates the impact of exposure to prenatal maternal stress on BMI and central adiposity in children at age 13(1/2) years: Project Ice Storm. Epigenetics. 2015;10(8):749–761. 10. Torche F, Kleinhaus K. Prenatal stress, gestational age and secondary sex ratio: the sex-specific effects of exposure to a natural disaster in early pregnancy. Hum Reprod. 2012;27(2):558–567. 11. Wang PX, Wang JJ, Lei YX, et al. Impact of fetal and infant exposure to the Chinese Great Famine on the risk of hypertension in adulthood. PLoS One. 2012;7(11):e49720. 12. van Ewijk RJ, Painter RC, Roseboom TJ. Associations of prenatal exposure to Ramadan with small stature and thinness in adulthood: results from a large Indonesian population-based study. Am J Epidemiol. 2013;177(8):729–736. 13. Hohwu L, Li J, Olsen J, et al. Severe maternal stress exposure due to bereavement before, during and after pregnancy and risk of overweight and obesity in young adult men: a Danish National Cohort Study. PLoS One. 2014;9(5):e97490. 14. Hayes L, Bell R, Robson S, et  al. Association between physical activity in obese pregnant women and offspring health. Pregnancy Hypertens. 2014;4(3):234. 15. Guenard F, Deshaies Y, Cianflone K, et al. Differential methylation in glucoregulatory genes of offspring born before vs. after maternal gastrointestinal bypass surgery. Proc Natl Acad Sci USA. 2013;110(28):11439–11444. 16. Hanson MA, Gluckman PD. Early developmental conditioning of later health and disease: physiology or pathophysiology? Physiol Rev. 2014;94(4):1027–1076. 17. Dasinger JH, Alexander BT. Gender differences in developmental programming of cardiovascular diseases. Clin Sci (Lond). 2016;130(5):337–348. 18. Tomat AL, Salazar FJ. Mechanisms involved in developmental programming of hypertension and renal diseases. Gender differences. Horm Mol Biol Clin Investig. 2014;18(2):63–77. 19. Aiken CE, Swoboda PP, Skepper JN, et  al. The direct measurement of embryogenic volume and nucleo-cytoplasmic ratio during mouse pre-implantation development. Reproduction. 2004;128(5):527–535. 20. Sato E, Xian M, Valdivia RP, et al. Sex-linked differences in developmental potential of single blastomeres from in vitro-fertilized 2-cell stage mouse embryos. Horm Res. 1995;44(Suppl 2):4–8. 21. Lowe R, Gemma C, Rakyan VK, et  al. Sexually dimorphic gene expression emerges with embryonic genome activation and is dynamic throughout development. BMC Genomics. 2015;16:295. 22. Kobayashi S, Isotani A, Mise N, et al. Comparison of gene expression in male and female mouse blastocysts revealed imprinting of the X-linked gene, Rhox5/Pem, at preimplantation stages. Curr Biol. 2006;16(2):166–172. 23. Gebert C, Wrenzycki C, Herrmann D, et  al. DNA methylation in the IGF2 intragenic DMR is re-established in a sex-specific manner in bovine blastocysts after somatic cloning. Genomics. 2009;94(1):63–69.

303

24. Dobbs KB, Rodriguez M, Sudano MJ, et  al. Dynamics of DNA methylation during early development of the preimplantation bovine embryo. PLoS One. 2013;8(6):e66230. 25. Bermejo-Alvarez P, Rizos D, Rath D, et al. Epigenetic differences between male and female bovine blastocysts produced in vitro. Physiol Genomics. 2008;32(2):264–272. 26. Trevino LS, Wang Q, Walker CL. Phosphorylation of epigenetic “readers, writers and erasers“: implications for developmental reprogramming and the epigenetic basis for health and disease. Prog Biophys Mol Biol. 2015;118(1–2):8–13. 27. Serrano A, Decara JM, Fernandez-Gonzalez R, et al. Hyperplastic obesity and liver steatosis as long-term consequences of suboptimal in vitro culture of mouse embryos. Biol Reprod. 2014;91(2):30. 28. Fernandez-Gonzalez R, Moreira P, Bilbao A, et  al. Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. Proc Natl Acad Sci USA. 2004;101(16):5880–5885. 29. Sampino S, Zacchini F, Swiergiel AH, et al. Effects of blastomere biopsy on post-natal growth and behavior in mice. Hum Reprod. 2014;29(9):1875–1883. 30. Hansen PJ, Dobbs KB, Denicol AC, et  al. Sex and the preimplantation embryo: implications of sexual dimorphism in the preimplantation period for maternal programming of embryonic development. Cell Tissue Res. 2016;363(1):237–247. 31. Valdivia RP, Kunieda T, Azuma S, et al. PCR sexing and developmental rate differences in preimplantation mouse embryos fertilized and cultured in vitro. Mol Reprod Dev. 1993;35(2):121–126. 32. Hansen PJ, Dobbs KB, Denicol AC, et  al. Sex and the preimplantation embryo: implications of sexual dimorphism in the preimplantation period for maternal programming of embryonic development. Cell Tissue Res. 2015. 33. Gardner DK, Larman MG, Thouas GA. Sex-related physiology of the preimplantation embryo. Mol Hum Reprod. 2010;16(8):539–547. 34. Okamoto I, Heard E. Lessons from comparative analysis of X-chromosome inactivation in mammals. Chromosome Res. 2009;17(5):659–669. 35. van den Berg IM, Laven JS, Stevens M, et  al. X chromosome inactivation is initiated in human preimplantation embryos. Am J Hum Genet. 2009;84(6):771–779. 36. Epstein CJ, Smith S, Travis B, et  al. Both X chromosomes function before visible X-chromosome inactivation in female mouse embryos. Nature. 1978;274(5670):500–503. 37. Aiken CE, Ozanne SE. Sex differences in developmental programming models. Reproduction. 2013;145(1):R1–13. 38. Mittwoch U. The elusive action of sex-determining genes: mitochondria to the rescue? J Theor Biol. 2004;228(3):359–365. 39. Aiken CE, Cindrova-Davies T, Johnson MH. Variations in mouse mitochondrial DNA copy number from fertilization to birth are associated with oxidative stress. Reprod Biomed Online. 2008;17(6):806–813. 40. Aiken CE, Tarry-Adkins JL, Penfold NC, et al. Decreased ovarian reserve, dysregulation of mitochondrial biogenesis, and increased lipid peroxidation in female mouse offspring exposed to an obesogenic maternal diet. FASEB J. 2015 41. Fowler PA, Filis P, Bhattacharya S, et  al. Human anogenital distance: an update on fetal smoke-exposure and integration of the perinatal literature on sex differences. Hum Reprod. 2016;31(2):463–472. 42. Gray LE, Ostby J, Furr J, et al. Effects of environmental antiandrogens on reproductive development in experimental animals. Hum Reprod Update. 2001;7(3):248–264. 43. Beverly BE, Lambright CS, Furr JR, et al. Simvastatin and dipentyl phthalate lower ex vivo testicular testosterone production and exhibit additive effects on testicular testosterone and gene expression via distinct mechanistic pathways in the fetal rat. Toxicol Sci. 2014;141(2):524–537.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

304

20.  INTRAUTERINE PROGRAMMING OF SEXUAL DIMORPHISM

44. Furr JR, Lambright CS, Wilson VS, et  al. A short-term in vivo screen using fetal testosterone production, a key event in the phthalate adverse outcome pathway, to predict disruption of sexual differentiation. Toxicol Sci. 2014;140(2):403–424. 45. Zhao B, Li L, Liu J, et al. Exposure to perfluorooctane sulfonate in utero reduces testosterone production in rat fetal Leydig cells. PLoS One. 2014;9(1):e78888. 46. Steckler T, Wang J, Bartol FF, et al. Fetal programming: prenatal testosterone treatment causes intrauterine growth retardation, reduces ovarian reserve and increases ovarian follicular recruitment. Endocrinology. 2005;146(7):3185–3193. 47. Rae M, Grace C, Hogg K, et  al. The pancreas is altered by in utero androgen exposure: implications for clinical conditions such as polycystic ovary syndrome (PCOS). PLoS One. 2013;8(2): e56263. 48. Connolly F, Rae MT, Spath K, et al. In an Ovine Model of Polycystic Ovary Syndrome (PCOS) Prenatal Androgens Suppress Female Fetal Renal Gluconeogenesis. PLoS One. 2015;10(7):e0132113. 49. Hogg K, Wood C, McNeilly AS, et al. The in utero programming effect of increased maternal androgens and a direct fetal intervention on liver and metabolic function in adult sheep. PLoS One. 2011;6(9):e24877. 50. Connolly F, Rae MT, Bittner L, et al. Excess androgens in utero alters fetal testis development. Endocrinology. 2013;154(5):1921–1933. 51. Jahagirdar V, Quadros PS, Wagner CK. Endogenous oestradiol regulates progesterone receptor expression in the brain of female rat fetuses: what is the source of oestradiol? J Neuroendocrinol. 2008;20(3):359–365. 52. Pei M, Matsuda K, Sakamoto H, et al. Intrauterine proximity to male fetuses affects the morphology of the sexually dimorphic nucleus of the preoptic area in the adult rat brain. Eur J Neurosci. 2006;23(5):1234–1240. 53. Bermejo-Alvarez P, Rizos D, Lonergan P, et  al. Transcriptional sexual dimorphism during preimplantation embryo development and its consequences for developmental competence and adult health and disease. Reproduction. 2011;141(5):563–570. 54. Sokal R, Tata LJ, Fleming KM. Sex prevalence of major congenital anomalies in the United Kingdom: a national population-based study and international comparison meta-analysis. Birth Defects Res A Clin Mol Teratol. 2014;100(2):79–91. 55. Catalano R, Bruckner T, Anderson E, et  al. Fetal death sex ratios: a test of the economic stress hypothesis. Int J Epidemiol. 2005;34(4):944–948. 56. Brown CJ, Greally JM. A stain upon the silence: genes escaping X inactivation. Trends Genet. 2003;19(8):432–438. 57. Wells JC. Natural selection and sex differences in morbidity and mortality in early life. J Theor Biol. 2000;202(1):65–76. 58. Gabory A, Attig L, Junien C. Sexual dimorphism in environmental epigenetic programming. Mol Cell Endocrinol. 2009;304(1–2):8–18. 59. Eogan MA, Geary MP, O'Connell MP, et al. Effect of fetal sex on labour and delivery: retrospective review. BMJ. 2003;326(7381):137. 60. Sheiner E, Levy A, Katz M, et al. Gender does matter in perinatal medicine. Fetal Diagn Ther. 2004;19(4):366–369. 61. Gortner L, Shen J, Tutdibi E. Sexual dimorphism of neonatal lung development. Klin Padiatr. 2013;225(2):64–69. 62. Rosenfeld CS. Sex-specific placental responses in fetal development. Endocrinology. 2015;156(10):3422–3434. 63. Georgiades P, Ferguson-Smith AC, Burton GJ. Comparative developmental anatomy of the murine and human definitive placentae. Placenta. 2002;23(1):3–19. 64. Clifton VL. Review: sex and the human placenta: mediating differential strategies of fetal growth and survival. Placenta. 2010;31(Suppl):S33–S39. 65. Sood R, Zehnder JL, Druzin ML, et al. Gene expression patterns in human placenta. Proc Natl Acad Sci USA. 2006;103(14): 5478–5483.

66. Cvitic S, Longtine MS, Hackl H, et  al. The human placental sexome differs between trophoblast epithelium and villous vessel endothelium. PLoS One. 2013;8(10):e79233. 67. Wang Y, Pringle KG, Sykes SD, et al. Fetal sex affects expression of renin-angiotensin system components in term human decidua. Endocrinology. 2012;153(1):462–468. 68. O'Connell BA, Moritz KM, Walker DW, et al. Sexually dimorphic placental development throughout gestation in the spiny mouse (Acomys cahirinus). Placenta. 2013;34(2):119–126. 69. Gabory A, Roseboom TJ, Moore T, et  al. Placental contribution to the origins of sexual dimorphism in health and diseases: sex chromosomes and epigenetics. Biol Sex Differ. 2013;4(1):5. 70. Mao J, Zhang X, Sieli PT, et  al. Contrasting effects of different maternal diets on sexually dimorphic gene expression in the murine placenta. Proc Natl Acad Sci USA. 2010;107(12):5557–5562. 71. Reynolds CM, Vickers MH, Harrison CJ, et al. Maternal high fat and/or salt consumption induces sex-specific inflammatory and nutrient transport in the rat placenta. Physiol Rep. 2015;3:5. 72. Mando C, Calabrese S, Mazzocco MI, et al. Sex specific adaptations in placental biometry of overweight and obese women. Placenta. 2016;38:1–7. 73. Muralimanoharan S, Guo C, Myatt L, et al. Sexual dimorphism in miR-210 expression and mitochondrial dysfunction in the placenta with maternal obesity. Int J Obes (Lond). 2015;39(8):1274–1281. 74. Binder NK, Beard SA, Kaitu'u-Lino TJ, et al. Paternal obesity in a rodent model affects placental gene expression in a sex-specific manner. Reproduction. 2015;149(5):435–444. 75. Gabory A, Ferry L, Fajardy I, et  al. Maternal diets trigger sexspecific divergent trajectories of gene expression and epigenetic systems in mouse placenta. PLoS One. 2012;7(11):e47986. 76. Fleming TP, Velazquez MA, Eckert JJ. Embryos, DOHaD and David Barker. J Dev Orig Health Dis. 2015;6(5):377–383. 77. Eckert JJ, Porter R, Watkins AJ, et  al. Metabolic induction and early responses of mouse blastocyst developmental programming following maternal low protein diet affecting life-long health. PLoS One. 2012;7(12):e52791. 78. Steier JA, Bergsjo PB, Thorsen T, et al. Human chorionic gonadotropin in maternal serum in relation to fetal gender and utero-placental blood flow. Acta Obstet Gynecol Scand. 2004;83(2):170–174. 79. Jaskolka D, Retnakaran R, Zinman B, et al. Sex of the baby and risk of gestational diabetes mellitus in the mother: a systematic review and meta-analysis. Diabetologia. 2015;58(11):2469–2475. 80. Di Renzo GC, Rosati A, Sarti RD, et al. Does fetal sex affect pregnancy outcome? Gend Med. 2007;4(1):19–30. 81. Aiken CE, Ozanne SE. Sex differences in developmental programming models. Reproduction. 2013;145(1):R1–R13. 82. Chin EH, Christians JK. When are sex-specific effects really sexspecific? J Dev Orig Health Dis. 2015;6(5):438–442. 83. Kilkenny C, Browne WJ, Cuthill IC, et al. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. J Pharmacol Pharmacother. 2010;1(2):94–99. 84. Cao X, Laplante DP, Brunet A, et  al. Prenatal maternal stress affects motor function in 5(1/2)-year-old children: project ice storm. Dev Psychobiol. 2014;56(1):117–125. 85. Wainstock T, Shoham-Vardi I, Glasser S, et al. Fetal sex modifies effects of prenatal stress exposure and adverse birth outcomes. Stress. 2015;18(1):49–56. 86. Barrett ES, Swan SH. Stress and androgen activity during fetal development. Endocrinology. 2015;156(10):3435–3441. 87. Barrett ES, Parlett LE, Sathyanarayana S, et  al. Prenatal exposure to stressful life events is associated with masculinized anogenital distance (AGD) in female infants. Physiol Behav. 2013;114-115:14–20. 88. Clifton VL. Sexually dimorphic effects of maternal asthma during pregnancy on placental glucocorticoid metabolism and fetal growth. Cell Tissue Res. 2005;322(1):63–71.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

89. Turcotte-Tremblay AM, Lim R, Laplante DP, et  al. Prenatal maternal stress predicts childhood asthma in girls: project ice storm. Biomed Res Int. 2014;2014:201717. 90. Li J, Olsen J, Vestergaard M, et  al. Attention-deficit/hyperactivity disorder in the offspring following prenatal maternal bereavement: a nationwide follow-up study in Denmark. Eur Child Adolesc Psychiatry. 2010;19(10):747–753. 91. O'Connor TG, Heron J, Golding J, et  al. Maternal antenatal anxiety and children's behavioural/emotional problems at 4 years. Report from the Avon Longitudinal Study of Parents and Children. Br J Psychiatry. 2002;180:502–508. 92. Barrett ES, Redmon JB, Wang C, et al. Exposure to prenatal life events stress is associated with masculinized play behavior in girls. Neurotoxicology. 2014;41:20–27. 93. Van den Bergh BR, Van Calster B, Smits T, et al. Antenatal maternal anxiety is related to HPA-axis dysregulation and self-reported depressive symptoms in adolescence: a prospective study on the fetal origins of depressed mood. Neuropsychopharmacology. 2008;33(3):536–545. 94. Watson JB, Mednick SA, Huttunen M, et al. Prenatal teratogens and the development of adult mental illness. Dev Psychopathol. 1999;11(3):457–466. 95. Dunn GA, Morgan CP, Bale TL. Sex-specificity in transgenerational epigenetic programming. Horm Behav. 2011;59(3):290–295. 96. Barker DJ, Osmond C. Low birth weight and hypertension. BMJ. 1988;297(6641):134–135. 97. Zimmermann E, Gamborg M, Sorensen TI, et al. Sex differences in the association between birth weight and adult type 2 diabetes, Diabetes. 2015;64(12):4220–4225. 98. Richardson LJ, Hussey JM, Strutz KL. Origins of disparities in cardiovascular disease: birth weight, body mass index, and young adult systolic blood pressure in the national longitudinal study of adolescent health. Ann Epidemiol. 2011;21(8):598–607. 99. Lawlor DA, Ebrahim S, Davey Smith G. Is there a sex difference in the association between birth weight and systolic blood pressure in later life? Findings from a meta-regression analysis. Am J Epidemiol. 2002;156(12):1100–1104. 100. Tobi EW, Lumey LH, Talens RP, et  al. DNA methylation differences after exposure to prenatal famine are common and timing- and sex-specific. Hum Mol Genet. 2009;18(21):4046–4053. 101. Matthews SG, Phillips DI. Transgenerational inheritance of stress pathology. Exp Neurol. 2012;233(1):95–101. 102. Severino GS, Fossati IA, Padoin MJ, et  al. Effects of neonatal handling on the behavior and prolactin stress response in male and female rats at various ages and estrous cycle phases of females. Physiol Behav. 2004;81(3):489–498. 103. Garcia-Caceres C, Lagunas N, Calmarza-Font I, et  al. Gender differences in the long-term effects of chronic prenatal stress on the HPA axis and hypothalamic structure in rats. Psychoneuroendocrinology. 2010;35(10):1525–1535. 104. Mueller BR, Bale TL. Sex-specific programming of offspring emotionality after stress early in pregnancy. J Neurosci. 2008;28(36):9055–9065. 105. Li M, Reynolds CM, Sloboda DM, et  al. Maternal taurine supplementation attenuates maternal fructose-induced metabolic and inflammatory dysregulation and partially reverses adverse metabolic programming in offspring. J Nutr Biochem. 2015;26(3):267–276. 106. Kim DW, Young SL, Grattan DR, et  al. Obesity during pregnancy disrupts placental morphology, cell proliferation, and inflammation in a sex-specific manner across gestation in the mouse. Biol Reprod. 2014;90(6):130. 107. Dahlhoff M, Pfister S, Blutke A, et al. Peri-conceptional obesogenic exposure induces sex-specific programming of disease susceptibilities in adult mouse offspring. Biochim Biophys Acta. 2014;1842(2):304–317.

305

108. Guo C, Li C, Myatt L, et al. Sexually dimorphic effects of maternal nutrient reduction on expression of genes regulating cortisol metabolism in fetal baboon adipose and liver tissues. Diabetes. 2013;62(4):1175–1185. 109. Adam CL, Bake T, Findlay PA, et al. Impact of birth weight and gender on early postnatal hypothalamic energy balance regulatory gene expression in the young lamb. Int J Dev Neurosci. 2013;31(7):608–615. 110. Duffield JA, Vuocolo T, Tellam R, et al. Intrauterine growth restriction and the sex specific programming of leptin and peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA expression in visceral fat in the lamb. Pediatr Res. 2009;66(1):59–65. 111. Woods LL, Ingelfinger JR, Nyengaard JR, et  al. Maternal protein restriction suppresses the newborn renin-angiotensin system and programs adult hypertension in rats. Pediatr Res. 2001;49(4):460–467. 112. Woods LL, Ingelfinger JR, Rasch R. Modest maternal protein restriction fails to program adult hypertension in female rats. Am J Physiol Regul Integr Comp Physiol. 2005;289(4):R1131–R1136. 113. Loria AS, Yamamoto T, Pollock DM, et  al. Early life stress induces renal dysfunction in adult male rats but not female rats. Am J Physiol Regul Integr Comp Physiol. 2013;304(2):R121–R129. 114. Behan AT, van den Hove DL, Mueller L, et al. Evidence of femalespecific glial deficits in the hippocampus in a mouse model of prenatal stress. Eur Neuropsychopharmacol. 2011;21(1):71–79. 115. Biala YN, Bogoch Y, Bejar C, et  al. Prenatal stress diminishes gender differences in behavior and in expression of hippocampal synaptic genes and proteins in rats. Hippocampus. 2011;21(10):1114–1125. 116. Goyal R, Wong C, Van Wickle J, et al. Antenatal maternal protein deprivation: sexually dimorphic programming of the pancreatic renin-angiotensin system. J Renin Angiotensin Aldosterone Syst. 2013;14(2):137–145. 117. Moritz KM, Cuffe JS, Wilson LB, et al. Review: sex specific programming: a critical role for the renal renin-angiotensin system. Placenta. 2010;31(Suppl)):S40–S46. 118. Aiken CE, Tarry-Adkins JL, Ozanne SE. Suboptimal nutrition in utero causes DNA damage and accelerated aging of the female reproductive tract. FASEB J. 2013;27(10):3959–3965. 119. Katkhuda R, Peterson ES, Roghair RD, et  al. Sex-specific programming of hypertension in offspring of late-gestation diabetic rats. Pediatr Res. 2012;72(4):352–361. 120. Ojeda NB, Hennington BS, Williamson DT, et  al. Oxidative stress contributes to sex differences in blood pressure in adult growth-restricted offspring. Hypertension. 2012;60(1):114–122. 121. Allison BJ, Kaandorp JJ, Kane AD, et al. Divergence of mechanistic pathways mediating cardiovascular aging and developmental programming of cardiovascular disease. FASEB J. 2016 122. Aiken CE, Tarry-Adkins JL, Ozanne SE. Transgenerational developmental programming of ovarian reserve. Sci Rep. 2015;5:16175. 123. Intapad S, Tull FL, Brown AD, et al. Renal denervation abolishes the age-dependent increase in blood pressure in female intrauterine growth-restricted rats at 12 months of age. Hypertension. 2013;61(4):828–834. 124. Alexander BT. Placental insufficiency leads to development of hypertension in growth-restricted offspring. Hypertension. 2003;41(3):457–462. 125. Tao H, Rui C, Zheng J, et al. Angiotensin II-mediated vascular changes in aged offspring rats exposed to perinatal nicotine. Peptides. 2013;44:111–119. 126. Darnaudery M, Maccari S. Epigenetic programming of the stress response in male and female rats by prenatal restraint stress. Brain Res Rev. 2008;57(2):571–585. 127. Schulz KM, Pearson JN, Neeley EW, et al. Maternal stress during pregnancy causes sex-specific alterations in offspring memory

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

306

128.

129.

130.

131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

144.

20.  INTRAUTERINE PROGRAMMING OF SEXUAL DIMORPHISM

performance, social interactions, indices of anxiety, and body mass. Physiol Behav. 2011;104(2):340–347. Glover V, Hill J. Sex differences in the programming effects of prenatal stress on psychopathology and stress responses: an evolutionary perspective. Physiol Behav. 2012;106(5):736–740. Kreider ML, Levin ED, Seidler FJ, et al. Gestational dexamethasone treatment elicits sex-dependent alterations in locomotor activity, reward-based memory and hippocampal cholinergic function in adolescent and adult rats. Neuropsychopharmacology. 2005;30(9):1617–1623. Garcia AP, Palou M, Priego T, et  al. Moderate caloric restriction during gestation results in lower arcuate nucleus NPYand alphaMSH-neurons and impairs hypothalamic response to fed/fasting conditions in weaned rats. Diabetes Obes Metab. 2010;12(5):403–413. Drake AJ, van den Driesche S, Scott HM, et al. Glucocorticoids amplify dibutyl phthalate-induced disruption of testosterone production and male reproductive development. Endocrinology. 2009;150(11):5055–5064. Macleod DJ, Sharpe RM, Welsh M, et  al. Androgen action in the masculinization programming window and development of male reproductive organs. Int J Androl. 2010;33(2):279–287. Scott HM, Mason JI, Sharpe RM. Steroidogenesis in the fetal testis and its susceptibility to disruption by exogenous compounds. Endocr Rev. 2009;30(7):883–925. Pallares ME, Adrover E, Baier CJ, et  al. Prenatal maternal restraint stress exposure alters the reproductive hormone profile and testis development of the rat male offspring. Stress. 2013;16(4):429–440. Kapoor A, Matthews SG. Testosterone is involved in mediating the effects of prenatal stress in male guinea pig offspring. J Physiol. 2011;589(Pt 3):755–766. Engelbregt MJ, Houdijk ME, Popp-Snijders C, et al. The effects of intra-uterine growth retardation and postnatal undernutrition on onset of puberty in male and female rats. Pediatr Res. 2000;48(6):803–807. McPherson NO, Fullston T, Aitken RJ, et  al. Paternal obesity, interventions, and mechanistic pathways to impaired health in offspring. Ann Nutr Metab. 2014;64(3–4):231–238. Radford EJ, Ito M, Shi H, et al. In utero effects. In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism. Science. 2014;345(6198):1255903. Meunier L, Siddeek B, Vega A, et  al. Perinatal programming of adult rat germ cell death after exposure to xenoestrogens: role of microRNA miR-29 family in the down-regulation of DNA methyltransferases and Mcl-1. Endocrinology. 2012;153(4):1936–1947. Rodgers AB, Morgan CP, Leu NA, et  al. Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress. Proc Natl Acad Sci USA. 2015;112(44):13699–13704. Rodriguez-Gonzalez GL, Reyes-Castro LA, Vega CC, et  al. Accelerated aging of reproductive capacity in male rat offspring of protein-restricted mothers is associated with increased testicular and sperm oxidative stress. Age (Dordr). 2014;36(6):9721. Vickers MH, Clayton ZE, Yap C, et al. Maternal fructose intake during pregnancy and lactation alters placental growth and leads to sex-specific changes in fetal and neonatal endocrine function. Endocrinology. 2011;152(4):1378–1387. Guzman C, Garcia-Becerra R, Aguilar-Medina MA, et  al. Maternal protein restriction during pregnancy and/or lactation negatively affects follicular ovarian development and steroidogenesis in the prepubertal rat offspring. Arch Med Res. 2014;45(4):294–300. Bernal AB, Vickers MH, Hampton MB, et  al. Maternal undernutrition significantly impacts ovarian follicle number and

145.

146.

147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

157.

158.

159.

160.

161. 162.

increases ovarian oxidative stress in adult rat offspring. PLoS One. 2010;5(12):e15558. Sloboda DM, Howie GJ, Pleasants A, et  al. Pre- and postnatal nutritional histories influence reproductive maturation and ovarian function in the rat. PLoS One. 2009;4(8):e6744. Chernoff N, Gage MI, Stoker TE, et  al. Reproductive effects of maternal and pre-weaning undernutrition in rat offspring: age at puberty, onset of female reproductive senescence and intergenerational pup growth and viability. Reprod Toxicol. 2009;28(4):489–494. Leese HJ, Hugentobler SA, Gray SM, et al. Female reproductive tract fluids: composition, mechanism of formation and potential role in the developmental origins of health and disease. Reprod Fertil Dev. 2008;20(1):1–8. Martin JR, Lieber SB, McGrath J, et  al. Maternal ghrelin defic� ciency compromises reproduction in female progeny through altered uterine developmental programming. Endocrinology. 2011;152(5):2060–2066. Torrens C, Poston L, Hanson MA. Transmission of raised blood pressure and endothelial dysfunction to the F2 generation induced by maternal protein restriction in the F0, in the absence of dietary challenge in the F1 generation. Br J Nutr. 2008;100(4):760–766. Hemmings DG, Veerareddy S, Baker PN, et al. Increased myogenic responses in uterine but not mesenteric arteries from pregnant offspring of diet-restricted rat dams. Biol Reprod. 2005;72(4):997–1003. Gapp K, Jawaid A, Sarkies P, et al. Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice. Nat Neurosci. 2014;17(5):667–669. Zambrano E, Martinez-Samayoa PM, Bautista CJ, et  al. Sex differences in transgenerational alterations of growth and metabolism in progeny (F2) of female offspring (F1) of rats fed a low protein diet during pregnancy and lactation. J Physiol. 2005;566(Pt 1):225–236. Reyes-Castro LA, Rodriguez-Gonzalez GL, Chavira R, et  al. Paternal line multigenerational passage of altered risk assessment behavior in female but not male rat offspring of mothers fed a low protein diet. Physiol Behav. 2015;140:89–95. Pembrey ME, Bygren LO, Kaati G, et  al. Sex-specific, maleline transgenerational responses in humans. Eur J Hum Genet. 2006;14(2):159–166. Kaati G, Bygren LO, Pembrey M, et  al. Transgenerational response to nutrition, early life circumstances and longevity. Eur J Hum Genet. 2007;15(7):784–790. Roseboom TJ, van der Meulen JH, Ravelli AC, et al. Blood pressure in adults after prenatal exposure to famine. J Hypertens. 1999;17(3):325–330. Veenendaal MV, Painter RC, de Rooij SR, et al. Transgenerational effects of prenatal exposure to the 1944-45 Dutch famine. BJOG. 2013;120(5):548–553. Pinheiro AR, Salvucci ID, Aguila MB, et  al. Protein restriction during gestation and/or lactation causes adverse transgenerational effects on biometry and glucose metabolism in F1 and F2 progenies of rats. Clin Sci (Lond). 2008;114(5):381–392. Benyshek DC, Johnston CS, Martin JF. Glucose metabolism is altered in the adequately-nourished grand-offspring (F3 generation) of rats malnourished during gestation and perinatal life. Diabetologia. 2006;49(5):1117–1119. Jimenez-Chillaron JC, Isganaitis E, Charalambous M, et  al. Intergenerational transmission of glucose intolerance and obesity by in utero undernutrition in mice. Diabetes. 2009;58(2):460–468. Trivers RL, Willard DE. Natural selection of parental ability to vary the sex ratio of offspring. Science. 1973;179(68):90–92. Mathews F, Johnson PJ, Neil A. You are what your mother eats: evidence for maternal preconception diet influencing foetal sex in humans. Proc Biol Sci. 2008;275(1643):1661–1668.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

163. Rickard IJ, Russell AF, Lummaa V. Producing sons reduces lifetime reproductive success of subsequent offspring in preindustrial Finns. Proc Biol Sci. 2007;274(1628):2981–2988. 164. Meikle DB, Drickamer LC. Food availability and secondary sex ratio variation in wild and laboratory house mice (Mus musculus). J Reprod Fertil. 1986;78(2):587–591. 165. Rosenfeld CS, Grimm KM, Livingston KA, et al. Striking variation in the sex ratio of pups born to mice according to whether maternal diet is high in fat or carbohydrate. Proc Natl Acad Sci USA. 2003;100(8):4628–4632.

307

166. Almond D, Edlund L. Trivers-Willard at birth and one year: evidence from US natality data 1983-2001. Proc Biol Sci. 2007;274(1624):2491–2496. 167. Bruckner T, Catalano R. The sex ratio and age-specific male mortality: evidence for culling in utero. Am J Hum Biol. 2007;19(6):763–773.

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C H A P T E R

21 Immune Response—The Impact of Biological Sex and Gender Sabine Oertelt-Prigione Charité – Universitätsmedizin, Berlin, Germany

O U T L I N E 21.1 Introduction

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21.2 Sex and Genetics in the Immune Response

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21.3 Sex and Hormones in the Immune Response 311 21.3.1 Hormonal Effects on the Immune Cells 312 21.3.2 Hormonal Stages and the Immune Response 313 21.4 Gender in the Immune Response

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21.4.1 Exposure to Viral and Microbial Agents 21.4.2 Exposure to Nonmicrobiological Agents 21.4.3 Food Intake and Food Variety 21.5 Health Care Access

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References 318

21.1 INTRODUCTION The immune responses of men and women display many striking differences that have been neglected for decades. Different cell numbers and cellular activation patterns,1 distinct responses to viral and bacterial infection,2 different susceptibility to autoimmune diseases,3 and different inflammatory profiles in many common diseases, e.g., cardiovascular, metabolic, and oncologic, have been described. Basic biomedical research commonly focuses on biological aspects to explain these differences, with a special interest in genetics and hormonal differences. The role of gender, i.e., the impact of sociocultural and environmental factors on the development of the immune system, is frequently neglected. Since both biological sex and gender are essential to the development of an immune response and influence each other, both will be described in this chapter. The extent of research conducted in these fields and the methodology employed varies considerably, highlighting not only the different investigative approaches but also the challenges in synthesizing results in a truly interdisciplinary fashion. Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00023-1

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21.2  SEX AND GENETICS IN THE IMMUNE RESPONSE When analyzing the impact and involvement of gender and biological sex in any disease the approaches can include an analysis of the role of the autosomes or the involvement of the sex chromosomes (Table 21.1). Although there is a known interaction between the sex chromosomes and autosomes,4 their influence is generally analyzed separately and their interaction is dissected to a limited degree. The exploration of the contribution of genetics to sex differences in the field of immunology focuses primarily on the contribution of the sex chromosomes (principally the X chromosome, and to a lesser degree the Y chromosome). While autosomal differences appear to play a role, the investigation of their contribution is limited. The X chromosome harbors about 1000 genes, many of which have a function related to the immune response.5 This is demonstrated not only by the existence of X-linked diseases, which are phenotypically expressed almost exclusively in males, but also by the association of many of

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TABLE 21.1  Sex and Genetics in the Immune Response (Putative) Mechanism

Effects

X-linked diseases

●

Affect almost exclusively males Phenotypic expression due to mutation on X chromosome

●

X inactivation escape

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One of the two X chromosomes in females in not fully silenced

X monosomy

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Skewed X inactivation

●

Y chromosome

●

Sex differences in epigenetics

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Complete loss of one of the two X chromosomes in females ● Possible pathogenetic mechanism due to loss of contribution from other X chromosome (see X inactivation escape) Preferential inactivation of one of the two parental chromosomes

Contribution of haplotypes to disease susceptibility ● Disease expression due to peripheral loss of Y chromosome Different methylation patterns of susceptibility genes in females and males

these genes with general dysfunctions of the immune response. Examples of X-linked immunodeficiencies include the IPEX syndrome,6 a disease characterized by immunodysregulation, polyendocrinopathy, and enteropathy, which is caused by a dysfunction of the FoxP3 gene, a prominent regulator of immune tolerance; the X-linked hyper-IgM syndrome,7 linked to a defect of the CD40L gene leads to an increase in the production of all immunoglobulin classes and increased susceptibility for opportunistic infections; the Wiskott-Aldrich syndrome,8 linked to a mutation in the WAS gene, that presents with eczema, thrombocytopenia, autoimmune features, and an increased incidence of malignancies; X-linked thrombocytopenia and myelodysplasia,9 which expresses as a male-specific clotting defect and is also related to the WAS gene and severe combined immunodeficiency; or SCIDX1, a disease that affects the common interleukin chain, CD132, and leads to dysfunction of multiple interleukin receptors and to defects in the development of T lymphocytes and NK cells.10 A unique example of X-linked deficiencies is represented by the DDX3 (DEAD-box helicase 3) gene which is encoded by homologs on both the X and the Y chromosome. Interestingly, only the gene on the X chromosome is translated and has been critically linked to type-I IFN production.11 All of these diseases manifest most commonly in men who have only one copy of the X chromosome and to a lesser or absent degree in women.

Next we will discuss the direct association of X chromosomal genes with the immune function; their transcriptional profiles should also be considered. Carrel and colleagues12 have demonstrated the phenomenon of X inactivation escape in a landmark paper. Contrary to the widespread belief that there is always complete inactivation of one of the two copies of the X chromosome in female somatic cells, the authors described the escape of several genes on the supposedly inactivated X chromosome which were actively transcribed. The escape mechanism appears to display tissue-specific variability with some cells displaying higher rates of inactivation escape than others, e.g., fibroblasts12 compared to lymphoblastoid cell lines.13 In addition to X chromosomal inactivation escape, there may be a complete loss of one of the X chromosomes—X monosomy. Loss of an X chromosome has been associated with premature ovarian failure14 and more importantly with the Turner syndrome.15 Both of these conditions are known to be associated with autoimmune diseases and immunologic dysfunctions, ranging from autoimmune thyroiditis16 to systemic sclerosis and primary biliary cirrhosis.17 The phenomenon could not be confirmed in cells from patients with systemic lupus erythematosus.18 The underlying pathogenetic mechanism has not been clarified; however, it is assumed that loss of one X either leads to a reduced X chromosome transcription—given the contribution of the second X in women—or that preferential loss of one of the two chromosomes could generate an unfavorable mosaic which leads to overexpression of a potentially harmful X chromosome copy. In fact, a possible skewing of X inactivation has been reported with increasing age.19 The presence of two X chromosomes might be generally relevant for health and longevity, as other animal species with an inverse distribution of the sex chromosomes display a consequent survival pattern. Specifically, in birds, the male has two copies of one sexual chromosome (Z as an analogous to the human X) and the female has discordant sex chromosomes (Z and W, analogous to the X and Y chromosomes, respectively, in human); interestingly the mortality patterns in birds are inverted compared to humans, with males living longer than females.20 The availability of two analogous chromosomes allows for a potentially skewed inactivation, which could be potentially protective if the second X chromosome does not harbor any disease-related mutations. However, skewed X inactivation has also been associated with several autoimmune diseases, among others autoimmune thyroid disease,21 systemic sclerosis,22 juvenile idiopathic arthritis,23 and Sjögren’s syndrome,24 possibly due to the uncovering of susceptibility genes due to inactivation. The role of the Y chromosome in immunology has had less attention especially when it comes to research on human cells and samples. The X and Y chromosomes

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originated from a common precursor but over time evolved into two distinct sets of chromosomes.25 The Y chromosome harbors about 78 genes, which are believed mostly to be involved in governance and maintenance of male sex.26 A possible involvement of Y chromosomal genes in immune function has been postulated; however, this was subsequently linked to the presence of translocated genes from the X chromosome rather than due to independent genes on the Y chromosome.27 Nonetheless, recently, a role for the Y chromosome as a potential influencer of methylation in both animal models and humans has been proposed28 as well as the ability of genes on its long arm to influence the immune response, specifically the number of B cells and NK cells.29 Further research into this exciting novel area is needed. A recent study from the United Kingdom has demonstrated how distinct haplotypes of the Y chromosome are associated with different susceptibility to cardiovascular disease. The difference was unrelated to traditional cardiovascular risk factors but associated with different expression of immune-related genes in macrophages.30 Analogous to the phenomenon of X monosomy, the loss of the Y chromosome in peripheral cells might be a mechanism associated with autoimmunity as recently demonstrated in primary biliary cirrhosis, Hashimoto’s Thyroiditis, and Grave’s Disease.31,32 The modulatory role of epigenetics has gained much attention in the last decade. Sex specificity in methylation appears likely among other factors due to the presence of estrogen responsive elements in the promotor region of the methyltransferases. Tobi et  al. have described sex-specific methylation patterns of several metabolically relevant genes,33 a finding confirmed by larger studies on sex differences in X chromosomal and autosomal methylation patterns and diabetes.34 These methylation patterns might be acquired as early as in utero and could represent a connection between fetal exposure to stress, malnutrition, environmental triggers, etc. and adult susceptibility to disease.35 This is especially relevant in the case of gender-sensitive medicine, since most environmental triggers are related to gender as described in the latter sections of this chapter. The association between epigenetics and potential sex differences in the brain is also commanding much attention,36 highlighting the potential importance of this research approach for many different specialties.

21.3  SEX AND HORMONES IN THE IMMUNE RESPONSE

the human organism both with regard to sexual function and to many other bodily systems.37 Estrogens have been investigated and described in the most detail. They interact with two distinct types of receptors, estrogen receptor alpha (ERa) and estrogen receptor beta (ERb), and exert their function through binding of hormone/ receptor complexes to estrogen response elements (EREs) in promotor or enhancer regions of genes. This ligation can than enhance or reduce translation. Indirect responses have also been described by means of interaction of the complex with other unrelated transcription factors.38 Progesterone also links to two distinct types of receptor, progesterone receptor A (PRA) and progesterone receptor B (PRB), with PRA representing a shorter version of the PRB receptor.39 The functions of the receptor are elicited through direct binding or indirect cascades. The androgen receptor, notably encoded by a gene on the X chromosome, is engaged by testosterone and 5alpha-dihydrotestosterone and binds to a response element (ARE—androgen response element) in the promotor region.40 No indirect action has been described for androgens this far. Historically, estrogens have been described as enhancers of the immune response, while progesterone and androgen reportedly decreased it. However, this paradigm has frequently been questioned through the identification of biphasic effects as well as complex and multilayered effects of the steroid hormones. In the following section, the available knowledge about hormonal effects on immune cells will be summarized (Table 21.2) as well as the impact of different hormonal stages on the development of an immune response.

TABLE 21.2  Sex and Hormones in the Immune Response Immune cell

Estrogen

T lymphocytes

●

Macrophages

●

IFN-gamma/ IL-2 +/● IL-4 +/● IL-10 o

Progesterone IL-4 +

●

TNF +/ IL-1 +/● IL-6 –

TNF o

●

●

B lymphocytes

●

Ig M/ Ig G production + ● Ig class shift +

Granulocytes

●

NK(T)

●

Ig M/ Ig G production-

●

Mobility +/-

●

Mobility +/-

Maturation +

●

Apoptosis + IFN gamma -

●

The role of steroid hormones, namely estrogen, progesterone, and testosterone, on the immune response has been described to various degrees in the past. Steroid hormones are known to display diversified effects on

Testosterone

Dendritic cells

IL-6 + IL-8 + ● IFN-alpha + ● ●

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Mobility o

●

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21.3.1  Hormonal Effects on the Immune Cells 21.3.1.1 T Lymphocytes T lymphocytes are central elements in cell-mediated immunity. They develop in the thymus and then migrate to the periphery to aid in the recruitment of other immune cells, to develop cellular memory of previous infection and to directly destroy certain types of cells, like virus-infected cells or tumor cells. They are classified into several subsets ranging from T helper cells to memory T cells, cytotoxic T cells, and suppressor cells. Regulatory T cells constitute a specific subset of T cells, which exert immunomodulatory functions and are of particular relevance to the maintenance of immunological tolerance and the development of autoimmunity. Most investigation of the role of T lymphocytes has focused on the relationship between the hormonal stimulation of the cells and their responses in terms of cytokine production. The limitations of many of these studies lie in the fact that different cell populations, different experimental protocols, and different study subjects have been analyzed making adequate comparisons difficult. Conflicting reports about the effects of estrogens on Th1 cytokines, such as IFN gamma and IL-2,37,41 exist. In addition to fluctuations during the female fertile years, they appear to increase after menopause and steadily decrease thereafter. The production of Th2 cytokines, such as IL-4 and IL-10, appears unaffected by the influence of estrogens.42,43 In fact, IL-4 levels have been described as both affected and unaffected by hormones, including as a consequence of hormonal replacement therapy.44 While some cytokines fluctuate during the menstrual cycle, as described more in detail later in the chapter, IL-10 does not differ between women and men nor during the menstrual cycle.37 While most experiments have been performed in peripheral blood cells, the analysis of distinct T cell subsets might offer additional insight into their behavior. In fact, resident T lymphocytes in the peritoneal and pleural cavities appear to be higher in number in women compared to men and display a modulatory effect on the cytokine production by macrophages.45 21.3.1.2 Macrophages Macrophages are the scavenger cells of the immune system; they have the ability to engulf all kinds of other cells and cellular debris. They can be both circulating entities as well as resident cells within specific tissues, where they differentiate further according to the local needs and most likely insults. In addition to their scavenger abilities, they also function as antigen-presenting cells, recruiting T lymphocytes and propagating the immune response. The study of the effect of hormones on macrophages also focuses primarily on different cytokine production

after direct stimulation. For example, the production of TNF-alpha after estrogen stimulation has been described as biphasic,46 while testosterone does not appear to have any influence on its concentration. However, as described with T lymphocytes, differences in experimental protocols hinder a robust comparison between experimental results. In the case of IL-1 production, the effect of estrogen has been reported both to increase47 or decrease48 its production. In the case of IL-6, experimental results appear to coincide; in fact, its production appears clearly inhibited by estrogens, not only in macrophages,49 but also in osteoblasts50 and bone marrow cells.51 Next to the direct induction profiles, a potentially compensatory genetic mechanism has also been proposed. In fact, in birds sex-specific upregulation of IFN-gamma receptor genes has been reported as a mechanism to potentially compensate for sex-specific differences in the production of the cytokine.52 IL-12 appears downregulated in monocytes from pregnant women in their third trimester suggesting a suppressive role by estrogen and progesterone at elevated concentrations.53 The sex-specific role of different estrogen receptors in the activation of macrophages by LPS has been recently described. Specifically, the ERa appeared more relevant than its beta counterpart in eliciting the effect.54 This points to a novel area of investigation of the immune response, taking into consideration how different ER receptors might be contributing to or be the cause of the reported contradictory effects. 21.3.1.3  B Lymphocytes B lymphocytes have multiple functions in the immune system. Next to being the central element of humoral immunity and responsible for producing and secreting specific antibodies, they also function as antigen-presenting cells to other immune cells and actively secrete cytokines. Estrogen has been reported to increase the survival of B cells in both humans and animal models and protect their progenitors from apoptosis55,56 in animal models. In addition to their effect on B cell longevity, an increased production of antibodies by B lymphocytes in women compared to men has been established for many decades.57 In addition to these physiological differences, the ability of female patients to produce increased levels of autoantibodies compared to males when affected by autoimmune disease has also been established.58 Estrogen induces polyclonal activation of B cells, increasing the production of IgM and IgG in peripheral blood cells. Specifically, the effects of estrogens and testosterone display opposing effects on IgM and IgG production, with estrogens increasing it in both males and females59 and androgens decreasing it.60 The effect of estrogens on antibody class shift has been reported in animal models. Specifically, in a mouse model of arthritis a switch from IgG1 to an IgG2 subtype has been

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21.3  Sex and Hormones in the Immune Response

reported, i.e., a shift from a more complement-binding isoform to a less binding one.61 Hence, in addition to modulating disease due to increased cell numbers and survival, estrogen might also be directly involved in the modulation of disease severity. 21.3.1.4 Granulocytes Granulocytes or polymorphonuclear lymphycytes are characterized by the presence of granuli in their cytoplasm. They are classified into distinct subgroups and act through phagocytosis and release of the products of their cytoplasmic granuli into the bloodstream or environment. Granulocyte function has been scantly investigated. Neutrophil apoptosis appears decreased in women compared to men.62 Estrogens and progesterone have conflicting effects on their mobility, while testosterone did not appear to affect that phenomenon. The data concerning the effects of hormones on free radical production by granulocytes have been contradictory.62,63 Overall, very little research is devoted to the detection of the functions of these cells, especially with regard to sex differences. 21.3.1.5  Natural Killer Cells NK cells function as cytotoxic lymphocytes and are essential elements in the innate immune response. They are also involved in the adaptive immune response, mostly in the immunity against cancer cells. The activity of natural killer cells appears to decrease in females in the periovulatory period compared to males; these effects could not be identified in the follicular phase.64 Absolute differences among the activity of NK cells in fertile females and males were also confirmed in other studies, yet the menstrually-related fluctuations could not be confirmed.65 A recent study by Al-Attar and colleagues identified more vigorous NK cell responses in elderly women compared to men; a phenomenon associated with an apparently distinct maturation pattern in the elderly subjects.66 Women with premature menopause display higher numbers of NK cells.67 Direct effects of progesterone, estrogen, and testosterone on NK cell activity could not be confirmed in vitro, although some reports point to a biphasic effect of estrogen depending on the concentration investigated. Progesterone has been reported to induce NK apoptosis and inhibit their IFNgamma production.68 NKT cells also appear to be generally lower in numbers in males compared to females, especially with older age.69 Bernin recently described not only numeric differences in NKT cells between males and females, but also a higher inducibility of female NKTs by apha-GalCer compared to males, leading to an overall increased production of cytokines.70 Overall, NK and NKT cells appear more numerous and more active in fertile women compared to men, yet these differences might disappear postmenopausally.

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21.3.1.6  Dendritic Cells Dendritic cells represent the primary antigen-presenting cells of the immune system. They exert their function in peripheral tissues and within the lymph nodes after contact with an antigen, which is then presented to T and B cells. While the effect of hormones on dendritic cells in animal models has been described in detail (for review Ref. 71) information about their effects on human cells is still limited. Exposure of immature dendritic cells to estrogens in vitro increased their stimulatory capacity for T lymphocytes and their production of proinflammatory cytokines, namely of IL-6, IL-8, and MCP-1.72 These findings in humans are also confirmed in animal models, where differentiation of DCs from bone marrow could be enhanced by estrogen as well as their upregulation of MHC-II.73 Plasmocytoid dendritic cells in females appear to produce more elevated levels of IFN-alpha after TLR7 induction compared to males.74 In a recent study Griesbeck and colleagues identified a novel explanatory mechanism for this phenomenon. The differences appear to correlate with an increased basal expression of IFN regulatory factor 5 (IRF) in females compared to males which leads to increased response after TLR7 stimulation.75 Given the association of TLR7 with several sex differences in immune function, this mechanism appears most promising for dissection of the process and therapeutic exploitation. Interferon production by dendritic cells has also been correlated with contributing X chromosomal gene dosage as well as estrogen concentrations.76 These recent investigations point toward an increasing interest in the topic for human subjects and its translational aspects.

21.3.2  Hormonal Stages and the Immune Response 21.3.2.1  Puberty and Menstrual Cycle Puberty represents a turning point for hormonal development and correlates with changes in the immune system, which have been most prominently described in correlation with an increase in the incidence of autoimmune diseases. Interestingly, while the prepubescent incidence of most autoimmune diseases, although relatively rare, displays limited sex differences, the incidence increases more in girls compared to boys after puberty up until the premenopausal period. This has been described in lupus erythematosus, where 1:2 male:female ratios have been reported before puberty and up to 1:9 thereafter; the ratio commonly described in studies of the adult population.77 The same pattern has been described in the case of multiple sclerosis, where sex differences in incidence appear minimal or absent before puberty and show an increased incidence in women in their fertile years.78,79

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Hormonal fluctuations during the menstrual cycle have been reported to be associated with cellular modifications and, in turn, with disease severity during the follicular and luteal phase.80 Most diseases display increased symptoms and are most difficult to manage clinically in the luteal phase of the cycle, right before menstruation starts. This has been described for autoimmune diseases, such as multiple sclerosis and systemic lupus erythmatosus, but also for asthma and diabetes. Next to the direct influence of fluctuating hormonal levels on physiologic functions, their impact on the immune cells during the cycle appears most relevant. These differences have been described in most detail for regulatory T cells which display striking differences in their activity profiles throughout the cycle.68,81 In the follicular phase, they appear most active and then decrease from the periovulatory period up until menstruation, most likely to foster the tolerogenic environment, which would be necessary in case of successful conception. Proinflammatory cytokine production has also been reported as most active in the follicular phase, with more elevated concentrations of IL-1 and reduced ones in the luteal phase.48 Information on cellular number fluctuation is more controversial with partially conflicting reports on most cell populations. Whether these variations should be considered in animal experiments is a matter of debate; currently the data available point toward not considering these variations; for one thing, there is a very short cycle in rodents, which last about four days compared to the average 28 of a human female. Such short cycles appear less likely to affect cellular populations in a significant manner across the cycle.82 21.3.2.2 Pregnancy Pregnancy represents a physiological hormonal surge that is limited to one sex. During pregnancy women experience a sharp rise in estriol, progesterone, prolactin, and alpha-fetoprotein,83 all of which have been connected to immune functions. While most of the hormonal levels decrease after delivery, prolactin remains elevated, as does oxytocin. The elevation of steroid hormones during pregnancy has been postulated to contribute to the establishment of tolerance toward the implanted fetus; without this immunological shift, the risk for miscarriage would be significantly increased. Pregnancy is characterized by an elevation of regulatory T cells (T regs)84 and a downregulation of Th17 cells, promoting a favorable environment for implantation and pregnancy progression. Fluctuations in regulatory T cell levels have been described during the menstrual cycle68 and appear to correlate with hormonal levels. The same effect of estrogens might affect T reg function during pregnancy, since low levels of estrogen correlate with lower numbers of T regs in patients with missed abortion.85

Pregnancy exerts different effects in patients with autoimmune disease. Pregnancy has been reported to (1) trigger first time appearance of autoimmune disease, as well as to (2) induce flares of disease or to (3) reduce its activity. First time appearance of autoimmune disease during or right after pregnancy is a relatively rare event, which has, however, been described for diabetes86 and thyroiditis.87 Worsening or improvement of autoimmune disease during pregnancy has been primarily associated with a Th1 or Th2 typology of disease. Lupus erythematosus, which displays a relevant Th2 component, tends to worsen during pregnancy with common flares of different severity.88 Th1 diseases like rheumatoid arthritis and multiple sclerosis89 have been reported to improve during pregnancy with a remission of symptoms, which tend to reappear postpartum. Assisted reproductive technologies also appear to affect the course of autoimmune disease; nonetheless, it is not clear whether this depends on the disease or on the technique itself. In fact, worsening has been described mostly for lupus erythematosus,90 the disease that most commonly intensifies during natural pregnancy. 21.3.2.3 Menopause Menopause associates with the natural decrease of all circulating female steroid hormones and leads to a significant remodeling of cellular functions within the body. It also correlates with a general increase in the incidence of many diseases, which has prompted researchers to focus on the potentially protective role of estrogens and progesterone for many years. The decrease in estrogen production is the most striking phenomenon associated with the menopausal transition; this change is however not steady and linear but rather erratic, challenging the correlation of acute immune changes with hormonal levels. The cellular changes in menopause have been investigated less frequently as stand-alone phenomena and mostly in correlation with their effect on autoimmune diseases. Menopause appears to affect autoimmune diseases in different ways. Rheumatoid arthritis has been reported to worsen after menopause and lead to increased physical symptoms and progressive disability in patients.91 This is in striking contrast with the description of the effect of menopause on systemic lupus erythematosus and recapitulates the differences these two diseases also display during pregnancy and the menstrual cycle. In fact, patients with lupus erythematosus display fewer postmenopausal flares. However, disease progression is not necessarily halted given the more intense organ damage during flares.92 Generally, lupus erythematosus is most frequently diagnosed during a woman’s fertile years but if diagnosed in the perimenopausal period it appears generally less severe.93 Whether this difference is due to a less aggressive form of disease

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21.4  Gender in the Immune Response

in the postmenopausal period or simply due to reduced overall duration of disease and hence to less cumulative disability, is not known.

21.4  GENDER IN THE IMMUNE RESPONSE In addition to the biological factors that affect the immune system, social, economic, cultural, and structural aspects also influence the immune response in a gender-specific manner (Table 21.3). All antigens we encounter influence our immune system, hence, gendered and sex-specific behavior will modulate its development through selective exposure. Environmental exposure, social interactions, and health care availability are as meaningful as the antigen processing process itself toward the establishment of an orchestrated immune response and should be considered accordingly. The narrow focus on biological differences offers detailed mechanistic information on the process itself, but the dissection of sex differences in immunology will only be adequate if the impact of factors beyond biology are adequately considered.

TABLE 21.3  Gender Aspects and the Immune Response Nonsex-related factors Viral and microbial

Nonmicrobial agents

Food intake and food variety

Access to health care

Influences Generally more vigorous response in females Anatomical and physiological factors influence incidence ● Gender aspects in access/negotiation of, e.g., contraception ● Gender differences in access to therapy and different incidence of side effects ● Gendered behavior in, e.g., exposure patterns ● ●

Different professional exposure Different exposure and effects of environmental pollution ● Different exposure in the home to, e.g., cooking fumes ● ●

Access to food Food choices ● Access to nutrients (e.g., micronutrients, proteins) ● Sex differences in the gut microbiota ● Access to clean water, water procurement procedures ● ●

Lack of engagement with health care offer Sex/gender differences in access to acute/ chronic health care offer ● Resource allocation ● Unequitable access to out-of-pocket services ● Travel times to health care facilities ● ●

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21.4.1  Exposure to Viral and Microbial Agents Differences in the immune response to bacterial and viral agents in women and men have been reported for decades,94 yet the mechanism of these different interactions were long unknown. Even today, information is limited to few diseases and is seldom rooted in current experiments. Most of this information derives from smaller studies conducted several years ago. Given the current rise in microbial resistance, the emergence of new microbial and viral pathogens, and the gendered pattern of some of these infections, this is a field warranting further exploration in the future. In general, women have been reported to display more vigorous immune responses toward infectious agents compared to men, potentially leading to more tissue damage, but also to more rapid clearance of the infectious agent from the organism. Next to these mostly biological sex differences, gender also plays a role in the exposure to infectious agents. For example, sexually transmitted viruses and bacteria might affect women to a more significant degree than men given the anatomical differences that predispose to longer persistence of the organism in the female reproductive tract. Also, pregnancy might affect sensitivity to infection, given the pro-tolerogenic environment associated with carrying a child.95 Materno–fetal transmission might affect the child in utero differently, and susceptibility to infection might be different in young boys compared to young girls. Access to diagnosis and therapy is determined by gender in many areas of the world, especially when families have to prioritize among household members for out-of-pocket services. Sex differences have been reported in the immune response to viral, bacterial, and protozoan agents and will be briefly summarized as follows: Sex differences in the response to viral infections are characterized by generally more favorable outcomes in women. Sex differences in the viral response have been reviewed in detail elsewhere2 and will be briefly summarized here. Higher prevalence rates for HIV as well as an increased incidence of comorbidities in women have been reported, especially in the countries with the highest burden of infection.96 Prevalence is due to both gendered factors such as limited opportunity for infection control due to limited negotiating power and limited availability of female condoms and microbicides, as well as sex-related anatomical factors.97 The role of an increased responsiveness of female plasmacytoid dendritic cells to the HI virus98 has been reported as well as the heightened response to TLR7 engagement.99 Increased incidence of unwanted side effects to antiretroviral therapy has been reported more frequently in women and might be a reason for increased discontinuation of therapy in females.100

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Hepatitis B virus (HBV) is characterized by a more vigorous viral response in females, which leads to both increased tissue damage, but also higher clearance rates of the virus and lower incidence of chronic hepatitis compared to males.101 Additionally, after HBV vaccination, women tend to develop a more vigorous response and more persistent immunity compared to males.102 Persistence of the virus is consistently longer in males compared to females and is associated with a higher incidence of HBV-associated hepatocarcinoma.103,104 Androgens have also been identified as direct modulators of HBV infection, both through modulation of the host response and through binding of androgen responsive elements in the HBV virus.105 Hepatitis C affects men more frequently than females worldwide, and, in addition, differences in progression rates have been reported with women displaying slower progression compared to men.106 This phenomenon correlates with fertility status: while women in their fertile years display slower progression rates, that of postmenopausal women is equal to that of males.107 From a therapeutic standpoint, the combination therapy of interferon with ribavirin led to a slightly increased incidence of side effects such as anemia, depression, and thyroid disease in women.108 Use of the novel antiviral agents, however, appears to have eliminated these differences.109 Susceptibility to parasitic infections also display sex differences. In fact, males display greater resistance to Trichomonas vaginalis110 and Toxoplasma gondii,111 while women have been reported to display greater resistance to Leishmaniasis,112 Trypanosomiasis,113 and Schistosoma mansoni.114 Leishmania infection is generally favored by a Th2 shift in T helper profiles115 and this appears to be induced by testosterone engagement.116 In male rodents this shift led to an increased expression of IL-4, IL-10, TNF-alpha, and -beta, while the opposite was identified in female animals.117 In humans, correlation of infection with age was also described for Leishmaniasis with young boys being significantly more affected than girls and a progressive leveling of this trend with increasing age.118 Compared to information on sex differences in viral and parasitic infection, surprisingly little is known about sex differences in bacterial infections. The available information is frequently outdated and composed of studies with limited power. Sex differences have been reported in the incidence of tuberculosis, with higher prevalence rates in males.119 Although this might be attributable in part to differences in health care access and reporting, many scholars confirm the reliability of this prevalence.120 A role has been postulated for steroid hormones in the susceptibility and severity of TB.121 However, little current information exists on the role of biological sex differences in tuberculosis and the area needs further exploration, especially if tuberculosis

therapy is to become more successful and potentially ridden with fewer side effects in the future. Tuberculosis also displays a delayed gendered component due to its frequent association with HIV infection, especially in Sub-Saharan Africa. As described above, women carry the highest burden of the HIV infection in this region and, since tuberculosis represents one of the primary opportunistic infections for HIV-positive patients, its rise, especially in the female population, has to be expected. Some common infections do not only display gender-specific patterns but also an association with age. For example, Streptococcal pharyngitis frequently has a slightly higher incidence in boys122 as well as rheumatic fever, which also affects boys more frequently than girls.123 Pertussis, on the other hand, appears to affect females more severely than males and most importantly in the prevaccination period.124 These trends recapitulate the findings described for autoimmune diseases and puberty and further underline a possible role for hormones in disease susceptibility.

21.4.2  Exposure to Nonmicrobiological Agents Next to the exposure to microbial agents, nonmicrobial antigens represent an area of relevant research in autoimmunity. Chemicals, biologicals, and heavy metals exemplify antigens to which people are frequently exposed in the workplace; hence, gender differences in occupational choices can significantly impact their effect on the immune response. Examples go from the exposure to wood dust, mining dust, and asbestos by workers in traditionally male professions, to the exposure to nail polish, cosmetics, and hair products by women more traditionally employed in the beauty industry. The mechanisms behind the immunological process leading from chemical or metal exposure to autoimmunity are not well understood. The role of chemicals and metals as immunomodulators has been previously reported, e.g., in the association between autoimmune thyroiditis and increased lymphocyte reactivity to mercury and nickel125 or in the description of the protective role of zinc in tumor immunology.126 None of these studies has evaluated whether these substances carry a sex-specific effect and the impact of gender roles on the exposure has not been investigated. Environmental pollution represents another source of potential trigger, which might lead to possible sex differences in expression of disease. The association between allergies and environmental pollution has been frequently reported and sex differences can be identified in incidence patterns. Asthma affects young boys more frequently than young girls and environmental pollution in the form of exhaust fumes, but also indoor smoke, indoor air pollution, and pest contamination, plays a role in predisposing children to the development of the

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21.5  Health Care Access

disease.127 The same triggers might also induce atopic dermatitis, which is more common in females, because of increased oxidative stress in the skin.128 Next to potential exposures in the workplace, the home environment might promote contact with potentially immunomodulatory antigens. Furniture and paint might be a source of exposure; even toys might have heavy chemicals in them exposing young children to potentially harmful immunomodulators. In addition to these sources, ambient smoke represents a significant source of pollution in low- and middle-income countries, which predominantly affects women since they are more likely to spend time in household cooking. Biomass fuels are especially involved in the alteration of the innate immune response and represent a specific threat to young children.129

21.4.3  Food Intake and Food Variety Access to food does not represent a problem in developed countries but still has a wide impact in middle- and especially low-income countries. The access to food is not just relevant in quantitative terms, but also in qualitative ones, since the type of food consumed, its caloric density, and richness in nutrients, vitamins, and essential minerals significantly affect the health of the consumer. Even in developed countries where quantitative food availability is a generally minor issue, the quality of the consumed food might have a potentially harmful effect. For example, the harmful roles of refined sugar as well as trans fats as potential immunomodulators and risk factors for the development of cardiovascular, metabolic, and oncologic disease are suspected and are currently under investigation.130,131 Consumed food not only affects body weight and composition; it will also affect the human microbiome.132 Sex differences in the gut microbiome have been previously described.133 Given the likely role of the microbiota in the development of the immune system these differences might be very significant toward the sexspecific development of autoimmunity—with or without an interaction with hormones—as some scholar have started to investigate.134,135 Not only does the microbiota differ between the sexes, diet does also appear to affect it differently.136 Food shortage or lack of nutrients also have a meaningful impact on human health. Food availability has a gendered component due to prioritization in eating patterns in households in many areas of the world, with women eating after men and children.137 This might not only lead to undernourishment but also significant lack of, e.g., protein intake, since these food components might be eaten by the first in line. Lack of protein can lead to a vast number of diseases and is amenable to prevention.138 The role of micronutrients for the immune

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system has been widely investigated. The role of vitamins as immunomodulators and relevant molecules for adequate functioning of the immune system has been reported. Vitamin D and the vitamin B complex have been associated with the immune function, eliciting functions that range from protection from pathogen infection to protection from food allergy.139–141 Micronutrients such as zinc, copper, and iron have all been reported as immunomodulators142 and given gender-specific eating patterns might lead to relevant differences in the immune response. Lastly, access to clean water represents another source of potentially gendered exposure to antigens. In most developing countries, women are in charge of fetching water from public sources and, hence, more intensely exposed to water-borne parasites than men. While these infections can be prevented with adequate handling practices, women are frequently not aware of them or the necessary tools are not available. From a gendered perspective, women are also more likely to expose children to these infections, since they generally carry the whole burden of child-rearing and have their children share and/or accompany them during their working duties.

21.5  HEALTH CARE ACCESS Health care access represents a critical gender issue in many parts of the world. It can be limited both by lack of knowledge about when to ask for medical advice as well as by limited resources to health care services themselves. In developed countries lack of clarity about when to seek medical advice or delay in health seeking behavior represent the most relevant causes for reduced access to health care services. Health seeking behavior displays gendered patterns all over the world,143 with men accessing health care in a delayed manner compared to women for chronic diseases. For acute conditions, on the other hand, provision of care appears generally more rapid for men than for women.144,145 This might be due to patterns of subjective decision making, engagement of health care providers by a third party, and rapidity of response by health care providers. While delayed access due to personal choice might affect individuals all over the world, family prioritization for out-of-pocket services and the inability to travel to access health care facilities disproportionately affects women in many developing countries.146,147 Out-ofpocket expenses for health care are a disproportionate burden on families in low-income countries and this burden might be more relevant when the pharmaceuticals needed are immunomodulators, since many of these drugs pertain to the most expensive brands of pharmaceuticals. Immunomodulatory drugs, chemotherapeutics,

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and last-generation antibiotics require expenses beyond the affordability of many families, who will prioritize accordingly. Travel times to health care facilities also represent a significant burden. The inability to travel due to family care and chores can affect chronic treatments, such as, e.g., directly observed tuberculosis therapy,148 as well as acute care, e.g., due to childbirth.149 Avoidable causes of gender-specific morbidity and mortality could be avoided if these issues were tackled from a policy perspective.

21.6  RELEVANCE FOR CLINICAL PRACTICE The previous sections have highlighted the complex interaction between sex and gender and the immune system. In terms of clinical practice, clinicians should be aware of the possible sex differences inherent in autoimmunity and inflammation, but, most importantly, should develop a sensitive approach to the gendered factors that additionally affect both vulnerability and response to disease. As we have pointed out, both factors might interact and contaminate each other, making a clear distinction between the biological and the sociocultural factually impossible in many instances. Nonetheless, a thorough clinical history, especially if inflammatory processes are relevant to disease expression, should include at least some of the aspects mentioned to be complete. While some might not be relevant in all instances, their systematic inclusion into patient–doctor communication will also convey to the patient the confidence that their physician is approaching their disease in a holistic manner.

References 1. Oertelt-Prigione S. The influence of sex and gender on the immune response. Autoimmun Rev. 2012;11(6–7):A479–A485. 2. Klein SL. Sex influences immune responses to viruses, and effic� cacy of prophylaxis and treatments for viral diseases. Bioessays. 2012;34(12):1050–1059. 3. Ngo ST, Steyn FJ, McCombe PA. Gender differences in autoimmune disease. Front Neuroendocrinol. 2014;35(3):347–369. 4. Silkaitis K, Lemos B. Sex-biased chromatin and regulatory crosstalk between sex chromosomes, autosomes, and mitochondria. Biol Sex Differ. 2014;5(1):2. 5. Fish EN. The X-files in immunity: sex-based differences predispose immune responses. Nat Rev Immunol. 2008;8(9):737–744. 6. Xavier-da-Silva MM, Moreira-Filho CA, Suzuki E, Patricio F, Coutinho A, Carneiro-Sampaio M. Fetal-onset IPEX: report of two families and review of literature. Clin Immunol. 2015;156(2):131–140. 7. Schneider LC. X-linked hyper IgM syndrome. Clin Rev Allergy Immunol. 2000;19(2):205–215. 8. Sasahara Y. WASP-WIP complex in the molecular pathogenesis of Wiskott-Aldrich syndrome. Pediatr Int. 2016;58(1):4–7. 9. Balduini CL, Savoia A. Genetics of familial forms of thrombocytopenia. Hum Genet. 2012;131(12):1821–1832.

10. Rans TS, England R. The evolution of gene therapy in X-linked severe combined immunodeficiency. Ann Allergy Asthma Immunol. 2009;102(5):357–362. quiz 63-5, 402. 11. Soulat D, Burckstummer T, Westermayer S, et  al. The DEADbox helicase DDX3X is a critical component of the TANKbinding kinase 1-dependent innate immune response. EMBO J. 2008;27(15):2135–2146. 12. Carrel L, Willard HF. X-inactivation profile reveals extensive variability in X-linked gene expression in females. Nature. 2005;434(7031):400–404. 13. Johnston CM, Lovell FL, Leongamornlert DA, Stranger BE, Dermitzakis ET, Ross MT. Large-scale population study of human cell lines indicates that dosage compensation is virtually complete. PLoS Genet. 2008;4(1):e9. 14. Persani L, Rossetti R, Cacciatore C. Genes involved in human premature ovarian failure. J Mol Endocrinol. 2010;45(5):257–279. 15. Sybert VP, McCauley E. Turner’s syndrome. N Engl J Med. 2004;351(12):1227–1238. 16. Invernizzi P, Miozzo M, Selmi C, et al. X chromosome monosomy: a common mechanism for autoimmune diseases. J Immunol. 2005;175(1):575–578. 17. Invernizzi P, Miozzo M, Battezzati PM, et  al. Frequency of monosomy X in women with primary biliary cirrhosis. Lancet. 2004;363(9408):533–535. 18. Invernizzi P, Miozzo M, Oertelt-Prigione S, et  al. X monosomy in female systemic lupus erythematosus. Ann N Y Acad Sci. 2007;1110:84–91. 19. Chagnon P, Provost S, Belisle C, Bolduc V, Gingras M, Busque L. Age-associated skewing of X-inactivation ratios of blood cells in normal females: a candidate-gene analysis approach. Exp Hematol. 2005;33(10):1209–1214. 20. Vicoso B, Kaiser VB, Bachtrog D. Sex-biased gene expression at homomorphic sex chromosomes in emus and its implication for sex chromosome evolution. Proc Natl Acad Sci USA. 2013;110(16):6453–6458. 21. Brix TH, Knudsen GP, Kristiansen M, Kyvik KO, Orstavik KH, Hegedus L. High frequency of skewed X-chromosome inactivation in females with autoimmune thyroid disease: a possible explanation for the female predisposition to thyroid autoimmunity. J Clin Endocrinol Metab. 2005;90(11):5949–5953. 22. Uz E, Loubiere LS, Gadi VK, et al. Skewed X-chromosome inactivation in scleroderma. Clin Rev Allergy Immunol. 2008;34(3):352–355. 23. Uz E, Mustafa C, Topaloglu R, et  al. Increased frequency of extremely skewed X chromosome inactivation in juvenile idiopathic arthritis. Arthritis Rheum. 2009;60(11):3410–3412. 24. Ozcelik T. X chromosome inactivation and female predisposition to autoimmunity. Clin Rev Allergy Immunol. 2008;34(3):348–351. 25. Cortez D, Marin R, Toledo-Flores D, et  al. Origins and functional evolution of Y chromosomes across mammals. Nature. 2014;508(7497):488–493. 26. Bachtrog D. Y-chromosome evolution: emerging insights into processes of Y-chromosome degeneration. Nat Rev Genet. 2013;14(2):113–124. 27. Santiago-Raber ML, Kikuchi S, Borel P, et  al. Evidence for genes in addition to Tlr7 in the Yaa translocation linked with acceleration of systemic lupus erythematosus. J Immunol. 2008;181(2):1556–1562. 28. Case LK, Wall EH, Dragon JA, et al. The Y chromosome as a regulatory element shaping immune cell transcriptomes and susceptibility to autoimmune disease. Genome Res. 2013;23(9):1474–1485. 29. Sun SL, Horino S, Itoh-Nakadai A, et al. Y chromosome-linked B and NK cell deficiency in mice. J Immunol. 2013;190(12):6209–6220. 30. Charchar FJ, Bloomer LD, Barnes TA, et al. Inheritance of coronary artery disease in men: an analysis of the role of the Y chromosome. Lancet. 2012;379(9819):915–922.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

31. Lleo A, Oertelt-Prigione S, Bianchi I, et  al. Y chromosome loss in male patients with primary biliary cirrhosis. J Autoimmun. 2013;41:87–91. 32. Persani L, Bonomi M, Lleo A, et al. Increased loss of the Y chromosome in peripheral blood cells in male patients with autoimmune thyroiditis. J Autoimmun. 2012;38(2-3):J193–J196. 33. Tobi EW, Lumey LH, Talens RP, et  al. DNA methylation differences after exposure to prenatal famine are common and timingand sex-specific. Hum Mol Genet. 2009;18(21):4046–4053. 34. Hall E, Volkov P, Dayeh T, et al. Sex differences in the genomewide DNA methylation pattern and impact on gene expression, microRNA levels and insulin secretion in human pancreatic islets. Genome Biol. 2014;15(12):522. 35. Lane RH. Fetal programming, epigenetics, and adult onset disease. Clin Perinatol. 2014;41(4):815–831. 36. McCarthy MM, Nugent BM. At the frontier of epigenetics of brain sex differences. Front Behav Neurosci. 2015;9:221. 37. Bouman A, Schipper M, Heineman MJ, Faas MM. Gender difference in the non-specific and specific immune response in humans. Am J Reprod Immunol. 2004;52(1):19–26. 38. An J, Ribeiro RC, Webb P, et  al. Estradiol repression of tumor necrosis factor-alpha transcription requires estrogen receptor activation function-2 and is enhanced by coactivators. Proc Natl Acad Sci USA. 1999;96(26):15161–15166. 39. Kastner P, Krust A, Turcotte B, et  al. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J. 1990;9(5):1603–1614. 40. Gelmann EP. Molecular biology of the androgen receptor. J Clin Oncol. 2002;20(13):3001–3015. 41. Giron-Gonzalez JA, Moral FJ, Elvira J, et al. Consistent production of a higher TH1:TH2 cytokine ratio by stimulated T cells in men compared with women. Eur J Endocrinol. 2000;143(1):31–36. 42. Cioffi M, Esposito K, Vietri MT, et al. Cytokine pattern in postmenopause. Maturitas. 2002;41(3):187–192. 43. Kamada M, Irahara M, Maegawa M, et al. Transient increase in the levels of T-helper 1 cytokines in postmenopausal women and the effects of hormone replacement therapy. Gynecol Obstet Invest. 2001;52(2):82–88. 44. Kumru S, Godekmerdan A, Yilmaz B. Immune effects of surgical menopause and estrogen replacement therapy in peri-menopausal women. J Reprod Immunol. 2004;63(1):31–38. 45. Scotland RS, Stables MJ, Madalli S, Watson P, Gilroy DW. Sex differences in resident immune cell phenotype underlie more efficient acute inflammatory responses in female mice. Blood. 2011;118(22):5918–5927. 46. Rogers A, Eastell R. The effect of 17beta-estradiol on production of cytokines in cultures of peripheral blood. Bone. 2001;29(1):30–34. 47. Ruh MF, Bi Y, D’Alonzo R, Bellone CJ. Effect of estrogens on IL-1beta promoter activity. J Steroid Biochem Mol Biol. 1998;66(4):203–210. 48. Polan ML, Daniele A, Kuo A. Gonadal steroids modulate human monocyte interleukin-1 (IL-1) activity. Fertil Steril. 1988;49(6):964–968. 49. Kim MS, Chae HJ, Shin TY, Kim HM, Kim HR. Estrogen regulates cytokine release in human mast cells. Immunopharmacol Immunotoxicol. 2001;23(4):495–504. 50. Cheung J, Mak YT, Papaioannou S, Evans BA, Fogelman I, Hampson G. Interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin production by human osteoblastic cells: comparison of the effects of 17-beta oestradiol and raloxifene. J Endocrinol. 2003;177(3):423–433. 51. Gordon CM, LeBoff MS, Glowacki J. Adrenal and gonadal steroids inhibit IL-6 secretion by human marrow cells. Cytokine. 2001;16(5):178–186.

319

52. Garcia-Morales C, Nandi S, Zhao D, et al. Cell-autonomous sex differences in gene expression in chicken bone marrow-derived macrophages. J Immunol. 2015;194(5):2338–2344. 53. Elenkov IJ, Wilder RL, Bakalov VK, et al. IL-12, TNF-alpha, and hormonal changes during late pregnancy and early postpartum: implications for autoimmune disease activity during these times. J Clin Endocrinol Metab. 2001;86(10):4933–4938. 54. Campesi I, Marino M, Montella A, Pais S, Franconi F. Sex differences in estrogen receptor alpha and beta levels and activation status in LPS-stimulated human macrophages. J Cell Physiol. 2016. 55. Grimaldi CM, Cleary J, Dagtas AS, Moussai D, Diamond B. Estrogen alters thresholds for B cell apoptosis and activation. J Clin Invest. 2002;109(12):1625–1633. 56. Hill L, Jeganathan V, Chinnasamy P, Grimaldi C, Diamond B. Differential roles of estrogen receptors alpha and beta in control of B-cell maturation and selection. Mol Med. 2011;17(3–4):211–220. 57. Rowley MJ, Mackay IR. Measurement of antibody-producing capacity in man. I. The normal response to flagellin from Salmonella adelaide. Clin Exp Immunol. 1969;5(4):407–418. 58. Whitacre CC, Reingold SC, O’Looney PA. A gender gap in autoimmunity. Science. 1999;283(5406):1277–1278. 59. Kanda N, Tamaki K. Estrogen enhances immunoglobulin production by human PBMCs. J Allergy Clin Immunol. 1999;103(2 Pt 1):282–288. 60. Kanda N, Tsuchida T, Tamaki K. Testosterone inhibits immunoglobulin production by human peripheral blood mononuclear cells. Clin Exp Immunol. 1996;106(2):410–415. 61. Latham KA, Zamora A, Drought H, et  al. Estradiol treatment redirects the isotype of the autoantibody response and prevents the development of autoimmune arthritis. J Immunol. 2003;171(11):5820–5827. 62. Molloy EJ, O’Neill AJ, Grantham JJ, et al. Sex-specific alterations in neutrophil apoptosis: the role of estradiol and progesterone. Blood. 2003;102(7):2653–2659. 63. Cassidy RA. Influence of steroids on oxidant generation in activated human granulocytes and mononuclear leukocytes. Shock. 2003;20(1):85–90. 64. Souza SS, Castro FA, Mendonca HC, et al. Influence of menstrual cycle on NK activity. J Reprod Immunol. 2001;50(2):151–159. 65. Yovel G, Shakhar K, Ben-Eliyahu S. The effects of sex, menstrual cycle, and oral contraceptives on the number and activity of natural killer cells. Gynecol Oncol. 2001;81(2):254–262. 66. Al-Attar A, Presnell SR, Peterson CA, Thomas DT, Lutz CT. The effect of sex on immune cells in healthy aging: Elderly women have more robust natural killer lymphocytes than do elderly men. Mech Ageing Dev. 2016;156:25–33. 67. Giglio T, Imro MA, Filaci G, et  al. Immune cell circulating subsets are affected by gonadal function. Life Sci. 1994;54(18):1305–1312. 68. Arruvito L, Giulianelli S, Flores AC, et al. NK cells expressing a progesterone receptor are susceptible to progesterone-induced apoptosis. J Immunol. 2008;180(8):5746–5753. 69. Kee SJ, Park YW, Cho YN, et al. Age- and gender-related differences in circulating natural killer T cells and their subset levels in healthy Korean adults. Hum Immunol. 2012;73(10):1011–1016. 70. Bernin H, Fehling H, Marggraff C, Tannich E, Lotter H. The cytokine profile of human NKT cells and PBMCs is dependent on donor sex and stimulus. Med Microbiol Immunol. 2016. 71. Kovats S, Carreras E. Regulation of dendritic cell differentiation and function by estrogen receptor ligands. Cell Immunol. 2008;252(1–2):81–90. 72. Bengtsson AK, Ryan EJ, Giordano D, Magaletti DM, Clark EA. 17beta-estradiol (E2) modulates cytokine and chemokine expression in human monocyte-derived dendritic cells. Blood. 2004;104(5):1404–1410.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

320

21.  SEX DIFFERENCES IN THE IMMUNE RESPONSE

73. Paharkova-Vatchkova V, Maldonado R, Kovats S. Estrogen preferentially promotes the differentiation of CD11c+ CD11b(intermediate) dendritic cells from bone marrow precursors. J Immunol. 2004;172(3):1426–1436. 74. Seillet C, Laffont S, Tremollieres F, et  al. The TLR-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor alpha signaling. Blood. 2012;119(2):454–464. 75. Griesbeck M, Ziegler S, Laffont S, et  al. Sex Differences in Plasmacytoid Dendritic Cell Levels of IRF5 Drive Higher IFNalpha Production in Women. J Immunol. 2015;195(11):5327–5336. 76. Laffont S, Rouquie N, Azar P, et al. X-Chromosome complement and estrogen receptor signaling independently contribute to the enhanced TLR7-mediated IFN-alpha production of plasmacytoid dendritic cells from women. J Immunol. 2014;193(11):5444–5452. 77. Tedeschi SK, Bermas B, Costenbader KH. Sexual disparities in the incidence and course of SLE and RA. Clin Immunol. 2013;149(2):211–218. 78. Chitnis T. Role of puberty in multiple sclerosis risk and course. Clin Immunol. 2013;149(2):192–200. 79. Ramagopalan SV, Valdar W, Criscuoli M, et  al. Age of puberty and the risk of multiple sclerosis: a population based study. Eur J Neurol. 2009;16(3):342–347. 80. Oertelt-Prigione S. Immunology and the menstrual cycle. Autoimmun Rev. 2012;11(6–7):A486–A492. 81. Prieto GA, Rosenstein Y. Oestradiol potentiates the suppressive function of human CD4 CD25 regulatory T cells by promoting their proliferation. Immunology. 2006;118(1):58–65. 82. Prendergast BJ, Onishi KG, Zucker I. Female mice liberated for inclusion in neuroscience and biomedical research. Neurosci Biobehav Rev. 2014;40:1–5. 83. Tan EK, Tan EL. Alterations in physiology and anatomy during pregnancy. Best Pract Res Clin Obstet Gynaecol. 2013;27(6):791–802. 84. Somerset DA, Zheng Y, Kilby MD, Sansom DM, Drayson MT. Normal human pregnancy is associated with an elevation in the immune suppressive CD25+ CD4+ regulatory T-cell subset. Immunology. 2004;112(1):38–43. 85. Cao W, Xu W, Chen T, et al. CD4CD25FoxP3 regulatory T cells and cytokines interact with estradiol in cases of missed abortion. Exp Ther Med. 2014;7(2):417–422. 86. Inagaki T, Nishii Y, Suzuki N, et al. Fulminant diabetes mellitus associated with pregnancy: case reports and literature review. Endocr J. 2002;49(3):319–322. 87. Radetti G, Zavallone A, Gentili L, Beck-Peccoz P, Bona G. Foetal and neonatal thyroid disorders. Minerva Pediatr. 2002;54(5):383–400. 88. Stojan G, Baer AN. Flares of systemic lupus erythematosus during pregnancy and the puerperium: prevention, diagnosis and management. Expert Rev Clin Immunol. 2012;8(5):439–453. 89. Borchers AT, Naguwa SM, Keen CL, Gershwin ME. The implications of autoimmunity and pregnancy. J Autoimmun. 2010;34(3):J287–J299. 90. Casoli P, Tumiati B, La Sala G. Fatal exacerbation of systemic lupus erythematosus after induction of ovulation. J Rheumatol. 1997;24(8):1639–1640. 91. Kuiper S, van Gestel AM, Swinkels HL, de Boo TM, da Silva JA, van Riel PL. Influence of sex, age, and menopausal state on the course of early rheumatoid arthritis. J Rheumatol. 2001;28(8):1809–1816. 92. Urowitz MB, Ibanez D, Jerome D, Gladman DD. The effect of menopause on disease activity in systemic lupus erythematosus. J Rheumatol. 2006;33(11):2192–2198. 93. Boddaert J, Huong DL, Amoura Z, Wechsler B, Godeau P, Piette JC. Late-onset systemic lupus erythematosus: a personal series of 47 patients and pooled analysis of 714 cases in the literature. Medicine (Baltimore). 2004;83(6):348–359.

94. Klein SLR, Roberts CW. Sex Hormones and Immunity to Infection. Heidelber: Springer; 2010. 95. Munoz-Suano A, Hamilton AB, Betz AG. Gimme shelter: the immune system during pregnancy. Immunol Rev. 2011;241(1):20–38. 96. Hegdahl HK, Fylkesnes KM, Sandoy IF. Sex Differences in HIV Prevalence Persist over Time: Evidence from 18 Countries in Sub-Saharan Africa. PLoS One. 2016;11(2):e0148502. 97. Aziz M, Smith KY. Treating women with HIV: is it different than treating men? Curr HIV/AIDS Rep. 2012;9(2):171–178. 98. Meier A, Chang JJ, Chan ES, et al. Sex differences in the Toll-like receptor-mediated response of plasmacytoid dendritic cells to HIV-1. Nat Med. 2009;15(8):955–959. 99. Berghofer B, Frommer T, Haley G, Fink L, Bein G, Hackstein H. TLR7 ligands induce higher IFN-alpha production in females. J Immunol. 2006;177(4):2088–2096. 100. Belden KA, Squires KE. HIV infection in women: do sex and gender matter? Curr Infect Dis Rep. 2008;10(5):423–431. 101. Blumberg BS. Sex differences in response to hepatitis B virus. I. History. Arthritis Rheum. 1979;22(11):1261–1266. 102. Bock HL, Kruppenbacher J, Sanger R, Hobel W, Clemens R, Jilg W. Immunogenicity of a recombinant hepatitis B vaccine in adults. Arch Intern Med. 1996;156(19):2226–2231. 103. Wang SH, Yeh SH, Lin WH, Wang HY, Chen DS, Chen PJ. Identification of androgen response elements in the enhancer I of hepatitis B virus: a mechanism for sex disparity in chronic hepatitis B. Hepatology. 2009;50(5):1392–1402. 104. Wu CF, Yu MW, Lin CL, et al. Long-term tracking of hepatitis B viral load and the relationship with risk for hepatocellular carcinoma in men. Carcinogenesis. 2008;29(1):106–112. 105. Wang SH, Yeh SH, Chen PJ. The driving circuit of HBx and androgen receptor in HBV-related hepatocarcinogenesis. Gut. 2014;63(11):1688–1689. 106. Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet. 1997;349(9055):825–832. 107. Villa E, Karampatou A, Camma C, et  al. Early menopause is associated with lack of response to antiviral therapy in women with chronic hepatitis C. Gastroenterology. 2011;140(3):818–829. 108. Dan AA, Martin LM, Crone C, et  al. Depression, anemia and health-related quality of life in chronic hepatitis C. J Hepatol. 2006;44(3):491–498. 109. Lawitz E, Mangia A, Wyles D, et  al. Sofosbuvir for previously untreated chronic hepatitis C infection. N Engl J Med. 2013;368(20):1878–1887. 110. Petrin D, Delgaty K, Bhatt R, Garber G. Clinical and microbiological aspects of Trichomonas vaginalis. Clin Microbiol Rev. 1998;11(2):300–317. 111. Liesenfeld O, Nguyen TA, Pharke C, Suzuki Y. Importance of gender and sex hormones in regulation of susceptibility of the small intestine to peroral infection with Toxoplasma gondii tissue cysts. J Parasitol. 2001;87(6):1491–1493. 112. Karami M, Doudi M, Setorki M. Assessing epidemiology of cutaneous leishmaniasis in Isfahan, Iran. J Vector Borne Dis. 2013;50(1):30–37. 113. Brabin L, Brabin BJ. Parasitic infections in women and their consequences. Adv Parasitol. 1992;31:1–81. 114. Degu G, Mengistu G, Jones J. Some factors affecting prevalence of and immune responses to Schistosoma mansoni in schoolchildren in Gorgora, northwest Ethiopia. Ethiop Med J. 2002;40(4):345–352. 115. Alexander J, Bryson K. T helper (h)1/Th2 and Leishmania: paradox rather than paradigm. Immunol Lett. 2005;99(1): 17–23.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

116. Liu L, Wang L, Zhao Y, Wang Y, Wang Z, Qiao Z. Testosterone attenuates p38 MAPK pathway during Leishmania donovani infection of macrophages. Parasitol Res. 2006;99(2):189–193. 117. Lezama-Davila CM, Isaac-Marquez AP, Barbi J, Oghumu S, Satoskar AR. 17Beta-estradiol increases Leishmania mexicana killing in macrophages from DBA/2 mice by enhancing production of nitric oxide but not pro-inflammatory cytokines. Am J Trop Med Hyg. 2007;76(6):1125–1127. 118. Guerra-Silveira F, Abad-Franch F. Sex bias in infectious disease epidemiology: patterns and processes. PLoS One. 2013;8(4):e62390. 119. Borgdorff MW, Nagelkerke NJ, Dye C, Nunn P. Gender and tuberculosis: a comparison of prevalence surveys with notification data to explore sex differences in case detection. Int J Tuberc Lung Dis. 2000;4(2):123–132. 120. Rhines AS. The role of sex differences in the prevalence and transmission of tuberculosis. Tuberculosis (Edinb). 2013;93(1):104–107. 121. Svanberg L. Effects of estrogen deficiency in women castrated when young. Acta Obstet Gynecol Scand Suppl. 1981;106:11–15. 122. Lin MH, Fong WK, Chang PF, Yen CW, Hung KL, Lin SJ. Predictive value of clinical features in differentiating group A beta-hemolytic streptococcal pharyngitis in children. J Microbiol Immunol Infect. 2003;36(1):21–25. 123. Breda L, Miulli E, Marzetti V, Chiarelli F, Marcovecchio ML. Rheumatic fever: a disease still to be kept in mind. Rheumatology (Oxford). 2013;52(5):953. 124. Celentano LP, Massari M, Paramatti D, Salmaso S, Tozzi AE. Resurgence of pertussis in Europe. Pediatr Infect Dis J. 2005;24(9):761–765. 125. Hybenova M, Hrda P, Prochazkova J, Stejskal V, Sterzl I. The role of environmental factors in autoimmune thyroiditis. Neuro Endocrinol Lett. 2010;31(3):283–289. 126. Bao B, Thakur A, Li Y, et  al. The immunological contribution of NF-kappaB within the tumor microenvironment: a potential protective role of zinc as an anti-tumor agent. Biochim Biophys Acta. 2012;1825(2):160–172. 127. Milligan KL, Matsui E, Sharma H. Asthma in Urban Children: Epidemiology, Environmental Risk Factors, and the Public Health Domain. Curr Allergy Asthma Rep. 2016;16(4):33. 128. Ahn K. The role of air pollutants in atopic dermatitis. J Allergy Clin Immunol. 2014;134(5):993–999. discussion 1000. 129. Lee A, Kinney P, Chillrud S, Jack D. A Systematic Review of Innate Immunomodulatory Effects of Household Air Pollution Secondary to the Burning of Biomass Fuels. Ann Glob Health. 2015;81(3):368–374. 130. Calder PC. Functional Roles of Fatty Acids and Their Effects on Human Health. JPEN J Parenter Enteral Nutr. 2015;39(1 Suppl):18S–32S. 131. Freitas-Simoes TM, Ros E, Sala-Vila A. Nutrients, foods, dietary patterns and telomere length: Update of epidemiological studies and randomized trials. Metabolism. 2016;65(4):406–415. 132. Tilg H, Moschen AR. Food, immunity, and the microbiome. Gastroenterology. 2015;148(6):1107–1119.

321

133. Haro C, Rangel-Zuniga OA, Alcala-Diaz JF, et  al. Intestinal Microbiota Is Influenced by Gender and Body Mass Index. PLoS One. 2016;11(5):e0154090. 134. Markle JG, Frank DN, Mortin-Toth S, et  al. Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity. Science. 2013;339(6123):1084–1088. 135. Yurkovetskiy L, Burrows M, Khan AA, et  al. Gender bias in autoimmunity is influenced by microbiota. Immunity. 2013;39(2):400–412. 136. Bolnick DI, Snowberg LK, Hirsch PE, et al. Individual diet has sex-dependent effects on vertebrate gut microbiota. Nature communications. 2014;5:4500. 137. Messer E. Intra-household allocation of food and health care: current findings and understandings--introduction. Soc Sci Med. 1997;44(11):1675–1684. 138. Batool R, Butt MS, Sultan MT, Saeed F, Naz R. Protein-energy malnutrition: a risk factor for various ailments. Crit Rev Food Sci Nutr. 2015;55(2):242–253. 139. Maiese K, Chong ZZ, Hou J, Shang YC. The vitamin nicotinamide: translating nutrition into clinical care. Molecules. 2009;14(9):3446–3485. 140. Suaini NH, Zhang Y, Vuillermin PJ, Allen KJ, Harrison LC. Immune Modulation by Vitamin D and Its Relevance to Food Allergy. Nutrients. 2015;7(8):6088–6108. 141. Trochoutsou AI, Kloukina V, Samitas K, Xanthou G. Vitamin-D in the Immune System: Genomic and Non-Genomic Actions. Mini Rev Med Chem. 2015;15(11):953–963. 142. Mocchegiani E, Costarelli L, Giacconi R, et  al. Micronutrientgene interactions related to inflammatory/immune response and antioxidant activity in ageing and inflammation. A systematic review. Mech Ageing Dev. 2014;136-137:29–49. 143. Deeks A, Lombard C, Michelmore J, Teede H. The effects of gender and age on health related behaviors. BMC Public Health. 2009;9:213. 144. Melloni C, Newby LK. Sex differences in medical care after acute myocardial infarction: what can be done to address the problem? Womens Health (Lond). 2009;5(4):339–341. 145. Smith MA, Lisabeth LD, Bonikowski F, Morgenstern LB. The role of ethnicity, sex, and language on delay to hospital arrival for acute ischemic stroke. Stroke. 2010;41(5):905–909. 146. Onah MN, Govender V. Out-of-pocket payments, health care access and utilisation in south-eastern Nigeria: a gender perspective. PLoS One. 2014;9(4):e93887. 147. Roy K, Howard DH. Equity in out-of-pocket payments for hospital care: evidence from India. Health Policy. 2007;80(2):297–307. 148. Khan MS, Sismanidis C, Godfrey-Faussett P. Factors influencing sex differences in numbers of tuberculosis suspects at diagnostic centres in Pakistan. Int J Tuberc Lung Dis. 2012;16(2):172–177. 149. Afsana K. The tremendous cost of seeking hospital obstetric care in Bangladesh. Reprod Health Matters. 2004;12(24):171–180.

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22 G­eo­epidemiology and the Impact of Sex on Autoimmune Diseases Ana Lleo Humanitas Clinical and Research Center, Milan, Italy

O U T L I N E 22.3.6 Autoimmunity and Pregnancy 22.3.7 Gut Microbiota

Abbreviations 323 22.1 Introduction

323

22.2 Geoepidemiology of Autoimmune Diseases 22.2.1 Genetic Predisposition 22.2.2 Environmental Factors

324 325 326

22.3 Sex Prevalence 22.3.1 Sex Chromosomes 22.3.2 Fetal Microchimerism in Autoimmunity 22.3.3 X-linked Genes and X-chromosome Encoded Micro RNAs 22.3.4 Epigenetics of the X-chromosome 22.3.5 Sex Hormones

326 326 326 326 327 327

22.4 The Influence of Gender

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22.5 Sex Prevalence in Autoimmune Diseases: Is There a Prognostic Role for Sex? 22.5.1 Systemic Lupus Erythematosus 22.5.2 Rheumatoid Arthritis 22.5.3 Primary Biliary Cholangitis 22.5.4 Multiple Sclerosis 22.5.5 Type 1 Diabetes Mellitus

328 328 329 329 329 329

22.6 Concluding Remarks and Future Directions

330

References 330

Abbreviations ADs Autoimmune Diseases SLE Systemic Lupus Erythematosus PBC Primary Biliary Cholangitis PSS Primary Sjögren’s Syndrome GWAS Genome Wide Association Studies HLA Human Leukocyte Antigens RA Rheumatoid arthritis MS Multiple sclerosis T1D Type 1 Diabetes ER Estrogen Receptor APC Antigen Presenting Cell DC Dendritic Cell HPA Hypothalamic-Pituitary-Adrenal FMC Fetal Microchimerism XCI X-chromosome Inactivation Treg Regulatory T FOXP3 Forkhead box P3 miRNAs microRNAs Th17 T helper type 17 Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00022-X

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22.1 INTRODUCTION Autoimmune diseases (ADs), a group of 70 different diseases, affect 5%–8% of the western population and are the fifth leading cause of mortality among young women.1–3 Geoepidemiology, the study of the distribution of the determinants of disease gradients across different regions and populations, suggests that incidence and prevalence of ADs are growing worldwide. Moreover, the frequency of ADs differs from country to country and geographic aggregates have been delineated.2 The importance of geoepidemiology does not lie solely in geographically delineating the burden of each disease, but also in advancing our understanding of its etiology, the mechanisms of immune disregulation, and triggering elements, all of which might lead to diagnosis and treatment.2 Unfortunately large, international

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© 2017 Elsevier Inc. All rights reserved.

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epidemiological investigations are lacking in the majority of ADs; on the other hand epidemiological data have been recently generated in countries with no previously available information. The variability that these disorders present, in terms of targeted tissues, age of onset, and response to immunosuppressive treatments, are key elements in order to properly understand their geoepidemiology. The one feature shared by the majority of these conditions, however, is the predominance in the female sex with over 80% of patients being women.4,5 The unbalanced sex ratio also varies significantly across the clinical spectrum. The more conspicuous sex differences are observed in Sjogren’s syndrome, systemic lupus erythematosus (SLE), and primary biliary cholangitis (PBC).4 Even though the female predisposition to AD has been known for over a century, the precise cause of this bias remains unknown and relatively few hypotheses have been proposed. Epidemiological studies have led to the identification of both genetic factors and environmental elements that are sex-specific; these might act as additional players in tolerance breakdown, to explain both ADs onset and the female predominance. Among sex-related factors that have been proposed, sex hormones and reproductive history, fetal microchimerism (FMC), X chromosome inactivation, and X chromosome abnormalities (both genetic and epigenetic) have had the most attention. However, none of these hypotheses have thus far gathered enough convincing support and in most cases data are conflicting. Moreover, differences between males and females are generally analyzed from a biological point of view,

and gender aspects are seldom incorporated. Sex and gender interact at several levels to determine the illness and effectiveness of the immune response and should be considered in concert whenever possible. Further investigation into both dimensions and especially their interaction could broaden our knowledge about sex and gender differences in immunology substantially and potentially identify unknown pathogenic pathways and novel therapeutic approaches for both women and men.

22.2  GEOEPIDEMIOLOGY OF AUTOIMMUNE DISEASES Avoiding methodological errors is essential in order to compare epidemiological data across diverse world areas, both in a specific time period and over time. Indeed, incidence and prevalence of most ADs are not homogeneous: their frequency differs from country to country, and geographic aggregates have been delineated. Some of the most common limitations entailed in geoepidemiology refer to methodological issues (e.g., comparison of data derived from large vs small studies, community-based studies versus hospital-based ones); however, temporal considerations (e.g., diagnostic advances, lack of up-todate data, improved standard of living), and socioeconomic factors (e.g., access to medical care, availability of diagnostic procedures, medical expertise) are key issues in the solidity of epidemiological studies. An illustrative example is summarized in Table 22.1; geoepidemiology of PBC has been studied across many

TABLE 22.1  Selected Studies of Prevalence, Incidence, and Sex Ratio in Primary Biliary Cholangitis Incidence (million/year)

Sex (M:F)

54

1:10

6

13.3

1:6

7

154

19

1:9

8

225

22

3.3

1:13

9

1995

69

27

2.3

1:22

10

Newcastle, UK

1997

160

240

22

1:10

11

Norway

1998

21

146

16

1:9

12

Minnesota, USA

2000

46

402

27

1:8

13

Newcastle, UK

2001

770

251

31

1:10

14

Victoria, Australia

2004

249

51

–

1:9

15

Japan

2005

9761

78

–

1:9

16

Ontario, Canada

2009

137

227

30

1:5

17

Denmark

2011

722

115

11.2

1:4

18

Lombardia, Italy

2011

2970

160

16.7

1:2

18

Patients (n)

Prevalence (per million)

Area

Year

Europe

1984

569

23

Sweden

1985

111

151

Newcastle, UK

1989

347

Ontario, Canada

1990

Estonia

References

Modified from Lleo A, Jepsen P, Morenghi E, et al. Evolving Trends in Female to Male Incidence and Male Mortality of Primary Biliary Cholangitis. Sci Rep. 2016;6:25906

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22.2  Geoepidemiology of Autoimmune Diseases

decades in several countries, leading to divergent results even in the same geographical area (i.e., Ontario, Canada) when considering two different periods of time.9,17 Indeed, previous epidemiological research on PBC, mainly European, has reported incidence rates ranging from 2 to 49 cases per million/year and prevalence of between 19 and 402 cases per million. The major strength of the most recent studies18,19 is the accurately defined numerator and denominator populations by using a validated method for case definition. Moreover, the use of administrative databases limits the selection bias inherent in studies restricted to a few health care providers or tertiary referral centers. They cannot be freely compared to other epidemiological studies,15,20,21 even those performed in the same region,9 due to the use of a completely different method. Using an assortment of particular geoepidemiology tools can merge a specific variety of etiopathogenic factors. In particular, methods for characterizing genetic risk include comparative study of ethnic differences and matching genetic data with geographical disease gradients. In contrast, environmental influences are typically unraveled by gradients—latitudinal, rural-urban or global—temporal phenomena—cyclic and secular—and lifestyle differences. Finally there are methods that could possibly distinguish genetic from environmental factors, which are migration studies and hot-spots.2 Of course, most of the methods interact and overlap inside scientific work and are only artificially separated. Indeed, only by combining as many as possible of the available approaches, may the full target of ascertaining genetic and environmental mechanisms of autoimmunity be reached. Improved longitudinal data are needed to make robust causal determinations about ADs. However, solid comparisons between locally-enrolled patient series can only be based on universal case-finding methods and diagnostic criteria. The first problem in studying the epidemiology of ADs is the difficulty in defining diagnostic criteria. In addition, while extensive information is collected at national level, better international coordination of survey design and implementation would help facilitate and improve cross-country comparisons. Meta-analyses do not appear to be the best method to describe clinical phenomena any longer, due to the inequities of methodology in different studies (discrepancies in diagnostic/inclusion criteria, variability of case-ascertainment methods, no standardization, etc.). These biases are likely to be overcome by an emerging tool in the hands of the clinical epidemiologist: the large administrative database. This type of data source can really improve our knowledge, not only applied to ADs, but in each field of clinical epidemiology; it is population-wide, inexpensive, and independent of selection biases.19,22

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22.2.1  Genetic Predisposition The importance of genetic factors in AD susceptibility is supported by familiar clustering, variable prevalence in different ethnic groups, associations with HLA haplotypes or single nucleotide polymorphisms, and concordance rates in monozygotic and dizygotic twins.23 It is well accepted that genetic polymorphisms contribute to autoimmune susceptibility, and thereby underlie ethnic differences in the presentation of the disease, or the severity of its symptoms.1,24 Indeed, monogenic ADs are extremely rare.25 Nevertheless, ADs are recognized to be the consequence of the interaction between polygenic risk factors and environmental factors.26 For long time, the genetic factors that regulate normal immune responses have been suspected to have a role in loss of tolerance. In addition to genes encoding MHC antigens, genes affecting antigen processing and presentation, lymphocyte proliferation and differentiation, genes encoding immunoglobulins, T cell receptors for antigen, and the molecules that control immune destruction have all been considered.24,27 Genome-wide association studies (GWAS), currently available for the most common ADs,2,28–34 have recently provided new insights into the genetic susceptibility to autoimmune disorders and the biological pathways necessary for the maintenance of immune homeostasis. However the most up-to-date results point out that genetic patterns associated with ADs are strongly variable:multiple genes are involved in determining disease susceptibility, and only few diseases share common genetic risks. Identification and characterization of the common variants in ADs have been more challenging because of their relatively small individual contributions to disease risk. Indeed, reported genetic associations show limited clinical significance, since they only account for a minority of patients.35–38 After the first wave of GWAS data, several general conclusions are warranted. First, genetic predisposition to each AD is based on multiple genes, and most of the associations disclosed by GWAS are relatively modest. Second, the HLA region has been confirmed as one of the major components of the genetic architecture of most ADs. Third, multiple AD clearly share some genetic variants, thus suggesting that many of these diseases have common pathogenic pathways. On the other hand, the lack of common genetic variants suggests the concomitant presence of specific mechanisms. However, the data now available (even if partial) indicate that autoimmunity results from a wide range of different pathways with large genetic heterogeneity that only in part concern immune-related genes and strongly suggest new hypotheses. Finally, the causative genetic variants at the base of the associations have not been identified in most ADs.

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22.2.2  Environmental Factors The role of environmental factors in the development of autoimmunity has been extensively demonstrated.2,28,39 Sex hormones have been the first proposed candidates to have important roles in the sex bias observed in autoimmunity. However, evidence implicates several other factors, which include socioeconomic status, infectious agents, environmental pollutants, vitamin D (dependent on sunlight exposure), nutrition, drugs, as well as physical and psychological stresses in the pathogenesis of autoimmunity.19,40–43 Moreover, environmental factors should be taken into consideration when talking about the latitudinal gradient of ADs; indeed some environmental factors, i.e., infectious agents as well as chemical compounds,44 have different geographical distributions. Tobacco smoking affects both innate and adoptive immunity, leading to the production of antibodies recognizing citrullinated proteins in RA or by the elevation of anti-dsDNA titers in SLE.45 Another emerging environmental factor involved in autoimmunity seems to be ultraviolet radiation, which had already been established to be intricately linked to the functions of the skin’s immune system. In predisposed patients, UV light can turn pathogenic inflammatory pathways on, leading to either atopy or autoimmune disorders. In other individuals, the same UV radiation is used as a phototherapy to switch off pathogenic immune aggression in the skin. These widely different features are a direct result of UV light’s ability to ionize molecules in the skin and alter its chemical composition, sometimes in essential ways (e.g., formation of previtamin D(3) from 7-dehydrocholesterol). In other cases there may be potential injury (like bondage of the DNA of adjacent pyrimidine bases). Cells often avoid malignant transformation by undergoing apoptosis, but that also potentially yields autoreactive immune responses by exposing the cell’s nuclear antigens.42

22.3  SEX PREVALENCE 22.3.1  Sex Chromosomes Major clues, including the observation that several genes crucial for the maintenance of immune function and tolerance map on the X-chromosome, have suggested a key role of sex chromosomes in ADs. In fact, specific mutations of X-chromosome genes cause immunodeficiency syndromes characterized by different degrees of severity.46 Further, constitutive X monosomy or major structural abnormalities of the X chromosome— as observed in Turner’s syndrome47—lead to common autoimmune features.48 X chromosome inheritance displays a peculiar pattern compared to autosomal chromosomes, since women are functional mosaics for X-linked genes. Although one

of the two X chromosomes in females is almost completely inactive, approximately 15% of X-linked genes escape inactivation in healthy women.49 Importantly, women with ADs have a significantly higher frequency of peripheral blood cells with a single X chromosome (i.e., X monosomy) compared to healthy women. This was observed in diseases with different organ specificities, such as scleroderma and autoimmune thyroid disease50 or PBC.51 Moreover, the lost X chromosome is preferentially parentally inherited.52 Other authors have suggested that women affected with specific femalepreponderant ADs manifest a skewed XCI pattern in their peripheral white blood cells, as supported by data in scleroderma and autoimmune thyroid diseases,53,54 while in other ADs, i.e., PBC, such preferential inactivation has not been demonstrated.52

22.3.2  Fetal Microchimerism in Autoimmunity It has been hypothesized that female predominance in AD might be a consequence of the presence in affected women of allogenic male fetal cells even decades after pregnancy, i.e., FMC. The first report of FMC in autoimmunity was in systemic sclerosis by Nelson and colleagues who found an increased level of male DNA in women with scleroderma compared to controls.55 However, other studies have failed to reproduce these findings.56 FMC has been investigated in different ADs, such as systemic sclerosis, SLE, autoimmune thyroid diseases, PBC, and juvenile inflammatory myopathies. In PBC a number of reports failed to confirm a role for FMC.57 Cumulatively, available data on the role of FMC in AD are still controversial and, while some studies lend support to the concept that FMC is involved in the pathogenesis of selected AD, studies also indicate FMC is not uncommon in healthy, i.e., unaffected, individuals.58

22.3.3  X-linked Genes and X-chromosome Encoded Micro RNAs The X-chromosome encodes a number of immuneassociated genes, including CD40L, CXCR3, FOXP3, TLR7, IL2RG, and IL9R, among others.59 The role of intracellular TLRs in the development of autoimmunity has been extensively studied.60 Briefly, it is well accepted that TLR7 and TLR9 are among the critical players during the development of lupus-like autoimmunity. However, they play different roles in the pathogenesis of murine lupus. TLR7 deficiency determines a partial protection from lupus in mice while TLR9 deficient mice show a worsening of the disease.61,62 More recent studies suggest that TLR9 signaling plays a protective role, involving a suppression of the TLR7dependent anti-RNP antibodies production.63

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22.3  Sex Prevalence

CD4+CD25+ regulatory T (Treg) cells play a pivotal role in the maintenance of immune homeostasis, where the X-linked master transcription factor forkhead box P3 (FOXP3) determines Treg cell development and function. Genetic deficiency, or mutation, of FOXP3 leads to an early onset, highly aggressive, and often fatal multiorgan AD.64 Finally, microRNAs (miRNAs) may also be governed by sex differences, and therefore contribute to susceptibility to autoimmunity. It has been reported that miRNAs are differentially expressed between males and females;65 however, it is not clear what drives that differential expression. The X chromosome is enriched in miRNAs66: about 7% (113 miRNAs) of human miRNAs are encoded on the X chromosome, while only two miRNAs have been reported on the Y chromosome. Although the functions of the majority of X-linked miRNAs remain unknown, some are reported to play a role in the regulation of immune responses or are associated with ADs.67–70

22.3.4  Epigenetics of the X-chromosome Epigenetic mechanisms act at the interface of genetic and environmental influences on human phenotype and disease risk by altering gene expression levels without altering DNA sequence or chromosome structure. In the case of ADs, epigenetic mechanisms have been implicated in the pathogenesis of SLE71,72 and type I diabetes.73 We had previously explored the epigenetic component of the X chromosome in PBC74,75 and demonstrated that CXCR3 promoter is demethylated in CD4+ T cells from PBC patients, leading to a significantly higher expression of CXCR3 in the same cell subtype.76 Moreover, CXCR3 expression levels in CD4+ T cells were significantly associated with duration of disease. However, whether CXCR3 may contribute to the female susceptibility to PBC warrants further investigation.

22.3.5  Sex Hormones The effects of sex hormones on the immune function were first based on the reported role of estrogens in lymphocyte maturation, activation, and synthesis of antibodies and cytokines.77–79 Estrogens, androgens, and prolactin have been implicated as the main factors in autoimmunity triggering and development, due to their ability to modulate the immune response.80,81 Sex hormones may also directly influence the homing of lymphocytes to a target organ and the process of antigen presentation,82 thus influencing the organ specificity of ADs as well as the breakdown of tolerance. Moreover, it has been recently demonstrated that estrogens play a crucial role in regulating the secretion of the autoimmune regulator (AIRE), a key thymic transcription factor in the development of central tolerance.83

327

Estrogens stimulate Th2 lymphocytes to secrete IL-4, IL-5, IL-6, and IL-10, are involved in B cell maturation, and promote the survival and activation of autoreactive B cells.84 Interestingly, estrogens and prolactin per se share the potential to break tolerance and lead to the appearance of DNA-reactive B cells.85 Estrogens are known to have a biphasic effect. Lower levels facilitate the immune response while higher levels suppress it. It has been demonstrated, e.g., that estrogens generally stimulate IFN-γ secretion but the peak enhancement is at lower levels of estrogen concentration.86 Importantly, the ability of estradiol and ER signaling to augment type I Interferon pathways may help to explain the sex bias in SLE, an AD characterized by high levels of IFN.87 The effect of estrogens on the secretion of TNF-αβ is also biphasic, with enhancement occurring at low and inhibition at high concentrations.88 These data indicate that estrogen is capable of modulating both pro- and anti inflammatory activities of CD4+ T cells and thus has the potential to influence the outcome of CD4+ T cell-mediated immune responsiveness. Sex hormones also influence the hypothalamicpituitary-adrenal (HPA) axis and are able to modulate the stress response. In fact, women have higher cortisol concentrations compared to men.89 Complex threeway interactions between these systems appear to be involved in the gender dimorphism of the immune system. Glucocorticoid response to stress, including immune challenge, is strongly inhibited by androgens and enhanced by estrogens. At the same time, glucocorticoids suppress the production of sex hormones and the effect of these hormones in tissues contributing therefore to the immune response itself.89,90 Possible interactions at the cell and gene level, with mutual antagonism or synergy between cortico- and gonadal steroids, open new exciting hypotheses that await clarification. Finally, a correlation between apoptosis and ADs, possibly through the ineffective removal of apoptotic cells, is well accepted.91 Both estrogens and prolactin may regulate immune cell survival through the Fas/ FasL system which is influenced in terms of FasL expression in monocytes,92 yet whether these two hormones play a direct role remains to be determined.

22.3.6  Autoimmunity and Pregnancy During pregnancy fetal antigens challenge the maternal immune system and lead to key modifications of the immune balance, even in healthy woman, in order to assure fetal tolerance, and therefore safety. For example, T helper type 17 (Th17) and Treg cells are active players in the establishment of tolerance and defense. The immune response is indeed characterized by a fine balance between Treg and Th17 response. However, during

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pregnancy Treg cells will dominate Th17 cells to guarantee fetal survival.93 Autoimmunity stigmata, in the absence of clinically manifest AD, can affect every aspect of pregnancy. A large variety of autoantibodies have been investigated for their potential association with unexplained infertility often with inconclusive results.94 Antiphospholipid syndrome and antithyroid antibodies have been implicated in pregnancy losses. Numerous studies have investigated the association between thyroid autoimmunity and miscarriage, demonstrating that presence of thyroid autoantibodies—even in the absence of clinical or subclinical thyroid dysfunction—increases the risk of miscarriage, particularly of pregnancy losses occurring in the first trimester.95 There appears to be considerable geographic variation in the prevalence of thyroid autoantibodies. One of the most severe complications of pregnancy is preeclampsia, which affects 1–5% of pregnancies worldwide in otherwise healthy women. The frequency is much higher in a variety of ADs, e.g., T1D, SLE, systemic sclerosis, RA, and other connective tissue diseases.96 Of note, fertility does not appear to be impaired in most ADs, except in women treated with cytotoxic agents. Nonetheless, pregnancies in most ADs are still classified as high risk because of the potential for major complications. These complications include disease exacerbations during gestation and increased perinatal mortality and morbidity in most ADs. Fetal mortality is a characteristic of the antiphospholipid syndrome.

22.3.7  Gut Microbiota The gastrointestinal tract hosts an extensive microbial community with which dynamic interactions have been established over centuries of coevolution. Gut-associated immune cells and innate receptors expressed in gut epithelia interact with commensal bacteria and their products, thereby promoting the proper development of the mucosal immune system and host homeostasis. Many studies have demonstrated that host–microbiota interactions play a key role during local and systemic immunity and that some ADs are associated with variations and/ or specific characteristics of the microbiota. Indeed, an effect of the microbiota has been also reported for the models of ankylosing spondylitis,97 RA,98 and Omenn syndrome;99 however, no effect has been described in the severity or the prevalence of lupus in GF MRLlpr mice,100 suggesting that the role of microbiota in ADs remains controversial and needs deeper elucidation.

22.4  THE INFLUENCE OF GENDER Differences between women and men can be identified at many levels of the immune response and might

affect its eventual outcome. The human immune system manifests some degree of sexual dimorphism with basic immune responses differing between females and males. In general terms, women have an enhanced antibody production and increased cell-mediated responses following immunization,101 while men produce a more intense inflammatory response to infectious organisms.102 Further, women have higher CD4+ T cell counts than men which contributes to an increased CD4/CD8 ratio,103 higher levels of plasma IgM,104 and greater Th1 cytokine production.105 The significance of these changes remains poorly defined since, with the obvious exception of ADs, there do not appear to be significant differences in susceptibility to infection or inflammation degrees between sexes. In addition to the influence of biological differences, gender specific psychosocial, cultural, and economic factors106 have a great influence on the immune response.107–109 Gender aspects are seldom included in studies of the immune response. Gender interacts with genetics in the development and outcomes of the immune response.107 Differences in societal roles will determine different exposure patterns to antigens. In addition, micronutrient intake as well as access to preventive health care facilities will be affected. Outcomes are also significantly influenced by gender. Gender differences will potentially limit access to antibiotics and immunomodulating agents and modify the immune response. Environmental factors during pregnancy can act as both protectors and risk factors for the developing fetus. Sex and gender interact at several levels in order to determine the immune response; further investigation into the sex–gender interaction will lead to the identification of unknown pathogenic pathways and novel therapeutic approaches for both men and women.

22.5  SEX PREVALENCE IN AUTOIMMUNE DISEASES: IS THERE A PROGNOSTIC ROLE FOR SEX? The most striking sex differences in ADs are observed in Sjogren’s syndrome, SLE, PBC, autoimmune thyroid disease, and scleroderma in which 80% of the patients are women. On the other hand, RA, MS, and myasthenia gravis have a lower female prevalence; nevertheless, 60% of the patients are women.23 As well as prevalence, the severity of symptoms and the degree of disability in ADs, may also differ between males and females. However, this is not easily defined.

22.5.1  Systemic Lupus Erythematosus SLE is an autoimmune disorder involving several organs and apparatus, with multifaceted clinical signs

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22.5  Sex Prevalence in Autoimmune Diseases: Is There a Prognostic Role for Sex?

and symptoms and immunological alterations. It occurs predominantly in female patients,108 with a sex ratio ranging from 5:1110 to 25:1.111 Annual incidence and prevalence vary from 2 and 19 to 8 and 241 per 100,000, respectively.112 Although accurate current data on its incidence and prevalence are largely lacking, there are numerous indications that SLE is far less common in Europeans and their descendants compared to all other ethnicities. The clinical manifestations of the disease show geographic or ethnic variation, generally being less severe in patients of European ancestry than in African, Asian, certain “Hispanic” or mestizo, and various indigenous populations. In particular, renal involvement is far more common in non-European patients. Genetic as well as environmental, sociodemographic, and sociocultural factors are likely to contribute to the differences in the incidence and clinical expression of SLE.109 Crosslin et al. recently showed greater disease severity in male SLE patients; indeed, male patients were more likely to suffer renal and cardiovascular comorbidities while female patients were more likely to suffer from urinary tract infections, hypothyroidism, depression, esophageal reflux, asthma, and fibromyalgia.113

22.5.2  Rheumatoid Arthritis Rheumatoid arthritis (RA) is a chronic rheumatic disease featured by bone and joint destruction with a particular “pattern” of damage. Individuals with RA have an increased risk of death for any cause.1 Incidence and prevalence of RA vary across different populations depending upon study methodology, and disease defining criteria. In North America and Northern Europe, the estimated incidence of RA is around 20–50 cases per 100,000 population/year and prevalence is at 0.5–1.1%. Lower presence of RA has been reported in Mediterranean countries, and scarce information is available about the disease in developing and Third World countries. Some experts reported declining incidences and prevalences in the last 50 years.114–117 Female-to-male ratio is usually documented to be between 2:1118 and 4:1.119 RA is a multifactorial disease that results from interactions between genetic and environmental factors. The chief genetic factors involved in RA are the presence of HLA-DRB1 and the tyrosinephosphatase gene PTPN22. Considering how environment influence may be implicated in the development of RA, smoking is demonstrated to have the strongest association with RA predisposition and is also a predictor of worse prognosis.1

22.5.3  Primary Biliary Cholangitis PBC is considered a disease predominantly affecting women with female:male ratios of up to 10:1 and is

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therefore a prime example of the characteristic sexual dimorphism in autoimmunity. There is a wide variation among studies of incidence and prevalence rates, with rates increasing over time.120–122 Carbone et  al. in a recent study from the United Kingdom, showed that male sex was an independent predictor of nonresponse on multivariate analysis to ursodeoxycholic acid (the only approved therapy).123 The same study also reported that men were less likely to be symptomatic, which may be a cause of delay in diagnosis.123 Moreover, we have recently compared the overall survival, incidence, and prevalence of PBC in two well-defined population-based studies over a recent decade, considering also sex ratios and mortality. Our data indicate for PBC a sex ratio significantly lower than previously cited, a reversal of the usual latitudinal difference in prevalence, and a surprisingly higher overall mortality for male patients.18

22.5.4  Multiple Sclerosis Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system characterized by demyelinzation and, in most cases, by a chronic course with relapses and remissions. Like the majority of complex diseases, pathogenesis is associated with exposure to environmental factors in a field of genetic susceptibility. The epidemiology of MS has been widely analyzed, in particular the geographic distribution of the disease, the influence of immigration and age on disease incidence, clustering, and epidemics.124 The disease is irregularly distributed worldwide, and its prevalence varies from 100 cases per 100,000 in temperate areas, especially those with large populations of Northern European origin, including North America and Oceania.125 Female patients are more affected than male, with a F/M ratio of 4/1. Many hypothetical risk factors are discussed, such as ultraviolet radiation, vitamin D, Epstein–Barr virus and infectious mononucleosis, other infectious agents and noninfectious factors. Two different hypotheses, the hygiene hypothesis and the prevalence hypothesis, were proposed to explain these environmental risk factors for MS. The epidemiological parameters, matched with pathological and immunological data, may contribute to the debate about how much the immune system contributes to the pathogenesis of MS, so it can be considered an AD, or a neurodegenerative disease sustained by a persistent or latent infection.124

22.5.5  Type 1 Diabetes Mellitus Type 1 diabetes (T1D) is a chronic AD characterized by total abolition of insulin secretion, resulting from the progressive destruction of pancreatic islet beta cells by the

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immune system. Environmental factors and genetic susceptibility are thought to be the pathogenic triggers. The major genetic influence comes from HLA complex loci, in particular HLA class II. The worldwide incidence of T1D varies by at least 100-fold, being highest in Finland, Sardinia, and Italy—(around 40 per 100,000 people/year) and lowest in Venezuela and China (0.1–4.5 per 100,000/ year).126 Annual incidence has been increasing worldwide at a rate of approximately 3% per year. Of note, genetics is likely to explain some of the topographic variability in T1D prevalence, but it cannot account for its rapidly increasing incidence. Childhood-onset T1D is generally characterized by a male:female ratio close to 1,127 whereas adult-onset disease shows a male predominance (approximately 1.5:1).128 Moreover, the changing ratio between newly diagnosed children with high-risk genotypes, over low-risk ones, suggests that environmental pressures are now able to switch T1D on in individuals who wouldn’t otherwise have developed the disease. Although comparative studies between countries and regions with low and high-incidence rates have suggested that higher socioeconomic status and degree of urbanization are associated to the increasing incidence of T1D, the findings are not firmly conclusive. Considerable geographic variation also exists in morbidity and mortality as well as causes of death. The role of genetics in prognosis as well as susceptibility to nephropathy, retinopathy, and other diabetic complications largely remain to be explored.109

22.6  CONCLUDING REMARKS AND FUTURE DIRECTIONS Geoepidemiological studies and clustering analyses are essential tools to define the associated risk of single environmental and genetic factors. The field of autoimmunity is clearly showing how the findings of basic science influence routine clinical practice in terms of diagnostic procedures, clinical management, and prediction of outcome. However, continuous effort should be aimed at collecting additional information on the incidence and prevalence of these rare conditions in additional geographical regions, not only in the so-called developed countries. If hormones are playing a central role in the pathogenesis, population studies based on measurement of hormone levels in a healthy cohort should be taken into consideration, thus comparing cluster areas. Second, we are convinced that the study of female autoimmunity cannot be complete without a careful evaluation of male cases which should be collected through a multicenter effort to achieve sufficient numbers of individuals. Third, the role of sex hormones, particularly estrogens, should be reevaluated with more modern research tools. Ultimately, the study of sex differences in autoimmunity will help toward a better

definition of the mechanisms leading to the widely different clinical features of AD, permitting a clearer delineating of cases more likely to progress and/or present major complications, thus enabling the development of more focused and novel therapeutic approaches.

References 1. Tobon GJ, Youinou P, Saraux A. The environment, geo-epidemiology, and autoimmune disease: rheumatoid arthritis. Autoimmun Rev. 2010;9(5):A288–A292. 2. Shapira Y, Agmon-Levin N, Shoenfeld Y. Defining and analyzing geoepidemiology and human autoimmunity. J Autoimmun. 2010;34(3):J168–J177. 3. Moroni L, Bianchi I, Lleo A. Geoepidemiology, gender and autoimmune disease. Autoimmun Rev. 2012;11(6–7):A386–A392. 4. Cooper GS, Stroehla BC. The epidemiology of autoimmune diseases. Autoimmun Rev. 2003;2(3):119–125. 5. Eaton WW, Rose NR, Kalaydjian A, Pedersen MG, Mortensen PB. Epidemiology of autoimmune diseases in Denmark. J Autoimmun. 2007;29(1):1–9. 6. Triger DR, Berg PA, Rodes J. Epidemiology of primary biliary cirrhosis. Liver. 1984;4(3):195–200. 7. Lofgren J, Jarnerot G, Danielsson D, Hemdal I. Incidence and prevalence of primary biliary cirrhosis in a defined population in Sweden. Scand J Gastroenterol. 1985;20(5):647–650. 8. Myszor M, James OF. The epidemiology of primary biliary cirrhosis in north-east England: an increasingly common disease? Q J Med. 1990;75(276):377–385. 9. Witt-Sullivan H, Heathcote J, Cauch K, et  al. The demography of primary biliary cirrhosis in Ontario, Canada. Hepatology. 1990;12(1):98–105. 10. Remmel T, Remmel H, Uibo R, Salupere V. Primary biliary cirrhosis in Estonia. With special reference to incidence, prevalence, clinical features, and outcome. Scand J Gastroenterol. 1995;30(4):367–371. 11. Metcalf JV, Bhopal RS, Gray J, Howel D, James OF. Incidence and prevalence of primary biliary cirrhosis in the city of Newcastle upon Tyne, England. Int J Epidemiol. 1997;26(4):830–836. 12. Boberg KM, Aadland E, Jahnsen J, Raknerud N, Stiris M, Bell H. Incidence and prevalence of primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis in a Norwegian population. Scand J Gastroenterol. 1998;33(1):99–103. 13. Kim WR, Lindor KD, Locke 3rd GR, et  al. Epidemiology and natural history of primary biliary cirrhosis in a US community. Gastroenterology. 2000;119(6):1631–1636. 14. Prince MI, Chetwynd A, Diggle P, Jarner M, Metcalf JV, James OF. The geographical distribution of primary biliary cirrhosis in a well-defined cohort. Hepatology. 2001;34(6):1083–1088. 15. Sood S, Gow PJ, Christie JM, Angus PW. Epidemiology of primary biliary cirrhosis in Victoria, Australia: high prevalence in migrant populations. Gastroenterology. 2004;127(2):470–475. 16. Sakauchi F, Mori M, Zeniya M, Toda G. A cross-sectional study of primary biliary cirrhosis in Japan: utilization of clinical data when patients applied to receive public financial aid. J Epidemiol. 2005;15(1):24–28. 17. Myers RP, Shaheen AA, Fong A, et al. Epidemiology and natural history of primary biliary cirrhosis in a Canadian health region: a population-based study. Hepatology. 2009;50(6):1884–1892. 18. Lleo A, Jepsen P, Morenghi E, et al. Evolving Trends in Female to Male Incidence and Male Mortality of Primary Biliary Cholangitis. Sci Rep. 2016;6:25906. 19. Ensenberger MG, Thompson J, Hill B, et  al. Developmental validation of the PowerPlex 16 HS System: an improved

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

16-locus fluorescent STR multiplex. Forensic Sci Int Genet. 2010;4(4):257–264. 20. Gershwin ME, Selmi C, Worman HJ, et  al. Risk factors and comorbidities in primary biliary cirrhosis: a controlled interview-based study of 1032 patients. Hepatology. 2005;42(5): 1194–1202. 21. James OF, Bhopal R, Howel D, Gray J, Burt AD, Metcalf JV. Primary biliary cirrhosis once rare, now common in the United Kingdom? Hepatology. 1999;30(2):390–394. 22. Lleo A, Oertelt-Prigione S, Bianchi I, et al. Y chromosome loss in male patients with primary biliary cirrhosis. J Autoimmun. 2013;41:87–91. 23. Whitacre CC. Sex differences in autoimmune disease. Nat Immunol. 2001;2(9):777–780. 24. Invernizzi P. Future directions in genetic for autoimmune diseases. J Autoimmun. 2009;33(1):1–2. 25. Cheng MH, Anderson MS. Monogenic autoimmunity. Annu Rev Immunol. 2012;30:393–427. 26. Marson A, Housley WJ, Hafler DA. Genetic basis of autoimmunity. J Clin Invest. 2015;125(6):2234–2241. 27. Jacobson EM, Tomer Y. The CD40, CTLA-4, thyroglobulin, TSH receptor, and PTPN22 gene quintet and its contribution to thyroid autoimmunity: back to the future. J Autoimmun. 2007;28(2-3):85–98. 28. Shapira Y, Agmon-Levin N, Shoenfeld Y. Geoepidemiology of autoimmune rheumatic diseases. Nat Rev Rheumatol. 2010;6(8):468–476. 29. Hom G, Graham RR, Modrek B, et al. Association of systemic lupus erythematosus with C8orf13-BLK and ITGAM-ITGAX. N Engl J Med. 2008;358(9):900–909. 30. Reddy MVPL, Wang H, Liu S, et al. Association between type 1 diabetes and GWAS SNPs in the southeast US Caucasian population. Genes Immun. 2011;12(3):208–212. 31. Liu X, Invernizzi P, Lu Y, et  al. Genome-wide meta-analyses identify three loci associated with primary biliary cirrhosis. Nat Genet. 2010;42(8):658–660. 32. Lewis SN, Nsoesie E, Weeks C, Qiao D, Zhang L. Prediction of disease and phenotype associations from genome-wide association studies. PloS One. 2011;6(11):e27175. 33. Strange A, Capon F, Spencer CC, et  al. A genome-wide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1. Nat Genet. 2010;42(11):985–990. 34. de Boer YS, van Gerven NM, Zwiers A, et  al. Genomewide association study identifies variants associated with autoimmune hepatitis type 1. Gastroenterology. 2014;147(2): 443–452. e5. 35. Altshuler D, Daly MJ, Lander ES. Genetic mapping in human disease. Science. 2008;322(5903):881–888. 36. Mackay IR. Clustering and commonalities among autoimmune diseases. J Autoimmun. 2009;33(3–4):170–177. 37. Gregersen PK, Olsson LM. Recent advances in the genetics of autoimmune disease. Annu Rev Immunol. 2009;27:363–391. 38. Youinou P, Pers JO, Gershwin ME, Shoenfeld Y. Geo-epidemiology and autoimmunity. J Autoimmun. 2010;34(3):J163–J167. 39. Gianchecchi E, Fierabracci A. Gene/environment interactions in the pathogenesis of autoimmunity: new insights on the role of Toll-like receptors. Autoimmun Rev. 2015;14(11):971–983. 40. Tyl RW, Marr MC, Brown SS, Dolbow EA, Myers CB. Validation of the intact rat weanling uterotrophic assay with notes on the formulation and analysis of the positive control chemical in vehicle. J Appl Toxicol. 2010;30(7):694–698. 41. Myers RP, Shaheen AA, Fong A, et  al. Validation of coding algorithms for the identification of patients with primary biliary cirrhosis using administrative data. Can J Gastroenterol. 2010;24(3):175–182.

331

42. Grams ME, Plantinga LC, Hedgeman E, et al. Validation of CKD and related conditions in existing data sets: a systematic review. Am J Kidney Dis. 2011;57(1):44–54. 43. Raichur S, Fitzsimmons RL, Myers SA, et al. Identification and validation of the pathways and functions regulated by the orphan nuclear receptor, ROR alpha1, in skeletal muscle. Nucleic Acids Res. 2010;38(13):4296–4312. 44. Selmi C. The worldwide gradient of autoimmune conditions. Autoimmun Rev. 2010;9(5):A247–A250. 45. Myers JR. Validation of a DNA quantitation method on the Biomek(R) 3000. J Forensic Sci. 2010;55(6):1570–1575. 46. Valiaho J, Riikonen P, Vihinen M. Novel immunodeficiency data servers. Immunol Rev. 2000;178:177–185. 47. Ranke MB, Saenger P. Turner’s syndrome. Lancet. 2001;358(9278):309–314. 48. Milkiewicz P, Heathcote J. Can Turner syndrome teach us about the pathogenesis of chronic cholestasis? Hepatology. 2004;40(5):1226–1228. 49. Carrel L, Willard HF. X-inactivation profile reveals extensive variability in X-linked gene expression in females. Nature. 2005;434(7031):400–404. 50. Invernizzi P, Miozzo M, Selmi C, et  al. X chromosome monosomy: a common mechanism for autoimmune diseases. J Immunol. 2005;175(1):575–578. 51. Invernizzi P, Miozzo M, Battezzati PM, et  al. Frequency of monosomy X in women with primary biliary cirrhosis. Lancet. 2004;363(9408):533–535. 52. Miozzo M, Selmi C, Gentilin B, et al. Preferential X chromosome loss but random inactivation characterize primary biliary cirrhosis. Hepatology. 2007;46(2):456–462. 53. Ozbalkan Z, Bagislar S, Kiraz S, et  al. Skewed X chromosome inactivation in blood cells of women with scleroderma. Arthritis Rheum. 2005;52(5):1564–1570. 54. Brix TH, Knudsen GP, Kristiansen M, Kyvik KO, Orstavik KH, Hegedus L. High frequency of skewed X-chromosome inactivation in females with autoimmune thyroid disease: a possible explanation for the female predisposition to thyroid autoimmunity. J Clin Endocrinol Metab. 2005;90(11):5949–5953. 55. Nelson JL, Furst DE, Maloney S, et  al. Microchimerism and HLA-compatible relationships of pregnancy in scleroderma. Lancet. 1998;351(9102):559–562. 56. Murata H, Nakauchi H, Sumida T. Microchimerism in Japanese women patients with systemic sclerosis. Lancet. 1999;354(9174):220. 57. Schoniger-Hekele M, Muller C, Ackermann J, et al. Lack of evidence for involvement of fetal microchimerism in pathogenesis of primary biliary cirrhosis. Dig Dis Sci. 2002;47(9):1909–1914. 58. Nelson JL. The otherness of self: microchimerism in health and disease. Trends Immunol. 2012;33(8):421–427. 59. Libert C, Dejager L, Pinheiro I. The X chromosome in immune functions: when a chromosome makes the difference. Nat Rev Immunol. 2010;10(8):594–604. 60. Jimenez-Dalmaroni MJ, Gerswhin ME, Adamopoulos IE. The critical role of toll-like receptors--From microbial recognition to autoimmunity: a comprehensive review. Autoimmun Rev. 2016;15(1):1–8. 61. Lau CM, Broughton C, Tabor AS, et  al. RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement. J Exp Med. 2005;202(9): 1171–1177. 62. Christensen SR, Kashgarian M, Alexopoulou L, Flavell RA, Akira S, Shlomchik MJ. Toll-like receptor 9 controls antiDNA autoantibody production in murine lupus. J Exp Med. 2005;202(2):321–331. 63. Nickerson KM, Christensen SR, Shupe J, et  al. TLR9 regulates TLR7- and MyD88-dependent autoantibody production and

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

332

22.  GEOEPIDEMIOLOGY, SEX AND AUTOIMMUNE DISEASES

disease in a murine model of lupus. J Immunol. 2010;184(4): 1840–1848. 64. Nie J, Li YY, Zheng SG, Tsun A, Li B. FOXP3( + ) Treg Cells and Gender Bias in Autoimmune Diseases. Front Immunol. 2015;6:493. 65. Morgan CP, Bale TL. Sex differences in microRNA regulation of gene expression: no smoke, just miRs. Biol Sex Differ. 2012;3(1):22. 66. Pinheiro I, Dejager L, Libert C. X-chromosome-located microRNAs in immunity: might they explain male/female differences? The X chromosome-genomic context may affect X-located miRNAs and downstream signaling, thereby contributing to the enhanced immune response of females. Bioessays. 2011;33(11):791–802. 67. Simpson LJ, Ansel KM. MicroRNA regulation of lymphocyte tolerance and autoimmunity. J Clin Invest. 2015;125(6):2242–2249. 68. Grigoryev YA, Kurian SM, Hart T, et al. MicroRNA regulation of molecular networks mapped by global microRNA, mRNA, and protein expression in activated T lymphocytes. J Immunol. 2011;187(5):2233–2243. 69. Fulci V, Scappucci G, Sebastiani GD, et  al. miR-223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis. Hum Immunol. 2010;71(2):206–211. 70. Wang P, Gu Y, Zhang Q, et al. Identification of resting and type I IFN-activated human NK cell miRNomes reveals microRNA-378 and microRNA-30e as negative regulators of NK cell cytotoxicity. J Immunol. 2012;189(1):211–221. 71. Strickland FM, Richardson BC. Epigenetics in human autoimmunity. Epigenetics in autoimmunity - DNA methylation in systemic lupus erythematosus and beyond. Autoimmunity. 2008;41(4):278–286. 72. Zhao M, Liu S, Luo S, et al. DNA methylation and mRNA and microRNA expression of SLE CD4+ T cells correlate with disease phenotype. J Autoimmun. 2014;54:127–136. 73. Stefan M, Zhang W, Concepcion E, Yi Z, Tomer Y. DNA methylation profiles in type 1 diabetes twins point to strong epigenetic effects on etiology. J Autoimmun. 2014;50:33–37. 74. Selmi C, Feghali-Bostwick CA, Lleo A, et  al. X chromosome gene methylation in peripheral lymphocytes from monozygotic twins discordant for scleroderma. Clin Exp Immunol. 2012;169(3):253–262. 75. Lleo A, Liao J, Invernizzi P, et  al. Immunoglobulin M levels inversely correlate with CD40 ligand promoter methylation in patients with primary biliary cirrhosis. Hepatology. 2012;55(1):153–160. 76. Lleo A, Zhang W, Zhao M, et  al. DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis. Clin Epigenetics. 2015;7(1):61. 77. Medina KL, Kincade PW. Pregnancy-related steroids are potential negative regulators of B lymphopoiesis. Proc Natl Acad Sci USA. 1994;91(12):5382–5386. 78. Medina KL, Garrett KP, Thompson LF, Rossi MI, Payne KJ, Kincade PW. Identification of very early lymphoid precursors in bone marrow and their regulation by estrogen. Nat Immunol. 2001;2(8):718–724. 79. Grimaldi CM, Cleary J, Dagtas AS, Moussai D, Diamond B. Estrogen alters thresholds for B cell apoptosis and activation. J Clin Invest. 2002;109(12):1625–1633. 80. Cutolo M, Sulli A, Straub RH. Estrogen metabolism and autoimmunity. Autoimmun Rev. 2012;11(6–7):A460–A464. 81. Gonzalez DA, Diaz BB, Rodriguez Perez Mdel C, Hernandez AG, Chico BN, de Leon AC. Sex hormones and autoimmunity. Immunol Lett. 2010;133(1):6–13.

