Prenatal Diagnostic Testing for Genetic Disorders: The revolution of the Non-Invasive Prenatal Test [1st ed. 2023] 3031317572, 9783031317576

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Prenatal Diagnostic Testing for Genetic Disorders The revolution of the Non-Invasive Prenatal Test Gian Carlo Di Renzo Editor

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Prenatal Diagnostic Testing for Genetic Disorders

Gian Carlo Di Renzo Editor

Prenatal Diagnostic Testing for Genetic Disorders The revolution of the Non-Invasive Prenatal Test

Editor Gian Carlo Di Renzo Centre for Perinatal & Reproductive Medicine University of Perugia Perugia, Umbria, Italy PREIS International and European School of Perinatal Neonatal and Reproductive Medicine Firenze, Italy

ISBN 978-3-031-31757-6    ISBN 978-3-031-31758-3 (eBook) https://doi.org/10.1007/978-3-031-31758-3 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed 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

In the past century, numerous discoveries in the fields of genetics and technology and their applications to prenatal medicine have brought about a considerable revolution in prenatal diagnosis. The detection and prevention of prenatal disease has unveiled the mystery surrounding the fetus and the dimension of inviolability that characterized it in the past. Discoveries with respect to the uterine world have contributed to an increase in scientific awareness of the fact that many problems of the child and of the adult have developed before birth. The introduction of methods of indirect (biochemical) and direct (biophysical) evaluation of the fetus have allowed adequate investigation, undertaken with the use of an every-growing number of techniques based on fetal material sampling (amniocentesis, chorionic villus sampling, cordocentesis). It has been possible to benefit not only from sophisticated diagnostics but also from intrauterine medical and/or surgical treatment, in some conditions. It was only in the mid-1960s that amniocentesis began to be used to detect fetal disease by spectrophotometric analysis of bilirubin on amniotic fluid, evaluating in this way fetal severity of RhD isoimmunization. In the same period, for the first time, chromosomal gender was determined on amniocytes through detection of Barr chromatin. However, in the 1970s the use of amniocentesis was very limited because it carried not only a high risk of abortion and prohibitive costs, but also because only women in their late-reproductive years were considered at high risk. Only at the end v

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of the 1980s was a new approach (besides amniocentesis) of screening without any risk, that considered not only maternal age but also concentration of some factors of fetal–placental origin present in maternal peripheral blood, brought into use. In the 1990s, screening by a combination of maternal age and fetal nuchal translucency (NT) thickness at 11–14 weeks of gestation was introduced, and subsequently it has been shown that the combination of maternal age, fetal NT, and maternal serum biochemistry (free β-hCG and pregnancy associated plasma protein (PAPP-A)) in the first trimester has allowed the identification of about 85–90% of trisomy 21-affected fetuses. However, in the time period of the mid-1980s, the discovery of the presence of fetal cells in maternal blood during pregnancy and their isolation for genetic analysis produced a great revolution in prospective prenatal diagnosis. Various types of fetal cells have been identified in maternal blood, and various researchers have been occupied in the study of the identification, selection, and genetic analysis of fetal cells amenable for prenatal genetic testing. Another possibility for non-invasive prenatal diagnosis began in 1997, when Dennis Lo and coworkers in Oxford showed the presence of cell-free fetal DNA (cfDNA) in maternal plasma and serum. From the studies following this discovery, it has been proved that fetal DNA is present in the maternal circulation from the first weeks of gestation and in major amounts with respect to that recovered from fetal cells. Moreover, free fetal DNA has the advantage that it degrades within a few hours of delivery, and therefore it cannot interfere with prenatal diagnosis of subsequent pregnancies. Since then, non-invasive prenatal testing (NIPT) for fetal aneuploidy using cfDNA has been widely integrated into routine obstetrical care. Initially, cfDNA tests focused on chromosomal aberrations addressed by conventional prenatal screening methods, namely trisomy 21, trisomy 18, and trisomy 13. The scope soon expanded to include sex chromosome aneuploidy and microdeletion panels. Recently, genome-wide analysis has become available, expanding the scope of NIPT to address rare autosomal trisomies and large chromosomal imbalances. Because the technical ability to test for a condition does not necessarily correspond with a clinical benefit to a population or to individual pregnant women, the benefits and harms of screening programs must be carefully weighed before implementation. At the current time, scientific evidence regarding clinical performance of expanded cell-free DNA panels is lacking. Expanded cell-free DNA menus therefore create a dilemma for diagnosis, treatment, and counseling of patients which is a matter of big debate nowadays and calls for an ethical and effective expansion of the test. Introduced for the first time in 2011, in a span of a decade the nowadays called NIPT (which refers practically only to the prenatal tests based on cfDNA) has expanded widely although irregularly in all the world rising many problems related to it applicability (particularly in low-middle income countries), costs, reliability (this pertaining mainly to the several in house tests flourished like mushrooms in many parts of the world), pre- and post-test counseling, ethical dilemmas, equity in access, and finally the “patentability” of the tests. All these aspects required a

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clarification, and this is why this book has been conceived, in order to give the reader an adequate comprehensive view of the techniques, the problems, and the potential solutions through the help of the most renowned experts in the field. Despite this undisputed success, research is continuously looking towards the identification of a cellular protein marker to identify the “best” fetal cell in maternal circulation amenable to prenatal genetic tests; researchers are conducting gene expression experiments, by using innovative technologies like microarrays and proteomic studies to detect differences in gene and/or protein expression between fetal and maternal cells. From this picture it is evident that although the goals achieved (use of free fetal DNA for the diagnosis of fetal chromosomes and genes in the clinical routines of many countries) are highly significant, so much has yet to be done, and research should always be focused on further improvement of technologies to help non-­ invasive prenatal diagnosis become the new “gold standard” which will surely change the history of maternal–fetal medicine. I am particularly indebted to all expert authors who accepted to contribute with up-to-date chapters to several aspects of this important and widely debated topic and to the Publishers, which have elegantly edited a timely publication. Hope it will interest and help many readers in the world to have a clear picture of the “new” status and potentialities of the prenatal genetic diagnosis. Perugia, Italy Detroit, MI, USA

Gian Carlo Di Renzo

Contents

1

Introduction����������������������������������������������������������������������������������������������    1 Y. M. Dennis Lo

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A Brief History of Noninvasive Prenatal Diagnosis and Its Forecast����������������������������������������������������������������������    3 Gian Carlo Di Renzo, Arun Meyyazhagan, and Valentina Tsibizova

Part I Clinical Genetics 3

The Nexus Between Chromosomal Abnormalities and Single Gene Disorders������������������������������������������������������������������������������   25 Arun Meyyazhagan and Gian Carlo Di Renzo

4

Clinical Implications of Chromosomal Polymorphisms in Congenital Disorders ����������������������������������������������   57 Arun Meyyazhagan, Haripriya Kuchi Bhotla, Manikantan Pappuswamy, Valentina Tsibizova, Karthick Kumar Alagamuthu, and Gian Carlo Di Renzo

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Placental Genetics. Fetus-Placental Discrepancies: Challenges in Prenatal Genetic Diagnosis ��������������������������������������������   67 Miriam Turiel-Miranda and Jose Luis Bartha

6

Underpinnings of the Conundrum Between Genetic Screening and Testing������������������������������������������������������������������������������   79 David W. Britt, Shara M. Evans, and Mark I. Evans

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 Epidemiology of Birth Defects in Twins������������������������������������������������   99 Petya Chaveeva, Maria Mar Gil, and Kypros Herodotos Nicolaides

8

 Screening of Aneuploidies in Twin Pregnancies������������������������������������  115 María Mar Gil, Petya Chaveeva, and Kypros Herodotos Nicolaides

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Contents

Part II Noninvasive Diagnosis 9

 Congenital Anomalies: The Role of Ultrasound������������������������������������  129 Valentina Tsibizova, Tatyana Pervunina, Veronika Artemenko, Arun Meyyazhagan, and Graziano Clerici

10 Customary  Complications and Screening Techniques of Early Pregnancy ������������������������������������������������������������������������������������������������  143 Arun Meyyazhagan, Haripriya Kuchi Bhotla, Manikantan Pappuswamy, and Gian Carlo Di Renzo 11 First  Trimester Screening for Common and Rare Chromosomal Abnormalities as Well as for Major Defects: Which Tests Should Be Combined?����������������������������������������  153 Karl Oliver Kagan 12 The  Technology of Cell-Free Fetal DNA-­Based NIPT��������������������������  165 Karen White, Bowdoin Su, Renee Jones, Emilia Kostenko, and Francesca Romana Grati 13 The  Technologies: Comparisons on Efficiency, Reliability, and Costs��������������������������������������������������������������������������������������������������  183 Zhijie Yang, Youxiang Wang, and Gian Carlo Di Renzo 14 Pre and Posttest Counseling������������������������������������������������������������������  217 Dick Oepkes 15 cffDNA  Testing in IVF Pregnancies�������������������������������������������������������  237 Emilia Mateu-Brull, Nuria Balaguer, María Gómez-López, Carlos Simón, and Miguel Milán 16 “ RATs”: Rare Autosomal Trisomies and Their Relevance in cfDNA Testing��������������������������������������������������������������������  249 Francesca Romana Grati and Peter Benn 17 Rapid  Detection of Foetal Mendelian Disorders: Thalassaemia and Sickle Cell Syndrome ����������������������������������������������  265 Karen Lim and Mahesh Choolani 18 Noninvasive  Antenatal Screening for Fetal RhD in RhD-Negative Women to Guide Targeted Anti-D Prophylaxis������������  277 C. Ellen van der Schoot and Dick Oepkes 19 Genome-Wide  Cell-Free Fetal DNA-Based Prenatal Testing: Limits and Perspectives ��������������������������������������������������������������������������  291 Elisa Bevilacqua and Jacques Jani

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Part III Clinical Setting and Trends 20 Developing  and Delivering a Clinical Service for the Non-invasive Prenatal Diagnosis of Monogenic Conditions����������������  305 Elizabeth Scotchman, Joseph Shaw, Natalie Chandler, and Lyn S. Chitty 21 Counseling  in a Changing World of Genetics����������������������������������������  321 C. M. Katia Bilardo 22 Maternal  Secondary Genomic Findings Detected by Fetal Genetic Testing��������������������������������������������������������������������������������  333 Amy Turriff and Diana W. Bianchi 23 Prenatal  Genome-Wide Sequencing for the Investigation of Fetal Structural Anomalies: Is There a Role for Noninvasive Prenatal Diagnosis?������������������������������������������������������������  357 Elizabeth Wall, Stephanie Allen, James S. Castleman, and Mark D. Kilby 24 Cross-cultural  Perspectives on Noninvasive Prenatal Testing ������������  379 Hazar Haidar, Marie-Christine Roy, Anne-Marie Laberge, and Vardit Ravitsky 25 International  Guidelines for Implementation of NIPT/cffDNA Testing ������������������������������������������������������������������������������������������������������  389 Maria José Rego de Sousa, Margarida Sousa, and Maria Grasielle Cruz 26 Overview  and Historical Perspective of Preimplantation Genetic Testing ����������������������������������������������������������������������������������������  429 Joe Leigh Simpson, Svetlana Rechitsky, and Anver Kuliev

Contributors

Karthick Kumar Alagamuthu  Department of Biotechnology, Selvamm Arts and Science College (Autonomous), Namakkal, India Margarida  Albuquerque  Prenatal Unit, Centro de Medicina Laboratorial Germano de Sousa, Lisbon, Portugal Stephanie  Allen  West Midlands Regional Genetics Laboratory, Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, UK Veronika Artemenko  Almazov National Research Centre, Laboratory of Maternal Fetal Medicine, St. Petersburg, Russia CEMER Eureopan Centre for Medicine and Research, Perugia, Italy Nuria  Balaguer  Prenatal Diagnosis Department, Igenomix Spain Lab S.L.U., Paterna, Spain José Luis Bartha  Departamento de Obstetricia y Ginecología, Clínica Universitaria de Navarra, Pamplona, Spain Peter  Benn  Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, CT, USA Elisa  Bevilacqua  Department of Women and Child Health Sciences and Public Health, Fondazione Policlinico Agostino Gemelli IRCCS, Rome, Italy Diana  W.  Bianchi  Prenatal Genomics and Fetal Therapy Branch, Center for Precision Health Research, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA Katia  Bilardo  Fetal Medicine, Department of Obstetrics and Gynaecology, Amsterdam UMC, Amsterdam, The Netherlands David W. Britt  Fetal Medicine Foundation of America, New York, NY, USA James S. Castleman  West Midlands Fetal Medicine Centre, Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, UK xiii

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Contributors

Sookhim Chan  Atila Biosystems Inc., Mountain View, CA, USA Natalie Chandler  North Thames Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust, London, UK Petya Chaveeva  Fetal Medicine Unit, Dr. Shterev Hospital, Sofia, Bulgaria School of Medicine, Medical University of Pleven, Pleven, Bulgaria Fetal Medicine Research Institute, King’s College Hospital, London, UK Xin Chen  Atila Biosystems Inc., Mountain View, CA, USA Yifan Chen  Atila Biosystems Inc., Mountain View, CA, USA Lyn  S.  Chitty  North Thames Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust, London, UK Genetic and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, London, UK Mahesh  Choolani  Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore Department of Obstetrics and Gynaecology, National University Hospital, Singapore, Singapore Graziano  Clerici  CEMER Eureopan Centre for Medicine and Research, Perugia, Italy Grasielle  Cruz  Prenatal Unit, Centro de Medicina Laboratorial Germano de Sousa, Lisbon, Portugal Maria del Mar Gil  Fetal Medicine Research Institute, King’s College Hospital, London, UK Obstetrics and Gynecology Department, Hospital Universitario de Torrejón, Madrid, Spain School of Medicine, Universidad Francisco de Vitoria (UFV), Madrid, Spain Maria  José  Rego  de Sousa  Prenatal Unit, Centro de Medicina Laboratorial Germano de Sousa, Lisbon, Portugal Gian Carlo Di Renzo  Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Italy PREIS International and European School of Perinatal, Neonatal and Reproductive Medicine, Firenze, Italy Department of Obstetrics and Gynecology, I.M. Sechenov First State University of Moscow, Moscow, Russia Department of Obstetrics and Gynecology and Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Umbria, Italy Mark I. Evans  Fetal Medicine Foundation of America, New York, NY, USA

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Comprehensive Genetics, PLLC, New York, NY, USA Department of Obstetrics and Gynecology, Icahn School of Medicine at Mt. Sinai, New York, NY, USA Shara M. Evans  Fetal Medicine Foundation of America, New York, NY, USA Department of Maternal Child Health, Gillings School of Public Health University of North Carolina at Chapel Hill, Chapel Hill, NC, USA María  Gómez-López  Prenatal Diagnosis Department, Igenomix Spain Lab Paterna, Valencia, Spain Francesca Romana Grati  Unit of Research and Development, Cytogenetics and Medical Genetics TOMA, Advanced Biomedical Assays, Impact Lab, Busto Arsizio, Varese, Italy Cytogenetics and Molecular Genetics, TOMA Advanced Biomedical Assays S.P.A., Busto Arsizio, Italy Hazar Haidar  Ethics Programs, Department of Letters and Humanities, University of Quebec at Rimouski, Rimouski, QC, Canada Jacques  Jani  Department of Obstetrics and Gynaecology, University Hospital Brugmann, Université Libre de Bruxelles, Brussels, Belgium Bo Jiang  Atila Biosystems Inc., Mountain View, CA, USA Renee Jones  Roche Sequencing Solutions, San Jose, CA, USA Karl  Oliver  Kagan  Department of obstetrics and Gynaecology, University of Tuebingen, Tübingen, Germany Mark D. Kilby  West Midlands Fetal Medicine Centre, Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, UK Emilia Kostenko  Roche Sequencing Solutions, San Jose, CA, USA Haripriya Kuchi Bhotla  Department of Life Sciences, CHRIST (Deemed to be University), Bengaluru, Karnataka, India Anver Kuliev  Department of Human and Molecular Genetics, Florida International University, Miami, FL, USA Reproductive Genetic Innovations, Chicago, IL, USA Anne-Marie Laberge  Department of Social and Preventive Medicine, School of Public Health, University of Montreal, Montreal, QC, Canada Medical Genetics, Department of Pediatrics, and Research Center, Centre Hospitalier Universitaire Sainte-Justine, Montreal, QC, Canada Jialuo Li  Atila Biosystems Inc., Mountain View, CA, USA

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Contributors

Karen Lim  Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore Dennis Y. M. Lo  Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, SAR, China Emilia Mateu-Brull  Prenatal Diagnosis Department, Igenomix Spain Lab S.L.U., Valencia, Spain Arun Meyyazhagan  Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Italy PREIS International and European School of Perinatal, Neonatal and Reproductive Medicine, Firenze, Italy Perinatology Research Branch, Wayne State University, Detroit, MI, USA Department of Life Sciences, CHRIST (Deemed to be University), Bengaluru, Karnataka, India Department of Obstetrics and Gynecology and Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Umbria, Italy Y. M. Dennis Lo  Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong SAR, Spain Miguel  Milán  Prenatal Diagnosis Department, Igenomix Spain Lab S.L.U., Paterna, Spain Kypros H. Nicolaides  Fetal Medicine Research Institute, King’s College Hospital, London, UK Dick Oepkes  Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands Manikantan Pappuswamy  Department of Life Sciences, CHRIST (Deemed to be University), Bengaluru, Karnataka, India Tatyana Pervunina  Almazov National Research Centre, Laboratory of Maternal Fetal Medicine, St. Petersburg, Russia Vardit Ravitsky  Department of Social and Preventive Medicine, School of Public Health, University of Montreal, Montreal, QC, Canada Svetlana  Rechitsky  Department of Human and Molecular Genetics, Florida International University, Miami, IL, USA Reproductive Genetic Innovations, Chicago, FL, USA Marie-Christine Roy  Department of Social and Preventive Medicine, School of Public Health, University of Montreal, Montreal, QC, Canada Elizabeth  Scotchman  North Thames Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust, London, UK

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Joseph Shaw  North Thames Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust, London, UK Yining Shi  Atila Biosystems Inc., Mountain View, CA, USA Carlos  Simón  Igenomix S.L., Obstetrics and Gynecology, Valencia University, Valencia, Spain Beth Israel Deaconess Medical Center, Harvard University, Boston, MA, USA Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, USA Joe  Leigh  Simpson  Department of Human and Molecular Genetics, Florida International University, Miami, FL, USA Department of Obstetrics and Gynecology, Florida International University, Miami, FL, USA Reproductive Genetic Innovations, Chicago, IL, USA Bowdoin Su  Roche Sequencing Solutions, San Jose, CA, USA Valentina  Tsibizova  PREIS School International and European School of Perinatal, Neonatal and Reproductive Medicine, Florence, Italy Institute of Perinatology and Pediatrics, Almazov National Medical Research Centre, St. Petersburg, Russia CEMER European Centre for Medicine and Research, Perugia, Italy Miriam  Turiel-Miranda  Departamento de Obstetricia y Ginecología, Clínica Universitaria de Navarra, Pamplona, Spain Amy Turriff  Prenatal Genomics and Fetal Therapy Branch, Center for Precision Health Research, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA Ellen van der Schoot  Department of Experimental Immunohematology, Sanquin Research, Amsterdam, The Netherlands Landsteiner Laboratory, Amsterdam University Medical Centre, AMC, University of Amsterdam, Amsterdam, The Netherlands Elizabeth Wall  Clinical Genetics Service, Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, UK Rong Wang  Atila Biosystems Inc., Mountain View, CA, USA Youxiang Wang  Atila Biosystems Inc., Mountain View, CA, USA Karen White  Roche Sequencing Solutions, San Jose, CA, USA Zhijie Yang  Atila Biosystems Inc., Mountain View, CA, USA Han Zhang  Atila Biosystems Inc., Mountain View, CA, USA

Chapter 1

Introduction Y. M. Dennis Lo

It has been 24 years since the first report of cell-free fetal DNA in maternal plasma [1]. This book is thus very timely to take stock of what has been achieved as the field enters its silver jubilee. The development of noninvasive prenatal testing (NIPT) has demonstrated the importance of investing in basic scientific research while we are pushing forward translational and clinical research. For example, without information regarding the concentrations of cell-free fetal DNA during different gestational ages [2], it would be difficult to design NIPT for chromosomal aneuploidies and monogenic diseases in which the diagnostic accuracy is closely governed by the fetal DNA fraction. As another example, without understanding the clearance of cell-free fetal DNA following parturition [3], it would be difficult to use NIPT with confidence in multiparious pregnant women. As circulating fetal DNA comes from the placenta, knowledge about placental biology would ultimately help us to understand more about the production, release, and biophysical characteristics of cell-free fetal DNA. In this regard, I am especially pleased to see a chapter on the placenta in this volume.

2022 Lasker-DeBakey Clinical Medical Research Award. Y. M. D. Lo (*) Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_1

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I am very glad to see that my many collaborators and friends have contributed to this volume. I had the privilege of collaborating with Prof. Kypros Nicolaides, Prof. Ranjit Akolekar, and Prof. Rossa Chiu on the first trial of the use of NIPT for trisomy 21 [4]. I am also eager to read the chapter by Prof. Lyn Chitty on the development of noninvasive preantal diagnosis of monogenic diseases. Prof. Chitty had previously collaborated with Prof. Allen Chan in my group on the use of digital PCR for this purpose [5]. I note the very interesting chapter by Dr. Diana Bianchi on incidental findings in NIPT, which is related to my other interest on cancer liquid biopsies [6, 7]. I had the good fortune of collaborating with Dr. Bianchi on observing aberrant concentration of cell-free fetal DNA in maternal plasma and serum in trisomy 21 [8]. I also look forward to reading the chapters written by many friends of mine including Prof. Di Renzo, Prof. Yuval Yaron, Dr. Mark Evans, Prof. Carlos Simon, Prof. Mahesh Choolani, and Prof. Joe-Leigh Simpson, among others. Finally, I am glad to see that in addition to science and clinical applications, this volume has also covered the ethics, societal, and cultural aspects of NIPT. With the rapid developments of science and technology, it is especially important to put such developments in the context of the human society and their ethical, legal, and societal impacts.

References 1. Lo YMD, et al. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997;350:485–7. https://doi.org/10.1016/S0140-­6736(97)02174-­0. 2. Lo YMD, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet. 1998;62:768–75. https://doi. org/10.1086/301800. 3. Lo YMD, et  al. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet. 1999b;64:218–24. https://doi.org/10.1086/302205. 4. Chiu RWK, et  al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ. 2011;342:c7401. https://doi. org/10.1136/bmj.c7401. 5. Barrett AN, et al. Digital PCR analysis of maternal plasma for noninvasive detection of sickle cell anemia. Clin Chem. 2012;58(6):1026–32. https://doi.org/10.1373/clinchem.2011.178939. 6. Chan KCA, et al. Analysis of plasma Epstein–Barr virus DNA to screen for nasopharyngeal cancer. N Engl J Med. 2017;377:513–22. https://doi.org/10.1056/NEJMoa1701717. 7. Sun K, et  al. Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments. Proc Natl Acad Sci U S A. 2015;112:E5503–12. https://doi.org/10.1073/pnas.1508736112. 8. Lo YMD, et  al. Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem. 1999a;45:1747–51. https://doi.org/10.1093/ clinchem/45.10.1747.

Chapter 2

A Brief History of Noninvasive Prenatal Diagnosis and Its Forecast Gian Carlo Di Renzo, Arun Meyyazhagan, and Valentina Tsibizova

Highlights • Noninvasive prenatal testing (NIPTS) ascertains specific fetal genetic anomalies as early as 10 weeks of gestation. • No adverse effect and risk of miscarriage. • Higher sensitivity for trisomies, especially T21, 13, and 18. • NIPT uses cell free fetal DNA (cffDNA) from maternal blood. • Ultrasound markers such as nuchal translucency and biochemical profiling (β-hCG, PAPP-A, inhibin A) can screen for risk in pregnant women to have a genetically abnormal fetus. • Low percentage of cffDNA, link to specific risk factors, and technical drawbacks may limit the results of the NIPT. • Confirmation of any test should be done by classical invasive techniques (amniocentesis, chorionic villi sampling).

G. C. Di Renzo (*) Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Italy PREIS School International and European School of Perinatal, Neonatal and Reproductive Medicine, Florence, Italy Department of Obstetrics, Gynaecology and Perinatology, I.M. Sechenov First State University of Moscow, Moscow, Russia e-mail: [email protected] A. Meyyazhagan Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Italy PREIS School International and European School of Perinatal, Neonatal and Reproductive Medicine, Florence, Italy V. Tsibizova PREIS School International and European School of Perinatal, Neonatal and Reproductive Medicine, Florence, Italy © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_2

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2.1 Introduction Noninvasive prenatal diagnosis, nowadays mostly commonly called noninvasive prenatal testing (NIPT) which utilizes cell-free DNA (cfDNA) circulating in maternal blood from the beginning of pregnancy, is the method most utilized to ascertain fetal genetic abnormalities, mostly aneuploidies, as early as the end of first trimester (10 weeks onwards) in pregnant women. Since the initial utilization of cfDNA for Down syndrome detection, the technology has been exploited for the detection of other trisomies and chromosome abnormalities. Technological advances have also allowed the identification of smaller anomalies in the genome such as copy number variants (CNVs) [1] and in genes causing single gene disorders [1]. NIPT is considered the final innovation in prenatal diagnosis, aiming to assist practitioners and future parents in managing pregnancy, making informed, conscious choices, and counselling on their in-utero child. Figure 2.1 illustrates the basic protocol followed during NIPTs. Since NIPT is a noninvasive method, the risk of miscarriage and adverse outcomes related to diagnostic tests is nil as it is done only using maternal blood samples. By the fact that it can be used in the early stages of pregnancy, NIPT is considered one of the best screening methods with zero risk to fetus and mother other than being efficient and cost-worthy [2]. Studies have shown higher sensitivity and specificity (>99%) for Down syndrome compared to previous screening modalities with progressively lower performance for other

Fig. 2.1  The outline of the protocol followed for noninvasive prenatal diagnosis (NIPTs). (Concerning the specific laboratory test there is a specific chapter in the book)

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genetic conditions [3, 4]. NIPT is estimated to be performed in over 61 countries and is expected to become a $6.0 billion market in the next 5  years [5]. Debate still continues on the practical implications of cfDNA screening in pregnancy generally related to knowledge gaps among both professionals and patients, implementation models, and in many cases increases in cost related to large-scale screening programs. There is increasing interest in understanding the social and ethical issues related to expansion of NIPT applications in terms of patient’s decision management, possible financial stress, patient anxiety, views regarding pregnancy termination [6], along with the apprehension of medical professionals concerning the increased use of NIPTs [2]. Though most women and public members view NIPT as a positive tool for prenatal care beyond Down syndrome, parents raise a similar objection about financial issues, accessibility, and information management. Table  2.1 shows the assumptions and reality of NIPTs seen in the world. It can be observed that the utilization of NIPTs in routine clinical practices is still observed as an absence of conformations on both professional and patients’ view. In this chapter, we described the technology’s evolution and its importance in the setting of prenatal noninvasive diagnosis.

Table 2.1  Assumptions related to NIPT made by the common people and clinicians versus its reality S. no. Characteristics 1. Performance period 2.

Risk to fetus

3.

Origin of the cells {cell free fetal (cff)-DNA} Follow-up study

4.

5. 6.

Time duration of the test Reliability

7.

Fetus state

8.

Output attainment/ results

Presumptions Performed in earlier gestational time Zero risk to the fetus

Reality/truth True, it can be performed from 9 to 10 weeks of gestation True as the maternal serum is utilized for evaluation Derived from the trophoblast cells

Expected to be originated from the fetus It doesn’t require an False, the confirmation of the anomalies in invasive approach case of positive screening is done by amniocentesis or chorionic villus sampling Quick and easily Long and technically complex for most of obtainable the tests Reliable for all 99% Reliable for trisomy 21, but genetic conditions progressively lower predictive value for other genetic disorders Fetus is not at all True, since it is only a screening test harmed Obtained faster Results take at least a week, and chances of repeating the test are higher in case of low cff-DNA percentage

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2.2 Chronological Footprints in Prenatal Diagnosis The field of noninvasive prenatal screening and diagnosis has undergone enormous progress over the past six decades. Compared to the direct analysis of fetal cells from amniocentesis or chorionic villus sampling, noninvasive approaches using maternal blood or ultrasound have the great advantage of posing no risk of miscarriage to the pregnancy. Subsequently, noninvasive tests, including serum analyte screening and cell-free DNA screening, were developed for purposes of screening for genetic abnormalities in pregnancy.

2.2.1 Main Steps Before the Ultrasound Introduction in Obstetrics The practice of tapping amniotic fluid has been around for at least a century. Third-­ trimester transabdominal amniocentesis was reported by Prochownick et al. [7] and Schatz [8]. Menees et al. [9] reported sampling of amniotic fluid by transabdominal needling; radio-opaque contrast was injected to outline the fetus and placenta. Fuchs and Riis [10] in their seminal article in “Nature” documented the first application of amniotic fluid analysis in 1956 to determine the fetal sex, where amniotic fluid cells are screened for the Barr body. In 1961, Liley [11] in Auckland, New Zealand, published the well-known correlation between the deviation of the spectral absorption curve of amniotic fluid resulting from bilirubin and the severity of rhesus isoimmunization. After Liley’s findings, amniocentesis became commonplace in obstetrics. Steele and Breg’s 20 study on amniotic fluid karyotyping was ground breaking. Nadler published a study on enzyme testing using fetal cells from amniotic fluid the same year where he reported one of the earliest cases of Trisomy 21. His researchers used lab-grown cells to examine chromosomes. In 1972, Milunsky and Littlefield [12] wrote about infant metabolic abnormalities. Brock and Sutcliffe in UK and contemporarily Hino et al. from Japan reported in 1972 that pregnant women with neural tube defects had increased alpha-fetoprotein (AFP) levels in the amniotic fluid [13]. Nadler and Gerbie did a seminal publication about “Role of amniocentesis in intrauterine diagnosis of genetic abnormalities” in 1970, and this was the real impetus for genetic amniocentesis and diagnosis. Mohr introduced antenatal genetic diagnosis utilizing chorionic villi in 1968 [14]. In this study, he did transcervical chorion biopsy under 5  mm endoscopic vision, but the first effective prenatal diagnostic use of chorionic villi biopsy was reported in 1975 from the Tietung Hospital in Anshan, China [15], where fetal sex was determined for sex preselection. This was published in an issue of the Chinese Medical Journal, which reported on 100 patients who had transcervial chorionic villus biopsy with 3 mm metal cannula blind aspirations. Upon introduction into the cervical canal and when gentle resistance was felt, a smaller internal tube was advanced and placental material was suctioned. There were six erroneous diagnoses

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out of 99 successful attempts made with four pregnancies lost. US researchers were unable to repeat these results; therefore, first-trimester prenatal diagnosis at that time was shelved. The first attempts at fetoscopy were carried out by Westin in 1954 [16] using a 10 mm diameter hysteroscope introduced through the cervix of patients who were to have therapeutic abortions between 14 and 18 weeks. The term “fetoscopy” was introduced by Scrimgeour in 1973 [17] when he described exposing the uterus at laparotomy and introduced a 2.2 mm needlescope to view the amniotic cavity and fetus. Valenti [18, 19] used the “endoamnioscope” and reported the first aspiration of fetal blood and the successful diagnosis of hemoglobinopathy https://www.ob-­ ultrasound.net/hobbins.html.

2.2.2 Main Steps After Ultrasound Introduction in Obstetrics Ultrasound and molecular genetics revived the demand for early antenatal diagnosis. Steele and Breg [20] cultured fetal cells from amniotic fluid and paved the way to karyotyping in 1966. All these above works showcased amniocentesis by the 1970s as the dominant prenatal genetic testing method and was then considered as standard practice as per Nadler and Gerbie’s mentioned publication in 1970 [21]. Development in the amniocentesis field was advanced by utilizing ultrasound to guide the needle as per the Bang and Northeved protocol in 1972 [22]. Hobbins and Mahoney [23] at Yale and Patrick et  al. [24] from Canada did ground-breaking work with sonography, before performing fetoscopy. These studies reported on the use of an ultrasonic scan to precisely map the placenta and fetal position, as well as to quantify the exact distance between the front abdominal wall and the chorionic plate. In 1977, Hobbins and Mahoney reported successful exclusion of beta-thalassemia and diagnosis of sickle cell disease without apparent risks to the mother and fetus of the procedure. Other important investigators included Alter et al. [25] and Fairweather et al. [26] in London. Rodeck and Campbell [27] reported the diagnosis of spina bifida using ultrasonography. In 1979, the same group reported a success rate of obtaining pure blood samples at 94% in 51 attempts of cord blood sampling. They introduced the method of aspirating blood from one of the larger vessels at the base of the cord. Most investigators started utilizing this technique for the diagnosis of hemoglobinopathies. Kazy et al. [28] reported fetal sexing and enzyme assay on chorion samples at 6–12 weeks’ gestation in the USSR in their first report of ultrasonographic guidance during chorionic villi sampling. Niazi et al. developed better methods for growing fibroblasts from trophoblast villi in 1981. Ward in London performed transcervical chorionic sampling using an ultrasonic catheter with syringe suction in 1983. The Brambati group in Milan showed in 1983 that ultrasonic guidance increased the recovery rate of obtaining chorionic material from 75 to 96%. Brambati used a 1.5  mm polyethylene tube with a soft stainless-steel obturator, which afterwards became the most frequent device utilized for chorionic villi sampling (CVS). In

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1983, the Simoni and Brambati [29] in Milan published a procedure for direct chromosomal and biochemical analysis on first trimester chorionic villi (‘direct prep’). Chorionic villi are aspirated or biopsied from the chorion frondosum at the placental disc. The inner cytotrophoblastic layer underneath the outer syncytiotrophoblast layer comprises actively mitotic cells in fresh villus samples. In 1984, Smidt-Jensen and Hahnemann [30, 31] in Copenhagen devised transabdominal fine-needle villus aspiration. With less infection problems and failed samplings requiring reinsertions, this approach became much more common than its transcervical equivalent. The Brambati and Simoni group in Milan and the Golbus group in San Francisco disclosed other ultrasonic approaches and adjustments in late 1980s. In 1986, the Golbus group reported early experience with 1000 CVS cases and showed a 3.8% fetal loss rate and 1.7% incidence of chromosomal mosaicism not subsequently confirmed in the fetus. After fetoscopy, ultrasound-guided pure fetal blood sampling, or cordocentesis, was pioneered in France in 1983 by Fernand Daffos [32]. Pure fetal blood was aspirated in-utero at around 18 weeks from the umbilical vein near the placental insertion of the cord using 20 G needle under ultrasound guidance. The procedure was also popularized around the same time in UK by Campbell and Rodeck at King’s College Hospital. The Hobbins group at Yale described their technique in 1985 and called the procedure percutaneous umbilical blood sampling (PUBS). This replaced blood sampling via fetoscopy which the group had pioneered 10 years before. In 1988, Nicolini, working with Rodeck at the Queen Charlotte’s Maternity Hospital in London, first described fetal blood sampling from the intrahepatic portion of the umbilical vein in the fetus, as an alternative procedure in cases where cord needling was unsuccessful. In the late 1980s, fetoscopy was mainly reserved for tissue or organ sampling, and fetal blood sampling came to always be done via the ultrasonic-guidance transabdominal needle procedure. The commonest indication of fetal blood sampling had evolved to become one of quick confirmations of suspected abnormal karyotype in the late second trimester, when a chromosomal abnormality has been suggested by fetal anatomic abnormalities seen at ultrasound anatomy. The introduction of nuchal translucency (NT) revolutionized the field of prenatal screening in the 1990s and became an important ultrasound maker for first trimester chromosomal screening [33]. Nicolaides at King’s College London and Ville in France popularized this technique in United Kingdom and in Europe after 1991. Then it became a standard screening modality for fetal aneuploidy screening in perinatal centers around the world. Spencer et al. [34] showed that fetal NT at 10–14 weeks can be used in conjunction with the biochemical marker’s beta-human chorionic gonadotrophin (-hCG) and pregnancy-associated plasma protein A (PAPP-A) to enhance aneuploidy screening in the first trimester. Rapid analytical techniques for the measurement of biochemical markers have enabled the development of a one stop clinic for assessment of risk for fetal anomalies (OSCAR), during which a patient can receive pretest counselling, a biochemical assessment, an ultrasound assessment, and a combined risk estimation all within the span of a hour (Spencer [35, 36]).

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2.2.3 Cell Free Fetal DNA Kazakov et al. [37] initially reported the presence of extracellular DNA in the blood of pregnant women during the first trimester. Extracellular free-floating DNA found in maternal blood can come from trophoblast cells, lymphocytes, or decidual cells, and its importance in pregnancy is highlighted in this section. The first definitive report of the finding of cell-free fetal DNA (cff-DNA) in maternal plasma and serum was published by Lo et al. [38]. The field of prenatal screening saw a new phase in 1997 after the identification of cff-DNA and the demonstration of increasing concentrations with advancing age in the maternal serum and plasma [38]. These findings were key to the development of an alternative noninvasive fetal aneuploidy screening method which came to be known as noninvasive prenatal testing (NIPT). These early findings suggested that its use as a practical pregnancy screening test as cff-DNA is cleared immediately after pregnancy (Fig. 2.2). However, it took until 2008 to demonstrate that trisomy could be identified in maternal blood by applying next-generation sequencing (NGS) [39, 40]. Fan and Chiu, with their colleagues, demonstrated the possibility of screening for trisomy 21 (T21) using the sequencing of cff-DNA, with a low false positive rate in pregnant women. This opened-up a race to develop the best NIPT tests using NGS to offer a more accurate and safer method aiming to avoid invasive test follow-up. Since the introduction of cff-DNA prenatal screening in 2011, in the subsequent 4 years more than two million NIPTs have already been performed [41]. Currently,

Fig. 2.2  Nowadays, the screening test performed during pregnancy comprises primarily noninvasive tests in the early weeks of gestation; invasive techniques are used where there are abnormalities with the presence of soft ultrasound markers, abnormal biochemical analysis, or positive NIPT

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alongside the evolution of genetic testing generally, there is a growing need for the reassurance of the health of the fetus, both from natural conceptions and in vitro fertilization techniques. Genetic tests have also been developed with the aim to discover recessive disease carrier status among couples in order to assess the preconception fetal risk of inheriting recessive conditions [42]. Alternatively, blastocyst DNA analysis for “diagnosis” before implantation can also be utilized using in vitro techniques [1, 43]. Preimplantation genetic diagnosis, however, does not preclude the standard antenatal screening or diagnostic tests [44]. The history of prenatal diagnosis tells a dynamic story of how widespread invasive prenatal diagnostic testing gradually changed to NIPT and in all its main chronological stages with many pioneering clinicians and scientists involved (Fig. 2.3).

Fig. 2.3  Pioneers in prenatal diagnosis. Top left to right: Thomas Orville Menees; Albert William Liley; Mark W Steele; Fritz Friedrich Fuchs; John Hobbins; Charles Rodeck. Second row left to right: Aubrey Milunsky; Jeremiah Mahoney; Henry Nadler; Albert B Gerbie; Jan Gunnar Faye Mohr; John Scrimgeour. Third row left to right: Fernand Daffos; Stuart Campell; Bruno Brambati; Umberto Nicolini; Kypros Nicolaides; Leonard Herzenberg; Fourth row left to right: Vasiliy Ivanovich Kazakov; Dennis Lo; Kevin Spencer; Christina Fan; Rossa Chiu; Diana W.  Bianchi; Fifth row left to right: André Boué; Jens Bang; Howard Cuckle; Nicholas (Nick) Wald

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2.3 Types of NIPTs Utilized to Date Whole genome analysis or targeted genomic analysis of cfDNA fragments [also using single nucleotide polymorphisms (SNPs)] is the basic methodology utilized for cfDNA-based NIPT [45]. Massive parallel shotgun sequencing (MPSS) or chromosomal selective sequencing (CSS) is used to analyze the millions of fragmented sequences of cff-DNA in MPSS and further assign them to original chromosomes for quantification. Usually, trisomic fetuses have higher quantities of cfDNA in maternal blood compared to euploid fetuses [46]. However, the CSS sequence of the specific region of chromosomes X, Y, 13, 18, and 21 is utilized with the advantage of cost-cutting, but a higher failure report than MPSS [1]. Table 2.2 enlists the commonly used practices of prenatal screening methods. Likewise, the utilization of multiplex PCR to detect SNPs aids in distinguishing the maternal and fetal fragments along with quantifying fetal fraction (FF) in CSS or PCR with a failure rate of 4% [51]. As an alternative to sequencing cfDNA fragments, quantification using microarray has been implemented for several years as accurate and faster alternative to CSS and reduces a certain amount of variability of the assay [52]. Additionally, digital PCR based on counting single molecule strategy has been validated for T21. This technology claims to be cost efficient and rapid in comparison to next-generation sequencing (NGS) with the limitations of requiring a sufficient quantity of cff-DNA and a single set of the probe to detect low degree mosaicism and chromosomal morphological anomalies like balanced translocation [53]. NGS is preferred for its ability to detect micro-duplications/deletions at a resolution limited to approximately 7–8 megabases. More recently, probe peptide nucleic acid (PNS)-based real time PCR has been demonstrated as an alternative to NGA for the detection of common fetal trisomies in cff-DNA. This technology utilizes PCR probes that generate differences in the melting points between perfect and mismatched sequences. As the probes are synthesized DNA analogues, they have uncharged backbone to make them effective for hybridization. The technique has also been used for finding resistance of Helicobacter pylori towards clarithromycin and to determine microsatellite instability in colorectal cancer [54, 55]. Other techniques, like locating the methylated regions in DNA and microRNA analysis in plasma, are also suggested for noninvasive maternal blood testing. Since both methods have practical limitations like pretreatment with methylation-­sensitive restriction enzymes and presence of low levels of microRNAs in early pregnancies, they are not used generally [55, 56]. RT-PCR-based NIPT is typically faster and can be generally done at low cost when compared with NGS. Massive parallel sequencing is the general and most widely used procedure for analyzing chromosomal defects from maternal plasma cfDNA, but reports have also demonstrated the use of nanopore sequencers. Belova with his colleagues in 2020 constructed long chimeric reads using the short cff-DNA from pregnancy to identify anomalies like T21, 18, 13 with higher specificity and sensitivity [57]. NIPT, as some authors designate it, has been successfully used to detect fetal monogenetic disorders from maternal plasma cfDNA using direct haplotyping for a

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Table 2.2  Biochemical and ultrasound prenatal screening methods Trimesters First (11–13 weeks)

Technique Nuchal translucency (NT) + biochemical markers Methods of screening Maternal age (MA) + fetal NT MA + serum free β-hCG and PAPP-A MA + NT + free β-hCG and PAPP-A (combined test) Combined test + nasal bone or tricuspid flow or ductus Second Biochemical marker screening (16–20 weeks) Methods of screening MA + serum AFP, hCG (double test) MA + serum AFP, free β-hCG (double test) MA + serum AFP, hCG, uE3 (triple test) MA + serum AFP, free β-hCG, uE3 (triple test) MA + serum AFP, hCG, uE3, inhibin A (quadruple test) MA + serum AFP, serum AFP, free β-hCG, uE3, inhibin A (quadruple test) First and second Integrated prenatal screening (IPS) IPS without inhibin A, serum IPS Contingent screening

Sequential screening

First, second, and third (5 weeks onwards)

Ultrasound scan

Common interpretation Thick nuchal fold + mothers’ advance age + increased β-hCG = trisomies (Orlandi et al. [47])

Unconjugated estriol + hCG = triple screen or with inhibin A as quadruple screen to ascertain the risk of neural tube defects (NTD), T21, T18 (Dey et al. [48])

PAPP-A + NT + quadruple screen for trisomies’ risk detection (Wald et al. [49])

Alternative for IPS risk >1/50—invasive test should be done for aneuploidies (Wright et al. [50]) Chosen from first trimester screening and offered invasive diagnostics Low PPV = T21 suspicion Soft markers + ultrasound image + prior screening reports = risk of aneuploidies (Dey et al. [48])

limited range of single gene disorders such as haemophilia, cystic fibrosis, adrenal hyperplasia, Ellis van-creveld syndrome, Duchenne muscular dystrophy, and thalassemia. To address the difficult problem of detecting abnormalities in triplet-repeat genetic disorders, linked read libraries and targeted sequencing with the utilization of Bayesian analysis has been successfully used in a limited series to predict the variant status in fetuses with both Huntington’s disease and muscular dystrophy type 1 [58].

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2.4 NIPT Challenges in Diagnosing Monogenic Disease Though NIPT is a screening test for aneuploidy, there is still an opportunity to diagnose monogenic diseases. The common diagnosis includes determining of RhD status of the fetus in RhD-negative mothers, determining the sex in sex-linked disorder cases, and risk of pregnancy in case of de novo, dominant and recessive states. Known family history or abnormal sonography results are the main indicators for the anomalies. Through sequence analysis of exome, the presence of monogenic disorders can be predicted even after obtaining normal karyotype through the invasive procedure and abnormal sonography (Vora et al. [59], [60]). Utilizing technologies like NGS in these situations can overcome the challenge by diagnosing early by conducting noninvasive prenatal sequencing using cff-DNA for multiple Mendelian monogenic disorders and utilizing the haplotyping strategy to obtain reliable output [61]. The relative mutational dosage (RMD) assesses the parental genotype for mutational studies. The relative haplotype dosage (RHDO) checks the parental haplotype specific to heterozygous polymorphisms associated with mutation using proband within the family as reference or by analyzing the parental read sequences. RMD counts specific mutational sites were as RHDO compared with maternal plasma for multiple single nucleotide polymorphisms to increase the reproducibility and accuracy [62]. Studying the father’s haplotype can also help diagnose paternal inheritance not seen in the mother genome, like in cystic fibrosis [63]. The UK offers noninvasive diagnosis using cfDNA to detect autosomal dominant, autosomal recessive with paternal inheritance exclusion, or X-linked inherited disorders without prior invasive test confirmation [64]. NGS has the advantage of assessing several variants in a single panel and also assessing the single mutation site in comparison to indirect analysis considering the small quantity of cff-DNA in maternal serum and the presence of transcriptome and methylome of the fetus also aids in detecting the health status of fetus and mom [65, 66]. The PPV in the case of the cfDNA analysis for microdeletion syndromes using whole genome analysis has been quite low; a large data group showed about 13% PPV overall for common microdeletion syndromes like Prader-Willi/Angelman syndrome, del11p36, DiGeorge syndrome, and Cri-du-chat may be due to their lower prevalence and absence of risk factors association like maternal age is a major factor for T21. Wapner and his colleagues suggested that a high negative predictive index for microdeletion syndromes using cfDNA could be reassuring, but still requires large-scale clinical validation and there remains significant technical challenges [67]. Developing technologies can allow us to quantify single molecules with more specificity like different types of PCR, NGS, and nanopore can help us to assess over- and under-expressed fetal alleles in the cff-DNA obtained from the maternal plasma/serum. As this sample requires to accurately quantify the fetal load (total cff-DNA), it can specifically locate the mutated or over/under-expressed allele. Techniques like matrix-assisted laser desorption time of flight spectrometry (MALDI-TOF MS) can also allow us to find the polymorphism in the sequences and are highly amended in NIPTs to detect aneuploidies with cff-DNA above 4% to

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obtain reliable result (Lench et al. [68]). Utilization of circulating fetal trophoblastic cells for NIPTs to detect monogenic disease is also on talk specifically for diseases majorly with triplet repeat expansions or point mutations like cases through microsatellite multiplex PCR, whole genome sequencing succeeded with mini-­exome sequencing [69].

2.5 NIPT Limitation and Quality Assurance Though NIPT is highly accurate for screening, they pose several limitations that make the technology as it stands not diagnostic. Hence, a robust quality assessment is required to maintain minimum criteria for conducting and reporting the results [70]. For instance, measuring the fetal fraction is a prime analytical and quality assurance metric in screening with cfDNA. A low fraction is associated with higher maternal body mass index, early gestation age, drug usage, smoking, and other elements known to reduce the placental size [71]. About 4% is applied as the threshold for fetal fraction quality, and the results below are inconclusive. NIPTs are usually utilized for assessing the trisomies, and other chromosomal rearrangements are not deduced. Hence, cfDNA screening after the detection of structural anomalies should not be used as the incidence of chromosomal rearrangement is higher in the pregnant cohort [72]. Conflicting NIPT results can be due to many potential reasons, one being “vanishing twin”—spontaneous abortion in early pregnancy due to aneuploid twins, the maternal serum still possesses the cff-DNA released by the placenta after aneuploid fetal demise, and this can be attributed to false euploid twin report in NIPT [73]. Likewise, abnormalities in maternal cell lines like either mosaic or constitutional or malignant cytogenic anomalies can contribute to conflicting results due to circulating cell-free tumor DNA.  Hence, women with known malignancies shouldn’t be tested with NIPTs as the results may be difficult to resolve due to circulating tumor DNA [41, 74]. Lastly, cff-DNA might not represent the fetus genotype, and NIPT can give inaccurate reports due to confined placental mosaicism in case of aneuploidy or complete discordance between placental and fetal genotype. Depending on the predominant placental cell line, conflicting positive or negative results can be obtained [75].

2.6 The Search of the Fetal Cells in Maternal Blood In the middle of the 1980s, researchers made the startling finding that fetal cells could be found in the blood of pregnant mothers. Isolating these cells and putting them through genetic analysis sparked a major paradigm shift in the field of prospective prenatal diagnosis. Lymphocytes, trophoblasts, erythroblasts, granulocytes,

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hematopoietic stem cells and progenitor cells, and mesenchymal stem cells are some of the types of fetal cells that have been found in maternal blood. Other forms of fetal cells seen in maternal blood include lymphocytes and erythroblasts [76]. Numerous researchers have been actively engaged in the investigation of the identification, selection, and genetic analysis of fetal cells. In the course of their work, they have brought to light issues that are brought about either by the lack of a particular fetal antigen or by the small number of fetal cells that are present in maternal blood. The techniques of fluorescence-activated cell sorting (FACS), magnetic activated cell sorting (MACS), and ISET technology (isolation of epithelial tumor cells), which have so far been used to trophoblasts, are the methods that are utilized to circumvent these challenges [76]. In light of the fact that enrichment techniques yield fetal cell samples that are nevertheless tainted by maternal cells, the scientific community has been concentrating on the investigation of a particular fetal cell marker. In the past, the most common types of fetal cell markers were fetal hemoglobin, which served as a proteic marker, and the Y chromosome, which served as a genetic marker. It has been demonstrated through research that fetal hemoglobin is not a particularly useful marker. This is not only due to the fact that it is found in maternal cells, but also due to the fact that it is an intracellular marker. Since it is an intracellular marker, the procedures for enrichment and identification involve cell fixation, which hinders the subsequent expansion of the fetal cells in  vitro. The identification of fetal cells based on the Y chromosome through the use of cytogenetic (fluorescence in situ hybridization—FISH) and biomolecular (polymerase chain reaction—PCR) techniques is an efficient method, but it is only applicable to women who are carrying male fetuses [76]. At the end of the 1990s and at the beginning of the 2000s, efforts were made to collect a sufficient number of these fetal cells for use in prenatal diagnosis. However, this presented a big challenge because there are few fetal cells available, and there is no unique marker that can be used to distinguish fetal cell types in blood samples that are uncontaminated by maternal blood cells. Because these cells can be encouraged to divide and proliferate in vitro to create fetal colonies, isolating hematopoietic stem cells of fetal origin in the maternal blood was seen as an alternate method. Using a combined methodological approach, Tilesi et al. [77] were able to enrich and propagate in vitro fetal CD34+ stem progenitor cells. CD34+ cells were obtained and enriched from pregnant women in the early second trimester of their pregnancies using a magnetic cell-sorting technique. These cells were then compared to CD34+ cells recovered from the blood of nonpregnant women. According to the findings of the study, fetal progenitor cells could be successfully cultivated and detected using an appropriate integrated methodological approach. Then Coata et al. [78] described a technique for the enrichment and analysis of fetal CD34+ stem cells following culture in order to assess whether or not it is possible to use the technique for prenatal diagnosis. Diagnosis of genetic abnormalities using fetal CD34+ stem cells in maternal circulation was investigated along with evidence that these cells do not affect diagnosis in subsequent pregnancies. Because the existence of fetal microchimerism does not impact fetal diagnosis in ongoing

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pregnancies, noninvasive prenatal testing of fetal aneuploidies can make use of fetal hematopoietic CD34+ cells. This is because fetal aneuploidies are genetic conditions that impair cell division [79]. Despite the encouraging results obtained by various groups on the isolation of fetal cells from maternal blood, the rapid introduction of free fetal DNA-based techniques for NIPT shortly determined the abandonment by many investigators of this area of research. Only recently some investigators, based also on the advances of biotechnologies, have reconsidered to pursue research in isolating fetal cells (mainly trophoblast cells) from maternal blood for prenatal diagnostic purposes [80].

