Clinical Management of Infertility: Problems and Solutions (Reproductive Medicine for Clinicians, 2) [1st ed. 2021] 9783030718374, 9783030718381, 3030718379

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Reproductive Medicine for Clinicians

Joseph G. Schenker Andrea R. Genazzani John J. Sciarra · Liselotte Mettler Martin H. Birkhaeuser   Editors

Clinical Management of Infertility Problems and Solutions

Reproductive Medicine for Clinicians Series Editors Joseph G. Schenker Department Obstetrics and Gynecology Hebrew University Jerusalem Israel John J. Sciarra Department of Obstetrics and Gynecology Northwestern Medical Faculty Foundation Chicago USA Liselotte Mettler Obstetrics & Gynaecology, House 24 University Hospital Schleswig-Holst Kiel Germany Andrea R. Genazzani International Society of Gynecological Endocrinology Pisa Italy Martin H. Birkhaeuser Professor emeritus of Gynaecological Endocrinology and Reproductive Medicine University of Bern Bern Switzerland

This series will focuses on and presents developments in knowledge and practice within all aspects of reproductive medicine. It will help to cover the important gap between the new possibilities offered by the most recent investigations and technical developments and the application in clinical practice. The series will be a useful tool for professionals and practitioners in the fields of Gynecology, Obstetrics, and Human Reproduction. Trainees interested in the most complete information on the developments of reproductive medicine will benefit as well. More information about this series at http://www.springer.com/series/15751

Joseph G. Schenker  •  Andrea R. Genazzani John J. Sciarra  •  Liselotte Mettler Martin H. Birkhaeuser Editors

Clinical Management of Infertility Problems and Solutions

Editors Joseph G. Schenker Department Obstetrics and Gynecology Hebrew University Jerusalem Israel

Andrea R. Genazzani Department of Obstetrics and Gynecology University of Pisa Pisa Italy

John J. Sciarra Department of Obstetrics and Gynecology Northwestern Medical Faculty Foundation Chicago USA

Liselotte Mettler Obstetrics and Gynaecology University Hospital Schleswig-Holst Kiel Germany

Martin H. Birkhaeuser Department of Gynaecological Endocrinology and Reproduction University Hospital of Bern Bern

Switzerland

ISSN 2523-3599     ISSN 2523-3602 (electronic) Reproductive Medicine for Clinicians ISBN 978-3-030-71837-4    ISBN 978-3-030-71838-1 (eBook) https://doi.org/10.1007/978-3-030-71838-1 © International Academy of Human Reproduction 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Foreword

The International Academy of Human Reproduction is delighted to announce the publication of the second volume of the series titled: Reproductive Medicine for Clinicians, which is published by our loyal partner: Springer. This series focuses on and presents developments in knowledge and practice within all aspects of reproductive medicine. The contents include original articles, reviews, and views arranged in five sections: 1 . Ethical issues in Human Reproduction 2. Clinical Challenges: Patients and Therapies 3. Fertility Preservation and Cryopreservation 4. Prenatal Testing 5. Recurrent Implantation Failure and Pregnancy Loss The chapters are written by established pioneers and experts in human reproduction and it is with great appreciation and gratitude that we thank them for their enormous contribution to this volume. The main objectives of the Academy are to extend the knowledge in all aspects of human reproduction, to encourage clinical experience and promote scientific thoughts and investigation, and to consider the ethical and social implications of the current practice of human reproduction. The fellows of the academy are elected based on their significant contribution to the field and must be acknowledged as world leaders in the discipline. The members of the academy are selected from among applicants from the fields of clinical medicine, medical education, medical and biological sciences, and other fields related to reproductive health and medicine. They are elected based on their singular and significant contributions to the field. Starting in 1974 in Rio de Janeiro, the Academy has held successful congresses every 3 years in Europe, Asia, Africa, the Americas, and Australia. Our congresses promote excellence in reproduction and aims to bridge the gap between the expansion of information and its implementation in clinical practice. During the COVID-19 pandemic, we have continued to exchange knowledge and research among members and the greater human reproduction community. Webinar sessions and our planned congresses in Jerusalem and Columbia, planned for 2021, will contribute to similar publications to this one. v

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Foreword

The series Reproductive Medicine for Clinicians is a useful tool for professionals and practitioners in the fields of gynecology, obstetrics, and human reproduction. Trainees interested in the most complete information on the developments of reproductive medicine will benefit as well. On behalf of the International Academy of Human Reproduction (IAHR), I trust you will support the sustainability of this high-quality book series devoted to human reproduction. Jerusalem, Israel

Joseph G. Schenker

Preface

This volume of Reproductive Medicine for Clinicians focuses on and presents developments in knowledge and practice within aspects of reproductive medicine. The contents include original articles, reviews, and views that will cover the clinical science and medical aspects of reproductive physiology, pathology, and endocrinology. Clinical aspects of infertility treatment, genetic diagnosis, fertility preservation, reproductive surgery, ethics, and social issues are discussed. Religious groups are active in influencing the public regarding bioethical positions, and this is particularly evident with issues concerning procreation, abortion, and infertility therapy. Therefore, it is important to those who practice reproductive techniques to learn about different religious perspectives related to reproductive-­ health problems. The attitude toward reproductive practice varies among the main monotheistic religions. An important concern in clinical practice today is the dilemma faced in the use of imaging especially in the field of infertility. As assisted reproductive technologies become more common, more complex, and more expensive, the concerns to the appropriate use of imaging become more timely and important every day. A social trend toward delaying childbearing has been observed in women of reproductive age. As a consequence, these women may be affected by age-related infertility when they decide to conceive, and fertility preservation techniques can be obtained through the so-called social egg freezing. There is a debate about whether it is morally permissible at all, the extent to which it should be permitted legally or even supported, and whether it is ethically desirable. Cross-border reproductive care (oocyte, sperm, and embryo donation as well as surrogacy) with patients traveling even to other continents to get treatment involves medical, social, ethical, and legal aspects. The legal restrictions vary widely in different regions and countries due to cultural differences, religious beliefs as well as variation in access to advanced reproductive technologies. Successful live births following uterine transplantation were achieved. It appears to be a viable option for women with uterine cause of infertility. The process is associated with significant risk, with the potential for complications in both donors and recipients, and a considerable risk of graft failure. Uterine transplantation remains an experimental procedure that requires the study and resolution of ethical, technical, financial, and social issues. vii