82. Nalbandian G, Kovats S. Understanding sex biases in immunity: effects of estrogen on the differentiation and function of antigen-presenting cells. Immunol Res. 2005;31(2):91–106. 83. Dragin N, Bismuth J, Cizeron-Clairac G, et al. Estrogen-mediated downregulation of AIRE influences sexual dimorphism in autoimmune diseases. J Clin Invest. 2016;126(4):1525–1537. 84. Jeganathan V, Peeva E, Diamond B. Hormonal milieu at time of B cell activation controls duration of autoantibody response. J Autoimmun. 2014;53:46–54. 85. Grimaldi CM, Jeganathan V, Diamond B. Hormonal regulation of B cell development: 17 beta-estradiol impairs negative selection of high-affinity DNA-reactive B cells at more than one developmental checkpoint. J Immunol. 2006;176(5): 2703–2710. 86. Siracusa MC, Overstreet MG, Housseau F, Scott AL, Klein SL. 17beta-estradiol alters the activity of conventional and IFNproducing killer dendritic cells. J Immunol. 2008;180(3):1423–1431. 87. Weckerle CE, Franek BS, Kelly JA, et  al. Network analysis of associations between serum interferon-alpha activity, autoantibodies, and clinical features in systemic lupus erythematosus. Arthritis Rheum. 2011;63(4):1044–1053. 88. Gilmore W, Weiner LP, Correale J. Effect of estradiol on cytokine secretion by proteolipid protein-specific T cell clones isolated from multiple sclerosis patients and normal control subjects. J Immunol. 1997;158(1):446–451. 89. Wilder RL. Neuroendocrine-immune system interactions and autoimmunity. Annu Rev Immunol. 1995;13:307–338. 90. Rotondi M, Falorni A, De Bellis A, et  al. Elevated serum interferon-gamma-inducible chemokine-10/CXC chemokine ligand-10 in autoimmune primary adrenal insufficiency and in vitro expression in human adrenal cells primary cultures after stimulation with proinflammatory cytokines. J Clin Endocrinol Metab. 2005;90(4):2357–2363. 91. Lleo A, Battezzati PM, Selmi C, Gershwin ME, Podda M. Is autoimmunity a matter of sex? Autoimmun Rev. 2008;7(8):626–630. 92. Mor G, Sapi E, Abrahams VM, et al. Interaction of the estrogen receptors with the Fas ligand promoter in human monocytes. J Immunol. 2003;170(1):114–122. 93. Figueiredo AS, Schumacher A. The T helper type 17/regulatory T cell paradigm in pregnancy. Immunology. 2016;148(1):13–21. 94. Cervera R, Balasch J. Bidirectional effects on autoimmunity and reproduction. Hum Reprod Update. 2008;14(4):359–366. 95. Liu H, Shan Z, Li C, et al. Maternal subclinical hypothyroidism, thyroid autoimmunity, and the risk of miscarriage: a prospective cohort study. Thyroid. 2014;24(11):1642–1649. 96. Borchers AT, Naguwa SM, Keen CL, Gershwin ME. The implications of autoimmunity and pregnancy. J Autoimmun. 2010;34(3):J287–J299. 97. Rehakova Z, Capkova J, Stepankova R, et  al. Germ-free mice do not develop ankylosing enthesopathy, a spontaneous joint disease. Hum Immunol. 2000;61(6):555–558. 98. Abdollahi-Roodsaz S, Joosten LA, Koenders MI, et al. Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis. J Clin Invest. 2008;118(1):205–216. 99. Rigoni R, Fontana E, Guglielmetti S, et al. Intestinal microbiota sustains inflammation and autoimmunity induced by hypomorphic RAG defects. J Exp Med. 2016;213(3):355–375. 100. Maldonado MA, Kakkanaiah V, MacDonald GC, et  al. The role of environmental antigens in the spontaneous development of autoimmunity in MRL-lpr mice. J Immunol. 1999;162(11):6322–6330. 101. Weinstein Y, Ran S, Segal S. Sex-associated differences in the regulation of immune responses controlled by the MHC of the mouse. J Immunol. 1984;132(2):656–661.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

102. Bouman A, Heineman MJ, Faas MM. Sex hormones and the immune response in humans. Hum Reprod Update. 2005;11(4):411–423. 103. Amadori A, Zamarchi R, De Silvestro G, et  al. Genetic control of the CD4/CD8  T-cell ratio in humans. Nat Med. 1995;1(12):1279–1283. 104. Butterworth M, McClellan B, Allansmith M. Influence of sex in immunoglobulin levels. Nature. 1967;214(5094):1224–1225. 105. Huygen K, Palfliet K. Strain variation in interferon gamma production of BCG-sensitized mice challenged with PPD II. Importance of one major autosomal locus and additional sexual influences. Cell Immunol. 1984;85(1):75–81. 106. Holdcroft A. Integrating the dimensions of sex and gender into basic life sciences research: methodologic and ethical issues. Gend Med. 2007;4(Suppl B):S64–S74. 107. Oertelt-Prigione S. The influence of sex and gender on the immune response. Autoimmun Rev. 2012;11(6–7):A479–A485. 108. Borchers AT, Naguwa SM, Shoenfeld Y, Gershwin ME. The geoepidemiology of systemic lupus erythematosus. Autoimmun Rev. 2010;9(5):A277–A287. 109. Borchers AT, Uibo R, Gershwin ME. The geoepidemiology of type 1 diabetes. Autoimmun Rev. 2010;9(5):A355–A365. 110. Nossent JC. Systemic lupus erythematosus on the Caribbean island of Curacao: an epidemiological investigation. Ann Rheum Dis. 1992;51(11):1197–1201. 111. Alarcon GS, McGwin Jr. G, Petri M, Reveille JD, RamseyGoldman R, Kimberly RP. Baseline characteristics of a multiethnic lupus cohort: PROFILE. Lupus. 2002;11(2):95–101. 112. Lim SS, Drenkard C. Epidemiology of systemic lupus erythematosus: capturing the butterfly. Curr Rheumatol Rep. 2008;10(4):265–272. 113. Crosslin KL, Wiginton KL. Sex differences in disease severity among patients with systemic lupus erythematosus. Gend Med. 2011;8(6):365–371. 114. Symmons DP, Barrett EM, Bankhead CR, Scott DG, Silman AJ. The incidence of rheumatoid arthritis in the United Kingdom: results from the Norfolk Arthritis Register. Br J Rheumatol. 1994;33(8):735–739. 115. Guillemin F, Briancon S, Klein JM, Sauleau E, Pourel J. Low incidence of rheumatoid arthritis in France. Scand J Rheumatol. 1994;23(5):264–268. 116. Pedersen JK, Kjaer NK, Svendsen AJ, Horslev-Petersen K. Incidence of rheumatoid arthritis from 1995 to 2001: impact of ascertainment from multiple sources. Rheumatol Int. 2009;29(4):411–415.

333

117. Alamanos Y, Voulgari PV, Drosos AA. Incidence and prevalence of rheumatoid arthritis, based on the 1987 American College of Rheumatology criteria: a systematic review. Semin Arthritis Rheum. 2006;36(3):182–188. 118. Costenbader KH, Chang SC, Laden F, Puett R, Karlson EW. Geographic variation in rheumatoid arthritis incidence among women in the United States. Arch Intern Med. 2008;168(15):1664–1670. 119. Carmona L, Villaverde V, Hernandez-Garcia C, Ballina J, Gabriel R, Laffon A. The prevalence of rheumatoid arthritis in the general population of Spain. Rheumatology (Oxford). 2002;41(1):88–95. 120. Zhang H, Carbone M, Lleo A, Invernizzi P. . Geoepidemiology, Genetic and Environmental Risk Factors for PBC. Dig Dis. 2015;33(Suppl 2):94–101. 121. Podda M, Selmi C, Lleo A, Moroni L, Invernizzi P. The limitations and hidden gems of the epidemiology of primary biliary cirrhosis. J Autoimmun. 2013;46:81–87. 122. Boonstra K, Beuers U, Ponsioen CY. Epidemiology of primary sclerosing cholangitis and primary biliary cirrhosis: a systematic review. J Hepatol. 2012;56(5):1181–1188. 123. Carbone M, Mells GF, Pells G, et  al. Sex and age are determinants of the clinical phenotype of primary biliary cirrhosis and response to ursodeoxycholic acid. Gastroenterology. 2013;144(3):560–569. e7; quiz e13-4. 124. Milo R, Kahana E. Multiple sclerosis: geoepidemiology, genetics and the environment. Autoimmun Rev. 2010;9(5):A387–A394. 125. Rosati G. The prevalence of multiple sclerosis in the world: an update. Neurol Sci. 2001;22(2):117–139. 126. DIAMOND Project Group. Incidence and trends of childhood Type 1 diabetes worldwide 1990–1999. Diabet Med. 2006;23(8):857–866. 127. Karvonen M, Pitkaniemi M, Pitkaniemi J, Kohtamaki K, Tajima N, Tuomilehto J. Sex difference in the incidence of insulindependent diabetes mellitus: an analysis of the recent epidemiological data. World Health Organization DIAMOND Project Group. Diabetes Metab Rev. 1997;13(4):275–291. 128. Kyvik KO, Nystrom L, Gorus F, et  al. The epidemiology of Type 1 diabetes mellitus is not the same in young adults as in children. Diabetologia. 2004;47(3):377–384. 129. Kovats S. Estrogen receptors regulate innate immune cells and signaling pathways. Cell Immunol. 2015;294(2):63–69.

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23 Gender and Gene Regulation in Human Immunity Michelle R. Longmire and Howard Chang Stanford University, Stanford, CA, United States

O U T L I N E 23.1 Introduction

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23.2 Gender and Immunity

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23.3 What Is Known About Gender and Gene Regulation

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23.4 Differential Differences in Gene Regulation in Immunity and Immune Disease

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23.5 Summary

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23.2  GENDER AND IMMUNITY

From response to vaccinations to autoimmune disease, gender differences in immunity have been long recognized in clinical medicine.1–8 The primary genetic difference between males and females is the complement of sex chromosomes. The X chromosome contains a far higher number of genes than the Y chromosome, and it has long been thought that the second X in females was effectively silenced to provide genetic equity or dosage compensation. Emerging technologies are telling an increasingly fascinating story and calling to question long held assumptions about the silenced X chromosome, suggesting that the X may be a regulator of immune system function in health and disease. It has been long postulated that epigenetic or regulatory differences between male and female immune cells may underlie immune difference and autoimmune susceptibility9 and emerging science suggests that sexchromosome and autosomal gene regulation may contribute to immune variation and provide insight into gender differences in health and disease. Herein we review recent findings on gender differences in immunity, with a focus on gene regulation and human T cells.10

The distinct female predominance of autoimmune disease provides insight into gender-specific differences in human immunity. About 8% of the general population suffers from one or more autoimmune diseases and there is a strong gender bias, as an estimated 75% of autoimmune disease affects women more than men.11 Further, specific types of autoimmune disease show dramatic female predominance. For example, systemic lupus erythematosus, hyperthyroidism, and primary biliary cirrhosis have been reported to have a disease occurrence that is 9:1 female to male.12 Genetic studies have only identified a limited number of causal genes and there is a high degree of discordance in autoimmune disease in monozygotic twins.13–15 One possible explanation is that a combination of genetic predisposition and various environmental factors influence gene regulation and tip the immune balance away from self-tolerance. This leads to the dynamic phenotype observed in autoimmune diseases with variations in the age of onset, recurrence or flaring, severity of symptoms, length of remissions, and response to drug treatments.15 Epigenetic mechanisms regulate gene expression and are sensitive to external

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stimuli and thus bridge the gap between underlying genetics and environmental factors.15 Gender-specific differences in immune regulation may combine with both genetic predisposition as well as environmental exposures and lead to the development of disease in one gender, even given a similar environmental milieu. Recent advances in epigenomic technologies allow us to gain greater insight into the relationship between gender and immune regulation. The ability to create epigenomic maps from cells without expansion is very important in advancing our understanding of genderspecific immune regulation, specifically with regard to autoimmunity. These techniques provide data that are felt to be more reflective of the regulome in the human system. New technologies provide more detailed and allelic assessments of gene expression, chromatin landscape, and chromatin marks, with far fewer cells.9,16 Specifically, the assay for transposase-accessible chromatin (ATAC)-seq method surveys accessible chromatin genome-wide and enables mapping of chromatin landscape from single cells, opening the door to understanding gene regulation from small populations of cells harvested directly from human tissue, without expansion. ATAC-seq has also shed insight into X chromosome gene regulation and revealed differences in chromatin accessibility in regions of the X chromosome thought to be inaccessible.10 This powerful new technique is shedding light into the dynamic nature of gene regulation and the role of noncoding regions in immunity and autoimmune disease.

23.3  WHAT IS KNOWN ABOUT GENDER AND GENE REGULATION To understand the impact of gender on immunity, let us first consider gene regulation of the sex chromosomes themselves. Notably, a large number of immune-related genes are located on the X chromosome.17 The impact of gender on gene regulation has been long recognized through X chromosome inactivation and X chromosome escape. In order to compensate for the presence of two X chromosomes, mammalian females randomly silence one of two X chromosomes in each cell, thus balancing the lack of the second X chromosome in the male of the species.9 However, a subset of genes on the inactivated X “escape” inactivation (XCI) and are actively expressed, resulting in higher levels of the gene product expressed compared to male counterparts.9 XCI is controlled by several long noncoding RNAs (lncRNAs), including XIST, which is transcribed from and mediates the epigenetic silencing of the inactive X chromosome (Xi).10 Thus, the active X chromosome (Xa) and Xi harbor distinct chromatin modifications and gene expression patterns. A subset of X-linked genes escape from XCI

in a tissue-specific fashion through poorly understood mechanisms, leading to differential X-linked expression in male versus female cells within the tissues.10 The differential levels of gene product related to XCI “escapees,” in females versus males provides a possible source of gender-based differential gene regulation. Current estimates suggest that 12–20% of human X-linked genes escape and are expressed from both the Xa and Xi.16 The expression of X-linked genes may be affected by X-linked micro-RNAs (miRNAs), which are small noncoding RNAs that regulate gene expression at a posttranscriptional level and play a role in maintaining immunological homeostasis.18 Interestingly, there are disproportionately more miRNAs located on the X chromosome than on any autosomal chromosome and the X chromosome contains 10% of the 800 miRNAs in the genome, whereas the Y chromosome contains only two miRNAs.18 Gender-specific regulomes may arise from the effect of sex-specific hormones, random XCI, X-linked escapee genes, or other factors.18 Utilizing ATAC-seq, a detailed survey of the chromatin landscape of Xa and Xi, is possible. Using this technique, comparison between males and females demonstrates that autosomal and genes subject to XCI have similar accessibility while escape genes have twice as much accessibility in females as males.10 The data demonstrate that escaped gene regions have accessible chromatin on both Xa and Xi, and previously unknown regions of accessibility in both coding and noncoding regions have been identified. ATAC-seq performed on CD4 T cells from the blood of healthy female donors revealed 43 elements associated with 17 coding genes (e.g., EIF1AX and KDM6A) and 3 noncoding genes known to escape XCI, as well as elements associated with 7 escapees predicted by others19 and 12 XCI escapees that had not been reported previously. Comparison of the regions of ATAC-seq signal to mRNA data, measuring genome-wide mRNA levels of CD4+ T cells from health donors, validated 16 out of 17 known XCI genes and 6 of 7 novel XCI escapees predicted by ATAC-seq that had well-measured transcripts. It is noteworthy that escapee genes that were not validated tended to have lower ATAC-seq signals and mRNA expression compared to those that were validated, which may be below the detection confidence of array technology. Comparison of gender-specific regulomes provides new insights that were not possible from gene expression measurements. Regulome maps are derived from regions of open chromatin directly and not gene products, ATAC-seq provides insights into noncoding genomic regions which are felt to contain potent regions of gene regulation and are increasingly felt to play an important role in immunity and autoimmune disease.20–22 For example, accessible elements that escape XCI are more likely to be found at promoters and introns of known

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23.4 Differential Differences in Gene Regulation in Immunity and Immune Disease

escapee genes but not at intergenic distal regions. Further, ATAC-seq data provides evidence of genderspecific regulatory landscapes of XCI escapees. This new understanding of the chromatin landscape calls to question long-standing assumptions of XCI escape. XCI has traditionally been considered simply a failure of Xi silencing; hence, it was believed that the regulatory pattern on the Xa will simply be duplicated on the Xi for escapee genes. However, this model of XCI escape lacks direct evidence and using ATAC-seq, Xi-specific regulatory elements on XCI escapees that have signal only in female and not in male cells have been identified, suggesting that the role of XCI or lack thereof may not be related to dosage compensation alone. Take, e.g., FIRRE, a recently described X-linked lncRNA that escapes XCI and is involved in chromosome topological organization.23 RNA in situ hybridization documented two equivalent RNA foci in female cells and one focus in male cells.23 FIRRE contains a series of putative intronic enhancers embedded throughout its locus, as documented by the enhancer-associated modification histone H3 lysine 27 acetylation (H3K27ac) in a survey of mixed-sex cells.24 Intriguingly, regulome maps of CD4 T cells generated with ATAC-seq suggest that two FIRRE enhancers in intron 2 are active in male cells, whereas over a dozen enhancers in introns 2–12 are active only in female cells.10 This regulatory divergence implies gender-specific regulation, allele-specific regulation of FIRRE on Xa versus Xi, or combinations of both strategies. Further advances in allele-specific technology will provide important insights into the regulome of the active versus inactive X, amplifying our understanding of maternal and paternal driven regulation.

23.4  DIFFERENTIAL DIFFERENCES IN GENE REGULATION IN IMMUNITY AND IMMUNE DISEASE In addition to the action of the sex chromosomes, global genomic regulation is also influenced by gender.10 Gender-based differences in T cell production of IFNgamma and interleukin-2 (IL-2) have been documented in healthy children25,26 and IFN-gamma is also known to be affected by sex hormones.18 Gender-specific differences in gene regulatory networks generated from CD4 T cells with ATAC-seq reveal a set of active transcription factor (TF) regulators with cognate sites on each gene that differ in male versus female samples with several hundred genes demonstrating significant differences in their predicted regulatory network. Ranking autosomal genes by gender-specific regulatory variance revealed that the top divergent genes include many genes with well-known and important functions in immune

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function or development, including FGL2, GZMK, IFNG, CRTAM, CARD16, FYN, IL2, and IL6. Further, mRNA levels of a subset of these also showed significant differential expression between males versus females.10 Among the regulatory networks with differential activity identified across autosomes using ATAC-seq, FGL2 showed the greatest gender-specific variance in chromatin accessibility across the genome, with nearly twice that of the next most variable gene. FGL2 had a greater promoter ATAC-seq signal in male than in female T cells, and analysis of specific TF signals revealed that interferon-regulatory factor (IRF) family members and NHLH1 TFs are bound in male, but not female, T cells.10 Notably, FGL3 encodes a fibrinogen-like protein secreted by regulatory T cells and other cells that have immunosuppressive activity27 and emerging data suggest that Fgl2 may also have immunosuppressive activity.28 A mouse knockout model showed that Fgl2 is required for Treg function and prevention of spontaneous autoimmunity.29,30 Similarly, CRTAM encodes a T cell adhesion molecule that has been recently recognized to critically control the differentiation of CD4+ cells into inflammatory Th17 cells.31 Thus, gender-specific regulation of FGL2 and CRTAM may contribute in part to genderlinked differences in autoimmune disease.10 A look at the top 1000 differentially regulated genes reveals genes that are significantly enriched for biological functions in the defense response, i.e., the response to viruses, the formation of the immune complex, and in the different aspects of inflammatory disease. It is important to note that analysis of the regulatory elements of many well-expressed genes, such as housekeeping genes, were also surveyed in these experiments and did not show gender-specific differences, suggesting that subsets of physiologic function and human biology may not be subject to gender regulation while others, such as immunity, may be more heavily influenced by sex. TF regulators are felt to have potent effects on the regulome and are interesting to consider with regard to gender as these relatively small noncoding genomic regions can have a wide reaching regulatory impact. Examination of TF regulators that exhibit gender-specific activity using ATAC-seq are few in number in comparison to the 1000 target genes identified; only a handful of TFs with gender-associated divergence have been described. Among the most divergent is ESR2 (encoding estrogen receptor beta); its differential is a consequence of the female hormone estrogen.10 Additionally, ATACseq analysis revealed two notable gender-divergent regulators that are IRF family members, encoding well-studied TFs activated by interferon signaling and other signals in innate and adaptive immunity responses that can cooperate or compete with other TFs to exert regulatory effects.10,32 Therefore, while a large number of genes are likely to be differently regulated, the

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differential regulation is associated with a small number of TF motifs, which showed differential activity across many genes. The findings suggest that a small number of regulators may impact a large number of target genes to yield the observed male–female divergence in the regulome.10 The IFNG locus emerged as a prime example of the intersection of predicted divergence of both regulators and target genes. IFNG was also identified as an important divergent gene using ATAC-seq analysis. IFNG encodes interferon gamma and is a key regulator of immune response and Th1 cell differentiation. Multiple studies have documented gender-specific association of allelic variants at IFNG regulatory elements or IFNG protein levels with human disease.33 For example, IFNG variants are associated with multiple sclerosis34 and asthma35 in males, but not females, although their mechanisms are not known. NeST (also known as IFNG-AS1 or TMEVPG1) is located proximal to IFNG and encodes an lncRNA that is required to program active chromatin state and promote expression of IFNG.36 NeST is convergently transcribed relative to IFNG, and a long isoform of NeST is transcribed through the IFNG promoter. NeST itself is induced by Th1 polarization,37 and murine NeST was first discovered as a genetic locus that controlled pathogen resistance and immune-mediated disease.38 Interestingly, ATAC-seq reveals that IFNG and NeST show gender-specific regulation. A cluster of elements nearest to 5′ of IFNG is equally active in male versus female cells, but high-resolution analysis indicated that IRF family members were more abundant or had a greater impact in males, but NF-YA and CST6 were more potent in females.10 Males also have higher TF frequency of NeST.10 These results suggest that positive regulatory loops comprising NeST, IRF, and IFNG may differ in a gender-specific fashion. Consistent with this idea, careful genetic analyses showed that NeST locus mutation has stronger pathogenic impact in male than in female mice.38 The fact that unbiased examination of target genes and regulators using ATAC-seq nominates IFNG suggests that interferon signaling is a major gender-specific regulatory feature in T cells.39 Gene regulation is impacted by a number of factors, ranging from environmental exposures to inherited genetic variation. Inherited genetic predisposition may combine with environmental exposure to determine the risk of disease.15 This combination of genetic risk and environmental exposure may be particularly relevant to autoimmune disease given the lack of strong genetic evidence of disease causality and the disease discordance observed in monozygotic twins.40 One method of examining the intersection of genetic risk and the dynamic chromatin landscape of the immune system is to analyze regulome variation across genomic regions

containing single nucleotide polymorphisms linked to human diseases.10 There is a relatively short list of SNPs associated with autoimmune disease and few confer substantial disease risk.8,41 Utilizing ATAC-seq, it is possible to examine chromatin accessibility of CD4+ T cells in regions of SNPs and such analysis reveals that active regulatory elements are enriched in SNPs as a class compared to the remainder of the genome.10 The biological significance of the variable regulatory elements, and variability in chromatin accessibility across individuals and by gender was recently demonstrated to be a novel feature associated with the cause of disease.10 Examining the intersection of chromatin accessibility with a recently identified set of autoimmune casual SNPs reveals that these regions are strongly enriched in CD4+ T cell ATAC-seq peaks. These variants are an interesting subset of SNPs to consider with regard to gender and immune regulation as they were identified utilizing a fine-mapping algorithm, which then integrated algorithm-generated predictions with transcription and cis-regulatory element annotations, derived by mapping RNA and chromatin in primary immune cells, including resting and stimulated CD4+ T cell subsets, regulatory T cells, CD8+ T cells, B cells, and monocytes. Roughly 60% of the causal variants mapped to immune cell enhancers.42 Many of these variants gain histone acetylation and transcribe enhancer-associated RNA upon immune stimulation.42 These variants tend to occur near binding sites for master regulators of immune differentiation and stimulusdependent gene activation, but only 10–20% directly alter recognizable TF binding motifs.42 Rather, most noncoding risk variants, including those that alter gene expression, affect noncanonical sequence determinants not well-explained by current gene regulatory models.42 Analysis of ATAC-seq peaks that show interindividual variation are enriched for disease or eQTL SNPs compared to invariant open chromatin sites in CD4+ T cells.10 Autoimmune causal SNPs are most significantly enriched in variable peaks compared to invariant open chromatin sites, including causal variants for type 1 diabetes, rheumatoid arthritis, lupus erythematosus, Crohn’s disease, and vitiligo. In negative controls, generic SNP sets from genome-wide association studies of all diseases or expressive quantitative trait loci (which are not T cell-specific) show no significant enrichment. Collectively, these results illustrate the biological significance of the variable regulatory elements, and variability in chromatin accessibility across individuals emerges as a novel feature associated with locations of causal disease SNPs. Variable elements are, by definition, “noisy” and capable of being readily switched on or off—properties that may enable even a single-nucleotide mutation to change its activity.42

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REFERENCES

23.5 SUMMARY Our understanding of the impact of gender on gene regulation in immunity is rapidly advancing due to the emergence of new technologies that provide insight into the role of the regulome in human immunity. Recent research suggests that gender may influence genetic regulation of the immune system. These insights may help to explain observed differences in immune response in males versus females as well as shed light on the female predisposition to autoimmune disease. Moving forward, the authors hope that such advances will inform better therapies for autoimmune disease and immune system related disorders. With the emergence of personalized medicine, the regulome will be critical to the identification of individually optimized therapies. This will be especially important for diseases driven by a confluence of genetic predisposition and environmental factors, such as those of the immune system. Future research related to gender-specific immune regulation should seek to better clarify the intersection of gender, environmental exposure, and disease development. The authors predict that assays providing insights into the immune system regulome will shape diagnostics and therapeutics alike and individualize patient care. Future research should produce more effective and safer therapies to improve the lives of women and men who suffer from immune related disease.

References 1. Gobert M, Lafaille JJ. Maternal-fetal immune tolerance, block by block. Cell. 2012;150(1):7–9. 2. Oertelt-Prigione S. The influence of sex and gender on the immune response. Autoimmun Rev. 2012;11(6–7):A479–A485. 3. Bogdanos DP, et al. Twin studies in autoimmune disease: genetics, gender and environment. J Autoimmun. 2012;38(2–3):J156–J169. 4. Gleicher N, Barad DH. Gender as risk factor for autoimmune diseases. J Autoimmun. 2007;28(1):1–6. 5. Ji J, Sundquist J, Sundquist K. Gender-specific incidence of autoimmune diseases from national registers. J Autoimmun. 2016;69:102–106. 6. Knudsen GP. Gender bias in autoimmune diseases: X chromosome inactivation in women with multiple sclerosis. J Neurol Sci. 2009;286(1–2):43–46. 7. Ngo ST, Steyn FJ, McCombe PA. Gender differences in autoimmune disease. Front Neuroendocrinol. 2014;35(3):347–369. 8. Ponomarenko MP, et  al. Candidate SNP markers of genderbiased autoimmune complications of monogenic diseases are predicted by a significant change in the affinity of TATA-binding protein for human gene promoters. Front Immunol. 2016;7:130. 9. Balaton BP, Brown CJ. Escape Artists of the X Chromosome. Trends Genet. 2016;32(6):348–359. 10. Qu K, et al. Individuality and variation of personal regulomes in primary human T cells. Cell Syst. 2015;1(1):51–61. 11. Rubtsova K, Marrack P, Rubtsov AV. TLR7, IFNgamma, and T-bet: their roles in the development of ABCs in female-biased autoimmunity. Cell Immunol. 2015;294(2):80–83.

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12. Invernizzi P, et al. X monosomy in female systemic lupus erythematosus. Ann N Y Acad Sci. 2007;1110:84–91. 13. Goodnow CC, et  al. Cellular and genetic mechanisms of self tolerance and autoimmunity. Nature. 2005;435(7042):590–597. 14. Rioux JD, Abbas AK. Paths to understanding the genetic basis of autoimmune disease. Nature. 2005;435(7042):584–589. 15. Gupta B, Hawkins RD. Epigenomics of autoimmune diseases. Immunol Cell Biol. 2015;93(3):271–276. 16. Balaton BP, Cotton AM, Brown CJ. Derivation of consensus inactivation status for X-linked genes from genome-wide studies. Biol Sex Differ. 2015;6:35. 17. Bianchi I, et  al. The X chromosome and immune associated genes. J Autoimmun. 2012;38(2-3):J187–J192. 18. Rubtsova K, Marrack P, Rubtsov AV. Sexual dimorphism in autoimmunity. J Clin Invest. 2015;125(6):2187–2193. 19. Zhang Y, et al. Genes that escape X-inactivation in humans have high intraspecific variability in expression, are associated with mental impairment but are not slow evolving. Mol Biol Evol. 2013;30(12):2588–2601. 20. Witte S, O’Shea JJ, Vahedi G. Super-enhancers: Asset management in immune cell genomes. Trends Immunol. 2015;36(9):519–526. 21. Shih HY, et al. Transcriptional and epigenetic networks of helper T and innate lymphoid cells. Immunol Rev. 2014;261(1):23–49. 22. Vahedi G, et  al. Super-enhancers delineate disease-associated regulatory nodes in T cells. Nature. 2015;520(7548):558–562. 23. Hacisuleyman E, et al. Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre. Nat Struct Mol Biol. 2014;21(2):198–206. 24. Consortium EP. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489(7414):57–74. 25. Eyrich M, et al. Immune function in children under chemotherapy for standard risk acute lymphoblastic leukaemia - a prospective study of 20 paediatric patients. Br J Haematol. 2009;147(3):360–370. 26. Wiegering V, et al. Age-related changes in intracellular cytokine expression in healthy children. Eur Cytokine Netw. 2009;20(2):75–80. 27. Marsden PA, et al. The Fgl2/fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis. J Clin Invest. 2003;112(1):58–66. 28. Xu L, et  al. Inhibitory function of Tregs via soluble FGL2 in chronic hepatitis B. J Huazhong Univ Sci Technol Med Sci. 2012;32(4):540–545. 29. Liu H, et  al. The FGL2-FcgammaRIIB pathway: a novel mechanism leading to immunosuppression. Eur J Immunol. 2008;38(11):3114–3126. 30. Shalev I, et al. Targeted deletion of fgl2 leads to impaired regulatory T cell activity and development of autoimmune glomerulonephritis. J Immunol. 2008;180(1):249–260. 31. Cortez VS, et al. CRTAM controls residency of gut CD4+CD8+ T cells in the steady state and maintenance of gut CD4+ Th17 during parasitic infection. J Exp Med. 2014;211(4):623–633. 32. Ikushima H, Negishi H, Taniguchi T. The IRF family transcription factors at the interface of innate and adaptive immune responses. Cold Spring Harb Symp Quant Biol. 2013;78:105–116. 33. Li Pira G, et al. Validation of a miniaturized assay based on IFNg secretion for assessment of specific T cell immunity. J Immunol Methods. 2010;355(1–2):68–75. 34. Kantarci OH, et al. Interferon gamma allelic variants: sex-biased multiple sclerosis susceptibility and gene expression. Arch Neurol. 2008;65(3):349–357. 35. Loisel DA, et  al. IFNG genotype and sex interact to influence the risk of childhood asthma. J Allergy Clin Immunol. 2011;128(3):524–531. 36. Gomez JA, et al. The NeST long ncRNA controls microbial susceptibility and epigenetic activation of the interferon-gamma locus. Cell. 2013;152(4):743–754.

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37. Hu G, et al. Expression and regulation of intergenic long noncoding RNAs during T cell development and differentiation. Nat Immunol. 2013;14(11):1190–1198. 38. Bihl F, Brahic M, Bureau JF. Two loci, Tmevp2 and Tmevp3, located on the telomeric region of chromosome 10, control the persistence of Theiler’s virus in the central nervous system of mice. Genetics. 1999;152(1):385–392. 39. Kantarci OH, et  al. IFNG polymorphisms are associated with gender differences in susceptibility to multiple sclerosis. Genes Immun. 2005;6(2):153–161.

40. Ballestar E. Epigenetics lessons from twins: prospects for autoimmune disease. Clin Rev Allergy Immunol. 2010;39(1):30–41. 41. Sirota M, et  al. Autoimmune disease classification by inverse association with SNP alleles. PLoS Genet. 2009;5(12):e1000792. 42. Farh KK, et  al. Genetic and epigenetic fine mapping of causal autoimmune disease variants. Nature. 2015;518(7539):337–343.

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24 Sex and Gender Specific Aspects—From Cells to Cardiovascular Disease Vera Regitz-Zagrosek1,2 1

Charité Universitätsmedizin, Berlin, Germany, 2DZHK partner site Berlin, Berlin, Germany

O U T L I N E 24.1 Introduction

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24.2 Contribution of Sex Chromosomes

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24.3 Contribution of Sex Hormones and Their Receptors

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24.4 Epigenetic Modifications 24.4.1 DNA and Histone Modifications 24.4.2 Noncoding RNAs, MicroRNA

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24.5 Differentiation of Chromosomal and Sex Hormone effects

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24.6 Sex Differences in Cardiovascular Cell Functions 345 24.6.1 Calcium Homeostasis 345 24.6.2 Energy Metabolism 346 24.6.3 Apoptosis and Autophagy 346 24.6.4 Inflammatory Pathways or Mechanisms 347 24.6.5 Myocardial Hypertrophy 347 24.6.6 Fibrosis 347

24.1 INTRODUCTION Most cardiovascular diseases (CVD) differ in women and men in their epidemiology, manifestation, pathophysiology, treatment, and outcome.1,2 We discuss biological mechanisms of sex differences (SD) in CVD. We discuss SD in genetic mechanisms, epigenetic mechanisms, and sex hormones, as reviewed recently.3–5 We describe the interaction of sex hormones with intracellular signaling relevant for cardiovascular cells and the cardiovascular system. We discuss resulting SD in cell Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00008-5

24.7 Sex Differences in Clinical Syndromes 24.7.1 Sex and Gender 24.7.2 Arterial Hypertension 24.7.3 Ischemic Heart Disease 24.7.4 Treatment and Outcomes 24.7.5 Takotsubo Cardiomyopathy 24.7.6 Aortic Stenosis 24.7.7 Mitral Valve Disease: Mitral Valve Prolapse and Mitral Regurgitation 24.7.8 Arrhythmia 24.7.9 Cardiomyopathies 24.7.10 Heart Failure

347 347 348 349 351 351 352 353 353 353 354

24.8 Conclusions and Future Directions of Research 356 Acknowledgment 357 References 357

function and animal models for adaptation of the CV system to internal and external stress, such as genetic variation, exercise, ischemia, pressure overload, hormonal disturbances, inflammation, over- and undernutrition, and pregnancy. We give an overview on SD in clinical syndromes, in ischemic disease, valvular heart disease, arrhythmia, cardiomyopathy, heart failure (HF), and hypertension and here we include the effect of gender—differences between women and men, arising from attitudes toward disease and access to health care. Finally we make suggestions for future research directions.

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24.2  CONTRIBUTION OF SEX CHROMOSOMES Among the most evident reasons for SD are differences in sex chromosome complement. Normally, all female cells exhibit XX genotype and all males XY. In order to compensate for the second X chromosome in female cells, nature invented X-inactivation and silences the second X chromosome in female cells, providing the same dosage of X chromosomal genes in male and female cells. However this mechanism is not completely efficient and a number of genes escape X-inactivation. The number of these genes is tissue-specific and speciesspecific, differs in mice and man, and is greater in man than in mice (about 15% in men and 3% in mice).6 The sex chromosome dosage is relevant for the cardiovascular system. This is clearly documented in clinical defects that are associated with an aberrant number of sex chromosomes, i.e., the Turner syndrome or Klinefelter syndrome. Women with Turner syndrome and a XO genotype quite frequently exhibit characteristic cardiovascular phenotypes. Males with Klinefelter syndromes, i.e., an XXY karyotype or a mosaic, exhibit defects in brain development and body composition and development. The X chromosome is a large chromosome with approximately 1500 genes that are involved in the function of the brain, the heart, and the immune system. However, not many gene defects as causes for human diseases have been located to the X chromosome

(Fig. 24.1). This is astonishing but probably at least partially due to the fact that the X chromosome was frequently excluded from whole genome sequencing approaches. More complex technological and analytical approaches are needed to include the single X chromosome into analysis where all other chromosomes come in pairs. However more recent studies clearly document the relevance of X chromosomal genes for human disease.7,8 The Y chromosome expresses many fewer genes than the X chromosome. Nevertheless some of these are of cardiovascular relevance and may also lead to SD in phenotypes. Gene variance on the Y chromosome has been linked to lipid metabolism disturbances, to SD in hypertension, and to a particularly severe form of coronary artery disease (CAD) in men: left main coronary artery stenosis. The manifestation of all these syndromes is more frequent in men than in women and is independent of the testosterone levels.9–11 These most recent data convincingly show that Y chromosomal gene variance is associated with CVD in man. Surprisingly some autosomal gene variants also exert their effect in a sex-specific manner. This has been shown for adiposity and obesity. A large number of recently identified autosomal gene variants12 exhibited significant sexual dimorphism with stronger effects in women. These genes were associated with obesity and metabolism. It is still completely unclear why these genetic polymorphisms do have a sexually dimorphic effect. Other examples have been found where common

FIGURE 24.1  Presentation of all genome white loci for association of variants with human disease. Different disease groups are encoded in the same color. The sex chromosomes have significantly less variants than all 22 autosomes. The NHGRI-EBI Catalog of published genome-wide association studies. Available at: www.ebi.ac.uk/gwas. Accessed (25.02.16).

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24.3  Contribution of Sex Hormones and Their Receptors

genetic polymorphism are associated with changes in physiology only in men: This is the case for Chymase (CMA1) gene that is only associated with left ventricular (LV) hypertrophy in men and not in women. Variants in the bradykinin type one receptor have also been associated with SD in the cardiovascular system.13,14

24.3  CONTRIBUTION OF SEX HORMONES AND THEIR RECEPTORS Sex hormones are synthesized during embryogenesis under the influence of the sex chromosomes and impact the development of the embryo. During embryogenesis most of them are synthesized in the gonads but later in life extragonadal synthesis takes place in many organs like the heart and brain or adipose tissue. The most important sexual hormones are androgens, estrogens, and progesterone but many other hormones and metabolites also exist. We focus in this chapter on the hormones that have clearly described actions in the cardiovascular system. The most important female sexual hormone is estrogen, mainly in form of its metabolite 17beta-estradiol (E2). Other metabolites are estrone and estriol. The latter two are weaker compounds and their effects are less well studied. Estrogen is produced mainly in the ovaries, however it can be generated in a number of organs and

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most interestingly it can be produced as a result of aromatase activity in fat and in the cardiovascular system, in women and men. Bone, brain, heart vasculature, and adipose tissue are the major extragonadal loci of estrogen action. Local estrogen synthesis can be significantly increased by disease states like inflammation and obesity. Extragonadal synthesis of estrogen in the adipose tissue from testosterone can play a major role in the pathophysiology of diseases in elderly men. Obese men can reach the same estrogen levels as postmenopausal women. Indeed, altered estrogen levels in men (very high levels as well as very low levels) have been associated with disease states including HF.15 Other sexual hormones with effects in the cardiovascular system, such as progesterone, are less well described in their actions. However, effects on cardiac rhythm and cardiac growth have been mentioned in the literature. Estrogen and androgens bind to a family of hormone receptors, i.e., androgen receptors, estrogen receptors subtypes alpha and beta, progesterone receptors, and GPR30. Estrogen, androgen, and progesterone receptors are mainly nuclear receptors. This means that after being activated by binding their ligand they bind to the DNA, either as homo or heterodimers, to initiate transcription. In all cases a large number of cofactors are assembled that significantly contribute to the specific actions of these receptors (Fig. 24.2). Cofactor composition of a cell is crucial for the effects of estrogen and androgen receptors.

FIGURE 24.2  Overview on estrogen receptor signaling in a cardiomyocyte with its membrane bound signaling pathways and nuclear encoded effects. Curr Opin Pharmacol. 2007 Apr;7(2):130–139.

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Estrogen and androgen receptors can also be located in the plasma membrane. They can bind their ligands there and activate intracellular signaling pathways including mTOR signaling, ERK signaling, p38 MAPK, JNK, β-catenin, GSK3β, and PI3K (phosphoinositide 3-kinase) signaling.16,17 These mechanisms induce rapid alterations of calcium handling, mitochondrial function, and ion channels, among others. These effects must be distinguished from the longer lasting effects on gene transcription that sex hormone receptors exert by binding to the DNA. Testosterone is the best known circulating androgen. It has some highly effective metabolites, like dihydrotestosterone, which has a higher affinity for the androgen receptor than testosterone itself, and it has been assumed that it represents an amplifying mechanism. It appears that increased levels of dihydrotestosterone are found in hypertrophied human hearts.18 So far no consistent positive effect of androgens, testosterone, or dihydrotestosterone on the human heart have been shown. Several studies in HF tried to use these hormones, however longterm use was associated with myocardial hypertrophy and HF.19–21 Testosterone has also been used in women based on its vasodilatory effects. However, in pharmacological doses no consistent beneficial effect could be substantiated. To understand SD due to sex hormones in animal models, ovariectomy (OVX), orchiectomy, as well as hormone substitution protocols have been used. E2 administration was shown to reduce infarct size and to improve postischemic myocardial function in rabbits, mice, and rats.5 Hearts from OVX mice and rats exhibited a greater infarct size, impaired functional recovery and adverse remodeling, which were reversed by E2 administration in physiological doses. E2 supplementation also exerted antihypertrophic effects in different pressure overload models and ERα inhibited myocardial fibrosis in female mice under pressure overload.22,23 In contrast, the removal of androgens through orchiectomy in males prevented adverse myocardial remodeling and dysfunction in response to several stressors. Collectively, the data suggest that estrogen and androgen levels play a major role in cardiac pathophysiology in both sexes. To verify which sex hormone receptors, ERα or ERβ, mediate the beneficial effects of E2, selective agonists have been used. A number of studies point to a cardioprotective effect of ERα agonists. Comparing ERα agonism and ERβ agonism in the same setting showed that both conferred protecttion following ischemia/reperfusion. To obtain more mechanistic evidence for the role of sex hormone receptors in disease, models with genetic deletion or overexpression of sex hormone receptors have been developed. Using mice with genetic deletion of ERα (ERKO) and/or ERβ (BERKO) verified the role of

ERα and ERβ in mediating beneficial effects of E2 during cardiac hypertrophy.24–27 Other studies determined the role of ERα or ERβ in mediating the beneficial actions of E2 in ischemia/reperfusion injury.23 These studies did not achieve a clear consensus regarding the specific role of a specific ER in protection against cardiac injury, but they agree that both ERs are involved in the cardiac effects of E2. Our own studies with a unique cardiacspecific overexpressing model of ERα showed that this receptor protects the heart against ischemic injury, showing that cardiomyocyte-specific ERα induces neovascularization and attenuates profibrotic gene expression through paracrine actions.28 Most recently, ERβ has also been overexpressed in cardiomyocytes.29 ERβ-OE led to improved survival, reduced maladaptive remodeling, improved cardiac function, and reduced HF development after MI in both sexes. Altogether, these animal models convincingly support the contribution of sex hormones and their receptors to cardiovascular function in health and disease.

24.4  EPIGENETIC MODIFICATIONS 24.4.1  DNA and Histone Modifications The next group of variations that are particularly important to characterize SD are epigenetic modifications. They represent how the environment, stress, toxins, nutrition, talk to our genome and modify us as human beings. The same or similar mechanisms are present in most experimental animals and can be studied there. Epigenetic modifications can occur directly at the level of DNA, as methylation of cytosines in the primary DNA sequence. Next, they can occur as histone modifications including acetylation. Epigenetic modifications can have very different lifetimes, going from rather short durations to lifelong changes. Some epigenetic modifications pass through the germ cells and can be inherited.30,31 Epigenetic modifications in the embryo can affect the developing individual.32 Interestingly enough, epigenetic modifications can occur in a sexspecific manner. One of the reasons may be that some DNA methylations or demethylations are controlled by estrogen.33 In the Rotterdam study, a number of significant DNA methylation loci were identified that were changed by famine exposure during pregnancy. Frequently the methylation was stronger in men than in women.34 If individuals from these Dutch cohorts that were subjected to famine of their mothers in utero were examined at the age of 59 years, increased systolic blood pressure and even hypertension were observed in the affected individuals.35 However, more data are needed to make conclusions on the role of epigenetics on SD in clinical conditions.

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24.6  Sex Differences in Cardiovascular Cell Functions

DNA and histone modifications may have sexually dimorphic effects. We, as others, are studying the DNA binding sites of estrogen receptors and concurrent histone modifications. The hypothesis is that sex-specific binding of cofactors may play a major role in DNA and histone modifications. We suspect that in particular CREB binding protein (CBP) and E1A binding protein p300 (EP 300)36 are involved. DNA or histone modifications influence cardiac hypertrophy and fibrosis.37–39 SD of these mechanisms may be inferred through the fact that some of the demethylases as well as some methylases belong to the genes that escape X inactivation. Imbalance in these genes may be a first step to introduce sex-specific DNA opening or closing for transcriptional processes. First experimental studies in rat models suggest that they are involved in prohypertrophic mechanisms.

24.4.2  Noncoding RNAs, MicroRNA A number of different noncoding RNA species have recently been discovered. Their role in regulating the genome is still largely unknown. Long noncoding RNA as well as small noncoding RNA or microRNA belong to these noncoding RNA species. MicroRNAs are small noncoding DNA sequences that control gene expression and protein synthesis by pairing to 3-untranslated region of specific messenger RNA. Several hundred different microRNA have been described in target organs and in the cardiovascular system. They regulate cardiac hypertrophy and fibrosis.40–42 We recently identified a number of microRNAs that regulate cardiovascular physiology in a sex-specific manner.43 Some of the very important microRNAs related to cardiac fibrosis (miRNA-21, -24, -27, -106) have been found to be regulated by estrogen receptor beta. These microRNA control genes are specifically linked to cardiac fibrosis like RASA1, RASA2, and Spry1. Our results strongly suggest that these microRNAs do play a major role in the control of cardiac function by estrogen and estrogen receptors. We also identified a number of cardiac microRNAs that are regulated by estrogen, estrogen receptors, and sex, that are related to cardiac energy metabolism.44 A number of these microRNA also regulate pressure overload in a sex-specific manner. These pressure overload induced microRNAs control mitochondrial function and fibrosis, MAPK signal transcription, and the organization of the extracellular matrix. Estrogen receptor beta controls the regulation of these microRNA in both sexes. Interaction with pressure overload or stress however will lead to regulation only in male genes. Therefore these microRNAs are interesting targets to modify the stress response of the heart in a sex-specific manner and and estrogendependent manner. Estrogen receptor beta may be an interesting target in these processes.

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24.5  DIFFERENTIATION OF CHROMOSOMAL AND SEX HORMONE EFFECTS Since all cells in the human or in an animal body express X and Y or XX chromosomes and also synthesize sex hormones it is quite challenging to distinguish between the effects of sex hormones and sex chromosomes in the experimental situation. To achieve this goal a sophisticated model system has been developed that is based on the translocation of the sex determining region sry from the Y chromosome to an autosome (chromosome 18) in mice. Breeding animals with such a genetic variation will lead to four types of animals: XX male and XX female phenotype, XY female and XY male phenotype. Breeding these animals and subjecting them to gonadectomies to eliminate the effect of sexual hormones will lead to experimental conditions where the effect of sex chromosome complement and effect of sex hormones can be separated. For example, it has been shown that the number of X chromosomes correlates with the development of obesity and protects against the development of myocarditis. The four core genotype model can also be used to distinguish between the activational and organizational effects of sex hormones. Activational effects of sex hormones are those that are the result of the presence of the sex hormones and their activation of signaling pathways or gene transcription. In contrast, activational effects of sex hormones are those that are due to DNA, chromatin, or histone modifications that have been introduced at a given time point and last for significant periods of life of an animal. These effects are not bound to the direct activity of sex hormones and persist after gonadectomy.45

24.6  SEX DIFFERENCES IN CARDIOVASCULAR CELL FUNCTIONS Alterations in calcium handling, in energy metabolism, in inflammation, hypertrophy, and fibrosis interact and together lead to SD in cardiac function (Fig. 24.3).

24.6.1  Calcium Homeostasis Significant SD and sex hormone related differences have been shown for expression and regulation of calcium channels. Female mice and rat exhibit smaller calcium inward currents and a lower sarcoplasmic calcium load than males. This is particularly evident after betaadrenergic stimulation.46 It has been assumed that these differences are related to estrogen since ovariectomy increases sarcoplasmic reticulum calcium content and calcium transients.47 Calcium content depends also on the estrous cycle.48 It has also been observed that females

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FIGURE 24.3  Role of sex and sex hormones in fibrosis metabolism, and inflammation leading to contractile dysfunction and heart failure under stress.

have smaller calcium transients and smaller changes in cardiomyocyte shortening.46 Female hearts have reduced calcium entry after ischemic injury49 and this may be associated with NO-mediated changes of the L-type calcium channel.50 These protective mechanisms in female cells may be due to downregulation of the L-type calcium channel by estrogen receptor alpha, or by inhibition of this channel. Estrogen also regulates the myofilament sensitivity to calcium.50,51 A number of ovariectomy experiments and estrogen substitution experiments confirmed these findings.51,52 Estrogen also acts on the ATP sensitive potassium channels (KATP) in the sarcolemma and mitochondria. Activation of these channels may contribute to the protection of the female heart by estrogen against ischemia/reperfusion injury. Testosterone seems to regulate L- and T-type calcium channels in the opposite direction from estrogens. Testosterone also shortens the QT interval whereas estrogen prolongs it.53,54 Genetic studies support the protection of the female heart against disturbances of calcium handling. Knockout of the Fkbp1b gene, a gene that regulates sarcoplasmic calcium release via ryanodine receptors, causes myocardial hypertrophy in male mice only.55 Similarly, phospholamban deletion is more harmful to male than to female mice.56 These cellular differences in calcium handling may underlie some of the clinically observed differences between women and men in arrhythmia.

24.6.2  Energy Metabolism Female animals maintain energy metabolism in HF and under stress, ischemic or other forms of stress, better than males. Balance of lipid and carbohydrate use coordinated by muscle carnitine palmitoyltransferase and pyruvate dehydrogenase are involved in these processes.57 Furthermore, production of reactive oxygen species (ROS) under ischemic conditions and other forms of

stress appears less pronounced in the female than in male hearts. Also degradation of free radicals, ROS by manganese superoxide dismutase and glutathione peroxidase is more pronounced in female than in male hearts.58 Estrogen also controls mitochondrial energy metabolism. Estrogen controls the expression of mitochondrial genes and induces posttranslational modification of mitochondrial proteins. The role of estrogen has been documented by ovariectomy and estrogen replacement.59,60 Control of central activators of mitochondrial function and gene transcription, like PPARγ and PGC-1 alpha, may play a major role in these mechanisms.61–64 Estrogen is particularly involved in the control of synthesis of respiratory proteins and the tricarboxylic citric acid cycle.65,66 This is a major mechanism explaining female and estrogen-mediated protection against ischemic cardiovascular stress.67,68 The effects may be important in cardiomyocytes and vascular smooth muscle cells as well. SD have also been observed in cardiac lipid and carbohydrate metabolism. Membrane fatty acid transporter CD36 as well as fatty acid transport proteins FATP69 are controlled by estrogen. Estrogen reduces free fatty acid levels. This estrogen regulation of LDL, HDL, and cholesterol level is relevant in several cell types including macrophages, vascular smooth muscle cells, and myocytes.70 It has been suggested that female sex and/or estrogens also lead to enhanced myocardial fatty acid oxidation in women.71

24.6.3  Apoptosis and Autophagy Rates of apoptosis in stressed hearts are always greater in males than in females. Similar observations have been made in rodent hearts as well as in human aging and human HF.72,73 Signaling protein kinase B or Akt may play a role in these phenomena.74 Regulation of the antiapoptotic protein Bcl2 and proapoptotic protein

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24.7  Sex Differences in Clinical Syndromes

Bax in a sex-specific manner may contribute. Inhibition of TNF-alpha and p53 also contribute to reduction of apoptosis by estrogens.75 Autophagy may protect cells from induction of apoptosis. Females exhibit higher levels of autophagy than males. However, data are still to some degree contradictory.76,77

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of matrix turnover, inflammation, and activation of the renin–angiotensin system in the male hearts and less downregulation of oxidative metabolism in the female hearts.84 This pattern is similar to that in human aortic stenosis (AS), as discussed below. Thus, significant SD exist in mouse models of pressure overload and mimic the events in the human heart.

24.6.4  Inflammatory Pathways or Mechanisms Inflammation and autoimmunity are regulated in sexspecific, sex hormone-dependent manner. Activation of cardiac macrophages, cytokine production, and chemokine productions depend on sex hormones. Testosterone and male sex appear to drive macrophages into polarization versus proinflammatory M1 phenotype, whereas female sex and estrogens drive them toward the polarization into the antiinflammatory phenotype. Thus, female cells produce less TNF-alpha, IL1, and IL6.78,79 Overexpression of TNF-alpha may produce higher mortality and lead to a stronger development of HF in male than in female hearts.80 Cardioprotective effects of estrogen have been described and been linked to the inhibition of neutrophil infiltration in an estrogen receptor-dependent manner after ischemia/reperfusion.81 Estrogen receptor beta is also involved in the inhibition of the inflammatory response after cardiac injury.82 Again, gonadectomy experiments showing that ovariectomized rats have a lower increase in the pro-inflammatory cytokine TNF-alpha, support the lower degree of inflammation in female hearts. In our own hands, in a pressure overload model26 estrogen receptor beta was needed to inhibit inflammatory pathways and NF-κB regulation.82 Nevertheless estrogen receptor alpha is also involved.83 In contrast, observations on the impact of testosterone on inflammation are more inconsistent and have not yet been clarified to the same level as those of estrogen.

24.6.5  Myocardial Hypertrophy SD exist in physiological and pathological myocardial hypertrophy and are modulated by estrogen. Exercise in mice leads to more adaptive myocardial hypertrophy in female than in male mice.27 If pressure overload is induced in mice by constriction of the thoracic aorta, males develop an unfavorable form of myocardial hypertrophy, i.e., more eccentric hypertrophy and fibrosis, whereas females develop more concentric myocardial hypertrophy and better energy metabolism. Contractile reserve and calcium handling are better preserved in females than in males subjected to pressure overload. Furthermore, males develop HF at an earlier stage than females.26 A more pronounced increase in myocardial dilatation and fibrosis exists in male than in female animals. The genomic response to pressure overload differed significantly between the sexes, with stronger activation

24.6.6 Fibrosis Major SD occur in cardiac fibrosis. They are similar in mice and men. The changes and differences may explain some SD in human disease.84–86 Estrogen and androgen receptors control collagen synthesis in a sex-specific manner. In general, estrogen inhibits cardiac fibrosis in females but may to some degree enhance it in males.85,87 This happens partially by effects on gene transcription and partially by reduced activation of estrogen receptor alpha and MAP kinase pathway signaling17 (Fig. 24.4). The fact that estrogen inhibits the progesterone receptor which has also profibrotic effects may also contribute to SD.88 Profibrotic microRNAs like miRNA-21, -24, -27 may also be involved in stimulation and inhibition of fibrosis in a sex-specific manner. Their activity seems to be partially controlled by estrogen receptor beta.43

24.7  SEX DIFFERENCES IN CLINICAL SYNDROMES 24.7.1  Sex and Gender In a number of human diseases affecting the cardiovascular system, significant differences between women and men exist. These are in part due to the biological mechanisms described above. It must however be mentioned that in the human the sociocultural dimension “gender” also plays a major role. Gender summarizes all the differences between women and men that are due to attitudes, behaviors, sociocultural expectations of friends, family, the society, and ourselves, leading to our behavior as women or men. Gender roles influence our awareness of disease, attitudes toward disease, manifestation of disease symptoms, communication of pain, access to the health care system, attitudes of doctors toward patients, interpretation of signs and symptoms of disease, and much more (Fig. 24.5). Perception of stress differs between women and men and will lead to different reactions of the cardiovascular system toward stressors. The sexually dimorphic manifestation of the Takotsubo syndrome, which is a stress-induced cardiomyopathy almost exclusively occurring in women, gives an optimal example for the importance of these mechanisms. Different percentages of myocardial infarction (MI) due to physical or emotional stress in women and men also are a good example.

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FIGURE 24.4  Sex-specific and estrogen mediated gene regulation lead to sex differences in cardiac fibrosis.

FIGURE 24.5  Complex interaction of sex and gender during lifetime.

Some scientists would prefer to clearly separate the effects of biology, the sex effects, the effects of gender, and the sociocultural mechanisms of disease. However, in the field of medicine this is not always possible since the environment, by influencing nutrition, stress, or behavior, also impacts epigenetic remodeling and thereby the biology of our bodies. Most environmental stresses leave their traces in epigenetic modifications. As a consequence, in the human situation it is very difficult to clearly separate the effects of sex and gender in medicine and gender medicine has to cover both aspects.

24.7.2  Arterial Hypertension 24.7.2.1 Epidemiology In most Western countries, hypertension is much more frequent in young men compared with young women, whereas in the elderly, the ratio switches and women are more frequently affected.89 24.7.2.2 Pathophysiology SD in hypertension have frequently been related to SD in the renin–angiotensin system. Animal models

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FIGURE 24.6  Mechanisms of ischemic heart disease in women. Adapted from EUGenMed Cardiovascular Clinical Study Group, Regitz-Zagrosek V, Oertelt-Prigione S, et al. Gender in cardiovascular diseases: impact on clinical manifestations, management, andoutcomes. Eur Heart J. 2016; 37(1):24–34.

with modifications of the renin–angiotensin system, such as the spontaneously hypertensive rat, exhibit in general significant SD with more severe hypertension and more severe end-organ damage in the males. This argues for a stronger role of this system in men than in women. In agreement with this hypothesis, hypertensive LV hypertrophy can be treated less well in women than in men with inhibitors of the renin–angiotensin system, angiotensin converting enzyme inhibitors (ACEI).90 Other pathophysiological mechanisms with sex-specific expression are related to SD in the endothelial system and in the Nitric Oxide (NO) system. Also beta-adrenergic stimulation appears to exhibit significant differences in women and men. Hypertensive women frequently exhibit more vascular and also more myocardial stiffness than men at comparable age probably due to structural remodeling.3 As a consequence of the greater stiffness of the vascular system, women more frequently exhibit isolated systolic hypertension in comparison with men. 24.7.2.3 Diagnosis The diagnosis of hypertension and related end-organ damage in women and men is generally the same.91 However, 24-h blood pressure readings may be recommended over isolated blood pressure assessment, in particular in women who are prone to developing white coat hypertension leading to an overdiagnosis of hypertension if only office blood pressures readings are used.91

24.7.2.4 Treatment and Outcomes Most antihypertensive drugs work well in women and men. However, for some unknown reasons, women are treated much more frequently with diuretics than men. Women are also more frequently treated with calcium antagonists. However, some of these, like amlodipine, may have more severe side effects in women. Women have a greater tendency to develop cough with ACE inhibitors which does not occur with angiotensin receptor blockers. Under treatment with beta blockers, women develop more adverse effects if beta blockers are used that are metabolized through CYP2D6, which is less active in women than in men, leading to enhanced blood levels of the these substances. No SD have been described so far for angiotensin receptor blockers and direct renin inhibitors. Hypertensive women have a higher rate of developing stroke in comparison with men.92 As in normal controls, LV function is greater in hypertensive women compared with hypertensive men. Nevertheless hypertensive women develop systolic HF or HF with preserved ejection fraction more frequently than hypertensive men.3

24.7.3  Ischemic Heart Disease 24.7.3.1 Epidemiology Ischemic heart disease (IHD) which is one of the most frequent syndromes of the Western world is most frequently due to atherosclerosis in men but not necessarily in women (see below) (Fig. 24.6). It develops earlier in

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men than in women and affects mainly the epicardial coronary arteries, designated as CAD. As a consequence, MI in general appears 10 years earlier and is associated with a more widely distributed CAD in men than in women. Sex distribution changes over lifetime: acute coronary syndromes (ACS) and MI occur 3–4 times more often in men than women below age 60, but after 75 years, women represent the majority of patients. Due to unfavorable lifestyle changes over the past decades the number of ACS in women below 60 years has doubled and in women below 50 years has even tripled.93 While Western countries have seen a general decline in prevalence and mortality from CAD in recent decades, the development seems less beneficial in younger women with some studies even suggesting trends toward an increase.94 Risk factors for IHD are similar in women and men but some differences exist.95 Diabetes mellitus—type 2, type 1, and gestational diabetes—emerged as a major risk factor that worsens IHD outcome more in women than in men. There is a threefold excess fatal IHD risk in women with diabetes compared with nondiabetic women, with a higher adjusted hazard ratio of fatal IHD in women compared with men with diabetes. MI also occurs earlier in women with diabetes compared with men, and is associated with higher mortality. Reasons are unclear; impact of sexual hormones, or lifestyle have both been implicated.96 A higher number of comorbidities such as obesity and inflammatory disease may contribute. There is also a gender difference in the HF risk induced by diabetes, as documented in the Framingham Heart Study. HF risk was twofold higher in men (p60 years of age with aortic stenosis. Am J Cardiol. 1994;74(8):794–798. Cramariuc D, Rieck AE, Staal EM, et al. Factors influencing left ventricular structure and stress-corrected systolic function in men and women with asymptomatic aortic valve stenosis (a SEAS Substudy). Am J Cardiol. 2008;101(4):510–515. Cramariuc D, Rogge BP, Lonnebakken MT, et al. Sex differences in cardiovascular outcome during progression of aortic valve stenosis. Heart. 2015;101(3):209–214. Carroll JD, Carroll EP, Feldman T, et  al. Sex-associated differences in left ventricular function in aortic stenosis of the elderly. Circulation. 1992;86(4):1099–1107. Douglas PS, Otto CM, Mickel MC, Labovitz A, Reid CL, Davis KB. Gender differences in left ventricle geometry and function in patients undergoing balloon dilatation of the aortic valve for isolated aortic stenosis. NHLBI Balloon Valvuloplasty Registry. Br Heart J. 1995;73(6):548–554. Villari B, Campbell SE, Schneider J, Vassalli G, Chiariello M, Hess OM. Sex-dependent differences in left ventricular function and structure in chronic pressure overload. Eur Heart J. 1995;16(10):1410–1419. National Research Council. Women’s Health Research: Progress, Pitfalls, and Promise.. Washington, DC: The National Academies Press; 2010. Smith CR, Leon MB, Mack MJ, et al. Transcatheter versus surgical aortic-valve replacement in high-risk patients. New Engl J Med. 2011;364(23):2187–2198. Stangl V, Baldenhofer G, Knebel F, et  al. Impact of gender on three-month outcome and left ventricular remodeling after transfemoral transcatheter aortic valve implantation. Am J Cardiol. 2012;110(6):884–890. Ribeiro HB, Webb JG, Makkar RR, et al. Predictive factors, management, and clinical outcomes of coronary obstruction following transcatheter aortic valve implantation: insights from a large multicenter registry. J Am Coll Cardiol. 2013;62(17):1552–1562. Zhao ZG, Liao YB, Peng Y, et  al. Sex-related differences in outcomes after transcatheter aortic valve implantation: a systematic review and meta-analysis. Circ Cardiovasc Interv. 2013;6(5): 543–551. Stangl V, Baldenhofer G, Laule M, Baumann G, Stangl K. Influence of sex on outcome following transcatheter aortic valve implantation (TAVI): systematic review and meta-analysis. J Interv Cardiol. 2014;27(6):531–539. Marijon E, Uy-Evanado A, Reinier K, et  al. Sudden cardiac arrest during sports activity in middle age. Circulation. 2015;131(16):1384–1391. Reinoehl J, Frankovich D, Machado C, et al. Probucol-associated tachyarrhythmic events and QT prolongation: importance of gender. Am Heart J. 1996;131(6):1184–1191. Lehmann MH, Hardy S, Archibald D, MacNeil DJ. JTc prolongation with d, l-sotalol in women versus men. Am J Cardiol. 1999;83(3):354–359. Benjamin EJ, Levy D, Vaziri SM, D’Agostino RB, Belanger AJ, Wolf PA. Independent risk factors for atrial fibrillation in a

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population-based cohort. The Framingham Heart Study. JAMA. 1994;271(11):840–844. Albert CM, McGovern BA, Newell JB, Ruskin JN. Sex differences in cardiac arrest survivors. Circulation. 1996;93(6):1170–1176. AVID. A comparison of antiarrhythmic-drug therapy with implantable defibrillators in patients resuscitated from nearfatal ventricular arrhythmias. The Antiarrhythmics versus Implantable Defibrillators (AVID) Investigators. New Engl J Med. 1997;337(22):1576–1583. Benjamin EJ, Wolf PA, D’Agostino RB, Silbershatz H, Kannel WB, Levy D. Impact of atrial fibrillation on the risk of death: the Framingham Heart Study. Circulation. 1998;98(10):946–952. Kannel WB, Wilson PW, D’Agostino RB, Cobb J. Sudden coronary death in women. Am Heart J. 1998;136(2):205–212. Buxton AE, Lee KL, Fisher JD, Josephson ME, Prystowsky EN, Hafley G. A randomized study of the prevention of sudden death in patients with coronary artery disease. Multicenter unsustained tachycardia trial investigators. New Engl J Med. 1999;341(25):1882–1890. Rautaharju PM, Zhou SH, Wong S, et  al. Sex differences in the evolution of the electrocardiographic QT interval with age. Can J Cardiol. 1992;8(7):690–695. Salama G, Bett GC. Sex differences in the mechanisms underlying long QT syndrome. Am J Physiol Heart Circ Physiol. 2014;307(5):H640–H648. Pham TV, Sosunov EA, Gainullin RZ, Danilo Jr. P, Rosen MR. Impact of sex and gonadal steroids on prolongation of ventricular repolarization and arrhythmias induced by I(K)-blocking drugs. Circulation. 2001;103(17):2207–2212. Zhang Y, Ouyang P, Post WS, et  al. Sex-steroid hormones and electrocardiographic QT-interval duration: findings from the third National Health and Nutrition Examination Survey and the Multi-Ethnic Study of Atherosclerosis. Am J Epidemiol. 2011;174(4):403–411. Kurokawa J. Sex and gender aspects in antiarrhythmic therapy. In: Regitz-Zagrosek V, ed. Sex and Gender Differences in Pharmacology. Heidelberg: Springer Verlag; 2012:237–263. McMurray JJ, Adamopoulos S, Anker SD, et al. ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. Eur Heart J. 2012;33(14):1787–1847. Seeland U. Sex and gender differences in cardiovascular drug therapy. In: Regitz-Zagrosek V, ed. Sex and Gender Differences in Pharmacology. Heidelberg: Springer Verlag; 2012:211–236. Bagger JP, Baandrup U, Rasmussen K, Moller M, Vesterlund T. Cardiomyopathy in western Denmark. Br Heart J. 1984;52(3): 327–331. Gillum RF. Idiopathic cardiomyopathy in the United States, 1970-1982. Am Heart J. 1986;111(4):752–755. Codd MB, Sugrue DD, Gersh BJ, Melton 3rd LJ. Epidemiology of idiopathic dilated and hypertrophic cardiomyopathy. a population-based study in Olmsted County, Minnesota, 1975-1984. Circulation. 1989;80(3):564–572. Regitz-Zagrosek V. Therapeutic implications of the genderspecific aspects of cardiovascular disease. Nat Rev Drug Discov. 2006;5(5):425–438. Sliwa K, Hilfiker-Kleiner D, Mebazaa A, et al. EURObservational Research Programme: a worldwide registry on peripartum cardiomyopathy (PPCM) in conjunction with the Heart Failure Association of the European Society of Cardiology Working Group on PPCM. Eur J Heart Fail. 2014;16(5):583–591. Bauersachs J, Arrigo M, Hilfiker-Kleiner D, et al. Current management of patients with severe acute peripartum cardiomyopathy: practical guidance from the Heart Failure Association of the

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

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176.

177.

178.

179.

180.

181.

182.

183.

184.

185.

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European Society of Cardiology Study Group on peripartum cardiomyopathy. Eur J Heart Fail. 2016;18(9):1096–1105. Yancy CW, Jessup M, Bozkurt B, et al. ACCF/AHA guideline for the management of heart failure: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation. 2013;128(16):1810–1852. Authors/Task Force members Elliott PM, Anastasakis A, et  al. ESC Guidelines on diagnosis and management of hypertrophic cardiomyopathy: the Task Force for the Diagnosis and Management of Hypertrophic Cardiomyopathy of the European Society of Cardiology (ESC). Eur Heart J. 2014;35(39):2733–2779. Levy D, Kenchaiah S, Larson MG, et al. Long-term trends in the incidence of and survival with heart failure. New Engl J Med. 2002;347(18):1397–1402. Cleland JG, Swedberg K, Follath F, et al. The EuroHeart Failure survey programme -- a survey on the quality of care among patients with heart failure in Europe. Part 1: patient characteristics and diagnosis. Eur Heart J. 2003;24(5):442–463. Gori M, Lam CS, Gupta DK, et  al. Sex-specific cardiovascular structure and function in heart failure with preserved ejection fraction. Eur J Heart Fail. 2014;16(5):535–542. Lam CS, Carson PE, Anand IS, et  al. Sex differences in clinical characteristics and outcomes in elderly patients with heart failure and preserved ejection fraction: the Irbesartan in Heart Failure with Preserved Ejection Fraction (I-PRESERVE) trial. Circ Heart Fail. 2012;5(5):571–578. Mentz RJ, Kelly JP, von Lueder TG, et  al. Noncardiac comorbidities in heart failure with reduced versus preserved ejection fraction. J Am Coll Cardiol. 2014;64(21):2281–2293. Luchner A, Brockel U, Muscholl M, et  al. Gender-specific differences of cardiac remodeling in subjects with left ventricular dysfunction: a population-based study. Cardiovasc Res. 2002;53(3):720–727. Regitz-Zagrosek V, Seeland U. Cardiovascular therapy in women and men. In: Regitz-Zagrosek V, ed. Sex and Gender Differences in Pharmacoloy. Heidelberg: Springer Verlag; 2012. Regitz-Zagrosek V, Brokat S, Tschope C. Role of gender in heart failure with normal left ventricular ejection fraction. Prog Cardiovasc Dis. 2007;49(4):241–251. Donekal S, Venkatesh BA, Liu YC, et al. Interstitial fibrosis, left ventricular remodeling, and myocardial mechanical behavior

186.

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188.

189.

190.

191.

192.

193.

194.

195.

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in a population-based multiethnic cohort: the Multi-Ethnic Study of Atherosclerosis (MESA) study. Circ Cardiovasc Imaging. 2014;7(2):292–302. Regitz-Zagrosek V, Erdmann J, Wellnhofer E, Raible J, Fleck E. Novel mutation in the alpha-tropomyosin gene and transition from hypertrophic to hypocontractile dilated cardiomyopathy. Circulation. 2000;102(17):E112–E116. Agvall B, Dahlstrom U. Patients in primary health care diagnosed and treated as heart failure, with special reference to gender differences. Scand J Prim Health Care. 2001;19(1):14–19. Rathore SS, Wang Y, Krumholz HM. Sex-based differences in the effect of digoxin for the treatment of heart failure. New Engl J Med. 2002;347(18):1403–1411. Bristow MR, Saxon LA, Boehmer J, et al. Cardiac-resynchronization therapy with or without an implantable defibrillator in advanced chronic heart failure. New Engl J Med. 2004;350(21):2140–2150. Moss AJ, Hall WJ, Cannom DS, et al. Cardiac-resynchronization therapy for the prevention of heart-failure events. New Engl J Med. 2009;361(14):1329–1338. Arshad A, Moss AJ, Foster E, et  al. Cardiac resynchronization therapy is more effective in women than in men: the MADITCRT (Multicenter Automatic Defibrillator Implantation Trial with Cardiac Resynchronization Therapy) trial. J Am Coll Cardiol. 2011;57(7):813–820. Zusterzeel R, Selzman KA, Sanders WE, et al. Cardiac resynchronization therapy in women: US Food and Drug Administration meta-analysis of patient-level data. JAMA Intern Med. 2014;174(8):1340–1348. Regitz-Zagrosek V, Petrov G, Lehmkuhl E, et al. Heart transplantation in women with dilated cardiomyopathy. Transplantation. 2010;89(2):236–244. Weymann A, Patil NP, Sabashnikov A, et al. Gender differences in continuous-flow left ventricular assist device therapy as a bridge to transplantation: a risk-adjusted comparison using a propensity score-matching analysis. Artif Organs. 2015;39(3):212–219. Oertelt-Prigione S, Gohlke BO, Dunkel M, Preissner R, RegitzZagrosek V. GenderMedDB: an interactive database of sex and gender-specific medical literature. Biol Sex Differ. 2014;5:7. Ludwig S, Oertelt-Prigione S, Kurmeyer C, et  al. A Successful strategy to integrate sex and gender medicine into a newly developed medical curriculum. J Women’s Health. 2015;24(12):996–1005.

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C H A P T E R

25 Estrogen: Impact on Cardiomyocytes and the Heart Jin Kyung Kim The University of California Irvine Medical Center, Orange, CA, United States

O U T L I N E 25.1 Introduction

25.3.4 Estrogen and ERs in Cardiomyocyte Apoptosis 369 25.3.5 Estrogen and ERs in Cardiac Regeneration 370 25.3.6 Estrogen and ERs in Cardiac Hypertrophy 371 25.3.7 Estrogen and ERs in Cardiac Electrophysiology and Contractility 372

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25.2 Estrogen on the Heart: Clinical Considerations 364 25.2.1 Hormone Replacement Therapy in Heart Disease 364 25.2.2 The Timing Hypothesis 365 25.3 Estrogen on Cardiomyocytes: Scientific Data 25.3.1 Estrogen Synthesis and Estrogen Receptors in the Heart 25.3.2 ER Genetics and CVD 25.3.3 Estrogen and ERs in Cardiac Metabolism

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25.1 INTRODUCTION Cardiovascular disease (CVD) is the leading cause of mortality in the United States and other developed nations.1,2 Death from coronary artery disease (CAD), also referred to as ischemic heart disease (IHD) or coronary heart disease (CHD), constitutes the largest portion of mortality from CVD. Although CAD affects both men and women, there are distinct gender differences observed in the manifestation of the disease and associated morbidities. These gender differences are increasingly recognized by the medical community and the lay public, but the underlying mechanisms remain poorly understood. One of the notable gender-specific distinctions in heart disease is that premenopausal women have a significantly lower incidence of CAD, compared to age-matched men, but that protection is lost after menopause. The lower incidence of CAD in premenopausal Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00016-4

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women was documented as early as the 1930s by several case series studying myocardial infarction (MI), in which the mechanism for the gender-difference in the time of onset of the disease was postulated to be related to the “endocrine make-up of men and women.”3–5 Then, in 1976, the Framingham Heart Study group published a landmark article that firmly established the association between menopause and increased risk of CHD.6 Reported in the article were the following seminal observations: (1) men over 30 years old develop CHD at more than twice the rate of age-matched women, and experience a higher incidence of the more serious clinical manifestations of MI, including sudden cardiac death (SCD). The incidence of CAD increases roughly 20 years later in women compared with men. (2) Traditional risk factors associated with CHD, such as hypertension or hyperlipidemia, alone did not account for the gender difference. Diabetes was the only risk factor that virtually eliminated

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the female advantage in CVD morbidity and mortality; and (3) there was a highly significant, twofold increase in the relative risk of CHD in postmenopausal women, compared to age-matched premenopausal group. As a result of these early observations, it was postulated that the endogenous female hormone, estrogen, might render women resistant to IHD until menopause, at which point there is a gradual loss of protection as the hormone level drops close to that of men.

25.2  ESTROGEN ON THE HEART: CLINICAL CONSIDERATIONS 25.2.1  Hormone Replacement Therapy in Heart Disease Over the years following the Framingham paper, the hypothesis that estrogen may be cardioprotective has been extensively studied and supported by a plethora of controlled experiments using animal and cultured cell models, as well as observational, population-based investigations. A majority of these reports demonstrate estrogen to have antiatherogenic, antiarrhythmogenic, antiinflammatory effects in various experimental settings, as discussed later in the chapter.7–10 Given the strength of evidence linking estrogen to cardioprotection, and the accelerated risk of CAD after menopause, it was postulated that hormone replacement therapy (HRT) would prevent the increased rate of heart disease in postmenopausal women. This theory was put to test in the Women’s Health Initiative (WHI).11 WHI was a large, prospective, randomized, placebo-controlled clinical trial funded by the National Institutes of Health (NIH) to address CVD, cancer, and osteoporosis—conditions common in postmenopausal women. It enrolled 161,808 women aged 50–79, including 68,132 women to randomized clinical trials to investigate the role of HRT, dietary modification, and calcium/vitamin D supplement, as well as 93,676 women in an observational study to identify predictors of disease. Of these different arms of WHI, by far the most anticipated were the results from the two interventional arms testing HRT as a preventive therapy to reduce CVD in postmenopausal women— 16,608 women with an intact uterus in the estrogen plus progestin versus placebo group and 10,739 in the estrogen alone versus placebo group. Unexpectedly, the first wave of results published from the trial showed no overall benefit of HRT in the primary prevention of CVD.11,12 In the estrogen plus progestin versus placebo group, the number of women experiencing CHD events (nonfatal MI and CHD death) was 37 per 10,000 personyears in the estrogen plus progestin group, compared to 30 per 10,000 person-years in placebo, reaching nominal statistical significance (at the 0.05 level), with a wide

adjusted 95% confidence interval (CI) of 0.82–2.13.11 No significant differences were observed in CHD deaths or revascularization procedures. Most of the excess was in nonfatal MI. In the estrogen alone versus placebo group, the CHD rate in the estrogen only group, compared with placebo, was 49 versus 54 per 10,000 person-years, with the hazard ratio (HR) of 0.91 and CI of 0.75–1.12.12 This was not thought to be significant statistically, but a high dropout rate of 53.8% was noted. The original findings of this landmark trial prompted a nationwide reluctance to use estrogen to prevent heart disease in postmenopausal women. However, the study design was harshly criticized by many researchers and clinicians, who questioned the baseline characteristics of the study population, the validity of the data analyses, and conclusions which were strikingly at odds with the collective scientific literature supporting the beneficial cardiovascular effects of estrogen. In fact, there are several explanations for the discrepancy between the results of WHI and the data from numerous scientific studies attesting to estrogenmediated cardioprotection. They are as follows: (1) the estrogen used in WHI, oral conjugated equine estrogen 0.625 mg per day, is not the same as endogenous human estrogen. The formulation used in WHI consisted of a heterogeneous group of estrogens, including at least 10 equine estrogenic molecules. Many of these exogenous substances are not secreted by human ovaries, and their effects on estrogen receptor (ER) activation and tissue-specific consequences remain largely unknown. Furthermore, the actions of these equine hormones on the human heart are poorly characterized; (2) HRT used in WHI contained doses of estrogens which produced supra-physiological plasma levels of the hormone, which may have negatively impacted the benefit-risk ratio; (3) the estrogen plus progestin arm of the trial used medroxyprogesterone, which has been shown to oppose the protective action of human endogenous estrogen on vascular endothelium, coronary artery dilation, and protection from ischemic injury7,8,13; (4) there were also concerns about oral administration of the hormones, especially in high doses, since the orally given drug is metabolized in the liver and induces activated protein C (APC) resistance associated with increased blood coagulation and C-reactive protein (CRP), a proinflammatory and pro-atherosclerotic marker.14 More recent studies testing the efficacy of HRT in ameliorating CVD thus examine formulations to avoid the prothrombotic posthepatic effect by including transdermal or vaginal routes of administration and/or the use of much lower doses of the hormone15–17; (5) while one of the principal intents of WHI was to see the effectiveness of HRT in the primary prevention of CAD, the majority of the study population was well past menopause (average age at enrollment was 63.3 years), and two-thirds of

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25.2  Estrogen on the Heart: Clinical Considerations

the subjects were obese and receiving treatment for high blood pressure. Older age, obesity, and hypertension are well-known risk factors for CAD. Given the chronic, insidious natural history of atherosclerosis, silent CAD may already have been present in many of these high risk patients who had been without endogenous estrogen for many years prior to enrollment, keeping in mind that the average age of menopause in the United States is 51 years of age.18 This would mean an estrogen-depleted state of longer than 10 years for many of the study participants prior to the start of WHI. As estrogen does not eliminate preexisting atherosclerosis, administration of the hormone in women likely to have CAD is not ideal for primary prevention.19 Thus, the baseline characteristics of the study population were suboptimal for the purpose of testing the primary prevention of CVD by estrogen; (6) WHI was not designed to address whether endogenous human estrogen might be effective in reducing complication/sequelae of acute coronary ischemia, i.e., acute MI or unstable angina. In various scientific models in which the effect of estrogen in acute ischemia was tested, the most potent form of human estrogens, 17β-estradiol (E2), was proven to be effective in reducing infarct size and negative sequelae of ischemic stress, including fatal arrhythmia, heart failure, or mortality; (7) with two-thirds of the study population in their 60s and high drop-out rates, the HRT treatment arm of WHI is estimated to be more than 10-fold underpowered to detect a change in cardiovascular events in younger, perimenopausal patients, a group most likely to draw benefit from a primary prevention therapy against the development of CVD during the menopausal transition.20 The answer to the question of why the WHI did not observe a preventive effect of estrogen in postmenopausal women is complicated. Because of the scope and expense of the study, a similar trial is not likely to be repeated using a better selection of preparation, route of administration and clinical subjects—the WHI was a $625 million study that enrolled 161,808 women. A small number of earlier trials testing HRT in secondary prevention of CAD, namely Heart and Estrogen/progestin Replacement Study (HERS) and HERS II, also failed to show overall benefit in reducing rates of primary CHD events or secondary cardiovascular events.21 This null finding may, in fact, be related in part to the short duration of the trial. These studies were also much smaller in scale (a total of 2763 postmenopausal women), compared to WHI, with a patient population that was not only older (mean age of 66.7 years old) but had documented atherosclerotic heart disease. Considering what is now known about the actions of estrogen, which does not effectively eradicate already existing atherosclerotic plaques, the reported outcome is not surprising.

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25.2.2  The Timing Hypothesis Estrogen improves many of traditional risk factors for CAD. Endogenous E2 or judiciously applied exogenous hormone lowers blood pressure,22 improves the serum lipid profile,23 retards progression of atherogenesis,10 reduces risk of developing diabetes,24 and improves perfusion of the myocardium by inhibiting coronary vasoconstriction.25 Putting together these salutary effects of estrogen with the observation that the onset of CAD accelerates after the loss of endogenous E2 through menopause, and that estrogen does not reverse preexisting CAD, it was reasoned that the group that would derive the most benefit from hormone replacement might be younger, perimenopausal women without established CVD. Subsequent analyses of the WHI subgroups, in fact, support this thought, demonstrating positive cardiovascular benefits in younger cohorts in whom HRT was started soon after menopause, i.e., when women had a shorter period of lacking endogenous estrogen before starting hormone replacement.26,27 The results of these post hoc analyses and what is already known about the actions of estrogen on atherogenesis gave rise to the timing hypothesis, which states that there may exist a critical perimenopausal period during which HRT could benefit women by mitigating the onset or progression of atherosclerotic heart disease. When administered beyond this time window, hormone therapy is unlikely to reverse already established atherosclerosis, and may even expose the patient to an increased incidence of MI. In support of the timing hypothesis, the recent trials, such as the Kronos Early Estrogen Prevention Study (KEEPS) and the Early Versus Late Intervention Trial With Estradiol (ELITE) testing low dose hormones in perimenopausal women, revealed that not only was HRT safe when started soon after menopause, but imaging end points determined by carotid artery intima-media thickness were consistent with slowing of atherosclerosis, accompanied by improvement of several markers of cardiovascular risk, including low- and high-density lipoprotein cholesterol levels and CRP.15,16 Because these two trials were not powered to detect statistical differences in cardiovascular mortality rates, whether the lower doses of nonoral HRT started early in menopause will reduce CV mortality risk remains to be confirmed. Nevertheless, a large, observational study involving 195,756 women showed that the use of vaginal estrogen was accompanied by significant decreases in the risk of death from CHD or stroke, with the highest risk reduction seen in women aged 50–59 years.17 These results corroborate the subgroup analyses of the WHI trials, indicating that HRT may reduce total mortality when initiated soon after menopause. The WHI suggests that HRT nonsignificantly reduce total mortality by 30% when initiated

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in women younger than 60 years.27 When data from both arms of HRT were combined, that reduction was statistically significant. Thus, the relevance of HRT on cardioprotection is far from being clearly defined, and remains a topic of continuing debate and controversy. Currently, the guidelines and recommendations from medical societies emphasize that individualization is of key importance in the decision to use HRT, incorporating the woman’s quality of life priorities and her personal risk factors.28,29 The 2016 recommendations from the International Menopause Society conclude that, for women starting HRT T, was associated with an increase in adverse outcomes among men and a decreased risk of outcomes in women. This finding suggests a role of cardiac aromatase in estrogen-mediated cardioprotection. E2 works through ER. Two classic receptor subtypes are ERα and ERβ, encoded by ESR1 and ESR2, respectively. They belong to the nuclear receptor superfamily and modulate transcriptional processes of target genes by binding to the ER element and recruiting coactivators. E2 is involved in the regulation of a wide range of genes, including those known to participate in mitochondrial function, redox homeostasis, carbohydrate metabolism, lipogenesis, and extracellular matrix integrity.45,46 Ligand-activated ER also work by a rapid, nongenomic mechanism at the plasma membrane level.47 ERα and ERβ are widely expressed in different tissue types in addition to uterus and ovary. Both receptor subtypes are expressed in the neonatal and adult heart.48–50 The receptors are found in the hearts of both genders and present in both atrial and ventricular cells.51 The cardiac ER regulates gene expression and posttranslational modifications via genomic and nongenomic mechanisms.52 Evidence on the whole demonstrates that estrogen-activated ER (E2/ER) effects antiapoptotic, prohypertrophic, antiinflammatory, vasodilatory, and angiogenic processes in the cardiovascular system.52 Within a cardiomyocyte, the functions of ER subtypes

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25.3  Estrogen on Cardiomyocytes: Scientific Data

do not overlap significantly. Rather, each receptor subtype contributes to distinct aspects of estrogen signaling related in part to their subcellular localization. While both are seen in the cytosol of the cardiomyocyte, ERα is also present in the plasma membrane where it regulates rapid, nongenomic signaling. Evidence for the presence of ERβ in the cardiomyocyte plasma membrane is lacking.51 Though both receptor subtypes are found in the mitochondria of cardiomyocytes and participate in the E2-initiated regulation of mitochondrial functions, the types of target molecules and extent of control exerted by each ER subtype are divergent.45,53–55 Gene expression profiling via microarray revealed that there was little overlap between the sets of genes regulated by ERα and ERβ, and that the direction of gene regulation by each ER subtype was differential. In addition to the classical ERα and ERβ, G proteincoupled receptor 30 (GPR30), previously thought to be an orphan receptor, is now recognized as an ER involved in rapid, nongenomic E2 signaling at the plasma membrane, and has been renamed G-protein coupled estrogen receptor (GPER).56,57 GPER is present in both male and female hearts and shown to be cardioprotective in its function under ischemic and hypertensive stress.58,59 Although the mechanism of the E2/GPER-mediated cardioprotection is still not clear, studies examining the role of GPER during ischemia reperfusion (I/R) injury suggest that the receptor inhibits the mitochondria permeability transition pore (mPTP) opening, a crucial step in programmed cell death or apoptosis, and activates the phospho-inositide-3 kinase (PI3K)-dependent prosurvival pathway.60 In addition, GPER activation acutely lowered blood pressure in animal models, and caused vasodilation of both rodent and human arterial blood vessels.61 The hemodynamic effect was abrogated in a genetic model of GPER loss-of-function, which was also associated with increased visceral obesity. These findings suggest that cardioprotection by GPER involves direct cytoprotection within the heart as well as mediation of vasoreactivity and metabolism. In sum, these ER are expressed and functional in cardiomyocytes, and orchestrate a myriad of estrogenspecific genomic and nongenomic responses in the heart.

25.3.2  ER Genetics and CVD More than 300 genes are reported to be associated with risk of CVD, and genome-wide association studies (GWAS) identified at least 33 genetic variants linked to increased risk for CAD and MI.62,63 While genetic factors are well-accepted to play a role in CAD risk, detailed genetics-based mechanistic work on gender differences has only begun to emerge. GWAS recently identified rs715 (localizing to carbamoyl-phosphate synthase 1, or CPS1) to have a strikingly significant and protective

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association with decreased risk of CAD only in women.64 CPS1 encodes a mitochondrial enzyme that catalyzes the rate-limiting step in the urea cycle. As aforementioned, all ER subtypes are found in mitochondria, and in the light of this new finding on rs715, a novel area of scientific inquiry may be considered to assess whether or not female cardioprotection is in part related to an interaction between estrogen and CPS1. A point of convergence between the E2/ER and CPS1 pathways has not been reported yet, but would be a potentially fruitful area of research. Establishing causality between the genetic polymorphisms, phenotypic variations, and downstream functionality within the context of disease susceptibility and prognosis is a challenging, multilayered issue that requires coordinated analyses of genomic, epigenomic, proteomic, and clinical data profile. In many cases of identified genetic variations, clinical implications of such polymorphisms are not clearly borne out, as there is seldom a linear narrative between the variant and endpoint disease risk. In general, however, mutations in the ER genes leading to changes in target gene expression and signaling are believed to alter the extent of estrogenspecific regulation on the heart, as is often the case with the ER polymorphism in cancer.65 Certain genetic variants in both ERα and ERβ can confer susceptibility to CVD.66–71 Patients with the ESR1 TA repeat polymorphism were found to exhibit higher left ventricular (LV) mass.68 It should be noted that increased LV mass portends adverse cardiovascular outcomes and is associated with an increased incidence of CVD, mortality from CVD, and mortality from all causes.72 Furthermore, several studies have identified polymorphisms in the ESR1 and ESR2 gene (coding for ERα and ERβ, respectively) that confer gender-differential risk of CVD. For ERβ, two polymorphisms of the receptor gene, ESR2 rs1256031 and ESR2 rs1256059, were strongly associated with increased LV mass and thickness in women with hypertension, independent of variation in blood pressure, plasma lipoprotein levels, or hyperglycemia, while there was no such association found in men.69 An ESR2 single nucleotide polymorphism (SNP), rs1271572 T allele, was significantly more common in those who developed MI, and this association was found to be limited to men only.66 Interestingly, another group reported essentially the reverse, concluding that the same SNP was associated with risk of MI in women but not men.70 The reason for the discordant findings remains unclear, but the distinctions of different genetic make-up and heritage (Spanish vs North American) and the ethnicitydependent variant frequencies may be responsible in these population-based studies. Several ESR1 variants are also ascribed to gender-specific risk of IHD. The CC genotype of –397T/C ERS1 gene contributed to higher risk of CHD only in men.67 Postmenopausal women

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with ESR1 variants, c.454–397 T allele and c.454–351 A allele, had an increased risk of MI and IHD, independent of known cardiovascular risk factors.71 In men, no such association was observed. In a telling case of the close relationship between ERα and atherosclerosis, a man who lacked functional ERα had premature CAD, hinting that the loss of functional ERα may put men at risk of CAD.73 Together, these studies represent an increasing number of cases supporting ER polymorphism as part of the genetic basis underlying sexual dimorphism in heart disease. Moreover, there may be an ER-related genetic mechanism behind the varying responses to HRT. In women with CAD taking 0.625 mg of oral conjugated equine estrogen per day or estrogen plus 2.5 mg of medroxyprogesterone acetate per day, an ESR1 polymorphism (IVS1–401 C/C genotype) was linked to an augmented response of the HDL cholesterol subfraction, HDL3, which is strongly associated with coronary events.74,75 This pattern was evident in both women receiving estrogen and those receiving estrogen plus progestin, and was preserved across racial and ethnic groups. However, not all studies of systems genetics yield a positive correlation between ER polymorphism and risk of heart disease. In a meta-analysis of 14 GWAS, the pertinence of ERα genetic variations in CAD risk was disputed, though the strength of the relationship is likely variant- and population-specific.76,77 There are also study limitations inherent to meta-analysis drawing conclusions extrapolated from heterogeneous groups of population-based studies. Data are currently lacking for GPER polymorphism and cardiac risk, although a certain GPER variant is implicated in risk of cancer.78 Over the recent years marked by groundbreaking advances in human genetics, evidence has been emerging that links ERα and ERβ polymorphisms to gender-specific risk of CVD. Though some of the data are inconsistent, the balance of accumulated findings suggests a substantive interaction between the ER polymorphisms and risk of IHD. Further research is needed to provide cogent biological mechanisms of how the specific genetic variations translate to associated risk. Eventual development and application of therapy tailored to the genetic basis of the individual will be a powerful clinical tool in the coming era of personalized medicine.

25.3.3  Estrogen and ERs in Cardiac Metabolism Estrogen plays a pivotal role in control of energy balance and glucose homeostasis via a diverse set of mechanisms.79 Clinically, this is manifest by development of altered metabolism, increased abdominal adipose tissue, and dyslipidemia in postmenopausal women.80 Animal

models of estrogen depletion are similarly notable for marked weight gain and a dysregulated metabolic state, which are reversed by E2 replacement.81 Of the three known ER, evidence points to ERα as the major factor in E2-specific regulation of energy homeostasis. This has been studied with experimental models of ERα knockout (KO) mice. These animals, both male and female, develop adipocyte hyperplasia, insulin resistance, and reduced energy expenditure, much like humans lacking ERα or aromatase.82,83 Furthermore, these in vivo findings are supported by recent innovative studies elucidating the underlying molecular mechanism of E2/ER-mediated regulation of cardiac energy metabolism. Until recently, most of the mechanistic studies examining E2 effects on cellular energetics have been conducted using nonmyocardial cells. However, new studies directly examine the role of estrogen in the regulation of cardiac metabolism. E2 supplementation significantly improved myocardial ATP levels and mitochondrial respiratory function in the heart.84 ERα was indispensable for normal glucose uptake in the murine heart.85 Cardiomyocyte-specific deletion of ERα changed sex-dependent metabolic gene expression and diverse transcriptional networks in cardiomyocytes.46 Of note, one study reported a novel finding of ERα-mediated regulation of glucose transporter type 4 (GLUT4) expression in mouse gastrocnemius muscle, while another demonstrated ERα-dependent glucose uptake via GLUT4 translocation in breast cancer cells.86,87 GLUT4 is a key molecule in insulin-sensitive glucose transport, and is expressed in the heart and adipose tissue in addition to skeletal muscle.88 Therefore, the newly discovered relationship between ERα and GLUT4 provides an important clue to the molecular mechanisms behind the E2-mediated regulation of glucose metabolism in cardiomyocytes. It is also important to remember that ERα is present and functional in mitochondria, the site of cellular energy production and redox regulation. Estrogen via its receptors has significant influence on maintaining mitochondrial integrity and function, thereby protecting the cardiomyocyte from oxidative stress and metabolic derangement when under ischemic or hypoxic insult.54,89–91 Recently, estrogen receptor-related receptor alpha (ERRα) working in concert with peroxisome proliferator-activated receptor-γ (PPAR γ) coactivator-1 alpha (PGC-1α) was found integral to cardiomyocyte cellular metabolism.92 Considering the high homology between ERα and ERRα subunits and the evidence of cross-talk between the two receptors, it is conceivable that ERRα may indirectly modulate E2/ERα signaling in cardiac metabolism or that E2/ERα signaling in turn affects ERRα activity.93 This may serve as yet another mechanism by which E2/ER mediates pathways involved in cardiac bioenergetics.

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In contrast to the consistent line of evidence affirming ERα activity in energy metabolism, the role of ERβ in metabolic regulation is less evident, and available data are somewhat conflicting. Lack of ERβ did not affect total body fat or lipoprotein levels,94 and the selective ERβ agonist diarylpropionitrile (DPN) did not alter food intake or body weight gain in animal models.95 However, another study showed that ligand-dependent ERβ activation efficiently treated high-fat diet-induced obesity, repressed several genes involved in the lipogenesis pathways, and inhibited PPARγ, a known proadipogenic transcription factor, and its transactivator, PGC-1α.96 Other studies also support the involvement of ERβ in control of cellular metabolism in mitochondria.55,97 In a coronary I/R injury model using cardiac-specific ERα and ERβ KO mice, the expression of several fatty acid metabolism genes in the heart was differentially altered by ERβ, when compared to ERα or wild-type mice.98 ERβ was reported to regulate mitochondrial respiratory complex IV activity in rat hearts after trauma-hemorrhage,99 with ERβ-specific cardioprotection via upregulation of PGC-1α.100 Some literature suggests that selective activation of ERβ modulates GLUT4 expression, although the extent of regulation is tissue-specific and limited to noncardiac cells in these cases.101,102 Experimental models also support a discrete role for GPER in protecting the body from obesity, insulin resistance, dyslipidemia, and a pro-inflammatory state, the conditions defining the metabolic syndrome, a risk factor for CVD.61,103 In the GPER KO mice model, receptor function is germane to estrogen-mediated glucose homeostasis in the pancreas.104 While these findings collectively provide a valuable insight into the role of GPER and potential for novel therapeutic targets, more preclinical studies are needed to gain a complete picture of the GPER signaling pathways in cardiac metabolism. Much of the conclusions on the E2/ER input in cardiomyocyte energetics were based on the manipulation of experimental animals or cultured cells. These are clearly invaluable research tools to shed light on the E2-specific mechanisms in the fuel homeostasis of the cardiomyocyte. However, it is of vital importance to translate these preclinical findings to relevant patient-based studies to cement the concepts delineated in mechanistic experiments. To date, there is one active clinical trial looking into the role of the estrogen-containing compound in insulin sensitivity and metabolism listed at clinicaltrials. gov, an NIH-sponsored online database of clinical studies performed worldwide. There is currently no populationbased study examining the role of specific ER subtypes in cardiac metabolism at clinicaltrials.gov. While specific loss- or gain-of-function useful to dissect the causal relationship of E2/ER in cardiac metabolism may not be practical in clinical studies, alternate noninvasive

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methods taking advantage of the contemporary imaging technology may be applicable to assess E2 signaling in cardiac energy milieu.105 Researchers used positron emission tomography (PET)-based imaging to show that women taking estrogen had evidence of increased myocardial fatty acid utilization when compared to men.106 Another study investigating a perioperative nutritional status was able to use 18F-fluorodeoxyglucose PET to assess myocardial glucose metabolism in patients with CAD.107 It is now feasible to map myocardial creatine kinase metabolism through chemical exchange saturation transfer in vivo, or to rapidly quantify human myocardial lipid levels using a commercial proton magnetic resonance spectroscopy.108,109 A combination of cutting edge imaging modalities with the intervention of E2 or ER subtype-specific modulators will provide a powerful investigative approach to further our understanding on the role of E2/ER in cardiac metabolism. A handful of such ER modulators have been developed for clinical purposes and are being tested in research, albeit mainly related to cancer and not yet in cardiomyocyte energy dynamics.110–113

25.3.4  Estrogen and ERs in Cardiomyocyte Apoptosis Most of cardiomyocytes in the human adult heart are believed to be terminally differentiated. In the early postnatal stage, human cardiomyocytes are arrested in the polyploid phase sans cytokinesis or karyokinesis, exhibiting nonproliferative cell cycles with increased DNA content without completing cell division.114,115 The adult heart is larger in size than the infant heart not because of an increased number of cardiomyocytes, but because of the increasing size of individual cells and changes in the extracellular matrix of the myocardium including increases in the connective tissue cells.114 Although a small percentage of cardiomyocytes in the adult heart renews, the rate of regeneration is low (1% turning over annually at the age of 20, gradually decreasing to 0.3% at the age of 75) and not deemed sufficient to restore the myocardium and cardiac function fully in the aftermath of severe injury.116–118 Therefore, it is imperative to curtail the loss of myocardial cells in the face of ischemic stress triggering extensive cell death. MI, heart failure, or severe coronary ischemia with or without reperfusion injury are all well-known triggers of cardiomyocyte apoptosis, or programmed cell death, a highly regulated and energy-requiring process.119–121 Apoptosis was shown to be a major factor contributing to infarct size, accounting for 86% of total cell loss in early stages of MI.122 Given its role in myocardial damage, its well-characterized molecular steps, and the unique features that distinguish it from necrosis, the apoptotic process is a desirable therapeutic target to

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be considered in an attempt to attenuate the extent of ischemia-related cardiac injury. Generally, the apoptotic processes are divided into the “extrinsic pathway” involving “death receptors,” such as tumor necrosis factor (TNF) and Fas receptors, or the “intrinsic pathway” involving mitochondria.123,124 How much each pathway contributes to myocardial apoptosis following an ischemia-related insult is not known, but there is evidence that the two pathways ultimately intersect at the mitochondria.125 Thus, mitochondria is not only the powerhouse of cellular energy regulation in the cardiomyocyte, but the command center of cell fate in normal physiology or under pathological conditions. Estrogen inhibits cardiac apoptosis. The antiapoptotic effect of estrogen on cardiomyocytes was first reported by Pelzer et al. in 2000.126 Since then, several cytoprotective or prosurvival molecular pathways have been characterized to be part of estrogen signaling in the inhibition of myocyte apoptosis. They include downregulation of NF-κB,127 activation of PI3K/Akt signaling,128 inhibition of apoptosis signal-regulating kinase 1 (ASK1) activity,91 upregulating corticotropin-releasing hormone receptor type 2,129 and more recently, promotion of p38β activity leading to inhibition of p53 and subsequent mitigation of mitochondrial redox response.54,90,130 Both the intrinsic and extrinsic apoptotic pathways are prevented by E2.131 Together, the data from both in-vitro and invivo experiments employing a variety of apoptotic triggers have consistently shown that E2 reduces cardiac apoptosis, suggesting that this property of estrogen may be an essential part of gender-specific cardioprotection against IHD. Both ERα and ERβ are involved in E2-specific mitigation of cardiomyocyte apoptosis. Both receptor subtypes are present in cardiomyocyte mitochondria, a major site of apoptotic signaling as mentioned above.48,53,55 Specific animal models studying either receptor subtype have demonstrated that cardioprotection is achieved by each of the tested receptor subtypes, leaving open the question of which subtype ultimately is accountable for the E2-mediated inhibition of cell death.132,133 One study did perform a head-to-head comparison between ERα and ERβ by using wild-type (WT), ERαKO, and ERβKO mice in an ex-vivo model of global I/R.98 In this study, ERβKO female hearts exhibited significantly less functional recovery than WT, while the extent of I/R injury was similar between ERαKO and WT female hearts, suggesting that ERβ plays a larger protective role in the female heart.98 Nonetheless, the literature to date yields no final consensus on which ER receptor has a dominant role in cardioprotection. This may be due to heterogeneity in the experimental models, end points, and doses of ER agonists from one report to another, as well as selection bias inherent in the study designs. In-vitro studies using cultured cardiomyocytes have produced

similarly mixed results.134,135 To this end, a possibility of ERs working as heterodimers has been considered. In a classically accepted paradigm of steroid hormones, E2-activated estrogen receptor undergo a conformational change and form homodimers to function as transactivators. However, in-vitro work has shown that heterodimers of ERα/β are transcriptionally active in noncardiac cells.136,137 Equivalent data using an in-vivo model have not been reported. Available information on functionally active ERα/β heterodimers in the adult heart so far is limited and mainly deduced from an in-vitro study using cultured neonatal murine cardiomyocytes.138 The third ER, GPER, was shown to be cytoprotective also by inhibiting myocyte apoptosis, a mechanism likely responsible for the previous observations that activation of this receptor subtype led to improved cardiac function and reduction in infarct size of both male and female murine hearts after I/R injury ex vivo.58,60,139 Further investigations are needed to define the contribution of each ER subtype in estrogen-mediated cardioprotection in heart disease and to confirm a functional pool of ER heterodimers in vivo.

25.3.5  Estrogen and ERs in Cardiac Regeneration As discussed above, the data consistently support the ability of estrogen to protect the heart by attenuating myocardial cell death. Under ischemic stress, the E2-treated cardiomyocyte is resistant to apoptotic triggers, leading to the organ-level protection seen in terms of better functional recovery and less infarct size, lending support to the idea that E2 is cytoprotective and improves viability of existing cardiomyocytes. There is now a new line of evidence to add another mechanism to explain the hormone’s positive actions on the heart: stimulation of myocyte regeneration. In the last decade or so, work in cardiac regenerative medicine has escalated at unprecedented speed. While the issues such as identifying the exact source of cardiac stem cells (CSC) or fine-tuning optimal techniques of generating mature myocytes from pluripotent progenitor cells are yet to be settled,140–142 there has been, nevertheless, remarkable progress in the field, culminating in the harvest of human resident cardiac cells that are positive for c-kit (stem cell-related surface antigen), selfrenewing, and multipotent.143–145 When these cells were treated with E2 and infused into isolated mouse hearts, there was a more robust production of CSC-derived protective factors and improved cardiac function, as well as better cardiomyocyte survival after acute ischemia.146 Similar observations were made with mesenchymal stem cells (MSC), in which pretreatment with E2 resulted in heightened paracrine production, leading to improved myocardial function and viability after the infusion of

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these E2-treated MSC to the heart.147 E2 also promotes mouse embryonic stem cell proliferation via p44/42 mitogen-activated protein kinases (MAPKs), cyclindependent kinase (CDK)2 and CDK4, and upregulation of c-fos, c-jun, and c-myc proto-oncogene expression.148 Both ERα and ERβ contribute to E2-mediated endothelial progenitor cell activation and tissue incorporation, leading to preservation of cardiac function after MI.149,150 Of the two classical ER subtypes, ERα may have more profound and direct effects on cardiac progenitor cells in situ, as the receptor was upregulated in postinfarct c-kit+precursor cells accumulating in the peri-infarct myocardium and supported proliferation of undifferentiated myoblast cells.151 ERα stimulation by E2 and propyl pyrazole triol (PPT, ERα-specific agonist) reduced apoptosis and increased survival of adult myocytes cocultured with postinfarct cardiac c-kit+cells, while ERβ-selective agonist, DPN, had no effect.151 Thus, it is becoming apparent that E2 signaling augments the regenerative capability of the stem cells to achieve improved recovery of cardiac function post ischemic injury. Research on the role of estrogen in cardiac regeneration is still in its nascent stage, but these encouraging early findings provide a unique insight into how E2 protects the heart, and may prove a focus of avant-garde therapeutic strategies in designing gender-specific treatment of IHD. Cardiac repair via stem cells after myocardial injury is one of the most intense areas of clinical investigation today. In the last several years, after much preclinical work has been done on testing different types of progenitor cells, delivery routes, and pathologies suited for stem cell therapy, a number of small-scale clinical trials applying stem cells to the diseased heart have been published. The results are certainly promising but not definitive.152–157 However, given what is known about the ability of E2 to boost the regenerative potential of various stem cell lines, an adjunct therapy designed to activate ER at the time of stem cell implantation may provide a novel approach to complement the primary regenerative therapy, analogous to the measurable clinical benefit derived from adjunct antiplatelet, antithrombotic agents used in conjunction to the primary mechanical revascularization therapy as a mainstay treatment for an acute MI.

25.3.6  Estrogen and ERs in Cardiac Hypertrophy An increase in LV mass is strongly associated with a higher incidence of cardiovascular death.72 From regulating the normal postnatal cardiac growth, to modulating the physiological response to exercise, to mitigating maladaptive left ventricular hypertrophy (LVH) to pressure overload, estrogen is involved in a broad array of left ventricular hypertrophic signaling. Similarly, E2 has

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been shown to ameliorate right ventricular hypertrophy and attenuate the increase in RV afterload in the experimental model of pulmonary artery hypertension.158 Estrogen antagonizes abnormal cellular hypertrophy of cardiomyocytes treated with angiotensin II in experimental models, and the hormone supplement is associated with improved LV geometry in human subjects.159,160 As is the case with the antiapoptotic actions of E2, both ER isoforms are implicated in the E2-mediated antihypertrophic effects against pathological triggers.161,162 The purported molecular mechanisms by which estrogen impacts cardiomyocyte hypertrophy and LVH include atrial natriuretic factor (ANF)-mediated autocrine/ paracrine effects,159 calcineurin degradation,163 mTOR signaling,164 p38 MAPK pathways,165 and regulation of cardiomyocyte histone deacetylases.166 Under normal physiological conditions, however, the development of exercise-induced cardiac hypertrophy was shown to be dependent on ERβ in an experimental animal model utilizing WT as well as ERα- and ERβ-deleted mice.167 The study also demonstrated that ERβ was at the center of the gender-specific regulation of mitochondrial signaling molecules and mitochondrial size distribution in the physiological hypertrophic response to exercise. On the other hand, estrogenic regulation for normal myocardial development was shown to be primarily via ERα, upregulating Igf1 and Myocd, genes for insulin-like growth factor 1 and myocardin, respectively, required for normal cardiac growth and cardiogenesis.168 Myocardin-null mutant mice exhibit myocardial hypoplasia, defective atrial and ventricular chamber maturation, heart failure associated with blocked cardiomyocyte proliferation, and an increase in programmed cell death, leading to embryonic lethality at midgestation.169 One of the ER coactivators, steroid receptor coactivator 3 (SRC3), is also a transcriptional factor for myocardin, providing a converging site of action for estrogen in the transactivation of myocardin essential for normal cardiac development.170 One of the more recent discoveries in the study of cardiac hypertrophy is the identification of endonuclease G (endoG), a mitochondrial-localized nuclease expressed in cardiomyocytes, as a central link between maladaptive LVH and mitochondrial processes unrelated to apoptosis.171 Inhibition of endoG expression directly induced cultured myocyte hypertrophy in vitro, and Endog loss-of-function in vivo resulted in cardiomyocyte hypertrophy and increased LV mass following angiotensin II stimulation. Additionally, endoG in this context was shown to be a transcriptional target of ERR-α and PGC-1α, the previously discussed master regulators of mitochondrial function and cardiac metabolism. Given the homology between ERR-α and ERs, E2 participation is possible in the regulation of endoG via cross-talk between the two related receptors. However, as of this

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writing, evidence for a direct interaction between estrogen and EndoG is scant, and there is none pertaining to the hormone’s previously described role in the heart, though one study showed a correlation between E2 administration and EndoG release in the central nervous system.172 Considering the strong physiological data on the role of E2 in LVH, exploring a potential relationship between the E2/ER and EndoG may serve useful to broaden our current understanding of the estrogenmediated myocardial hypertrophic response.

25.3.7  Estrogen and ERs in Cardiac Electrophysiology and Contractility The differences in the electrophysiological properties of the heart between men and women have long been recognized, and are exemplified by distinct surface electrocardiogram (ECG) findings of healthy men and women. Women are more likely to have a faster resting heart rate, shorter QRS duration, and longer QTc interval.173,174 The prognostic significance of this difference in repolarization is that women are at twofold increased risk, compared to men, for developing drug-related torsade de pointes (TdP). Women also have shorter sinus node recovery times after overdrive pacing. The biological basis for these gender differential properties in cardiac conduction is still not fully understood, but the evidence to suggest a role of sex hormones is strong. To this end, it has been reported that the gender differences in QTc are absent in newborns, become apparent at puberty, and fade somewhat after the fifth decade.175 QTc also varies within a menstrual cycle.176,177 Furthermore, during simultaneous atrioventricular pacing, atrial refractoriness is shortened significantly in postmenopausal women and men but not in premenopausal women.178 The incidence of atrial fibrillation (AF) is low in young women and higher in men and postmenopausal women.179 Although incidence of lone AF is higher in men than women, women are at higher risk of mortality and stroke from AF, compared with their male counterparts. On the other hand, men are more likely to die from SCD from ischemia-induced arrhythmia.180 Taken together, these gender differences in clinical presentation implicitly support the idea that sex hormones and hormonal fluctuation may account for at least part of gender-specific electrophysiology and the associated different risks and outcomes of disturbances in cardiac rhythm. At the cellular and organ level, a large body of evidence illustrates how estrogen directly impacts the action potential property of the cardiomyocyte. In one of the earliest animal models of I/R-induced ventricular arrhythmia, estrogen supplement lowered the incidence of lethal ventricular tachyarrhythmias.9 Of note, this

finding may explain the fact that women are better protected from SCD related to fatal ventricular tachyarrhythmia in the setting of IHD. Subsequent bench research has provided details to substantiate the E2-specific antiarrhythmic effects and to delineate molecular mechanisms involved. They demonstrate that the E2 actions within the heart elicit changes in the activity of Ca2+-activated K++ (KCa) channels and endothelial [Ca2+]i, via rapid, nongenomic signaling, as well as endothelium-independent effect on BK channels.181–183 Additional mechanistic studies offer more supportive data and affirm the antiarrhythmic effects of estrogen during I/R, reporting that the E2-mediated augmentation of endogenous nitric oxide (NO) release and Na++/H++ exchanger (NHE1) inhibition constitute part of the estrogen signaling pathways to suppress ischemia-induced arrhythmia and limit myocardial injury.181,184 The heart is a unique organ in that it requires coupling of its electrophysiological properties with mechanical contraction for normal performance. Studies show that estrogen is important in the maintenance of normal excitation-contraction (E-C) coupling in the female heart. The resting LV ejection fraction is higher in women than men.185 In animal models, ovariectomy leads to significant contractile dysfunction of the heart, which is reversed by E2 supplement, and in postmenopausal women, hormone supplement greatly improves the LV myocardial performance index and exercise performance.186,187 Together, these findings underscore the intimate relationship between E2 and LV function in healthy women. There are significant gender differences in the parameters of cardiac E-C coupling displayed at the organ and cellular level.188 Some of these differences are attributed to the regulation of calcium homeostasis by E2/ER in the heart. E2 modulates cardiomyocyte L-type Ca2+ channel activity, a major determinant in cardiac excitability and contractility, and alters the membrane density and expression of both L-type Ca2+ channels and low-voltage-activated Ca(V)3.2  T-type calcium channels.189–191 The latter are key components of spontaneous pacemaker activity and have also been implicated as part of arrhythmogenic foci associated with the negative remodeling post injury.190,192 Modulation of these cardiac ion channels by E2 expands the mechanistic explanations of how gender differential presentations seen in clinical electrophysiology are mediated by the hormone’s action on the inherent conduction and contractile properties of cardiomyocytes. Which cognate ERs partake(s) in the E2-mediated control of cardiac E-C coupling is still unclear. One study negated the role of ERα or ERβ in this context, while others support the involvement of the classic ER subtypes in regulating electrophysiological and contractile function

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in cardiomyocytes.193–195 The rapid effects on contractility and conduction by the hormone were attributed to the membrane-associated ER.196 A comprehensive approach using the ER subtype-selective agonists (PPT for ERα, DPN for ERβ, and G1 for GPER) and genetic deletion models indicated only ERβ as having positive effects on E-C coupling and myocyte contractility of the female heart. By contrast, the selective ERα agonist PPT suppressed contractility in a dose-dependent manner and completely inhibited the E2 effects, and the GPR30 agonist G1 had no effect on myocyte contractility. ERβ deficiency led to a significant prolongation of the QTc and ventricular repolarization, and a significantly lower expression of Kv4.3 channel in female mice with chronic MI.197 ERβ also effectively suppressed reperfusion-induced ventricular arrhythmias, while ERα had no effect in an in-vivo simulation of reperfusion injury.198 Although these data overall advocate for the ERβ subtype to mediate cardiomyocyte Ca2+ handling and inotropy, there is some inconsistency in the published literature. A study using the whole cell patchclamp technique on isolated ventricular cardiomyocytes of WT, ERαKO or ERβKO mice showed that the effect of estrogens on Ca2+ current (ICaL) was not dependent on ERα or ERβ,195 leaving open a possibility of participation by GPER, another membrane-associated ER. This notion is supported by the latest data demonstrating the involvement of GPER in calmodulin (CaM) upregulation and endothelial Ca2+ homeostasis.199,200 In the vast, complex network of ion channels involved in cardiomyocyte conduction and contraction, the direction of the E2 signaling is likely to be ER subtype- and target channel-specific. Further investigations are needed to clarify the contribution of each ER subtype in cardiomyocyte E-C coupling and arrhythmogenicity. Regardless, cumulative findings from both clinical and basic science research support the direct role of estrogen in the electrophysiological functions and contractility of the heart.

25.4 CONCLUSION This chapter highlights the key actions of estrogen on the heart and cardiomyocytes in the hope that the biological principles underlying gender differences in heart disease can be better understood and further investigated in the future. It is also important to keep in mind that gender dichotomy in CVD exists beyond the molecular and physiological workings of the hormone discussed here. There are significant gaps in our understanding of differences in the gender-specific consequences of many facets of CVD. Women with CVD are still underdiagnosed, hampered by underrecognition of often atypical symptoms despite the fact that an approximately equal

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number of men and women live with CVD.2,201 Even when appropriately diagnosed, women are less likely to be offered evidence-based therapies.202,203 Women suffer higher complication rates from mechanical or pharmacological cardiac interventions, compared to men.204–206 Advances in understanding etiology and pathophysiology of cardiac disorders well-known to have striking female affinity, such as Takotsubo stress cardiomyopathy, have been slow to come. Finally, there is a persistent underrepresentation of women in clinical trials.207 Fortunately, these disparities have begun to be acknowledged by the medical and scientific community in recent years, resulting in steady progress toward intensification of research, advocacy for gender balance in scientific pursuits and clinical practice, and the application of new knowledge to patient care. The NIH now requires that grant applications include plans for the balance of male and female cells and animals in preclinical studies.208 As of January 25, 2016, scientists who apply for federal research grants at NIH are expected to account for the possible role of sex as a biological variable in vertebrate animal and human studies.209 Moreover, for the first time since 1985, the gap in CVD mortality between men and women has finally closed, according to the 2016 Heart Disease and Stroke Statistics from the American Heart Association. This milestone is reached after dedicated years of strong advocacy, national and community-based awareness campaigns on gender disparity, and rigorous evidence-based application of knowledge derived from modern day research, all of which must continue. In conclusion, gender differences in CVD have long eluded a satisfactory cohesive explanation. While basic science and clinical investigations are ongoing and necessary to further our understanding, the efficacy of estrogen and its receptors in cardioprotection is acknowledged by a large volume of existing clinical and experimental studies. E2 and its receptors play a critical role in a complex network of genomic and nongenomic pathways that regulate cardiac metabolism, cytoprotection, cardiomyocyte regeneration, LV remodeling, and the electrophysiological and contractile function of the heart. Insights gained from future research on the effects of estrogen in cardiomyocytes and the heart will be indispensable to designing therapies that are both personalized and gender-specific, and will significantly impact our prevention, diagnosis, and gender-specific treatment of CVD.

Funding Sources This work was supported by a grant from the National Heart, Lung, Blood Institute at the National Institutes of Health (R01HL111180 to J.K.K).

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Disclosures No conflicts of interest to disclose.

References 1. The top 10 causes of death. The World Health Organization March 2014 [cited Fact sheet N°310]. Available from:. 2. Mozaffarian D, Benjamin EJ, Go AS, et  al. Heart disease and stroke statistics--2015 update: a report from the American Heart Association. Circulation. 2015;131:e29–322. 3. Bean WB. Infarction of the heart: a morphological and clinical appraisal of three hundred cases part I. Predisposing and precipitating conditions. Am Heart J. 1937;14:684–702. 4. Conner L, Holt E. The subsequent course and prognosis in coronary thrombosis. Am Heart J. 1930;5:705–719. 5. Master AM, Dack S, Age HL J. Sex and hypertension in myocardial infarction due to coronary occlusion. Arch Intern Med. 1939;64:767–786. 6. Kannel WB, Hjortland MC, McNamara PM, Gordon T. Menopause and risk of cardiovascular disease: the Framingham study. Ann Intern Med. 1976;85:447–452. 7. Booth EA, Lucchesi BR. Medroxyprogesterone acetate prevents the cardioprotective and anti-inflammatory effects of 17betaestradiol in an in vivo model of myocardial ischemia and reperfusion. Am J Physiol Heart Circ Physiol. 2007;293:H1408–H1415. 8. Levine RL, Chen SJ, Durand J, Chen YF, Oparil S. Medroxyprogesterone attenuates estrogen-mediated inhibition of neointima formation after balloon injury of the rat carotid artery. Circulation. 1996;94:2221–2227. 9. McHugh NA, Cook SM, Schairer JL, Bidgoli MM, Merrill GF. Ischemia- and reperfusion-induced ventricular arrhythmias in dogs: effects of estrogen. Am J Physiol. 1995;268:H2569–H2573. 10. Resanovic I, Rizzo M, Zafirovic S, et al. Anti-atherogenic effects of 17beta-estradiol. Horm Metab Res. 2013;45:701–708. 11. Rossouw JE, Anderson GL, Prentice RL, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women’s Health Initiative randomized controlled trial. JAMA. 2002;288:321–333. 12. Anderson GL, Limacher M, Assaf AR, et  al. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial. JAMA. 2004;291:1701–1712. 13. Miyagawa K, Rosch J, Stanczyk F, Hermsmeyer K. Medroxyprogesterone interferes with ovarian steroid protection against coronary vasospasm. Nat Med. 1997;3:324–327. 14. Turgeon JL, McDonnell DP, Martin KA, Wise PM. Hormone therapy: physiological complexity belies therapeutic simplicity. Science. 2004;304:1269–1273. 15. Harman SM, Black DM, Naftolin F, et  al. Arterial imaging outcomes and cardiovascular risk factors in recently menopausal women: a randomized trial. Ann Intern Med. 2014;161:249–260. 16. Hodis HN, Mack WJ, Shoupe D, et  al. Methods and baseline cardiovascular data from the Early versus Late Intervention Trial with Estradiol testing the menopausal hormone timing hypothesis. Menopause. 2015;22:391–401. 17. Mikkola TS, Tuomikoski P, Lyytinen H, et  al. Vaginal estradiol use and the risk for cardiovascular mortality. Hum Reprod. 2016;31:804–809. 18. Treloar AE. Menstrual cyclicity and the pre-menopause. Maturitas. 1981;3:249–264. 19. Hodis HN, Mack WJ, Azen SP, et al. Hormone therapy and the progression of coronary-artery atherosclerosis in postmenopausal women. N Engl J Med. 2003;349:535–545.

20. Naftolin F, Taylor HS, Karas R, et  al. The Women’s Health Initiative could not have detected cardioprotective effects of starting hormone therapy during the menopausal transition. Fertil Steril. 2004;81:1498–1501. 21. Grady D, Herrington D, Bittner V, et  al. Cardiovascular disease outcomes during 6.8 years of hormone therapy: Heart and Estrogen/progestin Replacement Study follow-up (HERS II). JAMA. 2002;288:49–57. 22. Dubey RK, Oparil S, Imthurn B, Jackson EK. Sex hormones and hypertension. Cardiovasc Res. 2002;53:688–708. 23. Fahraeus L. The effects of estradiol on blood lipids and lipoproteins in postmenopausal women. Obstet Gynecol. 1988;72:18S–22S. 24. Rossi R, Origliani G, Modena MG. Transdermal 17-beta-estradiol and risk of developing type 2 diabetes in a population of healthy, nonobese postmenopausal women. Diabetes Care. 2004;27:645–649. 25. Collins P, Rosano GM, Sarrel PM, et al. 17 beta-Estradiol attenuates acetylcholine-induced coronary arterial constriction in women but not men with coronary heart disease. Circulation. 1995;92:24–30. 26. Manson JE, Allison MA, Rossouw JE, et al. Estrogen therapy and coronary-artery calcification. N Engl J Med. 2007;356:2591–2602. 27. Rossouw JE, Prentice RL, Manson JE, et al. Postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause. JAMA. 2007;297:1465–1477. 28. The North American Menopause Society The 2012 hormone therapy position statement of: The North American Menopause Society. Menopause. 2012;19:257–271. 29. Baber RJ, Panay N, Fenton ATIWG 2016 IMS recommendations on women’s midlife health and menopause hormone therapy. Climacteric. 2016;19:109–150. 30. Mosca L, Benjamin EJ, Berra K, et al. Effectiveness-based guidelines for the prevention of cardiovascular disease in women-2011 update: a guideline from the American Heart Association. J Am Coll Cardiol. 2011;57:1404–1423. 31. Chakrabarti S, Morton JS, Davidge ST. Mechanisms of estrogen effects on the endothelium: an overview. Can J Cardiol. 2014;30:705–712. 32. Stocco C. Tissue physiology and pathology of aromatase. Steroids. 2012;77:27–35. 33. Tsugaya M, Harada N, Tozawa K, et al. Aromatase mRNA levels in benign prostatic hyperplasia and prostate cancer. Int J Urol. 1996;3:292–296. 34. Trotter A, Maier L, Grill HJ, Wudy SA, Pohlandt F. 17Betaestradiol and progesterone supplementation in extremely lowbirth-weight infants. Pediatr Res. 1999;45:489–493. 35. Ling S, Komesaroff P, Sudhir K. Cellular mechanisms underlying the cardiovascular actions of oestrogens. Clin Sci (Lond). 2006;111:107–118. 36. Hong Y, Li H, Yuan YC, Chen S. Molecular characterization of aromatase. Ann N Y Acad Sci. 2009;1155:112–120. 37. Naftolin F, Ryan KJ, Petro Z. Aromatization of androstenedione by limbic system tissue from human foetuses. J Endocrinol. 1971;51:795–796. 38. Nawata H, Tanaka S, Takayanagi R, et  al. Aromatase in bone cell: association with osteoporosis in postmenopausal women. J Steroid Biochem Mol Biol. 1995;53:165–174. 39. Ackerman GE, Smith ME, Mendelson CR, MacDonald PC, Simpson ER. Aromatization of androstenedione by human adipose tissue stromal cells in monolayer culture. J Clin Endocrinol Metab. 1981;53:412–417. 40. Bulun SE, Simpson ER. Competitive reverse transcription-polymerase chain reaction analysis indicates that levels of aromatase cytochrome P450 transcripts in adipose tissue of buttocks, thighs, and abdomen of women increase with advancing age. J Clin Endocrinol Metab. 1994;78:428–432.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

375

REFERENCES

41. Harada N, Utsumi T, Takagi Y. Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and promoters, and switching of tissue-specific exons 1 in carcinogenesis. Proc Natl Acad Sci USA. 1993;90:11312–11316. 42. Grohe C, Kahlert S, Lobbert K, Vetter H. Expression of oestrogen receptor alpha and beta in rat heart: role of local oestrogen synthesis. J Endocrinol. 1998;156:R1–R7. 43. Jazbutyte V, Stumpner J, Redel A, et  al. Aromatase inhibition attenuates desflurane-induced preconditioning against acute myocardial infarction in male mouse heart in vivo. PLoS One. 2012;7:e42032. 44. Beitelshees AL, Johnson JA, Hames ML, et  al. Aromatase gene polymorphisms are associated with survival among patients with cardiovascular disease in a sex-specific manner. PLoS One. 2010;5:e15180. 45. O’Lone R, Knorr K, Jaffe IZ, et  al. Estrogen receptors alpha and beta mediate distinct pathways of vascular gene expression, including genes involved in mitochondrial electron transport and generation of reactive oxygen species. Mol Endocrinol. 2007;21:1281–1296. 46. Devanathan S, Whitehead T, Schweitzer GG, et  al. An animal model with a cardiomyocyte-specific deletion of estrogen receptor alpha: functional, metabolic, and differential network analysis. PLoS One. 2014;9:e101900. 47. Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, Shaul PW. Estrogen receptor alpha mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest. 1999;103:401–406. 48. Grohe C, Kahlert S, Lobbert K, et  al. Cardiac myocytes and fibroblasts contain functional estrogen receptors. FEBS Lett. 1997;416:107–112. 49. Mahmoodzadeh S, Eder S, Nordmeyer J, et al. Estrogen receptor alpha up-regulation and redistribution in human heart failure. FASEB J. 2006;20:926–934. 50. Taylor AH, Al-Azzawi F. Immunolocalisation of oestrogen receptor beta in human tissues. J Mol Endocrinol. 2000;24:145–155. 51. Lizotte E, Grandy SA, Tremblay A, Allen BG, Fiset C. Expression, distribution and regulation of sex steroid hormone receptors in mouse heart. Cell Physiol Biochem. 2009;23:75–86. 52. Knowlton AA, Lee AR. Estrogen and the cardiovascular system. Pharmacol Ther. 2012;135:54–70. 53. Jazbutyte V, Kehl F, Neyses L, Pelzer T. Estrogen receptor alpha interacts with 17beta-hydroxysteroid dehydrogenase type 10 in mitochondria. Biochem Biophys Res Commun. 2009;384:450–454. 54. Liu H, Yanamandala M, Lee TC, Kim JK. Mitochondrial p38beta and manganese superoxide dismutase interaction mediated by estrogen in cardiomyocytes. PLoS One. 2014;9:e85272. 55. Yang SH, Liu R, Perez Jr. EJ, et  al. Mitochondrial localization of estrogen receptor beta. Proc Natl Acad Sci USA. 2004;101:4130–4135. 56. Funakoshi T, Yanai A, Shinoda K, Kawano MM, Mizukami Y. G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane. Biochem Biophys Res Commun. 2006;346:904–910. 57. Meyer MR, Haas E, Prossnitz ER, Barton M. Non-genomic regulation of vascular cell function and growth by estrogen. Mol Cell Endocrinol. 2009;308:9–16. 58. Deschamps AM, Murphy E. Activation of a novel estrogen receptor, GPER, is cardioprotective in male and female rats. Am J Physiol Heart Circ Physiol. 2009;297:H1806–H1813. 59. Jessup JA, Lindsey SH, Wang H, Chappell MC, Groban L. Attenuation of salt-induced cardiac remodeling and diastolic dysfunction by the GPER agonist G-1 in female mRen2. Lewis rats. PLoS One. 2010;5:e15433. 60. Bopassa JC, Eghbali M, Toro L, Stefani E. A novel estrogen receptor GPER inhibits mitochondria permeability transition pore

61.

62.

63. 64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

opening and protects the heart against ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol. 2010;298:H16–H23. Haas E, Bhattacharya I, Brailoiu E, et  al. Regulatory role of G protein-coupled estrogen receptor for vascular function and obesity. Circ Res. 2009;104:288–291. Pranavchand R, Reddy BM. Current status of understanding of the genetic etiology of coronary heart disease. J Postgrad Med. 2013;59:30–41. Roberts R, Stewart AF. Genes and coronary artery disease: where are we? J Am Coll Cardiol. 2012;60:1715–1721. Hartiala JA, Tang WH, Wang Z, et  al. Genome-wide association study and targeted metabolomics identifies sex-specific association of CPS1 with coronary artery disease. Nat Commun. 2016;7:10558. Barone I, Brusco L, Fuqua SA. Estrogen receptor mutations and changes in downstream gene expression and signaling. Clin Cancer Res. 2010;16:2702–2708. Domingues-Montanari S, Subirana I, Tomas M, Marrugat J, Senti M. Association between ESR2 genetic variants and risk of myocardial infarction. Clin Chem. 2008;54:1183–1189. Kunnas T, Silander K, Karvanen J, Valkeapaa M, Salomaa V, Nikkari S. ESR1 genetic variants, haplotypes and the risk of coronary heart disease and ischemic stroke in the Finnish population: a prospective follow-up study. Atherosclerosis. 2010;211:200–202. Leibowitz D, Dresner-Pollak R, Dvir S, Rokach A, Reznik L, Pollak A. Association of an estrogen receptor-alpha gene polymorphism with left ventricular mass. Blood Press. 2006;15:45–50. Peter I, Shearman AM, Vasan RS, et  al. Association of estrogen receptor beta gene polymorphisms with left ventricular mass and wall thickness in women. Am J Hypertens. 2005;18:1388–1395. Rexrode KM, Ridker PM, Hegener HH, Buring JE, Manson JE, Zee RY. Polymorphisms and haplotypes of the estrogen receptorbeta gene (ESR2) and cardiovascular disease in men and women. Clin Chem. 2007;53:1749–1756. Schuit SC, Oei HH, Witteman JC, et al. Estrogen receptor alpha gene polymorphisms and risk of myocardial infarction. JAMA. 2004;291:2969–2977. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med. 1990;322:1561–1566. Sudhir K, Chou TM, Chatterjee K, et  al. Premature coronary artery disease associated with a disruptive mutation in the estrogen receptor gene in a man. Circulation. 1997;96:3774–3777. Herrington DM, Howard TD, Hawkins GA, et  al. Estrogenreceptor polymorphisms and effects of estrogen replacement on high-density lipoprotein cholesterol in women with coronary disease. N Engl J Med. 2002;346:967–974. Robins SJ, Collins D, Wittes JT, et  al. Relation of gemfibrozil treatment and lipid levels with major coronary events: VA-HIT: a randomized controlled trial. JAMA. 2001;285:1585–1591. Lucas G, Lluis-Ganella C, Subirana I, et al. Post-genomic update on a classical candidate gene for coronary artery disease: ESR1. Circ Cardiovasc Genet. 2011;4:647–654. Wei CD, Zheng HY, Wu W, et  al. Meta-analysis of the association of the rs2234693 and rs9340799 polymorphisms of estrogen receptor alpha gene with coronary heart disease risk in Chinese Han population. Int J Med Sci. 2013;10:457–466. Giess M, Lattrich C, Springwald A, Goerse R, Ortmann O, Treeck O. GPR30 gene polymorphisms are associated with progesterone receptor status and histopathological characteristics of breast cancer patients. J Steroid Biochem Mol Biol. 2010;118:7–12. Gupte AA, Pownall HJ, Hamilton DJ. Estrogen: an emerging regulator of insulin action and mitochondrial function. J Diabetes Res. 2015;2015:916585.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

376

25.  THE ROLE OF ESTROGEN IN THE HEART

80. Carr MC. The emergence of the metabolic syndrome with menopause. J Clin Endocrinol Metab. 2003;88:2404–2411. 81. Wallen WJ, Belanger MP, Wittnich C. Sex hormones and the selective estrogen receptor modulator tamoxifen modulate weekly body weights and food intakes in adolescent and adult rats. J Nutr. 2001;131:2351–2357. 82. Heine PA, Taylor JA, Iwamoto GA, Lubahn DB, Cooke PS. Increased adipose tissue in male and female estrogen receptoralpha knockout mice. Proc Natl Acad Sci USA. 2000;97:12729–12734. 83. Ribas V, Nguyen MT, Henstridge DC, et al. Impaired oxidative metabolism and inflammation are associated with insulin resistance in ERalpha-deficient mice. Am J Physiol Endocrinol Metab. 2010;298:E304–E319. 84. Chen Y, Zhang Z, Hu F, et  al. 17beta-estradiol prevents cardiac diastolic dysfunction by stimulating mitochondrial function: a preclinical study in a mouse model of a human hypertrophic cardiomyopathy mutation. J Steroid Biochem Mol Biol. 2015;147:92–102. 85. Arias-Loza PA, Kreissl MC, Kneitz S, et al. The estrogen receptoralpha is required and sufficient to maintain physiological glucose uptake in the mouse heart. Hypertension. 2012;60:1070–1077. 86. Barros RP, Machado UF, Warner M, Gustafsson JA. Muscle GLUT4 regulation by estrogen receptors ERbeta and ERalpha. Proc Natl Acad Sci USA. 2006;103:1605–1608. 87. Garrido P, Moran J, Alonso A, Gonzalez S, Gonzalez C. 17betaestradiol activates glucose uptake via GLUT4 translocation and PI3K/Akt signaling pathway in MCF-7 cells. Endocrinology. 2013;154:1979–1989. 88. James DE, Brown R, Navarro J, Pilch PF. Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein. Nature. 1988;333:183–185. 89. Rattanasopa C, Phungphong S, Wattanapermpool J, Bupha-Intr T. Significant role of estrogen in maintaining cardiac mitochondrial functions. J Steroid Biochem Mol Biol. 2015;147:1–9. 90. Liu H, Pedram A, Kim JK. Oestrogen prevents cardiomyocyte apoptosis by suppressing p38alpha-mediated activation of p53 and by down-regulating p53 inhibition on p38beta. Cardiovasc Res. 2011;89:119–128. 91. Satoh M, Matter CM, Ogita 2nd H, et al. Inhibition of apoptosisregulated signaling kinase-1 and prevention of congestive heart failure by estrogen. Circulation. 2007;115:3197–3204. 92. Ramjiawan A, Bagchi RA, Albak L, Czubryt MP. Mechanism of cardiomyocyte PGC-1alpha gene regulation by ERRalpha. Biochem Cell Biol. 2013;91:148–154. 93. Giguere V, Yang N, Segui P, Evans RM. Identification of a new class of steroid hormone receptors. Nature. 1988;331:91–94. 94. Ohlsson C, Hellberg N, Parini P, et al. Obesity and disturbed lipoprotein profile in estrogen receptor-alpha-deficient male mice. Biochem Biophys Res Commun. 2000;278:640–645. 95. Roesch DM. Effects of selective estrogen receptor agonists on food intake and body weight gain in rats. Physiol Behav. 2006;87:39–44. 96. Yepuru M, Eswaraka J, Kearbey JD, et  al. Estrogen receptor{beta}-selective ligands alleviate high-fat diet- and ovariectomyinduced obesity in mice. J Biol Chem. 2010;285:31292–31303. 97. Simpkins JW, Yang SH, Sarkar SN, Pearce V. Estrogen actions on mitochondria--physiological and pathological implications. Mol Cell Endocrinol. 2008;290:51–59. 98. Gabel SA, Walker VR, London RE, Steenbergen C, Korach KS, Murphy E. Estrogen receptor beta mediates gender differences in ischemia/reperfusion injury. J Mol Cell Cardiol. 2005;38:289–297. 99. Hsieh YC, Yu HP, Suzuki T, et al. Upregulation of mitochondrial respiratory complex IV by estrogen receptor-beta is critical for inhibiting mitochondrial apoptotic signaling and restoring cardiac functions following trauma-hemorrhage. J Mol Cell Cardiol. 2006;41:511–521.