2.7 Concluding Remarks The primary predictor for any screening test or NIPT for fetal aneuploidy is the advancing age of the mother, history of chromosomal alterations in the family, ultrasound results showing fetal abnormalities, or a previous child with a chromosomal aberration. Advancing technologies and studies in prenatal screening have given varying approaches for the reassurance of a fetus without aneuploidy to expectant parents. Due to practical implementation and technical issues, NIPTs are still considered screening procedures, not diagnostic tools. Growing accuracy of these NIPTs and their diffusion in assessing the monogenic disorders can help in their implementation. Proper guidance and educational support are necessary to ensure providers as trained and are of prime importance for the evolving prenatal field. Issues like the advantages and disadvantages of varying screening techniques and understanding the analyzing population and abnormal test results are of prime importance. Advances in the isolation of fetal cells from maternal blood and the analysis of the fetal genome at the single-cell level are also seen as potential next steps in the future of NIPT. With progress in areas of cell fixation, whole genome amplification and single-cell sequencing various areas that were confined to the research realm are making their way into clinical diagnostics in the not too distant future. With the improvement of NIPT technology, and thus the identification of a higher resolution of fetal genomic abnormalities, the complexity of counselling will also increase. Above all, patient education and counselling will remain as a mandatory component of formulating guidelines worldwide and demand the increased need for and accessibility of genetic counsellors and fetal medicine specialists. So-called “whole genome” or “genome-wide” cfDNA analyses claim to look beyond specific microdeletions to CNVs throughout the genome, including deletions and duplications, as well as rare autosomal trisomies (RATs) involving chromosomes other than 21, 18, or 13. The majority of these conditions are exceedingly rare to the point where the prevalence has not even been defined. It is extremely difficult to make the argument that these conditions are a significant enough health problem to warrant screening. Despite the publication of proof of concept and analytical validation studies regarding cfDNA testing for panels of microdeletions and genome-wide cfDNA analysis, the clinical performance of these tests has not been

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described. Due to the rarity of individual CNVs and RATs, determination of sensitivity and specificity in a prospective, blinded validation study would require enrolment of a prohibitively large number of patients. There have been retrospective reports of laboratory experience, but these do not report outcome data for all subjects, precluding determination of the detection rate (sensitivity) of the test and providing specificity that may be biased by ascertainment With the expansion of cfDNA test menus, the clinical relevance of included conditions and validation data decreases and the false positive rates increase, undermining the main achievement of NIPT: the significant reduction of the invasive testing rate. Counselling becomes more challenging with an increase in the complexity of testing options and the potential of identifying conditions with an uncertain prognosis. There is also the potential to identify previously undiagnosed genetic changes in the pregnant woman. It is already felt that there is limited time and resources to adequately counsel patients about their options for prenatal screening. Broadening the scope of cfDNA testing may undermine productive decision-making. Another aspect that deserves attention is the psychological burden that unexpected findings pose to women that chose cfDNA to avoid an invasive procedure, but may end-up in undergoing having it for conditions of uncertain significance that may cast a shadow on their emotional experience around the pregnancy. The test based on cff-DNA is superior and more accurate than any other prenatal screening methods and will quickly spread in high income countries and low to middle-income countries for the forthcoming years. Although the extremely low false positive rate is a distinct and important advantage, it still remains a screening test and not a diagnostic one. The widespread introduction of NIPT is changing the scenario and the consequences of prenatal screening and diagnosis. Genetic counselling should accompany the application of the test. Nations should implement regulations and oversight to ensure that NIPT fits into existing legal frameworks. All stakeholders should have a voice in crafting policies to ensure the ethical and equitable use of NIPT across the world. Acknowledgements  The authors would like to thank Dr. Thomas Musci, MD, San Francisco, USA, for useful comments on the manuscript.

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4. Valderramos SG, Rao RR, Scibetta EW, Silverman NS, Han CS, Platt LD.  Cell-free DNA screening in clinical practice: abnormal autosomal aneuploidy and microdeletion results. Am J Obstet Gynaecol. 2016;215(5):626.e1–626.e10. 5. BIS Research. Global noninvasive prenatal testing (NIPT) market: focus on tests, methods, platforms, applications, 26 countries’ analysis, patent landscape, and competitive landscape— analysis and forecast, 2018–2028. New York: Globe Newswire; 2019. 6. Bowman-Smart H, Savulescu J, Mand C, Gyngell C, Pertile MD, Lewis S, Delatycki MB. Is it better not to know certain things? Views of women who have undergone noninvasive prenatal testing on its possible future applications. J Med Ethics. 2019;45(4):231–8. 7. Prochownick L. Beiträge zur Leptre vom Fruchtwasser und seiner Entsehung, Arch. Gynaekol. 1877;11:304. 8. Schatz F. Eine besondere Art von ein seitiger Polyhydramnie mit anderseitiger Oligohydramnie bei einaguen Zwillingen, Arch. Gynaekol. 1882;19:329. 9. Menees TO, Millar JD, Holly LE.  Amniography. Preliminary report. Am J Roentgenol. 1930;24:353–66. 10. Fuchs F, Riis P. Antenatal sex determination. Nature (London). 1956;177:330. 11. Liley AW. Liquor amnii analysis in management of pregnancy complicated by rhesus sensitization. Am J Obstet Gynecol. 1961;82:1359–70. 12. Milunsky A, Littlefield JW. The prenatal diagnosis of inborn errors of metabolism. Am Rev Med. 1972;23:57. 13. Hino M, Koki Y, Nishi S.  Nimpu ketsu naka no alphafetoprotein. Igaku No Ayumi. 1972;82:512–3. 14. Mohr J. Foetal genetic diagnosis: development of techniques for early sampling of foetal cells. Acta Pathol Microbiol Scand. 1968;73:7377. 15. Department of Obstetrics and Gynaecology, Tietung Hospital. Fetal sex prediction by sex chromatin of chorionic villi cells during early pregnancy. Chin Med J. 1975;1:117–26. 16. Westin B. Hysteroscopy in early pregnancy. Lancet. 1954;11:872. 17. Scrimgeour JB. Other techniques for antenatal diagnosis. In: Emery AEH, editor. Antenatal diagnosis of genetic disease. Edinburgh: Churchill Livingstone; 1973. p. 49. 18. Valenti C.  Endoamnioscopy and fetal biopsy: a new technique. Am J Obstet Gynecol. 1972;114:561. 19. Valenti C. Antenatal detection of hemoglobinopathies. Am J Obstet Gynecol. 1973;115:851. 20. Steele MW, Breg WR Jr. Chromosome analysis of human amniotic fluid cells. Lancet. 1966;1:383. 21. Nadler HL, Gerbie AB. Role of amniocentesis in the intra-uterine diagnosis of genetic defects. N Engl J Med. 1970;282:596. 22. Kelley K.  Amniocentesis prior to 1980. In: Embryo project encyclopedia. Tempe: Arizona State University; 2010. http://embryo.asu.edu/handle/10776/2072. 23. Hobbins JC, Mahoney MJ.  Fetoscopy in continuing pregnancies. Am J Obstet Gynecol. 1977;129:440. 24. Patrick JE, Perry TB, Kinch FAH.  Fetoscopy and fetal blood sampling: a percutaneous approach. Am J Obstet Gynecol. 1974;119:539. 25. Alter BP, Modell CB, Fairweather D, Hobbins JC, Mahoney MJ, Frigoletto FD, Sherman AS, Nathan DG. Prenatal diagnosis of hemoglobinopathies: a review of 15 cases. N Engl J Med. 1976;295:1437. 26. Fairweather DVI, Ward RHT, Modell B. Obstetrics aspects of midtrimester fetal blood sampling by needling or fetoscopy. Br J Obstet Gynaecol. 1980;87:87. 27. Rodeck CH, Campbell S. Sampling pure fetal blood by fetoscopy in second trimester of pregnancy. Br Med J. 1978b;2:728. 28. Kazy Z, Stygar AM, Bakharev VA. Chorionic biopsy under immediate realtime (ultrasonic) control. Orv Hetil. 1980;121:2765.

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29. Simoni G, Brambati B, Danesino C, Rossella F, Terzoli GL, Ferrari M, Fraccaro M. Efficient direct chromosome analyses and enzyme determinations from chorionic villi samples in the first trimester of pregnancy. Hum Genet. 1983;63:349–57. https://doi.org/10.1007/BF00274761. 30. Hahnemann N. Early prenatal diagnosis: a study of biopsy techniques and cell culturing from extra-embryonic membrane. Clin Genet. 1974;6:294–306. 31. Smidt-Jensen S, Hahnemann N, Jensen PKA, et al. Experience with find needle biopsy in the first trimester—an alternative to amniocentesis. Clin Genet. 1984;26:272. 32. Daffos F, Cappella-Pavlovsky M, Forestier F.  Fetal blood sampling via the umbilical cord using a needle guided by ultrasound. Report of 66 cases. Prenat Diagn. 1983;3:271–7. 33. Nicolaides KH, Azar G, Byrne D, Mansur C, Marks K. Fetal nuchal translucency: ultrasound screening for chromosomal defects in first trimester of pregnancy. BMJ. 1992;304:867–9. https://doi.org/10.1136/bmj.304.6831.867. 34. Spencer K, Ong C, Skentou H, Liao AW, Nicolaides KH. Screening for trisomy 13 by fetal nuchal translucency and maternal serum free beta-hCG and PAPP-A at 10–14 weeks of gestation. Prenat Diagn. 2000;20:411–6. 35. Spencer K, Liao AW, Skentou H, Cicero S, Nicolaides KH. Screening for triploidy by fetal nuchal translucency and maternal serum free beta-hCG and PAPP-A at 10–14 weeks of gestation. Prenat Diagn. 2000a;20:495–9. 36. Spencer K, Ong C, Skentou H, Liao AW, Nicolaides KH. Screening for trisomy 13 by fetal nuchal translucency and maternal serum free beta-hCG and PAPP-A at 10–14 weeks of gestation. Prenat Diagn. 2000b;20:411–6. 37. Kazakov VI, Bozhkov VM, Linde VA, Repina MA, Mikhaĭlov VM. Vnekletochnaia DNK v krovi beremennykh zhenshchin [Extracellular DNA in the blood of pregnant women]. Tsitologiia. 1995;37(3):232–6. 38. Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, et  al. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997;350:485–7. 39. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L.  Quake SR noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci U S A. 2008;105:16266–71. https://doi.org/10.1073/pnas.0808319105. 40. Chiu RWK, Allen Chan KC, Gao Y, Lau VYM, Zheng W, Leung TY, Foo CHF, Xie B, Tsui NB, Lun FM, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci U S A. 2008;105:20458–63. https://doi.org/10.1073/pnas.0810641105. 41. Bianchi DW, Chudova D, Sehnert AJ, Bhatt S, Murray K, Prosen TL, Garber JE, Wilkins-Haug L, Vora NL, Warsof S, et al. Noninvasive prenatal testing and incidental detection of occult maternal malignancies. JAMA. 2015;314:162–9. https://doi.org/10.1001/jama.2015.7120. 42. Singer A, Sagi-Dain L. Impact of a national genetic carrier-screening program for reproductive purposes. Acta Obstet Gynecol Scand. 2020;99:802–8. https://doi.org/10.1111/aogs.13858. 43. Coonen E, Van Montfoort A, Carvalho F, Kokkali G, Moutou C, Rubio C, De Rycke M, Goossens V. ESHRE PGT consortium data collection XVI–XVIII: cycles from 2013 to 2015. Hum Reprod Open. 2020;2020:hoaa043. https://doi.org/10.1093/hropen/hoaa043. 44. Kimelman D, Pavone ME.  Noninvasive prenatal testing in the context of IVF and PGT-­ A.  Best Pract Res Clin Obstet Gynaecol. 2020;70:51–62. https://doi.org/10.1016/j. bpobgyn.2020.07.004. 45. Cariati F, D’Argenio V, Tomaiuolo R.  Innovative technologies for diagnosis and screening of genetic diseases in antenatal age. J Lab Precis Med. 2020;5:6. https://doi.org/10.21037/ jlpm.2019.11.02. 46. Chitty LS, Lo YM. Noninvasive prenatal screening for genetic diseases using massively parallel sequencing of maternal plasma DNA. Cold Spring Harb Perspect Med. 2015;5:a023085. 47. Orlandi F, Damiani G, Hallahan TW, Krantz DA, Macri JN. First-trimester screening for fetal aneuploidy: biochemistry and nuchal translucency. Ultrasound in obstetrics & gynecology: the official journal of the International Society of Ultrasound in Obstetrics and Gynecology. 1997 10(6):381–6.

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48. Dey AC, Shahidullah M, Mannan MA, Noor MK, Saha L, Rahman SA. Maternal and neonatal serum zinc level and its relationship with neural tube defects. Journal of Health, Population, and Nutrition. 2010;28:343–50. 49. Wald NJ, Rodeck C, Hackshaw AK, Walters J, Chitty L and Mackinson AM. First and second trimester antenatal screening for Down’s syndrome: the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). J Med Screen 2003;10:56–104. 50. Wright D, Bradbury I, Benn P, Cuckle H, Ritchie K. Contingent screening for Down syndrome is an efficient alternative to non-disclosure sequential screening. Prenatal diagnosis, 2004;24(10):762–6. 51. Gil MM, Galeva S, Jani J, Konstantinidou L, Akolekar R, Plana MN, Nicolaides KH. Screening for trisomies by cfDNA testing of maternal blood in twin pregnancy: update of the Fetal Medicine Foundation results and meta-analysis. Ultrasound Obstet Gynecol. 2019;53:734–42. 52. Stokowski RP, Wang E, White K, Batey A, Jacobsson B, Brar H, Balanarasimha M, Hollemon D, Sparks A, Nicolaides K, et al. Clinical performance of noninvasive prenatal testing (NIPT) using targeted cell-free DNA analysis in maternal plasma with microarrays or next g­ eneration sequencing (NGS) is consistent across multiple controlled clinical studies. Prenat Diagn. 2015;35:1243–6. https://doi.org/10.1002/pd.4686. 53. Benn P, Cuckle H, Pergament E. Noninvasive prenatal testing for aneuploidy: current status and future prospects. Ultrasound Obstet Gynecol. 2013;42:15–33. https://doi.org/10.1002/ uog.12513. 54. Jang M, Kwon Y, Kim H, Kim H, Min BS, Park Y, Kim TI, Hong SP, Kim WK. Microsatellite instability test using peptide nucleic acid probe-mediated melting point analysis: a comparison study. BMC Cancer. 2018;18:1218. 55. Kim SY, Lee SM, Kim SM, et al. Novel method of real-time PCR-based screening for common fetal trisomies. BMC Med Genet. 2021;14:195. https://doi.org/10.1186/s12920-­021-­01039-­1. 56. Zednikova I, Chylikova B, Seda O, Korabecna M, Pazourkova E, Brestak M, Krkavcova M, Calda P, Horinek A. Genome-wide miRNA profiling in plasma of pregnant women with Down syndrome fetuses. Mol Biol Rep. 2020;47(6):4531–40. 57. Belova V, Plakhina D, Evfratov S, Tsukanov K, Khvorykh G, Rakitko A, Konoplyannikov A, Ilinsky V, Rebrikov D, Korostin D. NIPT technique based on the use of long chimeric DNA reads. Genes. 2020;11:590. https://doi.org/10.3390/genes11060590. 58. Liautard-Haag C, Durif G, VanGoethem C, et al. Noninvasive prenatal diagnosis of genetic diseases induced by triplet repeat expansion by linked read haplotyping and Bayesian approach. Sci Rep. 2022;12:11423. https://doi.org/10.1038/s41598-­022-­15307-­2. 59. Vora NL, Powell B, Brandt A, et al. Prenatal exome sequencing in anomalous fetuses: new opportunities and challenges. Genetics in medicine: official journal of the American College of Medical Genetics. 2017;19(11):1207–16. 60. Sarno L, Maruotti GM, Izzo A, Mazzaccara C, Carbone L, Esposito G, Di Cresce M, Saccone G, Sirico A, Genesio R, et al. First trimester ultrasound features of X-linked Opitz syndrome and early molecular diagnosis: case report and review of the literature. J Matern Fetal Neonatal Med. 2019;21:1–5. https://doi.org/10.1080/14767058.2019.1677594. 61. Chiu EKL, Hui WWI, Chiu RWK. cfDNA screening and diagnosis of monogenic disorders— where are we heading? Prenat Diagn. 2018;38:52–8. https://doi.org/10.1002/pd.5207. 62. Di Maggio F, Borrillo F, Cariati F, Tomaiuolo R, D’Argenio V. Glossary of molecular biology and clinical molecular biology. Part III: Molecular diagnostics. Biochim Clin. 2020;44:174–8. 63. Guissart C, Debant V, Desgeorges M, Bareil C, Raynal C, Toga C, Pritchard V, Koenig M, Claustres M, Vincent MC. Noninvasive prenatal diagnosis of monogenic disorders: an optimized protocol using MEMO qPCR with miniSTR as internal control. Clin Chem Lab Med. 2015;53:205–15. https://doi.org/10.1515/cclm-­2014-­0501. 64. Jenkins LA, Deans ZC, Lewis C, Allen S. Delivering an accredited noninvasive prenatal diagnosis service for monogenic disorders and recommendations for best practice. Prenat Diagn. 2018;38:44–51. https://doi.org/10.1002/pd.5197.

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65. Scotchman E, Shaw J, Paternoster B, Chandler N, Chitty LS. Noninvasive prenatal diagnosis and screening for monogenic disorders. Eur J Obstet Gynecol Reprod Biol. 2020;253:320–7. https://doi.org/10.1016/j.ejogrb.2020.08.001. 66. Wong FCK, Lo DYM. Prenatal diagnosis innovation: genome sequencing of maternal plasma. Annu Rev Med. 2016;67:419–32. https://doi.org/10.1146/annurev-­med-­091014-­115715. 67. Wapner RJ, Babiarz JE, Levy B, Stosic M, Zimmermann B, Sigurjonsson S, Wayham N, Ryan A, Banjevic M, Lacroute P, et al. Expanding the scope of noninvasive prenatal testing: detection of fetal microdeletion syndromes. Am J Obstet Gynecol. 2015;212(332):e1–9. https://doi. org/10.1016/j.ajog.2014.11.041. 68. Lench N, Barrett A, Fielding S, McKay F, Hill M, Jenkins L, White H, Chitty LS. The clinical implementation of non-invasive prenatal diagnosis for single-gene disorders: Challenges and progress made. Prenat Diagn 2013;33:555–62. 69. Cayrefourcq L, Vincent MC, Pierredon S, et  al. Single circulating fetal trophoblastic cells eligible for non-invasive prenatal diagnosis: the exception rather than the rule. Sci Rep. 2020;10:9861. 70. Deans ZC, Allen S, Jenkins L, Khawaja F, Gutowska Ding W, Patton SJ, Chitty LS, Hastings RJ.  Ensuring high standards for the delivery of NIPT worldwide: development of an international external quality assessment scheme. Prenat Diagn. 2019;39:379–87. https://doi. org/10.1002/pd.5438. 71. Kuhlmann Capek M, Chiossi G, Singh P, Monsivais L, Lozovyy V, Gallagher L, Kirsch N, Florence E, Petruzzi V, Chang J, et al. Effects of medication intake in early pregnancy on the fetal fraction of cell-free DNA testing. Prenat Diagn. 2019;39:361–8. https://doi.org/10.1002/ pd.5436. 72. Al Toukhi S, Chitayat D, Keunen J, Roifman M, Seaward G, Windrim R, Ryan G, Van Mieghem T. Impact of introduction of noninvasive prenatal testing on uptake of genetic testing in fetuses with central nervous system anomalies. Prenat Diagn. 2019;39:544–8. https://doi. org/10.1002/pd.5466. 73. Alberry M, Maddocks D, Jones M, AbdelHadi M, Abdel-Fattah S, Avent N, Soothill PW. Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast. Prenat Diagn. 2007;27:415–8. https://doi.org/10.1002/pd.1700. 74. Lenaerts L, Van Calsteren K, Che H, Vermeesch JR, Amant F. Pregnant women with confirmed neoplasms should not have noninvasive prenatal testing. Prenat Diagn. 2019;39:1162–5. https://doi.org/10.1002/pd.5544. 75. Shaw J, Scotchman E, Chandler N, Chitty LS. PREIMPLANTATION GENETIC TESTING: noninvasive prenatal testing for aneuploidy, copy-number variants and single-gene disorders. Reproduction. 2020;160(5):A1–A11. https://rep.bioscientifica.com/view/journals/rep/160/5/ REP-­19-­0591.xm 76. Di Renzo GC, Picchiassi E. Are we facing a revolution in non-invasive prenatal genetic diagnosis? J Matern Fetal Neonatal Med. 2006;19(4):195–8. 77. Tilesi F, Coata G, Pennacchi L, Lauro V, Tabilio A, Di Renzo GC. A new methodology of fetal stem cell isolation, purification, and expansion: preliminary results for noninvasive prenatal diagnosis. J Hematother Stem Cell Res. 2000;9(4):583–90. 78. Coata G, Tilesi F, Fizzotti M, Lauro V, Pennacchi L, Tabilio A, Di Renzo GC. Prenatal diagnosis of genetic abnormalities using fetal CD34+ stem cells in maternal circulation and evidence they do not affect diagnosis in later pregnancies. Stem Cells. 2001;19(6):534–42. 79. Coata G, Picchiassi E, Centra M, Fanetti A, Maulà V, Benedetto C, Di Renzo GC. Persistence of male hematopoietic CD34+ cells in the circulation of women does not affect prenatal diagnostic techniques. Am J Obstet Gynecol. 2009;200(5):528.e1–7. 80. Weymaere J, Vander Plaetsen AS, Van Den Branden Y, Pospisilova E, Tytgat O, Deforce D, Van Nieuwerburgh F.  Enrichment of circulating trophoblasts from maternal blood using filtration-­based Metacell technology. PLoS One. 2022;17(7):e0271226.

Part I

Clinical Genetics

Chapter 3

The Nexus Between Chromosomal Abnormalities and Single Gene Disorders Arun Meyyazhagan and Gian Carlo Di Renzo

Abbreviations aCGH Array comparative genomic hybridization ACOG American College of Obstetricians and Gynecologists AD Autosomal dominant disorders AR Autosomal recessive disorders ATP Adenosine triphosphate cfRNA Cell-free RNA CMA Chromosomal microarray analysis CNVs Copy number variations CVS Chorionic villi sampling DMD Duchenne muscular dystrophy DNA Deoxy ribo nucleic acid ES Exome sequencing mtDNA Mitochondrial DNA NBS Newborn infant screening NGS Next-generation sequencing NIPT Non-invasive prenatal screening PGT Preimplantation genetic testing PKU Phenylketonuria PND Prenatal determination

A. Meyyazhagan · G. C. Di Renzo (*) Department of Obstetrics and Gynecology and Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Umbria, Italy PREIS International and European School of Perinatal, Neonatal and Reproductive Medicine, Firenze, Italy e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_3

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Single-gene disorders Single nucleotide polymorphism Untranslating regions X-linked dominant X-linked recessive

3.1 Introduction About 3 × 109 nucleotides constitute the genome in humans and results in 50,000–100,000 genes on 46 chromosomes to produce proteins, which act as the blueprint of a human body [1]. The central region of the cell comprises stick-shaped forms called chromosomes hosting these genes in the nucleus. Every gene is designated with a specific function within the human body, and any abnormality at either genetic or chromosomal level can lead to jeopardized bodily function and ultimately to malaise condition. Hence, genetics play a primary role in predicting and identifying the risk in patients to either counsel them or guide them for future parenthood. Blooming genetics techniques and deeper knowledge allow physicians and clinicians to get better practical outputs by reducing the social burden in the world. Extra care by physicians will provide pragmatic heights in this field. Genetic advances play a significant role in clinical medicines due to big proportions of genetic disorders compromising the total world diseases in pediatric and adult populations. Each chromatid is obtained from each parent and consists of genes from both maternal and paternal sides, and these genes form the segment of deoxy ribo nucleic acid (DNA), which translates into the end product, proteins. The genes which code for proteins are known as genotypes. These genotypes result in different physical results in an organism known as phenotype. Sometimes the genes are not expressed for the specific phenotype, i.e. in recessive form. Usually, all the changes do not lead to pathological conditions; they are known as single nucleotide polymorphisms (SNP). In general, alteration in the DNA genes culminating in the production and functioning of abnormal protein is termed mutation. Similar mutations in the gene do not necessarily correlate with the physical findings, and it differs from individual to individual, which is collectively known as gene expression. The position of genes on chromosomes is referred to as allele, and while inheritance, one allele is transmitted to the fetus from each parent of each locus. If there is destruction or disruption on the DNA, the repair genes present on DNA are correct. Often mutations in these repairing genes result in higher chances of disease phenotypes. Chromosomes are the blueprint of every organism as they have the genes necessary for bodily function and known as an individual’s genetic map/programming code of functioning obtained from each parent. They were inheriting faulty genes from any parent resulting in a diseased condition, including developmental declination both physically and mentally. Sometimes the abnormality in the structure of genes and chromosomes can result from lifestyle habits and result in faulty phenotypes like cancers, irrespective of locations.

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Every gene is either coded or non-coded region for the translation of proteins with coding regions called exons comprising of the vital information necessary for the translation process to obtain the sequences of amino acids (proteins), and the noncoding region constitutes the introns and the 3′–5′ untranslating regions (UTRs) necessary for the splicing mechanism, transcripts expression for each tissue, and some other functions. Most frequent alterations are seen in the noncoding zone of the DNA, which usually do not translate into protein sequences and do not affect an individual pathologically. Contrarily, sometimes these alterations can be virulent and can lead to significant effects in a few rare population groups. According to the genetic structure, only about 1% of the human genome constitute the coding region and any variation, either deletion, insertion, missense, substitution, etc., in these parts reports for about 85% of the pathological phenotypes [2]. They are analyzing and spotting these variant genes for respective diseases aids in diagnosing the patient’s condition at the molecular level and can be done sometimes in prenatal stages to reduce the social, mental, and economic trauma in parents [3]. Conventionally, the defects in the genes like Mendelian disorders solemnly rely on analytical studies of chromosome linkages, mapping the autozygotic genes responsible for the disease phenotype and giving a detailed insight into the disease. Though conventional strategies help locate the responsible genes for rare diseases, it is filled with few shortcomings like not identifying the causative genes in case of disease caused by variations in multiple genes, any de novo gene alterations [4].

3.2 Genetic Disorders and Types Alterations in one small part or whole of the gene in chromosome DNA can lead to genetic conditions like defects and disorders due to abnormal protein structure or functioning due to deviation from the normal sequences observed in healthy individuals. The alterations can be minuscule, like change in a single nucleotide comprising the gene in the DNA to huge like addition or removal of the whole gene present in a chromosome. In general, alteration in one single gene is termed a monogenic disorder, as alterations in a group of genes/more than one gene are termed multifactional inherited disorders. Sometimes the combo of alteration in genes and environmental factors can also induce genetic disorders like conditions by changing the structure or number of the genes constituting the chromosomes. Deciphering the human genome secret, that is, the human’s whole genetic composition, has made it possible to unveil the genes responsible for almost all the known diseases in the world to date. Few pathological conditions are due to mutations present in parent’s genetic composition and are inherited to children from birth, like sickle cell disease. While other forms of diseases are acquired due to mutations in the individual’s genetic composition at any stage of a person’s life due to one’s lifestyle and the environment one resides in. These mutations are not passed

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from parents to offsprings and are not inheritable. These can randomly affect individual like cancers, few forms of neurofibromatosis. Below are the various kinds of genetic disorders seen in the family of patients by their physicians:

3.2.1 Chromosome Disorders Chromosomes are the structures present in each cell’s nucleus and comprise the DNA containing the nucleotides A, T, C, G to form the genes and encodes for the proteins. Humans contain 23 pairs of chromosome in each cell, and any change in their number, shape, or arrangement can lead to disorders. A frequency of about 0.2% is observed in the population, resulting in a chromosome change. Generally, the number of chromosomes is due to either producing too few or more chromosomes than usual due to mitotic nondisjunction causing disrupted assembling of the chromosomes somehow. 3.2.1.1 Alteration in the Number of Chromosomes Universally, every normal human contains 23 pairs, i.e. 46 chromosomes and if any mistake occurs in the cellular division process, especially at its chromatid formation phase turns out to lose an entire chromosome; as a result, it losses the genes on the chromosome and the corresponding protein and hence loss of phenotype. Some of the common functional loss is related to the growth of the bodily organs, size or developmental retardation based on mental and physical aspects and majorly jeopardies the body’s essential functions corresponding to the lost genes. Likewise, the change can occur while the gametes (the reproductive cells), namely the sperm in men and eggs in women, are formed in the developing fetus or after birth. This process leads to the addition/subtraction of chromosomes leading to an aneuploidy state (Fig. 3.1a, c). The most prevalent form of aneuploidy observed in the human population is trisomy, an extra chromosome in 23 pairs. The typical example for trisomy is Down’s syndrome caused in people with one extra chromosome in the 21st pair, making 47 chromosomes in total. The trisomy-prone person shows developmental and mental retardation with alterations in physical, especially facial, features due to the presence of a whole chromosome set in the third chromosome in the 21st pair. Another form of aneuploidy observed more often is monosomy, as the word indicates mono, meaning one (Fig. 3.1b). Hence, the loss of one chromosome from the pairs leads to the decreased count of chromosome in total. The highly prevalent monosomy conditions are Turner’s syndrome, explicitly affecting women who are more prone to this syndromic conditions due to loss of one x chromosome from the sex chromosome pair, resulting in short stature and underdeveloped secondary sexual characters with mental retardations. In total, the count of chromosomes is 45 in monosomy conditions [5].

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Fig. 3.1  Abnormalities in the chromosomal structure

3.2.1.2 Abnormalities in Chromosomal Structures Retaining the three prime features of chromosomes even after alteration could lead to a phylogenetic inheritance pattern while the cell is dividing. The progeny cells may or might not retain all the genes from the dividing cell’s chromosomes. The three prime features include the origin of replication, centromere, and the telomere region in the chromosome. When a chromosome loses a segment of DNA, it is correlated with the loss of genes in the segmented region and produces malformed structures causing abnormal shaping of the chromosomes due to these missing genes. We can observe small spaces or breaks in the DNA strand, making the chromosomic region disappearing at that particular location. Below are the possible reasons to alter the structure of chromosomes. Deletions Losing or deleting a fraction of DNA from a chromosome, as shown in Fig. 3.1a, is called a deletion. This can occur at any location of any chromosome with different dimensions. Depending on the deleted genes, the phenotypic characteristics are seen in the individual. In case of the genes associated with the development of the neural building and connections, formation is deleted. The person shows learning disabilities from a diminutive form like dyslexia to higher forms like cri du chat with other motor deficits and likely to have some other health problems associated with the heart, lungs, eyes, kidneys, or liver. The pathological seriousness is linked with the size of the deleted segment comprising of the genes necessary for bodily function.

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Duplications In some cases, the whole chromosome replicates itself and leads to a considerable quantity of chromosome materials, i.e. the genes shown in Fig. 3.1b. The addition of a significant number of genes through this copied chromosome could lead to hyper-instruction in the body and leads to confusion in the bodily functions due to rapid and higher gene expression. These alterations can induce developmental and learning disorders along with other physiological conditions. Conditions like Pallister–Killian syndrome occur due to chromosome set in excess at 12 chromosome region due to duplication. The person shows extreme intellectual disability, irregular muscle tone, prominent forehead and coarse facial features, stiffness in joints, seizures, seizures, cataracts, hearing loss, etc. Inversions The chromosomal part changes its orientation resulting in a change in the nucleotide sequence on the gene in the chromosomal region (Fig.  3.1c). Generally, the orientation is upside down or in the opposite direction than the usual (Inverted). This structural abnormality on the chromosomes can be passed along the generations with 50–50 plausibility of inducing or noninducing the congenital disabilities. The majority of inversion cases show normal bodily function, but in few cases, congenital anomalies, growth decline, cancers, Walker–Warburg syndrome, or acute leukaemia like conditions can be observed in inversion cases, especially on chromosome 9. Translocations When a chromosome is shifted from one locus to another locus in the sister chromatid region, it is termed translocation. Sometimes, chromosomal rearrangement can occur within the same chromosome or from one chromosome to another (Fig. 3.1d). There are two types of translocation process: Balanced Translocation  The condition where the exchange of the segment of DNA is equal between the chromosome with no loss or gain of the segments of DNA (genes). This is known as balanced and healthy translocation, but the parent with this type of alteration might risk dangling chromosomes to their progeny and lead to developmental problems. The best example for translocation condition includes Down’s syndrome. Robertsonian Translocation  Universally observed chromosomal reorganization involves mainly two acrocentric chromosomes. These chromosomes unite and consist of a single centromere and can increase the risk of Patau syndrome and Downs by creating genetic imbalance.

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Unbalanced Translocation  Normal and balanced translocated chromosomes in either parent can increase the chances of progeny consisting of an extra chromosome loss or presence, resulting in de novo translocation in children with parents containing normal chromosome. Developmental and intellectual disabilities are observed in the child. Rings The chromosomal ends break and rejoin, forming a ring-like shape in few conditions, as seen in Fig. 3.1e. Usually, it results from the deletion of the chromatid ends in the same chromosome, making the ends sticky and fusing with the remaining part forming a circular structure. The effect of the circular-shaped chromosome relies on the deleted portion of the chromosome, based on the loss of the genetic sequences. They can occur on any chromosome, and the phenotypes are varying from individual to individual. In general, growth deficiency with some malformations is observed in patients with ring chromosomes

3.2.2 Mendelian Disorders Also known as single-gene disorders (SGD), defects arise due to alterations in the alleles in one locus or one single gene. These defective genes are carried from one generation to another in either dominant form, recessive form of X-linked in either dominant or recessive way with a frequency of about 0.35% in the world population. 3.2.2.1 Autosomal Dominant Autosomes are the somatic/body chromosomes involved in inducing the genetic effect. Generally, the 22 pairs of chromosomes are inherited from each one while forming a zygote and record almost all the genes in our body (Fig. 3.2). The dominance pattern inheritance conveys that a single defective gene is sufficient to comprehend the pathology in a person. Dominance is only dependent on the function of the inherited gene products. One mutant copy results in autosomal dominant disorders (AD), and through ontological studies using pedigree analysis tool, it is observed that one parent is affected with the disorder, which is transmitted to the progeny [6]. With a probability of 50% of inheritance of the mutated allele in AD pattern, the penetrance is declined, which means inheritance of the mutated allele in all the progeny to develop the disorder is very limited. Disorders like Von Willebrand disease, multiple hereditary exostoses (a highly penetrant autosomal dominant disorder), Marfan syndrome, Huntington’s disease [6], hereditary nonpolyposis colorectal cancer, acute intermittent porphyria, tuberous sclerosis, neurofibromatosis type 2, and neurofibromatosis type 1 are inherited in AD pattern.

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Fig. 3.2  Pictorial representation of autosomal dominant and recessive inheritance

3.2.2.2 Autosomal Recessive Autosomes exhibit this pattern of inheritance, and to cause the disease phenotype, the affected individual must possess both the mutated gene alleles. The presence of defective alleles in both the parents is necessary, and these are transmitted to the next generation to cause the malaise (Fig. 3.2). Hence, autosomal recessive (AR) inheritance patterns occur when both the parents have at least one autosome with a defective allele to pass it to the successive generation. The individual with a single faulty allele is known as the “carrier” as they do not have the disease, and these carriers have a higher risk of passing the disease to their progeny. In conclusion, for a child to have the disease, both the parents must be a carrier or affected. Mostly the AR disorders are due to the presence of non-function genes involved in the inheritance pattern resulting in the production of truncated or non-­translational products (proteins) and switch off the genetic expression. In some cases, a single copy of the working allele counterbalances the production of proteins to maintain the body’s normal functioning without inducing the effect of the disease. Few AR disorders include sickle cell disease, spinal muscular atrophy, phenylketonuria (PKU), Tay–Sachs disease, Niemann–Pick disease, thalassaemia, albinism, medium-­ chain acyl-­ CoA dehydrogenase deficiency, cystic fibrosis [7–10], and Roberts syndrome. In some cases, wet and dry earwax is seen as a phenotypical characteristic [11].

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3.2.2.3 X-Linked Disorders The presence of abnormal genes in one of the sex chromosomes, i.e. X, is known as X-linked disorders. These disorders can be either X-linked dominant (XLD) or recessive (XLR). A higher rate of XLR disorders is seen in the human population with a greater significance rate clinically, especially in Fragile X syndrome families. XLR disorders are highly prevalent in males, and females, in general, are protected from the XLR disorders in case of having one perfectly functioning X allele (as they have 2 copies of the X allele). The functional X allele compensates for the dysfunction allele, whereas males do not possess different allele and hence affected, and they are the ones who can act as carrier/effectors to transmit the defective X allele to their daughters in a successive generation. This leads to having the female progeny as carriers of the XLR disorders traits, whereas males without any effects. Females with one faulty X chromosome will not give any trait to her, but she will be the carrier for transmitting the defective allele to her children. Usually, the affected males have carrier mothers, and the carrier mother will also have half of the daughter progeny as a carrier. The following are a few XLR disorders/pedigrees: 1. Colour blindness 2. Duchenne muscular dystrophy (DMD) 3. Fabry’s disease 4. Glucose-6-phosphate dehydrogenase deficiency 5. Hemophilia A&B 6. Hypohidrotic ectodermal dysplasia 7. Lesch–Nyhan disease 8. Mucopolysaccharidosis

3.2.3 Multifactorial Disorders Mutations in several genes lead to pathological condition and are often due to epigenetic causes. The interactions between the various environmental factors and the bodily genes are interlinked, and any minuscule change in any of these conditions results in a pool of pathologies. However, the interaction between them is to be analysed to its fullest to get a deeper insight into the pathological conditions. The disorders include glycaemia, cancer, diabetes mellitus, obesity, and other health conditions occurring in the patient’s life. The risk assessment is done empirically with an estimation of 5% frequency in the general population. This is also known as a polygenic, complex disorder associated with varied genes, lifestyle, and environmental factors. These disorders often run in the family and do not follow inheritance patterns. Being challenging to study and treat

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complex disorders, they are still in an expedition state. Worldwide, researchers are continuously engaged in unravelling the causative genetic factors for these highly prevalent complex disorders.

3.2.4 Somatic Genetic Disorders Any alterations in the somatic/body cells and which are not involved in the inheritance are known to induce somatic genetic disorders. Generally, malignancies are reported due to these mutations and lay a cause for genetic predisposition for a disorder. This type of mutation is acquired by the cell when it is undergoing division and cannot occur in the germinal cells like the gametes inherited from parents to progeny. These mutations result from frequent exposure to harmful chemicals and radiation coming under environmental factors. It is possible to attain the mutation at any stage of the cellular division since the zygote formation like during the first cleavage or following stages till the individual is formed. All the descendants of the mutated cell pose the same mutation and can lead to the pathological condition. This mutation can lead to various other pathophysiological states like cancers. Most of the cancers are due to the aggregation of mutations in the somatic cells.

3.2.5 Mitochondrial Disorders Mutations in the genetic composition of the cell’s powerhouse in the mitochondria are inherited from only the mother’s side. These are biological organelles seen in all the cells of our body and are the site for producing energy to maintain all the biochemical reactions occurring in our body. The mutations occurring in the gene sequences in the DNA of mitochondria are limited to the organelle itself. Hence, these mutations in the mitochondria lead to insufficient or no production of adenosine triphosphate (ATP) required for essential bodily function and can affect any part or organ of the body. The symptoms of the disorders caused by this mutation depend on the organ or part of the body involved in the mutations. The mitochondrial DNA (mtDNA) genetics differ from nuclear DNA in a unique way [12] as only maternal DNA is involved in inheritance despite the entry of few sperm mitochondria in egg [13]. In general, mtDNA is transmitted from oocyte to their offspring and consecutively transferred from the daughter to successive generation [14, 15]. Usually, mitochondria are polyploidic cells, and every cell in human contains about hundreds of mitochondria; each mitochondrion possesses 2–10 DNA molecules. These DNA molecules are randomly moved to daughters. The genotype of mitochondria comprises single DNA and known as homoplasmy condition. A random mutation in the DNA of the mitochondria leads to a transitory condition called heteroplasmy, comprising both the wild type and mutant form expressed together in intracellular regions of the cells. Being polyploidic,

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mtDNA is stochastically spread in the daughter cells [16]. This process leads to extreme mutational loads seen in successive generations in families with mtDNA heteroplasmy and shows remarkable phenotypic variations in the mitochondrial disorders. The characters linked with mtDNA are based on DNA segregation’s threshold effectiveness and polyploidy [17]. Usually, crossing a certain threshold of mutated gene copies leads to deleterious effects without bothering the co-existing wild mtDNA in heteroplasmic mutations conditions. This leads to misfunctioning of the cells and leads to pathological phenotypes [18]. Impressive mutations in mitochondrial disorders are available on http://www.mitomap.org/ to give deeper insight into the genotypic and phenotypic characteristics in mitochondrial disorders (Fig. 3.3). Variations in the clinical phenotypes of mtDNA depend on many facts like the type of mutation to induce intrinsic malaise. It depends on the affected gene, load of mutation, and its distribution in the tissue and organs involved in the energy assimilation of the mutated region mitochondria. Usually, the central and parasympathetic nervous systems, the ocular and auditory zones, muscular system, circulatory and endocrine system, and kidney and liver are the hot spots for OXPHOS failure. Zeviani and Carelli [19] listed the 15-year study on mtDNA mutations to link the

Fig. 3.3  Mitochondrial genome

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cellular and molecular mechanism for specific manifestation observed, yet it is to be deciphered clearly. mtDNA mutations are classified as a rearrangement on a vast scale either with partial deletion or duplication of the whole segment, and sometimes the mutations restricted to one locus are known as an inherited point mutation. Both forms of mutations show characteristic clinical disorders and manifestation with large-scale mutations occurring in sporadic inheritance pattern and point mutation as maternal inheritance.

3.3 Difference Between Chromosomal Abnormality and Single Gene Defect Chromosomes are the forms seen in the central part of every cell called the nucleus and are composed of segments of DNA containing the nucleotides forming the genetic codons and produce proteins for the body function. The DNA comprises about 70,000 genes in every cell in the human body in normal condition except in the reproductive cells, the sperm, and the ovum as they contain only one chromosome from each pair, i.e. 23 chromosomes in them. X and Y are two types of sex chromosome and responsible for the gender of the zygote, XX as female and XY as males. Diseased conditions in an average person can induce changes in the segments of DNA in one or more chromosomes. If the defect is due to mutation limited to one nucleotide in the genetic codon, it is known as single gene defects and does not alter the structure or number of the chromosomes. Similarly, a person can have no defect in the genes, but might have an extra or lesser copy of chromosomes containing the genes, leading to chromosomal abnormalities. These chromosomal abnormalities are seen in both auto and allosomes in the human body and affect the chromosome’s number and structure. Massive abnormalities like aneuploidies can be easily viewed under the microscope while analyzing the chromosomes, also known as karyotyping. Minute abnormalities on chromosome require extensive and specialized tests to precisely scan the whole genome to locate the missing or extra bits in chromosomes. In some cases, the mutations are restricted to genes in specific locations with specific functions. These small regions cannot induce any external alterations in the chromosomes and are not detected while analyzing the chromosomes during karyotyping. This mutation sometimes gives no clinical manifestation, while few can give mild to severe clinical symptoms like cystic fibrosis, sickle cell anaemia, or muscular dystrophy. In medicine, now scientists are unravelling the genes linked to disorders, yet it is puzzling how most of the mutations are occurring. The mutations are expected to appear spontaneously either while getting exposed to various environmental factors to destroy or alter the genes, and they are termed as mutagens. Harmful radiations like ultraviolet, gamma, and a few drugs and chemicals are known as mutagens to induce defects during birth. If the mutations occur in the gametes, it is successively passed to the child from the parent, whereas

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mutations of other chromosomal genes are usually not transmitted to the progeny and induce serious effect; both the copies of abnormal genes are present in most of the cases.

3.4 Factors Affecting the Phenotypes of Single Gene Disease Phenotype The externally visible features seen as a result of gene expression are known as phenotype, and the genetic codon responsible for this feature is termed “genotype”. The external features mainly involve easily perceptional features like skin colour, eyes, its size, shape, height, weight, behaviour, temperament, health condition, and history of disease in the family. There is a strong link between genotype and phenotype, and it would be possible to predict phenotype based on genotype. Different phenotypes can be seen under the same genetic constitution. The environment we live in also shapes and affects our growth and health and deploying or employing the genes. Studies on genetically identical individuals have shown a light on the role of the environment in the modulation of gene and producing a different phenotype. The phenotypic variations seen in a genotype for a pathology depend on the person’s environment. In recent times the disease aetiology is linked with environmental factors, and they are seen as partners in crime in the disease world by directly interacting with each other. Hence, analyzing this direct link is challenging as well as beneficial to study human ailments. Analyzing the intricate mechanism linking the genotype and phenotype is the next priority to identify the disorders as it influences the DNA-based testing involving diagnosis, therapy, or counselling the proband. The postgenomic era baffles medical practitioners and researchers with the diverse phenotypical manifestations observed due to single gene alteration. With advances in medicine, we can attain tremendous knowledge in understanding the diseases at their genetic levels. With the ability to find the alterations to the level of DNA segments, we can delineate clinical phenotypes in most cases and fail occasionally.

3.4.1 Many Genes and Environmental Influences Affect Phenotypes The epigenetic factors are the environmental agents capable of inducing mutations in the cells; factors like diet, the concentration of oxygen, temperature, luminance, and other mutagens can alter the normal human genome and lead to the production of damaged protein-producing varied phenotypes in humans as it is known that genes carry the set of information throughout generations and help in the development of the body. The phenotype can be altered either at the embryonic stage or

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while growing when exposed to environmental agents capable of inducing mutations. The environmental factors vary from person to person due to different lifestyle and feeding habits, illness, stress levels, and regional climate. Altogether, environmental agents can produce the final phenotype an individual displays. It is always said as nature versus nurture, i.e. the genes vs environment plays a significant role in an individual’s life. In general, monozygotic/identical twins are studies to find a correlation between the genotype and phenotype. Genotype is a whole genetic identity that can be inherited. It is a unique genome that can be disclosed through genomic sequencing. It is mediated by a single gene, a cluster of genes, or a collection of genes carried by a person. Individual traits are determined by genes, which are DNA sequences that code for protein creation. All cellular operations, including mitosis, meiosis, DNA replication, protein synthesis, molecule transportation, and so on, are controlled by the genetic code contained in DNA.  Not all phenotypes, however, are caused by genotype. Most phenotypes are influenced by one’s genotype and the environment in which one has spent his or her life, incorporating all that has occurred. We frequently think of “nature” as one’s unique genotype and “nurture” as the environment in which one has grown. The phenotype of an organism is determined by the genetic composition and is changed by the environment in which it is exposed through numerous epigenetic processes. It encompasses all of the organism’s properties, including qualities at numerous levels of biological organization, such as morphology, physiology, cellular features, biochemical pathways, gene expression, and individual behaviour and trait evolution [20]. A different version of an organism’s feature, a phenotypic trait (simply trait or character state), is an evident and measurable property exhibited in an observable form. Eye colour (blue, brown, green, and hazel) is an example of a phenotypic trait with polygenetic inheritance. The phenotypic trait may be inherited from parents or determined by environmental variables; some are determined by genotype [20] and others by environmental variables. Mutations can cause distinct genes or alleles to be passed down through generations, resulting in distinct phenotypic features. Though the environment might influence the phenotype, the heritability of a phenotypic trait is defined as the fraction of total phenotypic variation revealed solely by genetic variation [21]. The number of genotypic variants, environmental factors, and potential phenotypes that can be combined is impossible to anticipate. The intricate interactions of several genetic loci with various environmental signals need to develop unique approaches for analyzing these scenarios, such as evaluating thousands of genes simultaneously under various environmental conditions. While these methods may never predict an exact phenotype, it is evident that we must include gene–environment interactions when studying biology and human disease. Because of the human genome sequence, the generation of widespread markers of genetic variation, and the development of new technologies that allow investigators to associate disease phenotypes with genetic loci, researchers have made significant progress in studying polygenic and other complex human diseases. Finally, our phenotypes are influenced by environmental circumstances throughout our lifetimes, and it is this ongoing interaction between genetics and the environment that distinguishes us all.

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3.5 Mendelian Disorder: Are Truly SGD? Single gene defects are also known as Mendelian disorders, where a mutation in a single gene leads to the manifestation of the disease phenotype. These are also known as monogenic disorders. A classic example of a single gene defect is Sickle cell anaemia caused by a single mutation in the beta-globin gene on the chromosome and mentioned the different types of SGD in Table 3.1 [22]. Mendelian disorders are monogenic, but they do not follow one gene-one trait relationship in most cases. The most common cause of heterogeneity within a gene is allelic heterogeneity—where multiple deleterious variants in the same gene cause the same disease phenotype. More than 1500 different pathogenic variants can cause, e.g. Haemophilia A, whereas, in the case of cystic fibrosis, more than 1900 mutations are known. However, about 90% of mutations in cystic fibrosis patients of Northern European descent [23, 24]. Mendelian diseases are rare, but collectively, they are a significant cause of morbidity and mortality. To date, more than 3700 genes causing SGD have been identified, but many diseases remain to be characterized. An online catalogue of human genes and genetic disorders (Online Mendelian Inheritance in Man: OMIM) indexed nearly 4971 phenotypes, out of which only about 3379 SGD have been characterized [25, 26]. Mendel’s experiments are well-known. The anatomical structure of DNA carries the chemical information that allows the exact transmission of genetic information from one cell to its daughter cell and from one generation to the next. Some alterations in the DNA modify its gene function, and error Table 3.1  Single gene disorders with their inheritance pattern S. no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

12. 13.

Single gene disorders Stickler syndrome Marshall syndrome Weissenbacher–Zweymuller syndrome Otospondylomegaepiphyseal dysplasia Congenital myotonic dystrophy Spondyloepiphyseal dysplasia, congenita Treacher Collins syndrome Van der Woude syndrome Cerebrocostomandibular syndrome Toriello–Carey syndrome (corpus callosum, agenesis of, with facial anomalies and Robin sequence) Carey–Fineman–Ziter syndrome (myopathy, congenital nonprogressive with Moebius and Robin sequences) Arthrogryposis multiplex congenita with whistling face Otopalatodigital syndrome type I

14. 15.

Catel–Manzke syndrome Osteopathia striata with cranial sclerosis

11.

Inheritance AD AD AD AD AD AD AD AD AD AR AR AR X-linked dominant X-linked X-linked dominant

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may occur, which is expressed phenotypically. Let us talk more about DNA and genetic information. The narrow hereditary bridge between the parents and the offsprings is now well understood in terms of molecular information, where code has now been revealed to the inquiring mind. Identifying the gene responsible for these diseases provides support in molecular diagnosis of patients, testing mutation carriers in families, and prenatal testing [3]. Conventional strategies for identifying the causative gene responsible for Mendelian disease rely on chromosomal analysis, linkage analysis, autozygosity mapping, and other association studies. Even after a successful history of identifying candidate gene for rare disorders, the conventional approaches have many shortcomings in situations such as multiple genetic variants, de novo variants, and sporadic disorders with very few available cases, which make these strategies not suitable for all studies [4]. The experiment is to be performed to understand and study the development process and expression of character breeding. All breeding experiments are performed by looking at naturally existing genetic differences in a species. Mendel mainly carries breeding experiments to find the structure carried from one generation to another and later termed DNA. Alterations in these DNA modify the function and phenotypes. Some SGD such as achondroplasia and DMD show slight variation in severity of the phenotype, even in unrelated individuals and can be considered to show true single-gene inheritance. However, disorders with stable mutations showing variable expressivity suggest the possible effects of modifying genes. There are also conditions in which only the susceptibility to the trait is inherited as a single-gene disorder. Examples include autosomal dominant familial cancers such as early-onset breast cancer and early onset colon cancer. Cancers are caused by a sequence of genetic changes (triggered by environmental factors) occurring in a clone of somatic cells in the affected tissue. Over time, these somatically inherited mutations, some of which may need to become homozygous, lead to uncontrolled cellular proliferation in the clone. The first key step in familial cancers is inherited through the germline, often as an autosomal dominant. This results in the whole sequence being completed more quickly, giving rise to an earlier onset of familial cancers than sporadic cancers. Some individuals who have inherited the susceptibility mutation may not be exposed to the factors that cause the complete subsequent sequence of somatic mutations and may never develop cancer and be non-penetrant for the trait. These healthy individuals can pass their susceptibility mutation to their offspring, who may not appreciate their own risk of developing cancer. Finally, mutations in some SGD may not be in a single-gene at all. In myotonic dystrophy, which is transmitted as an autosomal dominant single-gene disorder, some single-gene illnesses, such as achondroplasia and DMD, have little variation in phenotypic severity unrelated individuals and can be regarded as a true single-gene inheritance. Disorders with persistent mutations and variable expressivity, on the other hand, point to the possibility of changing genes. There are other situations where only the trait’s susceptibility is inherited as a single-gene disease. Autosomal dominant familial malignancies, such as early-onset breast cancer and early-onset colon cancer, are examples.

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Cancer is caused by a series of genetic alterations in a clone of somatic cells in the afflicted tissue. These somatically inherited mutations, some of which may need to become homozygous, cause the clone to proliferate uncontrollably over time. The first critical stage in familial malignancies is inherited through the germline, generally an autosomal dominant trait. As a result, the entire process is completed faster, resulting in a younger age of onset in family malignancies than sporadic malignancies. Some people who have inherited the susceptibility mutation may never get cancer and so be non-penetrant for the trait because they are not exposed to the stimuli that produce the whole sequence of somatic mutations. These healthy people can pass on their cancer susceptibility mutation to their children, who may be unaware of their own cancer risk. Finally, some SGD may not be caused by changes in a single gene. Myotonic dystrophy is an autosomal dominant single-gene condition that affects the muscles [27].

3.6 Assessment for SGD The hereditary premise is undoubtedly known for some single-gene issues, and the infection is causing gene variations related to genetic testing. Individuals are tried for two fundamental reasons: to see whether they have a specific hereditary issue or see whether they are carriers.

3.6.1 Making a Diagnosis A specialist may arrange to test for a particular issue or set of problems dependent on an individual’s side effects or attributes. If manifestations will, in general, show up further down the road, an individual may wish to be tried when they are more youthful, particularly if they realize the issue runs in their family, and if there is something they can do to forestall or postpone coming about clinical issues. Numerous hereditary problems are distinguished in infants through infant genetic screening, even before manifestations show up. Large numbers of infant screening tests take a gander at synthetics in the blood that indicate an issue. A positive screening result is typically trailed by genetic testing on a DNA test.

