Animal Models for Endometriosis: Evolution, Utility and Clinical Relevance [1st ed.] 9783030518554, 9783030518561

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Advances in Anatomy, Embryology and Cell Biology

Kathy L. Sharpe-Timms  Editor

Animal Models for Endometriosis Evolution, Utility and Clinical Relevance

Advances in Anatomy, Embryology and Cell Biology publishes critical reviews and state-ofthe-art research in the areas of anatomy, developmental and cellular biology. Founded in 1891, this book series has a long standing tradition of publishing focused and condensed information on a given topic with a special emphasis on biomedical and translational aspects. The series is open to both contributed volumes (each collecting 7 to 15 focused reviews written by leading experts) and single-authored or multi-authored monographs (providing a comprehensive overview of their topic of research). Advances in Anatomy, Embryology and Cell Biology is indexed in BIOSIS, Journal Citation Reports/Science Edition, Medline, SCImago, SCOPUS, Science Citation Index Expanded (SciSearch), Zoological Record. Manuscripts should be addressed to Editor-in-Chief Prof. Dr. PETER SUTOVSKY, Division of Animal Sciences and Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, Columbia, MO, USA e-mail: [email protected] Series Editors Prof. Dr. FRANCISCO CLASCÁ, Department of Anatomy, Histology and Neurobiology,  Universidad Autónoma de Madrid, Madrid, Spain e-mail: [email protected] Prof. Dr. Z. KMIEC, Department of Histology and Immunology, Medical University of Gdansk,  Gdansk, Poland e-mail: [email protected] Prof. Dr. HORST-WERNER KORF, Anatomy and Brain Research Center, Department for Anatomy 1, Heinrich Heine University Düsseldorf, Düsseldorf, Germany e-mail: [email protected] Prof. Dr. MICHAEL J. SCHMEISSER, Institute of Microscopic Anatomy and Neurobiology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany e-mail: [email protected] Prof. Dr. BALJIT SINGH, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada e-mail: [email protected] Prof. Dr. JEAN-PIERRE TIMMERMANS, Laboratory of Cell Biology and Histology/Core Facility Biomedical Microscopic Imaging, Department of Veterinary Sciences, University of Antwerp, Wilrijk, Belgium e-mail: [email protected] Prof. Dr. SVEN SCHUMANN, Inst, f. Mikroskop. Anatomie u. Neurobio, Johannes Gutenberg University of Mainz, Mainz, Rheinland-Pfalz, Germany e-mail: [email protected]

232 Advances in Anatomy, Embryology and Cell Biology

Editor-in-Chief P. Sutovsky Series Editors F. Clascá • Z. Kmiec • H.-W. Korf • M.J. Schmeisser • B. Singh • J.-P. Timmermans • S. Schumann More information about this series at http://www.springer.com/series/102

Kathy L. Sharpe-Timms Editor

Animal Models for Endometriosis Evolution, Utility and Clinical Relevance

Editor Kathy L. Sharpe-Timms Division of Reproductive & Perinatal Research The University of Missouri School of Medicine Columbia, MO, USA

ISSN 0301-5556     ISSN 2192-7065 (electronic) Advances in Anatomy, Embryology and Cell Biology ISBN 978-3-030-51855-4    ISBN 978-3-030-51856-1 (eBook) https://doi.org/10.1007/978-3-030-51856-1 © Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Each of the chapters of this book were written by authors whose lives have been influenced by endometriosis, where we, family members, or friends have suffered from severe pain, infertility, or both. We empathize with our sisters and friends who have had their ovary(ies), uterus, or entire reproductive track removed and the horror of a 24-year-old woman engaged to be married undergoing a hysterectomy due to unbearable pain from the effects of endometriosis. We deeply feel the anguish and heartache of women and their partners who are trying unsuccessfully to conceive, undergoing multiple attempts at in vitro fertilization and embryo transfer, at a time when many of their friends are establishing their families. The existence of endometriosis has been known since at least the nineteenth century and this condition affects 10% of women, yet despite the physical, social, and psychological consequences of endometriosis, financial funding is far less than for any comparable condition with similar health impacts. Whether guided by our personal background life experiences, mentors whose lives have also been influenced by endometriosis, and/or our deeply rooted research passion of a condition stemming from the uterine endometrium and transforming into a pathological estrogen-dependent, neuroinflammatory disease, many of us have studied endometriosis for 25 to 30 years or more. We are determined to identify and understand the pathophysiological mechanisms related to the establishment of endometriotic lesions in the peritoneal cavity and how the presence of ectopic endometrium inflicts such dreadful pain and infertility. This information offers hope to eliminate non-effective invasive surgical approaches or medical cessation of the menstrual cycle which is counterproductive to fertility. Tragically, endometriosis is unjustly lumped together with “just another woman’s female disease.” We aim to provide new information such that novel, non-­ surgical, non-hormonal methods for management of endometriosis, and perhaps a prevention or cure, may be developed. It is the goal of this book to provide a background, current knowledge, and promising methods to advance management of endometriosis. Columbia, MO, USA