100. Hsieh YC, Choudhry MA, Yu HP, et  al. Inhibition of cardiac PGC-1alpha expression abolishes ERbeta agonist-mediated cardioprotection following trauma-hemorrhage. FASEB J. 2006;20:1109–1117. 101. Barros RP, Gabbi C, Morani A, Warner M, Gustafsson JA. Participation of ERalpha and ERbeta in glucose homeostasis in skeletal muscle and white adipose tissue. Am J Physiol Endocrinol Metab. 2009;297:E124–E133. 102. Ruegg J, Cai W, Karimi M, et  al. Epigenetic regulation of glucose transporter 4 by estrogen receptor beta. Mol Endocrinol. 2011;25:2017–2028. 103. Sharma G, Hu C, Brigman JL, Zhu G, Hathaway HJ, Prossnitz ER. GPER deficiency in male mice results in insulin resistance, dyslipidemia, and a proinflammatory state. Endocrinology. 2013;154:4136–4145. 104. Sharma G, Prossnitz ER. Mechanisms of estradiol-induced insulin secretion by the G protein-coupled estrogen receptor GPR30/GPER in pancreatic beta-cells. Endocrinology. 2011;152:3030–3039. 105. Taegtmeyer H, Young ME, Lopaschuk GD, et  al. Assessing Cardiac Metabolism: A Scientific Statement From the American Heart Association. Circ Res. 2016;118(10):1659–1701. 106. Herrero P, Soto PF, Dence CS, et al. Impact of hormone replacement on myocardial fatty acid metabolism: potential role of estrogen. J Nucl Cardiol. 2005;12:574–581. 107. Visser M, Davids M, Verberne HJ, et al. Nutrition before, during, and after surgery increases the arginine:asymmetric dimethylarginine ratio and relates to improved myocardial glucose metabolism: a randomized controlled trial. Am J Clin Nutr. 2014;99:1440–1449. 108. Haris M, Singh A, Cai K, et  al. A technique for in vivo mapping of myocardial creatine kinase metabolism. Nat Med. 2014;20:209–214. 109. Rial B, Robson MD, Neubauer S, Schneider JE. Rapid quantification of myocardial lipid content in humans using single breathhold 1H MRS at 3 Tesla. Magn Reson Med. 2011;66:619–624. 110. Fenner A. Prostate cancer: GTx-758 reduces testosterone. Nat Rev Urol. 2014;11:422. 111. Hazim S, Curtis PJ, Schar MY, et al. Acute benefits of the microbial-derived isoflavone metabolite equol on arterial stiffness in men prospectively recruited according to equol producer phenotype: a double-blind randomized controlled trial. Am J Clin Nutr. 2016;103:694–702. 112. Roman-Blas JA, Castaneda S, Cutolo M, Herrero-Beaumont G. Efficacy and safety of a selective estrogen receptor beta agonist, ERB-041, in patients with rheumatoid arthritis: a 12-week, randomized, placebo-controlled, phase II study. Arthritis Care Res (Hoboken). 2010;62:1588–1593. 113. van Vollenhoven RF, Houbiers JG, Buttgereit F, In ‘t Hout J, Boers M, Leij S, et  al. The selective estrogen receptor alpha agonist Org 37663 induces estrogenic effects but lacks antirheumatic activity: a phase IIa trial investigating efficacy and safety of Org 37663 in postmenopausal female rheumatoid arthritis patients receiving stable background methotrexate or sulfasalazine. Arthritis Rheum. 2010;62:351–358. 114. Adler CP, Friedburg H. Myocardial DNA content, ploidy level and cell number in geriatric hearts: post-mortem examinations of human myocardium in old age. J Mol Cell Cardiol. 1986;18:39–53. 115. Laflamme MA, Murry CE. Heart regeneration. Nature. 2010;473:326–335. 116. Bergmann O, Bhardwaj RD, Bernard S, et al. Evidence for cardiomyocyte renewal in humans. Science. 2009;324:98–102. 117. Mollova M, Bersell K, Walsh S, et al. Cardiomyocyte proliferation contributes to heart growth in young humans. Proc Natl Acad Sci USA. 2013;110:1446–1451.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

118. Murry CE, Reinecke H, Pabon LM. Regeneration gaps: observations on stem cells and cardiac repair. J Am Coll Cardiol. 2006;47:1777–1785. 119. Saraste A, Pulkki K, Kallajoki M, Henriksen K, Parvinen M, Voipio-Pulkki LM. Apoptosis in human acute myocardial infarction. Circulation. 1997;95:320–323. 120. Olivetti G, Abbi R, Quaini F, et  al. Apoptosis in the failing human heart. N Engl J Med. 1997;336:1131–1141. 121. Scarabelli TM, Gottlieb RA. Functional and clinical repercussions of myocyte apoptosis in the multifaceted damage by ischemia/reperfusion injury: old and new concepts after 10 years of contributions. Cell Death Differ. 2004;11(Suppl 2):S144–S152. 122. Kajstura J, Cheng W, Reiss K, et  al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest. 1996;74:86–107. 123. Crow MT, Mani K, Nam YJ, Kitsis RN. The mitochondrial death pathway and cardiac myocyte apoptosis. Circ Res. 2004;95:957–970. 124. Regula KM, Kirshenbaum LA. Apoptosis of ventricular myocytes: a means to an end. J Mol Cell Cardiol. 2005;38:3–13. 125. Khosravi-Far R, Esposti MD. Death receptor signals to mitochondria. Cancer Biol Ther. 2004;3:1051–1057. 126. Pelzer T, Schumann M, Neumann M, et  al. 17beta-estradiol prevents programmed cell death in cardiac myocytes. Biochem Biophys Res Commun. 2000;268:192–200. 127. Pelzer T, Neumann M, de Jager T, Jazbutyte V, Neyses L. Estrogen effects in the myocardium: inhibition of NF-kappaB DNA binding by estrogen receptor-alpha and -beta. Biochem Biophys Res Commun. 2001;286:1153–1157. 128. Patten RD, Pourati I, Aronovitz MJ, et  al. 17beta-estradiol reduces cardiomyocyte apoptosis in vivo and in vitro via activation of phospho-inositide-3 kinase/Akt signaling. Circ Res. 2004;95:692–699. 129. Cong B, Zhu X, Cao B, Xiao J, Wang Z, Ni X. Estrogens protect myocardium against ischemia/reperfusion insult by upregulation of CRH receptor type 2 in female rats. Int J Cardiol. 2013;168:4755–4760. 130. Kim JK, Pedram A, Razandi M, Levin ER. Estrogen prevents cardiomyocyte apoptosis through inhibition of reactive oxygen species and differential regulation of p38 kinase isoforms. J Biol Chem. 2006;281:6760–6767. 131. Liou CM, Yang AL, Kuo CH, Tin H, Huang CY, Lee SD. Effects of 17beta-estradiol on cardiac apoptosis in ovariectomized rats. Cell Biochem Funct. 2010;28:521–528. 132. Wang M, Crisostomo P, Wairiuko GM, Meldrum DR. Estrogen receptor-alpha mediates acute myocardial protection in females. Am J Physiol Heart Circ Physiol. 2006;290:H2204–H2209. 133. Wang M, Crisostomo PR, Markel T, Wang Y, Lillemoe KD, Meldrum DR. Estrogen receptor beta mediates acute myocardial protection following ischemia. Surgery. 2008;144:233–238. 134. Liu CJ, Lo JF, Kuo CH, et al. Akt mediates 17beta-estradiol and/ or estrogen receptor-alpha inhibition of LPS-induced tumor necresis factor-alpha expression and myocardial cell apoptosis by suppressing the JNK1/2-NFkappaB pathway. J Cell Mol Med. 2009;13:3655–3667. 135. Cao J, Zhu T, Lu L, et  al. Estrogen induces cardioprotection in male C57BL/6J mice after acute myocardial infarction via decreased activity of matrix metalloproteinase-9 and increased Akt-Bcl-2 anti-apoptotic signaling. Int J Mol Med. 2011;28:231–237. 136. Li X, Huang J, Yi P, Bambara RA, Hilf R, Muyan M. Single-chain estrogen receptors (ERs) reveal that the ERalpha/beta heterodimer emulates functions of the ERalpha dimer in genomic estrogen signaling pathways. Mol Cell Biol. 2004;24:7681–7694. 137. Cowley SM, Hoare S, Mosselman S, Parker MG. Estrogen receptors alpha and beta form heterodimers on DNA. J Biol Chem. 1997;272:19858–19862.

377

138. de Jager T, Pelzer T, Muller-Botz S, Imam A, Muck J, Neyses L. Mechanisms of estrogen receptor action in the myocardium. Rapid gene activation via the ERK1/2 pathway and serum response elements. J Biol Chem. 2001;276:27873–27880. 139. Li WL, Xiang W, Ping Y. Activation of novel estrogen receptor GPER results in inhibition of cardiocyte apoptosis and cardioprotection. Mol Med Rep. 2015;12:2425–2430. 140. Leri A, Rota M, Pasqualini FS, Goichberg P, Anversa P. Origin of cardiomyocytes in the adult heart. Circ Res. 2015;116:150–166. 141. Sultana N, Zhang L, Yan J, et al. Resident c-kit( + ) cells in the heart are not cardiac stem cells. Nat Commun. 2015;6:8701. 142. Schwach V, Passier R. Generation and purification of human stem cell-derived cardiomyocytes. Differentiation. 2016. 143. Bearzi C, Rota M, Hosoda T, et  al. Human cardiac stem cells. Proc Natl Acad Sci USA. 2007;104:14068–14073. 144. D’Amario D, Cabral-Da-Silva MC, Zheng H, et al. Insulin-like growth factor-1 receptor identifies a pool of human cardiac stem cells with superior therapeutic potential for myocardial regeneration. Circ Res. 2011;108:1467–1481. 145. Kajstura J, Bai Y, Cappetta D, et al. Tracking chromatid segregation to identify human cardiac stem cells that regenerate extensively the infarcted myocardium. Circ Res. 2012;111:894–906. 146. Wang L, Gu H, Turrentine M, Wang M. Estradiol treatment promotes cardiac stem cell (CSC)-derived growth factors, thus improving CSC-mediated cardioprotection after acute ischemia/reperfusion. Surgery. 2014;156:243–252. 147. Erwin GS, Crisostomo PR, Wang Y, et al. Estradiol-treated mesenchymal stem cells improve myocardial recovery after ischemia. J Surg Res. 2009;152:319–324. 148. Han HJ, Heo JS, Lee YJ. Estradiol-17beta stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDKs as well as protooncogenes. Am J Physiol Cell Physiol. 2006;290:C1067–C1075. 149. Hamada H, Kim MK, Iwakura A, et al. Estrogen receptors alpha and beta mediate contribution of bone marrow-derived endothelial progenitor cells to functional recovery after myocardial infarction. Circulation. 2006;114:2261–2270. 150. Iwakura A, Shastry S, Luedemann C, et al. Estradiol enhances recovery after myocardial infarction by augmenting incorporation of bone marrow-derived endothelial progenitor cells into sites of ischemia-induced neovascularization via endothelial nitric oxide synthase-mediated activation of matrix metalloproteinase-9. Circulation. 2006;113:1605–1614. 151. Brinckmann M, Kaschina E, Altarche-Xifro W, et  al. Estrogen receptor alpha supports cardiomyocytes indirectly through post-infarct cardiac c-kit+ cells. J Mol Cell Cardiol. 2009;47: 66–75. 152. Fisher SA, Zhang H, Doree C, Mathur A, Martin-Rendon E. Stem cell treatment for acute myocardial infarction. Cochrane Database Syst Rev. 2015;9:CD006536. 153. Perin EC, Borow KM, Silva GV, et al. A Phase II Dose-Escalation Study of Allogeneic Mesenchymal Precursor Cells in Patients With Ischemic or Nonischemic Heart Failure. Circ Res. 2015;117:576–584. 154. San Roman JA, Sanchez PL, Villa A, et al. Comparison of Different Bone Marrow-Derived Stem Cell Approaches in Reperfused STEMI. A Multicenter, Prospective, Randomized, Open-Labeled TECAM Trial. J Am Coll Cardiol. 2015;65:2372–2382. 155. Nasseri BA, Ebell W, Dandel M, et al. Autologous CD133+ bone marrow cells and bypass grafting for regeneration of ischaemic myocardium: the Cardio133 trial. Eur Heart J. 2014;35:1263–1274. 156. Suncion VY, Ghersin E, Fishman JE, et al. Does transendocardial injection of mesenchymal stem cells improve myocardial function locally or globally?: an analysis from the Percutaneous Stem Cell Injection Delivery Effects on Neomyogenesis (POSEIDON) randomized trial. Circ Res. 2014;114:1292–1301.

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25.  THE ROLE OF ESTROGEN IN THE HEART

157. Malliaras K, Makkar RR, Smith RR, et  al. Intracoronary cardiosphere-derived cells after myocardial infarction: evidence of therapeutic regeneration in the final 1-year results of the CADUCEUS trial (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction). J Am Coll Cardiol. 2014;63:110–122. 158. Liu A, Schreier D, Tian L, et  al. Direct and indirect protection of right ventricular function by estrogen in an experimental model of pulmonary arterial hypertension. Am J Physiol Heart Circ Physiol. 2014;307:H273–H283. 159. Babiker FA, De Windt LJ, van Eickels M, et  al. 17beta-estradiol antagonizes cardiomyocyte hypertrophy by autocrine/ paracrine stimulation of a guanylyl cyclase A receptor-cyclic guanosine monophosphate-dependent protein kinase pathway. Circulation. 2004;109:269–276. 160. Light KC, Hinderliter AL, West SG, et al. Hormone replacement improves hemodynamic profile and left ventricular geometry in hypertensive and normotensive postmenopausal women. J Hypertens. 2001;19:269–278. 161. Pelzer T, Jazbutyte V, Hu K, et al. The estrogen receptor-alpha agonist 16alpha-LE2 inhibits cardiac hypertrophy and improves hemodynamic function in estrogen-deficient spontaneously hypertensive rats. Cardiovasc Res. 2005;67:604–612. 162. Jazbutyte V, Arias-Loza PA, Hu K, et al. Ligand-dependent activation of ER{beta} lowers blood pressure and attenuates cardiac hypertrophy in ovariectomized spontaneously hypertensive rats. Cardiovasc Res. 2008;77:774–781. 163. Donaldson C, Eder S, Baker C, et  al. Estrogen attenuates left ventricular and cardiomyocyte hypertrophy by an estrogen receptor-dependent pathway that increases calcineurin degradation. Circ Res. 2009;104:265–275. 11p following 75. 164. Gurgen D, Kusch A, Klewitz R, et al. Sex-specific mTOR signaling determines sexual dimorphism in myocardial adaptation in normotensive DOCA-salt model. Hypertension. 2013;61:730–736. 165. van Eickels M, Grohe C, Cleutjens JP, Janssen BJ, Wellens HJ, Doevendans PA. 17beta-estradiol attenuates the development of pressure-overload hypertrophy. Circulation. 2001;104:1419–1423. 166. Pedram A, Razandi M, Narayanan R, Dalton JT, McKinsey TA, Levin ER. Estrogen regulates histone deacetylases to prevent cardiac hypertrophy. Mol Biol Cell. 2013;24:3805–3818. 167. Dworatzek E, Mahmoodzadeh S, Schubert C, et  al. Sex differences in exercise-induced physiological myocardial hypertrophy are modulated by oestrogen receptor beta. Cardiovasc Res. 2014;102:418–428. 168. Kararigas G, Nguyen BT, Jarry H. Estrogen modulates cardiac growth through an estrogen receptor alpha-dependent mechanism in healthy ovariectomized mice. Mol Cell Endocrinol. 2014;382:909–914. 169. Huang J, Elicker J, Bowens N, et al. Myocardin regulates BMP10 expression and is required for heart development. J Clin Invest. 2012;122:3678–3691. 170. Li HJ, Haque Z, Lu Q, Li L, Karas R, Mendelsohn M. Steroid receptor coactivator 3 is a coactivator for myocardin, the regulator of smooth muscle transcription and differentiation. Proc Natl Acad Sci USA. 2007;104:4065–4070. 171. McDermott-Roe C, Ye J, Ahmed R, et  al. Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function. Nature. 2011;478:114–118. 172. Pereira RT, Porto CS, Abdalla FM. Ovariectomy and 17betaestradiol replacement play a role on the expression of Endonuclease-G and phosphorylated cyclic AMP response element-binding (CREB) protein in hippocampus. Mol Cell Endocrinol. 2014;382:227–233. 173. Nakagawa M, Ooie T, Ou B, et al. Gender differences in autonomic modulation of ventricular repolarization in humans. J Cardiovasc Electrophysiol. 2005;16:278–284.

174. Bernal O, Moro C. [Cardiac arrhythmias in women]. Rev Esp Cardiol. 2006;59:609–618. 175. Rautaharju PM, Zhou SH, Wong S, et al. Sex differences in the evolution of the electrocardiographic QT interval with age. Can J Cardiol. 1992;8:690–695. 176. Endres S, Mayuga KA, Cristofaro A, Taneja T, Goldberger JJ, Kadish AH. Menstrual cycle and ST height. Ann Noninvasive Electrocardiol. 2004;9:121–126. 177. Rodriguez I, Kilborn MJ, Liu XK, Pezzullo JC, Woosley RL, Drug-induced QT. Prolongation in women during the menstrual cycle. JAMA. 2001;285:1322–1326. 178. Tse HF, Oral H, Pelosi F, Knight BP, Strickberger SA, Morady F. Effect of gender on atrial electrophysiologic changes induced by rapid atrial pacing and elevation of atrial pressure. J Cardiovasc Electrophysiol. 2001;12:986–989. 179. Michelena HI, Powell BD, Brady PA, Friedman PA, Ezekowitz MD. Gender in atrial fibrillation: ten years later. Gend Med. 2010;7:206–217. 180. Kannel WB, Wilson PW, D’Agostino RB, Cobb J. Sudden coronary death in women. Am Heart J. 1998;136:205–212. 181. Node K, Kitakaze M, Kosaka H, Minamino T, Funaya H, Hori M. Amelioration of ischemia- and reperfusion-induced myocardial injury by 17beta-estradiol: role of nitric oxide and calciumactivated potassium channels. Circulation. 1997;96:1953–1963. 182. White RE, Han G, Maunz M, et  al. Endothelium-independent effect of estrogen on Ca(2+)-activated K(+) channels in human coronary artery smooth muscle cells. Cardiovasc Res. 2002;53:650–661. 183. Rusko J, Li L, van Breemen C. 17-beta-Estradiol stimulation of endothelial K+ channels. Biochem Biophys Res Commun. 1995;214:367–372. 184. Anderson SE, Kirkland DM, Beyschau A, Cala PM. Acute effects of 17beta-estradiol on myocardial pH, Na+, and Ca2+ and ischemia-reperfusion injury. Am J Physiol Cell Physiol. 2005;288:C57–C64. 185. Lang RM, Badano LP, Mor-Avi V, et  al. Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. J Am Soc Echocardiogr. 2015;28(1–39):e14. 186. Ozdemir K, Celik C, Altunkeser BB, et al. Effect of postmenopausal hormone replacement therapy on cardiovascular performance. Maturitas. 2004;47:107–113. 187. Ribeiro Jr. RF, Pavan BM, Potratz FF, et al. Myocardial contractile dysfunction induced by ovariectomy requires AT1 receptor activation in female rats. Cell Physiol Biochem. 2012;30:1–12. 188. Parks RJ, Howlett SE. Sex differences in mechanisms of cardiac excitation-contraction coupling. Pflugers Arch. 2013;465:747–763. 189. Johnson BD, Zheng W, Korach KS, Scheuer T, Catterall WA, Rubanyi GM. Increased expression of the cardiac L-type calcium channel in estrogen receptor-deficient mice. J Gen Physiol. 1997;110:135–140. 190. Marni F, Wang Y, Morishima M, et al. 17 beta-estradiol modulates expression of low-voltage-activated Ca(V)3.2 T-type calcium channel via extracellularly regulated kinase pathway in cardiomyocytes. Endocrinology. 2009;150:879–888. 191. Nakajima T, Iwasawa K, Oonuma H, et al. Antiarrhythmic effect and its underlying ionic mechanism of 17beta-estradiol in cardiac myocytes. Br J Pharmacol. 1999;127:429–440. 192. Fareh S, Benardeau A, Thibault B, Nattel S. The T-type Ca(2+) channel blocker mibefradil prevents the development of a substrate for atrial fibrillation by tachycardia-induced atrial remodeling in dogs. Circulation. 1999;100:2191–2197. 193. El Gebeily G, El Khoury N, Mathieu S, Brouillette J, Fiset C. Estrogen regulation of the transient outward K(+) current involves estrogen receptor alpha in mouse heart. J Mol Cell Cardiol. 2015;86:85–94.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

194. Kulpa J, Chinnappareddy N, Pyle WG. Rapid changes in cardiac myofilament function following the acute activation of estrogen receptor-alpha. PLoS One. 2012;7:e41076. 195. Ullrich ND, Krust A, Collins P, MacLeod KT. Genomic deletion of estrogen receptors ERalpha and ERbeta does not alter estrogen-mediated inhibition of Ca2+ influx and contraction in murine cardiomyocytes. Am J Physiol Heart Circ Physiol. 2008;294:H2421–H2427. 196. Belcher SM, Chen Y, Yan S, Wang HS. Rapid estrogen receptor-mediated mechanisms determine the sexually dimorphic sensitivity of ventricular myocytes to 17beta-estradiol and the environmental endocrine disruptor bisphenol A. Endocrinology. 2012;153:712–720. 197. Korte T, Fuchs M, Arkudas A, et al. Female mice lacking estrogen receptor beta display prolonged ventricular repolarization and reduced ventricular automaticity after myocardial infarction. Circulation. 2005;111:2282–2290. 198. Wang Y, Wang Q, Zhao Y, et  al. Protective effects of estrogen against reperfusion arrhythmias following severe myocardial ischemia in rats. Circ J. 2010;74:634–643. 199. Tran QK, Firkins R, Giles J, et  al. Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-Coupled Estrogen Receptor 1. J Biol Chem. 2016. 200. Gao Q, Liu S, Guo F, et  al. Nonylphenol affects myocardial contractility and L-type Ca(2+) channel currents in a non-monotonic manner via G protein-coupled receptor 30. Toxicology. 2015;334:122–129. 201. Mosca L, Linfante AH, Benjamin EJ, et  al. National study of physician awareness and adherence to cardiovascular disease prevention guidelines. Circulation. 2005;111:499–510.

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202. Milcent C, Dormont B, Durand-Zaleski I, Steg PG. Gender differences in hospital mortality and use of percutaneous coronary intervention in acute myocardial infarction: microsimulation analysis of the 1999 nationwide French hospitals database. Circulation. 2007;115:833–839. 203. Ghanbari H, Dalloul G, Hasan R, et al. Effectiveness of implantable cardioverter-defibrillators for the primary prevention of sudden cardiac death in women with advanced heart failure: a meta-analysis of randomized controlled trials. Arch Intern Med. 2009;169:1500–1506. 204. Vaccarino V, Abramson JL, Veledar E, Weintraub WS. Sex differences in hospital mortality after coronary artery bypass surgery: evidence for a higher mortality in younger women. Circulation. 2002;105:1176–1181. 205. Reynolds HR, Farkouh ME, Lincoff AM, et al. Impact of female sex on death and bleeding after fibrinolytic treatment of myocardial infarction in GUSTO V. Arch Intern Med. 2007;167:2054–2060. 206. Lansky AJ, Pietras C, Costa RA, et al. Gender differences in outcomes after primary angioplasty versus primary stenting with and without abciximab for acute myocardial infarction: results of the Controlled Abciximab and Device Investigation to Lower Late Angioplasty Complications (CADILLAC) trial. Circulation. 2005;111:1611–1618. 207. Kim ES, Menon V. Status of women in cardiovascular clinical trials. Arterioscler Thromb Vasc Biol. 2009;29:279–283. 208. Clayton JA, Collins FS. Policy: NIH to balance sex in cell and animal studies. Nature. 2014;509:282–283. 209. Consideration of Sex as a Biological Variable in NIH-funded Research. 2015 [cited June 9, 2015]; NOT-OD-15-102. Available from:

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C H A P T E R

26 The Sexually Dimorphic Characteristics of the Pathophysiology and Treatment of Atrial Fibrillation Uma Mahesh R. Avula, Meghana Noonavath and Elaine Wan Columbia University Medical Center, New York, NY, United States

O U T L I N E 26.1 Prevalence of Atrial Fibrillation

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26.2 Electrophysiology of AF 26.2.1 Effects of Hormones on QT 26.2.2 Women With LQT 26.2.3 Atrial Arrhythmias During Pregnancy

382 382 383 383

26.3 Cellular Electrophysiology and Ionic Basis for Sex Differences 383 26.3.1 Estrogen’s Effect on Cardiac Ion Channels 384 26.3.2 Progesterone’s Effect on Cardiac Ion Channels 384

26.1  PREVALENCE OF ATRIAL FIBRILLATION Atrial fibrillation (AF) is the most common type of cardiac arrhythmia in clinical practice. AF affects 2–6 million men and women in the United States alone, a number projected to double in the next 25 years.1 In addition to compromising the quality of life, AF often also complicates the management of other chronic diseases.2 AF is known to increase the risk of cognitive impairment in the elderly,3 stroke,4 heart failure,5 and sudden death.6 In 2010, the annual new cases of AF were estimated to be 5 million with an increasing trend each year.7 As a result, there is a rapidly growing economic burden and health care resource usage often approximating to $6–26 billion annually with the major

Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00003-6

26.3.3 Testosterone’s Effect on Cardiac Ion Channels 384 26.4 Sex-Based Differences in Presentation, Treatment and Outcomes in Women With AF 385 26.5 Drug Therapy for AF

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26.6 Catheter Ablation for AF

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26.7 Conclusions

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References 387

cost-drivers being hospitalizations, stroke, and loss of productivity.8 Older age groups have higher rates of AF. Males in the age range of 75–79 years have a fivefold higher prevalence of AF compared to males in the range of 55–59 years old. Adjusted for age, risk of AF is 1.5 times higher in males than in females.9 Higher risk in males can be partly attributed to the increase in male hormones (androgens) after puberty, which shortens the heart’s QT interval.10 But the absolute number of women with AF is greater due to the fact that there are more women over 75 years of age compared to men in the general population.10 The increased prevalence of AF could be caused by external factors such as aging trends in the global population, increasing numbers of other cardiovascular diseases (such as hypertension and atherosclerosis) that lead to AF, improved awareness of

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AF symptoms, improved clinical diagnosis, and/or lack of definitive treatment.7 Geographically, AF has higher prevalence rates in the developed countries compared to developing countries.7 This is not necessarily an indication of the lower prevalence in developing or underdeveloped countries, and can be attributed to the lack of awareness and underdiagnosis.

26.2  ELECTROPHYSIOLOGY OF AF Knowledge of the detailed pathophysiology of AF is essential in order to formulate and improve therapeutic approaches. Current drug therapy for AF management has major limitations such as lack of efficacy and life threatening pro-arrhythmic events. These limitations are largely due to the lack of knowledge about the molecular basis of AF. Though the key components in the cellular milieu that transform normal sinus rhythm to arrhythmia is precisely unknown, reentry is generally accepted as the mechanism that sustains AF after the triggering event. Sustaining reentry depends on the tissue substrate and its properties, such as slowed conduction and shorter refractory periods. The triggering event (ectopic activity) is initiated by either calcium handling abnormality that cause delayed afterdepolarizations or prolonged action potential durations that cause early afterdepolarizations. Prolonged episodes of AF alter the ionic properties of the atrial myocardium and increase the likelihood of ectopic activity and reentry. Refractory period depends on the action potential duration (APD) and is the key factor governing the likelihood of reentry. Ion transport processes (involving various channels, exchangers, and pumps) determine APD. Any changes in these processes lead to alterations in action potential properties that influence the occurrence of AF. Sodium current (INa) governs the phase 0 (upstroke) of the APD and corresponds to the depolarization of the myocardium. It determines the conduction velocity and abnormalities in phase 0 contributes to the reentry. Studies on the ion channel remodeling leading to AF are mostly conducted in animal models. Yue et al. first demonstrated that atrial remodeling in the tachypaced dogs has AP properties similar to that of humans with AF.11 The group demonstrated the critical role played by ICa,L in remodeling and AP morphology in these tachypaced dogs. ICa,L density decreased over the duration of AF, thus shortening the APD. Ca2+-independent transient outward current (Ito) was reduced similarly, while T-type Ca2+ current (ICa,T), delayed rectifier (IKs, IKr, IKur), and inward rectifier (IK1) potassium currents and the Ca2+-dependent Cl−− current were not altered.11 In a sustained AF goat model, expression of the L-type Ca2+ channel, and of Kv1.5 was shown to be reduced,

and expression of Kv4.3 and Kv4.2 was not altered.12 INa and gap junctions determine conduction velocity in the heart,13 and in the dog model conduction velocity is decreased in the atria due to the reduction of INa current density.14 Prolonged tachypacing also caused a decrease in the amplitude of the Ca2+-transients, leading to decreased contractility.15 In humans with AF, shortening of APD, effective refractory period,16 and triangular action potentials17 were reported. Chronic AF decreased ICa,L densities while no such effect is seen in paroxysmal AF.18 Other changes observed include reduction in the Ito19 and increase in IK1 and IKACh.20 Data on remodeling of connexins in human AF is limited and one study pointed to the reduction in Cx43 expression.21

26.2.1  Effects of Hormones on QT The EKG is a simple and powerful tool used for diagnosis and prognosis in a wide range of cardiovascular diseases. An EKG is a representation of the electrical activity of the heart and consists of a P wave (atrial activation and recovery), QRS complex (ventricular activation), and T wave (ventricular recovery). The QT interval represents ventricular activity and is calculated from the beginning of the QRS complex to the end of the T wave. Corrected QT (QTc) is calculated by taking the heart rate into consideration using Bazett’s formula. Prolonged QTc interval (long QT syndrome) is an important marker to evaluate the risk of ventricular arrhythmias such as torsades de pointes (TdP). Long QT Syndrome (LQT) can be either hereditary or acquired. The hereditary form is a result of various genetic mutations on channels involved in the cardiac action potential22 while the acquired form is usually a reversible condition that is induced either by drugs such as antihistamines, antibiotics, antipsychotic drugs, and gastrointestinal prokinetics23 or metabolic conditions such as hypokalemia, hypomagnesemia, hypothyroidism, and hemodialysis. While these latter risk factors can cause LQT in both males and females alike, female gender is an independent risk factor for drug-induced LQT. Sex hormones such as estrogen, progesterone, and testosterone have been shown to affect the QTc interval. Bazett first described the gender difference in QTc interval, with women’s QTc interval 24 ms greater than that of men.24 The gender differences do not exist at birth and males tend to have a shorter QTc interval throughout puberty and adulthood.25,26 However, by the age of 50, this gender difference disappears.27 This is attributed to the testosterone levels decreasing in males after age 50, which is thought to be the reason for shorter ventricular repolarization. Several interventional studies have demonstrated this direct association. QTc intervals decreased with increased endogenous testosterone in the males from the Rotterdam study cohort and Study

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26.3  Cellular Electrophysiology and Ionic Basis for Sex Differences

of Health In Pomerania (SHIP).27 Zhang et  al. showed that middle-aged men with the highest testosterone levels had significantly shorter QTc intervals.28 A negative linear relationship between QTc and the level of testosterone was demonstrated in hypogonadic men after a single injection of testosterone. QTc interval tended to be shorter (mean difference of 13.6 ms, p = 0.0007) with the higher dose of testosterone in the injections.28 Geraldi et  al. studied QTc intervals in the hypogonadic males and compared them with the age matched controls. QTc was shown to have a higher prevalence in the hypogonodal men.29 All these results demonstrate that both endogenous and exogenous testosterone show similar effects on shortening QTc interval. The effect of endogenous estrogens on the QTc interval is conflicting. QTc in mice with endogenous estrogen is found to have a prolonged QTc compared with ovariectomized mice with no endogenous estrogen. QTc in ovariectomized mice with estradiol injections tend to revert to that of the normal mice.30 This is in contrast to what has been observed in humans. A study in premenopausal women before and after bilateral oophorectomy showed a significant decrease in estradiol postsurgery but there was no significant change in the QTc interval.31 Another study by Saba et al. showed that the QTc does not vary between premenopausal women (405 ±21 ms) and postmenopausal women even though the estrogen levels were significantly lower in the postmenopausal women (419 ±30 ms).32 Progesterone, like testosterone, shortens the APD and QTc interval in women.30 During the menstrual cycle, progesterone levels have the dominant effect on ventricular repolarization. Progesterone levels are higher in the luteal phase, during which the QTc interval is shorter in women.33 This is also evident from studies done on women under menopause hormone therapy. Women placed on estrogen alone therapy have QTc prolongation compared to women on estrogen plus progesterone therapy. In the latter case, estrogen and progesterone tend to have a counterbalancing effect on the QTc interval.34

26.2.2  Women With LQT More females than males have symptomatic LQT syndrome.35 Shorter QT interval in adult men (an effect of testosterone) may explain the lower incidence of related cardiac events in men compared to women. Hormonal changes, in addition to various physiological changes, occur throughout pregnancy. Lechmanova et  al. have shown prolongation of the QTc in pregnant women.36 In this study, EKGs were recorded in healthy pregnant women (36–40 weeks of gestation) and in healthy nonpregnant women. In addition, women with established LQTs have a higher risk of developing TdP in the first

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9 months after the pregnancy37 after which the risk level returns to baseline as in before the pregnancy.38 This higher risk of arrhythmias immediately after pregnancy can be partially explained by changes in heart rate. After delivery, heart rate returns to baseline and this protection of an increased heart rate is lost, resulting in prolonged QT intervals and thus increasing the risk of arrhythmias.39 In addition, the associated increase in stress and lack of sleep due to child care could increase the incidence of cardiac events postpartum. β-blocker use during pregnancy and postpartum was independently associated with the decrease in cardiac events.

26.2.3  Atrial Arrhythmias During Pregnancy During pregnancy, the incidence of supraventricular tachyarrhythmias (SVT) is increased. In a study on 60 consecutive women with SVT, it is shown that the risk of exacerbation and new onset of SVT is increased during pregnancy.40 The risk level however was not affected by the stage of pregnancy. The exact cause of increased arrhythmias during pregnancy is unknown, although changes in autonomic tone, hormonal levels, and hemodynamic alterations have all been suggested to be causes. Management of arrhythmias during pregnancy is often complicated by the possibility of fetal injury. Current antiarrhythmic drugs are not potent enough and can pass through the placenta to injure the fetus. Abstaining from treatment could also put the fetus at risk with the resultant maternal hypotension from hemodynamically significant arrhythmias. Extreme care in selecting the drugs is necessary and long-term drug treatment should be avoided except in the cases of severe arrhythmias.

26.3  CELLULAR ELECTROPHYSIOLOGY AND IONIC BASIS FOR SEX DIFFERENCES Gender differences in electrophysiological properties are well known. Healthy men and women tend to show notable differences in surface electrocardiograms such as in QTc interval, heart rate, and QRS interval.41 These variations can be partly attributed to the effect of sex hormones on cardiomyocytes at the cellular and ion channel level. In the early 1980s, McGill et  al. provided conclusive evidence that cardiac myocytes possess both androgen42 and estrogen receptors (ERs).43,44 They also demonstrated that these receptors’ binding is isoform specific—myocytes have receptors only for dihydrotestosterone (DHT) and not testosterone.45 These receptors are functional and, depending on the stimulus, can modulate gene expression and nongenomic signaling pathways.46

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26.3.1  Estrogen’s Effect on Cardiac Ion Channels Nuclear ERs come primarily in two types: ERα and ER β . In addition to their expression in the uterus, ERs are widely expressed in the heart.47 ERα is mainly located on the cardiomyocyte membrane.47 Both receptors are abundant in myocyte mitochondria and regulate mitochondrial function.48 ERs are translocated to nuclei after exposure to 17 β -estradiol.49 Estrogen also has a direct effect on the conduction properties of cardiac myocytes. Chronic estradiol treatment has modulatory effects on the coronary smooth muscle K++ channels50 and cardiac calcium channels.51 In a canine model of ventricular arrhythmias induced after ischemia/reperfusion, estrogen administration significantly reduced the arrhythmia burden.52 These antiarrhythmic properties are due to estrogen’s effect on opening KCa channels53 and inhibiting the Na++/H++ exchanger54 during ischemia/reperfusion injury. Estrogen also plays an important role in excitation–contraction coupling by regulating calcium homeostasis in the heart55,56 and regulating membrane density and expression of L-type Ca2+ channels on the cardiac myocytes.57,58 Ovariectomy caused significant myocardial dysfunction in rats.59 17 β -estradiol inhibited occurrence of early afterdepolarizations and depolarization-induced ectopic-triggered activity, such as in myocardial ischemia, potentially acting as an antiarrhythmic agent.58 Estrogen is shown to inhibit ICa,L in a voltage-dependent fashion. This is particularly important in myocardial infarction, in which myocytes in the ischemic zone are partially depolarized and estrogen prevents triggered activity from these partially-depolarized surviving ischemic myocytes.58 Further studies are needed to demonstrate the long-term effects of estrogen on the other cardiac channels.

26.3.2  Progesterone’s Effect on Cardiac Ion Channels Several clinical studies have shown the protective role of progesterone in LQTS-associated arrhythmias.33,38,60 This protective role is due to progesterone’s actions on cardiac repolarization. Progesterone modulates ion channels through a nongenomic pathway that induces NOS activation. It enhances IKs and inhibits ICa,L, which reduces the QT interval and thus is beneficial in patients with LQTS.61 Inhibitory action on ICa,L occurs only when ICa,L has been activated by sympathetic stimulation.62 This genomic and nongenomic regulation of cardiac ion channels by sex hormones may contribute to the development of gender differences and dynamic fluctuations of QTc interval and arrhythmic risk in women. Normal levels of circulating ovarian hormones influence baseline cardiac repolarization. Both estrogen and

progesterone exhibit a modulatory effect on cardiac repolarization either directly by altering potassium channel expression and conductance63,64 or indirectly by influencing autonomic tone.65 On the genomic level, estrogen downregulates the expression of IKr and IKs ion channels, thereby affecting cardiac repolarization and prolonging the QTc interval.66 Jiang et al. demonstrated in the isolated cardiac myocytes from guinea pigs that both estrogen and progesterone play an important role in regulating calcium levels in the sarcoplasmic reticulum (SR).67 Estrogen and progesterone have opposing effects on cardiac repolarization—estrogen prolongs the QTc interval while progesterone shortens the QTc interval. There is controversy regarding the effect of these hormones at their physiological concentrations (usually in the nanomolar range). Studies at micromolar concentrations34 have shown cardioprotective effects whereas studies at physiological nano molar concentrations did not show any significant cardioprotection.68 During the menstrual cycle, progesterone reverses the effect of estrogen-induced QTc prolongation.69

26.3.3  Testosterone’s Effect on Cardiac Ion Channels Testosterone acts similarly to progesterone by accelerating the repolarization and thus providing protection from drug-induced arrhythmias.61 McGill et al. first described the presence of androgen receptors in atrial and ventricular myocytes, leading to the discussion of sex hormones’ possible effect on myocardial function.42 Testosterone is generally considered to increase cardiovascular risk. In fact, recent studies have shown that the acute effects of testosterone are beneficial and are different from the effects of chronic testosterone exposure.70 Cardiac L-type calcium channels (ICa,L) have a major role in maintaining intracellular calcium homeostasis and thus play a critical role in the induction of arrhythmias. Chronic exposure of rat cardiac myocytes to testosterone for 24–30 h increased ICa,L, mainly by increasing the expression levels of the alpha 1C subunit of L-type calcium channel and open probability of the single channel. Frequency of calcium sparks increased without any increase in the SR calcium load. In contrast, acute treatment of the myocytes with testosterone caused a decrease in the ICa,L. These differences are thought to be due to activation of nuclear receptor-mediated pathways71 in chronic treatment and the direct blocking effect of acute treatment.70 Despite the well-known physiological distinctions and outcomes between men and women with CVD, sex-specific treatment strategies are not well-studied. Further research on sex hormones and their role in the heart is key to developing personalized and sex-specific therapies.

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26.4  Sex-Based Differences in Presentation, Treatment, and Outcomes in Women with AF

26.4  SEX-BASED DIFFERENCES IN PRESENTATION, TREATMENT, AND OUTCOMES IN WOMEN WITH AF Sex differences studies in the arrhythmias have received relatively less attention compared to coronary heart disease.72 Large registry studies including Registry on Cardiac Rhythm Disorders Assessing the Control of Atrial Fibrillation (RECORD AF)73 and EURO bservational Research Programme—Atrial Fibrillation General Registry Pilot Phase,74 focused on the AF symptoms, but there were no detailed analysis on sex differences in symptoms and quality of life. As discussed previously, women have lower incidence of AF, but the number of patients with AF above age 75 years is about same due to the fact that women outnumber men in the group with the highest percentage of AF.75,76 The few studies available report that women present with higher heart rates during AF than men.77 Women also have a higher incidence of paroxysmal AF after successful cardioversion, which is attributed mainly to the greater number of reported recurrent AF episodes.78 At the first diagnosis of AF, sex differences are quite evident, with females presenting with more symptomatic AF, hypertension, and other comorbid conditions such as diabetes and thyroid disease. However, after the diagnosis there is no sex-based difference in progression to permanent AF.79,80 Conflicting reports exist over the increased risk of stroke in women with AF than in men.81–83 In the Copenhagen City Heart Study, it was shown that AF is a greater risk factor for stroke and cardiovascular death in women than in men.84 The possible mechanisms for the increased risk of stroke is not clear85 but impaired renal function may partly explain the higher stroke rate in females, due to increased thrombosis.86 A study that analyzed Canadian Registry of Atrial Fibrillation (CARAF) data showed that despite its proven efficacy, warfarin is not commonly used in women with AF because of higher susceptibility to major bleeding (about 3.35 times more likely than men).80 Stroke Prevention in Atrial Fibrillation III (SPAF III) data identified women as a high risk group for bleeding complications, especially for women above 75 years of age.87 In the Swedish Atrial Fibrillation Cohort Study, the risk for stroke or systemic embolism was shown to be 20% higher in women.88 Thus, while considering anticoagulation treatment, female sex should be taken into account. In the Euro Heart survey report, it was noted that compared to men, women with AF had a lower quality of life with higher comorbidities and reported more palpitations, dyspnea, and fatigue. However, long-term quality of life changes and other morbidities and mortality were similar in both men and women.89 Data from the EORP-AF Pilot registry, managed by European cardiologists, showed females with

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AF have a higher proportion of ischemic heart failure and preserved ejection fraction heart failure compared to men.90 Symptomatic females with AF more often received rate control than rhythm control while asymptomatic females received mostly rate control management.90 Initial management of AF did not differ in men and women with AF. Digoxin is the first prescription at the baseline visit followed by antiarrhythmic drugs such as amiodarone, propafenone, and sotalol. Women are less likely to undergo catheter ablation for AF. Though permanent AF is less common in women, there is a higher rate of AV nodal ablation, which is usually a last resort for drug refractory and symptomatic AF.91 Arrhythmias such as AF, which causes palpitations may be associated with depression. However, the effects of depression on quality of life in men and women is not clearly understood.92 Clinicians should be aware of these sex differences while considering AF treatment strategies for women. The American Heart Association (AHA), in its 2011 guidelines, highlighted the need for reporting sex-specific analyses in cardiovascular interventions to aid in the development of future sex-specific treatment guidelines.93 AF management during pregnancy is a concern for the well-being of both mother and fetus. The onset of episodes during pregnancy can be either primary or a recurrence of previously diagnosed AF. Most of the primary episodes are benign and thus appropriate advice and reassurance is helpful. In the episodes that are highly symptomatic and occur in the setting of previous AF judicious use of antiarrhythmic therapy is required. It is a delicate balance to consider both the benefits of arrhythmia treatment and the maternal and fetal side effects of antiarrhythmic drugs. The first trimester, during which fetal organogenesis occurs, possesses a major risk for the use of any antiarrhythmic drug. If AF is associated with valvular disease such as severe mitral stenosis, it is advantageous to consider terminating AF using antiarrhythmic drugs to avoid the need for anticoagulation, because pregnancy in particular is a prothrombotic condition. Avoiding anticoagulation would reduce risk to the fetus and avoid the complications of pregnancy. Sotalol, atenolol, flecainide, or procainamide are the preferred drugs in such cases. These drugs should be administered in the lowest effective dose and constant monitoring of mother and fetus during the treatment is recommended. Electrical cardioversion is usually safe in pregnancy irrespective of the trimester. However, it has been reported that there is a remote possibility of cardioversion initiating fetal arrhythmias which require emergency cesarean section.94 There are also reported incidents where electrical cardioversion led to fetal death and contracted uterus.95 Hence

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electrical cardioversion is recommended only in facilities equipped to monitor fetal heart rate and perform emergency cesarean section. Ibutilide can be considered for pharmacological cardioversion and it is shown to be safe in pregnancy in a case report by Kockova et.al.96 Cardioverter-defibrillators (ICDs) may be another safe alternative and reports show that women with ICD during pregnancy did not risk fetal health.97

26.5  DRUG THERAPY FOR AF Therapy for AF includes antithrombotic drugs for stroke prevention in all patients with more than one moderate stroke risk factor according to the CHADS2 score, which allocated 1 point for congestive heart failure, hypertension, age >75 years, and diabetes mellitus, and 2 points for history of stroke. In 2009, the CHA2DS2-VASc score was developed after identifying other stroke risk factors in patients with AF. The CHA2DS2-VASc scoring system assigns 1 point for age 65–74, and 2 points for age ≥75, 1 point for female sex, 1 point for congestive heart failure, 1 point for hypertension, 2 points for history of stroke, transient ischemic attack, or history of thromboembolism, 1 point for vascular disease, and 1 point for diabetes mellitus. The study shows that a score of 0 is low risk for thromboembolic events, score of 1 intermediate risk (0.6% rate at 1 year), and greater than 1 high risk (3% rate at 1 year). It also highlights that female sex is an independent risk factor for an increased risk for stroke.85,98,99 As an alternative to oral anticoagulation, aspirin (81– 325 mg) can be used in low risk patients such as those with lone AF, who do not have comorbidities as listed above or those with contraindications to oral anticoagulation. Options for oral anticoagulants include warfarin, a vitamin K antagonist. However, newer anticoagulants, which do not necessitate frequent blood testing and adjustment of doses have increased in popularity in usage and prescribing preferences. Factor Xa inhibitors, such as rivaroxaban, apixaban, and edoxaban, have been increasingly used over the usage of direct thrombin inhibitors, such as dabigatran.100 Dabigatran was found to be equally effective as warfarin for stroke prevention in the RE-LY study, and is readily used in the United States and Europe. In the ROCKET AF study, rivaroxaban was shown to be noninferior to warfarin in the ROCKET AF trail. The ARISTOTLE study demonstrated that Apixaban was shown to prevent stroke and be noninferior to warfarin. All these novel oral anticoagulants in these studies have shown a trend of decreased intracranial and fatal bleeding compared to warfarin, which is important as the population ages and has increased risk factors for bleeding, such as advanced age, uncontrolled hypertension, coronary artery disease, stroke, anemia, and usage

of other antiplatelet agents. Since risk of stroke is higher in women, better understanding of treatments and their bleeding risks in women is important.98–100 Drug therapy for AF includes also rate control or rhythm control. Rate control focuses on slowing the ventricular rate during AF even though the patient remains in AF, whereas in rhythm control the strategy is to restore normal sinus rhythm. In the AFFIRM trial and RACE trial, survival of patients with AF were similar in the rate and rhythm control strategy. Sex did not predict any difference in response to either therapy. Medications used for rate control include agents acting on the atrioventricular node such as β blockers, calcium channel blockers, and/or digoxin. If ventricular rate is too slow on these agents, implantation of a pacemaker may be necessary. RACE II trial suggested that allowing for heart rates to 110 beats per minute may be allowable in patients with a preserved ejection fraction. Assessing adequacy of rate control may be accomplished using holter monitoring and stress testing.98,101 Compared to men, women have an increased risk of deleterious arrhythmias when treated with antiarrhythmics, since they have longer QT intervals and it is known that longer QT intervals increase the risk of TdP. In a study of d,l-sotalol women had 4.1% incidence of torsades compared to 1% for men. This gender specific increase was seen also in the SWORD trial and the DIAMOND-CHF trial, which evaluated usage of dofetilide. It has also been noted that women have a higher incidence of sick sinus syndrome due to antiarrhythmics such as flecainide, sotalol, and amiodarone, thus leading to increased rate of pacemaker implantation.66,90 The proarrhythmic risks of drugs may also be due to hormonal changes, especially when progesterone and estradiol levels abruptly decrease as in the postpartum period. Sole estrogen replacement hormonal therapy also is known to be proarrhythmic.98

26.6  CATHETER ABLATION FOR AF Catheter ablation for AF may be a curative therapy for patients who are refractory to pharmacological therapy. Restoration and maintenance of sinus rhythm after ablation has been known to improve quality of life, symptoms, exercise tolerance, and left ventricular function. Unfortunately the success of catheter ablation can be variable from 60–85%, and highly dependent on duration of AF and structural abnormalities such as increased atrial fibrosis and left atrial size. Paroxysmal AF is known to be more amenable to successful catheter ablation than permanent AF. Women tend to have longstanding AF, larger left atrial size, and a history of not responding well to antiarrhythmics; these factors are associated with poorer outcomes.85,102 Women are also

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referred three times less often than men for AF ablation which may reflect sex bias in referral patterns, even though women with AF report poorer quality of life and have a higher risk of stroke and increased mortality. AF ablation in women may also be more technically challenging as women often had a higher incidence of nonpulmonary vein sources such as from the superior vena cava compared to men. Women also suffer from higher procedural complications. Incidence of cardiac tamponade, femoral vascular complications, such as hematomas, and pseudoaneurysms are higher in women compared to men. In addition, women are often underrepresented in clinical trials of invasive procedures such as catheter ablation. Women who are enrolled in single center or multicenter trials have usually been less than 30%.98,103,104 The National Institutes of Health has recognized this deficiency and has begun to implement new strategies to advance studies in gender specific differences in basic science research as well as incorporate gender specific differences in design of new technologies, such as medical devices and drugs. A novel transgenic mouse model with spontaneous and sustained AF is the first step to understanding the pathophysiological differences in females and males with AF. In many diseases, the availability of a mouse model is critical for molecular research because of the ease of genetic engineering and the large number of preexisting genetically altered mice for cross-breeding. Studies of AF have been hindered by the lack of a mouse model that accurately recapitulates the spontaneous initiation and sustained periods of AF observed in humans. Most, if not all, studies use nonphysiological methods including highfrequency burst pacing to induce very short episodes in mice, typically lasting several seconds, of AF compared to hours, days, or longer for humans. Wan et al.105 generated transgenic (TG) mice with doxycycline-inducible and titratable, cardiac-specific expression of FLAGepitope-tagged human NaV1.5 with a mutation (F1759A) in the local anesthetic binding site,106 which causes window current and persistent Na++ current. We found that two founder lines with doxycycline-independent low expression of the mutant Na++ channels had the phenotype of atrial enlargement, cardiomyopathy, frequent relatively long episodes of spontaneous AF, and nonsustained polymorphic ventricular tachycardia, observed as early as 5 weeks of age. These mice phenocopied gain-offunction human SCN5A mutations that have been implicated in dilated cardiomyopathy and hypertrophy, and arrhythmias such as LQT, TdP, and AF.107 The sustained and spontaneous nature of the atrial arrhythmias enabled the exploration of mechanisms by which dysfunctional Na++ channel inactivation causes cardiomyopathy and arrhythmias. The study showed that the primary effects of incomplete NaV1.5 inactivation on cardiomyocyte electrophysiology, namely prolongation and dispersion

of the APD, and the secondary downstream effects on chamber enlargement, fibrosis, and mitochondrial necrosis/reactive oxygen species (ROS) synergistically cause the unique phenotype of spontaneous and prolonged episodes of AF in mice, mimicking human disease. In the vast majority of previously reported mouse models of AF, atrial arrhythmias could only be elicited by very aggressive burst pacing, suggesting that whereas there may be a substrate for atrial arrhythmia, this can be well tolerated and undetected in the absence of a triggering factor.108 AF is defined in these studies by a duration of at least 1 s and most previously reported mouse models demonstrated these relatively short episodes of AF.108 A new therapeutic approach for AF was identified by pharmacologically targeting the downstream effects of enhanced Na++ entry, using a relatively specific inhibitor of the Na++–Ca2+ exchanger (NCX), which significantly reduced AF burden.105 This model can be used for future further understanding of the gender specific differences in male and female mice with AF, and how those differences may reflect gender specific cardiac remodeling, structure, and electrophysiology in men and women with AF.

26.7 CONCLUSIONS AF is one of the most commonly diagnosed cardiac arrhythmias. The pathophysiology and treatment of AF is different in men compared to women, and hopefully further research will lead to better treatment of patients. Because there are significant differences in AF even at the cellular level in males and females, specialized regimens are essential for improving prognosis. Additional research into the effect of hormones on AF will lead to the development of more personalized and sex-specific therapies.

References 1. January C, Wann L, Alpert J, et al. Aha/acc/hrs guideline for the management of patients with atrial fibrillation: a report of the American College of Cardiology/American Heart Association task force on practice guidelines and the heart rhythm society [published online ahead of print March 28, 2014]. J Am Coll Cardiol. 2014;64:2246–2280. 2. Dorian P, Jung W, Newman D, et al. The impairment of healthrelated quality of life in patients with intermittent atrial fibrillation: implications for the assessment of investigational therapy. J Am Coll Cardiol. 2000;36:1303–1309. 3. Kilander L, Andrén B, Nyman H, Lind L, Boberg M, Lithell H. Atrial fibrillation is an independent determinant of low cognitive function a cross-sectional study in elderly men. Stroke. 1998;29:1816–1820. 4. Fuster V, Rydén LE, Cannom DS, et al. Acc/aha/esc 2006 guidelines for the management of patients with atrial fibrillation— executive summary: a report of the American College of Cardiology/American Heart Association task force on practice

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

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guidelines and the European Society of Cardiology Committee for practice guidelines (writing committee to revise the 2001 guidelines for the management of patients with atrial fibrillation) developed in collaboration with the European Heart Rhythm Association and the Heart Rhythm Society. J Am Coll Cardiol. 2006;48:854–906. 5. Wang TJ, Larson MG, Levy D, et al. Temporal relations of atrial fibrillation and congestive heart failure and their joint influence on mortality the Framingham Heart Study. Circulation. 2003;107:2920–2925. 6. Benjamin EJ, Wolf PA, D’Agostino RB, Silbershatz H, Kannel WB, Levy D. Impact of atrial fibrillation on the risk of death the Framingham Heart Study. Circulation. 1998;98:946–952. 7. Chugh SS, Havmoeller R, Narayanan K, et  al. Worldwide epidemiology of atrial fibrillation: a global burden of disease 2010 study. Circulation. 2013. http://dx.doi.org/10.1161/ CIRCULATIONAHA.113.005119 8. Kim MH, Johnston SS, Chu B-C, Dalal MR, Schulman KL. Estimation of total incremental health care costs in patients with atrial fibrillation in the united states. Circ Cardiovasc Qual Outcomes. 2011;4:313–320. 9. Benjamin EJ, Levy D, Vaziri SM, D'Agostino RB, Belanger AJ, Wolf PA. Independent risk factors for atrial fibrillation in a population-based cohort: the Framingham Heart Study. JAMA. 1994;271:840–844. 10. Wolbrette DL. Risk of proarrhythmia with class iii antiarrhythmic agents: sex-based differences and other issues. Am J Cardiol. 2003;91:39–44. 11. Yue L, Feng J, Gaspo R, Li G-R, Wang Z, Nattel S. Ionic remodeling underlying action potential changes in a canine model of atrial fibrillation. Circ Res. 1997;81:512–525. 12. Velden HM, Zee L, Wijffels MC, et  al. Atrial fibrillation in the goat induces changes in monophasic action potential and mrna expression of ion channels involved in repolarization. J Cardiovasc Electrophysiol. 2000;11:1262–1269. 13. Buchanan J, Saito T, Gettes LS. The effects of antiarrhythmic drugs, stimulation frequency, and potassium-induced resting membrane potential changes on conduction velocity and dv/ dtmax in guinea pig myocardium. Circ Res. 1985;56:696–703. 14. Gaspo R, Bosch RF, Bou-Abboud E, Nattel S. Tachycardiainduced changes in Na+ current in a chronic dog model of atrial fibrillation. Circ Res. 1997;81:1045–1052. 15. Sun H, Gaspo R, Leblanc N, Nattel S. Cellular mechanisms of atrial contractile dysfunction caused by sustained atrial tachycardia. Circulation. 1998;98:719–727. 16. Attuel P, Childers R, Cauchemez B, Poveda J, Mugica J, Coumel P. Failure in the rate adaptation of the atrial refractory period: Its relationship to vulnerability. Int J Cardiol. 1982;2:179–197. 17. Boutjdir M, Heuzey JY, Lavergne T, et al. Inhomogeneity of cellular refractoriness in human atrium: Factor of arrhythmia? Pacing Clin Electrophysiol. 1986;9:1095–1100. 18. Skasa M, Jüngling E, Picht E, Schöndube F, Lückhoff A. L-type calcium currents in atrial myocytes from patients with persistent and non-persistent atrial fibrillation. Basic Res Cardiol. 2001;96:151–159. 19. Workman A, Kane K, Rankin A. Cellular changes in action potentials and ion currents associated with chronic atrial fibrillation in humans. Eur Heart J. 2000;21 544-544. 20. Bosch RF, Zeng X, Grammer JB, Popovic K, Mewis C, Kühlkamp V. Ionic mechanisms of electrical remodeling in human atrial fibrillation. Cardiovasc Res. 1999;44:121–131. 21. Patel P, Jones D, Dupont E, Severs N, Peters N. Remodelling of atrial connexin43 expression in human atrial fibrillation. Eur Heart J. 1998;19:465. 22. Sauer AJ, Moss AJ, McNitt S, et al. Long qt syndrome in adults. J Am Coll Cardiol. 2007;49:329–337.

23. van Noord C, Eijgelsheim M, Stricker BH. Drug- and nondrug-associated qt interval prolongation. Br J Clin Pharmacol. 2010;70:16–23. 24. Bazett HC. An analysis of the time-relations of electrocardiograms. Heart. 1920;7:353–370. 25. Stramba-Badiale M, Spagnolo D, Bosi G, Schwartz PJ. Are gender differences in qtc present at birth? Misnes investigators. Multicenter Italian study on neonatal electrocardiography and sudden infant death syndrome. Am J Cardiol. 1995;75:1277–1278. 26. Rautaharju PM, Zhou SH, Wong S, et  al. Sex differences in the evolution of the electrocardiographic qt interval with age. Can J Cardiol. 1992;8:690–695. 27. van Noord C, Dorr M, Sturkenboom MC, et al. The association of serum testosterone levels and ventricular repolarization. Eur J Epidemiol. 2010;25:21–28. 28. Zhang Y, Ouyang P, Post WS, et  al. Sex-steroid hormones and electrocardiographic qt-interval duration: findings from the third national health and nutrition examination survey and the multi-ethnic study of atherosclerosis. Am J Epidemiol. 2011;174:403–411. 29. Pecori Giraldi F, Toja PM, Filippini B, et al. Increased prevalence of prolonged qt interval in males with primary or secondary hypogonadism: a pilot study. Int J Androl. 2010;33:e132–138. 30. Saito T, Ciobotaru A, Bopassa JC, Toro L, Stefani E, Eghbali M. Estrogen contributes to gender differences in mouse ventricular repolarization. Circ Res. 2009;105:343–352. 31. De Leo V, la Marca A, Agricola E, Morgante G, Mondillo S, Setacci C. Resting ecg is modified after oophorectomy and regresses with estrogen replacement therapy in premenopausal women. Maturitas. 2000;36:43–47. 32. Saba S, Link MS, Homoud MK, Wang PJ, Estes III NA. Effect of low estrogen states in healthy women on dispersion of ventricular repolarization. Am J Cardiol. 2001;87:354–356. A359–310. 33. Rodriguez I, Kilborn MJ, Liu XK, Pezzullo JC, Woosley RL. Druginduced qt prolongation in women during the menstrual cycle. JAMA. 2001;285:1322–1326. 34. Kadish AH, Greenland P, Limacher MC, Frishman WH, Daugherty SA, Schwartz JB. Estrogen and progestin use and the qt interval in postmenopausal women. Ann Noninvasive Electrocardiol. 2004;9:366–374. 35. Moss AJ, Schwartz PJ, Crampton Jr. RS, et  al. The long qt syndrome. Prospective longitudinal study of 328 families. Circulation. 1991;84:1136–1144. 36. Lechmanova M, Kittnar O, Mlcek M, et  al. Qt dispersion and t-loop morphology in late pregnancy and after delivery. Physiol Res. 2002;51:121–129. 37. Meregalli PG, Westendorp IC, Tan HL, Elsman P, Kok WE, Wilde AA. Pregnancy and the risk of torsades de pointes in congenital long-qt syndrome. Neth Heart J. 2008;16:422–425. 38. Seth R, Moss AJ, McNitt S, et  al. Long qt syndrome and pregnancy. J Am Coll Cardiol. 2007;49:1092–1098. 39. Rashba EJ, Zareba W, Moss AJ, et al. Influence of pregnancy on the risk for cardiac events in patients with hereditary long qt syndrome. Lqts investigators. Circulation. 1998;97:451–456. 40. Tawam M, Levine J, Mendelson M, Goldberger J, Dyer A, Kadish A. Effect of pregnancy on paroxysmal supraventricular tachycardia. Am J Cardiol. 1993;72:838–840. 41. Bernal O, Moro C. Cardiac arrhythmias in women. Rev Esp Cardiol (English Edition). 2006;59:609–618. 42. McGill H, Anselmo V, Buchanan J, Sheridan P. The heart is a target organ for androgen. Science. 1980;207:775–777. 43. McGill H, Sheridan P. Nuclear uptake of sex steroid hormones in the cardiovascular system of the baboon. Circ Res. 1981;48:238–244. 44. Stumpf WE, Sar M, Aumuller G. The heart: a target organ for estradiol. Science. 1977;196:319–321.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

45. Sheridan PJ, McGill HC, Aufdemorte TB, Triplett RG, Holt RG. Heart contains receptors for dihydrotestosterone but not testosterone: possible role in the sex differential in coronary heart disease. Anat Rec. 1989;223:414–419. 46. Knowlton A, Lee A. Estrogen and the cardiovascular system. Pharmacol Ther. 2012;135:54–70. 47. Lizotte E, Grandy SA, Tremblay A, Allen BG, Fiset C. Expression, distribution and regulation of sex steroid hormone receptors in mouse heart. Cell Physiol Biochem. 2009;23:075–086. 48. Yang S-H, Liu R, Perez EJ, et  al. Mitochondrial localization of estrogen receptor β. Proc Natl Acad Sci. 2004;101:4130–4135. 49. Grohé C, Kahlert S, Löbbert K, et al. Cardiac myocytes and fibroblasts contain functional estrogen receptors. FEBS Lett. 1997;416:107–112. 50. Harder DR, Coulson PB. Estrogen receptors and effects of estrogen on membrane electrical properties of coronary vascular smooth muscle. J Cell Physiol. 1979;100:375–382. 51. Ishii K, Kano T, Ando J. Sex differences in (3h) nitrendipine binding and effects of sex steroid hormones in rat cardiac and cerebral membranes. Jap J Pharmacol. 1988;46:117–125. 52. McHugh N, Cook S, Schairer J, Bidgoli M, Merrill G. Ischemia-and reperfusion-induced ventricular arrhythmias in dogs: effects of estrogen. Am J Physiol-Heart Circ Physiol. 1995;268:H2569–H2573. 53. Node K, Kitakaze M, Kosaka H, Minamino T, Funaya H, Hori M. Amelioration of ischemia-and reperfusion-induced myocardial injury by 17β-estradiol role of nitric oxide and calcium-activated potassium channels. Circulation. 1997;96:1953–1963. 54. Anderson SE, Kirkland DM, Beyschau A, Cala PM. Acute effects of 17β-estradiol on myocardial ph, na+, and ca2+ and ischemiareperfusion injury. Am J Physiol-Cell Physiol. 2005;288:C57–C64. 55. Parks RJ, Howlett SE. Sex differences in mechanisms of cardiac excitation–contraction coupling. Pflügers Arch-Eur J Physiol. 2013;465:747–763. 56. Farrell SR, Ross JL, Howlett SE. Sex differences in mechanisms of cardiac excitation-contraction coupling in rat ventricular myocytes. Am J Physiol-Heart Circ Physiol. 2010;299:H36–H45. 57. Johnson BD, Zheng W, Korach KS, Scheuer T, Catterall WA, Rubanyi GM. Increased expression of the cardiac l-type calcium channel in estrogen receptor–deficient mice. J Gen Physiol. 1997;110:135–140. 58. Nakajima T, Iwasawa K, Oonuma H, et al. Antiarrhythmic effect and its underlying ionic mechanism of 17β-estradiol in cardiac myocytes. Br J Pharmacol. 1999;127:429–440. 59. Ribeiro Jr R, Pavan BM, Potratz FF, et al. Myocardial contractile dysfunction induced by ovariectomy requires at1receptor activation in female rats. Cell Physiol Biochem. 2012;30:1–12. 60. Nakagawa M, Ooie T, Takahashi N, et  al. Influence of menstrual cycle on qt interval dynamics. Pacing clin electrophysiol. 2006;29:607–613. 61. Nakamura H, Kurokawa J, Bai C-X, et  al. Progesterone regulates cardiac repolarization through a nongenomic pathway an in vitro patch-clamp and computational modeling study. Circulation. 2007;116:2913–2922. 62. Furukawa T, Kurokawa J. Non-genomic regulation of cardiac ion channels by sex hormones. Cardiovasc Haematol Disord-Drug Targets (Formerly Curr Drug Targets-Cardiovasc Hematol Disord). 2008;8:245–251. 63. Saba S, Zhu W, Aronovitz MJ, et  al. Effects of estrogen on cardiac electrophysiology in female mice. J Cardiovasc Electrophysiol. 2002;13:276–280. 64. Korte T, Fuchs M, Arkudas A, et al. Female mice lacking estrogen receptor β display prolonged ventricular repolarization and reduced ventricular automaticity after myocardial infarction. Circulation. 2005;111:2282–2290. 65. Dart AM, Du X-J, Kingwell BA. Gender, sex hormones and autonomic nervous control of the cardiovascular system. Cardiovasc Res. 2002;53:678–687.

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66. Pham TV, Rosen MR. Sex, hormones, and repolarization. Cardiovasc Res. 2002;53:740–751. 67. Jiang C, Poole-Wilson PA, Sarrel PM, Mochizuki S, Collins P, MacLeod KT. Effect of 17β-oestradiol on contraction, ca2+ current and intracellular free ca2+ in guinea-pig isolated cardiac myocytes. Br J Pharmacol. 1992;106:739–745. 68. Kurokawa J, Tamagawa M, Harada N, et  al. Acute effects of oestrogen on the guinea pig and human ikr channels and drug-induced prolongation of cardiac repolarization. J Physiol. 2008;586:2961–2973. 69. Philp K, Hussain M, Byrne N, Diver M, Hart G, Coker SJ. Greater antiarrhythmic activity of acute 17β-estradiol in female than male anaesthetized rats: correlation with ca2+ channel blockade. Br J Pharmacol. 2006;149:233–242. 70. Er F, Michels G, Brandt MC, et  al. Impact of testosterone on cardiac l-type calcium channels and ca 2+ sparks: acute actions antagonize chronic effects. Cell calcium. 2007;41:467–477. 71. Golden K, Marsh J, Jiang Y. Testosterone regulates mrna levels of calcium regulatory proteins in cardiac myocytes. Horm metab res. 2004;36:197–202. 72. Wolbrette D, Patel H. Arrhythmias and women. Curr Opin Cardiol. 1999;14:36. 73. Ha AC, Breithardt G, Camm AJ, et al. Health-related quality of life in patients with atrial fibrillation treated with rhythm control versus rate control insights from a prospective international registry (registry on cardiac rhythm disorders assessing the control of atrial fibrillation: record-af). Circ: Cardiovas Qual Outcomes. 2014;7:896–904. 74. Lip GY, Laroche C, Ioachim PM, et al. Prognosis and treatment of atrial fibrillation patients by European cardiologists: one year follow-up of the eurobservational research programme-atrial fibrillation general registry pilot phase (eorp-af pilot registry). Eur Heart J. 2014;35:3365–3376. 75. Feinberg WM, Blackshear JL, Laupacis A, Kronmal R, Hart RG. Prevalence, age distribution, and gender of patients with atrial fibrillation: analysis and implications. Arch Intern Med. 1995;155:469. 76. Larsen JA, Kadish AH. Effects of gender on cardiac arrhythmias. J Cardiovasc Electrophysiol. 1998;9:655–664. 77. Hnatkova K, Waktare JE, Murgatroyd FD, Guo X, CAMM A, Malik M. Age and gender influences on rate and duration of paroxysmal atrial fibrillation. Pacing Clin Electrophysiol. 1998;21:2455–2458. 78. Suttorp MJ, Kingma JH, Koomen EM, van't Hof A, Tijssen JG, Lie KI. Recurrence of paroxysmal atrial fibrillation or flutter after successful cardioversion in patients with normal left ventricular function. Am J Cardiol. 1993;71:710–713. 79. Potpara TS, Marinkovic JM, Polovina MM, et al. Gender-related differences in presentation, treatment and long-term outcome in patients with first-diagnosed atrial fibrillation and structurally normal heart: the belgrade atrial fibrillation study. Int J Cardiol. 2012;161:39–44. 80. Humphries KH, Kerr CR, Connolly SJ, et  al. New-onset atrial fibrillation sex differences in presentation, treatment, and outcome. Circulation. 2001;103:2365–2370. 81. Cabin HS, Clubb KS, Hall C, Perlmutter RA, Feinstein AR. Risk for systemic embolization of atrial fibrillation without mitral stenosis. Am J Cardiol. 1990;65:1112–1116. 82. Moulton AW, Singer DE, Haas JS. Risk factors for stroke in patients with nonrheumatic atrial fibrillation: a case-control study. Am J Med. 1991;91:156–161. 83. Investigators AF. Risk factors for stroke and efficacy of antithrombotic therapy in atrial fibrillation. Analysis of pooled data from five randomized controlled trials. Arch Intern Med. 1994;154:1449. 84. Friberg J, Scharling H, Gadsbøll N, Truelsen T, Jensen GB. Comparison of the impact of atrial fibrillation on the risk of

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

390

85.

86.

87.

88.

89.

90.

91.

92.

93.

94. 95.

26.  The Sexually Dimorphic Characteristics of the Pathophysiology and Treatment of Atrial Fibrillation

stroke and cardiovascular death in women versus men (the copenhagen city heart study). Am J Cardiol. 2004;94:889–894. Cove CL, Albert CM, Andreotti F, Badimon L, Van Gelder IC, Hylek EM. Female sex as an independent risk factor for stroke in atrial fibrillation: possible mechanisms. Thromb Haemost. 2014;111:385–391. Guo Y, Wang H, Zhao X, et al. Relation of renal dysfunction to the increased risk of stroke and death in female patients with atrial fibrillation. Int J Cardiol. 2013;168:1502–1508. Cheung R. Patients with nonvalvular atrial fibrillation at low risk of stroke during treatment with aspirin. Stroke prevention in atrial fibrillation iii study. JAMA. 1998. Friberg L, Rosenqvist M, Lip GY. Evaluation of risk stratification schemes for ischaemic stroke and bleeding in 182 678 patients with atrial fibrillation: the swedish atrial fibrillation cohort study. Eur Heart J. 2012;33:1500–1510. Dagres N, Nieuwlaat R, Vardas PE, et al. Gender-related differences in presentation, treatment, and outcome of patients with atrial fibrillation in Europe: a report from the euro heart survey on atrial fibrillation. J Am Coll Cardiol. 2007;49:572–577. Lip GY, Laroche C, Boriani G, et  al. Sex-related differences in presentation, treatment, and outcome of patients with atrial fibrillation in europe: a report from the euro observational research programme pilot survey on atrial fibrillation. Europace. 2015;17:24–31. Tsadok MA, Gagnon J, Joza J, et  al. Temporal trends and sex differences in pulmonary vein isolation for patients with atrial fibrillation. Heart Rhythm. 2015;12:1979–1986. von Eisenhart Rothe A, Hutt F, Baumert J, et al. Depressed mood amplifies heart-related symptoms in persistent and paroxysmal atrial fibrillation patients: a longitudinal analysis—data from the German competence network on atrial fibrillation. Europace. 2015;17(9):1357–1362. Mosca L, Benjamin EJ, Berra K, et al. Effectiveness-based guidelines for the prevention of cardiovascular disease in women— 2011 update: a guideline from the American Heart Association. J Am Coll Cardiol. 2011;57:1404–1423. Adamson DL, Nelson-Piercy C. Managing palpitations and arrhythmias during pregnancy. Heart. 2007;93:1630–1636. Barnes EJ, Eben F, Patterson D. Direct current cardioversion during pregnancy should be performed with facilities available

96.

97.

98.

99.

100.

101.

102.

103.

104. 105.

106.

107. 108.

for fetal monitoring and emergency caesarean section. BJOG. 2002;109:1406–1407. Kockova R, Kocka V, Kiernan T, Fahy GJ. Ibutilide-induced cardioversion of atrial fibrillation during pregnancy. J Cardiovasc Electrophysiol. 2007;18:545–547. Natale A, Davidson T, Geiger MJ, Newby K. Implantable cardioverter-defibrillators and pregnancy a safe combination? Circulation. 1997;96:2808–2812. Cha Y-M, Lloyd MA, Birgersdotter-Green UM. Arrhythmias in Women : Diagnosis and Management. Oxford University Press, New York. Patel D, Mohanty P, Di Biase L, et al. Outcomes and complications of catheter ablation for atrial fibrillation in females. Heart Rhythm. 2010;7:167–172. Heidbuchel H, Verhamme P, Alings M, et al. Advisors. Updated European Heart Rhythm Association practical guide on the use of non-vitamin-k antagonist anticoagulants in patients with nonvalvular atrial fibrillation: executive summary. Eur Heart J. 2016 Chinitz JS, Vaishnava P, Narayan RL, Fuster V. Atrial fibrillation through the years: contemporary evaluation and management. Circulation. 2013;127:408–416. Shah SV, Kruse J, Andrei AC, et  al. Gender differences in outcomes after surgical ablation of atrial fibrillation. J Thorac Cardiovasc Surg. 2016;151(391–398):e392. Roten L, Rimoldi SF, Schwick N, et  al. Gender differences in patients referred for atrial fibrillation management to a tertiary center. Pacing Clin Electrophysiol. 2009;32:622–626. Sra J. Atrial fibrillation ablation complications. J interv card electrophysiol. 2008;22:167–172. Wan E, Abrams J, Weinberg RL, et  al. Aberrant sodium influx causes cardiomyopathy and atrial fibrillation in mice. J Clin Invest. 2016;126:112–122. Ragsdale DS, McPhee JC, Scheuer T, Catterall WA. Molecular determinants of state-dependent block of na+ channels by local anesthetics. Science. 1994;265:1724–1728. Remme CA, Bezzina CR. Sodium channel (dys)function and cardiac arrhythmias. Cardiovasc Ther. 2010;28:287–294. Riley G, Syeda F, Kirchhof P, Fabritz L. An introduction to murine models of atrial fibrillation. Frontiers in physiology. 2012;3:296.

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C H A P T E R

27 Gender Differences in Chronic Obstructive Pulmonary Disease— Current Knowledge and Deficits Santosh K. Nepal1 and Shambhu Aryal2 1

Carilion Roanoke Memorial Hospital, Roanoke, VA, United States, 2Duke University, Durham, NC, United States

O U T L I N E Abbreviations 391

27.7 Gender Differences in Treatment 27.7.1 Smoking Cessation 27.7.2 Pharmacotherapy 27.7.3 Oxygen Therapy 27.7.4 Pulmonary Rehabilitation 27.7.5 Lung Transplantation

395 395 395 396 396 396

27.1 Introduction

392

27.2 Gender Differences in the Prevalence of COPD

392

27.3 Gender Differences in the Etiological Risk Factors of COPD 27.3.1 Smoking 27.3.2 Other Risk Factors

392 392 393

27.8 Gender Differences in Morbidity and Mortality 396

27.4 Gender Differences in Genetic Susceptibility 393

27.9 Gender Differences in Comorbidities of COPD

397

27.5 Gender Differences Clinical Presentation

394

27.10 Conclusion

398

27.6 Gender Differences in Diagnosis and Health Care Delivery

395

References 398

Abbreviations AATD Alpha Anti-Trypsin Deficiency ACOS Asthma COPD Overlap Syndrome ATS American Thoracic Society BODE Body mass index, Airflow obstruction, Dyspnea, Exercise CCI Charlson Comorbidity Index CI Confidence Interval COPD Chronic obstructive pulmonary disease DLCO Diffusion Capacity of Carbon Monoxide DNA DeoxyriboNucleic Acid ELCIPSE Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points FEV1 Forced Expiratory Volume in the first second

Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00009-7

GOLD Global Initiative for Chronic Obstructive Lung Disease HRQoL Health Related Quality of Life ISHLT International Society for Heart and Lung Transplantation OHT Orthotopic Heart Transplantation OR Odds ratio MMRC Modified Medical Research Council NHIS National Health Information Survey PCO2 Partial Pressure of Carbon Dioxide PFT Pulmonary Function Test PO2 Partial Pressure of Oxygen PR Pulmonary Rehabilitation TORCH TOwards a Revolution in COPD Health UPLIFT Understanding Potential Long-term Impacts on Function with Tiotropium

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© 2017 Elsevier Inc. All rights reserved.

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27.1 INTRODUCTION Chronic obstructive pulmonary disease (COPD) is a very common disease; it is currently the third leading cause of death worldwide.1 COPD was historically considered a disease of men but lost this attribute in the year 2000 when for the first time, female deaths outnumbered male deaths.2 Surveillance data on COPD from the first decade of the 21st century show a decreasing prevalence of COPD and a decrease in mortality rate in men as opposed to an increase in prevalence of the disease with unchanged mortality in women.3 The increasing incidence of COPD in females and the morbidity and mortality from it has been attributed to the increasing trend of smoking in females in the last 50 years. However, the majority of recent work suggests that secular trends in tobacco consumption are only a part of the puzzle in the differences noted in the two genders and there are other environmental as well as biologic and hormonal factors of significant importance contributing to the differences. The increase in interest on the gender differences of COPD has led to new understanding on the differential susceptibility of the two sexes to various risk factors including smoking, differences in the pattern of presentation, disease outcomes, and the impact of therapeutic interventions. This chapter will summarize what we know and do not know about those gender differences.

27.2  GENDER DIFFERENCES IN THE PREVALENCE OF COPD Despite COPD being an extremely prevalent disease, the measurement and monitoring of the true burden of illness remains difficult.4 Prevalence estimates of COPD have relied on self-report, administrative databases, or lung function testing in large, population-based samples. Each method may contribute to under or overestimates of COPD in men and women. Women tend to have a higher prevalence of COPD compared with men by self-report, whereas the opposite pattern is seen when using billing data from administrative health service databases.5 Surveillance data on COPD from 1999 to 2011 showed a decreasing prevalence of COPD in the males from 4.6% to 4.3% whereas there was an increase in the females to 7% from 6.7% as shown in Fig. 27.1.3 Hospitalization rates for COPD have been approximately equal for men and women since 1995.6 Data on COPD prevalence from other developed nations shows a similar trend to the United States, while that from developing nations still show the trend seen in the United States a few decades ago with higher prevalence in the males.4

FIGURE 27.1  Age-adjusted prevalence (%) of self-reported physician-diagnosed COPD among adults aged 25 years, by sex and year— United States, National Health Interview Survey, 1999–2011.

27.3  GENDER DIFFERENCES IN THE ETIOLOGICAL RISK FACTORS OF COPD 27.3.1 Smoking Cigarette smoking is the best-recognized risk factor for COPD with 80% of deaths related to COPD attributed to smoking,7 although only 25% of smokers get COPD.8 Worldwide, there are five times more male smokers than females.9 In the United States, the prevalence of smoking among males is 18.8% as opposed to 14.8% in females with an overall decrease in smoking prevalence from 20.9% in 2005 to 16.8% in 2014.10 Smoking in women is no longer a social stigma as it was in the past. The roles of female empowerment and advertising strategies in past decades definitely played a role in increasing smoking in women.11 Unfortunately, women seem to be more vulnerable to cigarette smoke. This has been demonstrated by several studies which show an accelerated decline of Forced Expiratory Volume in (FEV1) in female smokers compared to male smokers12–17; there is only one published study which reports a different result.18 Cigarette smoking may be more damaging to teenage girls than boys as shown by a study that found out that the decline in growth rate with smoking was more pronounced in girls than boys.19 The increased susceptibility of females to tobacco smoking is attributed to multiple factors including relatively short and narrow airways in females, differences in the metabolism of toxins present in cigarette smoke, and possibly differences in immunological reaction to the toxins.20 Estrogen appears to induce the cytochrome-450 enzyme system thus activating the metabolism of chemicals present in the cigarette smoke including nicotine and cotinine to more metabolically active forms which are

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27.4  Gender Differences in Genetic Susceptibility

more damaging; some of these products cause lung damage by forming reactive oxygen species (ROS). The ROS may bind to deoxyribonucleic acid (DNA) within local cells, leading to the formation of DNA adducts, which impair cellular replication and repair and thus increasing the risk for not only COPD but also lung cancer.21 Besides nicotine, there are likely non-nicotinic factors responsible for habit formation in women. A recent study by Cosgrove et al.22 suggests that male smokers seem to have a rapid dopaminergic response in the ventral striatum as they smoke, as opposed to females who do not seem to have that surge with smoking. It appears that women tend to smoke for different reasons than men, i.e., they use it to cope with stress and to regulate mood. The authors of the study proposed that the locus of smoking reinforcement for women may be found in the dorsal striatum as opposed to the ventral striatum in men, and that the same loci are critical for habit formation as was shown by studies done by Porrino et al. in 2004 and Yin et  al. in 2006.23,24 Furthermore, in addition to nicotine effect in women, there is significant role of non-nicotinic factors in smoking habituation in female as discussed in the smoking cessation section later in the chapter.

27.3.2  Other Risk Factors A significant proportion of people with COPD are nonsmokers. Data from 14 countries in the Burden of Obstructive Lung Disease (BOLD) study to describe characteristics of COPD in never-smokers showed that although never-smokers were less likely to have COPD and had less severe COPD than ever-smokers, neversmokers nonetheless comprised 23.3% (240/1031) of those classified with GOLD stage II + COPD (defined as COPD with FEV1 of 50–80% predicted). Importantly, twothirds of these subjects with COPD were female.25 This result is similar to prior population-based studies, which showed the incidence of COPD cases in nonsmokers to be a quarter to a third of total COPD cases.26–29 Lamprecht et al. have shown that there is a significant risk of underdiagnosis and delay in treatment in this group.25 In addition to cigarette smoking, other risk factors for COPD include advancing age, environmental tobacco smoke exposure also known as passive smoking, air and industrial pollution, biomass fuel exposure, childhood respiratory illnesses, and possible genetic susceptibility. Women in general have a longer life expectancy than men and thus would be at an increased risk of development of COPD. Also, with the increased involvement of women in the previously male dominated occupations with known occupational exposures, women are increasingly being exposed to the same risk. The risk of development of COPD with those occupational exposures appears to be higher for females similar to the observation noted with smoking. The study by Lamprecht

393

et al. described above did suggest that risk of COPD for women exposed for more than 10 years to organic dust was higher than for men.25 Biomass fuel is increasingly recognized as a significant risk factor for COPD. Close to 50% of the world’s population uses biomass fuel as their primary source of domestic energy for cooking, home heating, and lighting, ranging from very little in developed countries to more than 80% in China, India, and sub-Saharan Africa. The overall risk of COPD in women exposed to indoor air pollution from domestic solid fuel use, especially wood, was consistently higher (OR, 3.2; 95% CI, 2.3–4.8) than in men (OR, 1.8; 95% CI, 1.0–3.2), who were likely less exposed.30 This is going to be increasingly important with increasing immigration to the United States from many regions of the world where the use of biomass fuels is significant.

27.4  GENDER DIFFERENCES IN GENETIC SUSCEPTIBILITY Severe alpha-1-antitrypsin deficiency is the only proven genetic risk factor for COPD. There is no sex predilection for this disease although the time from symptom onset to diagnosis has been found to be longer in females.31 Genetic predisposition to COPD was suggested by a study done by Silverman et  al. which showed a high relative risk of COPD amongst the first degree relatives of the probands with early onset COPD.32 The COPD Gene Study by Foreman et  al. on 2500 smokers from African and European ancestry with severe COPD (defined as COPD with FEV1 less than 50% predicted) also suggested increased genetic predisposition of females to COPD by showing a higher proportion of females (66%) in early onset (defined as less than 55 years old) severe COPD compared to late onset disease where the proportion of females was only 43%. As noted in the study, the early onset group did have lesser exposure to cigarette exposure compared to the late onset (42 pack years vs 61 pack year) group. However, a definite genetic locus is yet to be identified.33 Furthermore, the role of estrogen on telomere length and its effect on disease process including lung disease may be of significance. One hypothesis suggests that the action of estrogen on estrogen-response-element present in the telomerase reverse transcriptase stimulates telomerase activity, and the addition of telomere repeats to the ends of chromosomes. With age and decreasing hormone activity in females, telomeres progressively shorten and thus diminish the advantage of protection from age-related biological processes such as COPD.34 We also suggest that the two sexes may differ in lung microbiomes related to anatomic, physiologic, and hormonal differences, which may explain some of the

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27.  GENDER DIFFERENCES IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE—CURRENT KNOWLEDGE AND DEFICITS

differential susceptibility of sexes to COPD; this is a potential area for future research.

27.5  GENDER DIFFERENCES CLINICAL PRESENTATION Cough, shortness of breath, and sputum production are the three cardinal symptoms of COPD, while emphysema and chronic bronchitis are the two broad phenotypes of COPD. Burrows et al. suggested the gender differences in the phenotypic expression of COPD as early as 1987; according to their study, chronic asthmatic bronchitis was more common in women, whereas emphysema was more common in men.35,36 National Health Information Survey (NHIS) data showed similar findings. This trend, however, seems to be changing as after 2011 more women than men are diagnosed with COPD.36 In the past, COPD with chronic asthmatic bronchitis with bronchial responsiveness was considered to portend a better prognosis than COPD without

bronchial hyperresponsiveness35 but recent data suggest the contrary with bronchial hyperresponsiveness being a predictor of mortality.37 In this more recent study, 87% of the female smokers with mild to moderate COPD demonstrated bronchial hyperresponsiveness versus 63% males, thus suggesting women in general might be at a survival disadvantage. Table 27.138 summarizes the differences seen in the clinical presentation between the two sexes. Women seem to be more symptomatic with comparable FEV1 and also have more dyspnea than cough or sputum production. Men on the other hand, are more likely to report cough although men and women are equally likely to report sputum. Overall, females have worse symptom related quality of life compared to men. In one study, women reported more exacerbations and worse dyspnea as well as overall worse health status scores compared with men for the same level of obstruction.39 Some of these differences in clinical presentation may be due to differences in physiological reserve attributed to sex-related differences in airway anatomy although

TABLE 27.1  Gender Differences in Chronic Obstructive Pulmonary Disease Symptoms Symptoms

Study and year

Study design

Summary

Dyspnea

de Torres and colleagues (2005)40

Cross-sectional; 53 men and women with COPD recruited from pulmonary clinic and matched on lung function

Women with COPD report more dyspnea on the ATS-MMRC scale than men (p = .0003)

Watson and colleagues (2004)41

Cross-sectional; randomly sampled population-based telephone survey; self-report of COPD diagnosis

Women with self-reported COPD report severe dyspnea on the MRC scale more frequently than men (p < .05)

Di Marco and colleagues (2006)42

Case-control; patients with COPD attending a pulmonary clinic compared with nonCOPD patients on prevalence of symptoms

Women with COPD report more severe dyspnea on the MRC scale for a given lung function compared with men (p = .003)

Chronic cough

Watson and colleagues (2006)43

Longitudinal study; placebo arm of randomized clinical trial of budesonide

Higher prevalence of “being woken by an attack of coughing” (p < .001) and “chronic cough during winter” (p = .03) in women compared with men

Sputum production

Cydulka and colleagues (2005)44

Secondary analysis of prospective cohort of patients presenting to emergency with an acute exacerbation of COPD

Men more likely to report productive cough on most days compared with women (p = .02)

Watson and colleagues (2004)41

Cross-sectional; randomly sampled population-based telephone survey; self-report of COPD diagnosis

Men and women equally likely to report sputum

Depression

Di Marco and colleagues (2006)42

Case-control; patients with COPD attending a pulmonary clinic compared with nonCOPD patients on prevalence of symptoms

High prevalence of depression compared with controls; women had higher levels of depression and worse symptom-related quality of life compared with men

Anxiety and fatigue

Di Marco and colleagues (2006)42

Case-control; patients with COPD attending a pulmonary clinic compared with nonCOPD patients on prevalence of symptoms

High prevalence of anxiety compared with controls; women had higher levels of anxiety compared with men

Gift and Shepard (1999)45

Cross-sectional study of patients attending a pulmonary clinic

Women and men were similar in their psychological symptoms except for anxiety, which was higher in women. Women reported greater fatigue

Reprinted with permission of the American Thoracic Society. Copyright © 2016 American Thoracic Society. ATS-MMRC, American Thoracic Society–modified Medical Research Council; COPD, chronic obstructive pulmonary disease; MRC, Medical Research Council.

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27.7  Gender Differences in Treatment

other factors like psychosocial factors often play important roles. In a study done by O’Donell et al., dyspnea in females did not correlate with partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), or diffusion capacity of carbon monoxide (DLCO) as much as it did with these factors in males.46 Another study on 56 females and 56 males by Torres et al. showed that Pi (mouth occlusion pressure)/Pmax (maximum inspiratory pressure) ratio was the only independent respiratory factor associated with the Modified Medical Research Council (MMRC) score in females when other variables including FEV1, DLCO, nutritional status, and oxygenation were matched indicating a heightened central respiratory drive.47 Besides this, the possibility of less reactive pulmonary vasculature due to decreasing estrogen with advancing age may explain the increase in dyspnea at relatively lower level of physiological stress.48

27.6  GENDER DIFFERENCES IN DIAGNOSIS AND HEALTH CARE DELIVERY Although the year 2000 marked the year when number of females dying from COPD exceeded that of men, and there is a clear epidemiological trend of increasing burden of COPD in females, there still appears to be a bias among health care providers leading to underdiagnosis of COPD in females. A study by Chapman et  al. in the United States and Canada showed that when an identical scenario was presented to a treating physician, it was more likely for the physician to suspect COPD and order pulmonary function tests (PFT) in males compared to females.49 Unfortunately, this bias appears to persist even a decade later as shown by a study of more than 3500 patients by Anocochea et  al. in which there was significant underdiagnosis of women with COPD compared to men.50 Furthermore, there appears to be differences in the sexes in care of COPD. A 2012 study showed that diagnostic delay was reported more frequently by women who also experienced more difficulty reaching their physicians and not being able to get enough time with their physicians.51 Women were also more likely to choose alternative nonevidence based therapy in the same study. Those results on the bias in diagnosis of COPD as well as perception of COPD care suggest that there are probably many factors behind the differences in the outcomes of COPD in the two genders including maintaining disease control, avoiding hospitalizations and exacerbations, and preventing deaths from COPD. With the dawn of Precision Medicine, understanding genderspecific issues in diagnosis and delivery of care of COPD would help us improve outcomes both at individual and population levels.

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27.7  GENDER DIFFERENCES IN TREATMENT Although there has been an increased recognition of gender-related factors in the natural history of COPD over the last couple of decades, there are not many studies looking at the impact of various treatment modalities in the two sexes. Major landmark studies like Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE),52 TOwards a Revolution in COPD Health (TORCH),53 and Understanding Potential Longterm Impacts on Function with Tiotropium (UPLIFT)54 were underrepresented by females (TORCH—male 75%, female 25%; ECLIPSE—male 65% and female 35%; and UPLIFT—male 75%, female 25%). At this time, until there are large prospective studies focused on the differential outcomes with different treatment modalities with regards to sexes, formal conclusions and recommendations cannot be made. However, in the section below, we will discuss what has been observed in the recent years.

27.7.1  Smoking Cessation Stopping cigarette smoking is the most important intervention in preventing progression of COPD. As noted by Cosgrove et al.,22 the reinforcement associated with smoking is different between females and males; whereas nicotine is important for habit formation in both sexes, other non-nicotinic factors including taste and smell of cigarette smoke are equally important in females.55 West et  al. showed that even the hand to mouth movement in the female smoker was a significant non-nicotinic factor and inhaler nicotine was better than patch and gum.56 What this means in terms of smoking cessation strategies is that nicotine replacement therapy does not work as well in females as in males, whereas non-nicotine therapy including varenicline and bupropion work equally well in both sexes.55 Females are less successful in quitting smoking despite better benefit from quitting smoking.57 This is attributed to multiple factors including less symptomatic benefit upon quitting, more anxiety, fear of weight gain, and need of more social support. This emphasizes the importance of behavioral and group therapy, weight reduction strategies, and addressing coexisting psychiatric illness before attempting cessation in women.

27.7.2 Pharmacotherapy Most pharmacotherapy studies for COPD have not been designed to assess treatment in men versus women and generally most trials have enrolled more men than women. Little is known about how differences in lung anatomy and physiology may affect dosage, delivery, and effectiveness of inhaled medications. While there

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27.  GENDER DIFFERENCES IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE—CURRENT KNOWLEDGE AND DEFICITS

is ongoing debate on whether to continue inhaled corticosteroids or not on stable patients in general, a study done by Schermer et al. showed a higher probability of respiratory deterioration (either symptomatic or exacerbations) when stopping inhaled steroids in females.58 There appears to be no or little data otherwise on how men and women respond to conventional medications for COPD including beta agonists, anticholinergics, inhaled and systemic steroids, or antibiotics.

27.7.3  Oxygen Therapy Oxygen is known to improve survival in patients with COPD with hypoxemia as evidenced by PaO2 of 55 mm Hg or less than 60 mm Hg in the presence of cor-pulmonale, polycythemia, or pulmonary hypertension when used more than 15 h a day.59,60 Data for the differences in benefits of oxygen therapy between the two sexes is mixed at best. A study done by Crockett et al. involving 505 patients (249 males and 256 females) showed a survival advantage for females using at least 19 h of supplemental oxygen per day.61 However, another prospective study by Machado et al. including 435 patients (184 women and 251 men) showed that women receiving oxygen therapy were more likely to die than men though the reasons were unclear.62 These conflicting results could be related to the differences in the group of populations included in the study, differences in selection and exclusion criteria, differences in the selected indications between sexes, and unknown compliances.

27.7.4  Pulmonary Rehabilitation Pulmonary rehabilitation (PR) is an integral part of COPD treatment and is shown to improve quality of life by decreasing respiratory symptoms and complications including rates of exacerbations and hospitalizations. A good PR program should not only include exercise but also emotional support and education, including smoking cessation and nutritional counseling. Despite the known benefit, a significant proportion of patients do not complete PR. In a recent study done by Brown et al. including 440 subjects, 41% of which were female, there was no difference in the sexes on the enrollment and adherence to PR program. The overall dropout rate was, however, 48% and smoking was a sole independent predictor for unsuccessful PR completion.63 As discussed earlier, smoking cessation may be relatively more difficult in females and thus this may ultimately decrease the completion rate of PR women. Gender difference with PR was shown to be significant on the long term only by a study by Foy et  al.; in this study, although both men and women reported improvement at 3 months, women lost this benefit over

time and at 18 months, only men reported sustained improvement.64

27.7.5  Lung Transplantation COPD (both non-AATD and AATD) is the most common indication for lung transplantation worldwide.65 The International Society for Heart and Lung Transplantation (ISHLT) guidelines of 2014 on lung transplantation for COPD includes progressive disease despite maximal treatment including medications, PR, and oxygen therapy; BODE index of 5–6; PCO2 >50 mm Hg or 6.6 kPa and/or PO2 35 days postinfection) ovariectomized mice with 17β-estradiol induced virus reactivation, as measured by increased viral loads, and that the reactivation was estrogen receptor dependent.130 Thus, gender susceptibility differences may also be due to the type of infection model and/or the genetics of the mouse strain used as another mouse study done in C57Bl/6 mice showed that females had higher levels of viral loads during acute infection (3–7 days postinfection) with HSV-1, but that males had higher levels of viral load during chronic infection (37 days postinfection).131 In contrast to HCV, influenza, and HSV-1, where males tend to have worse outcomes, for CMV infections females tend to have a higher prevalence of infection than males.13 Female bone marrow donors in Germany of child-bearing age,1 female solid organ donors in Hungary,132 and female kidney transplant patients in Hungary133 showed increased seropositivity to CMV compared to age-matched males. Additionally, the risk of abnormal brain development in congenitally

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29.6 Viruses

CMV-infected fetuses was twice as high in females than in males.3 Thus, female gender is considered to be a risk factor for CMV infection. Similarly, there seems to be a female predominance in human T lymphotropic virus-I (HTLV-I) infections134–136 and HTLV-II infections.137 Though, lower viral loads were found in females that were HTLV-II+ compared to males.138 HTLV-I is associated with adult T-cell lymphoma (ATL) and HTLV-1-associated myelopathy (HAM). The HTLV-II serotype is also associated with HAM. Interestingly, in Japanese patients with ATL, there seem to be more infections in men139,140 and increased mortality in men.140 Since it was reported that the HTLV-1 Tax protein could bind to the TNF-α promoter141 and increased TNF-α production could explain some of the characteristics of ATL, the authors determined if Tax binding to TNF-α was influenced by estrogen. They found that estradiol repressed Tax activation of TNF-α through either the estrogen receptor α or β.142 Thus, at least for ATL, estrogen seems to be protective for women. Additionally, host genetics may play a role in susceptibility as only the Japanese population seems to show a male susceptibility, possibly due to abnormal lymphocyte subsets in that population.143 Studies examining gender susceptibility differences in morbidity and mortality in infection with human immunodeficiency virus (HIV) have shown inconsistent results, possibly due to variable effects of sex hormones on replication or transmission of different HIV subtypes.144 In general, females show increased susceptibility to HIV infection,145,146 and increased progression to AIDS,147 which is likely exacerbated by the increased frequency of heterosexual transmission in the female reproductive tract.148 According to the review by Hladik and McElrath, the authors posit that the increased risk of infection is due to the larger surface area of the vaginal wall, which provides increased potential access, especially if the epithelial cell layer is breached during intercourse. This would align with the theory that infected semen persist for a long time in the vagina, another contributing factor to women’s increased susceptibility to HIV infection. Women may be more susceptible to HIV infection due to the presence of an activated subset of CD4+ T cells in the cervix that express multiple HIV susceptibility markers (α4β7, CCR5, IL-17A, and IFN-γ).149 As these T cells were completely depleted in HIV+ female sex workers, it suggests that these immune cells are key targets for HIV infection.149 A new animal model in rhesus macaques also showed that females progressed faster to AIDS and mounted an earlier and more robust proinflammatory response than males after infection with simian HIV-1.150 In addition, the authors found significant differences in the macaques gut microbiome, suggesting the gut microbiome may alter responses to HIV-1 infection.150 Other studies also found that the gut microbiome is critical for

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the development and shaping of the reproductive tract innate and adaptive immune responses,151,152 which may influence response to HIV infection.153 In contrast to the above studies, a recent study using meta-analyses of 115 studies found that males have an increased risk of progression to AIDS and mortality compared to females154,155 and that over a 10-year period, 80% of patients with acute HIV infection were male.156 Though some of that may be due to treatment/ health care compliance issues as 70% of patients that missed scheduled appointments with infectious disease practitioners were male.157 A possible explanation for the reported differences in susceptibility of males or females to HIV infection is that women have more favorable clinical and immunological patterns than men in early infection, due to an initial protective role of estrogen and/or progesterone, but once the infection is established these patterns are reversed with women faring worse than men overall.158 Similar to the inconsistent results of gender susceptibility to HIV infection, there are also inconsistent gender differences in response to antiretroviral therapy (ART). In general, women tend to fare better than men. One study found that virologic failure, lower immune reconstitution, and faster progression to death were increased in African men, suggesting that African men were more vulnerable to ART failure than women159 and that women had a more favorable outcome. In agreement with that study, a recent study analyzed protein differences in PBMCs in response to long-term ART (>5 years) in HIV+ versus HIV– patients. The authors found specific proteins that were gender-specific in HIV+ patients compared to HIV– patients and that overall women had a more favorable immunological pattern and recovery during ART than men.160 Similarly, another study found that males had a 20% increased risk of death compared to females after treatment and gained fewer CD4 T cells after treatment than women, suggesting women had a better immune response after treatment than men.161 A analogous finding was seen in women in sub-Saharan Africa that achieved better long-term immune response to ART, reaching CD4+ T cell levels associated with lower risks of AIDS-related morbidity and mortality quicker than men.162 However, the increased immune response in women with ART may explain why women also showed increased adverse reactions to ART.163 There are a number of studies demonstrating genderspecific differences in immune responses or adverse reactions after vaccination. This topic was reviewed by Cook in 2008164 and 2009165 and again by Klein in 2012166 and 2015.167 For the measles, mumps, rubella (MMR) vaccine, males tended to have better responses to the measles and rubella portion of the vaccine, but not the mumps portion. Measles is caused by the measles virus and is most common in children. Males were more likely to be

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infected than females in China over a 10-year period,168 but overall world mortality is increased in females.169–171 Females also tend to have higher antibody titers,172 more severe reactions to MMR vaccines, and increased mortality,170,173,174 possibly due to a decrease in their antibodydependent cellular cytotoxicity response.171,175 Females also showed higher antibody titers against vaccination with mumps,176 though there was a higher prevalence in unvaccinated males in Turkey.177 Interestingly, there was an association with both the DRB1 HLA haplotypes and single nucleotide polymorphisms in the IL-12 cytokine receptor with mumps-specific lymphoproliferation, though the authors did not test if these haplotypes or SNPs were associated with one gender more than the other.176 For rubella virus infections, more males than females were infected in a recent outbreak in Japan178 and India.179 For rubella virus vaccination, males had earlier onset and higher levels of IgG, IgM and IgA antibodies compared to females180,181 and higher levels of lymphoproliferation than females,180 indicating more cell-mediated immunity at 2 and 4 weeks after vaccination. However, females had higher antibody titers 3 years after vaccination. This enhanced immunostimulation may explain the increased arthritis or arthralgia in females after rubella infection.182 In summary, gender differences to infections with viruses seem to be more complex than infections with bacteria, fungi, or parasites and whether males or females fare better depends on both the interaction and the genetic background of the host and pathogen. And while the actions of the sex hormones on the host immune response have been tested in multiple systems, given the geneticdependent differences seen in outcomes, it is crucial that studies be done looking at the effects of sex hormones on the different genetic backgrounds of the pathogens themselves. Aside from the few studies done on the effects of sex hormones on HCV and HIV, nothing is known. Those types of studies will better delineate the specific molecular mechanisms that are driving the observed gender susceptibility differences in viral infections.

29.7 PRIONS Prion diseases or transmissible spongiform encephalopathies are a group of fatal, progressive, and untreatable neurodegenerative diseases that can occur in human and animal hosts and involve the conversion of the normal host PrPC to the abnormal form, PrpSc. Prion diseases can, and most often do occur spontaneously. They can also be inherited or acquired via infection, typically by ingesting meat products containing the already transformed PrpSc.183 Despite method of occurrence or host species, prion diseases all have long, clinically

asymptomatic incubation periods. Gender differences exist in the incubation times of transmissible spongiform encephalopathies.184 Clinical studies of Creutzfeldt-Jacob disease (CJD) indicate that females show an earlier onset (1–2 years) but longer survival rates (4–5 months).183 Comprehensive mouse studies looking at 12 inbred lines of mice inoculated with different types of prions show that sex can have a significant difference on incubation time of the disease.184 However, while profound, these differences are highly specific depending on the mouse and prion strain used. The reason for the sex difference as it relates to the prion disease incubation period is yet unknown. Males and females show no difference in endogenous levels of PrpSc. Castration of males in mouse studies negate the sexual dimorphism in some groups but not others which suggests that androgens alone are an insufficient explanation.184 In addition to incubation period, there appear to be sex differences during the active portion of prion diseases.185 Both the innate and memory response in scrapie-infected mice was significantly more depressed in females than in males. Researchers reported decreased lymphocyte response 3 months postscrapie infection in females when exposed to LPS, while male mice immune responses appeared normal. Further, proinflammatory cytokines were decreased in females postinfection (IL-6, IFN-γ). This may relate to hormones affecting cellular proliferation. In conclusion, prion diseases are complex and while gender differences exist in both human and mouse models, the trend is unclear. In humans, females tend to show an earlier onset but typically have longer survival times.183 Incubation periods vary significantly with gender in mice but depend on the specific prion protein involved and likely the genetic background of the host.184 Additionally, because prion diseases are 100% lethal, it makes long-term studies impossible. Specific molecular mechanisms relating to gender are currently unknown.

29.8 SUMMARY In 2011 when we wrote the last review,13 our main conclusion was that males or females fared better depending on the interaction with the specific microbe. In the last 5 years it has become apparent that sexually dimorphic susceptibility to infection is becoming better recognized and much more research has been done to try to explain those differences. In general, females have an advantage in bacterial, fungal, and parasitic infections, due to protective effects of estrogen while males generally fare worse, likely due to suppressive effects of testosterone (Table 29.1). Viral infections are more mixed, likely due to specific genetic interactions between the host and pathogen (Table 29.1).

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29.8 SUMMARY

TABLE 29.1  Sex Differences in Bacterial, Fungal, Parasitic, and Viral Infections

Host species

Experiences more severe outcome

Direct evidence of sex hormone involvement

Known molecular pathway

Reference

Periodontal disease caused by Porphyromonas gingivalis

Humans

Males

Yes

No

18–20,45

Bacterial sepsis

Humans, mice

Males

Yes

No

16,21,186

Pneumonia caused by Streptococcus pneumoniae

Humans, mice

Males

Yes

Yes

6,27,113

Infections due to air pollution

Humans, mice

Females

Yes

No

25,26,187

Mycobacterium tuberculosis

Humans, mice

Males

Yes

No

29–31,155

Clostridium difficile

Humans

Males

No

No

32–34

Cryptococcus neoformans

Humans, mice

Males

Yes

No

37–39

Paracoccidioides brasiliensis

Humans, mice

Males

Yes

No

36,40,41,188

Candida albicans

Humans

Females

No

No

35,43–45

Humans, mice

Males

Yes

No

48,55

Organism species/disease BACTERIA

FUNGI

PARASITES Malaria caused by Plasmodium spp. Cutaneous Leishmaniasis

Males

Yes

No

57–62

Toxoplasmosis

Humans, mice

Females

Yes

No

59,84,86,87

Trichuris muris

Mice

Males

Yes

No

69,70

Schistosoma

Humans, mice

Males

Yes

No

72–75

Entamoeba histolytica

Humans, mice

Males

Yes

No

77,78,80,82

Hepatitis C

Humans, mice

Males

Yes

Yes

4,88,91,95,102,105,124,164,166

Influenza

Humans, mice

Males

Yes

No

111,112,114,115,117,189

Herpes

Humans, mice

Females (acute infections)

Yes

No

119,127,128,131,166

VIRUSES

Males (chronic infections) Cytomegalovirus (CMV)

Humans

Females

No

No

1,3,125,132

Human T lymphotropic virus (HTLV)

Humans

Females

Yes

Yes

134,136,137,140,142

Measles

Humans

Females

No

No

169,171,173

Mumps

Humans

Males

No

No

167,173,177

Rubella

Humans

Males

No

No

164,173,178–180

Human Immunodeficiency VIrus (HIV)

Humans

Yes

No

2,7,147,154,157–159,163

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29.  GENDER DIFFERENCES IN INFECTION

However, it is also clear that in many instances, researchers attribute much of the gender differences to different effects of sex hormones on the immune response of the host and not necessarily to how sex hormones affect the pathogen. Since gender susceptibility to infection is really about the interaction between host and pathogen, with both sides contributing to disease,190 we have to determine how sex hormones affect both the host and the pathogen sides of the equation. Therefore, further research is needed to better delineate the role of sex hormones on the pathogens themselves. In addition, in very few instances is it known the specific molecular mechanisms which might explain the observed gender-specific differences to infection. This information has to be acquired before physicians can start to differentially treat males and females for infections that exhibit differences in gender susceptibility. The sexual dimorphism observed in humans during an infection is as complex as the species itself. Some research has begun to clarify the complexity, but as a whole, specific molecular mechanisms that might explain the observed gender differences from both the host and pathogen sides of the equation are sorely lacking. Thus, further research is needed to clarify those mechanisms.

References 1. Enders G, Daiminger A, Lindemann L, et  al. Cytomegalovirus (CMV) seroprevalence in pregnant women, bone marrow donors and adolescents in Germany, 1996-2010. Med Microbiol Immunol. 2012;201(3):303–309. 2. Muenchhoff M, Goulder PJ. Sex differences in pediatric infectious diseases. J Infect Dis. 2014;209(Suppl 3):S120–S126. 3. Picone O, Costa JM, Dejean A, Ville Y. Is fetal gender a risk factor for severe congenital cytomegalovirus infection? Prenat Diagn. 2005;25(1):34–38. 4. van Lunzen J, Altfeld M. Sex differences in infectious diseasescommon but neglected. J Infect Dis. 2014;209(Suppl 3):S79–S80. 5. Giefing-Kroll C, Berger P, Lepperdinger G, Grubeck-Loebenstein B. How sex and age affect immune responses, susceptibility to infections, and response to vaccination. Aging Cell. 2015;14(3):309–321. 6. Yang Z, Huang YC, Koziel H, et  al. Female resistance to pneumonia identifies lung macrophage nitric oxide synthase-3 as a therapeutic target. eLife. 2014:3. 7. Solianik R, Skurvydas A, Vitkauskiene A, Brazaitis M. Genderspecific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses. Cryobiology. 2014;69(1):26–33. 8. Candore G, Balistreri CR, Listi F, et al. Immunogenetics, gender, and longevity. Ann N Y Acad Sci. 2006;1089:516–537. 9. Dariotis JK, Chen FR, Granger DA. Latent trait testosterone among 18-24 year olds: methodological considerations and risk associations. Psychoneuroendocrinology. 2016;67:1–9. 10. Barton M. Cholesterol and atherosclerosis: modulation by oestrogen. Curr Opin lipidol. 2013;24(3):214–220. 11. Vincent CM, Gwynne DT. Sex-biased immunity is driven by relative differences in reproductive investment. Proc Biol Sci. 2014;281:1790.

12. Moreno-Garcia M, Vargas V, Ramirez-Bello I, HernandezMartinez G, Lanz-Mendoza H. Bacterial Exposure at the Larval Stage Induced Sexual Immune Dimorphism and Priming in Adult Aedes aegypti Mosquitoes. PloS One. 2015;10(7):e0133240. 13. McClelland EE, Smith JM. Gender specific differences in the immune response to infection. Arch Immunol Ther Exp. 2011;59(3): 203–13. 14. Challis J, Newnham J, Petraglia F, Yeganegi M, Bocking A. Fetal sex and preterm birth. Placenta. 2013;34(2):95–99. 15. Kim-Fine S, Regnault TR, Lee JS, et  al. Male gender promotes an increased inflammatory response to lipopolysaccharide in umbilical vein blood. J Matern Fetal Neonatal Med. 2012;25(11):2470–2474. 16. Imahara SD, Jelacic S, Junker CE, O’Keefe GE. The influence of gender on human innate immunity. Surgery. 2005;138(2):275–282. 17. Bouman A, Schipper M, Heineman MJ, Faas MM. Gender difference in the non-specific and specific immune response in humans. Am J Reprod Immunol. 2004;52(1):19–26. 18. Lin TH, Lung CC, Su HP, et al. Association between periodontal disease and osteoporosis by gender: a nationwide populationbased cohort study. Medicine. 2015;94(7):e553. 19. Duan X, Gleason RC, Li F, et al. Sex dimorphism in periodontitis in animal models. J Periodontal Res. 2016;51(2):196–202. 20. Shiau HJ, Reynolds MA. Sex differences in destructive periodontal disease: exploring the biologic basis. J Periodontol. 2010;81(11):1505–1517. 21. Angele MK, Pratschke S, Hubbard WJ, Chaudry IH. Gender differences in sepsis: cardiovascular and immunological aspects. Virulence. 2014;5(1):12–19. 22. Drechsler S, Weixelbaumer K, Raeven P, et  al. Relationship between age/gender-induced survival changes and the magnitude of inflammatory activation and organ dysfunction in posttraumatic sepsis. PloS One. 2012;7(12):e51457. 23. Newsome CT, Flores E, Ayala A, Gregory S, Reichner JS. Improved antimicrobial host defense in mice following poly-(1,6)-beta-Dglucopyranosyl-(1,3)-beta-D-glucopyranose glucan treatment by a gender-dependent immune mechanism. Clin Vaccine Immunol. 2011;18(12):2043–2049. 24. Mikerov AN, Haque R, Gan X, Guo X, Phelps DS, Floros J. Ablation of SP-A has a negative impact on the susceptibility of mice to Klebsiella pneumoniae infection after ozone exposure: sex differences. Respir Res. 2008;9:77. 25. Spencer-Hwang R, Soret S, Ghamsary M, et al. Gender Differences in Respiratory Health of School Children Exposed to Rail YardGenerated Air Pollution: The ENRRICH Study. J Environ Health. 2016;78(6):8–16. 26. Demoulin-Alexikova S, Plevkova J, Mazurova L, et  al. Impact of Air Pollution on Age and Gender Related Increase in Cough Reflex Sensitivity of Healthy Children in Slovakia. Front Physiol. 2016;7:54. 27. Durrani F, Phelps DS, Weisz J, et  al. Gonadal hormones and oxidative stress interaction differentially affects survival of male and female mice after lung Klebsiella pneumoniae infection. Exp Lung Res. 2012;38(4):165–172. 28. Kadioglu A, Cuppone AM, Trappetti C, et  al. Sex-based differences in susceptibility to respiratory and systemic pneumococcal disease in mice. J Infect Dis. 2011;204(12):1971–1979. 29. Nhamoyebonde S, Leslie A. Biological differences between the sexes and susceptibility to tuberculosis. J Infect Dis. 2014;209(Suppl 3):S100–S106. 30. de Faria Gomes NM, da Mota Bastos MC, Marins RM, et  al. Differences between Risk Factors Associated with Tuberculosis Treatment Abandonment and Mortality. Pulm Med. 2015;2015:546106.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

31. Dale K, Tay E, Trevan P, Denholm JT. Mortality among tuberculosis cases in Victoria, 2002-2013: case fatality and factors associated with death. Int J Tuberc Lung Dis. 2016;20(4):515–523. 32. Lessa FC, Mu Y, Winston LG, et  al. Determinants of Clostridium difficile Infection Incidence Across Diverse United States Geographic Locations. Open Forum Infect Dis. 2014;1(2) ofu048. 33. Khanafer N, Toure A, Chambrier C, et al. Predictors of Clostridium difficile infection severity in patients hospitalised in medical intensive care. World J Gastroenterol. 2013;19(44):8034–8041. 34. Zarowitz BJ, Allen C, O’Shea T, Strauss ME. Risk Factors, Clinical Characteristics, and Treatment Differences Between Residents With and Without Nursing Home- and Non-Nursing HomeAcquired Clostridium difficile Infection. J Manag Care Spec Pharm. 2015;21(7):585–595. 35. Kali A, Charles MP, Noyal MJ, Sivaraman U, Kumar S, Easow JM. Prevalence of Candida co-infection in patients with pulmonary tuberculosis. Australas Med J. 2013;6(8):387–391. 36. de Oliveira HC, Assato PA, Marcos CM, et  al. Paracoccidioideshost Interaction: An Overview on Recent Advances in the Paracoccidioidomycosis. Front Microbiol. 2015;6:1319. 37. Casadevall AaP A and Perfect AR. Cryptococcus neoformans. Washington, DC: Asm Press; 1998. 38. Maduro AP, Goncalves L, Inacio J, Faria NC, Martins ML, Teles FR. HIV/AIDS-Associated Cryptococcosis in Portugal Spanning the Pre- to Post-HAART Era: A Retrospective Assessment at the Genotypic Level Based on URA5-RFLP. Curr Microbiol. 2015;71(4):449–457. 39. McClelland EE, Hobbs LM, Rivera J, et al. The role of host gender in the pathogenesis of Cryptococcus neoformans infections. PloS One. 2013;8(5):e63632. 40. Shankar J, Restrepo A, Clemons KV, Stevens DA. Hormones and the resistance of women to paracoccidioidomycosis. Clin Microbiol Rev. 2011;24(2):296–313. 41. Restrepo A, Benard G, de Castro CC, Agudelo CA, Tobon AM. Pulmonary paracoccidioidomycosis. Semin Respir Crit Care Med. 2008;29(2):182–197. 42. Shankar J, Wu TD, Clemons KV, Monteiro JP, Mirels LF, Stevens DA. Influence of 17beta-estradiol on gene expression of Paracoccidioides during mycelia-to-yeast transition. PloS One. 2011;6(12):e28402. 43. Ashman RB, Papadimitriou JM. Production and function of cytokines in natural and acquired immunity to Candida albicans infection. Microbiol Rev. 1995;59(4):646–672. 44. Nebavi F, Ayala FJ, Renaud F, et  al. Clonal population structure and genetic diversity of Candida albicans in AIDS patients from Abidjan (Cote d’Ivoire). Proc Nat Acad Sci USA. 2006;103(10):3663–3668. 45. Javed F, Klingspor L, Sundin U, Altamash M, Klinge B, Engstrom PE. Periodontal conditions, oral Candida albicans and salivary proteins in type 2 diabetic subjects with emphasis on gender. BMC Oral Health. 2009;9:12. 46. Marin DE, Taranu I, Pascale F, et al. Sex-related differences in the immune response of weanling piglets exposed to low doses of fumonisin extract. Br J Nutr. 2006;95(6):1185–1192. 47. Bhandari N, He Q, Sharma RP. Gender-related differences in subacute fumonisin B1 hepatotoxicity in BALB/c mice. Toxicology. 2001;165(2-3):195–204. 48. Cucunuba ZM, Guerra A, Rivera JA, Nicholls RS. Comparison of asymptomatic Plasmodium spp. infection in two malariaendemic Colombian locations. Trans R Soc Trop Med Hyg. 2013;107(2):129–136. 49. Haque U, Sunahara T, Hashizume M, et al. Malaria prevalence, risk factors and spatial distribution in a hilly forest area of Bangladesh. PloS One. 2011;6(4):e18908.

429

50. Pathak S, Rege M, Gogtay NJ, et  al. Age-dependent sex bias in clinical malarial disease in hypoendemic regions. PloS One. 2012;7(4):e35592. 51. Stirnadel HA, Al-Yaman F, Genton B, Alpers MP, Smith TA. Assessment of different sources of variation in the antibody responses to specific malaria antigens in children in Papua New Guinea. Int J Epidemiol. 2000;29(3):579–586. 52. Hu D, Huang Y. [Epidemiological analysis of malaria situation in Shuyang County, Jiangsu Province from 2004 to 2013]. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2015;27(4):419–421. 53. Houben CH, Fleischmann H, Guckel M. Malaria prevalence in north-eastern Nigeria: a cross-sectional study. Asian Pac J Trop Med. 2013;6(11):865–868. 54. Park SH, Lee MJ, Baek JH, Lee WC. Epidemiological aspects of exotic malaria and dengue fever in travelers in Korea. J Clin Med Res. 2011;3(3):139–142. 55. Cernetich A, Garver LS, Jedlicka AE, et al. Involvement of gonadal steroids and gamma interferon in sex differences in response to blood-stage malaria infection. Infect Immun. 2006;74(6):3190–3203. 56. Krucken J, Dkhil MA, Braun JV, et  al. Testosterone suppresses protective responses of the liver to blood-stage malaria. Infect Immun. 2005;73(1):436–443. 57. Murback ND, Hans Filho G, Nascimento RA, Nakazato KR, Dorval ME. American cutaneous leishmaniasis: clinical, epidemiological and laboratory studies conducted at a university teaching hospital in Campo Grande, Mato Grosso do Sul, Brazil. An Bras Dermatol. 2011;86(1):55–63. 58. Marlow MA, da Silva Mattos M, Makowiecky ME, et  al. Divergent profile of emerging cutaneous leishmaniasis in subtropical Brazil: new endemic areas in the southern frontier. PloS One. 2013;8(2):e56177. 59. Bernin H, Lotter H. Sex bias in the outcome of human tropical infectious diseases: influence of steroid hormones. J Infect Dis. 2014;209(Suppl 3):S107–S113. 60. Armijos RX, Weigel MM, Izurieta R, et al. The epidemiology of cutaneous leishmaniasis in subtropical Ecuador. Trop Med Int Health. 1997;2(2):140–152. 61. Travi BL, Osorio Y, Melby PC, Chandrasekar B, Arteaga L, Saravia NG. Gender is a major determinant of the clinical evolution and immune response in hamsters infected with Leishmania spp. Infect Immun. 2002;70(5):2288–2296. 62. Lezama-Davila CM, Isaac-Marquez AP, Barbi J, Oghumu S, Satoskar AR. 17Beta-estradiol increases Leishmania mexicana killing in macrophages from DBA/2 mice by enhancing production of nitric oxide but not pro-inflammatory cytokines. Am J Trop Med Hyg. 2007;76(6):1125–1127. 63. Lezama-Davila CM, Isaac-Marquez AP, Barbi J, Cummings HE, Lu B, Satoskar AR. Role of phosphatidylinositol-3-kinase-gamma (PI3Kgamma)-mediated pathway in 17beta-estradiol-induced killing of L. mexicana in macrophages from C57BL/6 mice. Immunol Cell Biol. 2008;86(6):539–543. 64. Bryson KJ, Millington OR, Mokgethi T, McGachy HA, Brombacher F, Alexander J. BALB/c mice deficient in CD4 T cell IL-4Ralpha expression control Leishmania mexicana Load although female but not male mice develop a healer phenotype. PLoS Negl Trop Dis. 2011;5(1):e930. 65. Lynch DM, Green EA, McFadzean JA, Pugh IM. Trichuris trichiura infestations in the United Kingdom and treatment with difetarsone. Br Med J. 1972;4(5832):73–76. 66. Lowry M, O’Callaghan JM, Furnell MJ, Curtin M. Trichuris tricuria infestation in Limerick. J Ir Med Assoc. 1968;61(369):75–79. 67. Pham-Duc P, Nguyen-Viet H, Hattendorf J, et al. Ascaris lumbricoides and Trichuris trichiura infections associated with wastewater and human excreta use in agriculture in Vietnam. Parasitol Int. 2013;62(2):172–180.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

430

29.  GENDER DIFFERENCES IN INFECTION

68. Gyorkos TW, Maheu-Giroux M, Casapia M, Joseph SA, CreedKanashiro H. Stunting and helminth infection in early preschoolage children in a resource-poor community in the Amazon lowlands of Peru. Trans R Soc Trop Med Hyg. 2011;105(4):204–208. 69. Hepworth MR, Grencis RK. Disruption of Th2 immunity results in a gender-specific expansion of IL-13 producing accessory NK cells during helminth infection. J Immunol. 2009;183(6):3906–3914. 70. Hepworth MR, Hardman MJ, Grencis RK. The role of sex hormones in the development of Th2 immunity in a genderbiased model of Trichuris muris infection. Eur J Immunol. 2010;40(2):406–416. 71. Mbanefo EC, Huy NT, Wadagni AA, Eneanya CI, Nwaorgu O, Hirayama K. Host determinants of reinfection with schistosomes in humans: a systematic review and meta-analysis. PLoS Negl Trop Dis. 2014;8(9):e3164. 72. Nicolls DJ, Weld LH, Schwartz E, et al. Characteristics of schistosomiasis in travelers reported to the GeoSentinel Surveillance Network 1997-2008. Am J Trop Med Hyg. 2008;79(5):729–734. 73. Martins-Melo FR, Pinheiro MC, Ramos Jr. AN, Alencar CH, Bezerra FS, Heukelbach J. Trends in schistosomiasis-related mortality in Brazil, 2000-2011. Int J Parasitol. 2014;44(14):1055–1062. 74. Nakazawa M, Fantappie MR, Freeman Jr. GL, et al. Schistosoma mansoni: susceptibility differences between male and female mice can be mediated by testosterone during early infection. Exp Parasitol. 1997;85(3):233–240. 75. Boissier J, Chlichlia K, Digon Y, Ruppel A, Mone H. Preliminary study on sex-related inflammatory reactions in mice infected with Schistosoma mansoni. Parasitol Res. 2003;91(2):144–150. 76. Fallon PG, Richardson EJ, Jones FM, Dunne DW. Dehydroepiandrosterone sulfate treatment of mice modulates infection with Schistosoma mansoni. Clin Diagn Lab Immunol. 1998;5(2):251–253. 77. Acuna-Soto R, Maguire JH, Wirth DF. Gender distribution in asymptomatic and invasive amebiasis. Am J Gastroenterol. 2000;95(5):1277–1283. 78. Blessmann J, Van Linh P, Nu PA, et al. Epidemiology of amebiasis in a region of high incidence of amebic liver abscess in central Vietnam. Am J Trop Med Hyg. 2002;66(5):578–583. 79. Helk E, Bernin H, Ernst T, et al. TNFalpha-mediated liver destruction by Kupffer cells and Ly6Chi monocytes during Entamoeba histolytica infection. PLoS Pathog. 2013;9(1):e1003096. 80. Lotter H, Jacobs T, Gaworski I, Tannich E. Sexual dimorphism in the control of amebic liver abscess in a mouse model of disease. Infect Immun. 2006;74(1):118–124. 81. Lotter H, Gonzalez-Roldan N, Lindner B, et al. Natural killer T cells activated by a lipopeptidophosphoglycan from Entamoeba histolytica are critically important to control amebic liver abscess. PLoS Pathog. 2009;5(5):e1000434. 82. Lotter H, Helk E, Bernin H, et  al. Testosterone increases susceptibility to amebic liver abscess in mice and mediates inhibition of IFNgamma secretion in natural killer T cells. PloS One. 2013;8(2):e55694. 83. Beverley JK, Fleck DG, Kwantes W, Ludlam GB. Age-sex distribution of various diseases with particular reference to toxoplasmic lymphadenopathy. J Hyg (Lond). 1976;76(2):215–228. 84. Roberts CW, Cruickshank SM, Alexander J. Sex-determined resistance to Toxoplasma gondii is associated with temporal differences in cytokine production. Infect Immun. 1995;63(7):2549–2555. 85. Walker W, Roberts CW, Ferguson DJ, Jebbari H, Alexander J. Innate immunity to Toxoplasma gondii is influenced by gender and is associated with differences in interleukin-12 and gamma interferon production. Infect Immun. 1997;65(3):1119–1121. 86. Xiao J, Viscidi RP, Kannan G, et al. The Toxoplasma MAG1 peptides induce sex-based humoral immune response in mice and distinguish active from chronic human infection. Microbes Infect. 2013;15(1):74–83.

87. Liesenfeld O, Nguyen TA, Pharke C, Suzuki Y. Importance of gender and sex hormones in regulation of susceptibility of the small intestine to peroral infection with Toxoplasma gondii tissue cysts. J Parasitol. 2001;87(6):1491–1493. 88. Bakr I, Rekacewicz C, El Hosseiny M, et al. Higher clearance of hepatitis C virus infection in females compared with males. Gut. 2006;55(8):1183–1187. 89. Inoue G, Horiike N, Michitaka K, Onji M. Hepatitis C virus clearance is prominent in women in an endemic area. J Gastroenterol Hepatol. 2000;15(9):1054–1058. 90. Yamakawa Y, Sata M, Suzuki H, Noguchi S, Tanikawa K. Higher elimination rate of hepatitis C virus among women. J Viral Hepat. 1996;3(6):317–321. 91. Poynard T, Ratziu V, Charlotte F, Goodman Z, McHutchison J, Albrecht J. Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis c. J Hepatol. 2001;34(5):730–739. 92. Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet. 1997;349(9055):825–832. 93. Gordon SC, Lamerato LE, Rupp LB, et al. Prevalence of cirrhosis in hepatitis C patients in the Chronic Hepatitis Cohort Study (CHeCS): a retrospective and prospective observational study. Am J Gastroenterol. 2015;110(8):1169–1177. quiz 78. 94. Hayashida K, Shoji I, Deng L, Jiang DP, Ide YH, Hotta H. 17betaestradiol inhibits the production of infectious particles of hepatitis C virus. Microbiol Immunol. 2010;54(11):684–690. 95. Golden-Mason L, Stone AE, Bambha KM, Cheng L, Rosen HR. Race- and gender-related variation in natural killer p46 expression associated with differential anti-hepatitis C virus immunity. Hepatology. 2012;56(4):1214–1222. 96. Xue XX, Gong JM, Tang SD, et  al. Single nucleotide polymorphisms of toll-like receptor 7 in hepatitis C virus infection patients from a high-risk Chinese population. Inflammation. 2015;38(1):142–151. 97. Cussigh A, Falleti E, Fabris C, et  al. Interleukin 6 promoter polymorphisms influence the outcome of chronic hepatitis C. Immunogenetics. 2011;63(1):33–41. 98. Naugler WE, Sakurai T, Kim S, et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science. 2007;317(5834):121–124. 99. Harrison-Findik DD. Gender-related variations in iron metabolism and liver diseases. World J Hepatol. 2010;2(8):302–310. 100. Courselaud B, Troadec MB, Fruchon S, et al. Strain and gender modulate hepatic hepcidin 1 and 2 mRNA expression in mice. Blood Cells Mol Dis. 2004;32(2):283–289. 101. Fujita N, Sugimoto R, Takeo M, et  al. Hepcidin expression in the liver: relatively low level in patients with chronic hepatitis C. Mol Med. 2007;13(1–2):97–104. 102. Latour C, Kautz L, Besson-Fournier C, et  al. Testosterone perturbs systemic iron balance through activation of epidermal growth factor receptor signaling in the liver and repression of hepcidin. Hepatology. 2014;59(2):683–694. 103. Guo W, Bachman E, Li M, et  al. Testosterone administration inhibits hepcidin transcription and is associated with increased iron incorporation into red blood cells. Aging cell. 2013;12(2):280–291. 104. White DL, Tavakoli-Tabasi S, Kuzniarek J, Pascua R, Ramsey DJ, El-Serag HB. Higher serum testosterone is associated with increased risk of advanced hepatitis C-related liver disease in males. Hepatology. 2012;55(3):759–768. 105. Ahmed NH, El-Abaseri TB, El-Sayed HF, El-Serafi TI. Female Sex Hormones Pattern and Its Relation to Disease Severity and Treatment in Pre- and Postmenopausal Patients with Chronic Hepatitis C Virus (Genotype 4) Infection. Int J Chronic Dis. 2015;2015:927974.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

106. Villa E, Karampatou A, Camma C, et  al. Early menopause is associated with lack of response to antiviral therapy in women with chronic hepatitis C. Gastroenterology. 2011;140(3):818–829. 107. Bernabucci V, Villa E. The role played by gender in viral hepatitis. Scand J Clin Lab Invest Suppl. 2014;244:90–94. 108. Codes L, Asselah T, Cazals-Hatem D, et  al. Liver fibrosis in women with chronic hepatitis C: evidence for the negative role of the menopause and steatosis and the potential benefit of hormone replacement therapy. Gut. 2007;56(3):390–395. 109. Cai L, Gao C, Tang S, et al. Sex-specific association of estrogen receptor 2 polymorphisms with hepatitis C virus infection outcomes in a high-risk Chinese Han population. Infect Genet Evol. 2014;28:118–124. 110. Wu H, Xu L, Chen J, et al. Association of estrogen receptor beta variants and serum levels of estradiol with risk of colorectal cancer: a case control study. BMC Cancer. 2012;12:276. 111. Bonmarin I, Belchior E, Bergounioux J, et al. Intensive care unit surveillance of iinfluenza infection in France: the 2009/10 pandemic and the three subsequent seasons. Euro Surveill. 2015;20(46) 112. Larrauri A, de Mateo S, Spanish Influenza Sentinel Surveillance S. Characterisation of swabbing for virological analysis in the Spanish Influenza Sentinel Surveillance System during four influenza seasons in the period 2002-2006. Euro Surveill. 2007;12(5):E5–E6. 113. Crighton EJ, Elliott SJ, Moineddin R, Kanaroglou P, Upshur RE. An exploratory spatial analysis of pneumonia and influenza hospitalizations in Ontario by age and gender. Epidemiol Infect. 2007;135(2):253–261. 114. Hoffmann J, Otte A, Thiele S, Lotter H, Shu Y, Gabriel G. Sex differences in H7N9 influenza A virus pathogenesis. Vaccine. 2015;33(49):6949–6954. 115. Shah NS, Greenberg JA, McNulty MC, et  al. Severe Influenza in 33 US Hospitals, 2013-2014: Complications and Risk Factors for Death in 507 Patients. Infect Control Hosp Epidemiol. 2015;36(11):1251–1260. 116. Klein SL, Pekosz A. Sex-based biology and the rational design of influenza vaccination strategies. J Infect Dis. 2014;209(Suppl 3):S114–S119. 117. Furman D, Hejblum BP, Simon N, et al. Systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination. Proc Nat Acad Sci USA. 2014;111(2):869–874. 118. Zivkovic I, Bufan B, Petrusic V, et  al. Sexual diergism in antibody response to whole virus trivalent inactivated influenza vaccine in outbred mice. Vaccine. 2015;33(42):5546–5552. 119. Torcia MG, Nencioni L, Clemente AM, et  al. Sex differences in the response to viral infections: TLR8 and TLR9 ligand stimulation induce higher IL10 production in males. PloS One. 2012;7(6):e39853. 120. Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001;19:683–765. 121. Karnam G, Rygiel TP, Raaben M, et  al. CD200 receptor controls sex-specific TLR7 responses to viral infection. PLoS Pathog. 2012;8(5):e1002710. 122. Robinson DP, Lorenzo ME, Jian W, Klein SL. Elevated 17betaestradiol protects females from influenza A virus pathogenesis by suppressing inflammatory responses. PLoS Pathog. 2011;7(7):e1002149. 123. Klein SL, Passaretti C, Anker M, Olukoya P, Pekosz A. The impact of sex, gender and pregnancy on 2009 H1N1 disease. Biol Sex Differ. 2010;1(1):5. 124. Perez CM, Marrero E, Melendez M, et al. Seroepidemiology of viral hepatitis, HIV and herpes simplex type 2 in the household population aged 21-64 years in Puerto Rico. BMC Infect Dis. 2010;10:76.

431

125. Rabenau HF, Lennemann T, Kircher C, et  al. Prevalence- and gender-specific immune response to opportunistic infections in HIV-infected patients in Lesotho. Sex Transm Dis. 2010;37(7):454–459. 126. Solomon L, Cannon MJ, Reyes M, et al. Epidemiology of recurrent genital herpes simplex virus types 1 and 2. Sex Transm Infect. 2003;79(6):456–459. 127. Han X, Lundberg P, Tanamachi B, Openshaw H, Longmate J, Cantin E. Gender influences herpes simplex virus type 1 infection in normal and gamma interferon-mutant mice. J Virol. 2001;75(6):3048–3052. 128. Brown AS, Davis JM, Murphy EA, et al. Gender differences in macrophage antiviral function following exercise stress. Med Sci Sports Exerc. 2006;38(5):859–863. 129. Brown AS, Davis JM, Murphy EA, et al. Susceptibility to HSV-1 infection and exercise stress in female mice: role of estrogen. J Appl Physiol (1985). 2007;103(5):1592–1597. 130. Vicetti Miguel RD, Sheridan BS, Harvey SA, Schreiner RS, Hendricks RL, Cherpes TL. 17-beta estradiol promotion of herpes simplex virus type 1 reactivation is estrogen receptor dependent. J Virol. 2010;84(1):565–572. 131. Burgos JS, Ramirez C, Sastre I, Alfaro JM, Valdivieso F. Herpes simplex virus type 1 infection via the bloodstream with apolipoprotein E dependence in the gonads is influenced by gender. J Virol. 2005;79(3):1605–1612. 132. Varga M, Gorog D, Kari D, et al. Cytomegalovirus seroprevalence among solid organ donors in Hungary: correlations with age, gender, and blood group. Transplant Proc. 2011;43(4):1233–1235. 133. Varga M, Rajczy K, Telkes G, et al. HLA-DQ3 is a probable risk factor for CMV infection in high-risk kidney transplant patients. Nephrol Dial Transplant. 2008;23(8):2673–2678. 134. Birmann BM, Breen EC, Stuver S, et  al. Population differences in immune marker profiles associated with human T-lymphotropic virus type I infection in Japan and Jamaica. Int J Cancer. 2009;124(3):614–621. 135. Lu SC, Kao CL, Chin LT, et al. Seroprevalence and demographic characteristics of HTLV-I among blood donors in Taiwan: 19961999. Int J Hematol. 2001;74(3):333–337. 136. Melbye M, Poulsen AG, Gallo D, et  al. HTLV-1 infection in a population-based cohort of older persons in Guinea-Bissau, West Africa: risk factors and impact on survival. Int J Cancer. 1998;76(3):293–298. 137. Liu H, Leung P, Glynn S, Murphy EL. Human T-lymphotropic virus type II RFLP subtypes a0 and b4/b5 are associated with different demographic and geographic characteristics in the United States. Virology. 2001;279(1):90–96. 138. Murphy EL, Lee TH, Chafets D, et  al. Higher human T lymphotropic virus (HTLV) provirus load is associated with HTLV-I versus HTLV-II, with HTLV-II subtype A versus B, and with male sex and a history of blood transfusion. J Infect Dis. 2004;190(3):504–510. 139. Hisada M, Okayama A, Tachibana N, et  al. Predictors of level of circulating abnormal lymphocytes among human T-lymphotropic virus type I carriers in Japan. Int J Cancer. 1998;77(2):188–192. 140. Hisada M, Okayama A, Spiegelman D, Mueller NE, Stuver SO. Sex-specific mortality from adult T-cell leukemia among carriers of human T-lymphotropic virus type I. Int J Cancer. 2001;91(4):497–499. 141. Albrecht H, Shakhov AN, Jongeneel CV. Ttrans activation of the tumor necrosis factor alpha promoter by the human T-cell leukemia virus type I Tax1 protein. J Virol. 1992;66(10):6191–6193. 142. Tzagarakis-Foster C, Geleziunas R, Lomri A, An J, Leitman DC. Estradiol represses human T-cell leukemia virus type 1 Tax activation of tumor necrosis factor-alpha gene transcription. J Biol Chem. 2002;277(47):44772–44777.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

432

29.  GENDER DIFFERENCES IN INFECTION

143. Prince HE. American blood donors seropositive for human T-lymphotropic virus types I/II exhibit normal lymphocyte subsets. Transfusion. 1990;30(9):787–790. 144. Ragupathy V, Devadas K, Tang S, et al. Effect of sex steroid hormones on replication and transmission of major HIV subtypes. J Steroid Biochem Mol Biol. 2013;138:63–71. 145. Hegdahl HK, Fylkesnes KM, Sandoy IF. Sex Differences in HIV Prevalence Persist over Time: Evidence from 18 Countries in Sub-Saharan Africa. PloS One. 2016;11(2):e0148502. 146. Magadi MA. Understanding the gender disparity in HIV infection across countries in sub-Saharan Africa: evidence from the Demographic and Health Surveys. Sociol Health Illn. 2011;33(4):522–539. 147. Farzadegan H, Hoover DR, Astemborski J, et  al. Sex differences in HIV-1 viral load and progression to AIDS. Lancet. 1998;352(9139):1510–1514. 148. Hladik F, McElrath MJ. Setting the stage: host invasion by HIV. Nat Rev Immunol. 2008;8(6):447–457. 149. McKinnon LR, Nyanga B, Chege D, et al. Characterization of a human cervical CD4+ T cell subset coexpressing multiple markers of HIV susceptibility. J Immunol. 2011;187(11):6032–6042. 150. Ren W, Ma Y, Yang L, et al. Fast disease progression in simian HIV-infected female macaque is accompanied by a robust local inflammatory innate immune and microbial response. AIDS. 2015;29(10):F1–F8. 151. Brotman RM, Ravel J, Bavoil PM, Gravitt PE, Ghanem KG. Microbiome, sex hormones, and immune responses in the reproductive tract: challenges for vaccine development against sexually transmitted infections. Vaccine. 2014;32(14):1543–1552. 152. Mirmonsef P, Gilbert D, Zariffard MR, et al. The effects of commensal bacteria on innate immune responses in the female genital tract. Am J Reprod Immunol. 2011;65(3):190–195. 153. Nguyen PV, Kafka JK, Ferreira VH, Roth K, Kaushic C. Innate and adaptive immune responses in male and female reproductive tracts in homeostasis and following HIV infection. Cell Mol Immunol. 2014;11(5):410–427. 154. Abioye AI, Soipe AI, Salako AA, et al. Are there differences in disease progression and mortality among male and female HIV patients on antiretroviral therapy? A meta-analysis of observational cohorts. AIDS Care. 2015;27(12):1468–1486. 155. Agaba PA, Digin E, Makai R, et al. Clinical characteristics and predictors of mortality in hospitalized HIV-infected Nigerians. J Infect Dev Ctries. 2011;5(5):377–382. 156. Kuruc JD, Cope AB, Sampson LA, et al. Ten Years of Screening and Testing for Acute HIV Infection in North Carolina. J Acquir Immune Defic Syndr. 2016;71(1):111–119. 157. Nagata D, Gutierrez EB. Characteristics of HIV patients who missed their scheduled appointments. Rev Saude Publica. 2015:49. 158. Cabrera-Munoz E, Hernandez-Hernandez OT, Camacho-Arroyo I. Role of estradiol and progesterone in HIV susceptibility and disease progression. Mini Rev Med Chem. 2012;12(11):1049–1054. 159. Boulle C, Kouanfack C, Laborde-Balen G, et  al. Gender Differences in Adherence and Response to Antiretroviral Treatment in the Stratall Trial in Rural District Hospitals in Cameroon. J Acquir Immune Defic Syndr. 2015;69(3):355–364. 160. Zhang L, Wang Z, Chen Y, et al. Label-free proteomic analysis of PBMCs reveals gender differences in response to long-term antiretroviral therapy of HIV. J Proteomics. 2015;126:46–53. 161. Maskew M, Brennan AT, Westreich D, McNamara L, MacPhail AP, Fox MP. Gender differences in mortality and CD4 count response among virally suppressed HIV-positive patients. J Womens Health (Larchmt). 2013;22(2):113–120. 162. Maman D, Pujades-Rodriguez M, Subtil F, et al. Gender differences in immune reconstitution: a multicentric cohort analysis in sub-Saharan Africa. PloS One. 2012;7(2):e31078.