3.6.2 Identifying Carrier Status Genetic testing can likewise uncover whether an individual is a carrier of a hereditary problem. A carrier does not have the actual problem, yet they have an expanded danger of having a youngster with the issue. Progressively usually, even individuals with no known family background of a hereditary issue learn of their carrier status through genetic testing before they attempt to have youngsters. A few specialists

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and hereditary guides suggest explicit hereditary tests dependent on their patients’ ethnic foundations. Notwithstanding, for some reasons, they are getting bound to suggest comprehensive transporter screening, a solitary test that takes a gander at many qualities. Also, direct-to-purchaser hereditary testing units give some data about carrier status for probably the most well-known reasons for hereditary problems (Table 3.2). Carriers of a hereditary issue who wish to have a kid can utilize preimplantation hereditary testing to extraordinarily build their odds of having a sound child. Truly, evaluating single quality issues started in 1963 with the newborn infant screening (NBS) for PKU. Infant screening is the most significant populace-based hereditary screening program in the USA [28]. Advances in molecular technology have made both infant screenings for babies influenced with severe disorders and carrier screening of people in danger of posterity with more perplexing and generally accessible hereditary issues. “Conventional” carrier screening depends on the family background of a particular hereditary issue or evaluating for messes with expanded carrier recurrence dependent on ethnic/racial foundation. Because of the more delicate methodologies used for NBS, carrier status may likewise be distinguished. Innate oddities happen in 2–4% of all infants and cause 20.4% perinatal passing [28]. A few methods have been created and utilized over Table 3.2  Different types of assessment in genetic disorders Assessment Nuchal translucency/free beta/PAPP AFP/free Beta screen Triple screen

Gestation weeks 10–13.5

13–22

15–20

Quadruple screen 15–20

Amniocentesis

14– 16 weeks

Chorionic villus sampling Percutaneous umbilical blood sampling

8 weeks 18 weeks

Detection rate Description 91% A screen uses ultrasound to measure nuchal Translucency in addition to the free beta hCG and pregnancy-associated plasma protein A 80% This test measures the alpha fetoprotein produced by the fetus, and free beta hCG, produced by the placenta 75% This test measures the maternal serum alpha-­ fetoprotein, human chorionic gonadotropin, and unconjugated estriol 79% This test measures the maternal serum alpha feto protein, estriol, human chorionic gonadotropin, and high inhibin-alpha 80 This test measures the maternal sample of amniotic fluid that has been withdrawn from the mother’s uterus by a needle. The cells are then cultured, and chromosomal, biochemical, or DNA analyses are conducted, depending on the condition for which the procedure is being performed 85 CVS can be performed transcervically or transabdominally 80 The diagnosis of toxoplasmosis, the procedure allows access to the fetal circulation for evaluation of hematologic abnormalities and diagnosis of some inborn errors of metabolism

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the long run to identify chromosomal changes or single-gene variations to advance the regenerative result. Because of the advances in sub-molecular biology techniques as mentioned in Fig. 3.4, the number and nature of tests accessible for antenatal ID of congenital infections significantly expanded; the choices now accessible reach from non-invasive prenatal screening (NIPT) to the targeted assessment of at-risk couples [29]. Likewise, the hereditary carrier screening, a choice preceding origination, permits each accomplice to acquire data about their genotype corresponding to autosomal recessive, dominant, or X-linked diseases; in this way, the purpose of genetic carrier screening is to design following conceptive decisions (i.e., conventional prenatal determination (PND), egg or sperm donation or adoption, preimplantation hereditary finding). Antenatal hereditary tests expect to give data on the genotype of the embryo [preimplantation genetic testing (PGT)] or fetus (customary PND, NIPT). These tests are willful and should just be attempted after genetic counselling, guiding the test’s idea, potential outcomes, and alternatives available [29]. Antenatal analytic systems are set off by parent’s risk factors or potentially congenital infections recently distinguished in the family and ultrasound modifications perceived in the fetus. The result of preimplantation or pre-birth hereditary finding is educated conceptive dynamic beginning from the examination of family ancestry, ethnic beginning, past obstetric history, and guardians’ transporter status, up to molecular

Fig. 3.4  Assessment of single-gene disorders by using next generation sequencing

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biology investigation on an early stage of fetal biological example [30]. The most well-known genetic conditions for which antenatal hereditary tests are required incorporate cystic fibrosis, thalassemia, DMD/Becker muscular dystrophy, and Delicate X disorder: these tests permit to distinguish the genetic changes described in their parents. Subsequently, a shortfall of those particular hereditary modifications does not bar the likelihood that the child may have others [30].

3.6.3 Antenatal Genetic Diagnosis Antenatal hereditary diagnosis is characterized as a cycle that detects or distinguishes early-stage and fetal infections. Furthermore, results give parents and specialists data focused on improving pregnancy results and the prosperity of the family [30]. Since the end of pregnancy was a choice if there should be an adverse result of the pre-birth hereditary conclusion, numerous endeavours have been made to get a finding quickly as time permits. Without a doubt, preimplantation hereditary analysis conventions have been created as an option in contrast to traditional prenatal hereditary determination [30].

3.6.4 Genetic Counselling and Family History Partner/couples recognized as being at risk of transmitting an inherited condition based on a three-generation pedigree study, ethnic origin, personal symptoms, or obstetrical/past medical history should get genetic counselling, ideally before pregnancy (intellectual disability, muscular dystrophy, or bleeding disorders). It is necessary to offer information on carrier screening and prenatal/preimplantation genetic diagnosis. Information about ethnicity may aid in the selection of appropriate screening studies. When a family history raises the possibility of a specific hereditary condition, a thorough investigation with genetic counselling is required. As part of the informed consent procedure, direct gene mutation or expanded next-­ generation sequencing (NGS) testing should be mentioned.

3.6.5 Single Gene Abnormalities Are Screened for In Utero Many patients are unaware that genetic screening for SGD is available before their first prenatal appointment and may confuse it with aneuploidy screening. If a patient and their spouse are both carriers of the same illness, a prenatal diagnosis to test the fetus for the illness may be offered. Rapid testing of the infant with cord blood

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testing is another option. If a couple is at risk for a problem tested on the newborn screening panel, they may choose to screen for the disease at birth with a cord blood sample rather than waiting several weeks for the newborn screening test results. In this situation, it is critical to think about early screening and testing so that all reproductive alternatives are available. The risks, advantages, and limitations of chorionic villi sampling (CVS) and amniocentesis should be reviewed as part of standard counselling. When administered by a skilled physician, the American College of Obstetricians and Gynecologists (ACOG) practice guide Prenatal Diagnostic Testing for Genetic Disorders claims a loss rate of 0.1–0.3% [31]. Direct mutation identification for diagnostic testing, neonatal and carrier screening, prenatal diagnosis, and pharmacogenomics is now possible because of recent advances in molecular technologies.

3.6.6 SGD Diagnosis Variants in a single gene are known to produce single-gene diseases. The Online Mendelian Inheritance in Man website (OMIM, http://omim.org/) has a human genes and disorders database. Single gene illnesses include cystic fibrosis, sickle cell anaemia, Fragile X syndrome, spinal muscular dystrophy, and alpha-1-­ antitrypsin deficiency in humans. These disorders have autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, and mitochondrial inheritance patterns. Contiguous gene deletions/duplications or copy number variations (CNVs) in the chromosome. These studies include karyotyping, gene mutation testing, gene panel sequencing, clinical exome, whole-exome, and whole-genome sequencing. The complete genome is routinely screened for copy number changes using chromosomal microarray analysis (CMA) tools such as array comparative genomic hybridization (aCGH) and SNP arrays. The American College of Medical Genetics recommended CMA as a first-line diagnostic test for patients with developmental delays and intellectual disabilities in 2010. Single gene diseases identifiable by molecular analysis allow an ever-increasing number of genetic diseases to be identified in the prenatal period. The diagnostic technique is based on either direct mutation identification or linkage analysis; in either case, genotypic characterization of the parents and the family index case is required. A detailed genetic consultation must precede the molecular diagnosis to alert the couple about the consequences of possible results, as well as difficulties and requirements [30, 32]. There are two diagnostic alternatives for couples at risk of passing on a hereditary illness to their children: PGT or PND. These two diagnostic procedures have the same diagnostic goal, but they differ in diagnostic duration, sample method, and laboratory operations. The most modern PND procedures for detecting chromosomal changes during pregnancy are based on echography combined with karyotype and microarray [33]. Karyotyping allows for the analysis of

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all 23 pairs of chromosomes, including sex chromosomes, using cultured fetal cells collected from either chorionic villus collection or amniocentesis. As a result, the benefit is that it allows for the detection of chromosomal abnormalities such as trisomy, monosomy, massive deletions and duplications, translocations, inversions, and mosaic; however, the disadvantages are that it necessitates cell culture and that the analysis takes a long time (up to 2 weeks). When ultrasonography changes are discovered, the microarray-based chromosomal examination has proved to have a better sensitivity; consequently, it is recommended as a first-level diagnostic [34]. This technique enables the detection of chromosomal aneuploidy, microdeletions, and microduplications inside a chromosome that is too small to be detected by a standard karyotype. Chromosome microarray can be performed on various systems, including aCGH.  Compared to traditional cytogenetics, the development of microarray-based methods has enhanced the diagnostic yield of prenatal diagnostics [35, 36]. However, 70–80% of fetuses with ultrasonography abnormalities are still undiagnosed molecularly [35, 36]. The cytogenetic techniques outlined above are unable to identify changes at the single nucleotide level. As a result, molecular biology techniques can be used to study specific illness-causing genes and emphasize the causative mutations already found in the family in the case of an inherited disease [32, 37]. Both prenatal and preimplantation genetic diagnosis has these limitations, which is to be expected [30]. The limitations of current prenatal diagnostic strategies have spurred the need for more sensitive tools, and the recent discovery of novel sequencing technologies is ushering PND into a new era [38]. Indeed, like other molecular diagnostics domains, NGS is transforming PND: Prenatal exome sequencing (ES) is now an option, at least in certain circumstances, in addition to targeted sequencing of single disease-­ causing genes or small groups of genes of interest. Finally, Ribo nucleic acid (RNA)-sequencing may provide new insights into human development during pregnancy and novel non-invasive biomarkers to monitor placental functions through maternal plasma cell-free RNA (cfRNA), which has been used for research but is not yet applicable in clinical settings due to data interpretation difficulties. Finally, genome sequencing has been used for research, but is not yet applicable in clinical settings due to data interpretation difficulties; finally, genome sequencing has been used for research, but is not yet applicable in clinical settings due to data interpretation difficulties [29, 38, 39]. This method has the potential to reveal new insights into embryonic development and elucidate the mechanisms underlying specific diseases of interest, but it is currently only useful for research and is limited by the scarcity of tissues (Table 3.3).

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Table 3.3  Genetical assessment tools S. no. Approach 1. Karyotyping

Advantages Easy to perform Can detect balanced chromosomal events

2.

Linkage analysis

Easy to perform Ideal for any type of inherited disease

3.

Homozygosity Ideal for recessive mapping monogenic disorder gene identification Small families can be studied

4.

Copy number variations analysis

5.

Candidate Requires no mapping gene approach Biological functionbased study to identify disease causing gene

6.

Whole exome Exome wide base sequencing pair level resolution Detects most types of genomic variations present in and around exonic regions Can identify causative variant using single or few patients or family members

7.

Whole genome sequencing

Genome wide, high resolution for CNVs

Genome wide base pair level resolution Detects all types of genomic variations Can identity causative variant using a single patient

Disadvantages Low resolution Only detects large chromosomal aberrations Mutation detection is not possible Requires large families Identifies large linked loci Disease gene identification requires combination of different approaches Helpful only in cases of consanguineous families Identifies large loci Combinational approach required for identification of disease causing gene Only investigates disorders due to CNVs Cannot detect balanced translocations No resolution at base pair Level For gene identification combinational approach Required Thorough knowledge required about disease pathogenicity and biological processes Only few successful cases are known Limited detection of noncoding variants Unable to detect variants deep intronic and non-coding genomic regions Poor resolution for CNVs and structural variations Coverage variability issue due to enrichment process Expensive than other conventional methods Data analysis is complex Expensive than exome Sequencing Generates large number of variants, thus difficult to analyse significance of each variant

Example Genetic disorders like down syndrome, Edwards syndrome, etc. Duchene muscular dystrophy (DMD), to identify chromosome regions likely to harbor genes for the trait Hereditary retinal dystrophies

Multiple hereditary diseases, dosage imbalance of one or more genes

Associate-specific genetic variations with particular diseases

Neurodegenerative disorders, Parkinson diseases

Rare genetic disorders

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3.7 Chromosomal Anomalies Seen in SGD Based on the Involved Genes Researchers around the globe are actively engaged in assessing the polygenicity of human diseases complex in nature as the knowledge on the human genome is expanding, and new techniques make it feasible to locate the gene loci for respective genes for easy diagnosing and prognosing. Polygenetic variation is widely seen in most diseases, with few disorders caused explicitly due to mutations in the single gene and are known as SGD. Through the recent genetical advances, it is possible to point out the molecular functioning of the pathological conditions, especially the SGD. The mutations in one gene are usually observed in phylogenic form; means it runs in families and can lead to dominance form in favourable conditions like consanguineous marriages or to recessive or sex-linked either in recessive or dominant forms. The inheritance pattern is seen and understood by plotting the affected member’s pedigree analysis chart to view other carriers/affected individuals (Table 3.4). Table 3.4  Types of disorders and their inheritance S. no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Disorders Complex I deficiency Combined hypolipidemia Dilated cardiomyopathy Progeroid syndrome Retinitis pigmentosa Miller syndrome Sensory neuropathy with dementia and hearing loss Autoimmune lymphoproliferative Syndrome Nonsyndromic hearing loss Perrault syndrome Chondrodysplasia and abnormal Joint development Kabuki syndrome Hajdu–Cheney syndrome Hyperphosphatasia mental retardation syndrome Skeletal dysplasia Osteogenesis imperfecta Schinzel–Giedion syndrome Congenital chloride diarrhea Kaposi sarcoma Nonsyndromic mental retardation

Inheritance AR AR AD AR AR AR AD AR AR AR AR AD AD AR AR AR AD AR AR AR

3  The Nexus Between Chromosomal Abnormalities and Single Gene Disorders Table 3.4 (continued) S. no. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

Disorders Spinocerebellar ataxia Nonsyndromic mental retardation Autism Amyotrophic lateral sclerosis Sensenbrenner syndrome Cerebral cortical malformations Inflammatory bowel disease Phenylketonuria (PKU) Cystic fibrosis Sickle-cell anemia Albinism, oculocutaneous, type II Huntington’s disease Myotonic dystrophy type 1 Neurofibromatosis, type 1 Polycystic kidney disease 1 and 2 Hemophilia A Muscular dystrophy, Duchenne type Hypophosphatemic rickets, X-linked dominant Rett’s syndrome Spermatogenic failure Achondroplasia Cystic fibrosis Duchenne muscular dystrophy Hypercholesterolemia Fragile X syndrome Gaucher’s disease Glucose 6-phosphate dehydrogenase deficiency Hemochromatosis Holoprosencephaly Huntington disease (also Huntington chorea) Marfan syndrome Myotonic dystrophy Neurofibromatosis I Osteogenesis imperfecta Phenylketonuria Polycystic kidney disease Sex reversal Tay–Sachs disease Thalasemias Xeroderma pigmentosum

Inheritance AD AD AD AD AR AR AD AR AR AR AR AD AD AD AD X-linked recessive X-linked recessive X-linked dominant X-linked dominant Y-linked AD AR X-linked recessive AD X-linked dominant AR X-linked recessive AR AD AD AD AD AD AD AR AD Various AR AR AR

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3.7.1 Autosomal Recessive Disorders (AR) AR occur when both genes or alleles at a given autosomal (autosome refers to chromosome numbers 1 through 22 and excludes the sex chromosomes) are abnormal. To induce recessive phenotype disorders, the chromosome pair must have abnormalities in both the alleles or chromosomes in a pair must have a mutation in both genes. If one gene is abnormal and the other gene is typical for an autosomal recessive disorder, the individual will be unaffected by the disorder. However, this individual will be a carrier of the defective form of the gene and pass it on to their offspring. If both partners are carriers, one-fourth of their offspring are expected to be affected. AR commonly affect males and females with the same frequency and severity. The rarer the disorder, the more likely the partners are related. AR are often more severe, less variable and less age-dependent than ‘autosomal dominant disorders’. A characteristic pedigree or family tree and some examples are given below: 1. 2. 3. 4. 5. 6. 7. 8.

Albinism Cystic fibrosis Fanconi anaemia Galactosemia PKU Tay–Sachs disease Werdnig–Hoffmann-disease Haemoglobinopathies

3.7.2 Autosomal Dominant Disorders AD occur when either gene of a pair of the autosomal chromosome is abnormal. If an individual is born with an abnormal form of a gene that causes autosomal dominant disorders, he or she will be affected and can also pass the defect to his or her offspring. The trait is inherited from one parent, and one half of the offspring are affected. Isolated cases without a family history of the disorder may arise from new mutations with a 50% risk to their offspring. An autosomal dominant trait affects males and females with the same frequency and severity. AD are often associated with malformations and are more variable but usually less severe than autosomal recessive disorders. A characteristic pedigree and some examples of AD are given below: 1. Achondroplasia 2. Huntington disease 3. Myotonic dystrophy 4. Neurofibromatosis 5. Polycystic kidney disease 6. Osteogenesis imperfecta

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3.7.3 X-Linked Genetic Disorders Genetic disorders due to alterations in the allosomes, especially in the X chromosome, are termed X-linked genetic disorders. Humans have two sex chromosomes, X and Y, and XX chromosomes give female progeny, and XY gives male progeny. Genetic disorders due to X-linked pattern of inheritance affects differentially in males and females. Like other autosomal disorders, X-linked disorders are also seen to follow the dominance and recessive pattern of transmission from one generation to another. Though dominant X-linked is rare as the parent must have the disorder confirm to be dominant in inheritance, the parents usually could not conceive easily to have progeny.

3.7.4 X-Linked Dominant Mutations in the X-chromosome genes can transmit disease from parent to child with a single chromosome, which is rare (Fig. 3.5), and significantly fewer disorders are seen to follow these inheritance patterns, namely X-linked hypophosphatemic rickets. Irrespective of gender, all are affected equally with this disorder, with higher severity in males than females. Other disorders include Klinefelter syndrome (44+ XXY), Aicardi syndrome, incontinentia pigmenti type 2, and Rett syndrome. These disorders sometimes show higher severity in females too.

Fig. 3.5  Schematic representation of X-linked dominant inheritance

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3.7.5 X-Linked Recessive Mutations in the X chromosomes and the alleles’ needs to express their pathophysiology are called X-linked recessive disorder. This disorder is mainly seen in males than females due to the presence of other X chromosomes in females to compensate the defective with the higher disparity of transmission between the genders; the recessive disorders linked to X-chromosomes include haemophilia A, Lesch–Nyhan syndromes, DMD, as well as not so critical phenotypic conditions like male pattern baldness and red-green colour blindness. In some cases, females are also targeted to have X-linked recessive conditions (Fig. 3.6). A similar condition is seen due to the inactivation of the X chromosome either by skewed or monosomy, as seen in Turner syndrome.

3.7.6 Y-Linked Disorders Genetic diseases linked to mutant alleles on the Y chromosome in humans are called Y-linked diseases—The characteristic example of a Y-linked trait is hairy pinna of the ear, an exclusive character, seen in males. One of the known Y-linked diseases, Ichthyosis Hystrix syndrome, was later proved autosomal.

3.7.7 Codominance The presence of gene products from both the alleles in a locus in a heterozygous state in equal amount leads to a condition known as codominance. Different gene products in the same locus will involve varied cellular processing and various

Fig. 3.6  Pictorial representation of X-linked recessive inheritance

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metabolic activities and enzymes involved in processing the proteins. The presence of the AB allele in its heterozygous form in the typical locus of blood typing is the best and typical example of codominance presenting both antigens (A&B) in the locus. These antigens are produced by the enzymatic role of two glycosyltransferases encoded by allele A and B. Beta-thalassemia minor is another codominance case comprising of mutation in the β-chain of haemoglobin [40].

3.7.8 Mitochondrial Alterations in the mitochondria genome show the alternative side of the non-­ Mendelian inheritance pattern known as maternal inheritance. These mutations are passed from the mother to the progeny. These alterations hinder mitochondria’s functioning and compromise cellular processes like ATP production. A wide range of varying severity and penetration is observed in the disorders caused due to mitochondrial mutations combined effect called heteroplasmy (combination of standard and mutated DNA of mitochondria in a cell) plus environmental and genetic interactions. At present, about 50 genetic disorders are listed due to alterations in the genetic constitution of mitochondria.

3.8 Chromosomal Abnormality or Instability as Prognostic Markers in SGD Solving the clinical problems using cytogenetic studies is considered a primitive diagnostic application to analyse the pathological states already known. The application of cytogenetic testing is beneficial in estimating congenital aberration conditions and abnormalities in the sexual and somatic developmental states, mental retardations, spontaneous recurring abortions, and infertilities. Through the cytogenetical assessments, we can elucidate the effects on few environmental droghers like viruses, drugs, ionizing radiation effects on human health, and few disease pathologies. This shows the role of cytogenetics in the medical field, and it is an integral part of both theoretical and practical aspects of the medical field. A wide range of efficacious strategies is used to analyse the underlying cause of Mendelian disorder at the molecular level. The strategies involve mapping the region of the chromosome causing the pathology, followed by identifying the alterations on the specific chromosome for the candidate gene responsible for the function [41]. Abnormalities and instability of chromosomes are hallmarks to illuminate the pathogenesis of the malaises and their progression [42]. Most of the pathologies, specifically SGD, are due to change in the chromosome structure due to deletion, substitution, duplication, or losing its allele pairs. These conditions are also known to predispose to pathological conditions and help in assessing or predicting the potential disorders and known as biomarkers [43].

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Apart from the conventional cytogenetics methods to identify the disease at its stratification and through gene expressional studies recently, we can obtain potential markers at the genetic level to obtain more information about the disease and predict the treatment outcomes. We can identify the persons prone to malaise and its relapse through markers and assess the therapy for the prone person.

3.9 Conclusion Chromosomal anomalies are renowned prognosis markers for a wide array of diseases and allow the clinicians to identify the proband and the degree of therapy and surveillance required. Ascertaining the frequency of alterations at the genetic level helps in giving a clear picture of disease pathology. Through exosome sequencing, the diagnosis is made a little easier with a significant impact in medicine. In the future, computational studies, i.e. systems diagnostics, could integrate data between a single patient and all other patients plus healthy individuals to identify more single-­gene diseases for better diagnostics and deeper insight into these diseases. Rapidly increasing research wealth on human genomic studies can provide promising diagnostics upliftment and lead to personalized medicine for SGD. Acknowledgements  We appreciate the diagram and editing for this book chapter by Dr. Kathika Pushparaj, Assistant Professor, Department of Zoology, Avinashilingam Institute of Home Science and Higher Education for Women, Coimbatore—641043, India.

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9. Weatherall DJ. The thalassemias: disorders of globin synthesis. In: Williams hematology. 9th ed. New York: McGraw Hill; 2015. p. 725. 10. Woolf LI. The heterozygote advantage in phenylketonuria. Am J Hum Genet. 1986;38(5):773–5. 11. Yoshiura K, Kinoshita A, Ishida T, Ninokata A, Ishikawa T, Kaname T, et al. A SNP in the ABCC11 gene is the determinant of human earwax type. Nat Genet. 2006;38(3):324–30. 12. Zeviani M, Spinazzola A, Carelli V.  Nuclear genes in mitochondrial disorders. Curr Opin Genet Dev. 2003;13(3):262–70. 13. Schwartz M, Vissing J.  Paternal inheritance of mitochondrial DNA.  N Engl J Med. 2002;347(8):576–80. 14. Giles RE, Blanc H, Cann HM, Wallace DC.  Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci U S A. 1980;77(11):6715–9. 15. Ankel-Simons F, Cummins JM.  Misconceptions about mitochondria and mammalian fertilization: implications for theories on human evolution. Proc Natl Acad Sci USA. 1996;93(24):13859–63. 16. Jenuth JP, Peterson AC, Fu K, Shoubridge EA.  Random genetic drift in the female germline explains the rapid segregation of mammalian mitochondrial DNA.  Nat Genet. 1996;14(2):146–51. 17. Jenuth JP, Peterson AC, Shoubridge EA. Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice. Nat Genet. 1997;16(1):93–5. 18. Thorburn DR, Dahl HH. Mitochondrial disorders: genetics, counseling, prenatal diagnosis and reproductive options. Am J Med Genet. 2001;106(1):102–14. 19. Zeviani M, Carelli V. Mitochondrial disorders. Curr Opin Neurol. 2003;16(5):585–94. 20. Ralston A, Shaw K. Environment controls gene expression: sex determination and the onset of genetic disorders. Nat Educ. 2008;1(1):20. 21. Martin T, Bell J, Spector T. Twin studies and epigenetics. Amsterdam: Elsevier; 2015. https:// doi.org/10.1016/B978-­0-­08-­097086-­8.82051-­6. 22. Genetic Alliance, District of Columbia Department of Health. Understanding genetics: a District of Columbia guide for patients and health professionals. Washington (DC): Genetic Alliance; 2010. 23. Ashavaid TF, Raghavan R, Dhairyawan P, Bhawalkar S. Cystic fibrosis in India: a systematic review. J Assoc Physicians India. 2012;60:39–41. 24. Loo TW, Bartlett MC, Clarke DM. Introduction of the most common cystic fibrosis mutation (Delta F508) into human P-glycoprotein disrupts packing of the transmembrane segments. J Biol Chem. 2002;277(31):27585–8. 25. Amberger JS, Bocchini CA, Schiettecatte F, Scott AF, Hamosh A. Online Mendelian Inheritance in Man (OMIM®), an online catalog of human genes and genetic disorders. Nucleic Acids Res. 2015;43(Database issue):D789–98. 26. McKusick VA. Mendelian inheritance in man and its online version. OMIM Am J Hum Genet. 2007;80(4):588–604. https://doi.org/10.1086/514346. 27. Al-Turkmani MR, Deharvengt SJ, Lefferts JA. Chapter 31: Molecular assessment of human diseases in the clinical laboratory. In: Coleman WB, Tsongalis GJ, editors. Essential concepts in molecular pathology. Amsterdam: Elsevier; 2020. 28. Murphy SL, Mathews TJ, Martin JA, Minkovitz CS, Strobino DM. Annual summary of vital statistics: 2013–2014. Pediatrics. 2017;139(6):e20163239. 29. Cariati F, D'Argenio V, Tomaiuolo R. The evolving role of genetic tests in reproductive medicine. J Transl Med. 2019;17(1):267. https://doi.org/10.1186/s12967-­019-­2019-­8. 30. Cariati F, Savarese M, D'Argenio V, Salvatore F, Tomaiuolo R.  The SEeMORE strategy: single-­tube electrophoresis analysis-based genotyping to detect monogenic diseases rapidly and effectively from conception until birth. Clin Chem Lab Med. 2017;56(1):40–50. 31. American College of Obstetricians and Gynecologists. Practice Bulletin No. 162. Prenatal diagnostic testing for genetic disorders. Obstet Gynecol. 2016;127:e108e22. 32. Tomaiuolo R, Nardiello P, Martinelli P, Sacchetti L, Salvatore F, Castaldo G. Prenatal diagnosis of cystic fibrosis: an experience of 181 cases. Clin Chem Lab Med. 2013;51(12):2227–32.

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Chapter 4

Clinical Implications of Chromosomal Polymorphisms in Congenital Disorders Arun Meyyazhagan, Haripriya Kuchi Bhotla, Manikantan Pappuswamy, Valentina Tsibizova, Karthick Kumar Alagamuthu, and Gian Carlo Di Renzo

4.1 Introduction Trivial alterations in the banding karyotype are seen in every people and remain constant in a few individuals. These alterations are known as chromosomal polymorphism (CPM) (Table 4.1). CPM comprises heterochromatin zones abundant in Arun Meyyazhagan and Haripriya Kuchi Bhotla have equally contributed as first authors. A. Meyyazhagan Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Italy Department of Life Sciences, CHRIST (Deemed to be University), Bengaluru, Karnataka, India PREIS International and European School of Perinatal, Neonatal and Reproductive Medicine, Firenze, Italy H. Kuchi Bhotla · M. Pappuswamy Department of Life Sciences, CHRIST (Deemed to be University), Bengaluru, Karnataka, India V. Tsibizova Institute of Perinatology and Pediatrics, Almazov National Medical Research Centre, Saint-Petersburg, Russian Federation K. K. Alagamuthu Department of Biotechnology, Selvamm Arts and Science College (Autonomous), Namakkal, India G. C. Di Renzo (*) Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Italy Department of Obstetrics and Gynecology, I.M. Sechenov First State University of Moscow, Moscow, Russia PREIS International and European School of Perinatal, Neonatal and Reproductive Medicine, Firenze, Italy e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_4

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Table 4.1  The polymorphic regions seen in chromosomes process of CMP formation S. no. 1. 2. 3. 4.

Polymorphic regions Chromosomal bands Fragile sites Major structural variants Nucleolus organizing region

Description Region of heterochromatin with highly repetitive DNA Nonstained chromatid region due to breaks Inversions, duplications, deletions Genes for 18s and 28sr-RNA regions are present in the acrocentric chromosomes

repetitive satellite deoxyribose nucleic acid (DNA) located at noncoding regions in both short and long arms of chromosomes (mostly chromosomes 1, 9, and 16) [1]. Chromosomes belonging to groups D and G, specifically 13, 14, 15, 21, and 22, show polymorphism around the short arm, stalk, and satellite region; likewise, inversions in pericentric chromosomes, either on the short or long arm of chromosomes, 1, 2, 9, and Y. CPMs are usually phenotypically silent. At the same time, they can pose significant alterations causing cancers, reproductive disorders, congenital defects, and mental retardations [2]. The difference observed in a population in the size and staining of the chromosomal regions is called chromosomal variation or CPM. Though being phenotypically silent, these variations can lead to clinical manifestation or reproductive anomalies associated with the individual’s internal and external surroundings [3]. These polymorphisms can be due to the duplication or deletion of tiny DNA fragments and lead to nearly 300 defects. Chromosomal abnormalities are seen in 0.25% of live-born infants with chromosome 9 pericentric inversion as balanced chromosomal alteration seen in both affected and normal children [4]. CPMs are the inducers of reproductive separation by triggering a chain of error throughout meiosis, either as pairing or segregation error or during crossing over. These meiosis defects have deleterious effects on an individual’s life [5]. CPMs lead to congenital abnormalities and deficiency in the person’s growth and intellect. About 15% of pregnancies recognized by clinics terminate due to fetal loss, and among this death, cytogenetic errors are vital players for spontaneous abortion or stillbirths [6]. These anomalies differ from duplication to rearrangement of the whole genome, like fine-scale insertion and base pairs’ composition alterations. Classically, CPM refers to structural abnormalities such as translocation of the whole arm, fusion or fission, transpositions, shift in centromere, reduction or amplification of heterochromatin region, and inversions in the chromosomes. However, current advances have shown the abnormalities at a submicroscopic state like DNA sequence level [7]. The polymorphisms of a chromosome are majorly due to breaks in DNA. This can threaten cells if left unrepaired and results in instability of the genome, eventually causing alteration in chromosomes and cell death. The anomalies in a chromosome are majorly due to double-stranded (ds)DNA breaks. About 5000 single-strand DNA breaks are seen per nucleus in every cell division, and among them, approximately 1% convert into dsDNA breaks. The formation of CMP is generally explained through two theories, namely breakage and reunion theory and the theory of exchange.

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4.1.1 Breakage and Reunion Theory (Darlington, 1935) The broken strands formed during the cell division unite and rejoin to produce the native structure of the chromosome through restoration.

4.1.2 The Exchange Theory (Sutton and Boveri, 1902) According to exchange theory, the dsDNA breaks and joins with other breaks of other chromosomes during cell division. These breaks in the dsDNA could be restored and mended in three ways • Homologous recombination repairing to fix the original sequence in case of a single breakpoint. • Nonhomologous DNA ends joining mends the small alterations like substitution, insertion, or deletion of base pairs with two breakpoints. • Single-stranded annealing forms the interstitial deletions, and repairs break either one or two in eukaryotes and mammalian cells [8]. Additionally, the CMPs can occur by varying chemical and physical components like ionization rays, endogenous reactive oxygen species, replication errors, and topoisomerases (Fig. 4.1).

Fig. 4.1  The two theories explaining the formation chromosomal polymorphism

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Table 4.2  The incidence and causes of congenital anomalies in general Common congenital anomalies causes Single gene defect Chromosomal alteration Epigenetic and maternal causes

Prevalence per 1000 live births (%) 17 10 4–10

4.2 Incidence of CPMs in Congenital Disorders About 15% of clinically acknowledged pregnancies lead to the death of a fetus, and karyotypic anomalies are seen in most fetal death and spontaneous abortions compared to stillbirth [6]. CPMs rely on evaluating the tissue type, the study population, and the diagnostic method. Chromosome microarray highlights more CPMs than the standard karyotyping methodology, though it misses the chromosome rearrangement balance [9]. According to the world health organization (WHO), the prevalence of congenital anomalies is recorded as 3%, about three million births per year [10], with a minute difference between the countries. Reports from the developed countries showed the prevalence rate as 45–50 in 1000 live birth, and Middle Eastern and African countries showed the prevalence of congenital anomalies as 20–30 among 1000 live births, with the higher death rate in lower- and middle-­ income countries. Congenital heart disease is the highly prevalent congenital anomaly, and Down syndrome is the prevalent chromosomal polymorphism anomaly [11]. Congenital anomalies were linked to consanguineous marriage as CPMs were linked to advanced maternal and paternal age [12]. Higher maternal age is an established risk component for nondisjunction meiosis during chromosome segregation and leads to aneuploidy [13] (Table 4.2).

4.3 Detection of CPMs The anomalies and aberrations in the chromosome are identified using some conventional methods. These are the popular methods:

4.3.1 Karyotyping The laboratory procedure used to diagnose the numerical and structural abnormalities and the related disorders use the peripheral blood, amniotic fluid, tissue, bone marrow, or the cord blood to culture lymphocytes in a medium. The lymphocytes are arrested in the metaphase stage of cell division by adding colchicine followed by hypotonic cellular fixation with methanol and acetic acid at a 3:1 ratio on a glass slide before Giemsa staining. Chromosomes are distinguished and labelled using varying banding techniques. However, the most common one is the GTG banding

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procedure, which classifies chromosomes based on size, centromere position, banding pattern, and AT-rich heterochromatin region stained darkly and euchromatin region stained lightly. Based on the international system of human cytogenetic nomenclature 2020, the reporting of the chromosomes is done by mentioning the total number of autosomes flowed by sex chromosomes for normal human karyotype. + + and − signs show gain or loss of the chromosomes. The suffix del, dup, and inv. are used in brackets next to the chromosomal number for deletion, duplication, and inversions in structural aberrations. Likewise, suppose the rearrangement is seen in-between inside the chromosomes. In that case, they are represented by a semicolon—;. A female patient with Down syndrome and deletion of chromosome 9 is shown as 47, XX, +21; del(9). Micro aberrations in the chromosomes lesser than 5 Mb are not easily detected in karyotyping; likewise, loss of heterozygosity and homozygosity is not detected by karyotyping [14].

4.3.2 Fluorescent In Situ Hybridization (FISH) The FISH methodology utilizes fluorescence probes to identify specific target regions in DNA to observe under a fluorescent microscope. These are the following probes used in diagnostics: 1. Probes specific to locus: the probe targets a particular gene of a specific chromosome. 2. Probe for the whole chromosome: numerous samples in smaller numbers are used with fluorescent dyes to bind at specific chromosome segments to form a chromosome map, leading to immediate detection of abnormalities like translocation. 3. Probes for centromere repeats: chromosome number is identified by repeated binding sequences required for prenatal diagnosis. This type of FISH minimizes the turnaround time (TAT) associated with minor aberrations and helps find the mosaicism proportions [15].

4.3.3 Chromosome Microarray A microarray of chromosomes is highly cost-effective based on genome alteration or single nucleotide variants. It gives higher resolution results in prenatal tests to give defects like microdeletions, aneuploidies, and duplications of the entire chromosome on a single chromosome microchip. This is a powerful tool for detecting the imbalance in the genes and has revolutionized the current era of cytogenetics by identifying the loss or gain of copy number variants of DNA in various hereditary disorders [15].

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Fig. 4.2  The various classical and modern methods used till date to detect the presence of chromosomal polymorphism and their association with various diseases

4.3.4 Quantitative Fluorescence Polymerase Chain Reaction (QF-PCR) This is a prenatal diagnostic method to identify chromosomes 13, 18, 21, and sex chromosome aneuploidies. This method is rapid and automated because the fetal cells are not cultured. The DNA is extracted from amniotic fluid, chronic villus samples, or tissues and the DNA obtained is polymerized using the fluorescent primers to analyze the aberration. This method requires a small sample and less labor, and less time (Fig. 4.2).

4.4 Studies Related to CPMs Generally, the disease caused in patients is majorly due to genetic factors and includes both inherited/de novo Mendelian disorders and chromosomal polymorphisms. These genetic disorders are caused by alterations in nucleotide bases, genome structure, or genome copy numbers on a molecular basis. Most of the alterations are too minute to view under a light microscope. Hence, techniques like microarray analysis and sequencing analysis are required to detect the disorders [16]. Below are the CPMs in a few congenital disorders.

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4.4.1 Cerebral Palsy (CP) CPMs are seen in CP patients. Some show loss or gain of the genetic material in the segmental form to be observed in cytogenetic examination in a few DECIPHER and Clin Var databases. These segmental losses are generally duplication or deletion identified through chromosomal microarray assay or the sequencing of exomes. Indels are the smaller genomic loss and gain (90%. It is less reliable for all other tested conditions, with much lower PPV’s— which may even be unavailable for some very rare conditions If the NIPT screening test result If the NIPT screening test is negative, is negative, no further action is diagnostic testing is recommended under two conditions: 1-Confirmed or required questionable sonographic or biochemical/ NT results; 2-normal sonographic results but wish to exclude a microarray abnormalities or placental mosaicism If the NIPT screening test result If the NIPT screening test has a positive test, the offering of confirmatory diagnostic is positive, there is a clear screening is almost always obligatory indication that the fetus has a genetic problem, so no further testing is necessary NIPT, either in its normal or expanded NIPT is a test that can exclude versions, cannot exclude all possible all genetic problems, just like diagnostic tests. It follows that genetic conditions. Diagnostic tests provide definitive evidence of genetic conditions there is no need for follow-up for many more conditions and are diagnostic testing recommended for all positive NIPT screening tests and negative NIPT where higher risk is suspected NIPT is a quick test that is easy Neither NIPT as a screening test nor CVS/ AC or more recent diagnostic tests are easy to understand, so that it can be to interpret. There should be a standard of used effectively by OBGYN’s demonstrated competence with both tests with no special training to properly discuss their interpretation and implications (continued)

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Table 6.1 (continued) Dimension Implications for discussing potentially life-changing implications of having an affected child Implications for women below the age of 35

Biased presentation NIPT provides all the information necessary

Actual case Only diagnostic tests provides a degree of certainty that permits a grounded, informed discussion of longer-term implications. No test can find “everything.”

NIPT provides all the information that is necessary

There are several rare microdeletion syndromes that are individually very rare but as a group are significantly more frequent than their risk for common trisomies. For “younger” women, microarrays can find 10X the number of significant abnormalities.

This table borrows heavily from Liehr (2021) [31] Table 6.1. It also incorporates reasoning and examples from Dondorp et al. (2015), Evans et al. (2016), Henry et al. (2008), and others *

It is important to note that the level of commercialization is a contextual factor, having implications for what patients know in advance of their sessions, how medical professionals think about NIPT, and how the interaction between patients and medical professionals plays out. The level of commercialization is not completely exogenous since it is at least partially under the control of both providers and patients. However, we agree with Liehr (2021) and others that it may influence, in sometimes subtle ways, how NIPT is presented in a positive light and how more definitive testing is downplayed in importance and/or portrayed as being procedurally riskier than it actually is in well-trained hands. The explosive growth of NIPT and the consequent reduction in diagnostic testing has been documented in too many different situations not to be real [35]. Opinion is mixed, however, whether the trend will moderate. The long-term trend towards increased accuracy and scope of NIPT seems empirically grounded and certain to continue [35]. Eventually, marketing claims may become less exaggerated and more realistic as general knowledge and experience of physicians increase regarding NIPT, guidelines become more specific, and the threat of lawsuits regarding substantially erroneous claims or negligent care becomes more apparent [36]. There will be continuing challenges as the possibilities of genetic analysis become more far-reaching. Thus, at the national level, “nuanced and contextualized discussions of sociotechnical implications are indispensable as countries cope with decisions regarding what uses of NIPT they wish to allow or fund” [28, 37]. A second contextual factor that must be brought into the discussion is the level of sociopolitical conservatism that exists in the geographic area in which screening and diagnostic tests are being given. The level of conservatism is sufficiently important that it may be used to differentiate countries from one another. The concept applies within countries such as the USA for understanding the kinds of tests that may be offered and the nature of the dialog that might be expected during counseling sessions [28, 31, 37]. Since at the present time, most of the findings from

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prenatal screening and diagnosis necessarily include discussions about decisions about abortion, it is important to understand that states and nations in which abortion is banned or severely restricted will have a different counseling process in vivo—no matter what professional association guidelines indicate should be best practice—than those in which abortion is more readily and operationally available [38, 39]. In more conservative jurisdictions there are generally fewer geneticists and genetic counselors, and such professionals are likely to be confined to larger metropolitan areas. As such, even with telemedical advances in such prenatal counseling, assuring equity of access is a continuing, daunting problem [38, 39]. The counseling process itself may also vary from area to area, as more conservative states may either explicitly or implicitly limit the kinds of information that can be shared, how it may be shared, and what sorts of options can even be discussed [40]. Further, in such areas, “women may be pushed to use illegal means or may suffer psychological or physical harms from lack of access to safe termination,” reinforcing already existing inequalities of access [40].

6.4 Individual Factors Much of the analysis of factors associated with the acceptance of screening and diagnosis has justifiably focused on characteristics of individuals [41]. Information on individual characteristics serves to immediately expose the fragility of the assumption that counseling sessions can have “one size fits all” uniformity. The problem is that we cannot learn much about patients and how they are going to react to counseling by examining one characteristic at a time. Bluntly, patients are composites of characteristics who have a multifaceted identity and a differentiated biographical situation even in the same culture. Combinations of attributes and values are important, and this concept is something that is increasingly being shared among researchers and practitioners [39–42]. We have studied a parallel dynamic which shows up at the individual level for women carrying multifetal pregnancies who have come into our center to discuss whether they want to reduce the number of fetuses they are carrying and if so, how far to reduce [43]. These are very stressful decisions, as is the case for higher-risk women facing the possibility of genetic anomalies in the fetus(es) they are carrying, and we believe the logic is transferrable. For women educated in the sciences, engineering, or the law, a “medical frame” that makes possible a realistic assessment and balancing of risks is quite likely, and for women with such a biography, the decision to reduce is stressful but relatively “straightforward” [44]. It would be a mistake, however, to think that the impact of more advanced education can be appreciated without also knowing the job/career status of these women and the importance to them of having a career. This is certainly not a new dilemma for women in more developed societies, but as a higher percentage of women become educated, have careers (and often delaying having children because of that), and have mothers and other female family members who have had or wanted to have careers, more models for trying to negotiate such a balance of family and career are feasible. The anxiety

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here is acute, but there are, at least, culturally supported templates for such balancing acts for those with a “lifestyle frame.” Such decisions, however, become at once both more anxiety-laden and stressful as the level of personal religious involvement becomes more salient. For such women, religious authorities are typically more credible than medical authorities, and the sense of having few, if any, options is an almost crushing burden. Women with such a “conservatism frame” find the prospect of reduction as an option for creating a safe passage for their hoped-for children excruciatingly hard. Consequently, or experience over the past 35  years suggests they are much less likely to reduce unless they are carrying typically four or more fetuses, and they will usually reduce only to three in an attempt to avoid losing the entire pregnancy [45]. As appreciation of such situations has increased both in medical and popular media, even more religious patients are considering reduction or termination options much more frequently than we saw 20–30 years ago. By extension, the same biases may occur in the presentation and acceptance of potentially life-changing and difficult-to-hear genetic information. Chen et  al. (2018), also working within a combinatorial framework, developed a typology in which two dimensions of what patients bring with them (as forms of capital) are considered in conjunction with one another [42]. The first is “intellectual capital” (the ability to understand medical information) and the second is “values capital” (the strength and clarity of their moral values). Their definition of intellectual capital is very close to our conception of a medical frame. Where we differ is with respect to what they call “moral capital.” In our opinion, this concept permits greater insight by being decomposed so that the substantive nature of the values becomes evident. Strength and clarity could apply to any number of things, including intellectual capital. More specifically, having strong and clear conservative values is very different from having strong and clear liberal values in terms of implications for tests that are chosen (if any), the anxiety and stress of having to personally confront such information, and the people/institutions that will be involved in the decision process. Following from our findings regarding the reduction of multiples, those with a more conservative orientation should be less likely to have either screening or diagnostic testing (and more likely to stop after screening if they do choose to engage at all), more likely to choose pregnancy continuation rather than abortion or reductions for multiples, and more likely to involve others (or perhaps even cede to others) in the decision process. These same ideas regarding strength and clarity should be applied to the degree to which women feel as if having a career is a strong part of their identity. Increasingly as women have become more involved in the labor force and have careers, they may have a greater need for certainty in making decisions about balancing children with special needs and the time and effort required by a career. They may experience somewhat less stress, but only if they have a strong medical frame. They may have difficulty making a decision (depending on their level of resources and level of familial support), and they may be more likely to keep the decision limited to the immediate family [42]. In short, a three-dimensional typology with capacity to understand risk information, the intensity of commitment to a career, and the intensity of commitment to conservative moral values constitutes the three dimensions—each of which can vary in their inherent strength and level of familial and other support (Fig. 6.4). Such an

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Fig. 6.4  Medical frames become more intense as education, particularly education that is based on analysis of facts, so that there is an ability to understand and openness to consider risk and options. Conservatism frames become more intense as the commitment to a socially and politically conservative moral stance increases. Lifestyle frames become more intense as the commitment to the role of women expands beyond only having and raising children to include commitment to a career or other activities increase

approach could easily become elaborated by academics studying how women and their partners construct identities or realities around such decisions, or numericized to yield coordinate scores that could be interpreted by some AI algorithms. We offer it simply as a corrective to thinking about patients in purely additive statistical terms and in hopes that it will aid practitioners in thinking about the kinds of strategies that may be useful at the local level in different cultures and with women with different biographies and values. Briefly examining Fig. 6.4, there are two colored icons roughly identifying two different spatial areas that have elements that are somewhat mutually reinforcing, suggesting some coherence to the identities that they feel might describe them, and some social acceptance of these identities that may (if they have had the leverage to engage in such an effort) have come from years of sifting through friends and coworkers to find support for their identities. The rounded blue icon represents a hypothetical woman who has strong liberal moral values coupled with strong commitments to a career and a high capacity to understand and accept the implications of risk-related information because of her education and training. Granted that these sessions are potentially highly stressful, such a woman would probably be more likely to be able to understand the objective risks of her condition, relatively open to consideration of options, and with proper support, able to stay in the situation emotionally and intellectually and make an informed choice about taking the screening test and even considering more invasive options initially or in sequence. A problem for providers—especially those without some special training, which appears to constitute a fairly large chunk of providers presenting and interpreting the test [40]

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may be that such a woman might become dissatisfied with how relatively unexpert the provider is or may just be blithely ignorant of a simplistic explanation that misses important contextual issues. The square red icon in Fig. 6.4 represents a hypothetical woman coming from a very different set of circumstances, someone without much education especially in the sciences or Law, combined with career interests that are modest at best and strong conservative moral values. Such a woman may not want to take the screening test at all and would have a difficult time comprehending the risks associated with the genetic makeup of the fetus(es) she is carrying and their implications. The challenge for the provider is to find a way to present enough information for the woman to make an informed choice without shutting down and essentially leaving the situation emotionally and intellectually, perhaps by leaving the burden of choice “in God’s hands.” Focusing only on the implications of the frame combinations’ becoming less mutually supportive, the possibilities for increased anxiety and tension escalate. So, for example, a woman with strong conservative moral values may also be well-­ educated and be intending to have a career. In such a situation, the dissonance among these frames may create much more anxiety and stress than would be the case with a different configuration. However, this is only half of the story, because in a counseling session, there are at least two people involved, patient and provider.

6.5 The Dyadic Counseling Session The essential common perspective that we bring to this review is to reinforce the assumption that medical care interactions involving the communication of risk cannot be understood without understanding the larger context within which women and their partners are making such decisions. A genetic counseling session at a minimum consists of two people, a patient and a provider [1]. Much progress has been made in the analysis and specification of how prenatal screening and genetic counseling sessions are being refined and elaborated [46]. Thoughtful discussions of context make an important point of reflection for such analyses [40]. Vassy et al. make a strong case that the nature of the national system reaches down several levels and shapes how it is that screening and diagnostic tests are presented with nuanced shades of positive or neutral sentiment by providers [28]. Decisions regarding testing are taking place in a situation composed of a pregnant women at a certain level of risk (e.g., determined variously by family history, genetic screening tests, maternal age, previous chromosomal problems, abnormal nuchal translucency, and structural abnormalities) whose wholeness cannot be captured by single attributes and a provider of some sort (fertility specialist, OBGYN, geneticist, MFM, midwife, genetic counsellor, and so on) who, similarly, cannot be portrayed as a onedimensional entity. So, following the general assumption that there will be a differential impact on the interactive decision process in different settings [5, 29, 39, 47], the norms, values, knowledge of testing and resources that could support

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alternative decisions, and empathic and social skills of both parties, as well as the characteristics of the area in which the interaction is taking place need to be considered in trying to understand and facilitate the degree of informed choice of the patient. In most professional guidelines [48, 49], there are implicit assumptions that have been developed to guide the conduct of counseling sessions regarding genetic tests. Obvious differences across patients, contextual resources, and cultures can be downplayed so as to arrive at a set of guidelines that will do pretty well serving everyone in their catchment areas. Even as such, a “one-size-fits-all” assumption of an underlying realist posture is easily disproven and readily acknowledged to be false. Those who follow the UNESCO statement of cultural diversity [50] differ in this respect because they are confronted by enormous diversity not only with respect to the kinds of support available in different societies for raising a child with disabilities, but also with respect to the legal constraints placed upon abortion and the availability of safe options for abortion. Analyzing how external influences affect patient and provider opinions and actions is difficult enough. Moving to the consideration of the interaction between them in routine medical visits, or something more formal that is supposed to achieve informed consent regarding tests or procedures, is immensely difficult. Some of the threats to sessions’ breaking down are internal; others are attributable to the systems within which the sessions are embedded. Conducting sessions “in the best of circumstances” implies patients who are open to considering risk statistics and able to understand them coupled with a medical professional who is both knowledgeable about risks and long-term implications as well as skilled at developing and maintaining rapport with their patients. In such circumstances, the presentation of what is essentially bad or life-changing news may still break down the communication order that may have been established between the medical professional and the patient. Bad news generates both emotion and coping mechanisms that undermine rapport and interfere with the presentation of information, ironically at the very time that a close bond and good communication are most desperately needed [51]. The continual evolution of counseling guidelines and training (especially in the area of training with respect to the delivery of bad news), coupled with additional resources devoted to continuing training of providers regarding advances and increasing complexity of screening and diagnostic tests, should be able to mitigate internal problems [1]. However, the need for continuing resources and attention that must be devoted to such efforts cannot be overstated, as the complexity and rate of change are only going to increase. One of the external contextual factors that may intrude is the sociopolitical conservatism of the area in which these sessions are conducted. If the area is liberal and cosmopolitan, the main concern may be that the screening tests take on a taken-for-­ granted credibility of being routine care, thus potentially undermining the very idea of informed choice [38]. Through our work with women carrying multiples, we were able to document different ways in which breakdowns may manifest themselves across people (between physician and patient and among physicians), places (fertility clinic, high-risk-care clinic, and hospital), and time (prepregnancy counseling with obstetricians and fertility specialists, early counseling and sonographs,

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discovery of the presence of multiples, and ongoing care options). So, for example, risk information that goes beyond the possibility of early delivery with increasing numbers of fetuses being carried can be very disturbing to some patients. Such may manifest as: discussions of risk and options may be extremely truncated as patients may not want to receive disturbing information that forces them to at least consider reduction as a possibility. Physicians may avoid confronting the facts of the situation since they are so used to having patients resist what they are being told and the selection of specialists for referral may not be on grounds of specialized knowledge (as opposed to how close to the patient the specialist is located or a physician who is likely to tell a patient what she wants to hear) [51]. Furthermore, sometimes records regarding the emerging and continuing problems have not been transferred properly from one clinic and physician to another, and preparations for delivery may as a result be underdeveloped and inappropriate given the risks inherent for a particular pregnancy. Further, clinics have refused to treat those who are seeking to reduce and/or treat them as if they were morally corrupt. There have been attempts by various licensing/regulatory agencies and medical staffs to sanction those physicians who have attempted to help women with certain reduction wishes (we had one such documented case in a Southern US state). There are countries (and now multiple states within the USA) where abortion is illegal, and obstetricians may not even have heard of selective reduction as an option for multiples (one such documented case in our sample). In short, the communication of risk information and options that should be realistically considered appears to be all-to-likely to break down in conservative countries and states, creating multiple and expanded versions of what Press and Browner (1993) called collective fictions (in which both parties to the communication exchange are consciously or unconsciously trying to avoid thinking of the pregnancy as being at great risk) [52]. In an analogous manner, more conservative areas (both in the USA and abroad in both developed and less well-developed areas) may become problematic with respect to several issues. Autonomy with respect to decision making, admittedly a Westernized concept, is a reflection of the relative power of women in their families. In more conservative areas, husbands and other relatives may have a greater say in such decisions. Providers as members of the local culture may be more likely to engage in a collective fiction with the family. The loser in such situations are those women with educational and career aspirations. In our view, this does not relieve the professional of the ethics burden of presenting information regarding procedural and genetic risks and discussing the longer-term implications of different choices. Second, more conservative areas are much more likely to have extreme constraints placed upon the possibility of obtaining a legal, safe abortion. This puts similar distortion pressure on the posture of both the provider and the patient and, save for those who have the money and freedom to go elsewhere, the possibility of informed choice may become almost a charade. Those most at risk here are women with the least financial resources. The potential intrusiveness of high levels of commercialization of NIPT cross cuts the contextual variation that exists in sociopolitical conservatism. Where the

6  Underpinnings of the Conundrum Between Genetic Screening and Testing Table 6.2  Drivers of patient’s choice

Nothing Religiosity “Red state”

NIPT Know fetal sex Avoid procedure

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Diagnostic procedure Prior poor outcome “Blue state” Want most information

belief exists that providers have what might politely be called “too close a connection” with testing companies, the level of trust of providers to have the interests of their patients as a top priority may be weakened [40]. This also now applies in the United States to health care systems that dictate referral patterns such that patients are directed to lesser qualified providers because they are in “the system,” and they are never told of higher quality, but unaffiliated providers. This may operate whether patients are inclined toward expecting guidance and yielding to it or patients are expecting a complete and thorough presentation and analysis of the specifics of their case. Granted that many of the biases in presentation cited in Tables 6.1 and 6.2 are anecdotal clinical observations, the prospects for biases appear too great to be ignored. In sum, informed consent may become problematic, and the process may become full of dilemmas in any number of ways. Some of these are traceable to women and their circumstances (which must be considered together), to providers and their circumstances (which must be considered together), and the cultural and the medical and nonmedical resource characteristics of the areas in which service is being provided [40]. With specific reference to the conduct of genetic information sharing at a moment in time when there have been revolutionary advances in the development of noninvasive screening tests, a key practical question involves the drop-off in invasive testing and how that phenomenon is related to the factors that may undermine informed consent in a counseling session. To simplify the discussion somewhat, we limit this discussion to the use of NIPT as a first-tier screening test for women who for a variety of reasons may be identified as being at higher risk. There are several points that have been raised regarding the impact of NIPT on the chances of seeking greater certainty through more invasive testing (Table 6.1): the more favorable and complete NIPT is presented as being and the more unfavorable and risky diagnostic tests are presented, the smaller the percentage of women who will likely choose to get more definitive information on their condition and that of their fetus. Guidelines, reviews of best practice, provider education, and specialization, workshops and grand rounds, as well as the chastening presence of litigation brought against providers and lab-test companies are part of a system that serves to shape a level of professionalism and uniformity in pretest counselling sessions. Such a system will be at least partially self-regulating, such as what happened in fertility clinics during a period in which women were choosing to have larger numbers of embryos implanted to increase the chances of a successful pregnancy. The side effect that there were larger numbers of women carrying four and five fetuses (thereby increasing their risk) eventually reversed as success rates (and knowledge in general) improved, suits were brought, guidelines were changed, and public attention was mobilized—all serving to provide negative feedback loops that kept the system from spiraling out of control [25, 44].