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Adolescent gynecology is dealing with gynecological issues in girls from neonatal period till the completion of adolescence. Characterized by dynamic changes in anatomy and physiology of the reproductive system. Understanding the hormonal and metabolic homeostasis peculiar to this period enables correct approach to many disorders which may be an important hallmark for later reproductive health. Ovulation induction by pulsatile administration of GnRH is a relatively cheap method with a high success rate and a very low risk for multiples and hyperstimulation. It represents the only physiological substitution of the missing hypothalamic hormone and re-establishes successfully follicular maturation and ovulation. A chapter consecrated to the circadian rhythm analyses all the aspects of the biological clock in relation to human reproduction. Polycystic ovary syndrome (PCOS) sounds like it is exclusively a disease of the ovaries, but it is not. While PCOS does affect the ovaries and ovulation, it is actually a full-body endocrine and metabolic disorder. Recent studies demonstrated in PCOS patients that insulin resistance and compensatory hyperinsulinemia might have a certain grade of epigenetic origins that might be implemented by familial predisposition to specific dismetabolic diseases such as diabetes. The direct hysteroscopic vision of the uterine cavity gives today optimal diagnostic and therapeutic possibilities of treatment. Management of patients with endometriosis should always be individualized based on patient’s age, main complaints, clinical presentation, and desire for future pregnancy. Treatment of endometriosis includes different medical and surgical approaches. Since in most cases endometriosis begins at an early reproductive age, preservation of fertility should be the top priority of treatment. Whereas the therapeutic possibilities in female infertility have advanced in big steps, male infertility stayed behind. Only in the 1990s, significant developments in diagnostic techniques and assisted reproductive technologies (ART), the introduction of intracytoplasmic (ICSI) has made it possible for couples with severe male factor infertility to have their own genetic children. A considerable number of young women are diagnosed with breast cancer during their reproductive life. Within this group, most cancer cases require cytotoxic chemotherapy and/or hormone therapy, which are responsible for a decrease in the patients’ reproductive function, along with their age. At present, the most widespread techniques to preserve fertility in women diagnosed with breast cancer are oocyte and embryo cryopreservation, immature oocyte retrieval and in vitro maturation, and ovarian tissue cryopreservation depending on the presence of a partner or according to legislative issues. Ovarian tissue cryopreservation (OTCP) aims to provide a chance for future fertility for young women and pre-pubertal girls who are at major risk for significant ovarian injury and sterility, most commonly as a result of radiation/chemotherapy-­ induced loss of ovarian follicular reservoir. A social trend toward delaying childbearing has been observed in women of reproductive age. As a consequence, these women may be affected by age-related infertility when they decide to conceive, and fertility preservation techniques can be obtained through the so-called social egg freezing. There is a debate about whether

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it is morally permissible at all, the extent to which it should be permitted legally or even supported, and whether it is ethically desirable. Modern methods such as noninvasive testing of cell-free DNA in the maternal plasma (NIPT), targeted cell-free DNA testing, or other noninvasive genetic techniques such as TRIC (trophoblast retrieval and isolation from the cervix) are discussed in an extensive review and compared with the benefits and risks of classical invasive methods. One of the interesting developments in perinatal medicine is the Kurjak Antenatal Neurodevelopmental Test (KANET). KANET gives the clinician a new imaging tool to evaluate fetal behavior and to identify those fetuses at high risk for neurodevelopmental abnormalities. Recurrent implantation failure (RIF) is deduced when good quality embryos fail to implant after a number of in vitro fertilization (IVF) treatment cycles. Several etiologies related to either maternal, embryonal, or paternal factors have been attributed to RIF. A couple presenting with RIF should have a systematic and through investigation. Treatment should be personalized accordingly, with novel methods and tools for better selection of embryos. Recurrent pregnancy loss (RPL) affects approximately 2–5% of couples, depending on the definition of recurrent pregnancy loss. Etiology can be varied and in many cases the cause remains unexplained. The therapeutic approach should be a personalized depending on an accurate diagnosis, specific therapy, rather than treating RPL as homogeneous condition. Jerusalem, Israel Pisa, Italy  Chicago, IL, USA  Kiel, Germany  Bern, Switzerland 

Joseph G. Schenker Andrea R. Genazzani John J. Sciarra Liselotte Mettler Martin H. Birkhaeuser

Contents

Part I Ethical Issues in Human Reproduction  Human Reproduction: Religious Perspectives������������������������������������������������   3 Joseph G. Schenker  Ethics in Clinical and Imaging Practice in Reproductive Medicine ������������  19 Sanja Kupesic Plavsic and Sushila Arya Reproduction at Advanced Parental Age��������������������������������������������������������  29 Eran Altman and Yoel Shufaro  Cross-Border Reproductive Care: Current State of the Art��������������������������  53 Tomer Singer, Liron Bar-El, Laurence B. McCullough, and Frank A. Chervenak Uterine Transplantation������������������������������������������������������������������������������������  69 Victor Gomel Part II Clinical Challenges: Patients and Therapies  Pediatric and Adolescent Gynecology Physiology and Pathology ����������������  83 Dvora Bauman  Biological Clock in Human Reproduction������������������������������������������������������ 101 Dov Feldberg  Ovulation Induction by Pulsatile Administration of GnRH: State of the Art �������������������������������������������������������������������������������������������������� 107 Martin H. Birkhaeuser  Pathogenesis of PCOS: From Metabolic and Neuroendocrine Implications to the Choice of the Therapeutic Strategy�������������������������������� 131 Alessia Prati, Andrea R. Genazzani, and Alessandro D. Genazzani  Mysteries of the Uterine Cavity������������������������������������������������������������������������ 155 Liselotte Mettler and Ibrahim Alkatout Endometriosis: Therapeutic Approach������������������������������������������������������������ 165 Leila Adamyan xi