163. Prins M, Meyer L, Hessol NA. Sex and the course of HIV infection in the pre- and highly active antiretroviral therapy eras. AIDS. 2005;19(4):357–370. 164. Cook IF. Sexual dimorphism of humoral immunity with human vaccines. Vaccine. 2008;26(29–30):3551–3555. 165. Cook IF. Sex differences in injection site reactions with human vaccines. Hum Vaccin. 2009;5(7):441–449. 166. Klein SL. Sex influences immune responses to viruses, and effic� cacy of prophylaxis and treatments for viral diseases. Bioessays. 2012;34(12):1050–1059. 167. Klein SL, Marriott I, Fish EN. Sex-based differences in immune function and responses to vaccination. Trans R Soc Trop Med Hyg. 2015;109(1):9–15. 168. Wang X, Boulton ML, Montgomery JP, et  al. The epidemiology of measles in Tianjin, China, 2005-2014. Vaccine. 2015;33(46):6186–6191. 169. Garenne M. Sex differences in measles mortality: a world review. Int J Epidemiol. 1994;23(3):632–642. 170. Knudsen KM, Aaby P, Whittle H, et al. Child mortality following standard, medium or high titre measles immunization in West Africa. Int J Epidemiol. 1996;25(3):665–673. 171. Atabani S, Landucci G, Steward MW, Whittle H, Tilles JG, Forthal DN. Sex-associated differences in the antibody-dependent cellular cytotoxicity antibody response to measles vaccines. Clin Diagn Lab Immunol. 2000;7(1):111–113. 172. Green MS, Shohat T, Lerman Y, et  al. Sex differences in the humoral antibody response to live measles vaccine in young adults. Int J Epidemiol. 1994;23(5):1078–1081. 173. Khalil MK, Al-Mazrou YY, Al-Ghamdi YS, Tumsah S, Al-Jeffri M, Meshkhas A. Effect of gender on reporting of MMR adverse events in Saudi Arabia. East Mediterr Health J. 2003;9(1–2):152–158. 174. Shohat T, Green MS, Nakar O, et al. Gender differences in the reactogenicity of measles-mumps-rubella vaccine. Isr Med Assoc J. 2000;2(3):192–195. 175. Forthal DN, Landucci G, Habis A, et  al. Age, sex, and household exposure are associated with the acute measles-specific antibody-dependent cellular cytotoxicity antibody response. J Infect Dis. 1995;172(6):1587–1591. 176. Ovsyannikova IG, Jacobson RM, Dhiman N, Vierkant RA, Pankratz VS, Poland GA. Human leukocyte antigen and cytokine receptor gene polymorphisms associated with heterogeneous immune responses to mumps viral vaccine. Pediatrics. 2008;121(5):e1091–e1099. 177. Kanra G, Isik P, Kara A, Cengiz AB, Secmeer G, Ceyhan M. Complementary findings in clinical and epidemiologic features of mumps and mumps meningoencephalitis in children without mumps vaccination. Pediatr Int. 2004;46(6):663–668. 178. Sugishita Y, Shimatani N, Katow S, Takahashi T, Hori N. Epidemiological characteristics of rubella and congenital rubella syndrome in the 2012-2013 epidemics in Tokyo, Japan. Jpn J Infect Dis. 2015;68(2):159–165. 179. Gupta SN, Gupta N, Gupta S. A Mixed Outbreak of RubeolaRubella in District Kangra of Northern India. J Family Med Prim Care. 2013;2(4):354–359. 180. Mitchell LA, Zhang T, Tingle AJ. Differential antibody responses to rubella virus infection in males and females. J Infect Dis. 1992;166(6):1258–1265. 181. Mitchell LA. Sex differences in antibody- and cell-mediated immune response to rubella re-immunisation. J Med Microbiol. 1999;48(12):1075–1080. 182. Tingle AJ, Allen M, Petty RE, Kettyls GD, Chantler JK. Rubellaassociated arthritis. I. Comparative study of joint manifestations associated with natural rubella infection and RA 27/3 rubella immunisation. Ann Rheum Dis. 1986;45(2):110–114. 183. Pocchiari M, Puopolo M, Croes EA, et  al. Predictors of survival in sporadic Creutzfeldt-Jakob disease and other

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

human transmissible spongiform encephalopathies. J Neurol. 2004;127(Pt 10):2348–2359. 184. Akhtar S, Wenborn A, Brandner S, Collinge J, Lloyd SE. Sex effects in mouse prion disease incubation time. PloS One. 2011;6(12):e28741. 185. Cho IS, Spinner DS, Kascsak RJ, et al. Altered lymphocyte proliferation and innate immune function in scrapie 139A- and ME7-infected mice. Viral immunol. 2013;26(3):192–200. 186. Wehrenpfennig P, Drechsler S, Weixelbaumer KM, Bahrami S, Osuchowski MF. Mouse model of posttraumatic abdominal sepsis: survival advantage of females over males does not depend on the cecum size. Eur surg Res. 2014;52(1-2):83–89.

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187. Koehoorn M, Karr CJ, Demers PA, Lencar C, Tamburic L, Brauer M. Descriptive epidemiological features of bronchiolitis in a population-based cohort. Pediatrics. 2008;122(6):1196–1203. 188. Pinzan CF, Ruas LP, Casabona-Fortunato AS, Carvalho FC, Roque-Barreira MC. Immunological basis for the gender differences in murine Paracoccidioides brasiliensis infection. PloS One. 2010;5(5):e10757. 189. Gabriel G, Arck PC. Sex, immunity and influenza. J Infect Dis. 2014;209(Suppl 3):S93–S99. 190. Casadevall A, Pirofski LA. The damage-response framework of microbial pathogenesis. Nat Rev Microbiol. 2003;1(1):17–24.

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C H A P T E R

30 Gender-Based Differences in Mortality in Indian Children Aged 5 to 14 years Claire K. Nguyen and Shaun K. Morris 1

The Hospital for Sick Children, Toronto, ON, Canada

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30.1 INTRODUCTION India is a country with a high burden of mortality. In 2015 an estimated 1.2 million of the world’s 5.9 million deaths in those under 5 years of age occurred in India. India has a greater absolute number of child deaths than any other country, and accounted for 19% of the world’s deaths in 2013.1 The major causes of mortality in Indian children under 5 years include pneumonia, diarrheal illnesses, and infectious disease.2,3 Further contributors to mortality include injury and noncommunicable diseases.4 Female children in India are at particular risk with relatively increased mortality rates.4 We know that there are major gender-based differences in deaths in those younger than 5 years.4–6 Indeed, there has been a growing recognition of the gender differentials in mortality in Indian children. Some of the findings are astounding: the authors report a five-fold difference in postneonatal mortality from pneumonia between girls in central India and boys in south India, and a four-fold difference in the number of deaths from diarrhoeal disease between girls in central India and boys in west India.7

The increased mortality seen in girls is especially notable because there is some evidence with certain

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pathogens that susceptibility is higher in males due to greater Th1 immune responses in females. Ultimately, however, the interplay between gender, hormones, and immunity is complex and greatly confounded by external biologic (e.g., nutrition) and nonbiologic (e.g., health seeking behavior) and gender-based differences in immune response clearly cannot explain a fivefold difference in mortality. The population in India is not homogenous and is distributed across a wide geography and range of socioeconomic statuses. The relatively high national mortality rate has important implications for health policy and health initiatives. While there has been progress in reducing child mortality (average rates of decline over the past two decades was 3.7% per year), there continues to be a lack of clarity around causes of and risk factors for deaths, particularly in rural areas due to modeling and data collection limitations.1 Little is known about mortality in the 5 to 14 years age group (middle childhood) and the causes of death in this group, despite much work internationally related to under-5 child mortality. To date, in developing countries, children older than 5 years are rarely represented in research. Though there is currently limited published data related to ages 5 through 14 years, it would be logical to speculate that the same health issues affecting

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those under 5 years of age, including those related to gender, carry on through childhood. However, focused research on these middle childhood years is needed to fully understand whether this assumption is valid. The literature around gender differences in child mortality in India is minimal. Only one study concerning children aged 5 to 14 years was identified. Thus, while this chapter reviews literature pertaining to genderbased differences in all children, most data concerns children under 5 years of age. In the absence of data in the middle childhood years, we believe it is most likely that many of the gender-based factors affecting younger children carry forward to affect older children.

30.2  BY THE NUMBERS Gender-based mortality differences in India arise even prior to birth where an apparent preference for boys in some segments of the population is a well acknowledged phenomenon and has led to dramatic shifts in child sex ratios. Jha et  al. examined sex ratios by birth order among 250,000 births in three rounds of the National Family Health Survey from 1990 to 2005.8 They found that birth ratios favoring boys, particularly after a firstborn girl, had increased over the study time period and presumably reflects selective termination of pregnancy. Our analyses find that selective female abortion in India has grown in the last two decades and accounts for most of the large and growing imbalance between the number of girls to boys at ages 0–6 years. Sex ratios for births that followed a firstborn girl fell sharply from 1990 to 2005, even though sex ratios for all births (regardless of birth order) did not.8

Sex ratio at birth and in the population is usually constant in human populations aside from small excesses in male births after war.9 The normal sex ratio at birth ranges from 1.05 to 1.06 or 105 to 106 boys for each 100 girls born.10,11 However, sex ratios at birth in India have been demonstrated to be skewed in favor of boys outside of the normal range. It appears that sex-selective abortion is positively correlated with distorted sex ratios commencing at birth. This distorted sex ratio is compounded by increased mortality amongst young females. The 2011 India census data showed a child sex ratio of children aged 0 to 6 years at 0.919 or 919 girls for every 1000 boys.12 Females typically have a biological advantage over males when it comes to survival in childhood. Many reasons have been postulated for this advantage including an X-linked genetic trait, physiologic benefits of estrogen, and differences in immune response, however, none of these have been conclusively proven. However, with the combination of a skewed sex ratio at birth and a

higher mortality in female children in India, the 2011 Census data revealed 7.1 million fewer girls than boys aged 0–6 years which was an increase from the 6 million fewer girls in 2001.8 The child sex ratio fell between the study period by 1.9% in the decade starting in 1991 and by a further 1.4% the following decade (see Fig. 30.1) resulting in even fewer girls relative to boys. Further, Jha et al. noted that child sex ratios favoring boys are positively correlated with increased education and income, possibly because of financial access to ultrasound and abortion services.8 One additional explanation for the greater disturbance in sex ratios in recent years may be related to increased access to antenatal gender identification abroad. The government website which publishes Census data online attributes the declining female birth ratio to violence. The major cause of the decrease of the female birth ratio in India is considered to be the violent treatments meted out to the girl child at the time of the birth.12

The skewed sex ratio favoring boys in India is in contrast to worldwide trends. Globally, Alkema et  al. note that girls from 0 to 5 years have survival advantages over boys which typically results in a mortality sex ratio greater than one, i.e., more girls than boys survive.13 Additionally, the survival advantage usually increases as total mortality declines with childhood age.13 In a study of global mortality sex ratios, Alkema et al. found that India had the highest excess female mortality for children aged 1–4 years in 2012. Further, they found that India had the largest excess female under-5 years mortality rate in the world, followed by two other South Asian countries, Afghanistan and Pakistan.13 Rao, Director of the Indian Institute of Public Health in Hyderabad in 2009 stated: A preference for sons has resulted in an adverse gender ratio which is worse in the economically better off states. This ratio has, disturbingly, worsened over time, and now stands at about 93.5 girls per 100 boys, in sharp contrast to a natural gender ratio at birth of 105 boys per 100 girls.14

In discussing how to counteract sex-selective abortions, Jha et al. comment that the Pre-Natal Techniques (PNDT) Act, 1996, enacted to thwart prenatal sex selection, was likely largely ineffective as the majority of health providers were unregulated and not subject to enforcement under the Act. Despite the wide acknowledgment by policy makers in India that sex-selective abortions occur, it has remained difficult to enforce legislation designed to curb its incidence.8 Much has been written about the missing women in India. Brooks considers the origins and consequences of the “missing women” problem. He postulates that “Son preferences and associated cultural practices like

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FIGURE 30.1  Relative changes in percent in child sex ratio of girls to boys at ages 0–6, between 2001 and 2011 for the districts of India. Reprinted from Jha P, Kumar R, Ram F, Ram U, Aleksandrowicz L, Bassani D, Chandra S, Banthia J. Trends in selective abortion of female foetuses in India: analysis of nationally representative birth histories from 1990–2005 and census data from 1991–2011. Lancet 2011;377(9781):1921–1928, Copyright (2011), with permission from Elsevier.

patrilineal inheritance, patrilocality and the Indian Hindu dowry system arise among the wealthy and powerful elites for reasons consistent with models of sex-biased parental investment.” He goes on to describe that son preference and its impact on sex ratios manifests in a number of ways.

ages 1 and 5 y was more than 30% higher than boys’. While the estimates suggest that the sex ratio of child mortality may have increased somewhat in India since the 1980s, girls remain disadvantaged in mortality, compared both to the sex differences found in other parts of the developing world and to the historical experience of developed countries at the same level of mortality.19

Most dramatically, female infants are sometimes killed at birth upon the wishes of the parents or their family. But the systematic neglect of daughters and women whose families don’t feed, immunize, seek medical attention and care for them as well as they do for brothers and husbands has long been the main cause of elevated female mortality.16

There are data that suggest infants and children under 5, are at higher risk for death in families in which there is domestic violence against women. Silverman et  al. found that:

Multiple studies and authors describe the phenomena of female infanticide, sex-selective abortion, and differential treatment of girls.8,9,15,16 And although the under-5 mortality rate has declined in general over the past decades, females do not benefit from the same rate of decline as males.17 In a study examining mortality in northern India, mortality rates among girls remained significantly higher than boys during the postnatal period (29–365 days of age) (160–200% higher) as well as into childhood (160–230% higher).18 The girls’ survival disadvantage was particularly acute in the 1- to 4-year age group. In the 2000s, the ratio of male to female child mortality was estimated at 74 … meaning that girls’ mortality between

Infants and young children in India were found to suffer significantly greater risk of death in families in which mothers had experienced spousal violence from their husbands. Furthermore, the effect of such gender-based violence was profoundly gendered; infant girls and children bear a far greater share of the mortality burden associated with IPV. In contrast, IPV was not significantly associated with infant boys and child mortality in adjusted analyses. To our knowledge, investigators have not studied mortality rates in children older than 5 in families with intimate partner violence.20

Life expectancy is another method by which to examine mortality. The measurement of life expectancy in India also shows a differential between females and males. Until 1980, women in India were disadvantaged with respect to life expectancy at age 0, despite the overall trend of a longer life expectancy of women globally.19

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For the first time, in the years 1981–1985 females showed a life expectancy that exceeded males.19 Dubey, Ram, and Ram indicate, however, that the increased overall female life expectancy is misleading as Indian women younger than 20 years of age continue to be disadvantaged as a result of socioeconomic discrimination.19 They further argue that examining life expectancy at ages 0, 1, and 5 years allows for a clearer picture of gender differentials that might be obscured when only examining life expectancy at age 0. The Indian life tables reveal that a newly born child has shorter longevity than does a child who has survived to age one and/or five years. The life expectancy at age zero for women is three years greater than that for men in 2009 and at the same time the U5MR for female children was 10% higher than the U5MR for male children during the same year implying that the life expectancy at age zero alone may hide the widespread gender discrimination and disadvantages for female children at early ages of life.20

Dubey et al. note that life expectancy should decline over time with the maximum value at age 0. In India’s past, life expectancies at ages 1 and 5 outstripped that at age 0, attributable to high mortality rates at early ages, particularly during infancy.19 Further, they discuss the concept of crossover (where longevity at age 0 crosses over, or is greater than, the longevity at another time interval). Unfortunately, females in India had a delayed crossover by 4 to 5 years as compared to males. In a population like India where gender discrimination at early ages in life are extensively and commonly prevalent, there is a need to consider other indicators of mortality in addition to life expectancy at age zero … despite higher life expectancy at age zero for female, crossover for women got delayed by almost 4 to 5 years reinstating the fact that the women in India are grossly disadvantaged during the early ages of lives. In this context, relative values of life expectancies at age zero, one and five become important.

30.3  PREVIOUS RESEARCH Health data related to children aged 5–14 in developing countries is scarce. We searched for publications from various electronic courses, including Cochrane Library, PubMed, MEDLINE, and CINAHL. Search terms were intentionally kept broad and included: child, pediatric, pediatric, India, mortality, death, sex, gender. One example of the challenge in searching for relevant data is exemplified by advanced search parameters that were limited in refining by age. For example, in PubMed, advanced search parameters were limited to birth to 18 years or birth to 23 months. Despite this, studies addressing child mortality and gender in India were reviewed and only one paper directly addressing this age group was located.

Morris et al., using a representative sample of deaths, explored the issue of mortality attributable to infectious disease in children in India aged 5 through 14 years. Through the use of verbal autopsy assessment of almost 4000 children in that age bracket, it was found that 60% of mortality in the age group was attributable to infectious disease and further, that mortality was 50% higher in girls than boys for the top two causes of diarrheal disease and pneumonia. In their analysis, Morris et al. found that girls in the Central, East, and Northeast regions of India were at a much higher risk of mortality as compared to their male or other geographically located counterparts. In particular, for diarrhea and pneumonia, mortality of girls was nearly 50% higher than boys and the increased mortality rates held true for all-cause mortality (see Table 30.1). It was postulated that the gender differential may have been attributable to parents being less likely to immunize, seek medical attention, and/or being less likely to use appropriate antibiotic therapy for sick female children, as described in other research.21–23 Excess female mortality is related in part to vaccination policy.6,24 In India, girls are immunized less than boys thus leaving them more susceptible to vaccine-preventable deaths at all ages.21 Unfortunately, females are also less likely to be fed a nutritious diet as compared to boys when their mothers were not literate.21 A girl between her first and fifth birthday in India or Pakistan has a 30–50% higher chance of dying than a boy. This neglect may take the form of poor nutrition, lack of preventive care (specifically immunisation), and delays in seeking health care for disease.25

Additionally, we know that care-seeking is influenced by the gender of the child.26–28 In a study conducted in Southern India, Pillai et al. found that care was sought for illness significantly more often when the child was a boy.29 Nalhotra N, Upadhyay30 also found gender-based difference in care-seeking for diarrheal illnesses in children less than 5 years of age. In a study examining how providers in an Indian NICU reach end-of-life decisions, gender bias was a common variable in treatment decisions. Nevertheless, we observed that, after birth, gender was a regular concern for providers. Informants felt a strong duty to protect female newborns against culturally entrenched discrimination yet often felt powerless to do much to change longstanding prejudices: “So the gender bias is the biggest problem in this place. I feel so bad when I see a baby (girl) that is kicking around, having a good prognosis and the parents say that, ‘No, no, no, we want to take this baby home,’ and I know that the chance of survival (at home) is very small, but I have to accept.” Informants reported the manifestation of gender bias in numerous ways: families were observed to be less interested in intensive care, in buying basic medicines, and coming to follow-up outpatient appointments after discharge when their infant was female.31

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30.4 Future Directions

TABLE 30.1  Estimated Total Deaths and Mortality Rates from Diarrhea and Pneumonia in Children Aged 5 to 14 Years

Source: Morris SK, Bassani DG, Awasthi S, et al. Diarrhea, pneumonia, and infectious disease mortality in children aged 5 to 14 years in India. PLoS One. 2011;6(5):e20119.

In examining HIV-infected children brought to hospital for treatment, Rajasekaran et  al. found that an increasing proportion of the patients were girls (38.2% in 2002 and 44.9% in 2004).32 However, compared to the population proportion, significantly more boys were brought in for treatment. Willis et al. found that financial expenditure for health care in the neonatal period was fourfold higher in households with males compared to females.33 Further, female newborns used cheaper public care providers.33 Willis et  al. concluded “…during the neonatal period, households tended to neglect girls compared to boys in the care-seeking process. Neglectful care-seeking behavior occurred in newborn care as early as the time of recognition of initial illness.”33 Kamath discussed the health state of the girls in India succinctly.

Debata, Deswal, Kumath examined unnatural deaths (deaths due to murder, suicide, traffic accident, industrial accident, or other accident like drowning, falling, snake bite, sun stroke, etc.) in a study of 434 postmortems in children aged 1 to 19 years.34 They found that unnatural deaths in those less than 5 years differed significantly from older children, and that those aged 11 to 19 were the most vulnerable. The most common cause of death for all age groups was flame burns. The findings in this study suggest that for deaths not attributable to infectious disease, those aged 5 through 14 years of age may, in fact, have substantially different trends compared with children less than 5 years of age.

The female child’s status in India indicates the general attitude of the society towards women. Girls in the country are at a higher risk of malnutrition and growth retardation. Many reports highlight that girls are offered less food – both in terms of quantity and quality – than boys, especially in Northern India. This in turn leads to anemia and poor weight gain during pregnancy, perpetuating the cycle of intrauterine growth retardation and malnutrition. A status report brought out by “Save the Children” highlighted that India has the largest gender survival gap in the world. Indian girls are 61% more likely than boys to die between the ages of 1 and 5. Gender-based discrimination has also been documented in care-seeking during common illnesses, including diarrhea and acute respiratory infections.29

A variety of organizations have been actively working to improve the health and well-being of children in India. Particular attention has increasingly been paid to female children older than 5 years. UNICEF has targeted the health of females from birth through to motherhood and has acknowledged the unique needs of older female children. “Complications in pregnancy and childbirth are among the leading cause of death for adolescent girls.”35 Further, UNICEF has created programming that directly targets adolescent empowerment, health, and nutrition. In one of its programs, school-aged girls are specifically targeted for inclusion.

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UNICEF has also been working with girls’ collectives such as Meena Manch in states including Bihar, West Bengal and Uttar Pradesh to build self-confidence among girls, create awareness about the importance of education and attending school regularly and desired hygiene and sanitation practices, and develop leadership qualities and team spirit. There is evidence that involvement in these collectives has helped delay the marriage age of participants’ peers and others in the local community and increased the flow of children withdrawing from work and enrolling and regularly attending school.36

The WHO has acknowledged that maternal health has a direct impact on child health. Poor access to health and nutrition services for mothers and children is at the root of these high mortality rates. One out of every five children under age of 5 years is wasted and 43% are underweight for their age, outcomes which are closely related the nutritional status of their mothers. Children whose mothers are underweight (with a body mass index less than 18.5 kg/m2) are much more likely than other children to be stunted, wasted and underweight.37

The preferential treatment of males has significant implications for the future health and well-being of all Indian persons. While all children should have access to adequate nutrition, housing, education, and health care, the health of girls is of particular importance as they will bear future populations. It is important to ensure female health is optimized throughout childhood and prior to pregnancy in order to optimize health for future child-bearing. Given the paucity of literature in children greater than 5 years of age, it is essential that further study be conducted to explore the unique needs of this age group. Study in children aged 5 to 14 years in India is of particular importance to understand how risks faced by young female children in the country with more deaths than any other may extend into older age groups.

References 1. GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;385(9963):117–171. 2. Black EB, Morris SS, Bryce J. Where and why are 10 million children dying every year? Lancet. 2003;361:2226–2234. 3. Million Death Study Collaborators. Causes of neonatal and child mortality in India: a nationally representative mortality survey. Lancet. 2010;376(9755):1853–1860. 4. Morris SK, Bassani DG, Awasthi S, et al. Diarrhea, pneumonia, and infectious disease mortality in children aged 5 to 14 years in India. PLoS One. 2011;6(5):e20119. Epub 2011 May 24. Erratum in: PLoS One. 2013;8(11). 5. Jagnoor J, Bassani DG, Keay L, et  al. MDS Collaborators. Unintentional injury deaths among children younger than 5 years of age in India: a nationally representative study. Inj Prev. 2011;17:151–155.

6. Corsi DJ, Bassani DG, Kumar R, et  al. Gender inequity and ageappropriate immunization coverage in India from 1992 to 2006. BMC Int Health Hum Rights. 2009(Suppl 1):S3. 7. Gakidou E, Lopez AD. What do children die from in India today? Lancet. 2010;376(9755):1810–1811. 8. Jha P, Kumar R, Ram F, et al. Trends in selective abortion of female foetuses in India: analysis of nationally representative birth histories from 1990-2005 and census data from 1991-2011. Lancet. 2011;377(9781):1921–1928. 9. Hesketh T, Xing ZW. Abnormal sex ratios in human populations: causes and consequences. Proc Nat Acad Sci. 2006;103(36): 13271–13275. 10. Egan JF, Campbell WA, Chapman A, Shamshirsaz AA, Burram P, Benn PA. Distortions of sex ratios at birth in the United States; evidence for prenatal gender selection. Prenat Diagn. 2011;31(6): 560–566. 11. Warade Y, Blasarkar G, Bandekar P. A study to review sex ratio at birth and analyze preferences for the sex of the unborn. J Obstet Gynaecol India. 2014;64(1):23–26. 12. Census of India 2011. New Delhi: Registrar General & Census Commissioner of India. < http://www.census2011.co.in/sexratio. php>; 2011 Accessed 15.05.16. 13. Alkema L, Chao F, You D, Pdersen J, Sawyer C. National, regional, and global sex ratios of infant, child, and under-5 mortality and identification of countries with outlying ratios: a systematic assessment. Lancet Glob Health. 2014;2:e521–e530. 14. Rao M. Tackling health inequalities in India. Perspect Public Health. 2009;129(5):205–206. 15. Brooks R. “Asia’s missing women” as a Problem in Applied Evolutionary Psychology. Evol Psychol. 2012;12(5):910–925. 16. Srivastava VK, Arora N. Child survival and equity. Indian J Public Health. 2007;51(2):83–85. 17. Mohanty SK, Rajbhar M. Fertility transition and adverse child sex ratio in districts in India. J Biosoc Sci. 2014;46(6):753–771. 18. Krishnan A, Ng N, Byass P, Pandav CS, Kapoor S. Sex-specific trends in under-five mortality in rural Ballabgarh. Indian Pediatr. 2014;51:48–51. 19. Dubey M, Ram U, Ram F. Threshold levels of infant and underfive mortality for crossover between life expectancies at ages zero, one and five in India: a decomposition analysis. PLoS One. 2015;10(12):e0143764. 20. Silverman JG, Decker MR, Cheng DM, et al. Gender-based disparities in infant and child mortality based on maternal exposure to spousal violence: the heavy burden borne by Indian girls. Arch Pediatr Adolesc Med. 2011;165(1):22–27. 21. Borooah VK. Gender bias among children in India in their diet and immunisation against disease. Soc Sci Med. 2004;58(9): 1719–1731. 22. Pande RP, Yazbeck AS. What’s in a country average? Wealth, gender, and regional inequalities in immunization in India. Soc Sci Med. 2003;57(11):2075–2088. 23. Srivastava SP, Nayak NP. The disadvantaged girl child in Bihar: study of health care practices and selected nutritional indices. Indian Pediatr. 1995;32(8):911–913. 24. Hirve S, Bavdekar A, Juvekar S, Benn CS, Nielsen J, Aaby P. Nonspecific and sex-differential effects of vaccinations on child survival in rural western India. Vaccine. 2012;30(50):7300–7308. 25. Fikree FF, Pasha O. Role of gender in health disparity: the South Asian context. BMJ. 2004;328:823–826. 26. El Arifeen S. Excess female mortality in infants and children. Lancet Glob Health. 2014;2(9):e491–e492. 27. Khera R, Jain S, Lodha R, Ramakrishnan S. Gender bias in child care and child health: global patterns. Arch Dis Child. 2014;99: 369–374. 28. Kamath SS. Journey of a girl child in India during health and disease. Indian Pediatr. 2015;52:835.

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REFERENCES

29. Pillai RK, Williams SV, Glick HA, Polsky D, Berlin JA, Lowe RA. Factors affecting decisions to seek treatment for sick children in Kerala, India. Soc Sci Med. 2003;57(5):783–790. 30. Nalhotra N, Upadhyay RV. Why are there delays in seeking treatment for childhood diarrhoea in India. Acta Paediatr. 2013;102(9):e413–e418. 31. Miljeteig I, Sayeed SA, Jesani A, Johansson KA, Norheim OF. Impact of ethics and economics on end-of-life decisions in an Indian neonatal unit. Pediatrics. 2009;124(2):e322–e328. 32. Rajasekaran S, Jeyaseelan L, Raja K, Ravichandran N. Demographic and clinical profile of HIV infected children accessing care at Tambaram, Chennai, India. Indian J Med Res. 2009;129(1):42–49. 33. Willis JR, Kumar V, Mohanty S, et  al. Gender differences in perception and care-seeking for illness of newborns in rural Uttar Pradesh, India. J Health Popul Nutr. 2009;27(1):62–71.

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34. Debata PK, Deswal S, Kumath M. Causes of unnatural deaths among children and adolescents in northern India – A qualitative analysis of post-mortem data. J Forensic Leg Med. 2014;26:53–55. 35. Unicef. Innovative approaches improve maternal and child health care in Madya Pradesh, India. < http://www.unicef.org/health/ india_70766.html>; Accessed 25.06.16. 36. Unicef. Gender Education. < http://unicef.in/Whatwedo/16/ Gender-and-Inclusion>; Accessed 28.06.16. 37. WHO (2014). Improving maternal and child health in India. SERP’s Nutrition Day Care Centers mobile app. WHO/ RHR/13.27. ; Accessed 25.06.16.

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C H A P T E R

31 Consideration of Biological Sex in Translating Regenerative Stem Cell Therapies Galina Shapiro1, Gadi Pelled1,2 and Dan Gazit1,2 1

Hebrew University of Jerusalem, Jerusalem, Israel, 2Cedars-Sinai Medical Center, Los Angeles, CA, United States

O U T L I N E 31.1 Introduction

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31.6 Muscle-Derived Stem Cells

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31.2 Embryonic Stem Cells

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31.7 Endothelial Progenitor Cells

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31.8 Neural Stem Cells

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31.4 Mesenchymal Stem Cells

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31.9 Current Challenges and Future Prospects

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31.5 Hematopoietic Stem Cells

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References 453

31.1 INTRODUCTION Regenerative cell therapies are emerging as the next major development in medicine.1–3 These treatments represent a medical paradigm shift from palliation to the repair or replacement of cells, tissues or organs to restore function. We will focus on stem cell (SC) therapies, defined as any treatment based on viable human SCs, including autologous (patient-derived cells) and allogeneic (donor-derived cells) therapies. Historically, SCs have been clinically used since the 1950s4 with the first successful bone marrow transplantations in the late 1960s.5,6 However, since the late 1990s there has been a significant leap in basic and clinical research with the isolation, generation, and application of multiple SCs including: adult SCs, embryonic SCs7 (ESC) and induced pluripotent SCs8 (iPSC). Their ability to self-renew and potential to differentiate into multiple cell types has led to many clinical trials for tissue regeneration,

Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00019-X

immune modulation, and cancer therapy among other indications.9 The development of a SC therapy involves evaluation of several factors including cell type, source, isolation, culture, and differentiation methods and the appropriate analytical tests to evaluate identity, purity, and potency. Then a mechanism of action is identified and safety concerns are addressed, including: undesirable in vivo cell differentiation, unwanted cell migration, uncontrolled proliferation, immunogenicity, graft versus host reactions. Altogether, safety and efficacy of a candidate product are established by preclinical research, usually both in vitro and in vivo. Then translational research can be undertaken. The challenges surrounding commercial viability and clinical uptake are related to difficulties in establishing clinical utility and cost-effectiveness. This requires combined efficient quality bioprocessing and scale-up approaches for scaled production at an affordable price. However, a potentially crucial translational

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aspect—which has been mostly overlooked until recently—is biological sex. Until the end of the 20th century, scientists assumed without any direct evidence that women were essentially identical to men except for their reproductive biology.10 The last two decades of the 20th century saw the development of a growing awareness that the physiology and the experience of the same diseases were significantly different for men and women.11 It became clear that what was true of males could not be assumed to be true of females at the cost of many lives with 8 out of 10 drugs withdrawn from the US market between 1997 and 2000 because of life-threatening health effects with “greater health risks for women than for men.” Furthermore, gender bias in basic research, commonly manifested as failing to use appropriate samples of male and female cells, tissues, and animals, yields faulty results. Although the American congress passed the 1993 National Institutes of Health (NIH) Revitalization Act12 mandating the inclusion of women as subjects in clinical research, the NIH has only recently published a notice “expecting that sex as a biological variable will be factored into research in vertebrate animal and human studies.”13,14 Controlling for gender bias could save lives and billions of dollars spent on failed drug development. According to an Institute of Medicine report,15 sex is a narrow term defining living things as male and female based on the composition of sex chromosomes and the presence of reproductive organs. Yet, it is indisputable that biological sex is not cleanly divided into male and female with the spectrum including diverse discordances between chromosomal sex and genital anatomy and variations on sexual identity and behavior. As the processes underlying biological sex determination are becoming clearer so is the categorization of humans who are the subjects of clinical investigation into “male” and “female” becoming more complex. In spite of this, the need for inclusion of males and females in preclinical and clinical investigation is a fundamentally important first step in authenticating the importance of sex in molding phenotype. Including sex as a biological variable could help build a knowledge base that better informs the design of clinical research and trials in humans.16 From basic research to clinical care, studying both sexes should be a guiding principle across the biomedical research continuum. Basic research at the molecular to cellular level should include the evaluation of sex-variable SC intrinsic properties including: gene expression and its epigenetic regulation, isolation or generation techniques, culturing techniques, self-renewal, clonogenicity, proliferation, homing and mobilization, trophic factor expression, differentiation, and immune modulation. Then an added level of complexity should account for sex-dependent SC extrinsic variables influencing SC behaviors such as:

(1) the hormonal environment including the biological effect of hormones and the elucidation of its mediating receptors and signaling pathways; (2) unique hormonal states: menstrual cycle phases, pregnancy, and postmenopause; and (3) immunological and environmental factors like parity and aging. Finally, efficacy and safety studies should be designed to include all combinations of donor/recipient sexes and report any sex-specific side effects. In this review, we focus on the recent advances in understanding the implications of biological sex on translational regenerative SC therapies.

31.2  EMBRYONIC STEM CELLS ESCs are obtained from the inner mass of a blastocyst. They are capable of differentiating into cells belonging to all three germ layers, including: endoderm, mesoderm, and ectoderm.7 Due to their pluripotent differentiation ability, ESCs are promising potential regenerative therapeutic agents for a wide range of pathologies.17 Yet, only a small number of clinical trials involving human ESCs (hESCs) have enrolled patients for conditions including: spinal cord injury, macular dystrophy, and type I diabetes mellitus.18,19 First evidence of safety, graft survival, and biological activity of subretinal transplantation of hESC-derived retinal pigment epithelium into 18 patients with macular dystrophy was recently published.19 No evidence of serious safety issues was found and in 17 out of 18 patients there was an improvement in eye function. While these are highly promising results, there has yet to be any demonstration of hESC clinical efficacy; which requires larger robust phase 2 studies. Taken together with the ethical controversy surrounding ESC research and the recent discovery of iPSCs, the clinical role of ESC is under debate.20 The factors limiting the rather inefficient derivation of hESCs are not fully understood. A significant increase (76%) in female ESC lines was attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of hESC lines.21 It appears that male ESCs are more vulnerable than female cells, leading to the favoring of female ESC growth in culture conditions. This could be explained by a difference in the epigenetic state such as aberrant X chromosome inactivation (XCI), X chromosome erosion,22 and/ or overexpression of critical metabolic X-linked genes.23 It is interesting to note that, in contrast to humans, the maintenance of stable female murine ESC lines is more difficult because of frequent losses of an active X chromosome possibly due to global hypomethylation.24 More recently, it has been suggested that the female sex bias may be explained by genetic associations.25 Findlay et  al. demonstrated that the minor allele of the human single nucleotide polymorphism (SNP) rs2231947 found

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31.3  Induced Pluripotent Stem Cells

within the NODAL gene locus is underrepresented in male but not female hESC lines. This SNP was highly functional in hESC lines, controlling the alternative splicing of the NODAL gene, a known regulator of pluripotency and cell fate. ESCs appear to differ not only in derivation efficiency but also in gene expression. By comparing the gene expression profiles of male and X-inactivated female human pluripotent SCs, Ronen et al. proposed that the presence of the Y chromosome and specifically SRY may drive sex-dependent gene expression leading to sexspecific differences in the growth and differentiation of pluripotent SC.26 More than 200 autosomal genes were differentially expressed between male and female ESCs including genes involved in steroid metabolism pathways. However, not only genetic factors but also epigenetic factors, such as differentially methylated regions, could contribute to sexual dimorphism in ESCs.27 Yet it appears that cell sex alone does not fully explain the differences in ESC line propensity to differentiate into specific lineages.28 The most studied extrinsic sex difference between ESCs is probably the effect of sex hormones. Androgen receptor (AR) is known to be expressed in the inner cell mass and ESCs.29 Androgen stimulation induced AR upregulation and ESC differentiation,30 and vice versa, antiandrogen treatment inhibited differentiation and stimulated ESC growth. This suggests that androgens via AR promote ESCs differentiation but suppress ESC self-renewal. However, ESC proliferation is unaffected by androgen treatment. Androgen and AR signaling have also been found to play an indirect protective role in oxidative stress-induced apoptosis in ESCs.17 Finally, estrogens have been found to stimulate ESC expression of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ), increase expression of the proto-oncogenes, and increase proliferation.31 Estradiol and progesterone had a positive effect on proliferation of ESC derived committed precursors and subsequent differentiation to dopaminergic neurons.32,33 Nonetheless, it is not clear whether these differences exist in vivo as well.

31.3  INDUCED PLURIPOTENT STEM CELLS iPSCs, derived from transcription factor-mediated reprogramming, are pluripotent SCs with molecular and functional properties similar to ESCs.34 iPSCs offer a pluripotent autologous cell source for replacement therapy, and patient-specific iPSCs can serve as in vitro models for disease mechanism studies and drug screening. The enormous regenerative potential of iPSCs has led to a first clinical trial in less than 20 years from

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their discovery.3 The safety data from this trial and the use of newer “zero-footprint” reprogramming techniques could provide an important stepping stone for advancing the clinical use and research of iPSC.35 Yet, this promise is obscured by recent findings of genetic and epigenetic variations in iPSCs.36 Variations between ESCs and iPSCs, and between different iPSC lines, are thought to be due to incomplete reprogramming, suggesting that some lines may not be suitable for human therapy. Further research is needed to fully characterize the reprogramming process and understand how to produce iPSCs consistently enough to meet the high quality and safety requirements for use in the clinic. The most studied sex-specific aspect of the iPSC phenotype is XCI. This is innately unique to female somatic cells, which achieve X chromosome dosage compensation through this process. Interestingly, female human iPSCs (hiPSCs), unlike murine iPSCs, retain an inactive X chromosome in spite of reprograming.37 Nonetheless, reprogramming and long-term culture can induce abnormalities in the XCI status.22,38 While early passage hiPSCs maintain XCI (XaXi), higher passage cells have been reported to undergo X reactivation resulting in the presence of two active X chromosomes (XaXa),39 followed by inactivation of either of the X chromosomes upon differentiation. Others have shown that high passage hiPSCs lose XIST expression and undergo repressive chromatin modifications followed by erosion of the gene silencing on the inactivated X (XaXe).40 X erosion (Xe) appears to have a complex effect on gene expression with some loci remaining inactive,39,41 while others undergo increased gene expression.40 Interestingly, at least some male hiPSCs were found to have patterns of aberrant differential gene expression resembling female hiPSC with deranged XCI.39 This implies epigenetic instability may not be simply avoidable by using male hiPSC, but may instead be symptomatic of a more global underlying epigenetic derangement. Importantly, XaXe cells appear to have a growth advantage and gradually take over the hiPSC population. This advantage is likely due to the enhanced expression of oncogenes in XaXe hiPSC lines.41,42 Consistent with the potential cancer-like properties, XaXe hiPSCs show inefficient differentiation when subjected to differentiation cues.42 In addition, the eroded state of Xe is also passed onto differentiated cells that likewise lacked normal, expected XIST expression and therefore erosion of dosage compensation in these cells is irreversible.39,40 This is of particular concern given recent evidence that targeted deletion of Xist in female hematopoietic SCs (HSCs) led to rapid development of a highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome.43

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31.4  MESENCHYMAL STEM CELLS Mesenchymal stem cells (MSCs) are a highly promising adult SC population with a multitude of potential applications in regenerative medicine. The International Society for Cellular Therapy defined MSCs as plastic adherent, CD73, CD90, and CD105 expressing cells capable of differentiating into osteoblasts, adipocytes, and chondroblasts.44 MSCs have been isolated from virtually all postnatal organs and tissues. As many as 610 MSC-based clinical trials, either complete or ongoing, are currently registered in the database of the US NIH.45 MSCs have consistently demonstrated therapeutic capabilities far beyond their differentiation capacities.46 The mechanisms by which MSCs exert these actions include: immunomodulation, trophic and proangiogenic functions and migration to injury sites.47 In addition, MSC have also been proposed as cellular vehicles for therapeutic gene delivery.48,49 In spite of the paucity of long-term safety data, MSC administration is widely considered a feasible and safe procedure with no adverse events reported.50 As gender could be a factor in reaching desired cell numbers, research optimizing sex-dependent extraction and culturing of MSCs is of major importance. For example, human adipose MSCs (ASCs) underwent most effective digestion when adipose tissue was incubated with 0.2% collagenase for 1 h for males, whereas overnight digestion was more effective for tissue from females.51 Gender-optimized protocols resulted in a higher human ASC yield from female adipose tissue. Furthermore, in males the highest yielding collection site was the abdomen, whereas in females the biopsy region did not influence cell recovery. Then again, in human bone marrow-derived MSCs (BMSCs), no gender differences in BMSCs frequency were found regardless of whether bone marrow samples were first processed for mononuclear cell isolation or used directly.52 Interestingly, Seeback et  al.53 found gender differences in human BMSC (hBMSC) reservoir and proliferation capacity in trauma patients after injury severity-based subgroup analysis. On the other hand, lower BMSC frequencies in rodent females were found54 and even suggested to be responsible for the suboptimal healing of a segmental bone defect model.55 The inconsistency in sex-dependent MSC yield in different investigations may reflect differences in species, source tissue, or culturing and aspiration technique. BM aspiration is especially prone to causing variability in MSC concentration as it is highly dependent on the surgical aspiration technique and dilution with blood.56 More work should be done to ensure that the maximum yield of MSCs is extracted from both male and female patients. Studies related to the gender effects on the selfrenewal potential of BMSCs remain unclear with one

study showing gender has little effect on the BMSCs self-renewal.57 Yet, others were able to show that the male sex has suppressive effects on the generation of ASCs, which have been shown to possess characteristics similar to BMSCs.58 The latter was also supported by studies using AR knockout mice17 and AR overexpressing transgenic mice.59 Diminished male derived BMSC self-renewal in these studies was suggested to be mediated by epidermal growth factor receptor activation and AKT and Erk1/2 signaling. Since AR signals appear to reduce self-renewal, perhaps AR antagonists could be used therapeutically to enhance MSC self-renewal. Female hBMSC had higher clonogenic activity and exhibited enhanced expression of the surface antigens, CD119 and CD130.60 These cells were also smaller, divided more rapidly, and were more frequent in hBMSC preparations from younger female donors. Some researchers have suggested that the increased proliferation rate in female BMSCs is a result of sex steroid regulation and specifically estradiol. Estradiol was found to stimulate hBMSC proliferation.61 For rat BMSCs (rBMSCs), the optimal proliferation-enhancing dose and steroid combination varied depending on the gender of donor.62 In addition, estrogen has been discovered recently to inhibit and delay MSC senescence by upregulation of telomerase activity.63 These results strongly indicate that steroid hormones can serve as effective stimulators to facilitate MSC capacity. Importantly, gender-related MSC proliferation differences appear to be species- and strain-specific, warranting more research before transnationally useful conclusions could be drawn.54 The donor variation in yield, growth, differentiation, and in vivo regeneration of MSCs is a bottleneck for standardization of therapeutic protocols. Gender appears to play a complex role in MSC differentiation modulation. For example, human and rabbit ASCs isolated from males were found to be more osteogenic in vitro than those isolated from females.64,65 Also, in the male but not female human ASCs, the superficial abdominal fat derived cells differentiated faster and more efficiently than those of the deep fat derived cells. While most of the published data compares male and female MSCs with respect to osteogenic and adipogenic differentiation, some evidence of donor and host66 sex-linked MSC differentiation differences was also published for chondrogenic67 and neurogenic differentiation.68 Few straightforward associations between gender and hBMSC osteogenic differentiation were found.60 Interestingly osteogenic differentiation capacity was reported to be an age effect after sex-based subgroup analysis. Leskelä et al.69 suggested an increasing osteogenicity in female hBMSCs with age based on increased secretion of amino-terminal propeptide of type I procollagen concentration, specific alkaline phosphatase (ALP) activity and Ca concentration. While Muschler et  al.70

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31.4  Mesenchymal Stem Cells

reported the opposite, a decrease of ALP activation based on ALP expressing colony forming unit counts. In vitro osteogenic differentiation of murine BMSCs (mBMSCs) appears to be highly donor age-dependent. 1-month-old female mBMSCs were less osteogenic than male mBMSCs,54 3-month-old and 6-month-old female mBMSCs were not significantly different then male mBMSCs, and 9-month-old female mBMSCs were less osteogenic than male mBSCs.71 In addition, female mBMSCs showed greater levels of adipocyte-related genes than male mBMSCs in all tested age groups. Female mBMSCs were also significantly more sensitive to rosiglitazone-induced suppression of osteogenesis. Female mBMSCs grown in estrogen-stripped medium showed similar responses to rosiglitazone as mBMSCs grown in serum containing estrogen. mBMSCs from female mice that had undergone ovariectomy before sexual maturity also were sensitive to rosiglitazone-induced effects on osteogenesis. These results suggest that female MSCs are more sensitive to modulation of differentiation by PPARγ and that these differences are intrinsic to the sex of the animal from which the mBMSCs came. The conclusion that osteogenic capacity is sexdependent is further supported by the fact that steroid regulation of MSC osteogenic differentiation is also sexdependent. Progesterone increased proliferation and differentiation of female rBMSCs but not male rBMSCs.72 Also, glucocorticoid receptor silencing in MSCs upregulated the expression of fibroblast growth factor-2 and Sox-11 of human MSC.73 MSC with endogenous GC blocked in vitro had greater proliferation rates, were more osteogenic, and significantly less adipogenic. Furthermore, extracellular62 and intracellular74 estradiol delivery effectively enhanced the osteogenic differentiation of MSCs. Yet different effective doses to maximally improve osteogenic differentiation for rBMSC were found for male and female donors.62 The estradiol concentrations to produce peak Osteocalcin amounts in both males and females were consistent with that of BMP-7 expression and steroid receptor expression in male and female rBMSCs. The function of BMP-7 including bone formation and cancer cell proliferation is estrogen-dependent.75 Leskela et al. found that estradiol increased calcium deposition significantly by hBMSCs of both sexes but ALP activity only in the male hBMSCs.76 Not only estradiol but also phytoestrogens such as resveratrol and genistein, have been shown to enhance osteogenic differentiation and repress adipogenic differentiation of hMSC, possibly through the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway.77 Estradiol-induced osteogenic differentiation could be mediated by ERα and ERβ.76 Interestingly, osteogenic differentiation seems to increase the mRNA expression of all estrogen receptors (ER) present in MSCs with the exception of ERβ4 isoform that seems to be mainly

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expressed in undifferentiated MSC.78 Such changes in ER profile during differentiation of MSC suggest different effects of and sensitivity to estrogens depending on the differentiation lineage and stage. ER gene polymorphism may also account for certain interindividual variability in responses to estradiol stimulations.76 Testosterone, on the other hand, had no effect on ALP activity or calcium deposition in either sex. More recent studies have revealed that hMSC express key enzymes needed for intracrine conversion of dehydroepiandrosterone to estrogens or testosterone.79 Interestingly, the expression of these converting enzymes was also found to be differentiation-dependent. This suggests the interesting possibility that hMSC may themselves locally adjust sex steroid levels. Therefore, providing an additional local mechanism for MSCs to modulate differentiation in an intra-, auto-, or paracrine manner. Immunomodulatory properties of MSC through indirect effects on immune cells and tissue SC populations may be an important aspect of their regenerative ability. Gender-related differences in MSC immunomodulation and trophic factor secretion have recently come to attention. Naïve female hBMSCs under standard culture conditions showed increased expression of IFN-γR1 and IL-6β, and were more potent in T cell proliferation suppression.60 Naïve rBMSCs grown on complex microstructured titanium surfaces showed sex-dependent expression of TGF-b1 and VEGF-A.80 In addition, testosterone but not estrogen decreased VEGF secretion in rBMSCs in a dose-dependent manner.81 MSCs were also shown to be sexually dimorphic when stressed in vitro.82 Endotoxin provoked significantly more antiinflammatory VEGF production in female mBMSCs versus male mBMSCs. Hypoxia and hydrogen pyroxide exposure also caused significantly more VEGF production in female mBMSCs compared to male mBMSCs. Female mBMSCs expressed significantly less inflammatory tumor necrosis factor alpha and IL-6 than male mBMSCs after acute endotoxin and hypoxia. A follow-up study showed that the release of TNF, IL-6, and VEGF, in male but not female mBMSCs, were mediated by TNFR1.83 TNFR1 was also noted to mediate male but not female mBMSC apoptosis. A similar investigation demonstrated that TNFR2 is a significant regulator of VEGF and IGF-1 production in male but not female MSCs.84 Ex vivo, better functional recovery, decreased levels of TNF, and more VEGF were observed subsequent to intracoronary infusion of female mBMSCs into isolated rat hearts after ischemia compared to male mBMSC transplantation.85 Moreover, estradiol stimulated male MSC production of VEGF in vitro86 and hearts infused with estradiol-treated MSCs exhibited greater functional recovery after ischemia/reperfusion injury compared to those infused with untreated MSCs.44 Given that the

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female mBMSCs were removed during the in vivo estrous cycle, these observed responses may reflect the inherent chronic effects of estrogen.85 Furthermore, female mBMSCs offered a greater protective advantage in a rat endotoxic injury model compared with male mBMSCs.87 Female mBMSCs treatment resulted in greater preservation of cardiac function potentially facilitated by an alteration of the myocardial apoptotic profile as indicated by increased normalization of the myocardial Bcl-xL/Bax ratio. MSCs are promising therapeutic agents for organ protection, but further study of the sex-dimorphic expression of trophic factors and immunomodulation is necessary to maximize this protection. By manipulating the mechanisms that BMSCs use to produce growth factors, we may be able to engineer SCs to produce maximum growth factors during therapeutic use.

31.5  HEMATOPOIETIC STEM CELLS Circulating blood cells are formed in bone marrow through hematopoiesis. Very small embryonic-like SCs (VSELs) are the most primitive pluripotent SCs in the bone marrow that self-renew and give rise to HSCs under stress. HSCs further divide and differentiate to maintain homeostasis. HSCs are multipotent, capable of differentiating into the cells of all blood lineages: erythrocytes, platelets, neutrophils, eosinophils, basophils, monocytes, T and B lymphocytes, natural killer cells, and dendritic cells.88 In fact, the discovery of HSCs is greatly responsible for the paradigmatic use of SCs as a means of replacing injured or diseased tissue as early as the late 1940s.89 Currently, HSC transplantation (HSCT) is the only SC therapy in standard medical practice treating hematologic malignancies and acquired bone marrow failure.90 There are over 24 million registered adult HSC donors, and the numbers of unrelated donor HSCTs are increasing. Yet, the optimal strategy for prioritizing among comparably human leukocyte antigen (HLA) matched potential donors has not been established. Peripheral blood SCs are nowadays the most common source of CD34+ cells used in HSCT. Interestingly, female donors are less likely to meet the requested cell dose after Granulocyte colony-stimulating factor induced CD34+ SC mobilization and subsequent harvest by apheresis.91 Since CD34+ cell dose is a critical factor in the outcome of unrelated peripheral blood SCs transplantation, female donors appear less attractive.92 Furthermore, HSC donation procedure generally causes more harm to female donors as demonstrated by a higher rate of acute toxicities, pain, fatigue, the need for a central venous catheter placement, avascular osteonecrosis necessitating total arthroplasty, prolonged hospitalization after peripheral blood SC or bone marrow collection.93

Not only the biological sex of the HSC donor but also the sex of the HSC recipient appears to impact HSCT success. A study of 1386 patients undergoing allogeneic HSCT at a single medical center showed that sex matching between donors and recipients correlated with better overall survival, although HSCs from male donors were associated with better long-term survival.94 On the other hand, when HLA-identical sibling donor HSCT was used to treat multiple myeloma, cells from female donors produced better outcomes.95 Women patients who receive female HSCs have lower mortality than women patients treated with male HSCs. For male patients with multiple myeloma, the sex of donor cells did not significantly influence overall mortality, but did influence modes of mortality: men treated with male HSCs were more likely to die from myeloma relapse, whereas men treated with female HSCs were more likely to die from nonrelapse-related causes, such as graft-versus-host disease (GvHD).95 More recently, Kollman96 et  al. found that survival after HLA-matched HSCT for hematologic malignancy was better after HSCT of grafts from young HLA matched donors. Other donor characteristics, such as sex and parity, were not associated with survival. Taken together, these studies emphasize the need for more research on the role of sex in donor selection for HSCT. Currently it is still unclear whether donor/recipient sex itself should be considered a prognostic factor. If so, it appears that disease-specific research should be carried out. Female donor/male recipient (FDMR) transplants are often considered unfavorable compared with all other sex combinations. Stern et  al.97 analyzed donor/ recipient sex combinations in more than 50,000 patients treated with allogeneic HSCT. They found FDMR to be associated with increased chronic GvHD, increased late transplant-related mortality, and decreased survival irrespective of underlying disease.98 These data support the current practice of avoiding female donors for male patients, if possible.99 Interestingly, FDMR patients had lower relapse rates especially in patients with high-risk disease or advanced disease stage. However, this was not enough to offset the mortality associated with higher GvHD incidence and transplant-related mortality.95,97,100 These gender differences may be explained by the mismatch in minor histocompatibility antigens present on the Y chromosome (HY). For example, HY-specific T cells of female donor origin are known to accumulate in male GvHD-affected skin101 and HSCT patient-derived HY-specific T cell clones cause severe tissue damage in vitro when tested in skin explant assays.102 These observations point out that HY is a clinically relevant transplantation antigen. One of the most disputed non-HLA factor influencing HSCT outcome is donor parity.103 Several studies have reported that recipients of grafts prepared from

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female donors who had undergone multiple pregnancies prior to donation display a significantly higher rate of GvHD.96 Possibly reflecting maternal alloimunization following placental cell exchange. While one-third of the mothers are sensitized to the paternal antigens of the child, some of the mothers become tolerant to them, as reflected by the dominant presence of circulating cytotoxic HY-specific T cells104 or HY-specific T regulator cells, respectively.105,106 The latter are predominantly present in women who do not have male offspring, including both nulliparous and parous donors with female offspring.105 Thus, female HSCT donors form a highly heterogeneous population in terms of their HY-specific T cell repertoire. As these cells persist for a long time postpartum,107 they may end up in cellular products prepared from female donors. However, all donors, regardless of sex and parity, have been exposed during fetal life and by breastfeeding to microchimeric maternal cells and molecules, which may induce long-lasting T regulator cells.108 Hence, alloimmune status should probably be taken into account with all donors. Furthermore, the pre-HSCT established alloimmune repertoire of a female donor may be advantageous in particular HSCT settings including: T regulatory prevention of GvHD109 and better HLA mismatched grafts.110 Hormonal environment is probably the most studied sex-dependent intrinsic SC variable influencing HSC behavior. HSCs express sex hormone receptors111 and sex hormones in suboptimal doses of hematopoietic cytokines and growth factors are known to enhance clonogenic growth of human HSCs. On the other hand, sex steroid ablation induces hematopoietic and lymphoid recovery by functionally enhancing both HSC selfrenewal and propensity for lymphoid differentiation.112 The terminal differentiation and function of some hematopoietic cells are regulated by sex hormones,113 but HSC function is thought to be similar in both sexes. Estrogen is the primary female sex hormone and plays a key role in various physiological events such as pregnancy, menstrual cycle, vasculogenesis, and bone formation. Estrogen appears to engage HSCs both directly and indirectly through alterations in the HSC niche. Estrogen has a direct effect on HSCs via ERα promoting HSC self-renewal, causing female HSCs to proliferate much more frequently than those of their male counterparts.114 Conditional deletion of ERα from HSCs reduced HSC division in female, but not male, mice and attenuated the increases in HSC division, HSC frequency, and erythropoiesis during pregnancy. An indirect estrogen effect on HSCs appears to involve T-cells stimulated by CD40 ligand to secrete Wnt10b.115 Furthermore, a recent study showed that the epigenetic changes intrinsic to the HSCs cooperate with the extrinsic effect of female hormones to regulate the efficiency of HSC engraftment and reconstitution.116

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Not only estrogen but also follicular stimulating hormone (FSH) therapy resulted in statistically significant enhancement in peripheral blood number of both VSELs and HSCs.117 FSH therapy mobilizes VSELs and HSCs into peripheral blood that on one hand supports their developmental origin from germ lineage, and on the other hand FSH can become a promising candidate tool for mobilizing HSCs and SCs with VSEL phenotype in clinical settings.

31.6  MUSCLE-DERIVED STEM CELLS Skeletal muscle is derived from mesodermal progenitors that proliferate, differentiate, fuse, and mature into skeletal muscle fibers, a process known as myogenesis.118 Importantly, a subpopulation of these cells enters a quiescent state, making up the adult muscle-derived stem cell population (MDSCs, also known as satellite cells). MDSCs demonstrate high self-renewal, long-term proliferation capacities, and promote tissue repair by secreting trophic factors.119–121 Altogether, these characteristics give adult skeletal muscle its high regenerative capacity in response to injury or trauma. Importantly, MDSCs are multipotent cells, capable of differentiating into muscle, fat, bone, and cartilage.122 They have been used in preclinical studies and clinical trials to regenerate a variety of tissues following acute injury or chronic disease, such as muscular dystrophies,123 bone and cartilage injuries,124,125 and peripheral nerve damage.126 MDSC cell lines display variability in regenerative ability. Using a murine muscular dystrophy model, Deasy et al.123 demonstrated that MDSCs isolated from a female mouse had a significantly stronger regenerative effect than MDSCs isolated from a male mouse. This effect was independent of other variables such as immune response and exogenous estrogenic effects. Even though all MDSCs could differentiate into dystrophin-expressing fibers in vitro, only female-derived MDSCs could robustly regenerate muscle in vivo. Furthermore, not only the sex of the donor cell but also the sex of the recipient animal was found to significantly impact the regenerative outcome. Female recipients showed superior regeneration to that of male recipients regardless of donor cell sex. Further experiments using immune deficient mice suggest that the effect of host sex but not the effect of cell sex was immune-modulated.123 Even when sex was a statistically significant predictor of SC behavior, not all cell lines are alike within a sex. Mouse MDSCs show significant variation in regeneration potential within a single sex. Stolting et  al.127 showed that MDSCs from young female donors provided a higher yield of faster-growing cells in vitro with an optimum contractile output in vivo. In contrast, elderly- and male-derived cells grown under expansion conditions differentiate faster in vitro.

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MDSCs can improve bone healing in critical-sized defects.124 While normal skeletal bone formation, growth, and maintenance are influenced by sex hormones,128–130 no known studies have examined host sex or sex hormones in the context of a critical-sized cranial defect model. Yet, of the 7367 genes expressed in murine skeletal muscle, 55.4% of these are sexually dimorphic.131 Interestingly, four of these sexually dimorphic skeletal muscle genes are also known to be involved in BMPinduced endochondral ossification.132 MDSCs isolated from males engineered to overexpress BMP4 were superior with regards to in vitro osteogenic differentiation including staining for ALP, ALP activity, and ALP and Runx2 gene expression.133 Furthermore, in vivo implantation of BMP4 overexpressing MDSCs showed that male MDSCs produce denser and more consistent volumes of ectopic bone, while female MDSCs produce variable volumes of ectopic bone.133 MDSCs are not only sexually dimorphic but also osteogenically and chondrogenically dependent on host sex.134 Male hosts, whether unaltered or castrated, form significantly larger volumes of MDSCmediated ectopic bone and cranial defect healing model than female hosts, either unaltered or ovariectomized. Interestingly, in the healing model but not in the ectopic bone model, volume differences were found between hosts of the same sex. In both models, these differences were attributed to varying rates of endochondral bone formation in male and female hosts. As far as chondrogenic differentiation, male MDSCs show superior differentiation in a micromass culture system in vitro and are also better at repairing a created osteochondral defects.135

31.7  ENDOTHELIAL PROGENITOR CELLS Despite the improvements in acute care and the impact of primary and secondary prevention, the epidemic of cardiovascular disease is global. Approximately 1 million myocardial infarctions occur per year in the United States and 5 million patients have heart failure, with a 20% annual mortality rate.136 Moreover, cardiac transplantation will not fill the need given that donors are lacking and xenotransplantation remains experimental. In cardiovascular medicine, innovation in SC biology has created curative solutions for the treatment of cardiomyopathy. Multiple cell-based platforms have been developed, harnessing the regenerative potential of various natural and bioengineered sources.137 Specifically, diverse subsets of endothelial progenitor cells (EPCs) have been used for the treatment of ischemic diseases in clinical trials.138–141 EPCs, discovered in 1997, proliferate, migrate, and differentiate into mature endothelial cells.142,143 EPCs can be isolated from bone marrow, adipose tissue, cord blood, and other adult tissues.

Numeric and functional impairments of EPCs are associated with increased cardiovascular and cerebrovascular morbidity and mortality.144 However, it is not completely clear whether differences in EPC number contribute to the gender-related differences in the prevalence of cardiovascular events. Some found no sex-difference in EPC number in middle-age adults,145 while others found more circulating EPCs in fertile women than in men.146 These conflicting results may be attributed to the use of different surface markers for EPC detection, subgroup analysis, and sample sizes. Female EPCs have a superior proangiogenic potential in vivo,146 are more clonogenic, and superiorly adherent.147 When male and female mice with severe dietinduced atherosclerosis were given bone marrow cells from either male or female donors, EPCs proliferated to a greater extent in female recipients. Furthermore, only female donor cells administered to male recipients reduced the plaque burden,148 suggesting that in females the mechanism of plaque development and regression involves cells other than EPCs. Interestingly, EPCs vary in phase with menstrual cycle in ovulatory women and directly respond to estrogen via ER with increased proliferation and adhesion.146 Cyclic EPC mobilization in fertile women could provide a pool of cells for endometrial homeostasis and result in higher circulating EPC levels, representing a cardiovascular protection mechanism in this population. This suggests that exogenous estrogen could be therapeutically used to mobilize EPCs to treat ischemic injury. A number of preclinical studies demonstrated this approach could be used to treat myocardial infarction,115,149,150 vascular injury,115,151 and limb ischemia.152 Notably, estrogen was shown to augment EPC function by various mechanisms including: endothelial nitric oxide synthase-mediated augmentation of matrix metalloproteinase-9 expression,150 negative modulation of proinflammatory mediator expression and the resultant chemotaxis for neutrophils,151 upregulation of EPC C-X-C chemokine receptor type 4 expression,115 and upregulation of EPC expressed vascular endothelial growth factor.149 Nonetheless, long-term estrogen hormone replacement therapy has shown mixed cardiovascular outcomes and current US Preventive Services Task Force recommends against estrogen for the prevention of chronic conditions.153 While estrogen is a potent stimulator of EPCs, androgens may improve the biology of these cells as well, inducing endothelial protection in coronary heart disease. Several studies showed that androgens augment the proliferation, migration, adhesion, colony formation activity, and angiogenesis of EPCs through an AR-dependent pathway and PI3K/Akt and Erg1 signaling pathways.154 AR was found essential for mobilization but not angiogenic differentiation needed for robust revascularization

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31.8  Neural Stem Cells

in response to ischemia in both male and female mice.155 Additionally, untreated hypogonadotropic hypogonadal men have a low number of circulating EPCs that could increase significantly after testosterone treatment, suggesting that androgens are positively correlated with the numbers of circulating EPCs.156 On the other hand, a subsequent study by Fadini et al. indicated that androgens exert no direct effects on the expansion and adhesion of circulating humane male EPCs whereas they positively modulate early “monocytic” EPCs.157 Moreover, rats’ castration was followed by a decrease in circulating EPCs, but androgen replacement failed to restore EPC levels. Prompting the hypothesis that circulating EPC levels are more directly associated with estradiol, rather than androgen levels. Therefore, the exact relationship of andogens and EPCs is elusive and remains to be deeply investigated.

31.8  NEURAL STEM CELLS Many neurological conditions are the consequence of neuronal loss caused by acute or chronic injury. It has long been assumed that the adult mammalian brain has very limited regenerative capacities. Nonetheless, some functional restoration of neural networks after stroke158 and functional compensation in subclinical Parkinson’s disease159 are well known. These repair mechanisms have clear limitations, causing adult brain injury to often result in permanent functional impairment. For example, 8 out of 10 of the most debilitating disorders are neurological according to a global study conducted by the World Health Organization.160 Without effective regenerative treatments, the neurological burden of disease is expected to increase as the world population ages. However, since the generation of neurons and astrocytes from isolated neural SCs (NSCs) in the early 1990s161 many SC-based therapies have been researched.162 NSCs are the precursors of neurons and glia capable of selfrenewal and generating all differentiated neural cells of the CNS. Currently, NSCs are in clinical trials for stroke, amyotrophic lateral sclerosis (ALS), and Parkinson’s disease among other diseases.163 Mitotic and neurogenic differences occur in a sex- and age-dependent manner in NSCs.164 Sexual dimorphism in the neurogenic capacity was observed in cultured NSCs derived from the subventricular zone of adult rats NSCS (rNSCs).165,166 The in vitro fate of rNSCs was affected by sex and aging. Young male rNSCs mainly expressed markers of neuronal and oligodendroglial fate, whereas young female rNSCs underwent astroglial differentiation. In a different study, following transplantation of young and old rNSCs of both genders into rats of both sexes and age categories (young and old), young recipient rats were noted to have a poorer cell survival rate

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while also displaying improved neurogenesis.167 Older recipient animals had better cell survival both with cells derived from younger, same-sex rats and cells derived from older, opposite-sex rats. This indicates the potential for benefit utilizing both autologous and allogenic transplantation while accentuating the significance of the age and gender of both donor and recipient. Estrogen has been studied for neuroprotection for over 20 years.168 Estradiol is known to induce NPC proliferation in vitro,169 in vivo,169 in utero,170 during pregnancy,171 and after brain injury.172 Estradiol also affects NPC survival and NPC differentiation. Yet, while estradiol was reported to differentiate human NSCs into dopaminergic neurons in vitro and support their survival in vivo,173 administration of estradiol to embryonic rNSCs increased the fraction of oligodendrocytes via membrane-associated ER.174 Estradiol altered neurogenesis in female ovariectomized, but not male castrated rats.175 Undifferentiated NSC displayed sexual dimorphism in the expression of steroid receptors and metabolizing enzymes.165,166 Specifically, ERα was overexpressed in young female-derived cells, whereas ERβ was the more prevalent receptor in young male cells. No differences were found between progesterone receptors and glucocorticoid receptor expression in NSCs for both sexes and across ages. The expression of ERα and ERβ was shown to increase during aging for both sexes. Finally, aromatase, a key enzyme in the conversion of testosterone to estradiol, was expressed in male NSCs but not female NSCs. This suggests that male NSCs can metabolize testosterone to produce estradiol, thus providing them with the ability to alter their local environment and modulate endogenous neurogenesis in a different manner than female NSCs may do. Progesterone, like estrogen, is a gonadal steroid hormone that has been extensively studied for its neuroprotective effects.176 Progesterone mediates rNPC proliferation and concomitant regulation of mitotic cell cycle genes via a progesterone receptor membrane component/ERK pathway.177 A subsequent study showed that several clinically relevant progestins significantly increased rNPC proliferation in vitro, whereas others were without effect or even inhibited rNPC proliferation.178 Importantly, proliferative progestins in vitro were also neuroprotective in vivo via activation of MAPK to promote proliferation. In combination with estradiol some progestins significantly increased proliferation while others significant increased in apoptosis. Neurogenic response to clinical progestins varies dramatically. Progestin impact on the regenerative capacity of the brain has clinical implications for contraceptive and hormone therapy formulations prescribed for preand postmenopausal women. Ransome et  al. used male and female adult subventricular murine NSCs to further examine direct responses

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to principal sex hormones.179 Testosterone, estradiol, and progesterone were unable to support neurosphere growth in the absence of other growth factors. Testosterone, estradiol, or progesterone induced cell proliferation irrespective of sex in an Erk-dependent manner without affecting subsequent neuronal differentiation. AR inhibition but not aromatase inhibition reduced basal and testosterone-induced neurosphere growth in females, while only concurrent inhibition of AR and aromatase produced the same effect in males. This sex-specific effect was supported by higher aromatase expression in male neurospheres compared to females. Oxidative stress impaired neurosphere growth and upregulated aromatase expression in both sexes. However, under oxidative stress letrozole significantly exacerbated impaired neurosphere growth in males only. While both estradiol and testosterone could prevent oxidative stress-induced growth reduction in both sexes, the effects of testosterone were dependent on innate aromatase activity. Sexual dimorphism in NSCs could be the result of sex steroids, but it might also be caused by the intrinsic differences in the sex chromosomes in male and female NPCs. The human Y chromosome encodes 27 different proteins,180 eight of which are expressed in the male brain. It is, therefore, very conceivable that these male proteins could have a male-specific effect on the brain, independent of any gonadal hormone influence.181 For example, the male-specific SRY protein has been known to upregulate the expression of Sox9,182 which is also expressed in NSCs in the CNS and recognized as a key transcription factor involved in altering neurogenic potential in these cells.183 Further profiling the expression of steroid receptors and Sox proteins would help determine the molecular mechanism of sexual dimorphism in NSCs. Some of the sex-dependent differences known for NSCs were also investigated in the context of ALS. ALS is a fatal neurodegenerative disease, which selectively affects motor neurons throughout the CNS. A genetic link to point mutations in the superoxide dismutase 1 (SOD1) gene has been shown in familial ALS and used to generate rodent ALS models. Males have been shown to be at a higher risk for ALS than females. Possible reasons for the ALS gender difference include underlying differences between the male and female nervous systems and different abilities to repair damage.184 Studies using SOD1G93A mice suggested that gonadal steroids are involved in the occurrence and disease progression of ALS. Ovariectomy accelerated ALS progression, reducing female life span, and thus making it comparable to that of the male transgenic mice.185 Treatment of ovariectomized females with estradiol significantly improved the disease course. On the other hand, a study using SOD1G93A rats found no significant effect of gonadectomy on disease onset and progression

in spite of sexual dimorphism in intact and gonadectomized rats.186 Dehydroepiandosterone treatment did not alter disease progression or survival in SOD1G93A rats. Sexual dimorphism in ALS could also be caused by intrinsic cellular sex differences and the SOD1G93A transgene. Fetal male and female rNSCs derived from CNS of SOD1G93A transgenic rats and wild type (WT) rats were compared in vitro.187 WT male rNSCs were more proliferative and neurogenic then WT female rNSCs. Yet, SOD1G93A overexpression significantly reduced only male cell proliferation and neurogenesis. There was no sex-based difference in rNSC sensitivity to oxidative stress, rather increased cytotoxicity by SOD1G93A overexpression occurred, especially in male rNSCs. Finally, ALS sexual dimorphism could also be a result of host differences. For example, female SOD1G93A mice responded better to intrathecal human spinal cord-derived NSCs treatment and other types of cell therapy than males.188

31.9  CURRENT CHALLENGES AND FUTURE PROSPECTS Taking into account sex differences when researching and developing regenerative therapies should become common practice. Future SC strategies should be based on research that has considered sex as a biological variable throughout the biomedical development spectrum to maximize positive outcomes and avoid gender bias. Gender-aware research could be viewed as a part of a greater movement toward enhancing rigor, transparency, and reproducibility in translational research headed by the NIH.189 Sex differences should be thought of as crucial experimental design elements along with blinding, randomization, replication, and sample-size calculation. Furthermore, all of the above should be clearly reported, otherwise both sexes will continue to receive suboptimal care. Currently, clinical trials appear to be less at risk for rigor and transparency concerns as they are already governed by various regulations that stipulate rigorous design and independent oversight.189 Yet, underrepresentation of women and lack of sex-specific data analysis in clinical trials is still very much the norm,190 perpetuating their vulnerability to adverse drug effects and disparities in medical outcomes. While sex is acknowledged as a variable that should be considered in clinical trials, gender remains mostly overlooked. Even if it may be technically difficult to separate the two, controlling for gender could add valuable information for personalizing regenerative therapies. For example, women experience more pain after HSC donation.93 This difference could be explained by cultural gender differences in reporting pain by patients, cultural gender differences in pain perception by physicians,

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REFERENCES

or a physiological sex difference in pain sensation.191 Additionally, sex-based differences in responses to pain interventions could also be the result of sex and/or gender differences.192 Investigating the mechanisms underlying these differences is the only way to truly optimize patient care. Studies should be designed to answer research questions in a sex- and gender-specific manner whenever possible.193 Researchers should account for sex and gender in study design, data collection, data analysis, and finally report of clinical research findings. Preclinical research is considered to be currently more susceptible to not considering sex as a biological variable.194 Testing only in male animals and not reporting cell sex,195 greatly limits the implications of results from such studies.196 Nonetheless, currently available evidence suggests that many aspects of the SC phenotype are sexually dimorphic, as reviewed here. Key SC properties including proliferation, differentiation, mobilization, and trophic factor expression have all been shown to be sex-specific. These differences have been found to be regulated by genetic, epigenetic, and hormonal mechanisms. In addition to cell-based differences, the interaction between donor cell sex and recipient sex was also found to be an important predictor of both therapeutic success and adverse events. Importantly, it appears that no single SC type has been fully characterized in term of sex differences even in the major aspects mentioned above. Even for the more studied SCs, such as MSCs and HSCs, it is difficult to reach translational conclusions as differences in methodology including the choice of species, strains, and techniques confound sex differences. Furthermore, as life span increases, sex-associated differences in SC aging should become of greater interest. At this point, however, not only has very limited work has been done to directly address this question, but also only about 50% of the papers published in 2014 reported both the sex and age of mice.197 Going forward, translational SC research should include sex as a critical variable in experiments, which can be translated to improve health outcomes for men and women.198 On the other hand, overemphasizing sex differences should also be avoided. For example, multivariate studies including sex as one variable among many should be done to test for interactions between sex and other outcome predictors.199 Otherwise, therapeutically relevant variability may be falsely attributed to sex. By reporting the effect size of a sex difference and p values for outcomes, the significance of a sex difference may be critically evaluated and appropriately interpreted.200 However, even without a measurable sex difference, both sexes should be included in all experiments as sex is a complex variable. Integrating sex as a biological variable into biomedical research holds the promise of advancing regenerative SC therapies to the clinic.

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References 1. Ratcliffe E, Glen KE, Naing MW, Williams DJ. Current status and perspectives on stem cell-based therapies undergoing clinical trials for regenerative medicine: case studies. Br Med Bull. 2013;108:73–94. 2. Trounson A, DeWitt ND. Pluripotent stem cells progressing to the clinic. Nat Rev Mol Cell Biol. 2016;17(3):194–200. 3. Kimbrel EA, Lanza R. Current status of pluripotent stem cells: moving the first therapies to the clinic. Nat Rev Drug Discov. 2015;14(10):681–692. 4. Thomas ED, Lochte Jr. HL, Lu WC, Ferrebee JW. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. New Engl J Med. 1957;257(11):491–496. 5. Bach FH, Albertini RJ, Joo P, Anderson JL, Bortin MM. Bonemarrow transplantation in a patient with the Wiskott-Aldrich syndrome. Lancet. 1968;2(7583):1364–1366. 6. Gatti RA, Meuwissen HJ, Allen HD, Hong R, Good RA. Immunological reconstitution of sex-linked lymphopenic immunological deficiency. Lancet. 1968;2(7583):1366–1369. 7. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et  al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282(5391):1145–1147. 8. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–676. 9. Li MD, Atkins H, Bubela T. The global landscape of stem cell clinical trials. Regen Med. 2014;9(1):27–39. 10. Legato MJ. Gender-specific medicine in the genomic era. Clin Sci. 2016;130(1):1–7. 11. Miller VM. Why are sex and gender important to basic physiology and translational and individualized medicine? Am J Physiol Heart Circ Physiol. 2014;306(6):H781–H788. 12. Congress U. National Institutes of Health revitalization act of 1993. Public Law. 1993:103–143. 13. National Institutes of Health (NIH). Consideration of Sex as a Biological Variable in NIH-funded Research. 2015. 14. Clayton JA, Collins FS. Policy: NIH to balance sex in cell and animal studies. Nature. 2014;509(7500):282–283. 15. Exploring the biological contributions to human health: does sex matter? J. Womens Health Gend Based Med 2001;10(5): 433–439. 16. Clayton JA. Studying both sexes: a guiding principle for biomedicine. FASEB J. 2016;30(2):519–524. 17. Huang CK, Luo J, Lee SO, Chang C. Concise review: androgen receptor differential roles in stem/progenitor cells including prostate, embryonic, stromal, and hematopoietic lineages. Stem Cells. 2014;32(9):2299–2308. 18. Simonson OE, Domogatskaya A, Volchkov P, Rodin S. The safety of human pluripotent stem cells in clinical treatment. Ann Med. 2015;47(5):370–380. 19. Schwartz SD, Regillo CD, Lam BL, et  al. Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet. 2015;385(9967):509–516. 20. Kobold S, Guhr A, Kurtz A, Loser P. Human embryonic and induced pluripotent stem cell research trends: complementation and diversification of the field. Stem Cell Reports. 2015;4(5):914–925. 21. Ben-Yosef D, Amit A, Malcov M, et al. Female sex bias in human embryonic stem cell lines. Stem Cells Dev. 2012;21(3):363–372. 22. Silva SS, Rowntree RK, Mekhoubad S, Lee JT. X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells. Proc Natl Acad Sci USA. 2008;105(12):4820–4825. 23. Kochhar HP, Peippo J, King WA. Sex related embryo development. Theriogenology. 2001;55(1):3–14.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

454

31.  BIOLOGICAL SEX IN STEM CELL THERAPIES

24. Zvetkova I, Apedaile A, Ramsahoye B, et al. Global hypomethylation of the genome in XX embryonic stem cells. Nat Genet. 2005;37(11):1274–1279. 25. Findlay SD, Postovit LM. Brief Report: Common Genetic Variation in Chromosome 10 q22.1 Shows a Strong Sex Bias in Human Embryonic Stem Cell Lines and Directly Controls the Novel Alternative Splicing of Human NODAL which is Associated with XIST Expression in Female Cell Lines. Stem cells. 2016;34(3):791–796. 26. Ronen D, Benvenisty N. Sex-dependent gene expression in human pluripotent stem cells. Cell Rep. 2014;8(4):923–932. 27. Ito S, Hirabayashi K, Moriishi K, et al. Novel sex-dependent differentially methylated regions are demethylated in adult male mouse livers. Biochem Biophys Res Commun. 2015;462(4):332–338. 28. Osafune K, Caron L, Borowiak M, et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat Biotechnol. 2008;26(3):313–315. 29. Chang CY, Hsuuw YD, Huang FJ, et al. Androgenic and antiandrogenic effects and expression of androgen receptor in mouse embryonic stem cells. Fertil Steril. 2006;85(Suppl 1):1195–1203. 30. Goldman-Johnson DR, de Kretser DM, Morrison JR. Evidence that androgens regulate early developmental events, prior to sexual differentiation. Endocrinology. 2008;149(1):5–14. 31. Wong CK, So WY, Law SK, et al. Estrogen controls embryonic stem cell proliferation via store-operated calcium entry and the nuclear factor of activated T-cells (NFAT). J Cell Physiol. 2012;227(6):2519–2530. 32. Diaz NF, Diaz-Martinez NE, Camacho-Arroyo I, Velasco I. Estradiol promotes proliferation of dopaminergic precursors resulting in a higher proportion of dopamine neurons derived from mouse embryonic stem cells. Int J Dev Neurosci. 2009;27(5):493–500. 33. Diaz NF, Diaz-Martinez NE, Velasco I, Camacho-Arroyo I. Progesterone increases dopamine neurone number in differentiating mouse embryonic stem cells. J Neuroendocrinol. 2009;21(8):730–736. 34. Stadtfeld M, Hochedlinger K. Induced pluripotency: history, mechanisms, and applications. Genes Dev. 2010;24(20):2239–2263. 35. Rao MS, Malik N. Assessing iPSC reprogramming methods for their suitability in translational medicine. J Cell Biochem. 2012;113(10):3061–3068. 36. Liang G, Zhang Y. Genetic and epigenetic variations in iPSCs: potential causes and implications for application. Cell Stem Cell. 2013;13(2):149–159. 37. Pomp O, Dreesen O, Leong DF, et  al. Unexpected X chromosome skewing during culture and reprogramming of human somatic cells can be alleviated by exogenous telomerase. Cell Stem Cell. 2011;9(2):156–165. 38. Wutz A. Epigenetic alterations in human pluripotent stem cells: a tale of two cultures. Cell Stem Cell. 2012;11(1):9–15. 39. Anguera MC, Sadreyev R, Zhang Z, et al. Molecular signatures of human induced pluripotent stem cells highlight sex differences and cancer genes. Cell Stem Cell. 2012;11(1):75–90. 40. Mekhoubad S, Bock C, de Boer AS, Kiskinis E, Meissner A, Eggan K. Erosion of dosage compensation impacts human iPSC disease modeling. Cell Stem Cell. 2012;10(5):595–609. 41. Tchieu J, Kuoy E, Chin MH, et al. Female human iPSCs retain an inactive X chromosome. Cell Stem Cell. 2010;7(3):329–342. 42. Bruck T, Yanuka O, Benvenisty N. Human Pluripotent Stem Cells with Distinct X Inactivation Status Show Molecular and Cellular Differences Controlled by the X-Linked ELK-1 Gene. Cell Rep. 2013;4(2):262–270. 43. Yildirim E, Kirby JE, Brown DE, et al. Xist RNA is a potent suppressor of hematologic cancer in mice. Cell. 2013;152(4):727–742. 44. Dominici M, Le Blanc K, Mueller I, et  al. Minimal criteria for defining multipotent mesenchymal stromal cells. The

International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315–317. 45. National Institutes of Health (US). < https://clinicaltrials.gov/ ct2/results?term=mesenchymal+stem+cell>; Accessed 15.5.16. 46. D’souza N, Rossignoli F, Golinelli G, et al. Mesenchymal stem/ stromal cells as a delivery platform in cell and gene therapies. BMC Med. 2015:13. 47. Squillaro T, Peluso G, Galderisi U. Clinical Trials With Mesenchymal Stem Cells: An Update. Cell Transplant. 2016;25(5):829–848. 48. Shapiro G, Kallai I, Sheyn D, et al. Ultrasound-mediated transgene expression in endogenous stem cells recruited to bone injury sites. Polym Adv Technol. 2014;25(5):525–531. 49. Sheyn D, Shapiro G, Tawackoli W, et  al. PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries. Mol Ther. 2016;24(2): 318–330. 50. Escacena N, Quesada-Hernandez E, Capilla-Gonzalez V, Soria B, Hmadcha A. Bottlenecks in the Efficient Use of Advanced Therapy Medicinal Products Based on Mesenchymal Stromal Cells. Stem Cells Int. 2015;2015:895714. 51. Faustini M, Bucco M, Chlapanidas T, et al. Nonexpanded mesenchymal stem cells for regenerative medicine: yield in stromal vascular fraction from adipose tissues. Tissue Eng Part C Methods. 2010;16(6):1515–1521. 52. Jones E, Schafer R. Where is the common ground between bone marrow mesenchymal stem/stromal cells from different donors and species? Stem Cell Res Ther. 2015;6:143. 53. Seebach C, Henrich D, Tewksbury R, Wilhelm K, Marzi I. Number and proliferative capacity of human mesenchymal stem cells are modulated positively in multiple trauma patients and negatively in atrophic nonunions. Calcif Tissue Int. 2007;80(4):294–300. 54. Zanotti S, Kalajzic I, Aguila HL, Canalis E. Sex and genetic factors determine osteoblastic differentiation potential of murine bone marrow stromal cells. PloS One. 2014;9(1):e86757. 55. Strube P, Mehta M, Baerenwaldt A, et  al. Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity. Bone. 2009;45(6):1065–1072. 56. Hernigou P, Homma Y, Flouzat Lachaniette CH, et al. Benefits of small volume and small syringe for bone marrow aspirations of mesenchymal stem cells. Int Orthop. 2013;37(11):2279–2287. 57. Dexheimer V, Mueller S, Braatz F, Richter W. Reduced reactivation from dormancy but maintained lineage choice of human mesenchymal stem cells with donor age. PloS One. 2011;6(8):e22980. 58. Fossett E, Khan WS. Optimising human mesenchymal stem cell numbers for clinical application: a literature review. Stem Cells Int. 2012;2012:465259. 59. Semirale AA, Zhang XW, Wiren KM. Body composition changes and inhibition of fat development in vivo implicates androgen in regulation of stem cell lineage allocation. J Cell Biochem. 2011;112(7):1773–1786. 60. Siegel G, Kluba T, Hermanutz-Klein U, Bieback K, Northoff H, Schafer R. Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells. BMC Med. 2013;11:146. 61. DiSilvio L, Jameson J, Gamie Z, Giannoudis PV, Tsiridis E. In vitro evaluation of the direct effect of estradiol on human osteoblasts (HOB) and human mesenchymal stem cells (h-MSCs). Injury. 2006;37(Suppl 3):S33–S42. 62. Hong L, Sultana H, Paulius K, Zhang G. Steroid regulation of proliferation and osteogenic differentiation of bone marrow stromal cells: a gender difference. J Steroid Biochem Mol Biol. 2009;114(3–5):180–185.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

63. Cha Y, Kwon SJ, Seol W, Park KS. Estrogen receptor-alpha mediates the effects of estradiol on telomerase activity in human mesenchymal stem cells. Mol Cells. 2008;26(5):454–458. 64. Aksu AE, Rubin JP, Dudas JR, Marra KG. Role of gender and anatomical region on induction of osteogenic differentiation of human adipose-derived stem cells. Ann Plast Surg. 2008;60(3):306–322. 65. Dudas JR, Losee JE, Penascino VM, et  al. Leporine-derived adipose precursor cells exhibit in vitro osteogenic potential. J Craniofac Surg. 2008;19(2):360–368. 66. Morita E, Watanabe Y, Ishimoto M, et al. A novel cell transplantation protocol and its application to an ALS mouse model. Exp Neurol. 2008;213(2):431–438. 67. Payne KA, Didiano DM, Chu CR. Donor sex and age influence the chondrogenic potential of human femoral bone marrow stem cells. Osteoarthritis Cartilage. 2010;18(5):705–713. 68. Yuan J, Yu JX, Ge J. Sexual dimorphism on the neurogenic potential of rhesus monkeys mesenchymal stem cells. Biochem Biophys Res Commun. 2010;396(2):394–400. 69. Leskela HV, Risteli J, Niskanen S, Koivunen J, Ivaska KK, Lehenkari P. Osteoblast recruitment from stem cells does not decrease by age at late adulthood. Biochem Biophys Res Commun. 2003;311(4):1008–1013. 70. Muschler GF, Nitto H, Boehm CA, Easley KA. Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors. J Orthop Res. 2001;19(1):117–125. 71. Bragdon B, Burns R, Baker AH, et  al. Intrinsic Sex-Linked Variations in Osteogenic and Adipogenic Differentiation Potential of Bone Marrow Multipotent Stromal Cells. J Cell Physiol. 2015;230(2):296–307. 72. Ishida Y, Heersche JN. Progesterone stimulates proliferation and differentiation of osteoprogenitor cells in bone cell populations derived from adult female but not from adult male rats. Bone. 1997;20(1):17–25. 73. Hong L, Wei N, Joshi V, et al. Effects of glucocorticoid receptor small interfering RNA delivered using poly lactic-co-glycolic acid microparticles on proliferation and differentiation capabilities of human mesenchymal stromal cells. Tissue Eng Part A. 2012;18(7-8):775–784. 74. Hong L, Krishnamachari Y, Seabold D, Joshi V, Schneider G, Salem AK. Intracellular release of 17-beta estradiol from cationic polyamidoamine dendrimer surface-modified poly (lactic-coglycolic acid) microparticles improves osteogenic differentiation of human mesenchymal stromal cells. Tissue Eng Part C, Methods. 2011;17(3):319–325. 75. Moazzaz P, Gupta MC, Gilotra MM, et al. Estrogen-dependent actions of bone morphogenetic protein-7 on spine fusion in rats. Spine. 2005;30(15):1706–1711. 76. Leskela HV, Olkku A, Lehtonen S, et  al. Estrogen receptor alpha genotype confers interindividual variability of response to estrogen and testosterone in mesenchymal-stem-cell-derived osteoblasts. Bone. 2006;39(5):1026–1034. 77. Dai Z, Li Y, Quarles LD, et  al. Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation. Phytomedicine. 2007;14(12):806–814. 78. Heim M, Frank O, Kampmann G, et  al. The phytoestrogen genistein enhances osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells. Endocrinology. 2004;145(2):848–859. 79. Sillat T, Pollanen R, Lopes JR, et al. Intracrine androgenic apparatus in human bone marrow stromal cells. J Cell Mol Med. 2009;13(9B):3296–3302. 80. Olivares-Navarrete R, Hyzy SL, Boyan BD, Schwartz Z. Regulation of Osteoblast Differentiation by Acid-Etched and/

455

or Grit-Blasted Titanium Substrate Topography Is Enhanced by 1,25(OH)2D3 in a Sex-Dependent Manner. Biomed Res Int. 2015;2015:365014. 81. Ray R, Herring CM, Markel TA, et  al. Deleterious effects of endogenous and exogenous testosterone on mesenchymal stem cell VEGF production. Am J Physiol Regul Integr Comp Physiol. 2008;294(5):R1498–R1503. 82. Crisostomo PR, Wang M, Herring CM, et  al. Sex dimorphisms in activated mesenchymal stem cell function. Shock. 2006;26(6):571–574. 83. Crisostomo PR, Wang M, Herring CM, et al. Gender differences in injury induced mesenchymal stem cell apoptosis and VEGF, TNF, IL-6 expression: role of the 55 kDa TNF receptor (TNFR1). J Mol Cell Cardiol. 2007;42(1):142–149. 84. Markel TA, Crisostomo PR, Wang M, et  al. TNFR1 signaling resistance associated with female stem cell cytokine production is independent of TNFR2-mediated pathways. Am J Physiol Regul Integr Comp Physiol. 2008;295(4):R1124–R1130. 85. Crisostomo PR, Markel TA, Wang M, Lahm T, Lillemoe KD, Meldrum DR. In the adult mesenchymal stem cell population, source gender is a biologically relevant aspect of protective power. Surgery. 2007;142(2):215–221. 86. Erwin GS, Crisostomo PR, Wang Y, et al. Estradiol-treated mesenchymal stem cells improve myocardial recovery after ischemia. J Surg Res. 2009;152(2):319–324. 87. Manukyan MC, Weil BR, Wang Y, et  al. Female stem cells are superior to males in preserving myocardial function following endotoxemia. Am J Physiol Regul Integr Comp Physiol. 2011;300(6):R1506–R1514. 88. Becker AJ, Mc CE, Till JE. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature. 1963;197:452–454. 89. Jacobson LO, Marks EK, et al. The role of the spleen in radiation injury. Proc Soc Exp Biol Med. 1949;70(4):740–742. 90. Gratwohl A, Baldomero H, Aljurf M, et  al. Hematopoietic stem cell transplantation: a global perspective. JAMA. 2010;303(16):1617–1624. 91. Billen A, Madrigal JA, Szydlo RM, Shaw BE. Female donors and donors who are lighter than their recipient are less likely to meet the CD34+ cell dose requested for peripheral blood stem cell transplantation. Transfusion. 2014;54(11): 2953–2960. 92. Pulsipher MA, Chitphakdithai P, Logan BR, et al. Donor, recipient, and transplant characteristics as risk factors after unrelated donor PBSC transplantation: beneficial effects of higher CD34+ cell dose. Blood. 2009;114(13):2606–2616. 93. Pulsipher MA, Chitphakdithai P, Logan BR, et  al. Acute toxicities of unrelated bone marrow versus peripheral blood stem cell donation: results of a prospective trial from the National Marrow Donor Program. Blood. 2013;121(1):197–206. 94. Pond GR, Lipton JH, Messner HA. Long-term survival after blood and marrow transplantation: comparison with an ageand gender-matched normative population. Biol Blood Marrow Transplant. 2006;12(4):422–429. 95. Gahrton G, Iacobelli S, Apperley J, et  al. The impact of donor gender on outcome of allogeneic hematopoietic stem cell transplantation for multiple myeloma: reduced relapse risk in female to male transplants. Bone Marrow Transplant. 2005; 35(6):609–617. 96. Kollman C, Spellman SR, Zhang MJ, et al. The effect of donor characteristics on survival after unrelated donor transplantation for hematologic malignancy. Blood. 2016;127(2):260–267. 97. Stern M, Brand R, de Witte T, et  al. Female-versus-male alloreactivity as a model for minor histocompatibility antigens in hematopoietic stem cell transplantation. Am J Transplant. 2008;8(10):2149–2157.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

456

31.  BIOLOGICAL SEX IN STEM CELL THERAPIES

98. Gratwohl A, Stern M, Brand R, et al. Risk score for outcome after allogeneic hematopoietic stem cell transplantation: a retrospective analysis. Cancer. 2009;115(20):4715–4726. 99. Stern M, Brand R, de Witte T, et  al. Female-versus-male alloreactivity as a model for minor histocompatibility antigens in hematopoietic stem cell transplantation. Am J Transplant. 2008;8(10):2149–2157. 100. Kongtim P, Di Stasi A, Rondon G, et  al. Can a female donor for a male recipient decrease the relapse rate for patients with acute myeloid leukemia treated with allogeneic hematopoietic stem cell transplantation? Biol Blood Marrow Transplant. 2015;21(4):713–719. 101. Kim YH, Faaij CM, van Halteren AG, et al. In situ detection of HY-specific T cells in acute graft-versus-host disease-affected male skin after sex-mismatched stem cell transplantation. Biol Blood Marrow Transplant. 2012;18(3):381–387. 102. Dickinson AM, Wang XN, Sviland L, et al. In situ dissection of the graft-versus-host activities of cytotoxic T cells specific for minor histocompatibility antigens. Nat Med. 2002;8(4):410–414. 103. van Halteren AG, Dierselhuis MP, Netelenbos T, Fechter M. Donor parity no longer a barrier for female-to-male hematopoietic stem cell transplantation. Chimerism. 2014;5(2):56–58. 104. Lissauer D, Piper K, Goodyear O, Kilby MD, Moss PA. Fetalspecific CD8+ cytotoxic T cell responses develop during normal human pregnancy and exhibit broad functional capacity. J Immunol. 2012;189(2):1072–1080. 105. Dierselhuis MP, Jankowska-Gan E, Blokland E, et al. HY immune tolerance is common in women without male offspring. PloS One. 2014;9(3):e91274. 106. van Halteren AG, Jankowska-Gan E, Joosten A, et  al. Naturally acquired tolerance and sensitization to minor histocompatibility antigens in healthy family members. Blood. 2009;114(11):2263–2272. 107. Verdijk RM, Kloosterman A, Pool J, et  al. Pregnancy induces minor histocompatibility antigen-specific cytotoxic T cells: implications for stem cell transplantation and immunotherapy. Blood. 2004;103(5):1961–1964. 108. Dierselhuis MP, Blokland EC, Pool J, Schrama E, Scherjon SA, Goulmy E. Transmaternal cell flow leads to antigen-experienced cord blood. Blood. 2012;120(3):505–510. 109. Fozza C, Dazzi F. Regulatory T cells in stem cell transplantation: main characters or walk-on actors? Crit Rev Oncol Hematol. 2012;84(1):18–25. 110. Ichinohe T, Maruya E, Saji H. Long-term feto-maternal microchimerism: nature’s hidden clue for alternative donor hematopoietic cell transplantation? Int J Hematol. 2002;76(3):229–237. 111. Abdelbaset-Ismail A, Suszynska M, Borkowska S, et al. Human haematopoietic stem/progenitor cells express several functional sex hormone receptors. J Cell Mol Med. 2016;20(1):134–146. 112. Khong DM, Dudakov JA, Hammett MV, et al. Enhanced hematopoietic stem cell function mediates immune regeneration following sex steroid blockade. Stem Cell Rep. 2015;4(3):445–458. 113. Zoller AL, Kersh GJ. Estrogen induces thymic atrophy by eliminating early thymic progenitors and inhibiting proliferation of beta-selected thymocytes. J Immunol. 2006;176(12):7371–7378. 114. Nakada D, Oguro H, Levi BP, et  al. Oestrogen increases haematopoietic stem-cell self-renewal in females and during pregnancy. Nature. 2014;505(7484):555–558. 115. Li H, Liu J, Ye X, et  al. 17beta-Estradiol enhances the recruitment of bone marrow-derived endothelial progenitor cells into infarcted myocardium by inducing CXCR4 expression. Int J Cardiol. 2013;162(2):100–106. 116. Vasanthakumar A, Zullow H, Lepore JB, et al. Epigenetic Control of Apolipoprotein E Expression Mediates Gender-Specific Hematopoietic Regulation. Stem Cells. 2015;33(12):3643–3654.

117. Zbucka-Kretowska M, Eljaszewicz A, Lipinska D, et al. Effective Mobilization of Very Small Embryonic-Like Stem Cells and Hematopoietic Stem/Progenitor Cells but Not Endothelial Progenitor Cells by Follicle-Stimulating Hormone Therapy. Stem Cells Int. 2016;2016:8530207. 118. Laker RC, Ryall JG. DNA Methylation in Skeletal Muscle Stem Cell Specification, Proliferation, and Differentiation. Stem Cells Int. 2016;2016:5725927. 119. Qu-Petersen Z, Deasy B, Jankowski R, et  al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol. 2002;157(5):851–864. 120. Deasy BM, Gharaibeh BM, Pollett JB, et  al. Long-term selfrenewal of postnatal muscle-derived stem cells. Mol Biol Cell. 2005;16(7):3323–3333. 121. Usas A, Maciulaitis J, Maciulaitis R, Jakuboniene N, Milasius A, Huard J. Skeletal muscle-derived stem cells: implications for cell-mediated therapies. Medicina. 2011;47(9):469–479. 122. Jankowski RJ, Deasy BM, Huard J. Muscle-derived stem cells. Gene Ther. 2002;9(10):642–647. 123. Deasy BM, Lu A, Tebbets JC, et al. A role for cell sex in stem cellmediated skeletal muscle regeneration: female cells have higher muscle regeneration efficiency. J Cell Biol. 2007;177(1):73–86. 124. Wright V, Peng H, Usas A, et  al. BMP4-expressing musclederived stem cells differentiate into osteogenic lineage and improve bone healing in immunocompetent mice. Mol Ther. 2002;6(2):169–178. 125. Matsumoto T, Cooper GM, Gharaibeh B, et  al. Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle-derived stem cells expressing bone morphogenetic protein 4 and soluble Flt-1. Arthritis Rheum. 2009;60(5):1390–1405. 126. Lavasani M, Thompson SD, Pollett JB, et  al. Human musclederived stem/progenitor cells promote functional murine peripheral nerve regeneration. J Clin Invest. 2014;124(4):1745–1756. 127. Stolting MN, Hefermehl LJ, Tremp M, Azzabi F, Sulser T, Eberli D. The role of donor age and gender in the success of human muscle precursor cell transplantation. J Tissue Eng Regen Med. 2014. 128. Riggs BL, Khosla S, Melton 3rd LJ. Sex steroids and the construction and conservation of the adult skeleton. Endocrine Rev. 2002;23(3):279–302. 129. Vanderschueren D, Vandenput L, Boonen S, Lindberg MK, Bouillon R, Ohlsson C. Androgens and bone. Endocrine Rev. 2004;25(3):389–425. 130. Abu EO, Horner A, Kusec V, Triffitt JT, Compston JE. The localization of androgen receptors in human bone. J Clin Endocrinol Metab. 1997;82(10):3493–3497. 131. Yang X, Schadt EE, Wang S, et al. Tissue-specific expression and regulation of sexually dimorphic genes in mice. Genome Res. 2006;16(8):995–1004. 132. Clancy BM, Johnson JD, Lambert AJ, et  al. A gene expression profile for endochondral bone formation: oligonucleotide microarrays establish novel connections between known genes and BMP-2-induced bone formation in mouse quadriceps. Bone. 2003;33(1):46–63. 133. Corsi KA, Pollett JB, Phillippi JA, Usas A, Li G, Huard J. Osteogenic potential of postnatal skeletal muscle-derived stem cells is influenced by donor sex. J Bone Miner Res. 2007;22(10):1592–1602. 134. Meszaros LB, Usas A, Cooper GM, Huard J. Effect of host sex and sex hormones on muscle-derived stem cell-mediated bone formation and defect healing. Tissue Eng Part A. 2012;18(17–18):1751–1759. 135. Matsumoto T, Kubo S, Meszaros LB, et al. The influence of sex on the chondrogenic potential of muscle-derived stem cells:

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

136.

137.

138.

139.

140.

141.

142.

143. 144.

145.

146.

147.

148.

149.

150.

151.

152.

implications for cartilage regeneration and repair. Arthritis Rheum. 2008;58(12):3809–3819. Balistreri CR, Buffa S, Pisano C, Lio D, Ruvolo G, Mazzesi G. Are Endothelial Progenitor Cells the Real Solution for Cardiovascular Diseases? Focus on Controversies and Perspectives. Biomed Res Int. 2015;2015:835934. Gersh BJ, Simari RD, Behfar A, Terzic CM, Terzic A. Cardiac cell repair therapy: a clinical perspective. Mayo Clinic Proc. 2009;84(10):876–892. Wollert KC, Meyer GP, Lotz J, et  al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet. 2004;364(9429):141–148. Schachinger V, Tonn T, Dimmeler S, Zeiher AM. Bone-marrowderived progenitor cell therapy in need of proof of concept: design of the REPAIR-AMI trial. Nat Clin Pract Cardiovascr Med. 2006;3(Suppl 1)):S23–S28. Fuchs S, Kornowski R, Weisz G, et  al. Safety and feasibility of transendocardial autologous bone marrow cell transplantation in patients with advanced heart disease. Am J Cardiol. 2006;97(6):823–829. Perin EC, Dohmann HF, Borojevic R, et  al. Improved exercise capacity and ischemia 6 and 12 months after transendocardial injection of autologous bone marrow mononuclear cells for ischemic cardiomyopathy. Circulation. 2004;110(11 Suppl 1):II213–II218. Asahara T, Murohara T, Sullivan A, et  al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275(5302):964–967. Urbich C, Dimmeler S. Endothelial progenitor cells functional characterization. Trends Cardiovasc Med. 2004;14(8):318–322. Werner N, Kosiol S, Schiegl T, et  al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med. 2005;353(10):999–1007. Stauffer BL, Maceneaney OJ, Kushner EJ, et  al. Gender and Endothelial Progenitor Cell Number in Middle-Aged Adults. Artery Res. 2008;2(4):156–160. Fadini GP, de Kreutzenberg S, Albiero M, et al. Gender differences in endothelial progenitor cells and cardiovascular risk profile: the role of female estrogens. Arterioscler Thromb Vasc Biol. 2008;28(5):997–1004. Hoetzer GL, MacEneaney OJ, Irmiger HM, et al. Gender differences in circulating endothelial progenitor cell colony-forming capacity and migratory activity in middle-aged adults. Am J Cardiol. 2007;99(1):46–48. Zenovich AG, Panoskaltsis-Mortari A, Caron GJ, et al. Sex-based differences in vascular repair with bone marrow cell therapy: relevance of regulatory and Th2-type cytokines. Transplant Proc. 2008;40(2):641–643. Hamada H, Kim MK, Iwakura A, et al. Estrogen receptors alpha and beta mediate contribution of bone marrow-derived endothelial progenitor cells to functional recovery after myocardial infarction. Circulation. 2006;114(21):2261–2270. Iwakura A, Shastry S, Luedemann C, et al. Estradiol enhances recovery after myocardial infarction by augmenting incorporation of bone marrow-derived endothelial progenitor cells into sites of ischemia-induced neovascularization via endothelial nitric oxide synthase-mediated activation of matrix metalloproteinase-9. Circulation. 2006;113(12):1605–1614. Miller AP, Feng W, Xing D, et al. Estrogen modulates inflammatory mediator expression and neutrophil chemotaxis in injured arteries. Circulation. 2004;110(12):1664–1669. Ruifrok WP, de Boer RA, Iwakura A, et  al. Estradiol-induced, endothelial progenitor cell-mediated neovascularization in male mice with hind-limb ischemia. Vasc Med. 2009;14(1):29–36.

457

153. Nelson HD, Walker M, Zakher B, Mitchell J. Menopausal hormone therapy for the primary prevention of chronic conditions: a systematic review to update the U.S. Preventive Services Task Force recommendations. Ann Int Med. 2012;157(2):104–113. 154. Ye Y, Li X, Zhang Y, Shen Z, Yang J. Androgen Modulates Functions of Endothelial Progenitor Cells through Activated Egr1 Signaling. Stem Cells Int. 2016;2016:7057894. 155. Yoshida S, Aihara K, Ikeda Y, et al. Androgen receptor promotes sex-independent angiogenesis in response to ischemia and is required for activation of vascular endothelial growth factor receptor signaling. Circulation. 2013;128(1):60–71. 156. Foresta C, Zuccarello D, De Toni L, Garolla A, Caretta N, Ferlin A. Androgens stimulate endothelial progenitor cells through an androgen receptor-mediated pathway. Clin Endocrinol. 2008;68(2):284–289. 157. Fadini GP, Albiero M, Cignarella A, et al. Effects of androgens on endothelial progenitor cells in vitro and in vivo. Clin Sci. 2009;117(10):355–364. 158. Pedersen PM, Jorgensen HS, Nakayama H, Raaschou HO, Olsen TS. Aphasia in acute stroke: incidence, determinants, and recovery. Ann Neurol. 1995;38(4):659–666. 159. Zigmond MJ, Abercrombie ED, Berger TW, Grace AA, Stricker EM. Compensations after lesions of central dopaminergic neurons: some clinical and basic implications. Trends Neurosci. 1990;13(7):290–296. 160. Menken M, Munsat TL, Toole JF. The global burden of disease study: implications for neurology. Arch Neurol. 2000;57(3):418–420. 161. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992;255(5052):1707–1710. 162. Martinez-Morales PL, Revilla A, Ocana I, et al. Progress in stem cell therapy for major human neurological disorders. Stem Cell Rev. 2013;9(5):685–699. 163. Trounson A, McDonald C. Stem Cell Therapies in Clinical Trials: Progress and Challenges. Cell Stem Cell. 2015;17(1):11–22. 164. Lecanu L. Sex, the underestimated potential determining factor in brain tissue repair strategy. Stem Cells Dev. 2011;20(12):2031–2035. 165. Waldron J, McCourty A, Lecanu L. Aging differentially affects male and female neural stem cell neurogenic properties. Stem Cells Cloning. 2010;3:119–127. 166. Waldron J, McCourty A, Lecanu L. Neural stem cell sex dimorphism in aromatase (CYP19) expression: a basis for differential neural fate. Stem Cells Cloning. 2010;3:175–182. 167. Waldron J, Lecanu L. Age and sex differences in neural stem cell transplantation: a descriptive study in rats. Stem Cells Cloning. 2011;4:25–37. 168. Engler-Chiurazzi EB, Singh M, Simpkins JW. From the 90s to now: a brief historical perspective on more than two decades of estrogen neuroprotection. Brain Res. 2016;1633:96–100. 169. Pawluski JL, Brummelte S, Barha CK, Crozier TM, Galea LA. Effects of steroid hormones on neurogenesis in the hippocampus of the adult female rodent during the estrous cycle, pregnancy, lactation and aging. Front Neuroendocrinol. 2009;30(3):343–357. 170. Martinez-Cerdeno V, Noctor SC, Kriegstein AR. Estradiol stimulates progenitor cell division in the ventricular and subventricular zones of the embryonic neocortex. Eur J Neurosci. 2006;24(12):3475–3488. 171. Oboti L, Ibarra-Soria X, Perez-Gomez A, et  al. Pregnancy and estrogen enhance neural progenitor-cell proliferation in the vomeronasal sensory epithelium. BMC Biol. 2015;13:104. 172. Suzuki S, Gerhold LM, Bottner M, et al. Estradiol enhances neurogenesis following ischemic stroke through estrogen receptors alpha and beta. J Comp Neurol. 2007;500(6):1064–1075.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

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173. Kishi Y, Takahashi J, Koyanagi M, et al. Estrogen promotes differentiation and survival of dopaminergic neurons derived from human neural stem cells. J Neurosci Res. 2005;79(3):279–286. 174. Okada M, Makino A, Nakajima M, Okuyama S, Furukawa S, Furukawa Y. Estrogen stimulates proliferation and differentiation of neural stem/progenitor cells through different signal transduction pathways. Int J Mol Sci. 2010;11(10):4114–4123. 175. Barker JM, Galea LA. Repeated estradiol administration alters different aspects of neurogenesis and cell death in the hippocampus of female, but not male, rats. Neuroscience. 2008;152(4):888–902. 176. Singh M, Su C. Progesterone-induced neuroprotection: factors that may predict therapeutic efficacy. Brain Res. 2013;1514:98–106. 177. Liu L, Wang J, Zhao L, et al. Progesterone increases rat neural progenitor cell cycle gene expression and proliferation via extracellularly regulated kinase and progesterone receptor membrane components 1 and 2. Endocrinology. 2009;150(7):3186–3196. 178. Liu L, Zhao L, She H, et al. Clinically relevant progestins regulate neurogenic and neuroprotective responses in vitro and in vivo. Endocrinology. 2010;151(12):5782–5794. 179. Ransome MI, Boon WC. Testosterone-induced adult neurosphere growth is mediated by sexually-dimorphic aromatase expression. Front Cell Neurosci. 2015;9:253. 180. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, et  al. The malespecific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature. 2003;423(6942):825–837. 181. Xu J, Burgoyne PS, Arnold AP. Sex differences in sex chromosome gene expression in mouse brain. Hum Mol Genet. 2002;11(12):1409–1419. 182. Sekido R, Lovell-Badge R. Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer. Nature. 2008;453(7197):930–934. 183. Wegner M, Stolt CC. From stem cells to neurons and glia: a Soxist’s view of neural development. Trends Neurosci. 2005;28(11):583–588. 184. McCombe PA, Henderson RD. Effects of gender in amyotrophic lateral sclerosis. Gend Med. 2010;7(6):557–570. 185. Choi CI, Lee YD, Gwag BJ, Cho SI, Kim SS, Suh-Kim H. Effects of estrogen on lifespan and motor functions in female hSOD1 G93A transgenic mice. J Neurol Sci. 2008;268(1-2):40–47. 186. Hayes-Punzo A, Mulcrone P, Meyer M, McHugh J, Svendsen CN, Suzuki M. Gonadectomy and dehydroepiandrosterone (DHEA) do not modulate disease progression in the G93A mutant SOD1 rat model of amyotrophic lateral sclerosis. Amyotrop Lateral Scler. 2012;13(3):311–314.

187. Li R, Strykowski R, Meyer M, Mulcrone P, Krakora D, Suzuki M. Male-specific differences in proliferation, neurogenesis, and sensitivity to oxidative stress in neural progenitor cells derived from a rat model of ALS. PloS One. 2012;7(11):e48581. 188. Knippenberg S, Rath KJ, Boselt S, et al. Intraspinal administration of human spinal cord-derived neural progenitor cells in the G93A-SOD1 mouse model of ALS delays symptom progression, prolongs survival and increases expression of endogenous neurotrophic factors. J Tissue Eng Regener Med. 2015. 189. Collins FS, Tabak LA. Policy: NIH plans to enhance reproducibility. Nature. 2014;505(7485):612–613. 190. Phillips SP, Hamberg K. Doubly blind: a systematic review of gender in randomised controlled trials. Glob Health Action. 2016;9:29597. 191. Leopold SS, Beadling L, Dobbs MB, et  al. Fairness to all: gender and sex in scientific reporting. Clin Orthop Related Res. 2014;472(2):391–392. 192. El-Shormilisy N, Strong J, Meredith PJ. Associations between gender, coping patterns and functioning for individuals with chronic pain: a systematic review. Pain Res Manag. 2015;20(1):48–55. 193. Mazure CM, Jones DP. Twenty years and still counting: including women as participants and studying sex and gender in biomedical research. BMC Womens Health. 2015;15:94. 194. Ritz SA, Antle DM, Cote J, et al. First steps for integrating sex and gender considerations into basic experimental biomedical research. FASEB J. 2014;28(1):4–13. 195. Taylor KE, Vallejo-Giraldo C, Schaible NS, Zakeri R, Miller VM. Reporting of sex as a variable in cardiovascular studies using cultured cells. Biol Sex Differ. 2011;2:11. 196. Arain FA, Kuniyoshi FH, Abdalrhim AD, Miller VM. Sex/gender medicine. The biological basis for personalized care in cardiovascular medicine. Circ J. 2009;73(10):1774–1782. 197. Florez-Vargas O, Brass A, Karystianis G, et al. Bias in the reporting of sex and age in biomedical research on mouse models. Elife. 2016:5. 198. McCullough LD, de Vries GJ, Miller VM, Becker JB, Sandberg K, McCarthy MM. NIH initiative to balance sex of animals in preclinical studies: generative questions to guide policy, implementation, and metrics. Biol Sex Differ. 2014;5:15. 199. Stanford. ; Accessed 6.12.16. 200. Klein SL, Schiebinger L, Stefanick ML, et al. Opinion: Sex inclusion in basic research drives discovery. Proc Natl Acad Sci USA. 2015;112(17):5257–5258.

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C H A P T E R

32 Adipose-Derived Stem Cells in Regenerative Medicine Hiroshi Mizuno1, Morikuni Tobita1, Rei Ogawa2, Hakan Orbay3,4, Juri Fujimura2, Shimpei Ono2, Natsuko Kakudo5, Kenji Kusumoto5 and Hiko Hyakusoku2 1

Juntendo University School of Medicine, Tokyo, Japan, 2Nippon Medical School, Tokyo, Japan, 3University of California, Davis, CA, United States, 4University of California, Sacramento, CA, United States, 5Kansai Medical University, Moriguchi, Japan

O U T L I N E 32.6.1 Adipose Tissue 32.6.2 Musculoskeletal Tissue 32.6.3 Nerve Tissue 32.6.4 Cardiovascular Tissue 32.6.5 Liver Tissue 32.6.6 Skin and Wound Healing 32.6.7 Periodontal Tissue

32.1 Introduction—Approaches of Adipose-Derived Stem Cells for Cell-based Therapeutics 460 32.2 Localization and Isolation of Adipose-Derived Stem Cells From Adipose Tissue 32.2.1 Functional and Content of Adipose Tissue 32.2.2 Localization of Adipose-Derived Stromal/Stem cells 32.2.3 Isolation for ASCs 32.3 Cellular Characterization and Soluble Factors 32.3.1 Cell Surface Marker Expression 32.3.2 Soluble Factor Secretion 32.3.3 Immunomodulatory Effects of ASCs

460 460 461 461 462 462 463 463

32.4 The Proliferation Capacity of Adipose-Derived Stem Cells 463 32.4.1 Proliferation Capacity of ASCs 463 32.4.2 ASC Growth Stimulation Factors 464 32.4.3 Culturing ASCs for Clinical Application 464 32.5 Differentiation Capacity of Adipose-Derived Stem Cells 32.5.1 Ectodermal Lineage Cells 32.5.2 Mesodermal Lineage Cells 32.5.3 Endodermal Lineage Cells

465 466 467 469

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Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00050-4

32.7 Ongoing Clinical Trials and Future Direction of Adipose-Derived Stem Cells in Regenerative Medicine 32.7.1 Acute Myocardial Infarction and Chronic Heart Diseases 32.7.2 Thoracic Repair 32.7.3 Bone Regeneration and Repair 32.7.4 Adipose Tissue Regeneration 32.7.5 Chronic Intractable Fistula 32.7.6 Cutaneous Wound Healing 32.7.7 Stress Urinary Incontinence Repair 32.7.8 Immunomodulatory Effects and Graft Versus Host Diseases 32.7.9 Safety Concern and Future Direction of ASCs in Regenerative Medicine 32.8 Conclusions

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32.1 INTRODUCTION—APPROACHES OF ADIPOSE-DERIVED STEM CELLS FOR CELL-BASED THERAPEUTICS There are a variety of life-threatening diseases such as organ failure, tissue loss due to trauma, cancer abrasion, and congenital structural anomalies. Although most of these disorders can be treated by current clinical technologies, including organ transplantation, autologous tissue transfer, and the use of artificial materials, there are potential problems, e.g., organ shortage, damage to healthy parts of the body during treatment, allergic reactions, and immune rejection. Recent developments in the emerging field of regenerative medicine may allow the eventual replacement of organs and the repair of tissue damage. In general, the fields of regenerative medicine and tissue engineering require a reliable source of stem cells together with appropriate biomaterial scaffolds and cytokine growth factors. A stem cell is characterized by its ability to self-renew and to differentiate along multiple lineage pathways. A major advantage of the stem cell approach is the supply of an unlimited number of cells having the potential to become a functional organ. Stem cell candidates include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and postnatal adult stem cells. Although the therapeutic potential of ESCs and iPSCs is enormous due to their autoreproducibility and pluripotentiality, there are still some limitations to their practical use, e.g., cell regulation, genetic manipulation, and ethical considerations.1,2 In contrast, postnatal adult stem cells are, by their nature, immunocompatible, and there are no ethical issues related to their use. Mesenchymal stem cells (MSCs), isolated from bone marrow stroma, are representative of adult stem cells, and possess adipogenic, osteogenic, chondrogenic, myogenic, and neurogenic potential in vitro.3–5 However, recently, MSCs with similar characteristics to bone marrowderived MSCs have been isolated from different tissue sources, including trabecular bone, muscle, periosteum, synovial membrane, articular cartilage, skin, pericytes, peripheral blood, deciduous teeth, periodontal ligament, and umbilical cord. Although the stem cell populations derived from these sources are valuable, common problems include low numbers of harvested cells and limited amounts of harvested tissues. In fact, the population of bone marrow-derived MSCs within the bone marrow stromal cell population is limited and decreases with aging (approximately 1:10,000 cells in newborns to 1:2,000,000 cells in octogenarians). Such a limited supply of stem cells means that ex vivo expansion would be required to obtain a sufficient quantity of cells for clinical applications, which, in turn, would mean extended incubation periods with the potential risk of contamination.

Adipose tissue is present in abundance in many depots throughout the body where it plays a major role in energy balance and displays strong endocrine functions.6 According to the American Society for Aesthetic Plastic Surgery, nearly half million elective liposuction surgeries are performed each year in the United States.7 Since such adipose tissue is a source of stem cells, termed adipose-derived stem cells or ASCs,8 it is regarded as one of the most promising sources for future cell-based therapies and regenerative medicine for the following reasons: (1) when compared to other stem cell populations and sources, ASCs can be isolated easily and obtained in higher yields; for instance, 1 g of adipose tissue yields approximately 5 × 103 stem cells, which is 500-fold greater than the number of MSCs present in 1 g of bone marrow9,10; (2) while ASCs are probably not capable of producing all of the differentiated cells in an organism, unlike ESCs and iPSCs, ASCs avoid the ethical considerations surrounding the use of ESCs and the genetic manipulations necessary to create iPSCs; (3) minimally invasive liposuction can be used to harvest the adipose tissue. This chapter is divided into sections describing the ASC isolation procedure, the underlying biology of ASCs, their current clinical applications, and the future of ASCs in regenerative medicine.

32.2  LOCALIZATION AND ISOLATION OF ADIPOSE-DERIVED STEM CELLS FROM ADIPOSE TISSUE 32.2.1  Functional and Content of Adipose Tissue Adipose tissue is composed mainly of fat cells organized into lobules, and is a highly complex tissue consisting of mature adipocytes that constitute more than 90% of the tissue volume,11 and a stromal vascular cell fraction (SVF), which includes preadipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells, resident monocytes/macrophages, and lymphocytes. Unlike most adult tissues, which are normally stable in size, adipose tissue can grow and regress throughout adulthood. In conjunction with this, while the vasculatures of most adult tissues are quiescent, adipose tissue vasculature is active during adipose tissue expansion. Specifically, it has been shown that adipose tissue angiogenesis often precedes adipogenesis12 while the inhibition of angiogenesis results in adipose tissue degeneration.13 Adipose vasculature contains cell populations with extensive proliferative capacities and high differentiation potentials.14 Anatomically, adipose tissue is located at visceral, subcutaneous, intermuscular, and intramuscular sites in

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32.2  Localization and Isolation of Adipose-Derived Stem Cells From Adipose Tissue

mammals. Subcutaneous white adipose tissue is found in the abdomen, hip, thigh, and gluteal locations, and is mostly composed of mature adipocytes each filled with a single lipid droplet that can vary in size. Adipose tissue functions, not only as an energy storage organ, but also as the largest endocrine organ in the body, releasing various adipokines such as leptin and adiponectin.11 Simultaneously, adipose tissue is also able to influence neuroendocrine, endothelial, immunological, hematological, angiogenic, and vascular functions in an endocrine, paracrine, and autocrine manner.6,15,16 Finally, differentiation potential of ASCs toward some mesodermal lineages depends on the gender. Detailed descriptions are made in the following sections.

32.2.2  Localization of Adipose-Derived Stromal/Stem cells The phenotype of bone marrow-derived MSCs has been well documented; however, the phenotypic characterization of ASCs is still in its infancy, and all attempts to establish an exact definition of the ASC phenotype and to discriminate clearly between these cells and fibroblasts have been unsuccessful to date.16 Therefore, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy has proposed a minimum set of four criteria to define human MSCs17: 1. MSCs have to be plastic-adherent when maintained under standard culture conditions. 2. MSCs must have the ability for osteogenic, adipogenic, and chondrogenic differentiation. 3. MSCs must express CD73, CD90, and CD105. 4. MSCs must not express the hematopoietic lineage markers c-kit, CD14, CD11b, CD34, CD45, CD19, CD79, and the human leukocyte antigen (HLA)-DR. In addition, cell sorting was used to demonstrate that cells carrying MSC markers express the same phenotype as pericytes (CD146+, CD34−, CD45−, CD56−). Recently, SVF, isolated from adipose tissue, was found to contain an abundance of CD34+ cells. CD34+ cells are widely distributed among adipocytes and are predominantly associated with vascular structure.13,18 Histological analysis of adipose tissue revealed that CD34+ cells from freshly isolated SVF were CD31–/ CD144−. Recently published data reported that ASCs were CD31–/CD34+, and that the nonendothelial population of these cells occupied a pericytic position.18 However, although CD34+/CD31–/CD144− cells also coexpressed the pericytic markers, chondroitin, sulfate proteoglycan, CD140a, and CD140b, these cells did not express another pericytic marker, 3G5.14 Anatomically, ASCs can be obtained easily by liposuction from several regions of the body including hip,

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thigh, and abdominal regions, and can be grown under standard culture conditions. However, since fat tissues have metabolic characteristics, such as lipolytic activity, fatty acid composition, and gene expression profiles, that differ depending on their anatomical locations, the source of subcutaneous adipose tissue grafts (abdominal subcutaneous vs peripheral-subcutaneous) could have a profound influence on the long-term characteristics of the fat graft.16 Furthermore, ASCs isolated from visceral adipose tissues in rabbits, were reported to have greater osteogenic potential than ASCs isolated from subcutaneous adipose tissue.19 With respect to depot-related differences of ASCs, some researchers have indicated that ASCs, harvested from superficial abdominal regions, are significantly more resistant to apoptosis than ASCs harvested from the upper arm, medial thigh, trochanteric, and superficial deep abdominal depots.20,21

32.2.3  Isolation for ASCs ASCs originate from the SVF of adipose tissue. Freshly isolated SVF cells are a heterogeneous mixture of endothelial cells, smooth muscle cells, pericytes, fibroblasts, mast cells, and preadipocytes.22 Culturing these cells under standard conditions eventually results in the appearance of a relatively homogenous population of mesodermal or mesenchymal cells.8 Generally, excised fat pads are first minced and washed extensively to remove contaminating hematopoietic cells. Then, the tissue fragments are incubated with collagenase and the digest is centrifuged, thereby separating the floating population of mature adipocytes from the pelleted SVF cells.8 A consensus isolation procedure has not yet been formulated/regulated; therefore, the fact that different isolation procedures can affect cells in different ways must be considered. Furthermore, not only can cell viability and differentiation capacity be affected by different isolation techniques, different collagenase batches and centrifugation speeds can also result in the isolation of different cell subsets. Thus, a detailed molecular characterization of the isolated cells has to be performed each time.16 The frequency of proliferating MSCs and the population doubling time are also dependent on the surgical procedure used, with some advantages reported for resection and tumescent liposuction compared with ultrasound-assisted liposuction.23 In a comparison study with aspirated and excised adipose tissue, Eto et  al. reported that aspirated derived adipose tissue had significant tissue damage and a deficiency in the yield of progenitor cells in humans.11 Moreover, the attachment and proliferation capacity are more pronounced in ASCs derived from younger donors than in those derived from older donors, while the differentiation capacity is maintained with aging.24

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Initially, fragments of human tissue were minced by hand; however, with the development of liposuction surgery, this procedure has been simplified. Routinely 107 adipose stromal/stem cells have been isolated with greater than 95% purity from 300 mL of lipoaspirate. In one study comparing lipoaspirate-derived ASCs and bone marrow-derived MSCs from the same patient, no significant differences were observed regarding the yield of adherent stromal cells, growth kinetics, cell senescence, multilineage differentiation capacity, or gene transduction efficiency.25 Recently, ASC isolation procedures were examined for greater collection efficiencies, and improved techniques for isolating viable populations of MSCs from lipoaspirate saline fractions within 30 min, without the use of regents such as collagenase, were described.26 In general, the isolation of ASCs involves a couple of hours of intense effort, requiring specialized equipment and reagents. However, because adding reagents during the isolation process may influence the characterization and differentiation of ASCs, isolation methods need to be examined carefully before clinical use.

32.3  CELLULAR CHARACTERIZATION AND SOLUBLE FACTORS The characterization of ASCs remains a challenge because the white adipose tissues that contain ASCs are distributed all over the body and there is evidence that the differentiation potential of ASCs is dependent on the anatomic location of the fat and the donor’s gender and age.27 Ogawa et  al. showed that ASCs, harvested from female green fluorescent protein (GFP) transgenic mice, are significantly more adipogenic than those harvested from male mice, as assessed by PPAR-γ2 gene expression using real-time PCR.28 Additionally, human ASCs from male donors appear to possess greater osteogenic differentiation potential than ASCs isolated from females.29 Aksu et al. revealed that male ASCs from both superficial and deep adipose layers differentiate at a faster rate and more effectively than female ASCs from both layers as confirmed by Alkaline phosphatase staining and Alizarin red staining.29 The same study also showed that osteogenic differentiation of ASCs from male donors differs according to the harvest site; ASCs isolated from superficial adipose layers differentiate quicker, and to a greater extent, than ASCs isolated from the deep adipose layers of male abdominoplasty specimens.27 Moreover, different ASC-harvesting techniques greatly affect the resulting proportions of various cell types that accompany the ASC population, including leukocytes, red blood cells, endothelial cells, fibroblasts, pericytes, preadipocytes, and mature adipocytes. Contamination with red blood cells is not a problem as

they can be readily dissolved by lytic buffers. However, when lipoaspiration is used to harvest the fat rather than simple excision of solid fats, the resulting intraoperative bleeding elevates the proportion of leukocytes in the preparation. Moreover, liposuction aspirates separate into fatty and fluid portions, both of which contain stem cells. In the paper by Yoshimura et  al., the fatty portion stem cells were called PLA cells (processed lipoaspirate cells), and these were found to differ in cell surface marker phenotype from the LAF (liposuction aspirate fluid) cells.30 Another important issue is that, compared to the in vivo gene expression profile of ASCs in the body, the ASC transcriptome changes constantly as a result of being harvested, isolated, cultured, and passaged. In particular, cell culture conditions markedly affect the gene expression profiles of ASCs, especially the medium and the mechanophysiological environment (e.g., threedimensional (3D) culture, the imposition of mechanical force on the cells, and the degree of oxygenation). Clearly, the effects of donor traits, fat location, harvest methods, and culture conditions on ASC characteristics should be more clearly elucidated.

32.3.1  Cell Surface Marker Expression Compared to ASCs from later passages, freshly isolated and early passage ASCs express higher levels of CD117, HLA-DR, and stem cell-associated markers such as CD34 along with lower levels of stromal cell markers such as CD13, CD29, CD44, CD63, CD73, CD90, CD105, and CD166.27,30–33 While the consequences of the decrease in CD34 expression are not clear, several studies have shown that CD34-positive ASCs have a greater proliferative capacity while CD34-negative ASCs are more plastic.27,34 ASCs share many cell surface markers in common with pericytes and MSCs.27 The pericyte markers that ASCs express include smooth muscle β-actin, plateletderived growth factor (PDGF) receptor-β, and neuroglial proteoglycan 2 (NG2).27,35 The markers that ASCs have in common with MSCs include CD13, CD29 (β1 integrin), CD44, CD58, CD90 (Thy-1), CD105 (endoglin), and CD166. Several commercial FACS-based kits have been developed to isolate human ASCs. One of these is the StemPro Human Adipose-derived Stem Cell Kit (Invitrogen, CA, USA), which uses the following markers to isolate ASCS: positive markers CD29, CD44, CD73, CD90, CD105, and CD166, and negative markers CD14, CD31, CD45, and Lin1. Over 95% and less than 2% of the resulting ASCs bear the positive and negative markers, respectively. Another commercial kit is the Human Adipose-Derived Stem Cells kit (ADSC) (Lonza, Cologne, Germany), which is similar to the Invitrogen kit except that an additional

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32.4  The Proliferation Capacity of Adipose-Derived Stem Cells

positive marker, CD13, is used and the Lin1 negative marker is not used. A third kit, Human Mesenchymal Stem Cells from Adipose Tissue (hMSC-AT) (PromoCell, Heidelberg, Germany), uses CD44 and CD105 as positive markers and CD31 and CD45 as negative markers. Over 95% of the resulting ASCs are positive and negative for these markers, respectively. To simplify the isolation of ASCs from fat or early passages of cell cultures, and to provide a standard ASC cell surface marker profile, it is recommended that, as with the hMSC-AT kit, only CD44 and CD105 should serve as positive markers and only CD31 and CD45 should serve as negative markers. Thus, more than 95% of the ASCs that are to be used for experiments should be CD44+ CD105+ CD31− CD45− cells.

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proliferation, which indicates that they are poorly immunogenic. Moreover, coculture with ASCs suppressed the mixed lymphocyte reaction, even when they were not treated with IFN-γ.36 By using a transwell system, Cui et al. then showed that the immunosuppressive effect of ASCs was not dependent on cell–cell contact.36 To define the soluble factors that mediate the immunomodulatory properties of ASCs, the passaged ASCs that were cocultured with mixed lymphocytes were analyzed and it was found that the coculture significantly increased their secretion of prostaglandin E2 (PGE2) but not TGF- or HGF. These observations indicate that in vitro-expanded ASCs retain their low immunogenicity and immunosuppressive properties, and that PGE2 may be the main soluble factor that mediates the in vitro inhibition of the allogeneic lymphocyte reaction.36

32.3.2  Soluble Factor Secretion Analyses of the human ASC secretome reveal that cultured ASCs at relatively early passages secrete hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β, insulin-like growth factor (IGF)-1, basic fibroblast growth factor (bFGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, interleukins 6, 7, 8, and 11, adiponectin, angiotensin, cathepsin D, pentraxin, pregnancy zone protein, retinol-binding protein, and CXCL12.15,36–40 It has also been shown that the ASC secretome can be modulated by exposure to different agents.15 For example, HGF expression is increased after the cells are exposed to bFGF, epidermal growth factor (EGF), or ascorbic acid.15,41 Thus, it may be that when ASCs are transplanted into inflammatory or ischemic regions, they actively secrete these growth factors, thereby significantly promoting wound healing and tissue repair.

32.4  THE PROLIFERATION CAPACITY OF ADIPOSE-DERIVED STEM CELLS

32.3.3  Immunomodulatory Effects of ASCs

32.4.1  Proliferation Capacity of ASCs

ASCs have been shown to be capable of modulating the effector functions of immune cells and thus may be useful in therapies for immune-mediated diseases. This concept has already been translated into the clinical situation as MSCs have been used to treat severe graft versus host disease (GVHD) that is refractory to conventional therapies.42 Thus, ASCs are also expected to be useful for treating GVHD. The mechanisms by which ASCs modulate immune responses remains to be elucidated but one study has examined this issue. Cui et  al. showed that passaged ASCs expressed HLA class I but not class II molecules, and that high levels of HLA I expression can be induced by treatment with interferon-gamma (IFN-γ).36 In addition, they found that coculture with the passaged (IFNγ-treated or untreated) ASCs did not elicit lymphocyte

The proliferative capacity of ASCs is greater than that of bone marrow-derived MSCs obtained from the same patient.25 The doubling times of ASCs during the logarithmic phase of growth range from 40 to 120 h,8,25 and depend on donor age, type (white or brown tissue), and location (subcutaneous or visceral) of the adipose tissue, culture conditions, the surgical procedure performed, plating density, and media formulations.9,31 The survival of ASCs can be extended by overexpression of the catalytic subunit of the human telomerase gene.45 The younger the donor, the greater will be the proliferation and adhesion of the ASCs;24 however, with passaging, these cells gradually lose their proliferative capacity. The senescence of ASCs, which is based on β-galactosidase activity, is similar to that of bone marrow-derived MSCs.25

ASCs can differentiate into fat, bone, cartilage, tendon, and skeletal muscle when cultivated under lineage-specific conditions. The majority of ASCs exhibit fibroblastic morphological features and expand easily under standard tissue culture conditions.8 Approximately 8 × 108 ASCs can be obtained from 300 mL of liposuctioned fat aspirates,43 while 1 g of adipose tissue contains approximately 5 × 103 ASCs,9,10 which is 500-fold greater than the number of MSCs present in 1 g of bone marrow.44 Because ASCs can be conveniently isolated and show extensive proliferative capacities in vitro, they are a promising source of human stem cells for use in regenerative medicine. This section highlights the in vitro proliferation capacity of ASCs and discusses the factors involved in promoting ASC proliferation.

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In general, it is considered that ASCs are stable throughout long-term culture.46 In a previous study, even in the ASCs that had passed more than 100 population doublings, no abnormalities in the karyotype or diploid karyotype were observed.47 However, Rubio et al. showed that human ASCs undergo malignant transformation when passaged over a period of more than 4 months.48 These results indicate that careful manipulation of ASCs and long-term observation of the patients after clinical application are essential. Moreover, the above-described phenomenon implies that freshly isolated ASCs might be safer and more effective for clinical use than cultured ASCs.9

32.4.2  ASC Growth Stimulation Factors 32.4.2.1 Cytokines The proliferation of ASCs can be stimulated using bFGF or FGF-2, EGF, PDGF, IGF-1, TNF-α, and Oncostatin M. Of these cytokines, FGF-2 is an especially effective growth-stimulating factor and acts to ensure the long-term propagation and self-renewal of ASCs via the extracellular signal-related kinase 1/2 signaling pathway.49 Under the microscope, ASCs grown in the presence of FGF-2 were found to be small and compact, with glossy nuclei.50 These morphological changes were accompanied by changes in the cell proliferation ability as well. ASCs cultured with FGF-2 and EGF grew more rapidly than those cultured with either one of the factors.51 On the other hand, it was found that the proliferation of ASCs could be stimulated using PDGF, which acts by activating JNK,52 or using oncostatin M, which acts via the activation of the microtubule-associated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and the JAK3/STAT1 pathways.53 32.4.2.2  Human Blood Biomaterials Current culture techniques include the use of fetal bovine serum (FBS) and fetal calf serum (FCS), which contain highly variable and undefined components that

can cause adverse reactions in patients. In recent years, alternative factors have been investigated to eliminate the use of animal products. Kocaoemer et  al. showed that ASCs cultured with human serum or thrombin-activated platelet-rich plasma (PRP) display proliferative rates significantly higher than those of ASCs cultured with FCS.54 Moreover, Kakudo et  al. reported that the addition of 5% activated PRP to the medium maximally promoted the proliferation of ASCs, but this effect was not observed with 20% activated PRP.55 Blande et  al. reported that the addition of autologous human platelet lysate to animal serum-free medium resulting in higher the proliferation of the ASCs compared to that observed with the addition of FBS.56 Clearly, the establishment of safe and efficient protocols for ASC expansion without the use of animal products is necessary to avoid crossspecies contamination and virus or protein transmission. 32.4.2.3  Other Factors The proliferation of ASCs can be also stimulated by using other reagents including sphingosine-1-phosphate (S1P),57 and 5-azacytidine.58 Lee et al. showed that the proliferation of ASCs under hypoxic conditions (2% O2) was higher than that of ASCs grown under normoxic conditions.59 In contrast, Wang et al. reported that ASCs showed a lower proliferation rate under hypoxic conditions (5% O2).60 Cellular responses to hypoxic stress depend greatly on the duration of culture, concentration of the cells, and environmental conditions under which the cells are cultured.