Kathy L. Sharpe-Timms

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Contents

 Introduction to Preclinical Evidence from Animal Models of Endometriosis����������������������������������������������������������������������������������������������������   1 Kathy L. Sharpe-Timms and Julie A. W. Stilley  Identifying Mechanisms of Endometriosis-­Associated Reduced Fecundity in a Rat Model: Novel Insights toward Understanding Human Infertility ����������������������������������������������������������������������������������������������   9 Kathy L. Sharpe-Timms, Henda Nabli, and Julie A. W. Stilley  Endometriosis-Associated Pain – Do Preclinical Rodent Models Provide a Good Platform for Translation?������������������������������������������������������  25 Erin Greaves, Matthew Rosser, and Philippa T. K. Saunders  Environmental Endocrine Disruptors and Endometriosis����������������������������  57 Jelonia T. Rumph, Victoria R. Stephens, Anthony E. Archibong, Kevin G. Osteen, and Kaylon L. Bruner-Tran  Deciphering the Role of miRNAs in Endometriosis Pathophysiology Using Experimental Endometriosis Mouse Models����������������������������������������  79 Ayushi Vashisht, Zahraa Alali, and Warren B. Nothnick  What Have We Learned from Animal Models of Endometriosis and How Can We Use the Knowledge Gained to Improve Treatment of Patients?��������������������������������������������������������������������������������������  99 Philippa T. K. Saunders

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Introduction to Preclinical Evidence from Animal Models of Endometriosis Kathy L. Sharpe-Timms and Julie A. W. Stilley

Contents 1  Historical Aspects of Endometriosis 2  Biological and Epidemiological Aspects of Endometriosis in Women 3  Endometriosis-Associated Infertility 4  Pain in Endometriosis 5  Endocrine-Disrupting Chemicals (EDCs) 6  Epigenetic Modifications in Endometriosis 7  The Price of Endometriosis 8  The Need for Improved Management of Endometriosis 9  Moving Towards Understanding of Endometriosis Through Animal Models 10  Summary

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Abstract  Endometriosis, the presence and growth of uterine endometrial glandular epithelial and stroma cells outside the uterine cavity, causes pain and infertility in women and girls of reproductive age. As randomized, double-blinded, controlled studies of endometriosis in women are impractical and at times ethically prohibitive, animal models for endometriosis arose as an important adjunct to gain mechanistic insights into the etiology and pathophysiological mechanisms of this perplexing disorder. A more thorough understanding of endometriosis in women may help develop novel noninvasive diagnostics, classification systems, therapeutic

K. L. Sharpe-Timms (*) Division of Reproductive & Perinatal Research, The University of Missouri School of Medicine, Columbia, MO, USA e-mail: [email protected] J. A. W. Stilley Department of Obstetrics, Gynecology and Women’s Health, University of Missouri School of Medicine, Columbia, MO, USA Division of Animal Science, College of Agriculture, Food, and Natural Resources, The University of Missouri, Columbia, MO, USA Department of Emergency Medicine, University of Missouri School of Medicine, Columbia, MO, USA e-mail: [email protected] © Springer Nature Switzerland AG 2020 K. L. Sharpe-Timms (ed.), Animal Models for Endometriosis, Advances in Anatomy, Embryology and Cell Biology 232, https://doi.org/10.1007/978-3-030-51856-1_1

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regimes, and even preventative methods for the management of endometriosis. This chapter is intended to introduce a brief historical background, biological and epidemiological aspects, the major symptoms, the effects of endocrine-disrupting chemicals, and an example of an epigenetic factor of endometriosis in women. Keywords  Endometriosis · Animal models · Infertility · Pain · Endocrine-­ disrupting chemicals · microRNAs · Management of endometriosis

1  Historical Aspects of Endometriosis Endometriosis is a gynecological disorder affecting ~10% of women and girls of reproductive age worldwide. Early reports describing endometriosis were written in the mid- to late-nineteenth century. Historically, the conditions described as adenomyosis externa, ovarian endometrioma, and peritoneal endometriosis were regarded as the same disease. In 1855, Rokitansky described the occurrence of epithelial glands and stroma like those of the endometrial lining of the uterus, growing elsewhere in the peritoneal cavity. Yet then and now it is unclear how the endometrium-­like lesions became established outside of the uterus; several theories have been postulated. In 1927, Sampson was the first to demonstrate specific endometrial attributes like desquamation at menses and decidualization in pregnancy, leading to his publication of the Sampson’s Theory of Retrograde Menstruation and Implantation.

2  B  iological and Epidemiological Aspects of Endometriosis in Women Biologically, endometriosis is defined by the presence and growth of uterine endometrial glandular epithelial and stroma cells outside the uterine cavity, which are called endometriotic lesions. Grossly, endometriosis may appear as small reddish petechiae, white cystic vesicles, brownish diffuse peritoneal defects, or classic bluish-­black puckered defects in the peritoneum. The lesions are primarily found on the ovaries and in the posterior cul-de-sac, and while most frequently found in the peritoneal cavity, endometriotic lesions have been observed and histologically confirmed in atypical places throughout the body. Epidemiologically, traits strongly associated with a greater risk for endometriosis include menarche at an early age, short menstrual cycle length, and lean body size. Greater parity on the other hand has been associated with a lower risk of endometriosis. In addition, characteristics such as physical activity, dietary factors, and

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lactation have been less consistent, partially because of the need for rigorous data collection and a longitudinal study design. Yet to date, a cause and effect relationship between endometriosis, reduced fecundity, and/or pain has not been established. Infertility and pain associated with endometriosis can be even more puzzling as not every patient may experience the same symptoms and all patients do not respond to therapies in the same way. Hence difficulty understanding endometriosis has led to knowledge gaps and the need for personalized medicine is evident.