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 ART and Male Infertility���������������������������������������������������������������������������������� 179 Mausumi Das and Hananel E. G. Holzer  Laparoscopy and Gynecological Cancers�������������������������������������������������������� 197 Liselotte Mettler and Ibrahim Alkatout Part III Fertility Preservation and Cryopreservation  Breast Cancer and Fertility Preservation�������������������������������������������������������� 213 S. Grewe and K. Diedrich  Fertility Preservation Using Cryopreserved-Stored Ovarian Tissue: Where Are We Today? �������������������������������������������������������������������������������������� 223 Dror Meirow Elective Oocyte Cryopreservation�������������������������������������������������������������������� 235 Shmuel Herzberg and Tal Imbar  Medical and Elective (Social) Egg Freezing: Key Insights from Women’s Perspectives������������������������������������������������������������������������������ 247 Marcia C. Inhorn, Daphna Birenbaum-Carmeli, and Pasquale Patrizio Part IV Prenatal Testing  Noninvasive Prenatal Testing (NIPT): Past, Present, and Future ���������������� 259 Alexander Scharf and Wolfgang Holzgreve  Kurjak Antenatal Neurodevelopmental Test (KANET): A Useful Tool for Fetal Neurodevelopmental Assessment���������������������������������������������� 271 Asim Kurjak, Milan Stanojevć, Lara Spalldi Barišić, and Erden Radončić Part V Recurrent Implantation Failure and Pregnancy Loss Recurrent Implantation Failure ���������������������������������������������������������������������� 305 Natali Schachter-Safrai, Alex Simon, and Neri Laufer Recurrent Pregnancy Loss�������������������������������������������������������������������������������� 315 Howard Carp

Part I Ethical Issues in Human Reproduction

Human Reproduction: Religious Perspectives Joseph G. Schenker

Introduction It is important to those who practice reproductive techniques to learn about different religious perspectives related to reproductive-health problems. Religious groups are active in influencing the public with bioethical positions, and this is particularly evident with issues concerning procreation, abortion, and infertility therapy. The developments in reproductive medicine raise new ethical questions for different religions that do not always have clear answers. Religious leaders in some countries still exert a powerful influence on the development and practice of reproductive technology. In some countries, religious groups’ main influence will stem from their direct influence on medical protocols. Therefore, it is important for practitioners in the field of reproductive medicine to understand attitudes toward reproduction that derive from different religions. According to the Web site Adherents.com, the numbers of adherents in the “Big Five” world religions are as follows: Judaism (14 million, 0.22%), Christianity (2.1 billion, 34%), Islam (1.5 billion, 24%), Hinduism (900 million, 14%), and Buddhism (376 million, 6%) [1].

The Jewish Law: Halakha The world is derived from the Hebrew root that means “to go or to walk” [2]—is the collective body of Jewish religious law, including the biblical law (the 613 “mitzvoth,” or commandments) and the later Talmudic and rabbinic law as customs and traditions.

J. G. Schenker (*) Department Obstetrics and Gynecology, Hebrew University, Jerusalem, Israel e-mail: [email protected] © International Academy of Human Reproduction 2021 J. G. Schenker et al. (eds.), Clinical Management of Infertility, Reproductive Medicine for Clinicians 2, https://doi.org/10.1007/978-3-030-71838-1_1

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In principle, the halakhic literature is composed of two divisions: 1. The Written Law—the Torah—the first five books of the Scripture, which are the origin of authority. 2. The dominant parts of the Oral Law are the Mishnah, the Talmud, the post-­ Talmudic codes, and the responsa. Torah  The foundation of the Written Law and the origin of authority is the Torah, the first five books of the Scripture. As stated above, the Written Law is defined as the origin of authority. This definition is derived from the ancient tradition and the belief concerning the original revelation on Mount Sinai, when God ascribed the Torah to Moses and to the Jewish people, the recipients. The Torah is not an ordinary text of law. It is an expression of God’s revelation, teaching, and guidance for man. The attitude to the Torah is therefore as to a unique and holy divine text, which includes moral values as well as practical laws. The Oral Law interprets, expands, and elucidates the written Torah and regulates new rules and customs. Its authority is derived from the written Torah. The Mishnah  This early textbook was compiled systematically by numerous scholars over a few centuries. Its final form was established early in the third century. The Mishnah includes early traditional and original interpretations of the written Torah, ancient regulations that are not written in the Torah, and postbiblical regulations. The Mishnah consists of six major section orders that cover all aspects of human life: Zeraim (laws regarding agriculture), Moed (laws regarding holidays), Nashim (laws regarding women and family life), Nezikin (civil law), Kodashim (laws regarding the Temple), and Toharot (laws regarding ritual purity). Two of the orders, Nashim and Toharot, are relevant to Judaic family practices. The Talmud  For approximately three centuries after the final compilation of the Mishnah, the great interpreters studied the six orders to the Mishnah and wrote a monumental composition, the Talmud. The great interpreters (Amoraim) included within the Talmud commentaries and interpretative studies of the Mishnah and the Midrashim, or investigations, and established regulations and new customs. The Amoraim in Babylon composed the Babylonian Talmud, while the Amoraim in the Holy Land composed the Jerusalem Talmud. During the period of Jewish sovereignty in Judea, laws were made by the Sanhedrin, a body of 71 leaders gathered for this purpose, and by local courts with 23 judges. After the fall of the Second Temple, halakha became primarily the creation of rabbinic Judaism.

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Post-Talmudic codes  An enormous amount of Talmudic knowledge was essential for accurate ruling. These post-Talmudic codes were compiled with the intention of assisting access to the laws, regulations, and customs of the Talmudic halakha. Up to the sixteenth century, different scholars summarized and reviewed the halakhic conclusions of the Talmud in the post-Talmudic codes. Among the scholars were Rashi (1040–1105), Rabbi Moshe Ben Nachman (1195–1270), and Rabbi Menachem Ben Shlomo Hameiri (1249–1316). The most prominent post-Talmudic codes are the Sheilot, Halakhot, Maimonides, the Piskey Harosh, and the Shulchan Aruch. Responsa  The various attitudes of rabbinic scholars about the way halakha should be applied in a changing world are analyzed and discussed with regard to the legal codes. Throughout the ages, written opinion has been given by qualified authorities to questions about aspects of Jewish law. Responsa is a term usually confined to written replies given to questions on all aspects of Jewish law by qualified authorities from the time of the later Geonim to the present day. About 1000 volumes, containing more than half a million separate responsa, have appeared in print. Contemporary rabbinic scholars deal with new problems that arise with the investigation and treatment of infertility. The responsa of later rabbinic authorities are often short monographs in which every text remotely relevant to the point at issue is quoted or discussed. Different groups have developed among Jews from ancient times and especially in the modern era. The Orthodox, Reform, and Conservative movements are the three major ones today. Orthodoxy is the only movement formally and legally recognized by Israel; non-­ Orthodox movements have remained largely a feature of Judaism in the Diaspora.