32.4.3  Culturing ASCs for Clinical Application Recently, the serum-free culture of ASCs in the presence of growth-stimulating factors has been explored.61 There may be many unknown factors present in sera and the compositional differences between lots may adversely influence culture stability. Thus, the challenge is to culture ASCs in a serum-free environment with only known components (Fig. 32.1).

FIGURE 32.1  ASCs observed by phase-contrast microscopy. ASCs cultured in a DMEM medium supplemented with 10% FBS (basal medium) (A); ASCs cultured in a basal medium supplemented with 10 ng/mL FGF-2 (B). ASCs have a fibroblast-like morphology. ASCs grown with FGF-2 were small and compact, and the nuclei were glossy (bar, 100 m).

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FIGURE 32.2  A schema of the workflow for extracting and culturing ASCs for clinical application. ASCs are extracted from the patient’s own fat tissue for mass culture with various growth-stimulating factors. Currently, culture media with animal serum, such as FBS and FCS, are routinely used for ASCs. However, culture media with various cytokines and the patient’s autologous blood biomaterials, such as PRP, are desired for future clinical application.

A scheme of the workflow for purifying and culturing ASCs for clinical application is shown in Fig. 32.2. ASCs are purified from the patient’s fat tissue for mass culture with various growth-stimulating factors. The ASCs are grown in the presence of FGF-2, a growth factor that promotes the self-renewal of ASCs,49 and with the patient’s autologous sera or PRP, etc. To culture stem cells, it is critical to examine whether their differentiation potential is maintained during mass culture. For example, it has been demonstrated that FGF-2 allows cell growth while maintaining the potential of ASCs to differentiate into adipose tissue cells.50 This means that (1) FGF-2 could be used to generate a large amount of ASCs exhibiting adipose differentiation potential from a small amount of fat tissue; (2) FGF-2 could be useful in clinical applications requiring the regeneration of adipose tissue. In clinical applications, a safer and more rapid expansion method is required in view of time and cost requirements.

32.5  DIFFERENTIATION CAPACITY OF ADIPOSE-DERIVED STEM CELLS About 200 kinds of cells are known to exist in the human body, which means, broadly speaking, that there are 200 human genome expression patterns. However, there are likely to be many subtypes of these patterns. For example, the genome expression pattern of the myocytes of skeletal muscles varies depending on their location in

FIGURE 32.3  Human adipose-derived stem cells (ASCs).

the body. In addition, researchers are starting to realize that the location of fibroblasts also alters their phenotype. Moreover, since differentiated (mature) cells differentiate from stem cells via many stages, there are likely to be thousands of different cells in the human body. Thus, the classification of human cells remains to be fully defined. This section describes the differentiation capacity of ASCs (Fig. 32.3). While ASCs have been traditionally considered as adipose tissue fibroblasts, it seems that ASCs actually have a tremendous capacity to differentiate into various types of cells, although it is not believed that they can regenerate all 200 of the major cell types.

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32.5.1  Ectodermal Lineage Cells 32.5.1.1  Epithelial Cells There are many kinds of epithelial cells in the human body. These include keratinocytes, basal cells, squamous cells, salivary gland cells, sweat gland cells, mammary gland cells, sebaceous gland cells, lacrimal cells, and prostate cells. A few studies describing the epithelial differentiation of ASCs have been reported, as follows. With regard to the in vitro epithelial differentiation of ASCs, Brzoska et al. reported that ASCs cultured in monolayers with retinoic acid-expressed cytokeratin 18,62 but not other cytokeratins. Long et  al. reported that when ASCs were treated by EGF in the presence of a fibrin matrix, this resulted in the differentiation of a superficial layer of cells that expressed the epithelial marker proteins E-cadherin and cytokeratin 8.63 However, when the ASCs were cultured in monolayers with EGF alone, this differentiation into epithelial cells was not observed, although the cells did exhibit increased cell proliferation and chemotaxis.64 Thus, it appears that the in vitro epithelial differentiation of ASCs requires EGF, retinoicacid, and 3D culture. In terms of the in vivo epithelial differentiation of ASCs, Li et  al. reported that when C57BL/6 mice with ischemia-reperfusion (I/R) kidneys were injected intrarenally with ASCs, the ASCs differentiate into renal tubular epithelium.65 Thus, it seems that ASCs can differentiate into various types of epithelial cells if certain conditions like a 3D environment and growth factors are met. 32.5.1.2  Pigmented Cells Pigment cells consist of retinal pigment cells and melanocytes. Vossmerbaeumer et  al. reported that in vitro culture with vasoactive intestinal peptide (VIP) induced ASCs to develop retinal pigment cell phenotypes, as indicated by the expression of retinal pigment epithelium (RPE) markers, namely bestrophin, cytokeratins 8 and 18, and RPE 65.66 Unlike retinal pigment cells, melanocytes are derived from neural crest cells, which means they have an ectodermal lineage. While several conference papers have reported the melanocyte differentiation of ASCs, similar observations have not been reported in medical journals. Further studies are needed before it is known whether ASCs can differentiate into functional melanocytes. 32.5.1.3  Neural Cells Zuk et  al. reported that when ASCs were cultured in induction medium containing β-mercaptoethanol, they differentiated into cells with small cell bodies and multiple processes that morphologically resembled neurons.46 It was also shown that the majority of these cells expressed neuron-specific enolase (NSE), and that

a subpopulation was positive for the neuronal nuclear antigen neuN. However, expression of glial cell markers, galactocerebroside, or glial fibrillary acidic protein (GFAP) was not observed. This study thus suggests that ASCs are able to assume an early neuronal or neural precursor phenotype. When Safford et  al. treated human and murine ASCs with a complex mixture of chemical reagents, the murine ASCs expressed both neuronal (neuN) and glial (GFAP) cell markers, while the human ASCs expressed the neuronal markers neuN, intermediate filament M, and nestin.67 When the murine cells were subjected to a more extensive characterization, it was found that they also expressed more mature markers such as microtubule-associated protein (MAP-2) and tau.68 In addition, immunocytochemistry revealed that a minority of these induced cells expressed the neurotransmitter GABA and the neuronal receptors NMDAR1 and 2. After these early reports, chemically-induced ASCs isolated from humans, mice, and rats were shown to express other neuronal (neurofilament-70 and the NGF receptor trkA) and glial cell (S100 and CNPase) markers69,70 (Fig. 32.4). Moreover, induced human ASCs were found to exhibit the functional coordinated activity of voltage-gated Na+ and K+ channels.71 ASCs were also found to be able to differentiate into specific glial cell phenotypes since Kingham et  al. reported that rat ASCs treated with a mixture of glial growth factors differentiated into Schwann cells.72 In addition, when Xu et al. used a similar inducing system, the ASCs differentiated into cells that were immunopositive for nestin and the Schwann cell markers p75, GFAP, and S-100.73 Moreover, Wang et  al. reported that ASCs cocultured with olfactory ensheathing glial cells (OEGCs) differentiated into OEGCs themselves.74 Notably, OEGCs are also known as olfactory ensheathing cells (OEC),

FIGURE 32.4  Neural cell differentiation of human ASCs.

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32.5  Differentiation Capacity of Adipose-Derived Stem Cells

which can be taken from the lining of the nose and are expected to be useful for treating spinal cord injuries. Thus, it appears that ASCs can differentiate into glial cells as well as neurons. However, additional research into the functions of these ASC-derived neural cells and the additional modifications that are needed to sustain their phenotypes for long periods of time is needed.

32.5.2  Mesodermal Lineage Cells

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grafting with the grafting of fat enriched in ASCs found that the latter provided superior outcomes.76 However, it appears that this was due to better de novo angiogenesis, which means that ASCs mainly provide angiogenic factors rather than differentiating into adipocytes. Thus, one of the challenges for the future is to determine how the adipogenic differentiation capability of ASCs can be used in clinical situations.

32.5.2.1 Adipocytes Zuk et al.8 were the first group to describe the mesodermal multilineage potential of human ASCs. Since then, it has been believed that the differentiation of ASCs toward adipocytes is a necessary and sufficient condition of ASCs. However, it is not necessarily true that ASCs are the precursor cells of adipocytes, since the white adipose tissue that contains ASCs also has many cell types apart from preadipocytes and mature adipocytes, including fibroblasts, endothelial cells, pericytes, and macrophages. Moreover, recent reports have revealed that ASCs are located in the perivascular areas of adipose tissues,35 they differentiate readily into endothelial cells,75 and that they secrete angiogenic factors,38 which suggests that ASCs may actually primarily differentiate into endodermal-lineage cells. Several factors been reported to induce ASCs to differentiate into adipocytes, namely dexamethasone, isobutyl methylxanthine, indomethacin, insulin, and thiazolidinedione8,28,46 (Fig. 32.5). Indeed, it seems that the conditions needed for the in vitro adipogenic differentiation of ASCs are well-established. It would be highly useful if ASCs could be used clinically to promote the complete regeneration of healthy vascularized adipose tissue that completely fills a defect.20 Indeed, trials comparing the performance of simple fat

32.5.2.2 Osteocytes In vitro studies have shown that ASCs can differentiate into osteogenic lineages8,46 (Fig. 32.6). In these papers, ascorbic acid, bone morphogenetic protein (BMP)-2 and -4, dexamethasone, and 1,25dihydroxy vitamin D3 served as the differentiating factors. By contrast, the conditions needed for in vitro osteoclast differentiation have not yet been established. This may relate to the possibility that osteoblasts and osteoclasts are of different lineage. Indeed, it may be that the monocyte phagocytic system is the precursor of osteoclasts.77 It has been suggested that mechanotransduction plays a crucial role in clinical bone repair and regeneration. For example, distraction osteogenesis is specifically characterized by osseous regeneration that occurs primarily via intramembranous ossification, which is stimulated by several mechanotransductive pathways.78 In the integrin-mediated, ERK 1/2-dependent mechanotransduction pathway, ERK 1/2 is a potential central mediator that acts as a signaling convergence point and regulates the osteogenic differentiation of MSCs during distraction.79,80 Thus, it may be that adjusting the mechanophysiological environment (e.g., by using bioreactors) could promote the osteogenic differentiation of ASCs in vitro.81 Our studies have revealed that murine ASCs can promote bone marrow regeneration.82 Although

FIGURE 32.5  Adipocyte differentiation of human ASCs.

FIGURE 32.6  Osteocyte differentiation of human ASCs.

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FIGURE 32.7  Chondrocyte differentiation of human ASCs.

hematopoietic cells were not differentiated from ASCs, it was suggested that hematopoietic cell niche was regenerated by ASCs. That ASCs could be used for hematopoietic system regeneration in vivo is highly interesting from a clinical perspective and warrants further research. 32.5.2.3 Chondrocytes The chondrogenic potential of ASCs has been investigated extensively8,46,83–94 (Fig. 32.7). A comparison of ASCs and bone marrow-derived MSCs revealed that MSCs are more potent than ASCs in terms of cartilage regeneration.87 Moreover, in the presence of TGFβ1, MSCs produced more collagen types II and X than ASCs.89 These findings clearly suggest that in terms of chondrogenic-specific genes and the production of extracellular matrix (ECM), the expression profiles of MSCs and ASCs differ significantly.95 In these studies, ascorbic acid, BMP 6, dexamethasone, insulin, and TGFβ1 served as inductive factors. It is clear that hydrostatic pressure and shear stresses affect the development and maintenance of chondrocytes, particularly those in articular cartilage. These mechanical forces may also be able to regulate chondrocyte differentiation, maturation, and tissue formation.91,95 Thus, it makes sense that those stresses are included when manipulating chondrogenesis in vitro. Our group was the first to show that when human ASCs were seeded into a collagen sponge and incubated with TGF-β1 under cycles of hydrostatic pressure followed by static culture, the chondrogenesis of human ASCs improved.91 Specifically, compared to ASCs cultured under static conditions, the ASCs cultured under cyclic hydrostatic pressure accumulated more collagen types II and X and expressed higher levels of col1, col2, and sox9 mRNAs. Therefore, hydrostatic pressure may be highly useful for cartilage tissue engineering using ASCs. 32.5.2.4  Skeletal Myocytes A few articles have described the myogenic differentiation of ASCs in vitro and in vivo. Essentially, the

myogenic differentiation of ASCs can be induced by culture in medium supplemented with horse serum and/ or under reduced serum conditions or by coculture with skeletal myoblasts.96–99 It has been reported that during their myogenic differentiation, ASCs develop into multinucleated myotubes and express several myogenic markers, including MyoD1, myogenin, desmin, Troponin-T, skeletal muscle myosin, and actin.100 Our group has observed that early passages of ASCs are more likely to differentiate into skeletal myotubes than ASCs from later passages.99 Subsequently, detailed studies that support our observation were published by two independent groups.96,98 In particular, the study by Vieira et  al.98 reported that earlier passages of ASCs placed in control culture plates at high density fused spontaneously with myotube-like cells. In addition, they showed that when human ASCs were cocultured with human Duchenne muscular dystrophy (DMD) myoblasts or myotubes, they participated in myotube formation and were able to restore dystrophin expression in DMD cells.98 Thus, ASCs have been demonstrated to contribute to the restoration of muscular function via the direct differentiation and/or cellular fusion with myocytes derived from both normal and pathological muscular tissue. 32.5.2.5 Cardiomyocytes Several papers have described the in vitro differentiation of ASCs toward cardiomyocytes.101–106 Palpant et  al. have also reported that the cardiac myocyte differentiation of ASCs is augmented by noncanonical Wnt agonists, canonical Wnt antagonists, and cytokines.103 Moreover, they stated that the ASCs that are capable of cardiac lineage differentiation can be enriched by selection for the stem cell-specific membrane markers Sca1 and c-kit.103 This study, and the others cited above, also suggest that transferrin, IL-3, IL-6, VEGF, laminin, 5-azacytidine, and TGF-β are cardiomyocyte-inducing factors. Zhu et al.105 have also reported a coculture method using neonatal rat cardiomyocytes. ASC-derived differentiated myocytes have been shown to bear morphological characteristics that are consistent with the cardiac phenotype, including the presence of a sarcomeric ultrastructure.104,106 They also express cardiomyocyte lineage markers such as MEF2c, GATA4, Nkx2.5, ANP, β-MHC, MLC2v, MLC2a, and the L-type calcium channel.106 In addition, Palpant et  al.103 reported that cardiomyocyte-differentiated ASCs bear the functional properties of myocytes and calcium-handling kinetics that indicate the presence of a developing sarcoplasmic reticulum. 32.5.2.6  Endothelial Cells In general, the serial passage of ASCs is associated with a fall in the expression of the stem cell-associated marker CD34 and a concomitant rise of the levels of

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32.6  In vivo Tissue/Organ Regeneration by Adipose-Derived Stem Cells

the stromal markers CD13, CD29, CD44, CD63, CD73, CD90, CD166, and CD105.27 While the implications of the decreasing CD34 expression remain unclear, some studies have shown that CD34+ ASCs are more proliferative, while CD34– ASCs possess greater plasticity.27,34 The fact that ASCs express CD34 suggests that these cells may have angiogenic potential. This means that ASCs could differentiate directly into endothelial cells and/ or could secrete angiogenic factors under certain circumstances. Indeed, when ASCs are plated in semisolid media, most align and express von Willebrand factor.75 Moreover, when ASCs are injected into an ischemic area, angiogenesis is increased.107,108 In vitro studies have shown that proprietary medium (EGM-2-MV; Cambrex) containing ascorbate, EGF, bFGF, and hydrocortisone can induce the differentiation of ASCs toward endothelial cells.38,75 However, it has also been shown that in clinical situations, the direct injection of ASCs into the recipient site can induce endothelial cell regeneration, even though most of the injected cells are dead at that time. Thus, at this stage, it is still unclear whether the ex vivo differentiation of ASCs toward endothelial cells is actually needed. In any case, further development of both in vitro and in vivo techniques of undifferentiated ASC transfer is warranted.

32.5.3  Endodermal Lineage Cells 32.5.3.1 Hepatocytes Several reports have shown that ASCs have the potential to differentiate into hepatocytes.109–111 These studies employed HGF and fibroblast growth factors (FGF)-1 and 4 as induction factors. ASCs that were differentiated in this manner synthesized urea, maintained glycogen stores, and incorporated into the liver microarchitecture in vivo.109–111 It seems that the expression of liver-selective transcription factors (such as HNFs, CCAAT⁄enhancer-binding proteins, and GATA-binding proteins) is essential for the induction of liver development and differentiation from ASCs.111 From the clinical perspective, hepatic diseases are generally caused by heterogeneous inflammation and fibrosis.111 In the future, ASCs can be used to reduce inflammation and treat fibrosis of the liver by directly differentiating into hepatocytes or by secreting factors such as angiogenic, antiapoptotic, antiinflammatory, and antifibrotic factors. 32.5.3.2  Pancreatic Cells The exposure of ASCs to nicotinamide, activin-A, exendin-4, HGF, and petagastrin induces them to differentiate into functional, pancreatic-like ASCs that are capable of insulin, glucagon, and somatostatin secretion.112–114 During the proliferation period, the cells express the stem cell markers nestin, ABCG2, SCF, and

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Thy-1, as well as the pancreatic endocrine transcription factor Isl-1.113 Culture in these defined conditions induces the cells to differentiate into a pancreatic endocrine phenotype within 3 days.113

32.6  IN VIVO TISSUE/ORGAN REGENERATION BY ADIPOSE-DERIVED STEM CELLS Over the past decade, tissue regeneration techniques using multipotent stem cells, present within adipose tissue, have been developed and much progress has been made in the field of regenerative medicine. SVF cells from adipose tissue can be used directly or can be cultured in plastic ware to select and expand an adherent population known as ASCs. Compared with bone marrow-derived MSCs, ASCs have an equal potential to differentiate into cells and tissues of mesodermal origin, such as adipocytes, cartilage, bone, and skeletal muscle.16 Further, the isolation and culture of ASCs with multipotent differentiation capacity have been described at the single cell level.115 However, as there are numerous reports describing the culture of adult MSCs from bone marrow, umbilical cord, and adipose tissue, for use in regenerative medicine in the clinical arena, Gimble et  al. have suggested that stem cells for regenerative medicinal applications should, ideally, meet the following criteria116,117: 1. Be present in abundant quantities (millions to billions of cells). 2. Be harvestable by a minimally invasive procedure. 3. Be able to differentiate along multiple cell lineage pathways in a controllable and reproducible manner. 4. Be safely and effectively transplantable to either an autologous or allogeneic host. 5. Be manufactured in accordance with current Good Manufacturing Practice guidelines. Adipose tissue potentially fulfill all of these criteria extremely effectively when compared with other stem/ stromal cell sources.9 With the developments in various medical fields, adipose tissue is now recognized as an accessible, abundant, and reliable source of stem cells for future clinical situations. Practically, SVF or ASCs can be applied in a autologous basis. However, in case cultured ASCs are used in an allogeneic fashion, the sex of the donor and recipient may be considered to be matched particularly for the purpose of either adipogenesis or osteogenesis. Numerous research groups are now performing in vivo preclinical studies on the use of ASCs in regenerative medicine. Thus, this section discusses the current state of ongoing preclinical animal studies within the context of future applications of ASCs in regenerative medicine.

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32.6.1  Adipose Tissue Fat graft survival is limited and thus is associated with an inconsistent clinical outcome. The transplanted fat graft loses volume over time due to tissue resorption that can result in the loss of anywhere between 20% and 90% of the original graft volume.118 This loss of volume prevents the stable filling necessary for long-term repair of soft tissue defects.20 Graft loss is mainly attributed to a lack of vascularization within the new tissue, with improper nutrition leading to cell necrosis. Since the long-term outcomes from fat grafting remain unpredictable in the hands of most practitioners, novel strategies for enhancing adipose tissue regeneration, including cell therapies for soft tissue defect repair, are being investigated. Recently, the use of ASCs combined with free fat grafting has been reported in the field of adipose tissue engineering.119 This approach can accomplish several things: (1) the secretion of angiogenic factors from ASCs; (2) the prevention of apoptosis of the grafted fat pad; and (3) an increase in the rate of angiogenesis and adipocyte differentiation.

32.6.2  Musculoskeletal Tissue The musculoskeletal system includes bone, cartilage, skeletal muscle, and tendons. Bone tissue regeneration using cell-based therapies is an attractive and active area of research in the field of regenerative medicine. Studies have accumulated over the past decade, and results have demonstrated that bone marrow-derived MSCs have the potential to augment in vivo osseous healing in both animal models and humans.120,121 Lee et  al. published the first report on the in vivo bone forming capacity of ASCs.122 Subsequently, it has been observed that ASCs retain their proliferative capacity independent of the age of the donor from which the cells are harvested. In addition, Cowan et  al. demonstrated the ability of ASCs to regenerate bone in critical-sized calvarial defects in an in vivo study.123 Meanwhile, there have been reports of bone regeneration using ASCs with numerous scaffolds, such as hydroxyapatite, beta-tricalcium phosphate (β-TCP), polylactides (PLA), polyglycolides (PGA), polylactic-co-glycolic (PLGA), and anorganic bovine bone (ABB), together with growth factors including dexamethasone and the BMP family, in vivo. Recently, Tajima et al. have demonstrated that the transplantation of admixture of ASCs and PRP had dramatic effects on bone regeneration in a rat cranial defect model without any scaffolds.124 In a large animal model, an interesting report described the combination of autologous ASCs and BMP-2 with porcine allogeneic mandible bone.125 These constructs were inserted into the rectus abdominis muscle with insertion of the superficial inferior epigastric vascular pedicle into the medullary cavity. The result

showed that the allografts were absorbed completely and replaced with new, full-thickness, cancellous bone after 7–8 weeks implantation. Cartilage has unique histological characteristics due to its abundant ECM, sparse cell density, and large water content. These characteristics allow restoration of the cartilage after compression or extension following tissue deformation. In articular cartilage, chondrocytes and adjacent connective tissue cells receive stresses, including hydrostatic pressure, distortional stress, and changes in osmotic pressure due to weight bearing and joint loading. The effects of these stresses have been shown to alter the production of cartilage-specific proteoglycans (aggrecan), hyaluronan, and collagen type II.91,95 In the field of cartilage tissue research, various procedures have been developed using MSCs and cell culture technology. The pellet culture technique results in the formation of 3D structures and mimics precartilage condensation, while cell-to-cell interactions among chondrocytes are known to be important in preventing dedifferentiation.126 Johnstone et al. previously demonstrated that stem cells plated at high density could accelerate chondrogenic differentiation in vitro.127 Although it is still difficult to construct three-dimensional neocartilage from ASCs both in vitro and in vivo, direct transplantation of ASCs, preinduced toward chondrogenesis, into a cartilage defect area, such as an articular surface or an intervertebral disk, have resulted in cartilage regeneration, as evidenced by the expression of type II collagen and aggrecan.128,129 ASCs are capable of direct differentiation into a myogenic lineage, fusion to existing but damaged myocytes, release of paracrine cytokines/factors, and/or scavenging of reactive oxygen species.100 In one study on myogenic differentiation from ASCs in a dystrophy mouse model, local injected ASCs were incorporated into skeletal muscle fibers at up to 20% of the total area in vivo, and it was demonstrated that approximately 10% of the myofibers from ASC-transplanted muscle expressed dystrophin.96

32.6.3  Nerve Tissue The ability of ASCs to differentiate into neural cell types and to release a number of soluble factors that stimulate neural tissue regeneration has opened up potential applications for these abundantly available stem cells in the treatment of various neurological disorders.130 Neural stem cells in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) are the principal targets of cell-based therapies. In the CNS, it has been clearly demonstrated that certain types of stem cells such as neural stem cells, bone marrow-derived MSCs, ESCs, and iPSCs have the ability to differentiate into neurons. It has been suggested that

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32.6  In vivo Tissue/Organ Regeneration by Adipose-Derived Stem Cells

CNS regeneration could be achieved using a combination of various methods and materials that are now available.131–134 Nakada et al. demonstrated the regeneration of central nervous tissue using ASCs with a collagen scaffold implanted in a cerebral cortex defect in a rat model.135 The results revealed that 0.2% of the implanted ASCs were positive for antihuman/rat MAP-2 antibody within the collagen scaffold implanted area. In the regeneration of PNS, di Summa et  al. investigated fibrin nerve conduits seeded with ASCs and implanted into sciatic nerve defects in a rat model. The ASC-seeded conduits showed enhanced axonal regeneration in the implanted site.136 However, in general, the percentage of transplanted cells that survive in the nervous system are reported to range between 0.2% and 38%135,137,138; thus the development of new strategies to improve stem cell survival both in the PNS and in the CNS is still a major issue.

32.6.4  Cardiovascular Tissue In adult life, the regenerative potential of cardiac tissue is limited and is not sufficient to prevent the degeneration that occurs in pathological conditions such as myocardial infarction.139 Research into the repair of cardiovascular tissue is a high priority in the field of regenerative medicine because heart disease is still the leading cause of death in the United States. In an in vitro study, Planat-Bénard et al. showed that murine ASCs expressed the cardiac-specific transcription factors, Nkx2.5, GATA4, and MEF2C, the structural cardiac protein βMHC, and the late-stage cardiac specification proteins MLC-2v, MLC-2a, and ANP.104 Based on previous results, numerous investigators have examined the effects of ASCs in the treatment of acute myocardial infarction and chronic heart failure in vivo using several procedures, including: (1) direct injection into the cardiac wall; (2) intracoronary administration by catheter; and (3) patch graft of an ASC sheet onto the cardiac muscle. The discovery that ASCs secrete significant quantities of angiogenic and antiapoptotic factors, including VEGF and HGF,38 led to a series of in vivo studies that focused on evaluating their therapeutic potential.140 Recently, it has been shown that human ASCs were superior to human MSCs in mediating angiogenesis in a mouse ischemic hind limb model.141 In addition, Hong et  al. demonstrated vascular growth and cardiac tissue rescue by human ASCs. Intramuscular, as well as intravenous, injections of approximately 5 × 105 human ASCs successfully revascularized ischemic hind limbs of immunocompromised mice.140,142 Recently Traktuev et al. demonstrated the ability of human ASCs to cooperate with human endothelial progenitor cells (EPCs) to form a robust vascular network in an ischemic mouse model,143 and suggested that ASCs were highly effective when used in combination with human EPCs.

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32.6.5  Liver Tissue Liver transplantation is the only effective treatment for severe liver injuries. However, because of organ rejection and lack of donors, alternative strategies are urgently needed. The hepatogenic differentiation capacity of MSCs harvested from bone marrow, umbilical cord blood, and adipose tissue, has been confirmed in many independent studies.144,145 The possibility for their future application in the therapy of liver diseases is very promising; however, transdifferentiation is inefficient, and the functioning of these induced hepatocyte-like cells is low compared with that of real hepatocytes.146 Recently, Banas et al. evaluated the therapeutic potential of ASCderived hepatocyte-like cells after transplantation into mice with liver injury.110 The results suggested that ASCderived hepatocytes could restore liver functions such as ammonia and purine metabolism after transplantation into mice with acute liver failure.

32.6.6  Skin and Wound Healing Intractable skin ulcers due to ischemia, diabetes, radiation, and decubitus represent significant problems with few solutions. ASCs are known to participate in neovascularization and the healing process of skin, directly and indirectly,147 and their role in skin rejuvenation is also under active investigation.148 Preclinical studies in a murine model have shown that the topical administration of autologous ASCs in conjunction with a type I collagen sponge matrix into a diabetic animal accelerated the healing of diabetic ulcers.149 It has been speculated that the healing mechanism is due to the release of several growth factors such as VEGF and HGF, and to the subsequent angiogenesis and proliferation of keratinocytes or dermal fibroblasts. In the field of wound healing using cell therapy, numerous preclinical studies have been initiated in murine or rat models. In a recent study, the authors highlighted the significance of direct ASC implantation following the injection of ASCs directly into the wound area in a murine model, which promoted greater wound healing compared with the control group.150 Lu et al. and Uysal et  al. reported that ASCs injected into skin flaps can extend flap survival directly by direct differentiation into endothelial cells, and indirectly, by secreting angiogenic growth factors, which enhance angiogenesis.107,151

32.6.7  Periodontal Tissue Key factors in achieving successful periodontal regeneration include the correct recruitment of cells to the site and the production of a suitable ECM consistent with the periodontal tissue. It is difficult to regenerate periodontal tissue because the microvascular system is sparse at

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the surface of the dental root. Thus, the combination of stem cells, growth factors, and scaffolds is also necessary for securing vascular regeneration in periodontal tissue as in other tissues.152 Preclinical data for periodontal tissue regeneration with ASCs demonstrated that mature periodontal tissue can be regenerated in a rat and canine model 2–3 months after ASC implantation.153,154 These findings showed that ASCs differentiated into periodontal tissues directly, and revealed a potential use for ASCs in treating oral disease. Recently the use of human ASCs in periodontal tissue regeneration has been investigated in a nude rat model, and the results showed that the alveolar bone regeneration was greater with human ASCs than in the other control groups.

32.7  ONGOING CLINICAL TRIALS AND FUTURE DIRECTION OF ADIPOSE-DERIVED STEM CELLS IN REGENERATIVE MEDICINE Adult stem cells hold tremendous promise for the treatment of diseases and injuries, including cardiovascular disease, chronic wounds, diabetes, cancer, neurological disorders, soft tissue reconstruction, and many others. Generally, the use of adult stem cells, including ASCs, in cell-based therapy is considered safer and more practical than either ESCs or iPSCs, as adult stem cells are autologous, immunocompatible, and do not pose ethical considerations or involve genetic manipulation. As with bone marrow-derived MSCs, it was believed that the major value of ASCs was related to their capacity for differentiation into different mature lineages. In recent years, however, it has been shown that the secretome of ASCs, which consists of proteins and growth factors secreted into the extracellular milieu, has a beneficial impact on different organs/systems within the human body. Based on such fundamental knowledge, successes with ASCs in preclinical models have led to their use in human trials to treat several disorders. In this section, ongoing clinical trials that have been published in peer-reviewed journals are described, together with future directions for ASCs in regenerative medicine.

32.7.1  Acute Myocardial Infarction and Chronic Heart Diseases Based on the various preclinical studies,102,155–157 clinical trials may be expected to show whether ASCs are efficacious in human patients. Currently, two firstin-man clinical trials using ASCs are underway in the field of cardiovascular diseases, in which ASCs are being used to treat both ST-elevation myocardial infarction (ClinicalTrials.gov identifier: NCT00442806)

and nonrevascularizable ischemic heart disease (ClinicalTrials.gov identifier: NCT00426868). For acute myocardial infarction, ASCs were delivered to patients with ST-elevation and left ventricular ejection fraction impairment by intracoronary infusion after appropriate infarct-related artery repair with stent implantation. So far, ASCs have demonstrated a similar effect to bone marrow-derived MSCs.158,159 Another clinical trial is currently ongoing for patients with end-stage coronary artery disease that is not amenable for revascularization, and with moderate to severe left ventricular dysfunction. In this trial, autologous ASCs were delivered via transendocardial injections after LV electromechanical mapping. Although the results have yet to be published, these clinical trials represent the first attempts to elucidate the role of ASCs for cardiac repair in humans.

32.7.2  Thoracic Repair Although there have been some preclinical studies on the treatment of thoracic and lung disease using ASCs,160,161 there is currently only one clinical report demonstrating that autologous ASCs can repair tracheomediastinal fistula.162 In this study, a patient with a tracheomediastinal fistula that resulted from tracheal cancer laser therapy received autologous ASCs with fibrin glue bronchoscopically. This treatment resulted in the successful closure of the fistula as evidenced by reepithelization and neovascularization.

32.7.3  Bone Regeneration and Repair The only clinical study on ASC-mediated bone repair to be published was a case report describing the repair of a posttraumatic calvarial defect with autologous ASCs in a child.163 In this report, a 7-year-old girl, who had wide calvarial defects after severe head injury with multifragment calvarial fractures, received decompressive craniectomy for refractory intracranial hypertension followed by resection of the reimplanted bone due to chronic infection. Thereafter, the patient received revision surgery in which, not only autologous cancellous bone from her ilium, but also ASCs were simultaneously engrafted onto the critical calvarial defect. A postoperative CT scan showed new bone formation and near complete calvarial continuity. The authors of this clinical trial speculated that ASCs, integrated with the cancellous bone, augmented bone regeneration from the transplanted bone itself and from the surrounding calvaria through the direct effect of ASC osteogenic differentiation and the indirect effect of angiogenesis or growth factor secretion by the implanted ASCs. Currently, several scaffolds are being used in preclinical bone engineering studies, including hydroxyapatite,

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32.7  Ongoing Clinical Trials and Future Direction of Adipose-Derived Stem Cells in Regenerative Medicine

PLA, PGA, PLGA, ABB, and composite scaffolds.123,164–167 Moreover, exogenous administration of bone morphogenetic protein-2 (BMP-2) is also promising for bone tissue engineering and regeneration. Taken together, the various studies demonstrate that ASCs have potential clinical applications for bone tissue regeneration and engineering.

32.7.4  Adipose Tissue Regeneration The regeneration and engineering of adipose tissue, a representative soft tissue, is promising for breast reconstruction postmastectomy, breast augmentation for cosmetic purposes, and soft tissue augmentation and the improvement of contour deformities due to trauma, cancer abrasion, and congenital anomalies in the field of plastic, reconstructive, and esthetic surgery. Recently, however, a trend has developed in soft tissue augmentation that uses ASCs combined with free-fat grafting in a technique called cell-assisted lipotransfer (CAL).119,168 In theory, this approach could accomplish several desired effects: (1) the direct differentiation of ASCs into adipocytes as a reservoir for adipose tissue turnover; (2) the direct differentiation of ASCs into endothelial cells, resulting in an increase in blood supply to the grafted fat tissue and thereby a decrease in the rate of graft resorption; (3) the release of angiogenic growth factors by ASCs and the induction of angiogenesis; (4) protection of the graft from ischemic reperfusion injury by ASCs; and (5) the acceleration of wound healing at the recipient site. In clinical trials, CAL has been used in breast reconstruction after partial mastectomy, in breast augmentation for cosmetic purposes, and in the correction of hemifacial atrophy.76,169 Currently, there are several hundred cases where CAL has been performed for breast augmentation, in which the resorption rate is estimated at 20–40%.

32.7.5  Chronic Intractable Fistula Even though preclinical studies for this condition have not been described, ASCs have also been demonstrated to be useful in the treatment of intractable enterocutaneous fistula and rectovaginal fistula as a result of Crohn’s disease. The fistulas were healed following direct injection of ASCs into the tract wall together with a fibrin glue sealant, and no adverse effects were observed.170,171 Interestingly, culture-expanded ASCs were more effective than freshly isolated stromal vascular fraction including ASCs. Further understanding of the role of the ASC versus the cells in the SVF is needed. To investigate further the effectiveness and safety of ASCs in the treatment of complex fistulas, a phase II,

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multicenter, randomized, controlled trial with ASCs for the treatment of Crohn’s disease and non-Crohn’s disease-related perianal fistula was recently completed.172 Results showed that: (1) the rate of fistula healing was 71% in the ASC in the fibrin glue group compared with 16% in the fibrin glue alone group; (2) the proportion of patients with healing was similar in Crohn’s and nonCrohn’s subgroups; and (3) quality of life scores were higher in patients who received ASCs than in those who received fibrin glue alone. Although the mechanism of such healing remains elusive, this novel approach of using ASCs for the treatment of complex fistulas may be widely applicable.

32.7.6  Cutaneous Wound Healing Intractable skin ulcers as a result of ischemia, diabetes, radiation, and decubitus represent significant challenges with few treatment solutions. Historically, Rigotti et al. examined the use of autologous lipoaspirates in the treatment of tissue damaged by radiotherapy postmastectomy.173 In this study with 20 patients, the lipoaspirate helped to regenerate healthy tissue at the damaged sites, with 19 out of 20 patients demonstrating improved healing. In this series, ASCs were not isolated and used to enrich the lipoaspirate. However, based on the understanding of ASC biology, it is likely that the beneficial effects of the lipoaspirate injections were due to the SVF, rather than to the mature adipocytes. Some preclinical studies were conducted coincidentally to determine the effect of ASCs in skin wound healing, and suggested that ASCs used with a type I collagen sponge matrix, have therapeutic potential in intractable cutaneous healing and repair in both chemically-induced ulcers and diabetic ulcers.149,174 From these studies, it has been speculated that ASC-mediated healing is a result of the release of several growth factors, such as VEGF and HGFs, and the subsequent angiogenesis and proliferation of keratinocytes or dermal fibroblasts. Recently, similar methods using either an acellular dermal matrix or synthetic dermal substitutes have been applied clinically for wound bed preparation. Jeong demonstrated a successful case in which autologous ASCs, seeded onto synthetic dermal substitutes, were delivered to the bone-exposed wound to prepare the granulation tissue for subsequent skin grafting.175 Recently, there have been an increasing number of research papers implicating the synergistic effect of ASCs combined with PRP. The therapeutic effects of PRP are believed to occur through the provision of concentrated levels of platelet-derived growth factors such as PDGF-BB, TGF-β1, VEGF, and EGF.176 Adipose stem cells, supplied with PRP, have been shown to contribute to the repair of injured tissue by supporting neovascularization

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through the enhancement of angiogenic stimuli, and by stabilizing newly formed vessels.177 It is also reported that PRP increases the proliferation of ASCs and dermal fibroblasts in vitro by releasing PDGF-AB and TGF-β1.55 As such, injection of adipose stem cells, with or without PRP, could be an efficacious method in future clinical treatments of cutaneous lesions. Finally, exposure to radiation causes cellular damage, and the histopathology of radiation injury in local tissue is basically progressive, obliterative endarteritis, which leads to severe tissue ischemia. There have been various studies demonstrating the beneficial effects of human MSCs, including ASCs, on radiation-induced complications.178,179 A clinical study on radiation-induced tissue damage using human adipose stem cells showed progressive improvement of tissue hydration and new vessel formation.173,175

32.7.7  Stress Urinary Incontinence Repair Several reports of preclinical studies have demonstrated that ASCs could be used to treat stress-induced urinary incontinence.180,181 The direct injection of ASCs into the urethral wall, or intravenous injection in a urinary incontinence model induced several effects: (1) an increased thickness of the urethral mucosa, confirmed histologically by the expression of both smooth muscle actin and elastin; (2) a significant increase in maximal bladder capacity and leak-point pressure; and (3) an eventual decreased rate of abnormal voiding. Although the concept was different, unique clinical trials for stress-induced urinary incontinence and postradical prostatectomy have been conducted with successful outcomes.182 In these trials, the isolated ASCs and adipose tissue were injected transurethrally into the rhabdosphincter and submucosal space of the urethra, respectively. Results showed significant improvement, as evidenced by increasing maximum urethral closing pressure, functional pressure profile, and blood flow at the treated area.

32.7.8  Immunomodulatory Effects and Graft Versus Host Diseases There have been data indicating that bone marrowderived MSCs are effective for steroid-resistant acute graft-versus-host disease (GVHD), a life-threatening complication following allogeneic transplantation with hematopoietic stem cells. Le Blanc et al. conducted multicenter phase II clinical trials for the treatment of GVHD with MSCs, derived using the European Group for Blood and Marrow Transplantation ex vivo expansion procedure, and concluded that infusion of MSCs, expanded in vitro, irrespective of the donor, might be an effective therapy for patients with steroid-resistant, acute GVHD.183

Based on such evidence, ASCs have been considered asalternative candidates for acute GVHD treatment. Fang et  al. demonstrated success in the treatment of steroid-resistant acute GVHD with ASCs.184 Although two patients died due to multiorgan failure and relapse of leukemia, the remaining four patients survived in a good clinical condition. However, as none of the preclinical studies investigating the use of ASCs as a therapy for GVHD have been published in peer-reviewed journals, further studies are needed to examine the mechanism by which ASCs may attenuate this disease and to determine the long-term effects of such treatment.

32.7.9  Safety Concern and Future Direction of ASCs in Regenerative Medicine As indicated in the previous sections, human adipose tissue presents an appealing source of stem cells for mesenchymal tissue regeneration and engineering since human adipose tissue is ubiquitous and easily obtainable in large quantities under local anesthesia with little patient discomfort. However, there are still several concerns to be addressed. Generally, the use of adult stem cells, including ASCs, in cell-based therapy is considered safer and more practical than either ESCs or iPSCs, as adult stem cells are autologous, immunocompatible, and do not elicit any ethical controversies or involve genetic manipulations. Another issue to be addressed, with the use of ASCs, is cellular transformation. In particular, it has been reported that the long-term culture of MSCs prior to their use results in significant changes in cell cycle kinetics, a decrease in telomerase activity, karyotype abnormalities, and a potential risk of their transformation into malignant cells.117,185 However, compared with other sources of stem cells, it is unlikely that there will be a need for long-term culture for ASC expansion in vitro, as adipose tissue is plentiful and can be harvested in larger quantities than other adult stem cell populations. Second, there is still a lack of information as to the optimal dose of ASCs in any of the target disorders. Moreover, the timing, frequency, and route of administration of ASCs still remains elusive, particularly when treating GVHD, brain infarction, and pulmonary emphysema since ASCs administrated intravenously can become trapped in the lung microcirculation.186 Longer follow-up animal experiments and multicenter randomized clinical trials are necessary to clarify such controversies. Third, it was reported that the number of adult stem cells decreases with age.21,187 As with stem cells from other sources, ASCs may have a limited potential for practical use although it does appear that some of the functions of ASCs seem to be preserved.24

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REFERENCES

32.8 CONCLUSIONS During the past decade, both preclinical studies and clinical trials using ASCs have been conducted in various medical fields, from cardiovascular research to applications for corneal diseases. ASCs are classified as adult multipotent stem cells and, as such, their multipotency is limited compared with ESCs and iPSCs. In addition, relatively few trials in a limited number of research areas have been conducted to assess the therapeutic potential of ASCs compared with the large number of published preclinical studies. However, ASCs have practical advantages in clinical medicine because adipose tissue, the primary source of ASCs, is abundant and easy to obtain with few donor site morbidities. Further preclinical and clinical studies are needed to determine whether ASC-based therapies can fulfill expectations and be used successfully to treat disorders for which current medical and surgical therapies are either ineffective or impractical.

References 1. Lenoir N. Europe confronts the embryonic stem cell research challenge. Science. 2000;287:1425–1427. 2. Young FE. A time for restraint. Science. 2000;287:1424. 3. Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science. 1998;279:1528–1530. 4. Pittenger MF, Mackay AM, Beck SC, et  al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–147. 5. Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 2000;61:364–370. 6. Kershaw EE, Flier JS. Adipose tissue as an endocrine organ. J Clin Endocrinol Metab. 2004;89:2548–2556. 7. Housman TS, Lawrence N, Mellen BG, et  al. The safety of liposuction: results of a national survey. Dermatol Surg. 2002;28:971–978. 8. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211–228. 9. Mizuno H. Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review. J Nippon Med Sch. 2009;76:56–66. 10. Strem BM, Hicok KC, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med. 2005;54:132–141. 11. Eto H, Suga H, Matsumoto D, et al. Characterization of structure and cellular components of aspirated and excised adipose tissue. Plast Reconstr Surg. 2009;124:1087–1097. 12. Crandall DL, Hausman GJ, Kral JG. A review of the microcirculation of adipose tissue: anatomic, metabolic, and angiogenic perspectives. Microcirculation. 1997;4:211–232. 13. Rupnick MA, Panigrahy D, Zhang CY, et  al. Adipose tissue mass can be regulated through the vasculature. Proc Natl Acad Sci U S A. 2002;99:10730–10735. 14. Lin CS, Xin ZC, Deng CH, Ning H, Lin G, Lue TF. Defining adipose tissue-derived stem cells in tissue and in culture. Histol Histopathol. 2010;25:807–815.

475

15. Salgado AJ, Reis RL, Sousa NJ, Gimble JM. Adipose tissue derived stem cells secretome: soluble factors and their roles in regenerative medicine. Curr Stem Cell Res Ther. 2010;5:103–110. 16. Schaffler A, Buchler C. Concise review: adipose tissue-derived stromal cells--basic and clinical implications for novel cell-based therapies. Stem Cells. 2007;25:818–827. 17. Dominici M, Le Blanc K, Mueller I, et  al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–317. 18. Traktuev DO, Merfeld-Clauss S, Li J, et  al. A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circ Res. 2008;102:77–85. 19. Peptan IA, Hong L, Mao JJ. Comparison of osteogenic potentials of visceral and subcutaneous adipose-derived cells of rabbits. Plast Reconstr Surg. 2006;117:1462–1470. 20. Brayfield CA, Marra KG, Rubin JP. Adipose tissue regeneration. Curr Stem Cell Res Ther. 2010;5:116–121. 21. Schipper BM, Marra KG, Zhang W, Donnenberg AD, Rubin JP. Regional anatomic and age effects on cell function of human adipose-derived stem cells. Ann Plast Surg. 2008;60:538–544. 22. Pettersson P, Cigolini M, Sjostrom L, Smith U, Bjorntorp P. Cells in human adipose tissue developing into adipocytes. Acta Med Scand. 1984;215:447–451. 23. Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, et al. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy. 2006;8:166–177. 24. Shi YY, Nacamuli RP, Salim A, Longaker MT. The osteogenic potential of adipose-derived mesenchymal cells is maintained with aging. Plast Reconstr Surg. 2005;116:1686–1696. 25. De Ugarte DA, Morizono K, Elbarbary A, et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs. 2003;174:101–109. 26. Francis MP, Sachs PC, Elmore LW, Holt SE. Isolating adiposederived mesenchymal stem cells from lipoaspirate blood and saline fraction. Organogenesis. 2010;6:11–14. 27. Bailey AM, Kapur S, Katz AJ. Characterization of adiposederived stem cells: an update. Curr Stem Cell Res Ther. 2010;5:95–102. 28. Ogawa R, Mizuno H, Watanabe A, Migita M, Hyakusoku H, Shimada T. Adipogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice-including relationship of sex differences. Biochem Biophys Res Commun. 2004;319:511–517. 29. Aksu AE, Rubin JP, Dudas JR, Marra KG. Role of gender and anatomical region on induction of osteogenic differentiation of human adipose-derived stem cells. Ann Plast Surg. 2008;60:306–322. 30. Yoshimura K, Shigeura T, Matsumoto D, et al. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol. 2006;208:64–76. 31. Mitchell JB, McIntosh K, Zvonic S, et  al. Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers. Stem Cells. 2006;24:376–385. 32. Varma MJ, Breuls RG, Schouten TE, et  al. Phenotypical and functional characterization of freshly isolated adipose tissuederived stem cells. Stem Cells Dev. 2007;16:91–104. 33. Mizuno H, Tobita M, Uysal AC. Concise review: Adiposederived stem cells as a novel tool for future regenerative medicine. Stem Cells. 2012;30:804–810.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

476

32.  Adipose-Derived Stem Cells in Regenerative Medicine

34. Suga H, Matsumoto D, Eto H, et al. Functional implications of CD34 expression in human adipose-derived stem/progenitor cells. Stem Cells Dev. 2009;18:1201–1210. 35. Amos PJ, Shang H, Bailey AM, Taylor A, Katz AJ, Peirce SM. IFATS collection: The role of human adipose-derived stromal cells in inflammatory microvascular remodeling and evidence of a perivascular phenotype. Stem Cells. 2008;26:2682–2690. 36. Cui L, Yin S, Liu W, Li N, Zhang W, Cao Y. Expanded adiposederived stem cells suppress mixed lymphocyte reaction by secretion of prostaglandin E2. Tissue Eng. 2007;13:1185–1195. 37. Kilroy GE, Foster SJ, Wu X, et  al. Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors. J Cell Physiol. 2007;212:702–709. 38. Rehman J, Traktuev D, Li J, et  al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation. 2004;109:1292–1298. 39. Sadat S, Gehmert S, Song YH, et al. The cardioprotective effect of mesenchymal stem cells is mediated by IGF-I and VEGF. Biochem Biophys Res Commun. 2007;363:674–679. 40. Zvonic S, Lefevre M, Kilroy G, et  al. Secretome of primary cultures of human adipose-derived stem cells: modulation of serpins by adipogenesis. Mol Cell Proteomics. 2007;6:18–28. 41. Sugiura K, Taketani S, Yoshimura T, et al. Effect of hepatocyte growth factor on long term hematopoiesis of human progenitor cells in transgenic-severe combined immunodeficiency mice. Cytokine. 2007;37:218–226. 42. Yanez R, Lamana ML, Garcia-Castro J, Colmenero I, Ramirez M, Bueren JA. Adipose tissue-derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft-versus-host disease. Stem Cells. 2006;24:2582–2591. 43. Mizuno H, Hyakusoku H. Mesengenic potential and future clinical perspective of human processed lipoaspirate cells. J Nippon Med Sch. 2003;70:300–306. 44. Fraser JK, Wulur I, Alfonso Z, Hedrick MH. Fat tissue: an underappreciated source of stem cells for biotechnology. Trends Biotechnol. 2006;24:150–154. 45. Jun ES, Lee TH, Cho HH, Suh SY, Jung JS. Expression of telomerase extends longevity and enhances differentiation in human adipose tissue-derived stromal cells. Cell Physiol Biochem. 2004;14:261–268. 46. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13:4279–4295. 47. Rodriguez AM, Elabd C, Delteil F, et al. Adipocyte differentiation of multipotent cells established from human adipose tissue. Biochem Biophys Res Commun. 2004;315:255–263. 48. Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res. 2005;65:3035–3039. 49. Zaragosi LE, Ailhaud G, Dani C. Autocrine fibroblast growth factor 2 signaling is critical for self-renewal of human multipotent adipose-derived stem cells. Stem Cells. 2006;24:2412–2419. 50. Kakudo N, Shimotsuma A, Kusumoto K. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells. Biochem Biophys Res Commun. 2007;359:239–244. 51. Hebert TL, Wu X, Yu G, et  al. Culture effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on cryopreserved human adipose-derived stromal/stem cell proliferation and adipogenesis. J Tissue Eng Regen Med. 2009;3: 553–561. 52. Kang YJ, Jeon ES, Song HY, et al. Role of c-Jun N-terminal kinase in the PDGF-induced proliferation and migration of human adipose tissue-derived mesenchymal stem cells. J Cell Biochem. 2005;95:1135–1145.

53. Song HY, Jeon ES, Jung JS, Kim JH. Oncostatin M induces proliferation of human adipose tissue-derived mesenchymal stem cells. Int J Biochem Cell Biol. 2005;37:2357–2365. 54. Kocaoemer A, Kern S, Kluter H, Bieback K. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells. 2007;25:1270–1278. 55. Kakudo N, Minakata T, Mitsui T, Kushida S, Notodihardjo FZ, Kusumoto K. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts. Plast Reconstr Surg. 2008;122:1352–1360. 56. Blande IS, Bassaneze V, Lavini-Ramos C, et  al. Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate. Transfusion. 2009;49:2680–2685. 57. He X, H’Ng SC, Leong DT, Hutmacher DW, Melendez AJ. Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells. J Mol Cell Biol. 2010;2:199–208. 58. Rodriguez-Serrano F, Alvarez P, Caba O, et  al. Promotion of human adipose-derived stem cell proliferation mediated by exogenous nucleosides. Cell Biol Int. 2010;34:917–924. 59. Lee EY, Xia Y, Kim WS, et al. Hypoxia-enhanced wound-healing function of adipose-derived stem cells: increase in stem cell proliferation and up-regulation of VEGF and bFGF. Wound Repair Regen. 2009;17:540–547. 60. Wang DW, Fermor B, Gimble JM, Awad HA, Guilak F. Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells. J Cell Physiol. 2005;204:184–191. 61. Parker AM, Shang H, Khurgel M, Katz AJ. Low serum and serum-free culture of multipotential human adipose stem cells. Cytotherapy. 2007;9:637–646. 62. Brzoska M, Geiger H, Gauer S, Baer P. Epithelial differentiation of human adipose tissue-derived adult stem cells. Biochem Biophys Res Commun. 2005;330:142–150. 63. Long JL, Zuk P, Berke GS, Chhetri DK. Epithelial differentiation of adipose-derived stem cells for laryngeal tissue engineering. Laryngoscope. 2010;120:125–131. 64. Baer PC, Schubert R, Bereiter-Hahn J, Plosser M, Geiger H. Expression of a functional epidermal growth factor receptor on human adipose-derived mesenchymal stem cells and its signaling mechanism. Eur J Cell Biol. 2009;88:273–283. 65. Li K, Han Q, Yan X, Liao L, Zhao RC. Not a process of simple vicariousness, the differentiation of human adipose-derived mesenchymal stem cells to renal tubular epithelial cells plays an important role in acute kidney injury repairing. Stem Cells Dev. 2010;19:1267–1275. 66. Vossmerbaeumer U, Ohnesorge S, Kuehl S, et al. Retinal pigment epithelial phenotype induced in human adipose tissue-derived mesenchymal stromal cells. Cytotherapy. 2009;11:177–188. 67. Safford KM, Hicok KC, Safford SD, et  al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun. 2002;294:371–379. 68. Safford KM, Safford SD, Gimble JM, Shetty AK, Rice HE. Characterization of neuronal/glial differentiation of murine adipose-derived adult stromal cells. Exp Neurol. 2004;187:319–328. 69. Fujimura J, Ogawa R, Mizuno H, Fukunaga Y, Suzuki H. Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice. Biochem Biophys Res Commun. 2005;333:116–121. 70. Ning H, Lin G, Lue TF, Lin CS. Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells. Differentiation. 2006;74:510–518. 71. Anghileri E, Marconi S, Pignatelli A, et  al. Neuronal differentiation potential of human adipose-derived mesenchymal stem cells. Stem Cells Dev. 2008;17:909–916.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

72. Kingham PJ, Kalbermatten DF, Mahay D, Armstrong SJ, Wiberg M, Terenghi G. Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro. Exp Neurol. 2007;207:267–274. 73. Xu Y, Liu L, Li Y, et al. Myelin-forming ability of Schwann celllike cells induced from rat adipose-derived stem cells in vitro. Brain Res. 2008;1239:49–55. 74. Wang B, Han J, Gao Y, et al. The differentiation of rat adiposederived stem cells into OEC-like cells on collagen scaffolds by co-culturing with OECs. Neurosci Lett. 2007;421:191–196. 75. Planat-Benard V, Silvestre JS, Cousin B, et  al. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation. 2004;109: 656–663. 76. Yoshimura K, Sato K, Aoi N, Kurita M, Hirohi T, Harii K. Cellassisted lipotransfer for cosmetic breast augmentation: supportive use of adipose-derived stem/stromal cells. Aesthetic Plast Surg. 2008;32:48–55. discussion 6-7. 77. van Furth R. The origin of phagocytic cells in the joint and bone. Scand J Rheumatol Suppl. 1981;40:13–20. 78. Huang C, Ogawa R. Mechanotransduction in bone repair and regeneration. FASEB J. 2010;24:3625–3632. 79. Rhee ST, El-Bassiony L, Buchman SR. Extracellular signalrelated kinase and bone morphogenetic protein expression during distraction osteogenesis of the mandible: in vivo evidence of a mechanotransduction mechanism for differentiation and osteogenesis by mesenchymal precursor cells. Plast Reconstr Surg. 2006;117:2243–2249. 80. Tong L, Buchman SR, Ignelzi Jr. MA, Rhee S, Goldstein SA. Focal adhesion kinase expression during mandibular distraction osteogenesis: evidence for mechanotransduction. Plast Reconstr Surg. 2003;111:211–222. discussion 23-4. 81. van Griensven M, Diederichs S, Roeker S, et  al. Mechanical strain using 2D and 3D bioreactors induces osteogenesis: implications for bone tissue engineering. Adv Biochem Eng Biotechnol. 2009;112:95–123. 82. Ogawa R. The importance of adipose-derived stem cells and vascularized tissue regeneration in the field of tissue transplantation. Curr Stem Cell Res Ther. 2006;1:13–20. 83. Afizah H, Yang Z, Hui JH, Ouyang HW, Lee EH. A comparison between the chondrogenic potential of human bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) taken from the same donors. Tissue Eng. 2007;13:659–666. 84. Awad HA, Wickham MQ, Leddy HA, Gimble JM, Guilak F. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials. 2004;25:3211–3222. 85. De Ugarte DA, Alfonso Z, Zuk PA, et  al. Differential expression of stem cell mobilization-associated molecules on multilineage cells from adipose tissue and bone marrow. Immunol Lett. 2003;89:267–270. 86. Dragoo JL, Samimi B, Zhu M, et al. Tissue-engineered cartilage and bone using stem cells from human infrapatellar fat pads. J Bone Joint Surg Br. 2003;85:740–747. 87. Hennig T, Lorenz H, Thiel A, et  al. Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6. J Cell Physiol. 2007;211:682–691. 88. Huang JI, Zuk PA, Jones NF, et  al. Chondrogenic potential of multipotential cells from human adipose tissue. Plast Reconstr Surg. 2004;113:585–594. 89. Knippenberg M, Helder MN, Zandieh Doulabi B, Wuisman PI, Klein-Nulend J. Osteogenesis versus chondrogenesis by BMP-2 and BMP-7 in adipose stem cells. Biochem Biophys Res Commun. 2006;342:902–908.

477

90. Ogawa R, Mizuno H, Watanabe A, Migita M, Shimada T, Hyakusoku H. Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice. Biochem Biophys Res Commun. 2004;313:871–877. 91. Ogawa R, Mizuno S, Murphy GF, Orgill DP. The effect of hydrostatic pressure on three-dimensional chondroinduction of human adipose-derived stem cells. Tissue Eng Part A. 2009;15:2937–2945. 92. Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum. 2005;52:2521–2529. 93. Segawa Y, Muneta T, Makino H, et al. Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles. J Orthop Res. 2009;27:435–441. 94. Xu Y, Malladi P, Wagner DR, Longaker MT. Adipose-derived mesenchymal cells as a potential cell source for skeletal regeneration. Curr Opin Mol Ther. 2005;7:300–305. 95. Ogawa R, Mizuno S. Cartilage regeneration using adiposederived stem cells. Curr Stem Cell Res Ther. 2010;5:129–132. 96. Di Rocco G, Iachininoto MG, Tritarelli A, et  al. Myogenic potential of adipose-tissue-derived cells. J Cell Sci. 2006;119: 2945–2952. 97. Lee JH, Kemp DM. Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions. Biochem Biophys Res Commun. 2006;341:882–888. 98. Vieira NM, Brandalise V, Zucconi E, et  al. Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro. Biol Cell. 2008;100:231–241. 99. Mizuno H, Zuk PA, Zhu M, Lorenz HP, Benhaim P, Hedrick MH. Myogenic differentiation by human processed lipoaspirate cells. Plast Reconstr Surg. 2002;109:199–209. discussion 10-1. 100. Mizuno H. The potential for treatment of skeletal muscle disorders with adipose-derived stem cells. Curr Stem Cell Res Ther. 2010;5:133–136. 101. Fraser JK, Schreiber R, Strem B, et al. Plasticity of human adipose stem cells toward endothelial cells and cardiomyocytes. Nat Clin Pract Cardiovasc Med. 2006;3(Suppl 1):S33–S37. 102. Miyahara Y, Nagaya N, Kataoka M, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med. 2006;12:459–465. 103. Palpant NJ, Yasuda S, MacDougald O, Metzger JM. Noncanonical Wnt signaling enhances differentiation of Sca1+ /c-kit+ adipose-derived murine stromal vascular cells into spontaneously beating cardiac myocytes. J Mol Cell Cardiol. 2007;43:362–370. 104. Planat-Benard V, Menard C, Andre M, et  al. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res. 2004;94:223–229. 105. Zhu Y, Liu T, Song K, Ning R, Ma X, Cui Z. ADSCs differentiated into cardiomyocytes in cardiac microenvironment. Mol Cell Biochem. 2009;324:117–129. 106. Palpant NJ, Metzger JM. Aesthetic cardiology: adipose-derived stem cells for myocardial repair. Curr Stem Cell Res Ther. 2010;5:145–152. 107. Lu F, Mizuno H, Uysal CA, Cai X, Ogawa R, Hyakusoku H. Improved viability of random pattern skin flaps through the use of adipose-derived stem cells. Plast Reconstr Surg. 2008;121:50–58. 108. Moon MH, Kim SY, Kim YJ, et al. Human adipose tissue-derived mesenchymal stem cells improve postnatal neovascularization in a mouse model of hindlimb ischemia. Cell Physiol Biochem. 2006;17:279–290.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

478

32.  Adipose-Derived Stem Cells in Regenerative Medicine

109. Aurich H, Sgodda M, Kaltwasser P, et  al. Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo. Gut. 2009;58: 570–581. 110. Banas A, Teratani T, Yamamoto Y, et al. Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure. J Gastroenterol Hepatol. 2009;24:70–77. 111. Ishikawa T, Banas A, Hagiwara K, Iwaguro H, Ochiya T. Stem cells for hepatic regeneration: the role of adipose tissue derived mesenchymal stem cells. Curr Stem Cell Res Ther. 2010;5:182–189. 112. Chandra V, G S, Phadnis S, Nair PD, Bhonde RR. Generation of pancreatic hormone-expressing islet-like cell aggregates from murine adipose tissue-derived stem cells. Stem Cells. 2009;27:1941–1953. 113. Kim SC, Han DJ, Lee JY. Adipose tissue derived stem cells for regeneration and differentiation into insulin-producing cells. Curr Stem Cell Res Ther. 2010;5:190–194. 114. Timper K, Seboek D, Eberhardt M, et  al. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells. Biochem Biophys Res Commun. 2006;341:1135–1140. 115. Rodriguez AM, Elabd C, Amri EZ, Ailhaud G, Dani C. The human adipose tissue is a source of multipotent stem cells. Biochimie. 2005;87:125–128. 116. Gimble JM. Adipose tissue-derived therapeutics. Expert Opin Biol Ther. 2003;3:705–713. 117. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007;100:1249–1260. 118. Cherubino M, Marra KG. Adipose-derived stem cells for soft tissue reconstruction. Regen Med. 2009;4:109–117. 119. Zhu M, Zhou Z, Chen Y, et  al. Supplementation of fat grafts with adipose-derived regenerative cells improves long-term graft retention. Ann Plast Surg. 2010;64:222–228. 120. Connolly JF. Clinical use of marrow osteoprogenitor cells to stimulate osteogenesis. Clin Orthop Relat Res. 1998:S257–S266. 121. Ohgushi H, Goldberg VM, Caplan AI. Repair of bone defects with marrow cells and porous ceramic. Experiments in rats. Acta Orthop Scand. 1989;60:334–339. 122. Lee JA, Parrett BM, Conejero JA, et al. Biological alchemy: engineering bone and fat from fat-derived stem cells. Ann Plast Surg. 2003;50:610–617. 123. Cowan CM, Shi YY, Aalami OO, et  al. Adipose-derived adult stromal cells heal critical-size mouse calvarial defects. Nat Biotechnol. 2004;22:560–567. 124. Tajima S, Tobita M, Orbay H, Hyakusoku H, Mizuno H. Direct and indirect effects of a combination of adipose-derived stem cells and platelet-rich plasma on bone regeneration. Tissue Eng Part A. 2015;21:895–905. 125. Runyan CM, Jones DC, Bove KE, Maercks RA, Simpson DS, Taylor JA. Porcine allograft mandible revitalization using autologous adipose-derived stem cells, bone morphogenetic protein-2, and periosteum. Plast Reconstr Surg. 2010;125:1372–1382. 126. Han Y, Wei Y, Wang S, Song Y. Cartilage regeneration using adipose-derived stem cells and the controlled-released hybrid microspheres. Joint Bone Spine. 2010;77:27–31. 127. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res. 1998;238:265–272. 128. Dragoo JL, Carlson G, McCormick F, et  al. Healing full-thickness cartilage defects using adipose-derived stem cells. Tissue Eng. 2007;13:1615–1621. 129. Ganey T, Hutton WC, Moseley T, Hedrick M, Meisel HJ. Intervertebral disc repair using adipose tissue-derived stem and regenerative cells: experiments in a canine model. Spine (Phila Pa 1976). 2009;34:2297–2304.

130. Erba P, Terenghi G, Kingham PJ. Neural differentiation and therapeutic potential of adipose tissue derived stem cells. Curr Stem Cell Res Ther. 2010;5:153–160. 131. Ellis-Behnke RG, Liang YX, You SW, et al. Nano neuro knitting: peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision. Proc Natl Acad Sci U S A. 2006;103:5054–5059. 132. McDonald JW, Becker D, Holekamp TF, et  al. Repair of the injured spinal cord and the potential of embryonic stem cell transplantation. J Neurotrauma. 2004;21:383–393. 133. Okano H, Ogawa Y, Nakamura M, Kaneko S, Iwanami A, Toyama Y. Transplantation of neural stem cells into the spinal cord after injury. Semin Cell Dev Biol. 2003;14:191–198. 134. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676. 135. Nakada A, Fukuda S, Ichihara S, et al. Regeneration of central nervous tissue using a collagen scaffold and adipose-derived stromal cells. Cells Tissues Organs. 2009;190:326–335. 136. di Summa PG, Kingham PJ, Raffoul W, Wiberg M, Terenghi G, Kalbermatten DF. Adipose-derived stem cells enhance peripheral nerve regeneration. J Plast Reconstr Aesthet Surg. 2010;63:1544–1552. 137. Heine W, Conant K, Griffin JW, Hoke A. Transplanted neural stem cells promote axonal regeneration through chronically denervated peripheral nerves. Exp Neurol. 2004;189:231–240. 138. Shimizu S, Kitada M, Ishikawa H, Itokazu Y, Wakao S, Dezawa M. Peripheral nerve regeneration by the in vitro differentiatedhuman bone marrow stromal cells with Schwann cell property. Biochem Biophys Res Commun. 2007;359:915–920. 139. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation. 1990;81:1161–1172. 140. Hong SJ, Traktuev DO, March KL. Therapeutic potential of adipose-derived stem cells in vascular growth and tissue repair. Curr Opin Organ Transplant. 2010;15:86–91. 141. Kim Y, Kim H, Cho H, Bae Y, Suh K, Jung J. Direct comparison of human mesenchymal stem cells derived from adipose tissues and bone marrow in mediating neovascularization in response to vascular ischemia. Cell Physiol Biochem. 2007;20:867–876. 142. Cao Y, Sun Z, Liao L, Meng Y, Han Q, Zhao RC. Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem Biophys Res Commun. 2005;332:370–379. 143. Traktuev DO, Prater DN, Merfeld-Clauss S, et  al. Robust functional vascular network formation in vivo by cooperation of adipose progenitor and endothelial cells. Circ Res. 2009;104:1410–1420. 144. Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest. 2002;109:1291–1302. 145. Seo MJ, Suh SY, Bae YC, Jung JS. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem Biophys Res Commun. 2005;328:258–264. 146. Lue J, Lin G, Ning H, Xiong A, Lin CS, Glenn JS. Transdifferentiation of adipose-derived stem cells into hepatocytes: a new approach. Liver Int. 2010;30:913–922. 147. Shingyochi Y, Orbay H, Mizuno H. Adipose-derived stem cells for wound repair and regeneration. Expert Opin Biol Ther. 2015;15:1285–1292. 148. Charles-de-Sa L, Gontijo-de-Amorim NF, Maeda Takiya C, et al. Antiaging treatment of the facial skin by fat graft and adiposederived stem cells. Plast Reconstr Surg. 2015;135:999–1009. 149. Nambu M, Kishimoto S, Nakamura S, et al. Accelerated wound healing in healing-impaired db/db mice by autologous adipose

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

32.8 Conclusions

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tissue-derived stromal cells combined with atelocollagen matrix. Ann Plast Surg. 2009;62:317–321. Lim JS, Yoo G. Effects of adipose-derived stromal cells and of their extract on wound healing in a mouse model. J Korean Med Sci. 2010;25:746–751. Uysal AC, Mizuno H, Tobita M, Ogawa R, Hyakusoku H. The effect of adipose-derived stem cells on ischemia-reperfusion injury: immunohistochemical and ultrastructural evaluation. Plast Reconstr Surg. 2009;124:804–815. Tobita M, Mizuno H. Periodontal disease and periodontal tissue regeneration. Curr Stem Cell Res Ther. 2010;5:168–174. Tobita M, Uysal AC, Ogawa R, Hyakusoku H, Mizuno H. Periodontal tissue regeneration with adipose-derived stem cells. Tissue Eng Part A. 2008;14:945–953. Tobita M, Uysal CA, Guo X, Hyakusoku H, Mizuno H. Periodontal tissue regeneration by combined implantation of adipose tissue-derived stem cells and platelet-rich plasma in a canine model. Cytotherapy. 2013;15:1517–1526. Bai X, Yan Y, Song YH, et al. Both cultured and freshly isolated adipose tissue-derived stem cells enhance cardiac function after acute myocardial infarction. Eur Heart J. 2010;31:489–501. Schenke-Layland K, Strem BM, Jordan MC, et  al. Adipose tissue-derived cells improve cardiac function following myocardial infarction. J Surg Res. 2009;153:217–223. Valina C, Pinkernell K, Song YH, et al. Intracoronary administration of autologous adipose tissue-derived stem cells improves left ventricular function, perfusion, and remodelling after acute myocardial infarction. Eur Heart J. 2007;28:2667–2677. Meliga E, Strem BM, Duckers HJ, Serruys PW. Adipose-derived cells. Cell Transplant. 2007;16:963–970. Mizuno H. Adipose-derived stem and stromal cells for cellbased therapy: current status of preclinical studies and clinical trials. Curr Opin Mol Ther. 2010;12:442–449. Shigemura N, Okumura M, Mizuno S, Imanishi Y, Nakamura T, Sawa Y. Autologous transplantation of adipose tissuederived stromal cells ameliorates pulmonary emphysema. Am J Transplant. 2006;6:2592–2600. Suzuki T, Kobayashi K, Tada Y, et al. Regeneration of the trachea using a bioengineered scaffold with adipose-derived stem cells. Ann Otol Rhinol Laryngol. 2008;117:453–463. Alvarez PD, Garcia-Arranz M, Georgiev-Hristov T, GarciaOlmo D. A new bronchoscopic treatment of tracheomediastinal fistula using autologous adipose-derived stem cells. Thorax. 2008;63:374–376. Lendeckel S, Jodicke A, Christophis P, et  al. Autologous stem cells (adipose) and fibrin glue used to treat widespread traumatic calvarial defects: case report. J Craniomaxillofac Surg. 2004;32:370–373. Betz OB, Betz VM, Abdulazim A, et  al. The repair of criticalsized bone defects using expedited, autologous BMP-2 geneactivated fat implants. Tissue Eng Part A. 2009;16:1093–1101. Di Bella C, Farlie P, Penington AJ. Bone regeneration in a rabbit critical-sized skull defect using autologous adipose-derived cells. Tissue Eng Part A. 2008;14:483–490. Jeon O, Rhie JW, Kwon IK, Kim JH, Kim BS, Lee SH. In vivo bone formation following transplantation of human adiposederived stromal cells that are not differentiated osteogenically. Tissue Eng Part A. 2008;14:1285–1294. Pieri F, Lucarelli E, Corinaldesi G, et al. Dose-dependent effect of adipose-derived adult stem cells on vertical bone regeneration in rabbit calvarium. Biomaterials. 2010;31:3527–3535. Matsumoto D, Sato K, Gonda K, et al. Cell-assisted lipotransfer: supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection. Tissue Eng. 2006;12:3375–3382.

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169. Yoshimura K, Sato K, Aoi N, et al. Cell-assisted lipotransfer for facial lipoatrophy: efficacy of clinical use of adipose-derived stem cells. Dermatol Surg. 2008;34:1178–1185. 170. Garcia-Olmo D, Herreros D, De-La-Quintana P, et al. Adiposederived stem cells in Crohn’s rectovaginal fistula. Case Report Med. 2010;2010:961758. 171. Garcia-Olmo D, Herreros D, Pascual M, et  al. Treatment of enterocutaneous fistula in Crohn’s Disease with adiposederived stem cells: a comparison of protocols with and without cell expansion. Int J Colorectal Dis. 2009;24:27–30. 172. Garcia-Olmo D, Herreros D, Pascual I, et al. Expanded adiposederived stem cells for the treatment of complex perianal fistula: a phase II clinical trial. Dis Colon Rectum. 2009;52:79–86. 173. Rigotti G, Marchi A, Galie M, et al. Clinical treatment of radiotherapy tissue damage by lipoaspirate transplant: a healing process mediated by adipose-derived adult stem cells. Plast Reconstr Surg. 2007;119:1409–1422. discussion 23-4. 174. Nambu M, Ishihara M, Nakamura S, et  al. Enhanced healing of mitomycin C-treated wounds in rats using inbred adipose tissue-derived stromal cells within an atelocollagen matrix. Wound Repair Regen. 2007;15:505–510. 175. Jeong JH. Adipose stem cells and skin repair. Curr Stem Cell Res Ther. 2010;5:137–140. 176. Eppley BL, Woodell JE, Higgins J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing. Plast Reconstr Surg. 2004;114:1502–1508. 177. Blanton MW, Hadad I, Johnstone BH, et  al. Adipose stromal cells and platelet-rich plasma therapies synergistically increase revascularization during wound healing. Plast Reconstr Surg. 2009;123:56S–64S. 178. Akita S, Akino K, Hirano A, Ohtsuru A, Yamashita S. Mesenchymal stem cell therapy for cutaneous radiation syndrome. Health Phys. 2010;98:858–862. 179. Francois S, Mouiseddine M, Mathieu N, et  al. Human mesenchymal stem cells favour healing of the cutaneous radiation syndrome in a xenogenic transplant model. Ann Hematol. 2007;86:1–8. 180. Fu Q, Song XF, Liao GL, Deng CL, Cui L. Myoblasts differentiated from adipose-derived stem cells to treat stress urinary incontinence. Urology. 2010;75:718–723. 181. Lin G, Wang G, Banie L, et al. Treatment of stress urinary incontinence with adipose tissue-derived stem cells. Cytotherapy. 2010;12:88–95. 182. Yamamoto T, Gotoh M, Hattori R, et  al. Periurethral injection of autologous adipose-derived stem cells for the treatment of stress urinary incontinence in patients undergoing radical prostatectomy: report of two initial cases. Int J Urol. 2010;17:75–82. 183. Le Blanc K, Frassoni F, Ball L, et  al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet. 2008;371:1579–1586. 184. Fang B, Song Y, Liao L, Zhang Y, Zhao RC. Favorable response to human adipose tissue-derived mesenchymal stem cells in steroid-refractory acute graft-versus-host disease. Transplant Proc. 2007;39:3358–3362. 185. Izadpanah R, Kaushal D, Kriedt C, et  al. Long-term in vitro expansion alters the biology of adult mesenchymal stem cells. Cancer Res. 2008;68:4229–4238. 186. Jones BJ, McTaggart SJ. Immunosuppression by mesenchymal stromal cells: from culture to clinic. Exp Hematol. 2008;36:733–741. 187. El-Ftesi S, Chang EI, Longaker MT, Gurtner GC. Aging and diabetes impair the neovascular potential of adipose-derived stromal cells. Plast Reconstr Surg. 2009;123:475–485.

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33 Sex, Gender, and Pain Roger B. Fillingim University of Florida, Gainesville, FL, United States

O U T L I N E 33.1 Introduction

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33.4 Sex Differences in Experimental Pain Responses 485 33.5 Mechanisms Contributing to Sex Differences in Pain 33.5.1 Biological Mechanisms 33.5.2 Psychosocial Mechanisms

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33.1 INTRODUCTION Pain is a universal experience, yet surprisingly difficult to define. While many definitions and descriptions of pain have been proffered, the most widely accepted definition is that provided by the International Association for the Study of Pain (IASP). IASP defines pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.”1 This brief definition conveys several different truisms regarding pain that warrant mention. First, while many think of pain as a sensory experience, the perception of pain has both sensory and emotional components. Notably, these two aspects of pain can vary independently, and are subserved by distinct neural pathways.2 Second, while pain is often a consequence of tissue damage, either can occur without the other. Thus, in many cases pain and tissue damage are poorly correlated, which explains the frequent failure of diagnostic and treatment procedures focused excessively on peripheral tissues as the source of pain. Third, implicit in this definition is that pain is Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00038-3

33.5.3 Mood and Affect 33.5.4 Coping and Catastrophizing 33.5.5 Early Life Adversity 33.5.6 Gender Roles 33.5.7 Biopsychosocial Interactions

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a personal and subjective experience. Indeed, the IASP Taxonomy Committee elaborated that their “definition avoids tying pain to the stimulus. Activity induced in the nociceptor and nociceptive pathways by a noxious stimulus is not pain, which is always a psychological state….” Therefore, despite great interest in developing biomarkers for pain, the gold standard for measuring pain is self-report. While acute pain is a universal experience and an important warning sign that promotes survival, chronic pain represents a pathological state in which pain persists beyond the normal healing time and loses its adaptive function. Chronic pain is arguably the most prevalent and costly public health issue in the developed world. Depending on the definition of chronic pain, up to onethird of adults report chronic pain, with 10% of adults endorsing high impact pain, which adversely affects quality of life and motivates health care seeking.3,4 Moreover, in the United States alone, the societal costs of pain are estimated at between $500 and $635 billion, exceeding the combined costs of cancer, AIDS, and heart disease.3 Chronic pain conditions are also leading causes

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of disability. Low back pain is responsible for more years lived with disability than any other condition worldwide, and chronic pain conditions account for three of the top four causes of years lived with disability.5 The enormous magnitude of chronic pain as a public health problem disproportionately affects some population groups. In particular, women are more commonly affected than men. This chapter will focus on sex differences in pain. Specifically, after briefly discussing some important conceptual issues, I will summarize findings regarding sex differences in clinical pain. These clinical findings have motivated considerable research examining sex differences in responses to experimentally induced pain, which I will also review. Then I will discuss biological and psychosocial mechanisms that may contribute to sex differences in pain, followed by a presentation of findings regarding sex differences in pain treatment responses. The chapter will conclude with consideration of important future directions.

33.2  CONCEPTUAL ISSUES Before discussing existing findings related to sex, gender, and pain, it is important to provide some context by highlighting several important conceptual issues. First, while the terms sex and gender are often used interchangeably, they have distinct meanings, which has important applications to the study of sex-related influences on pain. Sex refers to the biological categorization of male versus female based on anatomy and chromosomal complement. Gender, while related to sex, also incorporates psychosocial influences and the individual’s identification with and adherence to socially sanctioned gender norms and behaviors. Observed differences in pain responses between males and females inevitably reflect the influence of a combination of both sex (i.e., biological) and gender (i.e., psychosocial), though the proportional contributions and interactions among the sex and gender variables are typically unmeasured and poorly understood. We will return to this distinction between biological and psychosocial influences later in the chapter, when discussing the biopsychosocial model of pain. Another important issue to consider is whether the sex difference under study is quantitative or qualitative in nature. As related to pain, quantitative differences emerge when females are found to have a greater magnitude of pain than males, or vice versa. This is by far the most common type of sex difference reported in the literature, as will be discussed below. For example, epidemiological findings regarding potential sex differences in the prevalence of specific pain conditions represent quantitative sex differences, which can have profound implications for public health and resource allocation. However, it is also important to recognize qualitative

sex differences, wherein the biological and/or psychosocial mechanisms underlying pain responses are fundamentally different in women versus men. For example, a recent study reported that experimental nerve injury produced a similar magnitude of mechanical hypersensitivity in females and male mice; however, the hypersensitivity was mediated by activation of microglia in male but not female mice.6 This type of qualitative difference has several important implications. First, had these authors only studied quantitative sex differences, this would have been a null finding, as female and male mice showed similar magnitudes of hypersensitivity. Second, had the authors only included male mice in their study (as is typical), the mechanisms underlying pain hypersensitivity after nerve injury would have been incompletely understood. Third, these qualitative sex differences suggest that different treatments may need to be developed for females and males in order to reverse hypersensitivity after nerve injury. Additional examples of qualitative sex differences will be highlighted below.

33.3  SEX DIFFERENCES IN CLINICAL PAIN Abundant epidemiological evidence addresses whether there are sex differences in the prevalence of chronic pain. Before summarizing the findings, several methodological issues should be noted, as these can influence interpretation of the findings. Of course, epidemiologic data are influenced by multiple aspects of research design, including sampling methods, sample size and characteristics (e.g., age, race, socioeconomic status), response rates, and data analytic approaches. However, the case definition of chronic pain represents one of the most significant sources of variability in population-based studies. For example, multiple methods have been applied to define general chronic pain. One common approach is to ask respondents if they have experienced pain on more days than not over the last 6 months, to which a “yes” response qualifies as chronic pain. However, others have simply asked whether the individual is often troubled by pain, while another approach has been to ask whether the person has any of a number of chronic pain conditions (e.g., headache, back pain, neck pain, abdominal pain), and any affirmative response comprises chronic pain. These different approaches to classification of chronic pain produce variability in estimates of prevalence, which may influence the direction and/or magnitude of sex differences in pain prevalence. For example, if a respondent were asked “has a doctor ever told you that you have one of the following conditions,” this could bias the findings in the direction of increased frequency in females, given that women seek health care more often than men.

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10 Excess female prevalence (%)

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Case classification of chronic pain in epidemiological studies also suffers from the tendency to emphasize presence versus absence of pain with far less consideration for the severity and impact of pain. For example, the IOM Report previously reported that 100 million adults in the United States have chronic pain.3 While this number appears accurate based on other epidemiologic evidence, a significantly smaller proportion of people report pain that substantially impacts quality of life and function.4,7 Recognizing this, the recently released National Pain Strategy recommended a set of screener questions that can be used to identify “highimpact chronic pain,” based on pain severity and interference of pain with occupational and social activities. Characterizing the severity and impact of chronic pain has important implications for health care policy, since health care utilization is greatest (and arguably least effective) for those with high-impact pain. Population-based studies that have estimated the prevalence of general chronic pain have consistently reported greater chronic pain prevalence among women compared to men.8–11 As we summarized in a review article several years ago, across 10 population-based studies from across the world, prevalence of pain in women ranged from 11% to 59% in women and from 10% to 49% in men. All studies reported higher prevalence in women, with the excess female prevalence ranging from 1% to 14%. Since that time, additional findings have corroborated these results. For example, using data from Washington state, Nahin4 found that significantly more women (57.3%) than men (53.3%) reported “at least some pain” over the previous 3 months. Moreover, among non-Hispanic whites and blacks who preferred English as a language, women had higher rates of more severe pain. Data from the National Health Interview Survey (NHIS) reported that a significantly greater proportion of women (21.6%) than men (16.2%) endorsed persistent pain, defined as pain on most days or every day for the past 3 months.12 Thus, though the magnitude of the sex difference varies across studies, there is a highly consistent pattern of results showing that women more frequently endorse general chronic pain than men. Sex differences in the prevalence of specific types of chronic pain conditions have also been investigated. Data from such studies were recently summarized by Mogil,8 who computed the excess prevalence in females of several different chronic pain conditions, including: back pain, migraine, musculoskeletal pain, neuropathic pain, oral pain, osteoarthritis pain, and widespread pain. Out of 47 comparisons, only one showed an excess prevalence of pain among men, one showed no sex difference, and 45 showed an excess prevalence in women (computed as the prevalence in men subtracted from the prevalence in women). The data are summarized in Fig. 33.1, which shows that across all of the pain

FIGURE

33.1 Excess female prevalence of different pain conditions. Excess female prevalence was computed by subtracting the percentage of men who have the condition from the percentage of women with the condition. Source: Data are from Mogil JS. Sex differences in pain and pain inhibition: multiple explanations of a controversial phenomenon. Nat Rev Neurosci 2012;13(12):859–866.

conditions studied, the average excess prevalence in females was 5.5%. More recent findings have produced similar results. A World Health Organization study of adults 50 and older from six low- and middle-income countries revealed a higher prevalence of back pain in the past month among women (34.9%) versus men (24.2%), and men and women were overrepresented in the low and high pain severity groups, respectively.10 A recent Canadian study reported significantly greater prevalence of both knee and hip osteoarthritis among women.13 A meta-analysis of studies examining the prevalence of chronic widespread pain (CWP) found that rates of CWP were on average twice as high among women compared to men.14 Fibromyalgia (FM) is widely known to show female predominance15; however, a recent study demonstrates that the magnitude of the sex difference in FM prevalence varies greatly depending on the diagnostic criteria used.16 These authors determined the population prevalence of FM based on: (1) the 1990 American College of Rheumatology (ACR) Criteria,17 which required widespread pain (pain on both sides of the body and above and below the waist, as well as axial skeletal pain) plus pain in response to digital palpation (4 kg of force) in 11 of 18 tender points; (2) the 2010 ACR Preliminary Diagnostic Criteria,18 which implemented a widespread pain index score and replaced the tender point criterion with presence of nonpain symptoms (e.g., fatigue, cognitive symptoms, waking unrefreshed, somatic symptoms); and (3) a modified version of the 2010 ACR Criteria that used patient self-report rather than clinician examination for ascertainment of symptoms.19 The female-to-male ratios for FM prevalence based on the

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different diagnostic criteria the 1990 ACR Criteria, the preliminary 2010 ACR criteria, and the modified 2010 ACR criteria were 13.7, 4.8, and 2.3, respectively. This reveals a dramatic influence of diagnostic criteria on the magnitude of the sex difference. In particular, the presence of the tender point count appears to greatly bias the diagnosis toward women. Thus, taken together the available evidence demonstrates that most common chronic pain conditions are more frequent among women, though the size of the sex difference varies across conditions and even within conditions depending on classification methods. There are pain conditions that show excess male prevalence (e.g., cluster headache); however, these are far less common pain disorders. The above findings address sex differences irrespective of age, and given that pain prevalence varies across the lifespan, it is possible that sex differences might show different patterns in different age groups. Regarding pain in children, a systematic review found that most pain conditions are more prevalent in girls than boys, and girls are more likely to report multiple pain conditions.20 The studies reviewed included children from ages 2 to 18. Most pain conditions showed increasing prevalence with age among children, except for abdominal pain, which appeared to be more common in younger children. Some evidence suggests that the greater prevalence of pain in girls is more common among older children, particularly for headache and musculoskeletal pain. At the other end of the age spectrum, sex differences in pain prevalence among older adults vary across pain conditions. For example, the female predominance of headache, abdominal pain, and temporomandibular disorder (TMD) that is observed in early adulthood and middle age becomes far less pronounced among older adults. In contrast, women continue to experience higher rates of joint pain and neuropathic pain in late life.21 Thus, sex differences in pain prevalence occur across the lifespan, but the patterns can vary depending on age. The above findings address sex differences in pain prevalence, but it is also important to consider whether the severity and impact of pain differ for women and men. In addition to ratings of pain intensity, other measures of pain severity should be considered, including the bodily extent of pain, the number of comorbid pain conditions, and the duration of pain (i.e., transition from acute to chronic pain). Regarding ratings of pain among people with existing chronic pain conditions, most studies have examined patients in the clinical setting, which introduces a selection bias, since people seek health care primarily when they have relatively intense pain. These studies report variable findings regarding sex differences in pain severity, with some reporting significantly greater pain among women, while other report no sex differences in pain (e.g., Ref. 9,22). A less biased

approach to addressing this question resides in studies of community-based samples. For example, using data from the Multicenter Osteoarthritis Study (MOST), Glass and colleagues reported higher pain levels in women versus men across all grades of radiographic knee OA severity.23 However, the magnitude of these differences was small, and some became nonsignificant after controlling for potential confounding factors. We recently reported no sex differences in clinical pain severity in a community-based sample of patients with knee OA; however, women reported pain in more body sites than did men.24 A recent population-based Scandinavian study reported that women were more likely to report higher levels of pain associated with musculoskeletal conditions compared to men.25 In a study of young adults in Portugal, while FM prevalence was not significantly different between women and men, women reported significantly higher pain and nonpain symptoms of FM and greater widespread pain compared to men.26 Plesh and colleagues27 reported that among individuals with TMD, women were significantly more likely to have multiple comorbid pain conditions (e.g., headache/migraine, neck pain, low back pain, joint pain) compared to males. Also, a recent telephone survey of adults using chronic opioid therapy revealed that compared to men women had poorer global pain status (a metric that combined pain intensity with pain-related interference, pain impact, and activity limitation).28 In a study exploiting data from electronic health records from more than 11,000 patients who were seeking medical care, Ruau and colleagues29 found that ratings of disease-associated pain were significantly higher among women compared to men, though these differences were quite small in magnitude. Taken together these studies reflect the overall pattern in the literature that among people with existing chronic pain, the burden of pain appears to be higher among women than men. In addition to these findings regarding chronic pain severity, considerable research has addressed sex differences in acute pain severity. This is of particular importance, since more than 100 million surgical and other invasive procedures are performed in the United States each year,30 virtually all of which elicit pain.31 Moreover, severity of acute pain predicts postprocedural morbidity and costs and is a consistent indicator of risk for development of chronic pain.32 A previous systematic review found mixed evidence regarding sex differences in postoperative pain severity, with some studies showing greater pain in females, others showing less pain, and some studies showing no sex differences in pain.33 This variability in findings could be due to differences in the types of surgery, perioperative pain management protocols, or other methodological details. Tighe and colleagues34 examined more than 300,000 postoperative

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33.4  Sex Differences in Experimental Pain Responses

pain scores available in an electronic health record and found a small but statistically significant sex difference in postoperative pain ratings, with average pain for females being 4.11/10 and for males, 3.74/10. Notably, these authors also examined the frequency of severe pain events (i.e., pain ratings between 7 and 10), which they found to occur significantly more frequently among women than men. Given that acute pain severity predicts greater risk of chronic postoperative pain, some studies have examined sex as a risk factor for the latter. One systematic review of multiple types of surgeries found sex not to be a predictor of chronic postsurgical pain.35 Similarly, for total knee arthroplasty, which is among the most common elective surgical procedures, women appear to be at no greater risk for persistent pain than men.36 These findings from studies of postoperative pain suggest that while women may experience slightly greater acute pain, the risk of chronic postoperative pain does not differ as a function of sex. There is an abundant literature that addresses myriad aspects of sex differences in clinical pain. To briefly summarize, most common chronic pain conditions occur more frequently among women than men. Data regarding the severity of chronic and acute pain are less compelling. While some evidence indicates that chronic and acute pain are more severe among women (e.g., higher pain ratings, more comorbid pain conditions), these differences are less consistent and smaller in magnitude than findings regarding pain prevalence.

33.4  SEX DIFFERENCES IN EXPERIMENTAL PAIN RESPONSES Sex differences in clinical pain could arise due to numerous factors, including possible differences in disease processes and/or responses to treatment. In order to evaluate sex differences in pain while removing the influence of such clinical variables, researchers have investigated whether females and males respond differently to carefully controlled experimentally induced pain. These approaches are often referred to as quantitative sensory testing (QST), which refers to “a group of procedures that assess the perceptual responses to systematically applied and quantifiable sensory stimuli for the purpose of characterizing somatosensory function or dysfunction.”37 QST uses multiple stimulus modalities (e.g., vibration, thermal, pressure, electrical) that induce both painful and nonpainful perceptual experiences. Also, numerous measures are available to quantify the individual’s response to the stimulus. For example, pain threshold refers to the minimum stimulus intensity required to elicit pain, while pain tolerance refers to the maximum stimulus intensity an individual is

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willing to experience. Another common measure of pain responsivity involves delivery of standardized stimulus intensities and asking the person to rate the intensity of pain evoked by each stimulus (i.e., suprathreshold pain ratings). In recent years, QST studies have increasingly incorporated more dynamic protocols, such as temporal summation of pain, which refers to increased pain in response to repeated, rapid application of a series of painful stimuli.38 Temporal summation reflects a transient form of central sensitization and is the most commonly used QST measure of pain facilitation. Another dynamic QST measure is conditioned pain modulation (CPM), which assesses pain inhibitory capacity by quantifying the extent to which the painfulness of a stimulus applied to one body site is attenuated by concurrent application of a painful stimulus at a remote site.39 Thus, QST approaches can be used to assess general pain sensitivity (e.g., pain threshold), pain facilitation (e.g., temporal summation), and pain inhibition (e.g., CPM), and all of these approaches have been applied to the study of sex differences in pain perception. Over the past 25 years, several review articles have addressed the literature regarding sex differences in experimental pain responses. In a qualitative review, we40 reported that women exhibit greater experimental pain sensitivity than males, and this sex difference emerges for multiple stimulus modalities and at multiple body sites, suggesting that this is a generalized phenomenon rather than a site- or modality-specific one. A subsequent meta-analysis reported that sex differences in pain threshold and pain tolerance showed moderate effect sizes across multiple stimulus modalities.41 In 2009, we updated our previous review, including studies that had been published in the intervening years, and concluded “current human findings regarding sex differences in experimental pain indicate greater pain sensitivity among females compared with males for most pain modalities, including more recently implemented clinically relevant pain models such as temporal summation of pain and intramuscular injection of algesic substances.”9 In contrast, Racine and colleagues42 subsequently stated that “10 years of laboratory research have not been successful in producing a clear and consistent pattern of sex differences in human pain sensitivity, even with the use of deep, tonic, long-lasting stimuli, which are known to better mimic clinical pain.” Interestingly, Mogil8 examined the very studies upon which Racine and colleagues based their conclusion and presented compelling evidence that these studies indeed demonstrate significantly greater sensitivity among females. More recently, Hashmi and Davis43 have argued that sex differences in pain sensitivity “do represent trends, but, in most cases, the findings are more nuanced than the conclusions.” How is it that several review articles,

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each ostensibly examining the same literature, seemingly come to quite different conclusions? And what is the truth about sex differences in experimental pain sensitivity? To answer the second question first, the findings regarding sex differences in responses to experimentally induced pain are remarkably consistent in their direction—females consistently show greater pain sensitivity than men. This is true across virtually all stimulus modalities, all body regions, and all measures of pain sensitivity (i.e., pain threshold, pain tolerance, and suprathreshold pain ratings). In addition, women consistently show greater temporal summation of pain than men, including responses to heat,44 cutaneous mechanical,45 and pressure stimuli.46 Regarding pain inhibitory responses, the findings vary depending on the method of assessing pain inhibition; men appear to show greater CPM,47 but other methods of pain inhibition show variable results.9 Among children, the direction of sex differences is generally similar to that of adults; however, the magnitude of the differences appears to be considerably smaller, and the presence of sex differences is impacted by developmental stage.48 While the direction of the sex difference in experimental pain sensitivity is inarguable, as previous authors have noted,42 it is indeed true that the magnitude of the sex difference varies across studies. Multiple methodological factors likely contribute to this variability in effect size. For example, stimulus modality and parameters are important. As Hashmi and Davis49 reported, females show greater temporal summation in response to brief, repeated thermal stimuli, but females also show greater adaptation and habituation in response to sustained thermal stimuli. Thus, though both sets of studies involve dynamic heat stimuli, different stimulus parameters can dramatically influence the observed sex difference. Characteristics of the study sample can also impact the magnitude of the sex difference. It is important to recognize that sex represents only one of many individual difference factors that can substantively impact pain responses; therefore, the distribution of these other individual difference variables in the study sample can impact the magnitude of the sex difference.50 For example, other demographic factors, such as age and race/ethnicity, are strongly associated with experimental pain responses, and they could potentially interact with sex to influence pain perception. Hence, the age and racial/ethnic makeup of the study sample could impact the sex difference. In addition, myriad other biopsychosocial factors can affect pain sensitivity, and these influences (whether measured or unmeasured) could either magnify or attenuate the observed sex difference. Finally, there is some evidence that characteristics of the experimenter can impact experimental pain responses, which may contribute to the variability in magnitude of the effect across studies.

33.5  MECHANISMS CONTRIBUTING TO SEX DIFFERENCES IN PAIN Undoubtedly more important than explaining the variable effect sizes from studies of sex differences in pain sensitivity is elucidating their mechanisms and clinical relevance, a sentiment expressed by multiple authors.8,42,43 Numerous lines of evidence support the general clinical relevance of QST findings, including: (1) QST distinguishes patients with chronic pain from pain-free controls and has been useful in subgrouping chronic pain patients; (2) overlapping biopsychosocial mechanisms appear to influence both clinical pain and QST responses (e.g., genetics, demographic factors, psychological processes); (3) QST measures have predicted future development or severity of clinical pain; and (4) QST measures can predict responses to pain treatment and are sensitive to treatment outcomes.37 However, minimal direct evidence addresses whether sex differences in experimental pain sensitivity are associated with sex differences in clinical pain. Nonetheless, experimental pain measures provide the opportunity to investigate sex-related pain mechanisms under more highly controlled and systematic experimental conditions than are possible with studies of clinical pain. Those interested in understanding the mechanisms contributing to sex differences in pain should approach this from the perspective of the biopsychosocial model, which posits that the experience of pain is sculpted by complex and dynamic interactions among biological, psychological, and social factors.51 This model applies quite well to the study of sex differences in pain. Indeed, the terms sex and gender embody the biopsychosocial model as they refer to primary biological versus psychosocial influences, respectively, that contribute to sexrelated outcomes. Importantly, the factors that impact pain, including sex differences therein, are almost never purely “biological” or “psychosocial.” Rather, biological processes (e.g., neural activation, inflammation) directly impact psychological function (e.g., mood/affect, pain coping), and vice versa. Thus, when I refer to biological and psychosocial factors, this is primarily a distinction of convenience, which is based largely on the methods we use to measure these factors.

33.5.1  Biological Mechanisms A variety of biological processes likely contribute to sex differences in pain, with sex hormones representing potentially important factors. The epidemiology of some pain conditions implicates sex hormones, particularly estrogens. For example, the female predominance of several pain conditions emerges postpuberty and diminishes later in life, presumably postmenopausally.21

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33.5 Mechanisms Contributing to Sex Differences in Pain

Moreover, use of exogenous estrogens increases risk for some pain conditions among women.9 Endogenous hormonal fluctuations across the female menstrual cycle may also impact pain. For example, dysmenorrhea and menstrual migraine are by definition influenced by the menstrual cycle. Moreover, in a recent review article, Hassan and colleagues52 reported menstrual cycle influences on symptoms in several chronic pain conditions, including FM, TMD, migraine, and irritable bowel syndrome (IBS), with the greatest symptomatology occurring during cycle phases marked by low estrogen levels. Studies of experimental pain in humans have demonstrated changes in pain sensitivity across the menstrual cycle, with women showing greatest sensitivity to pain during premenstrual and menstrual phases of the cycle53; however, these effects are quite variable and small in magnitude.54,55 Interestingly, one study that showed no menstrual cycle effects on pain perception found that variability in hormonal levels across the menstrual cycle was associated with pain responses among healthy women. Specifically, higher testosterone levels predicted lower pain sensitivity, while higher estradiol levels predicted greater pain sensitivity.56 Thus, individual differences in hormonal activity could contribute to the inconsistent findings regarding menstrual cycle effects on pain responses. Thus, sex hormones appear to influence pain responses; however, the direction and magnitude of these effects are quite variable. This is likely because multiple factors can impact hormonal modulation of pain responses, including: (1) the timing and doses of hormones; (2) the type of pain being influenced; (3) the particular combinations of hormones (e.g., balance of estrogens, progestins, and testosterone); and (4) the hormones’ site(s) of action (e.g., peripheral, spinal, supraspinal). It is also important to recognize that sex hormones produce both organizational (long-term developmental influences) and activational (transient effects of hormonal fluctuations in adulthood) effects, and the former are quite difficult to study in humans.57 Sex hormones could potentially impact pain responses via both peripheral and central nervous system effects. For example, estrogens can heighten inflammatory responses, which can induce both peripheral and central sensitization.9 However, it should be noted that estrogenic influences on inflammation are highly complex and depend on the levels of estrogens and their sites of action.9 Sex hormones can also directly modulate central nociceptive processing via effects on multiple neurotransmitter systems. As we previously reviewed, estrogens can influence the endogenous opioid system as well as dopaminergic, serotonergic, and NMDA-receptor mediated neurotransmission.9 Additional biological mechanisms beyond sex hormones contribute to sex differences in pain responses. Sex differences in inflammatory responses have been

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well documented,58 and in addition to inflammatory conditions that include pain as a primary symptom (e.g., rheumatoid arthritis), increasing evidence implicates low-grade systemic inflammation as a contributor to chronic pain and increased pain sensitivity. Specifically, inflammation can sensitize peripheral nociceptors, but also activates microglia in the central nervous system, which can produce central pain sensitization.59 In humans, endotoxemia produced by injection of lipopolysaccharide (LPS) induces heightened pain sensitivity and impaired pain inhibition, and these effects emerged only among women in one study,60 while studies have reported no sex differences.61,62 Thus, systemic inflammation enhances pain sensitivity; however, the extent to which inflammatory processes differentially affect pain responses in women versus men remains unclear. The endogenous opioid system is the most studied pain modulatory system, and both preclinical and human studies demonstrate sex differences in endogenous opioid function. For example, rodent studies have demonstrated sexual dimorphism in the circuitry of the periaqueductal gray (PAG) and rostral ventromedial medulla (RVM), the major brainstem circuit that mediates endogenous and exogenous opioid analgesia.63 Moreover, inflammatory pain preferentially activated this descending PAG-RVM circuit in male animals.63 In humans, sustained muscle pain produced greater brain mu-opioid receptor activation among males than females.64 These findings imply sex differences in functioning of the endogenous opioid system, which may contribute to sex differences in pain. These findings are indirectly corroborated by evidence of sex-dependent associations between a variant of the mu-opioid receptor gene and pain responses. While two studies have shown that the A118G variant is protective against pain in men but not women,65,66 others have shown that the G-allele was associated with improved pain outcomes among women after motor vehicle accidents, but only those who reported high levels of peritraumatic distress.67 Thus, the endogenous opioid system seems to differentially influence pain in females and males, but the exact nature of these contributions remains to be determined. Because clinical and experimental pain studies in humans rely primarily on self-report to assess pain responses, sex differences in pain reporting could be an important contributor to sex differences in experimental pain sensitivity. That is, women may be more likely to report pain than men, which could result in lower pain thresholds and tolerance and higher suprathreshold pain ratings. While pain is by definition a subjective experience, measurable only by self-report, brain imaging is being increasingly used to assess pain-related cerebral responses, which are thought to reflect the neural processes contributing to the pain experience. Thus, sex differences in pain-related brain measures would reflect

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evidence of neural structure and function potentially contributing to sex differences in pain. As we previously reviewed,9 several studies have examined sex differences in brain activation in response to different experimental pain modalities. While there is considerable overlap in pain-induced brain responses between women and men, multiple studies have shown sex differences in activation across different brain regions, including the insula, cingulate cortex, prefrontal cortex, and the amygdala. While most of these studies showed greater activation of these brain regions among females, some have shown greater activation in males. More recently, in response to a visceral pain stimulus (rectal distension), women showed greater activation in the dorsolateral prefrontal cortex and the middle temporal gyrus during pain anticipation, and in response to pain women showed greater activation in the cerebellum and medial frontal gyrus.68 In a sample of older adults, Monroe and colleagues69 reported that in response to mild thermal pain females exhibited less deactivations than males in bilateral dorsolateral prefrontal cortex, somatomotor area, rostral anterior cingulate cortex (rACC), and dorsal ACC. However, at moderate pain detection levels, males showed greater activation in ipsilateral primary (SI) and secondary somatosensory cortices (SII) and posterior insula. Taken together, these findings from brain imaging studies indicate sex differences in neural responses to pain stimuli; however, the pattern of results varies across studies, due in part to differences in stimulus modalities and intensities. In addition to pain-induced cerebral activation, several studies have used neuroimaging to examine sex differences in resting state functional and structural brain measures as they relate to pain. For example, in a community-based sample of adults with joint pain, women showed lower gray matter volume in several brain regions compared to men, and heat pain thresholds were correlated with gray matter volumes (hippocampus, thalamus, anterior cingulate cortex) only among women.70 In a study of pediatric migraine patients and healthy controls, female migraineurs had more gray matter in the primary somatosensory cortex (S1), supplementary motor area, precuneus, basal ganglia, and amygdala compared to male migraineurs and to healthy controls.71 Also, female migraineurs showed greater resting state functional connectivity between the right precuneus and the left putamen, right caudate, left thalamus, and left amygdala compared with male migraineurs and healthy controls. This suggests that both structural and functional brain correlates of migraine headache differ for girls and boys. Among healthy adults, Galli and colleagues72 found the association of resting state networks with heat pain sensitivity to be sex-dependent. Specifically, increased connectivity within the visual resting state network correlated with

pain measures among men, while for women, decreased connectivity between this network and parietal and prefrontal brain regions was related to pain ratings. Also, Coulombe et  al.73 reported sex differences in the functional connectivity of the PAG with other brain regions, which could contribute to sex differences in endogenous pain modulation. In sum, these findings provide some evidence that resting state networks and structural brain changes may be associated with pain responses in a sexdependent manner.

33.5.2  Psychosocial Mechanisms The experience of pain is strongly influenced by multiple psychosocial variables. Indeed, it is well recognized that people with chronic pain report higher levels of psychological symptoms compared to pain-free individuals; however, psychological functioning is also a risk factor for future development of chronic pain.74,75 Moreover, psychological factors are associated with severity of acute pain after surgery as well as with experimental pain sensitivity.76 In the general population, various aspects of psychological functioning differ substantially for women versus men; therefore, psychosocial factors represent potentially important contributors to sex differences in pain responses, as has been recently reviewed in substantial detail.9,77 Below I will highlight several psychosocial variables that are relevant to sex differences in pain: mood and affect, pain coping and catastrophizing, early life adversity (ELA), and gender roles. Before discussing these specific variables, it is important to consider that there are two primary ways in which these variables could contribute to sex differences in pain. First, if depression were associated with increased risk for pain, and women experience higher levels of depression, then depression could partially or completely mediate sex differences in pain. That is, even if depression is equally related to pain in women and men, if women experience more depression, this could explain their higher levels of pain. Second, it is possible that a given psychosocial variable could be differentially related to pain in women versus men. For example, even if men and women reported similar levels of psychosocial stress, this variable could contribute to sex differences in pain if the relationship between stress and pain were stronger in women than in men.

33.5.3  Mood and Affect The two most commonly studied mood states in the context of pain are depression and anxiety, both of which contribute to increased risk for clinical pain. Regarding depression, clinical depression is more prevalent among women than men, and in the general population women report higher levels of sad or depressed mood compared

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to men.78 Among people with depression, a greater proportion of women than men report somatic symptoms, including pain.79 In a sample of immigrants with chronic back pain, depression was more common among women; however, this sex difference was accounted for by the presence of multiple pain sites in women.80 In contrast, in a study of older Irish adults, chronic pain more strongly predicted depression among men.81 Like depression, anxiety is more prevalent among women,78 and a recent study of older individuals with widespread musculoskeletal pain found that women were at increased risk for persistent anxiety.82 Interestingly, as we and others have previously reviewed, anxiety may be more strongly related to experimental pain sensitivity among women than men,9,83 although a more recent study found similar associations between anxiety and pain sensitivity across sex.84 These findings suggest that higher rates of both anxiety and depression among women could contribute to sex differences in pain; however, evidence that depression or anxiety shows sexdependent associations with pain is mixed.

33.5.4  Coping and Catastrophizing Pain coping refers to cognitive and behavioral strategies individuals employ to manage pain and reduce its negative impact on quality of life. Of course, coping strategies can be adaptive or maladapative. The former includes active coping approaches such as behavioral activation, calming self-statements, and distraction, while the latter refers to passive coping approaches, the most studied of which is pain catastrophizing.85 Pain catastrophizing is a negative cognitive and attentional approach to pain, which is characterized by magnification, rumination, and helplessness.86 Abundant research demonstrates that pain coping influences pain-related outcomes. Active coping strategies consistently predict improved pain-related adjustment, whereas catastrophizing is associated with increased pain severity, greater disability, and poorer psychological outcomes.87 Moreover, considerable evidence suggests that women and men cope differently with pain. Women have been found to employ a broader repertoire of pain coping strategies than men, which includes active coping approaches (e.g., behavioral activation, positive selfstatement, seeking social and emotional support) and higher levels of catastrophizing.88,89 In adults with osteoarthritis, Keefe and colleagues found that women reported greater pain, disability, and pain behavior, and these sex differences were mediated by higher levels of pain catastrophizing among women.90 These investigators also found that catastrophizing was more strongly related to negative mood among men than women with osteoarthritis pain.89 Among healthy individuals, Edwards and colleagues91 found that women reported

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higher pain catastrophizing, higher daily pain, and greater sensitivity to experimental pain. Also, catastrophizing mediated the sex difference in daily pain but not the sex difference in experimental pain sensitivity. These findings indicate consistent sex differences in pain coping, which may mediate sex differences in clinical pain; however, whether catastrophizing and pain coping contribute to sex differences in experimental pain sensitivity remains unclear.

33.5.5  Early Life Adversity ELA (e.g., physical or sexual abuse, trauma, parental neglect, social stress) contributes to multiple adverse health outcomes, including chronic pain. Indeed, ELA has been associated with multiple chronic pain conditions, particularly those characterized by altered central pain processing.92 Females more often experience ELA, hence, this represents a potential contributor to sex differences in pain. In addition, preclinical models have demonstrated sex-specific effects of ELA on pain responses and pain-related neural circuitry, particularly in models of visceral pain.93,94 Also, in humans, ELA was associated with altered resting state connectivity in salience networks in both male and female patients with IBS; however, only in males did ELA predict increased connectivity of the cerebellar network, which has been associated with fear perception, and physical and psychological pain.95 It is also important to note that ELA could drive other risk factors that may influence sex differences in pain. Specifically, ELA increases risk for depression and anxiety, which as discussed above may contribute to increased pain in women. Further, ELA may sex-dependently impact psychological responses to chronic pain. In a preclinical study, ELA produced heightened thermal and mechanical hypersensitivity after nerve injury in both female and male mice; however, ELA induced increased depression-like behaviors after nerve injury only in females.96 Thus, not only might ELA contribute to sex differences in pain due to its greater frequency in women, but the pain-related neurobiological and psychological consequences of ELA may also be sex-specific.

33.5.6  Gender Roles Gender roles refer to different patterns of cognitive and behavioral responses that are believed to be culturally and socially sanctioned for women versus men. These social roles that are perceived to be more appropriate for women are described as feminine, while those for men are considered masculine. One school of thought has posited that sex differences in pain are largely mediated by gender roles, in that females are socialized to attend to and report pain, whereas males are socialized to suppress pain responses.97 This line of

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thinking implies that gender roles influence pain reporting, such that observed sex differences in pain responses reflect biases in self-report or behavioral expression of pain rather than actual differences in the pain experience. Considerable research has explored the relationship between experimental pain responses and gender roles. Global gender role measures are weakly associated with pain responses, such that higher masculinity and lower femininity predict higher pain threshold and tolerance.98 But these correlations are small in magnitude. Pain-specific gender role measures have been developed, which assess individuals’ perceptions of their own pain sensitivity relative to the average man or women as well as their willingness to report pain compared to the average man or woman.99 Studies using these measures suggest that individuals who consider themselves less sensitive to pain have higher thresholds and tolerances, while those who report greater willingness to report pain provide higher ratings of painful stimuli.98 These correlations between pain-specific gender role measures and pain responses are moderate in magnitude. The influence of experimenter gender has also been examined as a reflection of gender role influences on pain, and these studies have shown mixed results. Most studies have shown no interactive effects of experimenter and participant gender on pain responses. When experimenter gender was emphasized by including attractive experimenters stereotypically attired to accentuate their masculine or feminine traits, some studies have found that male participants report less pain in the presence of a female versus a male experimenter.100,101 Taken together, these findings suggest that gender roles have small to moderate influences on experimental pain responses. Several important conceptual issues need to be considered when interpreting findings related to gender roles and pain. First, the prevailing view is that gender roles reflect cognitive and behavioral patterns that result from social learning—i.e., we teach girls to overexpress pain and boys to under-express it. However, in addition to social learning, gender-related attitudes and behaviors are strongly influenced by prenatal and neonatal hormonal events.102 Thus, to some extent gender roles reflect organizational hormonal effects, which could potentially overlap with the organizational hormonal influences on pain responses. This highlights the important possibility that rather than representing social influences on pain expression, gender roles may actually reflect the impact of sex-related neurobiological influences on pain processing. Second, the strongest associations of gender roles with pain responses come from studies that have used pain-specific measures of gender roles. These measures actually reflect gender-related constructs combined with individuals’ evaluations of their own pain sensitivity and pain reporting behavior, which complicates interpretation of the findings.

33.5.7  Biopsychosocial Interactions The above discussion highlights multiple biological and psychosocial mechanisms that contribute to sex differences in pain. The biopsychosocial model emphasizes that biological and psychosocial factors interact to influence pain, and sex-related examples of such interactions have been reported. The most studied genetic factor in pain research has been the catechol-O-methyltransferase (COMT) gene, which codes for COMT, an enzyme that metabolizes catecholamines. People with genotypes characterized by low COMT activity have been found to be more pain sensitive and at higher risk for clinical pain.103 Interestingly, some findings have demonstrated sex-specific effects of COMT genotype on pain, such that low COMT activity is more strongly associated with increased pain among females.104 Recently, Meloto and colleagues105 demonstrated that COMT and stress interacted to influence pain in a sex-dependent manner. Specifically, among low stress women and men, an increasing number of low COMT haplotypes predicted increased pain after motor vehicle accidents. However, in high stress men, an increasing number of low COMT activity haplotypes predicted reduced pain, an effect not present in females. Another example of a sex × stress × gene interaction involved the gene encoding the vasopressin-1a receptor.106 Mouse studies revealed an association of Avpr1a with nociceptive responses to inflammatory pain. Subsequent human studies found no association of a single nucleotide polymorphism (SNP, rs10877969) in the promoter region of the human AVPR1A gene with responses to capsaicin. However, additional data analysis that incorporated both stress level and sex in the model demonstrated a significant three-way interaction, such that among males reporting high stress, AVPR1A genotype significantly affected pain ratings.106 This AVPR1A SNP was not related to pain response in males reporting low stress or in females regardless of stress level. Interestingly, similar interactions between genotype, sex, and stress were observed in the efficacy of desmopressin (a stable vasopressin analog) against capsaicin pain. These two sets of reveal sex-related biopsychosocial interactions on pain responses, clearly demonstrating the importance of considering sex when investigating the individual and combined effects of biological and psychosocial factors on pain.

33.6  SEX DIFFERENCES IN PAIN TREATMENT Beyond sex differences in the prevalence or severity of pain, sex differences in pain treatment and responses thereto have been the subject of considerable research.

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Patient sex/ gender

Biopsychosocial pain mechanisms

Patient-provider interactions Pain treatment responses

FIGURE 33.2  Pathways whereby patient sex/gender can influence pain treatment. This conceptual model shows that patient sex/ gender could directly influence treatment outcomes. Also, two indirect paths are also plausible: (1) patient sex/gender could influence pain– provider interactions, thereby potentially impacting both the treatment that is delivered and the patient’s response to that treatment; (2) patient sex/gender could influence biological or psychosocial mechanisms, which could in turn influence treatment response.

Fig. 33.2 presents a conceptual model of pathways whereby the sex/gender of the patient could influence pain treatment. There is a potential direct pathway, whereby patient sex influences the response to pain treatment, presumably because the impact of the active ingredient of the treatment is sexually dimorphic (e.g., hormonal influences on drug effects). However, there are also indirect pathways. Specifically, patient sex/gender could impact patient–provider interactions as well as patient and/or provider decisions regarding paint treatments. This could not only lead to differences in the provision of pain treatment for female versus male patients, but may also alter patient responses to those treatments, since patient provider interactions could influence nonspecific aspects of treatment, including compliance and expectations. A second indirect pathway exists in that the biological and psychosocial mechanisms that treatments affect may have sex-dependent influences on pain outcomes. For example, if a given psychological intervention altered brain circuits that contribute more robustly to pain among women, this treatment would likely be more effective for women. Regarding patient–provider interactions, considerable evidence demonstrates that the gender of the patient interacts with the gender of the physician to impact the nature of these interactions.107 This raises the question whether women and men receive different treatments for pain. That is, is there a gender bias in pain treatment? While several oft-cited studies have reported that women are at risk for under treatment of pain, a systematic review concluded that women and men often receive different pain treatments; however, this disparity does not consistently show bias against women or men.108 This pattern was recently corroborated by a study of veterans, which demonstrated that chronic pain care for females overall was more consistent with

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clinical practice guidelines; however, women were also more likely to be prescribed sedatives, which is inconsistent with guidelines.109 Notably, biases in pain treatment typically result from complex interactions among multiple factors, including both patient and provider characteristics. A recent study showed that after reviewing a patient video, providers (including specialist physicians and medical students) judged female patients to have less pain and be more likely to exaggerate their pain.110 This effect was greater in students than physicians. Moreover, providers were more likely to prescribe analgesics for male patients and psychological treatment for female patients. Also, a recent study using computerized patient avatars to depict patients with chronic low back pain demonstrated that female patients were more likely to be prescribed antidepressants and mental health treatments; however, this effect only emerged for female providers.111 These studies suggest that genderrelated biases in pain treatment exist; however, these biases are influenced by multiple contextual factors that interact with the patient’s sex. Whether pain treatments are differentially effective for women versus men has received substantial empirical attention. Regarding opioid analgesia, Niesters and colleagues112 conducted a meta-analysis of studies examining sex differences in opioid analgesia in acute clinical pain as well as experimental pain. For mu-opioid agonists, there was modest evidence of lower postoperative opioid consumption for females versus males, and this effect was most pronounced for studies of morphine administered via patient controlled analgesia (PCA). The authors surmised that PCA studies typically evaluate opioid consumption over a longer period of time, which may better reveal sex differences as morphine has a slower onset but longer duration of action in women. It is important to note that these findings reflect differences in opioid consumption rather than analgesic efficacy. Interestingly, in experimental studies, which tend to be shorter in duration and also directly assess analgesic efficacy, women showed greater mu-opioid analgesia, and these effect sizes were larger than for the clinical studies. With mixed action opioids (e.g., pentazocine, nalbuphine, butorphanol), clinical studies in acute pain models demonstrate substantially greater analgesic responses among women. In contrast, experimental studies demonstrated no sex differences in analgesia whatsoever. These divergent findings may reflect the nature of the pain models, as the clinical models that revealed more robust analgesia in women all involved inflammation, while the experimental pain models were definitively noninflammatory. It is important to note that compared to men women experience more side effects from acute opioid administration, including nausea and vomiting and adverse cognitive-affective effects.113 These findings suggest that for acute clinical and experimental pain,

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women appear to show better mu-opioid analgesia, and mixed action opioid confer greater analgesia for women in postoperative pain models but not experimental pain. However, the greater side effects among women suggest that the therapeutic window may be similar across sex. The use of opioids for chronic noncancer pain has generated considerable controversy and public health concern in recent years. Little evidence has addressed sex differences in the efficacy of opioids for chronic pain. Because of the greater prevalence of chronic pain, women are more likely to be prescribed opioids than men; however, females are more likely to use prescription opioids even when accounting for presence of chronic pain.114,115 Also, as noted above, among people with chronic pain using long-term opioids, women were at greater risk for unfavorable chronic pain status.28 However, men are at increased risk for dose escalation and opioid-related death.116 Moreover, nonmedical use of opioids appears to be higher among males.117 Thus, while opioid use is more common among women with chronic pain, some adverse outcomes are more frequent in males, but sex differences in the efficacy of opioids in this population are poorly understood. Limited research has examined sex differences in responses to other pharmacologic pain treatments. In a previous human laboratory study males exhibited greater analgesia than females for ibuprofen tested against electrical pain118; however, a subsequent analysis of ibuprofen for postoperative pain showed no sex differences in analgesia.119 Interestingly, a recent study demonstrated that when they expected to receive ibuprofen, males showed greater analgesic responses whether they received ibuprofen or placebo, suggesting greater expectation-induced analgesia in males.120 Likewise, other studies of laboratory pain models have reported greater placebo analgesic responses among males than females.121,122 Others have reported greater placebo responses among women,123 and a recent study of children showed that girls experienced more robust placebo analgesia than boys.124 These potential sex differences in placebo analgesia have important implications for interpretation of data from clinical trials, and they may also reflect differences in endogenous analgesia mechanisms between females and males. A few studies have reported sex differences in outcomes from nonpharmacologic pain treatments. As we previously described, studies of rehabilitation and interdisciplinary pain care have shown variable findings regarding sex differences.9 Some studies indicate that women show greater improvements, some show similar treatment-related improvement across sex, while others find that men experience better treatment outcomes. The small number of studies and differences in patient populations, interventions, and outcome variables likely contribute to the inconsistent results.

33.7  CONCLUSIONS AND FUTURE DIRECTIONS While the literature reviewed above reveals considerable variability in findings regarding sex differences in pain, several conclusions can be confidently drawn. First, women bear a greater burden of clinical pain than men. Specifically, the prevalence of chronic pain, broadly defined, is consistently higher among women, and many of the most common chronic pain conditions are also more common among women. Evidence regarding sex differences in clinical pain severity is less consistent; however, the trend is toward greater pain severity among women. Sex differences in responses to experimental pain show a highly consistent pattern of results, with women reporting lower pain thresholds and tolerances and providing higher ratings of painful stimuli. While this pattern is quite consistent, the magnitude of these differences varies considerably across studies and across stimulus modalities. Multiple mechanisms contribute to the observed sex differences in pain, including sex hormones, endogenous opioids, brain responses, mood and affect, pain coping, and gender roles. Notably, several studies have demonstrated that biological and psychosocial factors interact to influence sex differences in pain responses. Thus, sex differences in pain are best conceptualized in the context of the biopsychosocial model of pain. Finally, sex differences in responses to pain treatments have been investigated. Women appear to exhibit greater analgesic responses to mu-opioid agonists and to mixed action opioids; however, these findings vary across pain models, specific medications, and modes of drug delivery. Sex differences in responses to other pain treatments have received limited attention.

33.7.1  Future Directions The past 20 years have witnessed tremendously increased preclinical and clinical research addressing sex differences in pain, yet this burgeoning knowledge base has produced minimal impact on assessment and treatment of pain in humans. To remedy this shortcoming, I offer several recommendations to guide future research on sex differences in pain. First, preclinical research needs to incorporate sex differences as a matter of course. The National Institutes of Health (NIH) recently announced a policy requiring investigators to include both sexes in their preclinical studies, unless there was compelling justification for not doing so.125 There has been considerable backlash against this policy from the preclinical research community, though proponents have convincingly argued that the benefits of the policy could be transformative.126 Indeed, the historical absence of sex differences in preclinical research may help explain why

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REFERENCES

the robust sex differences that have been observed have not led to substantive clinical impact. That is, because preclinical discovery research has almost completely excluded sex as a potential factor, the opportunity to identify targets, and/or to conduct early tests of compounds that may show sex-specific effects has been lost. Moreover, it seems plausible that some compounds that were deemed not worth pursuing based on studies in male rodents might have produced more promising results in the female animals that were never tested. Thus, incorporating both sexes into preclinical research would allow investigators to identify sex-specific targets and molecules, thereby seeding the pipeline with the potential to produce sex-tailored therapies. Second, future research should focus on elucidating biopsychosocial mechanisms driving sex differences in pain, in lieu of additional descriptive studies that demonstrate yet again that males and females are different. This recommendation will be best served by translational approaches that take novel findings from preclinical studies and test their relevance in humans, as in previous studies.106,127 Where possible, mechanistic research should incorporate study designs that model interactions among biological and psychosocial factors, as this is a critical tenet of the biopsychosocial model that is often overlooked. Importantly, quantitative sex differences in pain are not a prerequisite to elucidating sex-specific pain mechanisms, as qualitative sex differences can arise even when males and females show similar pain responses. For example, we previously showed that the melanocortin-1-receptor gene influenced analgesic responses to kappa-opioid agonists among females only, even though no overall sex difference in analgesia emerged.127 Hence, analyses to identify sex-specific mediators of pain and treatment responses can be highly informative, even when no quantitative sex difference is observed. Third, clinical studies, particularly clinical trials, should routinely include analyses of sex differences, even if these findings are presented in supplementary tables. Very limited information regarding sex differences in the efficacy and/or adverse effects of pain treatments is available, despite the fact that both sexes have been included in clinical trials for many years. Had these data been presented in publications, meta-analyses could handily be carried out to identify any potential sex differences. Even knowing that no sex differences whatsoever are present for a given treatment would be important information. The growth in knowledge over the past two decades combined with the continuing increase in interest regarding sex differences bodes well for the future of research on sex differences in pain. Building on the existing research base by following the above recommendations holds the potential to substantively impact pain treatment among women and men in the foreseeable future.

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References 1. Merskey H, Bogduk N. Classification of Chronic Pain. Seattle, WA: IASP Press; 1994. 2. Rainville P, Duncan GH, Price DD, Carrier B, Bushnell MC. Pain affect encoded in human anterior cingulate but not somatosensory cortex. Science. 1997;277(5328):968–971. 3. Institute of Medicine (US) Committee on Advancing Pain Research, Care, and Education Relieving Pain in America: A Blueprint for Transforming Prevention, Care, Education, and Research. Washington, DC: National Academies Press; 2011. 4. Nahin RL. Estimates of pain prevalence and severity in adults: United States, 2012. J Pain. 2015;16(8):769–780. 5. Global Burden of Disease Study 2013 Collaborators Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;386(9995):743–800. 6. Sorge RE, Mapplebeck JC, Rosen S, et al. Different immune cells mediate mechanical pain hypersensitivity in male and female mice. Nat Neurosci. 2015;18(8):1081–1083. 7. Blyth FM, March LM, Brnabic AJ, Jorm LR, Williamson M, Cousins MJ. Chronic pain in Australia: a prevalence study. Pain. 2001;89(2-3):127–134. 8. Mogil JS. Sex differences in pain and pain inhibition: multiple explanations of a controversial phenomenon. Nat Rev Neurosci. 2012;13(12):859–866. 9. Fillingim RB, King CD, Ribeiro-Dasilva MC, Rahim-Williams B, Riley III JL. Sex, gender, and pain: a review of recent clinical and experimental findings. J Pain. 2009;10(5):447–485. 10. Stewart Williams J, Ng N, Peltzer K, et  al. Risk factors and disability associated with low back pain in older adults in low- and middle-income countries. results from the WHO Study on Global AGEing and Adult Health (SAGE). PLoS One. 2015;10(6):e0127880. 11. Herin F, Vezina M, Thaon I, Soulat JM, Paris C, group E. Predictive risk factors for chronic regional and multisite musculoskeletal pain: a 5-year prospective study in a working population. Pain. 2014;155(5):937–943. 12. Kennedy J, Roll JM, Schraudner T, Murphy S, McPherson S. Prevalence of persistent pain in the U.S. adult population: new data from the 2010 national health interview survey. J Pain. 2014;15(10):979–984. 13. Plotnikoff R, Karunamuni N, Lytvyak E, et  al. Osteoarthritis prevalence and modifiable factors: a population study. BMC Public Health. 2015;15:1195. 14. Mansfield KE, Sim J, Jordan JL, Jordan KP. A systematic review and meta-analysis of the prevalence of chronic widespread pain in the general population. Pain. 2016;157(1):55–64. 15. McBeth J, Jones K. Epidemiology of chronic musculoskeletal pain. Best Pract Res Clin Rheumatol. 2007;21(3):403–425. 16. Jones GT, Atzeni F, Beasley M, Fluss E, Sarzi-Puttini P, Macfarlane GJ. The prevalence of fibromyalgia in the general population: a comparison of the American College of Rheumatology 1990, 2010, and modified 2010 classification criteria. Arthritis Rheumatol. 2015;67(2):568–575. 17. Wolfe F, Smythe HA, Yunus MB, et  al. The American College of Rheumatology 1990 criteria for the classification of fibromyalgia. Report of the multicenter criteria committee. Arthritis Rheum. 1990;33(2):160–172. 18. Wolfe F, Clauw DJ, Fitzcharles MA, et al. The American College of Rheumatology preliminary diagnostic criteria for fibromyalgia and measurement of symptom severity. Arthritis Care Res. 2010;62(5):600–610. 19. Wolfe F, Clauw DJ, Fitzcharles MA, et al. Fibromyalgia criteria and severity scales for clinical and epidemiological studies: a

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modification of the ACR Preliminary Diagnostic Criteria for Fibromyalgia. J Rheumatol. 2011;38(6):1113–1122. 20. King S, Chambers CT, Huguet A, et  al. The epidemiology of chronic pain in children and adolescents revisited: a systematic review. Pain. 2011;152(12):2729–2738. 21. LeResche L. Epidemiologic perspectives on sex differences in pain. In: Fillingim RB, ed. Sex, Gender, and Pain. Seattle, WA: IASP Press; 2000:233–249. 22. Racine M, Castarlenas E, de la Vega R, et al. Sex differences in psychological response to pain in patients with fibromyalgia syndrome. Clin J Pain. 2015;31(5):425–432. 23. Glass N, Segal NA, Sluka KA, et al. Examining sex differences in knee pain: the multicenter osteoarthritis study. Osteoarthritis Cartilage. 2014;22(8):1100–1106. 24. Bartley EJ, King CD, Sibille KT, et  al. Enhanced pain sensitivity among individuals with symptomatic knee osteoarthritis: potential sex differences in central sensitization. Arthritis Care Res (Hoboken). 2016;68(4):472–480. 25. Wranker LS, Rennemark M, Berglund J. Pain among older adults from a gender perspective: findings from the Swedish National Study on Aging and Care (SNAC-Blekinge). Scand J Public Health. 2016;44(3):258–263. 26. Lourenco S, Costa L, Rodrigues AM, Carnide F, Lucas R. Gender and psychosocial context as determinants of fibromyalgia symptoms (fibromyalgia research criteria) in young adults from the general population. Rheumatology (Oxford). 2015;54(10):1806–1815. 27. Plesh O, Adams SH, Gansky SA. Temporomandibular joint and muscle disorder-type pain and comorbid pains in a national US sample. J Orofac Pain. 2011;25(3):190–198. 28. LeResche L, Saunders K, Dublin S, et  al. Sex and age differences in global pain status among patients using opioids long term for chronic noncancer pain. J Womens Health (Larchmt). 2015;24(8):629–635. 29. Ruau D, Liu LY, Clark JD, Angst MS, Butte AJ. Sex differences in reported pain across 11,000 patients captured in electronic medical records. J Pain. 2012;13(3):228–234. 30. Cullen KA, Hall MJ, Golosinskiy A. Ambulatory surgery in the United States, 2006. Natl Health Stat Rep. 2009(11):1–25. 31. Gerbershagen HJ, Aduckathil S, van Wijck AJ, Peelen LM, Kalkman CJ, Meissner W. Pain intensity on the first day after surgery: a prospective cohort study comparing 179 surgical procedures. Anesthesiology. 2013;118(4):934–944. 32. Kehlet H, Jensen TS, Woolf CJ. Persistent postsurgical pain: risk factors and prevention. Lancet. 2006;367(9522):1618–1625. 33. Ip HY, Abrishami A, Peng PW, Wong J, Chung F. Predictors of postoperative pain and analgesic consumption: a qualitative systematic review. Anesthesiology. 2009;111(3):657–677. 34. Tighe PJ, Riley 3rd JL, Fillingim RB. Sex differences in the incidence of severe pain events following surgery: a review of 333,000 pain scores. Pain Med. 2014;15(8):1390–1404. 35. Hinrichs-Rocker A, Schulz K, Jarvinen I, Lefering R, Simanski C, Neugebauer EA. Psychosocial predictors and correlates for chronic post-surgical pain (CPSP) - a systematic review. Eur J Pain. 2009;13(7):719–730. 36. Lewis GN, Rice DA, McNair PJ, Kluger M. Predictors of persistent pain after total knee arthroplasty: a systematic review and meta-analysis. Br J Anaesth. 2015;114(4):551–561. 37. Cruz-Almeida Y, Fillingim RB. Can quantitative sensory testing move us closer to mechanism-based pain management? Pain Med. 2014;15(1):61–72. 38. Yarnitsky D, Granot M, Granovsky Y. Pain modulation profile and pain therapy: between pro- and antinociception. Pain. 2014;155(4):663–665. 39. Yarnitsky D, Arendt-Nielsen L, Bouhassira D, et  al. Recommendations on terminology and practice of psychophysical DNIC testing. Eur J Pain. 2010;14(4):339.

40. Fillingim RB, Maixner W. Gender differences in the responses to noxious stimuli. Pain Forum. 1995;4(4):209–221. 41. Riley JL, Robinson ME, Wise EA, Myers CD, Fillingim RB. Sex differences in the perception of noxious experimental stimuli: a meta-analysis. Pain. 1998;74:181–187. 42. Racine M, Tousignant-Laflamme Y, Kloda LA, Dion D, Dupuis G, Choiniere M. A systematic literature review of 10 years of research on sex/gender and experimental pain perception - part 1: are there really differences between women and men? Pain. 2012;153(3):602–618. 43. Hashmi JA, Davis KD. Deconstructing sex differences in pain sensitivity. Pain. 2014;155(1):10–13. 44. Fillingim RB, Maixner W, Kincaid S, Silva S. Sex differences in temporal summation but not sensory-discriminative processing of thermal pain. Pain. 1998;75(1):121–127. 45. Sarlani E, Grace EG, Reynolds MA, Greenspan JD. Sex differences in temporal summation of pain and aftersensations following repetitive noxious mechanical stimulation. Pain. 2004;109(1-2):115–123. 46. Graven-Nielsen T, Vaegter HB, Finocchietti S, Handberg G, Arendt-Nielsen L. Assessment of musculoskeletal pain sensitivity and temporal summation by cuff pressure algometry: a reliability study. Pain. 2015;156(11):2193–2202. 47. Popescu A, LeResche L, Truelove EL, Drangsholt MT. Gender differences in pain modulation by diffuse noxious inhibitory controls: a systematic review. Pain. 2010;150(2):309–318. 48. Boerner KE, Birnie KA, Caes L, Schinkel M, Chambers CT. Sex differences in experimental pain among healthy children: a systematic review and meta-analysis. Pain. 2014;155(5):983–993. 49. Hashmi JA, Davis KD. Women experience greater heat pain adaptation and habituation than men. Pain. 2009;145(3):350–357. 50. Fillingim RB. Individual differences in pain responses. Curr Rheumatol Rep. 2005;7(5):342–347. 51. Engel GL. The need for a new medical model: a challenge for biomedicine. Science. 1977;196(4286):129–136. 52. Hassan S, Muere A, Einstein G. Ovarian hormones and chronic pain: A comprehensive review. Pain. 2014;155(12):2448–2460. 53. Riley III JL, Robinson ME, Wise EA, Price DD. A meta-analytic review of pain perception across the menstrual cycle. Pain. 1999;81:225–235. 54. Sherman JJ, LeResche L. Does experimental pain response vary across the menstrual cycle? A methodological review. Am J Physiol Regul Integr Comp Physiol. 2006;291(2):R245–R256. 55. Iacovides S, Avidon I, Baker FC. Does pain vary across the menstrual cycle? A review. Eur J Pain. 2015;19(10):1389–1405. 56. Bartley EJ, Palit S, Kuhn BL, et  al. Natural variation in testosterone is associated with hypoalgesia in healthy women. Clin J Pain. 2015;31(8):730–739. 57. Craft RM. Modulation of pain by estrogens. Pain. 2007;132: S3–S12. 58. Derry HM, Padin AC, Kuo JL, Hughes S, Kiecolt-Glaser JK. Sex Differences in Depression: Does Inflammation Play a Role? Curr Psychiatry Rep. 2015;17(10):78. 59. Ren K, Dubner R. Interactions between the immune and nervous systems in pain. Nat Med. 2010;16(11):1267–1276. 60. Karshikoff B, Lekander M, Soop A, et al. Modality and sex differences in pain sensitivity during human endotoxemia. Brain Behav Immun. 2015;46:35–43. 61. Karshikoff B, Jensen KB, Kosek E, et al. Why sickness hurts: A central mechanism for pain induced by peripheral inflammation. Brain Behav Immun. 2016;57:38–46. 62. Wegner A, Elsenbruch S, Rebernik L, et  al. Inflammationinduced pain sensitization in men and women: does sex matter in experimental endotoxemia? Pain. 2015;156(10):1954–1964. 63. Loyd DR, Murphy AZ. The neuroanatomy of sexual dimorphism in opioid analgesia. Exp Neurol. 2014;259:57–63.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

64. Zubieta JK, Dannals RF, Frost JJ. Gender and age influences on human brain mu-opioid receptor binding measured by PET. Am J Psychiatry. 1999;156(6):842–848. 65. Fillingim RB, Kaplan L, Staud R, et al. The A118G single nucleotide polymorphism of the mu-opioid receptor gene (OPRM1) is associated with pressure pain sensitivity in humans. J Pain. 2005;6(3):159–167. 66. Olsen MB, Jacobsen LM, Schistad EI, et al. Pain intensity the first year after lumbar disc herniation is associated with the A118G Polymorphism in the opioid receptor mu 1 gene: evidence of a sex and genotype interaction. J Neurosci. 2012;32(29):9831–9834. 67. Linnstaedt SD, Hu J, Bortsov AV, et  al. mu-Opioid receptor gene A118 G variants and persistent pain symptoms among men and women experiencing motor vehicle collision. J Pain. 2015;16(7):637–644. 68. Benson S, Kotsis V, Rosenberger C, et al. Behavioural and neural correlates of visceral pain sensitivity in healthy men and women: does sex matter? Eur J Pain. 2012;16(3):349–358. 69. Monroe TB, Gore JC, Bruehl SP, et  al. Sex differences in psychophysical and neurophysiological responses to pain in older adults: a cross-sectional study. Biol Sex Differ. 2015;6:25. 70. de Kruijf M, Bos D, Huygen FJ, et  al. Structural brain alterations in community dwelling individuals with chronic joint pain. AJNR Am J Neuroradiol. 2016;37(3):430–438. 71. Faria V, Erpelding N, Lebel A, et al. The migraine brain in transition: girls vs boys. Pain. 2015;156(11):2212–2221. 72. Galli G, Santarnecchi E, Feurra M, et  al. Individual and sexrelated differences in pain and relief responsiveness are associated with differences in resting-state functional networks in healthy volunteers. Eur J Neurosci. 2016;43(4):486–493. 73. Coulombe MA, Erpelding N, Kucyi A, Davis KD. Intrinsic functional connectivity of periaqueductal gray subregions in humans. Hum Brain Mapp. 2016;37(4):1514–1530. 74. Linton SJ. A review of psychological risk factors in back and neck pain. Spine. 2000;25(9):1148–1156. 75. Gatchel RJ, Peng YB, Peters ML, Fuchs PN, Turk DC. The biopsychosocial approach to chronic pain: scientific advances and future directions. Psychol Bull. 2007;133(4):581–624. 76. Mavros MN, Athanasiou S, Gkegkes ID, Polyzos KA, Peppas G, Falagas ME. Do psychological variables affect early surgical recovery? PloS One. 2011;6(5):e20306. 77. Racine M, Tousignant-Laflamme Y, Kloda LA, Dion D, Dupuis G, Choiniere M. A systematic literature review of 10 years of research on sex/gender and pain perception - part 2: do biopsychosocial factors alter pain sensitivity differently in women and men? Pain. 2012;153(3):619–635. 78. Altemus M, Sarvaiya N, Neill Epperson C. Sex differences in anxiety and depression clinical perspectives. Front Neuroendocrinol. 2014;35(3):320–330. 79. Marcus SM, Kerber KB, Rush AJ, et al. Sex differences in depression symptoms in treatment-seeking adults: confirmatory analyses from the Sequenced Treatment Alternatives to Relieve Depression study. Compr Psychiatry. 2008;49(3):238–246. 80. Taloyan M, Lofvander M. Depression and gender differences among younger immigrant patients on sick leave due to chronic back pain: a primary care study. Prim Health Care Res Dev. 2014;15(1):5–14. 81. Regan CO, Kearney PM, Savva GM, Cronin H, Kenny RA. Age and sex differences in prevalence and clinical correlates of depression: first results from the Irish Longitudinal Study on Ageing. Int J Geriat Psychiatry. 2013;28(12):1280–1287. 82. Rzewuska M, Mallen CD, Strauss VY, Belcher J, Peat G. Oneyear trajectories of depression and anxiety symptoms in older patients presenting in general practice with musculoskeletal pain: A latent class growth analysis. J Psychosom Res. 2015;79(3):195–201.