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6.6 Public Health Perspectives There are multiple conceptualizations as to the underpinnings of changes in the practice of genetic screening and testing. The public health community traditionally is known for focusing on the implementation of techniques to improve care. There is, in fact, a specific discipline of “implementation science” which has its own literature and paradigms for how to improve care [47]. There is also a field of Health Technology Assessment that seeks to provide useful, multidisciplinary assessments at several stages in the development of new technologies [48–52]. The major challenge for both systematic implementation and assessment in balancing innovation and quality on several dimensions is the market. As with NIPT, the market is great at innovation, but at what cost? [51]. Having a great program is useless, however, if too many patients cannot access it. For example, having a great first trimester screening program such as nuchal translucency and follow-up CVS for high-risk patients is of little use if patients do not come for care until 20 weeks gestation. This was exactly our problem when we were at Wayne State University/Hutzel hospital in the 1990s, and the average first visit of our clinic patients was near 20 weeks [53]. Clinic patients were much less likely to be able to make multiple visits which as a direct effect resulted in exasperating the disparities in care provided by both race and class. Likewise, the type of insurance the patient has might require prior authorization which usually cannot be done immediately—again building in delay and again reducing access to care and procedures if indicated [54]. In order to make important options available to patients, serious changes had to be made to the entire process of patient entry into care. The idea of reconceptualizing the entire flow, going back to the beginning, relies on what is referred to as basic systems theory and relies heavily on the work of Donella Meadows, a foundational contributor to the literature [54, 55]. A full description is beyond the scope of this piece, but a few key points are important. For example, very often too much effort is put on rescuing a process gone wrong at the very end rather than changing the thinking at the beginning (such as the NT example above). Using that example, there are patients who have an abnormal NT and need a CVS. A program that had built into its structure the ability to perform the CVS on the spot would have much higher uptake of that than if the patient has to make a second appointment which might entail her having to get another day off from work and/or arrange baby-sitting for other children. Again, this exposes a disparity issue. A second aspect using systems thinking is the fundamental understanding that system creates what it was built to create and not necessary what was designed to create. As such, systems change always involves unintended outcomes. The interactions of design, population, resources, severity of the problem, public and professional support to change, and resistance to such changes as public health change becomes more and more politicized are so complex that it is impossible to predict

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all of the downstream effects [56, 57]. Our example of the unintended consequences of fertility clinic’s transferring excess numbers of embryos (a convenient fiction, as clinics and patients have a joint interest in success rates) is relevant here, as the system did adjust due to the operation of several negative feedback loops. Similarly, we expect that NIPT will continue to grow, but we hope it will do so with fewer instances of excessive claims due to negative feedback loops presented by increased numbers of suits and investments in training and practice. This will, however, be a continuing challenge given the rate of change in genetic testing and the increasing complexity of making such knowledge clinically useful.

6.7 The Changing Landscape with Restrictions on Access to Abortion Recent United States Supreme Court rulings and the reasoning posited for them will have thrown the United States into legal and political chaos. It will take several years to fully play out, but it is clear that the situation in varying states within the US will be dramatically different. In the liberal (“blue”) states, there should be little to no change. In the conservative (“red”) states, patient who cannot afford to get themselves to blue states for care will be forced to carry even known genetically abnormal babies or those conceived by rape or incest. What will happen to the use and follow-up to genetic screening will remain to be seen. We hypothesize that the use of screening and testing will go down in the short term as both providers and patients may pretend there is nothing to see because nothing can be done. All of this is pure speculation, however, but the challenge to professional associations, clinicians, and policy makers will be enormous.

6.8 Conclusions As the pace and scope of new technologies expand geometrically, it is ever more critical than in the past to try to keep “the ball down the middle of the fairway,” i.e., to keep from getting into trouble because of unanticipated developments in its implementations. The only way to maximize such guidance is to have a nuanced, culturally and sociopolitically sensitive understanding of the drivers of why and how the medical community reacts and adopts or not new approaches which change the fundamental approach to particular problems. Genetics is at the forefront of technological advances in the practice of medicine. The time to think about establishing such a framework for evaluation is now.

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References 1. Shane Michaels H, Nazareth S, Tambini L. Genetic counseling in Evans MI. In: Johnson MP, Yaron Y, Drugan A, editors. Prenatal diagnosis: genetics, reproductive risks, testing, and management. New York: McGraw Hill Publishing Co.; 2006. p. 71–8. 2. Pryde PG, Odgers AE, Isada NB, Johnson MP, Evans MI. Determinants of parental decision to abort (DTA) or continue for non-aneuploid ultrasound detected abnormalities. Obstet Gynecol. 1992;80:296–9. 3. Evans MI, Pryde PG, Evans WJ, Johnson MP. The choices women make about prenatal diagnosis. Fetal Diagn Ther. 1993;8(suppl 1):70–80. 4. De Jong A, Dondorp WJ, Frints SGM, De Die-Smulders EM, De Wert GMWR. Advances in prenatal screening: the ethical dimension. Nat Rev Genet. 2011;12:657–63. 5. Dondorp W, de Wert G, Bombard Y, on behalf of the European Society of Human Genetics and the American Society of Human Genetics, et al. Non-invasive prenatal testing for aneuploidy and beyond: challenges of responsible innovation in prenatal screening. Eur J Hum Genet. 2015;23(11):1438–50. 6. Gregg AR, Aarabi M, Klugman S, Leach NT, Bashford MT, Goldwaser T, et  al. Screening for autosomal recessive and X-linked conditions during pregnancy and preconception: a practice resource of the American College of Medical Genetics and Genomics. Genet Med. 2021;23:1793–806. 7. Braun K, Könninger S.  Realizing responsibility. Institutional routines, critical intervention, and the “big” questions in the controversy over non-invasive prenatal testing in Germany. New Genet Soc. 2018;37(3):248–67. 8. ACOG Committee Opinion. Informed consent and shared decision making in obstetrics and gynecology. Obstet Gynecol. 2021;137:e34–41. 9. Evans MI. Overcoming militant mediocrity. Am J Obstet Gynecol. 2008;198:656–61. 10. Hook EB, Cross PK, Schreinemachers DM. Chromosomal abnormality rates at amniocentesis and in live born infants. JAMA. 1983;249:2034–8. 11. Brock DJ, Sutcliffe RG. Alpha-fetoprotein in the antenatal diagnosis of anencephaly and spina bifida. Lancet. 1972;2:197–9. 12. Merkatz IR, Nitowsky FM, Macri JN, Johnson WE.  An association between low maternal serum alpha-fetoprotein and fetal chromosome abnormalities. Am J Obstet Gynecol. 1984;148:886–94. 13. Chik L, Spencer K, Johnson MP, Ayoub M, Krivchenia EL, Dombrowski MP, Evans MI. Precise Gaussian distribution functions of maternal serum AFP and free bHCG for trisomy 21 (T21) biochemical screening. Am J Obstet Gynecol. 1997;177:882–6. 14. Nicolaides KH, Bindra R, Heath V, Cicero S. One-stop clinic for assessment of risk of chromosomal defects at 12 weeks of gestation. J Matern Fetal Neonatal Med. 2002;12:9–18. 15. Evans MI, Hallahan TW, Krantz D, Galen RS. Meta-analysis of first trimester down syndrome screening studies: free beta hCG significantly outperforms intact hCG in a multi-marker protocol. Am J Obstet Gynecol. 2007;196:198–205. 16. Henry GP, Britt DW, Evans MI. Screening advances and diagnostic choice: the problem of residual risk. Fetal Diagn Ther. 2008;23:308–15. 17. Evans MI, Drugan A, Koppitch FC, Zador IE, Sacks AJ, Sokol RJ. Genetic diagnosis in the first trimester: the norm for the 1990s. Am J Obstet Gynecol. 1989;160:1332–9. 18. Firth HV, Boyd P, Chamberlain P, et al. Severe limb abnormalities after chorion villus sampling at 56–66 days’ gestation. Lancet. 1991;337:726. 19. Froster UG, Jackson L. Limb defects and chorionic villus sampling: results from an international registry, 1992–1994. Lancet. 1996;347(9000):489–94. 20. Bianchi DW, Simpson JL, Jackson LG, Elias S, Holzgreve W, Evans MI, et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. Prenat Diagn. 2002;22:609–15.

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21. Pollack A. Biotech company fires chief and others over handling of data. New York: New York Times; 2009. p. 29. 22. Ehrich M, Deciu C, Zwiefelhofer T, Tynan JA, Casasan L, Tim R, et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting. Am J Obstet Gynecol. 2011;204:205e1–11. 23. Evans MI, Wapner RJ, Berkowitz RL. Non invasive prenatal screening or advanced diagnostic testing: caveat emptor. Am J Obstet Gynecol. 2016;215:298–305. 24. Evans MI, Evans SM, Bennett TA, Wapner RJ. The price of abandoning diagnostic testing for cell free fetal DNA screening. Prenat Diagn. 2018;38:243–5. 25. Evans MI, Andriole S, Curtis J, Evans SM, Kessler AA, Rubenstein AF.  The epidemic of abnormal copy number variants missed because of reliance upon reliance upon noninvasive prenatal screening. Prenat Diagn. 2018;38:730–4. 26. Carayon P, Bass E, Bellandi T, Gurses A, Hallback S, Mollo V. Sociotechnical systems analysis in health care: a research agenda. IIE Trans Healthc Syst Eng. 2011;1(1):145–60. 27. Ravitsky V, Roy M-C, Haidar H, et al. The emergence and global spread of noninvasive prenatal testing. Annu Rev Genom Hum Genet. 2021;22:309–38. 28. Vassy C, Rosman S, Rousseau B. From policy making to service use. Down’s syndrome antenatal screening in England, France and The Netherlands. Soc Sci Med. 2014;106:67–74. 29. Avgidou K, Papageroghiou A, Bindra R, Spencer K, Nicolaides KH. Prospective first trimester screening for trisomy 21 in 30,546 pregnancies. Am J Obstet Gynecol. 2005;192:1761–7. 30. Crombag NM, Vellinga YE, Kluijfhout SA, et al. Explaining variation in Down’s syndrome screening uptake: comparing The Netherlands with England and Denmark using documentary analysis and expert stakeholder interviews. BMC Health Serv Res. 2014;14:437–58. 31. Liehr T. Non-invasive prenatal testing, what patients do not learn may be due to lack of specialist genetic training by gynecologists and obstetricians. Front Genet. 2021;12:682980. https:// doi.org/10.3389/fgene.2021.682980. 32. Lewis C, Silcock C, Chitty LS. Non-invasive prenatal testing for Down’s Syndrome. Pregnant women’s views and likely uptake. Public Health Genom. 2013;16(5):223–32. 33. Haidar H, Vanstone M, Laberge A-M, et al. Cross-cultural perspectives on decision making regarding noninvasive prenatal testing: a comparative study of Lebanon and Quebec. AJOB Empir Bioeth. 2018;9(2):99–111. 34. Beaudet AL. Using fetal cells for prenatal diagnosis: history and recent progress. Am J Med Genet Part C Semin Med Genet. 2016;172C:123–7. 35. Dar P, Jacobsson B, MacPherson C, Egbert M, Malone F, Wapner RJ, et al. Cell-free DNA screening for trisomies 21, 18, 13  in pregnancies at low and high risk for aneuploidy with genetic information. Am J Obstet Gynecol. 2022;227(2):259.e1–259.e14. https://doi. org/10.1016/j.ajog.2022.01.019. 36. Kliff S, Bhatia A. When they warn of rare disorders, these prenatal tests are usually wrong. New York: New York Times; 2022. p. 1. 37. O’Brien BM, Dugoff L. What education, background and credentials are required to provide genetic counseling? Semin Perinatol. 2018;42:290–5. 38. Britt DW, Van Voris S, Jamil S, Gebb J, Rosner M, Evans MI. The impact of area conservatism on deviation from best practice: women choosing to undergo selective reduction. Int J Health Welln Soc. 2017;7:115–40. 39. Britt DW, Norton JD, Lowery C. Equity in the development of telemedical sites in an Arkansas high-risk pregnancy program. J Telemed Telecare. 2005;11:242–5. 40. Mozersky J, Ravitsky V, Rapp R, Michie M, Chandrasekharan S, Allyse M. Toward an ethically sensitive implementation of noninvasive prenatal screening in the global context. Hast Cent Rep. 2017;47(2):41–9. https://doi.org/10.1002/hast.690. 41. Di Mattei V, Ferrari F, Perego G, Tobia V, Mauro F, Candiani M. Decision-making factors in prenatal testing: a systematic review. Health Psychol Open. 2021;8(1):2055102920987455. https://doi.org/10.1177/2055102920987455.

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42. Chen A, Tenhunen H, Torkki P, Heinonen S, Lillrank P, Stefanovic V. Considering medical risk information and communicating values: a mixed-method study of women’s choice in prenatal testing. PLoS One. 2017;12(3):e0173669. 43. Evans MI, Andriole SA, Britt DW. Fetal reduction—25 years’ experience. Fetal Diagn Ther. 2014;35:69–82. 44. Britt DW, Evans WJG, Mehta SS, Evans MI.  Framing the decision: determinants of how women considering MFPR as a pregnancy-management strategy frame their moral dilemma. Fetal Diagn Ther. 2004;19:232–40. 45. Evans MI, Fletcher JC, Zador IE, Newton BW, Struyk CK, Quigg MH.  Selective first trimester termination in octuplet and quadruplet pregnancies: clinical and ethical issues. Obstet Gynecol. 1988;72:35–8. 46. Schmidlen T, Sturm AC, Hovick S, Scheinfeldt L, Scott Roberts J, Morr L, McElroy J, Toland AE, Christman M, O'Daniel JM, Gordon ES, Bernhardt BA, Ormond KE, Sweet K.  Operationalizing the reciprocal engagement model of genetic counseling practice: a framework for the scalable delivery of genomic counseling and testing. J Genet Couns. 2018;27(5):1111–29. https://doi.org/10.1007/s10897-­018-­0230-­z. 47. Peterson H, Haidar J, Fixsen D, Ramaswamy R, Weiner BJ, Leatherman S.  Implementing innovations in global women’s children’s and adolescents’ health: realizing the potential for implementation science. Obstet Gynecol. 2018;131:423–30. 48. ACOG Committee Opinion. Consumer testing for disease risk. Obstet Gynecol. 2021;137:e1–5. 49. Bean LJH, Scheuner MT, Murray MF, Biesecker LG, Green RC, Monoghan KG, et al. DNA-­ based screening and personal health: a points to consider statement for individuals and health care providers from the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2021;23:979–98. 50. UNESCO. Universal declaration on cultural diversity. Paris: UNESCO; 2002. 51. Frankel R.  Communicating with patients: research shows it makes a difference. Deerfield: MMI Risk Management Resources; 1994. 52. Press N, Browner CH. ‘Collective fictions’ similarities in reasons for accepting MSAFP screening among women of diverse ethnic and social class backgrounds. Fetal Diagn Ther. 1993;8:97–106. 53. Evans MI, Johnson MP. The history of fetal diagnosis and therapy: the Wayne State experience. Fetal Diagn Ther. 2002;17:321–30. 54. Meadows D.  Thinking in systems [edited by Diana Wright, Sustainability Institute]. White River Junction: Chelsea Green Publishing; 2008. 55. Meadows D. Thinking in systems—a primer. London: Earthscan Publishing; 2001. 56. Petersen HB, d’Arcangus C, Haider J, Curtis KM, Merialdi M, Gulmezoglu AM, et  al. Accelerating science driven solutions to challenges in global reproductive health: a new framework for moving forward. Obstet Gynecol. 2011;117:720–6. 57. Petersen HB, Haider J, Merialdi M, Guzmeroglu L, Fajans PJ, Mbizvo MT, et al. Preventing maternal and newborn deaths globally: using innovation and science to address challenges in implementing life-saving interventions. Obstet Gynecol. 2012;120:636–42.

Chapter 7

Epidemiology of Birth Defects in Twins Petya Chaveeva

, Maria Mar Gil

, and Kypros Herodotos Nicolaides

7.1 Introduction Fetal structural defects are more common in twins than in singleton pregnancies. Fetal structural defects in twins could be further divided into those that are specific for monochorionic twins and those that are similar for both twins and singleton pregnancies. The prevalence of structural defects per fetus in dizygotic, thus dichorionic twins, is the same as in singletons, whereas the rate in monozygotic twins is increased 2–3 times [1, 2]. Twins could be concordant (both fetuses being affected) or discordant (only one fetus is affected) for any given defect. Concordance of congenital malformations according to zygosity has been reported in a population of 3386 twin gestations where the rate was higher in MZ twins than in DZ twins, and second, malformations of the central nervous system, cardiovascular system, cleft lip/palate, and urinary system were specifically more common in MZ twins. The twinning process itself, the haemodynamic imbalance, and genetic and environmental factors could explain the higher prevalence of defects in MZ twins. These P. Chaveeva Fetal Medicine Unit, Dr. Shterev Hospital, Sofia, Bulgaria School of Medicine, Medical University of Pleven, Pleven, Bulgaria Fetal Medicine Research Institute, King’s College Hospital, London, UK M. M. Gil (*) Fetal Medicine Research Institute, King’s College Hospital, London, UK Obstetrics and Gynecology Department, Hospital Universitario de Torrejón, Madrid, Spain School of Medicine, Universidad Francisco de Vitoria (UFV), Madrid, Spain e-mail: [email protected] K. H. Nicolaides Fetal Medicine Research Institute, King’s College Hospital, London, UK e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_7

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underlying factors are responsible for midline defects such as neural tube defects, cardiac defects, abdominal wall defects, vascular disruptive disorders resulting in brain lesions, limb reduction defects, some cardiac defects, and urinary system defects. The intrauterine demise of one twin of the monochorionic pair is associated with ischemia, bleeding, or infarction that could either lead to the immediate death of the co-twin or brain injury or postnatal complications such as aplasia cutis congenita [3]. This chapter will discuss the epidemiology of structural defects in twin pregnancies. Further, we will describe the importance of the first-trimester scan in determining chorionicity and amnionicity, detecting structural defects, and using early markers to identify pregnancies at high risk for either structural defects or adverse pregnancy outcomes. Finally, we will discuss the unique complications related to monochorionic monozygotic twins. Screening for chromosomal abnormalities will be discussed in a separate chapter.

7.1.1 Zygosity and Chorionicity 7.1.1.1 Zygosity Twin pregnancies are different in relation to zygosity and chorionicity. Dizygotic twins (DZ) occur when there is a fertilization of two gametes that produce two placentas, two amniotic sacs, and two embryos. Monozygotic twin pregnancies (MZ) have different types of chorionicity depending on the stage at which there was a cleavage to form the two embryos. Suppose the cleavage occurs within 3 days after fertilization on the morula stage. In that case, the pregnancy will be monozygotic dichorionic, and if the cleavage occurs after the fourth day but before the eighth day of fertilization, the pregnancy is defined as monochorionic diamniotic. When the blastocyst is divided into the latest stage, this is the third type of monozygotic pregnancy which is monochorionic monoamniotic. Further splitting of the early embryo beyond the 13th day or after the formation of the embryonic disk classified the fourth type of monozygotic pregnancy as monoamniotic conjoined twins [4]. 7.1.1.2 Chorionicity The major impact on pregnancy outcome is associated with chorionicity rather than zygosity, and the best time to determine the pregnancy as monochorionic or dichorionic is by first trimester ultrasound examination [5]. At the time of the 11–14 weeks scan, the amnion and chorion have not fused yet, and therefore the presence of the lambda and T sign can be visualized by ultrasound. The thick 3 layers: two chorionic membranes in the middle and two amniotic membranes on each side, are recognized by ultrasound examination as a lambda sign in dichorionic twins and only two thin amniotic membranes as a T sign in monochorionic diamniotic twins (Fig. 7.1).

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b

Fig. 7.1 (a) Ultrasound image of a dichorionic diamniotic twin pregnancy. The arrow shows the lambda sign: a thick layer of fused chorionic membranes with 2 thin amniotic layers. (b) Ultrasound image of a monochorionic diamniotic twin pregnancy. The arrow shows the T sign: the fusion of two thin amniotic layers

In cases where the lambda sign is absent, the intertwin membrane should be specifically searched for to identify monoamniotic twins, which are the rare monochorionic twins. In this case, additional findings such as close insertions of the umbilical cords into the placenta and proximity of the two fetuses should give the clue of diagnosis [6].

7.2 First-Trimester Ultrasound Examination in Twin Pregnancies After the introduction of the nuchal translucency (NT) thickness as a marker for chromosomal abnormalities at 11–13  weeks, ultrasound examination in the first trimester has been adopted worldwide as a routine scan during pregnancy. The implication of the first-trimester ultrasound in multiple pregnancies is first to determine chorionicity and amnionicity, second, to propose correct management planning for these pregnancies, and third, to identify the subgroup of pregnancies that will benefit from prenatal treatment or invasive testing [7–9]. A large study reported the pregnancy outcome of 6225 twin pregnancies without structural and chromosomal abnormalities at 11–13 weeks. The study identified that about 80% of the twin pregnancies presenting for a scan in the first trimester are diagnosed by ultrasound assessment as dichorionic, 20% as monochorionic diamniotic, and less than 1% as monochorionic monoamniotic. The adverse pregnancy outcome has been reported to differ among all three types of twins. As such, the risk of death between the first trimester and 24 weeks is about 8% for a monochorionic diamniotic pair, which is 4-times higher than the 2% risk of a dichorionic twin pregnancy and very much increased for a monoamniotic twin pregnancy of 20%. Although most deaths occur before 24  weeks, even after viability monochorionic twins remain at

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significantly increased risk for perinatal mortality and morbidity, and this excess can, to a great extent, be explained by the development of twin-to-­twin transfusion syndrome (TTTS), selective fetal growth restriction (sFGR), and twin anemia-polycythemia sequence (TAPS). The preterm birth rate before 34 weeks of gestation is 10 times higher in DC twins versus singleton pregnancies and 15 times higher in MC twins. Therefore, twin pregnancies have a contribution of 15% for all preterm births. The risk of small for gestational age in one or both babies is about 30% among the three groups of twins 10. The same is true for preeclampsia (PE), where the rate of preterm PE (the one that, due to its severity, requires delivery before 37 weeks) is 9 times higher in twins compared to singletons [10]. The adverse pregnancy outcome is an independent risk factor for prematurity and the consequences for the long-term outcome.

7.3 First Trimester Markers for Structural Defects 7.3.1 First-Trimester Detection of Fetal Structural Defects A recent large series showed the incidence of structural defects in twins at 11–13 weeks based on chorionicity. The results showed that the rate of structural defects was higher in MC (2.8%) than in DC (1.3%) twins. Ultrasound examination in the first trimester performed by standardized protocols can detect 35.6% of defects, including 27.1% of those in fetuses of DC twin pregnancies and 52.6% of those in MC twin pregnancies. This study demonstrated that the pattern of detectability (i.e., always detectable, never detectable, or sometimes detectable) is the same as in singleton pregnancies. The major fetal defects, including acrania, alobar holoprosencephaly, encephalocele, pentalogy of Cantrell, exomphalos, body-stalk anomaly, twin reversed arterial perfusion (TRAP) sequence, and conjoined twins, are always detectable (Fig. 7.2). However, many other anomalies, such as cleft lip, diaphragmatic hernia, structural heart defects, and renal agenesis, may also be detectable in the first trimester but with less accuracy. This study reported the detection of about 40% of the cases with open spina bifida, 33% of the cases with cleft lip and palate, 50% of the cases with congenital diaphragmatic hernia, 52% of the major congenital heart defects (CHD) like tetralogy of Fallot, hypoplastic left heart syndrome, arch defects, tricuspid atresia or complex heart defects, 71% of the lower urinary tract obstruction, 67% of the cases with lethal skeletal dysplasia, fetal akinesia deformation sequence, or absence of extremities. On the other hand, anomalies such as severe ventriculomegaly, hypoplastic cerebellum or vermis, congenital pulmonary airway malformations, atrioventricular or ventricular septal defect, transposition of the great arteries, aortic or pulmonary stenosis/atresia, double or right aortic arch, arrhythmia, rhabdomyoma, hemivertebra, duodenal atresia or bladder exstrophy, defects of digits, deformities of wrists or talipes, lymphangioma or sacrococcygeal teratoma were not detected in the first trimester. The most likely explanation is that most of these defects are not yet visible in the first trimester [12].

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7.3.2 Nuchal Translucency Thickness (NT) An increased NT thickness is a marker not only for chromosomal abnormalities, but also for structural defects. The incidence of structural defects and increased NT has been examined in singleton and twin pregnancies [11, 12]. A large study reported a population of 12,732 fetuses from twin gestations where 5.0% had fetal NT ≥95th percentile and 1.4% had NT ≥99th percentile. The incidence of fetal NT ≥95th percentile was higher in those with than in those without a defect (16.5% vs. 4.5% in DC twins and 19.2% vs. 5.9% in MC twins) and this was also true for NT ≥99th percentile (8.3% vs 1.0% in DC twins and 15.4% vs. 2.0% in MC twins). The overall incidence of increased NT ≥95th percentile and structural defects was reported at about 17% for those with structural defects and 4.7% for those without defects [12].

7.3.3 Crown-Rump Length (CRL) In both DC and MC twin pregnancies, the incidence of CRL discordance ≥10% and ≥15% is higher in those with than in those without defects. In the total population of twin pregnancies, the percentage with a fetal defect is about 9.0% in those with CRL discordance ≥10% and 2.6% in those with CRL discordance 99

NT nuchal translucency, DC dichorionic, MC monochorionic

False positive rate (%) 65 8 6 (DC), 9 (MC) 5 0.05

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8.1.1 Screening for Aneuploidies in Twin Pregnancies Prenatal diagnosis is more challenging in twin pregnancies than in singleton pregnancies for several reasons, including: (1) the techniques of invasive testing may give uncertain results or may be linked to higher miscarriage risks; (2) the fetuses may be discordant for the abnormality; (3) the various screening methods described for singletons need specific corrections and their performance may be lower; and (4) the number of affected cases studied in multiple pregnancies is much lower than that in singletons; therefore, estimates of test performance will always be less accurate [8–14]. The two fetuses in a monozygotic pregnancy are genetically identical (with few exceptions resulting from post-zygotic alterations); hence, they are either affected or unaffected by the trisomy [15]. Contrarily, dizygotic pregnancies, which are always genetically different, have two fetuses where one is likely to have the aneuploidy and the other likely not. Dizygotic pregnancies will receive an individual risk for each fetus, with the risk of aneuploidy for each fetus being more or less independent of the risk for the other (although NT measurements in both fetuses are somewhat correlated) [16]. On the other hand, when screening monozygotic pregnancies, an overall risk assessment is provided for the entire pregnancy. Yet only DNA fingerprinting, which necessitates amniocentesis, chorionic villus sample, or cordocentesis, can determine zygosity. As all monochorionic (MC) twins are monozygotic and around 90% of dichorionic (DC) twins are dizygotic, chorionicity can be indirectly identified by ultrasound and is employed as a proxy for zygosity in prenatal diagnosis. Dizygotic twins are always DC. Contrarily, depending on when the embryo splits, one-third of monozygotic twins are DC and two-thirds are MC [17]. The risk of structural abnormalities is higher in MC twins (as it was previously discussed in Chap. 7), but the risk of aneuploidy is the same as the risk in singleton pregnancies [17–19]. Contrarily, in dizygotic pregnancies, the risk of structural abnormalities and the maternal age-related risk for chromosomal abnormalities are the same for each twin as in singleton pregnancies. As a result, there is roughly twice-as-high likelihood that at least one fetus will be affected by a chromosomal defect as compared to singleton pregnancies [17]. In twin pregnancies, fetal NT thickness and maternal age can be used to effectively screen for trisomies. The higher false positive rate associated with NT screening in MC pregnancies is due to discordant NT thickness among fetuses, which is thought to be an early sign of twin–to–twin transfusion syndrome [20]. Compared to singleton pregnancies, twin pregnancies have different blood concentrations of the biochemical markers utilized for risk assessment in the first or second trimester, reflecting the existence of two fetuses as opposed to one. Moreover, the chorionicity of the fetus affects the concentration of these serum biomarkers. For instance, maternal blood concentrations in twin pregnancies are around double those in singleton pregnancies at 11–13 weeks of gestation, with a more noticeable difference in DC twins than in MC twins [8–10]. As a result, chorionicity needs to be adjusted when determining the risk of aneuploidies in twin pregnancies using the

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first-trimester combined test or the second-trimester triple or quadruple test. It is important to remember that biochemical markers are not fetus-specific; therefore, a defect in one fetus may be covered up by the normal one and vice versa (Fig. 8.1). In cfDNA testing, the relative proportion of the fetal to maternal origin of the cfDNA determines the capacity to detect the slight increase in the amount of a particular chromosome in maternal plasma in a trisomic compared to a disomic pregnancy. Fetal fraction refers to the percentage of cfDNA from the fetus in the mother’s blood. It is more challenging to distinguish between aneuploid and euploid pregnancies when the fetal percentage is low. CfDNA testing is generally more complex in twin pregnancies than in singleton pregnancies because, although the two fetuses may be monozygotic, which means they are genetically identical, they may also be dizygotic, which means that only one fetus is likely to have the aneuploidy when it is present. As a result, in monozygotic twins, the mother’s blood has a distinct “fetal” genome, and the relative amount of DNA derived from each infant is unimportant because the overall fetal fraction suffices to produce a result. Contrarily, with dizygotic twins discordant for aneuploidy, each fetus must contribute sufficiently (typically about 4%) to the maternal circulation to detect the anomaly of the affected twin, which makes the analysis far more challenging. It is known that each fetus sends a unique amount of cfDNA into the mother’s bloodstream, differing by almost a twofold [12]. A high contribution from the disomic co-twin could result in a good overall fetal fraction, masking the co-twin’s abnormality and resulting in an incorrect result of low risk for aneuploidy (Fig.  8.1). Two approaches have been suggested to eliminate this potential error: first, calculate the fetal fraction for each twin and use the lower of the two to estimate the risk; second, raise the fetal fraction reporting threshold from 4 to 8%. Unfortunately, these methods will inescapably result in a higher percentage of unsuccessful tests, meaning not giving a conclusive result. The performance of cfDNA testing for trisomy 21  in twin pregnancies is comparable to that in singletons, although the number of affected cases studied is a

b

c

Fig. 8.1  Number of counts for chromosome 21 in singletons (a), monochorionic (MC) (b), and dichorionic (DC) (c) twin pregnancies. Full black diamonds represent maternal cfDNA units. Empty diamonds and full yellow diamonds represent fetal units. Full pink diamonds represent fetal trisomy 21 (extra material present) units. D21 disomy 21, T21 trisomy 21, MC monochorionic, DC dichorionic

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much less than that for singletons. Nevertheless, there is insufficient scientific evidence to adequately assess how well the test performs in cases other than trisomy 21 [12, 13].

8.2 Performance and Clinical Considerations of the Different Screening Methods 8.2.1 Screening by Maternal Age There is less direct data on the prior maternal age-specific risk for twin pregnancies than there is for singletons. A twin pregnancy would have twice the chance of having a fetus with trisomy 21 than a singleton pregnancy if the risk of trisomy 21 for each fetus were fully independent of the risk of the other. But the risk is just slightly less than double (around 1.6) because, first, monozygotic pregnancies are concordant for aneuploidy and, second, because NT values in euploid twins are somehow connected even after taking sonographer into account [16, 21]. Based on maternal age, the risk for aneuploidies for the entire pregnancy is the same in MC pregnancies as in singleton pregnancies since both fetuses are presumed to be genetically identical. However, the standard 35-year-old cutoff used to designate the high-risk group by maternal age alone in singleton pregnancies (risk cut-off: 1  in 250 at 12 weeks of gestation) has been estimated to correlate to roughly 32-years-old in a DC twin pregnancy (modeling charts). Screening for aneuploidies in twin pregnancies based solely on maternal age should be discouraged for a number of reasons, including firstly, a disproportionally higher screened positive rate of about 65% that results from lowering the cut-off to define the high-risk group in a patient population with an already higher proportion of elderly women, second, a higher miscarriage rate linked to invasive diagnostic procedures in these pregnancies [14], and third, the likely overestimation of the risk from theoretical models where the observed incidence of trisomy is lower than that predicted [22].

8.2.2 Screening by Nuchal Translucency In twin pregnancies, effective screening for chromosomal abnormalities is provided by a combination of maternal age and fetal NT thickness. The optimal time for measuring NT thickness is between 11+0 and 13+6 weeks of gestation, when the fetal crown-rump length ranges from 45 to 84 mm. Fetal NT thickness increases with crown-rump length and a measurement above the 95th percentile detects about 75–80% of trisomy 21 fetuses both in singleton and multiple pregnancies [9, 23]. Additionally, since NT thickness is independent of maternal age, data from these two parameters can be

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combined to increase the DR of trisomy 21 up to 80–85% at the same FPR of 5% in singleton pregnancies while achieving a similar DR in twins at the cost of a higher FPR of about 8%, probably as a result of the recipient’s fetal heart failure in MC pregnancies complicated by early twin–to–twin transfusion syndrome [10, 20, 23].

8.2.3 Screening by First Trimester Combined Test The addition of maternal serum biochemistry may enhance screening accuracy by fetal NT thickness and maternal age in the first trimester when adjustments for chorionicity are made as necessary. The levels of maternal serum free β-hCG and PAPP-A in DC pregnancies are twice as high as those in singleton pregnancies at 11+0 to 13+6 weeks’ gestation [8–10]. These levels are also higher in MC twins than in singletons, but lower than those in DC twins [8–10]. The likelihood ratios for PAPP-A and free β-hCG can be added to the first-­ trimester risk assessment by maternal age and fetal NT in DC twins, which can increase the DR for fetal trisomy 21 up to 90% and decrease the FPR to 6–8%, which is comparable to the screening performance of the first-trimester combined test in singleton pregnancies [10, 24]. However, the DR of fetal aneuploidy in MC twins, which is between 85 and 90% for trisomy 21, and the FPR, which stays at around 9%, do not seem to be massively improved by the addition of biochemical markers [6]. In MC twins, risks for trisomy 21 are first calculated for each fetus using maternal age, fetal NT thickness, and crown-rump length and serum levels of free β-hCG and PAPP-A corrected by chorionicity. The overall pregnancy risk is then estimated by taking the average of the two risks [9, 17, 21]. However, in DC twins, the risks for trisomy 21 are calculated in the same way, but provided for each fetus individually [9, 17, 21]. For the risk assessment, it is possible to apply the presumption that the two pregnancies are independent or, alternatively, to account for the correlation in NT values between fetuses [9, 21]. Although the estimated patient-specific risk is significantly impacted by this metric, screening performance as a whole will not be significantly affected [21].

8.2.4 Screening by Second-Trimester Serum Biomarkers Many second-trimester blood indicators have been added to enhance the effectiveness of screening for trisomy 21 by maternal age alone, beginning at 14 weeks of gestation. At a fixed FPR of 5% for all combinations, the DR for singleton pregnancies is approximately 60–65% when maternal age, serum alpha-fetoprotein, and free β-hCG are combined (double test), approximately 65–70% when unconjugated estriol is added (triple test), and approximately 70–75% when inhibin A is added (quadruple test) [25]. However, for the same FPR of 5%, the highest DR obtained by this approach in twin pregnancies is only about 45–50% [25].

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One limitation of screening for trisomy 21 by serum biomarkers alone in twins is that the pregnancy is classified as high-risk as a whole without any indication of which fetus may be affected, unlike ultrasound markers, which are fetus-specific.

8.2.5 Screening by Cell-Free DNA Testing Without a maximum gestational age restriction, analysis of cfDNA testing in maternal blood for screening of aneuploidies should not be carried out before 10 weeks of pregnancy. As several clinical validation and implementation studies have demonstrated over the past 15 years, it is currently advised to use this method as first-­ line screening or as contingent screening in response to the findings of another screening method that has already been used. The performance of cfDNA testing for trisomy 21 in twin pregnancies is comparable to that found in singletons, with a DR of approximately 99.0% and an FPR of 0.02% [13]. The same figures for trisomy 18 are 92.3% DR and 0.01% FPR, and for trisomy 13, they are 94.7% DR and 0.10% FPR, although the number of cases reported in the literature is still insufficient to properly evaluate the performance of cfDNA screening for these trisomies [13]. The use of cfDNA testing to screen other aneuploidies or genetic conditions is not sufficiently validated and, therefore, not recommended. Despite its effectiveness, cfDNA testing is still only a screening test; thus a high-­ risk result still has to be confirmed by invasive testing, and a negative result, while comforting, does not necessarily mean that the pregnancy is unaffected. The two main concerns arising from cfDNA screening for trisomy 21 in twin pregnancies relate to first, the limited available data, and second, the increased rate of tests that do not yield a result (no-result rate) in DC twins compared to singleton pregnancies (up to 5 times higher) [11]. Twinning and the requirement to be particularly careful with the minimal fetal fraction required to assure that accurate results are two of the key explanations for this increased no-result rate in DC twins. Moreover, the use of assisted reproductive techniques for pregnancy conception, which is much more common in twin than in singleton pregnancies, is another risk factor for an increased no-result rate that has repeatedly been linked to a lower fetal fraction and, consequently, to an increased no-result rate. Increasing maternal weight, nulliparity, certain ethnic origins, such as Afro-Caribbean or South Asian, and characteristics connected to a smaller placental size, such as earlier gestational age or low serum levels of free β-hCG and PAPP-A, are additional risk factors for not receiving a result following testing [11]. Likewise, aneuploidies related to a small placenta, such as trisomies 18 and 13 and digynic triploidy but not trisomy 21, have a lower fetal fraction. Therefore, they are also associated with an increased no-result rate. To rule out structural defects linked to any of the aforementioned conditions, careful ultrasound assessment of the fetal anatomy must be performed when a result from cfDNA testing is not obtained. If those are excluded, repeating the test is a sensible management option, and a result will be obtained in the second draw in about 60% of cases [11]. In those cases where

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Fig. 8.2  Pregnancy management according to cfDNA results. T21 trisomy 21

a result is not reported after the second testing, other risk factors or screening tests must be considered to help decide if invasive diagnostic testing is advisable (Fig. 8.2). No-result rate should therefore be included in the pretest counseling along with DR and FPR since it is a major element of the test screening performance.

8.2.6 Screening of Aneuploidies in the Presence of a Vanishing Twin Since placental materials from the interrupted pregnancy remain in the maternal circulation for several weeks, the presence of a vanishing twin may complicate screening utilizing maternal serum biomarkers or cfDNA tests [26–28]. The cfDNA fetal fraction of the deceased fetus increases in maternal blood after the demise, peaking about 7–9 weeks later, to decrease gradually over gestation to almost become undetectable [27]. cfDNA analysis detects the presence of DNA material from the vanished twin up to 15–16 weeks after demise [26, 27]. The performance of screening by cfDNA testing in the presence of a vanishing twin is uncertain; however, it may diminish DR and raise false positive and no-result rates due to the lingering material from the deceased twin in the maternal circulation. Without new scientific evidence, cfDNA testing should not be offered in vanishing-twin pregnancies.

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An alternative to cfDNA testing in these cases is the first-trimester combined test. Although it was generally recommended not to use serum biomarkers when there is a measurable fetal pole [29], it has been recently reported that, unlike PAPP-A levels which are consistently increased in vanishing-twin pregnancies even if only an empty sac is visible, free β-hCG levels are not significantly different from those in normal singleton pregnancies, regardless of the time the fetal demise occurred [30]. This knowledge may allow performing first-trimester screening by combining maternal age, fetal NT thickness, and free β-hCG in these pregnancies, increasing DR from 80 to 85% compared to screening by maternal age and NT alone at 5% FPR [30].

8.3 Conclusions • For dichorionic pregnancies, an individual risk is provided for each fetus, whereas for monochorionic pregnancies, an overall risk assessment is provided for the entire pregnancy. • The risk of aneuploidy in monochorionic pregnancies is comparable to the risk in singleton pregnancies; however, in dizygotic pregnancies, the likelihood that at least one fetus would have a chromosomal abnormality is around twice as high as in singletons. • The first-trimester combined test in twin pregnancies shows a similar detection rate to singleton pregnancies, of about 90%, but at a higher FPR (6% for dichorionic twins and 9% for monochorionic twins). Only a DR of about 45% at a 5% false positive rate may be achieved for trisomy 21 screening using the second-­ trimester quadruple test. • Maternal blood cfDNA analysis for trisomy 21 screening in twin pregnancies performs similarly to singleton pregnancies, with a DR of 99.0% at FPR of 0.02%. Information on further aneuploidies and genetic disorders is scarce. • It is advised to screen by maternal age and fetal NT thickness (+/− free β-hCG) in cases of vanishing twins with or without detectable fetal poles since maternal blood contamination by placental materials from the aborted pregnancy may alter serum biomarker levels and cfDNA analyses.

References 1. Santorum M, Wright D, Syngelaki A, Karagioti N, Nicolaides KH.  Accuracy of first-­ trimester combined test in screening for trisomies 21, 18 and 13. Ultrasound Obstet Gynecol. 2017;49:714–20. 2. Gil MM, Accurti V, Santacruz B, Plana MN, Nicolaides KH. Analysis of cell-free DNA in maternal blood in screening for aneuploidies: updated meta-analysis. Ultrasound Obstet Gynecol. 2017;50:302–14. 3. van der Meij KRM, Sistermans EA, Macville MVE, Stevens SJC, Bax CJ, Bekker MN, Bilardo CM, Boon EMJ, Boter M, Diderich KEM, de Die-Smulders CEM, Duin LK, Faas

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BHW, Feenstra I, Haak MC, Hoffer MJV, den Hollander NS, Hollink IHIM, Jehee FS, Knapen MFCM, Kooper AJA, van Langen IM, Lichtenbelt KD, Linskens IH, van Maarle MC, Oepkes D, Pieters MJ, Schuring-Blom GH, Sikkel E, Sikkema-Raddatz B, Smeets DFCM, Srebniak MI, Suijkerbuijk RF, Tan-Sindhunata GM, van der Ven AJEM, van Zelderen-Bhola SL, Henneman L, Galjaard RH, Van Opstal D, Weiss MM, Dutch NIPT Consortium. TRIDENT-2: National implementation of genome-wide non-invasive prenatal testing as a first-tier screening test in The Netherlands. Am J Hum Genet. 2019;105:1091–101. 4. Nicolaides KH, Syngelaki A, del Mar GM, Quezada MS, Zinevich Y. Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood. Fetal Diagn Ther. 2014;35:212–7. 5. Chitty LS, Mason S, Barrett AN, McKay F, Lench N, Daley R, Jenkins LA.  Non-invasive prenatal diagnosis of achondroplasia and thanatophoric dysplasia: next-generation sequencing allows for a safer, more accurate, and comprehensive approach. Prenat Diagn. 2015;35:656–62. 6. Smith-Jensen S, Permin M, Philip J, Lundsteen C, Zachary JM, Fowler SE, Grüning K. Randomised comparison of amniocentesis and transabdominal and transcervical chorionic villus sampling. Lancet. 1992;340:1237–44. 7. Evans MI, Goldberg JD, Dommergues M, Wapner RJ, Lynch L, Dock BS, et  al. Efficacy of second-trimester selective termination for fetal abnormalities: international collaborative experience among the world’s largest centers. Am J Obstet Gynecol. 1994;171:90–4. 8. Spencer K, Kagan KO, Nicolaides KH. Screening for trisomy 21 in twin pregnancies in the first trimester: an update of the impact of chorionicity on maternal serum markers. Prenat Diagn. 2008;28:49–52. 9. Spencer K.  Screening for trisomy 21  in twin pregnancies in the first trimester using free beta-hCG and PAPP-A, combined with fetal nuchal translucency thickness. Prenat Diagn. 2000;20:91–5. 10. Madsen HN, Ball S, Wright D, Tørring N, Petersen OB, Nicolaides KH, Spencer K. A reassessment of biochemical marker distributions in trisomy 21-affected and unaffected twin pregnancies in the first trimester. Ultrasound Obstet Gynecol. 2011;37:38–47. 11. Galeva S, Gil MM, Konstantinidou L, Akolekar R, Nicolaides KH. First-trimester screening for trisomies by cfDNA testing of maternal blood in singleton and twin pregnancies: factors affecting test failure. Ultrasound Obstet Gynecol. 2019;53:804–9. 12. Gil MM, Galeva S, Jani J, Konstantinidou L, Akolekar R, Plana MN, Nicolaides KH. Screening for trisomies by cfDNA testing of maternal blood in twin pregnancy: update of the Fetal Medicine Foundation results and meta-analysis. Ultrasound Obstet Gynecol. 2019;53:734–42. 13. Judah H, Gil MM, Syngelaki A, Galeva S, Jani J, Akolekar R, Nicolaides KH. Cell-free DNA testing of maternal blood in screening for trisomies in twin pregnancy: updated cohort study at 10–14 weeks and meta-analysis. Ultrasound Obstet Gynecol. 2021;58:178–89. 14. Gil MM, Rodríguez-Fernández M, Elger T, Akolekar R, Syngelaki A, De Paco MC, Molina FS, Gallardo Arocena M, Chaveeva P, Persico N, Accurti V, Kagan KO, Prodan N, Cruz J, Nicolaides KH. Risk of fetal loss after chorionic villus sampling in twin pregnancy derived from propensity score matching analysis. Ultrasound Obstet Gynecol. 2021;59(2):162–8. https://doi.org/10.1002/uog.24826. 15. De Paepe ME.  Chapter 5: Multiple gestation: the biology of twinning. In: Lockwood C, et al., editors. Creasy and Resnik’s maternal-fetal medicine: principles and practice. 8th ed. Amsterdam: Elsevier; 2018. p. 68–80.e2. 16. Wøjdemann KR, Larsen SO, Shalmi AC, Sundberg K, Tabor A, Christiansen M. Nuchal translucency measurements are highly correlated in both mono- and dichorionic twin pairs. Prenat Diagn. 2006;26:218–20. 17. Nicolaides KH. The 11–13 weeks scan. 2nd ed. New Delhi: The Fetal Medicine Foundation; 2004. https://www.fetalmedicine.org/education/the-­11-­13-­weeks-­scan 18. Baldwin VJ. Anomalous development of twins. In: Baldwin VJ, editor. Pathology of multiple pregnancy. New York: Springer; 1994. p. 169–97. 19. Burn J. Disturbance of morphological laterality in humans. Ciba Found Sym. 1991;162:282–96.

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20. Sebire NJ, Snijders RJ, Hughes K, Sepulveda W, Nicolaides KH. Screening for trisomy 21 in twin pregnancies by maternal age and fetal nuchal translucency thickness at 10–14 weeks of gestation. Br J Obstet Gynaecol. 1996;103:999–1003. 21. Wright D, Syngelaki A, Staboulidou I, de Cruz JJ, Nicolaides KH.  Screening for trisomies in dichorionic twins by measurement of fetal nuchal translucency thickness according to the mixture model. Prenat Diagn. 2011;31:16–21. 22. Audibert F, Gagnon A, Genetics Committee of the Society of Obstetricians and Gynaecologists of Canada; Prenatal Diagnosis Committee of the Canadian College of Medical Geneticists. Prenatal screening for and diagnosis of aneuploidy in twin pregnancies. J Obstet Gynaecol Can. 2011;33:754–67. 23. Nicolaides KH. Screening for fetal aneuploidies at 11–13 weeks. Prenat Diagn. 2011;31:7–15. 24. Prats P, Rodríguez I, Comas C, Puerto B. Systematic review of screening for trisomy 21 in twin pregnancies in first trimester combining nuchal translucency and biochemical markers: a meta-analysis. Prenat Diagn. 2014;34:1077–83. 25. Cuckle H. Down’s syndrome screening in twins. J Med Screen. 1998;5:3–4. 26. Niles KM, Murji A, Chitayat D.  Prolonged duration of persistent cell-free fetal DNA from vanishing twin. Ultrasound Obstet Gynecol. 2018;52:547–8. 27. Chen M, Su F, Wang J, Zhou L, Liu Q, Chai X, Yuan Y, Cen M, Wu Y, Wang Y, Chen F, Zhang Y, Chen D, Gao Y. Temporal persistence of residual fetal cell-free DNA from a deceased cotwin after selective fetal reduction in dichorionic diamniotic twin pregnancies. Prenat Diagn. 2021;41:1602–10. 28. Zou Y, Cui L, Xue M, Yan J, Huang M, Gao M, Gao X, Gao Y, Chen ZJ. Applications of noninvasive prenatal testing in vanishing twin syndrome pregnancies after treatment of assisted reproductive technology in a single center. Prenat Diagn. 2021;41:226–33. 29. Khalil A, Rodgers M, Baschat A, Bhide A, Gratacos E, Hecher K, Kilby MD, Lewi L, Nicolaides KH, Oepkes D, Raine-Fenning N, Reed K, Salomon LJ, Sotiriadis A, Thilaganathan B, Ville Y. ISUOG Practice Guidelines: role of ultrasound in twin pregnancy [Erratum in: Ultrasound Obstet Gynecol 2018; 52:140.]. Ultrasound Obstet Gynecol. 2016;47:247–63. 30. Chaveeva P, Wright A, Syngelaki A, Konstantinidou L, Wright D, Nicolaides KH.  First-­ trimester screening for trisomies in pregnancies with vanishing twin. Ultrasound Obstet Gynecol. 2020;55:326–31.

Part II

Noninvasive Diagnosis

Chapter 9

Congenital Anomalies: The Role of Ultrasound Valentina Tsibizova, Tatyana Pervunina, Veronika Artemenko, Arun Meyyazhagan, and Graziano Clerici

9.1 Introduction Prenatal ultrasound at 11–13  weeks’ gestation is used widely to determine gestational age, fetal number, multiple pregnancy and chorionicity, cardiac activity, placental location, diagnosis of significant fetal anomalies, and aneuploidy screening. Another recent indication that is likely to become widespread is screening for preterm preeclampsia because the preventive aspirin administration substantially reduces the risk for this pregnancy complication in a high-risk group [1]. As a screening tool in obstetrics, ultrasonography identifies fetal structural abnormalities in approximately 3–5% of pregnancies. Since the 1990s, prenatal risk of aneuploidy has consisted of maternal serum analyses with an evaluation of nuchal translucency (NT) in the first trimester. With noninvasive prenatal screening in 2011, the model of prenatal screening has evolved. Nevertheless, ultrasonography has an essential part in prenatal diagnosis [2].