3  Endometriosis-Associated Infertility Historically, endometriosis-associated infertility in women has been associated with subtle, explicit, or multifaceted abnormalities. In fact, endometriosis appears to affect every part of a woman’s reproductive organs and regulation of their normal process. Peritoneal fluid from women with endometriosis harbors inflammatory cytokines, immune cells, proteolytic enzymes and inhibitors, and numerous other substances that negatively impact sperm binding to the zona pellucida of the oocyte in vitro and elicit DNA fragmentation of sperm from healthy donors. A historical perspective and details of infertility in women plus animal models that have advanced our knowledge of possible mechanisms causing infertility are reviewed in Chap. 2.

4  Pain in Endometriosis Chronic pelvic pain is another significant problem in women with endometriosis. The possible sources of pain are diverse plus endometriosis is also associated with comorbidities that also cause pain such as back pain and bowel and bladder issues thereby adding to the challenge of correlating pain with disease presentation. Pain from peritoneal endometriosis does not correlate with stage of disease and pain severity. Ovarian endometriosis is not generally linked to any specific type of pain and may be confounded by pain from concurrent pelvic adhesions restricting the movement of other organs. Deep endometriotic lesions primarily develop within the pouch of Douglas and invade the muscle of the bowel wall or diaphragm and may extend toward the uterine artery or ureters. Deep lesions are strongly associated with severe dyspareunia and sexual dysfunction. Chapter 3 provides an excellent review of endometriosis-associated pain and preclinical rodent models as a platform for translation.

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5  Endocrine-Disrupting Chemicals (EDCs) Endocrine-disrupting chemicals are ubiquitous, exogenous chemicals that interfere with the endocrine system by mimicking or inhibiting the action of one or more of the endogenous hormones. Many EDCs also interfere with the normal functions of the immune system. Compounds with both endocrine- and immune-disrupting properties are of interest regarding the development of endometriosis. The EDCs are not new to our environment, but their unintended actions on the human reproductive system are now evident. For example, diethylstilbestrol (DES), a synthetic, highly potent estrogen was first manufactured in 1938. It was originally described as a treatment for a wide range of maladies from psychological disorders to pregnancy miscarriage. The US Food and Drug Administration (FDA) banned the use of DES during pregnancy in 1971 as not only was DES fail to prevent pregnancy loss but instead increased incidence of malformations of the uterine cavity and of a rare vaginal cancer. Research using nonhuman primates has shown that exposure to a compound called dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is associated with an increased prevalence and severity of endometriosis. Rodent animal studies have revealed that exposure to EDCs prior to puberty can lead to adult reproductive disease and dysfunction. Adult female mice exposed to dioxin were used to demonstrate a transgenerational occurrence of several reproductive diseases that have been linked to endometriosis in women. Exposure to a variety of EDCs has been found to cause reproductive tract anomalies. Sources of EDCs continue to be identified and outcomes following both adult and developmental exposure to EDCs are far-reaching. Chapter 4 provides a thorough description of multiple EDCs, consequences of exposure to EDCs, and animal models as tools to explore strategies for the prevention of adverse outcomes following EDC exposure.

6  Epigenetic Modifications in Endometriosis Epigenetics is the study of changes in gene activity which are not caused by changes in the genetic code or the DNA sequence. Epigenetics is the modification of gene expression that results in phenotypic changes. There are many types of epigenetic modifications, the more common being methylation, acetylation, and microRNAs (miRNAs). The field of epigenetic modifications is rapidly expanding and has transformed our knowledge of genomes. The role of miRNAs in the pathophysiology of women with endometriosis was first evaluated approximately a decade ago by identifying miRNAs which may be differentially expressed in endometrial versus endometriotic lesion tissue. Some miRNAs may serve as biomarkers for early disease detection. With advancements in this field coming from animal models, future research

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may provide evidence of their involvement at multiple levels in endometriosis pathology. Approaches may include the use of genetically modified mice, which over/under express specific miRNAs as well as examine the utility of miRNAs in endometriosis diagnosis and treatment. Chapter 5 provides an overview of our current knowledge on the role of micoRNAs (miRNAs) in modulating cell proliferation, apoptosis, inflammation, and angiogenesis in normal endometrial physiology as well as miRNA misexpression which may influence these cellular events in the pathophysiology of endometriosis.