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83. Jones A, Zachariae R. Gender, anxiety, and experimental pain sensitivity: an overview. J Am Med Womens Assoc. 2002;57(2):91–94. 84. Moore DJ, Eccleston C, Keogh E. Does sex moderate the relationship between anxiety and pain? Psychol Health. 2013;28(7):746–764. 85. Keefe FJ, Rumble ME, Scipio CD, Giordano LA, Perri LM. Psychological aspects of persistent pain: current state of the science. J Pain. 2004;5(4):195–211. 86. Sullivan MJ, Thorn B, Haythornthwaite JA, et  al. Theoretical perspectives on the relation between catastrophizing and pain. Clin J Pain. 2001;17(1):52–64. 87. Peres MF, Lucchetti G. Coping strategies in chronic pain. Curr PainHeadache Rep. 2010;14(5):331–338. 88. El-Shormilisy N, Strong J, Meredith PJ. Associations between gender, coping patterns and functioning for individuals with chronic pain: a systematic review. Pain Res Manag. 2015;20(1):48–55. 89. Keefe FJ, Affleck G, France CR, et al. Gender differences in pain, coping, and mood in individuals having osteoarthritic knee pain: a within-day analysis. Pain. 2004;110(3):571–577. 90. Keefe FJ, Lefebvre JC, Egert JR, Affleck G, Sullivan MJ, Caldwell DS. The relationship of gender to pain, pain behavior, and disability in osteoarthritis patients: the role of catastrophizing. Pain. 2000;87(3):325–334. 91. Edwards RR, Haythornthwaite JA, Sullivan MJ, Fillingim RB. Catastrophizing as a mediator of sex differences in pain: differential effects for daily pain versus laboratory-induced pain. Pain. 2004;111(3):335–341. 92. Jones GT. Psychosocial vulnerability and early life adversity as risk factors for central sensitivity syndromes. Curr Rheumatol Rev. 2016;12(2):140–153. 93. Holschneider DP, Guo Y, Mayer EA, Wang Z. Early life stress elicits visceral hyperalgesia and functional reorganization of pain circuits in adult rats. Neurobiol Stress. 2016;3:8–22. 94. Chaloner A, Greenwood-Van Meerveld B. Early life adversity as a risk factor for visceral pain in later life: importance of sex differences. Front Neurosci. 2013;7:13. 95. Gupta A, Kilpatrick L, Labus J, et al. Early adverse life events and resting state neural networks in patients with chronic abdominal pain: evidence for sex differences. Psychosom Med. 2014;76(6):404–412. 96. Nishinaka T, Kinoshita M, Nakamoto K, Tokuyama S. Sex differences in depression-like behavior after nerve injury are associated with differential changes in brain-derived neurotrophic factor levels in mice subjected to early life stress. Neurosci Lett. 2015;592:32–36. 97. Bernardes SF, Keogh E, Lima ML. Bridging the gap between pain and gender research: a selective literature review. Eur J Pain. 2008;12(4):427–440. 98. Alabas OA, Tashani OA, Tabasam G, Johnson MI. Gender role affects experimental pain responses: a systematic review with meta-analysis. Eur J Pain. 2012;16(9):1211–1223. 99. Robinson ME, Riley III JL, Myers CD, et al. Gender role expectations of pain: relationship to sex differences in pain. J Pain. 2001;2:251–257. 100. Gijsbers K, Nicholson F. Experimental pain thresholds influenced by sex of experimenter. Percept Mot Skills. 2005;101(3): 803–807. 101. Levine FM, De Simone LL. The effects of experimenter gender on pain report in male and female subjects. Pain. 1991;44:69–72. 102. Berenbaum SA, Beltz AM. Sexual differentiation of human behavior: effects of prenatal and pubertal organizational hormones. Front Neuroendocrinol. 2011;32(2):183–200. 103. Diatchenko L, Slade GD, Nackley AG, et  al. Genetic basis for individual variations in pain perception and the development of a chronic pain condition. Hum Mol Genet. 2005;14(1):135–143.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

496

33.  SEX, GENDER, AND PAIN

104. Belfer I, Segall SK, Lariviere WR, et  al. Pain modality- and sex-specific effects of COMT genetic functional variants. Pain. 2013;154(8):1368–1376. 105. Meloto CB, Bortsov AV, Bair E, et  al. Modification of COMTdependent pain sensitivity by psychological stress and sex. Pain. 2016;157(4):858–867. 106. Mogil JS, Sorge RE, LaCroix-Fralish ML, et al. Pain sensitivity and vasopressin analgesia are mediated by a gene-sex-environment interaction. Nat Neurosci. 2011;14(12):1569–1573. 107. Bertakis KD. The influence of gender on the doctor-patient interaction. Patient Educ Couns. 2009;76(3):356–360. 108. LeResche L. Defining gender disparities in pain management. Clin Orthop Relat Res. 2011;469:1871–1877. 109. Oliva EM, Midboe AM, Lewis ET, et  al. Sex differences in chronic pain management practices for patients receiving opioids from the Veterans Health Administration. Pain Med. 2015;16(1):112–118. 110. Schafer G, Prkachin KM, Kaseweter KA, de CWAC. Health care providers' judgments in chronic pain: the influence of gender and trustworthiness. Pain. 2016;157(8):1618–1625. 111. Hirsh AT, Hollingshead NA, Matthias MS, Bair MJ, Kroenke K. The influence of patient sex, provider sex, and sexist attitudes on pain treatment decisions. J Pain. 2014;15(5):551–559. 112. Niesters M, Dahan A, Kest B, et al. Do sex differences exist in opioid analgesia? A systematic review and meta-analysis of human experimental and clinical studies. Pain. 2010;151(1):61–68. 113. Riley III JL, Hastie BA, Glover TL, Fillingim RB, Staud R, Campbell CM. Cognitive-affective and somatic side effects of morphine and pentazocine: side-effect profiles in healthy adults. Pain Med. 2010;11(2):195–206. 114. Samuelsen PJ, Svendsen K, Wilsgaard T, Stubhaug A, Nielsen CS, Eggen AE. Persistent analgesic use and the association with chronic pain and other risk factors in the population-a longitudinal study from the Tromso Study and the Norwegian Prescription Database. Eur J Clin Pharmacol. 2016;72(8):977–985. 115. Wright EA, Katz JN, Abrams S, Solomon DH, Losina E. Trends in prescription of opioids from 2003–2009 in persons with knee osteoarthritis. Arthritis Care Res (Hoboken). 2014;66(10):1489–1495.

116. Kaplovitch E, Gomes T, Camacho X, Dhalla IA, Mamdani MM, Juurlink DN. Sex differences in dose escalation and overdose death during chronic opioid therapy: a population-based cohort study. PLoS One. 2015;10(8):e0134550. 117. Back SE, Payne RL, Simpson AN, Brady KT. Gender and prescription opioids: findings from the National Survey on Drug Use and Health. Addict Behav. 2010;35(11):1001–1007. 118. Walker JS, Carmody JJ. Experimental pain in healthy human subjects: gender differences in nociception and in response to ibuprofen. AnesthAnalg. 1998;86(6):1257–1262. 119. Averbuch M, Katzper M. A search for sex differences in response to analgesia. Arch Intern Med. 2000;160(22):3424–3428. 120. Butcher BE, Carmody JJ. Sex differences in analgesic response to ibuprofen are influenced by expectancy: a randomized, crossover, balanced placebo-designed study. Eur J Pain. 2012;16(7):1005–1013. 121. Aslaksen PM, Bystad M, Vambheim SM, Flaten MA. Gender differences in placebo analgesia: event-related potentials and emotional modulation. Psychosom Med. 2011;73(2):193–199. 122. Compton P, Charuvastra V, Ling W. Effect of oral ketorolac and gender on human cold pressor pain tolerance. Clin Exp Pharmacol Physiol. 2003;30(10):759–763. 123. Pud D, Yarnitsky D, Sprecher E, Rogowski Z, Adler R, Eisenberg E. . Can personality traits and gender predict the response to morphine? An experimental cold pain study. Eur J Pain. 2006;10(2):103–112. 124. Krummenacher P, Kossowsky J, Schwarz C, et al. Expectancyinduced placebo analgesia in children and the role of magical thinking. J Pain. 2014;15(12):1282–1293. 125. Clayton JA, Collins FS. Policy: NIH to balance sex in cell and animal studies. Nature. 2014;509(7500):282–283. 126. Klein SL, Schiebinger L, Stefanick ML, et al. Opinion: Sex inclusion in basic research drives discovery. Proc Natl Acad Sci USA. 2015;112(17):5257–5258. 127. Mogil JS, Wilson SG, Chesler EJ, et al. The melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans. Proc Natl Acad Sci USA. 2003;100 4867-762.

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34 Sex and Gender Differences in Trauma Victims Presenting for Treatment Dorte M. Christiansen1,2 1

University of Aarhus, Aarhus, Denmark, 2University of Southern Denmark, Odense, Denmark

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34.5 Gender Differences in Trauma-Related Symptoms and Disorders 34.5.1 Posttraumatic Stress Disorder (PTSD) 34.5.2 Psychological Symptoms 34.5.3 Physical Symptoms

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34.1 INTRODUCTION Marked gender differences have been identified in the morphology, neurochemistry, hard-wiring, and functional outcomes of the male and female brain.1 In fact, even when there are no gender differences in the outcome of specific brain processes, such as cognition or problem solving, there may be underlying procedural differences, such as different mechanisms or pathways being used by men and women to reach the same goal.1 Gonadal hormones, such as estrogen and testosterone, play an important role in the development of differences in brain structures and functions and affect the lives of men and women in different ways. For example, estrogen is thought to have a very strong protective effect on disease prevention, especially on disorders of the

Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00043-7

34.6 Gender Differences in Treatment of Trauma-Related Disorders 501 34.6.1 Gender Differences in Psychopharmacology 502 34.6.2 Gender Differences in Psychotherapy 503 34.6.3 Clinical Implications 504 34.7 Discussion 505 34.7.1 Gender Bias in Research 505 34.7.2 Sex Versus Gender Differences 506 34.7.3 Gender Differences in a Clinical Context 508 34.8 Conclusion

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central nervous system (CNS), due to its neuroprotective and neuroactivating properties in women.1 In addition to the higher concentrations of estrogen in women, the specific effects of estrogen and other hormones may also have gender-specific effects.1,2 Gender differences have the potential to impact all aspects of human functioning, including differences in the types of stressors to which men and women are exposed, how they respond to such stressors, and in long-term outcome. For example, gonadal hormones influence reactivity of the hypothalamic-pituitary-adrenal (HPA) axis, glucocorticoid feedback sensitivity, and brain GABA content, all of which are implicated both in the acute response to a traumatic stressor and in the subsequent development of several psychiatric disorders such as posttraumatic stress disorder (PTSD), anxiety, and depression.3,4 Short-term

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fluctuations in gonadal hormone levels, such as those associated with the female menstrual cycle, occur at different intensities in both men and women, but the most dramatic changes are associated with onset of puberty in both boys and girls and with pregnancy, birth, lactation, and menopause in women, and possibly andropause in men. This chapter will focus on gender differences in how humans are affected by trauma and how these differences affect the clinical presentation and treatment of men and women in the health care system. Whereas much of the research included in this chapter has examined differences related to biological sex rather than social gender, the term gender will be used here to cover both constructs in acknowledgment of the importance of cultural influences on genomic expression.

34.2  PSYCHOLOGICAL TRAUMA IN A MEDICAL SETTING In many cases, the first contact with health professionals that a person has following a traumatic incident is at the hospital. The physician’s goal is first and foremost to ensure survival and to repair physical damage. Thus, the psychological aspects of the trauma that the patient has recently gone through are often overlooked in the hospital and other medical settings. This leaves the patient vulnerable to develop PTSD without having discussed the psychological impact of the trauma with anyone. For example, the common occurrence and medical simplicity of miscarriage may cause medical personnel to overlook the significant impact that early pregnancy loss can have on the woman and her partner, resulting in many parents feeling unsupported and misunderstood.5 This is problematic because trauma victims are generally much more likely to seek help for their physical rather than their psychological symptoms.6 This tendency may be more pronounced in male compared to female trauma victims, as women are generally more likely than men to seek treatment from mental health services.3 Thus, if hospital staff and other medical personnel overlook important psychological symptoms of severe trauma, the patient is at risk of developing severe and chronic symptoms, including PTSD, anxiety disorders, and depression, as well as physical symptoms and diseases commonly seen in the aftermath of trauma. Furthermore, there is some support that hostile or unsupportive social interactions in the acute aftermath of trauma may serve to increase symptoms of psychological and biological stress, perhaps especially in women, whereas positive social interactions help the CNS and the HPA axis return to base levels.7 Ultimately, untreated psychological trauma can lead to severe psychological suffering and suicide. For example, although the psychological impact

of miscarriage is often overlooked by health care personnel and by the support systems of parents, women who have suffered a miscarriage are approximately three times as likely to commit suicide within a year as women of the same age who have not suffered such a loss.5

34.3  GENDER DIFFERENCES IN TRAUMA EXPOSURE Men and boys tend to be exposed to more potentially traumatic events (PTEs) than women and girls, with an estimated 61% of men and 51% of women reporting exposure to at least one PTE.8,9 Men are generally more likely than women to report exposure to accidents, nonsexual interpersonal violence, combat and war, disasters, severe illness, unspecified injuries, and witnessing the death or serious injury of others. In contrast, women are more likely to be exposed to sexual trauma and betrayal trauma, where the individual is victimized by a trusted person, such as a spouse, a caregiver, or a teacher.8,10,11 Traumatic exposure is not only common in adults but also in children. In some parts of the world with high levels of war, conflict, or extreme poverty, childhood exposure to traumatic events represents the norm, rather than the exception, and the same is found in certain underprivileged environments of high-income Western countries where crime rates are high and families are struggling financially. In fact, more than 50% of all rapes occur in girls under the age of 18.12 Many traumatic events go unnoticed by society. Whereas exposure to war-zone stressors and rape are relatively widely acknowledged as causes for PTSD, other much more common events, such as pregnancy loss or loss of a loved one, appear to account for an even higher number of cases.13,14 In contrast, sexual trauma which is considered the most toxic single PTE8 is not as commonly reported as death of a loved one, and subsequently results in a comparably lower number of PTSD cases overall. However, when traumatic exposure and conditional risk of PTSD are considered separately for men and women, the picture changes somewhat. Conditional risk refers to the risk of developing PTSD specifically among those exposed to a PTE, as opposed to population-based risk, and can be calculated in relation to different specific events. As sexual trauma is much more common in women, it is accountable for a much higher proportion of PTSD cases in women compared to men, even though the conditional risk associated with sexual trauma does not appear to differ much between the genders.11 As a consequence, approximate half of all women suffering from PTSD nominate sexual assault or sexual abuse as their most distressing traumatic experience.8

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34.5  Gender Differences in Trauma-Related Symptoms and Disorders

34.4  GENDER DIFFERENCES IN THE ACUTE RESPONSE TO TRAUMA There is abundant preclinical and clinical research documenting the existence of gender differences in brain structures, pathways, and functions crucial to the human stress response.15–17 Therefore, it should come as no surprise that men and women respond differently to endogenous and exogenous threats.1 Many of the hormones and neurotransmitters working together to activate the acute stress response appear to be directly affected by both male and female gonadal hormones.15,18,19 In spite of this, research on this topic has traditionally focused on men. As a consequence, the most widely accepted model of trauma response is based on the male stress response. The fight-or-flight model proposes that when exposed to a stressor, the sympathetic nervous system (SNS) is immediately activated and releases epinephrine and norepinephrine into the bloodstream, thus preparing the body for either fight or flight. SNS activation further triggers activation of the HPA axis, which releases the more slowly working stress hormones, including cortisol. This model of fight-or-flight behavior in the face of a traumatic stressor has been extensively studied but mostly in male populations.2 Although the fightor-flight response is also present in women, there is some support that women are more likely to respond to traumatic stressors with a different response, i.e., the “tend-and-befriend” response.20 In contrast to the fight-or-flight response, the tend-and-befriend response is hypothesized to be associated with a suppression of HPA axis reactivity and is thought to be regulated by the parasympathetic nervous system (PNS).2 It is beyond the scope of this chapter to explain the neuropsychological processes behind the different responses, as these have been explained elsewhere.2,7,18,21 The tend-and-befriend theory suggests that, whereas men generally respond to stressful events with physiological hyperarousal and an increase in aggressive behaviors, women are more likely to group together and seek social support and to experience dissociative responses.2,20 The tend-andbefriend theory has primarily been tested in preclinical studies. Whereas some human studies have found that women and infant girls are more likely to display behavior consistent with the tend-and-befriend response and that infant boys display more fight-or-flight behavior in response to fearful stimuli, this has not been examined specifically in response to traumatic events.2 Even if future research does not support the tend-andbefriend response as the dominant stress response in women, there is a vast amount of research documenting psychophysiological gender differences in the human response to trauma. Any theory attempting to account for the human stress response must encompass observed gender differences in

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behavior and biology, such as the impact of gonadal hormones on important brain functions, including the HPA axis, the paraventricular nucleus (PVN), and glucocorticoid release, all of which appear to be more vulnerable to the dysregulating effects of stress in women compared to men.15,19,22 The importance of gonadal hormones is further supported by findings that prolonged use of oral contraceptives can alter the reactivity of the HPA axis.3 Prolonged activation of the HPA axis exhausts the stress response system and prevents its return to homeostasis. Because of this, exposure to severe or prolonged traumatic stress can have long-term detrimental consequences, such as a dysregulated HPA-axis response and alterations in the structure and functioning of the hippocampus and frontal cortex, which may cause behavioral disturbances, symptom development, and deficits in memory and learning.16,22,23 Very little is known about how acute stress responses, that may initially be adaptive, become maladaptive over time and lead to PTSD and other types of trauma-related symptomatology described in the following section. However, it is likely that gender differences in the initial stress response go on to affect the development and perpetuation of these disorders, possibly through sensitization processes.2 For this reason, gender differences in the acute response to traumatic stress are crucial to understanding gender differences in trauma sequelae, including in the development of PTSD.

34.5  GENDER DIFFERENCES IN TRAUMA-RELATED SYMPTOMS AND DISORDERS Stress and especially traumatic stress have deteriorating effects on both general health and quality of life and are considered major factors in the development of several psychiatric disorders, including anxiety disorders, major depression (MDD), and especially PTSD.19,24 Prolonged or intense exposure to severe stressors has been found to result in a dysregulated HPA-axis and to produce functional alterations in certain brain regions that are highly implicated in the development of psychiatric disorders, such as the hippocampus, amygdala, and prefrontal cortex.19 It is thus likely that these changes are responsible for the increased prevalence of anxiety and depression following traumatic exposure.19 It has been suggested that gender differences in the neuroendocrine, genomic, and behavioral responses to stress may account for the gender differences in these disorders.1 In addition, gonadal hormones also appear to have a continuous impact on symptoms of PTSD, depression, and anxiety with the intensity of symptoms in women being higher in the luteal phase of the menstrual cycle.24,25 Especially estrogen, progesterone,

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estradiol, and testosterone appear to have either direct or indirect impact on symptom severity.3,19 The interactions between gonadal hormones and psychological symptoms are extremely complex, as a specific hormone may have anxiolytic effects under certain circumstances and anxiogenic effects under others.19 Overall, however, the fluctuations in gonadal hormones throughout the female menstrual cycle appear to decrease the stability of the homeostatic system, making women more susceptible to stress than men.27 In fact, low levels of both estradiol and progesterone have been linked to symptom exacerbation in several psychiatric disorders, including PTSD, panic disorder, bipolar disorder, depression, eating disorders, and alcohol/substance use disorders.26 There are some indications that female gonadal hormones do not only affect symptom reporting, but that women exposed to trauma during the phases of their menstrual cycle when estradiol and progesterone levels are particularly high have an increased risk of developing PTSD.26 Thus, gonadal hormones are likely to affect not only gender differences in the risk of developing PTSD and other trauma-related disorders but also the severity of symptoms over time. Particularly interesting in the current context is the fact that fluctuations of fearrelated symptoms across the menstrual cycle appear to be more pronounced in women with PTSD compared to trauma-exposed women without PTSD.26

34.5.1  Posttraumatic Stress Disorder (PTSD) PTSD and acute stress disorder (ASD) are the only two psychiatric disorders to be directly linked to one or more specific traumatic event(s). As ASD is limited to 1 month of duration, it is not relevant to discuss here. PTSD was first introduced in the third version of the diagnostic and statistical manual of mental disorders (DSM-III) in 1980. From 1980 until the introduction of the fifth version of the diagnostic system (DSM-5) in 2013, there has been some variation and a lot of scientific debate concerning what exactly constitutes a traumatic event. Apart from this, the diagnosis has remained mostly unchanged in its requirement of symptoms relating to reexperiencing, avoidance, and arousal, persisting for more than 1 month following a severe traumatic incident. When the DSM-5 took over from the DSM-IV, the biggest change involved the introduction of a fourth symptom cluster requiring the existence of persistent and negative alterations in mood or cognitions.28–30 In spite of men being exposed to more PTEs, PTSD is more prevalent in women with a lifetime prevalence in the United States of 10.4% in women and 5.0% in men.8 The average conditional risk following traumatic exposure is 20.4% in women compared to 8.2% in men.8 In addition to women being more at risk of developing

PTSD, symptoms appear to be more severe and of longer duration in women compared to men.11,13 It is thus a well-established finding in PTSD research that women are approximately twice as likely to develop PTSD following exposure to a variety of PTEs.2,11 The 2:1 prevalence of PTSD in women compared to men has not changed as a consequence of the modifications made to the PTSD diagnosis in the DSM-5, with women in particular reporting higher levels of negative beliefs, diminished interest, restricted affect, and sleep disturbance compared to men.31 Once PTSD has developed, male and female PTSD patients tend to have more similarities than differences and generally present with similar symptom profiles.2 However, when it comes to comorbid symptoms and disorders there are several significant gender differences.

34.5.2  Psychological Symptoms Men with PTSD are generally more likely than women with the disorder to report high levels of anger, substance abuse, and antisocial behavior, whereas women have higher levels of self-blame, BPD, eating disorders, self-injurious behavior, suicide attempts, impulse control disorders, and persistent dissociation.2,32 Dissociation is thought to affect the general PTSD profile so much that a specific diagnostic category has been created for the dissociative subtype of PTSD.33 In addition, PTSD is associated with increased prevalence of a wide range of axis I and II disorders. These include major depressive disorder, dysthymia, alcohol use disorder, illegal drug use, generalized anxiety disorder (GAD), panic disorder, agoraphobia, somatoform disorder, specific phobia, eating disorder, and social phobia along with paranoid, schizoid, schizotypal, histrionic, narcissistic, borderline, antisocial, avoidant, obsessive-compulsive, and dependent personality disorders.34,35 In particular, depression, illegal drug use, panic disorder, agoraphobia, and borderline personality disorder (BPD) are much more common in PTSD populations than in the general population.34 Whereas PTSD is associated with an increased prevalence of these disorders in both men and women,34 women primarily report internalizing symptoms and men primarily report externalizing symptoms.36 Furthermore, the general constellation of disorders has been found to differ between the two genders. One study found that comorbidity in patients with PTSD was generally distributed across three major groups.35 One group consisting almost exclusively of women was made up of PTSD patients with high levels of hyperarousal combined with comorbid dysthymia, anxiety disorders, and suicidal behavior. A second group dominated by men consisted of PTSD patients with comorbid antisocial personality disorder, alcohol and substance abuse, and suicidal behavior. It is interesting

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34.6  Gender Differences in Treatment of Trauma-Related Disorders

that suicidal behavior which can be argued to represent both internalizing and externalizing behavior was included in both these comorbidity groups. The final and largest group consisted of patients with generally low comorbidity rates and was not dominated by either gender.35 It was found that comorbid anxiety disorders were especially associated with low recovery rates and a more chronic course of PTSD,35 which may help explain why women tend to have more long-lasting PTSD than men. A high number of comorbid disorders is generally associated with a more severe clinical profile, a worse prognosis, greater impairment, and higher disability levels.35 Whereas the research reviewed here has generally treated other trauma-related symptoms as comorbid with PTSD, it is important to notice that most of these disorders are associated with traumatic exposure in general, and not only with PTSD. However, when PTSD is present, it is generally considered the primary diagnosis. There is not sufficient research available to document whether differences exist in the clinical profile of other disorders, such as MDD or GAD, depending on whether they have developed subsequent to a traumatic event or not. Thus, at present it appears that traumatic exposure is simply another risk factor in the etiology of these other disorders. It is thus the presence of PTSD that makes these patients different from other patients in the health care system suffering from psychiatric disorders.

34.5.3  Physical Symptoms In addition to trauma-related psychological symptoms posing a problem in their own right, such symptoms and traumatic exposure may further exacerbate symptoms and suffering associated with medical illnesses, as well as possibly cause new ones to appear. For example, adult and childhood exposure to trauma has been linked to a general deterioration of physical health which can be measured by both morbidity and mortality rates.12,37 Trauma-exposed populations have higher prevalence of respiratory disease, peptic ulcers, arthritis, cardiovascular disease, stroke, renal disease, musculoskeletal problems, autoimmune disorders, and neurological problems than nontrauma exposed populations.6,37–39 In addition, traumatic exposure has been associated with unexplained and chronic pain conditions, including back pain, joint pain, headache, and neck pain.40 The association between traumatic exposure and these disorders is to some extent mediated by PTSD with some disorders being more prevalent in PTSD populations only.39 For example, patients with PTSD have a twofold risk of being diagnosed with a nonpsychiatric health condition compare to people without PTSD even after controlling for other more traditional risk factors, such as age, socioeconomic status and MDD.37

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Whereas gender differences are not as well-established in the physical sequelae of trauma as is the case with trauma-related psychological symptoms, there is some support that traumatic exposure is also more strongly associated with the development of physical symptoms in women compared to men. For examples, combat exposure in veterans more strongly predicted poor post-war health in women compared to men.40 In contrast, another study found that women’s higher reporting of pain was not significantly different from that of men and that gender did not consistently moderate associations between traumatic exposure and pain in male and female veterans.40 Women have been found to report more somatization and health-related complaints than men.2 It is thus possible that the inconsistent findings related to gender differences in trauma-related pain are caused by an increased level of functional pain syndromes and the increased tendency of women to report pain compared to men. There are several ways in which traumatic exposure and PTSD may increase the risk of physical disorders. First of all, PTSD has been associated with registered biological changes, such as changes in HPA reactivity and hippocampal volume.41 In addition, traumatic exposure, especially when combined with PTSD, may increase maladaptive health behavior, such as increased smoking, and alcohol/substance abuse, reduced physical exercise, health care visits, and compliance with medical treatments.38 Such negative health behavior may be particularly common among men, as risky health behavior has been linked to higher levels of masculinity.38 Furthermore, especially in young women, PTSD may be associated with risky sexual behavior.12 This may further increase the risk of sexual (re-)traumatization, sexually transmitted diseases, and teen pregnancy, all of which may have further deteriorating effects on health and quality of life.

34.6  GENDER DIFFERENCES IN TREATMENT OF TRAUMA-RELATED DISORDERS It has been argued that the variables involved in the development of chronic PTSD are highly likely to also affect treatment response.42 As women are consistently found to be more likely than men to develop PTSD when exposed to a traumatic event, it is surprising how little attention has been paid to gender as a potential moderator of treatment outcomes. Multiple factors of both biological, interpersonal, intrapersonal, and social origin have been found to affect HPA axis functioning.2 This influence of multiple factors on HPA reactivity is particularly important to keep in mind when considering interventions for trauma-related disorders, as it implies

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that HPA dysfunctions can also be treated through either pharmacological, psychotherapeutic, or possibly even social interventions. This section on gender differences in treatment will therefore focus on gender differences in the efficacy of both psychopharmacological and psychotherapeutic interventions.

34.6.1  Gender Differences in Psychopharmacology The goal of clinical pharmacology has been described as optimizing the use of medication in order to enhance therapeutic effects while minimizing side effects and other negative outcomes.43 This makes it extremely important to examine the impact of gender on treatment outcomes, as failing to do so may prevent its optimization in both men and women. In fact, it has been proposed that gender differences in the systems, hormones, and transmitters targeted by psychotropic medication are so widespread that pharmacotherapy needs to be gender-based to effectively treat stress- and traumarelated psychiatric disorders in both men and women.15 Gender differences in the efficacy of psychopharmacology is particularly relevant to examine because there are large gender differences in the amount of psychopharmacology being prescribed, with 24% of American women receiving some type of psychotropic medication compared to only 15% of men.44 These gender differences are particularly pronounced in prescriptions of anxiolytic and antidepressive medication.44,45 It is unclear whether the increased prescription of psychotropic medication to women compared to men can be fully accounted for by gender differences in symptom prevalence.44 It is possible that other factors, such as differences in help-seeking behavior or widespread bias in the doctors prescribing these medications, contribute to gender differences in the use of psychopharmacology. It has been reported that privately insured women with PTSD are 1.5 times more likely to receive psychotropic medications compared to men with the same diagnosis.44 Similarly, female veterans with PTSD have been reported to be more likely to receive medication including selective serotonin reuptake inhibitors (SSRIs), selective norepinephrine reuptake inhibitors (SNRIs), benzodiazepines, and atypical antipsychotic medication than their male colleagues with PTSD.44 The only drug more commonly prescribed to men than women was prazosin. Prazosin is used to block the effects of adrenalin and its increased use in men may thus be contributed to an attempt to treat excessive aggressive behavior in male veterans with PTSD. In both genders higher levels of disability and comorbidity were associated with increased prescriptions of all medications.44 Gender differences in comorbidity and/or demographic factors could largely account for differences in prescription of SSRIs, SNRIs,

and atypical antipsychotic drugs. However, these factors could not fully account for gender differences in the prescription of benzodiazepines. A further very concerning finding reported in this study was that benzodiazepines were less commonly prescribed to male veterans with substance use disorders but more commonly prescribed to female veterans with substance use disorders, in spite of the fact that benzodiazepines are in themselves highly addictive and should thus not be given to patients with such problems.44 It is unknown whether this problem is unique to veterans or to the unique study population or whether it represents a more general problem in female trauma survivors with dual diagnoses. Men and women have been found to respond differently to several kinds of medication used to treat a variety of diseases.46 In addition, gender differences have been reported in a number of variables that are highly likely to influence how men and women respond to different types of pharmacotherapy used to treat trauma-related disorders. These include gender differences in the serotonergic, dopaminergic, and norepinephrine systems along with differences in pharmacokinetics, pharmacodynamics, and metabolism.3,4,47 In addition gonadal hormones affect several neurobiological systems implicated in trauma-related disorders and targeted in pharmacological treatment, including treatment with SSRIs and benzodiazepines.15,48 Postmenopausal women have been reported to respond less favorably to antidepressant therapy than premenopausal women, suggesting that female gonadal hormones may have a significant impact on response to antidepressants.4 There are even preliminary results that estrogen may in itself have antidepressive effects and may potentially reduce PTSD symptoms in women.24,49 Whereas research has been conducted on gender differences in response to antidepressants, less focus has been paid to potential gender differences in anxiolytic medication. However, the effects of certain benzodiazepines have been found to be moderated by use of oral contraceptives,47 implicating that gonadal hormones may directly affect treatment with anxiolytic medication. Together these findings highlight the importance of examining gender differences in psychotropic medication prescribed to trauma patients. Although findings are very inconclusive and several studies have failed to report any gender effects, some studies have reported significant gender differences in the efficacy of different types of antidepressants. SSRIs have been found to be more effective in treating depression in women compared to men,4,50 and one study reported a similar gender difference in patients being treated for panic disorder.3 However, whether these gender differences are also present in trauma-exposed populations and in relation to symptoms other than depression and panic is presently unknown. In addition, there is some support for the suggestion that although

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34.6  Gender Differences in Treatment of Trauma-Related Disorders

gender differences are smaller, women tend to benefit more from treatment with SNRIs and monoamine oxidase inhibitors (MAOIs) compared to men.4 In contrast to these findings, there is some support that tricyclic antidepressants (TCAs) and tetracyclic antidepressants (TeCAs) may be more effective in treating depression in men compared to women,4,47,51 although it is unknown whether these gender differences are specific to depression. There is also preliminary support for a potential gender difference in response to PTSD treatment with propranolol, suggesting that this beta-blocker significantly decreases PTSD severity in men but increases PTSD severity in women.52 Finally, there is limited support for the assumption that women treated with antipsychotics tend to need higher dosages than men.47 Although this has not been examined specifically in relation to PTSD or trauma, it may be relevant in the current context because antipsychotics are sometimes prescribed to patients with PTSD and other trauma-related disorders. It has been reported that women generally have a 1.5– 1.7-fold greater risk of adverse reactions to a number of different medications compared to men and there have been cases where prescription drugs have been removed from the market because side effects and health issues specific to women were only discovered after these drugs had been approved based on research conducted on primarily or exclusively male populations.43,46 Such findings highlight the importance of not only considering symptom reduction but also gender differences in other consequences of therapy, such as side effects. In addition, there is some support that women are more likely to drop out from treatment with TCAs and that men are more likely to drop out from treatment with SSRIs.3 Such knowledge is highly relevant as it may keep one gender from obtaining the full effect of the medication.

34.6.2  Gender Differences in Psychotherapy Treatments focusing specifically on the traumatic event(s) and using exposure to desensitize the fear response to traumatic reminders are generally considered superior to other interventions in treating PTSD.53 As a consequence, trauma-focused cognitive behavioral therapy (CBT) has been more extensively studied than other treatments, also in relation to gender. There are a number of reasons why men and women could be expected to respond differently to CBT. Research suggests that extinction of previously conditioned fear responses in women are directly affected by levels of estrogen, estradiol, and progesterone.24,48 As trauma-focused CBT is specifically aimed at extinguishing conditioned fear through repeated exposure, this finding implies not only that men and women may respond differently to this type of therapy, but also that menstrual cycle might

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be a relevant factor to consider when initiating CBT in women. In addition, several gender differences have been reported in relation to learning and conditioning that are likely to be of great clinical relevance in relation to both the development and treatment of PTSD and anxiety disorders following exposure to traumatic stress.24 Finally, it has been suggested that variables that are unequally associated with the development of PTSD and other trauma-related disorders in men and women are extremely important to examine, as they may cause gender differences in the specific symptoms and disorders that are developed following traumatic exposure, how such disorders are expressed, and most importantly how they are treated in men and women.2,54 A number of studies have examined gender differences in the effect of psychotherapy and have reported an absence of such differences, in many cases likely because of low power and especially a very low number of male participants.55–58 In contrast, a number of studies have reported significant gender differences in outcomes of psychotherapy. One study comparing imaginal exposure and cognitive restructuring to eye movement desensitization and reprocessing (EMDR) found that female gender significantly predicted better outcome on several PTSD measures.59 Similarly, a recent Norwegian study of refugees and asylum seekers with PTSD found that narrative exposure therapy and an unspecified control condition (therapy as usual) generally resulted in better outcomes in women compared to men.60 However, wartime offender status was associated with poor outcome and as this group consisted primarily of men, it is not possible to separate the impact of past offender status from that of gender.60 Finally, one study comparing imaginal exposure to cognitive therapy found that gender was the strongest predictor of treatment outcome with women benefitting more from therapy.42 Unfortunately neither of these studies were able to conduct gender-specific analyses and it is thus unknown whether the reported gender differences were general or limited to one of the treatment conditions. Women have also been reported to benefit more from therapy focusing on verbal processing of traumatic content61,62 and possibly from cognitive processing therapy,32 although the latter was limited by low power and gender differences did not reach significance in spite of effect sizes ranging from –.29 to –.58. In accordance with these findings, a number of literature reviews report that women with PTSD respond better to psychotherapy in general and especially to trauma-focused therapy compared to men.2,57,63–65 This hypothesis has received some additional support from a meta-analysis from 2005 on psychotherapeutic treatment of PTSD.53 Although this meta-analysis did not include gender as a moderator, the authors did find that trauma type significantly affected treatment outcome. Studies focusing on

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combat exposure reported the lowest level of symptom improvement and studies focusing on sexual assault or abuse (in two cases combined with physical abuse) reported the highest average level of symptom improvement, with studies focusing on other traumas falling in between these two.53 This is very interesting in relation to gender differences, as these are the two most genderspecific types of traumatic exposure, suggesting that the results may in fact reflect gender differences. Finally, whereas drop-out rates are rarely examined separately for men and women or even separately for different interventions, some gender differences have been reported. Some studies have found that men are more likely to miss therapy sessions42 and to drop out, especially from exposure-based therapy compared to women.2,57,63,65 Furthermore, it has been suggested that women with PTSD may be particularly likely to avoid trauma-related triggers during the late luteal and early follicular phases of the menstrual cycle.26 It is possible that this may not only cause fluctuations in symptom levels but that it can also affect drop-out rates in relation to therapy, as women may be more likely especially to drop out of exposure-based therapy if it is initiated during these phases of the menstrual cycle.

34.6.3  Clinical Implications Very few studies have examined gender differences in treatment outcome following traumatic exposure. The few studies that have examined gender differences in psychotherapy used to treat PTSD patients are generally limited by low sample sizes and inadequate statistical power, as gender differences are very rarely a primary focus, resulting in a high risk of type II errors, where an actual gender difference is found to be nonsignificant due to low statistical power. The gender distribution in these studies is rarely equal and analyses are very rarely conducted separately for men and women. In contrast to studies examining the impact of psychotherapy, psychotropic interventions are generally not limited by small sample sizes. Thus, inconsistent findings regarding gender differences are less likely to be the result of type II error and are more likely to be attributable to confounding factors. Furthermore, the reliability of these studies is somewhat limited by the fact that very few studies have examined PTSD or focused specifically on traumaexposed patients. Gender differences reported in the use of antidepressants in patients with MDD may not be generalized to PTSD patients. Furthermore, as certain gender differences reported in relation to the acute stress response have only been documented in response to stressors of a traumatic nature,2 gender differences in the treatment of symptoms unrelated to trauma may not be relevant in patients with PTSD or other trauma-related disorders.

Whereas gender differences reported in studies of psychopharmacological treatments of general symptoms of anxiety or depression may not be 100% reliable in patients where symptoms are related to a traumatic experience, this is even less likely to be the case in relation to psychotherapy, where PTSD symptoms are best treated with specific trauma-focused interventions. When PTSD is present together with other comorbid disorders and symptoms, it is generally found that PTSD should be treated as the primary disorder, and that other symptoms will often subside as a consequence of a significant reduction in PTSD.56 Although whether or not other trauma-related symptoms are also best treated with trauma-focused psychotherapy, even when PTSD is not present, has not been studied, focusing on the trauma is likely to reduce the severity of all symptoms that have developed in response to the traumatic event(s) or have been exacerbated by exposure. In accordance with this, preliminary findings suggest that symptoms often found to be comorbid with PTSD, such as dissociation, self-injurious behavior, substance use, and major depression do not reduce the efficacy of prolonged exposure therapy.66 Instead, most of these symptoms generally decrease over the course of therapy along with symptoms of PTSD. The exception is very severe symptoms that may warrant disorder-specific approaches.66 However, whereas traumatic exposure serves as a risk factor for psychological symptoms, other than PTSD, it may do so without being a directly precipitating factor. Thus trauma-focused therapy may not necessarily be the most effective treatment for other trauma-related symptoms in the absence of PTSD. Finally, whereas PTSD is exclusively related to traumatic exposure, physical health symptoms are impossible to link directly to a specific incident, unless they are related to an injury inflicted at the time of the trauma. Furthermore, certain diseases, such as cardiovascular disease, may develop over several years, making the link to trauma even more difficult to establish. In contrast to the psychological symptoms, most somatic symptoms are less likely to respond well to trauma-focused therapy. The exception here may be somatization and other medically unexplained syndromes that may respond well to both treatment with SSRIs and CBT. However, to the degree that somatic symptoms are associated with PTSD or other trauma-related disorders, it is possible that they may be prevented by early intervention, or that even if trauma-focused treatment is received at a later stage, exacerbation of such physical symptoms may be prevented. Furthermore, treatment of psychological symptoms may indirectly alleviate some somatic symptoms by improving treatment compliance and healthrelated behavior. In general, very few studies have examined gender differences in relation to psychotherapeutic or

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34.7 Discussion

psychopharmacological treatment of patients with trauma-related disorders, and even fewer studies have actually been designed with this purpose in mind. There is a general lack of high-quality research in the field which results in an absence of knowledge regarding possible gender differences in treatment outcomes, compliance, side effects, drop-out rates, relapse, secondary outcomes, and other important aspects of psychotherapeutic and psychopharmacological interventions following trauma. In relation to treatment outcomes there is especially a need for more knowledge concerning long-term outcomes and relapse rates. Whereas proper follow-up intervals are important in all treatment effect studies, they may be particularly important when studying gender differences. One study found that maintenance of treatment gains was moderated by gender, even when initial outcomes were not.67 In addition, studies on gender differences in both psychotherapy and psychopharmacology only very rarely report any findings related to outcomes other than symptom reduction in PTSD, anxiety, and/or depression. This is problematic, as sometimes gender differences are found in secondary outcomes, such as anger, guilt, and dissociation, even when no reductions are found in PTSD symptoms.32 The potential for gender differences in treatment response is of huge theoretical and clinical importance. Knowledge of gender differences in the drop-out rates, side effects, and effectiveness associated with different interventions could have important implications for treatment decisions. Furthermore, if men and women are found to benefit from different interventions, this may significantly improve overall treatment effect for different trauma-related disorders and reduce the societal costs associated with chronic, treatment-resistant symptoms. In accordance with this, it has been suggested that identifying potential gender differences in the biological substrates and mechanisms directly involved in the etiology of anxiety disorders may help relieve some of the economic and personal costs associated with ineffective treatment strategies.17 This same argument can be used in relation to other trauma-related disorders, including PTSD.

34.7 DISCUSSION 34.7.1  Gender Bias in Research Biomedical research is characterized by an overrepresentation of male subjects because, among other reasons, it was feared that results may be confounded by surges in gonadal hormones related to the female menstrual cycle.1,46,68 As a consequence female subjects are underrepresented in both animal models of stress response and psychiatric disorders and in human studies on the

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brain structure and function associated with acute and prolonged exposure to stress. Similarly, the vast majority of both preclinical and clinical research on the brain’s involvement in learning, memory, fear conditioning, and fear extinction has been conducted on male populations, with less than 2% of studies being based on females.3 This gender bias is especially problematic in relation to the field of psychotraumatology and psychopharmacology, as most of the disorders found in the aftermath of trauma are more prevalent in women and women are generally prescribed more psychotropic medication. Thus, whereas animal models can be a valuable tool in understanding different disorders in humans, it may be misleading if the animal models are based on one sex, whereas the actual psychiatric disorders are more commonly found in the other. In addition, preclinical research has severe limitations in that results cannot automatically be assumed to be directly applicable to humans. This limitation affects all animal models of psychological and medical disorders but is especially problematic in models of gender differences, in that they can only be used in attempts to understand the impact of biological sex, but not the importance of social gender. In spite of these limitations, preclinical research may help identify potential targets for gender-specific psychopharmacological interventions,17 as long as it never stands alone and relevant human studies are conducted before any clinical implications of such studies are considered. Although men also experience some fluctuations of gonadal hormones, the surges of hormones associated with the female menstrual cycle may cause females to be more heterogeneous than males. Whereas this may make research with female subjects more complicated, it should never be used as an argument to exclude women in research or to only include women at specific stages of their menstrual cycle or at a specific reproductive stage. For example, it is possible that certain pharmacological and possibly even psychotherapeutic interventions are most effective if they are initiated at a specific stage in the female menstrual cycle or if they are not offered to women who are using oral contraceptives. In America, 1/4 of women between the ages of 15 and 51 use oral contraceptives and 1/3 of women between the ages of 50 and 65 use some kind of hormone replacement therapy (HRT).3 Thus, if hormonal fluctuations are thought to affect treatment trials for a specific pharmacological intervention, this is all the more reason to learn how the gonadal hormones affect the women in need of that particular treatment. In other words, if the menstrual cycle complicates the inclusion of female subjects, this makes the inclusion of females even more important, as real-life sex and gender differences that may fluctuate within the menstrual cycle, or be manipulated by the use of oral contraceptives or HRT, are more than likely to exist as well. Similarly, it is highly problematic that pregnant

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and breast-feeding women are hardly ever included in treatment trials for psychotropic medication.14 Whereas the ethical reasons behind this are important to consider, and expected benefits for the woman should always be weighed against potential harm to the fetus or child, this decision is impossible for both mothers and doctors to make without the necessary knowledge that comes from including these groups of women in treatment trials. For this reason, pregnant and breast-feeding women should be included in both psychotherapeutic and psychopharmacological treatment trials whenever there is not considered to be a strong basis for believing that the treatment might be harmful to mother or child. Regardless of whether research on specific disorders use primarily male or female participants, the tendency to overlook one gender may have important consequences. As mentioned previously, research on acute response to stress has primarily been conducted on men and male animals, and thus more is known about the male compared to the female stress response. In contrast, because women are more likely to suffer from most trauma-related disorders, research on risk and protective factors, symptom development, and particularly treatment outcomes tends to focus primarily on women. As a consequence, there is some support that more is known about factors associated with the development and maintenance of PTSD in women compared to men.2 Furthermore, whereas most of the psychotherapeutic treatment studies mentioned here were generally characterized by low sample sizes, they were especially limited by very low numbers of male participants. Ultimately, this increased focus on women in psychotraumatology research may lead to the widespread use of psychotherapeutic treatments that have been empirically validated in female trauma populations, but that may be less effective in male trauma victims. An additional problem in this area of research is the fact that even when both genders are included, gender-specific analyses are rarely conducted. An absence of gender-specific analyses has been reported in the fields of physiology, neuroscience, immunology, pharmacology, and psychotraumatology.2,46 This is a very serious limitation, because in addition to these studies severely underestimating the potential impact of gender on important brain functions and associations with biobehavioral outcome, they also mistakenly give the impression that the results apply to both men and women. This is overly simplistic at best, but at worst it may have damaging consequences for one or both genders. For example, if a treatment trial for a specific anxiolytic drug is found not to have any effect on men and women examined together, gender-specific analyses may reveal that the drug is ineffective in both genders, or that it has strong beneficial effects in one gender but harmful effects in the other. Even more problematic, if

one gender is severely underrepresented, as is often the case with women in psychopharmacological research and with men in psychotherapeutic research, studies may reveal good treatment outcomes even if the minority gender experiences mostly damaging effects. Thus, severe side effects may be overlooked along with potential important positive effects when men and women (or male and female animals) are indiscriminately analyzed together or when gender is simply treated as predictor variable. Even when men and women appear to share a similar outcome, they may follow different pathways to get there, and these different routes may have important implications for risk and protective factors associated with the specific outcome in question as well as for potential treatment.2 This strongly highlights the importance of systematically conducting and reporting gender-specific analyses. In fact, doing so ought to be the rule, rather than the exception, in all fields related to human and animal behavior and health. Fig. 34.1 shows an illustration of how gender affects psychotraumatology at every step and every association from even before traumatic exposure to treatment outcomes.

34.7.2  Sex Versus Gender Differences Psychotraumatology research has generally focused more on sex differences than on gender, as masculinity, femininity, sexuality, and other gender-role-related concepts are rarely considered.2 The emergence of gender differences in the physiological stress response very early in life along with the impact of hormones on HPA-axis functioning2 clearly indicate the involvement of biological sex in the differences between men and women discussed in this chapter. However, variance in the strength of gender differences across cultures clearly indicates that although some of the gender differences in the prevalence and severity of PTSD are universal and likely attributable to biological sex, social gender also influences trauma-related symptomatology in important ways.2,69 For example, gender-role-identity has been reported to moderate the association between stressor type and activation of the HPA-axis, causing men and women to respond differently to certain stressors.2 This further points to gender-identity being an important factor in the subsequent development of different types of symptomatology. Furthermore, whereas femininity is generally unassociated with anxiety symptoms, masculinity has been found to be significantly and negatively associated with symptom severity in both men and women across a number of anxiety disorders.3 Interestingly, in contrast to these findings, a study of male veterans found that identification with masculine gender-role was weakly but significantly associated with higher PTSD levels.38 However this study did not control for combat exposure, and thus it is unknown whether a

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34.7 Discussion

Gender Acute physiological response

Everyday functioning

Traumatic event(s)

Acute psychological response

Side-effects PTSD Anxiety/ depression Internalizing symptoms Externalizing symptoms Somatization Somatic symptoms

Drop-out-rates Primary treatment

Pharmacotherapy & Psychotherapy

Secondary treatment Long-term treatment

Compliance

Relapse

FIGURE 34.1  Gender differences in different levels of psychotraumatology . The figure illustrates the development from normal functioning to traumatic exposure, symptom development, treatment, and treatment outcomes. This process develops in a context of gender, as both biological sex and social gender influences every process and every association illustrated in the model. In addition to side effects and compliance associated with different treatments apparently being directly affected by gender, these two variables also affect the different treatment outcomes, an association that may again be influenced by gender.

higher degree of traumatic exposure in the participants with the highest level of conformity to traditional masculine gender-roles may account for the differences. The same study reported a moderate association between masculine gender-role stress and PTSD severity,38 which is consistent with findings in anxiety research.3 Together, these studies highlight the importance of taking both biological sex and social gender into account when attempting to understand how men and women are affected by one or more traumatic events. As suggested by Rodin and Ickovics, studies on human health must advance beyond the use of sex as a predictor variable and start conceptualizing gender as a dynamic construct that varies across ethnic groups and social class and works in complex interactions with other physiological and social factors.68 It is beyond the scope of this chapter to present and discuss different theories trying to explain how biological sex and social gender come together to influence how men and women experience and respond to trauma and are subsequently affected by it. However, it should be noted that the gender differences discussed in this chapter cannot simply be reduced to measurement error, methodological bias, reporting error, or differences in the specific traumatic events that men and women are exposed to, including childhood and adult sexual trauma.2,70 Instead, it is more likely that differences in genetic, hormonal, biochemical, cultural, and social factors all contribute to gender differences in the

occurrence of different trauma-related disorders and symptoms.16,70 One way through which sex and gender may come together to contribute to gender differences in trauma-related disorders can be illustrated in a diathesis-stress model. As described elsewhere specifically in relation to the development of anxiety disorders, women appear to possess an inherent preparedness to respond more strongly to traumatic stressors than men.3 This preparedness is likely associated with biologically grounded traits that are generally more common in women than men, such as neuroticism, anxiety sensitivity, and trait anxiety, along with increased HPA axis reactivity, limbic system hyperactivation, and similar biologically based systems preparing women to quickly register and react to potential threats.3 In contrast, the stress component of the diathesis–stress model may primarily be made up of gender differences. Gender-role and differences in the specific traumatic stressors that men and women are exposed to affect not only the risk of developing traumarelated disorders but also the specific symptom profiles that men and women express. One study examining the contribution of different factors to gender differences in PTSD severity found that a combination of risk factors more prevalent in women, some of which were hereditary and others that were associated with the acute trauma response and the posttraumatic environment, could account for 83% of the association between gender and PTSD severity.70 Whereas the authors argued that several variables not included in the study may further

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34.  SEX AND GENDER DIFFERENCES IN TRAUMA VICTIMS

contribute to women’s increased prevalence and severity of PTSD, this study generally supports the hypothesis that it is the interaction of numerous risk factors more prevalent in women along with protective factors more prevalent in men that are responsible for women’s higher risk of PTSD. These risk and protective factors represent the influence of both biological sex and social gender. Interestingly, most of these risk factors are not uniquely associated with PTSD but are also very likely to contribute to gender differences in the other types of posttraumatic sequelae discussed in this chapter.

34.7.3  Gender Differences in a Clinical Context The fact that patients suffering from PTSD tend to overuse physical health care combined with the high level of somatic symptoms experienced by this group suggests that PTSD patients are likely to be overrepresented in primary care. A systematic review of PTSD in primary care settings reported a prevalence of current PTSD ranging from 2% to 39%.37 Some trauma survivors come to see their general practitioner to seek help specifically with trauma-related symptoms. However, many patients do not realize that the symptoms they experience are in any way related to their traumas. For this reason, primary care clinics are important locations for identifying patients with PTSD so that they can get the help they need.37 Unfortunately PTSD is difficult to detect in this context, especially in patients who do not present with classic PTSD symptoms or even disclose their trauma history, and a review of PTSD in primary care found that detection rates of PTSD ranged from 0% to 61.5%.37 For this reason, educating primary care personnel about the importance and consequences of traumatic exposure is extremely important. It has been suggested that it may be beneficial to screen for PTSD in specific at-risk primary care patient groups.37 Such groups may include patients with medically unexplained pain, difficult-to-treat sleep problems, or comorbid psychiatric symptoms. Understanding the importance of trauma and how it affects symptom development in men and women may help health care personnel obtain a broader understanding of their patients’ symptom profiles and help guide treatment. The considerable burden that is placed on the health care delivery system by the group of patients with PTSD may be decreased if primary care personnel become better at recognizing and treating patients with PTSD,37 or even better referring them to mental health professionals who have specialized in psychotraumatology and who are equipped to offer the patients evidence-based treatment. This chapter has focused on mean gender differences in the population. In contrast, doctors and other health care personnel are not faced with mean gender

differences but with unique individuals who differ from other men and women in a multitude of ways. Thus, even though women are much more likely than men to suffer from most trauma-related disorders, several men do suffer from PTSD, dissociation, eating disorder, and somatization, whereas women can become both highly aggressive and dependent on alcohol and other substances following traumatic exposure. It is therefore very important when dealing with real trauma survivors that health care personnel are informed by prior research without letting such mean gender differences bias their evaluations of a specific patient’s symptom profile. Whereas this point may seem obvious, there is a disturbing number of examples of professionals letting their knowledge of general gender differences affect the diagnosis given to a specific person, and where a man and a woman presenting with identical symptom profiles are given completely different diagnoses.45 A number of variables that contribute to gender differences may be important to consider as they may also affect intragender variance in both symptom profiles and treatment response. There is some evidence that treatment efficacy is directly affected by the patient’s symptom severity.50 Thus women may in some cases respond more favorably to certain treatments than men due to their higher symptom levels. Another variable that is relevant to consider in a clinical context is comorbidity, as variables such as anger have been found to predict poor treatment outcome.57 As men tend to report significantly higher levels of anger in the aftermath of trauma compared to women, it is possible that anger may pose a partially gender-specific complication to male treatment seekers and that these men may benefit more from treatment if their anger issues are dealt with prior to the initiation of trauma-focused psychotherapy. Other variables that have a potential impact on treatment efficacy and that may both contribute to gender differences and intragender variance include treatment expectations, compliance, health behaviors, and hormones including menstrual cycle, reproductive status, and use of oral (and possibly other hormone-based) contraceptives in women. Specifically in relation to pharmacotherapy, other potentially confounding factors include weight, body fat, alcohol use, smoking, and use of other medication. Whereas these variables generally differ between men and women, they also vary within each gender, making them important to consider when deciding whether a specific patient should be prescribed a specific medication or offered a psychotherapeutic intervention. Whereas the impact of such gender differences can be controlled for in statistical analyses in attempts to separate the unique influence of gender, such variables may be highly relevant in a clinical context where the unique influence of gender is not separated from such widespread gender differences. In a clinical context it is

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REFERENCES

irrelevant whether a patient responds poorly to a prescribed anxiolytic drug because of her weight or her metabolism. What is relevant, however, is that when her general symptom level and degree of comorbidity are taken into account, she is more or less likely to respond positively to a specific medication than men with the exact same symptom profile. It may also be clinically relevant that by designing the initiation of treatment in accordance to her menstrual cycle, her chances of a positive outcome may be increased. Finally, a high proportion of treatment studies tend to use unnecessarily high exclusion rates of approximately 30%, which is associated with higher effect sizes.53 This suggests, that the relatively high effect sizes obtained in psychotherapeutic treatment studies may be more difficult to obtain in a real-life treatment context where patients do not fit so neatly into the inclusion criteria set up for randomized treatment trials. Such limitations to the ecological validity of treatment studies may not only affect the reliability of treatment effects but also of gender differences reported (or not reported) in these studies, as men and women may be disproportionally affected by the exclusion criteria. The fact that many comorbid symptoms respond well to trauma-focused psychotherapy clearly shows that refusing treatment to patients based exclusively on the presence of such symptoms is not likely to be ethically responsible.

34.8 CONCLUSION Gender differences affect every area of psychotraumatology from exposure to stressful and traumatic events to the acute and prolonged reactions to such events, including development, maintenance, and treatment of PTSD and other trauma-related disorders. Both biological sex differences and social gender differences are at work, influencing every aspect of psychotraumatology and affecting general health and quality of life via multiple processes. Among others, these include gonadal hormones, HPA axis reactivity, and gender-role identification. Trauma-exposed patients may present to primary care and other health care providers with a multitude of psychological and somatic symptoms and disorders. The findings presented in this chapter underscore the importance of educating health care providers in the importance of trauma and trauma-related disorders. More research is needed that examines gender differences at every step from trauma response to treatment outcomes, and that conducts gender-specific analyses to acknowledge the full potential of gender differences. Knowledge of gender differences in the development and treatment of PTSD and other disorders should be used to enlighten researchers and clinicians, but it is extremely important that health care personnel do not

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let such knowledge bias them in their treatment of a specific patient.

References 1. Gillies GE, McArthur S. Estrogen actions in the brain and the basis for differential action in men and women: a case for sexspecific medicines. Pharmacol Rev. 2010;62(2):155–198. 2. Christiansen DM, Elklit A. Sex differences in PTSD. In: Ovuga E, ed. Post Traumatic Stress Disorder in a Global Context. Rijeka: InTech; 2012:113–142. 3. Christiansen DM. Examining sex and gender differences in anxiety disorders. In: Durbano F, ed. A Fresh Look at Anxiety Disorders. Rijeka: InTech; 2015:17–49. 4. Sramek JJ, Cutler NR. The impact of gender on antidepressants. In: Neil JC, Kulkarni J, eds. Biological Basis of Sex Differences in Psychopharmacology. Berlin: Springer Verlag; 2011:231–249. 5. Lok IH, Neugebauer R. Psychological morbidity following miscarriage. Best Pract Res Clin Obstet Gynaecol. 2007;21(2):229–247. 6. Calhoun PS, Bosworth BH, Grambow SC, Dudley TK, Beckham JC. Medical service utilization by veterans seeking help for posttraumatic stress disorder. Am J Psychiatry. 2002;159:2081–2086. 7. Taylor SE. Tend and befriend: Biobehavioral bases of affiliation under stress. Curr Dir Psychol Sci. 2006;15:273–277. 8. Kessler RC, Sonnga A, Bromet E, Hughes M, Nelson CB. Posttraumatic stress disorder in the national comorbidity survey. Arch Gen Psychiatry. 1995;52:1048–1060. 9. Norris FH, Foster JD, Weisshaar DL. The epidemiology of sex differences in PTSD across developmental, societal, and research contexts. In: Kimerling R, Ouimette P, Wolfe J, eds. Gender and PTSD. New York: The Guilford Press; 2007:3–42. 10. Goldberg LR, Freyd JJ. Self-reports of potentially traumatic experiences in an adult community sample: gender differences and test-retest stabilities of the items in a brief betrayal-trauma survey. J Trauma Dissociation. 2006;7(3):39–63. 11. Tolin DF, Foa EB. Sex differences in trauma and posttraumatic stress disorder: A quantitative review of 25 years of research. Psychol Trauma. 2008;S(1):37–85. 12. Ginsberg DL. Women and Anxiety Disorders: Implications for Diagnosis and Treatment (monograph). New York: MBL Communications Inc; 2004. 13. Breslau N, Kessler RC, Chilcoat HD, Schultz LR, Davis GC, Andreski P. Trauma and posttraumatic stress disorder in the community. Arch Gen Psychiatry. 1998;55:626–632. 14. Christiansen DM. Posttraumatic stress disorder in parents following infant death: A systematic review. Clin Psychol Rev. 2017;51:60–74. 15. Bangasser DA, Valentino RJ. Sex differences in molecular and cellular substrates of stress. Cell Mol Neurobiol. 2012;32(5): 709–723. 16. Dalla C, Pitychoutis PM, Kokras N, Papadopoulou-Daifoti Z. Sex differences in response to stress and expression of depressive-like behaviours in the rat. In: Neill JC, Kulkarni J, eds. Biological Basis of Sex Differences in Psychopharmacology. Berlin: Springer-Verlag; 2011:97–118. 17. Donner NC, Lowry CA. Sex differences in anxiety and emotional behavior. Pflugers Arch. 2013;465(5):601–626. 18. Kudielka BM, Kirschbaum C. Sex differences in HPA axis responses to stress: a review. Biol Psychol. 2005;69(1):113–132. 19. McHenry J, Carrier N, Hull E, Kabbaj M. Sex differences in anxiety and depression: role of testosterone. Front Neuroendocrinol. 2014;35(1):42–57. 20. Taylor SE, Klein LC, Lewis BP, Gruenewald TL, Gurung RAR, Updegraff JA. Biobehavioral responses to stress in females: Tendand-befriend, not fight-or-flight. Psychol Rev. 2000;107(3):411–429.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

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34.  SEX AND GENDER DIFFERENCES IN TRAUMA VICTIMS

21. Neumann ID. Brain oxytocin: A key regulator of emotional and social behaviours in both females and males. J Neuroendocrinol. 2008;20:858–865. 22. DeSantis SM, Baker NL, Back SE, et al. Gender differences in the effect of early life trauma on hypothalamic-pituitary-adrenal axis functioning. Depress Anxiety. 2011;28(5):383–392. 23. Klein LC, Corwin EJ. Seeing the unexpected: how sex differences in stress responses may provide a new perspective on the manifestation of psychiatric disorders. Curr Psychiatry Rep. 2002;4:441–448. 24. Catuzzi JE, Beck KD. Anxiety vulnerability in women: a two-hit hypothesis. Exp Neurol. 2014;259:75–80. 25. Bryant RA, Felmingham KL, Silove D, Creamer M, O´Donnell M, McFarlane AC. The association between menstrual cycle and traumatic memories. J Affect Disord. 2011;131:398–401. 26. Nillni YI, Pineles SL, Patton SC, Rouse MH, Sawyer AT, Rasmusson AM. Menstrual cycle effects on psychological symptoms in women with PTSD. J Trauma Stress. 2015;28(1):1–7. 27. Altemus M. Sex differences in depression and anxiety disorders: potential biological determinants. Horm Behav. 2006;50(4):534–538. 28. American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders: DSM-III. 3rd ed. Washington, DC: Author; 1980. 29. American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders: DSM-IV-TR. 4th, text revision ed. Washington, DC: Author; 2000. 30. American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders: DSM-5. 5th ed. Washington, DC: Author; 2013. 31. Carragher N, Sunderland M, Batterham PJ, et  al. Discriminant validity and gender differences in DSM-5 posttraumatic stress disorder symptoms. J Affect Disord. 2016;190:56–67. 32. Galovski TE, Blain LM, Chappuis C, Fletcher T. Sex differences in recovery from PTSD in male and female interpersonal assault survivors. Behav Res Ther. 2013;51(6):247–255. 33. Association AP. Diagnostic and Statistical Manual of Mental Disorders: DSM-5. 5th ed. Washington, DC: Author; 2013. 34. Amstadter AB, Aggen SH, Knudsen GP, Reichborn-Kjennerud T, Kendler KS. Potentially traumatic event exposure, posttraumatic stress disorder, and Axis I and II comorbidity in a populationbased study of Norwegian young adults. Soc Psychiatry Psychiatr Epidemiol. 2013;48(2):215–223. 35. Müller M, Vandeleur C, Rodgers S, et al. Factors associated with comorbidity patterns in full and partial PTSD: findings from the PsyCoLaus study. Compr Psychiatry. 2014;55(4):837–848. 36. Pratchett LC, Pelcovitz MR, Yehuda R. Trauma and violence: are women the weaker sex? Psychiatr Clin North Am. 2010;33(2):465–474. 37. Greene T, Neria Y, Gross R. Prevalence, detection and correlates of PTSD in the primary care setting: a systematic review. J Clin Psychol Med Settings. 2016 38. Morrison JA. Masculinity moderates the relationship between symptoms of PTSD and cardiac-related health behaviors in male veterans. Psychol Men Masculin. 2012;13(2):158–165. 39. Spitzer C, Barnow S, Völzke H, John U, Freyberger HJ, Grabe HJ. Trauma, posttraumatic stress disorder, and physical illness: findings from the general population. Psychosom Med. 2009;71:1012–1017. 40. Driscoll MA, Higgins DM, Seng EK, et al. Trauma, social support, family conflict, and chronic pain in recent service veterans: does gender matter? Pain Med. 2015;16:1101–1111. 41. Yehuda R. Biology of posttraumatic stress disorder. J Clin Psychiatry. 2000;61:14–21. 42. Tarrier N, Sommerfield C, Pilgrim H, Faragher B. Factors associated with outcome of cognitive-behavioural treatment of chronic post-traumatic stress disorder. Behav Res Ther. 2000;38:191–202.

43. Soldin OP, Mattison DR. Sex differences in pharmacokinetics and pharmacodynamics. Clin Pharmacokinet. 2009;48:143–157. 44. Bernardy NC, Lund BC, Alexander B, Jenkyn AB, Schnurr PP, Friedman MJ. Gender differences in prescribing among veterans diagnosed with posttraumatic stress disorder. J Gen Intern Med. 2012;28(Suppl 2):S542–S548. 45. McGrath E, Keita GP, Stricklanol BR, Russo NF. . Women and Depression: Risk Factors and Treatment Issues. Washington, DC: American Psychological Association; 1990. 46. Beery AK, Zucker I. Sex bias in neuroscience and biomedical research. Neurosci Biobehav Rev. 2011;35(3):565–572. 47. Hamilton JA, Jensvold MF. Sex and gender as critical variables in feminist psychopharmacology research and pharmacotherapy. Women Ther. 1995;16(1):9–30. 48. Pineles SL, Nillni YI, King MW, et  al. Extinction retention and the menstrual cycle: different associations for women with posttraumatic stress disorder. J Abnorm Psychol. 2016;125:349–355. 49. Keating C, Tilbrook A, Kulkarni J. Oestrogen: an overlooked mediator in the neuropsychopharmacology of treatment response? Int J Neuropsychopharmacol. 2011;14(4):553–566. 50. Robinson DS. Clinical trials: problems with negative outcomes. Prim Psychiatry. 2005;12:21–22. 51. Keers R, Aitchison KJ. Gender differences in antidepressant drug response. Int Rev Psychiatry. 2010;22(5):485–500. 52. Nugent NR, Christopher NC, Crow JP, Browne L, Ostrowski S, Delahanty DL. The efficacy of early propranolol administration at reducing PTSD symptoms in pediatric injury patients: a pilot study. J Trauma Stress. 2010;23(2):282–287. 53. Bradley R, Greene J, Russ E, Dutra L, Westen D. A multidimensional meta-analysis of psychotherapy for PTSD. Am J Psychiatry. 2005;162:214–227. 54. Christiansen DM. Sex differences in PTSD: mediation and moderation effects. In: Martin CR, Preedy VR, Patel VB, eds. Comprehensive Guide to Post-Traumatic Stress Disorder. Switzerland: International Publishing; 2016. 55. Basoglu M, Salcioglu E, Livanou M. A randomized controlled study of single-session behavioural treatment of earthquakerelated post-traumatic stress disorder using an earthquake simulator. Psychol Med. 2007;37(2):203–213. 56. Blanchard EB, Hickling EJ, Devieni T, et al. A controlled evaluation of cognitive behaviorial therapy for posttraumatic stress in motor vehicle accident survivors. Behav Res Ther. 2003;41:79–96. 57. van Minnen A, Arntz A, Keijsers GPJ. Prolonged exposure in patients with chronic PTSD: predictors of treatment outcome and dropout. Behav Res Ther. 2002;40:439–457. 58. Basoglu M, Salcioglu E, Livanou M, Kalender D, Acar G. Singlesession behavioral treatment of earthquake-related posttraumatic stress disorder: a randomized waiting list controlled trial. J Trauma Stress. 2005;18(1):1–11. 59. Karatzias A, Power K, McGoldrick T, et al. Predicting treatment outcome on three measures for post-traumatic stress disorder. Eur Arch Psychiatry Clin Neurosci. 2007;257:40–46. 60. Stenmark H, Guzey IC, Elbert T, Holen A. Gender and offender status predicting treatment success in refugees and asylum seekers with PTSD. Eur J Psychotraumatol. 2014:5. 61. Gidron Y, Gal R, Givati G, Lauden A, Snir Y, Benjamin J. Interactive effects of memory structuring and gender in preventing posttraumatic stress symptoms. J Nerv Ment Dis. 2007;195(2):179–182. 62. Ironson G, O’Cleirigh C, Leserman J, et  al. Gender-specific effects of an augmented written emotional disclosure intervention on posttraumatic, depressive, and HIV-disease-related outcomes: a randomized, controlled trial. J Consult Clin Psychol. 2013;81(2):284–298. 63. Blain LM, Galovski TE, Robinson T. Gender differences in recovery from posttraumatic stress disorder: a critical review. Aggress Violent Beh. 2010;15(6):463–474.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

64. Cason D, Grubauch A, Reisick P. Gender and PTSD treatment: efficacy and effectiveness. In: Kimerling R, Ouimette P, Wolfe J, eds. Gender and PTSD. New York: The Guilford Press; 2002:305–334. 65. Lange A, Rietdijk D, Hudcovicova M, van de Ven JP, Schrieken B, Emmelkamp PM. Interapy: a controlled randomized trial of the standardized treatment of posttraumatic stress through the internet. J Consult Clin Psychol. 2003;71(5):901–909. 66. van Minnen A, Harned SM, Zoellner L, Mills K. Examining potential contradindications for prolonged exposure therapy for PTSD. Eur J Psychotraumatol. 2012:3.

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67. Felmingham KL, Bryant RA. Gender differences in the maintenance of response to cognitive behavior therapy for posttraumatic stress disorder. J Consult Clin Psychol. 2012;80(2):196–200. 68. Rodin J, Ickovics JR. Women´s health. Review and research agenda as we approach the 21st century. Am Psychol. 1990;45:1018–1034. 69. Norris FH, Perilla JL, Ibanez GE, Murphy AD. Sex differences in symptoms of posttraumatic stress: does culture play a role. J Trauma Stress. 2001;14:7–28. 70. Christiansen DM, Hansen M. Accounting for sex differences in PTSD: a multi-variable mediation model. Eur J Psychotraumatol. 2015;6:260–268.

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C H A P T E R

35 Women Do Worse Than Men—GenderSpecific Differences in Burn Patients Ines Ana Ederer1, Florian Hackl2 and Reinhard Pauzenberger1 1

Medical University Vienna, Vienna, Austria, 2Tufts School of Medicine, Brighton, MA, United States

O U T L I N E 35.1 Introduction

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35.2 Classification of Burn Injuries

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35.3 Scoring Systems in Burns

514

35.4 Epidemiology of Burn Injuries 35.4.1 Gender-Specific Burn Epidemiology 35.4.2 Gender Dimorphism in Burn Mortality

515 516 516

35.5 Causes for Gender-Specific Differences in Burn Mortality

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35.6 Alternative Reasons for Gender Dimorphism in Burns

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35.7 Conclusion

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35.8 Suggestions for Further Research

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References 521

35.1 INTRODUCTION Burn injury can affect the old and the young, males and females, and may occur at home or during work. The vast majority of burns are minor, although very painful. Nevertheless, burn injuries can be devastating even if only a small area is involved. Flames and fire, scalds, and contact burns represent the three most prevalent causes of severe burns and follow an age- and gender-related pattern.1–3 Accidents with fire and flames have been shown to mainly occur in the young male and elderly female population, as compared to scalds, which have more frequency in children and women.2,4 Facing the long-established assignment of gender-specific tasks in everyday life, this distribution seems anything but surprising. Female burn patients, however, have also been shown to be at increased risk for the development of hypertrophic scarring.5,6 The causal relationship for this gender-specific difference is not fully understood yet. Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00028-0

35.2  CLASSIFICATION OF BURN INJURIES Burn injuries can primarily be classified as thermal damage to the skin, but can affect other organs such as the lungs, potentially resulting in inhalational trauma (IHT). When damage is caused by hot liquids, hot solids, or fire these wounds are classified as thermal burns. Injuries due to radiation, electricity, or contact with chemicals (acids and bases) are also termed burns. Traditionally, burns can be categorized into three degrees according to the depth of the injury. First degree burns involve the epidermis only, resulting in an erythematous, warm and very painful wound since the sensory endings located in the dermal layer remain intact. Blistering is absent and healing will usually be completed in less than a week. Superficial second degree burns affect the entire epidermis and the portion of the dermis just above the basal layer. These pink, moist, and very painful lesions often result in blister formation.

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© 2017 Elsevier Inc. All rights reserved.

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35.  Women Do Worse Than Men—Gender-Specific Differences in Burn Patients

Complete regeneration usually occurs within 2 weeks without esthetic sequelae. Deep second degree burns extend into the reticular portion of the dermis and due to partial destruction of sensory endings the tactile perception is significantly decreased compared to superficial second degree burns. Blister formation may be possible; however, following debridement deep second degree burns appear pinkish to white, dry, and blanching of the skin under digital pressure is sluggish or absent. Excision and skin grafting might be necessary due to possible wound contraction and scarring. Finally, fullthickness or third degree burns are defined as lesions extending deep into subdermal structures, sometimes even including aponeurosis or bones. These lesions appear hard, brown-black, leathery, and insensate. Third degree burns generally require wide excision and skin grafting for optimal healing. When circumferential, often seen on upper and lower extremities, these wounds can become a source of peripheral ischemia requiring immediate surgical escharotomy or fasciotomy. During the initial triage process it is essential to evaluate the total size and depth of the burn injury. A widely used tool to estimate total body surface area (TBSA) burned is the rule of nines, introduced by Wallace in 1951.7–9 When calculating TBSA, however, only areas of partial and full-thickness burns are included, but not superficial dermal injuries limited to the epidermis.10 In the pediatric population the rule of nines has to be adjusted to account for different body surface proportions of infants and children compared to those of adults.7–9 The Lund and Browder chart takes into consideration age-dependent differences in body proportions, thus making it especially useful in the pediatric population.11 Alternatively patients’ hand surface area, accounting for approximately 1% TBSA, can be utilized to estimate the extent of the burn injury.8,9 Visual assessment and vascular evaluation are essential for determining burn depth. More advanced techniques including skin biopsies and laser Doppler, ultrasound, or fluorescence fluorimetry, intended to minimize observer subjectivity by visualizing dermal microcirculation, are rarely utilized in a clinical setting.8,9 Furthermore, accurate determination of burn depth and extension must be repeated over several days due to the dynamic nature of burn injuries and a less accurate assessment prior to the third day postinjury.8 According to Jackson, a burn wound can be characterized in three zones: the zone of coagulation (the central and most severely damaged area), the zone of stasis (initially viable but can advance to a zone of coagulation when wound perfusion is not provided), and the zone of hyperemia (characterized by vasodilatation and potential regeneration).12 These initial evaluations guide the decision making process regarding further treatment, fluid resuscitation, and the need for surgery.

35.3  SCORING SYSTEMS IN BURNS Patient mortality is still the primary objective outcome parameter in burn care. In general, scoring systems pose a central role in evidence-based medicine as they allow the evaluation of changes in terms of quality control in care and disease management.13 As a consequence, their use has led to major improvements in burn therapy due to interhospital comparison of patients outcome data.14 Scoring systems further allow the identification of patients at increased risk and can be directive for the decision making process and proper allocation of hospital resources.14,15 Apart from the Acute Physiology and Chronic Health Evaluation (APACHE) Scores, which are the most widely used mortality prediction models at intensive care units (ICU), the majority of current trauma scores are not applicable to burn injuries as they do not reflect burn victims’ situation with sufficient accuracy.16,17 For this reason and because of the quantifiable nature of burn injuries, especially as far as burn size and burn depth are concerned, specific scoring systems for burn injuries have been introduced. The literature on risk factors for mortality following burn injury is extensive. Therefore, general agreement exists only for a few prognostic determinants—namely for age, %TBSA, and the presence of IHT.18–21 The former two factors gained importance when the Baux score was first described in 1961.22 It is originally defined as the sum of age and %TBSA and still is a commonly used prediction model due to its simplicity.22 Interestingly, genderrelated differences in burn mortality were already noted quite early in the 1970’s when Tobiasen et al. developed a simple and clinically useful instrument to measure burn outcome: the Abbreviated Burn Severity Index, also known as ABSI.23 It was first published in 1982 and uses five variables (sex, age, inhalation injury, %TBSA burned, and full-thickness burn) to predict mortality (Table 35.1).23 Within constant development in burn care and the steadily improved survival after burn injuries, it has become necessary to question its predictive accuracy nowadays. According to a recent validation, however, even 30 years after its invention the ABSI can still be regarded as a valuable model for the prediction of mortality in burn patients and therefore finds broad acceptance in clinical practice.24 Nevertheless, numerous prognostic scoring systems based on a minimal set of variables including and weighing different risk factors have been developed.17 Although many of them are proven to reflect the risk of death following thermal injuries quite accurately, none of them can be nominated as the most valid one.17,20,25 What is even more surprising, the majority of these models does not reflect gender differences even though gender dimorphism in mortality has been noted in the majority of studies.

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35.4  Epidemiology of Burn Injuries

TABLE 35.1  Abbreviated Burn Severity Index (ABSI) Variable

Patient characteristic

Score

Female

1

Sex

Male

0

Age in years

0–20

1

21–40

2

41–60

3

61–80

4

81–100

5

Inhalation injury full

1

Thickness burn

1

TBSA (%)

1–10

1

11–20

2

21–30

3

31–40

4

41–50

5

51–60

6

61–70

7

71–80

8

81–90

9

91–100

10

81–90

9

Total score

Threat to life

Probability of death (%)

2–3

Very low

80

ABSI, Abbreviated Burn Severity Index; TBSA, total body surface area burned.

35.4  EPIDEMIOLOGY OF BURN INJURIES Despite advances in modern medicine, burn injuries still represent the fourth most common type of trauma, following traffic accidents, falls, and interpersonal violence.4,26,27 Globally, in 2013 an estimated number of 31 million patients received medical attention due to burn injuries and approximately 3 million of them required inpatient hospitalization.28 According to the World Health Organization, approximately 265,000 individuals worldwide die from burn injuries every year.29 The global incidence of burns shows significant geographical and socioeconomic variability especially

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when comparing high- and low-income countries. In the United States burn-related injuries represent about 2% of all trauma cases, similar to reports from Europe with an annual incidence between 2 and 29 per 100,000 individuals.2,30 Contrary to these results, the incidence of burn injuries requiring medical care is almost 20 times higher in Southeast Asia when compared to the Americas.31 Recent data from Taiwan showed that every year 671 per 100,000 in males and 853 per 100,000 in females received medical treatment as a result of burn injuries.32 Areas with the highest incidence rates, such as the Western Pacific Region, Southeast Asia, and the Eastern Mediterranean Region, correlate with a lack of prevention programs as well as limited accessibility to an infrastructure of satisfactory health care.29,31,33 In high-income countries, however, because of precautionary measures and higher standard of medical care a constantly decreasing number of burn injuries has been reported over the past decades.2,4,31 As for Slovakia, a reduction of 20% in burn incidence was noted between 1990 and 2004.2 Even if burns are sustained throughout the entire lifespan, the very young and the elderly population are most likely to sustain burn injuries.2,4,34 Lack of child supervision, low socioeconomic status, frailty, and comorbid illnesses occurring with age are only some factors placing people at the extremes of age at an increased risk.33 The growing insight into the pathophysiology of burn injuries has allowed major improvements in burn therapy, such as the surgical approach of early excision and immediate wound closure, adequate nutritional therapy, fluid resuscitation, and the prevention and treatment of infections.2,35 Once described as a medical field of “primitive care,” burn treatment has become highly sophisticated and even led to the installation of specialized centers consisting of a well-organized team of subspecialty experts from different health care professions.35,36 As a result, these advances have enabled the majority of patients—also referring to victims who sustain burns of up to 90% TBSA—to survive these dramatic injuries nowadays.19,37 This considerable increase in survival can be highlighted when compared with United States data from the 1940s, showing the comparable LD50 occurred at 20% TBSA.37 Recent data of the WHO further demonstrate an overall decrease in burn mortality of 6% between 1990 and 2010.31 Similar to burn incidence, however, survival estimates vary substantially among different patient populations all over the world. Whereas the risk of death following burns in the United States lies between 3% and 5%, low- and middle-income countries show quite different data.1,3,35 These regions account for over 90% of all fatal burnrelated injuries worldwide.4,29,33 Despite the progressively decreasing mortality rates mentioned above, recent studies in industrial

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35.  Women Do Worse Than Men—Gender-Specific Differences in Burn Patients

countries have shown that this trend has stagnated in past years.14,20,35 A review of results from the last 70 years conducted at the Massachusetts General Hospital highlighted that overall mortality of burn victims has remained unchanged at 4% since 1984, which indicates that current treatment approaches have reached their maximum in optimizing patients’ survival.35 Hence, sepsis and multiorgan failure (MOF), with renal failure as the most frequently encountered organ, are still the main causes of death in the setting of thermal injuries.19,38,39 Moreover, nonfatal burn injuries continue to be a leading cause of morbidity, including disfigurement and disability, often resulting in social rejection and discrimination.4,26,29,33 As a main reason for disabilityadjusted life-years (DALY) burn injuries have a considerable impact on health economic.26,29 For example, in Norway costs for inpatient burn management exceeded $14.7 million in 2007.29 To put it differently, severe nonfatal burns are among the most expensive traumatic injuries worldwide.40–42 This consideration includes the patients’ long hospitalization and rehabilitation period with respect to reintegration into society and returning to the workforce as well as the intensive follow-up due to long-term complications such as contractures.40–42

35.4.1  Gender-Specific Burn Epidemiology Gender-specific differences exist in burn incidence and vary by age, region, and socioeconomic status. In general, a disproportionately higher number of male cases has been described in the majority of current literature.1–4,33 These sources, however, are mostly limited to high-income countries and only partly reflect the current situation in other parts of the world. In the United States, the American Burn Association reports an overall incidence of 68% male versus 32% female burn patients.1 In contrast, a systematic review of unintentional burns in South Asia including India, Pakistan, Bangladesh, and Sri Lanka highlights male predominance in burn incidence only during early childhood.43 With increasing age, though, most serious burns affect females, implicating a shift in gender distribution which correlates with a change in traditional gender-specific roles in these regions.43 A vast majority of burn injuries and casualties from fire are caused unintentionally and tend to occur in a domestic setting.1,2,29,33,38 In lower-income countries, and rural communities in particular, mostly nonelectric appliances are used for cooking, heating, and lighting, which implicate an increased risk for burns. In addition, it is mostly women who engage in a majority of household activities in these regions. For these reasons burn injuries attributed to open flames account for 77% of all cases in Nepal and affect women more often than men (89 vs 69%, respectively).44 In the Western

countries, however, up to one-third of burn injuries are work-related which might explain the higher incidence observed in the young male population.1,2,4 With respect to the elderly, the literature demonstrates a conversion of the gender-specific incidence in high-income countries. The fact that the life expectancy of women exceeds that of men results in more females than males sustaining thermal injuries in later life.2,3,34 Finally, burns injuries may be caused intentionally, such as in suicide attempts or interpersonal violence.2,45 Only 1% of all suicide attempts in the United States and Europe are attributed to deliberate self-inflicted burning.45 In India, however, almost 40% of all burn cases occur as part of suicide attempts affecting women significantly more often than men with a preponderance of 90%.46

35.4.2  Gender Dimorphism in Burn Mortality Women in Southeast Asia face the highest risk to die from burn injuries worldwide.31 In 2012, 30% of all female deaths from unintentional burns happened in this region.31,44 Disregarding the regional aspect which can be partly contributed to the lack of infrastructure in the prehospital setting, the lower standard of medical care, and specific differences in burn etiology, there is increasing evidence for differences in burn mortality attributable to gender itself. In recent years, the relationship between gender and mortality has already been demonstrated for traumatic injuries with a more favorable outcome for (especially young) women and an increased mortality associated with the male gender.47–54 When it comes to burn injuries, however, the situation is reversed: female gender is associated with a more detrimental outcome in the majority of studies.55–58 In 2001, a retrospective investigation of data from 4096 patients showed an almost twofold increase in mortality for women following burn injury.59 Additionally, multivariate analysis in this study identified female sex as an important prognostic risk factor for outcome estimates, even though it might be less significant than other parameters such as age or %TBSA.59 Similar associations were made by the Burns Evaluation and Mortality Study (BEAMS), a recently published multicenter study conducted in Australia and New Zealand.60,61 Logistic regression and examination of demographic data from adult burn patients admitted to the ICU were used for the development of a discriminatory prediction model including female sex as a very strong risk factor for burn mortality. In fact, female burn patients were about three times more likely to die than similarly injured men in this study. An even stronger relationship, though, was established by a retrospective investigation of 540 burn patients in Iran in 2014.62 Women following severe burn injuries were identified to be approximately four times

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

35.5 Causes for Gender-Specific Differences in Burn Mortality

more likely to die than men.62 These results remained significant even after adjusting for confounding factors like %TBSA. Importantly, there was no significant relationship between intention, i.e., what caused the burn, and burn mortality despite the high incidence of suicidal burn cases accounting for about 30% of all burns injuries, which affected women six times more often than men and resulted in the most extensive burn injuries with a median TBSA of 62.5%.62 O’Keefe and colleagues reviewed burn registry data from a 10-year period in the United States and established a prediction model of burn mortality taking into account gender-specific differences.63 In their model, women aged 30–59 years had an approximately twofold higher mortality rate compared with male burn victims of the same age group.63 In the youngest and oldest age group no difference in mortality between men and women could be described.63 The different fatality rates between the two genders, however, could neither be described by an imbalance of burn etiology or burn severity, like greater burn size or burn depth in women, nor could they be attributed to a difference of complications, such as ARDS, pneumonia, or sepsis during the time of hospitalization.63 Data by McGwin et al. are consistent with the results mentioned above.25 When modified by age, gender dimorphism in burn mortality was limited to patients younger than 60 years, with women being 2.3 times more likely to die from burns than men of the same age group.25 These associations remained significant after adjusting for potentially confounding factors although demographic data identified women to be generally more likely to have preexisting medical conditions, to be older, and to be of the black race compared to male patients.25 Interestingly, mean %TBSA was slightly higher in men than women who died from burn injuries, but this difference did not reach statistical significance (48% vs 38%, respectively).25 Causes and timing of death did not differ among female and male nonsurvivors, though.25 A few years later, the same study group used a large patient data from the National Trauma Databank addressing more precisely the effect of age on the relationship between gender and burn mortality by dividing the study population into four different age groups.64 Gender dimorphism was persistent across all ages with mortality rates of 7.5% in men and 12.2% in women, respectively. Female nonsurvivors were generally older (68 years vs 54 years) and suffered from lower %TBSA than men (25% vs 35%).64 However, adjusting for confounding factors including age, race, comorbidity, injury type, inhalation injury, and %TBSA, the study showed disparity in gender dimorphism in burn mortality in certain age groups.64 Therefore, the adjusted association between gender and burn mortality only

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remained statistically significant among patients aged from 20 to 34 years.64 In addition, these observations found strong confirmation in an extensive investigation of nearly 50,000 patients in the United States, reconfirming gender-specific differences in burn mortality being most significant among women in their 30s and 40s with an approximately twofold increase, persistent even in adjusted analysis.65 Further studies, however, failed to consistently reproduce gender-specific differences in burn mortality with respect to age. Logistic regression analysis by Summers et al. confirmed female gender as an independently associated risk factor of burn mortality, but when adjusted for age, gender dimorphism persisted only in the pediatric group increasing the risk to succumb to burns up to 12-fold among girls compared to similarly injured boys.66 Contrary to these results, another study revealed survival disadvantage only among female patients older than 65 years.67 Some authors could not describe any gender-specific differences in burn mortality, neither in the young nor in the elderly population.68–71 Conflicting data and the lack of consensus in clinical studies, however, do not seem surprising when taking into account the very heterogeneous group of patients, thus making valid comparisons difficult (Table 35.2). Differences included variation in burn severity, sample size, and patients’ age ranges. The study time period sometimes covered 10 years or even more implicating that patients were exposed to different treatment protocols and current therapeutic approaches might not have been taken into account. Finally, all of these studies were characterized by a huge predominance of male patients who accounted for approximately 70% of all burn victims.

35.5  CAUSES FOR GENDER-SPECIFIC DIFFERENCES IN BURN MORTALITY Several experimental and clinical studies aimed to examine the underlying causes for the relationship between gender and burn mortality. It has been postulated that women’s adverse outcome might be due to gender-specific differences in metabolic and neuroendocrine responses following burn injury.63 Stress response to burn injury is indeed well known to be very severe resulting in a hypermetabolic state, which is not comparable to any other traumatic injury or disease.69 It is mainly characterized by massive oxygen and glucose consumption, augmented CO2 production, glycogenolysis, proteolysis, and lipolysis, resulting in a hyperdynamic catabolic status with enormous substrate and energy requirements.72 This burn-specific injury response occurs approximately five days postinjury and certain metabolic changes tend to continue up to several months, rather up to 3 years causing a considerable

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

TABLE 35.2  Summary of Clinical Studies Supporting Gender Dimorphism in Burn Mortality Author

Study period

O’Keefe et al. (2000)

1989–1998 4927 (all ages)

No patients

Gender Overall distribution (%) mortality (%) Gender-specific mortality

Risk factors of mortality

75 male

●

5.3

2.4-fold higher in women aged 30–59 years

Age Female sex ● IHT ● %FTSA ● %TBSA ●

De Souza (2002)

1991–1997 921 (all ages)

Muller et al. (2001)

67.1

8.4

Two-fold higher in women

Not evaluated

1972–1996 4094 (patients older 66 male than 10 years)

3.6

1.8-fold increase among females across all ages

●

McGwin et al. (2002)

1994–2000 1611 (patients older than 20 years)

76.3male

8.7

Unadjusted analysis: 7.2% in men versus 13.4% in women Modified by age: 2.3-fold higher in females up to the age of 60 exclusively

Not evaluated

George et al. (2005)

1994–2002 6236 (patients older 77.4 male than 20 years)

8.6

Unadjusted analysis: 7.5% in men and 12.23% in women Modified by age: about two-fold higher in females aged 20–34 years exclusively

Not evaluated

Sharma et al. (2005)

1993–2001 2111 (all ages)

5.3

Two-fold higher in women

70 male

Age Burn size ● Female sex ● IHT ●

Age >60 years Female sex ● Flame burns ● %TBSA >70 ● ●

Kerby et al. (2006)

1991–2001 49,079 (data from the NBR including all ages)

70.2 male

Not given

1.5-fold higher in women Stratified by age, strongest association for women aged 20–39 years, no difference in youngest and oldest age group

Pereira et al. (2006)

1989–2005 1674 (all ages, burns >20% TBSA) 179 autopsies

70.6 male

11

Stratified by age, higher risk only for women >65 years (80% vs 40% Not evaluated in men) Women aged >65 years did not show any improvement in mortality since 1989 (contrary to women 65% TBSA, no IHT)

Not evaluated

57.6 male

25.8

Four times higher in women in univariable logistic model, also after adjusting for %TBSA

15

Two-fold higher in females (even stronger association in patients with burns >60% TBSA and in the pediatric group)

19.4

1.8-fold higher in women

Age >60 years Burn size ● Female sex ● ●

Not evaluated

Female sex %TBSA

● ●

All studies follow inequalities in gender distribution respecting burn incidence and burn mortality. Studies were mostly conducted to determine risk factors for predicting mortality of death in order to establish applicable scoring systems in clinical practice.%FTSA, %full-thickness surface area burned; ICU, intensive care unit; IHT, Inhalational trauma; NBR, National Burn Repository.

35.5 Causes for Gender-Specific Differences in Burn Mortality

long-term mortality among burn patients.73,74 Therefore, gender-specific differences might not become apparent in the immediate time after injury but manifest themselves during the course of disease when it comes to the ability to tolerate prolonged critical illness.63 In this regard, women who suffered from burn injuries during childhood were associated with significantly increased long-term mortality compared with their male counterparts.73 Contrary to these results, Summers et al. argue that the differential trend in survival between male and female burn patients occurs immediately after the burn injury.66 Their findings, however, were limited to the pediatric group exclusively, and so were genderspecific differences in burn mortality.66 With regard to pediatric burn patients, Jeschke et al. do not favor metabolic changes being responsible for gender divergent outcome.69 In their study, burn-injured girls sustained decreased catecholamine and stress hormone levels associated with less inflammation and hypermetabolic response, leading to shortened hospital stay compared to boys.69 Improved outcome among girls was not reflected in mortality, though.69 In contrast, other studies among adult burn patients showed that women have a significant longer hospitalization period and an approximately twofold increased risk of dying from burns compared to similarly injured men.59,72 As there were mostly no differences in demographic or injury characteristics, a growing body of literature points out gender-specific alterations in cell-mediated immunity.25,64,65 It might contribute to the development of immunodysfunction, which is characterized by subsequent immunosuppression after an initial phase of an inflammatory cascade, leaving patients more susceptible to infections and sepsis.25,64,65 In general, the immune response following burns has been shown to be prolonged and more intense compared to other nonthermal injuries.75 In this context, experimental and clinical studies confirmed that cytokines and macrophages play a crucial role in burn-specific immune response and high levels of interleukin 6 (IL-6) correlate with increased patient mortality.76–78 Additionally, animal models of thermal injury successfully demonstrated gender-specific differences in cell-mediated immunity.77,79 After induction of thermal injury, male mice experienced rapid increases of IL-6 levels corresponding with the suppression of cell-mediated immune function within 24 h after injury.77 Over time, cytokine levels declined and returned to baseline, resulting in adequate immune function once again.77 In contrast, suppression of immune function was delayed in female mice because initially normal IL-6 levels rose significantly later in the postburn period.77 In summary, these observations suggest that gender dimorphism following burn injuries might be due to a delayed amplification in cell-mediated immunity among females. Assuming that cytokine

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release indeed differs temporarily, this effect should lead to a divergent timing of death between male and female patients. This association has not been confirmed yet. Moreover, gender-specific differences in immune response were identified to be reversible by administering 17β-estradiol to males, and in females by performing ovariectomy or administering estrogen receptor antagonist, which indicates that sex hormones are central mediators in the immune function following burns.80 Since several clinical studies emphasized that detrimental outcome in female patients was most obvious at the peak of their reproductive age, indeed, sex hormones could provide a reasonable explanation for why survival disadvantage affects women with high specificity in this age group only.25,64,65 This gender-specific interconnection between endocrine and immune system via an influence on humoral as well as cell-mediated immunity has already been demonstrated among trauma patients.81–83 Clinical studies and models of trauma-hemorrhage documented estrogen to have protective effects corresponding with enhanced immune function and survival advantage, while testosterone was associated with depressed cell-mediated immune response leading to immunosuppression and more detrimental outcome among men.84–89 In animal studies, these observations were most obvious in the follicular phase of the estrus cycle where estrogen levels are at their maximum.90 In diestrus mice, the protective influence of estrogen on the immune response was not that apparent and outcome was similar to that of male mice.90,91 The same applied to ovariectomied mice.90 In contrast, burn injured female mice showed a doubling of serum estrogen correlating with significantly increased levels of IL-6 and a higher risk of death compared to male mice.80 Supporting the detrimental role of estrogens in mediating postburn immune response, aromatase knockout mice subjected to a 15% TBSA burn showed significantly lower production of IL-6 and partial restoration of immune suppression than wild type mice.92 As already mentioned above, injury response to burns obviously differs remarkably from other nonthermal trauma. Nevertheless, it remains unclear why hormonal effects influence outcome that divergently in those types of injury, since there were also experimental studies which translated protective effects of estrogen to burn victims who simultaneously suffered from distant organ injury (lung, gut, brain injury, and cardiac dysfunction).93–96 The concept of hormonal influence with special regard to sex hormones seems very plausible since it is young women who appear to be most affected. In addition to this, assuming that high levels of estrogen indeed are responsible for the increased mortality in females, it remains unclear why gender-specific differences in survival exist among children—a period of life in which hormonal influences, especially in prepuberty, should

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35.  Women Do Worse Than Men—Gender-Specific Differences in Burn Patients

not be present to such an extent. In fact, it has been demonstrated that the majority of literature could either not detect any gender-related difference in this age group, or it highlighted the fact that boys exhibit an increased risk of dying compared to similarly injured girls.63,69,70,97–99 Furthermore, as the hormonal milieu changes with age, with special regards to females and the time after menopause, the hormonal theory would also explain why women possess an advantage over time and survival differences are not apparent among the elderly generation.25,63,68 Some literature, however, indicated contrary results with a significantly higher proportion of deaths only among women older than 65 years.67 At the same time, these results were not attributed to the influence of hormones but instead to physiologic changes of age such as worsening of microcirculation resulting in delayed wound healing and increased susceptibility to infections.67 This, however, would also affect men and cannot explain gender-related differences among the elderly. In addition, the unfavorable effects of estrogen might not be relevant to these observations since women older than 65 years can be assumed to be in a postmenopausal stage with significantly lower levels of sex hormones. Apparently, hormonal influence cannot explain all the data in current literature since the unfavorable gender dimorphism in burns obviously occurs among both premenopausal and postmenopausal women. Therefore, some age-independent factors such as genetic differences might also contribute to gender-related differences in burns.53,70,100 Finally, it would be imperative to measure circulating sex hormone levels. To the best of our knowledge, we could not find any clinical study which quantified hormone levels in burn patients although sex hormones were often discussed and held responsible for gender-related divergent outcomes. Only in this manner, however, will it be possible to establish a causal relationship and fully evaluate the potential of the protective or detrimental properties of sex hormones in patients following severe burns.

35.6  ALTERNATIVE REASONS FOR GENDER DIMORPHISM IN BURNS Since most obvious explanations such as a greater burn size or the etiology of burn injury (flames vs scalds) have been excluded as the cause of the increased mortality among females in adjusted analyses, it remains possible that anatomical differences might be a factor in gender-related divergent mortality. Body composition, for example, and the proportion of body fat to muscle mass are well-known to be different between men and women and have been discussed to affect genderrelated outcomes following burns.59,61 One might also speculate about the role of specific differences in skin

characteristics in each gender. Anatomically, the subcutaneous adipose tissue and the epidermis are more than 10-fold thicker in women whereas the dermis layer is more prominent in men.101 Also, the vascular supply varies in the different skin layers and is generally lower in the adipose tissue than in others.101 For this reason, women—even though estrogen influences wound healing positively—tend to have longer donor site healing time and are at increased risk of skin graft failure, which leads to longer hospitalization and subsequent complications than men.61,67,101–103 The lack of reasonable explanations for the survival disparities among men and women regardless of age also suggests that nonphysiologic factors might contribute to gender-specific outcome differences following severe thermal injuries. For example, there is evidence that differences in access to trauma care exist in the prehospital setting, favoring men over women despite having similar severities of injury.104 This has been partly related to assumptions about the probability of benefiting from trauma care as well as to subconscious gender bias leading to perceived differences in injury severity.104,105 In addition to this, male patients have been identified to receive more interventions and resources than female patients in critical care.68,105 This observation could amplify the negative effects of estrogen in injury response, which have been demonstrated in the experimental setting. In other words, this imbalance in burn care could provide a reason why especially young women, as a result of both the inequality of resource stratification and hormonal influence, might be more likely to succumb to burn injuries. Ultimately, differences in management and burn care might further explain why gender dimorphism in burn patients persists among the elderly generation, which is not supposed to be as affected by hormonal influences anymore.

35.7 CONCLUSION Although the association between gender and mortality of patients following burn injuries has been repeatedly documented, only little is known about the possible mechanisms and pathways responsible for these genderbased divergent outcomes. Sex hormones might indeed influence immune function and physiological responses. This explanation, however, cannot entirely answer why women are more likely to succumb to burns when compared to similarly injured men; it suggests a multifactorial genesis which might also include genetic polymorphism or even gender-specific differences in burn care. Importantly, gender-related factors are often not included in the clinical setting. This emphasizes the need for more extensive and focused research in this area. In the future, these findings should be incorporated into

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

new therapeutic approaches to further decrease burn mortality and morbidity and their associated significant personal and public health implications.

35.8  SUGGESTIONS FOR FURTHER RESEARCH Do gender-related differences also become important for postburn complications? For example, are women more susceptible to develop sepsis after burns? ● Are there gender-specific causes of death after burn injury? ● Can differences in hormone levels indeed serve as an explanation for the higher mortality of women? ● Does hormone replacement therapy influence the survival of burn victims? ● Does body weight have an impact on genderspecific mortality in burn patients? ●

References 1. American Burn Association. Burn Incidence Fact Sheet. ; 2015 Accessed 13.02.16. 2. Brusselaers N, Monstrey S, Vogelaers D, Hoste E, Blot S. Severe burn injury in Europe: a systematic review of the incidence, etiology, morbidity, and mortality. Crit Care. 2010;14:R188. 3. Miller SF, Bessey P, Lentz CW, et  al. National burn repository 2007 report: a synopsis of the 2007 call for data. J Burn Care Res. 2008;29:862–870. discussion 871. 4. Peck MD. Epidemiology of burns throughout the world. Part I: Distribution and risk factors. Burns. 2011;37:1087–1100. 5. Lawrence JW, Mason ST, Schomer K, Klein MB. Epidemiology and impact of scarring after burn injury: a systematic review of the literature. J Burn Care Res. 2012;33:136–146. 6. Gangemi EN, Gregori D, Berchialla P, et  al. Epidemiology and risk factors for pathologic scarring after burn wounds. Arch Facial Plast Surg. 2008;10:93–102. 7. Wallace AB. The exposure treatment of burns. Lancet. 1951;1:501–504. 8. Kamolz LP, Herndon DN, Jeschke MG. Verbrennungen Diagnose, Therapie und Rehabilitation eines thermischen Traumas. Wien: Springer; 2009. 9. Hyakusoku H, Orgill DP, Téot L, Pribaz JJ, Ogawa R. Color atlas of burn reconstructive surgery. Berlin: Springer; 2010. 10. Thorne CH. Grabb and Smith’s Plastic Surgery, 6th edn. Philadelphia, PA: Lippincott Williams & Wilkins, 2007. 11. Hettiaratchy S, Papini R. Initial management of a major burn: II--assessment and resuscitation. BMJ. 2004;329:101–103. 12. Jackson DM. The diagnosis of the depth of burning. Br J Surg. 1953;40:588–596. 13. Brusselaers N, Hoste EAJ, Monstrey S, et  al. Outcome and changes over time in survival following severe burns from 1985 to 2004. Intensive Care Med. 2005;31:1648–1653. 14. Saffle JR. Predicting outcomes of burns. N Engl J Med. 1998;338:387–388. 15. Choinière M, Dumont M, Papillon J, Garrel DR. Prediction of death in patients with burns. Lancet. 1999;353:2211–2212. 16 Tanaka Y, Shimizu M, Hirabayashi H. Acute physiology, age, and chronic health evaluation (APACHE) III score is an

521

alternative efficient predictor of mortality in burn patients. Burns. 2007;33:316–320. 17. Sheppard NN, Hemington-Gorse S, Shelley OP, Philp B, Dziewulski P. Prognostic scoring systems in burns: a review. Burns. 2011;37:1288–1295. 18. Benmeir P, Sagi A, Greber B, et  al. An analysis of mortality in patients with burns covering 40 per cent BSA or more: a retrospective review covering 24 years (1964-88). Burns. 1991;17(5):402. 19. Bloemsma GC, Dokter J, Boxma H, Oen IMMH. Mortality and causes of death in a burn centre. Burns. 2008;34:1103–1107. 20. Ryan CM, Schoenfeld DA, Thorpe WP, Sheridan RL, Cassem EH, Tompkins RG. Objective estimates of the probability of death from burn injuries. N Engl J Med. 1998;338(6):362. 21. Colohan SM. Predicting prognosis in thermal burns with associated inhalational injury: a systematic review of prognostic factors in adult burn victims. J Burn Care Res. 2010;31:529–539. 22 Baux S. Contribution a l’etude du traitement local des brulures thermigues etendues. These; Paris, 1961. 23. Tobiasen J, Hiebert JM, Edlich RF. The abbreviated burn severity index. Ann Emerg Med. 1982;11:260–262. 24. Forster NA, Zingg M, Haile SR, Künzi W, Giovanoli P, Guggenheim M. 30 years later--does the ABSI need revision? Burns. 2011;37:958–963. 25. McGwin Jr G, George RL, Cross JM, Reiff DA, Chaudry IH, Rue 3rd. LW. Gender differences in mortality following burn injury. Shock. 2002;18:311–315. 26. Global Burden of Disease Study 2013 Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;386:743–800. 27. Eurostat. Health Statistics: Atlas on Mortality in the European Union. Luxembourg: Office for Official Publications of the European Communities; 2009:143–152. 28. Haagsma JA, Graetz N, Bolliger I, et  al. The global burden of injury: incidence, mortality, disability-adjusted life years and time trends from the Global Burden of Disease study 2013. Inj Prev. 2015;22:4–5. 29. World Health Organization WHO. Burns Fact Sheet N°365. ; 2014 Accessed 13.02.16. 30. American College of Surgeons. National Trauma Databank Annual Report. ; 2014 Accessed 20.04.16. 31. World Health Organization WHO. The Global Burden of Disease: 2004 Update. World Health Organ. Geneva. ; 2008 Accessed 28.03.16. 32. Chen S-H, Chen Y-C, Chen T-J, Ma H. Epidemiology of burns in Taiwan: a nationwide report including inpatients and outpatients. Burns. 2014;40:1397–1405. 33. Forjuoh SN. Burns in low- and middle-income countries: a review of available literature on descriptive epidemiology, risk factors, treatment, and prevention. Burns. 2006;32:529–537. 34. Keck M, Lumenta DB, Andel H, Kamolz LP, Frey M. Burn treatment in the elderly. Burns. 2009;35:1071–1079. 35. Tompkins RG. Survival from burns in the new millennium: 70 years’ experience from a single institution. Ann Surg. 2015;261:263–268. 36. Al-Mousawi AM, Mecott-Rivera GA, Jeschke MG, Herndon DN. Burn teams and burn centers: the importance of a comprehensive team approach to burn care. Clin Plast Surg. 2009;36:547–554. 37. Klein MB, Goverman J, Hayden DL, et  al. Benchmarking outcomes in the critically injured burn patient. Ann Surg. 2014;259: 833–841.

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38. Dokter J, Felix M, Krijnen P, et al. Mortality and causes of death of Dutch burn patients during the period 2006-2011. Burns. 2015;41:235–240. 39. Kallinen O, Maisniemi K, Böhling T, Tukiainen E, Koljonen V. Multiple organ failure as a cause of death in patients with severe burns. J Burn Care Res; 2012;33:206–211. 40. Sánchez J-LA, Perepérez SB, Bastida JL, Martínez MM. Costutility analysis applied to the treatment of burn patients in a specialized center. Arch Surg. 2007;142:50–57. discussion 57. 41. Hop MJ, Wijnen BFM, Nieuwenhuis MK, et al. Economic burden of burn injuries in the Netherlands: A 3 months follow-up study. Injury. 2016;47:203–210. 42. George S, Javed M, Hemington-Gorse S, Wilson-Jones N. Epidemiology and financial implications of self-inflicted burns. Burns. 2015;42:196–201. 43. Golshan A, Patel C, Hyder AA. A systematic review of the epidemiology of unintentional burn injuries in South Asia. J Public Health (Oxf). 2013;35:384–396. 44. Sharma NP, Duke JM, Lama BB, et al. Descriptive epidemiology of unintentional burn injuries admitted to a tertiary-level government hospital in Nepal: gender-specific patterns. Asia Pac J Public Health. 2015;27:551–560. 45. Peck MD. Epidemiology of burns throughout the World. Part II: intentional burns in adults. Burns. 2012;38:630–637. 46. Kumar S, Ali W, Verma AK, Pandey A, Rathore S. Epidemiology and mortality of burns in the Lucknow region, India--a 5 year study. Burns. 2013;39:1599–1605. 47. Frink M, Pape H-C, van Griensven M, Krettek C, Chaudry IH, Hildebrand F. Influence of sex and age on mods and cytokines after multiple injuries. Shock. 2007;27:151–156. 48. George RL, McGwin G, Windham ST, et al. Age-related gender differential in outcome after blunt or penetrating trauma. Shock. 2003;19:28–32. 49. Guidry CA, Swenson BR, Davies SW, et al. Sex- and diagnosisdependent differences in mortality and admission cytokine levels among patients admitted for intensive care. Crit Care Med. 2014;42:1110–1120. 50. Haider AH, Crompton JG, Oyetunji T, et al. Females have fewer complications and lower mortality following trauma than similarly injured males: a risk adjusted analysis of adults in the National Trauma Data Bank. Surgery. 2009;146:308–315. 51. Mostafa G, Huynh T, Sing RF, Miles WS, Norton HJ TM. Genderrelated outcomes in trauma. J Trauma. 2002;53:430–434. discussion 434–5. 52. Yang K-C, Zhou M-J, Sperry JL, et al. Significant sex-based outcome differences in severely injured Chinese trauma patients. Shock. 2014;42:11–15. 53. Sethuraman KN, Marcolini EG, McCunn M, Hansoti B, Vaca FE, Napolitano LM. Gender-specific issues in traumatic injury and resuscitation: consensus-based recommendations for future research. Acad Emerg Med. 2014;21:1386–1394. 54. Deitch EA, Livingston DH, Lavery RF, Monaghan SF, Bongu A, Machiedo GW. Hormonally active women tolerate shock-trauma better than do men: a prospective study of over 4000 trauma patients. Ann Surg. 2007;246:447–453. discussion 453–5. 55. De-Souza DA, Manço ARX, Marchesan WG, Greene LJ. Epidemiological data of patients hospitalized with burns and other traumas in some cities in the southeast of Brazil from 1991 to 1997. Burns. 2002;28:107–114. 56. Germann G, Barthold U, Lefering R, Raff T, Hartmann B. The impact of risk factors and pre-existing conditions on the mortality of burn patients and the precision of predictive admissionscoring systems. Burns. 1997;23:195–203. 57. Vico P, Papillon J. Factors involved in burn mortality: a multivariate statistical approach based on discriminant analysis. Burns. 1992;18:212–215.

58. Sharma PN, Bang RL, Ghoneim IE, Bang S, Sharma P, Ebrahim MK. Predicting factors influencing the fatal outcome of burns in Kuwait. Burns. 2005;31:188–192. 59. Muller MJ, Pegg SP, Rule MR. Determinants of death following burn injury. Br J Surg. 2001;88:583–587. 60. Moore EC, Pilcher D, Bailey M, Cleland H. Women are more than twice as likely to die from burns as men in Australia and New Zealand: an unexpected finding of the Burns Evaluation And Mortality (BEAM) Study. J Crit Care. 2014;29:594–598. 61. Moore EC, Pilcher DV, Bailey MJ, Stephens H, Cleland H. The Burns Evaluation and Mortality Study (BEAMS): predicting deaths in Australian and New Zealand burn patients admitted to intensive care with burns. J Trauma Acute Care Surg. 2013;75:298–303. 62. Fazeli S, Karami-Matin R, Kakaei N, Pourghorban S, SafariFaramani R, Safari-Faramani B. Predictive factors of mortality in burn patients. Trauma Mon. 2014;19:e14480. 63. O’Keefe GE, Hunt JL, Purdue GF. An evaluation of risk factors for mortality after burn trauma and the identification of gender-dependent differences in outcomes. J Am Coll Surg. 2001;192:153–160. 64. George RL, McGwin G, Schwacha MG, et  al. The association between sex and mortality among burn patients as modified by age. J Burn Care Rehabil. 2005;26:416–421. 65. Kerby JD, McGwin Jr G, George RL, Cross JA, Chaudry IH, Rue 3rd. LW. Sex differences in mortality after burn injury: results of analysis of the National Burn Repository of the American Burn Association. J Burn Care Res. 2006;27:452–456. 66. Summers JI, Ziembicki JA, Corcos AC, Peitzman AB, Billiar TR, Sperry JL. Characterization of sex dimorphism following severe thermal injury. J Burn Care Res. 2014;35:484–490. 67. Pereira CT, Barrow RE, Sterns AM, et  al. Age-dependent differences in survival after severe burns: a unicentric review of 1,674 patients and 179 autopsies over 15 years. J Am Coll Surg. 2006;202:536–548. 68. Chang EJ, Edelman LS, Morris SE, Saffle JR. Gender influences on burn outcomes in the elderly. Burns. 2005;31:31–35. 69. Jeschke MG, Mlcak RP, Finnerty CC, et  al. Gender differences in pediatric burn patients: does it make a difference? Ann Surg. 2008;248:126–136. 70. Steinvall I, Fredrikson M, Bak Z, Sjoberg F. Mortality after thermal injury: no sex-related difference. J Trauma. 2011;70: 959–964. 71. Kobayashi K, Ikeda H, Higuchi R, et  al. Epidemiological and outcome characteristics of major burns in Tokyo. Burns. 2005;31:S3–11. 72. Barret JP, Herndon DN. Modulation of inflammatory and catabolic responses in severely burned children by early burn wound excision in the first 24 hours. Arch Surg. 2003;138:127–132. 73. Duke JM, Rea S, Boyd JH, Randall SM, Wood FM. Mortality after burn injury in children: a 33-year population-based study. Pediatrics. 2015;135:e903–e910. 74. Jeschke MG, Gauglitz GG, Kulp GA, et al. Long-term persistance of the pathophysiologic response to severe burn injury. PLoS One. 2011;6:e21245. 75. Mace JE, Park MS, Mora AG, et  al. Differential expression of the immunoinflammatory response in trauma patients: burn vs. non-burn. Burns. 2012;38:599–606. 76. Drost AC, Burleson DG, Cioffi WG, Jordan BS, Mason AD, Pruitt BA. Plasma cytokines following thermal injury and their relationship with patient mortality, burn size, and time postburn. J Trauma. 1993;35:335–339. 77. Gregory MS, Faunce DE, Duffner LA, Kovacs EJ. Gender difference in cell-mediated immunity after thermal injury is mediated, in part, by elevated levels of interleukin-6. J Leukoc Biol. 2000;67:319–326.

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REFERENCES

78. Schwacha MG, Chaudry IH. The cellular basis of post-burn immunosuppression: macrophages and mediators. Int J Mol Med. 2002;10:239–243. 79. Plackett TP, Gamelli RL, Kovacs EJ. Gender-based differences in cytokine production after burn injury: a role of interleukin-6. J Am Coll Surg. 2010;210:73–78. 80. Gregory MS, Duffner LA, Faunce DE, Kovacs EJ. Estrogen mediates the sex difference in post-burn immunosuppression. J Endocrinol. 2000;164:129–138. 81. Croce MA, Fabian TC, Malhotra AK, Bee TK, Miller PR. Does gender difference influence outcome? J Trauma. 2002;53:889–894. 82. Verthelyi D. Sex hormones as immunomodulators in health and disease. Int Immunopharmacol. 2001;1:983–993. 83. Angele MK, Schwacha MG, Ayala A, Chaudry IH. Effect of gender and sex hormones on immune responses following shock. Shock. 2000;14:81–90. 84. Sperry JL, Minei JP. Gender dimorphism following injury: making the connection from bench to bedside. J Leukoc Biol. 2008;83:499–506. 85. Kawasaki T, Chaudry IH. The effects of estrogen on various organs: therapeutic approach for sepsis, trauma, and reperfusion injury. Part 2: liver, intestine, spleen, and kidney. J Anesth. 2012;26:892–899. 86. Kawasaki T, Chaudry IH. The effects of estrogen on various organs: therapeutic approach for sepsis, trauma, and reperfusion injury. Part 1: central nervous system, lung, and heart. J Anesth. 2012;26:883–891. 87. Angele MK, Schneider CP, Chaudry IH. Bench-to-bedside review: latest results in hemorrhagic shock. Crit Care. 2008;12:218. 88. Sheth SU, Palange D, Xu D-Z, et  al. Testosterone depletion or blockade in male rats protects against trauma hemorrhagic shock-induced distant organ injury by limiting gut injury and subsequent production of biologically active mesenteric lymph. J Trauma. 2011;71:1652–1658. 89. Knöferl MW, Diodato MD, Angele MK, et  al. Do female sex steroids adversely or beneficially affect the depressed immune responses in males after trauma-hemorrhage? Arch Surg. 2000;135:425–433. 90. Knöferl MW, Jarrar D, Angele MK, et al. 17 beta-Estradiol normalizes immune responses in ovariectomized females after traumahemorrhage. Am J Physiol Cell Physiol. 2001;281:C1131–C1138. 91. Zellweger R, Wichmann MW, Ayala A, Stein S, DeMaso CM, Chaudry IH. Females in proestrus state maintain splenic immune functions and tolerate sepsis better than males. Crit Care Med. 1997;25:106–110.

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92. Plackett TP, Oz OK, Simpson ER, Kovacs EJ. Lack of aromatase improves cell-mediated immune response after burn. Burns. 2006;32:577–582. 93. Ananthakrishnan P, Cohen DB, Xu DZ, Lu Q, Feketeova E, Deitch EA. Sex hormones modulate distant organ injury in both a trauma/hemorrhagic shock model and a burn model. Surgery. 2005;137:56–65. 94. Ozveri ES, Bozkurt A, Haklar G, et  al. Estrogens ameliorate remote organ inflammation induced by burn injury in rats. Inflamm Res. 2001;50:585–591. 95. Gatson JW, Maass DL, Simpkins JW, Idris AH, Minei JP, Wigginton JG. Estrogen treatment following severe burn injury reduces brain inflammation and apoptotic signaling. J Neuroinflammation. 2009;6:30. 96 Yao X, Wigginton JG, Maass DL, et al. Estrogen-provided cardiac protection following burn trauma is mediated through a reduction in mitochondria-derived DAMPs. Am J Physiol Heart Circ Physiol. 2014;306:H882–H894. 97. Barrow RE, Herndon DN. Incidence of mortality in boys and girls after severe thermal burns. Surg Gynecol Obstet. 1990;170:295–298. 98. Barrow RE, Przkora R, Hawkins HK, Barrow LN, Jeschke MG, Herndon DN. Mortality related to gender, age, sepsis, and ethnicity in severely burned children. Shock. 2005;23:485–487. 99. Karimi H, Motevalian SA, Momeni M, Safari R, Ghadarjani M. Etiology, Outcome and Mortality Risk Factors in Children Burn. Surg Sci. 2015;6:42–49. 100. Sperry JL, Nathens AB, Frankel HL, et al. Characterization of the gender dimorphism after injury and hemorrhagic shock: are hormonal differences responsible? Crit Care Med. 2008;36:1838–1845. 101. Mohammadi AA, Pakyari MR, Seyed Jafari SM, et  al. Effect of burn sites (upper and lower body parts) and gender on extensive burns’ mortality. Iran J Med Sci. 2015;40:166–169. 102. Gilliver SC, Ashworth JJ, Ashcroft GS. The hormonal regulation of cutaneous wound healing. Clin Dermatol. 2007;25:56–62. 103. Thornton MJ. The biological actions of estrogens on skin. Exp Dermatol. 2002;11:487–502. 104. Gomez D, Haas B, de Mestral C, et  al. Gender-associated differences in access to trauma center care: A population-based analysis. Surgery. 2012;152:179–185. 105. Samuelsson C, Sjöberg F, Karlström G, Nolin T, Walther SM. Gender differences in outcome and use of resources do exist in Swedish intensive care, but to no advantage for women of premenopausal age. Crit Care. 2015;19:129.

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C H A P T E R

36 Exercise Physiology in Men and Women Anne-Marie Lundsgaard*, Andreas M. Fritzen* and Bente Kiens University of Copenhagen, Copenhagen, Denmark

O U T L I N E 36.1 Introduction

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36.2 Body Composition

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36.3 Cardiovascular Differences and Maximal Oxygen Uptake

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36.4 Muscle Fiber Type Composition

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36.5 Anaerobic and Aerobic Exercise 528 36.5.1 Anaerobic Potential 528 36.5.2 Aerobic Carbohydrate and Lipid Oxidation During Exercise 529 36.6 Substrate Metabolism During Exercise 530 36.6.1 Amino Acid Oxidation During Exercise 530 36.6.2 Lipid Energy Sources Utilized During Exercise 530 36.6.3 Glucose Metabolism 533 36.6.4 Glycogen Stores and Glycolytic Capacity 533 36.7 ATP Resynthesizes in Skeletal Muscle

36.8 Estrogen and its Impact on Metabolism

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36.9 Gender Differences in Metabolism During Recovery From Exercise

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36.10 Nutritional Implications in Relation to Exercise 536 36.10.1 Energy Availability in Athletes 536 36.10.2 Dietary Macronutrient Composition 537 36.11 Concluding Highlights

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References 538

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36.1 INTRODUCTION Women and men exhibit many gender-specific anthropometric and physiologic characteristics, which may impact the response when the female or male body is subjected to increased metabolic stress in response to physical activity. In terms of aerobic exercise performance, it has been proposed that women may perform similarly or slightly *Funded by the Danish Diabetes Academy, supported by the Novo Nordisk Foundation Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00017-6

36.7.1 Mitochondrial Fatty Acid Transport and Beta-Oxidation 534 36.7.2 Tricarboxylic Acid Cycle and Oxidative Phosphorylation 534 36.7.3 Finetuning of Acetyl-coA Input to the Tricarboxylic Acid Cycle 535

better during long-term endurance exercise than men. Hence, it has been reported that the gender difference in running speed disappears at long running distances,1 and hence women have a reduced completion time and higher mean relative intensity during 90 km running, when men and women of comparable 42.2 km running performance were compared.2 Furthermore, when data from 14 US marathons were obtained from ~92,000 men and women, there was a greater slowing in the running pace of men than women during the second half of the marathon.3 These observations indicate an inherent gender difference in adaptations to endurance exercise.

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The area of substrate utilization and muscle metabolism during exercise has been the subject of genderspecific research in particular, and an increasing body of evidence suggests that there is a distinct gender dimorphism in the metabolic properties of skeletal muscle. Notably, when the human vastus lateralis muscle was subjected to microarray analysis, gender was reported to have a stronger influence on metabolic gene expression than age and even training status.4 This further suggests that the metabolic responses to exercise vary in women and men. Sex differences can be ascribed to both sex chromosome and sex hormone exposure, and it is clear that gender-specific features of exercise physiology result from both of these factors. Investigating primary gender differences in exercise physiology and substrate metabolism is difficult, as confounding variables like adiposity, fat distribution, hormonal fluctuations, and aerobic fitness level might complicate interpretations. Thus, proper matching of men and women is crucial to determine the effect of gender per se. The available evidence presented in this chapter is derived mainly from studies in healthy men and women, including untrained as well as already trained subjects. As the menopausal transition is proposed to have an impact on metabolism, conclusions are derived from studies in premenopausal women and age-matched men, while the effect of menopause on exercise physiology is covered in a separate section.

36.2  BODY COMPOSITION There is an obvious gender difference in muscle mass and adiposity. On average, women have around twothirds of the skeletal muscle mass of their male counterparts, as measured by MRI-scanning in ~470 men and women, and the gender difference in muscle mass seems to be greater in the upper body than the lower body.5 Notably, the gender difference in muscle mass remains after adjusting for body weight and height. This observation implies that women have a greater body fat mass than men. Actually, the body fat percentage for normalweight women is similar to that of men classified obese.6 The gender difference in fat mass is present already at birth7 and becomes more marked during puberty.8 Furthermore, varying within different age groups, a 6–12% higher body fat was observed in women, when a large cohort of 16,000 12–80 years old men and women was analyzed by bioelectrical impedance.9 Importantly, body fat is also distributed differently in men and women. Men have a higher amount of intra-abdominal (visceral) adipose tissue, whereas women have more subcutaneous fat, particularly in the gluteo-femoral region, as measured by computed

tomography (CT) scanning and magnetic resonance imaging (MRI).10,11 This sex difference in fat distribution, known as the android and gynoid distribution pattern, becomes prominent during puberty and ceases after menopause and has therefore been suggested to be sex-hormone dependent. It should be kept in mind though that despite variation within each gender, the subcutaneous fat depot comprises the majority of the total body fat, corresponding to ~80%. It is possible that an increased anabolic response to exercise may contribute to the observation of greater muscle mass in men than women. In the literature, it is apparent that men and women experience similar relative strength gains to resistance-type exercise training, but there appears to be less muscle hypertrophy with strength improvement in women when compared to men. Several studies have compared the rate of muscle protein synthesis in men and women by using a primed, constant labeled amino acid infusion technique to calculate the fractional turnover rate of muscle, by measuring the incorporation of tracer into muscle protein. Gender differences in protein turnover can then be investigated in both the postabsorptive basal state and in the response to anabolic stimuli, such as exercise. In the basal state, there does not appear to be a difference between men and women in muscle protein fractional synthesis rate.12–14 Only a few studies have evaluated basal muscle protein breakdown rate, and they reported that it is also similar between men and women.13,15 Thus, there does not seem to be any detectable gender difference in basal protein turnover in skeletal muscle. This is evident when expressed per unit of muscle mass, suggesting that the intracellular protein turnover is the same. However, it is obvious that total protein synthesis is higher in men on a whole body level, due to a greater total lean body mass (LBM). Thus when men and women ingested a standard diet with 0.86 g protein/kg body mass (BM)/day and nitrogen balance was assessed over 3 days, total protein turnover was greater in men than women.16 In response to resistance exercise, muscle protein synthesis rate has been investigated by infusion of a phenylalanine tracer during recovery in the fed state, and the increase in protein synthesis was reported to be similar between men and women, despite a 45-fold greater exercise-induced increase in testosterone during exercise in men.17 A similar increase in exercise-induced protein synthesis is also confirmed by another study showing that postexercise muscle protein synthesis rate increased similarly in men and women after resistance exercise, together with a similar increase in the mammalian target of rapamycin (mTOR) and p70S6 kinase (S6K1) phosphorylation,12 indicating that anabolic signaling in skeletal muscle was not subject to gender differences. Thus, the obvious gender difference in muscle

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36.3 Cardiovascular Differences and Maximal Oxygen Uptake

mass does not seem to result from differences in protein turnover rates in the period following exercise. Rather, there is a genetic influence, which indeed may involve the male-specific Y chromosome, though it is difficult to separate direct genetic effects from hormonal effects in adult humans. Therefore, differences in the hormonal milieu (i.e., testosterone levels) are hypothesized to be important for the regulation of a greater total muscle mass in men, besides the influence of physical activity patterns in men and women that over time may contribute to gender differences in lean mass.

36.3  CARDIOVASCULAR DIFFERENCES AND MAXIMAL OXYGEN UPTAKE During exercise, several physiological adjustments are made by the cardiovascular system to supply skeletal muscle with oxygen and energy substrates. The components of the cardiovascular system have several sexually dimorphic characteristics. The total blood volume is ~70 mL/kg BM in adult women and thus a little lower than in adult men, who have ~80 mL/kg BM. Exercise training is followed by an increase in blood volume of up to 20–25% compared to sedentary subjects,18 an adaptation which is evident in both genders. During the initial weeks of training there is an expansion of plasma volume, after which the greater blood volume is accounted for by an equal increase in plasma and red cell volume. In general, the greater body size of men is associated with a larger heart. Therefore, when comparing the hearts of men and women, investigators have tried to correct the measurements for body surface area. The majority of studies have observed a greater left ventricular mass in men than women, and a higher resultant stroke volume at rest in men than women.19 The stroke volume at rest is ~55 mL and ~65 mL in sedentary women and men, respectively. Some, but not all, studies report that resting heart rate is similar in women and men, with some studies showing that resting heart rate is higher in women. The inconsistent observations in regard to heart rate might be related to improper control of physical activity level, differences in emotional arousal, and subject age, which make accurate conclusions in this area rather difficult to obtain. As the cardiac output is given by the product of stroke volume and the heart rate, it follows that cardiac output is lower in women than men, with resting values of ~3.5 and ~5–6 L/min, respectively. This difference applies for both sedentary and active individuals. During dynamic exercise, cardiac output increases in direct proportion to the increase in oxygen uptake. The maximal heart rate is reported to be similar20 or slightly higher in men than women.21,22 The maximal stroke volume increases with

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exercise training in both women and men, but is lower in women than men at all exercise intensities.20 A lower maximal stroke volume combined with a similar or slightly lower maximal heart rate in women implies that maximal cardiac output is lower in women than men. In addition to this, women have a lower blood volume than men and a ~12% lower hemoglobin concentration.23 This further implies that the oxygen binding capacity is lower per unit of blood volume in women. The maximal oxygen uptake is determined by the maximal cardiac output and the maximal oxygen extraction by the tissues. The capacity for oxygen extraction in skeletal muscle will be dependent on microvascular blood flow in skeletal muscle and the ability to extract oxygen from the capillaries. The maximal oxygen uptake in L/min (hereafter referred to as VO2peak) is used as an assessment of physical fitness level. A ~30–40% higher VO2peak is observed in men compared with equally trained women, with reports of VO2peak values ~6 L/ min in elite male endurance athletes. The higher VO2peak in men is primarily related to their larger O2 transport organs, given the greater body size of men. Accordingly, when expressed per kg BM, VO2peak is ~10–20% lower in women than men.24 The persisting difference is mainly due to the lower muscle mass and also lower hemoglobin concentration, and hence hematocrit values, in women compared to men. Hence, a more proper way to express maximal oxygen uptake when comparing men and women is in mL/kg LBM/min. Thus, when maximal oxygen uptake is expressed relative to LBM and women and men are carefully matched in regard to training status and activity level, the gender difference in VO2peak becomes small and nonsignificant.25 Interestingly, most of the evidence on gender differences in the cardiovascular response has been documented at rest and during maximal exercise. However, in a recent study trained men and women were studied during submaximal exercise at 40% and 75% of peak workload.26 During the same relative workloads (and corrected for differences in BM) women demonstrated lower cardiac output as a result of lower stroke volume, as heart rate was almost similar between genders. When corrected for wattage (as exercise intensities were set at the same % of peak workload), cardiac output, heart rate, and stroke volume were higher in women than men, indicating that greater cardiac work is needed in women to meet the same physical work demand. Interestingly, as a compensatory mechanism for the lower stroke volume and hence cardiac performance, peripheral oxygen extraction (i.e., arteria-venous O2 difference) was higher in women. It has been suggested that the lower stroke volume in women during submaximal exercise could be the result of a blunted sympathetic response and a higher basal vasodilation, evidenced by lower catecholamine levels in women during submaximal exercise.26,27

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36.  Exercise Physiology in Men and Women

Taken together, the observation of lower VO2peak values in women than men seems to be related to a smaller ventricular mass and thereby stroke volume. This will in turn lead to a lower peak cardiac output during maximal exercise in women. This is coupled with lower blood volume and hemoglobin concentration, which will reduce the oxygen binding capacity. In combination with a higher ratio of fat to muscle mass, this may diminish women's ability to extract oxygen. However, this might to some extent be accounted for by different mechanisms in terms of peripheral oxygen extraction in skeletal muscle.

36.4  MUSCLE FIBER TYPE COMPOSITION Skeletal muscle in humans comprises three major fiber types: type I, IIA, and IIX. The morphology and enzymatic properties of the specific muscle fiber types depend on their myosin heavy chain (MHC) expression. The relative proportion of type I, IIA, and IIX fibers will thereby affect the total muscular capacity for oxidative versus glycolytic energy turnover and substrate storage. Gender differences in the morphology of skeletal muscle have mainly been investigated in the vastus lateralis muscle by the needle biopsy technique. The most extensively used technique is histochemical staining of a cross-section of the muscle biopsy by use of myosin adenosine-triphosphatase (ATP-ase) staining, by which type I, IIA, and IIX can be differentiated in human muscle. The technique also enables determination of the cross-sectional area (CSA) of each muscle fiber type, and hence its relative contribution to the total muscle area. Using this technique, gender differences in muscle morphology have been well documented. An early study, in which the fiber type distribution was investigated in m. biceps brachii and m. vastus lateralis, showed a higher type I/II fiber ratio in women than men.28 They also detected a larger size of type II fibers in men. Later, a higher number of type I muscle fibers was consistently observed in the vastus lateralis muscle in women compared to matched men. When expressed relative to area, the proportion of type I fibers has been described to be 27–35% greater in women, while the proportion of type IIA,29,30 or both IIA and IIX is reported to be greater in men.25 Hence, a greater muscle area is covered by type I fibers in women of untrained, moderately-, and endurance trained matched men and women.25 Furthermore, a larger individual CSA of type IIA,29 and both type IIA and IIX fibers,30 has been observed in men. Others have confirmed these observations of a greater size of type II fibers in men compared to women and hence a greater ratio of type II to I fibers in men, also using myofibrillar ATP-ase staining.31–34 Notably, these immunohistochemically findings are also reflected at the transcriptional

level of the MHC, as MHCI mRNA content is reported to be lower in the vastus lateralis muscle of men than women,35 while MHCIIA and -IIX mRNA levels are higher in men.36 It could be questioned whether the greater proportion of type II fibers in men makes them able to generate more tension during maximal contractions compared to women. At the whole muscle level men are able to generate a greater absolute force than women, but when maximal voluntary concentric strength is related to muscle CSA, there is no significant difference between genders, as reported for elbow flexion, knee extension, and knee flexion.33,37 This suggests that total muscle area, rather than muscle fiber composition, is primary for the greater maximal strength observed in men. Of note, the total muscle fiber number was estimated from biopsies and shown to be similar between genders in these studies, despite men having greater total muscle areas than women. This suggests that the greater absolute force production in men is due to greater fiber sizes. When single fibers were dissected from male and female skeletal muscle, and electrically stimulated to contract, the maximal active tension was greater in both type I and II fibers from male muscle, a gender difference that was eliminated when expressed per fiber CSA.38 The number of capillaries surrounding each muscle fiber is found to be similar in men and women, but due to a lower total amount of type II fibers and a smaller individual area of these, a greater capillary density per given muscle area is observed in women.29,30 This may have implications for the nutritive flow to the muscle fibers, i.e., oxygen and substrate delivery, and the implications of this will be discussed in the context of substrate utilization in Section 36.5.2.2.

36.5  ANAEROBIC AND AEROBIC EXERCISE During exercise, ATP is continuously degraded to ADP in skeletal muscle. The ensuing ATP resynthesis is generated anaerobically from glucose and glycogen and aerobically by the oxidation of lipids and carbohydrates.

36.5.1  Anaerobic Potential It has been documented that there is a greater anaerobic capacity in men than women during maximal intensity exercise, both when absolute and BM corrected estimates of anaerobic capacity are applied. The 30 s sprint test on a bicycle (the so-called Wingate test or modifications thereof) is designed to estimate anaerobic power and capacity. It has been reported that during a single 30 s sprint, there is a higher proportion of ATP regeneration via anaerobic metabolism in

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36.5  Anaerobic and Aerobic Exercise

men than women, suggesting a higher anaerobic component in men during maximal exercise.39 Furthermore, a greater anaerobic potential in men has been evidenced by greater postexercise disturbances of the acid–base balance and a greater increase in blood lactate concentrations in the blood of men than women during maximal exercise tests.40 In another observation from an incremental cycling maximal exercise test, the lactate threshold was similar between genders, but after this was reached women accumulated plasma lactate at a slower rate than men, with men having greater plasma lactate concentrations at exhaustion.41 These findings are further supported by observations of a lower increase in blood lactate accumulation in women than men after a single42 or several 30 s sprints on a cycle ergometer.43 The greater lactate accumulation in the circulation of men during intense exercise may reflect a greater glycolytic activity in muscle. Interestingly, the anaerobic performance during 3 × 30 s Wingate tests has been described to be highly correlated with the proportion of type II fibers and the activity of the enzyme phosphofructokinase (PFK) in both men and women.44 This goes well along with the greater proportion of type II fibers in men, and reports of increased capacity of the glycolytic enzymes in male muscle, which will be discussed in a subsequent paragraph.

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During exercise carbohydrate and lipids are the main energy sources. The respective contribution of lipid versus carbohydrates to fuel energy consumption can be measured by indirect calorimetry, which reflects whole-body substrate oxidation. Skeletal muscle substrate utilization can also be estimated more specifically by calculation of leg respiratory quotient (RQ), which reflects the arterialvenous O2 and CO2 exchange in blood obtained from an artery and vein across the muscle tissue. In practice, this method is difficult and requires proper handling of the blood samples. However, during steady-state whole-body exercise, it has been shown that the respiratory exchange ratio (RER) reflects substrate utilization well in skeletal muscle. Therefore, RER measurements are usually used for determination of substrate utilization during exercise.

the subjects prior to an exercise bout greatly influences substrate utilization during exercise, which is why it is important to standardize the diet before the test. A final factor to consider when comparing the metabolism between women and men during exercise is the possible effect of the female sex hormones on substrate utilization during exercise. It has become clear that 17-β estradiol (hereafter referred to as estrogen) has a wide spectrum of actions and that estrogen is implicated in the regulation of metabolism in skeletal muscle. In premenopausal women, the levels of female sex hormones undergo profound fluctuations during each menstrual cycle phase, with plasma estrogen concentrations varying from 10 to 300 pg/mL. In the early follicular phase, estrogen and progesterone concentrations in the blood are at their lowest, while estrogen concentration is peaking at the end of the follicular phase. Despite these changes in circulating estrogen, differences have not been reported for resting whole-body metabolic rate and RER,45 fasting plasma glucose and insulin,46 or fatty acid (FA) concentrations47 when the follicular and luteal phase are compared. This suggests that substrate metabolism at rest is not significantly affected by the menstrual cycle. There are conflicting findings with regard to substrate metabolism during aerobic exercise, concerning whether the time point in the menstrual cycle has an impact on substrate oxidation during exercise. Several studies do not find any change in substrate oxidation during exercise throughout the menstrual cycle,46,48,49 while others have suggested that women have higher lipid utilization in the luteal phase compared to the follicular phase.50–53 Thus, it is difficult to conclude whether there is an actual effect of menstrual cycle phase on substrate choice during exercise, which may be related to improper dietary control in the studies and differences in training status. In many gender-comparative studies, it has become the norm that women are subjected to experiments in the follicular phase (day 7–11), due to the lower levels of circulating female sex hormones during this period compared with the luteal phase. This minimizes the differences in the level of sex hormones between genders. However, there are also many gender-comparative studies, which do not consider menstrual cycle phase, and furthermore do not control for habitual activity level or diet.