Jews Law and Infertility Jewish attitudes toward infertility can be discerned from the fact that the first command from God to Adam was “Be fruitful and multiply” after he was created (Gen. 1:28). This commandment has been interpreted as an obligation on the part of the man to reproduce. Marriage is a legal contract between a man and a woman. The couple commit themselves to their mutual duties and create between them a binding religious relationship that also affects others. From a practical perspective, marriage is a mitzvah, or religious duty. It is also the proper framework in which to fulfill God’s command to be fruitful and multiply. Sex is part of human life. The Jewish approach to sex has

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always been free, healthy, and lacking frustration, and Jewish law recognizes sexual desire. Each partner has conjugal duties toward the other. According to Jewish law, the infertile couple should be diagnosed and treated as a single unit [3]. The medical treatment is different for men and women. When evaluating an infertile couple, one should first evaluate the female partner. If pathology is not found, one may proceed to investigate the male. The male factors that should be evaluated are inadequate or abnormal sperm production, ejaculation, or deposition of spermatozoa. The chief halakhic impediment to the standard method of extracting semen (masturbation) is that doing so anywhere but inside one’s wife is prohibited, because it is considered “wasting seed.” Examination of the semen for infertility workup is not included in the prohibition against “spilling one’s seed.” According to Jewish law, the preferred method of seminal fluid analysis should be the postcoital test. There are three basic principles, which, with certain restrictions, favor the permissibility of fertility treatment: 1 . The commandment to “Be fruitful and multiply” 2. The mitzvah gemilut hasadim (loving-kindness) 3. Family integrity In order to implement in vitro fertilization (IVF) and embryonic transfer in Israel, support of halakhic authorities is required. In Israel, special legal problems arise due to the exclusive jurisdiction in matters of personal status that is vested in the rabbinical courts [4]. Although Israeli law is secular, legislated by the Knesset, Israel’s parliament, matters of personal status are governed by halakhic law and enforced by special rabbinical courts. Matters concerning marriage, divorce, paternity, legitimacy, and bastardy and cases of Jewish identity are therefore adjudicated according to Judaic law by these rabbinical courts. The development of ARTs has made it necessary to consider the question of the beginning of life and the moral status of the embryo from different perspectives [5]. Procreation is acknowledged in the Bible to be the gift of God. The halakhic interpretation of when human life begins is extracted predominantly from the halakhic sources. The conclusion as to when human life begins can be obtained from the Torah’s stated position on the issue of abortion: The only indication considered for abortion is a hazard to the mother’s life. Otherwise, the destruction of an unborn child is a grave offence, although not murder. It can be viewed that the fetus is granted some recognition of human life, but it does not equal that of the mother’s and can be sacrificed if her life is in danger [5]. Artificial insemination by donor is unacceptable to most rabbinical authorities. Rabbis have been discussing the principles involving AID for many centuries. Their discussions are based on ancient sources in the Talmud and codes of Jewish law dating back to the fifth century. AID, according to the Jewish law, is prohibited for

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a variety of reasons (e.g., the possibility of incest, a lack of genealogical identity [6], and the problem of inheritance). The child conceived through AID is considered by many rabbinical scholars as having the status of mamzer, or bastard. Oocyte donation  In order to start the clinical trial of an oocyte-donation program in the Hadassah Medical Center at the Hebrew University of Jerusalem, the authorization of a chief rabbi was required. The trial resulted in the world’s first birth using oocyte donation [7]. In egg donation or embryo donation, the problem that arises is that of who should be considered the mother—the donor of the oocyte or the one in whose uterus the embryo develops, the one who gives birth. If one of the women is Jewish and the other is not, problems will arise, since according to Jewish law, the religious status of the child is determined by his mother—the one who gives birth. This interesting subject has an apparent precedent in the Rabbinical literature. According to ancient tradition found in the Talmud and the Midrashim, regarding the birth of Joseph by Rachel. The general opinion is that the gestational mother is regarded as a mother. The religious law decrees that only the offspring of a Jewish mother is regarded as a Jew. According to halakha, the donor should be a single woman. Surrogacy is a source of great controversy in society and in the medical profession. 1. Partial natural surrogacy—has been known for thousands of years: The husband of the infertile woman has intercourse with another woman (the surrogate mother), who donates her genetic material and the use of her womb. The child is then given to the man who donated the sperm and to his legal wife, without adoption procedures. The first mother to become a partial natural surrogate was Hagar: “Now Sarai, Abram’s wife, bore him no children. Sarai said unto Abram, go into my maid: it may be that I may obtain children by her. And Abram harkened to the voice of Sarai. And he went into Hagar, and she conceived” (Gen. 16:1–6). 2. Complete surrogacy—the sperm and the oocyte (genetic material) are of commissioner couple. The surrogate contributes the uterus and gives birth. There are three basic halakhic principles that, with certain restrictions, favor the acceptability of the practice of surrogacy: First, there is the commandment “Be fruitful and multiply.” Second, there is the gemilut hasadim, the mitzvah of benevolence, which originates in the verse “Love thy neighbor as thyself” (Lev. 19:8). In cases of personal ­distress (material, mental, or both), a Jew is duty bound to practice the mitzvah and help one’s neighbor. A childless couple falls within this category, in which a clear obligation exists to assist in every permissible way as long as no one else is thereby harmed. Third, domestic harmony and the integrity of the family are extremely important in Jewish law. Israeli legislation on surrogacy is partly based on halakha [8, 9].