7  The Price of Endometriosis There is a significant economic burden imposed by endometriosis. Costs of surgical removal of endometriotic lesions over a decade ago were over 17 billion dollars per year in the United States alone. Indirect costs from loss of work productivity due to debilitating chronic pelvic pain accounted for another almost 5 billion dollars lost in the United States. Over the past decade, others have published reviews updating cost estimates, direct and indirect cost burdens attributed to endometriosis surgeries as well as country-specific. Overall, there is no doubt that more effective, individualized treatments for endometriosis are greatly needed.

8  The Need for Improved Management of Endometriosis The classic duo of symptoms of endometriosis are pain and infertility. The abnormalities causing these symptoms in endometriosis are not known. Due to the complex characteristics of the disease, the mechanisms causing these problems are multifaceted. To date, treatments focusing on reducing pain and infertility associated with the disease often cause cessation or chemical alteration of the reproductive cycle. Curative treatments are unknown and many cause unwanted side effects. Further, many treatments are inappropriate for patients seeking treatment for infertility. Due to its diverse symptoms, the gaps in our knowledge about endometriosis are extremely difficult if not impossible to resolve relying solely on a patients’ clinical presentation. Due to the obvious ethical and practical considerations, it is not possible to perform a controlled study with repeated invasive surgeries and experimental techniques in human patients. Collectively, these and other complex factors have limited progress toward a more thorough understanding of endometriosis in women, which if known may help develop novel noninvasive diagnostics, classification systems, therapeutic regimes, and even preventative methods for the management of endometriosis. From these needs, the development of animal models for endometriosis arose.

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9  M  oving Towards Understanding of Endometriosis Through Animal Models Animal models for endometriosis (Fig. 1) permit studies of mechanisms and regulators and integration of cellular and molecular technologies in a controlled manner free from confounding influences such as individual patient variation, diet, environmental influences, confounding medications, or other comorbidities. While there are known disadvantages of extrapolating data across species, animal models may be used to study events involved in the pathogenesis and pathophysiologies of endometriosis or novel therapeutic approaches for this disorder that are not accessible in humans due to restrictions of both repeated surgeries and use of experimental drugs in women. In the 1920s, attempts at establishing animal models for endometriosis in the rabbit and in the monkey were reported. Since this pioneering work, a variety of endometriosis models have emerged in species including, but not limited to, rabbits, rats, mice, and primates, where autologous endometrial pieces are transferred back into the peritoneal cavity by injection or surgical attachment; cells from human endometrium have also been injected into animal host species. Hundreds of additional publications using these animal models for endometriosis abound in the literature searching with the keywords “endometriosis” and “animal models”. Primates of species that menstruate are good models for studying endometriosis as they menstruate and spontaneously develop endometriosis. However, the prohibitive cost of primates, training and special facilities needed, as well as ethical concerns with treating primates limit research with this model. Nonprimate animal models of induced endometriosis include, for example, rabbits, rats, hamsters, and mice. Rodent models are used to study in vivo mechanisms involved in endometriosis that nonhuman primate research has been unable to achieve. Rodent models are more cost-effective, more readily available, and provide an avenue to investigate multiple aspects of the disease at multiple time points. Facilities to care and house these animals are common and require less extensive management and handler training. Rodent models do not impose the same ethical restrictions as humans and primates.

10  Summary To elucidate mechanisms involved in the etiology and pathophysiology of endometriosis, hypothesis-driven, randomized, placebo-controlled experimental design is necessary. In addition, studies using appropriate tissues from women patients with and without endometriosis in combination with physiologically relevant in vitro and in vivo laboratory models are needed. Animal models of endometriosis have played a significant role in our understanding on how endometriosis causes infertility and pain. They permit studies of not only treatment effects but importantly they have

Preclinical Evidence from Endometriosis Animal Models Fig. 1  Animals models for human endometriosis. (a) Rabbits have been used as a model for endometriosis reproductive failures, yet as they do not spontaneously ovulate their utility is limited. (b) Murine models use a variety of strains of mice, care should be taken with interspecies comparisons. (c) Athymic “nude” mice permit studies of human endometrial and endometriotic tissues without immune rejection; yet their value is compromised without a function immune system which is deemed to play a role in endometriosis. (d) Green fluorescent mice express the GFP protein, fluorescent endometrium is transferred to normal mice helping identify the lesions. (e) Sprague-­ Dawley rats are used as endometriosis models in a variety of research; they are arguably the best established, most popular rat strain for modeling endometriosis. (f) The baboon, Papio anubis, is a primate species which menstruates and naturally develops endometriosis. All models have pros and cons; selection of a model must match the facets of each animal and the experimental goals. See individual chapters for additional details and citations

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helped expand our ability to study evolving and novel mechanisms involved in the etiology and pathophysiologies of this perplexing, multifactorial disease. The outcomes of these studies hold optimism for moving away from invasive surgical approaches and ineffective, noncurative medical treatments toward previously unknown, nonsurgical, nonchemical methods to identify, treat, and perhaps someday prevent and cure endometriosis in women. The material reported in this book provides a solid background of these aspects in endometriosis plus advances in our knowledge using outcomes which may only be accessible with the use of animal models for endometriosis.