36.5.2.1  Matching of Women and Men for Comparison of Substrate Utilization During Exercise When comparing the substrate utilization during exercise between women and men it is important to match the genders in accordance with cardiorespiratory fitness and training history. In addition, the workload during the exercise bout should be matched according to VO2peak relative to LBM. The dietary status of

36.5.2.2  Respiratory Exchange Ratio During Exercise Several studies, but not all, have shown that the relative fat oxidation during exercise is higher in women than in men. However, to do proper gender-comparative studies during exercise, the above mentioned requirements for matching of men and women must be considered. All these criteria were indeed fulfilled in study by Roepstorff et al.27 First, women and men did not differ

36.5.2  Aerobic Carbohydrate and Lipid Oxidation During Exercise

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in VO2peak per kg LBM, training history, and physical activity level, suggesting that women and men had similar training status. Also, the relative exercise intensity during 90 min of cycling exercise did not differ between women and men (60% of VO2peak), heart rate was identical, and so was the fiber type recruitment pattern during the exercise bout. In this well controlled study, fat oxidation was significantly higher during the submaximal exercise bout in women than in the matched men.29 In addition, when calorimetric data from 25 studies comparing substrate oxidation in men and women during endurance exercise (>60 min) were summarized in a systematic review, mean RER data indicate a greater relative fat oxidation in women compared to men (RER 0.87 vs 0.90).54 Lower RER values in women than men are observed for both untrained and trained subjects, and are maintained when untrained women and men complete a similar training regimen. In general, the relative fat utilization during exercise depends on exercise intensity. From RER measurements, the fat oxidation rate can be calculated from O2 and CO2 by use of stoichiometric calculations.55 During submaximal exercise, the absolute FA oxidation rate increases from low to moderate exercise intensities, whereafter it declines with increasing exercise intensities, thereby forming a bell-like curve. The top of the curve represents the maximal rate of fat oxidation and has been referred to as FATmax.56 In a large number of subjects (300 men and women) it was demonstrated by indirect calorimetry that the maximal fat oxidation rate was higher in women (8.3 ± 0.2 mg/kg fat free mass [FFM] ∙ per min) than in men (7.4 ± 0.2 mg/kg FFM∙per min) during submaximal incremental exercise tests on a treadmill. The intensity eliciting the maximal fat oxidation rate was 52% in women versus 45% of VO2peak in men, and hence higher in women.57 The findings indicate that men have an earlier shift in using carbohydrate as the predominant fuel at increasing exercise intensity. A later study, applying indirect calorimetry during submaximal incremental test on cycle ergometer, has confirmed that the intensity that elicits the maximal fat oxidation rate is higher in women than men (58% vs 50% of VO2peak).58 Thus the curve seems to be right-shifted in women. The higher fat oxidation in women than in men during submaximal exercise might be due to a better maintenance of cellular energy balance in skeletal muscle by women. Support for this notion are the findings of an increase in the AMP/ATP ratio in men, but not in women, during prolonged submaximal exercise at the same relative workload29 and a smaller ATP reduction in women than in men during repeated bouts of high intensity exercise.43 A better maintenance of muscle cellular energy balance during exercise in women than in men appears to be due to the sex-specific muscle morphology. As previously described, the proportion of the oxidative

type I muscle fibers is higher in women than in men, women have a smaller muscle fiber CSA, in particular of type II fibers, and women have a higher capillary density compared with men. Together, these intrinsic factors of female skeletal muscle favor an increased potential for improved oxidative substrate utilization, and thus the potential for enhanced FA oxidation. That the higher fat oxidation during exercise in women appears to be due to gender-specific muscle morphology is supported by the findings of a significant correlation between fat oxidation, the proportion of type I fibers, and capillary density.29

36.6  SUBSTRATE METABOLISM DURING EXERCISE 36.6.1  Amino Acid Oxidation During Exercise The relative contribution of protein to the energy delivery during aerobic exercise is small, and therefore often not considered when nonprotein RER values are calculated from gaseous exchange. Estimation of protein utilization is possible using amino acid tracers, and it has been estimated that protein oxidation comprises around 1–5% of total aerobic exercise energy expenditure, but will increase during prolonged exercise or if muscle glycogen stores are low before exercise.59 During exercise, the branched chain amino acids leucine, isoleucine, and valine are preferentially oxidized in skeletal muscle.60 As indirect evidence for amino acid oxidation during exercise, it has been shown that urinary urea excretion is greater in men than women on a day with exercise (15.1 km run) under conditions of a controlled diet.61 This observation of a lower increase in urea excretion in women than men in response to exercise has later been verified by several studies. When amino acid oxidation was directly measured by use of stable isotopes, leucine oxidation during submaximal endurance exercise was described to be ~70% greater in endurance trained men than women,16 and this higher leucine oxidation in men has also been confirmed by other research groups studying endurance exercise (50–60% of VO2peak) for 60–90 min.62 Thus, it is a consistent finding that men oxidize a greater amount of amino acids and lower amounts of lipids during exercise compared to women.

36.6.2  Lipid Energy Sources Utilized During Exercise Lipids utilized during exercise originate from three different sources: FA liberated from adipose tissue, FA liberated from hydrolysis of circulating triacylglycerol (TG), and FA liberated from intramyocellular triacylglycerol (IMTG), and it appears that there are gender differences in the utilization of these energy sources.

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36.6.2.1  Increased Sensitivity to Lipolytic stimuli in Female Adipose Tissue The concentration of plasma FA is of importance for lipid availability to skeletal muscle. Investigators have determined that women have a higher postprandial plasma FA concentration than men, which may be consistent with their larger relative fat mass. Indeed, in a large systematic review including 43 studies with reports of overnight-fasted plasma FA concentrations it was concluded that plasma FA concentration was higher in women than men (median 517  μ mol/L in women vs 434  μ mol/Lin men).63 This gender difference was confirmed when plasma FA kinetics were further investigated by applying 2.2-2H2-palmitate or U-13C-palmitate tracers, as a higher basal FA rate of appearance (Ra) was shown in women compared to men.64–66 Together, these findings demonstrate that women have higher fasting FA concentrations at rest, and thereby a higher FA availability per unit of their LBM. Exercise induces an increase in circulating catecholamines, which among other things stimulates lipolysis in adipose tissue through β1-, β2-, and β3-adrenoceptors and thereby enhances the plasma FA concentration to accommodate the increased FA use by the exercising muscles. When exercise was performed at the same relative intensity, it has been shown that men have higher circulating epinephrine and norepinephrine concentrations than women, irrespective of training status.27,67,68 Notably, women exhibit a higher lipolytic sensitivity to catecholamine as shown by a higher increase in plasma glycerol concentration than in men when infused to a similar plasma concentration of epinephrine and norepinephrine.69 This is likely related to a greater stimulation of lipolysis in subcutaneous adipose tissue, as the contribution of visceral adipose tissue on measures of whole body lipolysis is relatively small. In this context, it has been shown in vitro that epinephrine stimulates lipolysis in subcutaneous adipocytes from men to a lower extent than in those from women.70 In female adipocytes, a greater β-adrenergic efficiency was coupled with lower α2-adrenergic receptor activation (antilipolytic role). Thus, the greater sensitivity to lipolytic stimuli in women is reported to be partly related to lower adipose tissue antilipolytic α2 activation in women than men in response to epinephrine, which has recently been confirmed in an in vivo study.71 Despite lower epinephrine concentrations during exercise in women than in men, higher plasma concentrations of glycerol and Ra of glycerol have been observed in women compared with men during the same relative exercise intensity. This has been observed in untrained and moderately trained subjects during acute 60–90  min of submaximal exercise and after 7 weeks of endurance training.27,68,72 Importantly it

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should be noted that indices of greater lipolysis in women than men are still observed when absolute body fat mass is matched in women and men.73 The higher plasma glycerol concentration observed in untrained and moderately trained individuals was also followed by a higher arterial plasma FA concentration in females than in males when submaximal exercise was performed.72 On the other hand, in endurance trained women and men subjected to 90 min submaximal exercise at the same relative workload, the arterial glycerol concentration was similar between genders, as was the arterial plasma FA concentration and rate of appearance of FA expressed relative to LBM.74 The discrepancy between these findings appears attributable to differences in training status of the subjects. Notably, when uptake and oxidation of plasma FA was measured across the exercising leg in endurance trained individuals, no gender differences in the uptake and oxidation of plasma FA were obtained when exercise was performed at low (25% of VO2peak), moderate (60–65% of VO2peak), or high intensities (85% of VO2peak).74,75 Hence, the uptake of plasma FA during exercise does not appear to be greater in endurance trained women, despite a greater stimulation of lipolysis. 36.6.2.2  Intramyocellular Triacylglycerol Utilization The FA taken up into skeletal muscle can be esterified with glycerol to form triglycerides (TG), which is stored in lipid droplets. When cellular energy demands increase, IMTG can be hydrolyzed by lipolysis to yield FA available for oxidation. The Kiens group was the first to demonstrate that IMTG content is ~25–30% higher in women compared to men,25,74 and this finding was later supported by themselves and others.27,30,76,77 Gender differences in the concentration of TG in muscle have mainly been evaluated in the vastus lateralis muscle by biochemical analyses and histochemical Oil red O staining, but it has also been demonstrated by use of two-dimensional magnetic resonance spectroscopy (MRS) that women have higher content of intramyocellular lipid in the soleus muscle.78 It is well known that type I muscle fibers contain more IMTG than type II fibers,79 and since women usually have more type I fibers than matched men, this could partly explain the higher IMTG content on the level of whole muscle in women compared with men. However, it has also been documented that both type I and type II muscle fibers from women contain more TG compared with men. In the literature, IMTG concentrations have often been described to be negatively associated with whole-body insulin sensitivity. However, endurance trained athletes, having a high level of lipid oxidation, exhibit both a high content of IMTG and enhanced insulin sensitivity.80 The same scenario is present in trained women, who express

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high levels of IMTG and at the same time have higher insulin sensitivity compared to men.81 The observations of higher IMTG concentrations in female skeletal muscle in women could also in part be attributed to the higher basal plasma FA concentration in women than men, which increases the availability of FA to skeletal muscle. In skeletal muscle, uptake of FA is mediated by lipid binding proteins and passive diffusion.82 The fatty acid translocase CD36 (FAT/CD36) is the most studied FA transporter in skeletal muscle; others studied include the membrane bound FA binding protein (FABPpm), FA transport protein 1 (FATP1), and FA transport protein 4 (FATP4), all of which are involved in plasma membrane transport and handling of FA. In particular, FAT/CD36 has been suggested to be important for FA uptake into skeletal muscle during exercise.83–85 Notably, a higher mRNA and protein content of FAT/CD36 in skeletal muscle have been reported in women compared to men, irrespectively of training status.86 Although the mRNA content of the other FA transporters are observed to be higher in women than men, as demonstrated for FABPpm,86 FATP1,87 and also the cytosolic fatty acid binding protein (FABPc),54,88 there have to our knowledge not been any reports that the protein content of these are higher in women. Thus, only FAT/CD36 is confirmed to be higher at the protein level in women than men, an observation that indeed implies that women may have a greater capacity to increase FA transport into skeletal muscle. Hence, the greater IMTG concentrations in women may be linked to a more efficient FA uptake mediated by FAT/CD36, in combination with their higher plasma FA concentrations. Interestingly, during 60–90 min submaximal exercise at same relative workload women utilize IMTG to a larger extent than men, irrespective of training status.25,29,74 Thus, in these studies it was reported that IMTG content was reduced by ~25–35% in women, while breakdown of IMTG in matched men was barely detectable. Triacylglycerol breakdown in skeletal muscle (as in adipose tissue) is carried out by the adipose triglyceride lipase (ATGL), which hydrolyzes the first ester bond thereby releasing FA and forming diacylglycerol (DAG). DAG is hydrolyzed by hormone sensitive lipase (HSL), generating monoacylglycerol and another FA. In the last step monoacylglycerol is hydrolyzed by monoacylglycerol lipase (MGL).89 When total TG hydrolase activity was measured in skeletal muscle homogenates obtained at rest in the fasting state, a twofold higher activity was reported in women compared to men, with no differences in DAG hydrolase activity.90 These findings suggest that there is a higher maximal capacity for ATGL-mediated lipolysis in women than men. This is not linked to gender differences in the protein content of ATGL and its activator CGI-58, which is similar in matched men and women (Kiens, unpublished

observations). Regarding HSL, 90 min submaximal exercise increased HSL activity to a similar extent in women and men, despite a higher total protein content of HSL in women than men.27 However, the increased IMTG breakdown during submaximal exercise in women was not coupled with increased HSL activity. These findings point to a gender-specific regulation of ATGL phosphorylation and activity during exercise, which awaits further gender-comparative studies. It has been found by use of electron microscopy that IMTG in women is localized in a higher number of smaller lipid droplets compared to men.77 This morphologic characteristic might increase accessibility of lipases and proteins associated with the lipid droplets, and thereby increase the turnover of IMTG. Interestingly, lipid droplets in women are located closer to mitochondria after an exercise bout,76 a location which may increase susceptibility to oxidation. The phospholipid surface of lipid droplets is covered with a number of proteins involved in lipid metabolism and trafficking of the lipid droplets. In untrained men and women, matched for VO2peak/kg LBM, skeletal muscle protein expression of perilipin 2, 3, 4, and 5 (also known as ADRP, TIP47, S3-12, and OXPAT, respectively) was 1.5- to 2-fold higher in women.91 Of these, perilipin 3 may be considered important for lipid droplet lipolysis,92 and perilipin 5 has been described to interact with lipolytic key proteins as ATGL and its activator CGI-58.93 Furthermore, recent work also indicates that perilipin 5 mediates an interaction between lipid droplets and mitochondria.94 Taken together, smaller and more abundant lipid droplets and increased expression of perilipins in women are likely to increase association with lipases and eventually mitochondria during exercise, thereby increasing lipolytic turnover of IMTG in women compared with men. Whether the higher expressions of the perilipins are simply due to a higher content of lipid droplets in women remains to be elucidated. Studies have consistently shown that IMTG contributes to a larger extent as energy fuel in women. On the other hand, likely due to their lower intramuscular lipid levels, men may be more dependent on plasma lipids, which includes FA from circulating very-low density lipoprotein-triacylglycerol (VLDL-TG). A greater utilization of FA from VLDL-TG could be due to enhanced hydrolysis in the capillary bed of skeletal muscle, and here muscle lipoprotein lipase (LPL) is an important player. It is a well-known observation that trained subjects have a higher LPL activity in muscle in the resting fasted state compared to untrained subjects. This is evident in both men and women, with no gender difference in basal LPL activity.86 Muscle LPL activity in men may be greater than that of women during exercise. Hence, when LPL activity was measured immediately after 90 min exercise (85% of lactate threshold),

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there was an exercise-induced increase of 56% in men, while mLPL activity was similar to preexercise values in women.95 In support of this, it has also been shown in another study that muscle LPL activity is increased during exercise in men,96 though not compared to women in this study. Thus, it seems possible that men activate LPL in skeletal muscle more during submaximal exercise. An increase in muscle LPL-activity will increase lipolysis of circulating VLDL-TG and thereby release FA to be taken up by the surrounding tissue. In accordance, VLDL-TG might be a useful energy substrate in men during exercise.74

36.6.3  Glucose Metabolism When skeletal muscle contracts during exercise, intracellular signaling events lead to an increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane, in order to increase glucose uptake. A similar total protein content of GLUT4 in skeletal muscle of men and women has been observed,30 despite reports of higher GLUT4 mRNA content in women compared to men.54,97 Hexokinase II (HKII) is another key protein involved in glucose uptake, catalyzing the phosphorylation of glucose to glucose-6-phosphate (G6P) after entry into the skeletal muscle cells. In this way, HKII maintains the concentration gradient that facilitates the transport of glucose. Also, the addition of the phosphate group ensures that glucose is trapped within the muscle cells. For HKII, a 56% higher protein content has been demonstrated in women compared to men,98 which agrees with the finding of 2.4-fold higher HKII mRNA in female skeletal muscle.54 It can be hypothesized that an increased capacity for intracellular phosphorylation of glucose will facilitate its uptake and thereby contributes to an increased capacity for glucose uptake in women. During exercise, however, women have not been shown to rely more on plasma glucose than men, and thus it appears that women may not benefit from their increased hexokinase capacity, at least during submaximal aerobic exercise. In this regard, it should also be noted that when maximal HKII activity is evaluated in vitro in skeletal muscle biopsies of men and women, the activity was found to be similar.99 During 90 min submaximal aerobic exercise, glucose Ra and glucose rate of disappearance (Rd) were similar in untrained men and women both before and after a training period.72 Likewise, in endurance trained individuals no significant gender differences were observed during 90 min of submaximal exercise (60% of VO2peak) in glucose uptake across the exercising leg when expressed per kg lean leg mass.74 Even during more intense exercise (88% of VO2peak), glucose Rd expressed relative to LBM was similar between women and men.100

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36.6.4  Glycogen Stores and Glycolytic Capacity Glucose can be stored as glycogen in skeletal muscle. Depending on training status and carbohydrate intake, the total amount of glycogen in muscles can comprise ~350–500 g. In the resting fasting state, skeletal muscle glycogen content is not different between untrained and trained men and women, matched for training status and during conditions of a controlled diet.29,61 The similarity of the glycogen stores is confirmed by observations of a similar activity of the rate-limiting enzyme of glycogen synthesis, glycogen synthase (GS), when evaluated in vitro in skeletal muscle homogenates obtained at rest from matched men and women (Kiens, unpublished observations). During exercise, the greater catecholamine concentrations in men than women during submaximal as well as maximal intensity exercise may be speculated to increase glycogenolysis to a greater extent in men. There have, however, been divergent findings in regard to whether men use more or less muscle glycogen during exercise compared to women. Considering the studies that have directly assessed glycogen breakdown in skeletal muscle biopsies, there are some reports of men using more skeletal muscle glycogen than women. Hence, after submaximal treadmill running for 90 min at 65% of VO2peak, a 25% greater glycogen breakdown was observed in well-trained men compared to women,101 while submaximal bicycle exercise seemed to induce a similar glycogen breakdown in men and women.61,74 When it comes to more intense exercise, like 30 s bicycle sprinting, glycogen depletion was described to be 50% less in type I muscle fibers in women than in men.102 The discrepancies may be related to the type of exercise and fiber type recruitment, with the latter study demonstrating that the gender difference may be more pronounced during intense exercise. In support of a greater capacity for glycogenolysis in men, a higher maximal activity of glycogen phosphorylase (GP) has been reported in muscle homogenates from untrained men compared to women.103 In the muscle cells, G6P derived from either glycogen breakdown or glucose taken up from plasma is substrate for glycolysis, of which the end-product pyruvate can be converted to acetyl-coA which is a fuel for the tricarboxylic acid (TCA) cycle. Notably, the glycolytic capacity appears to be greater in men, due to several reports of higher maximal activity of enzymes important for glycolysis. Higher activities of PFK (third step in glycolysis), pyruvate kinase (PK) (final step in glycolysis), and lactate dehydrogenase (LDH) (interconversion of pyruvate and lactate) have been demonstrated in muscle homogenates from untrained men compared with women.103 These findings are supported in later studies, demonstrating a higher PFK, LDH, and also

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malate dehydrogenase (MDH) activity in muscles of men compared to women.44,104 The observations were made in homogenates from the vastus lateralis muscle, but in addition to these findings a higher maximal activity of LDH and PFK in men than women was also observed when the tibialis anterior muscle was used for comparison.105 These findings clearly indicate that men have a higher capacity for glycogenolysis and glycolytic flux during exercise. This might imply that men are better able to cope with intense myocellular energy demands. Along these lines, a lower ratio between β-hydroxy acyl-CoA dehydrogenase (HAD) activity and glycolytic enzyme activity in skeletal muscle was observed in men compared to women,103 which could indicate that male muscle has a higher potential for glycolysis rather than beta-oxidation. Considering the fact that type II fibers have a higher glycolytic potential compared to type I fibers,106 it is possible that the difference in glycolytic capacity between men and women is simply related to the higher relative amount of type II fibers in men. This has not been studied, but could be investigated using single-fiber analyses.

36.7  ATP RESYNTHESIZES IN SKELETAL MUSCLE Apparently a gender-specific difference exists in the utilization of energy substrates during exercise. The question is whether there may be gender-specific differences in the molecular machineries that resynthesize ATP from the different energy metabolites in skeletal muscle.

36.7.1  Mitochondrial Fatty Acid Transport and Beta-Oxidation In the cytosol of the skeletal muscle cells, the FA taken up from plasma or liberated from IMTG lipolysis is activated to fatty acyl-CoA by acyl-CoA synthase. Before oxidiation in the mitochondria, fatty acyl-CoA has to be converted to acylcarnitine to cross the outer mitochondrial membrane, a reaction catalyzed by carnitine palmitoyl transferase 1 (CPT1). CPT1 activity is reported to be a key regulator of FA oxidation in skeletal muscle. Free carnitine is required to create acylcarnitine, and it has been proposed that the absolute amount of free carnitine near CPT1 is vital for the regulation of FA oxidation during exercise.107 There is no gender difference in the level of muscle free carnitine at rest,108 while gender differences in muscle free carnitine during exercise have not been investigated. It has been observed that muscle CPT1 mRNA content is higher in women,54 a finding which was also retained

in myotubes obtained from women compared to men.109 However, CPT1 protein content and enzyme activity, measured in intact mitochondria isolated from vastus lateralismuscle biopsies, were reported to be similar in both untrained and trained men and women,110,111 and there does not seem to be a difference in the maximal capacity of CPT1. Within the mitochondria, the fatty acyl-CoA enters the beta-oxidation pathway, in which the acyl-CoA dehydrogenases catalyze the first oxidation reaction. Studies have shown that both long-chain acyl-CoA dehydrogenase (LCAD) mRNA,54 and very long- and medium-chain acyl-CoA dehydrogenase (VLCAD and MCAD) protein content are higher in skeletal muscle of women than men.112 Mitochondrial trifunctional protein α (TFPα) catalyzes the second (hydration) and third (oxidation) reaction for acyl-CoA substrates, while TFPβ catalyzes the fourth thiolysis reaction in the production of acetyl-coA. The mRNA as well as protein content of TFPα is reported to be higher in women,88,112 while TFPβ protein content seems to be similar in men and women.35 The HAD enzyme is part of TFPα and catalyzes the third reaction which leads to NADH. The maximal activity of HAD seems however to be similar in men and women.34,113 Collectively, women have a higher expression of FA oxidation enzymes responsible for FA oxidation compared with men, especially in the initial parts of the beta-oxidation, which together contribute to a higher capacity for generation of acetyl-coA from FA.

36.7.2  Tricarboxylic Acid Cycle and Oxidative Phosphorylation The acetyl-coA from glycolysis and beta-oxidation fuels the TCA cycle, which produces NADH coenzymes to fuel oxidative phosphorylation. The maximal in vitro activity of citrate synthase (CS), the first and rate-limiting enzyme in the TCA cycle, is similar in skeletal muscle from men and women,30,113 and hence there is a similar capacity of the TCA cycle between genders. As maximal CS activity has been found to be the best marker of mitochondrial content measured by electron microscopy in human skeletal muscle,114 these findings may also be indicative of a similar mitochondrial content in skeletal muscle in men and women. Exercise training can increase TCA cycle capacity, as it is well documented that endurance trained subjects have a higher CS activity compared to untrained subjects, and it has been shown that the activity of CS increases to a similar extent in women and men after a period with aerobic training.34 With regard to oxidative phosphorylation, the maximal activity of cytochrome C oxidoreductase (complex III) and cytochrome C oxidase (complex IV) are also reported to be similar in muscles of men and women.34 Along with these observations, when maximal ATP

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production during oxidative phosphorylation was measured in freshly isolated mitochondria from skeletal muscle, a similar ATP production rate was observed in sedentary men and women.115 Also, when mitochondrial respiration was analyzed in an oxygraph on muscle bundles, and complex I-, II-, III-, and IV-dependent respiration measured individually, no gender differences were observed.116 Together, the capacity for acetyl-coA flux through TCA and ATP production from oxidative phosphorylation appears to be similar in men and women, suggesting an equal capacity for myocellular energy generation from acetyl-coA. The propensity for a higher relative FA utilization in women than in men is therefore not due to a higher mitochondrial capacity in women but rather to an increased ratio of beta-oxidative capacity to glycolytic capacity in women, concomitant with increased cellular availability of FA from IMTG and plasma sources.

36.7.3  Finetuning of Acetyl-coA Input to the Tricarboxylic Acid Cycle Pyruvate dehydrogenase (PDH) is a convergence point in the regulation of the metabolic finetuning between glucose and FA oxidation. Hence, PDH converts pyruvate to acetyl-coA, and thereby increases the influx of acetyl-coA from glycolysis into the TCA cycle. Pyruvate dehydrogenase kinase 4 (PDK4) is a regulator of PDH, as it inhibits PDH activity, which in turn will increase the influx of acetyl-coA from beta-oxidation into the TCA cycle, thereby leading to enhanced FA oxidation and slowing of glycolysis or glycolytic intermediates to alternative metabolic pathways. A role for estradiol in the transcriptional regulation of PDK4 has been documented, suggesting gender-specific regulation of PDK4. Thus, when human primary myotubes obtained from women and men are incubated with 17-β estradiol, PDK4 mRNA content is increased in female myotubes,109 and a study in humans has shown that estrogen treatment during menopause led to an increase in PDK4 mRNA in skeletal muscle.117 The protein content of PDK4 in human skeletal muscle has not been subject to gendercomparative studies. However, it has been observed that PDH-E1α protein content is 25% lower in skeletal muscle of women than men and that PDK4 mRNA is higher in female skeletal muscle (Kiens, unpublished observations). These observations suggest that there is a lower requirement for PDH in female skeletal muscle, perhaps due to a lower glycolytic activity in women. A similar sexual dimorphism has been observed in the rat heart. Thus, the PDH complex was significantly higher expressed in male hearts compared with female rat hearts, with the gene expression of PDK4 being significantly higher in female heart.118 Taken together, these findings suggest that gender differences in PDK4 protein

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content and the regulation of PDH activity may contribute to explain the difference in the oxidative flux of glucose and FA in men and women in relation to exercise.

36.8  ESTROGEN AND ITS IMPACT ON METABOLISM The described gender differences in substrate metabolism as well as the molecular differences in glycolytic and beta-oxidative capacity are likely in part related to sex hormone effects. The greater relative lipid utilization during exercise in women has been speculated to be partly ascribed to estrogen actions. In support of a role for estrogen, differences in exercise substrate metabolism between women and men are not observed in childhood, but become evident with puberty.119 In addition, fat oxidation of postmenopausal women was 33% lower during same relative exercise at 50% of VO2 peak when compared to premenopausal women.120 It was also observed in a longitudinal study that resting fat oxidation was lowered by 32% in post- compared to premenopausal women, when subjected to 24 h whole room calorimetry.121 The estrogen receptors α (ERα) and β (ERβ) are both expressed in human skeletal muscle.122,123 By immunohistochemistry, it has been shown that ERα and ERβ are localized to the nuclei of the myofibers, with comparable expression levels in women and men.124 A nuclear localization of the receptors suggests a role in transcriptional regulation of muscle enzymes and proteins. ERα appears to be the most important isoform, as it is markedly higher expressed than ERβ in human skeletal muscle.123 Estradiol incubation of human myotubes increases only ERα mRNA, but not ERβ mRNA.125 Interestingly, both ERα and ERβ mRNA were reported to be three- to fivefold higher in endurance trained men compared to moderately active men, suggesting a role of these estrogen receptors in the adaptations to exercise in skeletal muscle.126 The ERα and ERβ receptors mainly function as transcription factors, while some estrogen actions in muscle may also be mediated by nongenomic effects, as extra nuclear estrogen receptors have been identified. In this context, there is evidence for a mitochondrial location of ERα in the C2C12 mouse skeletal muscle cell line, demonstrated by immunostaining of labeled estradiol binding to mitochondrial fractions.127 Hence, a role for estrogen and ERα in regulation of mitochondrial metabolism human muscle is suggested, but still unexplored. Today many women use oral contraceptives (OC) that modify hormonal status. The active estrogen is often ethinyl estradiol, which is reported to be the most potent of the estrogen agonists.128 OC use reduces natural estrogen production, and depending on OC type, three to five

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36.  Exercise Physiology in Men and Women

times more exogenous estrogen is provided compared with normal endogenous estrogen concentrations.129 Considering the role of estrogen in skeletal muscle substrate metabolism, the use of OC may indeed have implications for exercise metabolism. There is, however, a large variation in the type of OC that is administered (i.e., monophasic and triphasic), and the type and dose of estrogen and progesterone are also subject to large differences. These variations make interpretations of the existing studies more difficult, and thus the evidence for the effect of OC use on exercise metabolism in women is not clear. Several interventional or cross-sectional studies have investigated whether OC use has implications for substrate utilization and aerobic capacity. It has been suggested by some studies that inactive women on monophasic OC have greater FA concentrations, and hence greater relative lipid utilization during exercise compared to non-OC users. However, this has not been found in trained women, and several inconsistent findings require that further studies are needed in this area. It has also been reported that triphasic OC may have a negative impact on VO2peak,49 but this is not found with monophasic OC use. The available literature has been reviewed by Burrows and Peters,129 systematically taking into account the OC type. Taken together, wellcontrolled studies with a high sample size are required to gain further insight into this complex area.

36.9  GENDER DIFFERENCES IN METABOLISM DURING RECOVERY FROM EXERCISE While women rely more on lipids during exercise compared to men, the opposite scenario is evident in the period following exercise. Hence, a recent meta-analysis including 18 studies investigating substrate utilization in men and women during 2–22 h of recovery from 60–120 min endurance exercise at 28–75% of VO2peak, has reported a greater FA oxidation in men than women after exercise, when investigated by indirect calorimetry and in the postabsorptive state.130 Furthermore, when tracer analysis was added to the indirect calorimetry and applied during 3 h of recovery from moderate intensity exercise at 45% or 65% of VO2peak, a greater FA oxidation was confirmed in men compared to women.131 Thus, it seems to be well documented that women exhibit a greater oxidative utilization of glucose in the period following exercise than men. This is likely due to the reason that plasma FA are used for replenishment of the IMTG that was broken down during exercise, rather than being used to cover the oxidative needs during postexercise recovery. In men, IMTG stores are not depleted during exercise, and therefore it might be more beneficial to oxidize FA, while preserving glucose to resynthesize

glycogen in skeletal muscle. This reciprocal shift in preferential substrate utilization in recovery compared to the exercise situation may counterbalance the difference between the amount of glucose and FA used in response to exercise. It has been shown by use of 1H-NMR that the exercise-induced decrease (25%) in IMTG is fully replenished after 20 h on an eucaloric medium-fat diet (33 E% fat), but not on a low-fat diet (10 E%) fat, suggesting that sufficient exogenous dietary FA are needed in order to efficiently resynthesize IMTG stores.

36.10  NUTRITIONAL IMPLICATIONS IN RELATION TO EXERCISE 36.10.1  Energy Availability in Athletes For some athletes it is important to pay attention to their total energy intake. However, appetite is not a reliable indicator of energy needs in athletes, as it has been well documented that prolonged or heavy exercise suppresses ad libitum food intake. Therefore, energy intake can sometimes be lower than the athlete’s energy requirements, in particular during periods with a high training volume. The energy deficit produced during exercise does not induce compensatory responses in appetite, which has been described to be due to exercise-induced suppression of circulating acylated ghrelin,132 and concomitant increases in peptide YY (PYY), glucagon-like peptide-1 (GLP-1), and pancreatic polypeptide (PP) levels,133 which together will suppress appetite and hunger sensations. Some recent studies have added to the understanding of the impact of exercise on appetite in a well-controlled gender-comparative setup. When moderately active men and women exercised corresponding to 30% of daily energy expenditure at 70% of VO2peak, no differences in PYY3–36 and acylated ghrelin were observed, and both genders experienced a similar suppression of appetite and ad libitum energy intake. These findings were confirmed in another study, also in moderately active men and women, which exhibited similar appetite, acylated ghrelin, and PYY3–36 responses to an exercise-induced energy deficit from 60 min running at 70% of VO2peak.134 To date, no studies have compared the appetite hormone response to high-intensity exercise in well trained men and women. Despite these reports of a similar regulation of exercise-induced appetite suppression between genders, it has been frequently found that the energy intake of trained women is lower than the corresponding energy requirement. The term energy availability is defined as dietary energy intake minus exercise energy expenditure,135 and it has been reported that low energy availability in female athletes is prevalent during severe training periods and in sports where body weight has

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

36.10  Nutritional Implications in Relation to Exercise

implications for performance or esthetic appeal. Women undertaking heavy exercise training are indeed susceptible to periods of low energy availability, which results in hormonal disturbances and a high prevalence of menstrual disorders. Over time, low energy availability may result in disturbance of the gonadotropin releasing hormone (GnRH) pulse in the hypothalamus, which again disrupts luteinizing hormone pulsatility, with implications for ovarian function and estrogen homeostasis. The term “female athlete triad” encompasses the spectrum of restrained eating (low energy availability), menstrual dysfunction, and poor bone health, which will increase the risk of stress fractures, osteopenia, or osteoporosis later in life. It has been controversial whether the underlying culprit is insufficient body fat stores or the stress of exercise. However, it has become clear that the hormonal disturbances in women undergoing extensive training periods are due to the energy cost of exercise, and hence low energy availability that leads to luteal suppression and anovulation, and not the exercise in itself. In this context, it has been shown that hormonal disturbances occur when energy availability is reduced below a threshold between 20 and 30 kcal/kg LBM∙per day,135 and therefore the minimal energy requirement has been set to 30–45 kcal/kg LBM∙per day, plus the amount of energy needed for physical activity, to preserve normal reproductive function and skeletal health.136 On the other side of the spectrum, it has been proposed that women may be more resistant than men to exercise-induced weight loss, in part because women have a proportionally greater fat mass than men. Hence, this is in contrast to the conditions described above in well trained female athletes, and is more likely to be relevant for obese and untrained subjects. It has been speculated that this greater fat storage capacity is due to estrogenic actions in the hypothalamic regions that regulate food intake, energy expenditure, and white adipose tissue distribution.137 It has been shown by prediction equations that the energy cost associated with body weight loss is greater in women (30–32 MJ/kg BM) than men (21–23 MJ/kg BM),138 but in a recent review investigating exercise-induced weight loss in men and women, it was described that even though many of the studies reported a significantly higher weight loss in men than women, the effect size was very small and not of physiologic significance.139 Therefore, there does not appear to be a need for a gender-specific prescription when exercise-induced weight loss regimens are devised.

36.10.2  Dietary Macronutrient Composition The current guidelines for nutrition in sports have been composed without paying particular attention to whether there is a need of specific guidelines for the

537

female versus male athlete. Here, we offer a short summary of general advice on macronutrient composition, with the addition of a few studies that are relevant to whether or not there is a need for gender-specific nutritional prescriptions. The guidelines for carbohydrate intake are preferentially provided in grams relative to BM rather than as percentage of dietary energy. Currently, a carbohydrate intake range of 5–7 g/kg BM∙per day has been suggested for general training needs and 7–10 g/kg BM∙per day for the increased needs of endurance athletes.140 There does not appear to be any reason to make explicit recommendations for carbohydrate intake in women and men, except when it comes to carbohydrate loading as a performance enhancing strategy before long-term endurance exercise. In this context, the efficacy of carbohydrate loading has been proposed to be different in men and women. In an early study, it was shown that increasing dietary carbohydrate intake from 55 to 75 E% for 4 days increased skeletal muscle glycogen concentration and enhanced cycling performance in endurance trained men, but not in women.141 The lack of an increased glycogen content in muscle of women was attributed to a low total energy intake, which resulted in an absolute carbohydrate intake of less than 6.5 g/kg·per day. Hence, in a later follow-up study it was shown that increasing total energy intake, while maintaining 75 E% carbohydrate, resulted in a carbohydrate intake of 8 g/kg·per day, and increased muscle glycogen content in women.99 In regard to daily protein intake, the current US, Canadian, and Australian RDI state that a protein intake between 0.75 and 0.8 g/kg BM will meet the needs of 98% of the population. It has been proposed that this is an underestimation,142 and it is still debated what the requirements are in aerobically or resistance trained athletes. To determine dietary protein requirements in the endurance or resistance training state, nitrogen balance studies have been applied. On the basis of the existing evidence the American College of Sports Medicine Position Stand recommends that dietary protein requirements range from 1.2  to  2 g/kg BM∙per day in athletes to support metabolic adaptations, repair, and protein turnover. In support of this, it has been found that well trained women and men were both in a negative nitrogen balance, when ingesting 0.8 and 0.94 g/kg BM∙ per day.16 Furthermore, in a study measuring 72 h of nitrogen balance in endurance training women (~10 h/ week), it was estimated that 1.6 g/kg BM∙per day was needed to attain nitrogen balance.143 Most athletes can easily reach these protein requirements from their usual diet. The intake of dietary fat by athletes is in general proposed to be in accordance with the public health guidelines. However, athletes should be discouraged

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

538

36.  Exercise Physiology in Men and Women

from fat intakes or=twice/ night) had increased risks (OR1.65, 95%CI: 1.02–2.69).188 An initial paper from NHS data documented that the increased risk of breast cancer was there in night shift working women but only if they had worked that shift a minimum of 20 years (multivariate RR = 1.79; 95%CI: 1.06–3.01).189 This was replicated in a Swedish Cancer Registry study with a HR = 1.68 (95%CI: 0.98–2.88) for the group with >20 years of night work. Shorter duration of night work showed no significant risk.190 In addition to night shift work, longer major sleep periods of >9 h and not napping synergistically increased the risk of breast cancer in night shift workers from OR (95%CI) 1.34 (1.05–1.72) to OR (95%CI) 3.69 (1.94–7.02).178 40.6.2.5.2  Other Cancers Working the night shift for 20+ years also increases the risk of endometrial cancer. Multivariate adjusted relative risk (MVRR) was 1.47 (95%CI: 1.03–1.14) in a large

prospective cohort study. This association was even stronger for women who were obese.191 This increased risk does not seem to be related to duration of the major sleep period as a large cohort study the Women’s Health Initiative Observational Study has demonstrated.192 There is mixed data regarding ovarian cancer. Poole et  al. analyzed data from NHS and NHS2 studies and found no correlation with increased incidence of ovarian cancer and night shift work, regardless of the number of years spent working that shift.193 Two other studies, however, showed that rotating night shift work and the resulting circadian disruption do increase the risk of ovarian cancer. Bhatti et  al. demonstrated an OR = 1.24, (95%CI: 1.04–1.49) for invasive ovarian cancer and an OR = 1.48 (95%CI: 1.15–1.90) of borderline tumors for night shift work in a cohort of 1101 women. This risk was independent of the frequency and duration of night shift work.194 There is also increased mortality from ovarian cancer associated with swing shift work or night shift work in the American Cancer Society’s Cancer Prevention study. The RR was 1.27 (95%CI: 1.03–1.56)This was independent of sleep duration or complaints of insomnia.195 Lower melatonin levels appear to play a strong role in the pathogenesis of ovarian cancer, particularly in women who work night shift.196 Data from NHS and NHS2 studies also demonstrated a slight increase in the risk of colorectal cancer but only among women who worked the night shift 15 plus years. The MVRR was 1.35 (95%CI: 1.03–1.77).197 The risk of small cell lung cancer was also elevated in the same cohort but only among smokers who worked night shift 15+ years, RR = 1.61 (95%CI: 1.21–2.13; p trend 75% T1 high-grade disease

Kluth et al. 1.31 1.03 1.67 0.03

1.25 0.80 1.95 0.32

1.14 0.62 2.08 n.s.

916 T1 high-grade patients treated with transurethral resection with or without intravesical therapy

Boorijan et al.

1.18 0.85 1.63 0.33

0.94 0.79 1.11 0.44

1.4

UB

Mortality

Thomas et al.

1.37 1.03 1.83 0.03

Fernandez- 1.80 1.33 2.44 0.0001 Gomez et al.

1.01 0.58 1.76 0.98

1026 patients treated with BCG for nonmuscle invasive bladder cancer 1.34 1.04 1.72 0.03

1555 patients treated with transurethral resection with or without intravesical treatment for nonmuscle invasive bladder cancer 1491 patients treated with BCG for nonmuscle invasive bladder cancer

Kluth et al. 1.06 0.95 1.19 0.3

1.17 1.05 1.37 0.005

8102 patients treated with radical cystectomy

Messer et al.

1.25 1.09 1.44 0.002

4296 patients treated with radical cystectomy

Otto et al.

1.26 1.05 1.49 0.011

2483 patients treated with radical cystectomy

HR, hazard ratio (hazard ratios represent increased risk of women compared to men); 95% CI, 95% confidence interval; LB, lower bound; UB, upper bound.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

609

REFERENCES

Some studies could demonstrate that cancer-specific and overall survival is longer in women compared to men. Using the CORONA database, comprising 6234 patients treated with radical or partial nephrectomy, women had significantly better disease-specific (HR 0.75) and overall (HR 0.80) survival rates than their male counterparts.51 In a SEER-based study, Aaron et al. also reported a better overall survival in women compared to men; cancerspecific survival did, however, not differ significantly. Finally, a recent study by Onishi et al. demonstrated, that in 768 patients treated with nephrectomy for RCC, overall survival was not different between sexes. Only in the subgroup of patients with clear-cell RCC, women had a better overall survival compared to men (3565 vs 3961 days).52 Just as is the case with bladder cancer, much molecular and cohort-based research is needed to uncover the mechanisms of gender-specific differences in incidence, progression, and response to treatment. This avenue of research promises to open opportunities toward more accurate gender-specific prevention, screening, early detection, and therapy resulting in better survival. Until more clear data are generated to allow gender-specific clinical decision-making, gender can only be assessed as the consequence of exposure risk (Table 41.2).

12.

13.

14.

15.

16.

17.

18.

19.

References 1. Antoni S, Ferlay J, Soerjomataram I, et  al. Bladder Cancer Incidence and Mortality : A Global Overview and Recent Trends. Eur Urol. 2016:1–13. 2. Fajkovic H, Halpern JA, Cha EK, et  al. Impact of gender on bladder cancer incidence, staging, and prognosis. World J Urol. 2011;29:457–463. 3. Dobruch J, Daneshmand S, Fisch M, et al. Gender and Bladder Cancer : A Collaborative Review of Etiology, Biology, and Outcomes. Eur Urol. 2016;69:300–310. 4. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2015. CA Cancer J Clin. 2015;65:5–29. 5. Jemal A, Siegel R, Ward E, et al. Cancer Statistics, 2008. CA Cancer J Clin. 2008;58:71–96. 6. Mungan NA, Kiemeney LA, van Dijck JA, van der Poel HG, Witjes JA. Gender differences in stage distribution of bladder cancer. Urology. 2000;55:368–371. 7. Soave A, Dahlem R, Hansen J, Weisbach L. Gender-specific outcomes of bladder cancer patients : a stage-specific analysis in a contemporary, homogenous radical cystectomy cohort. Eur J Surg Oncol. 2015;41:368–377. 8. Kluth LA, Rieken M, Xylinas E, et  al. Gender-specific differences in clinicopathologic outcomes following radical cystectomy : an international multi-institutional study of more than 8000 patients. Eur Urol. 2014;66:913–919. 9. Najari BB, Rink M, Li PS, et  al. Sex Disparities in Cancer Mortality : The Risks of Being a Man in the United States. J Urol. 2013;189:1470–1474. 10. Mungan NA, Aben KK, Schoenberg MP, et  al. Gender differences in stage-adjusted bladder cancer survival. Urology. 2000;55:876–880. 11. Martin-doyle W, Leow JJ, Orsola A, Chang SL, Bellmunt J. Improving selection criteria for early cystectomy in highgrade T1 bladder cancer: a meta-analysis of 15, 215 patients.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

J Clin Oncol. 2015;33(6):643–650. http://dx.doi.org/10.1200/ JCO.2014.57.6967. Kluth LA, Fajkovic H, Xylinas E, Crivelli JJ. Female gender is associated with higher risk of disease recurrence in patients with primary T1 high-grade urothelial carcinoma of the bladder. World J Urol. 2013:1029–1036. Boorjian SA, Zhu F, Herr HW. The effect of gender on response to bacillus Calmette-Guerin therapy for patients with nonmuscle-invasive urothelial carcinoma of the bladder. BJU Int. 2010;106:357–361. Siegrist T, Savage C, Shabsigh A, Cronin A, Donat SM. Analysis of gender differences in early perioperative complications following radical cystectomy at a tertiary cancer center using a standardized reporting methodology. Urol Oncol. 2010;28:112–117. Liberman D, Lughezzani G, Sun M, et al. Perioperative mortality is significantly greater in septuagenarian and octogenarian patients treated with radical cystectomy for urothelial carcinoma of the bladder. Urology. 2011;77:660–666. Otto W, May M, Fritsche H-M, et al. Analysis of sex differences in cancer-specific survival and perioperative mortality following radical cystectomy: results of a large German multicenter study of nearly 2500 patients with urothelial carcinoma of the bladder. Gend Med. 2012;9:481–489. Messer JC, Shariat SF, Dinney CP, et al. Female gender is associated with a worse survival after radical cystectomy for urothelial carcinoma of the bladder : a competing risk analysis. Urology. 2014;83:863–868. Hartge P, Harvey EB, Linehan WM, et  al. Unexplained excess risk of bladder cancer in men. J Natl Cancer Inst. 1990;82: 1636–1640. Hemelt M, Yamamoto H, Cheng KK, Zeegers MPA. The effect of smoking on the male excess of bladder cancer: a meta-analysis and geographical analyses. Int J cancer. 2009;124:412–419. Chalasani V, Chin JL, Izawa JI. Histologic variants of urothelial bladder cancer and nonurothelial histology in bladder cancer. Can Urol Assoc J. 2009;3:S193–S198. Lucca I, Fajkovic H, Klatte T. Sex steroids and gender differences in nonmuscle invasive bladder cancer. Curr Opin Urol. 2014;24:500–505. Zhang Y. Understanding the gender disparity in bladder cancer risk: the impact of sex hormones and liver on bladder susceptibility to carcinogens. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2013;31(4):287–304. http://dx.doi.org/10.1080/1 0590501.2013.844755. Miyamoto H, Yao JL, Chaux A, et al. Expression of androgen and oestrogen receptors and its prognostic significance in urothelial neoplasm of the urinary bladder. BJU Int. 2012;109:1716–1726. Wu S, Chiang H, Ku W. Dual Roles of 17-β Estradiol in Estrogen Receptor-dependent Growth Inhibition in Renal Cell Carcinoma. Cancer Genomics Proteomics. 2016;230:219–230. Shen SS, Smith CL, Hsieh J-T, et al. Expression of estrogen receptors-alpha and -beta in bladder cancer cell lines and human bladder tumor tissue. Cancer. 2006;106:2610–2616. Bolenz C, Lotan Y, Ashfaq R, Shariat SF. Estrogen and progesterone hormonal receptor expression in urothelial carcinoma of the bladder. Eur. Urol. 2009;56:1093–1095. Lucca I, Klatte T, Fajkovic H, De Martino M, Shariat SF. Gender differences in incidence and outcomes of urothelial and kidney cancer. Nat Publ Gr. 2015;12:585–592. Boorjian S, Ugras S, Mongan NP, et al. Androgen receptor expression is inversely correlated with pathologic tumor stage in bladder cancer. Urology. 2004;64:383–388. Mir C, Shariat SF, van der Kwast TH, et  al. Loss of androgen receptor expression is not associated with pathological stage, grade, gender or outcome in bladder cancer: a large multi-institutional study. BJU Int. 2011;108:24–30.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

610

41.  Gender Differences in Bladder and Kidney Cancers

30. Lee JE, Hankinson SE, Cho E. Reproductive factors and risk of renal cell cancer: the Nurses’ Health Study. Am J Epidemiol. 2009;169:1243–1250. 31. Izumi K, Ito Y, Miyamoto H, Miyoshi Y, Ota J. Expression of androgen receptor in non-muscle-invasive bladder cancer predicts the preventive effect of androgen deprivation therapy on tumor recurrence. Oncotarget; 7: 14153–14160. 32. Johnson EK, Daignault S, Zhang Y, Lee CT. Patterns of hematuria referral to urologists: does a gender disparity exist? Urology. 2008;72:493–498. 33. Henning A, Wehrberger M, Madersbacher S, et al. Do differences in clinical symptoms and referral patterns contribute to the gender gap in bladder cancer? BJU Int. 2013;112:68–73. 34. Investigation ANC, Cohn JA, Vekhter B, Lyttle C, Steinberg GD, Large MC. Sex disparities in diagnosis of bladder cancer after initial presentation with hematuria: a nationwide claims-based investigation. Cancer. 2014;120:555–561. 35. Garcõ R, Puente D, Malats N, et al. Gender-related differences in clinical and pathological characteristics and therapy of bladder cancer. Eur Urol. 2003;43:53–62. 36. Thorstenson A, Hagberg O, Ljungberg B, et  al. Gender-related differences in urothelial carcinoma of the bladder : a population-based study from the Swedish National Registry of Urinary Bladder Cancer. Scand J Urol. 2016;50(4):292–297. http://dx.doi. org/10.3109/21681805.2016.1158207. 37. Haines L, Bamias A, Krege S, et  al. The impact of gender on outcomes in patients with metastatic urothelial carcinoma. Clin Genitourin Cancer. 2013;11:346–352. 38. Rose TL, Deal AM, Nielsen ME, Smith AB. Sex disparities in use of chemotherapy and survival in patients with advanced bladder. Cancer. 2016;122(13):2012–2020. http://dx.doi.org/10.1002/ cncr.30029 39. Znaor A, Lortet-tieulent J, Laversanne M, Jemal A. International variations and trends in renal cell carcinoma incidence and mortality. Eur Urol. 2015;67:519–530. 40. Hew MN, Zonneveld R, Kummerlin IPED, Opondo D, de la Rosette JJMCH, Laguna MP. Age and gender related differences in renal cell carcinoma in a European cohort. J Urol. 2012;188:33–38.

41. Hunt JD, Van Der Hel OL, Mcmillan GP, Boffetta P, Brennan P. Renal cell carcinoma in relation to cigarette smoking : Metaanalysis of 24 studies. Int J Cancer. 2005;108:101–108. 42. Renehan AG, Tyson M, Egger M, Heller RF, Zwahlen M. Bodymass index and incidence of cancer : a systematic review and meta-analysis of prospective observational studies. Lancet. 2007:569–578. 43. Macleod LC, Hotaling JM, Wright JL, et al. Risk factors for renal cell carcinoma in the VITAL study. J Urol. 2013;190:1657–1661. 44. Yu C-P, Ho J-Y, Huang Y-T, et al. Estrogen inhibits renal cell carcinoma cell progression through estrogen receptor-beta activation. PLoS One. 2013;8:e56667. 45. Zucchetto A, Talamini R, Dal Maso L, et al. Reproductive, menstrual, and other hormone-related factors and risk of renal cell cancer. Int J Cancer. 2008;123:2213–2216. 46. Beisland C, Medby PC, Beisland HO. Renal cell carcinoma: gender difference in incidental detection and cancer-specific survival. Scand J Urol Nephrol. 2002;36:414–418. 47. Aron M, Nguyen MM, Stein RJ, Gill IS. Impact of gender in renal cell carcinoma: an analysis of the SEER database. Eur Urol. 2008;54:133–140. 48. Chen J, Shi B, Zhang D, Jiang X, Xu Z. The clinical characteristics of renal cell carcinoma in female patients. Int J Urol. 2009;16:554–557. 49. Woldrich JM, Mallin K, Ritchey J, Carroll PR, Kane CJ. Sex differences in renal cell cancer presentation and survival : an analysis of the national cancer database, 1993–2004. J Urol. 2010;179:1709–1713. 50. Lipworth L, Morgans AK, Edwards TL, et  al. Renal cell cancer histological subtype distribution differs by race and sex. BJU Int. 2016;177(2):260–265. 51. May M, Aziz A, Zigeuner R, et  al. Gender differences in clinicopathological features and survival in surgically treated patients with renal cell carcinoma: an analysis of the multicenter CORONA database. World J Urol. 2013;31:1073–1080. 52. Onishi T, Oishi Y, Goto H, Yanada S, Abe K. Gender as a prognostic factor in patients with renal cell carcinoma. BJU Int. 2002;90:32–36.

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C H A P T E R

42 The Complex Challenge of Blood Pressure Regulation: Influences of Sex and Aging on Sympathetic Mechanisms Emma C. Hart1 and Nisha Charkoudian2 1

University Hospitals Bristol NHS Foundation Trust, Bristol, United Kingdom, 2University of Bristol, Bristol, United Kingdom

O U T L I N E 42.1 Summary

611

42.2 Introduction

612

42.3 Measuring SNA in Humans

612

42.4 The Sympathetic Regulation of BP: Short- and Long-Term BP Control

613

42.5 SNA and the Level of BP in Young Men and Women 42.5.1 Young Men, SNA and BP 42.5.2 Young Women, SNA and BP 42.5.3 Physiological Mechanisms

613 613 614 614

42.6 The Impact of Aging and the Menopause on BP Control 42.6.1 Aging and Women 42.6.2 Aging and Men

615 615 616

42.7 Sex Differences and the Reproductive Hormones in the Pathophysiology of Essential Hypertension 42.7.1 Hormone Replacement Therapy, SNA and BP

616

42.8 Overall Summary and Conclusions

618

617

Disclaimer 618 References 618

42.1 SUMMARY The regulation of arterial pressure is one of the most complex and integrated phenomena in human physiology. Central to this regulation is the sympathetic nervous system, which controls vascular tone, cardiac chronotropy and inotropy, and modulates renal and adrenal hormonal responses. Importantly, sympathetic regulation of arterial pressure is very different between men and women. In younger healthy people, women tend to have lower blood pressure and lower sympathetic neural

Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00032-2

activity compared to men. As a result, women have lower risk of hypertension and other cardiovascular diseases until around the age of menopause. After menopause, the risk of hypertension and its associated comorbidities increases sharply in women, and can be even greater than that in men (although risk of hypertension increases with each decade of age in both sexes). In this chapter, our goal is to provide a brief clinical update of mechanisms contributing to arterial pressure regulation in healthy men and women, and how these mechanisms change with age, menopause, and development of hypertension.

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© 2017 Elsevier Inc. All rights reserved.

612

42.  SEX DIFFERENCES IN BLOOD PRESSURE CONTROL

42.2 INTRODUCTION A consistent theme of advances in medical knowledge over the past decade is the idea of individualized medicine. Advances in understanding interindividual variability in almost every aspect of human physiology have led us away from “one size fits all” treatments to more comprehensive assessments of the unique biology and circumstances of the individual patient. In cardiovascular science and medicine, this has been particularly apparent in the striking advances in understanding of differences between men and women in both health and disease. Historically, hypertension was viewed as predominantly a male disease; the prevalence of hypertension is typically lower in young women compared to men of comparable age (65 years belonging to the Nijmegen Biomedical Study,96 the annual decline of estimated GFR was as small as 0.4 mL/min per year. Conversely, in a miscellaneous population of adults aged ≥ 65 years including participants with significant CV and renal morbidities, estimated creatinine clearance had an average 2.6 mL/min per year decline over a 3-year follow-up.97 Overall, these findings seem to indicate that renal function declines with aging but also show that in about one-third of elderly individuals the GFR remains curiously constant. The age-dependent decline in renal function is accelerated in individuals with preexisting CV disease and/ or risk factors for CV disease. In a large cohort study of elderly individuals, the components of the metabolic syndrome and insulin resistance predicted the risks of prevalent and incident CKD.106 Hypertension, a classical age-related disease, induces typical changes in renal function and structure and elevated blood pressure amplifies age-related atherosclerosis and vascular stiffness.107 The severity of systemic atherosclerosis, in turn, has been postulated to be one of the major triggers of age-related glomerulosclerosis and decline in renal function.108 Although the causal nature of the relationships between aging and hypertension, atherosclerosis, arterial stiffness, and renal dysfunction is reasonably well defined. In the healthy elderly cohort of the BLSA study blood pressure did not predict the age-dependent decline

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43.4 Structural and Functional Changes of the Aging Kidney and Impact of Gender

627

TABLE 43.1 Summary of Main Clinical Studies in Humans Analyzing the Impact of Gender on Age-Associated Changes in Renal Functionality and Structure Author (References)

Year

Study population and methods

Davies and Shock89

1950

●

Rowe et al.90

1976

●

Lindeman et al.91

1985

●

Neugarten et al.92

1999

●

Stachenfeld et al.93

2001

●

Rule et al.94

2004

●

Ble et al.95

2005

●

Wetzels et al.96

2007

●

Lauretani et al.97

2008

Poggio et al.98

2009

Rule et al.99

2010

Cross-sectional analysis of 70 men (aged 25–89 years) including healthy subjects and hospitalized patients. ● mGFR by inulin clearance. Cross-sectional analysis of 548 men (aged 20–80 years) selected from the Baltimore Longitudinal Study of Aging. ● eGFR by creatinine clearance. Prospective analysis of a cohort of 254 men (aged 20–80 years) without kidney disease selected from the Baltimore Longitudinal Study of Aging. ● eGFR by creatinine clearance. Autopsy specimens of 250 elderly individuals (aged up to 90 years). Before–after analysis of 16 healthy, nonsmoking old individuals (50% females), exposed to hypertonic saline infusion (HSI).

Retrospective analysis of 365 old potential living kidney donors. ● mGFR by iothalamate clearance, eGFR by MDRD and Cockroft-Gault formulas. Secondary analysis of a prospective, populationbased study (InCHIANTI) of 1005 older subjects aged over 65 years. ● Renal function assessed by serum creatinine and creatinine clearance (CrCl).

Cross-sectional analysis of an apparently healthy cohort of 3732 subjects (45% male) selected from the Nijmegen Biomedical Study, of whom 869 aged over 65 years. ● eGFR by MDRD. Cross-sectional and prospective analysis (3-yearfollow-up) of 931 adults (aged ≥65 years) from the InCHIANTI study. ● eGFR by Cockroft-Gault formula. ●

Cross-sectional analysis of 1057 (56% female) living kidney donors. ● mGFR by iothalamate clearance. ●

Cross-sectional analysis of 1203 adult living kidney donors. ● mGFR by iothalamate clearance, eGFR by MDRD and Cockroft-Gault formulas. ●

Findings Progressive linear decline (46%) in mGFR with age from 123 (at the age of 30) to 65 mL/min/1.73 m2 (at the age of 89).

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Progressive linear decline (31%) in eGFR from 140 (at the age of 30) to 97 mL/min/1.73 m2 (at the age of 80).

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eGFR decline of 0.63 mL/min per year in the age class 20–39 and 1.51 mL/min per year in the age class 40–80, respectively.

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No difference between genders in the development of glomerulosclerosis.

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No difference between genders in basal plasma arginine vasopressin (AVP) concentration. ● Men had greater plasma AVP sensitivity to changes in plasma osmolality during HSI compared to women. ● During HSI, the slope of the plasma AVP-plasma osmolality was greater in men than in women. ● During HSI, men excreted less Na+ and had greater systolic BP (132 vs 119 mmHg) than women. ●

Mean GFR decline was 4.6 versus 7.1 mL/min per decade in men versus women.

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In CrCl group of 30–60 mL/min, creatinine clearance values tended to be lower in women than in men, with a prevalence of 20% and 37.8% respectively for men and women; in the CrCl group ≤30 mL/min the prevalence was 1.6% and 3.3% respectively for men and women. ● The overall prevalence of anemia was 12.0% (11.5% in men and 12.5% in women) with a progressive increase with increasing age in both men and women. ●

eGFR declined by 0.4 mL/min per year. Progressive linear decline in mean eGFR from 100 (at the age 18–24) to 62 mL/min/1.73 m2 (at the age >85) in men. ● Progressive linear decline in mean eGFR from 91 (at the age 18–24) to 59 mL/min/1.73 m2 (at the age >85) in women. ● ●

Overall eGFR declined by 2.6 mL/min per year Higher creatinine clearance was associated with younger age and male sex

● ●

mGFR declined by 1.49 mL/min/1.73 m2 per decade. Rate of GFR decline was two-fold higher in donors aged over 45 as compared to younger donors. ● Women had 3.61 mL/min/1.73m2 higher mGFR than men ● ●

Progressive linear decline in eGFR with age, from 94 (at the age of 18–29) to 70 mL/min/1.73 m2 (at the age of 70–77) ● The prevalence of nephrosclerosis increased linearly from 2.7% for ages 18–29 years to 73% for ages 70–77 years and was similar between men and women (26% vs 29% after age-adjustment) ●

AVP, arginine-vasopressin; BP, blood pressure; CrCl, creatinine clearance; eGFR, estimated glomerular filtration rate; EPO, erythropoietin; HSI, hypertonic saline infusion; MDRD, modification of diet in renal disease; mGFR, measured glomerular filtration rate.

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TABLE 43.2 Main Structural and Functional Changes Which Characterize Renal Senescence MACROSCOPIC ↓ Kidney weight/mass Calcifications ● Renal masses ● Renal cysts ● ●

GLOMERULAR ↓ Number of glomeruli ↓ GFR ● Glomerulosclerosis ● Glomerular hyperthorphy ● Podocytes alterations ● ↑ Mesangial space ● ●

TUBULAR/INTERSTITIAL ● ● ● ● ● ● ● ● ●

↓ Tubular length Tubular atrophy Interstitial fibrosis ↑ Diverticula Impaired sodium balance Impaired fluid balance ↑ Potassium retention ↓ Capacity to dilute urines ↓ Capacity to lower urine pH

VASCULAR Aglomerular circulation Atherosclerosis ● Intimal/Medial hypertrophy ● ↑ Vessels tortuosity ● ↓ ERPF ● ↓ Capacity to lower urine pH ● ↑ Filtration fraction ● ↑ Postglomerular RVRs ● Impaired vasodilatory responses ● ●

ENDOCRINE ● ● ● ●

↓ Plasma RA and aldosterone ↑ EPO ↓ EPO response to anemia ↓ Vitamin D activation

EPO, erythropoietin; ERPF, effective renal plasma flow; GFR, glomerular filtration rate; RA, renin activity; RVRs, renovascular resistances.

in GFR.109 Similarly, carotid intima-media thickness, a well-established marker of systemic atherosclerosis, failed to predict changes in renal function in a community cohort in China.110 In a cohort of 2981 subjects aged 65–84 years with conserved renal function, GFR loss over time was associated with active smoking status, higher fibrinogen levels, diabetes and isolated systolic hypertension.111 These findings were in agreement with data from the Cardiovascular Health Study 112 in which smoking, systolic blood pressure, carotid intimamedia thickness and retinal microvascular disease were independent predictors of renal function decline over

time. Similar findings were reported in three other large community-based cohort studies.104,113,114 There is some evidence to suggest that gender may modulate somewhat the aging-mediated decline in renal function but observations, particularly in human studies, are conflicting. Despite the fact that nephron endowment is comparable in male and female mice and humans,115 rat females are protected from the age-dependent fall in GFR as compared to males.116 Such protection was partly attributed to the beneficial effects of estrogens on kidney tissue and their capacity to prevent glomerulosclerosis (see below), mostly based on the inhibition of mesangial matrix expansion and the antiproliferative action on mesangial cells.117 In addition, androgens exert detrimental effects on renal morphology, particularly by stimulating apoptosis and increasing mesangial matrix deposition118 and on renal vasculature, as demonstrated by the observation that castration in spontaneously hypertensive rats (SHR) prevents worsening in peripheral renal vasculature resistance.74 Nevertheless, under some circumstances, Dihydrotestosterone (DHT) may have a protective role on kidney function. In particular, in streptozocin-induced diabetic rats, DHT administered at low to very-low doses prevented development and worsening of albuminuria, the earliest sign of diabeticinduced renal damage.119 In a retrospective analysis of 365 potential living kidney donors,94 men at the age of 20 years had, on average, a higher estimated mean GFR than women (129 vs 123 mL/min), but exhibited a slower GFR decline in the mid-term (4.6 vs 7.1 mL/min per decade). Conversely, Berg et al.105 demonstrated that the physiological decline of renal function with aging is significantly slower in healthy females than in males (mean measured GFR loss of 1.4 vs 8.7 mL/min/1.73 m2 per decade) and these differences were detectable through the whole age spectrum (21.5–67.1 years, median 38.5 years). A thorough meta-analysis of 68 studies documented that the rate of renal function decline under pathological conditions is faster in men than in premenopausal women3 but this difference is attenuated after menopause.

43.4.2  Glomerular Alterations Glomerulogenesis in humans is mainly determined by genetic factors. Nevertheless, race, gender, and birth weight may influence the total number of glomeruli that, in the adult kidney, ranges from 330,000 to 1,100,000.120 During lifetime, the proportion of obsolete and hyaline glomeruli increases becoming as high as 70% in very old persons.99 Conversely, the overall number of functioning glomeruli decreases.121 Low glomerular density strongly predicts renal function decline in patients affected by glomerulonephritides122 and in elderly kidney donors

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glomerular density is inversely related to the percentage of sclerotic glomeruli.123 Age-related glomerulosclerosis is more prevalent in the subcapsular cortical zone.124 Although this kind of lesion is not pathognomonic of senescence, glomerulosclerosis purely attributable to aging could be suspected in the absence of other changes typically observed in diabetic and hypertensive patients and when the renal interstitium shows scarce cell infiltration. Experimental models in Munich-Wistar rats indicate that the number of normal glomeruli decreases with aging in male, but not in females.125 Conversely, the total volume of renal glomeruli tends to increase in both genders, although more prominently in male rats. Aged female C57 rats exhibit a significantly increased renal corpuscle diameter compared to males; yet, glomerular damage and interstitial fibrosis are more severe in males than in females.126 The impact of gender and sex hormones on the senescent glomerulus needs to be more investigated in humans. In a thorough analysis of 250 autopsy specimens of elderly individuals, Neugarten et  al.92 did not find differences between genders in the severity of glomerulosclerosis. Glomerular basement membrane thickening and extracellular mesangial matrix expansion are typical features of the aging glomeruli.127 Intrarenal arterial changes, such as intimal fibroplasia, usually accompany glomerular sclerosis.108 In rat models, aging induces hypertrophy in podocyte cells which may eventuate in apoptosis, podocytopenia and glomerulosclerosis.128 Human podocytes also are unable to undergo successful mitosis so that the number of these cells tends to decrease with age.129 Direct shunts between afferent and efferent arterioles in iuxtamedullary nephrons bypassing the glomerular skein represent an additional alteration commonly found in kidneys of elderly individuals.127 Dysregulated glomerular hemodynamics typical of renal aging, including segmentally increased glomerular plasma flow and intracapillary pressure, may accelerate glomerulosclerosis as a result of a “maladaptive” process.130 Long-term hyperperfused glomeruli become hypertrophic to maintain global GFR.131 However, glomerular hyperperfusion leads to glomerular hypertension, which is a powerful inducer of diffuse sclerosis.130 Glomerulosclerosis is known to occur faster and more intensely in males than in females and testosterone has been hypothesized to be a catalyst of progressive glomerulosclerosis associated with age.132 This hypothesis arises from the assumption that sclerosis is notably attenuated in kidney specimens from old castrated male rats with respect to controls.125 The mechanism by which androgens may increase susceptibility to age-related glomerular lesions seems to be unrelated to glomerular

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hypertension or hypertrophy,125 relying instead on their capacity for stimulating extracellular mesangial matrix production, mesangial expansion, and procollagen synthesis, as shown in models of subtotal nephrectomy.133 Testosterone is also endowed with other profibrotic actions, including enhanced synthesis of tumor necrosis factor-α (TNF-α) and interleukin-1β; moreover, male rat mesangial cells express higher baseline fibronectin mRNA levels as compared to female rat.134 Glomerular metalloproteinase (MMP) activity increases with age in intact females, but not in males, and castration of males normalizes the MMP activity and prevents glomerular lesions.5 Interestingly, female mice become susceptible to age-induced glomerulosclerosis after menopause135 and estrogen supplementation reverses glomerular sclerosis in male mice overexpressing TGF.136 Further evidence corroborates the hypothesis that estrogens may attenuate renal senescence. Activation of E2s in the kidney limits collagen synthesis by mesangial cells through modulation of the mitogen activated protein (MAP) kinase activity and the transcription factor AP-1117 and counteracts collagen synthesis by AT-II and TGF-β,45,117 hence limiting the progression of glomerulosclerosis. In addition, E2s are indirect regulators of mesangial cells proliferation by modulating the balance between growth promoters and growth inhibitors.137 In contrast with observations attributing positive effects of estrogens on aging-related glomerular injury, Sun et al.138 reported that ERα activation may have deleterious consequences, as demonstrated by the fact that aged ERα-null rats are somewhat protected by glomerular enlargement and matrix accumulation.

43.4.3  Tubulo-Interstitial Alterations As for glomeruli, the overall number of tubules decr­ eases with age139 and the age-dependent decline in renal mass and renal function rests more on tubulo-interstitial than on glomerular changes.140 From a microscopic point of view, involution in tubular length and volume and tubular atrophy are common findings of the aging kidney. In addition, tubular diverticula, expanded interstitial volume, infiltration of mononuclear cells, diffuse areas of fibrosis, and sparse areas of scarring are not infrequently found.141 Tubulo-interstitial fibrosis in the aging kidney is the main consequence of enhanced tubular apoptosis, altered regulation of the expression of metalloproteinases and TGF-β, activation of fibrosis- and hypoxia-related genes, and excessive collagen deposition with structural changes in the extracellular matrix.142 Such alterations may be inhibited or prevented by estrogens through activation of ERα.132 In contrast, androgens may accelerate tubular apoptotic processes. Testosterone increases proapoptotic signaling in proximal tubule cells 118 through

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the activation of inflammatory cytokines, such as c-Jun amino terminal kinase (JNK), and a downstream cascade involving the upregulation of an apoptosis-stimulating fragment and its ligands.49 Such activation can be prevented by the testosterone antagonist flutamide, which reduces JNK phosphorylation.49 Tubular diverticula are mostly localized in the distal convolute tubule and in the collecting duct and may evolve to simple renal cysts,143 a morphological alteration observed in about a half of persons ≥40 years, particularly in men.144 Tubular dilatation is usually accompanied by deposition of hyaline substance and basal membrane thickening. When extended, this process is known as “thyroidization” of the kidney, an involution pattern that physiological aging shares with ESKD.139 Alterations in tubular anatomy occur in parallel with functional involvement. Higher proximal sodium reabsorption and reduced distal fractional reabsorption are the mainstays of sodium balance regulation under steady-state conditions in the elderly.145 However, elderly people are more prone to volume depletion, dehydration and acute kidney injury as this functional resetting hampers the capacity to preserve sodium in response to low salt intake.146 Hyporeninemic hypoaldosteronism, (reduced aldosterone secretion due to inadequate activation of the renin–angiotensin system), is the main determinant of this phenomenon,147 and also explains the high incidence of nocturnal natriuresis found among old people.148 In addition, aged individuals show also a partial inability to eliminate sodium excess in response to salt load due to resistance to the actions of the atrial natriuretic peptide,149 an alteration predisposing to salt retention, CV congestion, and hypertension. Regulation of salt and water balance at the kidney level is influenced by androgen receptors8 and testosterone promotes fluid reabsorption in the proximal tubule, increasing blood pressure and extracellular volume.84 After acute hypertonic saline load, elderly men have lower sodium excretion rate than women with an increase in systolic blood pressure that is accompanied by a rightward shift of the pressure–natriuresis curve with the attempt of maintaining normal sodium balance.93 Stimulation of renal ERs produces significant effects on water and sodium control in the kidney. In mRen(2) Lewis strain rats, estrogen administrations leads to an increased sodium uptake at both the proximal and distal tubule level where ERs are known to abound.150 Aged oophorectomized rats manifest a severely reduced tubular fluid output and renal functional reserve which can be promptly reverted by estrogen administration.151 Young menopausal women not treated with hormonal replacement therapy respond to chronic salt load with a pronounced increase in renal proximal sodium reabsorption and with a marked right shift of the pressure–natriuresis

curve, compared to premenopausal women.152 Taken altogether, these observations point at a central role of estrogens in preserving renal (tubular) function during aging. Na-K ATPase activity is reduced in old persons as a consequence of tubular atrophy and tubular-interstitial scarring. This abnormality makes elderly subjects highly prone to hyperkalemia, particularly in the presence of other risk factors such as dehydration, metabolic acidosis, pathologically reduced renal function and administration of potassium-sparing drugs.153 Age impairs the capacity of diluting and concentrating urine in humans.154 Nocturia, a typical characteristic of old age, is partly related to such reduced concentrating ability155 while the impaired urine diluting capacity exposes elderly individuals to an increased risk of hyponatremia after water load.156 As shown in aging rat models, such abnormalities are principally related to a reduced expression of urea transporters in the inner medullary collecting ducts, a downregulation of vasopressin-2 receptors and a reduced expression of the water-channels aquaporin 2 and 3 in the collecting duct,157 which impair the ability of regulating urine concentration.158 Urine osmolarity is higher in adult men than in women although it decreases similarly during aging.159 Interestingly, in young male rats, testosterone administration induces an acute increase in water excretion.119 Under physiological conditions, vasopressin is the main regulator of extracellular fluid volume. In aged men the vasopressin system seems to be more sensitive to osmotic stimuli than in women, as demonstrated by the fact that higher osmotic loads are excreted through an increase in urine concentration rather than a decrease in urine volume.159 In addition, vasopressin secretion and circulating levels tend to be higher on average in elderly men as compared to women93 and men have also higher urine osmolarity than women on equal plasma vasopressin levels.160 Elderly subjects tend more than young individuals to develop acidosis in response to acid load mainly because of the incapacity to increase ammonia and H+ synthesis161 due to an impaired proton pump activity in the cortical collecting duct.162 Tubular-dependent metabolic acidosis is thought to be related to a series of pathological conditions, particularly in elderly females, including enhanced protein catabolism and muscle wasting, decreased citrate excretion, hypercalciuria, renal lithiasis, bone fractures and cardiomyopathy.163

43.4.4  Vascular Alterations From adulthood, renal plasma flow (RPF)89 and effective RPF (ERPF) steadily decline145 until the age of 80 years. Functional reduction in RPF mostly involve the

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43.4 Structural and Functional Changes of the Aging Kidney and Impact of Gender

renal cortex while blood flow in the medullary region is relatively more conserved.164 As a consequence, the contribution of iuxtamedullary glomeruli to the global filtration function increases.145 In healthy elderly people and in elderly subjects with CV comorbidities like heart failure or hypertension the GFR is relatively better preserved than ERPF due to an increase in postglomerular renovascular resistances (RVR).145 RPF and glomerular blood pressure control mostly rely on the regulation of resistances in afferent and efferent arterioles.165 Aging female Munich-Wistar rats have a more preserved GFR that is associated with small changes in RPF and glomerular blood pressure, facilitated by a more effective relaxation of both afferent and efferent renal arteriolar resistances.125 Conversely, male rats show a more pronounced increase in afferent and efferent resistances that leads to a fall in GFR consequent to a progressive decrease in RPF despite a relatively conserved glomerular pressure. Similar observations have been reported in humans. In healthy young adults, men exhibit higher values of GFR and RPF, while, by age 70, RPF tends to be similar among genders.105 In another study, a more significant decline in ERPF was demonstrated in males as early as 20 years of age, but not in females.105 Changes in ERPF are obviously related to structural changes in the renal vasculature, particularly at the postglomerular level.166 Structural abnormalities in renal vasculature recall those observed in vessels in other organ systems, such as intimal and medial hypertrophy, arteriolosclerosis, or even overt atherosclerotic plaques.164 In addition, as shown in postmortem angiograms and histology studies, increased tortuosity of preglomerular vessels, direct shunts between afferent and efferent vessels, wall thickening and narrowing of the vascular lumen of afferent arterioles may be frequent findings in the aging kidney.167 Microinfarctions triggered by cholesterol emboli are often observed along with atherosclerosis of the aorta and renal arteries, particularly in elderly patients with diabetes and hypertension and interlobular arteries in the elderly always show signs of fibro-intimal hyperplasia,164 a feature typically observed in chronic hypertensive patients regardless of age. No less important, the sensitivity of renal arterioles to endogenous and exogenous vasoactive substances and the reno-vascular response to vasodilatory agents become progressively altered as age progresses and may differ among genders.168 As alluded to earlier, androgens play a crucial role in regulating RVR, particularly by modulating renal sensitivity to angiotensin-II. Indeed, castration mitigates hypertension and moderates the renal vascular responses to angiotensin-II, while testosterone restores normal pressor and renal vascular responses.169 RVR is increased in aged male SHR as compared to younger animals but such an increase may be ameliorated by

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castration.170 In addition, interlobar arteries of old male rats show reduced responses to phenylephrine, probably consequent to a diminished alpha adrenergic receptor signaling for renal vasoconstriction.171 Renal vasculature of male aging rats is also more sensitive to the vasodilator effects of NO. After administration of angiotensin-II, renal perfusion pressure is more increased in males than in females but such difference can be attenuated by preventive blockade of NOS.172 Therefore, it can be speculated that, in aging male kidneys, the reduction in NO production may elicit the sensitivity to vasoconstrictive stimuli by angiotensin-II, which ultimately accelerates renal function deterioration.

43.4.5  Endocrine Alterations Vitamin D hydroxylation mostly occurs at the kidney level. This process is fundamental for making this compound biologically active. Vitamin D deficiency is prominent in old persons due to a reduced availability of the precursor 25-OH-vitamin D and due to a reduced ability of the aging kidney itself to convert 25-OH-vitamin D to 1,25 diOH vitamin D.173 Interestingly, a secondary analysis of the BELFRAIL study on persons aged >80 years with preserved renal function demonstrated that higher 25-OH-vitamin D levels are associated with active lifestyle and exposure to sunshine rather than renal function.174 The presence of CKD may aggravate vitamin D deficiency in elderly. Reduced 25-OH-vitamin D levels have been found to predict progression to dialysis and death in a large cohort study of elderly patients with impaired renal function.175 In postmenopausal women, the presence of an impaired renal function predicts the risk for bone fractures while vitamin D supplements reduce the incidence of falls.176 Such a protective effect has been attributed to an improvement in muscle strength promoted by upregulation of vitamin D receptors. EPO, the main hormone responsible for red blood cell production at the medullary level, is mainly produced by kidney interstitial cells. The lack of this hormone in CKD is the main trigger of normochromic-normocytic anemia that is exceedingly prevalent among renal patients. Healthy elderly have higher EPO production and higher circulating EPO levels as compared with younger persons, probably as a counterregulatory mechanism aiming at maintaining normal red blood cells mass in response to a higher turnover.177 On the other hand, EPO levels tend to be reduced in anemic elderly individuals, which suggests an impaired counterregulatory reaction to low hemoglobin.178 In a secondary analysis of the InCHIANTI study reduced EPO levels were also strongly associated with old age, the presence of anemia, and impaired renal function, particularly among elderly men.95

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Prostaglandins, prostacyclins, thromboxanes and leukotrienes, the so-called “autacoids,” are potent vasoactive agents which also modulate platelet aggregation. Although experimental inhibition of autacoids synthesis produces similar functional alterations in healthy elderly and young individuals, the production and biological activities of these compounds become progressively altered with aging.179 In kidneys of elderly individuals, PGF1-α production decreases with age while the synthesis of other prostaglandins (such as PGE2 and PGF2-α) is more preserved.180 In experimental models of sodium overload, old male rats as compared to younger animals show reduced PGF2-α and enhanced PGE2 production in kidney interstitial fluid.181 An imbalance in prostaglandin modulation has been assumed to explain the altered capacity of the aging kidney to respond to sympathetic stimulation, such as during mental stress, particularly in elderly persons with severe hypertension.182

43.5  FUTURE DIRECTIONS FOR DELAYING RENAL SENESCENCE Several plausible dietary and pharmaceutical approaches aiming at preserving kidney health and retarding organ senescence have been proposed and investigated in experimental and clinical studies. Observational evidence from the Nurses’ Health Study183 seems to indicate that low protein intake may slow down the age-dependent decline in renal function. Indeed, in a subgroup of women with conserved renal function, the change in GFR referable to protein intake excess was estimated to be 0.25 mL/min/1.73 m2 per each 10-g increase in protein intake over an 11-year observation period. Low salt diets are recognized to improve blood pressure control184; hence salt intake represents another important modifiable factor which may retard decline in renal function. Accordingly, in a small cohort of elderly hypertensive patients, renal function decline over time was strongly predicted by the average salt excretion and the baseline eGFR.185 Unfortunately, the observational nature of these findings limits the possibility of causal interpretations so that no recommendations for public health and clinical practice can be made in the absence of appropriate clinical trials confirming the benefits of such approaches. Other dietary interventions to delay systemic and kidney senescence have been extensively studied in experimental models. For instance, long-term administration of the NO precursor and ADMA antagonist L–arginine ameliorates proteinuria and improves renal function in aging rats.186 Isocaloric diets with low-AGE content reduce kidney and CV damage associated with age and extend life span in rats.187 Such effects could be further potentiated

by the chronic administration of powerful antioxidants, such as methylglyoxal.188 A diet enriched with other antioxidants (such as vitamin E), prevents accumulation of F2-isoprostanes and lipid peroxidation in aging kidneys, leading to a significant increase in the GFR.56 Caloric restriction delays age-associated structural changes in the kidney, such as glomerulosclerosis, tubular-interstitial fibrosis, vascular wall thickening and ischemic damage.189 These benefits go along with a series of well-known biological effects, including a reduced expression of the fibrogenic factor matrix-metalloproteinase-7 and a reduction in ceruloplasmin production, renal lipid accumulation and apoptosis.190 In models of experimental aging, such as the 24-month F344BN old rat, caloric restriction induces downexpression of the plasminogen activator inhibitor (PAI)-1, the vascular VEGF and other connective tissue growth factors, leading to a reduction of extracellular matrix accumulation and proteinuria.191 Caloric restriction also preserves renal SIRT-1 expression, a redox and energy state sensor with antiapoptotic and antifibrotic effects that is involved in the main cytoprotective mechanisms which may retard kidney aging.40 Although human studies demonstrating a beneficial effect of low-calories diets on renal function are lacking, long-term caloric restriction is efficacious in ameliorating hypertension and metabolic profile, retarding atherosclerosis and limiting the decline in diastolic function with age.192 Few pharmaceutical options have been tested so far for delaying renal senescence. Chronic treatments with angiotensin-converter enzyme inhibitors (ACEi) or angiotensin receptor-blockers (ARBs) are effective in reducing renal mitochondria damage, age-associated glomerulosclerosis, mesangial expansion, tubular-interstitial fibrosis and mononuclear cells infiltration.193 In aged SHR, selective ATR-1 blockade prevents renal damage by reducing oxidative stress and by increasing NO bioavailability.194 In experimental studies, peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists increase renal expression of Klotho and reduce oxidative load.195 This limits parenchymal sclerosis and cell senescence, thereby improving GFR and proteinuria. Selective estrogen receptor modulators (SERMs) are therapeutic agents reproducing several benefits exerted by estrogens on peripheral organs (mostly bone and vasculature) without mimicking their deleterious effects on the gonads. Interestingly, SERMs were as effective as estradiol in inhibiting collagen synthesis from renal cells in in vitro models196 and glomerulosclerosis is ameliorated by the SERM raloxifene in animal models of diabetes.197 These experimental observations set the stage for exploratory analyses in human trials on possible benefits of SERMs on kidney health, similarly to those exerted on

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REFERENCES

other organs. In the Raloxifene in Diabetic Nephropathy (RADIAN) study, 39 postmenopausal women with type-2 diabetes and signs of renal damage were randomized to receive either raloxifene or placebo for 6 months. Urinary protein excretion decreased in the patients receiving raloxifene but not in the placebo group.198 The Multiple Outcomes of Raloxifene Evaluation (MORE) study199 was a multicenter, randomized, double-blind trial of raloxifene versus placebo designed to assess the risk of fractures in 7705 (mostly) old postmenopausal women (aged 31–80 years) with osteoporosis. In a post hoc analysis of this trial,200 elderly women with conserved renal function at baseline had a slower yearly rate of creatinine increase and a significantly slower yearly rate of eGFR decrease over the 3-year follow-up. Raloxifene was also associated with significantly fewer kidney-related adverse events compared with placebo. Collectively, these findings suggest that raloxifene may be somewhat useful in retarding the age-associated decline in renal function also in humans. Future trials are currently ongoing to confirm this new, promising application of SERMs compounds.

43.6 CONCLUSIONS Renal aging is an intricate, multifactorial process. Establishing to what degree renal alterations in the elderly simply represent the impact of normal aging on the kidney as opposed to long-term exposure to chronic diseases remains problematic. Under both physiological and pathological conditions, the influence of sex hormones on kidney structure and functionality is pervasive. Hence, it is not surprising that gender may play a significant role also in modulating the sexually dimorphic phenotypic manifestations of renal aging. Recent advances in “omic” sciences are providing fundamental insights on renal aging which will help design targeted dietary and/or pharmaceutical treatments for delaying renal senescence. Appropriate lifestyle modifications, such as the adoption of low-AGEs and low-calories diets with a high content of antioxidants and, lastly, the administration of SERMs, currently represent the most promising approaches to be explored for preserving kidney health.

References 1. Carrero JJ. Gender differences in chronic kidney disease: underpinnings and therapeutic implications. Kidney Blood Press Res. 2010;33(5):383–392. 2. Huang JC, Lin HY, Lim LM, et al. Body mass index, mortality, and gender difference in advanced chronic kidney disease. PLoS One. 2015;10(5):e0126668. 3. Neugarten J, Acharya A, Silbiger SR. Effect of gender on the progression of nondiabetic renal disease: a meta-analysis. J Am Soc Nephrol. 2000;11(2):319–329.

633

4. Baylis C. Sexual dimorphism in the aging kidney: differences in the nitric oxide system. Nat Rev Nephrol. 2009;5(7):384–396. 5. Reckelhoff JF, Baylis C. Glomerular metalloprotease activity in the aging rat kidney: inverse correlation with injury. J Am Soc Nephrol. 1993;3(11):1835–1838. 6. Brandenberger AW, Tee MK, Lee JY, Chao V, Jaffe RB. Tissue distribution of estrogen receptors alpha (ER-alpha) and beta (ER-beta) mRNA in the midgestational human fetus. J Clin Endocrinol Metab. 1997;82(10):3509–3512. 7. Rogers JL, Mitchell AR, Maric C, Sandberg K, Myers A, Mulroney SE. Effect of sex hormones on renal estrogen and angiotensin type 1 receptors in female and male rats. Am J Physiol Regul Integr Comp Physiol. 2007;292(2):R794–R799. 8. Wilson CM, McPhaul MJ. A and B forms of the androgen receptor are expressed in a variety of human tissues. Mol Cell Endocrinol. 1996;120(1):51–57. 9. U.S. Census Bureau IDB. Mid-year population, by age and sex. . 10. Centers for Disease Control and Prevention NCfHS, National Vital Statistics System. National Vital Statistics Reports. 14, 2006. 11. Coresh J, Selvin E, Stevens LA, et al. Prevalence of chronic kidney disease in the United States. JAMA. 2007;298(17):2038–2047. 12. Magnason RL, Indridason OS, Sigvaldason H, Sigfusson N, Palsson R. Prevalence and progression of CRF in Iceland: a population-based study. Am J Kidney Dis. 2002;40(5):955–963. 13. Martin JE, Sheaff MT. Renal ageing. J Pathol. 2007;211(2):198–205. 14. Martin GM, Ogburn CE, Colgin LM, Gown AM, Edland SD, Monnat Jr. RJ. Somatic mutations are frequent and increase with age in human kidney epithelial cells. Hum Mol Genet. 1996;5(2):215–221. 15. Rodwell GE, Sonu R, Zahn JM, et al. A transcriptional profile of aging in the human kidney. PLoS Biol. 2004;2(12):e427. 16. Lin TH, Chiu HC, Wang CL, et  al. The Gly460Trp polymorphism of alpha-adducin gene as a predictor of renal function decline over 4 years of follow-up in an apparently healthy Chinese population. Transl Res. 2012;160(2):162–163. 17. Melk A, Mansfield ES, Hsieh SC, et al. Transcriptional analysis of the molecular basis of human kidney aging using cDNA microarray profiling. Kidney Int. 2005;68(6):2667–2679. 18. Kwon J, Lee B, Chung H. Gerontome: a web-based database server for aging-related genes and analysis pipelines. BMC Genom. 2010;11(Suppl 4):S20. 19. Kurosu H, Yamamoto M, Clark JD, et al. Suppression of aging in mice by the hormone Klotho. Science. 2005;309(5742):1829–1833. 20. Arking DE, Krebsova A, Macek Sr. M, et  al. Association of human aging with a functional variant of klotho. Proc Natl Acad Sci USA. 2002;99(2):856–861. 21. Kuro-o M, Matsumura Y, Aizawa H, et  al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997;390(6655):45–51. 22. Kuro-o M. Klotho as a regulator of oxidative stress and senescence. Biol Chem. 2008;389(3):233–241. 23. Koh N, Fujimori T, Nishiguchi S, et al. Severely reduced production of klotho in human chronic renal failure kidney. Biochem Biophys Res Commun. 2001;280(4):1015–1020. 24. Mitani H, Ishizaka N, Aizawa T, et  al. In vivo klotho gene transfer ameliorates angiotensin II-induced renal damage. Hypertension. 2002;39(4):838–843. 25. Mostoslavsky R, Chua KF, Lombard DB, et al. Genomic instability and aging-like phenotype in the absence of mammalian SIRT6. Cell. 2006;124(2):315–329. 26. Kume S, Uzu T, Horiike K, et  al. Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney. J Clin Invest. 2010; 120(4):1043–1055.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

634

43.  GENDER AT THE INTERFACE OF RENAL AGING

27. Herranz D, Serrano M. SIRT1: recent lessons from mouse models. Nat Rev Cancer. 2010;10(12):819–823. 28. Kapahi P, Chen D, Rogers AN, et  al. With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging. Cell Metab. 2010;11(6):453–465. 29. Zhuo L, Cai G, Liu F, et al. Expression and mechanism of mammalian target of rapamycin in age-related renal cell senescence and organ aging. Mech Ageing Dev. 2009;130(10):700–708. 30. Yumura W, Imasawa T, Suganuma S, et  al. Accelerated tubular cell senescence in SMP30 knockout mice. Histol Histopathol. 2006;21(11):1151–1156. 31. Chkhotua AB, Gabusi E, Altimari A, et al. Increased expression of p16(INK4a) and p27(Kip1) cyclin-dependent kinase inhibitor genes in aging human kidney and chronic allograft nephropathy. Am J Kidney Dis. 2003;41(6):1303–1313. 32. Melk A, Schmidt BM, Takeuchi O, Sawitzki B, Rayner DC, Halloran PF. Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney. Kidney Int. 2004;65(2):510–520. 33. Melk A. Senescence of renal cells: molecular basis and clinical implications. Nephrol Dial Transplant. 2003;18(12):2474–2478. 34. Jiang H, Ju Z, Rudolph KL. Telomere shortening and ageing. Z Gerontol Geriatr. 2007;40(5):314–324. 35. Westhoff JH, Schildhorn C, Jacobi C, et al. Telomere shortening reduces regenerative capacity after acute kidney injury. J Am Soc Nephrol. 2010;21(2):327–336. 36. Melk A, Ramassar V, Helms LM, et al. Telomere shortening in kidneys with age. J Am Soc Nephrol. 2000;11(3):444–453. 37. Delahunt B, Stehbens WE, Gilbert-Barness E, Shozawa T, Ruger BM. Progeria kidney has abnormal mesangial collagen distribution. Pediatr Nephrol. 2000;15(3–4):279–285. 38. Harvey SJ, Jarad G, Cunningham J, et al. Podocyte-specific deletion of dicer alters cytoskeletal dynamics and causes glomerular disease. J Am Soc Nephrol. 2008;19(11):2150–2158. 39. Wiggins JE, Patel SR, Shedden KA, et al. NFkappaB promotes inflammation, coagulation, and fibrosis in the aging glomerulus. J Am Soc Nephrol. 2010;21(4):587–597. 40. Bolignano D, Mattace-Raso F, Sijbrands EJ, Zoccali C. The aging kidney revisited: a systematic review. Ageing Res Rev. 2014;14:65–80. 41. Cardani R, Zavanella T. Age-related cell proliferation and apoptosis in the kidney of male Fischer 344 rats with observations on a spontaneous tubular cell adenoma. Toxicol Pathol. 2000;28(6):802–806. 42. Schmitt R, Cantley LG. The impact of aging on kidney repair. Am J Physiol Renal physiol. 2008;294(6):F1265–F1272. 43. Chou JS, Reiser IW, Porush JG. Aging and urinary excretion of epidermal growth factor. Ann Clin lab Sci. 1997;27(2):116–122. 44. Li Z, Chen X, Xie Y, et  al. Expression and significance of integrin-linked kinase in cultured cells, normal tissue, and diseased tissue of aging rat kidneys. J Gerontol Biol Sci Med Sci. 2004;59(10):984–996. 45. Neugarten J, Kasiske B, Silbiger SR, Nyengaard JR. Effects of sex on renal structure. Nephron. 2002;90(2):139–144. 46. Qiao X, Chen X, Wu D, et al. Mitochondrial pathway is responsible for aging-related increase of tubular cell apoptosis in renal ischemia/reperfusion injury. J Gerontol Biol Sci Med Sci. 2005;60(7):830–839. 47. Ding G, Franki N, Kapasi AA, Reddy K, Gibbons N, Singhal PC. Tubular cell senescence and expression of TGF-beta1 and p21(WAF1/CIP1) in tubulointerstitial fibrosis of aging rats. Exp Mol Pathol. 2001;70(1):43–53. 48. Zhang JH, Zhang Y, Herman B. Caspases, apoptosis and aging. Ageing Res Rev. 2003;2(4):357–366.

49. Verzola D, Villaggio B, Procopio V, et  al. Androgen-mediated apoptosis of kidney tubule cells: role of c-Jun amino terminal kinase. Biochem Biophys Res Commun. 2009;387(3):531–536. 50. Mendoza-Nunez VM, Ruiz-Ramos M, Sanchez-Rodriguez MA, Retana-Ugalde R, Munoz-Sanchez JL. Aging-related oxidative stress in healthy humans. Tohoku J Exp Med. 2007;213(3):261–268. 51. Frenkel-Denkberg G, Gershon D, Levy AP. The function of hypoxia-inducible factor 1 (HIF-1) is impaired in senescent mice. FEBS Lett. 1999;462(3):341–344. 52. Houben JM, Moonen HJ, van Schooten FJ, Hageman GJ. Telomere length assessment: biomarker of chronic oxidative stress? Free Radic Biol Med. 2008;44(3):235–246. 53. Lu C, He JC, Cai W, Liu H, Zhu L, Vlassara H. Advanced glycation endproduct (AGE) receptor 1 is a negative regulator of the inflammatory response to AGE in mesangial cells. Proc Natl Acad Sci U S A. 2004;101(32):11767–11772. 54. Semba RD, Ferrucci L, Fink JC, et al. Advanced glycation end products and their circulating receptors and level of kidney function in older community-dwelling women. Am J Kidney Dis. 2009;53(1):51–58. 55. Semba RD, Fink JC, Sun K, Windham BG, Ferrucci L. Serum carboxymethyl-lysine, a dominant advanced glycation end product, is associated with chronic kidney disease: the Baltimore longitudinal study of aging. J Ren Nutr. 2010;20(2):74–81. 56. Reckelhoff JF, Kanji V, Racusen LC, et  al. Vitamin E ameliorates enhanced renal lipid peroxidation and accumulation of F2-isoprostanes in aging kidneys. Am J Physiol. 1998;274(3 Pt 2):R767–R774. 57. Wiggins JE, Goyal M, Wharram BL, Wiggins RC. Antioxidant ceruloplasmin is expressed by glomerular parietal epithelial cells and secreted into urine in association with glomerular aging and high-calorie diet. J Am Soc Nephrol. 2006;17(5):1382–1387. 58. Long DA, Newaz MA, Prabhakar SS, et al. Loss of nitric oxide and endothelial-derived hyperpolarizing factor-mediated responses in aging. Kidney Int. 2005;68(5):2154–2163. 59. Xiong Y, Yuan LW, Deng HW, Li YJ, Chen BM. Elevated serum endogenous inhibitor of nitric oxide synthase and endothelial dysfunction in aged rats. Clin Exp Pharmacol Physiol. 2001; 28(10):842–847. 60. Thomas SE, Anderson S, Gordon KL, Oyama TT, Shankland SJ, Johnson RJ. Tubulointerstitial disease in aging: evidence for underlying peritubular capillary damage, a potential role for renal ischemia. J Am Soc Nephrol. 1998;9(2):231–242. 61. Reckelhoff JF, Hennington BS, Moore AG, Blanchard EJ, Cameron J. Gender differences in the renal nitric oxide (NO) system: dissociation between expression of endothelial NO synthase and renal hemodynamic response to NO synthase inhibition. Am J Hypertens. 1998;11(1 Pt 1):97–104. 62. Lyons D, Roy S, Patel M, Benjamin N, Swift CG. Impaired nitric oxide-mediated vasodilatation and total body nitric oxide production in healthy old age. Clin Sci (Lond). 1997;93(6):519–525. 63. Erdely A, Greenfeld Z, Wagner L, Baylis C. Sexual dimorphism in the aging kidney: Effects on injury and nitric oxide system. Kidney Int. 2003;63(3):1021–1026. 64. Baylis C. Sexual dimorphism, the aging kidney, and involvement of nitric oxide deficiency. Semin Nephrol. 2009;29(6):569–578. 65. Orshal JM, Khalil RA. Gender, sex hormones, and vascular tone. Am J Physiol Regul Integr Comp Physiol. 2004;286(2):R233–R249. 66. Amelina H, Cristobal S. Proteomic study on gender differences in aging kidney of mice. Proteome Sci. 2009;7:16. 67. Baylis C. Nitric oxide deficiency in chronic kidney disease. Am J Physiol Renal Physiol. 2008;294(1):F1–F9. 68. Reckelhoff JF, Kellum JA, Blanchard EJ, Bacon EE, Wesley AJ, Kruckeberg WC. Changes in nitric oxide precursor, L-arginine,

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

and metabolites, nitrate and nitrite, with aging. Life Sci. 1994;55(24):1895–1902. 69. Bode-Boger SM, Muke J, Surdacki A, Brabant G, Boger RH, Frolich JC. Oral L-arginine improves endothelial function in healthy individuals older than 70 years. Vasc Med. 2003;8(2): 77–81. 70. Scalera F, Borlak J, Beckmann B, et al. Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senescence. Arterioscler Thromb vasc Biol. 2004;24(10):1816–1822. 71. Kielstein JT, Bode-Boger SM, Frolich JC, Ritz E, Haller H, Fliser D. Asymmetric dimethylarginine, blood pressure, and renal perfusion in elderly subjects. Circulation. 2003;107(14):1891–1895. 72. Schulze F, Maas R, Freese R, Schwedhelm E, Silberhorn E, Boger RH. Determination of a reference value for N(G), N(G)-dimethylL-arginine in 500 subjects. Eur J Clin Invest. 2005;35(10):622–626. 73. Celermajer DS, Sorensen KE, Spiegelhalter DJ, Georgakopoulos D, Robinson J, Deanfield JE. Aging is associated with endothelial dysfunction in healthy men years before the age-related decline in women. J Am Coll Cardiol. 1994;24(2):471–476. 74. Reckelhoff JF. Gender differences in the regulation of blood pressure. Hypertension. 2001;37(5):1199–1208. 75. Weidmann P, Beretta-Piccoli C, Ziegler WH, Keusch G, Gluck Z, Reubi FC. Age versus urinary sodium for judging renin, aldosterone, and catecholamine levels: studies in normal subjects and patients with essential hypertension. Kidney Int. 1978;14(6):619–628. 76. Skott P, Ingerslev J, Damkjaer Nielsen M, Giese J. The reninangiotensin-aldosterone system in normal 85-year-old people. Scand J Clin Lab Invest. 1987;47(1):69–74. 77. Basso N, Paglia N, Stella I, et  al. Protective effect of the inhibition of the renin-angiotensin system on aging. Regul Pept. 2005;128(3):247–252. 78. Riordan JF, Angiotensin II. biosynthesis, molecular recognition, and signal transduction. Cell Mol Neurobiol. 1995;15(6):637–651. 79. Carey RM. Angiotensin receptors and aging. Hypertension. 2007;50(1):33–34. 80. Shan H, Bai X, Chen X. Angiotensin II induces endothelial cell senescence via the activation of mitogen-activated protein kinases. Cell Biochem Funct. 2008;26(4):459–466. 81. Benigni A, Corna D, Zoja C, et  al. Disruption of the Ang II type 1 receptor promotes longevity in mice. J Clin Invest. 2009;119(3):524–530. 82. Percy CJ, Power D, Gobe GC. Renal ageing: changes in the cellular mechanism of energy metabolism and oxidant handling. Nephrology. 2008;13(2):147–152. 83. Miller JA, Anacta LA, Cattran DC. Impact of gender on the renal response to angiotensin II. Kidney Int. 1999;55(1):278–285. 84. Quan A, Chakravarty S, Chen JK, et  al. Androgens augment proximal tubule transport. Am J Physiol Renal Physiol. 2004;287(3):F452–F459. 85. Zhou XJ, Rakheja D, Yu X, Saxena R, Vaziri ND, Silva FG. The aging kidney. Kidney Int. 2008;74(6):710–720. 86. Rao UV, Wagner Jr. HN. Normal weights of human organs. Radiology. 1972;102(2):337–339. 87. Gourtsoyiannis N, Prassopoulos P, Cavouras D, Pantelidis N. The thickness of the renal parenchyma decreases with age: a CT study of 360 patients. AJR Am J Roentgenol. 1990;155(3):541–544. 88. Lorenz EC, Lieske JC, Vrtiska TJ, et  al. Clinical characteristics of potential kidney donors with asymptomatic kidney stones. Nephrol Dial Transplant. 2011;26(8):2695–2700. 89. Davies DF, Shock NW. Age changes in glomerular filtration rate, effective renal plasma flow, and tubular excretory capacity in adult males. J Clin Invest. 1950;29(5):496–507.

635

90. Rowe JW, Andres R, Tobin JD, Norris AH, Shock NW. The effect of age on creatinine clearance in men: a cross-sectional and longitudinal study. J Gerontol. 1976;31(2):155–163. 91. Lindeman RD, Tobin J, Shock NW. Longitudinal studies on the rate of decline in renal function with age. J Am Geriatr Soc. 1985;33(4):278–285. 92. Neugarten J, Gallo G, Silbiger S, Kasiske B. Glomerulosclerosis in aging humans is not influenced by gender. Am J Kidney Dis. 1999;34(5):884–888. 93. Stachenfeld NS, Splenser AE, Calzone WL, Taylor MP, Keefe DL. Sex differences in osmotic regulation of AVP and renal sodium handling. J Appl Physiol (1985). 2001;91(4):1893–1901. 94. Rule AD, Gussak HM, Pond GR, et  al. Measured and estimated GFR in healthy potential kidney donors. Am J Kidney Dis. 2004;43(1):112–119. 95. Ble A, Fink JC, Woodman RC, et al. Renal function, erythropoietin, and anemia of older persons: the InCHIANTI study. Arch Intern Med. 2005;165(19):2222–2227. 96. Wetzels JF, Kiemeney LA, Swinkels DW, Willems HL, den Heijer M. Age- and gender-specific reference values of estimated GFR in Caucasians: the Nijmegen Biomedical Study. Kidney Int. 2007;72(5):632–637. 97. Lauretani F, Semba RD, Bandinelli S, et al. Plasma polyunsaturated fatty acids and the decline of renal function. Clin Chem. 2008;54(3):475–481. 98. Poggio ED, Rule AD, Tanchanco R, et al. Demographic and clinical characteristics associated with glomerular filtration rates in living kidney donors. Kidney Int. 2009;75(10):1079–1087. 99. Rule AD, Amer H, Cornell LD, et  al. The association between age and nephrosclerosis on renal biopsy among healthy adults. Ann Intern Med. 2010;152(9):561–567. 100. Smith H. The Kidney: Structure and Function in Health and Disease. New York, Oxford: Medical Publications; 1951. 101. Feinfeld DA, Guzik H, Carvounis CP, et al. Sequential changes in renal function tests in the old: results from the Bronx Longitudinal Aging Study. J Am Geriatr Soc. 1995;43(4):412–414. 102. Feinfeld DA, Keller S, Somer B, et al. Serum creatinine and blood urea nitrogen over a six-year period in the very old. Creatinine and BUN in the very old. Geriatr Nephrol Urol. 1998;8(3):131–135. 103. Fehrman-Ekholm I, Skeppholm L. Renal function in the elderly ( > 70 years old) measured by means of iohexol clearance, serum creatinine, serum urea and estimated clearance. Scand J Urol Nephrol. 2004;38(1):73–77. 104. Jiang S, Sun X, Gu H, et al. Age-related change in kidney function, its influencing factors, and association with asymptomatic carotid atherosclerosis in healthy individuals--a 5-year followup study. Maturitas. 2012;73(3):230–238. 105. Berg UB. Differences in decline in GFR with age between males and females. Reference data on clearances of inulin and PAH in potential kidney donors. Nephrol Dial Transplant. 2006;21(9):2577–2582. 106. Cheng HT, Huang JW, Chiang CK, Yen CJ, Hung KY, Wu KD. Metabolic syndrome and insulin resistance as risk factors for development of chronic kidney disease and rapid decline in renal function in elderly. J Clin Endocrinol Metab. 2012;97(4):1268–1276. 107. Tracy RE. The heterogeneity of vascular findings in the kidneys of patients with benign essential hypertension. Nephrol Dial Transplant. 1999;14(7):1634–1639. 108. Kasiske BL. Relationship between vascular disease and age-associated changes in the human kidney. Kidney Int. 1987;31(5):1153–1159. 109. Danziger RS, Tobin JD, Becker LC, Lakatta EE, Fleg JL. The ageassociated decline in glomerular filtration in healthy normotensive

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

636

43.  GENDER AT THE INTERFACE OF RENAL AGING

volunteers. Lack of relationship to cardiovascular performance. J Am Geriatr Soc. 1990;38(10):1127–1132. 110. Han L, Bai X, Lin H, Sun X, Chen XM. Lack of independent relationship between age-related kidney function decline and carotid intima-media thickness in a healthy Chinese population. Nephrol Dial Transplant. 2010;25(6):1859–1865. 111. Baggio B, Budakovic A, Perissinotto E, et  al. Atherosclerotic risk factors and renal function in the elderly: the role of hyperfibrinogenaemia and smoking. Results from the Italian Longitudinal Study on Ageing (ILSA). Nephrol Dial Transplant. 2005;20(1):114–123. 112. Edwards MS, Wilson DB, Craven TE, et al. Associations between retinal microvascular abnormalities and declining renal function in the elderly population: the Cardiovascular Health Study. Am J Kidney Dis. 2005;46(2):214–224. 113. Shlipak MG, Katz R, Kestenbaum B, Fried LF, Siscovick D, Sarnak MJ. Clinical and subclinical cardiovascular disease and kidney function decline in the elderly. Atherosclerosis. 2009;204(1):298–303. 114. Chonchol M, Gnahn H, Sander D. Impact of subclinical carotid atherosclerosis on incident chronic kidney disease in the elderly. Nephrol Dial Transplant. 2008;23(8):2593–2598. 115. Hoppe CC, Evans RG, Bertram JF, Moritz KM. Effects of dietary protein restriction on nephron number in the mouse. Am J Physiol Regul Integr Comp Physiol. 2007;292(5):R1768–R1774. 116. Baylis C, Corman B. The aging kidney: insights from experimental studies. J Am Soc Nephrol. 1998;9(4):699–709. 117. Zdunek M, Silbiger S, Lei J, Neugarten J. Protein kinase CK2 mediates TGF-beta1-stimulated type IV collagen gene transcription and its reversal by estradiol. Kidney Int. 2001;60(6):2097–2108. 118. Metcalfe PD, Leslie JA, Campbell MT, Meldrum DR, Hile KL, Meldrum KK. Testosterone exacerbates obstructive renal injury by stimulating TNF-alpha production and increasing proapoptotic and profibrotic signaling. Am J Physiol Endocrinol Metab. 2008;294(2):E435–E443. 119. Xu Q, Prabhu A, Xu S, Manigrasso MB, Maric C. Dosedependent effects of dihydrotestosterone in the streptozotocin-induced diabetic rat kidney. Am J Physiol Renal Physiol. 2009;297(2):F307–F315. 120. Nyengaard JR, Bendtsen TF. Glomerular number and size in relation to age, kidney weight, and body surface in normal man. Anat Rec. 1992;232(2):194–201. 121. Hoy WE, Douglas-Denton RN, Hughson MD, Cass A, Johnson K, Bertram JF. A stereological study of glomerular number and volume: preliminary findings in a multiracial study of kidneys at autopsy. Kidney Int Suppl. 2003;83:S31–S37. 122. Tsuboi N, Kawamura T, Miyazaki Y, Utsunomiya Y, Hosoya T. Low glomerular density is a risk factor for progression in idiopathic membranous nephropathy. Nephrol Dial Transplant. 2011;26(11):3555–3560. 123. Rule AD, Semret MH, Amer H, et  al. Association of kidney function and metabolic risk factors with density of glomeruli on renal biopsy samples from living donors. Mayo Clinic Proc. 2011;86(4):282–290. 124. Newbold KM, Sandison A, Howie AJ. Comparison of size of juxtamedullary and outer cortical glomeruli in normal adult kidney. Virchows Arch A Pathol Anat Histopathol. 1992;420(2):127–129. 125. Baylis C. Age-dependent glomerular damage in the rat. Dissociation between glomerular injury and both glomerular hypertension and hypertrophy. Male gender as a primary risk factor. J Clin Invest. 1994;94(5):1823–1829. 126. Yabuki A, Tanaka S, Matsumoto M, Suzuki S. Morphometric study of gender differences with regard to age-related changes in the C57BL/6 mouse kidney. Exp Anim. 2006;55(4):399–404.

127. Musso CG, Oreopoulos DG. Aging and physiological changes of the kidneys including changes in glomerular filtration rate. Nephron Physiol. 2011;119(Suppl 1):1–5. 128. Wharram BL, Goyal M, Wiggins JE, et  al. Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene. J Am Soc Nephrol. 2005;16(10):2941–2952. 129. Smeets B, Uhlig S, Fuss A, et al. Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells. J Am Soc Nephrol. 2009;20(12):2604–2615. 130. Brenner BM. Hemodynamically mediated glomerular injury and the progressive nature of kidney disease. Kidney Int. 1983;23(4):647–655. 131. Tan JC, Workeneh B, Busque S, Blouch K, Derby G, Myers BD. Glomerular function, structure, and number in renal allografts from older deceased donors. J Am Soc Nephrol. 2009;20(1): 181–188. 132. Gava AL, Freitas FP, Meyrelles SS, Silva IV, Graceli JB. Genderdependent effects of aging on the kidney. Braz J Med Biol Res. 2011;44(9):905–913. 133. Lombet JR, Adler SG, Anderson PS, Nast CC, Olsen DR, Glassock RJ. Sex vulnerability in the subtotal nephrectomy model of glomerulosclerosis in the rat. J Lab Clin Med. 1989;114(1):66–74. 134. Pawluczyk IZ, Tan EK, Harris KP. Rat mesangial cells exhibit sex-specific profibrotic and proinflammatory phenotypes. Nephrol Dial Transplant. 2009;24(6):1753–1758. 135. Zheng F, Plati AR, Potier M, et  al. Resistance to glomerulosclerosis in B6 mice disappears after menopause. Am J Pathol. 2003;162(4):1339–1348. 136. Blush J, Lei J, Ju W, Silbiger S, Pullman J, Neugarten J. Estradiol reverses renal injury in Alb/TGF-beta1 transgenic mice. Kidney Int. 2004;66(6):2148–2154. 137. Silbiger S, Lei J, Neugarten J. Estradiol suppresses type I collagen synthesis in mesangial cells via activation of activator protein-1. Kidney Int. 1999;55(4):1268–1276. 138. Sun J, Langer WJ, Devish K, Lane PH. Compensatory kidney growth in estrogen receptor-alpha null mice. Am J Physiol Renal Physiol. 2006;290(2):F319–F323. 139. Lindeman RD, Goldman R. Anatomic and physiologic age changes in the kidney. Exp Gerontol. 1986;21(4-5):379–406. 140. Nath KA. Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis. 1992;20(1):1–17. 141. Piepsz A. New renal scarring and age. Nuclear Med Commun. 2001;22(12):1273–1275. 142. Tanaka T, Kato H, Kojima I, et  al. Hypoxia and expression of hypoxia-inducible factor in the aging kidney. J Gerontol Biol Sci Med Sci. 2006;61(8):795–805. 143. Baert L, Steg A. Is the diverticulum of the distal and collecting tubules a preliminary stage of the simple cyst in the adult? J Urol. 1977;118(5):707–710. 144. Tada S, Yamagishi J, Kobayashi H, Hata Y, Kobari T. The incidence of simple renal cyst by computed tomography. Clin Radiol. 1983;34(4):437–439. 145. Fliser D, Franek E, Joest M, Block S, Mutschler E, Ritz E. Renal function in the elderly: impact of hypertension and cardiac function. Kidney Int. 1997;51(4):1196–1204. 146. Epstein M, Hollenberg NK. Age as a determinant of renal sodium conservation in normal man. J Lab Clin Med. 1976;87(3):411–417. 147. Crane MG, Harris JJ. Effect of aging on renin activity and aldosterone excretion. J Lab Clin Med. 1976;87(6):947–959. 148. Baylis C. Renal responses to acute angiotensin II inhibition and administered angiotensin II in the aging, conscious, chronically catheterized rat. Am J Kidney Dis. 1993;22(6):842–850.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

149. Ohashi M, Fujio N, Nawata H, et  al. High plasma concentrations of human atrial natriuretic polypeptide in aged men. J Clin Endocrinol Metab. 1987;64(1):81–85. 150. Brunette MG, Leclerc M. Effect of estrogen on calcium and sodium transport by the nephron luminal membranes. J Endocrinol. 2001;170(2):441–450. 151. Nielsen CB, Flyvbjerg A, Bruun JM, Forman A, Wogensen L, Thomsen K. Decreases in renal functional reserve and proximal tubular fluid output in conscious oophorectomized rats: normalization with sex hormone substitution. J Am Soc Nephrol. 2003;14(12):3102–3110. 152. Pechere-Bertschi A, Burnier M. Female sex hormones, salt, and blood pressure regulation. Am J Hypertens. 2004;17(10):994–1001. 153. Perazella MA, Mahnensmith RL. Hyperkalemia in the elderly: drugs exacerbate impaired potassium homeostasis. J Gen Inter Med. 1997;12(10):646–656. 154. Sands JM. Urine-concentrating ability in the aging kidney. Sci Aging Knowledge Environ. 2003;2003(24):PE15. 155. Kirkland JL, Lye M, Levy DW, Banerjee AK. Patterns of urine flow and electrolyte excretion in healthy elderly people. Br Med J. 1983;287(6406):1665–1667. 156. Zhou XJ, Saxena R, Liu Z, Vaziri ND, Silva FG. Renal senescence in 2008: progress and challenges. Int Urol Nephrol. 2008;40(3):823–839. 157. Tian Y, Serino R, Verbalis JG. Downregulation of renal vasopressin V2 receptor and aquaporin-2 expression parallels ageassociated defects in urine concentration. Am J Physiol Renal physiol. 2004;287(4):F797–F805. 158. Combet S, Geffroy N, Berthonaud V, et  al. Correction of age-related polyuria by dDAVP: molecular analysis of aquaporins and urea transporters. Am J Physiol Renal physiol. 2003;284(1):F199–F208. 159. Perucca J, Bouby N, Valeix P, Bankir L. Sex difference in urine concentration across differing ages, sodium intake, and level of kidney disease. Am J Physiol Regul Integr Comp Physiol. 2007;292(2):R700–R705. 160. Share L, Crofton JT, Ouchi Y. Vasopressin: sexual dimorphism in secretion, cardiovascular actions and hypertension. Am J Med Sci. 1988;295(4):314–319. 161. Luckey AE, Parsa CJ. Fluid and electrolytes in the aged. Arch Surg. 2003;138(10):1055–1060. 162. Nakhoul F, Carlos Z, Levin M, Gteen J, Winavet J, Better OS. Urinary acidification capacity in the elderly. Geriatr Nephrol Urol. 1995;5(3):149–155. 163. Alpern RJ. Trade-offs in the adaptation to acidosis. Kidney Int. 1995;47(4):1205–1215. 164. Hollenberg NK, Adams DF, Solomon HS, Rashid A, Abrams HL, Merrill JP. Senescence and the renal vasculature in normal man. Circ Res. 1974;34(3):309–316. 165. Brenner BM, Lawler EV, Mackenzie HS. The hyperfiltration theory: a paradigm shift in nephrology. Kidney Int. 1996;49(6):1774–1777. 166. Hoang K, Tan JC, Derby G, et  al. Determinants of glomerular hypofiltration in aging humans. Kidney Int. 2003;64(4):1417–1424. 167. Ljungqvist A, Lagergren C. Normal intrarenal arterial pattern in adult and ageing human kidney. A microangiographical and histological study. J Anat. 1962;96:285–300. 168. Fuiano G, Sund S, Mazza G, et al. Renal hemodynamic response to maximal vasodilating stimulus in healthy older subjects. Kidney Int. 2001;59(3):1052–1058. 169. Song J, Kost Jr. CK, Martin DS. Androgens augment renal vascular responses to ANG II in New Zealand genetically hypertensive rats. Am J Physiol Regul Integr Comp Physiol. 2006;290(6):R1608–R1615.

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170. Reckelhoff JF, Zhang H, Granger JP. Testosterone exacerbates hypertension and reduces pressure-natriuresis in male spontaneously hypertensive rats. Hypertension. 1998;31(1 Pt 2):435–439. 171. Passmore JC, Joshua IG, Rowell PP, Tyagi SC, Falcone JC. Reduced alpha adrenergic mediated contraction of renal preglomerular blood vessels as a function of gender and aging. J Cell Biochem. 2005;96(4):672–681. 172. Wangensteen R, Moreno JM, Sainz J, et  al. Gender difference in the role of endothelium-derived relaxing factors modulating renal vascular reactivity. Eur J Pharmacol. 2004;486(3):281–288. 173. Gallagher JC, Rapuri PB, Smith LM. An age-related decrease in creatinine clearance is associated with an increase in number of falls in untreated women but not in women receiving calcitriol treatment. J Clin Endocrinol Metab. 2007;92(1):51–58. 174. Van Pottelbergh G, Mathei C, Vaes B, Adriaensen W, Gruson D, Degryse JM. The influence of renal function on vitamin D metabolism in the very elderly. J Nutr Health Aging. 2013;17(2):107–111. 175. Ravani P, Malberti F, Tripepi G, et al. Vitamin D levels and patient outcome in chronic kidney disease. Kidney Int. 2009;75(1):88–95. 176. Gallagher JC, Rapuri P, Smith L. Falls are associated with decreased renal function and insufficient calcitriol production by the kidney. J Steroid Biochem Mol Biol. 2007;103(3-5):610–613. 177. Ershler WB, Sheng S, McKelvey J, et  al. Serum erythropoietin and aging: a longitudinal analysis. J Am Geriatr Soc. 2005;53(8):1360–1365. 178. Ferrucci L, Guralnik JM, Bandinelli S, et al. Unexplained anaemia in older persons is characterised by low erythropoietin and low levels of pro-inflammatory markers. Br J Haematol. 2007;136(6):849–855. 179. Qian H, Luo N, Chi Y. Aging-shifted prostaglandin profile in endothelium as a factor in cardiovascular disorders. J Aging Res. 2012;2012:121390. 180. Hornych A, Forette F, Bariety J, Krief C, Aumont J, Paris M. The influence of age on renal prostaglandin synthesis in man. Prostaglandins leukot Essent Fatty Acids. 1991;43(3):191–195. 181. Millatt LJ, Siragy HM. Age-related changes in renal cyclic nucleotides and eicosanoids in response to sodium intake. Hypertension. 2000;35(2):643–647. 182. Castellani S, Ungar A, Cantini C, et al. Impaired renal adaptation to stress in the elderly with isolated systolic hypertension. Hypertension. 1999;34(5):1106–1111. 183. Knight EL, Stampfer MJ, Hankinson SE, Spiegelman D, Curhan GC. The impact of protein intake on renal function decline in women with normal renal function or mild renal insufficiency. Ann Int Med. 2003;138(6):460–467. 184. Hasegawa E, Tsuchihashi T, Ohta Y. Prevalence of chronic kidney disease and blood pressure control status in elderly hypertensive patients. Intern Med. 2012;51(12):1473–1478. 185. Ohta Y, Tsuchihashi T, Kiyohara K, Oniki H. High salt intake promotes a decline in renal function in hypertensive patients: a 10-year observational study. Hypertension Res. 2013;36(2):172–176. 186. Reckelhoff JF, Kellum Jr. JA, Racusen LC, Hildebrandt DA. Long-term dietary supplementation with L-arginine prevents age-related reduction in renal function. Am J Physiol. 1997;272(6 Pt 2):R1768–R1774. 187. Cai W, He JC, Zhu L, et  al. Reduced oxidant stress and extended lifespan in mice exposed to a low glycotoxin diet: association with increased AGER1 expression. Am J Pathol. 2007;170(6):1893–1902. 188. Cai W, He JC, Zhu L, et al. Oral glycotoxins determine the effects of calorie restriction on oxidant stress, age-related diseases, and lifespan. Am J Pathol. 2008;173(2):327–336. 189. McKiernan SH, Tuen VC, Baldwin K, Wanagat J, Djamali A, Aiken JM. Adult-onset calorie restriction delays the

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

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190.

191.

192.

193.

194.

195.

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accumulation of mitochondrial enzyme abnormalities in aging rat kidney tubular epithelial cells. Am J Physiol Renal physiol. 2007;292(6):F1751–F1760. Lee JH, Jung KJ, Kim JW, Kim HJ, Yu BP, Chung HY. Suppression of apoptosis by calorie restriction in aged kidney. Expl Gerontol. 2004;39(9):1361–1368. Keenan KP, Coleman JB, McCoy CL, Hoe CM, Soper KA, Laroque P. Chronic nephropathy in ad libitum overfed SpragueDawley rats and its early attenuation by increasing degrees of dietary (caloric) restriction to control growth. Toxicol Pathol. 2000;28(6):788–798. Meyer TE, Kovacs SJ, Ehsani AA, Klein S, Holloszy JO, Fontana L. Long-term caloric restriction ameliorates the decline in diastolic function in humans. J Am Coll Cardiol. 2006;47(2):398–402. de Cavanagh EM, Piotrkowski B, Basso N, et al. Enalapril and losartan attenuate mitochondrial dysfunction in aged rats. FASEB J. 2003;17(9):1096–1098. Zhou XJ, Vaziri ND, Zhang J, Wang HW, Wang XQ. Association of renal injury with nitric oxide deficiency in aged SHR: prevention by hypertension control with AT1 blockade. Kidney Int. 2002;62(3):914–921. Yang HC, Deleuze S, Zuo Y, Potthoff SA, Ma LJ, Fogo AB. The PPARgamma agonist pioglitazone ameliorates aging-related progressive renal injury. J Am Soc Nephrol. 2009;20(11):2380–2388.

196. Neugarten J, Acharya A, Lei J, Silbiger S. Selective estrogen receptor modulators suppress mesangial cell collagen synthesis. Am J Physiol Renal Physiol. 2000;279(2):F309–F318. 197. Dixon A, Wells CC, Singh S, Babayan R, Maric C. Renoprotective effects of a selective estrogen receptor modulator, raloxifene, in an animal model of diabetic nephropathy. Am J Nephrol. 2007;27(2):120–128. 198. Hadjadj S, Gourdy P, Zaoui P, et  al. Effect of raloxifene -- a selective oestrogen receptor modulator -- on kidney function in post-menopausal women with Type 2 diabetes: results from a randomized, placebo-controlled pilot trial. Diabet Med. 2007;24(8):906–910. 199. Ettinger B, Black DM, Mitlak BH, et  al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators. JAMA. 1999;282(7):637–645. 200. Melamed ML, Blackwell T, Neugarten J, et  al. Raloxifene, a selective estrogen receptor modulator, is renoprotective: a posthoc analysis. Kidney Int. 2011;79(2):241–249.

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C H A P T E R

44 Gender Differences in Mobility of Elderly: Measurements and Interventions to Improve Mobility Emil Jovanov, Karen H. Frith, Priyanka Madhushri, Amy Hunter, Sharon Saunderson Coffey and Aleksandar Milenkovic The University of Alabama in Huntsville, Huntsville, AL, United States

O U T L I N E 44.1 Introduction

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44.4 Discussion

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44.1 INTRODUCTION Mobility is commonly defined as the ability to move in everyday life, but it can be more precisely defined as “the purposeful physical movements, including gross simple movements, fine complex movements, and coordination.”1 Mobility in daily life depends on the ability of the neural system to coordinate the movements of the musculoskeletal system. Functional mobility can be defined as the manner in which people are able to move around in the environment in order to participate in the Activities of Daily Living (ADLs) and move from place to place.2 Functional mobility is essential for any person in order to engage in physical activities at home, work, school, and in communities. This engagement contributes to better health and ultimately a better quality of life.

Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00054-1

References 651

Mobility is made up of many individual components that are both independent as well as interdependent. Mobility depends on factors such as flexibility of muscles, range of motion of joints, balance, and limb strength. From the physiological standpoint, mobility is a result of the effective interactive functioning of the musculoskeletal, neural, sensory, and cardiorespiratory systems. Common health conditions can disrupt these systems and impair mobility. Colon-Emeric and colleagues at Duke University Medical Center described the most common health conditions that may contribute to functional mobility decline or functional disability including cardiovascular disease, diabetes, obesity, cancer, dementia, changes in vision, and fractures.3 The authors state that the coexistence of two or more health conditions often contributes to a decrease in mobility. As the number of impairments increases from one to four,

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the percentage of persons reporting functional mobility decline increases exponentially (7 to 14% to 28 to 60%). Mobility and balance decline with age; beginning at the age of 40 years, balance, vision, and limb strength, all which affect mobility, begin to decline.4 Butler and colleagues studied age differences in seven tests of functional mobility. Their study included 50 participants aged 20–39 years and 684 older participants aged 75–98 years. After completing seven mobility measures on all participants, results showed that older participants performed significantly worse than younger participants in all the tests. Reduced mobility significantly increases the risk of falls. Studies suggest that more than 30% of Americans over 65 fall each year.5 Falls result in more than 2.5 million injuries treated in emergency departments annually.6 Every 13 s, an older adult is treated in the emergency department for a fall, and every 20 m an older adult dies from a fall.6 Falls are the third leading cause of worldwide chronic disability.5 More than 90% of hip fractures are caused by falls only.7 Furthermore, falls create fear of falling again and loss of self-confidence that in turn increase the probability of future falls.8 Falls among older adults are a major public health concern that contributes significantly to decreased mobility and financial burden.9 Loss of mobility and falls rob people of their overall health, leading to increased fragility and premature death.10 Falls significantly impede the life of aging adults by causing unnecessary, early dependence on caregivers and a reduced quality of life.11 In particular, falls reduce the participation of the elderly in social, cultural, and health related activities, all of which can lead to depression and withdrawal from meaningful activities. In essence, falls can inhibit the elderly person who will then have difficulty completing ADLs independently.12 The Center for Disease Control (CDC) created the STEADI (Stopping Elderly Accidents, Deaths & Injuries)

tool kit for health care providers.13 The CDC recommends the evaluation of gait, strength, and balance using three tests: Timed Up and Go (TUG) test,14 30-Second Chair Stand (30SCS) test,15 and the 4-Stage Balance Test (4SBT).16 The detailed description of the TUG test is given in Section 44.2.1. The 30SCS test measures the number of stand-ups a person can perform during a 30 s interval.17,18 The primary goal of this test is to measure lower extremity strength and endurance by correlating it with number of stand-ups in 30 s, but it can also indicate speed, balance, as well as mobility impairments.19–21 The test is conducted using a straight back chair with no arms, and a stopwatch. The subject sits in the middle of the chair with feet flat on the floor; hands placed on opposite shoulders and crossed at the wrists. The 4SBT test is used to assess static balance of elderly people during four progressively more challenging standing positions. In this test, the subject is not allowed to use any assistive devices and should keep his/her eyes open during the test positions. The four respective positions of the 4SBT are described as follows: (1) Feet together stand assumes feet are placed side by side; (2) Semitandem stand assumes that the instep of one foot is touching the big toe of the other foot; (3) Tandem stand assumes that one foot is placed in front of the other— heel of one foot touches the toe of other foot; and (4) One leg stand assumes standing on one leg. If the subject can hold a position for 10 s without moving his/her feet or needing support, the test advances to the next position. The standard test outcomes are times in seconds the subject is able to hold each of four positions. Table 44.1 shows objective scores of the standard mobility tests. Studies have consistently shown gender-related gaps in mobility and prevalence of falls.22,23 Men and women have disparities in their physical activities because of

TABLE 44.1  Objective Scores of the Standard Mobility Tests Test

Age

Men

Women

Below avg

4-Stage Balance Test

Above avg

10 s in each position

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different physical characteristics and social behavior. It has been shown that women are less physically active and have weaker lower body strength as compared to men.24–26 The mobility gap is particularly large in the countries where women have different roles and opportunities in society. 27–34 For example, housewives tend to have lower mobility leading to disability in later life.28 Women are at a higher risk for obesity during late life than men. This can be linked to their lower physical activity and lower metabolic rate than men.35,36 The higher prevalence of arthritis in women is also a reason for higher risk of mobility disability in old age.37 As a consequence, women are more likely than men to report a fall and seek medical care for fall-related injuries.38,39 However, fatal fall rates are higher in men as compared to women.40 Mechakra-Tahiri and colleagues conducted a study across 70 countries aimed at assessing the gender gap in mobility.41 Population-based data were used from the World Health Survey and included 276,647 adults aged 18 years and older that were recruited from six world regions. Mobility was measured using the World Health Survey that asked participants the level of difficulty they had in moving around to assess the level of difficulty in overall physical movement in the last 30-day on a twopoint scale: difficult or not difficult. Results from this study showed that women were more likely than men to report mobility difficulty (38% vs 27%, p < .0001). There were notable differences in reported mobility by geographic regions. The regions with different social roles and work opportunities between men and women (Morocco, Pakistan, Tunisia, United Arab Emirates) showed the largest gender gap in mobility, whereas the Western Pacific region (Australia, China, Lao People’s Democratic Republic, Malaysia, Philippines, Viet Nam) had the smallest gender gap in mobility. The researchers speculated that the differences in mobility across the regions of the world were related to the degree to which men and women had equal access to the same societal roles and work opportunities. The researchers recommended further studies to clarify the causes and nature of gender gaps in mobility.41 Several mobility studies have been conducted to quantify mobility gap between men and women.4,25,42 Butler et  al. performed a number of functional mobility tests on older adults and found that older women performed worse than older men in all of the tests. The functional tests include coordinated stability, near tandem balance, walking speed during 6-m walk, sitto-stand transition, alternatively placing left and right step, and stair ascent and descent.4 Steffen et  al. performed gender-related test among community-dwelling older adults, analyzing the TUG test, 6-min walk test, and gait speeds.42 They reported that the women were likely to have higher (11 ± 3 s) TUG times than men

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(10 ± 1 s) in the same age range of 80–89 years. The mean distance traveled by women was lower (392 ± 85 m) than men (417 ± 73 m) during 6-min walking test for the same subjects. The comfortable gait speed for men was higher (1.21 ± 0.18 m/s) than women (1.15 ± 0.21 m/s). The fast gait speed was also higher for men (1.65 ± 0.24 m/s) than for women (1.59 ± 0.28 m/s).42 In a retrospective study, Wei and Hester examined gender differences in falls associated with decreased mobility among adults treated in US emergency departments from 2007 to 2012.43 The study found that falls resulting in injuries increased with age for females aged ≥18 years. For males, these rates declined, reaching the lowest point at age 65–74 years, then increased again.43 In their study, Levielle and her colleagues began with the assumption found in other studies that gender differences in declining mobility seen in older adults exists and that the transitions that contribute to declining mobility include disability incidence, recovery from disability, and mortality as each variable is related to age.44 The sample size was 10,263 persons aged 65–95 years, and it included older men and women from communities of East Boston, Massachusetts, New Haven, Connecticut, and two counties in Iowa. The authors examined the gender differences in the prevalence of declining mobility in older adults according to the influences of three components: disability incidence, recovery from disability, and mortality.44 Outcomes showed that as women aged, their decline in mobility and prevalence of disability increased from 22% at age 70 to 81% at age 90 years. In the men, 15% had a decline in mobility at age 70 which increased to 57% at age 90. The researchers found a higher prevalence of disability in women compared to men. When disability from declining mobility occurs, elderly individuals can lose their independence leading to function-related dependence, pain, and insecurity. Aberg studied possible gender differences; she found that elderly women admitted for rehabilitation had a higher degree of function-related dependence, pain, and insecurity than men, but these features could be diminished with rehabilitation strategies.45 The results from this study confirm previous findings that older women were more likely to fall and cause disability or suffer from fall-related injuries than senior men.38,46 A study by Chang and Minh examined the prevalence of falls by gender and sociodemographic, lifestyle/behavioral, and medical factors.47 Interestingly, the authors also discovered that while women are at greater risk for falls, mortality rates from falls were found to be higher among men. Similar to other studies, this study found that fall prevalence increased with age among both genders. They conclude that this might contribute to age-induced declines in factors related to mobility such as decreased physical, sensory, and

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44.  Gender Differences in Mobility of Elderly: Measurements and Interventions to Improve Mobility

cognitive function, as well as an increase in the number of chronic health conditions.48,49 O and El Fakiri have investigated gender differences of risk factors affecting mobility and have found that ADL limitations and fear of falling appear to be stronger fall risk factors for men than for women.50 Stevens and colleagues found that of the 328 participants from Dane County, WI in the Safety Assessment for Elders (SAFE) study women aged 85 and older were most likely to be injured from a fall occurring at home.51 In contrast, a study conducted by Duckham and colleagues found that men had higher rates of outdoor falls, in locations of recreation, and on snowy or icy surfaces compared to women.52 Women in this study had higher rates of falls in the kitchen and while performing household activities. Despite the location of the falls, this study found in the participants of 65 years and older (N = 743), that both genders sustained equivalent injuries when a fall and immobility occurred in the outdoors, however, in all falls, women had twice as many injuries than did the men.52 Staying active as we age helps improve strength, balance, and overall mobility. It has been shown that regular exercise and physical activity promote mobility and protect against the negative effects of muscle and joint stiffness, muscle loss, bone density loss, and decrease in balance in both men and women.53 Staying active can also reduce the risk of cardiovascular disease, help fight depression, and aid in the protection of memory from cognitive decline, dementia, and Alzheimer’s disease.54,55 Several studies have recognized an association between physical activity levels at younger ages and maintenance of mobility in older age. For example, one study reviewed 679 participants whose average age was 80 years and found that those who regularly exercised from ages 20 to 64 had significantly better mobility in older age; physical activity was also linked to better grip strength and walking speed in older men but not in women.56 Individuals with weakness of lower extremities and balance problems are at risk for falls. Even individuals who have a history of falls benefit from progressive exercise and balance training program.57 It is important that exercise program focuses on increasing leg strength and improving balance, as exercise becomes progressively more challenging over time. The CDC has compiled findings from clinical trials of exercise and balance training, home-based modifications, and multifaceted interventions. Fall prevention strategies found to be effective include exercise and balance training.57 Exercise and balance strategies can be implemented as group-based exercise programs in the community or at home, but better outcomes are achieved in a community-based setting.58 In this chapter we present a smartphone-based TUG test for automated quantitative assessment of mobility that we developed, and the results of an intervention

and exercise program designed for older participants. Section 44.2 presents the automated quantitative assessment of mobility. In Section 44.3 we present the study of mobility of older adults participating in the Balance for Life intervention program that is designed to improve mobility and decrease falls of elderly. Section 44.4 discusses gender-specific effects of fall prevention program that utilizes our application suite for mobility assessment.

44.2  AUTOMATED ASSESSMENT OF MOBILITY The advancements in sensor and wearable technologies have created new opportunities for automatic assessment of mobility. The revolution in wearable technologies, smartphones, cloud computing, and advanced communication capabilities provide support for ubiquitous health and activity monitoring applications. There are a variety of commercially available wearable sensors including inertial, environmental, and optical sensors that can be used for activity logging during specialized mobility assessment tests or during ADLs. These sensors effectively assess and quantify mobility.59–66 To observe the body movements, a single or multiple inertial sensors can be mounted on the different locations of the body, typically on the chest, waist, or legs. The sensors can also be embedded in shoes to monitor activity throughout the day.67,68 The modern off-the-self smartphones include a number of built-in inertial and location sensors that can be utilized directly in activity monitoring applications. The rapid proliferation of smartphones and constant improvement of their performance as well as performance of sensors embedded in the smartphone, make it a very attractive and readily available option for quantification of mobility. The major smartphone operating systems such as Android and iOS provide built-in programming interfaces that enable continual sampling of sensor data and reliable data acquisition on the smartphone. The importance of mobile sensors inspired Apple to integrate the first motion coprocessor Apple M7 in their mobile devices in 2013.69 New generations of mobile processors (M8 and M9) are now a standard feature of Apple’s mobile devices. Their function is to collect sensor data from integrated accelerometers, gyroscopes, and compasses and offload the collecting and processing of sensor data from the main central processing unit. Assessment of physical activity became a standard feature of mobile operating systems (e.g., iOS automatically monitors the number of steps throughout the day). In addition, smartphone applications can process signals from a smartphone’s accelerometer and gyroscope to extract parameters that quantify mobility during tests.70–77 Wireless technologies such as Bluetooth and WiFi have

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44.2  Automated Assessment of Mobility

made wearable sensors and smartphones suitable for real time activity monitoring and improve user convenience during monitoring. We have been developing mobile health (mHealth) applications for automated ubiquitous monitoring of user’s during their daily activities.78 Recent developments of cloud computing make it even easier to provide support for data sharing and storage for longterm analysis. Instrumented mobility assessments using these technologies is a very active research topic that has the potential to provide insights about a subject’s mobility and health status during longitudinal monitoring.79

44.2.1  The Timed Up and Go Test The TUG is a frequently used clinical test for assessing balance, mobility, and fall risk in elderly population and for people with Parkinson’s disease.80,81 This is a simple test and easy to administer in a clinical setting and thus often used in screening protocols. The test is typically conducted by a nurse and the required equipment includes a standard armchair and a stopwatch. A marker is placed on the floor, 3 m away from the chair. Subjects wear regular footwear and can use a walking aid if needed. The subject is sitting back in the chair and the nurse explains the test. On the command “Go” the subject stands up from the chair, walks to the marker on the floor at his or her normal pace, turns around, walks back to the chair at his or her normal pace, and sits down again. Fig. 44.1 shows the different phases of the TUG test. The nurse starts time measurement on a stopwatch on the command “Go” and stops the stopwatch after the subjects has sat down in the chair. The test’s quantitative outcome is the time in seconds. An older adult who takes 12 s or more to complete the TUG test is considered at high risk of falling.14 The nurse fills a form with the TUG time. In addition, she qualitatively assesses the subject’s postural stability, gait, length stride, and sway and checks the following indicators if they apply: slow tentative pace, loss of balance, short strides, little or no

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arm swing, steadying self on walls, shuffling, en bloc turning, or improper use of assistive device. The standard mobility tests recommended by CDC are generally performed in a clinical setting, thus limiting the frequency of these tests and increasing the total costs of conducting the tests. In the near future, an increasing number of geriatric patients will need fall risk assessment because of the demographic change. The standard assessment is subjective and prone to errors. For example, if the subject loses balance a nurse would not be able to accurately measure the duration of the test. The standard tests use only limited parameters to quantify mobility and fall risks. New diagnostic procedures for mobility and fall risk assessment are needed to address these issues, and can be developed using available sensing and processing platforms. New systems would be able to monitor mobility at home as well as in clinics. The assessment parameters should be comprehensive for accurate assessment of fall risks and provide cost-effective and clinically applicable methods for accurate quantification of mobility.