V. Tsibizova (*) Almazov National Research Centre, Laboratory of Maternal Fetal Medicine, St. Petersburg, Russia CEMER Eureopan Centre for Medicine and Research, Perugia, Italy T. Pervunina · V. Artemenko Almazov National Research Centre, Laboratory of Maternal Fetal Medicine, St. Petersburg, Russia A. Meyyazhagan University of Perugia, Perugia, Italy Perinatology Research Branch, Wayne State University, Detroit, MI, USA G. Clerici CEMER Eureopan Centre for Medicine and Research, Perugia, Italy © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_9

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9.2 Marker of Chromosomal Abnormalities 9.2.1 Nuchal Translucency NT refers to the clear space in tissue folds behind a developing fetal neck. Its thickness is measured routinely during the first-trimester screening when the crown-­ rump length (CRL) is 45–84 mm, corresponding to approximately 11–14 weeks of gestation [3]. Enlarged NT is associated with increased risks for aneuploidy (trisomies 21, 18, 13), genetic syndromes, and structural abnormalities (particularly congenital heart disease), which, in turn, increase the risk for miscarriage, hydrops, fetal demise, and neonatal death. NT measurement normally increases with gestational age. The most commonly used thresholds for diagnosis of improved NT are the 95th and 99th percentiles. An increase of NT above them for gestational age measured by CRL increases the risk of aneuploidy. In one study, the incidence of chromosomal abnormalities with NT between 3.3 mm (the 95th percentile) and 3.4 mm, and NT 8.5 mm or more increased from 7 to 75%, respectively [4]. Moreover, most fetuses with trisomy 21 had NT less than 4.5 mm, most fetuses with trisomies 13 or 18 had NT between 4.5 and 8.4 mm, and those with Turner syndrome had 8.5 mm or more. Considering the importance of correct ultrasound NT measurement, a good midsagittal section of the fetus in the neutral position with only the fetal head and upper thorax in the image should be obtained [5]. The maximum thickness of the subcutaneous translucency between the skin and the soft tissue overlying the cervical spine should be measured [6]. The best interpretation of NT is combined with maternal age and -trimester serum markers to provide apatient-specific risk of having common autosomal trisomies. Importantly, fetuses with cystic hygroma or significantly enlarged NT are at particularly high risk of aneuploidy. A cystic hygroma is defined as a thin-walled, subcutaneous mass filled with lymphatic fluid and usually occurring in the posterior neck. Cystic hygromas may be septated or simple (non-septated). Aneuploidy appears to be more frequent with septated than simple cystic hygromas. Thus, the frequency of aneuploidy with septated and simple cystic hygromas was 57% and 21%, respectively [6]. The incidence of septated cystic hygroma in the first trimester is approximately 1 in 285 fetuses [2]. The prenatal presence of cystic hygroma is strongly associated with aneuploidy and has a significantly worse outcome than simple increased NT [2, 7, 8]. Cystic hygroma should be differentiated from increased NT. The mean size of first-trimester cystic hygromas has been reported to be 8 mm; when increased, NT tends to be smaller; cystic hygroma extends along the entire length of the fetus, whereas enlarged NT is visualized in the midline ligamentum nuchae [2, 7]. Furthermore, first-trimester nuchal septations visualization is a risk factor for chromosomal anomalies, even in the absence of increased NT [9]. Due to the clinical management guidelines 2016, in case of identified enlarged NT or a cystic hygroma during the ultrasound, the patient should be offered genetic counselling and diagnostic testing for aneuploidy and follow-up ultrasonography for fetal structural abnormalities [10].

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9.2.2 Additional Markers (Tricuspid Regurgitation, Ductus Venosus, Nasal Bone) The effectiveness of screening can be improved by assessing additional ultrasound markers, such as absent nasal bone, tricuspid regurgitation, ductus venosus flow in the first trimester. It is important to consider that screening for soft features is not a component of a basic obstetric ultrasound examination and reporting such findings to patients is controversial; such information requires further counselling and may lead to unnecessary invasive testing, which is costly and may result in complications associated with loss of a normal fetus. However, several ultrasonographic markers in one fetus significantly increase the risk of chromosomal defects [11]. The most useful marker is the fetal nasal bone, absent or hypoplastic, mostly in fetuses with trisomy 21. As an isolated marker in the first trimester, it has a detection rate (DR) of approximately 70% for trisomy 21 and DR of 32% for other chromosomal defects with a false-positive rate (FPR) depending on ethnic origin and CRL [12]. An absent nasal bone is much more frequent in fetuses with trisomies than with normal karyotypes. Moreover, this marker is more common in fetuses with Down syndrome than those with trisomy 13 or trisomy 18. Thus, in one review, the frequency of absent nasal bone at 11–13 gestational weeks in euploid fetuses was 2.5% compared to trisomies 13%, 18%, and 21–45%, 53%, and 60%, respectively [13]. The absence of the nasal bone combined with NT and maternal serum biomarkers improves the screening performance approximately to 90% for FPR of 5% [13, 14]. In the second trimester, the reported sensitivity of absent nasal bone for trisomy 21 varies widely, but is generally lower than that in the first trimester. For instance, in one systematic review and meta-analysis of nasal bone assessment in the second trimester, the absent nasal bone was in approximately 30–40% of fetuses with trisomy 21 and hypoplasia of the nasal bone was in about 50–60% of Down syndrome fetuses [15]. A regurgitant flow across the tricuspid valve (defined as velocity more than 60 cm/s for at least 50% of systole) is correlated with an increased risk for autosomal trisomies. Thus, tricuspid regurgitation for the euploid fetuses was 0.9%, and 55.7%, 33.3%, and 30% for the fetuses with trisomies 21, 18, and 13, respectively, and in 37.5% of those with Turner syndrome [16]. The role of the ductus venosus flow in improving risk assessment for chromosomal defects was observed. An absent or reversed a-wave in ductus venosus might be useful in suspicion of monosomy X with aneuploidy incidence 35.7% compared with euploidy 2.46% [17]. However, the assessment of the ductus venosus flow alone remains poor. The combined first-trimester screening can be improved by using ultrasound markers of the nasal bone, tricuspid regurgitation, and flow in the ductus venosus. Such an approach would enhance DR for common chromosomal anomalies while reducing the FPR from 4.8 to 3.4%, which lead to a lower number of unnecessary invasive diagnostic tests and subsequent complications [18].

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As mentioned before, ultrasonography in the first trimester has a crucial role in aneuploidy detection and major congenital malformations. Early detection of anomalies in the development of the fetus allows an earlier decision regarding the future of this pregnancy to be made, which is much less traumatic for both the physical and mental of the woman health.

9.3 Anomalies Identified in the First Trimester In recent years, advances in ultrasound technology have allowed fetal anatomic assessment and, in turn, genetic diagnoses to be made earlier in pregnancy. Major fetal abnormalities that can be easily identified on first-trimester ultrasound include hydrops, neural tube defects, anencephaly, alobar holoprosencephaly, body stalk anomaly, limb abnormalities, ectopia cordis, large omphalocele, large gastroschisis, megacystis, conjoined twins, and molar placenta [19]. At 11–13 weeks, we aimed to obtain: a transverse section of the head to demonstrate the skull, midline echo, and choroid plexuses, a midsagittal view of the face to demonstrate the nasal bone, midbrain, and brain stem, transverse views to demonstrate the orbits, upper lip, and palate, a sagittal section of the spine to illustrate the spine and overlying skin, a transverse section of the thorax and use of color Doppler to assess the four-­ chamber view of the heart and outflow tracts and record blood flow across the tricuspid valve, and transverse and sagittal sections of the trunk and extremities to demonstrate the stomach, kidneys, bladder, abdominal insertion of the umbilical cord, all the long bones, hands, and feet. Examination of the posterior fossa was included in the protocol only after 2011, and this was based on visual assessment rather than measurements of the brainstem and brainstem–occipital bone diameter [1].

9.4 Nonchromosomal Abnormalities Different anomalies may be recognized at different rates depending on the development of the organ system and, of course, of gestational ages, as shown by Syngelaki et  al. [20]. In the first trimester, we can identify all cases of acrania, alobar holoprosencephaly, encephalocele, tricuspid or pulmonary atresia, pentalogy of Cantrell, ectopia cordis, exomphalos, gastroschisis, and body-stalk anomaly and >50% of cases of open spina bifida, hypoplastic left heart syndrome, atrioventricular septal defect, complex heart defect, left atrial isomerism, lower urinary tract obstruction, absence of extremities, fetal akinesia deformation sequence, and lethal skeletal dysplasia (Table 9.1).

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Table 9.1  Modified from diagnosis of fetal nonchromosomal abnormalities in pregnancies undergoing routine ultrasound examination in the first trimester [20] Defect Central nervous system Acrania Alobar holoprosencephaly Encephalocele Open spina bifida Hypoplastic cerebellum vermis Agenesis of corpus callosum Schizencephaly Septo-optic dysplasia Microcephaly Sever ventriculomegaly Arachnoid cyst Brain hemorrhage Dural venous sinus thrombosis Craniosynostosis Occipital dermoid cyst Blake’s pouch cyst Brain tumor Face Anophthalmia/microphthalmia Dacryocystocele Cataract bilateral Cleft lip and palate Cleft lip only Cleft palate only Micrognatia Thorax Congenital diaphragmatic hernia Congenital pulmonary airway malformation Congenital high airway obstruction syndrome Mediastinal teratoma Pleural effusion Heart Tricuspid atresia Pulmonary atresia Polyvalvular dysplasia Hypoplastic left heart syndrome Atrioventricular septal defect

First trimester %

Second trimester %

Third trimester %

Postnatal %

100 100 100 59.3 13.3 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 40.7 86.7 96.2 66.7 100 11.1 77.8 35.7 50 100 50 100 100 0

0 0 0 0 0 3.8 33.3 0 88.9 22.2 64.3 50 0 50 0 0 100

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 34.6 0 0 14.3

100 0 0 65.4 85.7 0 85.7

0 100 0 0 0 0 0

0 0 100 0 14.3 100 0

29.2 0

58.3 90.7

8.3 9.3

4.2 0

0

100

0

0

0 0

100 67.7

0 33.3

0 0

100 100 100 92.5 90.9

0 0 0 7.5 9.1

0 0 0 0 0

0 0 0 0 0 (continued)

V. Tsibizova et al.

134 Table 9.1 (continued) Defect Complex heart defect Left atrial isomerism Tetralogy of Fallot Arch abnormality Tricuspid valve abnormality Transposition of great arteries Double/right aortic arch Aortic stenosis Pulmonary stenosis Common arterial trunk Ventricular aneurysm Arrythmia Cardiomyopathy Rhabdomyoma Ventricular septal defect Gastrointestinal tract Liver, spleen, gallbladder, mesenteric, or adrenal cyst Cloacal abnormality Meconium peritonitis Right-sided stomach Esophageal atresia Duodenal atresia Small-bowel obstruction Hirschsprung’s disease Imperforate anus Abdominal wall Exomphalos (bowel/liver) Gastroschisis Bladder exstrophy Genitourinary LUTO Bilateral renal agenesis Bilateral polycystic kidneys Unilateral pelvic kidney/agenesis Bilateral multicystic kidney Unilateral multicystic kidney Severe hydronephrosis Duplex kidney Horseshoe kidney Unilateral dilated ureter

First trimester % 60 57.1 39.3 31.6 25 13.3 15.6 0 0 0 0 0 0 0 0

Second trimester % 40 42.9 53.6 55.3 37.5 80 84.4 66.7 70 100 66.7 33.3 100 16.7 71.3

Third trimester % 0 0 3.6 10.5 25 0 0 16.7 20

Postnatal % 0 0 0 2.6 12.5 6.7 0 16.7 10

33.3 66.7 0 83.3 22.8

0 0 0 0 5.9

0

57.1

42.9

0

100 100 100 0 0 0 0 0

0 0 0 50 11.1 0 0 0

0 0 0 25 88.9 100 0 0

0 0 0 25 0 0 100 100

100 100 0

0 0 100

0 0 0

0 0 0

71.2 15.4 7.1 2.4 0 0 0 0 0 0

21.2 84.6 71.4 86.3 100 87.9 59.5 79.3 80 50

7.7 0 21.4 11.3 0 12.1 40.5 20.7 20 50

0 0 0 0 0 0 0 0 0 0

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Table 9.1 (continued) Defect Unilateral dilated cyst Ovarian cyst Ambiguous genitalia Hematocolpos Hypospadias Rectovaginal fistula Skeleton Absent hand, arm, leg, or foot Fetal akinesia deformation sequence Lethal skeletal dysplasia No-lethal skeletal dysplasia Abnormal digits Hemivertebra/scoliosis Talipes Tumor Sacrococcygeal teratoma Lymphangioma Testicular mass Thyroid goiter Other Body-stalk anomaly Pentalogy of Cantrell Ectopia cordis only Hydrops fetalis Multiply Pulmonary stenosis, microcephaly, micrognathia TOF, hemivertera, talipes Venntriculomegaly severe, cleft lip, and palate Diaphragmatic hernia, unilateral renal agenesis Cleft lip and palate, unilateral multicystic kidney Cleft lip and palate, megacystis, radial aplasia Cleft lip and palate, unilateral renal agenesis Complex heart defect, megacystis Data are given in %

First trimester % 0 0 0 0 0 0

Second trimester % 63.7 0 80 0 3.8 0

Third trimester % 36.4 100 0 100 0 0

Postnatal % 0 0 20 0 96.2 100

75 72.7 71.4 0 42.4 33.3 2.2

25 27.3 28.6 83.3 32.2 66.7 88.2

0 0 0 16.7 5.1 0 5.4

0 0 0 0 20.3 0 4.3

50 0 0 0

50 75 0 0

0 25 100 100

0 0 0 0

100 100 100 0

0 0 0 87.5

0 0 0 12.5

0 0 0 0

0

100

0

0

0 0

100 100

0 0

0 0

0

100

0

0

0

100

0

0

100

0

0

0

0

100

0

0

100

0

0

0

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9.4.1 Acrania The absence of cranial vault and cerebral hemispheres characterizes acrania. The prevalence of acrania is 1:1000 at 12 weeks gestation. At 12 weeks, acrania is suspected by the absence of the normally ossified skull and distortion of the brain (exencephaly). Association with chromosomal defects in isolated acrania is rare. The prognosis for this abnormality is severe and fatal, with death within the first week of life [21].

9.4.2 Holoprosencephaly The prevalence of about 1:10,000 is characterized by a spectrum of cerebral abnormalities resulting from incomplete cleavage of the forebrain. Usually, it is associated with trisomy 13. The risk of recurrence for sporadic, nonchromosomal holoprosencephaly is 6%. In the standard transverse view of the fetal head, a single dilated midline ventricle replaces the two lateral ventricles or partial segmentation of the ventricles. There are often associated facial defects, such as cyclopia, facial cleft, nasal hypoplasia, or proboscis [22].

9.4.3 Cystic Hygroma The prevalence is 1:800 pregnancies. It is characterized as bilateral symmetrical cystic structures located in the occipital-cervical region of the fetal neck. They are differentiated from nuchal edema by the presence of the nuchal ligament (midline septum) [23]. Cystic hygroma is caused by defects in the formation of the neck lymphatics. It is the most common form of lymphangioma (75% on the neck, 20% in the axillary region, and 5% on the chest wall, abdominal wall, and extremities). Chromosomal abnormalities, mainly Turner syndrome, are found in about 50% of cases of cystic hygroma [24]. Genetic syndromes are found in about 40% of cases. The most common are Noonan syndrome (autosomal dominant but >90% are due to de novo mutations; cystic hygromas, hypertelorism, pulmonary stenosis, fetal growth restriction), Multiple-pterygium syndrome (autosomal recessive; cystic hygromas, contractures in all joints, microcephaly, and micrognathia), Fryns syndrome (autosomal recessive; anophthalmia, facial cleft, micrognathia, ventriculomegaly, diaphragmatic hernia) and Neu-Laxova syndrome (autosomal recessive; hypertelorism, microcephaly, agenesis of the corpus callosum, contractures in the upper and lower limbs, fetal growth restriction) [25]. Hydrops (in addition to cystic hygromas, generalized edema, ascites, and pericardial or pleural effusions) occur in 60–80% of cases. In the case of hydrops, it

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is important to provide a detailed ultrasound examination, including echocardiography and invasive testing for karyotyping and array.

9.4.4 Major Cardiac Defects and Great Arteries The prevalence is 8–10:1000 birth. There is a high association between increased NT and cardiac defects in both chromosomally abnormal and euploid fetuses. Although high NT is not confined to specific cardiac defects, echocardiography has to be done in all high NT cases [26]. Fetal NT above 3.5 mm is found in about 1% of pregnancies. There is an increased risk for cardiac defects in these cases. Therefore, special attention should be placed on the examination of the heart and great arteries both at the time of the NT scan and in subsequent scans at 14–16 weeks and 20–22 weeks. The cardiac anomalies which can be detected by first-trimester screening are the following: 1. Atrioventricular septal defect 2. Ebstein anomaly 3. Hypoplastic left heart 4. Hypoplastic right heart 5. Disproportion of the ventricles 6. Double aortic arch 7. Right aortic arch

9.4.5 Diaphragmatic Hernia The prevalence is 1:4000 birth, usually sporadic—chromosomal defects association with Trisomy 18 (about 20%). Increased NT is present in about 40% of fetuses with a diaphragmatic hernia, and in such fetuses, the risk of neonatal death due to pulmonary hypoplasia increases. In the cases where the diaphragmatic hernia is associated with a good prognosis, the intrathoracic herniation of viscera may be delayed until the second or third trimesters of pregnancy. In such cases, the NT is normal [27].

9.4.6 Exomphalos Usually, at 8–10  weeks, all fetuses demonstrate herniation of the midgut that is visualized as a hyperechogenic mass in the base of the umbilical cord. Retraction into the abdomen occurs at 10–12 weeks, and it is completed by 11+5 weeks. The prevalence of exomphalos is about 1:1000, which is four times higher than live births. The condition is usually sporadic.

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The incidence of chromosomal defects, mainly trisomy 18, is about 60%, compared to about 30% at mid-gestation and 15% in neonates, because trisomy 18 is associated with a high rate of intrauterine death. The risk for chromosomal defects is higher if the exomphalos sac contains only the bowel rather than the liver. Increased NT is observed in about 85% of chromosomally abnormal and 40% of chromosomally normal fetuses with exomphalos [28].

9.4.7 Gastroschisis The prevalence of about 1:4000 is sporadic. The association with chromosomal abnormalities is rare. The evisceration of the intestine occurs through a small abdominal wall defect located just to the right of an intact umbilical cord. Prenatal diagnosis by ultrasound is based on the demonstration of the normally situated umbilicus and the herniated loops of the intestine, which are free-­ floating [29].

9.4.8 Megacystis The fetal bladder length is normally less than 6 mm at this gestation. Fetal megacystis in the first trimester, defined by a longitudinal bladder diameter of 7 mm or more, is found in about 1:1.500 pregnancies. When the longitudinal bladder diameter is 7–15  mm, the incidence of chromosomal defects, mainly trisomies 13 and 18, is about 24%. Still, there is spontaneous resolution of the megacystis in the chromosomally normal group in about 90% of cases. In contrast, in megacystis with bladder diameter greater than 15 mm, the incidence of chromosomal defects is about 11%. In the chromosomally normal group, the condition is invariably associated with progressive obstructive uropathy (LUTO). Megacystis is associated with increased NT, observed in about 75% of those with chromosomal defects and about 30% of those with normal karyotype [30].

9.4.9 Body Stalk Anomaly This lethal, sporadic abnormality occurs in about 1:10,000 fetuses at 11–13 weeks. The ultrasonographic features are major abdominal wall defect, severe kyphoscoliosis, and short umbilical cord with a single artery. Half of the fetal body is seen in the amniotic cavity, and the other half is in the celomic cavity, suggesting that early amnion rupture before obliteration of the celomic cavity is a possible cause of the syndrome.

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The fetal NT is increased in about 85% of the cases, but the karyotype is usually normal [31].

9.4.10 Spina Bifida In spina bifida, there is a failure to close the neural tube with secondary damage to the exposed nerves. The prevalence is about 1:1000 births, but this is subject to large geographical variations. Chromosomal abnormalities, single mutant genes, and maternal diabetes mellitus or ingestion of teratogens, such as antiepileptic drugs, are implicated in about 10% of the cases. However, the precise etiology for the majority of these defects is unknown. When a parent or previous sibling has had a neural tube defect, the risk of recurrence is 5%. Periconceptional supplementation of the maternal diet with folate reduces the risk of developing spina bifida by about 75% [32]. In the first trimester, the Arnold-Chiari II malformation is manifested in compression of the fourth ventricle (intracranial translucency). In the mid-sagittal view of the fetal face at 11–13 weeks, the lower part of the fetal brain between the sphenoid bone anteriorly and the occipital bone posteriorly can be divided into the brain stem in the front and a combination of the fourth ventricle and cistern magna in the back. In most open spina bifida, the diameter of the brain stem is increased, the distance between the brain stem and the occipital bone (BSOB) is decreased, and the ratio of the brain stem to BSOB is above 1.0.

9.4.11 Twin Pregnancy At the 11- to 13-week visit, we have to record maternal characteristics and perform an ultrasound scan for several reasons. First, to determine gestational age by measuring the CRL of the larger twin. Second, to assess chorionicity by the number of placentas and the presence or absence of the lambda sign at the intertwin membrane—placenta junction. Third, to measure fetal NT as part of screening for trisomies 21, 18, and 13. And last but not the least, to diagnose any fetal defects [20]. Conjoined twins are a rare complication of monoamniotic twinning. The true incidence of this complication is difficult to estimate because most women with the diagnosis choose not to continue the pregnancy, or an intrauterine demise ensues. Prognosis is mostly determined by the site fusion and the organs involved for ongoing pregnancies. Only a minority of twins that survive birth will be candidates for surgical separation. Because the ethical aspects of surgical separation are complex, a palliative care team is crucial for understanding the goals and preferences of the patient and her family [33]. To summarize, prenatal ultrasound in the first trimester can identify significant fetal anomalies, allowing the patient to decide on early pregnancy termination with minimal risk. Moreover, supplemented by the additional ultrasound markers listed

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above, first-trimester screening becomes more advanced. With the continued improvement of ultrasound technology, more fetal anatomy can be seen at earlier gestational ages (GAs). Recommendations for imaging parameters in the first trimester are evolving, leading to earlier identification of adverse pregnancy. However, there are limitations in evaluating certain organ structures, although high-frequency transducers make the task easier. If the organ is or has been developed at this stage of pregnancy, it can be seen. If there is a major malformation, it should also be seen. Achieving these goals may require a review of embryology to understand what is normal and abnormal and improved training programs in basic scanning in the first trimester. Although there is a gap in training and expertise at this point, one will never reach the detection rate that is possible unless one begins to look. The experience of the quality assurance programs for NT monitoring reveals the possibility of improved scanning over time with epidemiologic monitoring. Thus, it seems possible to expect an ever-higher level of performance in detecting fetal abnormalities in the first trimester. Based on current literature, it is clear that fetal anatomy is recognizable on a first-trimester scan. Nevertheless, given that certain structures are not yet fully formed at earlier GAs, the need for a second-trimester scan remains very relevant. There is an increasing need for enhanced imaging capabilities in the new era of prenatal diagnosis using cell-free DNA.  A noninvasive prenatal testing (NIPT) using cell-free DNA in maternal circulation has very high sensitivity and specificity for trisomy 21, with slightly lower sensitivity for trisomies 18 and 13. Nonetheless, it could not replace combined first-trimester screening. Moreover, the experience of some countries (for instance, the Netherlands) where ultrasound and biochemistry screening of the first trimester have been excluded and replaced by NIPT has shown increased obstetrics complications, abortions in advanced GA, and related adverse complications. This unfortunate experience has led to the conclusion that NIPT should not be used as a first-line test and substitute for combined first-trimester screening [34]. NIPT is currently considered a screening test in the intermediate-­ risk group. The presence of one “soft” ultrasound marker or case of contraindications to invasive diagnostics or in cases with advanced CRL (more than 84 mm) could not be evaluated during first-trimester screening. Thus, there must be a clear understanding that the value of NIPT lies only in supplementing ultrasound screening in the first trimester. Furthermore, all patients with a positive NIPT result should be advised to confirm it by an invasive diagnostic procedure (amniocentesis or chorionic villus biopsy) and subsequent karyotyping or microarray analysis, considered the gold standard diagnostic tests if termination of pregnancy is under consideration [35].

References 1. Salomon LJ, Alfirevic Z, Berghella V, Bilardo C, Hernandez-Andrade E, Johnsen S, et  al. Practice guidelines for performance of the routine mid-trimester fetal ultrasound scan. Ultrasound Obstet Gynecol. 2011;37(1):116–26.

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2. Kagan KO, Sonek J, Wagner P, Hoopmann M. Principles of first trimester screening in the age of noninvasive prenatal diagnosis: screening for chromosomal abnormalities. Arch Gynecol Obstet. 2017;296(4):645–51. 3. Malone FD, Ball RH, Nyberg DA, Comstock CH, Saade GR, Berkowitz RL, et  al. First-­ trimester septated cystic hygroma: prevalence, natural history, and pediatric outcome. Obstet Gynecol. 2005;106(6):1415–6. 4. Kagan KO, Avgidou K, Molina FS, Gajewska K, Nicolaides KH.  Relation between increased fetal nuchal translucency thickness and chromosomal defects. Obstet Gynecol. 2006;107(1):6–10. 5. Pellerito J, Bromley B, Allison S, Chauhan A, Destounis S, Dickman E, et al. AIUM-ACR-­ ACOG-SMFM-SRU practice parameter for the performance of standard diagnostic obstetric ultrasound examinations. J Ultrasound Med. 2018;37(11):E13–24. 6. Nicolaides KH, editor First-trimester screening for chromosomal abnormalities. Seminars in perinatology. Elsevier; 2005. 7. Graesslin O, Derniaux E, Alanio E, Gaillard D, Vitry F, Quéreux C, et al. Characteristics and outcome of fetal cystic hygroma diagnosed in the first trimester. Acta Obstet Gynecol Scand. 2007;86(12):1442–6. 8. Sanhal C, Mendilcioglu I, Ozekinci M, Yakut S, Merdun Z, Simsek M, et  al. Prenatal management, pregnancy and pediatric outcomes in fetuses with septated cystic hygroma. Braz J Med Biol Res. 2014;47:799–803. 9. Mack LM, Lee W, Mastrobattista JM, Belfort MA, Van den Veyver IB, Shamshirsaz AA, et al. Are first trimester nuchal septations independent risk factors for chromosomal anomalies? J Ultrasound Med. 2017;36(1):155–61. 10. Screening for fetal aneuploidy. Practice bulletin. Obstet Gynecol. 2016;127(5):e123–e137. 11. Breathnach FM, Fleming A, Malone FD, editors. The second trimester genetic sonogram. Am J Med Genet Part C Semin Med Genet. 2007;145C:62–72. 12. Cicero S, Rembouskos G, Vandecruys H, Hogg M, Nicolaides K. Likelihood ratio for trisomy 21  in fetuses with absent nasal bone at the 11–14-week scan. Ultrasound Obstet Gynecol. 2004;23(3):218–23. 13. Nicolaides KH.  Screening for fetal aneuploidies at 11 to 13 weeks. Prenat Diagn. 2011;31(1):7–15. 14. Rink BD, Norton ME, editors. Screening for fetal aneuploidy. Seminars in perinatology. Elsevier; 2016. 15. Moreno-Cid M, Rubio-Lorente A, Rodriguez M, Bueno-Pacheco G, Tenias J, Román-Ortiz C, et  al. Systematic review and meta-analysis of performance of second-trimester nasal bone assessment in detection of fetuses with Down syndrome. Ultrasound Obstet Gynecol. 2014;43(3):247–53. 16. Kagan K, Valencia C, Livanos P, Wright D, Nicolaides K. Tricuspid regurgitation in screening for trisomies 21, 18 and 13 and Turner syndrome at 11+ 0 to 13+ 6 weeks of gestation. Ultrasound Obstet Gynecol. 2009;33(1):18–22. 17. Wiechec M, Nocun A, Matyszkiewicz A, Wiercinska E, Latała E. First trimester severe ductus venosus flow abnormalities in isolation or combination with other markers of aneuploidy and fetal anomalies. J Perinat Med. 2016;44(2):201–9. 18. Ghaffari S, Tahmasebpour A, Jamal A, Hantoushzadeh S, Eslamian L, Marsoosi V, et  al. First-­trimester screening for chromosomal abnormalities by integrated application of nuchal translucency, nasal bone, tricuspid regurgitation and ductus venosus flow combined with maternal serum free β-hCG and PAPP-A: a 5-year prospective study. Ultrasound Obstet Gynecol. 2012;39(5):528–34. 19. Mei JY, Afshar Y, Platt LD. First-trimester ultrasound. Obstet Gynecol Clin. 2019;46(4):829–52. 20. Syngelaki A, Hammami A, Bower S, Zidere V, Akolekar R, Nicolaides K. Diagnosis of fetal non-chromosomal abnormalities on routine ultrasound examination at 11–13 weeks’ gestation. Ultrasound Obstet Gynecol. 2019;54(4):468–76. 21. Tercanli S, Contro E, Ghi T, Pilu G, Tutschek B.  Acrania, exencephaly, anencephaly, and encephalocele. Obstetric Imaging E-Book: Expert Radiology Series. 2012. p. 174.

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22. Vlasinova I, Grochova I, Gerychova R, Ventruba P. P 05.16: family case of holoprosencephaly. Ultrasound Obstet Gynecol. 2014;44(S1):211–2. 23. Hussamy DJ, Herrera CL, Twickler DM, Mcintire DD, Dashe JS. Number of risk factors in Down syndrome pregnancies. Am J Perinatol. 2019;36(1):79–85. 24. Witters G, Van Robays J, Willekes C, Coumans A, Peeters H, Gyselaers W, et al. Trisomy 13, 18, 21, Triploidy and Turner syndrome: the 5T’s. Look at the hands. Facts Views Vis Obgyn. 2011;3(1):15. 25. Moerman P, Fryns JP, Cornelis A, Bergmans G, Vandenberghe K, Lauweryns JM. Pathogenesis of the lethal multiple pterygium syndrome. Am J Med Genet. 1990;35(3):415–21. 26. Kumar M.  Screening and management of congenital anomalies. Management of high-risk pregnancy—a practical approach 2015. p. 17. 27. Pober BR, editor. Overview of epidemiology, genetics, birth defects, and chromosome abnormalities associated with CDH.  Am J Med Genet C Semin Med Genet. 2007;145C(2):158–171. 28. Souka A, Nicolaides K. Diagnosis of fetal abnormalities at the 10–14-week scan. Ultrasound Obstet Gynecol. 1997;10(6):429–42. 29. Kumar P. Gastroschisis. Congenital malformations. 2008. p. 247. 30. Fontanella F, Duin L, van Scheltema PNA, Cohen-Overbeek TE, Pajkrt E, Bekker M, et al. Antenatal workup of early megacystis and selection of candidates for fetal therapy. Fetal Diagn Ther. 2019;45(3):155–61. 31. Smrcek J, Germer U, Krokowski M, Berg C, Krapp M, Geipel A, et al. Prenatal ultrasound diagnosis and management of body stalk anomaly: analysis of nine singleton and two multiple pregnancies. Ultrasound Obstet Gynecol. 2003;21(4):322–8. 32. Onyekwum CC. An elaborate analysis of the correlation between air pollution and birth defect in different counties of Texas. Texas: A&M University-Kingsville; 2021. 33. Brock C, Johnson A. Diagnosis and management of conjoined twins. Twin and higher-order pregnancies, Springer; 2021. p. 287–299. 34. Togneri FS, Kilby MD, Young E, Williams D, Griffiths MJ, Allen SK. Implementation of cell-­ free DNA-based noninvasive prenatal testing in a National Health Service Regional Genetics Laboratory. Genet Res. 2019;101:e11. 35. Van Opstal D, Srebniak MI.  Cytogenetic confirmation of a positive NIPT result: evidence-­ based choice between chorionic villus sampling and amniocentesis depending on chromosome aberration. Expert Rev Mol Diagn. 2016;16(5):513–20.

Chapter 10

Customary Complications and Screening Techniques of Early Pregnancy Arun Meyyazhagan, Haripriya Kuchi Bhotla, Manikantan Pappuswamy, and Gian Carlo Di Renzo

10.1 Introduction The natural phenomenon of pregnancy and childbirth is a physiological process with the expectations of joy in all the families. Still, in some cases, it can cause suffering too in few families, either in the form of loss of the mother or child or developing long-term complications specifically seen extensively in lower-income countries [1]. Pregnancy constitutes a wide range of physiological alterations in the hematological indices, urinary tract functioning, and viral infections to cause sequelae in neonatal [2–4]. Identifying and diagnosing the indices mentioned above in the pregnancy course is quite difficult due to the scarcity of evidence to direct the obstetricians and consultants. Though the mortality of the mother has been seen to drop since 2000, still many regions have high mortality specifically, about 86% of the world maternal deaths are from Africans from the sub-Sahara region and South Asian regions with the significant complications of preeclampsia, gestational diabetes, and premature labor [5]. The nutritional profile of the expecting mother foresees the outcomes of perinatal and long-term effects in both the mother and fetus like obesity before pregnancy increases the chances of gestational diabetes mellitus (GDM), fetal developmental disorders, and the hypertensive syndrome, whereas in contrast underweight people are at the risk of delivering before term, giving birth to small for gestational age (SGA) newborns [6, 7]. Same way, women with poor weight addition could experience a wide range of complications like anemia, premature birth, lower weight at A. Meyyazhagan · G. C. Di Renzo (*) Department of Obstetrics and Gynecology and Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Italy e-mail: [email protected] H. Kuchi Bhotla · M. Pappuswamy Department of Life Sciences, CHRIST (Deemed to be University), Bengaluru, Karnataka, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_10

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Table 10.1  Highlights of few highly prevalent complications in pregnant women in the general population with no smoking/alcoholism Complications Hematological complications

Amniotic fluid complications

Urogynecology complications Viral infections

Disease/disorders • Anemia • Hypertension •  Gestation Diabetes Mellitus •  Thrombocytopenia’s and immune thrombocytopenia • Preeclampsia •  HELLP syndrome •  Preterm labor due to the higher pressure of the fluid •  Breathing difficulties in mother •  Fluids build up causing uncontrolled diabetes •  Birth defect •  Multiple pregnancy and growth declination, sometimes still birth •  Urinary tract infections (UTI’s) •  Retention of urine secondary to incarcerated gravid uterus •  Urological cancers (Rare) • Cytomegalovirus •  Hepatitis B &C virus •  Herpes simplex virus (HSV) •  Human immunodeficiency virus (HIV)

birth and SGA in contrast to women gaining excessive weight as they are prone to have GDM, gestational hypertension (GHp), higher chances of caesarean, and preeclampsia [6, 8, 9]. Regular antenatal examinations aim to point out the concurrent obstetric situations like thalassemia, vertical transmission of infections from mother like syphilis, and anemia like conditions [10]. Screening in the first trimester helps identify the complications, namely chromosomal anomalies of the later gestation period. Assessing prognostic biomarkers can help identify the most substantial cause for the complication like GDM, GHp, and restriction of fetal growth in prior gestational stages. For example, GHp or preeclampsia is analyzed in the initial terms of pregnancy using biomarkers like the mean value of maternal arterial pressure, Doppler indices of uterine artery, and markers for placental origin (Inhibin A) and functions (Plasma protein 13) [10, 11]. The following are the common complications observed during the early trimesters of the pregnancy, and we will discuss the clinical measures considered while tackling them (Table 10.1).

10.2 Few Hematological Complications 10.2.1 Anemia One of the highly prevalent hematological conditions in pregnant women is iron deficiency, aka anemia. As the pregnant women undergo several physiological alterations, they are prone to physiologic anemia in pregnancy, where the plasma volume elevates with respect to red cell mass, causing declination of the hemoglobin

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concentration, and if the hemoglobin concentration stoops down to 11 g/dL, it can be presumed to be due to iron deficiency and the women are prescribed to increase their iron intake [3]. Several studies have shown the association of severe anemia and unpleasant pregnancy with the recommendation of iron supplements closer to the dosage of dietary allowance with some controversial aspects of supplementations due to increased gestation time and birth weight [12, 13].

10.2.2 Thrombocytopenia and Immune Thrombocytopenia About 10% of pregnancy report for thrombocytopenia; though it is an often incidental feature, it is an important biomarker for the simultaneously existing gestational or systemic disorder. It requires interventions to protect the fetus [14]. Thrombocytopenia is confirmed with a peripheral smear to eliminate the possibilities of pseudo thrombocytopenia and platelet clumpings. Immune thrombocytopenia is determined later after checking for drug-induced thrombocytopenia, splenic sequestration, and cirrhosis [15]. De novo mutations or autosomal recessive patterns can cause immune thrombocytopenia in the absence of ancestral proofs. Moderate inherited thrombocytopenia and von Willebrand disease Type IIB are mainly observed during pregnancy, leading to thrombocytopenia in neonates. Hence, it is mandatory to assess platelet count before pregnancy and family/bleeding history must be extensively evaluated [16].

10.2.3 Preeclampsia and HELLP Syndrome Preeclampsia (PE) is seen in about 2–4% of pregnancies, one of the prime reasons for maternal discomfort and 14% mortality rate in expecting mothers [17]. Few studies have shown that the prediction of PE can be made in the first trimester by combining the history of the mother with some biophysical/chemical markers like blood pressure, ultrasound screening, uterine artery pulsatility index, biparietal diameter, nuchal translucency, fetal heartbeat, method of conception, mother’s BMI, parity, smoking habits, and gestational age [18, 19]. Screening PE at early pregnancy is about 41–96% sensitive compared with later stages [17]. Hemolysis, an elevated liver enzyme with lower platelets (HELLP) syndrome, is a type of preeclampsia among 0.9% pregnancies and 10–20% severe PE resulting from impaired placentation in the early stages of gestation [20].

10.2.4 Hypertension Almost 10% of pregnancies encounter hypertensive disorders in the form of placental abruptions, premature delivery, PE, HELLP, causing seizures, GDM, and sometimes death of fetus and mother [21]. If the systolic pressure is >140 mmHg or the

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diastolic pressure is >90  mmHg, it is considered hypertension during pregnancy [22]. Additionally, hypertension while pregnancy is linked with cardiovascular diseases like strokes, heart failure, and myocardial infarction. Medication and lifestyle alterations have been suggested to tackle this condition [23].

10.3 Some Urogynecology Complications The size of the kidney increases during pregnancy, and the plasma level rises by 60–80%, causing a 50% hike in glomerular filtration. These alterations result in declining levels of creatinine and urea in serum. Hydronephrosis is also seen in about 90% of pregnancies, specifically on the right side, thereby increasing urine volume by decreasing the detrusor muscles tone [4].

10.3.1 Urinary Tract Infections (UTI’s) Eight percent of pregnancies do struggle with UTI due to elevated stasis of urine and vesicoureteric reflux. UTIs might lead to premature labor/membrane rupture and low infants weight during birth. The high capacity of the bladder, urine stasis with ureters dilation, and partial urine unloading facilitate bacterial migration in the upper parts of the urinary tract. The causative reasons for UTIs while pregnancy involves: • • • • • •

Sickle cell disease Low socioeconomic status Urinary tract anatomic abnormality High maternal age Sexual activities Diabetes mellitus [24]

10.3.2 Retention of Urine Secondary to Incarcerated Gravid Uterus With the incidence rate of 20% of pregnancies, the retroverted uterus is corrected through progressing gestation as the uterus expels from the sacral hollow. Usually, 1 in 3000 pregnancies becomes complicated due to incarcerated uterus due to trapping of uterine fundus below sacral promontory and leads to enlargement of the uterus with the following risk factors:

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Inflammatory pelvic disease Pelvic surgery history Malformations in uterine Penetrating sacral promontory. Endometriosis [4]

10.3.3 Pregnancy and Urological Cancers Though rare renal carcinoma is seen in pregnancy after bladder cancer with the symptoms of loin or abdominal pains, distention’s, UTIs, and hematuria, these symptoms can be misconceived with pregnancy conditions. MRI can be used to find the lesions in case of doubt if hematuria is seen for a long time in the absence of renal stones or UTI as ionizing radiations in CT scan can be risky [25].

10.4 Gestational Diabetes Mellitus GDM is linked with short- and long-term health effects in pregnant mothers and offspring, as GDM is correlated with increased risk of hypertension disorders and premature delivery plus lifelong type 2 diabetes risk [26]. According to the American Diabetes Association, screening for diabetes must be done in the first prenatal visit in the suspected women. The fasting glucose in plasma is ≥5.1 mmol/L and classified as GDM in the early stages of pregnancy [27]. The risk factors for screening of GDM is listed as obesity, family history of GDM to find any first-degree relative, macrocosmic baby history, or any specific ethnic with higher diabetes prevalence [28]. Observational studies have given contemplating results for diagnosing and treating GDM to tackle the adverse pregnancy risks.

10.5 Viral Infections Yet another prevalent complication observed during pregnancy is viral infections ranging from fetal, obstetric, and neonate sequelae with the ability to transmit vertically from mother to neonates, sometimes leading to severe illness in fetus or congenital syndromes like herpes simplex virus (HSV), cytomegalovirus, human immunodeficiency virus (HIV), and hepatitis B&C [2, 29, 30]. These viral infections act as viral reservoirs in the fetus, and they are at higher risk for viral transmission throughout their life span. Few antiviral therapies like acyclovir and valacyclovir

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are administered to the mother with HSV and CMV to improve the health and decline the risk after peripartum [31]. Likewise, for Hepatitis B, drugs like tenofovir, telbivudine, and lamivudine are administered to decrease the vertical transmission.

10.6 Screening in the Early Phase of Pregnancy Parents are worried about the uncertainties linked with pregnancies, mainly the fetal health and many countries provide prenatal diagnosis programs—assess the fetus for chromosomal abnormalities and usually the risky groups include high maternal age, the pedigree of genetic disorder, and any abnormalities observed in ultrasound [32]. These screening helps to identify anomalies like trisomy’s, triploidy, and Turner’s. From 2011, the screening was done with noninvasive prenatal screening (NIPT) utilizing the cell-free DNA of the fetus and had no miscarriage risks with higher clinical advantages [33].

10.6.1 Sonography Since the last menstruation imaging, the 12–13  weeks of the pregnancy is done transvaginally to acquire real-time high-resolution images to ascertain the: gestational age, fetal anatomy. In addition, pelvic ultrasounds are used to find the intrauterine gestational sac to evaluate pregnancy viability. It is also used to assess pregnancy-related issues and ectopic pregnancy [34]. Women presenting characteristics of vaginal bleeding and pelvic/abdominal pain are routinely checked with transvaginal ultrasound to ascertain the presence or multiplicity of intrauterine pregnancy or its viability along with the stage in case of nonviable pregnancy abortion [35]. The ultrasonography can be utilized in combination with the evaluation of serum β-Human chorionic gonadotrophin (hCG) and can provide information relating to the confirmation and viability of the embryo to the clinicians with rapid information about the ectopic pregnancy.

10.6.2 Blood Tests Being a physiological process, pregnancy progresses with a significant change in the hormonal levels primarily initiating from the ovaries to the placenta. As soon as the conception begins, the first hormone released is hCG, followed by estrogen, progesterone, renin, prolactin and human placental lactogen, and sufficient circulation of thyroid hormones for normal reproductive maintenance [36]. Usually the initial antenatal screening includes testing for blood group and antibodies along

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Flowchart 10.1  The standard practices followed during the first 3 months of the pregnancy in the average population

with complete blood counts. The initial screening also consists of the rubella antibody levels, HIV, syphilis, and hepatitis B serology [37]. Additionally, tests for chlamydia and gonorrhoea must be considered for risk age groups below 25. Vitamin D levels must be assessed along with a glucose tolerance test to screen for GDM.  Most importantly, the levels of pregnancy-associated plasma protein-A (PAPP-A) must be used as it is linked with the complication of preeclampsia, intrauterine fetus death, small gestational age infants, and GDM. PAPP-A are metalloproteinase cleaving insulin growth factor binding domains and correlated with the dysfunction of the placenta [38] (Flowchart 10.1).

10.6.3 Cell-Free Fetal DNA (cfDNA) Screening cfDNA is a major prenatal test to screen for trisomies in both general and high-risk populations; due to its high cost, it is rarely utilized, and it seems to be necessary to find the benefiting patient like the one whose biochemical markers are wavering from the general populations’ average value [39]. Initially, this method was utilized as a second line test for pregnancies to identify the trisomies after analyzing with a conventional screening test. However, the cfDNA is being used in first-line screening tests in women with background risks [40].

10.7 Conclusion Screening pregnant women in their early trimesters can offer us a certain risk that the fetus carries. These tests constitute blood or tissue samplings to assess the growth pattern and any fetal/maternal health abnormalities. The care is based on

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Fig. 10.1  Illustrates the common complications most pregnant women face in their early phase and how they are diagnosed

routine checkups primarily for women at risk zones for any particular infection or an inheritable condition. The tests mainly analyze the situations occurring in the course of pregnancy or after delivery. The initial screening is done based on the mother’s routine blood tests and the fetal ultrasound in the first 3  months (1–13 weeks) of pregnancy. Majorly, the ultrasound analyses the thickening of the nuchal translucency of fetus and blood test constitutes the hormones, specifically the PAPP-A and hCG hormones. In addition, genetic screening is also done in the individual with abnormal results in the above tests to identify the anomalies and congenital disabilities. Based on the obtained results, the practitioner can help pregnant women further continue her pregnancy, provide any supplements, or terminate the pregnancy. They can also offer further genetic testing or counselling to the expected parents or help them with successive pregnancies (Fig. 10.1).

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23. Wu D-d, Gao L, Huang O, Ullah K, Guo M-X, Liu Y, Zhang J, Chen L, Fan J-X, Sheng J-Z, Lin X-H, Huang H-F.  Increased adverse pregnancy outcomes associated with stage 1 hypertension in a low-risk cohort. Hypertension. 2020;75:772–80. https://doi.org/10.1161/ HYPERTENSIONAHA.119.14252. 24. Betschart C, Albrich WC, Brandner S, et al. Guideline of the Swiss Society of Gynaecology and Obstetrics (SSGO) on acute and recurrent urinary tract infections in women, including pregnancy. Swiss Med Wkly. 2020;150:w20236. 25. Khochikar MV.  Management of urological cancers during pregnancy. Nat Rev Urol. 2010;7:195–205. 26. Zhang C, Catalano P. Screening for gestational diabetes. JAMA. 2021;326(6):487–9. https:// doi.org/10.1001/jama.2021.12190. 27. Hod M, Kapur A, Sacks DA, Hadar E, Agarwal M, Di Renzo GC, Roura LC, McIntyre HD, Morris JL, Divakar H.  The International Federation of Gynecology and Obstetrics (FIGO) initiative on gestational diabetes mellitus: a pragmatic guide for diagnosis, management, and care. Int J Gynecol Obstet. 2015;131:S173–211. 28. Webber J, Charlton M, Johns N. Diabetes in pregnancy: management of diabetes and its complications from preconception to the postnatal period (NG3). Br J Diabetes. 2015;15:107–11. 29. Saade G. The effect of treatment of maternal CMV infection on the development of placental syndrome. Am J Obstet Gynecol. 2020;222(1):S2–3. 30. McQuillan G, Kruszon-Moran D, Flagg EW, et al. Prevalence of herpes simplex virus type 1 and type 2  in persons aged 14–49: United States, 2015–2016. NCHS data brief, no 304. Hyattsville: National Center for Health Statistics; 2018. 31. Sheffield JS, Hollier LM, Hill JB, et al. Acyclovir prophylaxis to prevent herpes simplex virus recurrence at delivery: a systematic review. Obstet Gynecol. 2003;102(6):1396–403. 32. Di Mattei V, Ferrari F, Perego G, Tobia V, Mauro F, Candiani M.  Decision-making factors in prenatal testing: a systematic review. Health Psychol Open. 2021;8(1):205510292098745. https://doi.org/10.1177/2055102920987455. 33. Birko S, Ravitsky V, Dupras C, et al. The value of non-invasive prenatal testing: preferences of Canadian pregnant women, their partners, and health professionals regarding NIPT use and access. BMC Pregnancy Childbirth. 2019;19(1):1–11. 34. Murugan VA, Murphy BO, Dupuis C, Goldstein A, Kim YH. Role of ultrasound in the evaluation of first-trimester pregnancies in the acute setting. Ultrasonography. 2020;39(2):178–89. https://doi.org/10.14366/usg.19043. 35. Hendriks E, MacNaughton H, MacKenzie MC. First trimester bleeding: evaluation and management. Am Fam Physician. 2019;99:166–74. 36. Alkhatib A.  The role of laboratory medicine for health during pregnancy. EJIFCC. 2018;29(4):280–4. Published 2018 Dec 5. 37. Verlohren S, et al. Am J Obstet Gynecol. 2012;202(161):e1–e11. 38. Fruscalzo A, Cividino A, Rossetti E, et  al. First trimester PAPP-A serum levels and long-­ term metabolic outcome of mothers and their offspring. Sci Rep. 2020;10:5131. https://doi. org/10.1038/s41598-­020-­61830-­5. 39. Muller F, Dreux S, Czerkiewicz I, Bernard M, Guibourdenche J, Lacroix I, et al. Down syndrome maternal serum screening: results’ comments recommended by accredited biologists. J Gynecol Obstet Biol Reprod (Paris). 2014;43:671–9. 40. Carrara J, Vivanti A, Jani JC, et al. Usefulness and reliability of cell free fetal DNA screening for main trisomies in case of atypical profile on first trimester maternal serum screening. J Transl Med. 2019;17:398. https://doi.org/10.1186/s12967-­019-­02152-­7.

Chapter 11

First Trimester Screening for Common and Rare Chromosomal Abnormalities as Well as for Major Defects: Which Tests Should Be Combined? Karl Oliver Kagan

11.1 Introduction Screening for chromosomal abnormalities, especially for trisomy 21 has undergone a number of changes in the last 50 years [1]. While screening based on maternal age was the standard in the 1970s and 1980s, it was replaced by biochemical testing in the 1990s. In the last 20  years, combined first trimester screening (FTS) at 11–13 weeks’ gestation was used as standard and most effective method of screening. Today, cell-free DNA analysis (cfDNA) in the first trimester is the gold standard in screening for common trisomies—even as routine test for low-risk pregnancies [2]. There is an ongoing discussion if the spectrum of the cfDNA tests should be extended toward rare autosomal trisomies, microdeletions, and duplications or if even genome-wide cfDNA screening test should be carried out. There are numerous unsolved controversies about this development [2–5]. However, this discussion has raised the question of which diseases should fall within the scope of screening for fetal defects in general. The expansion of the spectrum of cfDNA testing in screening for chromosomal defects is an ideal example of this controversy. It is currently performed in accordance with the technical development of the test system and offered directly to pregnant women. However, it would be much more useful to focus on the frequency of fetal abnormalities. In this regard, neither the classical trisomies nor the rare chromosomal defects are at the top of the list of fetal problems. Severe defects occur in about 2.5% of all pregnancies, while the risk of classical trisomy or rare chromosomal disorder is about 0.3% each [6]. Therefore, instead of focusing on expanding cfDNA testing, more emphasis should be placed on how different screening tests can be combined to cover most of the relevant fetal problems.

K. O. Kagan (*) Department of obstetrics and Gynaecology, University of Tuebingen, Tübingen, Germany © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_11

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This chapter focuses on the common screening tests at 11–13 weeks and highlights the impact of these tests on screening for common trisomies, rare chromosomal abnormalities, and structural defects.

11.2 Risks for Fetal Defects and Chromosomal Abnormalities The Eurocat register summarizes the incidence of fetal defects in several European regions [6]. In summary, for the period 2013–2019, a fetal defect was observed in 263 of 10,000 births, thus in about 2.5%. Table  11.1 gives an overview of fetal defects according to different organ systems. In comparison, the overall incidence of chromosomal defects was 46.7/10,000 pregnancies (live births, stillbirths, and terminations) and the incidence of trisomy 21, 18, and 13 and Turner syndrome was 25.1/10,000; 6.3/10,000; 2.3/10,000; and 2.5/10,000, respectively. The incidence of common trisomies and Turner sums up to 0.36% [6]. These figures illustrate firstly that the risk of structural defects without genetic origin is much higher than the risk of chromosomal abnormality, and secondly that the risk of chromosomal abnormality is higher than the sum risk of common trisomies and Turner syndrome. Therefore, it is not appropriate to consider cfDNA testing for common trisomies as the most important test in pregnancy to rule out fetal defects. Table 11.1  Frequency of malformations depending on the organ system

Organ system CNS Eyes Ears, face, neck Facial clefts Heart Airways and lungs Digestive tract Abdominal wall Kidneys and urinary tract Genital Extremities Other/nongenetic syndromal diseases

Frequency (×/10,000) 26.6 4.2 1.9 14.5 81.7 4.1 19.6 6.7 36.4 21.2 39.3 2.0

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11.3 Combined First Trimester Screening (CFTS) 11.3.1 Common Trisomies CFTS is based on maternal and gestational age, fetal nuchal translucency thickness, and the serum markers free beta-hCG and PAPP-A [7, 8]. There are numerous studies that have investigated the effectiveness of CFTS for common trisomies. Table 11.2 summarizes the results of a study of more than 100,000 normal pregnancies and 654 fetuses with common trisomies [8]. The detection rate was about 92–96% for a false-positive rate of 4.6%. Test performance can be further increased by combining FTCS with additional markers such as the nasal bone and the ductus venosus and tricuspid blood flow [9–11]. The addition of these markers leads to a halving of the false-positive rate and a slight increase in the detection rate. However, it should be emphasized that, apart from the measurement of the pulsatility index of the ductus venosus, the additional markers represent dichotomous variables that can strongly influence the risk. In this respect, sufficient expertise is required for the assessment [12].