Identifying Mechanisms of Endometriosis-­ Associated Reduced Fecundity in a Rat Model: Novel Insights toward Understanding Human Infertility Kathy L. Sharpe-Timms, Henda Nabli, and Julie A. W. Stilley

Contents 1  2  3  4  5  6  7  8 

Introduction Anomalies in Reproductive Function Associated with Endometriosis The Hypothalamic-Pituitary-Ovarian (HPO) Axis Ovarian Follicle Development Ovulatory Disorders from Inadequate Matrix Remodeling Is Endometriosis a Familial Disease? Impact of Endometriosis on Male Fertility Transgenerational Subfertility in Female and Male Offspring in a Rat Model of Endometriosis 9  Discussion References

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Funding Source: National Institutes of Health National Institute of Child Health and Human Development R21 3625675 (to KST). K. L. Sharpe-Timms (*) Division of Reproductive & Perinatal Research, The University of Missouri School of Medicine, Columbia, MO, USA e-mail: [email protected] H. Nabli Department of Obstetrics, Gynecology and Women’s Health, University of Missouri School of Medicine, Columbia, MO, USA e-mail: [email protected] J. A. W. Stilley Department of Obstetrics, Gynecology and Women’s Health, University of Missouri School of Medicine, Columbia, MO, USA Division of Animal Science, College of Agriculture, Food, and Natural Resources, The University of Missouri, Columbia, MO, USA Department of Emergency Medicine, University of Missouri School of Medicine, Columbia, MO, USA e-mail: [email protected] © Springer Nature Switzerland AG 2020 K. L. Sharpe-Timms (ed.), Animal Models for Endometriosis, Advances in Anatomy, Embryology and Cell Biology 232, https://doi.org/10.1007/978-3-030-51856-1_2

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Abstract  The existence of endometriosis has been known since at least the nineteenth century, yet the lack of understanding of causes of infertility and therefore inadequate treatment approaches in endometriosis creates a significant challenge in reproductive medicine. Women worldwide suffer not only pain and infertility but also economical, societal, and physiological burdens. Studies of reproductive events in women are difficult to conduct due to a host of confounding personal and environmental factors and ethically limited due to the very nature of working with reproductive tissues and cells, especially embryos. Animal models are a viable adjunct to study mechanisms causing human reproductive anomalies and infertility in endometriosis. This chapter discusses reproductive anomalies causing infertility in endometriosis and well-established animal models which help decipher the problems and lead to heretofore unknown nonsurgical, nonhormonal methods to manage endometriosis in women. In addition, studies of effects of developmental exposure to endometriosis are revealing for the first time, in both female and male offspring, transgenerational subfertility in a rat model providing insights into the familial nature of endometriosis and possible epigenetic involvement. Keywords  Endometriosis · Developmental origins of health and disease · Female infertility · Male infertility · Developmental biology · Fecundity

1  Introduction The mere existence of an association between endometriosis and infertility was historically challenged (D’Hooghe et al. 2003; De Hondt et al. 2005). As new clinical and basic science studies are accumulating novel data, this opinion is being reversed as noted by the numerous review articles appearing in the literature (de Ziegler et al. 2010; Macer and Taylor 2012; Jin and Ruiz 2014; Surrey 2015; Tanbo and Fedorcsak 2017; Biobaku et al. 2018). The lack of understanding of the causes of reduced fecundity in endometriosis, however, remains one of the greatest challenges in reproductive medicine. Thorough reviews of cellular and molecular bases for endometriosis-associated infertility and other possible comorbidities have been published (Stilley et al. 2012; Giudice and Kao 2004).

2  A  nomalies in Reproductive Function Associated with Endometriosis Endometriosis-associated infertility in women has been associated with subtle, explicit, or multifaceted abnormalities (Cahill and Hull 2000; Pellicer et al. 1998; Garrido et al. 2000, 2002, 2003; Wunder et al. 2005), which are diverse, perplexing, and often confounding, thereby contributing to our lack of understanding of the endometriotic disease process. In fact, endometriosis appears to affect every part of

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a woman’s reproductive tract. Several factors are believed to contribute to subfertility in women with endometriosis including abnormalities in the hypothalamic-­ pituitary-­ovarian (HPO) axis (Cahill and Hull 2000; Pellicer et  al. 1998), the follicular and endocrine environment and hormone/receptor dysfunction (Pellicer et al. 1998; Garrido et al. 2000, 2003), oocyte quality (Wunder et al. 2005), embryonic quality (Garrido et al. 2000, 2002), endometrial function (Garrido et al. 2002; Lessey and Young 2019), embryo implantation (Pellicer et al. 1995; Radzinsky et al. 2019), and pregnancy outcomes (Farland et al. 2019; Leone Roberti Maggiore et al. 2016; Horton et  al. 2019). Peritoneal fluid (PF) harbors inflammatory cytokines, immune cells, proteolytic enzymes and inhibitors, and numerous other substances that negatively impact sperm binding to the zona pellucida of the oocyte in vitro (Muscato et  al. 1983; Coddington et  al. 1992) and elicit DNA fragmentation of sperm from healthy donors (Mansour et al. 2009). Abnormal genetic (Stefansson et al. 2002; Hansen and Eyster 2010) and epigenetic factors (Guo 2019; Borghese et al. 2017) are proposed to contribute to the risk of developing endometriosis. Endometriosis has an important genetic component with a heritability estimated at around 50% (Borghese et al. 2017). Both genetic and epigenetic variants are reportedly involved in endometriosis-associated infertility (Guo 2019). Borghese et al. (2017) published a review of insights into the genetic bases of endometriosis and a detailed overview of evidence of epigenetic alterations in endometriosis. In the future, these epigenetic markers may constitute therapeutic targets for pharmaceutical compounds able to modify the epigenetic code (Borghese et al. 2017). Addressing each of these anomalies in a single review would not be feasible. The remainder of this review will address anomalies of the hypothalamic-pituitary-­ ovarian axis, ovarian follicle development, and ovulatory dysfunction, with no intention of lessening the importance of oocyte quality, embryo development and implantation, hormone/receptor dysfunction, or the inflammatory milieu in the peritoneal cavity. Then, the familial aspects of endometriosis, endometriosis in men, and finally the latest information regarding transgenerational subfertility and addressing causes for these phenomena in both female and male offspring over three generations in a rat model for endometriosis will be presented.