44.2.2 iTUG Whereas the TUG test has been proven valuable in early assessment of balance and mobility, it is limited as its only outcome is the time to complete the test. An instrumented Timed Up and Go (iTUG) test was recently introduced.79,82 In this test, subjects are monitored by a dedicated device designed for gait and movement analysis that relies on inertial sensors, typically a three-dimensional accelerometer. The device is typically mounted on the subject’s lower back and can record x, y, and z acceleration components during the TUG test. The data are later analyzed off-line to parameterize the TUG test. A number of additional parameters can be derived that can better indicate gait and balance impairments, including duration of the Sit-to-Stand phase, Stand-toSit duration, the amplitude range of anterior–posterior acceleration, and others. iTUG has proven to be sensitive to pathologies79,82 and useful in fall risk prediction.83

FIGURE 44.1  Time Up and Go (TUG) test phases.

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FIGURE 44.2  System architecture of the Smart Time Up and Go (sTUG) application.

The iTUG relies on a specialized device for motion analysis and off-line data analysis to extract parameters of interest. Fortunately, modern smartphones integrate a growing number of inertial and location sensors, such as an accelerometer, gyroscope, and magnetometer (digital compass). The accelerometer measures proper acceleration and is typically used to keep the screen upright regardless of the smartphone orientation. The gyroscope measures angular movements, specifically the rotation around the x axis (a.k.a. roll), y axis (yaw), and z axis (pitch). The magnetic sensor is sensing the Earth’s magnetic field and is normally used to aid navigation by smartphone’s orientation determined relative to the Earth’s magnetic field. Major mobile operating systems, such as Android, iOS, and Windows 8 support frameworks for managing the sensors, including continual sampling, thus enabling a wide variety of new mobile sensing applications in different domains. Mellone et al. evaluated suitability of a smartphone’s built-in accelerometer for the iTUG.84 They compared the subject’s anteroposterior acceleration measured concurrently on a smartphone and a state-of-the-art device for movement analysis, and found the statistical agreement between the two. However, this study did not pursue a more ambitious goal of using smartphone application for quantifying the TUG test. Milosevic et  al.70,78 introduced an Android smartphone application called sTUG that stands for Smartphone Timed Up and Go (sTUG). The sTUG application completely automates the TUG test offering a modern, affordable, and easy to use information-technology based tool for continual mobility assessment in clinical and ambulatory settings.

44.2.3 sTUG We developed a sTUG application to automate and quantify the TUG test in clinical and home settings. Fig. 44.2 illustrates the architecture of the system. The system requires an Android smartphone with built-in accelerometer, gyroscope, and orientation sensors running Android 2.3 or above. Smartphone applications record and process signals from the sensors, extract parameters, and display results on the screen. The applications stop monitoring automatically after the completion of the test. They create test descriptors that include date and time when the tests are taken and all parameters that quantify the tests. The tests descriptors are stored in a text file on the smartphone and can be uploaded to an mHealth server via the Internet for long-term storage and analysis. The smartphone is mounted on the subject’s chest or back before the test starts. The smartphone is typically placed in a holder with two elastic textile straps, so it remains fixed relative to the trunk position during the test. Alternatively, the smartphone may be placed in a textile holder that is worn around the subject’s neck and attached to the clothes using a Velcro strap. The z axis corresponds to the sagittal axis, the y axis corresponds to the longitudinal axis, and the x axis corresponds to the frontal axis of the human body. On application start, it provides options to setup configuration and enter subject’s ID or create a new subject. The initial screen is presented in Fig. 44.3A. When the “Start” button on the screen is pressed, the application starts recording and processing data from in-built

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44.2  Automated Assessment of Mobility

(A)

(B)

(C)

FIGURE 44.3  sTUG Doctor smartphone application screens. (A) Application screen before the test is started; (B) Application screen with a selected set of parameters upon completion of a test; (C) Application screen showing a log with multiple measurements for a single subject.

inertial sensors to calculate TUG test parameters in real time. The application starts measuring time from the detection of a sit-to-stand transition during the test. The application stops automatically after it detects a standto-sit transition and important parameters are calculated and displayed instantly on the screen as shown in Fig. 44.3B. sTUG quantifies all the phases of the TUG test and extracted parameters are listed in Table 44.2. These parameters include the total TUG duration, sit-to-stand duration, stand-to-sit duration, maximum lean forward angle, maximum angular velocity, and the total number of steps taken during walking. The raw inertial sensor signal and calculated test parameters are saved on the smartphone and sent to the mHealth server together with subject ID for further analysis. This application also has an alternative display tab which stores all the recordings of a selected subject with exact date and time of the test along with summary parameters as shown in Fig. 44.3C. The calculation of parameters included in Table 44.1 is described in Ref.70 As an illustration, Fig. 44.4 shows raw acceleration and gyroscope signals collected by the sTUG application during a TUG test. The waveforms clearly show the transitions and the walking phase. When the “Start” button on the screen is pressed, the beginning timestamp is captured. The program then waits for the beginning of the sit-to-stand transition by searching for a change in the angular velocity around the frontal axis (the x component of the gyroscope). This change is caused by leaning forward as the subject prepares

to stand up. Additionally, acceleration thresholds are checked to ensure that the leaning forward signature is caused by an actual motion rather than by swinging forward while still in the sitting position. The angular velocity during the sit-to-stand transition has a distinct profile as it starts from zero, increases to reach the maximum, and drops to a zero at the end of the lean forward phase (LF). To determine the beginning of the transition, we first find the maximum angular velocity (v.LF) that is above a certain threshold, and then search backward to find the beginning of the slope. The maximum upper trunk angle (a.S2ST) is reached at the end of the lean forward phase. The time duration between the beginning of the transition and the end of the lean forward phase represents the duration of the lean forward phase (d.LF). The second phase of the sit-to-stand transition is characterized by a negative angular velocity as the subject moves into an upright position. The angular velocity reaches the minimum (i.e., v.LT), and then increases back to zero. The moment when it becomes positive is considered to be the end of the lift-up phase and the end of the sit-to-stand transition. By time stamping this moment, we calculate the duration of the lift-up phase (d.LT), and the total duration of sit-to-stand transition, d.S2ST = d.LF + d.LT.A stand-to-sit transition can also be divided into two separate phases, a prepare-to-sit (PS) and a sit-down (SD) phase. The angular velocity increases to the maximum and then drops back to zero as the subject leans forward in preparation to sit down. The moment when the angular velocity drops to zero marks

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the end of the PS phase and the beginning of the SD phase. By time-stamping these characteristic points we can determine the duration of the prepare-to-sit phase (d.PS). In the SD phase, the angular velocity is negative as the subject’s upper trunk moves back into the upright position. The moment it becomes positive marks the end of the SD phase and the entire stand-to-sit transition. By capturing the time stamp of this moment we calculate the duration of the sit-down phase (d.SD), and the entire stand-to-sit transition (d.ST2S = d.PS + d.SD). We calculate total walk time (d.WALK) by using captured timestamps and the number of steps (n.STEP) using acceleration and gyroscope threshold. We use magnetic sensor data to detect turning and the number of steps before turn (n.SBT).

TABLE 44.2 Parameters for TUG Characterization Parameter

Description

Units

d.TUG

Total duration of the TUG test (from “Go” to the completion of the test)

Seconds

d.S2ST

Total duration of the sit-to-stand transition; d.S2ST = d.LF + d.LT

Seconds

d.LF

Duration of the lean forward phase in the sit-to-stand transition

Seconds

d.LT

Duration of the lift-up phase in the sit-tostand transition

Seconds

d.WALK

Total time of walk

Seconds

d.ST2S

Duration of the stand-to-sit transition; d.ST2S = d.PS + d.SD

Seconds

d.PS

Duration of the prepare-to-sit phase in the stand-to-sit transition

Seconds

d.SD

Duration of the sit-down phase in the stand-to-sit transition

Seconds

a.S2ST

Maximum change of the trunk angle in the lean forward phase

Degrees

v.LF

Maximum angular velocity during the lean forward phase

Degrees/s

v.LT

Maximum angular velocity during the lift-up phase

Degrees/s

n.STEP

Total number of steps during walking phase

Steps

n.SBT

Total number of steps before turn

Steps

44.3  ASSESSMENT OF EFFECTIVENESS OF BALANCE FOR LIFE INTERVENTION PROGRAM ON MOBILITY The authors conducted a pilot study to investigate the effect of community-based exercise program on balance and gait in elderly individuals residing in a community. Our study was planned as a one group, repeated measures design, which included 20 participants recruited from two local churches in Huntsville, Alabama.85 These churches were affiliated with a nonprofit organization, The Center for Aging, established by a gerontologist.

Raw signal from accelerometer Acceleration [m/s2]

20

x y z

Sit-to-stand

10 0

–10

Walk time 0

2

4

6

8

Stand-to-sit 10

12

14

16

Angular velocity [rad/s]

Raw signal from gyroscope 4

x y z

Sit-to-stand

2 0 –2 Walk time –4

0

2

4

6

8 Time [s]

Stand-to-sit 10

12

14

FIGURE 44.4  Accelerometer, gyroscope signals (the x, y, z components) recorded during the TUG test.

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16

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44.3  Assessment of Effectiveness of Balance for Life Intervention Program on Mobility

TABLE 44.3 Raw Data From Participants Age

TUG 1 time [s]

TUG 2 time [s]

TUG 3 time [s]

Slope TUG [s/day]

Slope TUG rel [%/day]

4

86

16.33

14.71

13.87

–0.028

–0.170

2

5

79

16.59

13.34

10.92

–0.064

–0.385

3

6

74

14.76

13.43

8.38

–0.064

–0.431

4

8

83

15.52

12.95

12.68

–0.030

–0.193

5

9

85

16.76

15.61

12.00

–0.052

–0.313

6

11

80

15.11

11.12

10.46

–0.047

–0.314

7

12

89

14.98

11.99

14.08

–0.012

–0.079

8

15

81

17.69

13.33

9.95

–0.081

–0.456

9

17

72

11.10

10.18

10.89

–0.003

–0.027

10

23

77

17.62

14.26

10.3

–0.084

–0.475

11

24

87

20.96

16.62

15.03

–0.068

–0.323

12

26

72

19.19

12.08

8.45

–0.091

–0.472

13

39

89

15.55

13.73

10.36

–0.068

–0.439

14

40

76

18.15

14.44

11.08

–0.096

–0.526

18

81

13.15

11.14

9.25

–0.041

–0.311

16

21

84

25.47

19.81

14.70

–0.124

–0.486

17

22

77

37.39

18.96

20.17

–0.195

–0.522

18

25

88

21.22

16.68

14.93

–0.064

–0.302

19

27

72

17.98

16.46

10.25

–0.055

–0.307

20

38

91

16.71

17.2

7.60

–0.112

–0.670

Number

Gender

ID

1

Female

15

Male

The exercise program, called Balance for Life,86 is based on the recommendations from the CDC’s Compendium.57 All instructors were certified to deliver balance and fitness exercise. We collected demographic data including age and gender. We also performed an assessment of gait and balance based the CDC’s STEADI toolkit; we performed the TUG test using the smartphone application (sTUG).13 The measurements were made at baseline, 6 weeks, and 12 weeks. Attendance at Balance for Life classes was recorded to monitor for the effect of participation on gait and balance. A total of 20 participants were included in the study: 14 were women and 6 were men. Individual demographic data and raw scores on the TUG test using the sTUG application are presented in Table 44.3. The demographic and TUG times were summarized in Table 44.4. The majority of the sample was female (70%). Participants’ ages were 72–91 with a mean age of 81 (S.D. 6.16). Therefore, this sample was quite old. Participation in the exercise program was measured by using attendance rosters at the churches. The elderly individuals were asked to attend exercise 2 days per week

for 3 months. Their participation rate was a percentage of the number of sessions attended over the number of possible sessions in the same period of time. Table 44.4 shows that individuals, on average, participated 63% of the time at 6 weeks and 60% of the time at 12 weeks with a range of 25–92% at 6 weeks and 16–97% at 3 months. The descriptive statistics for participants’ TUG tests at baseline, 6 weeks, and 12 weeks are shown in Table 44.4. For the TUG, the number represents time measured in seconds. Therefore, the lower the numbers, the better the performance because it takes participants a shorter amount of time to stand up, walk 10 ft, return to the chair, and sit down. For all participants, there was a steady decline in the number of seconds from baseline (18.11 s) to 6 weeks (14.30 s) to 12 weeks (11.76 s). The sit-stand represents the transition of participants from sitting in a chair to standing before the first step in the TUG test. Similar to the interpretation of the total time for the TUG test, time for the sit-stand transition of participants with shorter times indicates better muscle strength compared to participants with longer transition time. The entire sample’s mean sit-stand time decreased from baseline (1.18 s) to 6 weeks (1.14 s), to 12 weeks (0.894 s).

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TABLE 44.4  Descriptive Statistics for Demographic and TUG Times Age

Participation at 6 weeks

Participation at 12 weeks

Total TUG 1

Total TUG 2

Total TUG 3

Sit-stand 1

Sit-stand 2

Sit-stand 3

N

20

20

20

20

20

20

20

20

20

Mean

81.15

0.63

0.60

18.11

14.30

11.77

1.18

1.14

0.894

Median

81.00

0.73

0.66

16.74

14.00

10.91

1.17

1.20

0.910

1.14

1.23

0.49a

Mode Standard deviation

72.00 6.158

0.22

0.24

0.23

0.24

a

11.10

5.485

a

a

10.18

7.60

2.562

2.991

.260

.364

0.206

Minimum

72.00

0.25

0.16

11.10

10.18

7.60

.75

.52

0.49

Maximum

91.00

0.92

0.97

37.39

19.81

20.17

1.62

1.85

1.24

a

Multiple modes exists. The smallest value is shown.

Acc 3D (m/s2)

Base line: STUG 14.73 seconds 20 15 10 5 0

5

10

15

10

15

Time [s]

Acc 3D (m/s2)

3 months later: STUG 8.79 seconds 20 15 10 5 0

5 Time [s]

FIGURE 44.5  Change of 3D acceleration after exercise; Subject ID 6, Female, Age 74.

Another way to understand the changes in the TUG test is to use the physical signals from the accelerometer in the sTUG application and graph them to demonstrate changes in the number of steps and the characteristics of the steps. We show a single participant’s TUG test in Fig. 44.5. The graph shows the participant improved total TUG time from 14.73 s to 8.79 s in 3 months. We can observe that the participant had cautious short steps at the baseline and longer and more powerful steps after 3 months of exercise. These changes were typical of participants in the sample. Because we were interested in gender differences in mobility, we ran an Analysis of Variance (ANOVA) with gender as the grouping variable and TUG time as the dependent variable. Table 44.5 shows the descriptive

statistics for males and females at baseline, 6 weeks, and 12 weeks. Males had longer average TUG times than females at each measurement point. Both males and females reduced the TUG time at each measurement point. Table 44.6 shows the results from the three ANOVA that were run to test for differences in TUG time at each measurement point. There were statistically significant differences in TUG time between males and females at baseline (F = 5.323; [df = 1,18]; p < .05), at 6 weeks, (F = 7.547; [df = 1,18]; p < .05), but not at 12 weeks (F = 1.065; [df = 1,18]; p > .05). This means that although males started with higher TUG times, by the end of the exercise program, both males and females had no statistically significant differences. Based on the results, an effect

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44.3  Assessment of Effectiveness of Balance for Life Intervention Program on Mobility

TABLE 44.5  TUG Descriptive Statistics for Males and Females Mean

Standard deviation

Standard error

Minimum

Maximum

6

21.9867

8.62516

3.52121

13.15

37.39

Female

14

16.4507

2.33509

0.62408

11.10

20.96

Total

20

18.1115

5.48475

1.22643

11.10

37.39

Male

6

16.3750

3.14476

1.28384

11.14

19.81

Female

14

13.4136

1.71879

0.45937

10.18

16.62

Total

20

14.3020

2.56173

0.57282

10.18

19.81

Male

6

12.8217

4.65734

1.90135

7.60

20.17

Female

14

11.3179

2.00111

0.53482

8.38

15.03

Total

20

11.7690

2.99130

0.66887

7.60

20.17

N TUG 1 time at baseline

TUG 2 time at 6 weeks

TUG 3 time at 12 weeks

Male

TABLE 44.6  TUG Test ANOVA Sum of squares TUG 1 time at baseline

TUG 2 time at 6 weeks

Mean square

F

Sig.

5.232

0.035

7.547

0.013

1.065

0.316

Between groups

128.716

1

128.716

Within groups

442.852

18

24.603

Total

571.568

19

Between groups

36.834

1

36.834

Within groups

87.834

18

4.881

124.687

19

9.498

1

9.498

Within groups

160.512

18

8.917

Total

170.010

19

Total TUG 3 time at 12 weeks

df

Between groups

size was calculated to determine the influence of exercise on TUG time. The results of the calculation showed the effect size as 0.56891, which was moderately large.87 We also wanted to examine TUG time over all three measurement points at the same time and to examine the interaction of TUG time with gender, age, and participation in group exercise. We used repeated measures ANOVA analysis to test for the main effect (TUG time) and the interactions (see Table 44.7). There was a statistically significant interaction effect with TUG time (in seconds) at baseline, 6 weeks, and 12 months (F = 5.901; df = 1; p < .05) and gender. However, there was no significant interactions between TUG time and age (F = 0.352; df = 1; p > .05), participation at 6 weeks and TUG (F = 0.301; df = 1; p > .05), or participation at 12 weeks and TUG (F = 2.407; df = 1; p > .05). We tested the sit-stand time of participants at baseline, 6 weeks, and 12 weeks in the same manner with ANOVA

TABLE 44.7  TUG Test Repeated Measures ANOVA df

Mean square

82.349

1

Age

8.018

Participation at 6 weeks

0.716

Participation at 12 weeks

Source

Type sum of squares

F

Sig.

82.349

3.617

0.077

1

8.018

0.352

0.562

1

0.716

0.031

0.862

54.791

1

54.791

2.407

0.142

Gender

134.349

1

134.349

5.901

0.028

Error

341.482

15

22.765

Intercept

and repeated measures ANOVA. Although there was a reduction in sit-stand time for participants, there were no statistically significant differences over time and no gender-based differences.

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44.  Gender Differences in Mobility of Elderly: Measurements and Interventions to Improve Mobility

0

Slope of improvement as a function of TUG time Male Female

slope [s/day]

–0.05

–0.1

–0.15

–0.2 10

15

20

25

30

35

40

TUG time [s]

FIGURE 44.6  Gender differences of the slope of improvement of TUG times as a function of the initial TUG time.

Another method of examining the progress of participants was to examine a slope of improvement of their TUG times. Fig. 44.6 presents gender-related differences in slope of the TUG time during participation in the fall prevention program. Larger negative values indicate more improvement (i.e., decrease of TUG times).

44.4 DISCUSSION The results from the pilot study showed that exercise reduced the time to complete the TUG test at 6 weeks and 12 weeks. Both males’ and females’ TUG test times were above the reference values,88 and participants were considered “at risk” for falls at baseline.14 By 6 weeks, females were approaching reference values, but males were still “at risk” for falls. At the final measurement (12 weeks), males, on average, were just slightly above reference values at 12.82 s, and females were below reference values at 11.31 s. Differences between the genders were statistically significant at baseline and 6 weeks with females having better TUG times than males; however, by 12 weeks, there were no significant differences in TUG times. Thus, exercise helped both genders, but males needed more improvement than females, and they were able to improve their balance and gait to be almost equal to females at the end of the 12 weeks of group-based exercise. Our study is consistent with other studies showing that exercise does improve mobility.53,56 The results of our pilot study need to be considered in light of the small sample size. With only 20 participants, and only six being male, the findings might not hold true in larger samples. The participants represented a very old group with a mean age of 81 who were still active

enough to live in the community, attend church, and attend community-based exercise classes. There were no data collected about exercise at home or medications used by participants. The examination of gender differences in mobility is an important line of inquiry for future research. The findings from our study show that elderly women had better TUG times than men. These findings are not consistent with other findings in the general mobility literature, which found men to have better mobility than women. However, because there can be differences in variables and measurements, the findings from our pilot study should only be compared to those studies that measured TUG times. Future studies of mobility using TUG tests need to include variables such as type of exercise, level of exercise, and duration of routine exercise. Because the elderly often have chronic illnesses and take multiple medications, data about these variables should be added to future studies as well. The measurement of TUG and other standardized tests of mobility including the 30SCS test and the 4SBT test can be added and measured with smartphone apps as those measurements become validated. Our team has a planned program of research to continue the development of technology to measure parameters that will quantify the effectiveness of interventions to improve mobility of older adults. For example, we are currently measuring the effectiveness of communitybased exercise using smartphone apps developed by the members of the research team (sTUG app, the 30SCS app, and the 4SBT test). In this study, we make measurements at shorter intervals to find the critical amount of time required for improvement in mobility. We have another research project that is longitudinal and more comprehensive; we enroll older adults who have already experienced a fall, reduce the number of medications taken, make home visits to reduce hazards, and encourage community-based exercise or teach home-based exercises. We assess mobility using the smartphone application suite at 3-month intervals for 12 months. At the conclusion of the study, we will compare participants’ results to the results of the matched control group (based on age and comorbidities) who did not participate in the mobility improvement intervention.

44.5 CONCLUSION This chapter discusses importance and characteristics of mobility in older adults and opportunities offered by recent technological advances, specifically through the use of a smartphone application for automated quantification of the standard Time Up and Go mobility test. We described the main application features and

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REFERENCES

benefits over the state-of-the-art practices. The smartphone application is used in a pilot study that aimed at quantifying the effects of group-based exercise on mobility of participants. Our plans for the future are to develop smartphone applications for other tests used in mobility assessment and deploy them in other studies. For example, continual assessment of mobility in less structured environments, such as participants’ homes, can prove useful to support aging in place.

References 1. Giddnes J. Concepts for Nursing Practice. 2 ed St. Louis, MO: Mosby; 2016.576. 2. Giannouli E, Bock O, Mellone S, Zijlstra W. Mobility in old age: capacity is not performance. BioMed Res Int. 2016;2016:1–8. 3. Colon-Emeric CS, Whitson HE, Pavon J, Hoenig H. Functional decline in older adults. Am Fam Physician. 2013;88:388–394. 4. Butler AA, Menant JC, Tiedemann AC, Lord SR. Age and gender differences in seven tests of functional mobility. J Neuroeng Rehabil. 2009;6:31. 5. Liu-Ambrose T, Davis JC, Hsu CL, Gomez C, Vertes K, Marra C, et al. Action seniors! - secondary falls prevention in communitydwelling senior fallers: study protocol for a randomized controlled trial. Trials. 2015;16:144. 6. Important Facts about Falls|Home and Recreational Safety|CDC Injury Center [Internet]. [cited 2016 Jun 2]. Available from: . 7. Choi W, Robinovitch S. Effects of pelvis impact angle and hip muscle forces on hip fracture risk during a fall using an advanced hip impact simulator. American Society of Biomechanics 39th Annual Meeting Abstracts. Phys Ther Fac Artic Res. 2015. Retrieved from: . 8. Delbaere K, Close JC, Brodaty H, Sachdev P, Lord SR. Fall risk and fear of falling in older people: the vigorous, the anxious, the stoic, and the aware. BMJ-Br Med J. 2016. . 9. WISQARS (Web-based Injury Statistics Query and Reporting System)|Injury Center|CDC [Internet]. [cited 2016 May 22]. Available from: . 10. Ensrud KE, Ewing SK, Taylor BC, et al. Comparison of 2 frailty indexes for prediction of falls, disability, fractures, and death in older women. Arch Intern Med. 2008;168(4):382–389. 11. De la Cuesta-Benjumea C, Roe B. Falls of older people living in the community- A critical review. CES Psicol. 2015;8(1):21–36. 12. Donoghue OA, Cronin H, Savva GM, O’Regan C, Kenny RA. Effects of fear of falling and activity restriction on normal and dual task walking in community dwelling older adults. Gait Posture. 2013;38(1):120–124. 13. STEADI Materials for Health Care Providers|STEADI - Older Adult Fall Prevention|CDC Injury Center [Internet]. [cited 2016 May 25]. Available from: . 14. CDC. The Timed Up and Go (TUG) Test [Internet]. [cited 2016 Jan 31]. Available from: . 15. CDC. 30 Second Chair Stand Test [Internet]. [cited 2016 Jan 31]. Available from: .

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16. CDC. The 4-Stage Balance Test [Internet]. [cited 2016 Jan 31]. Available from: . 17. Millor N, Lecumberri P, Gomez M, et al. Automatic evaluation of the 30-s chair stand test using inertial/magnetic-based technology in an older prefrail population. IEEE J Biomed Health Inform. 2013;17(4):820–827. 18. Csuka M, McCarty DJ. Simple method for measurement of lower extremity muscle strength. Am J Med. 1985;78(1):77–81. 19. Lord SR, Murray SM, Chapman K, Munro B, Tiedemann A. Sitto-stand performance depends on sensation, speed, balance, and psychological status in addition to strength in older people. J Gerontol A Biol Sci Med Sci. 2002;57(8):M539–M543. 20. Whitney SL, Wrisley DM, Marchetti GF, Gee MA, Redfern MS, Furman JM. Clinical measurement of sit-to-stand performance in people with balance disorders: validity of data for the FiveTimes-Sit-to-Stand Test. Phys Ther. 2005;85(10):1034–1045. 21. Buatois S, Miljkovic D, Manckoundia P, Gueguen R, Miget P, Vançon G, et al. Five times sit to stand test is a predictor of recurrent falls in healthy community-living subjects aged 65 and older. J Am Geriatr Soc. 2008;56(8):1575–1577. 22. Vellas BJ, Wayne SJ, Romero LJ, Baumgartner RN, Garry PJ. Fear of falling and restriction of mobility in elderly fallers. Age Ageing. 1997;26(3):189–193. 23. Deandrea S, Bravi F, Turati F, Lucenteforte E, La Vecchia C, Negri E. Risk factors for falls in older people in nursing homes and hospitals. A systematic review and meta-analysis. Arch Gerontol Geriatr. 2013;56(3):407–415. 24. Alvarado BE, Guerra RO, Zunzunegui MV. Gender differences in lower extremity function in latin american elders: seeking explanations from a life-course perspective. J Aging Health. 2007;19(6):1004–1024. 25. Guerra RO, Alvarado BE, Zunzunegui MV. Life course, gender and ethnic inequalities in functional disability in a Brazilian urban elderly population. Aging Clin Exp Res. 2008;20(1): 53–61. 26. Khadr Z, Yount K. Differences in self-reported physical limitation among older women and men in Ismailia, Egypt. J Gerontol B Psychol Sci Soc Sci. 2012;67(5):605–617. 27. Miszkurka M, Zunzunegui MV, Langlois EV, Freeman EE, Kouanda S, Haddad S. Gender differences in mobility disability during young, middle and older age in West African adults. Glob Public Health. 2012;7(5):495–508. 28. Onadja Y, Atchessi N, Soura BA, Rossier C, Zunzunegui M-V. Gender differences in cognitive impairment and mobility disability in old age: a cross-sectional study in Ouagadougou, Burkina Faso. Arch Gerontol Geriatr. 2013;57(3):311–318. 29. Rahman MO, Liu J. Gender differences in functioning for older adults in rural Bangladesh. The impact of differential reporting? J Gerontol A Biol Sci Med Sci. 2000;55(1):M28–M33. 30. Yount KM, Agree E. Differences in disability among older women and men in Egypt and Tunisia. Demography. 2005;42(1):169–187. 31. Zeki Al Hazzouri A, Mehio Sibai A, Chaaya M, Mahfoud Z, Yount KM. Gender differences in physical disability among older adults in underprivileged communities in Lebanon. J Aging Health. 2011;23(2):367–382. 32. Facts about Physical Activity|Physical Activity|CDC [Internet]. [cited 2016 May 19]. Available from: . 33. Stahl ST, Albert SM. Gender differences in physical activity patterns among older adults who fall. Prev Med. 2015;71:94–100. 34. Oman D, Reed D, Ferrara A. Do elderly women have more physical disability than men do? Am J Epidemiol. 1999;150(8):834–842. 35. Souza AMR, Fillenbaum GG, Blay SL. Prevalence and correlates of physical inactivity among older adults in Rio Grande do Sul, Brazil. PloS One. 2015;10(2):e0117060.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

652

44.  Gender Differences in Mobility of Elderly: Measurements and Interventions to Improve Mobility

36. Appelhans BM, Segawa E, Janssen I, et  al. Meal preparation and cleanup time and cardiometabolic risk over 14 years in the Study of Women’s Health Across the Nation (SWAN). Prev Med. 2015;71:1–6. 37. Whitson HE, Landerman LR, Newman AB, Fried LP, Pieper CF, Cohen HJ. Chronic medical conditions and the sex-based disparity in disability: the Cardiovascular Health Study. J Gerontol A Biol Sci Med Sci. 2010;65(12):1325–1331. 38. Stevens J, Sogolow E. Gender differences for non-fatal unintentional fall related injuries among older adults. Inj Prev. 2005;11(2):115–119. 39. Stevens JA, Ballesteros MF, Mack KA, Rudd RA, DeCaro E, Adler G. Gender differences in seeking care for falls in the aged Medicare population. Am J Prev Med. 2012;43(1):59–62. 40. Surveillance for Injuries and Violence Among Older Adults [Internet]. [cited 2016 May 27]. Available from: < https://www. cdc.gov/mmwr/preview/mmwrhtml/ss4808a3.htm > . 41. Mechakra-Tahiri SD, Freeman EE, Haddad S, Samson E, Zunzunegui MV. The gender gap in mobility: A global crosssectional study. BMC Public Health. 2012;12:598. 42. Steffen TM, Hacker TA, Mollinger L. Age- and gender-related test performance in community-dwelling elderly people: SixMinute Walk Test, Berg Balance Scale, Timed Up & Go Test, and gait speeds. Phys Ther. 2002;82(2):128–137. 43. Wei F, Hester AL. Gender difference in falls among adults treated in emergency departments and outpatient clinics. J Gerontol Geriatr Res. 2014;3:152. 44. Leveille SG, Penninx BW, Melzer D, Izmirlian G, Guralnik JM. Sex differences in the prevalence of mobility disability in old age: the dynamics of incidence, recovery, and mortality. J Gerontol B Psychol Sci Soc Sci. 2000;55:S41–S50. 45. Aberg AC. Gender comparisons of function-related dependence pain and insecurity in geriatric rehabilitation. J Rehabil Med. 2006;38:73–79. 46. Peel NM, Bartlett HP, McClure RJ. Healthy aging as an intervention to minimize injury from falls among older people. Ann N Acad Sci. 2007;1114:162–169. 47. Chang VC, Do MT. Risk factors for falls among seniors: implications of gender. Am J Epidemiol. 2015;181:521–531. 48. Ambrose AF, Paul G, Hausdorff JM. Risk factors for falls among older adults: a review of the literature. Maturitas. 2013; 75:51–61. 49. WHO|Falls Prevention in Older Age [Internet]. WHO. [cited 2016 May 22]. Available from: . 50. O YM, El Fakiri F. Gender differences in risk factors for single and recurrent falls among the community-dwelling elderly. SAGE Open. 2015;5(3). Available from: . 51. Stevens JA, Mahoney JE, Ehrenreich H. Circumstances and outcomes of falls among high risk community-dwelling older adults. Inj Epidemiol. 2014;1(1):5. 52. Duckham RL, Procter-Gray E, Hannan MT, Leveille SG, Lipsitz LA, Li W. Sex differences in circumstances and consequences of outdoor and indoor falls in older adults in the MOBILIZE Boston cohort study. BMC Geriatr. 2013;13:133. 53. Giné-Garriga M, Guerra M, Unnithan VB. The effect of functional circuit training on self-reported fear of falling and health status in a group of physically frail older individuals: a randomized controlled trial. Aging Clin Exp Res. 2013;25(3):329–336. 54. Defina LF, Willis BL, Radford NB, Gao A, Leonard D, Haskell WL, et al. The association between midlife cardiorespiratory fitness levels and later-life dementia: a cohort study. Ann Intern Med. 2013;158:162–168. 55. Rantanen T. Promoting mobility in older people. J Prev Med Public Health. 2013;46(Suppl 1):S50–S54.

56. Tikkanen P, Nykänen I, Lönnroos E, Sipilä S, Sulkava R, Hartikainen S. Physical activity at age of 20-64 years and mobility and muscle strength in old age: a community-based study. J Gerontol A Biol Sci Med Sci. 2012;67(8):905–910. 57. CDC Compendium of Effective Fall Interventions: What Works for Community-Dwelling Older Adults, 3rd Edition|Home and Recreational Safety|CDC Injury Center [Internet]. [cited 2016 May 25]. Available from: . 58. Lord SR, Tiedemann A, Chapman K, Munro B, Murray SM, Gerontology M, et al. The effect of an individualized fall prevention program on fall risk and falls in older people: a randomized, controlled trial. J Am Geriatr Soc. 2005;53(8):1296–1304. 59. Bosch S, Marin-Perianu M, Marin-Perianu R, Havinga P, Hermens H. Keep on moving! Activity monitoring and stimulation using wireless sensor networks Proceedings of the 4th European Conference on Smart Sensing and Context. Berlin, Heidelberg: Springer-Verlag; 2009.11.23 (EuroSSC’09). 60. Zampieri C, Salarian A, Carlson-Kuhta P, Aminian K, Nutt JG, Horak FB. The instrumented timed up and go test: potential outcome measure for disease modifying therapies in Parkinson’s disease. J Neurol Neurosurg Psychiatry. 2010;81(2):171–176. 61. Salarian A, Horak FB, Zampieri C, Carlson-Kuhta P, Nutt JG, Aminian K. iTUG, a sensitive and reliable measure of mobility. IEEE Trans Neural Syst Rehabil Eng Publ IEEE Eng Med Biol Soc. 2010;18(3):303–310. 62. Horak F, Zampieri C, Salarian A, Carlson-Kuhta P, Aminian K, Nutt J. Longitudinal monitoring of gait and mobility in Parkinson”s disease (PD) using an instrumented timed up and go test (iTUG). Mov Disord. 2009;24(Suppl 1):S403. 63. Salarian A, Zampieri C, Horak F, Carlson-Kuhta P, Aminian K. Objective evaluation of Get-up and Go test in patients with Parkinson’s disease using kinematic sensors. In: 2nd International Congress on Gait & Mental Function, Amsterdam, Netherlands, 2008. 64. Zampieri C, Salarian A, Carlson-Kuhta P, Aminian K, Nutt JG, Horak FB. An instrumented timed up and go test characterizes gait and postural transitions in untreated Parkinson’s disease. J Neurol Neurosurg Psychiatry [Internet]. 2009 Sep 2 [cited 2016 May 26]; Available from: . 65. Spain RI, St George RJ, Salarian A, Mancini M, Wagner JM, Horak FB, et al. Body-worn motion sensors detect balance and gait defic� cits in people with multiple sclerosis who have normal walking speed. Gait Posture. 2012;35(4):573–578. 66. Mancini M, Priest KC, Nutt JG, Horak FB. Quantifying freezing of gait in Parkinson’s disease during the instrumented timed up and go test. Conf ProcIEEE Eng Med Biol Soc. 2012;2012:1198–1201. 67. Faivre A, Dahan M, Parratte B, Monnier G. Instrumented shoes for pathological gait assessment. Mech Res Commun. 2004;31(5):627–632. 68. Mariani B, Jiménez MC, Vingerhoets FJG, Aminian K. On-shoe wearable sensors for gait and turning assessment of patients with Parkinson’s disease. IEEE Trans Biomed Eng. 2013;60(1):155–158. 69. Apple motion coprocessors. Wikipedia, the free encyclopedia [Internet]. 2016 [cited 2016 Jun 2]. Available from: . 70. Milosevic M, Jovanov E, Milenkovic A. Quantifying TimedUp-and-Go test: A smartphone implementation 2013 IEEE International Conference on Body Sensor Networks (BSN)20131.6 71. Madhushri P, Dzhagaryan AA, Jovanov E, Milenkovic A. A smartphone application suite for assessing mobility. 38th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC). Orlando, FL, 2016;3117-3110.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

72. Tacconi C, Mellone S, Chiari L. Smartphone-based applications for investigating falls and mobility. In: 2011 5th International Conference on Pervasive Computing Technologies for Healthcare (PervasiveHealth) and Workshops. Dublin, Ireland. 23–26 May 2011. 258–261. 73. Habib MA, Mohktar MS, Kamaruzzaman SB, Lim KS, Pin TM, Ibrahim F. Smartphone-based solutions for fall detection and prevention: challenges and open Issues. Sensors. 2014;14(4):7181–7208. 74. Mellone S, Tacconi C, Chiari L. Suitability of a Smartphone accelerometer to instrument the Timed Up and Go test: A preliminary study. Gait Posture. 2011;33:S50–S51. 75. Palmerini L, Mellone S, Rocchi L, Chiari L. Dimensionality reduction for the quantitative evaluation of a smartphonebased Timed Up and Go test. Conf Proc IEEE Eng Med Biol Soc. 2011;2011:7179–7182. 76. Mellone S, Tacconi C, Schwickert L, Klenk J, Becker C, Chiari L. Smartphone-based solutions for fall detection and prevention: the FARSEEING approach. Z Für Gerontol Geriatr. 2012;45(8):722–727. 77. Fontecha J, Navarro FJ, Hervás R, Bravo J. Elderly frailty detection by using accelerometer-enabled smartphones and clinical information records. Pers Ubiquitous Comput. 2012;17(6):1073–1083. 78. Milosevic M, Milenkovic A, Jovanov E. mHealth @ UAH: computing infrastructure for mobile health and wellness monitoring. XRDS Crossroads ACM Mag Stud. 2013;20(2):43–49. 79. Zampieri C, Salarian A, Carlson-Kuhta P, Nutt JG, Horak FB. Assessing mobility at home in people with early Parkinson’s disease using an instrumented Timed Up and Go test. Parkinsonism Relat Disord. 2011;17(4):277–280. 80. Thrane G, Joakimsen RM, Thornquist E. The association between timed up and go test and history of falls: the Tromsø study. BMC Geriatr. 2007;7:1.

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81. Podsiadlo D, Richardson S. The timed “Up & Go”: a test of basic functional mobility for frail elderly persons. J Am Geriatr Soc. 1991;39(2):142–148. 82. Weiss A, Herman T, Plotnik M, Brozgol M, Maidan I, Giladi N, et al. Can an accelerometer enhance the utility of the Timed Up & Go Test when evaluating patients with Parkinson’s disease? Med Eng Phys. 2010;32(2):119–125. 83. Marschollek M, Nemitz G, Gietzelt M, Wolf KH, Meyer Zu Schwabedissen H, Haux R. Predicting in-patient falls in a geriatric clinic: a clinical study combining assessment data and simple sensory gait measurements. Z Für Gerontol Geriatr. 2009;42(4):317–321. 84. Mellone S, Tacconi C, Chiari L. Validity of a smartphone-based instrumented timed up and go. Gait Posture. 2012;36(1):163–165. 85. Hunter A. An evidence-based fall prevention program to improve balance and gait in community-dwelling older adults. [Doctor of Nursing Practice Scholarly Practice Project]. University of Alabama in Huntsville. 2015. 86. Balance for Life|Center For Aging [Internet]. [cited 2016 25]. Available from: . 87. Becker L. Effect Size Calculators (Lee Becker)|University of Colorado Colorado Springs [Internet]. [cited 2016 25]. Available from: . 88. Bohannon RW. Reference values for the timed up and go test: a descriptive meta-analysis. J Geriatr Phys Ther. 2006;29(2):64–68.

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45 Personalized Medicine in Space Flight, Part I: Standard Clinical Approaches Michael A. Schmidt1,2, Thomas Goodwin1,3 and Marsh Cuttino4,5 1

Advanced Pattern Analysis & Countermeasures Group, Research Innovation Center, Colorado State University, Fort Collins, CO, United States, 2Sovaris Aerospace, LLC, Boulder, CO, United States, 3NASA, Johnson Space Center (Retired), Houston, TX, United States, 4Emergency Medicine Physician, Richmond, VA, United States, 5Orbital Medicine, Inc., Richmond, VA, United States

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45.3 Contextual Aspects of Commercial Space Flight 662 45.3.1 Categories of Space Travelers 662 45.3.2 Categories of Space Travel Based on Destination 662 45.3.3 Radiation Exposure 663 45.3.4 Orbital Habitats 664 45.3.5 Acceleration Forces in Space flight 665 45.3.6 Analogous Participant Environments 665 45.3.7 Exposure Conditions of the Space Environment 666 45.3.8 Pressurization in Space Flight 666

Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00067-X

45.3.9 Clearance to Fly Responsibilities 45.3.10 Informed Consent and Waivers

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45.4 Standard Clinical Approaches to Space Flight Medical Assessment 666 45.4.1 Recommended Clinical Assessment for Humans Entering Space 666 45.4.2 Preexisting Disease 667 45.4.3 Recommended Space Analog Testing for Medical Clearance 668 45.4.4 FAA Minimal Requirements for Space Flight Participants 668 45.4.5 Space Flight Disqualifying Medical Issues 668 45.5 Final Considerations

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References 669 Further Reading

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45.1 INTRODUCTION Space is among the most unique and challenging environments encountered by humans. Microgravity, isolation, confinement, radiation, and a broad range of environmental conditions can place considerable strain on humans in such environments. Human health, safety, and performance are key to mission success, and to the health and function of a space traveler upon return to Earth. A better understanding of individual differences may allow the development of individualized countermeasure packages that optimize the safety and performance of each person entering the space environment. To date, more than 600 civilians have registered to fly in space aboard commercial (private) suborbital space vehicles. This is more than the 530 professional astronauts that have flown in space, only 11% of whom have been women. Of these 530 people who have flown in space, approximately 130 have been NASA astronauts, of whom 80% have been men and 20% have been women.1 Private space organizations have made significant progress in achieving full launch capability, which includes suborbital and low earth orbit capability. The growth of the private space industry is further evidenced by the emergence of organizations like the Commercial Spaceflight Federation (CSF) and the Federal Aviation Administration (FAA) Office of Commercial Space Transportation, which are evolving their medical certification to include commercial space flight. The evolution of human space flight capability beyond that of government space programs greatly increases the likelihood that space flight participation will no longer be restricted to professional astronauts. This raises the probability that clinicians will encounter SFP in their clinical practices.

45.1.1  Personalized Medicine, Precision Medicine, and Gender-Specific Medicine The relative paucity of sex- and gender-specific data from space flight compels us to give significant attention to personalization. In this regard, it is useful to view our approach from the standpoint of two converging approaches: the application of standard space flight medicine and the application of personalized precision medicine. First, there are standard methods of clinical assessment and management that have been developed over years of aerospace medicine, which have proven essential in optimizing the safe entry and return of humans from space. These are and will remain the cornerstone of our clinical approach. Second, we have advanced a model of personalized precision medicine for space flight participation, which is

rooted in the fields of genomics, epigenomics, transcriptomics, proteomics, metabolomics, and microbiomics. From these disciplines, we have developed an evolving approach to human space flight that addresses molecular dynamics from the vantage point of essential inputs that govern the efficiency of molecular networks and the ever changing clinical phenotype. For this second purpose, it is important to clarify an evolving ontology. The terms personalized medicine, individualized medicine, and precision medicine are widely used to describe the use of advanced molecular profiling (and other methods of assessment) to understand the dynamics of an individual, which is then used to guide some form of tailored therapy. Here, we use the term personalized precision medicine. This is done to highlight the fact that our approach is necessarily tailored to the person, but that its precision is rooted in the use of molecular profiling to more precisely develop preventive measures, treatments, countermeasures, or training approaches tailored to the individual. This involves linking the molecular profile to the underlying metabolic networks and pathways that govern the physiologic phenotype. The sex- and gender-specific data from the current pool of professional astronauts are informative, but not sufficient to guide the kind of precision countermeasures that will 1 day be developed. Thus, our approach serves two purposes. The first is to tailor the countermeasures to the needs of an individual, but to do so in a manner that gives attention to the current understanding of sex- and genderspecific findings in space. These are actionable steps that can be deployed today. The second is to use our advanced molecular profiling approach to expand our understanding of this new, emergent group of SFP in the interest of advancing the field of space flight medicine. The first section herein, Sex-Specific Variation of Humans in Space, provides a brief overview of the current state of knowledge about sex and gender differences in the space flight environment. As noted, these data are somewhat limited by the fact that the number of females that have flown in space is substantially smaller than the number of males, which renders statistical comparisons difficult. While there is a lack of adequate data for quantitative comparisons in space, a helpful exercise is to examine the differences in function based on sex and gender on earth. This provides a useful, but also limited, insight into differences one may encounter in space. The remaining discussion will then shift to the general medical examination for SFP and to concepts of personalized medicine, regardless of gender. It is important to reiterate that most of human space flight to date has focused on professional astronauts who are part of government space programs. Moreover, career astronauts derive from a significantly different demographic than the new space participants. This

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45.2  Sex-Specific Variation of Humans in Space

limits the predictive accuracy of the data gleaned from space flight thus far. Most physicians in practice are unlikely to encounter professional astronauts, unless they are formally affiliated with a space program or unless a former astronaut is seeking medical care following retirement. The emergence of the commercial private space industry has led to a new class of space traveler. This includes space explorers, space workers, and space adventurers. Since the likelihood of the practicing physician interacting with a professional astronaut is low, this chapter will focus primarily on the commercial private space industry and the type of space traveler that may present in the primary care, human performance, or emergency setting. It is likely that the majority of early commercial SFP will have medical screening either by FAA aviation medical examiners or by aerospace physicians working in conjunction with the space flight providers. It is not the intent of this chapter to provide all the tools needed to provide medical clearance for an individual to participate in space flight operations, but instead enable the practitioner to know when additional expert evaluation might be required and to recognize key concerns. In Part II (see Chapter 46: Personalized Medicine in Space Flight, Part II: Personalized Precision Medicine Approaches) we outline a conceptual approach to personalized precision medicine applied to space flight. As the technological capabilities of Omics progresses, it is hoped that a personally tailored biophysical risk profile can be evaluated prior to the exposure to space flight, and specific countermeasures adapted to the individual will allow for protective and risk-lowering therapeutic options.

45.2  SEX-SPECIFIC VARIATION OF HUMANS IN SPACE 45.2.1  General Differences Between Men and Women in Space Flight This review focuses on key differences in the response to the space flight environment of six general systems, including but not limited to: the (1) cardiovascular response; the (2) immune response; the (3) sensorimotor response; the (4) musculoskeletal response; the (5) reproductive response; and the (6) behavioral response. It includes the experience and clinical history of some 530 humans in space across international space programs. Duration of exposure to the space environment in all cases is almost all less than 1 year and generally less than 6 months. Some of the major in-flight differences are listed in Table 45.1. Again, it should be noted that the number of men that have flown in space greatly outweighs the number of women, making robust statistical comparison difficult.

TABLE 45.1 Known In-flight Differences in Males and Females. Gender differences in immune, reproductive, and endocrine function are not shown here because of insufficient evidence in the spaceflight condition Male

System

Female

Less susceptible to orthostatic intolerance

Cardiovascular

More susceptible to orthostatic intolerance

More instances of visual impairment intracranial pressure (VIIP) Exhibit clinically significant visual impairment

Fewer cases of VIIP

Sensorimotor

Suffer more from hearing loss with advancing age, and display bias toward loss of hearing in the left ear Large Individual variability to muscle and bone loss

Do not exhibit clinically significant visual impairment Suffer less from hearing loss with advancing age, and do not display bias toward loss of hearing in the left ear

Musculoskeletal

More bone and muscle mass, on average.

Large Individual variability in muscle and bone loss Less bone and muscle mass, on average.

UTIs less common in males

Urinary

UTIs common in females

Exhibit a slight bias toward speed versus accuracy in response to alertness tests

Behavioral

Exhibit slight bias toward accuracy versus speed in response to alertness tests

With this in mind, as suborbital and space flight become increasingly feasible and more common, there is a concerted effort in the space flight community to increase the number of women participants and gather descriptive physiologic and molecular data on them.2 These data can then be used for more detailed analyses and further personalization. 45.2.1.1  Cardiovascular Response The cardiovascular system (CV) is the system most affected during short-duration space flight. The responses include alterations in the cardiac rate, blood pressure, and baroreceptor physiologic control.3 The initial and most significant stress on the CV occurs during acceleration stress. The initial stress of launch can introduce tachycardia, and pooling of the blood in the extremities with delayed vascular return depending on the orientation of the individual to the acceleration stress. During the microgravity portion of space flight operations, no significant arrhythmias have been observed. The

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deconditioning during prolonged microgravity would be expected to potentiate arrhythmias upon return to a high-G environment, but this has not been well studied.4 Centrifuge data have shown that even extremely healthy individuals can develop a cardiac arrhythmia under the conditions of extreme duress. In counterpoint, centrifuge studies and some individual examples have shown that even some individuals who would be expected to be at high risk for developing an arrhythmia have been able to tolerate the expected environment.5 One long-duration maladaptation on the CV system in reduced or zero gravity is termed visual impairment intracranial pressure (VIIP). In NASA risk assessments, VIIP is seen as the leading health risk. VIIP manifests pathophysiologically on a spectrum of symptoms from mild ocular changes, such as globe flattening, to optic disk edema, which is severe, clinically significant, and mission critical in some cases.6 Prolonged VIIP can cause loss of visual acuity, cognitive dysfunction, and potential neurological impairment longer-term. Interesting, all of the clinically significant VIIP cases thus far have been reported in men. Female astronauts have reported much milder visual symptoms (remains a statistically insignificant effect due to small female subject numbers7). Humans also experience orthostatic intolerance (OI). OI is the inability to stand without fainting for protracted periods of time. In contrast to VIIP incidence, a higher proportion of female astronauts have reportedly experienced OI to date. Bed rest studies indicate that the difference between the sexes can be attributed to reduced vascular leg compliance present in women.8 In addition to VIIP and OI, space flight-analog bed rest studies have indicated that women have a greater loss of plasma in-flight than men8 and exhibit an increased heart rate CV stress response, while men respond to CV stress with increased vascular resistance.9 45.2.1.2  Immunological Response Radiation, microgravity, isolation, physiological stress, and circadian misalignment have been shown to have profound effects on human immune function in space flight.10 Overall, a depression of the immune system in both males and females is seen.11 Prolonged exposures to these conditions increase the incidence of infectious diseases,12 hypersensitivities, autoimmunity,13 and malignancy.14 Commensal and pathogenic microflora from any of the human microbiomes are potentially harmful.15,16,17 A space traveler may also be exposed to the microflora from other travelers when enclosed in tight quarters. Preflight countermeasures can help to attenuate some of these exposures, such as screening the mission’s food supply for Salmonella and other common food pathogens preflight.18 Just as humans present different physiological responses to space than the response seen on earth,

commensal and pathogenic microflora elicit similar changes. Colonies of microflora have been observed to adapt to space flight conditions on space stations and are able to infect newly arriving individuals with potentially clinically significant infections.19 In space, bacteria often have increased growth rates, abnormal metabolic characteristics, and decreased susceptibilities to antibiotics.20 Viral particles exhibit similar changes in characteristics, as a result of microgravity. As such, astronauts from previous missions have experienced a wide range of infections, ranging from the mild to the debilitating. These include, but are not limited to, dental infections, upper respiratory infections, the chronic reactivation of latent viral infections, and incapacitating urinary tract infections.21,22 Interestingly, changes in commensal gut microbiome colony structure (and function) have links to immune function.23 Modulators of these changes may be flight duration, dietary intake, and the microflora of other astronauts.20 Notably, the link between microbiota and infectious disease has yet to be fully elucidated in the space flight environment. The immune response and immune dysfunction of humans exposed to the space flight environment are well known.24 Generally, there are changes in the concentrations and functionality of key constituents, cells, and signaling molecules. Changes in cytokine production, leukocyte subset distribution, antibody production and distribution, and the short- and longterm cell-mediated immune responses are all seen.10 The number of women that have flown in space is currently too small to make any meaningful comparisons and to draw conclusions. However, extrapolation from Earth-based studies of immune differential function indicate that women mount a more potent response to bacteria and viruses, are more resistant to infection, and once infected mount a more robust response.25 Consequently, this sensitive and robust defense activation renders females more susceptible to autoimmunity and radiation-induced cancers than men. The immune response differential has the potential to become additionally amplified in the space flight environment, but evidence-based dynamics remain elusive and are still largely unexplored. 45.2.1.3  Neurosensory Response Differences in the reported functionality of in-flight responses, gross anatomy, neuronal development and maintenance, neurochemical pathways, and stress responses inform the overall neurophysiological, sensorimotor, and sensory responses of men and women in space.26 A large body of the evidence helping to explain these differences in adaptation comes from the incidence and nature of disorders of the central and peripheral nervous systems. These include Alzheimer’s disease (AD), posttraumatic stress disorder (PTSD), multiple sclerosis

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(MS), attention deficit disorder (ADD), and anxiety and depressive disorders.27 In terms of visual acuity and processing, men tend to have a greater sensitivity for fine detail and fast moving stimuli. Conversely, females have a greater capacity for the processing and discrimination of color. Haptic task measures, such as touch-based and proprioceptionbased recognition of an object or the position of an object in space, tend to result in higher scores for males over their female counterparts.28 The gross anatomy of the vestibular system is different in males and females, with structures of the inner ear, such as the superior semicircular canal, being significantly larger in males.29 Women also have fewer myelinated axons in the vestibular nerve and are more likely to develop vestibular disorders, such as vertigo when subjected to high-G-force or microgravity conditions.30 Some evidence suggests that dedicated cognitive training can reduce sex differences in special task performance. However, the differences in anatomical structure, physiological function, and resulting disorders should be taken into consideration in all vestibular assessments of potential space flight participants while still on earth.31 Furthermore, it has been reported that women have a higher instance of entry and space motion sickness, as well as postflight vestibular instability and dysfunction (both short- and long-duration missions). This is based on the NASA Lifetime Surveillance of Astronaut (LSAH) database,11 a database that tracks NASA astronaut health longitudinally. The amygdala, the region of the brain associated with emotional material and fear learning, exhibits handedness of activity in men and women. Males process cues (e.g., visual cues) in the right amygdala, where women preferentially process the same material in the left amygdala.32–34 The in-flight functional consequences of this dimorphic response are presently unknown and are under investigation. Nevertheless, they could prove highly relevant in high-stress or long-duration space flight conditions, such as the isolation and duration present on a trip to Mars. General differences in the response to stimuli of somatosensation, such as touch, pressure, and temperature seem to be more intense in females than males. Nociception, the sensation of pain, is known to have many biases (social class, gender, ethnicity, and culture). However, it appears that women are more sensitive to painful stimuli than their male counterparts.28 A difference also exists in opioid receptor binding in several brain regions, such as the amygdala and thalamus, leading to varied responses to pain analgesics, such as morphine and fentanyl.35 Genetic factors also play a large role in the response of an individual to attempts at pain management via the cytochrome p450 genotype. Thus, the individual xenobiotic metabolic

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characteristics significantly overshadow the effects of any sex differences. Hearing and auditory function display gender and sex differences in the space flight environment, i.e., females exhibit better hearing thresholds than males. However, this difference does not seem to deviate from the patterns of hearing impairment in the general population, according to the LSAH database, which tracks former astronaut auditory function later into life.11 In general, male astronauts have more hearing loss at every age. This could be due to occupational hazards of many male astronauts, such as military and nonmilitary firearms usage (as many of the participants in the NASA astronaut program have a military background). Although these differences do exist, none seem to be gender-specific beyond what is normal in a control population of earth-bound men and women, where men have more hearing loss at every age.36 Accordingly, flight into space does not appear to cause any differential impairment of auditory function. 45.2.1.4  Musculoskeletal Response Men and women differ significantly in their muscle and skeletal mass. In most cases, and as a general trend, men have higher muscle and skeletal mass than women. What is less clear is the highly-variable individual response to unloading (micro and zero gravity) on the musculoskeletal system, as evidenced by past missions and prolonged duration missions on a space station. In-crew comparison can exhibit as much as a 10× difference among individuals. For instance, the loss of cancellous bone in the distal tibia for cosmonauts aboard the Mir space station ranged from 2% to 24% (these values range from negligible [2%] to the bone loss observed after a spinal cord injury [24%]37). The general response of muscle unloading in space flight is loss of muscle mass and muscle atrophy, with Type I muscle fibers experiencing preferential unloading in both males and females. In terms of sex and gender differences, little difference is observed in a side-by-side comparison of whole muscle atrophy in the two sexes for the first 14 days.38 At that point, the apparent loss of whole muscle volume39 and fiber area appears to be more prevalent in women.40 Past 30 days of unloading, there are little data to suggest any definitive trend. However, this question is relevant given the long-term duration of proposed exploratory missions. Interestingly, women exhibit greater impairment in neural activation of muscle fiber following short-duration unloading protocols,41 as well as slower strength recovery after time spent in low/zero gravity.42 In contrast, the loss of power and strength cannot fully be accounted for by whole muscle atrophy and decrease in fiber area. That is, the reduction of force and cross-sectional area in muscle exhibits similar patterns in both men and women,43,44 which argues for acquiring more definitive data from female subjects.

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Bone loss, as measured by bone mineral density (BMD), is related to muscle atrophy, genetics, nutritional, and physiological status. BMD differences in men and women have been well characterized on earth (women generally tend to exhibit more bone loss than men45). In bed rest studies, the gender difference is less well characterized, and is almost unknown in the space flight environment. One bed rest study found that women had significantly less bone loss than men, when measured at 10 bone sites,39 while another bed rest study found that men have less bone loss, as measured by total hip BMD after 60 days of bed rest.46 This leads to inconsistent conclusions about the overall trend in gender-specific differences in bone metabolism in space flight. Preclinical studies in animals suggest similar inconsistent conclusions related to bone loss and gender differences.47,48 Analysis of the trend in the current body of bone loss and spaceflight evidence leads to the conclusion that gender and sex differences are unclear, and the degree to which an individual will experience bone loss due to unloading is highly random and currently unknowable preflight. In this case, the molecular phenotype may be informative. Two further considerations that affect the musculoskeletal system in spaceflight are: (1) the influence of negative energy balance on muscle/bone loss (e.g. catabolizing muscle protein to compensate for caloric imbalance/deficiency) and (2) the risk of serious joint injury and the impact on articular cartilage due to bone/muscle loss. Whereas the evidence remains inconclusive for gender differences in bone and muscle loss as a result of negative energy balance, the effects on joint injuries are, in contrast, more clear. In-flight musculoskeletal injuries occur at a rate of 0.0021 per flight day for men and 0.015 per flight day in women.49 This gender difference in the incidence of musculoskeletal injuries may arise from a number of factors, they are: (1) preflight musculoskeletal status; (2) in-flight exercise regimen; (3) willingness to take risks; (4) genetics, (5) anatomy, and other factors. Relevant research questions yet to be determined are: (1) what is the time course of muscle and bone loss due to unloading; (2) does the initial state of muscle tone and mass and bone mass determine the rate or extent of loss; (3) is the rate of loss in long-term, spaceflight conditions linear and what events or countermeasures could potentially change this trajectory; (4) what secondary effects do muscle and bone loss have on connective and related tissues; and (5) how does the known musculoskeletal gender difference influence the questions above?50 The answer to these gender-, sex-, and individual-specific research questions is vital to successful countermeasure development and musculoskeletal stability on longduration missions.

45.2.1.5  Reproductive Response Potential risk factors to reproductive structures (endocrine and urogenital systems) in both men and women include, but are not limited to, exposure to radiation, stress, and microgravity. Radiation dose is dependent upon multiple factors, such as mission destination and duration, vehicle and habitat design, and solar conditions. The typical ISS mission (6 months) radiation dose is between 54 and 108 mSv depending on orbital altitude and patterns of solar activity.51 Short-duration suborbital flights would entail much less exposure and the dose would be analogous to a transatlantic flight. Longer duration missions, such as those to Mars, could be as much as 1800 mSv roundtrip,52 exceeding the acceptable lifetime radiation dose for humans of 1500 mSv. Reproductive tissues and processes, such as the gonads and spermatogenesis, are known to be highly sensitive to radiation exposure. Spaceflight has been associated with temporary infertility in both men and women in a dose-dependent manner.51 Microgravity, stress, and sleep disruption can likewise be traced to reproductive concerns. Spermatogenesis may be more affected by microgravity than radiation.53 Rat studies have indicated severe testicular and epididymal degeneration,54 although this research has not been replicated in a population of human subjects. Evidence is also lacking about the impact of space flight on the reproductive tissues of women. In mice, spaceflightinduced termination of menstrual cycling, loss of corpus luteum, and significant decrease of estrogen receptor mRNA in the uterus have been reported.55 Nevertheless, many men and women who have participated in spaceflight activities have returned to earth and been able to conceive children. Specific information concerning conceptions, pregnancy outcomes, and birth outcomes is unknown, but no adverse events in conception or child-bearing have been reported to date. The incidence of infertility following long-duration space flight is also not known. The endocrine functions of the hypothalamus–pituitary–adrenal (HPA) and hypothalamus–pituitary– gonadal (HPG) axes are known to be altered by real and simulated microgravity. In the hypothalamus of both males and females, microgravity stimulates an increase in the release of histamine, and decreases in serotonin, oxytocin, norepinephrine, and glutamate.56–60 Directly, these changes result in alterations in neuronal signaling and, potentially, indirectly in hormonal changes downstream of the HPA axis. In addition, the HPA axis also exhibits enhanced release of glucocorticoids, such as cortisol, in response to stressors encountered in-flight. Much of the HPG research thus far has focused on reduced levels of circulating testosterone in males.61 A postmission decrease has been reported in multiple cases, but there

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45.2  Sex-Specific Variation of Humans in Space

are no conclusive data to suggest this trend continues beyond a short postflight refractory period. In females, oxytocin, which plays a large part in the HPA axis of women to dampen the long-term stress response, decreases for a long period of time following space flight.62 When viewed in the convergent context, the female incidence of anxiety disorders and the lack of oxytocin in women postflight may promote increased anxiety. Furthermore, many women who participate in spaceflight are on oral contraceptives (OCs). These have some functional consequences on oxytocin release, but do not seem to significantly interfere with the female stress response in-flight. As a countermeasure, although OCs are not mandatory, flight surgeons have recommended 30–35 mcg OCs to provide (1) suppression of ovarian function to decrease cyst formation and (2) to decrease risk of estrogen-related bone loss (due to decreased concentrations of estrogen). It should be noted that OC formulations are constantly changing and evolving, and should therefore be prescribed after a cost–benefit analysis, as OCs are known to decrease circulating levels of hormones (testosterone, dihydrotestosterone, epiandrosterone, corticosteroid-binding globulin, prolactin, and sex-hormone binding globulin63,64). In females, there are several estrogen-regulated (estrogen receptor signaling) systems that are affected by space flight in the same way as aging. They are: (1) mood; (2) arterial vasodilation; (3) immunity; (4) cardioprotection; (5) growth and proliferation of breast tissue-a major risk factor for breast cancer; (6) production of liver proteins such as coagulation factors and hepatic lipoprotein receptors; (7) neuroprotection; (8) intraocular pressure; (9) skin aging; (10) maintenance of bone density and muscle mass; (11) putative reduction in risk of colon cancer; and (12) growth and differentiation of and water retention in primary sex organs—a risk factor for endometrial cancers.65 The convergent effects of variable estrogen levels are wide ranging and must be taken into account when considering the impact of spaceflight on females. 45.2.1.6  Behavioral Response Of the many exposures that pose a risk to the stability of a mission into space, behavioral adaptation and response is among the most deleterious and cognitive stability should be taken seriously. A risk assessment of individual and crew behavior must reflect upon mission duration, vehicle structure, proximity of the crew in relationship, temperament, and space. In addition, a multitude of unforeseeable mission-unique factors, myriad psychosocial stimuli, and psychological pressures are ever-present. In the highly trained astronaut population, there is a long history of problems in team cohesiveness and crew stability. Many of the Apollo missions, as well

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as missions of the Russian space program, have documented instances of infighting and cognitive difficulties in the crew.66 Individuals entering the spaceflight environment who are psychologically underprepared or are part of an ill-conceived team dynamic may present an obstacle to mission success. The sex-, gender-, individual-, and team-specific behavioral components of spaceflight can roughly be broken into three categories: Sleep, circadian rhythm, and neurobehavioral function, ● Social interactions within a group, work performance, and satisfaction, ● Stress and its role in clinical disorders. ●

These will be handled separately here, but are interdependent. An understanding of the competing dynamics from all three categories is needed for individual stability, team cohesion, and individual integration in spaceflight. No gender differences are readily apparent in terms of self-reported changes in sleep duration and quality, workload fatigue or stress, as collected in-flight and post-flight from astronauts on the ISS. However, the light–dark cycle of the ISS trajectory has been shown to be highly disruptive to astronaut sleep patterns and circadian rhythms, which often compels astronauts to take sleeping aids to maintain a normal sleep–wake rhythm.67 Ground analog studies of chronic sleep deprivation have found that men gain significantly more weight than women.68 Sleep deprivation in women seems to produce higher levels of leptin, and a less vigorous proinflammatory cellular immune response via monocyte production of IL-6 and TNFα.69,70 Women fall asleep faster and have better efficiency of sleep (more time asleep, less time awake per sleeping session69). Women also appear to have a propensity for morning wakefulness, where men appear to exhibit more late night wakefulness.71 Many of these findings may be marginally relevant to short-duration suborbital flights. However, on missions that require multiple days/months/years in a space flight environment, circadian misalignment or dysfunction, and the effects of sleep deprivation, such as delusions and hallucinations, have the potential to become amplified. This has consequences for daily task completion, personal morale and drive, cognitive and emotional stability, interpersonal relationships, team cohesion and cooperation, and alertness and decision making in solving problems or crises. In studies on personality and behavioral health in space, women score higher than men on expressivity (interpersonal orientation, sensitivity, and concern) and achievement (traits associated with taking work seriously and working hard), while men score higher on competitiveness (a desire to succeed in competitive

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interpersonal environment72). Astronauts with higher expressivity were rated higher by their peers on interpersonal and group skills in a group living environment.73 In an Antarctic analog mission, both men and women scored lower on neuroticism and higher on positive personality traits when compared to a group of their peers, but were not different from each other.74,75 In a team environment, women tend to assume more cooperative roles than men, and tend to exhibit more sensitivity to the welfare and goals of the group.76 Other studies have found that balanced teams (composed of equal parts of males and females) have higher levels of social compatibility.77 However, there are not enough participants in these studies to generalize the findings to larger populations. In terms of work performance and satisfaction, the evidence suggests that women have a more difficult interpersonal experience in polar work groups or expedition teams, and in many cases performance is affected.74,78–80 However, no definitive and consistent dynamic patterns of interaction have been elucidated. As teams become more heterogeneous (more data on female participants), studies must concentrate on interpersonal dynamics and reliable team cohesion metrics to optimize translation to and performance in the spaceflight environment. Earth-based studies indicate that anxiety disorders are twice as prevalent in women compared with men, and women with panic disorders have more frequent panic attacks than men.81 Women are more disposed to the development of PTSD in response to trauma, and to differential responses to medications for anxiety disorders, PTSD, and related comorbidities.82 It should also be noted that women have a higher propensity for depressive disorders, which could amplify in space.83 Women also present a unique set of issues surrounding menstruation and accompanying premenstrual disorders, which present a set of dynamics that could potentially amplify in space. However, in the spaceflight environment, there is limited evidence to suggest such sex differences are present, as astronauts undergo comprehensive psychological screening in order to reduce deleterious effects of psychological variance on mission success. Overall, the evidence suggests that when in an isolated, confined, and extreme environment, men and women respond similarly. Thus, attention should be paid to individual behavioral differences and performance, rather than focusing solely on sex differences. As a corollary to this statement, many of the men and women in these studies are highly trained professionals, and may have optimal or better than average behavioral stability. Notably, as the barriers to entry in space flight are lowered, differences in individual- and sexbased personality and team-oriented characteristics may become a more significant issue.

45.3  CONTEXTUAL ASPECTS OF COMMERCIAL SPACE FLIGHT 45.3.1  Categories of Space Travelers There are several ways to classify those entering space in the context of the emergent commercial space flight industry. For the purpose of the present discussion, we have classified those entering space as SFP to distinguish between private space travelers and career astronauts. SFP is a term preferred by NASA and the Russian Space Federation. The term astronaut is applied to those for whom space flight is a profession or a career. This includes pilots, engineers, space scientists, and those expected to experience sustained or repeated exposure to space travel. The term Space Flight Participant is applied to those who enter the space environment as adventurers or travelers, and do so on a limited basis (often only a single time in space). The US Federal Aviation Administration awards the title of Commercial Astronaut to trained crew members of privately funded spacecraft. The only people currently holding this title are Mike Melvill and Brian Binnie, the pilots of the Virgin Galactic SpaceShipOne. Undoubtedly, a new lexicon will develop around those who travel into space on commercial vehicles. We can envision such things as suborbital scientist, orbital scientist, orbital physician, and so on. For our purposes, the key is to understand (1) the frequency of entry into space, (2) the duration of each period of entry into space, and (3) the location or destination of each given entry into space. This forms the basis of how we approach the needs of a given SFP.

45.3.2  Categories of Space Travel Based on Destination 45.3.2.1 Suborbital Suborbital flight is defined as flight beyond the Kármán line—above the nominal edge of space at 100 km (62 mi) Earth altitude. The region above this line is called mesosphere (sometimes jokingly called the ignorosphere). This region is poorly understood (ignored), due to the fact that balloons lack the lift to go into this region and rockets merely pass through this region. 45.3.2.2  Low Earth Orbit (LEO) Low Earth orbit (LEO) is an orbit around Earth with an altitude between 160  km (99  miles) and 2000  km (1243 miles). Objects below roughly 160 km experience rapid orbital decay and loss of altitude. Low Earth Orbit is the region where the International Space Station resides. NASA is currently urging the commercial space

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45.3  Contextual Aspects of Commercial Space Flight

flight sector to take over the space station, as NASA focuses on exploration missions. 45.3.2.3  Beyond Low Earth Orbit (BLEO) Beyond low earth orbit is the region of space beyond 2000 km (1243 miles). This region includes the Moon and all space beyond. It has been 50 years since a human has flown BLEO.

45.3.3  Radiation Exposure The destination, frequency, and duration of an individual’s encounter with the space environment are crucial determinants of the nature and the extent of clinical examinations and countermeasuresa that are warranted (Table 45.2). Among the primary related concerns is space radiation exposure. The radiation exposure will depend on mission duration, orbital altitude, inclination,b vehicle shielding, vehicle orientation, SFP location within the vehicle, solar cycle, and solar activity (events) (Table 45.3). The general US population’s annual exposure to background radiation is generally less than 5 mSv per year.84 Exposure from a typical mid-latitude cross-country air flight is about 0.04 mSv. As a frame of reference, the International Commission on Radiological Protection (ICRP) recommends that work-related radiation exposure does not exceed 50 mSv per year, while the recommended limit for the general public is 1 mSv85 and the recommendation for pregnant women does not exceed 5 mSv for the term of the pregnancy. The cumulative experience of US astronauts has ranged from

TABLE 45.2  Examples of Commercial Space Flight by Destination and Duration Type of commercial flight

Flight duration range

Short commercial suborbital space flight

 2 hours

Short commercial orbital space flight

3–14 days

Short-term habitation in low earth orbit

15–60 days

Short-term habitation beyond low earth orbit

7–90 days

Long-term habitation or transit beyond low earth orbit

6 months to years

a

 For the purpose of our discussion, a countermeasure can be seen as a technology, method, or solution aimed at preventing an adverse outcome, usually in an extreme environment. b

 Inclination refers to the smaller angle between a reference plane and the orbital plane or axis of direction of an object in orbit around another object.

1 to 100 mSv. Mission-averaged rates have ranged from 0.1 to 4 mSv per day. During a nominal space environment (400 km altitude, with orbit inclination less than 30 degrees) under reasonable shielding, a week’s exposure would be on the order of 100 cross-country airplane flights, which is near the typical annual exposure for a US resident.86 Solar storms are much harder to predict and to quantify, since their effects vary depending on the timing of the event, peak of the event, altitude, and inclination of the spacecraft.87 For a space flight participant who embarks on a 2-h suborbital flight and is exposed to only 4 min of microgravity, extensive clinical workups beyond the standard requirements are probably not indicated. Such a mission would be focused primarily on short-term safety considerations and a basic medical examination (Tables 45.4–45.5). For a passenger who flies monthly on orbital or suborbital flights from New York to Hong Kong, the exposure to radiation begins to slowly accumulate over time. For a pilot who flies daily into the space environment, the radiation exposure may be of even greater importance. For instance, a suborbital pilot may participate in many weekly flights over many years. This would entail small individual exposures to radiation, but potentially significant cumulative longitudinal exposure to radiation. An SFP entering one of the orbital habitats would, of course, experience even greater exposures. In Part II (see Chapter 46: Personalized Medicine in Space Flight, Part II: Personalized Precision Medicine Approaches), we explore the personalized precision medicine approach to space flight with specific

TABLE 45.3  Effects of Acceleration on Human Physiology Acceleration risks

System

Arrhythmia

Cardiovascular

Injury to the heart or blood vessels

Cardiovascular

Eye injury

Ocular/neurologic

Loss of consciousness

Neurologic

Spinal column injury

Neurologic

Nerve injury

Neurologic

Seizure

Neurologic

Motion sickness

Vestibular

Subcutaneous hematoma

Vascular/skin

Peripheral edema

Vascular/skin

Petechiae

Vascular/skin

Musculoskeletal pain

Musculoskeletal

Hernia

Musculoskeletal

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TABLE 45.4  Potential Physiological Risk Development as a Function of Mission Duration

TABLE 45.5  Prospective Space Passengers Should Undergo a General Physical Examination Including101:

Mission duration

Risks

General physical examination components

30 days

Bone and muscle loss Cardiovascular deconditioning Nephrolithiasis Exacerbation of preexisting medical conditions radiation exposure Psychologic and behavioral concerns Postflight orthostatic intolerance—severe Postflight disequilibrium—severe Decreased immune function

attention to radiation exposure, DNA stability, DNA repair, molecular profiling, and countermeasures. While much additional work is required, this approach represents an emerging attempt to better understand factors that influence an individual’s radiation susceptibility with further attention to potential tailored countermeasures.

45.3.4  Orbital Habitats In the near future, there will be 1 week (or longer) space visits to orbiting hotels. Bigelow Aerospace, for instance, has two experimental orbiting habitats that have been in space for 10 years. A new private module was recently delivered to the ISS. A space flight participant going to one of these private space hotels would have greater radiation exposure, though still not as significant as those living on the ISS for months. There is

also a risk that an individual may be exposed to a solar event, such as a coronal mass ejection. This can sharply increase the radiation exposure in ways that are unpredictable and highly-variable. A private space scientist would face similar conditions on a Bigelow space habitat. It is expected that engineering firms and pharmaceutical companies will see benefit in experimentation and product development in microgravity, since materials behave differently than at 1 G Earth gravity. Thus, space scientists represent another category of space traveler who may encounter space for short durations or repeated missions. Space scientists may enter such a Bigelow orbiting module as soon as 2020. In testimony to the US Senate subcommittee on Commerce Science and Transportation, Michael N. Gold (D.C Chief of Business Operations Bigelow Aerospace) disclosed general plans for the operation of the Bigelow BA 330 expandable space habitat module. The business entities likely to use such a facility include big pharmaceutical companies, biotechnology companies, and academia. Gold revealed the desire to contract with companies, academics, and other countries to rent a section(s) of the immense BA330 (330 M3) in sections as small as 110 cubic meters (m3) for periods from 60 to 90 days (or as is needed for longer initiatives). To this end, private astronaut crews will be in orbit for months at a time conducting R&D at the behest of specific companies. In all likelihood, these will not be professionally trained astronauts, but highly skilled and educated scientists. This brings a completely new dynamic to space

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45.3  Contextual Aspects of Commercial Space Flight

habitation, and a driving need to ensure the health and welfare of the crews. One might suggest these individuals will return multiple times within short windows of time to conduct R&D for companies, thus furthering the need for unique mission-focused nutritional and physiological countermeasures.

45.3.5  Acceleration Forces in Space flight One of the unique aspects of space flight operations is the acceleration stress that is placed on people during launch and landing operations. Acceleration on the human body causes injury based on the force, direction, and duration of the exposures.c The human body can sustain astounding amounts of acceleration changes before injury occurs. Studies on professional athletes show that concussion injury occurred with G forces of 98 G delivered over 15 ms, while athletes with G-force accelerations of 60 G were uninjured.88 In launch and landing operations, there is generally a brief sustained G-force over several minutes, which ranges from 3 G for the Space Shuttle, and 7 G for the Soyuz, to 11 G for the Mercury Redstone rocket. These acceleration profiles place cardiovascular stress on participants. The actual G forces induced will be dependent upon the launch profile and orientation, and are expected to be dependent on the launch vehicle. Acceleration forces that occur in less than 200 ms are considered impacts. Increasing the duration of the acceleration exposure increases the body’s tolerance to the acceleration. The military has had significant interest in the effects of gravity and acceleration on human physiology, as in air combat, the acceleration forces from hard maneuvering can induce G-LOC: Gravity-induced Loss of Consciousness. Experience reveals that pilots would often lose consciousness when +Gz forces were sustained of 5–6 G in a sustained turn. With the development of advanced breathing techniques, it was found that male pilots could sustain increased G-Loads of up to 9 Gs in the +Gz direction.89 Studies of female pilots initially seemed to demonstrate that they had less acceleration tolerance.90 However, it was later determined that, with properly fitted G-suits, the acceleration tolerance was essentially equal.91 Studies also confirmed that the physiologic changes during menstruation did not affect c

 Acceleration is generally described as the force acting on the body with the directionality of that force designated as +/-. Gx is described as force acting on the body from chest to back. +Gx is the force that pushes the body back into the seat (chest to back), while –Gx describes the force from back to chest. Gy is a lateral force that acts from shoulder to shoulder. The +Gy axis extends out the left side, with the –Gy to the right. Gz is a gravitational force applied to the vertical axis of the body. A force from head to foot is termed (positive) +Gz. The force applied foot to head is termed (negative) –Gz.

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acceleration tolerance,92 clearing the way for women to participate in combat missions. This could rationally be extended to space flight operations. Indeed, flight operations were not changed for female NASA astronaut menstruation cycles (regardless of the questions of the media and the uncertainty of NASA engineers about how many tampons to send on the Space Shuttle93). Factors can be designed into space craft to improve the participant Gz tolerance, such as reclining seats. The improvement in G tolerance is enhanced by reducing the aortic valve/eye column height and can provide additional tolerance, though limitations such as difficulty inhaling limit G tolerance to 14–15 Gz maximum. These limits were explored historically by the Air Force’s John Stapp, who physically demonstrated that humans could survive deceleration forces of up to 46.2 G.94 Modern studies have demonstrated that many people with chronic illness are able to withstand acceleration forces that simulate current predicted flight profiles without difficulty.3,95 The effects of acceleration on human physiology center on the cardiac, pulmonary, musculoskeletal, neurovestibular, and psychological systems. In commercial aviation, the most common illness reported is gastrointestinal (22.3%), followed by cardiovascular (21.8%) and respiratory illnesses (10.2%).96,97 Deaths in commercial aviation are infrequent, around 0.3/million passengers,98 but do occur and will likely occur in SFP, as well.

45.3.6  Analogous Participant Environments While it is tempting to view the space environment as wholly unique, the space flight medical community also views it in the context of other travel and working environments. For example, at a recent meeting of the Next Generation Suborbital Researchers Conference (2016 NSRC), the panel framed the work of a scientist in a suborbital spacecraft as working in an “extension of the lab” or in “a novel laboratory field environment.” Many of the concerns for space flight are also present in the more mundane aerospace environment, long-duration naval deployments, polar research, as well as extreme travel and wilderness medicine. Thus, like a travel medicine specialist, human performance specialist, or occupational health specialist, each clinician must view the individual in the context of his or her unique environment. This requires becoming deeply familiar with the unique risks of a novel environment, with how the individual’s clinical state aligns with the unique environment, and with attention to the unique countermeasures for this environment. A deep sea diver or an Everest climber (whether a worker or a space flight participant) are useful comparators. For the radiation concern, a commercial airline pilot is a reasonable homolog, with the radiation concerns in suborbital space or beyond, requiring additional attention.

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45.3.7  Exposure Conditions of the Space Environment The general conditions of commercial space flight include, short-term acceleration (G loading), barometric pressure, microgravity, ionizing radiation, nonionizing radiation, noise, vibration, temperature, humidity, cabin air, pressurized flight suits, confinement, behavioral issues, and communications issues.

45.3.8  Pressurization in Space Flight It is expected that most commercial space flight providers will use pressurized cabin environments. In these cases, the space flight experience will have elements similar to a commercial transcontinental airplane flight, which is maintained above 10.91 psi, or equivalent to an altitude of 8000 ft. In commercial space flight conditions where a pressurized flight suit is required, individuals will be expected to have sufficient training and experience in a pressurized suit, since the experience of confinement within the space suit presents unique psychological issues for many people.

45.3.9  Clearance to Fly Responsibilities The FAA has jurisdiction over launch and landing operations of commercial spacecraft. Importantly, the FAA does not require that individual SFP receive a medical evaluation and clearance to fly by an aerospace medicine physician, unless they are a pilot or crew. Pilots and crew are required to have a Class II flight physical performed by a physician trained as an aviation medical examiner, though some consider a Class I flight physical to be preferable.d A commercial space flight working group has created a set of guidelines that can be used by space flight providers as appropriate to the risk profiles and safety requirements. The FAA has a document providing suggestions for medical clearance of passengers, including a list of conditions considered unsuitable for flight.e Some airlines have lists of medical conditions that are d

 Class I examinations are typically reserved for commercial airline transport pilots, but are requested by most First Officers and many business aviation flight crews. Class II examinations are required for general aviation pilots, including business aviation flight crew, crop dusters, aerial advertising pilots, charter pilots, or navigators and first officers of commercial airline flights. Class I FAA flight physicals include an EKG at age 35, and annually after age 40. They are performed more frequently than a Class II exam (every 6 months vs 12 months), and require an experienced FAA flight examiner. The physical standards for medical clearance are otherwise similar. e

 Flight Crew Medical Standards and Spaceflight Participant Medical Acceptance Guidelines for Commercial Space Flight. FAA Report, 6/30/2012

disqualifying for flight. All standards defer to an examining physician for individual cases and when to provide a medical waiver.99,100 The final decision on medical clearance falls to the examining physician and his or her medical judgment about the probability of an adverse event. Should an adverse event occur, there should be adequate emergency medical preparation to provide the appropriate medical response.

45.3.10  Informed Consent and Waivers It is assumed that a commercial space flight carries an unknown risk of injury or death. At the same time, the FAA has elected not to limit the ability of individuals to assume these risks, as with many other recreational activities, such as mountain climbing, parachuting, and scuba diving. Individuals are allowed to participate in hazardous activities, provided they are able to consent and are informed of the risk of participation. Therefore, the legal responsibility for informed consent lies with the space flight provider. This is governed by 14 CFR Part 460, Subpart B, Launch and Reentry with a Space Flight Participant and reads as follows: § 460.45 Operator informing space flight participant of risk. a. Before receiving compensation or making an agreement to fly a space flight participant, an operator must satisfy the requirements of this section. An operator must inform each space flight participant in writing about the risks of the launch and reentry, including the safety record of the launch or reentry vehicle type. An operator must present this information in a manner that can be readily understood by a space flight participant with no specialized education or training, and must disclose in writing: 1. For each mission, each known hazard and risk that could result in a serious injury, death, disability, or total or partial loss of physical and mental function. 2. That there are hazards that are not known. 3. That participation in space flight may result in death, serious injury, or total or partial loss of physical or mental function.

45.4  STANDARD CLINICAL APPROACHES TO SPACE FLIGHT MEDICAL ASSESSMENT 45.4.1  Recommended Clinical Assessment for Humans Entering Space Professional astronauts, whether part of a private or government space program, will generally undergo

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45.4  Standard Clinical Approaches to Space Flight Medical Assessment

more rigorous assessment and more detailed provision of countermeasures. This would include pilots, engineers, physicians, and scientists who enter the space environment repeatedly. A more comprehensive clinical approach is warranted in such circumstances. Space flight participants will undergo a much less rigorous screening process. The screening for a participant in a suborbital space flight will be primarily focused on the person’s ability to safely undergo the acceleration stress of the launch and landing operations. Longer-term effects and chronic illness would not be expected to have a significant impact upon launch operations, as long as they are well controlled and regulated. Conditions that would impede the ability to self-extricate or escape a disabled craft would require careful consideration and evaluation. They could be managed if there was a demonstration of the ability and the spacecraft operator made allowances. This would be similar to an FAA SODA (Statement of Demonstrated Ability). Orbital operations will require an increasingly comprehensive review and monitoring of the SFP health. Conditions requiring daily medications for control, such as insulin-dependent diabetes mellitus, will require much more preparation to prevent adverse outcomes. Screening should take into consideration the remoteness of orbital operations and the lack of medical facilities, as well as the extreme environment that must be transitioned to evacuate a patient back to Earth. The purpose of these evaluations and screenings is not to determine if a space flight participant is fit to go to space. Below is a representation of the minimal examination suggested for space flight participant, based on a recent report by the International Academy of Astronautics Study Group.101 For additional information, a 2012 report entitled “Flight Crew Medical Standards and Spaceflight Participant Medical Acceptance Guidelines for Commercial Space Flight” was released by the Center of Excellence for Commercial Space Transportation at the University of Texas Medical Branch. The guidelines summarized in this document provide additional insight into the clinical assessment for suborbital and orbital commercial SFP102 (Table 45.6–45.7).

45.4.2  Preexisting Disease It is beyond the scope of this chapter to fully characterize how to assess the fitness of an individual with preexisting disease to enter a given space environment. However, studies conducted by the National Aerospace Training and Research (NASTAR) Center provide a glimpse into how individuals with various clinical conditions respond to a known space analog condition on Earth.

TABLE 45.6 Recommended Medical Tests for Prospective Orbital Space Passengers. Prospective Space Passengers Should Complete the Following Medical Tests: Medical tests Routine hematology

Resting electrocardiogram

Tonometry

Clinical chemistry (serum)

Chest X-ray (PA & lateral)

Audiogram

Urinalysis

Corrected visual acuity

Pulmonary function testing and/ or methacholine provocation test (if clinically indicated)

TABLE 45.7  Questionnaire for Prospective Commercial Space Flight Participants Inventory of medical concerns for prospective commercial space flight participants Otitis, sinusitis, bronchitis, asthma, upper respiratory infections, or other respiratory disorders

Mental disorders, anxiety, or history of hyperventilation *Refer to section IIIB2

Severe hay fever or allergies

Attempted suicide

Dizziness or vertigo

Use of medications

Significant motion sickness requiring medication

Alcohol or drug dependence or abuse

Fainting spells or any other loss of consciousness

Date of last menstrual period, current pregnancy, recent postpartum (less than 6 weeks), or recent spontaneous or voluntary termination of pregnancy

Seizures, convulsions, epilepsy, stroke, muscular weakness, or paralysis

History of pneumothorax (collapsed lung)

Tuberculosis, hepatitis, AIDS, or other chronic infectious disorder

Kidney stones or blood in the urine

Surgery and/or other hospital admissions

Gallstones or gallbladder disease

Recent significant trauma

Diabetes

History of decompression sickness (DCS)

Cancer

Anemia or other blood disorders

History of radiation treatment or occupational exposure to radiation

Heart or circulatory disorders, including implanted pacemaker or defibrillator

Rejection for life or health insurance

Uncontrolled high or low blood pressure

History of disability requiring accommodation or functional impairment

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The primary objective of this research was to observe how those with disorders, such as cardiac disease, diabetes, hypertension, lung disease, spinal injuries (neck, back, etc.), or spinal disease responded to hypergravity exposure. Eighty-six participants (age 20–78) were exposed to controlled hypergravity, using a human centrifuge over a 2 day period at the NASTAR Center in Philadelphia, PA. They were monitored for subjective and objective symptoms of intolerance, including neurovestibular alterations, pulse oximetry, heart rhythm, heart rate, and blood pressure. In general, participants responded extremely well, with excellent tolerance of acceleration forces, regardless of medical history, medication use, age, and sex. Of significance was the finding that no adverse respiratory, cardiac, or cerebrovascular events occurred during the study. This included volunteers with medical histories that would have been disqualifying for selection as a career astronaut. Among the latter were those with diabetes that required indwelling insulin pumps. Others had cardiac dysrhythmia and implanted pacemakers. Those with musculoskeletal injuries requiring surgery or with long-term debility also tolerated the gravitational forces well.103 It is important to reiterate that this analog study was not conducted in space, which has a range of unique features, including launch (and reentry) acceleration forces, microgravity, radiation, isolation, and confinement. Nevertheless, the findings are encouraging, as we attempt to better understand the tolerances of humans not specifically trained for the space condition. Of additional note is the finding that orbital SFP have previously flown without incident, despite having multiple significant clinical morbidities.104

45.4.4  FAA Minimal Requirements for Space Flight Participants For crew members, those with safety-critical roles must possess and carry an FAA second-class airman medical certificate. According to US law, other Space Flight Participants (passengers) must: Sign informed consent after education about the risks ● Sign waiver of claims against the US Government ● Have training for emergency situations—smoke, fire, depressurization, emergency exit ● Meet security requirement—may not carry on board any explosives, firearms, knives, or other weapons. ●

45.4.5  Space Flight Disqualifying Medical Issues As of this writing, there are no specific required medical guidelines that would absolutely disqualify a person from flying into space. However, there are some basic guidelines for what would present an overwhelming risk of significant morbidity and mortality. Issues that would be disqualifying for space flight are those that would absolutely prevent the space flight participant from surviving the physiologic stresses of space flight, those with an anatomic deformity not consistent with survival during launch and landing operations, or those with a psychological construct not able to safely exist in a hazardous environment. To ensure safety of all participants, a robust waiver review process should be followed. For crew, this should be in accordance with FAA policy as outlined in 14 CFR Part 67. Some disqualifying conditions: Current pregnancy Individual below the age of legal consent (generally under age 18) ● Highly infectious conditions and communicable disease likely to be spread in confined quarters ● Recent surgery or trauma ● Conditions requiring medical devices incompatible with the operational environment of the spacecraft ● Uncontrolled psychiatric conditions that result in behavioral issues ● Functional defects or disabilities that interfere with the use of personal protective equipment ● Significant seizure disorder ● Known aneurysm ● Significant paralysis. ●

45.4.3  Recommended Space Analog Testing for Medical Clearance Some medical conditions may be cleared for space flight following special medical assessments in simulated space flight environments, including the use of an altitude chamber, high altitude mixed-gas (hypobaric) simulation, zero-G flight, and high-G centrifuge. The purpose is to run the potential space participant through a range of conditions that would identify novel clinical risks or disqualifying conditions. Using a flexible approach that applies aerospace medicine knowledge and experience-based medical risk analysis, it may be possible to permit special medical accommodations for prospective participants who have certain pathologies (including disabilities100). However, it should also be considered that spontaneous medical events have occurred in professional astronauts, even though each has undergone extensive screening, selection, and training.

●

45.5  FINAL CONSIDERATIONS Clinical care applied to the emergent field of commercial space exploration and commercial space flight

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occupational health presents a unique challenge to the clinical practice that is focused on Earth-based medicine. In the past, flight surgeons, aerospace medicine physicians, and emergency physicians with specialized training were those most likely to encounter individuals who will fly or who have flown in space. On the near horizon will be a new class of space flight aspirants who are looking for guidance on whether it is safe to fly, what special needs might require attention in order to fly, or to address the medical needs after one or many such flights. The question of who is best suited to attend to the medical needs of the commercial space flight community will be part of an evolving discussion. Previously, we described two closely aligned clinical approaches to supporting the space flight participant entering the commercial space flight environment. These are the standard clinical approach to space flight medical assessment and the personalized precision medicine approach to space flight medical assessment and countermeasures. In the case of the standard approach, it is expected that pre- and post-flight medical assessment of an individual will be conducted by a physician fully trained in aerospace medicine. In this context, it is likely that commercial space flight providers will have an approved list of aerospace medicine physicians available to consumers or employ physicians on staff that are authorized to provide such standard medical assessments. This will be a means to apply basic standards across commercial space flight platforms. Until the commercial space flight sector has had sufficient time to mature, this will reduce the likelihood that non-aerospace physicians will encounter pre- and pos-tflight participants. In the following chapter, we discuss the personalized precision medicine approach, where the situation is somewhat different. While the personalized medicine approach certainly falls within the purview of the aerospace medicine physician, it also falls within the training and expertise of a wide range of other medical professionals. An essential goal of anyone entering the space flight environment is to have the optimum experience, with optimum performance, and no adverse events (outcomes). The personalized precision medicine approach is focused solely on these same goals. Thus, it can be applied in advance of the space flight experience or at any time following the return from space. At the moment, this approach can be viewed as adjunctive to the standard approach. However, we expect that this personalized approach will 1 day be foundational to the standard aerospace medicine methodology. In the meantime, we anticipate that physicians in the community who have training and expertise in personalization, based on molecular phenotyping, will have the opportunity to benefit commercial SFP.

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References 1. WorldSpaceFlight.com. Astronaut and Cosmonayt Statistics 2016. . Accessed 1.11.16. 2. Mark S, Scott G, Donoviel D, et al. The impact of sex and gender on adaptation to space: executive summary. J Women’s Health. 2014;23(11):941–947. 3. Blue R, Pattarini J, Reyes D, et  al. Tolerance of centrifuge-simulated suborbital spaceflight by medical condition. Aviat Space Environ Med. 2014;85(7):721–729. 4. Eckberg D, Halliwill R, Beightol A, et al. Human vagal baroreflex mechanisms in space. J Physiol. 2010;588(7):1129–1138. 5. Blue R, Reyes D, Castleberry T, et al. Centrifuge-simulated suborbital spaceflight in subjects with cardiac implanted devices. Aerosp Med Hum Perform. 2015;86(4):410–413. 6. Mader T, Gibson C, Pass A. Optic disk edema, globe flattening, choroidal folds, and hyperopic shifts observed in astronauts after long-duration space flight. Opthamology. 2011;118:2058–2069. 7. Alexander D. Human Research Program Human Health Countermeasures Element Evidence Report: Risk of SpaceflightInduced Intracranial Hypertension and Vision Alterations. National Aeronautics and Space Administration 2012. Retrieved from . 8. Waters W, Ziegler M, Meck J. Postspaceflight orthostatic hypotension occurs mostly in women and is predicted by low vascular resistance. J Appl Physiol. 2002;92:586–594. 9. Martin E, Tan S, MacBride L, et  al. Sex differences in vascular and endothelial responses to acute mental stress. Clin Auton Res. 2008;18:339–345. 10. Sonnenfeld G, Shearer W. Immune function during space flight. Nutrition. 2002;18:899–903. 11. National Aeronautics and Space Administration (NASA). Lifetime Surveillance of Astronaut Health. Current Research. Houston, Texas. ; 2014 Accessed 15.09. 12. Goble F, Konopka E. Sex as a factor in infectious diseases. Trans NY Acad Sci. 1973;35:325–346. 13. Whitacre C. Sex differences in autoimmune disease. Nat Immunol. 2001:777–780. 14. Pierson D., Mehta S., Bruce R., et  al. Role of environmental factors in immunity and infectious disease risk. Paper presented at: International Conference on Environmental Systems, Rome, Italy, 2005. 15. Kim W, Tengra F, Young Z, et  al. Spaceflight promotes biofilm formation by Pseudomonas aeruginosa. PLoS One. 2013;8:e62437. 16. Crabbe´ A, Schurr M, Monsieurs P, et al. Transcriptional and proteomic response of Pseudomonas aeruginosa PAO1 to spaceflight conditions involves Hfq regulation and reveals a role for oxygen. Appl Environ Microbiol. 2011;77:1221–1230. 17. Gueguinou N, Huin-Schohn C, Bascove M, et  al. Could spaceflight-associated immune system weakening preclude the expansion of human presence beyond Earth’s orbit? J Leukoc Biol. 2009;86:1027–1038. 18. How to cite conference abstract: Ott C. Human immune function and microbial pathogenesis in human spaceflight. International Symposium on Microbial Ecology, Cancun, Mexico, 2004. 19. Ilyin V. Microbiological status of cosmonauts during orbital spaceflights on Salyut and Mir orbital stations. Acta Astronaut. 2005;56(9–12):839–850. 20. Nickerson C, Ott C, Wilson J, et al. Microbial responses to microgravity and other low-shear environments. Microbiol Mol Biol Rev. 2004;68:345–361. 21. Pierson D, McGinnis M, Viktorov A. Microbiological contamination. Nicogossian A, Mohler S, Gazenko O, Grigoryev A, editors.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

670

22.

23. 24.

25.

26. 27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

45.  Personalized Medicine in Space Flight, Part I: Standard Clinical Approaches

Space Biology and Medicine, Vol. 2. Washington, DC: American Institute of Aeronautics and Astronautics, Inc; 1994:77–93. Taylor G, Zaloguev S. Medically important microorganisms recovered from Apollo-Soyuz Test Project (ASTP) crewmembers. Life Sci Space Res. 1977;15:207–212. Thaiss C, Zmora N, Levy M, et  al. The microbiome and innate immunity. Nature. 2016;535:65–74. Crucian B, Sams C. Immune system dysregulation during spaceflight: clinical risk for exploration-class missions. J Leukoc Biol. 2009;86:1017–1018. Spencer M, Cherry J, Powell K, et  al. Antibody responses following Rubella immunization analyzed by HLA and ABO types. Immunogenetics. 1977;4:365–372. Cahill L. Why sex matters for neuroscience. Nat Rev Neurosci. 2006;7:477–484. Klein L, Corwin E. Seeing the unexpected: how sex differences in stress responses may provide a new perspective on the manifestation of psychiatric disorders. Curr Psychiatry Rep. 2006;4: 441–448. Schroeder J. Sex and gender in sensation and perception. In: Chrisler J, McCreary D, eds. Handbook of gender research in psychology. New York: Springer Science + Business Media; 2006: 235–257. Sato H, Sando I, Takahashi H. Computer-aided three dimensional measurement of the human vestibular apparatus. Otolaryngol Head Neck Surg. 1992;107:405–409. Moriyama H, Itoh M, Shimada K, et al. Morphometric analysis of fibers of the human vestibular nerve: sex differences. Eur Arch Otorhinolaryngol. 2007;264:471–475. Tremblay L, Elliott D, Starkes J. Gender differences in perception of self-orientation: software or hardware? Perception. 2004;33:329–337. Cahill L, Haier R, White N, et al. Sex-related difference in amygdala activity during emotionally influenced memory storage. Neurobiol Learn Memory. 2001;75:1–9. Cahill L, Uncapher M, Kilpatrick L, et al. Sex-related hemispheric lateralization of amygdala function in emotionally influenced memory: an FMRI investigation. Learn Memory. 2004;11:261–266. Canli T, Desmond J, Zhao Z, et  al. Sex differences in the neural basis of emotional memories. Proc Natl Acad Sci USA. 2002;99:10789–10794. Zubieta J, Dannals R, Frost J. Gender and age influences on human brain mu-opioid receptor binding measured by PET. Am J Psychiatry. 1999;156:842–848. Reschke M, Cohen H, Cerisano J, et  al. Sex and gender on adaptation to space: neurosensory systems. J Women’s Health. 2014;23(11):959–962. Vico L, Collet P, Guignandon A, et al. Effects of long-term microgravity exposure on cancellous and cortical weight bearing bones of cosmonauts. Lancet. 2000;355:1607–1611. Deschenes M, McCoy R, Mangis K. Factors relating to gender specificity of unloading-induced declines in strength. Muscle Nerve. 2012;46:210–217. Shackelford L, LeBlanc A, Driscoll T, et  al. Resistance exercise as a countermeasure to disuse-induced bone loss. J Appl Physiol. 2004;97(1985):119–129. Trappe S, Trappe T, Gallagher P, et al. Human single muscle fiber function with 84 day bed-rest and resistance exercise. J Physiol. 2004;557:501–513. Deschenes M, McCoy R, Holdren A, et al. Gender influences neuromuscular adaptations to muscle unloading. Eur J Appl Physiol. 2009;105:889–897. Clark B, Manini T, Hoffman R, et  al. Restoration of voluntary muscle strength after 3 weeks of cast immobilization is suppressed in women compared with men. Arch Phys Med Rehabil. 2009;90:178–180.

43. Trappe S, Creer A, Slivka D, et  al. Single muscle fiber function with concurrent exercise or nutrition countermeasures during 60 days of bed rest in women. J Appl Physiol. 2007;103:1242–1250. 44. Trappe S, Creer A, Minchev K, et al. Human soleus single muscle fiber function with exercise or nutrition countermeasures during 60 days of bed rest. Am J Physiol RegulIntegr Comp Physiol. 2008;294:R939–R947. 45. Fortes C, Goldberg T, Kurokawa C, et al. Relationship between chronological and bone ages and pubertal stage of breasts with bone biomarkers and bone mineral density in adolescents. J Pediatr (Rio J). 2014;90(6):624–631. 46. Beller G, Belavy’ D, Sun L, et  al. WISE-2005: Bed-rest induced changes in bone mineral density in women during 60 days simulated microgravity. Bone. 2011;49:858–866. 47. Squire M, Brazin A, Keng Y, et  al. Baseline bone morphometry and cellular activity modulate the degree of bone loss in the appendicular skeleton during disuse. Bone. 2008;42(2):341–349. 48. Hefferan T, Evans G, Lotinun S, et al. Effect of gender on bone turnover in adult rats during simulated weightlessness. J Appl Physiol. 2003;95:1775–1780. 49. Scheuring R, Mathers C, Jones J, et  al. Musculoskeletal injuries and minor trauma in space: incidences and injury mechanisms in U.S. astronauts. Aviat Space Environ Med. 2009;80:117–124. 50. Ploutz-Snyder L, Bloomfield S, Smith S, et al. Effects of sex and gender on adaptation to space: musculoskeletal health. J Women’s Health. 2014;23(11):963–966. 51. Cucinotta F, Kim M, Willingham V, et al. Physical and biological organ dosimetry analysis for International Space Station astronauts. Radiat Res. 2008;170:127–138. 52. Hassler D, Zeitlin C, Wimmer-Schweingruber R, et  al. Mars’ surface radiation environment measured with the mars science laboratory’s curiosity rover. Science. 2014;343:6169. 53. Nichiporuk I, Evdokimov V, Erasova V, et al. Male reproductive system in conditions of bed-rest in a headdown tilt. J Gravit Physiol. 1998;5:P101–P102. 54. Tash J, Johnson D, Enders G. Long-term (6-week) hindlimb suspension inhibits spermatogenesis in adult male rats. J Appl Physiol. 2002;92:1191–1198. 55. Tash J., Roby K., Gupta V., et  al. Long-term spaceflight impacts on male reproductive health. In: Fundamental Space Biology Division, ed. NASAPRS Task book. NASA, 2012. 56. Uno A, Takeda N, Horii A, et  al. Histamine release from the hypothalamus induced by gravity change in rats and space motion sickness. Physiol Behav. 1997;61:883–887. 57. Blanc S, Somody L, Gharib A, et  al. Counteraction of spaceflight-induced changes in the rat central serotonergic system by adrenalectomy and corticosteroid replacement. Neurochem Int. 1998;33:375–382. 58. Garcia-Ovejero D, Trejo J, Ciriza I, et al. Space flight affects magnocellular supraoptic neurons of young prepuberal rats: transient and permanent effects. Brain Res Dev Brain Res. 2001;130:191–205. 59. Kastello G, Sothmann M. Brain norepinephrine changes with simulated weightlessness and relation to exercise training. Physiol Behav. 1999;66:885–891. 60. Morita H, Abe C, Awazu C, et  al. Long-term hypergravity induces plastic alterations in vestibulo-cardiovascular reflex in conscious rats. Neurosci Lett. 2007;412:201–205. 61. Strollo F, Riondino G, Harris B, et  al. The effect of microgravity on testicular androgen secretion. Aviat Space Environ Med. 1998;69:133–136. 62. Heinrichs M, Baumgartner T, Kirschbaum C, et al. Social support and oxytocin interact to suppress cortisol and subjective responses to psychosocial stress. Biol Psychiatry. 2003;54:1389–1398. 63. Sulak P, Lippman J, Siu C, et  al. Clinical comparison of triphasicorgestimate/35 micrograms ethinyl estradiol and monophasic norethindrone acetate/20 micrograms ethinyl estradiol.

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

REFERENCES

Cycle control, lipid effects, and user satisfaction. Contraception. 1999;59:161–166. 64. Wiegratz I, Kutschera E, Lee J, et al. Effect of four oral contraceptives on thyroid hormones, adrenal and blood pressure parameters. Contraception. 2003;67:361–366. 65. Molina PE. Female reproductive system. In: Molina PE, ed. Endocrine Physiology. 3rd ed. New York: The McGraw-Hill Companies, Inc; 2010:215–254. 66. Collins D. Behavioral problems in early human spaceflight: USAF Human Resources Laboratory. Spaceflight Safety Mag. ; 2015. 67. Goel N, Bale T, Epperson C, et  al. Effects of sex and gender on adaptation to space: behavioral health. J Women’s Health. 2014;23(11):975–986. 68. Spaeth A, Dinges D, Goel N. Effects of sleep restriction on weight gain, caloric intake and meal timing in healthy adults. Sleep. 2013;36:981–990. 69. Simpson N, Banks S, Dinges D. Sleep restriction is associated with increased morning plasma leptin concentrations, especially in women. Biol Res Nurs. 2010;12:47–53. 70. Irwin MR, Carrillo C, Olmstead R. Sleep loss activates cellular markers of inflammation: sex differences. Brain Behavior Immunity. 2010;24(1):54–57.http://dx.doi.org/10.1016/j.bbi. 2009.06.001 71. Adan A, Natale V. Gender differences in morningness–eveningness preference. Chronobiol Int. 2002;19:709–720. 72. Musson D, Sandal G, Helmreich R. Personality characteristics and trait clusters in final stage astronaut selection. Aviat Space Environ Med. 2004;75:342–349. 73. McFadden T, Helmreich R, Rose R, et  al. Predicting astronaut effectiveness: a multivariate approach. Aviat Space Environ Med. 1994;65:904–909. 74. Sarris A. Personality, culture, fit, and job outcomes on Australian Antarctic stations. Environ Behav. 2006;38(3):356–372. 75. Steel GD, Suedfeld P, Peri A, Palinkas LA. People in high latitudes: The “Big-Five” personality characteristics of the circumpolar sojourner. Environ Behav. 1997;29:324–347. 76. Leon G. Men and women in space. Aviat Space Environ Med. 2005;76:B84–B88. 77. Kanas N. Psychosocial issues affecting crews during long-duration international space missions. Acta Astronaut. 1998;42:339–361. 78. Rosnet E, Jurion S, Cazes G, et  al. Mixed-gender groups: coping strategies and factors of psychological adaptation in a polar environment. Aviat Space Environ Med. 2004;75:C10–C13. 79. Grant I, Eriksen H, Marquis P, et  al. Psychological selection of Antarctic personnel: The “SOAP” instrument. Aviat Space Environ Med. 2007;78:793–800. 80. Wood J, Schmidt L, Lugg D, et al. Life, survival, and behavioral health in small closed communities: 10 years of studying isolated Antarctic groups. Aviat Space Environ Med. 2005;76:B89–B93. 81. Thieda K. Men’s anxiety vs. women’s anxiety. Psych Central ; 2011 Assessed 01.11.16. 82. Davidson J, Rothbaum B, van der Kolk B, Sikes C, Farfel G. Multicenter, double-blind comparison of sertraline and placebo in the treatment of posttraumatic stress disorder. Arch Gen Psychiatry. 2001;58:485–492. 83. Kessler RC. Epidemiology of women and depression. J Affect Disord. 2003;74:5–13. 84. National Council on Radiation Protection and Measurements. Ionizing radiation exposure of the population of the United States. Report Number 160. Bethesda, MD. Accessed 14.10.16. 85. International Comission on Radiological Protection. The 2007 Reccomandations of the International Comission on Radiological Protection. ICRP Publication 103. Ann ICRP 2007;37(2–4):1–332.

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86. Turner, R. Radiation health risks to the commercial space flight (suborbital and orbital). Washington, DC: Commercial Space Transportation Conference, Feburary 2012. 87. NASA Science. Who’s afraid of a Solar Flare? . Published October 7th, 2005. Assessed 15.10.16. 88. Pellmen E, et  al. Concussion in professional football: reconstruction of game impacts and injuries. Neurosurgery. 2003;53(4):799–814. 89. Burton R. Mathematical models for predicting G-duration tolerances. Aviat Space Environ Med. 2000 Oct;71(10):981–990. 90. Hearon C, et  al. Male/female SACM endurance comparison: support for the Armstrong Laboratory modifications to the CSU-13B/P anti-G suit. Aviat Space Environ Med. 1998;69(12):1141–1145. 91. Dooley J, et al. Accomodation of females in the high-G environment: the USAF Female Acceleration Tolerance Enhancement (FATE) Project. Aviat Space Environ Med. 2001;72(8): 739–746. 92. Heaps C, Fischer M. Female acceleration tolerance: affects of menstrual state and physical condition. Aviat Space Environ Med. 1997;68(6):525–530. 93. Ride S NASA Johnson Space Center Oral History Project 2002. ; Assessed 4.11.16. 94. Stapp J, Gell C. Human exposure to linear declarative force in the backward and forward facing seated positions. Mil Surg. 1951;109(2):106–109. 95. Blue R, Riccitello J, Tiard J, et al. Commercial spaceflight participant G-force tolerance during centrifuge-simulated suborbital flight. Aviat Space Environ Med. 2012;83(10):929–934. 96. Bagshaw M. Telemedicine in British Airways. J Telemed Telecare. 1996;2(1):36–38. 97. Johnston R. Clinical Aviation Medicine: safe travel by air. Clin Med. 2001;1(5):385–388. 98. Cummings R, Chapman P, Chamberlain D, et al. In-flight deaths during commercial air travel. How big is the problem? JAMA. 1988;259:1983–1988. 99. Cuttino M. Personal Communication, 2016. 100. Antuñano M, Baisden D, Davis J, et  al. Guidance for Medical Screening of Commercial Aerospace Passengers. Office of Aerospace Medicine, Federal Aviation Administration Report 2006. DOT/FAA/AM-06/1. ; Assessed October 2016. 101. Antuñano M, Gerzer R, Russomano T, et  al. Medical Safety Considerations for Passengers on Short-Duration Commercial Orbital Space Flights. International Academy of Astronautics Study Group. ; 2008. 102. Jennings R, Vanderploeg J, Antuñano M, et  al. Flight Crew Medical Standards and Spaceflight Participant Medical Acceptance Guidelines for Commercial Space Flight. Center for Excellence for Commercial Space Transportation. Published June 30th, 2012. ; Assessed September 2016. 103. Blue RS, Mathers CH, Castleberry TL, et al., (2014b). The human challenges of commercial spaceflight: an overview of medical research conducted by the University of Texas Medical Branch through the Federal Aviation Administration Center of Excellence for Commercial Space Transportation. New Space 2014;2(3). 104. Jennings R, Murphy D, Ware D, et al. Medical qualification of a commercial spaceflight participant: not your average astronaut. Aviat Space Environ Med. 2006;77(5):475–784.

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Further Reading 1. The Impact of Sex and Gender on Adaptation to Space: A NASA Decadal Review. J Women’s Health 2014;23(11) (Special Issue). 2. Mark S, Scott GBI, Donoviel DB, et al. The impact of sex and gender on adaptation to space: executive summary. J Women’s Health. 2014;23(11):941–947. 3. Platts SH, Noel BaireyMerz C, Barr Y, et al. Effects of sex and gender on adaptation to space: cardiovascular alterations. J Women’s Health. 2014;23(11):950–955. 4. Kennedy AR, Crucian B, Huff JL, et  al. Effects of sex and gender on adaptation to space: immune system. J Women’s Health. 2014;23(11):956–958.

5. Ploutz-Snyder L, Bloomfield S, Smith SM, Hunter SK, Templeton K, Bemben D. Effects of sex and gender on adaptation to space: musculoskeletal health. J Women’s Health. 2014;23(11):963–966. 6. Ronca AE, Baker ES, Bavendam TG, et al. Effects of sex and gender on adaptation to space: reproductive health. J Women’s Health. 2014;23(11):967–974. 7. Reschke MF, Cohen HS, Cerisano JM, et  al. Effects of sex and gender on adaptation to space: neurosensory systems. J Women’s Health. 2014;23(11):959–962. 8. Goel N, Bale T, Epperson C, et  al. Effects of sex and gender on adaptation to space: behavioral health. J Women’s Health. 2014;23(11):975–986.

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C H A P T E R

46 Personalized Medicine in Space Flight, Part II: Personalized Precision Medicine Approaches Michael A. Schmidt1,2, Thomas Goodwin1,3 and Marsh Cuttino4,5 1

Advanced Pattern Analysis & Countermeasures Group, Research Innovation Center, Colorado State University, Fort Collins, CO, United States, 2Sovaris Aerospace, LLC, Boulder, CO, United States, 3NASA, Johnson Space Center (Retired), Houston, TX, United States, 4Emergency Medicine Physician, Richmond, VA, United States, 5Orbital Medicine, Inc., Richmond, VA, United States

O U T L I N E 46.1 Introduction 46.2 Personalized Approaches to Space Flight Medical Assessment and Countermeasures 46.2.1 The Molecular Landscape Beneath the Clinical Phenotype

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46.3 Nonessential Inputs, Molecular Networks, and the Clinical Phenotype 676 46.3.1 Nonessential Inputs: Personalized Drug Therapeutics Based on Pharmacogenomics and Pharmacometabolomics 676

46.1 INTRODUCTION Systems biology can be described as the study of the interactions between the components of biological systems, and how these interactions give rise to function and behavior of that system. In human medicine, systems biology traditionally incorporates genomics, transcriptomics, proteomics, and metabolomics. More recent advances in the technology of the field has helped to widen this definition. It is now possible to incorporate exposomics, exomics, epigenomics, microbiomics, among others, to elucidate a more comprehensive picture of a biological system at any given moment in time. Data derived from these Omics disciplines are combined with metadata about an individual’s: Principles of Gender-Specific Medicine. DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00064-4

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(1) environment; (2) diet; (3) nutritional status; (4) psychosocial dynamics; (5) lifestyle; (6) medical treatments; (7) antecedents; (8) anthropometrics; and (9) broad information about the phenotype. Multivariate analysis and modeling methods are applied to a high dimensional data sets derived from analyses, in order to describe systems, derive meaning, and generate new hypotheses. Various phenotypic changes occur when humans enter the space environment or a space analog condition. The general phenotypes under observation are typically characterized as morphological, physiological, and behavioral. In such circumstances, attention is typically given to a broad range of clinical and physiologic measurements.

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It is important to recognize, however, that these morphological, physiological, and behavioral phenotypes are governed by an underlying molecular network of vast complexity, i.e., the molecular dynamics give rise to these phenotypes. Targeted molecular profiling can yield useful insights into these networks, but such profiling provides only a glimpse into such networks. Comprehensive omics profiling (Fig. 46.1) can provide a more robust examination of these networks for research purposes, but the complexity of such profiles presently limits their utility in the clinical setting. In order to reduce this complexity and enable the application of personalized precision medicine, it is important to focus on what is essential and actionable. For these purposes, we focus our clinical efforts on understanding the essential, conditionally essential, and nonessential inputs into the system.

46.2  PERSONALIZED APPROACHES TO SPACE FLIGHT MEDICAL ASSESSMENT AND COUNTERMEASURES 46.2.1  The Molecular Landscape Beneath the Clinical Phenotype 46.2.1.1  Essential Inputs and the Clinical Phenotype We have coined the term essential input metabolomics (EIM) and the umbrella term essential input omics (to include essential input-based epigenomics,

transcriptomics, proteomics, and phenomics3,9). Essential Inputs refers collectively to the class of small molecules, amino acids, vitamins, fatty acids, and trace elements that must be obtained from the diet. They cannot be synthesized by the human body. This also includes conditionally essential inputs that may become essential, as a result of genotype, disease, drug therapeutics, environmental conditions, physical loading (athletics, occupation, temperature, etc.), or dietary deficiencies of essential precursors. They are collectively referred to as inputs, because these are external components that input into the system to collectively influence all metabolic activities. These effects can be pronounced, regardless of the genotype of the individual. Essential input metabolomics recognizes that essential inputs shape the metabolome (and metabolic networks) and that these essential inputs must be considered when attempting to understand the metabolome (and the clinical phenotype). It is tempting to view these micronutrient essential inputs only in general ways and to underestimate the impact of relative states of insufficiency or excess. However, if one views the human in terms of integrated molecular networks and gives the requisite attention to the core metabolic steps in which these essential inputs participate, the true magnitude of small deficits in these inputs can be appreciated. As a point of reference, a flavin-containing cofactor, FAD or FMN (riboflavin-derived), is utilized by 151 (4%) of the 3870 enzymes cataloged in the ENZYME database. Pyridoxal-5-phosphate (vitamin B6) is utilized by

FIGURE 46.1  Molecular Landscape that Underlies the Morphological, Physiological, and Behavioral Phenotype. The progression from genome to phenotype is shown above (left to right), representing the extraordinary complexity of this landscape. Numbers in parentheses represent the estimated number of unique molecular forms found in each subdomain. Clinical assessment of humans entering the space environment frequently occurs at the physiological, morphological, and behavioral levels. More complex characterization of the molecular landscape in males and females entering space will add to our ability to optimize the space flight experience and enhance safety. A first step assessment of this landscape can be done by examining essential inputs.1,2

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46.2  Personalized Approaches to Space Flight Medical Assessment and Countermeasures

112 (3%) of the 3870 enzymes cataloged in the ENZYME database.4 To date, enzymatic databases list over 600 enzymes for which Mg2+ serves as cofactor, and another 200 in which Mg2+ may act as activator.5–7 This is well above the original estimates of 300 for Mg2+ participation originally reported by Ebel and Gunther.8 These numbers are often referred to as “participation numbers.” For each of the essential or conditionally essential inputs, a participation number exists that describes the number of enzymatic steps or processes in which the input is involved. Using an artificial neural network schematic representation (Fig. 46.2), we show how (one or more) inputs interact within the hidden layer (process layer), which leads to one or more outputs. The nature of the output is governed by the processes taking place in the hidden layer. Essential inputs in the form of essential or conditionally essential dietary elements enter the system (human) and participate in a vast number of

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molecular networks (hidden layer). This hidden layer contains metabolic pathways and molecular networks spanning a range of processes that vary considerably, based on the status of essential inputs (deficiency or excess), the number of converging essential inputs, and the genetic landscape (genome, epigenome) within this hidden layer, including the posttranslational modifications taking place in the proteome. Each essential input may participate in several hundred to over one thousand distinct metabolic processes.4 Our personalized medicine approach is rooted in the understanding that the efficiency of these networks strongly impacts health and performance. This represents a novel way of looking at individual SFP susceptibility, risk, and countermeasure development. In each case of the examples that follow, there are key small molecules (or elements) and gene variants that have the potential to significantly modify the human response in space.

FIGURE 46.2  A schematic representation of essential inputs and outputs that shape the clinical phenotype (A) Inputs interact within the hidden layer, which leads to one or more outputs. This representation mirrors an artificial neural network. (B) Essential inputs in the form of essential or conditionally essential dietary elements enter into and interact across vast molecular networks. The hidden layer contains metabolic pathways and molecular networks spanning a range of processes that vary considerably, based on the status of essential inputs (deficiency or excess), the number of converging essential inputs, the genetic landscape within this hidden layer, and environmental exposure. These inputs shape the physiologic, morphologic, and behavioral phenotypes (i.e., the clinical phenotype, symptom profile, and performance outputs1).

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46.3  NONESSENTIAL INPUTS, MOLECULAR NETWORKS, AND THE CLINICAL PHENOTYPE While essential inputs shape the molecular networks and clinical phenotype, nonessential inputs may also have far reaching effects. Nonessential inputs include nonessential food components (dietary flavonoids, protein, fat, carbohydrate, etc.), beverages (juices, soda pop, etc.), vaccines, drugs, and many others. For the purpose of this chapter, we focus on the interaction between prescription drugs, as one influential nonessential input for the SFP. Thus, the following sections will explore personalization based on pharmacogenomics and pharmacometabolomics, as well as personalization based on micronutrient essential inputs. The potential impact of these inputs on the individual response to space radiation is presented as an example. This is because the response to radiation is generally viewed more strongly from the perspective of radiation dose and duration, coupled with an oft-presumed normative response of the individual. In our view, the individual response to radiation may vary significantly, based on essential input status and genetics.9 We also explore the radiation exposure/response dynamic from the standpoint of function, rather than disease.

46.3.1  Nonessential Inputs: Personalized Drug Therapeutics Based on Pharmacogenomics and Pharmacometabolomics Any individual entering a space environment who relies upon medication (a nonessential input) for the management of a medical condition would benefit from a profile of his or her individual drug-metabolizing enzyme capacity. This is recommended in order to minimize adverse drug reactions when in the space environment and where medical attention will not be immediately available. This profile gives attention to two features: CYP450 SNPs and Phase II conjugating enzyme function. 46.3.1.1  Personalized Drug Therapeutics Based on Cytochrome P450 The first such characterization of individual space participant biotransformation capacity should be based on individual cytochrome P450 (CYP450) profiles. The CYP450 family is a major subset of drug-metabolizing enzymes. The CYP450 family of enzymes includes, but is not limited to, the following important genes10: CYP2D6 is also known as debrisoquine hydroxylase, which catalyzes the oxidation of approximately a quarter of all the commonly used therapeutic drugs in clinical practice today. For instance, codeine is metabolized by CYP2D6 to morphine. In such cases, enhanced CYP2D6

activity (i.e., in CYP2D6 ultra-rapid metabolizers) predisposes one to opioid intoxication.11 CYP2C19 (S-mephenytoin hydroxylase) acts on weakly or strongly basic drugs containing one hydrogen bond donor, or if there are functional groups containing carbon or sulfur, double bonded to oxygen present in the substrate. CYP2C19 is responsible for the metabolism of anticonvulsant drugs, proton pump inhibitors, and drugs that inhibit platelet function. CYP3A4 is involved in the oxidation of the largest range of substrates of all the CYPs. It is the most abundantly expressed P450 in human liver and it is known to metabolize more than 120 different drugs. Examples of CYP3A4 substrates relevant in human space flight include: acetaminophen, diazepam, erythromycin, lidocaine, lovastatin, and warfarin. CYP3A4 also is sensitive to enzyme induction, which tends to lower plasma concentrations of CYP3A4 substrates, resulting in reduced efficacy of the substrate. Some CYP450 genes are highly polymorphic, resulting in enzyme variants that may shape variance in drugmetabolizing capacities among individuals at Earth gravity (1 G), let  alone the microgravity environment. For context, it is estimated that genetics account for 20–95% of variability in drug disposition and effects.12 CYP450 metabolic capacities may be described as follows (Table 46.1)13: TABLE 46.1 Drug Metabolism Genotype and Endophenotype Characteristics Drug metabolism endophenotype

Genotype and endophenotype characteristics

Extensive metabolizers (EM; aka Normal Metabolizers)

Have two active CYP450 enzyme gene alleles, resulting in an active enzyme molecule

Intermediate metabolizers (IM)

Have one active and one inactive CYP450 enzyme gene allele May require lower dosage than normal, though pro-drugs may require higher dose

Poor metabolizers (PM)

Lack active CYP450 enzyme gene alleles May suffer more adverse events at usual doses of active drugs due to reduced metabolism and increased concentrations May not respond to administered pro-drugs that must be converted by CYP450 enzymes into active metabolites

Ultrarapid metabolizers (UM)

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Have 3 or more active CYP450 gene alleles May not reach therapeutic concentrations at usual, recommended doses of active drugs May suffer adverse events from pro-drugs that must be converted by CYP450 enzymes into active metabolites May require higher doses of prodrugs

46.3  Nonessential Inputs, Molecular Networks, and the Clinical Phenotype

For an individual on medication who is entering the space environment, it will be helpful to ensure that any medication ingested is compatible with his individual CYP450 profile. Where conflicts exist, alternative medications can be chosen that are metabolized via a different CYP pathway. While this approach is desirable for clinical care on Earth, it may be even more essential in space. This is because we know little about how drug metabolism in space differs from that on Earth and there is more risk if an adverse drug reaction occurs far from medical care. The US Food and Drug Administration (FDA) has approved a microarray device (AmpliChip) that can detect 29 variations in CYP2D6 and polymorphisms for the CYP2C19gene.14 Tests for CYP3A4, CYP3A5, and others are also available for clinical use. 46.3.1.2  Personalized Drug Therapeutics Based on Phase II Conjugation For many drugs, metabolism via CYP450 is the first phase of drug biotransformation, as noted. These same drugs frequently pass through a Phase II biotransformation reaction. Phase II drug metabolism reactions are generally characterized as conjugation reactions, wherein a small molecule is bound to the drug metabolite to improve solubility for eventual excretion. Characterization of space participant Phase II profile might consider at least three features. These are: (1) SNP variant profiles of Phase II conjugation enzymes; (2) adequacy of micronutrient cofactors of Phase II enzymes; and (3) adequacy of conjugation agents that directly bind drugs, as part of Phase II conjugation. Phase II profiles can be conducted for each space participant and, where the evidence is sufficient, be used to develop appropriate countermeasures. For the Phase II profile, assessment considerations include, but are not limited to: 1. SNP Variant Profiles of Phase II Conjugation Enzymes. For instance, UGT (UDP glucuronosyltransferases) is an enzymatic superfamily, which is involved in conjugation of endogenous compounds (bilirubin, steroidal hormones, thyroid hormones, biliary acids, vitamins) and exogenous compounds (drugs, carcinogens, and polluting dietary elements) that are transformed in N-, O-, S-, C-glucuronates. They are responsible for roughly 35% of Phase II reactions. Understanding genetic variants of Phase II enzymes will be helpful in designing individualized drug regimens.15 2. Adequacy of Nutrient Cofactors of Phase II Enzymes (e.g., riboflavin as cofactor for glutathione-Stransferase and glutathione reductase): Assess preflight status of all nutrient cofactors of drug biotransformation, using serum, plasma, or cells (RBC, WBC) to ensure optimum status for each

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individual SFP. If warranted by genotype, provide nutrient cofactor at dosage commensurate with the allelic variant (e.g., wild-type, heterozygote, homozygote). 3. Conjugation Agents that Directly Bind Drugs, as Part of Phase II Conjugation: Assess preflight status of key conjugation agents (e.g., glutathione, glycine, cysteine, glutamine, arginine, taurine, acetate) to ensure optimum status for that individual. This can be done by plasma amino acid profiles or, in the case of glutathione, white blood cells or whole blood. For example, in most cases of glutathione conjugation, more polar glutathione conjugates are eliminated into the bile or are subsequently subjected to other metabolic steps. This eventually leads to formation of mercapturic acids, which are excreted in urine. This process depletes glutathione stores, as GSH binds drugs through the conjugation of Phase I intermediate metabolites. This depletion can limit the amount of glutathione available for future drug metabolism reactions and, also, alter the REDOX balance of the cell. For instance, the analgesic acetaminophen is converted to the electrophilic N-acetyl-p-benzoquinone imine (NAPQI), which is conjugated for removal by glutathione. If glutathione is in poor supply, NAPQI exerts highly toxic effects by covalent reactions with proteins, such as those found in mitochondria. This can lead to liver damage.16 If glutathione depletion is identified through blood chemistry (low GSH or low GSH:GSSG ratio), premission glutathione or glutathione precursors can be provided at the dosage needed to assure optimum mission status. Stable glutathione precursors, such as N-acetylcysteine, can be provided on missions. 46.3.1.3  Differences in Drug Metabolism Based on Ethnicity Ethnic differences in CYP450 isoforms, such as CYP2D6 and CYP2C19, can be pronounced and warrant consideration when entering the space environment. For instance, most western populations are characterized by roughly 93% normal (or efficient) metabolizers, 7% poor metabolizers, and 1% ultrarapid metabolizers of CYP2D6. In contrast, only 1% of Asians are considered poor metabolizers of CYP2D6. Roughly 20% of Asians are poor metabolizers via CYP2C19, while only about 4% of Caucasians are considered poor metabolizers via this isoform.17 CYP3A5 expression also varies widely with ethnicity. For instance, more than 50% of African Americans express CYP3A5, while some 30% of Caucasians express this isoform. Understanding ethnic variance in CYP functionality has the potential to greatly enhance SFP safety and performance, by assuring that prescribed drugs

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work as expected and that the probability of adverse reaction to these drugs is minimized. 46.3.1.4  Differences in Drug Metabolism in Males and Females For the purposes of space flight safety, differences in drug metabolism between males and females can be viewed through the lens of adverse events. The US FDA maintains a voluntary database for reporting of adverse events (FDA Adverse Events Reporting System; AERS). According to AERS data and other data sources, women experience more adverse events than men. In addition, the adverse events experienced by women are of a more serious nature than those reported by men.18–22 From 1997 to 2000, the General Accounting Office (USA) reported that 8 of 10 drugs withdrawn were due to a greater risk of adverse events in women.23 Yu et  al. examined 668 drugs covering the most frequent 20 treatment regimens in the United States. Of these, 307 drugs showed sex differences in adverse events. Moreover, there were 736 unique drug-event combinations with significant sex differences. Of these, 332 reported drug-event combinations showed preference for female patients and 404 showed preference for male patients.24 While we discuss drug metabolizing enzymes here among clinical targets to personalize drug therapy, the reasons for different adverse drug events by sex are more complex. The diversity of responses is likely due to a convergence of factors, such as pharmacokinetic or pharmacodynamic factors, polypharmacy, differences in reporting patterns, different body composition, body size, or frequency of ingestion of drugs. Presently, little is known about sex differences in the drug response in space and this should be considered when an individual enters the space environment.

46.3.2  Essential Inputs: Small Molecules, DNA Repair, and Metabolic Networks Below, we explore three representative cases where essential inputs and their broad molecular networks converge to significantly impact how an individual might respond to the space environment. We have specifically chosen susceptibility to radiation-related adverse outcomes, since radiation is a driving concern in the space environment. However, it is important to recognize that these same inputs have effects across all physiologic systems. The primary examples cited below concern (1) onecarbon metabolism, (2) iron metabolism, and (3) magnesium metabolism. Each case represents a set of actionable targets, where clinical metrics and laboratory analytics are currently available to guide clinical decision making.

But these examples emphasize a greater principle, which is the importance of (1) essential inputs, (2) genetic predisposition, (3) the uniqueness of the environment, and (4) the need to consider the human in space from a multivariate, convergent variable point of view. 46.3.2.1  Essential Inputs: One-Carbon Metabolism One-carbon metabolism involves the transfer of methyl groups (CH3) from donors, such as folate, B12, choline, and trimethylglycine (betaine). Folate, B12, and choline are essential inputs that must be obtained from the diet. The conditionally essential input in this molecular network is trimethylglycine, which is also a direct methyl donor to homocysteine (Hcy). A deficit in methyl donors leads to a series of adverse events important to humans in space. The enzymes that govern one-carbon metabolism are produced from a series of methyl transfer genes. Common polymorphisms in key methyltransferase genes also lead to adverse events of importance. Frequently, convergence exists between altered micronutrient intake and genetic polymorphisms (Fig. 46.2). The potential implications for human space flight are only just emerging. 46.3.2.1.1  One-Carbon Metabolism and Neuro-ocular Health There is mounting evidence that specific health risks, such as neuro-ocular changes (and increased intracranial pressure), are related to disordered one-carbon metabolism involving folate, B12, and homocysteine. Approximately 20% of ISS crew members on space flight missions of 4 months or longer have experienced ocular changes and persistent visual problems on return to Earth. These were correlated with significantly elevated levels of homocysteine, methylmalonic acid, and cystathionine, along with reduced levels of folate and Vitamin B12.25 The metabolite data indicate that a significant proportion of crew members may have associated genetic traits that contribute to disturbed one-carbon metabolism. A follow-up study examined ocular changes and genetic polymorphisms in the one-carbon molecular network in 49 astronauts. Variants of methionine synthase reductase (MTRR) and serine hydroxymethyltransferase (SHMT1) were found associated with visual deficits, based on the number of G alleles of MTRR 66 (MTRR A66G) and C alleles of SHMT1 (SHMT1 C1420T;) Ref. 26. Specifically, all of the astronauts with the homozygous MTRR 66GG genotype exhibited choroidal folds and cotton wool spots. In contrast, none of the individuals with the MTRR 66AA genotype had evidence of choroidal folds or cotton wool spots after ISS missions. Of further significance, none of the astronauts with the

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46.3  Nonessential Inputs, Molecular Networks, and the Clinical Phenotype

SHMT1 1420TT genotype (wild-type) had evidence of disc edema after flight. While the link between space flight-induced ocular changes and altered one-carbon metabolism requires further investigation, the existence of such a link may have significant implications, given the altered fluid dynamics known to occur in space flight. Otto and colleagues have documented that 15 longduration crew members have experienced in-flight and postflight visual and anatomical changes including optic-disc edema, globe flattening, choroidal folds, and hyperopic shifts as well as documented postflight elevated intracranial pressure (ICP). In the postflight time period, some individuals have experienced transient changes while others have experienced changes that are persisting with varying degrees of severity. While the underlying etiology of these changes is unknown at this time, the NASA medical community suspects that the microgravity-induced cephalad-fluid shift and commensurate changes in physiology play a significant role. Furthermore, in retrospective examination of data, >60% of long-duration crew members (ISS/ MIR) and >25% of short-duration (Shuttle) crew members have reported subjective degradation in vision (based on debrief comments29,30). Decreased near-visual acuity was demonstrated in 46% of ISS/Mir and 21% of Shuttle crew members, resulting in a shift of up to 1–2 diopters in their refractive correction. It is also known that homocysteine (Hcy) is a causative agent in retinal ganglion cell death.31 These phenomena may have direct implications for the documented loss of vision that ISS astronauts and other long-durations flyers have experienced. 46.3.2.1.2  One-Carbon Metabolism, Chromosome Instability, and Space Radiation A set of convergent variants in one-carbon metabolism is known to strongly influence chromosome stability via direct effects on DNA. In nucleic acid synthesis, deoxythymidylate (dTMP) is synthesized from deoxyuridylate (dUMP), through one-carbon transfer from a methyl donor. The methyl donor is typically methylfolate, though methyl transfer is also strongly influenced by B12, betaine, and choline status (choline and betaine metabolism intersect with folate metabolism at the methylation of homocysteine to form methionine32). When methyl groups are unavailable from folate, conversion of uracil to thymine is reduced. This results in an alteration of the dTMP/dUMP ratio and in uracil accumulation in cell nuclei.33 Normally, when uracil appears in DNA, it is excised by uracil glycosylase enzymes. However, with excessive uracil accumulation, the process can lead to transient single-strand breaks in DNA. Two opposing singlestrand breaks can lead to double-strand chromosome breaks, which are more difficult to repair and can lead

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to genome instability. Common fingerprints of such changes include micronuclei, nucleoplasmic bridges, and nuclear buds.34 In a small (N=22) cross-sectional study of micronucleus frequency in erythrocytes of splenectomized subjects, Blount et  al. showed that the elevated micronucleus index was strongly associated with low levels of serum folate. In this study, uracil levels were found to be 70 times higher in subjects whose serum folate 280 µg/d) in nine postmenopausal women in a metabolic unit. These women showed a significant increase in micronucleus frequency in lymphocytes following depletion and a decrease following repletion. Micronucleus frequency in buccal cells decreased after the repletion phase. The depletion phase in this study also resulted in DNA hypomethylation, increased dUTP/dTTP ratio, and lowered NAD levels in lymphocytes.37 Fenech et  al. performed a randomized double-blind placebo-controlled dietary intervention study (N=31, 32 per group) to determine the effect of folate and vitamin B12 (B12) on DNA damage (micronucleus formation and DNA methylation) and plasmahomocysteine (Hcy) in young Australian adults aged 18–32 years. The dietary intervention involved supplementation with 700 µg folic acid and 7 µg vitamin B12 in wheat bran cereal for 3 months, followed by 2000 µg folic acid and 20 µg vitamin B12 via tablets for a further 3 months. This study revealed that micronucleated cell frequency is minimized when plasma homocysteine is below 7.5 mol/L, serum B12 is above 300 pmol/L, and red cell folate is above 700 nmol/L. The study also revealed that elevated plasma homocysteine may be a direct risk factor for chromosome damage.36 Milić et  al. studied 36 healthy males who were not vitamin deficient. They found a positive association between an increased number of micronuclei and lower plasma vitamin B12 concentrations, suggesting vitamin B12 levels are instrumental in maintaining DNA

PRINCIPLES OF GENDER-SPECIFIC MEDICINE

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46.  Personalized Medicine in Space Flight, Part II: Personalized Precision Medicine Approaches

integrity—not just in vitamin B12 deficient population, but also in a healthy one.38 Kapiszewska et  al. demonstrated that more than 400 pg/mL of vitamin B12 in plasma in subjects with a positive folate balance is critical for genomic stability, with uracil misincorporation into DNA being further related to the MTHFR genotype.39 The level of folate and B12 needed to prevent genome instability is significantly greater than the level needed to prevent anemia. Variants in one-carbon metabolism that induce uracil accumulation in DNA, defective DNA repair, and chromosome instability, may render folate-deficient cells more sensitive to the damaging effects of a second external stress.33 This raises particular concerns for SFP with methyl cycle defects who are exposed to ionizing radiation from a variety of sources, including galactic cosmic rays, solar protons, and high energy electrons and protons trapped by the Earth’s magnetic field (Van Allen Belts). To put this in perspective, it is estimated that, during a mission to Mars, every cell nucleus in an astronaut’s body would be hit by a proton or a secondary electron every few days, and by a high atomic number and energy (HZE) particle about once a month.40 Recent data from the RAD (Radiation Assessment Detector) experiment on the Mars Science Laboratory41 reveal that a during a 360-day Mars transit mission, an astronaut would receive a dose of about 662 millisieverts (mSv). This is just short of international space agencies career exposure limits of 1000 mSv, a limit that corresponds to a 3% risk of exposure-induced death from cancer.42 Levels of exposure for sensitive neural tissue like the hippocampus are independently set by NASA at 500 mSv per year and 1500 mSv for a career.43 These exposure data argue for a dedicated effort aimed at better characterization of all inherent influences on DNA stability and DNA repair prior to entering such radiation environments, as well as during such exposure. This is highlighted by concern about the extent to which one-carbon defects may actually mimic the degree of DNA damage one might encounter in the space radiation environment. For instance, Fenech et al. found that the chromosomal damage in cultured human lymphocytes, caused by reducing folate concentration from 120 to 12 nmol/L, is equivalent to that induced by an acute exposure to 0.2 Gy of low linear-energy-transfer ionizing radiation (e.g., X-rays), a dose of radiation that is 10 times greater than the annual allowed safety limit of exposure for the general population.44–46 Raising additional concern is the potential impact of one-carbon deficits and concomitant exposure to radiation in space. While this has not been studied in space, Earth-based ex vivo and in vitro studies are informative. Fenech et  al. studied the combined effect of folic acid deficiency and radiation exposure on genome stability,

using cultured lymphocytes of 12 human subjects with different MTHFR genotypes. Ex vivo cells were grown for 9 days, using different concentrations of folic acid (12, 24, and 120 nM folic acid), and exposed to 0.5 Gy of gamma rays. The effect of folic acid was highly significant (p50% of variance of both types of micronuclei. Also, nucleoplasmic bridges and buds were significantly increased under low folate supply. The increase in bridges was mainly observed in MTHFR 677TT cells, highlighting a significant effect of the MTHFR genotype (p