11.3.2 Other Chromosomal Abnormalities There are no reliable prospective studies that investigated the test performance of FTCS for other chromosomal abnormalities. Such a study would require a genetic test for all newborns, stillbirths, and terminations that could potentially detect rare abnormalities such as a whole exome analysis. Furthermore, since each of these chromosomal abnormalities is rare, such a study would require a very large number of participants. Therefore, any studies on this topic should be taken with caution. Although the group of other chromosomal anomalies is very heterogeneous, there are few better defined chromosomal disorders in this group such as sex chromosomal disorders and triploidy. In the large study of Santorum et al. which included more than 100,000 pregnancies, the authors reported a detection rate of 98.4% for monosomy X, 97.1% for triploidy, and 55% for other chromosomal abnormalities [8].

Table 11.2  Test performance of combined first trimester screening (CFTS) according to the study of Santorum et al. [8]

Screen positive rate (%) Karyotype Normal (n = 108,112) 4.6 Trisomy 21 (n = 432) 92.1 Trisomy 18 (n = 166) 96.4 Trisomy 13 (n = 56) 92.9

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With regard to triploidy, Engelbrechtsen et al. and Kagan et al. found in two similarly sized studies that 82–84% of affected fetuses had an increased CFTS risk of trisomy 21, 18 or 13 [13, 14]. Iwarsson et al. examined CFTS results for a large list of rare chromosomal abnormalities, excluding sex chromosome [15]. The detection rate by CFTS was 55%. Vogel et  al. studied CFTS results for sex chromosomal abnormalities in nearly 200,000 pregnancies [16]. The detection rate was about 80%. Other study groups have focused on the value of individual serum markers and nuchal translucency thickness. Miranda et al. studied 226 pregnancies with a NT thickness of 3.5 mm or more. In this group, 84 fetuses were found with genetic abnormalities [17]. A cfDNA analysis would have detected 68 (81.0%) cases. The remaining 19.0% would have remained undetected if only a cfDNA analysis had been performed without prior ultrasound examination. Most of these fetuses had sonographic abnormalities that would have required invasive examination. Petersen et al. focused on abnormal free beta-hCG and PAPP-A levels of less than 0.2 MoM [18]. The prevalence was 0.1 and 0.5%. An abnormal karyotype was found in 56.6% and 21.4% of these cases; they were classified as “atypical” and in 37.2% and 23.5%, respectively. The authors also investigated the impact of the nuchal translucency thickness. The nuchal translucency was above the 95th centile in 4.3% of the total population, of which 7.6% were chromosomally abnormal and 37.1% were found to have an atypical chromosomal abnormality. Berger et al. used a dataset of more than 450,000 FTCS examinations [19]. About 649 fetuses had a rare chromosomal abnormality, and 108 (16.6%) of these fetuses had a nuchal of 3.0 mm or more.

11.3.3 Structural Defects The relevant components of the CFTS test for the detection of structural defects are the nuchal translucency thickness and the additional ultrasound markers ductus venosus and tricuspid blood flow. The effectiveness of a detailed anatomical examination is described later. The relationship between increased NT and the frequency of structural malformations has been known for a long time. Baer et al. studied NT measurements in almost 76,000 patients, of whom 1379 had a major defect [20]. The risk increased by 1.6 and 3.1 times if the nuchal translucency thickness was above the 95th and 99th centile, respectively. Grande et al. studied the effectiveness of the nuchal translucency in a cohort of 13,700 normal pregnancies including 439 fetuses with nongenetic structural anomalies [21]. The detection rate of a fetal nuchal translucency thickness of 3.5 mm or more was 23%. In the cohort of the study from Syngelaki et  al. with 1720 fetuses with structural anomalies, the detection rate of a nuchal translucency thickness above the 95th centile was 12% [22]. Bardi et al. reported an increase in the prevalence of structural defects of 5.9% when the nuchal translucency was between the 95th–99th centile. The spectrum of structural anomalies and nongenetic syndromes known to be associated with an increased nuchal translucency thickness is diverse and was summarized by Souka et al. [23].

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The association between congenital heart defects and thickened fetal nuchal translucency is well documented. Minnella et al. observed that 37% of the fetuses with a cardiac defect had a nuchal translucency measurement above the 95th centile [24]. Chelemen et al. demonstrated, that the risk of heart defects increased with increasing nuchal translucency measurements from 2% for nuchal translucency measurements between 3.5 and 4.4 mm, to more than 10% for values above 5.5 mm [25]. Assessment of the tricuspid valve and ductus venosus (DV) with Doppler may further improve screening for heart defects. In the study by Minnella et al. which included 211 fetuses with heart defects, tricuspid regurgitation and reverse flow in the ductus venosus were present in 28–29% of cases [24]. Increased nuchal translucency above the 95th centile, tricuspid regurgitation or abnormal ductus venosus flow were present in 55% of the affected fetuses for a false-positive rate of 8.8%. Wagner et al. studied the waveform of the DV flow in normal fetuses and fetuses with cardiac defects [26]. If the ratio between the a- and the S-wave is used instead of the PIV, the detection rate can be increased by further 8%. Interestingly, biochemical markers such as PlGF and free beta-hCG are also altered in fetuses with cardiac defects [27]. However, these markers are not currently used in routine screening for structural defects.

11.4 Cell-Free DNA (cfDNA) Screening 11.4.1 Common Trisomies Many studies have examined the test performance of cfDNA screening for common trisomies. The results of the most recent meta-analysis of Demko et  al. are shown in Table 11.3 [28]. Test failure is among the most important limitations of cfDNA screening. Galeva et al. reported a test failure rate of 3.4% [29]. The test can be repeated 2 weeks after the first attempt and about two-thirds of the subsequent analyses are successful. The most important predictor of test failure is an increase in maternal weight. Unfortunately, fetal aneuploidy is also associated with test failure. Therefore, invasive testing or at least a detailed anatomical assessment is necessary, especially if the second attempt is also unsuccessful [30].

Table 11.3  Detection and false-positive rate of cfDNA screening for common trisomies (prospective validation studies)

Karyotype Trisomy 21 Trisomy 18 Trisomy 13

Detection rate (%) 98.0 92.8 93.2

False-positive rate (%) 0.09 0.09 0.08

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11.4.2 Other Chromosomal Abnormalities It is currently being discussed whether and to what extent the spectrum of cfDNA tests should be expanded. This includes screening for gonosomal disorders, rare trisomies, microdeletions/duplications, monogenic disorders, and structural chromosomal disorders. The problems with expansion in general have already been mentioned in the CFTS section. A detailed description of all available screening tests for other chromosomal abnormalities is beyond the scope of this article. The basic aim of extended cfDNA screening is to reduce the residual risk of genetic diseases. In a study of 7235 low-risk pregnancies, Maya et al. assessed the residual risk of genetic disease after using different cfDNA tests [31]. They screened for (a) trisomy 21, 18, and 13, (b) additional sex chromosome disorders, (c) additional common microdeletions/duplications (including 1p36.3-1p36.2, 4p16.3-4p16.2, 5p15.35p15.1, 15q11.2-15q13.1, and microdeletion 22q11.2), and (d) genome-wide ­ screening for structural chromosome disorders with more than seven megabase pairs. In total, 1.2% of the fetuses were found to have a pathogenic alteration. With the screening tests described above, the residual risks were 1.07%, 0.78%, 0.74%, and 0.68%, respectively. It can be seen that the residual risk is similar regardless of the screening test performed. The greatest reduction in the residual risk can be achieved by screening for gonosomal disorders. However, due to the generally good outcome of fetuses with sex chromosome disorders, this screening test should be questioned per se [32]. The German Society for Ultrasound in Medicine (DEGUM) has published recommendations for a balanced approach to cfDNA screening. These have been summarized under the “10 golden rules” [2]. These are as follows: • NIPT requires medical information and genetic counseling in accordance with the legal regulations. • NIPT currently allows reliable statements on the probability of trisomy 21, 18, 13, but no statements on structural malformations. However, these make up the majority of perinatally relevant anomalies. It is also not possible to detect most other chromosomal disorders and syndromal diseases. • NIPT requires a detailed ultrasound examination, ideally before blood collection and after 12 SSW. • In the case of a fetal defect or increased nuchal translucency, an invasive test (CVS or amniocentesis) is the method of choice to be able to detect chromosomal defects and to avoid unnecessary loss of time until the final diagnosis. • In the context of a NIPT examination, the fetal or pregnancy-specific proportion of cell-free DNA should always be specified. The “fetal fraction” is a quality parameter with a great influence on the test quality. • An inconclusive cfDNA test requires clarification. More chromosomal defects are found in this cohort, especially trisomies 13 and 18 as well as triploidies.

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• NIPT is a screening test. In the case of an abnormal NIPT, an invasive test should be offered. The indication for termination of pregnancy should not be based just on an abnormal cfDNA test. • NIPT for sex chromosome changes should not be routinely performed. • The use of cfDNA tests for rare autosomal aneuploidies, structural chromosomal defects, especially microdeletions and monogenetic diseases cannot be generally recommended at present. • In twin pregnancies, cfDNA testing has a higher failure rate. This is particularly true for pregnancies after assisted reproduction or in obese women.

11.4.3 Structural Defects CfDNA tests are not made to identify structural defects.

11.5 Detailed Anatomical Assessment In two meta-analyses by Karim et al., the detection rate of a detailed anatomical examination at 11–13 weeks was 46.1% for noncardiac defects and 55.8% for cardiac abnormalities, respectively [33, 34]. In comparison, the detection rate of the second trimester anomaly scan, which forms the basis of the screening for fetal defects, is also around 50% [22, 35]. In general, it is recommended to perform an invasive test in case of a structural anomaly because of the strong association between fetal defects and major chromosomal abnormalities [36, 37]. Therefore, a detailed anatomical examination in the first trimester could not only increase the overall detection rate of major defects throughout pregnancy, but also lead to an increase in the detection rate of chromosomal abnormalities [38].

11.5.1 Common Trisomies Regarding screening for trisomy 21, most studies have focused on the effectiveness of FTCS or cfDNA screening. Morlando et al. studied the karyotype of 110 fetuses with an atrioventricular septal defect, one of the cardinal symptoms of trisomy 21 [39]. The prevalence of trisomy 21 fetuses in this group was 41%. One could speculate that the detection of an AVSD in the first trimester might increase the overall CFTS test performance. Unfortunately, none of the studies systematically investigated whether there is an additional benefit in screening for trisomy 21. Wagner et al. examined the effectiveness of a detailed anatomical scan in addition to FTCS

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in screening for trisomy 18, 13, triploidy and monosomy X [40]. The detection rate of a screening strategy based on maternal age and nuchal translucency thickness was 75.8% for a false-positive rate of 3%. For the same false-positive rate, the detection rate increased to 94.5% when a detailed first trimester anomaly scan was included in the analysis.

11.5.2 Other Chromosomal Abnormalities Unfortunately, there are no acceptably large studies to adequately answer this research question. Syngelaki et al. focused on the risk of atypical chromosomal abnormalities in common anomalies such as holoprosencephaly, omphalocele, and megacystis. Of the fetuses with a chromosomal abnormality, 20.6%, 9.4% and 20% had a chromosomal abnormality that could not be detected by cfDNA analysis. The authors did not perform exome analysis, which may have further increased the proportion of chromosomal abnormalities [40].

11.6 Useful Combinations of ETS and cfDNA Analysis As shown, all three examinations, a first trimester anomaly scan, CFTS, and cfDNA analysis have their importance in screening for the most fetal relevant problems. CfDNA screening is certainly more suitable for the detection of common trisomies. However, due to the high prevalence of structural defects and the strong association between structural defects and chromosomal abnormalities, this test should always be combined with a detailed anomaly scan. In terms of cost-effectiveness, it may be better to start with CFTS and limit cfDNA screening to intermediate-risk pregnancies than to offer cfDNA screening for all pregnancies. Miltoft et  al. investigated a two-stage approach in which CFTS was first performed in all pregnant women, followed by cfDNA analysis for trisomy 21, 18, and 13 in a subcollective at risks between 1:100 and 1:1000 [41]. If the risk was less than 1:1000, no further testing was performed; if the risk was greater than 1:100 or the cfDNA analysis was abnormal, invasive testing was performed. This model, called contingent screening, was compared with classical CFTS with a risk cutoff of 1:300 to define the high-risk population. The study cohort consisted of 6449 pregnancies; 15 fetuses had trisomy 21. All affected pregnancies were detected by both screening methods. However, the false-positive rate of the two-stage model was only 1.2% compared to 3.0% for CFTS. Gil et al. took a similar approach. Pregnant women at risk between 1:101 and 1:2500 were offered cfDNA screening for CFTS [42]. In contrast to previous studies, women in the high-risk arm could also choose between invasive and cfDNA testing. The study included 11,692 pregnancies and 47 cases with trisomy 21. The

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approach resulted in a detection rate of 91.5%, with only 38% of women at risk greater than 1:100 opting for invasive testing. In the intermediate risk group, 91.5% of pregnant women opted for a cfDNA test. Overall, an invasive test was performed in 2.7% of cases. Sanchez-Duran et al. performed either CFTS or a quadruple test in the second trimester as the first-line screening test [43]. The cutoffs for additional cfDNA testing were 1:10 and 1:1500. Unfortunately, the study included only five cases with trisomy 21, but all were detected. The false-positive rate was 1.3%. An earlier study used prospectively collected CFTS results from nearly 87,000 pregnancies, including 324 with trisomy 21 [44]. The first-line risk was based on maternal age, fetal nuchal translucency, and the ductus venosus flow. It was postulated that cfDNA testing would be used in women with a risk between 1:100 and 1:2500, and that the detection and false-positive rates of cfDNA screening would be 99.0% and 0.08%, respectively. Using such an approach, the detection and false-­ positive rates would be 96.0% and 2.3%, respectively. If the upper threshold is raised to 1:10, the detection rate remains almost unchanged, but the false-positive rate drops to 0.8%. The proportion of women in the intermediate risk group who should be screened by cfDNA ranged from 11.4 to 29.9%.

11.7 Summary This overview highlights the importance of first trimester screening. Although cfDNA screening currently attracts much attention because of its high detection rate for common trisomies, its importance should not be overestimated. Structural abnormalities are much more common. Therefore, a detailed anomaly scan including the measurement of the nuchal translucency should always be part of any first trimester screening approach. In the case of a fetal malformation or increased nuchal translucency thickness, the risk of chromosomal abnormalities is high, regardless of whether it is a common or rare chromosomal disorder. Therefore, invasive screening should be considered in these cases.

References 1. Cuckle H, Maymon R.  Development of prenatal screening—a historical overview. Semin Perinatol. 2016;40:12–22. https://doi.org/10.1053/j.semperi.2015.11.003. 2. Kagan KO, Sonek J, Kozlowski P. Antenatal screening for chromosomal abnormalities. Arch Gynecol Obstet. 2022;305:825–35. https://doi.org/10.1007/s00404-­022-­06477-­5. 3. Christiaens L, Chitty LS, Langlois S. Current controversies in prenatal diagnosis: expanded NIPT that includes conditions other than trisomies 13, 18, and 21 should be offered. Prenat Diagn. 2021;41:1316–23. https://doi.org/10.1002/pd.5943. 4. Renzo GCD, Bartha JL, Bilardo CM.  Expanding the indications for cell-free DNA in the maternal circulation: clinical considerations and implications. Am J Obstet Gynecol. 2019;220:537–42. https://doi.org/10.1016/j.ajog.2019.01.009.

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5. Jani JC, Gil MM, Benachi A, et al. Genome-wide cfDNA testing of maternal blood. Ultrasound Obstet Gynecol. 2020;55:13–4. https://doi.org/10.1002/uog.21945. 6. EUROCAT prevalence data tables. http://www.eurocat-­network.eu/newprevdata/showPDF.asp x?winx=1416&winy=741&file=allsubgroups.aspx. Accessed 1 Jan 2023. 7. Kagan KO, Wright D, Baker A, et al. Screening for trisomy 21 by maternal age, fetal nuchal translucency thickness, free beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A.  Ultrasound Obstet Gynecol. 2008;31:618–24. https://doi.org/10.1002/ uog.5331. 8. Santorum M, Wright D, Syngelaki A, et al. Accuracy of first-trimester combined test in screening for trisomies 21, 18 and 13. Ultrasound Obstet Gynecol. 2017;49:714–20. https://doi. org/10.1002/uog.17283. 9. Kagan KO, Cicero S, Staboulidou I, et  al. Fetal nasal bone in screening for trisomies 21, 18 and 13 and Turner syndrome at 11-13 weeks of gestation. Ultrasound Obstet Gynecol. 2009;33:259–64. https://doi.org/10.1002/uog.6318. 10. Maiz N, Valencia C, Kagan KO, et al. Ductus venosus Doppler in screening for trisomies 21, 18 and 13 and Turner syndrome at 11-13 weeks of gestation. Ultrasound Obstet Gynecol. 2009;33:512–7. https://doi.org/10.1002/uog.6330. 11. Kagan KO, Valencia C, Livanos P, et al. Tricuspid regurgitation in screening for trisomies 21, 18 and 13 and Turner syndrome at 11 + 0 to 13 + 6 weeks of gestation. Ultrasound Obstet Gynecol. 2009;33:18–22. https://doi.org/10.1002/uog.6264. 12. Maiz N, Kagan KO, Milovanovic Z, et al. Learning curve for Doppler assessment of ductus venosus flow at 11 + 0 to 13 + 6 weeks’ gestation. Ultrasound Obstet Gynecol. 2008;31:503–6. https://doi.org/10.1002/uog.5282. 13. Engelbrechtsen L, Brøndum-Nielsen K, Ekelund C, et  al. Detection of triploidy at 11-14 weeks’ gestation: a cohort study of 198 000 pregnant women. Ultrasound Obstet Gynecol. 2013;42:530–5. https://doi.org/10.1002/uog.12460. 14. Kagan KO, Anderson JM, Anwandter G, et al. Screening for triploidy by the risk algorithms for trisomies 21, 18 and 13 at 11 weeks to 13 weeks and 6 days of gestation. Prenat Diagn. 2008;28:1209–13. https://doi.org/10.1002/pd.2149. 15. Iwarsson E, Conner P. Detection rates and residual risk for a postnatal diagnosis of an atypical chromosome aberration following combined first-trimester screening. Prenat Diagn. 2020;40:852–9. https://doi.org/10.1002/pd.5698. 16. Vogel I, Tabor A, Ekelund C, et  al. Population-based screening for trisomies and atypical chromosomal abnormalities: improving efficacy using the combined first trimester screening algorithm as well as individual risk parameters. Fetal Diagn Ther. 2019;45:424–9. https://doi. org/10.1159/000492152. 17. Miranda J, Miño FPY, Borobio V, et al. Should cell-free DNA testing be used in pregnancy with increased fetal nuchal translucency? Ultrasound Obstet Gynecol. 2020;55:645–51. https://doi.org/10.1002/uog.20397. 18. Petersen OB, Vogel I, Ekelund C, et  al. Potential diagnostic consequences of applying non-­invasive prenatal testing: population-based study from a country with existing first-trimester screening. Ultrasound Obstet Gynecol. 2014;43:265–71. https://doi. org/10.1002/uog.13270. 19. Berger VK, Norton ME, Sparks TN, et  al. The utility of nuchal translucency ultrasound in identifying rare chromosomal abnormalities not detectable by cell-free DNA screening. Prenat Diagn. 2020;40:185–90. https://doi.org/10.1002/pd.5583. 20. Baer RJ, Norton ME, Shaw GM, et al. Risk of selected structural abnormalities in infants after increased nuchal translucency measurement. Am J Obstet Gynecol. 2014;211:675.e1–675. e19. https://doi.org/10.1016/j.ajog.2014.06.025. 21. Grande M, Arigita M, Borobio V, et  al. First-trimester detection of structural abnormalities and the role of aneuploidy markers. Ultrasound Obstet Gynecol. 2012;39:157–63. https://doi. org/10.1002/uog.10070.

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22. Syngelaki A, Hammami A, Bower S, et al. Diagnosis of fetal non-chromosomal abnormalities on routine ultrasound examination at 11-13 weeks’ gestation. Ultrasound Obstet Gynecol. 2019;54:468–76. https://doi.org/10.1002/uog.20844. 23. Souka AP, Kaisenberg CSV, Hyett JA, et al. Increased nuchal translucency with normal karyotype. Am J Obstet Gynecol. 2005;192:1005–21. https://doi.org/10.1016/j.ajog.2004.12.093. 24. Minnella GP, Crupano FM, Syngelaki A, et al. Diagnosis of major heart defects by routine first-trimester ultrasound examination: association with increased nuchal translucency, tricuspid regurgitation and abnormal flow in ductus venosus. Ultrasound Obstet Gynecol. 2020;55:637–44. https://doi.org/10.1002/uog.21956. 25. Chelemen T, Syngelaki A, Maiz N, et  al. Contribution of ductus venosus Doppler in first-­ trimester screening for major cardiac defects. Fetal Diagn Ther. 2011;29:127–34. https://doi. org/10.1159/000322138. 26. Wagner P, Eberle K, Sonek J, et al. First-trimester ductus venosus velocity ratio as a marker of major cardiac defects. Ultrasound Obstet Gynecol. 2019;53:663–8. https://doi.org/10.1002/ uog.20099. 27. Fantasia I, Kasapoglu D, Kasapoglu T, et al. Fetal major cardiac defects and placental dysfunction at 11–13 weeks’ gestation. Ultrasound Obstet Gynecol. 2018;51:194–8. https://doi. org/10.1002/uog.18839. 28. Demko Z, Prigmore B, Benn P. A critical evaluation of validation and clinical experience studies in non-invasive prenatal testing for Trisomies 21, 18, and 13 and monosomy X. J Clin Med. 2022;11:4760. https://doi.org/10.3390/jcm11164760. 29. Galeva S, Gil MM, Konstantinidou L, et al. First-trimester screening for trisomies by cfDNA testing of maternal blood in singleton and twin pregnancies: factors affecting test failure. Ultrasound Obstet Gynecol. 2019;53:804–9. https://doi.org/10.1002/uog.20290. 30. Bardi F, Bet BB, Pajkrt E, et al. Additional value of advanced ultrasonography in pregnancies with two inconclusive cell-free DNA draws. Prenat Diagn. 2022;42:1358–67. https://doi. org/10.1002/pd.6238. 31. Maya I, Sheelo LS, Brabbing-Goldstein D, et al. Residual risk for clinically significant copy number variants in low-risk pregnancies, following exclusion of noninvasive prenatal screening–detectable findings. Am J Obstet Gynecol. 2022;226:562.e1–8. https://doi.org/10.1016/j. ajog.2021.11.016. 32. Samango-Sprouse CA, Porter GF, Lasutschinkow PC, et  al. Impact of early diagnosis and noninvasive prenatal testing (NIPT): knowledge, attitudes, and experiences of parents of children with sex chromosome aneuploidies (SCAs). Prenat Diagn. 2019;40:470–80. https://doi. org/10.1002/pd.5580. 33. Karim JN, Bradburn E, Roberts N, et  al. First-trimester ultrasound detection of fetal heart anomalies: systematic review and meta-analysis. Ultrasound Obstet Gynecol. 2022;59:11–25. https://doi.org/10.1002/uog.23740. 34. Karim JN, Roberts NW, Salomon LJ, Papageorghiou AT. Systematic review of first-trimester ultrasound screening for detection of fetal structural anomalies and factors that affect screening performance. Ultrasound Obstet Gynecol. 2017;50:429–41. https://doi.org/10.1002/ uog.17246. 35. Rydberg C, Tunón K. Detection of fetal abnormalities by second-trimester ultrasound screening in a non-selected population. Acta Obstet Gynecol Scand. 2017;96:176–82. https://doi. org/10.1111/aogs.13037. 36. Salomon LJ, Alfirevic Z, Audibert F, et  al. ISUOG updated consensus statement on the impact of cfDNA aneuploidy testing on screening policies and prenatal ultrasound practice. Ultrasound Obstet Gynecol. 2017;49:815–6. https://doi.org/10.1002/uog.17483. 37. Kozlowski P, Burkhardt T, Gembruch U, et al. DEGUM, ÖGUM, SGUM and FMF Germany recommendations for the implementation of first-trimester screening, detailed ultrasound, cell-­ free DNA screening and diagnostic procedures. Ultraschall Med. 2019;40:176–93. https://doi. org/10.1055/a-­0631-­8898.

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38. Kagan K, Tercanli S, Hoopmann M. Ten reasons why we should not abandon a detailed first trimester anomaly scan. Ultraschall Med. 2021;42:451–9. https://doi.org/10.1055/a-­1528-­1118. 39. Morlando M, Bhide A, Familiari A, et  al. The association between prenatal atrioventricular septal defects and chromosomal abnormalities. Eur J Obstet Gynecol Reprod Biol. 2017;208:31–5. https://doi.org/10.1016/j.ejogrb.2016.10.039. 40. Wagner P, Sonek J, Hoopmann M, et  al. First-trimester screening for trisomies 18 and 13, triploidy and Turner syndrome by detailed early anomaly scan. Ultrasound Obstet Gynecol. 2016;48:446–51. https://doi.org/10.1002/uog.15829. 41. Miltoft CB, Rode L, Ekelund CK, et al. Contingent first-trimester screening for aneuploidies with cell-free DNA in a Danish clinical setting. Ultrasound Obstet Gynecol. 2018;51:470–9. https://doi.org/10.1002/uog.17562. 42. Gil MM, Revello R, Poon LC, et al. Clinical implementation of routine screening for fetal trisomies in the UK NHS: cell-free DNA test contingent on results from first-trimester combined test. Ultrasound Obstet Gynecol. 2016;47:45–52. https://doi.org/10.1002/uog.15783. 43. Sánchez-Durán MÁ, García AB, Calero I, et al. Clinical application of a contingent screening strategy for trisomies with cell-free DNA: a pilot study. BMC Pregnancy Childbirth. 2019;19:274. https://doi.org/10.1186/s12884-­019-­2434-­0. 44. Kagan KO, Wright D, Nicolaides KH. First-trimester contingent screening for trisomies 21, 18 and 13 by fetal nuchal translucency and ductus venosus flow and maternal blood cell-free DNA testing. Ultrasound Obstet Gynecol. 2015;45:42–7. https://doi.org/10.1002/uog.14691.

Chapter 12

The Technology of Cell-Free Fetal DNA-­Based NIPT Karen White, Bowdoin Su, Renee Jones, Emilia Kostenko, and Francesca Romana Grati

12.1 Basis of cfDNA Testing Noninvasive prenatal testing (NIPT) for fetal genomic alterations is possible through the analysis of cell-free DNA (cfDNA) isolated from maternal plasma. Most of the cfDNA in circulation consists of fragments of DNA that have been released during the apoptosis of cells of hematopoietic origin [1, 2]. In pregnancy, a minority fraction, 10–20% in the first and second trimesters, is “fetal” cfDNA and derives from the cytotrophoblast cells of the placenta (reviewed by Chiu and Lo) [3]. The fragments of cfDNA of different origins vary in length. The fetal cfDNA has a broad size distribution with a higher frequency of shorter fragments (peaking at ~142 bp) compared to maternal cfDNA (peaking at ~166 bp) [4]. This is likely to be the result of nucleosome structure and more active processing of fetal DNA [3, 4]. The entire fetal genome is represented in cfDNA, generating potential for noninvasive fetal genetic testing [4]. Furthermore, cfDNA is cleared rapidly so fetal cfDNA does not persist to confound testing in subsequent pregnancies [5]. Fetal cfDNA testing cannot be performed free of maternal cfDNA because it is not possible to physically separate the DNA molecules from the two sources. Therefore, early work in NIPT relied on polymerase chain reaction (PCR) to detect paternally inherited sequences that are not present in maternal DNA. Targets were Y-chromosome sequences for fetal sex determination and RhD sequences for fetal genotyping in RhD negative women [6–8]. The major challenge for fetal aneuploidy screening with cfDNA was indeed the need to test fetal DNA against an

K. White · B. Su · R. Jones · E. Kostenko Roche Sequencing Solutions, San Jose, CA, USA F. R. Grati (*) Cytogenetics and Molecular Genetics, TOMA Advanced Biomedical Assays S.p.A., Busto Arsizio, Italy © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_12

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unavoidable maternal background. Advances in sequencing technology, which allow the generation of large amounts of sequence data, and sophisticated bioinformatics approaches enabled the inference of fetal copy number in a chromosomal region of interest [9, 10]. This was initially applied to trisomy 21 but is now used to screen for other trisomies, sex chromosome aneuploidy, and subchromosomal changes such as large partial imbalances, microdeletions, and microduplications. Although the initial cfDNA tests for fetal aneuploidy were sequencing-based, numerous approaches now exist. Any technology must contend with certain limitations due to biology. From a technical standpoint, the most important consideration is fetal fraction, the proportion of fetal cfDNA fragments. Fetal fraction varies widely between pregnancies but is positively associated with gestational age and negatively associated with maternal weight [11, 12]. Lower fetal fraction is associated with twin pregnancy, pregnancy achieved with in vitro fertilization (IVF), and some pregnancy-related or maternal conditions [13–16]. It varies also with fetal aneuploidy; for example, it is lower in pregnancies with trisomy 18 and 13 compared to unaffected pregnancies [11, 17]. As will be discussed below, cfDNA test methods should be able to measure fetal fraction as part of the workflow and consider the fetal fraction of a given sample in the interpretive algorithm.

12.2 Preanalytic Processing The collection and processing of samples influence the quality and yield of cfDNA [18]. Most importantly, lysis of maternal white blood cells can dilute a sample with maternal genomic DNA.  As the maternal DNA in a sample increases, there is a resultant decrease in the proportion of fetal DNA. Immediate processing will avoid this but is not practical in a standard clinical setting where samples must be transported to a testing laboratory. Protection from temperature extremes, minimizing transit time, and the use of specialized cfDNA tubes are optimal for specimen stability [19–22]. Once in the laboratory, plasma is separated using centrifugation, followed by cfDNA extraction, which will be discussed in a later chapter. It is possible to achieve some enrichment of fetal cfDNA by using magnetic beads or electrophoresis [23, 24].

12.3 Methods Common elements of all approaches include a method for quantitation and an interpretive algorithm to predict fetal copy number against the maternal background. The sequences analyzed may be targeted or genome-wide; the quantitation may use sequencing or another, fluorescence-based imaging. The algorithms used will depend on the approach.

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12.3.1 Genome-Wide Sequencing-Based Methods Genome-wide sequencing-based NIPT is often referred to in the literature as massively parallel shotgun sequencing (MPSS) but will be called genome-wide sequencing here to distinguish it from targeted sequencing-based methods. A genome-wide sequencing approach indiscriminately interrogates maternal and fetal cfDNA fragments and determines the base pair sequence, or “reads,” for each of the fragments with next generation sequencing. These reads can be mapped to the chromosome of origin using a human genome reference. The number of reads assigned to each chromosome are counted (hence the moniker “counting approach”). The method entails three basic steps that occur after DNA extraction: library preparation, sequencing, and analysis, as detailed below. There are different sequencing technologies available, but one popular method is an optical sequencing method known as sequencing by synthesis [25]. After cfDNA extraction, a library preparation step modifies the cfDNA fragments to prepare for sequencing. Unique “barcode” sequences are then added to fragments of each patient sample so the sequencing can be multiplexed, i.e., fragments derived from multiple patient samples are mixed and simultaneously sequenced. These barcoded single-stranded cfDNA fragments are placed on a glass surface called a “flow cell” where the DNA is amplified to create clusters of identical DNA fragments. In the sequencing step, DNA polymerases add fluorescent labeled nucleotides that are complementary to bases of the DNA templates of cluster and a camera records the unique color emitted as each base is added. This process is repeated, with each cycle adding one base. The images can then be decoded to generate reads, representing the sequences of the cfDNA fragments on the flowcell. “Paired end” sequencing is a variant of the typical sequencing step described above. Sequencing occurs twice, starting from each end of the cfDNA fragment (both 5′ and 3′ directions). This produces twice the number of reads but also uses more sequencing resources. Optical sequencing by synthesis has been most commonly used sequencing method thus far but there are other ways to capture sequence data. One method relies on semiconductor identification of the release of proton ions during DNA polymerization and has been applied to fetal cfDNA testing with comparable performance [26, 27]. A newer sequencing technology using protein nanopores to detect changes in electrical currents as nucleic acids pass through has been used for fetal cfDNA testing for monogenic disease [28], but not for fetal aneuploidy or copy number variants to date. After sequencing is complete, a demultiplexing step segregates the sequencing data for each sample based on the unique barcode sequences attached during library preparation. The sequence of each cfDNA fragment is aligned to a reference genome and mapped to a specific chromosome location. The ratio of read counts from the chromosome of interest is compared to counts from reference chromosomes or chromosome sequences that are assumed to be disomic (Fig. 12.1).

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Fig. 12.1  The principle of the genome-wide sequencing method. Most sequencing reads are from maternal cfDNA ( bars) with a minor proportion from fetal cfDNA ( bars). Fetal aneuploidy is detected by comparing the read counts from the chromosome of interest with the read counts from reference chromosomes or chromosome sequences. In a pregnancy with fetal trisomy 21, there will be more read counts from chromosome 21. The ratio will depend on fetal fraction. At 20% fetal fraction, the ratio of chromosome 21 to read counts from the reference will be 1.1:1

The output of this comparison is expressed as a z-score value. This is the deviation from the expected number of chromosome counts. Z-scores close to 0 would be indicative of disomy. Typically, a z-score cutoff of 3 is used [29]. Higher than 3 would be high-risk for trisomy; lower than −3 would be high-risk for monosomy, as with the case of monosomy X. While this approach was initially used to determine aneuploidy of an entire chromosome, it can also be used to identify segmental sub-chromosomal imbalances [30]. Each chromosome is subdivided into smaller regions, or “bins,” and counts are aggregated for each separate bin. Overrepresentation or underrepresentation of specific regions would suggest a duplication or deletion, respectively. For genome-wide sequencing, reliable detection of aneuploidy at lower fetal fractions and the detection of smaller chromosomal imbalances require more sequencing resources. When fetal fraction decreases, sequencing depth needs to be increased to achieve a comparable detection rate at a given false-positive rate [31]. Similarly, sequencing depth must increase to achieve a comparable performance when the size of the chromosomal imbalance to be detected decreases [31]. The use of more sequencing resources increases cost, increases turn-around-time, and decreases throughput. This must be balanced with the accuracy achieved, so tests are limited in their resolution of detection, in at least one case to copy number variants of 7 MB in size or larger [32].

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12.3.2 Targeted Methods Whereas there is some controversy about screening for genome-wide alterations, screening for trisomy 21, 18, and 13 is widely accepted as a clinical standard [33, 34]. The following targeted methods screen for these trisomies, sex chromosome aneuploidies, and recurrent microdeletions with a well-defined clinical phenotype. 12.3.2.1 Single-Nucleotide Polymorphism (SNP)-Based One targeted approach relies on changes in ratios of alleles at single-nucleotide polymorphisms (SNPs). The sequence of base pairs is mostly identical, or conserved, between all individuals. However, at each SNP locus, which occurs in roughly 1  in 1000 base pairs for each individual [35], there is a variation in the sequence at one base pair. These specific changes, or alleles, can be unique or common in a population. In cases of trisomy or monosomy, ratios of these alleles differ from those of disomy. Like genome-wide sequencing, cfDNA extraction, library preparation, sequencing, and analysis are still needed; however, there are differences in all but the sequencing step. cfDNA extraction occurs as in the genome-wide approach; however, to maximize performance and minimize the rate of no-results with a SNP approach, the maternal and paternal genotypes can also be analyzed [36, 37]. Maternal peripheral blood cells from the centrifuged sample are a convenient source of maternal DNA [36, 37]. Paternal DNA can be obtained from peripheral blood or buccal samples [36]. Target enrichment entails amplifying specific loci prior to sequencing. SNPs are carefully chosen using public SNP databases [38], selecting SNPs with high levels of heterozygosity (each of the alleles is present as close as possible to 50% of the time) [36]. At each SNP locus, one allele can be arbitrarily assigned as “A,” and the other as “B.” Having a high minor allele frequency (high levels of heterozygosity) is critical for determining copy number because a SNP locus can only be informative if there is a difference in the alleles of the mother and fetus at a given location [37, 39]. Figure 12.2 shows a typical distribution of fetal and maternal alleles at all of the SNPs in an instance of disomy (left panel) and trisomy (right panel). Given that the majority of cell-free DNA is derived from the mother, there will typically be three clusters of maternal allelic reads, two when the mother is homozygous (AA, shown in red, or BB, shown in blue) or one when she is heterozygous (AB, shown in green). The uppermost or lowermost labeled bands correspond to the instances when the homozygous mother is paired with a heterozygous fetus. These bands are shifted toward the middle of the plot in the case of trisomy because of the additional fetal allele. In addition, an additional band is seen in the middle cluster. In the analysis phase, the algorithm can identify ratio changes indicative of trisomy. Allelic ratios can also be used to identify monosomies [40] and microdeletions [41]. Fetal fraction can also be deduced by the distribution of the ratios [36,

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Fig. 12.2  The principle of the SNP-based method. The dots represent the proportion of A allele reads versus B allele reads from the sequencing data for every SNP locus. The pattern will typically be three clusters of allelic reads, two homozygous (AA and BB) and one heterozygous (AB) from the majority maternal cfDNA. The presence of fetal alleles will shift these clusters and generate two additional clusters in the case of disomy (a) and three additional clusters in the case of trisomy (b)

37, 42]. The algorithm chooses the mostly likely hypotheses (monosomic, disomic, or trisomic at a given fetal fraction) based on the data. For known twin gestations, it is possible to differentiate between monozygotic and dizygotic pregnancies [43]. Unlike genome-wide sequencing and other methods that rely on relative sequence quantities, the SNP method can identify triploidy [44, 45]. Unfortunately, discrimination between dizygotic twin gestations and triploidy is not possible [46]. Because the SNP method relies on parental genotyping, this method is challenged by pregnancies with multiple genotypes such as a twin pregnancy achieved with ovum donation [47]. Also, pregnancies with consanguineous parents will also have a higher no result rate because the number of informative SNPs will be reduced [48]. As with genome-wide sequencing, multiplexing is performed and sequencing depth, is crucial, especially at lower fetal fractions, to maintain accuracy [37]. Finally, as parental genotype affects accuracy it is higher with conditions that are paternally derived than it is with maternally derived conditions, especially at lower fetal fraction [37, 42, 49]. 12.3.2.2 Digital Analysis of Selected Regions (DANSR) Using a targeted approach, it is possible to combine aspects of the methods described above. One method combines a quantitative analysis, similar to that used by the genome-wide sequencing-based method, with a SNP-based analysis for fetal fraction assessment [50]. After cfDNA extraction, DANSR (digital analysis of selected regions), uses a large pool of oligonucleotide probes to simultaneously PCR-amplify

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two types of sequences. The first set of sequences are specific to the chromosome or chromosome region to be studied. The second set of sequences includes SNPs. Quantitation of the products of the first set allows the comparison of the region of interest to a disomic reference and the detection of a relative increase or decrease of sequences consistent with aneuploidy. Quantitation of the products of the second set allows the simultaneous assessment of fetal fraction using SNPs. Although the first-generation test applied sequencing for quantitation [50], the second-generation test uses a custom microarray [51]. As a result, some review articles in the scientific literature refer to the DANSR method as “targeted sequencing” and others refer to it as “microarray-based NIPT.” The microarrays are manufactured with fields of oligonucleotide sequences complimentary to the DANSR products. Identical subarrays on one microarray plate allow the processing of multiple independent samples simultaneously. After amplification and hybridization with a fluorescent probe, the imager quantifies the fluorescence intensity for all of the fields of all of the microarrays (Fig. 12.3). The analysis algorithm analyzes the fluorescent signal intensity data, essentially providing a representation of the concentration of each of the targeted sequences. Much like the SNP method, the algorithm establishes at which loci the SNPs are informative and estimates fetal fraction. For the chromosome-specific sequences, the algorithm estimates the relative concentrations of the chromosomes. The algorithm incorporates the estimated fetal fraction in the sample and determines the likelihood of obtaining the observed concentrations of the target sequences at the estimated fetal fraction from an aneuploid sample versus a euploid sample. This allows a better separation of trisomy or monosomy from disomy at lower fetal fractions [50]. An advantage of a targeted approach is that target sequences can be concentrated in the areas of interest. Putting more DANSR assays in small regions, such as the three megabase 22q11.2 deletion, achieves the equivalent of increasing the sequencing depth [52]. The use of two types of sets of target sequences allows more options than a SNPonly test, which requires a minimum number of informative SNPs in the tested region. Because SNPs are used only for fetal fraction determination, the algorithm can accommodate twin pregnancies, pregnancies with an egg donor and surrogate pregnancies. a

b

Fig. 12.3  Quantitation using microarray for the DANSR targeted approach. (a) DANSR products are captured by complementary probes in specific fields of a custom microarray. (b) After fluorescent labeling, the imager captures the signal intensity of each of the fields. The relative signal intensities will reflect the relative amounts of the targeted sequences. Each patient sample is run on a single array, with multiple microarrays on one plate

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12.3.2.3 Rolling-Circle Amplification One of the latest targeted approaches, introduced in 2018, uses an alternative to sequencing and replaces PCR with rolling circle amplification (RCA) with the aim of reducing cost with a less equipment-intensive process [53]. After cfDNA extraction from the maternal serum, targeted nucleic acid probes from the chromosomes-of-interest are used to capture specific fragments from the total cfDNA population. A series of enzymatic reactions individually ligate these fragments onto a longer oligonucleotide, which is then sealed into a circular structure. This process generates a collection of single-stranded, circular DNA templates containing sequences from the chromosome-of-interest. This approach is called rolling circle amplification because the next step involves duplication of each circular structure with DNA polymerase. The DNA “balls” are then fluorescently tagged to indicate the chromosome of origin and added to a microplate (Fig.  12.4). Visualization of the emissions from each fluorescent tag enables quantitation This approach assumes that the relative numbers of the fluorescent signals reflect the relative quantities of the chromosomes in the original maternal blood samples. Analysis of the fluorescent emission counts involves comparisons between chromosomes. Much like the genome-wide sequencing method described above, variations in the ratios are attributed to the fetal contribution and assessed as a z-score. The test does not measure fetal fraction. A previously published distribution of fetal fraction in a large clinical cohort in the UK [17] was used to inform assumptions in the calculations of required rolling circle products needed for the desired precision of the assay. The lack of PCR and sequencing for sample analysis suggest a less complex laboratory process. However, multiple enzymatic steps require quality control measures to ensure the visualization and subsequent analysis of the targeted sequences are precise. a

b

c

Fig. 12.4  Quantitation of rolling circle amplification products. (a) Target DNA sequences are amplified by rolling circle amplification. (b) The resulting balls labeled with chromosome-specific, fluorescent tags, and added to a microplate. (c) An imager captures the fluorescing molecules. The counts of these molecules represent the amounts of the target sequences present in the sample

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12.3.2.4 Future Possibilities The use of chip-based digital PCR (dPCR) of cfDNA for the detection of fetal aneuploidy was explored prior to sequencing [54, 55]. This is a variation of PCR, which allows the quantitation of very small amounts of DNA. cfDNA is diluted such that an average of one or fewer molecules is distributed into distinct compartments on a chip, generating compartments that contain zero or one copy of each DNA molecule. Using a PCR reaction with fluorescently labeled probes specific to the sequences of interest, the fluorescence is captured, and the number of compartments fluorescing allow sequence quantitation. More recently, groups have applied droplet dPCR, which works on the same principle but places the molecules into tens of thousands of water-in-oil droplets [56, 57]. dPCR for nucleic acid examination holds the promise of a faster, simpler, and more cost-effective methodology as compared to next-generation sequencing. However, challenges for applying this technology for aneuploidy screening remain, primarily the number of reactions necessary given the small proportion of fetal cfDNA. Detection of more than one condition in the same reaction also challenges this technology due the requirement of a number of fluorescent probes used simultaneously.

12.4 Fetal Fraction Estimation Fetal fraction is a critical factor in the ability of a test to differentiate between aneuploid and euploid samples [58]. Many laboratories use a fetal fraction threshold, which, if not met, yields an uninformative result. Some methodologies use fetal fraction as part of the interpretive algorithm. There are many methods to estimate fetal fraction. Y-chromosome and SNP-­ based methods can differentiate between fetal and maternal DNA sequences. Other indirect methods leverage different physical and molecular properties of fetal and maternal cfDNA. Because fetal fraction represents the proportion of fetal cfDNA present in a sample at the stage of the process when it is measured, both preanalytical specimen processing as well as biology and bioinformatic algorithms influence the result. Its measurement is therefore ideally performed as part of the laboratory workflow rather than as a separate assay.

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12.4.1 Chromosome-Based Y-chromosome sequences in maternal plasma are typically representative of fetal cfDNA sequences in pregnancies with a male fetus. Y-sequence quantitation, along with the determination of an underrepresentation of X-chromosome sequences, was the first method employed to determine fetal fraction and is widely used as a standard against which other methods are compared [10]. The advantage of this approach is that it can be used with any technology that quantifies Y-chromosome sequences. The obvious limitation is that Y chromosome assessment is not informative in pregnancies with female fetus. To overcome this, some laboratories use Y-chromosome analysis for male pregnancies in combination with indirect methods in female ones [59].

12.4.2 SNP-Based SNP-based fetal fraction determination is integral to the assay design in targeted methods such as the SNP-based and DANSR methods (described above). In addition to SNPs, use of insertion/deletion polymorphisms has also been described as part of a targeted method [60].

12.4.3 Indirect Methods In genome-wide sequencing-based tests, using SNPs is challenging due to the random sequencing of all cfDNA fragments. The depth of sequencing at any given locus is insufficient for determining the frequency of single alleles. Therefore, other methods of fetal fraction estimation relying on the different physical and molecular characteristics of fetal and maternal cfDNA are applied. The accuracy of all of these methods is limited by the fact that, despite different profiles, there is still considerable overlap between the two sources of cfDNA. Genes are differentially methylated in the cells of different tissues such the placenta and maternal peripheral blood cells. If methylation-sensitive restriction enzymes are used to digest hypomethylated cfDNA, differences in the quantity of cfDNA in specific differentially methylated regions can be used to determine fetal fraction [61]. Bisulfite sequencing can also determine differences in methylation profiles across the genome but is expensive [62]. Both methods require an additional assay outside the standard workflow. Another method, SeqFF, takes advantage of patterns of read counts across the genome [63]. The developers divided the genome into 50 kb bins and applied multivariate regression and a machine-learning model to generate an algorithm that uses sequence data to estimate fetal fraction. This is a widely used method for fetal

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fraction estimation in laboratories using genome-wide sequencing methodologies. The accuracy of this method is affected by read-depth and may need to be improved by training in different laboratory settings [64]. Fragment length can also be used to estimate fetal fraction, by determining the frequency of shorter fragments, which are more common in fetal cfDNA [65]. This can be done in laboratories that use paired-end sequencing. The use of electrophoresis has been described [66]; however, this would be performed presequencing or outside of the workflow. One group sought to develop a single-end sequencing method that leveraged the same differences in fragments related to nucleosome structure. They hypothesized that the different length fragments would be associated with a different pattern of sequences at the ends of the fragments and developed a nucleosome profile and algorithm that uses the frequency of read start positions to estimate fetal fraction [67].

12.5 Other Quality Metrics Each of the methods described leads to high-complexity assays that must detect subtle differences in sequence quantity of DNA present in very small amounts. Quality control measures, in addition to fetal fraction determination, are therefore critical. For example, total DNA input and DNA quality can cause variation in sequence counts or relative fluorescence intensity data. Variation has been shown to decrease the accuracy in sequencing-based-testing and should be assessed to ensure quality testing, regardless of the method [68]. Contamination with other sources of DNA can confound all DNA testing and is another example of a consideration for quality control. Although automated methods may mitigate the risk of cross-sample contamination, there is also the possibility of organ transplants and undisclosed egg donation contributing a third source of DNA [69, 70]. Contamination is detectable using SNP analysis.

12.6 Biological Considerations Unique biological situations create technical challenges for all cfDNA methods. In twin pregnancy, one or both twins could be aneuploid and the fetal fraction contribution from each twin could be unequal. When twins are discordant for aneuploidy, only a proportion of the fetal cfDNA will be aneuploid. Fetal fraction determination with SNPs provides an advantage, making it possible to take into account individual contributions and a lower fetal fraction contribution from one, possibly aneuploid, twin [43, 71]. The indirect methods of estimating fetal fraction can only determine the total fetal fraction of both twins.

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Pregnancies with a vanishing or demised twin add another layer of complexity. In a presumed singleton pregnancy, the SNP-based method can detect allelic ratios consistent with the additional chromosome complement from a vanishing demised twin [46]; however, aneuploidy assessment for the viable fetus in these cases is not performed. Testing in pregnancies conceived with a donor egg needs to be interpreted differently by any method using SNPs due to the additional genotype. Genome-wide sequencing methods do not need to make any adjustments. Mosaicism is a limitation for every methodology. Only a proportion of the fetal cfDNA may be aneuploid, challenging the limits of detection. Most significantly, mosaicism may be confined to the placenta or present in the fetus [72]. The likelihood of mosaicism for a given sample can be estimated by combining sequence quantitation with fetal fraction data but this calculation only represents the placenta and is not informative for the fetus [73]. Finally, maternal copy number variants (CNVs), such as deletions and duplications, can be a source of false-positive results if they are attributed to the fetus rather than the mother [74–76]. Targeted methods can avoid regions with common CNVs when designing the target sequences [77]. For the SNP-method and any method using fetal fraction as part of interpretation, maternal CNVs are identifiable using bioinformatics because they will cause an over- or underrepresentation of sequences that is inconsistent with the fetal fraction.

12.7 Conclusion The past decade has been a dynamic era with improvements in both technology and bioinformatics leading to a variety of different methods for fetal cfDNA aneuploidy screening. There has been an expansion from sequencing to other methods of quantitation, which has allowed for faster and more cost-effective tests. Bioinformatics improvements have led to increases in accuracy and improvements in managing the biological complexity of cfDNA. Resulting advances in the knowledge of cfDNA biology have enabled different approaches to fetal fraction determination and monitoring of test quality. Tests have also moved from screening for trisomy 21 and other whole chromosome aneuploidies to the interrogation of smaller segments of the fetal genome. In a relatively short time, these sophisticated methods have been developed and introduced into clinical markets around the world. The currently available tests are of immense value for clinical care; however, all of these methods have advantages and disadvantages. Communication to help individual clinicians, health systems, and policy makers understand the technology and its limitations should be a top priority for laboratories and the scientific community. Meanwhile, creative researchers will build upon the achievements of the past two decades and apply their ingenuity to further refine noninvasive fetal testing.

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Acknowledgments  The authors are grateful to Patrick Bogard for his input. Conflict of Interest  Francesca Romana Grati is full-time employee of TOMA laboratory, advisory board member for Roche and consultant and advisory board member of Menarini-Silicon Biosystems. Karen White, Bowdoin Su, Renee Jones, and Emilia Kostenko are full-time employees of Roche Sequencing Solutions.

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51. Juneau K, Bogard PE, Huang S, et  al. Microarray-based cell-free DNA analysis improves noninvasive prenatal testing. Fetal Diagn Ther. 2014;36(4):282–6. https://doi. org/10.1159/000367626. 52. Schmid M, Wang E, Bogard PE, et  al. Prenatal screening for 22q11.2 deletion using a targeted microarray-based cell-free DNA test. Fetal Diagn Ther. 2018;44(4):299–304. https:// doi.org/10.1159/000484317. 53. Dahl F, Ericsson O, Karlberg O, et  al. Imaging single DNA molecules for high precision NIPT. Sci Rep. 2018;8(1):4549. https://doi.org/10.1038/s41598-­018-­22606-­0. 54. Lo YMD, Lun FMF, Chan KCA, et  al. Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc Natl Acad Sci U S A. 2007;104(32):13116–21. https://doi. org/10.1073/pnas.0705765104. 55. Fan HC, Quake SR.  Detection of aneuploidy with digital polymerase chain reaction. Anal Chem. 2007;79(19):7576–9. https://doi.org/10.1021/ac0709394. 56. Tan C, Chen X, Wang F, et al. A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies. Analyst. 2019;144(7):2239–47. https://doi.org/10.1039/ c8an02018c. 57. Lee SY, Kim SJ, Han S-H, et  al. A new approach of digital PCR system for non-invasive prenatal screening of trisomy 21. Clin Chim Acta. 2018;476:75–80. https://doi.org/10.1016/j. cca.2017.11.015. 58. Canick JA, Palomaki GE, Kloza EM, Lambert-Messerlian GM, Haddow JE. The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies. Prenat Diagn. 2013;33(7):667–74. https://doi.org/10.1002/pd.4126. 59. Hestand MS, Bessem M, van Rijn P, et  al. Fetal fraction evaluation in non-invasive prenatal screening (NIPS). Eur J Hum Genet. 2019;27(2):198–202. https://doi.org/10.1038/ s41431-­018-­0271-­7. 60. Barrett AN, Xiong L, Tan TZ, et  al. Measurement of fetal fraction in cell-free DNA from maternal plasma using a panel of insertion/deletion polymorphisms. PLoS One. 2017;12(10):e0186771. https://doi.org/10.1371/journal.pone.0186771. 61. Nygren AOH, Dean J, Jensen TJ, et al. Quantification of fetal DNA by use of methylation-­ based DNA discrimination. Clin Chem. 2010;56(10):1627–35. https://doi.org/10.1373/ clinchem.2010.146290. 62. Jensen TJ, Kim SK, Zhu Z, et  al. Whole-genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains. Genome Biol. 2015;16(1):78. https://doi.org/10.1186/s13059-­015-­0645-­x. 63. Kim SK, Hannum G, Geis J, et al. Determination of fetal DNA fraction from the plasma of pregnant women using sequence read counts. Prenat Diagn. 2015;35(8):810–5. https://doi. org/10.1002/pd.4615. 64. Miceikaitė I, Brasch-Andersen C, Fagerberg C, Larsen MJ. Total number of reads affects the accuracy of fetal fraction estimates in NIPT. Mol Genet Genomic Med. 2021;9:e1653. https:// doi.org/10.1002/mgg3.1653. 65. Yu SCY, Chan KCA, Zheng YWL, et al. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing. Proc Natl Acad Sci U S A. 2014;111(23):8583–8. https://doi. org/10.1073/pnas.1406103111. 66. Bedon L, Vuch J, Monego SD, Meroni G, Pecile V, Licastro D. An online tool for fetal fraction prediction based on direct size distribution analysis of maternal cell-free DNA. Biotechniques. 2021;70(2):81–8. https://doi.org/10.2144/btn-­2020-­0143. 67. Straver R, Oudejans CBM, Sistermans EA, Reinders MJT.  Calculating the fetal fraction for noninvasive prenatal testing based on genome-wide nucleosome profiles. Prenat Diagn. 2016;36(7):614–21. https://doi.org/10.1002/pd.4816. 68. Blais J, Giroux S, Caron A, et al. Non-invasive prenatal aneuploidy testing: critical diagnostic performance parameters predict sample z-score values. Clin Biochem. 2018;59:69–77. https:// doi.org/10.1016/j.clinbiochem.2018.06.015.