3  The Hypothalamic-Pituitary-Ovarian (HPO) Axis The function of the HPO ovarian axis is reportedly disrupted in women with endometriosis (Cahill and Hull 2000; Bancroft et al. 1992), where the follicular phase of the menstrual cycle is longer in women endometriosis compared to women without this disease. The luteinizing hormone (LH) secretory pattern in women with endometriosis is abnormal, and the LH surge is delayed and of lower amplitude in endometriosis (Cahill and Hull 2000; Bancroft et al. 1992). There are also deficiencies in follicular LH receptors (Ronnberg et al. 1984). Anomalies of the HPO axis impair

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follicle growth, ovulation, and corpus luteum (CL) development in the ovary in women with endometriosis. Rats with endometriosis have significantly lower serum LH concentrations than controls (Stilley et al. 2009) like women, indicative of a similar pituitary-ovarian axis dysfunction. Others have used a rat model for endometriosis to test the effect of various compounds on the HPO axis and diminishing the endometriotic lesions (Cao et al. 2014; Harris et al. 2005).

4  Ovarian Follicle Development Normal follicular growth and development in women are controlled by a balance of hormones including folliicle stimulating hormone (FSH). These developing follicles produce estradiol (E2) which creates a feedback loop to the HPO axis, controlling output of gonadotropin-releasing hormone (GnRH) from the hypothalamus and FSH and eventually LH from the pituitary gland. As the follicles grow, a fluid-filled antrum forms, and follicular cells develop LH receptors readying the follicle for ovulation (Senger 2005). Molecular evidence supporting the idea that endometriosis can negatively influence growth, steroidogenesis, and the function of the ovarian granulosa cells (GCs) from women has been reviewed (Sanchez et al. 2016). They describe reduced cytochrome P450 aromatase expression, increased intracellular reactive oxygen species (ROS) generation, and altered WNT signaling characterize the GCs from women with endometriosis. Clear evidence for an increased level of GC apoptosis in association with the downregulation of pro-survival factors is also a factor. Lower levels of E2 and P4 have been noted in serum and urine of women with endometriosis (Brosens et  al. 1978; Williams et  al. 1986). As in women, rats with induced endometriosis (Endo) also have similar anomalies in folliculogenesis, fewer overall follicles, and fewer antral follicles than surgical controls (Shams) (Stilley et al. 2010; Moon et al. 1993; Pal et al. 1999; Sharpe-Timms 2002). The follicles of Endo rats produced lower concentrations of E2 and P4 than Shams and corresponding reduced serum concentrations of E2 and P4 compared to Sham rats (Pal et al. 1999). Oocytes trapped in a luteinizing unruptured follicle leading to a failure of ovulation are defined as luteinized unruptured follicle syndrome (LUFs). This phenomenon has been associated with endometriosis and infertility in women (Donnez and Thomas 1982; Mio et  al. 1992; Kaya and Oral 1999). And like women with endometriosis, rats with endometriosis have fewer CLs and an increased risk of luteinized unruptured follicles (LUFs) (Stilley et al. 2009, 2010; Moon et  al. 1993). Rats with endometriosis have fewer CLs than surgical controls (Stilley et al. 2009, 2010). The presence of lower serum P4 suggests that Endo rats may have a luteal phase defect as well and the luteal phase is shorter in rats than humans, even when normalized to cycle length (Sharpe-Timms 2002).