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Fetal reduction  The advances of infertility treatment in recent years, throughout use of ovulation-inducing drugs and of multiple embryo transfer in IVF, contributed to multiple birth. According to Jewish law, the fetus is regarded as a part of the mother’s body and not as a separate being until it begins to egress from the womb during parturition and attains the status of nephesh. Prior to this time, the fetus is not considered a person. In fact, until 40 days after conception, the fertilized egg is considered to be mere fluid [10]. The question of multiple fetal pregnancy reduction (MFPR) was debated in the responsa literature by rabbinical authorities. If the mother’s life is in danger, each fetus is a “rodef” and can be killed to save the mother. But if the danger is to the fetuses and not to the mother, each fetus is, with equal status for each role, both an aggressor and a victim. In this case, it might not be permissible to put aside one soul for the sake of another. Searching for a legal analogy for this situation, some rabbis focused on the case of a group of people who are in mortal danger and who can be saved by sacrificing one innocent member of the group. Most halakhic authorities agree that in case of multiple pregnancy, fetal reduction may be performed. The multiple pregnancy had a high risk of ending in miscarriage of all the fetuses, ruling that each fetus had the status of a “rodef” [11]. Gender preselection  Recent scientific advances have made highly reliable preconceptual sex selection possible by using preimplantation genetic diagnosis (PGD) or sperm separation by flow cytometry combined with AIH or IVF [12]. The requirement for a Jewish man to procreate by having a minimum of two children, a boy and a girl, is obligatory according to Jewish law. According to both schools, Shammai and Hillel, in order to fulfill the obligation of procreation, at least one son is required. Therefore, the application of sex preselection for nonmedical indications may be of practical importance. Cryopreservation  To date, successful results based on pregnancy rates and preservation of fertility for medical conditions have been obtained with cryopreserved spermatozoa, embryos, oocytes, and ovarian tissue [13]. Freezing of sperm and pre-embryos is permitted in Judaism only when all measures are taken to ensure that the father’s identity will not be lost. There are no restrictions of cryopreservation of oocytes and ovarian tissue. Posthumous reproduction was debated in the halakhic literature. Jewish law permits posthumous reproduction by using cryopreserved sperm and by sperm retrieval after death, to the extent that they are used within the context of a traditional marriage and the husband’s consents. Without the man’s consent, the procedure is forbidden. But if it is clearly known that he would have wanted the procedure done, there is no prohibition against performing postmortem sperm retrieval. There is a clear halakhic difference between unknown and uncertain paternity. With postmortem insemination using frozen sperm or with postmortem sperm retrieval, the biological father is known, and the controversy over paternity is strictly

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legal. Therefore, if a widow who was not pregnant at the time of her husband’s death was later inseminated with sperm he donated while still alive and becomes pregnant and gives birth, her deceased husband should be considered as having fulfilled the mitzvah of “pru urvu.” Pre-embryo research  According to the Talmud, during the first 40 days from fertilization until the completion of organogenesis, the embryo is defined, for the purpose of certain laws, as plain water. An embryo under the age of 40  days is not considered to be a person in any legal sense. According to this, pre-embryo research may be permissible if it is carried out in order to enable the sperm owner to have his own child. It is prohibited to use a preimplantation pre-embryo for research unless the research is essential for saving the pre-embryo’s life potential. The destruction or use of a preimplantation pre-embryo for research is forbidden as long as it has the potential to implant. The in vitro creation of preimplantation pre-embryos for research is allowed if there are real chances that the person providing the spermatozoa may benefit and have his own child as a result of this research. When this does not apply, the creation of a pre-embryo for research purposes is strictly forbidden. There is obviously a clear distinction between the preimplantation pre-embryo and the post-implantation embryo. However, the arbitrary 14-day limit is not recognized by Jewish law. Orthodox Jewish law does not forbid pre-embryo research, although some rabbinic authorities would permit it in spare embryos only, those left over in IVF treatment, and not in embryos created for the sole purpose of research. The view held by law in Israel is that pre-embryo research is forbidden [10, 14].

Christianity The Old and New Testaments comprise the scriptures that are sacred to Christians. The Old Testament emphasizes the agreement between God and his people and records Jewish history to illustrate how faithfully this agreement was observed. The New Testament contains promises made by God to humanity, as depicted in the teaching and experiences of Christ and his followers. Jesus Christ is viewed by Christians as the supreme revelation of God and as Lord of his followers. Three principal divisions comprise Christianity: the Roman Catholic Church, Protestant Churches, and Orthodox Catholic Churches. Christianity is particularly characterized by its universality and missionary activity. The most striking development in the evolution of Christianity from its Jewish origin was in its transition from a national religion (of the Jewish nation) to a universal religion. The church assumes a role inspired by a love for humankind in matters concerning reproduction and helps to define the rights and duties of members.

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Roman Catholic Church Roman Catholics base their beliefs on the Bible and the traditions of the church. Traditions are derived from declarations of church councils and popes in the form of dogmas. The Roman Catholic Church is a Christian church characterized by an episcopal hierarchy, with the pope as its head and belief in seven sacraments and the authority of tradition. The Catholic dogma contains three leading principles related to the status of the family, the child, and reproduction. The first principle commands the protection of the human being from the moment of its conception. The second principle is that procreation is inseparable from the physical union of the parents, and therefore, from the moral point of view, a child must be the fruit of marriage. Fidelity involves acknowledgment by spouses that they become parents only through one another and that their child is a living symbol of their love and a permanent sign of their conjugal union. The third principle is related to the personal norm of human integrity and dignity and should be taken into consideration in all medical decisions and especially in the field of infertility. The Vatican has had a clear position against assisted reproduction ever since 1956, when Pope Pius XII defined artificial fecundation as immoral and illegal because it affects human lives by separating procreation and sexual normal function. The Vatican’s instruction on respect for human life made an important contribution to discussions on the practice of new reproductive technologies. It was issued by the Congregation for the Doctrine of the Faith in February 1987 [15], signed by Cardinal Joseph Ratzinger, and approved by Pope John Paul II. Therefore, medical techniques used in assisted reproduction, such as embryo cryopreservation, embryo transfer (ET), application of embryonic stem cells, gamete manipulation, IVF, preimplantation genetic diagnosis, embryo editing, sex preselection, and surrogate motherhood, are not accepted by the Catholic Church. The Catholic Church has been opposed to contraception since at least the second century. All sex acts must be both unitive and procreative. In Pope Paul VI’s Humanae vitae in 1968, artificial contraception is considered intrinsically evil, but methods of natural family planning may be used, as they do not interrupt the natural way of conception. The official position of the Roman Catholic Church is that abortion under any circumstances, including abortion to save the life of the mother, should be prohibited.