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Chapter 13

The Technologies: Comparisons on Efficiency, Reliability, and Costs Zhijie Yang, Youxiang Wang, and Gian Carlo Di Renzo

Rong Wang Bo Jiang Sookhim Chan Jialuo Li Yining Shi Yifan Chen Xin Chen Han Zhang Atila Biosystems Inc. Mountain View, CA, USA

13.1 Introduction Noninvasive prenatal testing (NIPT) is a method used to determine the risk for the fetus being born with certain chromosomal abnormalities. The test normally only involves blood drawing from pregnant women, therefore, is noninvasive. The

Z. Yang · Y. Wang Atila Biosystems Inc., Mountain View, CA, USA G. C. Di Renzo (*) Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Italy Department of Obstetrics and Gynecology, IM Sechenov First State University, Moscow, Russia PREIS School (The Permanent International and European School of Perinatal, Neonatal and Reproductive Medicine), Florence, Italy e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_13

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chromosomal abnormalities include the most common aneuploidies, such as trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome), as well as the additional items, such as sex chromosome aneuploidies, rare autosomal aneuploidies, 22q11.2 microdeletion (DiGeorge syndrome), other microdeletions, and microduplications. The NIPT can also report fetal sex and fetal fraction. The NIPT analyzes small DNA fragments (99.9 >99.9 >99.9 96.40 74.10 95.50

95% CI 98.66–99.53% 94.20–98.15% 95.56–98.87% 96.1–99.6% 97.1–100% 91.4–100% 87.1–100% 82.3–99.4% 55.3–86.8% 92.7–97.3%

Specificity (%) 99.77 99.69 99.84 99.95 99.90 99.90 99.90 99.80 99.80 99.34

95% CI 98.92–99.91% 99.51–99.85% 99.77–99.93% 99.62–99.99% 99.63–99.97% 99.64–99.97% 99.64–99.97% 99.49–99.92% 99.49–99.92% 98.87–99.61%

74.1% and the specificity is 99.8% (Table 13.4). Only the concordance values are reported for sex chromosome aneuploidies, and they are 90.5% for Monosomy X, 100% for XXX, 100% for XXY, and 91.7% for XYY.

13.3.3 Harmony® Test and ClariTest™ Another widely used NIPT product is the Harmony® Test (ClariTest™), which was launched by Ariosa Diagnostics in 2012. In 2014, Ariosa Diagnostics was acquired by Roche, and in 2021, the US Ariosa centralized lab prenatal testing business was acquired by Opko subsidiary BioReference Laboratories. The ClariTest Core test offered by BioReference’s specialty health division Genpath uses the same core technology as the Harmony test [46, 47]. The Harmony Test/ ClariTest can be performed on pregnant women of any age and risk category with a gestation age as early as 10 weeks. The test can be used to screen singleton and egg donor/IVF pregnancies for common trisomies (T21, T18, and T13), Monosomy X (45, X, Turner syndrome), XXY (Dlinefelter syndrome), XXX (Trisomy X), XYY (Jacob syndrome), XXYY, and 22q11.2 syndrome (DiGeorge syndrome). The test is also available for twin pregnancies for only the common trisomies and fetal sex. Differing from other tests, the Harmony Test/ClariTest utilizes the DANSR™ platform and FORTE™ algorithm and has demonstrated great performance [48–54] and established a superior accuracy and reproducibility of the platform for fetal fraction assessment [55]. The DANSR platform is the abbreviation of digital analysis of selected regions platform [11], as shown in the Fig. 13.8. The DANSR is a targeted amplification technology and the amplification of the maternal blood cfDNA enables simultaneous quantification of hundreds of loci by catenating two locus-specific oligonucleotides via an intervening “bridge” oligo to form a PCR template. The selected loci have unique sequences, and the “bridge” oligo can largely help to improve the specificity.

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Fig. 13.8  The schematic of DANSR assay. Arrows and dots indicate 3′OH and 5′PO4 moieties, respectively. When the left, middle, and right ligation oligos hybridize to their cognate genomic DNA (gDNA) sequences, their termini form two nicks. Ligation of these nicks results in the creation of an amplifiable template using the indicated UPCR primers. UPCR with 96 distinct right UPCR primers enables pooling and simultaneous sequencing of 96 different UPCR products on a single lane. The UPCR primers also contain left and right cluster tail sequences that support cluster amplification. Cited from Sparks et al. [11]

The Harmony Test/ClariTest is performed on an automated instrument and the workflow is straightforward. The test requires approximately 4 mL of plasma collected in a cfDNA collection tube and the cfDNA is isolated using a commercially available cfDNA extraction kit. About 95 samples can be processed simultaneously to complete the DANSR amplification. Two DNA quantification methodologies, massively parallel sequencing (MPS) and DNA microarray quantification are utilized for cfDNA quantification analysis. Compared to the shotgun MPS, targeted MPS on DANSR products reduces the amount of sequencing required for NIPT and has the potential to dramatically reduce test costs. Furthermore, by designing loci with SNP on sex chromosomes, the test can also precisely measure and distinguish the fetal fractions. As to the sequencing step, the test also utilizes a microarrays-­based comparative genomic hybridization (CGH) method [56] and provides accurate cfDNA quantification. The microarray method can reduce the turnover time of the test, lower the assay variability, and additionally improve the fetal fraction precision. The FORTE™ algorithm, which stands for fetal-fraction optimized risk of trisomy evaluation algorithm, estimates the risk of aneuploidy using an odds ratio comparing a model assuming a disomic fetal chromosome and a model assuming a trisomic fetal chromosome [50]. FORTE will produce an individualized trisomy risk score for each sample. Results are available within 5–7 days. The report includes high-risk results for Trisomy 21, 18, 13, Monosomy X or 22q11.2 Deletion syndrome plus a positive predictive value (PPV), low-risk results plus a negative predictive value (NPV), and the fetal fraction. In the year 2014, The Harmony Test costs $95 for patients with insurance coverage or $795 out-of-pocket for those without qualifying insurance [30]. The Harmony Test/ClariTest has been intensively studied with over 60 peer-­ reviews publications. The performance characteristics are summarized in Table 13.5 [48–54].

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Table 13.5  The performance characteristics of the Harmony Test/ClariTest Disorder Trisomy 21 Trisomy 18 Trisomy 13 Monosomy X 22q11.2 microdeletion XXX/XXY/XYY/ XXYY

Number of samples 23,155

Sensitivity Specificity 99.3% 99.96% (418/421) (22,724/22,734) 22,399 97.4% 99.98% (147/151) (22,243/22,248) 14,243 93.8% 99.98% (30/32) (14,208/14,211) 1381 94.3% 99.8% (1291/1294) (82/87) 1953 75.2% 99.6% (1816/1824) (97/129) Other sex aneuploidies will be reported if detected. Limited data of these rarer aneuploidies preclude performance calculations

13.3.4 Panorama™ Test The Panorama™ Test, by Natera, was developed in 2012 based on the amplification and massively parallel sequencing (MPS) of single-nucleotide polymorphisms (SNPs) [57–59]. It can differentiate the genotypes between mother and fetus using a proprietary, patented algorithm called Next-generation Aneuploidy Test Using SNPs (NATUS). In general, the mother’s DNA is isolated and identified from her white blood cells. Then this information is used as a baseline to correct the maternal genotype, giving a more accurate fetal genotype (Fig. 13.9) [1]. The Panorama™ test can detect fetal Trisomy 21 (Down syndrome), Trisomy 18 (Edwards syndrome), Trisomy 13 (Patau syndrome), Monosomy X (Turner syndrome), XXY Syndrome (Klinefelter syndrome), Triple X syndrome, XYY Syndrome (Jacob’s Syndrome), and fetal sex if requested, as well as microdeletions (Fig. 13.10). Reliable and accurate results can be obtained for the samples with fetal fractions as low as 4%. This method is uniquely for the detection of triploidy [60], a condition that is estimated to happen in approximately 1:1500 pregnancies, because it is the only method independent of a reference chromosome. In validation studies, Panorama™ reported high sensitivity and specificity for detecting autosomal trisomies and fetal sex. For example, over 99% of sensitivity and specificity of Down Syndrome (Trisomy 21) and fetal sex were reported. Additionally, 91.7% sensitivity and over 99% specificity were obtained for Turner Syndrome (Monosomy X) (Levy B et al. Massively multiplexed targeted amplification and sequencing of SNPs as a method for identifying fetal chromosome disorders from cell-free DNA in maternal plasma. Annual Clinical Medical Genetics Meeting, March 19–23, 2013, in Phoenix, AZ.). In 2013, Samango-Sprouse and co-workers investigated the accuracy of SNP-based NIPT in detecting sex chromosome aneuploidies. They detected [45,X] with 91.7% sensitivity and 100% specificity, and [47,XXY], [47, XYY] with 99.78% average accuracy from the samples as early as 9.4 weeks of gestation [58]. In 2014, Dar P, Curnow KJ, and Gross SJ, et al. reported a study based on large-scale clinical reports using SNP-based NIPT. The clinical results yield an 82.9% PPV (positive predictive value) for all aneuploidies,

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Fig. 13.9  Scheme of how Panorama™ works. (Adapted from Megan P. Hall [1])

Fig. 13.10  The items of Panorama testing. (Adapted from Panorama Patient Brochure Jun. 7, 2022)

and a 90.9% PPV for trisomy 21, indicating the clinical performance of this method is consistent with the performance in validation studies [10]. So far, the sensitivities are improved to: Trisomy 21 (>99.9%), Trisomy 18 (98.2%), Trisomy 13 (>99%), Monosomy X (92.9%), 22q11.2 deletion syndrome (90%), and Microdeletion extended panel (1p36 deletion, 15q11–q13 deletions [Angelman syndrome and Prader-Willi syndrome], 5p deletion) (93.8 to >99%). False-positive rate was

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reported by Natera: Trisomy 21 (0%), Trisomy 18 (99% (99.2–99.9%) 91.7

Negative predictive value (NPV) (%) >99 >99

>99% (99.5–100%)

>99

90.9

and allows one lab technician to handle up to 20,000 samples per year. Apart from sample centrifuge steps, the hands-on time can be as little as 40 min, depending on the number of samples being tested. Performance of the Vanadis NIPT assay in sensitivity and specificity has been published in several publications [78–82]. The following table (Table 13.9) is a summary as cited from the website of https://www.nuh.nhs.uk/how-­accurate­is-­the-­vanadis-­nipt-­test-­for-­trisomy-­screening/.

13.4.3 PrenaTest and qNIPT PrenaTest by Eurofins LifeCodexx Eurofins Lifecodexx had a license agreement with Sequenom Inc. (USA) to use the essential patents for shotgun MPS-based NIPT test in Germany in 2011 and launched the first NIPT in Europe, the PrenaTest, that detects trisomy 21 in 2012 [83]. The PrenaTest was expanded to include more test items and by 2019, the PrenaTest is available for detection of trisomy 21, trisomy 18, trisomy 13, sex chromosomal aneuploidies (Turner, Triple X, Klinefelter, and XYY syndrome), 22q11.2 microdeletion (DiGeorge syndrome), and rare autosomal aneuploidies. The test is CE certified in 2012. In 2016, a qPCR-based NIPT is CE approved for the detection of fetal trisomy 21, the qNIPT PrenaTest [83, 84]. The quantitative real-time PCR-based assay determines the differences in methylation patterns of specific genomic regions in maternal DNA and fetal DNA and uses the information to determine fetal trisomy 21 status as illustrated in the Fig.  13.14 [84]. The qNIPT is a very cost-efficient solution and has a very short turnaround time compared to the NGS-based tests. As shown in the Table 13.10, a study in 2016 [84] on 966 plasma samples showed that the qNIPT demonstrated a positive percentage agreement (PPA; equates to sensitivity) of 100% (lower one-sided 95% confidence interval of 91.8%; n = 35/35) and a negative percentage agreement (NPA; equates to specificity; n = 931/931) of 100% compared to NGS-based PrenaTest®. The negative predictive value (NPV) of the smart qNIPT and confirmatory NGS testing was 100% (lower one-sided 95% confidence interval of 99.68%). The average fetal fraction of all examined blood samples was 8.1%. The qNIPT provided reliable test results in 54 blood samples with a fetal fraction below 4% and as low as 2.4%.

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Fig. 13.14  The qPCR-based NIPT test for trisomy 21. (Adapted from [84]) Table 13.10  The summary of study conducted in 2016 (Adapted from [84])

Performance characteristics Correctly classified samples Trisomy 21 positive Trisomy 21 negative Sensitivity (lower one-sided 95% CI) Specificity (lower one-sided 95% CI) NPV (lower one-sided 95% CI)

Statistics 966/966 (100%) 35/35 (100%) 931/931 (100%) 100% (91.88%) 100% (–) 100% (99.68%)

13.5 Other Types of NIPT Tests Most NIPT tests focus on the detection of chromosomal abnormalities, and in the other part, tests for monogenic disorder or diseases are developing and increasing rapidly [2]. Monogenic diseases are caused by a variant or an abnormality in one gene, such as deletions, insertions, substitutions of a base, etc. Monogenic diseases, based on their inheritance pattern, can be categorized into several major groups, autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, etc. Some of the more common monogenic disorders are listed in the Table 13.11 [85, 86]. The detection of a monogenic disorder employs the same process, such as cfDNA extraction, NGS technologies, and bioinformatics analysis. The presence or absence of mutations in the target gene is used to determine the monogenic disorder status [87–90]. Lo et al. [20] used relative haplotype dosage analysis (RHDO) to determine fetal genetic disorders. By building a genome-wide reference map, fetal haplotype can be deduced and evaluated. Other demonstrated approaches such as relative mutation dosage (RMD) [91] as well as the development of higher sensitive and accurate digital PCR methods also enable to detect inherited mutant alleles and de novo mutations [92]. Several examples of the monogenic tests are listed in the following. VERAgene [93], a product of NIPD Genetics, targets thousands of mutations to screen for 100 autosomal recessive and X-linked monogenic diseases. PreSeek™ NIPT from Baylor Genetics (Preseek Maternal) [94] targets 30 genes in which de novo variants

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Table 13.11  Common monogenic disorders, type of inheritance, and gene responsible Disease Albinism, oculocutaneous, type II Congenital deafness (nonsyndromic) Cystic fibrosis Duchenne muscular dystrophy Familial hypercholesterolemia Hemochromatosis Hemophilia A Huntington disease Hypercholesterolemia type B Hypophosphatemic rickets Myotonic dystrophy type 1 Neurofibromatosis, type 1 Phenylketonuria (PKU) Polycystic kidney disease 1 and 2 Rett’s syndrome Sickle cell anemia Spermatogenic failure, nonobstructive Tay-Sachs

Inheritance Autosomal recessive Autosomal recessive Autosomal recessive X-linked recessive Autosomal dominant Autosomal recessive X-linked recessive Autosomal dominant Autosomal dominant X-linked dominant Autosomal dominant Autosomal dominant Autosomal recessive Autosomal dominant X-linked dominant Autosomal recessive Y-linked Autosomal recessive

Gene responsible OCA2 GJB2 CFTR DMD LDL receptor HFE F8 HTT LDLR, APOB PHEX DMPK NF1 PAH PKD1, PKD2 MECP2 Beta-globin USP9Y HEXA

frequently occur. Vistara™ Single-Gene NIPT, product from Natera, tests for 25 genetic conditions showed good sensitivity and specificity [95]. Resura™ Prenatal Test, the NIPT product for monogenic disease from Progenity, uses ddPCR system to test pregnancies [96].

13.6 Summary In summary, NIPT tests have been available for a wide range of target disorders, with high sensitivity and specificity. The most widely used technologies in the NIPT are targeted or shotgun MPS, digital PCR, as well as digital imaging and others. As the technologies advance at a rapid pace, more diseases related to the fetal health will be available to general population at a lower cost.

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24. Dai P, Yang Y, Zhao G, Gu Z, Ren H, Hu S, Liu N, Jiao W, Li J, Kong X.  A dPCR-NIPT assay for detections of trisomies 21, 18 and 13 in a single-tube reaction-could it replace serum biochemical tests as a primary maternal plasma screening tool? J Transl Med. 2022;20:269. 25. Haidong W, Zhijie Y, Youxiang W, Renzo GCD, Picchiassi E, Tarquini F, Coata G, You W, Yu C.  Non-invasive prenatal testing of fetal aneuploidies using a new method based on digital droplet PCR and cell-free fetal DNA. Clinics in Mother and Child Health 2021;1–8. 26. Inc S Sequenom CMM’s MaterniT21 PLUS LDT now reporting on gender-specific chromosomal abnormalities. https://www.prnewswire.com/news-­releases/ sequenom-­c mms-­m aternit21-­p lus-­l dt-­n ow-­r eporting-­o n-­g ender-­s pecific-­c hromosomal-­ abnormalities-­189627021.html. Accessed 4 Sept 2022. 27. MaterniT 21 Plus | Labcorp. https://www.labcorp.com/pregnancy/maternit21-­plus. Accessed 4 Sept 2022. 28. Inc S Sequenom Laboratories expands international noninvasive prenatal testing services to broader population with VisibiliT™ test. https://www.prnewswire.com/news-­releases/ sequenom-­l aboratories-­expands-­i nternational-­n oninvasive-­p renatal-­t esting-­s ervices-­t o-­ broader-­population-­with-­visibilit-­test-­267914411.html. Accessed 4 Sept 2022. 29. Inc S Sequenom Laboratories launches MaterniT™ GENOME. https://www.prnewswire.com/ news-­releases/sequenom-­laboratories-­launches-­maternit-­genome-­300111863.html. Accessed 4 Sept 2022. 30. Agarwal A, Sayres LC, Cho MK, Cook-Deegan R, Chandrasekharan S. Commercial landscape of noninvasive prenatal testing in the United States. Prenat Diagn. 2013;33:521–31. 31. 451927: MaterniT21 PLUS Core (chr21,18,13,sex) | Labcorp. https://www.labcorp.com/ tests/451927/maternit21-­plus-­core-­chr21-­18-­13-­sex. Accessed 4 Sept 2022. 32. Jensen TJ, Zwiefelhofer T, Tim RC, et al. High-throughput massively parallel sequencing for fetal aneuploidy detection from maternal plasma. PLoS One. 2013;8:e57381. 33. McCullough RM, Almasri EA, Guan X, et al. Non-invasive prenatal chromosomal aneuploidy testing—clinical experience: 100,000 clinical samples. PLoS One. 2014;9:e109173. 34. Tynan JA, Kim SK, Mazloom AR, et al. Application of risk score analysis to low-coverage whole-genome sequencing data for the noninvasive detection of trisomy 21, trisomy 18, and trisomy 13. Prenat Diagn. 2016;36:56–62. 35. Dyr B, Boomer T, Almasri EA, Wardrop JL, Rafalko J, Chibuk J, McCullough RM. A new era in aneuploidy screening: cfDNA testing in >30,000 multifetal gestations: experience at one clinical laboratory. PLoS One. 2019;14:e0220979. 36. Lefkowitz RB, Tynan JA, Liu T, et al. Clinical validation of a noninvasive prenatal test for genomewide detection of fetal copy number variants. Am J Obstet Gynecol. 2016;215:227. e1–227.e16. 37. Inc VH Verinata Health announces successful introduction of its verifi™ prenatal test. https:// www.prnewswire.com/news-­releases/verinata-­health-­announces-­successful-­introduction-­of-­ its-­verifi-­prenatal-­test-­155906455.html. Accessed 6 Sept 2022. 38. Illumina to acquire Verinata for up to $450M. In: Genomeweb. 2013. https://www.genomeweb. com/clinical-­genomics/illumina-­acquire-­verinata-­450m. Accessed 6 Sept 2022. 39. Verifi and Verifi plus prenatal tests | Samples processed at CLIA-certified, CAP-accredited lab. https://www.illumina.com/clinical/illumina_clinical_laboratory/verifi-­prenatal-­tests.html. Accessed 6 Sept 2022. 40. Illumina launches the VeriSeq™ NIPT Solution in Europe. https://www.illumina.com/content/illumina-­marketing/amr/en_US/company/news-­center/press-­releases/2017/2260864. html?null. Accessed 6 Sept 2022. 41. VeriSeq NIPT Solution v2 | Comprehensive and reliable NIPT solution. https://www.illumina. com/products/by-­type/ivd-­products/veriseq-­nipt.html. Accessed 6 Sept 2022. 42. Non-Invasive Prenatal Testing (NIPT). 2. 43. Taneja PA, Snyder HL, de Feo E, Kruglyak KM, Halks-Miller M, Curnow KJ, Bhatt S. Noninvasive prenatal testing in the general obstetric population: clinical performance and counseling considerations in over 85 000 cases. Prenat Diagn. 2016;36:237–43.

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44. Smith-Gonzalez DJ, Smith DM, Doe J. Verifi Plus prenatal screening report. 2. 45. Pertile MD, Flowers N, Vavrek D, Andrews D, Kalista T, Craig A, Deciu C, Duenwald S, Meier K, Bhatt S. Performance of a paired-end sequencing-based noninvasive prenatal screening test in the detection of genome-wide fetal chromosomal anomalies. Clin Chem. 2021;67:1210–9. 46. Roche acquires Ariosa Diagnostics and enters the non-invasive prenatal test (NIPT) and cell-­ free DNA testing services markets. https://sequencing.roche.com/content/rochesequence/ en-­us/news-­overview/2014/roche-­acquires-­ariosa-­diagnostics-­and-­enters-­the-­non-­invasive-­pr. html. Accessed 4 Sept 2022. 47. BioReference Labs acquires US Ariosa Lab NIPT Business From Roche. In: Genomeweb. 2021. https://www.genomeweb.com/business-­news/bioreference-­labs-­acquires-­us-­ariosa-­lab-­ nipt-­business-­roche. Accessed 4 Sept 2022. 48. Stokowski R, Wang E, White K, et al. Clinical performance of non-invasive prenatal testing (NIPT) using targeted cell-free DNA analysis in maternal plasma with microarrays or next generation sequencing (NGS) is consistent across multiple controlled clinical studies. Prenat Diagn. 2015;35:1243–6. 49. Jones KJ, Wang E, Bogard P, White K, Schmid M, Stokowski R, Nicolaides KH.  Targeted cell-free DNA analysis with microarray quantitation for assessment of fetal sex and sex chromosome aneuploidy risk. Ultrasound Obstet Gynecol. 2018;51:275–6. 50. Norton ME, Brar H, Weiss J, et  al. Non-invasive chromosomal evaluation (NICE) study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol. 2012;207:137.e1–8. 51. Nicolaides KH, Syngelaki A, Ashoor G, Birdir C, Touzet G.  Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. Am J Obstet Gynecol. 2012;207:374.e1–6. 52. Gil MM, Galeva S, Jani J, Konstantinidou L, Akolekar R, Plana MN, Nicolaides KH. Screening for trisomies by cfDNA testing of maternal blood in twin pregnancy: update of the Fetal Medicine Foundation results and meta-analysis. Ultrasound Obstet Gynecol. 2019;53:734–42. 53. del Mar GM, Quezada MS, Bregant B, Syngelaki A, Nicolaides KH. Cell-free DNA analysis for trisomy risk assessment in first-trimester twin pregnancies. Fetal Diagn Ther. 2014;35:204–11. 54. Schmid M, White K, Stokowski R, Miller D, Bogard PE, Valmeekam V, Wang E. Accuracy and reproducibility of fetal-fraction measurement using relative quantitation at polymorphic loci with microarray. Ultrasound Obstet Gynecol. 2018;51:813–7. 55. Sparks AB, Struble CA, Wang ET, Song K, Oliphant A. Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. Am J Obstet Gynecol. 2012;206:319.e1–9. 56. Juneau K, Bogard PE, Huang S, Mohseni M, Wang ET, Ryvkin P, Kingsley C, Struble CA, Oliphant A, Zahn JM. Microarray-based cell-free DNA analysis improves noninvasive prenatal testing. Fetal Diagn Ther. 2014;36:282–6. 57. Zimmermann B, Hill M, Gemelos G, et al. Non-invasive prenatal aneuploidy testing at chromosomes 13, 18, 21, X, and Y, using targeted sequencing of polymorphic loci. Prenat Diagn. 2012;32:1233–41. 58. Samango-Sprouse C, Banjevic M, Ryan A, et  al. SNP-based non-invasive prenatal testing detects sex chromosome aneuploidies with high accuracy. Prenat Diagn. 2013;33:643–9. 59. Pergament E, Cuckle H, Zimmermann B, et al. Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort. Obstet Gynecol. 2014;124:210–8. 60. Curnow KJ, Wilkins-Haug L, Ryan A, Kırkızlar E, Stosic M, Hall MP, Sigurjonsson S, Demko Z, Rabinowitz M, Gross SJ. Detection of triploid, molar, and vanishing twin pregnancies by a single-nucleotide polymorphism-based noninvasive prenatal test. Am J Obstet Gynecol. 2015;212:79.e1–9. 61. Noninvasive prenatal testing for trisomies 21, 18, and 13, sex chromosome aneuploidies, and microdeletions: a health technology assessment. Ont Health Technol Assess Ser. 2019;19:1–166.

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62. Dar P, Jacobsson B, MacPherson C, et al. Cell-free DNA screening for trisomies 21, 18, and 13 in pregnancies at low and high risk for aneuploidy with genetic confirmation. Am J Obstet Gynecol. 2022;227:259.e1–259.e14. 63. Dar P, Jacobsson B, Clifton R, et al. Cell-free DNA screening for prenatal detection of 22q11.2 deletion syndrome. Am J Obstet Gynecol. 2022;227:79.e1–79.e11. 64. 5 Perinatal and genetic outcomes associated with no call cfDNA results in 18,496 pregnancies. Am J Obstet Gynecol. https://www.ajog.org/article/S0002-­9378(20)31483-­6/fulltext. Accessed 4 Sept 2022. 65. BGI NIFTY™ becomes first CFDA approved non-invasive prenatal genetic test. https://www. prnewswire.com/news-­releases/nifty-­becomes-­first-­cfda-­approved-­non-­invasive-­prenatal-­ genetic-­test-­265991971.html. Accessed 4 Sept 2022. 66. Introduction to the NIFTY test. In: The NIFTY™ test—a non-invasive prenatal test brought to by BGI diagnostics. https://www.niftytest.com/healthcare-­providers/intro-­to-­nifty/. Accessed 4 Sept 2022. 67. Clinical validation data for the NIFTY test. In: The NIFTY™ test—a non-invasive prenatal test brought to by BGI diagnostics. https://www.niftytest.com/healthcare-­providers/clinical-­ data/. Accessed 4 Sept 2022. 68. NIFTY-Brochure-Final.pdf. 69. NIFTY test methodology and sequencing technology. In: The NIFTY™ test—a non-invasive prenatal test brought to by BGI diagnostics. https://www.niftytest.com/healthcare-­providers/ nifty-­test-­methodology/. Accessed 4 Sept 2022. 70. Dan S, Wang W, Ren J, et al. Clinical application of massively parallel sequencing-based prenatal noninvasive fetal trisomy test for trisomies 21 and 18 in 11 105 pregnancies with mixed risk factors: clinical application of sequencing-based prenatal noninvasive fetal trisomy test. Prenat Diagn. 2012;32:1225–32. 71. Zhang H, Gao Y, Jiang F, et al. Non-invasive prenatal testing for trisomies 21, 18 and 13: clinical experience from 146 958 pregnancies. Ultrasound Obstet Gynecol. 2015;45:530–8. 72. Liang D, Cram DS, Tan H, et al. Clinical utility of noninvasive prenatal screening for expanded chromosome disease syndromes. Genet Med. 2019;21:1998–2006. 73. Quest diagnostics to enhance quality of noninvasive prenatal screening with QNatal Advanced™. In: Quest Diagnostics Newsroom. https://newsroom.questdiagnostics. com/2015-­05-­12-­Quest-­Diagnostics-­to-­Enhance-­Quality-­of-­Noninvasive-­Prenatal-­Screening-­ with-­QNatal-­Advanced. Accessed 4 Sept 2022. 74. Kim SY, Lee SM, Kim SM, et al. Novel method of real-time PCR-based screening for common fetal trisomies. BMC Med Genomics. 2021;14:195. 75. Hong S, Lee SM, Oh S, Kim SY, Jung YM, Kim SM, Park C-W, Jun JK, Kim BJ, Park JS. Simple and rapid detection of common fetal aneuploidies using peptide nucleic acid probe-­ based real-time polymerase chain reaction. Sci Rep. 2022;12:150. 76. Dahl F, Ericsson O, Karlberg O, et  al. Imaging single DNA molecules for high precision NIPT. Sci Rep. 2018;8:4549. 77. bro_vanadis-nipt_014094_01.pdf. 78. Ericsson O, Ahola T, Dahl F, et al. Clinical validation of a novel automated cell-free DNA screening assay for trisomies 21, 13, and 18 in maternal plasma. Prenat Diagn. 2019;39:1011–5. 79. Gormus U, Chaubey A, Shenoy S, et  al. Assessment and clinical utility of a non-next-­ generation sequencing-based non-invasive prenatal testing technology. Curr Issues Mol Biol. 2021;43:958–64. 80. Karlsson F, Ahola T, Dahlberg J, Prensky L, Moilanen H, Spalding H. Evaluation of repeat testing of a non-sequencing-based NIPT test on a Finnish general-risk population. Acta Obstet Gynecol Scand. 2021;100:1497–500. 81. Pavanello E, Sciarrone A, Guaraldo V, et  al. Cell-free DNA screening for fetal aneuploidy using the rolling circle method: a step towards non invasive prenatal testing simplification. Prenat Diagn. 2021;41:1694–700.

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Chapter 14

Pre- and Posttest Counseling Dick Oepkes

14.1 Introduction Virtually all pregnant women worry about the health of their baby. There is no need for professional to tell them that some babies are born with anomalies, this is common knowledge since the earliest times of mankind. Traditions such as “counting fingers and toes” at birth, or often-made remarks that it is not so relevant if it will be a boy or a girl, “as long as it is healthy” are well-known in many cultures. The first option to reliably predict presence or absence of a major disorder already early in pregnancy came with the introduction of amniocentesis. Karyotyping enabled counting of all chromosomes, mainly directed at detecting or excluding trisomy 21, the most common (often) nonlethal chromosomal disorder in live-born children. This invasive technique, within the early days a risk of 1% of causing a miscarriage, was offered to women of “advanced maternal age,” with a cutoff first of 40 years, then 38, 36, and 35 years when the technique became cheaper and safer. The relatively late gestational age at which the result became available (procedure at 16  weeks, with 2–3  weeks turnaround time) led to the introduction of chorionic villus sampling (CVS), possible from 10 to 11  weeks onwards. For many years, counseling about fetal testing on genetic disorders consisted of, first, asking the woman’s age (the screening test), and if above the local cutoff, discussing Down syndrome and outlining the differences between amniocentesis and CVS.  Apart from the gestational age at testing, a major difference between these two diagnostic tests was the testing only possible in amniotic fluid, of the alpha-fetoprotein (AFP) as a marker for spina bifida. We have come a long way. This is the area in obstetrics with by far the biggest advances of the past 50 years. Screening for genetic and structural anomalies is now D. Oepkes (*) Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_14

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offered to all women, regardless of age, with increasing reliability, and the disorders we can offer testing for are much more than trisomy 21 and spina bifida. These changes obviously have affected our counseling. As Chervenak and McCullough stated, in their in-depth analysis of professional ethics in this field, responsible counseling about fetal testing consists of unbiased presentation of results and counseling about all options [1]. This chapter describes current counseling practice for fetal genetic disorders, both pretest and posttest, focusing on generally accepted aspects, acknowledging the many differences that do exist between countries, societies, and cultures, and undoubtedly biased by the personal experience and opinion of the author.

14.1.1 What Is Counseling? Providing information is an essential part of a counseling session; however, it is only one aspect. Counseling is the art of helping someone, in this case a pregnant woman, to make a choice between various options. Autonomy of the pregnant woman is key, whatever decision she ultimately makes has to be her own decision: it will affect her life and that of her fetus, and of her future child. In most societies, it is accepted that until the umbilical cord is cut, the fetus is totally her responsibility and whatever happens to it can only be her choice. Obviously, some rare exceptions such as psychiatric disorders do exist. Most obstetric care professionals do involve the woman’s partner, often but not always the father of the fetus, in all aspects of the care, and most women prefer this as well. However, ultimately, her personal decision on in this case fetal testing is leading. In the remainder of this chapter, the focus lies on counseling the pregnant woman but it is clear that a partner is usually present and should be involved as well. Just providing information, even with all currently available tools such as websites and decision-aids, is not sufficient for making an informed decision. Most pregnant women do not have a university degree in medicine or genetics, and the amount and level of information that can be provided is by definition very, very limited. That is why counseling can be called an art. It requires in a short period of time getting a good sense of the woman’s health literacy, language abilities, advance knowledge, existing anxieties, and possible choices she already may have made. This in turn requires listening skills and empathy, not in fact different from many other aspects of health care, and something that is taught and trained during the many years of medical school, and refined in practice. Appropriate counseling, leading after an often amazingly short time of 20–30 min or even less, to a pregnant woman who makes a confident, well-informed autonomous decision does require training, experience, and a certain amount of talent. Some professionals are definitely better at it than others. Some colleagues complaining of “repeating the same story over and over” clearly miss the point. Good counselors therefore almost always enjoy this type of work. The patient leaving the room with the feeling of having made a very important, possibly life-changing decision, with the assistance of your information, empathy, and careful probing of the patients’ own views and values, and support for her final decision can be highly satisfying.

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14.1.2 Do All Pregnant Women Need to Be Counseled on Fetal Testing? Screening tests in general are commonly only allowed to be performed after informed consent. Consenting or not consenting to an offer of screening implies at least certain knowledge on the disease in question, and pros and cons of the (first) test. It is however difficult to force someone to read an information leaflet, or to listen to a professionals’ explanation of the various aspects of screening. This touches on an ethical principle described as “the right not to know.” In The Netherlands, the Health Council in its role as advisor on screening programs, considers this an important element of such programs. In clinical practice, this means that midwives and obstetricians first need to verify if the pregnant woman is at all interested in obtaining knowledge about screening options. It is allowed to send information brochures to all pregnant women, but a formal counseling session should only be scheduled after confirmation that the woman is interested. Declining the offer of counseling on screening may be related to previous experience, leading the woman to think she already knows all she needs to know and that she already has decided to undergo screening or not. In such a case, the professional may want to verify this, certain aspects may have changed over time and the woman may therefore miss essential information. Another not uncommon reason to decline is the already clear decision, often linked to religion, of the woman to never choose for the option to terminate the pregnancy. This leads for many of these women to the conclusion that screening for fetal disorders is therefore futile. For the health care professional, it may be worthwhile to at this point make clear that under no circumstances, a high-risk or abnormal test result will lead to any pressure to undergo further testing or to terminate the pregnancy. After the initial screening test, every further step in again an autonomous decision of the pregnant woman. The professional can consider to mention that it is not unusual for women to carry on with the pregnancy after an abnormal fetal genetic test, but that knowledge may help to get used to and accept the fact that a child will be born with a certain disorder. Still, it is important to maintain the principle that screening, and even information about screening, needs to voluntary.

14.2 Pretest Counseling 14.2.1 Preparing for a Counseling Session Since pregnant women actively make an appointment for consultation, there is the opportunity to send them a brochure or a link to a website with information on prenatal screening before their counseling visit. The majority will read and hopefully understand the information, making the counseling session much more efficient. Those of us professionals who have made the mistake of going into lengthy discussions on pros and cons of prenatal screening, and only afterwards find out that

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the fetus has no heartbeat, has anencephaly, or that the pregnancy is ectopic, or twins, or very different gestational age than expected know that it is absolutely essential to perform an ultrasound scan just prior to the counseling. There may be variation in logistic or financial issues influencing how to organize this, but ideally the counselor performs the scan at the start of the counseling session. Viability, a CRL measurement and exclusions of the events mentioned above takes only a few minutes. Pregnant women and partners hugely appreciate this, and during the scan one can already assess the level of understanding, language, and health literacy of the pregnant woman. Presence of the partner, of course only with permission of the pregnant woman, is preferred. An informed decision at the end of the session, the final goal of the counseling, that is shared by the partner, who also has had the opportunity to ask questions, is ideal. When there is doubt about the full autonomy of the pregnant woman, e.g., when the couple does not seem to agree or the partner appears to force the woman to decide something against her will, it is essential to try and talk to the woman in private. A detailed history, if not done appropriately beforehand, is important to before starting to talk about screening options. Occasionally, a family history of a genetic disorder may result in an indication for a specific diagnostic test that can include fetal chromosomal analysis, making a prior screening test superfluous. As was outlined above, not only fetal testing but also counseling on fetal genetic screening should be voluntary, in line with the ethical principle of “the right not to know.” This means asking consent to counsel the pregnant woman about screening.

14.2.2 Who Should Counsel Pregnant Women About Fetal Genetic Screening? Although a high level of knowledge on genetics, statistics, epidemiology, decision making, psychology, laboratory techniques, and invasive testing may be required to adequately counsel pregnant women about fetal genetic screening, it is not feasible to restrict this task to maternal-fetal medicine (MFM) specialists or geneticists. Since almost the entire pregnant population needs to be counseled, the same principle as for the first step in the testing cascade applies; this should be relatively easy and cheap. As we will see later on in this chapter however, even superficially discussing all relevant element required to reach an informed decision by the pregnant woman will take at least 20 min and often more. Most organizations have chosen for a program whereby the first counseling on fetal genetic screening is performed by midwives, genetic counselors or specialized nurses, usually with specific training and certification [2]. Obstetricians and family doctors may also be involved. There is always the option of referring women with difficult questions or special issues to an MFM or geneticist. Some countries have national requirements and training programs for counselors, including regular refresher courses to update knowledge in rapidly advancing field.

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14.2.3 The Counseling Session Fortunately, most pregnant women come to the session with at least some knowledge on the disorders that can be screened for, the type of tests, and often already an idea of what they want. Given the limited time for a counseling session, typically between 15 and 30 min, it is important to make full use of the prior knowledge. After the introduction, verification of the correct patient file on the computer screen, and clearly stating name and function of the counselor, the goal and outline of the session should be briefly explained. The goal is to reach a decision on whether or not, and if yes what type of fetal screening the woman would like, and to plan this next step. The outline is that first, an ultrasound is made to check viability and dating (unless very recently done), then to discuss the genetic disorders that can be tested for, the type of tests with their characteristics, and what will happen when the screening test is not reassuring. Then it would be a good time to ask if the woman has received, read, and understood the brochure and if she already has an idea of what she would like. Letting the woman talk for a few minutes about this will give an impression of her language use and health literacy. In the same time, as well as during the ultrasound, the counselor can get an impression of the level of anxiety. 14.2.3.1 The Conditions that Can Be Tested For The next step is to discuss, preferably interactively and with full awareness of the education level of the patient, the fetal conditions that can be tested for. (a) Down Syndrome. Many professionals think that the most important information the pregnant woman needs to understand is her individual risk for Down syndrome in her fetus or newborn. Almost all women already know that maternal age plays a role, but exact risk or odds is something the professional can calculate for her. A vast body of literature exists on risk estimates by maternal age, and modification of this risk by screening tests. Counselors often spend a fair amount of (precious) time talking about this risk calculation, and then try to make the woman understand that in the screening program, a cutoff is chosen above which she is considered to be of high risk, and below which she should not worry about Down syndrome. It is quite intriguing that worldwide, most women readily accept this concept. The cutoff principle, whether a certain maternal age or a calculation using various maternal serum and ultrasound tests, is based on a combination of financial and logistic considerations, and is of course purely artificial. A risk of one in 190 is not really different from one in 210. Women appear to accept that there is no room for individualization. Depending on all sorts of circumstances, such as issues with other children, plans for the future, financial situation, etc., one woman may accept a risk of one in 50 while another may at all cost want to be reassured despite having a risk of one in 500. This considerable counseling challenge virtually disappears in programs where NIPT of cfDNA testing is offered as the first-tier test.

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Unlike with all previous screening modalities, maternal age is no longer relevant. The NIPT test result will either be a very high risk (positive) or a very low risk (negative), still of course never 100% certain. This concept can be explained in a few minutes. Much more time becomes available for perhaps the most important part of the counseling; making sure the pregnant woman understands what the genetic disorder means for the fetus, newborn, child and adult, and its family. Although many women may say they know what Down syndrome is, it is important to verify this and explore knowledge gaps, or correct misunderstandings. Although it is outside the scope of this chapter to list all characteristics of Down syndrome, a few often encountered misunderstandings may be worth mentioning. Misunderstandings –– Down syndrome is a genetic syndrome caused by trisomy 21, the most common chromosomal anomaly in newborns, characterized by many symptoms but invariably by a sometimes mild, often moderate, and sometimes severe degree of mental handicap. However, many other causes, genetic and otherwise, for mental handicaps exist, and some pregnant women may use the term Down syndrome for all these causes. The major risk of this misunderstanding is that a test that can exclude Down syndrome is perceived as a reliable way to exclude all mental handicaps. –– Often, pregnant women and also health care professionals talk about the features of “children with Down syndrome,” thereby forgetting or ignoring the fact that with a life expectancy nowadays of about 55 years, a future child with Down syndrome will become an adult, with a whole list of additional concerns for the parents. –– Quite a number of women have heard or read about the cardiac defects that affect about half of the live-born Down syndrome children. Some ask for detailed ultrasound examination to detect or rule out the heart defect, typically an atrioventricular septum defect. However, medically this is not the most severe symptom of Down syndrome. If needed, the defect can often be successfully operated, and Down syndrome is no longer considered a contraindication to perform this surgery. The degree of mental retardation is unrelated to the presence or absence of a cardiac defect, and this impacts the person’s life to a much larger degree. In addition, many other medical issues are associated with Down syndrome, most of which cannot be diagnosed prenatally. To separately list and discuss these conditions, which include impaired hearing, vision, resistance against infection, gastrointestinal tract problems, epilepsy, diabetes, thyroid problems, leukemia, dementia, ­auto-­immune diseases, and testis carcinoma during a prenatal screening counseling session is not possible. A list may be given in a brochure or a website. It is important though, if the pregnant woman asks about the cardiac defect, to put this problem into perspective.

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–– It is interesting that in many countries, brochures and websites providing information directed at pregnant women to assist in decision making on Down syndrome screening appear to avoid hard data, and in describing features of Down syndrome, often tend to use language that softens the problems these individuals may have. Proper counseling means using objective, evidence-based data. In addition, since counseling is more than providing information, the woman should be assisted in interpreting these hard data to her own situation. This requires an interactive process, asking questions on her views on her life, her family life, and plans for the future, what it would mean, not only for the child (and future adult!) with the genetic disorder but also for herself and her family. A certain range of expected IQ often does not mean much to lay persons. Whether a child can learn to walk, to speak, to dress itself, go to a normal school or not, it may be able to care for itself and live on its own, to have children, that is what matters to pregnant women considering testing for fetal disorders. The Dutch brochure proving information on Down syndrome does not give any hard data. The language used appears to try and avoid any emphasis on the many problems related to trisomy 21. Under the heading: how does a child with Down syndrome develop? it says: “Children with Down syndrome develop more slowly and to a more limited extent than the average child. But this varies from child to child. It is difficult to predict how a child will develop. It is good to stimulate a child with Down syndrome right from birth. Parents can get support to help their child to develop well. Young children grow up in the family. Usually, they are able to go to a normal children’s day care center. Very occasionally, a special day care facility may be necessary. Most children with Down syndrome go to a normal primary school.” Under the heading: What do parents and siblings say? the governmental brochure says: “Almost all parents say that they love their son or daughter with Down syndrome very much. They are also proud of their child. Eight out of ten parents feel that their child has given them a more positive outlook on life.” (https://www.pns.nl/documenten/ information-­a bout-­p renatal-­s creening-­f or-­d own-­s yndrome-­e dwards-­ syndrome-­and-­pataus). And when discussing the various medical issues associated with trisomy 21, the brochure focuses on all the help from medical and other professionals that is available. The choice of the governmental screening committee to minimize or downplay the clinical picture of Down syndrome in the brochure undoubtedly has to do with the small but very active minority of people in the Dutch population who do not agree with the freedom for women to choose screening for Down syndrome. Partly, this group consists of people who are for religious reasons against any type of pregnancy termination, and partly of parents of children with Down syndrome who consider the disorder not a reason to screen fetuses for, at least not with the option to elect termination when trisomy 21 is found. It is therefore in the hands of the counselors to make sure that the pregnant woman, who actively requests counseling on fetal screening, understands what it means in reality to live

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with a child, and later and adult, with Down syndrome, both for that individual and for the family, obviously addressing the wide variation in the clinical picture. –– An often encountered question, and this is true for all types of counseling in medicine, is: what would you do? or what do other parents usually do? The first, personal question can almost always be avoided by explaining that your personal life and circumstances may be different from that of the patient, and therefore not so relevant for her. Whether or not the counselor is willing to share his or her own experience (e.g., “I understand your dilemma because my wife was given a risk for Down syndrome of one in 300 and we struggled with what to decide”) is a personal decision. However, patients and thus also pregnant woman seeking counseling on screening have the right to receive honest answers, and for instance have to be informed, when they ask this question, about the general uptake of screening and about the percentage of women that elect termination after a certain diagnosis. It is well known in the field of decision-making, not only on medical issues, that people take into account what other people do. They in general do not want to be the rare exception, but like to be reassured that their decision was the right one by knowing that most other people chose the same. (b) Trisomy 18 and 13 In the early days of prenatal screening, the focus was on detection of trisomy 21 and spina bifida. These were, and still are, relatively common, serious disorders that were immediately apparent at birth, and were not lethal and not curable. This meant life-long handicaps with major consequences for both the affected individual and the family. Almost all women receiving the diagnosis trisomy 21 or spina bifida requested termination of pregnancy. All of the more traditional screening tests for trisomy 21 (maternal age, serum screening, combined test), when positive, meant that the pregnant woman is also at increased risk for other trisomies, most notably trisomy 18 and 13. At first, this was considered an unintended additional or chance finding, usually leading to a welcome easier decision for termination since these anomalies are considered lethal. Most readers will know the story about how screening for spina bifida using maternal serum AFP actually led to the discovery of using this test for trisomy screening, due to a very low AFP level in a pregnant woman carrying a trisomy 18 fetus. Most full fetal autosomal trisomies result in an early miscarriage, but trisomy 18 and 13 may be associated with prolonged fetal survival, and in a minority of cases even newborn survival. Although originally not primarily the goal of the screening program, the detection of these severe anomalies was considered a beneficial side-effect of the program. At some point, the inventors of the first-trimester combined test had enough data to provide a separate algorithm for the prediction of trisomy 18 and 13. The fact that pregnant women would get two results from their first-trimester combined test, a trisomy 21 risk and a trisomy18/13 risk, meant that it became mandatory to explicitly and sepa-

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rately discuss these syndromes before testing. When cell-free DNA screening as a first-tier test became available, such as in The Netherlands and Belgium, and in many private clinics, separate risks were given for trisomy 21, 18, and 13, again meaning that these syndromes each needed to be part of pretest counseling. Later in this chapter the differences in test accuracy will be discussed. The general time scheduled for the pretest counseling is insufficient to list all possible anatomical and functional anomalies associated with Edwards and Patau syndrome. Brochures and website information are commonly provided with at least a summary of the most common anomalies; however, for decision making, it is likely sufficient to discuss that the majority of fetuses diagnosed with trisomy 18 or 13 in the first or early second trimester will not survive pregnancy, delivery or the first days after birth, due to a number of defects in most vital organs systems. The few survivors, of which most will turn out to be mosaics, are always severely mentally impaired, and unable to live without major medical support. Another fact that may be mentioned at this stage is that without genetic screening, nowadays almost all cases, especially of trisomy 13, will be picked up either by ultrasound done for other reasons or because of clinical symptoms, most notable fetal growth restriction. A clinically valid reason to consider screening for trisomy 18 and 13 is that this will prevent the still occurring unwanted situation of a (usually emergency) cesarean section for a growth restricted baby that is only diagnosed with (a lethal) trisomy after birth, leaving the woman with an unnecessary scar in her uterus. (c) Other Chromosomal Anomalies Although several different categories can be used, a common distinction is, apart from the abovementioned “common trisomies,” sex chromosome aneuploidies, rare autosomal trisomies, and subchromosomal/structural aberrations. Those who remember the era of maternal age-only screening will agree that in those days, it was worth mentioning the not uncommon finding after chorionic villus sampling and to a lesser extent amniocentesis, of sex chromosome aneuploidies (X0 and XXY especially) and mosaics. Again, a full description of the major features of Turner Syndrome and Klinefelter syndrome, or of the various forms of fetal and placental mosaic patterns cannot be part of a routine counseling session for low or average-risk women. Still, briefly mentioning these possibilities, and if needed, answering questions about them, was advised. Nuchal Translucency With the introduction of the nuchal translucency measurement as part of the first-­ trimester combined test, pretest counseling had to include some explanation about the rim of fluid in the fetal neck. A thickness within the normal limits would be used in the algorithm to calculate common trisomy risk, but in case of a large (common cutoff 3.5  mm) value would mean a direct indication for invasive testing, with Turner syndrome as one of the main possible abnormalities. Mentioning this diagnosis here would again mean a brief description of what this syndrome entails. If

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this is done correctly, it is important not to use the pediatric data on surviving children with Turner syndrome (“just” short stature, webbed neck, infertile) since the fetuses with a large nuchal translucency, cystic hygroma or hydrops early in pregnancy constitute the severe end of the spectrum They are highly likely to have serious heart defects and often die during pregnancy. Again, things have changed with the introduction of cell-free DNA screening. The cell-free DNA screening, as described elsewhere in this book, can be tailored to provide prespecified outcomes, e.g., on trisomy 21 only, on all three common trisomies, on trisomy 18 and 13 only, on the trisomies and on sex chromosomes, on a specific list of relatively common deletions and duplications or as whole-genome screening, looking at abnormal amounts of DNA throughout all chromosomes. The same principle applies; if a screening test is done specifically and intentionally for a certain anomaly or syndrome, the clinical features of the anomaly need to be described pretest to the pregnant woman. This may be possible within the limited time of pretest counseling visit with high-quality previsit information via websites, videos, and brochures, and a limited number of abnormalities, e.g., the common trisomies plus 22q deletion, and if parents elect to know the sex of the fetus, counseling on sex chromosome anomalies may be limited to stating that if an abnormal number of sex chromosomes are found, further posttest counseling will be provided. A longer list of specific deletions such as 5p min, 1p36, and uniparental disomy syndromes make detailed or even superficial counseling on clinical features virtually impossible in a routine setting. The only option is what counselors were used to in the days of maternal age-only screening: explaining to the pregnant woman that the laboratory technique chosen means that occasionally, some other than the common anomalies will be detected, that these anomalies range from harmless to lethal, and everything in between, or may even be unclear, and that in-depth counseling by a clinical geneticist will be provided on very short notice should such a finding occur. Obviously, such an approach is also needed when whole-genome testing is offered. For most experienced clinicians and counselors, this provides very little problem, and most pregnant women readily understand this concept. It is in fact similar to the counseling done prior to a “routine” ultrasound anatomy screening. Apart perhaps from mentioning spina bifida, pretest counseling for the 20-week scan cannot be expected to include a description of clinical features of all structural anomalies that may be found. Maternal Malignancy In very rare cases, in particular when using whole-genome cell-free DNA testing, an abnormal cell-free DNA pattern is found that may originate from a maternal tumor. Typically, anomalies are found in multiple chromosomes, with sometimes complex profiles. Laboratories and clinicians are now aware that this raises the suspicion of a maternal malignancy, such as breast cancer, ovarian cancer or hematological cancers. Alternatively, such patterns may arise from benign leiomyomas or fibroids. Such abnormal cell-free DNA patterns make interpretation for fetal risk impossible,