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5  Ovulatory Disorders from Inadequate Matrix Remodeling The process of normal ovulation in women with endometriosis is impaired. As mentioned, the altered LH surge may be involved. Others suggest that changes in proteolytic enzymes (Ebisch et  al. 2007; Smedts et  al. 2006; Birt et  al. 2013a), inflammatory cytokines (Garrido et  al. 2000; Pellicer et  al. 1998; Carlberg et  al. 2000), and vasculature (Pellicer et al. 1998; Garrido et al. 2000; Wunder et al. 2006), which are required for normal ovulation, are altered in the follicles of women with endometriosis. Collectively, these data provide evidence of mechanisms which could cause ovulatory dysfunction in endometriosis. The family of proteolytic enzymes called matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), are ubiquitous tissue remodeling components in both normal and pathological events in in the human body. The MMPs and TIMPs and their regulation have been of interest in the etiology and pathophysiologies of endometriosis for some time (Osteen et al. 2003; Bałkowiec et al. 2018; Tang et al. 2018). They have been studied extensively in humans and rats. Normal endometrium synthesizes and secretes MMPs and TIMPs during the cyclic tissue remodeling with menses. Endometriotic lesions produce and secrete MMPs and TIMPs in an autocrine pattern to facilitate establishment and transformation of the lesions through the various phenotypes and in a paracrine manner into the peritoneal cavity where TIMP1 bathes the ovarian tissue. Endometriotic-derived TIMP1 is one of the major proteins found in PF of women and rats with endometriosis (Stilley et al. 2009, 2010; Stilley and Sharpe-Timms 2012). TIMP1 is required for regulation of multiple MMPs as well as other biological functions in many cells of the body but especially for normal function of the ovaries during ovulation. Ovarian follicles of Endo rats have more TIMP1 localizing in the thecal layer, likely blocking matrix degradation of the follicle wall and ovulation and contributing to ovarian dysfunction in endometriosis (Stilley et al. 2009, 2010; Stilley and Sharpe-Timms 2012). To determine the direct effect of TIMP1 on endometriosis-­ associated subfertility, we performed three experiments (Stilley et al. 2010). We first cultured control rat embryos in the presence or absence of TIMP1 concentrations equal to those found in PF of rats with endometriosis. The embryos exposed to TIMP1 but not the control medium displayed morphological abnormalities including altered cytoplasm structure, poor blastomere adhesion, abnormal nucleoli, as well as anomalous nuclear envelope formation and an increase of TIMP1 localizing to the nucleus, possibly contributing to poor nuclear development. Second, intraperitoneal levels of TIMP1 were modulated in vivo in Sham and Endo rats. TIMP1-­ treated Sham rats had poorer oocyte quality, reduced embryo development, a reduction in follicles and corpora lutea (CLs) like those in women. Further, when Endo rats were treated in vivo with a TIMP1 function-blocking antibody, the phenotype of their oocytes and embryos was similar to that of Sham rats (Stilley 2010; Sharpe-Timms 2012). Endo rats treated in vivo with a TIMP1 functionblocking antibody also had increased numbers of follicles and CLs compared to Endoor TIMP1-treated Sham rats, at numbers equal to Sham rats treated with vehicle. The

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third experiment was done to test the specificity of TIMP1 to the endometriotic anomalies in PF. Control rats were given treatments of Endo PF with or without TIMP1 or Sham PF. Control rats treated with Endo PF had fewer follicles and CLs than control rats treated with either Endo PF without TIMP1 or Sham PF. Localization of TIMP1 in the ovary of control rats treated with Endo PF was increased compared to the control groups providing further evidence that PF TIMP1 can sequester into the ovary and cause disruption of the MMP/TIMP balance necessary for ovulation. TIMP1 may be a target for novel therapies to restore ovulatory function in women with endometriosis.

6  Is Endometriosis a Familial Disease? Also, of importance is the multigenerational impact of endometriosis on fertility. Reports describe familial clustering of endometriosis, yet a distinct genetic link, consistent between endometriosis studies, remains elusive (Stefansson et al. 2002; Hansen and Eyster 2010). There is a small body of literature describing subfertility in daughters developmentally exposed to their mothers with endometriosis (Gao et al. 2019; Vannuccini et al. 2016). One cohort study consisting of 3406 women born in Sweden (Gao et al. 2019) retrospectively collected data from archives of birth records, age of endometriosis diagnoses, as well as socioeconomic and reproductive characteristics from national and routine patient registers. They reported that women with endometriosis had a significantly lower birth weight for gestational age and were diagnosed with infertility before the diagnosis of endometriosis; no evidence was found that the number of births was associated with the inverse association between standardized birth weight and endometriosis. The second study was a case-control study of women without and with endometriosis answering questions concerning their mothers’ gestational data and their own perinatal and early postnatal lives (Vannuccini et al. 2016). Intrauterine developmental exposures, early neonatal exposures, and reportedly prematurity and formula feeding were risk factors for development of endometriosis in adulthood.  Our research has provided evidence supporting familial clustering of endometriosis suggesting developmental exposure to endometriosis may be responsible (Stilley et al. 2009; Birt et al. 2013b). These studies found similar anomalies such as reduced fecundity and abnormal eight-cell stage embryo morphology and gene expression in embryos in both the first and second generations exposed to endometriosis as embryos or primordial germ cells, respectively, but not in Sham surgery rats (Stilley et al. 2009; Birt et al. 2013b).