Eastern Orthodox Church The Eastern Orthodox Church, formally established in 1054 when it split from the Roman Catholic Church, consists mostly of several independent and self-governing churches. The most ancient self-governing churches are in Istanbul and Antakya in

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Turkey, as well as Alexandria, Damascus, and Jerusalem. The largest national churches are in Armenia, Bulgaria, Cyprus, Georgia, Greece, Romania, Russia, and Serbia. Orthodox Christianity claims to have fully preserved the traditions and doctrines of the original Christian church established by the apostles. The Eastern Orthodox Church’s stand on assisted reproductive practice is not as strict as the Roman Catholic Church’s, allowing the medical or surgical treatment of infertility, but it is against IVF and other ARTs, surrogate motherhood, donor sperm insemination (which it considers adultery), and embryo donation. The Greek Orthodox position on the ethics of assisted reproduction is similar to the Vatican’s. The Church cannot recommend it as the solution to infertility; instead, it proposes a non-secularized perception on life that guarantees simplicity, peace, abstinence, and mutual trust between spouses. It does not oppose resorting to medical help, but, at the same time, it suggests that men and women render their life into the hands of God [16]. The Russian Orthodox Church has condemned the practice of IVF methods. The Church’s position is based on the belief that “an embryo is a future human being and not just an accumulation of cells or a part of a mother’s body,” and it “defends the dignity of human life from the moment of its conception until the natural demise of a human” [17]. The Coptic Church accepts IVF only under the circumstances where the oocyte and sperm are taken from the husband and wife and fertilization occurred in vitro, with no doubt about gamete mixing. ET must be performed on the mother who is the source of the oocytes. All the steps of IVF should occur with the approval of the husband and wife, and the treating physician should be alert to the fact that no mixing of gametes should occur [18].

The Protestant Church The Protestant Church is one of the three major branches of Christianity, originating during the sixteenth-century Reformation. The term Protestant applies to the beliefs of Christians who do not adhere to Roman Catholicism or Eastern Orthodoxy. Protestantism resulted chiefly from the Reformation, a religious and political movement that began in Europe in 1517. At its foundation was protest against the bureaucracies and policies of the Roman Catholic Church. Protestantism is widespread mainly in Germany, the Netherlands, Switzerland, the United Kingdom and its former dominions (Australia, Canada, New Zealand), and the United States, as well as in the Scandinavian countries. It includes a number of autonomous churches and sects that differ somewhat in worship and organization but are linked by common origin and dogma. The three fundamental principles of Protestantism are:

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1 . The supremacy of the Bible 2. Justification by faith alone 3. The universal priesthood of believers Some Protestant denominations can be considered pro-life, while others may be considered pro-choice. Protestant churches have not only refrained from established doctrine about IVF but have shied away from public discussions of the topic. Moderate and liberal Protestant denominations mainly in the United States tend to affirm the right of individuals to discern for themselves how to make use of reproductive technologies. Conservative Protestantism positions on reproductive matters tend to include an active opposition to abortion, but assisted reproduction has not been much considered in formal church statements. In general, they “tend to approve of methods intended to correct physical problems that cause couples to be infertile, but they disapprove of methods that would violate the sanctity of the marriage bond by using donated sperm and eggs, as well as any method that would tamper with or discard a fertilized embryo.”

Anglican Church The Anglican Church is a body of churches in all parts of the world that are in communion with the Church of England. The establishment of an independent Church of England came during the reign of Henry VIII (1509–1547), when Pope Clement VII refused to approve the annulment of Henry’s marriage to Catherine of Aragon. There are 44 churches in the Anglican Communion, including the Anglican Church of Canada, the Scottish Episcopal Church, the Church in Wales, the Church of Ireland, and the Nippon Sei Ko Kwai Church in Japan. There is no single Anglican Church with universal juridical authority, as each national or regional church has full autonomy, and Anglicans do not refer to one central authority to make decisions regarding moral issues. The titular head of the Anglican Communion is the Archbishop of Canterbury, and its leadership consists of bishops, who, since 1868, have met once a decade within the framework of the Lambeth Conference. The Anglican Church allows assisted reproductive techniques, IVF, and ET and permits doctors to use sperm obtained after masturbation; however, it forbids gametal donation. The church, which believes moral status can be given only to an individual with a well-established personality, does not offer it to the embryo. Some Anglican Churches have varying opinions about gametal donation: There are those who think that the genetic origins of a child are fundamentally important and those who think that what is more important is a loved child in a stable relationship. Some think that if donation takes place within a stable marital relationship, it is good, while others hold that it threatens marriage, as understood by Christians, and that it should be strongly discouraged.

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There is unanimous agreement that surrogacy arrangements are unacceptable, and it is argued that while there is nothing wrong with adoption, it also states that the idea of surrogacy contracts entails legal complications and that it is an indignity for a woman to be paid for womb-letting services. The Church of England generally opposes abortion. In 1980 it stated that “In the light of our conviction that the fetus has the right to live and develop as a member of the human family, we see abortion, the termination of that life by the act of man, as a great moral evil.” But the Church also recognizes that in some instances abortion is “morally preferable to any available alternative.” The Anglicans were the first church to issue a statement in favor of contraception, which they did at the Lambeth Conference in 1930.

Islam Instructions that regulate everyday activity of life an observant Muslim should adhere to are called sharia. There are two sources of sharia: primary and secondary. The primary sources of sharia, in chronological order, are the Koran, the very words of God; the sunna and the hadith; the authentic traditions and sayings of Mohammed as collected by specialists in hadith; igmaa, the unanimous opinion of leading Islamic scholars; and qiyas (analogy), intelligent reasoning used to rule on events not mentioned by the Koran and the sunna by matching them against similar or equivalent events previously ruled on. The secondary sources of sharia are istihsan, the choice of one of several lawful options; views of Mohammed’s companions; current local customs, if lawful; public welfare; and rulings of previous divine religions if they do not contradict the primary sources of sharia. The sharia is not rigid. It is flexible enough to adapt to emerging situations in different times and places. It can accommodate different honest opinions as long as they do not conflict with the spirit of its primary sources and are directed to the benefit of humanity [19]. In the Muslim world, religion still has a powerful meaning and greatly influences behavior, practices, and policymaking in the Muslim countries and among the Muslim communities in non-Muslim countries.