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and the woman should be offered an invasive test, together with a full work-up to find the underlying cause of the pattern [3]. Most brochures and websites on fetal genetic screening briefly mention this rare possibility, and in pretest counseling it is certainly an option to mention it as well. If asked, the counselor should stress that NIPT for fetal screening is not a reliable test to find or rule out maternal cancer [4]. Summary In summary, when offering a screening test for specific fetal genetic anomalies, she has the right to be provided with objective, up-to-date information on the clinical features of the various syndromes. This is not the same as listing number of clinical features with their percentages. Women should, with the help of the counselor, try to understand what it would mean for the child (who, depending on the diagnosis, will become an adult and may have a life expectancy that may mean he or she will outlive the parents!) to have the disorder in question, and what it would mean, in daily life, for the parents and the family. This is particularly important in case she has the choice between restricted, extended or broad screening panels. This information should not be based only on pediatric data from long-term survivors, but needs to include fetal data, risk of fetal demise, growth restriction, and intrapartum problems. Given the complexity of the information, even when only a few common trisomies are tested for, written and digital information, free from value judgment, needs to be made available before the actual counseling session. When larger numbers of syndromes are tested for, or when the whole genome is scanned, the pregnant woman needs to be aware that she may receive a test result that requires significant posttest counseling before she can make further decisions, and that in complex cases, women sometimes experience doubt about their choice for screening. 14.2.3.2 Test Characteristics After discussing the various chromosomal anomalies that can be screened for, the test characteristics need to be clearly outlined to the pregnant woman. The details, the language, and the examples used must be adapted to the woman’s educational level. This is a skill all doctors, midwives, and counselors should have as part of their daily job. Even highly educated women may have difficulty grasping the meaning, for their personal situation, of terms and sensitivity, specificity, accuracy, etc. In the counseling, the professional should translate the published test characteristics (the ones that apply to the exact same test the woman may undergo, thus your own lab!) to the real-life situation of the individual pregnant woman; positive and negative predictive values, or even better the odds of being affected given a positive result. Decision aids with pictures are often recommended, and websites such as www.perinatalservicesbc.ca are helpful [5]. However, many graphic decision aids show a large number of normal or unaffected individuals, and a few dots or circles of another color, depicting the test-positive cases. When using such cards or

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graphics on a computer screen, it is worth outlining that the apparently normal cases are not all pregnancies with good outcome and healthy newborns! The number of serious problems happening to the “unaffected” group are much larger than the few that are actually affected by one or all of the common trisomies, and include genetic syndromes such as microdeletions and single-gene disorders, syndromes, and diseases of nongenetic or unknown origin, viral infections, and severe pregnancy complications that may lead to perinatal death or handicap. This message, that screening for a few specific chromosomal anomalies may take away only a limited proportion of all adverse outcomes of a pregnancy, needs to be clearly understood. In particular, the often-mentioned misconception that “Down syndrome” encompasses all forms of mental handicap (and that excluding Down syndrome means the child will have a normal-functioning brain) is worth eliminating. Translating test characteristics into meaningful examples is often easiest by using the most common scenarios. What may happen when the screening test is chosen? What if the test comes back positive, what if it is negative, and what happens when there is a test failure/unclear result? Here it is important to underline that for any additional step, such as invasive diagnostic testing and whether or not to terminate the pregnancy, additional counseling and freedom to make any decision at every step is part of the process. Obviously, after almost 10 years of using NIPT in the clinic, it is clear that false positives do occur, and that termination of pregnancy cannot be allowed without prior confirmation by an invasive test (with the possible exception of clear structural anomalies in case of trisomy 18 or 13, still with the requirement to confirm the diagnosis after termination). Scenarios Then several scenarios can be discussed in case the woman decides not to undergo the screening test. Most women consent to undergo a 20-week ultrasound to screen for structural anomalies. It is essential to outline that although many genetic syndromes are associated with major, ultrasound-detectable anatomic anomalies, the 20-week ultrasound is not a good test for trisomy 21, and even some cases of trisomy 18 can be missed. In addition, it may be worth mentioning that sometimes, clinical symptoms in the third trimester may lead to the suspicion, and when test the diagnosis, of a chromosomal anomaly. In many countries however, the law may prevent late termination of pregnancy especially for trisomy 21. One of the major advantages of NIPT over all previous screening tests is that when a result can be provided, it is either very reassuring (although no guarantee) or highly predictive of an abnormal karyotype. This saves a lot of time that in the past needed to be spent on trying to make the pregnant woman understand (and accept) the issue of intermediate risks and rigorous cutoffs for which the basis was hard to understand. Many women failed to understand (rightfully) why a risk of one in 280 was considered a low risk, with no indication for further testing, while one in 190 was a high risk, requiring a potentially hazardous invasive procedure. The absence of freedom of choice, at least in many public systems, was also hard to

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accept for some. One woman would be perfectly reassured receiving a one in 100 risk, while the other would lose months of sleep over a one in 1000 risk. Prevalence With the increasing options of tests in the screening panel, as outlined above, women should also be aware of the correlation between (esp. positive) predictive value and prevalence of each disorder. Recently, this discussion received world-wide media attention, after The New  York Times reported on a review from multiple studies which showed that when NIPT is used to test for uncommon things like Wolf-­ Hirschhorn syndrome (prevalence one in 20,000 births) or Cri-du-chat syndrome (one in 15,000), positive results are wrong 80 percent of the time or more (https:// www.nytimes.com/2022/01/01/upshot/pregnancy-­birth-­genetic-­testing.html). Correctly however, professional bodies such as the American College of Medical Genetics and Genomics quickly reacted by explaining the nature of screening tests in particular for more rare anomalies, for which a relatively low positive predictive value is known and accepted, and communicated with the pregnant women (https:// www.acmg.net/PDFLibrary/2022_ACMG_Response_NYT_NIPS.pdf). They also added that “current guidelines including those from ACMG and ACOG recommend noninvasive prenatal screening for common disorders, but not for less common syndromes caused by microdeletions, or missing pieces of chromosomes.” It may be good to mention, in case these issues come up in a counseling session, that in the past, screening for trisomy 21 was done by asking the woman’s age. That screening test was ‘wrong’ more than 99% of the time. Even recently, the first-­ trimester combined test, when giving a high-risk, thus positive result, defined as a risk of one in 200 or higher, was ‘wrong’ in the vast majority of cases. 14.2.3.3 Expectation Management When the pregnant woman, based on a reasonable understanding of what the anomalies screened for would mean for her unborn child, herself, and her family if she would choose not to screen, and with a good understanding of the test characteristics, elects to undergo the test, she needs to know a few more things. –– Failure rate including redraw rate A major disappointment and cause for complaints are tests that do not provide a result. The patient almost invariably thinks this means something is wrong, and anxiety increases. Because of the rarity of failures, pregnant women usually have not thought about what next. The lab and the doctor most often advise a redraw, which, depending on the possible cause of the failure may succeed in 50% of cases or more. It makes sense to make sure the patient has understood that there is a chance of failure, and this may be stressed more in women with an a priori increased risk of failure, such as early gestational age or obesity. The counselor should not be surprised when women do not like a redraw but ask for the certainty of an invasive test. This understandable reaction, a sign of

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loss of trust in the screening test, requires careful discussion of the likely still small chance of a positive test, to make sure the patient does not believe that the failed test increases her risk of a positive result. Encouraging her to have some more patience may be useful. Turnaround time Waiting for a test result is associated with anxiety, no matter how small the chance of an unfavorable result is. Experience with one-stop screening and diagnosis showed that, although professionals consider this the optimal service, may not be preferred by all patients. A few days between test and result are often acceptable, and may give some time to think about what to do in case of a positive result. In the Dutch Trident study, there was a clear increase in anxiety after day 11, this appeared to be the cut-off for an acceptable turnaround time [6]. Fortunately, almost all current screening and diagnostic tests now have results available within 3–5 days, at most one week. Communication of the most likely waiting time, and the still normal upper limit of the turnaround time is essential to avoid stress and anger. In addition, allowing the patient to contact the doctors’ office at the upper limit of the expected waiting time is advised. How the result is communicated There is a wide variety in the way labs or doctors communicate result. Low risk or negative results may be sent by mail, email or a phone call by a nurse or secretary. Any other result is preferably communicated by phone by a knowledgeable professional, who can answer the first few questions before arranging a clinic visit on short notice. Obviously, there can be cultural differences or logistic choices, making other arrangements acceptable. Key is to clearly communicate, pretest, how and by whom the patient is contacted, both in case of a reassuring result as in case of any abnormality. Who else gets her results In many health care systems, it is customary to copy lab results to the patients’ midwife and family doctor. This may not always be appreciated in case of potentially sensitive information. Thus, asking permission to share the results with other caregivers is advised. What happens when the result says “high risk” Although this is the topic of posttest counseling, it can be important to briefly make clear that undergoing a screening test in no way means that the pregnant woman made up her mind about decisions in case of a positive result. She has the right to be counseled about the meaning of the result and all her options, and only then she needs to decide what she prefers. It is not uncommon for patients to completely change their minds when actually confronted with a positive test result. Even the slightest hint of steering a patient in a certain direction needs to be avoided.

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14.3 Posttest Counseling 14.3.1 Who Should Provide Posttest Counseling? As soon as a pregnant woman has received the message that her screening test indicates a high risk, she has the right to an appointment with a health care professional who should have answers to all her questions, who has the knowledge and the experience to discuss all her options and who can arrange further testing on short notice. This includes in-depth knowledge of the condition for which the screening test was positive, as well as a good understanding, preferably experience, with the invasive diagnostic tests, their techniques, risks, and logistics. For the common trisomies, most obstetricians and maternal-fetal medicine specialists should have sufficient knowledge to provide the posttest counseling. More rare conditions likely require a clinical geneticist, who may or may not need to involve a maternal-fetal medicine colleague for details on the invasive tests. Quite a lot of women at this stage already ask detailed questions on how a termination of pregnancy is done. The counselor should have the required knowledge.

14.3.2 When to Schedule a Posttest Counseling Session? The number one criticism on screening programs is always the uncertainty, the waiting time between the first suspicious result and the final diagnosis. There seems to be, although studies are lacking, an advantage of having a little bit of time to let the message sink in. An appointment for counseling 1 or 2 days after communicating the screening result may be better than the same day, when the shock-effect can prevent taking in the information. In addition, the patient may have time to inform close family members for support. Every effort should be taken to organize the posttest counseling with both the pregnant woman and the partner present.

14.3.3 Content of the Counseling Posttest counseling has changed dramatically over the past years. In the days of maternal age screening, counseling women on the pros and cons of amniocentesis, CVS or no testing usually focused on the reassurance an invasive test could bring, for actually little risk. Currently, with the high accuracy of NIPT, the professional likely already adapts his or her language and body-language toward the likely unfavorable result of the invasive diagnostic test that will be offered.

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14.3.3.1 A Positive Screening Test Needs Confirmation In particular for a high trisomy 21 or 18 risk, the main message of the counseling is that due to some rare exceptions (false positives), it is necessary to confirm the screening test but that the patient should count on having an affected fetus. It would be totally unacceptable to run the risk of terminating a completely healthy fetus in this setting, where almost always the pregnancy and the future child are wanted. Since in most countries, pregnant women have the right to request abortion irrespective of whether the fetus is healthy or not, at least up to a certain gestational age, in rare cases a woman may choose not to wait for further testing but to ask for termination. A woman may use the positive screening test as an additional argument to elect abortion of a pregnancy she may not really have wanted. This would then fall under the regulations of “social” abortion, and women may self-refer to an abortion clinic. For the posttest counseling doctor, it would be good to clearly understand and be informed about the reasons for the (free) choice of a woman in such cases. This unfortunately rarely happens. Such cases should not be misinterpreted as failed counseling or irresponsible screening. 14.3.3.2 The Exception: Trisomy 13 This is not the case for trisomy 13, which is often not confirmed by invasive testing, unless ultrasound shows anomalies. Therefore, counseling after a positive NIPT for trisomy 13 should start by offering or even better, performing an ultrasound. If this shows a fetal death or anomalies (including a small fetus), invasive testing may be done as soon as possible. In very clear cases (severe anomalies or death), karyotyping can be done after termination. If there are no anomalies whatsoever on detailed ultrasound, including normal growth, NIPT may have been false positive, or there could be a low-level mosaic. Although there is still an indication for amniocentesis, it is certainly an option to wait for the 20-week scan to decide on invasive testing. 14.3.3.3 Amniocentesis, CVS, Ultrasound, No Testing? The same principles as outlined above apply; before choosing a test, the pregnant woman should understand what diseases or conditions are tested for, what the accuracy of the tests are, and in case of invasive tests, what risks are involved. In contrast to the decades of counseling done for advanced maternal age, where women could freely choose between amniocentesis or CVS if they wanted testing, a high-risk NIPT result now interferes with that freedom. There is increasing knowledge about the biology of the cell-free DNA fragments. Their placental origin is now clear, and for each particular chromosomal anomaly predicted by NIPT, there now is a preferred diagnostic test. Elsewhere in this book, the genetic background will be described. Guidelines are adapted all the time, with better understanding of the cell-­ free DNA origins in particular in relation to the risk of mosaicism. The posttest

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counselor needs to be aware of the latest insights, and although freedom of choice is still the underlying principle, the exact NIPT result may influence the test characteristics of the invasive diagnostic test [7]. Since the screening using NIPT is, at least for the common trisomies, highly accurate, there is, in the eye of many professionals, much less reason than in the past to worry about the inherent risks of the procedures. Pregnant women however may still fear the needle in their pregnant womb, irrespective of the (small) percentage of complications given by the counselor. The patient was just informed that sometimes, the NIPT is false positive therefore the fetus might be healthy. To lose a wanted, healthy fetus after this whole episode of testing would surely be a nightmare. The professional should try to understand this point of view, and not downplay the risks by quoting papers that suggest that the procedure-related risks of invasive testing are actually zero. An obstetrician suggesting that these procedures are free of risk has not performed enough procedures him- or herself. An issue that still could be of interest to the patient is the fact that the risk of fetal loss after an invasive diagnostic test is higher when the fetus has an abnormal karyotype. This is mainly important for women considering carrying keeping the pregnancy even though the fetus has Down syndrome. They may refrain from invasive testing. Some women request to wait for the 20-week anatomy scan to make a decision, even after a positive NIPT. A common misunderstanding is that a scan may be able to differentiate between milder or more severe forms of Down syndrome, e.g., by echocardiography. As was outlined above, the heart defect is relatively easy to correct surgically, the lower IQ is much more difficult, if at all, to improve. On the other hand, this request may be based on reluctance to decide quickly, some women need more time and waiting for a scan might give them what they need. There is never a reason to rush, apart from certain gestational age cutoffs for termination of pregnancy. An open mind of the counselor is also essential. The best is for the counseling session to start by inviting the woman to tell what she has been thinking since she received the positive screening result. After several minutes of free uninterrupted talking by the pregnant woman, and if possible, also by the partner, the counselor will likely have a feel for where the conversation will go. The counselor should ideally wait for the pregnant woman to come up with the topic of termination, otherwise there is a real risk that she gets the feeling that the counselor pushes her toward that goal. If the woman does not bring up termination, the counselor may tell her that it is good practice to list all options available, and then in a neutral fashion mention termination as just one of the several choices. 14.3.3.4 Psychological Support In all cases of posttest counseling, pregnant women should be offered help by a social worker or a psychologist. The anxiety and confusion of women when confronted with an abnormal test result originate in part from the fact that most did not seriously consider this to happen to then [8]. The emotional impact of both the

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decision-making process as well as the difficult time after the decision (whatever that decision is) is often huge, and may well require other care than what an obstetrician or geneticist can provide. 14.3.3.5 Additional Findings A still controversial issue is what to offer after a positive screening test for one of the common trisomies. It would make perfect sense to only test what was screened for, in practice meaning that a quick qf-pcr test for only chromosomes 21, 18, and 13 is done. However, in the field of prenatal diagnosis, there is the generally accepted feeling that once a patient undergoes a risky invasive test, she has the right to obtain all possible genetic information from the fetal (or placental) cell analysis. In the past, a karyotype would reveal numerical and gross structural abnormalities of all chromosomes, just because of the technique. Additional findings were not uncommon, and were considered beneficial although already in those days, some anomalies (e.g., sex chromosome mosaics) could lead to complex counseling. Current advanced options include high-resolution microarray and even whole-exome sequencing, which, apart from being more expensive and time-consuming, may lead to finding variants with a lot of uncertainties [9]. There appears to be some inequality in this system, where screening is done for a few specific disorders, and then to offer only the screenpositive group a full genetic analysis. Most potentially diagnosable genetic anomalies will go undetected this way. In part, this is a relic from past days of karyotyping as the only lab technique. The alternatives would be, as mentioned, to only test what is screened for, or to broaden the screening test to whole-genome testing. This is pioneered in a few studies, such as the Dutch Trident 2, but is a topic of heated debate [10, 11]. In the coming years, as will be discussed elsewhere in this book, we will need to make choices on the extent of screening programs, taking medical, ethical, legal, and economic aspects into consideration [11]. Personal choice and autonomy of pregnant women may prevail over rigid restrictions and guidelines. 14.3.3.6 Insurance Issues The American College of Obstetricians and Gynecologists Committee Opinion 693 recommends to warn patients that genetic testing could affect insurance premiums or eligibility for life or long-term care insurance [12].

14.3.4 Posttest Counseling After Final Diagnosis The same issues apply as discussed above. The counselor needs to have in-depth knowledge about the disorder of the fetus, and sufficient understanding about all the relevant details around termination of pregnancy, since this will most often (but

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definitely not always) be the next step. Professionals with both an obstetric and genetic training are obviously best suited, however, especially in Europe, these are not easy to find. If counseling about the two topics (the disorder and the termination) needs to be done by two physicians, the geneticist should come first. Ideally, both counseling sessions should be on the same or at most one day apart. Remember that there is no medical reason to rush, and that social work or a psychologist can add valuable support. This applies also to support by professionals from the religious group of which the pregnant woman may be a member. It is not uncommon for patients not to have a good understanding of the rules around termination of pregnancy with an abnormal fetus in their religious group. Two of the most asked questions in this counseling session, even before a termination is performed, are about the recurrence risk and about the interval advised before a next pregnancy. Although the professional might want to postpone this discussion to the follow-up visit several weeks after the termination, it is often very reassuring to already talk about these topics beforehand.

14.3.5 Family In a minority of cases, the fetal genetic diagnosis may affect other family members. This should be carefully discussed with a geneticist, who then needs to advise how to best communicate this with family members.

14.3.6 Counseling and the Internet Every pregnant woman coming to a posttest counseling session has looked up information on the web. Counselors should have a good understanding of what patients may find there, and should be willing to discuss any questions arising from Google searches and Wikipedia pages. If needed, one can go to the internet pages together with the patient during the consultation and discuss what is found. This may seem to take a lot of time, but in the end, it may overall be efficient. If the patient is left with a lot of questions, she may want to come back or make phone calls for more information at less convenient times.

14.3.7 Follow-Up Most commonly, just like after every childbirth, a follow-up visit is planned after a termination of pregnancy typically after 6 weeks. Since women after a termination may be more prone to psychological problems than after birth of a healthy child, contact at least by phone after 2  weeks is recommended, for early

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recognition of a pathological mourning process. At the 6-week follow-up, it is very important to discuss in detail what is possible or recommended in case of a next pregnancy. An early visit, at 7 or 8  weeks, may be useful to discuss possible changes in screening and diagnostic testing, since this field advances rapidly. Often, but not always, patients elect to skip the screening and go directly to diagnostic testing. Here it is important to outline that a next pregnancy is not only at possible (often small) increased risk of the same condition, but that the new fetus again has the risks associated with any pregnancy, possibly identified with standard screening.

References 1. Chervenak FA, McCullough LB. Professionally responsible counseling about fetal analysis. Obstet Gynecol Clin North Am. 2021;48:777–85. 2. Oxenford K, Daley R, Lewis C, Hill M, Chitty LS. Development and evaluation of training resources to prepare health professionals for counselling pregnant women about non-invasive prenatal testing for Down syndrome: a mixed methods study. BMC Pregnancy Childbirth. 2017;17:132. 3. Amant F, Verheecke M, Wlodarska I, et al. Presymptomatic identification of cancers in pregnant women during noninvasive prenatal testing. JAMA Oncol. 2015;1(6):814–9. 4. Lannoo L, Lenaerts L, Van Den Bogaert K, et  al. Non-invasive prenatal testing suggesting a maternal malignancy: what do we tell the prospective parents in Belgium? Prenat Diagn. 2021;41(10):1264–72. 5. Beulen L, van den Berg M, Faas B, Feenstra I, Hageman M, van Vugt JM, Bekker MN. The effect of a decision aid on informed decision making in the era of non-invasive prenatal testing: a randomised controlled trial. Ultrasound Obstet Gynecol. 2016;48:75. 6. Van Schendel RV, Page-Christiaens GCM, Beulen L, et  al. Women’s experience with non-­ invasive prenatal testing and emotional well-being and satisfaction after test-results. J Genet Counsel. 2017;26:1348–56. 7. Mardy AH, Norton ME. Diagnostic testing after positive results on cell-free DNA screening: CVS or amnio? Prenat Diagn. 2021;41(10):1249–54. 8. Vuorenlehto L, Hinnela K, Ayras O, Ulander V-M, Louhiala P, Kaijomaa M. Women’s experiences of counselling in cases of a screen-positive prenatal screening result. PLoS One. 2021;16(3):e0247164. 9. Klapwijk JE, Srebniak MI, Go ATJI, et al. How to deal with uncertainty in prenatal genomics: a systematic review of guidelines and policies. Clin Genet. 2021;100(6):647–58. 10. van der Meij KRM, Sistermans EA, Macville MV, Stevens SJ, Bax CJ, et al. TRIDENT-2: national implementation of genome-wide non-invasive prenatal testing as a first-tier screening test in the Netherlands. Am J Hum Genet. 2019;105:1091–101. 11. Ravitsky V, Roy M-C, Haidar H, Henneman L, Marshall J, Newson AJ, Ngan OMY, Nov-­ Klaiman T.  The emergence and global spread of noninvasive prenatal testing. Annu Rev Genom Hum Genet. 2021;22:309–38. 12. ACOG Committee Opinion Number 693. Counseling about genetic testing and communication of genetic test results. Obstet Gynecol. 2017;129:e96–e101.

Chapter 15

cffDNA Testing in IVF Pregnancies Emilia Mateu-Brull, Nuria Balaguer, María Gómez-López, Carlos Simón, and Miguel Milán

15.1 Introduction The incidence of aneuploidy in humans depends on its frequency at the time of conception, the rate of aneuploidy originated along with embryonic development, and the selection occurred from conception to the moment of birth. Data about these incidences come from studies along the developmental stages. At the time of conception, this estimation relies on human gametes study. After conception, data can be obtained from spontaneous abortions, stillbirths, and live births. Several studies indicated that approximately 0.3% of liveborn are aneuploid. In stillbirths, this percentage rises to 4% (over tenfold than for live births) and in spontaneous abortions to 35% (100-fold than for live births) [1]. These rates increase in pregnancies achieved by assisted reproduction (ART) up to 36–69% [2–9]. These figures reflect a higher rate of aneuploidies in the infertile population, as confirmed in other works [10–13]. Antenatal care programs have integrated prenatal screening in most developed countries worldwide. From the 1970s, second-trimester biochemical screening was utilized, by which up to 60% of Down syndrome (T21) pregnancies could be identified with a false-positive rate (FPR) of 5% [14]. Due to the relatively poor performance exhibited, there was a shift toward the currently combined first-trimester screening (cFTS). E. Mateu-Brull · N. Balaguer · M. Gómez-López · M. Milán (*) Prenatal Diagnosis Department, Igenomix Spain Lab S.L.U., Paterna, Spain e-mail: [email protected] C. Simón Igenomix S.L., Obstetrics and Gynecology, Valencia University, Valencia, Spain Beth Israel Deaconess Medical Center, Harvard University, Boston, MA, USA Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX, USA © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_15

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Nowadays, cFTS combines the maternal age-related risk with the analysis of maternal serum analytes [free beta-human chorionic gonadotropin (fβ-hCG) and pregnancy-associated plasma protein-A (PAPP-A)] and the measurement of the fetal nuchal translucency (NT) to produce an estimated risk for the most common aneuploidies: Trisomy (T) 21 (Down syndrome), T18 (Edwards syndrome), and T13 (Patau syndrome) [15]. The detection rate (DR) of the cFTS can be increased up to 95%, reducing the FPR to less than 3% by including additional ultrasound markers, such as fetal nasal bone, ductus venous blood flow, or tricuspid flow. By agreement, the maternal serum levels of PAPP-A and fβ-hCG are expressed as multiples of median (MoM) compared to the population control data. Thus, a high-risk or a screen-positive cFTS indicates that the adjusted risk derived from the ultrasonography and biochemistry measurements is higher than a certain threshold (typically 1 in 270). At the time of the prenatal screening, information about the method of conception is requested to determine whether a correction factor should be used to avoid a negative impact on the algorithm’s accuracy. Studies performed on a wide variety of cohorts have suggested the existence of differences in the marker measurement seen among in vitro fertilization (IVF) pregnancies compared with those conceived spontaneously [16]. Specifically, IVF conceptions experience higher FPR leading to increased uptake of invasive testing in this population. To pinpoint these drawbacks, cell-free fetal DNA (cffDNA) testing has gradually become a more reliable alternative to the cFTS, due to the high levels of accuracy shown in the detection of common trisomies. cffDNA analysis bases on the measurements of cell-free DNA molecules (cfDNA) found in the maternal bloodstream, containing the placental DNA released by the cytotrophoblast cells. On average, 10% of cfDNA in maternal plasma is of fetal origin. This percentage, commonly known as the fetal fraction (FF), varies between individuals and generally increases with gestational age. After delivery, cffDNA is completely cleared from the maternal circulation within hours. Therefore, cffDNA does not persist from one pregnancy to the next, so there is no risk of contamination in later cffDNA analyses. Up to date, since no increase in the fetal-placental mosaicism has been described in the ART population compared to spontaneous pregnancies [17], most of the confounding factors affecting the cFTS have been gradually overcome with the cffDNA testing (i.e., no need for algorithm corrections in case of preimplantational genetic testing for aneuploidies (PGT-A) cases, the availability of the donor age in egg donation cycles, the type of hormonal treatment, the type of embryo transfer, etc). However, despite this evident improvement, relatively few studies have evaluated the potential of cffDNA testing in ART pregnancies compared to the traditional cFTS. This chapter summarizes the existing knowledge regarding the cffDNA features in the IVF population (singleton, twin, vanishing twin pregnancies, and the PGT-A cohorts). Lastly, current advantages and limitations are discussed, emphasizing potential future perspectives.

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15.2 FF and cffDNA Testing Failure Rate in ART Versus Naturally Conceived Pregnancies The FF is a critical player in the success of cffDNA testing. The higher the fetal fraction, the more easily an aneuploidy can be detected for both partial and whole chromosomes. Every cffDNA analysis platform defines a threshold (typically 2–4% of FF) from which the test performance metrics are good enough for clinical practice. Test failure (or no-call) rate is a cffDNA analysis performance parameter associated with different factors, especially low FF. Failure rates can differ from platform to platform, acquiring values of 1.6, 3.6, and 6.4% for massive parallel sequencing (MPS), chromosome-specific sequencing (CSS), and single-nucleotide polymorphism-­based methods, respectively [18, 19]. Failure rates can decrease by repeating the analysis when those failures result from inherent sample characteristics such as a low FF [18]. Up to date, contradictory results exist concerning the FF and its impact on the cfDNA test performance metrics in women achieving a pregnancy via ART. Initially, comparisons of the mean levels of cffDNA between IVF and naturally conceived pregnancies showed no significant differences, suggesting its independence from the traditional screening markers (ɑ-fetoprotein, estriol, HCG, and inhibin A) [20]. Likewise, there have been no signs of cfDNA variations among euploid and aneuploid pregnancies after making a stratification by mode of conception (ART vs. naturally conceived pregnancies): Euploid (14.0% vs. 13.5%), aneuploid (13.0 vs. 14.5%) [21]. Contrary to this current of thought, other authors have suggested a significant FF decrease in ART-conceived pregnancies, especially for those classified as high risk for the T18 and T13 [22]. This observation is not surprising since it is well-known that those aneuploidies produce small fetuses and/or placentas, thus impacting the amount of cffDNA released to the maternal bloodstream. The first multivariate model intended to define the actual impact of ART in cffDNA testing performance stated that the FF reduction was significantly associated with the use of donor oocytes [23], discarding the embryo freezing as a possible cause for the FF value diminution. Later on, however, other studies evidenced that women receiving fresh embryo transfer (ET) in an exogenous stimulated cycle had a significantly lower FF (mean FF = 0.049) than those taking a frozen-thawed ET transfer in a modified natural cycle (mean FF = 0.063) [24]. Several hypotheses have been drawn to explain why ART treatments could affect FF. The most well-discussed is a possible increase in the maternal cfDNA fraction derived from a pronounced inflammation response and epithelial damage in ART-­ treated women [23]. Also, compromised placental formation following ovarian stimulation or deficient HLA compatibilities has been proposed as hypothetical reasons for the impaired FF in this obstetric population. Likewise, no correlations have been found between FF and maternal age, gestational age, PAPP-A (MoM), or the NT thickness [24].

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The discordant results regarding the informativeness rate in ART-treated women prove that a clear relationship between ART treatment and the FF estimation impairment has yet to be demonstrated. While most of the authors conclude there is no influence on the no-call rate in ART pregnancies [24, 25], others suggest a 3.8-fold increase in risk compared to natural conception [26].

15.3 Positive Predictive Value of cffDNA Analysis in ART Pregnancies The prevalence of a particular anomaly influences the positive predictive value (PPV) in prenatal screening in the population under evaluation. In general, ART-­ treated women are more elderly than natural-conceived pregnancies, making them a high-risk population for many morbidities, including fetal aneuploidy. Despite this generalized assumption, some studies agree that PPV for T21 is comparable between ART and spontaneous conceptions [23, 27]. Indeed, no false-positive have been identified for T21 screen-positive results, regardless of the mode of conception. Findings are discordant concerning the PPV for the T18. While certain studies describe a similar PPV of this condition in ART and natural-conceived pregnancies [27], others suggest poorer predictive values, up to twofold, in IVF conception (overall PPV: 28.6% vs. 73.4%) [23]. A similar but pronounced situation occurs for the T13. In most cases, the PPV calculation is impracticable for both conditions, most likely due to their low prevalence in the general population [25, 27]. Currently, few studies are drawing significant conclusions regarding the PPV for sex chromosome aneuploidies (SCAs) in the ART population. Despite this, a slight reduction has been suggested for the PPV in ART-treated women compared to natural-­conceived pregnancies (70% vs. 88%) [27].

15.4 cffDNA Analysis in Multiple Pregnancies Twin pregnancies are more frequent in the ART population than in the general population [28]. Although there is a tendency to transfer a single embryo in couples undergoing ART, the proportion of twin and multiple pregnancies is still significantly higher in the ART population. Evidence shows that cffDNA analysis can be performed in naturally conceived twin pregnancies, with high PPV values and low false-positive and -negatives rates [29–35]. Those figures are relevant since the risks associated with invasive test procedures are higher in twin pregnancies. To date, unexpected results exist in studies comparing the test performance in the ART population, considering that the total amount of cffDNA is more significant in twin pregnancies than in singletons [30, 36]. Thus, higher test failure rates and

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lower FF are more prevalent in some series comparing ART twin pregnancies with those naturally conceived [37–40]. Hence, in a study including a series of 515 twin pregnancies, the cffDNA test failure rate at first sampling was 1.7% in singletons and 5.6% in twins [37]. In addition, the median FF in twins (8.7%) was lower than in singleton pregnancies (11.7%). Furthermore, twin pregnancy, high body mass index, and IVF conception were significant predictors of test failure. In another casuistry, findings describe a DR of 100% for the T21 and 60% for T18–T13, respectively, with a false-positive rate of 0.25% in twin pregnancies after first or second sampling [39]. Similarly, more extensive studies confirmed that all T21 cases in twin pregnancies were confirmed by amniocentesis, giving a prevalence of 0.7% and a PPV of 100% for trisomy 21 [40]. No false positives or negatives were identified, and the failure rate was 0.9%. Later on, new evidence suggested a statistically significant reduction in the FF for ART twin pregnancies (mean FF = 0.129) compared to those with natural conception (mean FF  =  0.136). Logistic regression analysis for the cffDNA testing failure prediction in twins showed that only maternal weight significantly influenced it, being the type of conception statistically comparable [38]. Despite the lower FF and the higher failure test rate observed in the previous studies, better accuracy exists in the cffDNA analysis for common aneuploidies for patients with these types of pregnancies compared to conventional screening [32, 40–45]. However, clinicians and geneticists need to interpret cffDNA results with care. In monozygotic twins, the minimum FF to detect an aneuploidy would be the same as in singleton pregnancies since the two fetuses may have the same chromosomal complement. In dizygotic twins, only one fetus is usually affected [40], so each fetus must provide enough cffDNA to detect any aneuploidy.

15.5 cffDNA Analysis and Vanishing Twin Syndrome Vanishing twin (VT) is a spontaneous in utero fetal reduction with partial or complete disappearance during pregnancy. This phenomenon occurs in any week of pregnancy, but most commonly in the first trimester. At this stage of pregnancy, a high proportion of miscarriages are related to chromosomal aneuploidies. In dizygotic twin pregnancies, there are two living fetuses and two placentas that contribute in a similar way to the total amount of cffDNA. In VT syndrome, the amount of cffDNA from the demised fetus would be highly variable and strongly dependent on factors such as the gestational week at which the fetus arrested and the time of testing. Consequently, the presence of VT is a known factor affecting the accuracy of cfDNA analysis [41, 42, 46, 47]. VTs are more frequent in ART pregnancies due to a higher proportion of twin pregnancies linked to double or multiple embryo transferences [48]. Of all ART singletons born, between 10.4 and 30% originated from a twin gestation in early pregnancy [49]. The only study published to assess the clinical application of

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cffDNA analysis for VT pregnancies after ART describes a test failure rate of 1.4% after analyzing all the primary failed results with a second attempt [50]. Of the 548 samples completing the cffDNA testing, 12 samples had high-risk results, including nine high-risk cases for T21, two high-risk cases for T18, and one high-risk case for T13, giving a positive rate of 2.2%. Of these, 11 cases (92%) were false-positive, and one (8%) was a true positive, leading to a PPV of 8%. No false negatives were observed. The authors concluded that cffDNA analysis could be used in prenatal screening for pregnancies with a VT because of the similar PPVs to those from the conventional screening and the absence of false-negative cases. Although a VT reduces the cffDNA testing performance, certain timing of blood sampling allows a better classification of aneuploidy and sex calling [51]. The overall false-positive rate for VT pregnancies reduces from 2.6 to 0.8% whenever the test is performed from the 14th week of pregnancy. This diminution is especially important in VT pregnancies where the conventional screening has returned a high-­ risk result and for which invasive procedures would have been mandatory. In this specific subset of women, the cffDNA test returned a low-risk cffDNA result in more than 88% of pregnancies. This strategy reduces invasive procedures comparable to those previously reported for the general obstetric population for singleton pregnancies.

15.6 cffDNA Analysis in Patients Undergoing PGT-A All the international scientific societies recommend that prenatal genetic testing should be offered to all women when pregnancy ensues following PGT-A [52–54] because the latter is a screening test, and false-positive and false-negative results due to mosaicism can occur. Clinicians must present all the available prenatal testing procedures to all pregnant women, including prenatal invasive diagnostic tests, such as chorionic villus sampling and amniocentesis, and prenatal noninvasive diagnostic or screening tests, such as ultrasound scanning or cffDNA analysis. However, on some occasions, these societies recommend performing only ultrasound and amniocentesis (i.e., transfers of mosaic embryos). On the other hand, the international societies focused on prenatal diagnosis do not recommend performing two screening tests in the same pregnancy. After a PGT-A with the transference of euploid embryos, an ultrasound on 11–14th weeks and diagnostic genetic testing should be offered in case of structural abnormalities in the fetus [53, 55, 56]. Patients undergoing a transfer of an euploid embryo are at a relatively lower risk for fetal aneuploidy than the general population. Furthermore, in many cases, traditional prenatal genetic screening algorithms for trisomies 21, 18, and 13 are inappropriate for pregnancies conceived after PGT-A. However, some patients decline diagnostic testing because of the associated risk of miscarriage and choose prenatal screening instead. In these cases, cffDNA analysis could be an option after proper pretest genetic counseling, giving for and against

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arguments for this type of analysis in pregnancies achieved after an euploid embryo transfer. For pregnancies resulting from the transfer of a mosaic embryo, prenatal diagnostic testing with amniocentesis is highly recommended. Note that, in cases where ultrasound abnormalities are observed in the fetus, cffDNA analysis results should be interpreted with caution, regardless of whether they are natural or ART-­ conceived pregnancies with or without PGT-A. To our knowledge, only one study exists with a series of 1139 women undergoing PGT-A after a single euploid embryo transfer and cffDNA analysis. Among the patients included in the study, 1127 had a low-risk cffDNA analysis result. Four patients had no-call results. Three of them underwent diagnostic testing with normal results, and the other opted out of invasive testing. All four women with no-call results delivered phenotypically normal babies. A total of eight women had high-­risk cffDNA results. The prevalence of positive results following a euploid embryo transfer was 0.7% with these data. Seven women with a high-risk cffDNA analysis result underwent invasive diagnostic testing via amniocentesis. One woman opted out of invasive diagnostic testing, delivering a phenotypically normal baby. Six out of seven women had a diagnostic testing result demonstrating an euploid karyotype, concordant with their prior PGT-A results. One woman underwent amniocentesis after obtaining contradictory PGT-A and cffDNA testing results (46,XX and high/risk for monosomy X, respectively). The result confirmed Turner mosaicism (45,X karyotype in 80% of cells). The PPV of cffDNA analysis in this patient cohort was 12.5%, threefold higher than the cFTS [57].

Summary Box • cffDNA is currently the best screening method to seek for aneuploidies, even for ART patients. • Results regarding the effect of ART in FF values are contradictory with some studies showing no significant differences and others finding a statistically significant decreased FF in the ART population, both for singleton and twin pregnancies. • As unanimity, the FF reduction is subtle, often not affecting the rate of noninformative rates. • The PPV for trisomy 21 is similar regardless of the conception origin. • For conditions other than T21 (T13, T18, and SCAs), there may be a reduction of the PPV in the ART population compared to naturally conceived women. • The PPV described in the current scientific literature is still far superior to that obtained with conventional screening. Therefore, even though there may be a decrease in the reliability of the test, cffDNA testing continues to be the best option within prenatal screening alternatives.

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Palomaki GE.  Maternal plasma DNA testing for aneuploidy in pregnancies achieved by assisted reproductive technologies. Genet Med. 2014;16(5):419–22. 22. Revello R, Sarno L, Ispas A, Akolekar R, Nicolaides KH. Screening for trisomies by cell-free DNA testing of maternal blood: consequences of a failed result. Ultrasound Obstet Gynecol. 2016;47(6):698–704. 23. Lee TJ, Rolnik DL, Menezes MA, McLennan AC, da Silva Costa F. Cell-free fetal DNA testing in singleton IVF conceptions. Hum Reprod. 2018;33(4):572–8. 24. Talbot AL, Ambye L, Hartwig TS, Werge L, Sørensen S, Stormlund S, Prætorius L, Jørgensen HL, Pinborg A, Jørgensen FS.  Fetal fraction of cell-free DNA in pregnancies after fresh or frozen embryo transfer following assisted reproductive technologies. Hum Reprod. 2020;35(6):1267–75. 25. Costa JM, Letourneau A, Favre R, Bidat L, Belaisch-Allart J, Jouannic JM, Quarello E, Senat MV, Broussin B, Tsatsaris V, Demain A, Kleinfinger P, Lohmann L, Agostini H, Bouyer J, Benachi A. Cell-free fetal DNA versus maternal serum screening for trisomy 21 in pregnant women with and without assisted reproduction technology: a prospective interventional study. Genet Med. 2018;20(11):1346–53. 26. Galeva S, Gil MM, Konstantinidou L, Akolekar R, Nicolaides KH. First-trimester screening for trisomies by cfDNA testing of maternal blood in singleton and twin pregnancies: factors affecting test failure. Ultrasound Obstet Gynecol. 2019;53(6):804–9. 27. La Verde M, De Falco L, Torella A, Savarese G, Savarese P, Ruggiero R, Conte A, Fico V, Torella M, Fico A.  Performance of cell-free DNA sequencing-based non-invasive prenatal testing: experience on 36,456 singleton and multiple pregnancies. BMC Med Genet. 2021;14(1):93. 28. Nyboe Andersen A, Goossens V, Bhattacharya S, Ferraretti AP, Kupka MS, de Mouzon J, Nygren KG, European IVF-monitoring (EIM) Consortium, for the European Society of Human Reproduction and Embryology (ESHRE). Assisted reproductive technology and intrauterine inseminations in Europe, 2005: results generated from European registers by ESHRE: ESHRE.  The European IVF Monitoring Programme (EIM), for the European Society of Human Reproduction and Embryology (ESHRE). Hum Reprod. 2009;24(6):1267–87. 29. Bianchi DW, Platt LD, Goldberg JD, Abuhamad AZ, Sehnert AJ, Rava RP, MatErnal BLood IS Source to Accurately diagnose fetal aneuploidy (MELISSA) Study Group. Genome-­ wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol. 2012;119(5):890–901. 30. Canick JA, Kloza EM, Lambert-Messerlian GM, Haddow JE, Ehrich M, van den Boom D, Bombard AT, Deciu C, Palomaki GE. DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations. Prenat Diagn. 2012;32(8):730–4. 31. Dan S, Wang W, Ren J, Li Y, Hu H, Xu Z, Lau TK, Xie J, Zhao W, Huang H, Sun L, Zhang X, Liao S, Qiang R, Cao J, Zhang Q, Zhou Y, Zhu H, Zhong M, Guo Y, Lin L, Gao Z, Yao H, Zhang H, Zhao L, Jiang F, Chen F, Jiang H, Li S, Wang J, Duan T, Su Y. Clinical application of massively parallel sequencing-based prenatal noninvasive fetal trisomy test for trisomies 21 and 18 in 11,105 pregnancies with mixed risk factors. Prenat Diagn. 2012;32(13):1225–32. 32. Huang X, Zheng J, Chen M, Zhao Y, Zhang C, Liu L, Xie W, Shi S, Wei Y, Lei D, Xu C, Wu Q, Guo X, Shi X, Zhou Y, Liu Q, Gao Y, Jiang F, Zhang H, Su F, Ge H, Li X, Pan X, Chen S, Chen F, Fang Q, Jiang H, Lau TK, Wang W. Noninvasive prenatal testing of trisomies 21 and 18 by massively parallel sequencing of maternal plasma DNA in twin pregnancies. Prenat Diagn. 2014;34(4):335–40. 33. Lau TK, Jiang F, Chan MK, Zhang H, Lo PS, Wang W. Non-invasive prenatal screening of fetal Down syndrome by maternal plasma DNA sequencing in twin pregnancies. J Matern Fetal Neonatal Med. 2013;26(4):434–7. 34. Norton ME, Brar H, Weiss J, Karimi A, Laurent LC, Caughey AB, Rodriguez MH, Williams J, Mitchell ME, Adair CD, Lee H, Jacobsson B, Tomlinson MW, Oepkes D, Hollemon D, Sparks AB, Oliphant A, Song K. Non-invasive chromosomal evaluation (NICE) study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol. 2012;207(2):137.e131–8.

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Chapter 16

“RATs”: Rare Autosomal Trisomies and Their Relevance in cfDNA Testing Francesca Romana Grati and Peter Benn

16.1 Introduction The analysis of cell-free DNA to identify fetal trisomy 13, 18, and 21 is now well-­ established and widely used in clinical practice. These trisomies are the most common and the most clinically significant autosomal trisomies encountered in newborns and in first, and second trimester diagnostic testing. They are therefore referred to as the common autosomal trisomies (CATs). Rare autosomal trisomies (RATs) are defined as all other autosomal trisomies and are only encountered in livebirths when a normal cell line is also present (mosaicism). The noninvasive prenatal testing (NIPT) now in widespread clinical use is based on analysis of maternal plasma cell-free DNA (cfDNA) derived from the mother and the fetoplacental trophoblastic cell lineage [1–3]. An NIPT methodology that interrogates the cell-free DNA for imbalances throughout the genome (“genome-­ wide NIPT”) can be expected to identify all large chromosome imbalances that are present in the mother or in trophoblasts [4–6]. While the detection of chromosome abnormalities beyond CATs may initially seem to be advantageous, many of the additional findings, notably RATs, are associated with considerable clinical interpretation difficulties [7–9]. Before discussing RATs identified through NIPT, we present information on the association of RATs with spontaneous abortion and their challenge when RATs are encountered in CVS and amniotic fluid cells.

F. R. Grati (*) Unit of Research and Development, Cytogenetics and Medical Genetics TOMA, Advanced Biomedical Assays, Impact Lab, Varese, Italy P. Benn Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, CT, USA © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 G. C. Di Renzo (ed.), Prenatal Diagnostic Testing for Genetic Disorders, https://doi.org/10.1007/978-3-031-31758-3_16

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16.2 RATs in Spontaneous Abortions Autosomal aneuploidies (trisomy and monosomy) generally involve a catastrophic deregulation of the process that modulates embryo-fetal development. This change in the gene dosage is usually lethal [10–12]. More than 50% of spontaneous abortion tissues have a chromosomal abnormality and more than half of these are autosomal trisomies. Monosomies are much rarer but can occasionally be encountered. Although among RATs, any chromosome may be involved, there is a nonrandom distribution. For example, chromosomes 1 and 19 are rarely observed, while trisomy 16 is seen in 25% of cases. Although an association between the size of the chromosome involved in trisomy and the frequency of spontaneous abortions has long been known [13], recently, it was noted that a count of protein coding genes provides a better fit than chromosome size. Consequently, one can argue that aneuploid pregnancy loss is largely driven by the combined burden of multiple genes on each chromosome [14]. This is further supported by the evidence that, although chromosomes 13 and 18 are not the smallest autosomes, together with chromosome 21, they carry the least numbers of protein coding genes and therefore have the highest chance for survival to term. The frequency of autosomal trisomies in spontaneous abortions increases with maternal age [13, 15, 16]. When RATs are identified after the first trimester or in liveborn, they are only in a mosaic state with a karyotypically normal (disomic) co-existing cell line. In a mosaic, the disomic cell line would therefore appear to mitigate the lethal effect of the trisomy. This effect is likely to vary depending on the trisomy/disomy ratio in individual fetal organs and tissues [17]. The study of the blastocysts reveals a very high aneuploidy rate, up to almost 60% [18], and it has been estimated that more than 50% of spontaneous conceptions are lost before, or soon after, implantation. The progression of pregnancy is therefore accompanied by strong selective pressures that reduce the presence of aneuploidy either through early correction to disomy or through spontaneous fetal loss.

16.3 RATs in Prenatal Diagnosis In the first trimester chorionic villi (CV) analysis, when the cytogenetic approach includes the analysis of both placental layers (cytotrophoblast by direct preparation and mesenchyme by long-term culture), mosaicism (of any type) can be found in about 2% of CV samples. This can be classified into three types based on the distribution of the abnormal cell line: in type I, the abnormal cell line is detected in cytotrophoblast only; in type II in mesenchyme only; and in type III in both layers. The likelihood of fetal confirmation of a mosaicism in CV depends on several factors [19]:

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1. The combination of involved placental tissues (type I, II or III) 2. The distribution of the abnormal cell line in the affected placental tissue/s (abnormal nonmosaic or mosaic) 3. The type of chromosomal abnormality involved in mosaicism (trisomy, monosomy, triploidy, structural rearrangement or supernumerary marker chromosome) 4. The specific chromosome involved in the abnormality Generally, when a chromosomal mosaicism is detected in the CV, a confirmatory amniocentesis is usually recommended to verify whether the abnormal cell line involves fetal tissues or not and therefore to understand whether it is a true fetal mosaicism (TFM) or a mosaicism confined to the placenta (CPM). In this situation, chromosome analysis should be targeted to the particular abnormality under consideration. It should include the analysis of additional cell colonies [20], and/or interphase FISH [21, 22] and/or SNP-based chromosome microarray with careful attention to B-allele quantification [23, 24]. The mosaicisms in the CV can be classified using the six-group classification system for CPM and TFM depending on the distribution of the chromosomal anomaly in the two placental layers, and in the amniotic fluid (Table 16.1). The frequency of RATs in CV is 0.77%. For RATs only involving the cytotrophoblast (i.e., type I and III), the RAT frequency is 0.41% (Table 16.2). Taken singularly, each individual RAT is rare or ultra-rare (for example, approximately 0.10% of trisomy (T) 7, 0.002% of T19 and T6, and 0% for T1). The overall incidence of RATs in the cytotrophoblast is significantly higher than that of mosaic CATs (0.17%) and sex chromosome aneuploidies (SCAs; 0.22%) [15]. Considering mosaic aneuploidies identified in the cytotrophoblast (type I and III), the probability of confirmation in amniocytes is significantly lower for RATs (3%) versus common trisomies (20%) versus sex chromosome aneuploidies (31.5%) [15]. The low confirmation rate of RATs is likely to be related to the lethality of these trisomies or because some RATs arise in placental cell lineages, after the separation from cells destined to be fetal. Not all individual RATs have an equal risk of confirmation. T3 and T7, although very frequently found in the cytotrophoblasts, are almost never confirmed in amniocytes while others, for example, T14 and T20, have a very low risk of fetal confirmation. T4, T12, and T16 have a greater than 20% Table 16.1  Six-group classification system for confined placental mosaicism (CPM) and true fetal mosaicism (TFM) Type of fetoplacental mosaicism CPM I CPM II CPM III TFM IV TFM V TFM VI

Trophoblast (Dir) AbN N AbN AbN N AbN

Mesenchyme (LTC) N AbN AbN N AbN AbN

Amniocytes N N N AbN AbN AbN

CPM confined placental mosaicism, TFM true fetal mosaicism, Dir Direct preparation cytogenetic analysis, LTC long-term culture cytogenetic analysis, AbN abnormal, N normal

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Table 16.2  Incidence and rate of confirmation of mosaicism for rare autosomal trisomies (RATs), common autosomal trisomies (CATs), and sex chromosome aneuploidies (SCAs) Type of mosaicism RATs in CV RATs in cytotrophoblast (±mesenchyme) CATs in cytotrophoblast (±mesenchyme) SCAs in cytotrophoblast (±mesenchyme)

Incidence (%) | 95% CI 0.77 (0.7–0.84) 0.41 (0.36–0.47)

Rate of confirmation | 95% CI 2.9 (1.72–4.96) 3.0 (1.44–5.97)

0.17 (0.14–0.21)

20 (13.34–28.88)

0.22 (0.19–0.26)

31.5 (24.06–40.02)

CV chorionic villi, RATs rare autosomal trisomies, CATs common autosomal trisomies, SCAs sex chromosome aneuploidies

chance of confirmation. Finally, unlike CATs and SCAs, when RATs are confirmed as TFM, they are present only in a mosaic form in the amniotic fluid. It should be noted that although analysis of amniotic fluid cells sometimes allows fetal mosaicism to be confirmed, this additional testing can never exclude the abnormal cell line with certainty. A 10% risk of cryptic mosaicism has been estimated in cases where the analysis of amniotic fluid shows a normal karyotype in association with a mosaic CVS [25]. Cryptic mosaicism in amniotic fluid can be attributed to the origin of the amniocytes that are believed to be derived from fetal skin, urinary and respiratory tract, placenta, and membranes which are therefore not representative of all fetal tissues and organs [26]. Moreover, a very low level of true mosaicism may not lead to recognizable anatomic abnormality. These latter aspects add additional layers of complexity during genetic counseling of mosaic RATs. There has been a long-standing controversy over the question of whether CPM is associated with pregnancy complications. The literature has been conflicting, primarily because a TFM cannot be excluded with certainty, studies investigating clinical outcomes of CPMs had variable study structure and low statistical power, some studies did not include a systematic CV analysis on both placental layers, and fetal confirmation was not carried out in all cases. A recent retrospective multicentric study that included a large collection of clinical records of CPM for RATs (n = 106) and matched controls (n = 468), showed that clinical outcomes of pregnancies complicated with confined placental RATs (excluding T16) were not worse than pregnancies with a normal CVS karyotype [27]. The study could not investigate associations for every individual RAT involved due to insufficient power. However, data for T16 CPM did indicate a strong association with FGR, birthweight below the third percentile, preterm delivery, and low Apgar score (OR >8). There is also a substantial body of other evidence showing that CPM involving trisomy 16 is associated with FGR, prematurity, preeclampsia, and fetal abnormalities although the individual risk figures for each of these outcomes are unknown [28–32]. Most cases of trisomy 16 are thought to be of meiotic origin and an early postzygotic “trisomic rescue” is required for a viable pregnancy. This mechanism is generally associated

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with high levels of trisomy in trophoblasts, the occurrence of CPM type III (presence of abnormal cells in both placental layers), and a risk for fetal uniparental disomy (UPD), which is defined as a pair of homologous chromosomes derived from only one parent [17, 33]. The presence of fetal abnormalities in T16 CPM cases, despite an apparently normal fetal karyotype, is likely to be due to a hidden T16 cell line in fetal organs not represented in amniotic fluid or to an initial T16 cell line affecting early embryo development with the abnormal cell line no longer detectable later in pregnancy [16]. There is also evidence for an association between CPM type III and FGR or birthweight