7  Impact of Endometriosis on Male Fertility A literature review by Rei and colleagues (2018) found studies of endometriosis in men with a total of 16 cases previously reported in the literature (Rei et al. 2018). From these cases, they reported that endometriosis was most commonly found on

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the bladder, lower abdominal wall, and inguinal region. From the literature review, this publication provides possible prerequisites for the development of endometriosis in males including prolonged estrogen therapy, liver cirrhosis, chronic surgical inflammation, and obesity. Similar to the accompanying case report by Rei of endometriosis in an obese man (Rei et al. 2018), Schneider also proposed a link between estrogen production in obese men and endometriosis (Schneider et al. 1979). Two additional publications were found when searching for studies of subfertility in males following developmentally exposure to endometriosis. The first performed a preliminary analysis finding a possible twofold higher association of preconception maternal endometriosis and dihydrogesterone treatment in early pregnancy with the risk of isolated true undescended testes (Mavrogenis et  al. 2014). This population-based case-control study collected exposure data based from multiple sources; only prospective medically recorded data in the prenatal maternity logbooks were evaluated. We interpret these data with caution as it may be burdened by possible confounders like alteration of endocrine function, population selection bias, and retrospective reporting of maternal disease and hormone treatments by mothers introducing possible recall bias. Second, a Danish register-based study looked for an association between maternal endometriosis and occurrence of the cryptorchidism and hypospadias in sons. They did not find a higher occurrence of these anomalies in sons of women with endometriosis (Arendt et al. 2017). These two studies recognize the need for additional research to determine an association between maternal endometriosis and occurrence of the cryptorchidism and hypospadias in sons. In the general population, the reported prevalence of unexplained male infertility ranges from 6% to 37%, depending on how comprehensively the evaluation is performed (Moghissi and Wallach 1983; Collins and Crosignani 1992). In couples with infertility, Jarow (2007) reports that a male factor is the only cause for infertility in about 20% of couples and contributory in another 30–40% of couples; as such, a male factor is implicated in more than 50% of couples attempting to conceive (Jarow 2007). Yet the cause of this male factor infertility remains unknown and leads to unresolved angst in couples trying to achieve a pregnancy. We propose that some males with unexplained infertility have no idea they were developmentally exposed to endometriosis or that this might impact their own fertility.

8  T  ransgenerational Subfertility in Female and Male Offspring in a Rat Model of Endometriosis The goal of our recent studies was to identify the effects of in utero exposure to endometriosis leading to a novel, abnormal developmental phenotype and subfertility in three generations of female and male offspring. Identifying mechanisms behind these phenomena is paramount to the development of novel interventions to evolve from surgical or chemical obliteration of lesions or unsuccessful attempts at IVF. By comparing phenotypic differences between Endo and Sham offspring and

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K. L. Sharpe-Timms et al. F0 Female Sprague Dawley Rats Wild Type Females

Wild Type Males

Endo or Sham Surgery F1 Males

F1 Females ML-F2 FL-F2 Females Males

FL-F2 Females

ML-F2 Males

ML-F3 Males

ML-F3 F ML-F3 M

ML-F3 F

FL-F3 M

FL-F3 F

FL-F3 M

FL-F3 Females

Siblings Fig. 1  Three generations of rats were bred over 1.5 years. Female Sprague Dawley rats had either Endo or Sham surgeries. F1 (embryo exposure), F2 (germ cell exposure in F1s), and F3 (no direct exposure) generation male and female offspring were developed by breeding exposed males to WT females (left) and by exposed females to WT males (right). The siblings were not included in this study

their progeny over three generations, we anticipated defining specific, multigenerational phenotypes leading to reduced fertility in endometriosis. We also predicted reduced reproductive potential and fertility would be reflected in the analyses of size, weight, and development characteristics including aspects of sexual maturity. Using a well-established rat model for endometriosis developed by (Vernon and Wilson 1985) and used by Sharpe-Timms and colleagues for over 30 years (Stilley et al. 2009, 2010; Stilley and Sharpe-Timms 2012; Rigden et al. 2011; Birt et al. 2013a; Sharpe and Vernon 1993; Wright and Sharpe-Timms 1995; Sharpe et  al. 1990a, b; Sharpe-Timms et al. 2000, 2002, 2011; Piva et al. 2001; Cox et al. 2001), a founder generation (F0) of endometriosis (Endo) and surgical control (Sham) rats was developed. Three generations of Endo and Sham offspring (F1, F2, and F3 generation pups) were bred over 21 months (Fig. 1). By breeding F0 Endo and F0 Sham females to wild-type (WT) male breeders, the F1 generation female (Fig. 1, right) and male (Fig. 1, left) offspring were born. The F1 pups, female and male, were developmentally exposed to endometriosis as embryos. At sexual maturity, the F1 generation of female (female lineage, FL) and male (male lineage, ML) pups was independently bred to wild-type (WT) males and females, producing the F2 generation pups. The F2 pups had been exposed to endometriosis as germ cells in the F1 generation pups. The F2 FL and ML rats were then bred to WT rats developing the F3 FL and ML offspring, which had no direct exposure to endometriosis. For the three generations of the female and male lineages, both the breeding rates and pregnancy rates were lower in Endo rats compared to Sham rats (Fig. 2). The Endo rat litter size was smaller than Sham rat litter size for each of the three

Preclinical Endometriosis Subfertility in a Rat Model

P=0.014

P