Reproduction in Islam The primary sources of sharia have affirmed the importance of marriage, family formation, and procreation. A central feature of Muslim identity and family structure is authenticity of lineage. The Koran explicitly prohibits legal adoption. In Islam, treatment of infertility in married couples is encouraged, as it involves procreation and preservation of humankind childbirth. Child-rearing are regarded as family commitments of both partners, not just biological and social functions. ART

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was widely accepted only after prestigious scientific and religious bodies discussed broadly the issue [19, 20]. If assisted reproduction is indicated in a married couple as a necessary line of treatment, it is permitted within the validity of marriage contract with no mixing of sperm and oocyte [21]. If the marriage contract has come to an end because of divorce or death of the husband, ART cannot be performed on the female partner, even when using sperm cells from former husband. These guidelines are followed by most Sunni Muslims. The Shia guidelines, via fatwa from Ayatollah Ali Hosseini Khamenei in 1999, have opened the way to a third-party donation. This fatwa allows third-party participation, including egg donation, sperm donation, embryo donation, and surrogacy. Recently, there has been some concern about sperm donation among Shia, and most scholars today forbid sperm donation. Surrogacy is practiced among the Shia, whereas most Sunni do not accept it. The fatwa of the Islamic High Council of Mecca in 1984 allowed surrogacy by replacing the embryos inside the uterus of the second wife of the same husband who provided the spermatozoa. In 1985, however, the council withdrew its approval of surrogacy [22]. Multifetal pregnancy reduction may be performed. It is performed with the intention not to induce abortion but to preserve the life of remaining fetuses and minimize complications in the mother. Pregnancy in postmenopause is now possible, with the development of cryopreservation, using one’s own cryopreserved embryos. Oocytes and autografted cryopreserved ovaries may be permissible in exceptional cases. Sex preselection using preimplantation genetic technology (PGD) for nonmedical reasons such as sex selection or balancing sex ratio in the family may be applied in special cases [23]. Cryopreservation of oocytes, embryos, or ovarian tissue can be preserved by cryopreservation. The frozen embryos are the property of the couple alone and may be transferred to the same wife in a successive cycle, but only during the validity of the marriage contract [21]. Marriage ends at death or divorce, and procuring pregnancy in an unmarried woman after divorce or after the death of her husband is forbidden by religious principles of the children’s rights to be reared by two parents and by the child’s right to inheritance. Neither gametes nor gonadal tissues can be donated to another person during the lifetime or after the death of their owner.

Embryo Research According to Islamic teaching, organ differentiation occurs 42  days after fertilization.

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Enrollment of the fetus occurs after 120 days following fertilization, although some authorities consider it to occur as early as 42 days postfertilization. Embryo research, for advancement of scientific knowledge and benefit of humanity, is therefore allowed before 14  days after fertilization on surplus embryos donated for research with the free informed consent of the couple [24]. However these embryos should not be replaced in the uterus of the owner of the eggs or in the uterus of any other woman.

Hinduism Hinduism is a diverse body of religion, philosophy, and cultural practice predominant in India, characterized by a belief in reincarnation and a supreme being of many forms and natures. The religion is based in the teachings of the sacred Vedas. Hindu believers are governed by the three doctrines of dharma, or universal law; karma, or the cumulative effects of personal actions; and samsara, or the cycle of rebirth, liberation from which is the first goal of life. Hinduism has no single book, such as the Bible, that serves as the source of its doctrine, but it has many writings, all of which have contributed to its fundamental beliefs. A central belief of Hinduism is that an individual’s soul or self is eternal. In Hinduism, the soul is believed to be passed from one living being to another in a process called reincarnation. While views on the moral status of the human embryo differ, in traditional Hindu belief, conception is the beginning of a soul’s rebirth from a previous life. Some Hindu traditions place the beginning of personhood between 3 and 5 months of gestation, while a few believe that the soul’s rebirth can occur as late as the seventh month. Regarding fertility, the emphasis on reproduction is not just on having children but on having male offspring. There is a huge stigma attached to being infertile in Indian society, especially for the woman. In Indian society, men need children to have heirs and to prove their masculinity. Society puts pressure on woman to become pregnant and give birth even though the male may be the one who is infertile. In Indian society, there is a strong desire for a son to continue the family line and perform religious rituals for the salvation of departed souls. ARTs are acceptable in Hinduism because there is no single authority to accept or reject it on behalf of the faith. The most important condition is that the egg and sperm are from a legally married couple. In practice, artificial inseminations of donor and oocyte and embryo donation are performed with an anonymous donor. It is preferable that the sperm donor be a close relative of the husband.

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Modern Indians resort to ART, including oocyte and embryo donation, surrogacy, and sex preselection, enthusiastically. India became a leading country for reproductive tourism, especially for surrogacy. The reasons for the surrogacy boom in India are the relative low cost and easy availability of women for surrogacy, especially those from socioeconomically disadvantaged backgrounds [25].

Buddhism Buddhism, one of the major religions of the world, was founded in India about 500 BC by the Buddha. At various times, Buddhism has been a dominant religious, cultural, and social force in most of Asia, especially in China, India, Japan, Korea, Tibet, and Vietnam. In each area, Buddhism has combined with elements of other religions such as Hinduism and Shinto. All Buddhists have faith in the Buddha; his teaching, called the dharma; and the religious community he founded, called the sangha. The basis of what Buddha preached in the dharma is that existence is a continuing cycle of death and rebirth. Each person’s position and well-being in life are determined by his or her behavior in previous lives. Buddhists of all types in various countries are individualistic, and even their scriptures are not rigid. There is no central Buddhist authority to pronounce religious positions. They have no sacraments to administer or rites to perform for the people; every Buddhist is his or her own priest. Marriage within Buddhism does not have the high priority that it has in monotheistic religions. According to Buddhism, the three factors necessary for the rebirth of a human being are the female ovum, the male sperm, and the karma. This karma energy is sent forth by the dying individual at the moment of his or her death. Any technology that is used to achieve conception is morally acceptable, and treatment can be given to unmarried as well as to married women. IVF has been practiced in Japan since 1982 and is also practiced in other countries with Buddhist populations. In Buddhism, donation of sperm is not prohibited. In Buddhism, reincarnation is described as the rebirth of the self. These beliefs that the soul or the self is reborn may lead to a greater acceptance of cloning technology. To many Buddhists, cloning appears to be closely related to the transmigration of a person’s soul from one body to another or to a rebirth of the self.

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References 1. Adherents.com. The Adherents.com collection of religious adherent statistics. 9 Aug 2007. www.adherents.com/Religions_By Adherents.html. 2. Schenker JG, Halperin M.  Jewish family practice and their evolution. Global Bioeth. 1995;1:35–47. 3. Schenker JG.  Infertility evaluation and treatment according to Jewish law. Eur J Obstet Gynecol Reprod Biol. 1997;71:113–23. 4. Schenker JG, Frenkel DA. Medico-legal aspects of in vitro fertilization and embryo transfer practice. Obstet Gynecol Surv. 1987;42:405–10. 5. Schenker JG. The beginning of human life. J Assist Reprod Genet. 2008;25:271–6. 6. Schenker JG. Religious views regarding gamete donation. In: Seibel MM, Crockin SL, editors. Family building through egg and sperm donation. Boston: Jones and Bartlett; 1996. p. 238–50. 7. Navot D, Laufer N, Kopolovic J, et al. Artificially induced endometrial cycles and establishment of pregnancies in the absence of ovaries. N Engl J Med. 1986;314:806–11. 8. Knesset. Surrogacy Law, State of Israel. 1996. 9. Schenker JG.  Women’s reproductive health: monotheistic religions perspectives. Int J Gynaecol Obstet. 2000;70:77–86. 10. Eisenberg V, Schenker JG. The ethical, legal, and religious aspects of pre-embryo research. Eur J Obstet Gynecol Reprod Biol. 1997;75:11–24. 11. Schenker JG. Assisted reproductive practice: religious perspectives. Reprod Biomed Online. 2005;10:310–9. 12. Schenker JG. Gender selection. J Assist Reprod Genet. 2002;19:400–10. 13. Shufaro Y, Schenker JG.  Cryopreservation of human genetic material. Ann N Y Acad Sci. 2010;1205:220–4. 14. Schenker JG.  Jewish Law (Halakha) and Reproduction). In: Schenker JG, editor. Ethical dilemmas in assisted reproduction. Pub. DGRUYTER; 2011. 15. Congregation for the Doctrine of the Faith. Donum vitae: instruction on respect for human life in its origin and on the dignity of procreation: replies to certain questions of the day. Washington, DC: United States Catholic Conference; 1987. 16. Nikolaos M. The Greek Orthodox position on the ethics of assisted reproduction. Reprod Med Online. 2008;(Suppl 1):33. 17. Balashov N. Spokesman for the Russian Orthodox Church on IVF and Surrogacy Interfax-­ Religion. 6 Oct 2010. 18. Bishop Grigorios HG. The Christian opinion in in vitro fertilization coptic culture. Cairo, Egypt; 1988. 19. Gad El Hak AGE. In vitro fertilization and test tube baby in book of fatwa. Dar El Iftaa, Cairo. 1980;1225:1:115:3213–3228. 20. Serour GI. Islam and the four principles. In: Gillon R, editor. Principles of health care ethics. London: Wiley; 1994. 21. Serour GI, editor. Ethical implications of the use of ART in the Muslim World. Cairo: International Islamic Center for Population Studies and Research, Al-Azhar University; 1997. 22. Serour GI. In: Shenfield F, Sureau C, editors. Religious perspectives of ethical issues in ART: contemporary ethical dilemmas in assisted reproduction. London: Informa Healthcare; 2006. p. 99–114. 23. Dickens BM, Serour GI, Cook RJ, et al. Sex selection: treating different cases differently. Int J Gynaecol Obstet. 2005;90:171–7. 24. Serour GI, editor. Ethical implications of the use of ART in the Muslim World. Cairo: International Islamic Center for Population Studies and Research, Al-Azhar University; 2000. 25. Kumar A.  Ethical aspects of assisted reproduction—an Indian viewpoint. Reprod Biomed Online. 2007;14(1):140–2.

Ethics in Clinical and Imaging Practice in Reproductive Medicine Sanja Kupesic Plavsic and Sushila Arya

Introduction The professional responsibility model of clinical ethics governs the referral by the primary obstetrician-gynecologist of infertile patients for assisted reproduction. The professional responsibility model is based on three commitments: becoming and remaining scientifically and clinically competent, protecting and promoting the health-related and other interests of the patient, and acting in the best patient’s interest. The first commitment creates an ethical obligation to refer patients to competent specialists in reproductive medicine, such that the diagnosis and treatment for infertility can occur in a time-sensitive manner. Preconceptional care also allows optimizing the overall health for improved infertility treatment success and neonatal-obstetrical outcome. The reproductive endocrinologist has a range of treatment options, from ovulation induction (OI), intrauterine insemination (IUI), and in  vitro fertilization and embryo transfer (IVF-ET) to third-party reproduction (oocyte and sperm donation, gestational surrogacy). Ultrasound imaging is usually the first step in a comprehensive assessment of an infertile female patient, which enables identification of potential risks from treatment such as ovarian hyperstimulation syndrome and multiple pregnancies. The presence of hydrosalpinx and intracavitary abnormalities are associated with lower implantation potential, while structural uterine defects detected on a coronal plane by 3D ultrasound increase the risk of miscarriage and pregnancy complications. The second commitment creates an ethical obligation to prepare the patient for the referral process with questions she should ask about the risks and benefits of S. Kupesic Plavsic (*) Obstetrics and Gynecology, Faculty Development, Texas Tech University Health Sciences Center El Paso, El Paso, TX, USA S. Arya University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA © International Academy of Human Reproduction 2021 J. G. Schenker et al. (eds.), Clinical Management of Infertility, Reproductive Medicine for Clinicians 2, https://doi.org/10.1007/978-3-030-71838-1_2

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infertility treatment, success rates, and cost involved with the investigation and treatment. The moral duty of a reproductive endocrinologist is to objectively inform and educate the patient about the artificial reproductive techniques (ART), estimated success rates, and associated risks, based on medical history and diagnosis of infertility for the patient or couple. Management of an infertile couple should always be conducted on the basis of evidence-based judgments. Unrealistic expectations for the predictive accuracy of new imaging and ART should be avoided, and the usefulness of sophisticated ultrasound and laboratory equipment and tools should be appropriately assessed. The third commitment creates an ethical obligation to report inappropriate reproductive medicine services to professional associations and licensing authorities. In this chapter, we will review the most common ethical problems and dilemmas facing practicing clinicians in the field of clinical and imaging practice in human reproduction.

Need for Timely Referral Infertile patients typically present preconceptionally, enabling timely detection of the uterine, tubal, and ovarian conditions that may interfere with pregnancy, identification of risk factors related to pregnancy, and initiation of appropriate interventions (e.g., reinforcing folic acid supplementation). Ultrasound imaging is among the first tests performed in the initial work-up of an infertile couple, and the advanced high-resolution transvaginal ultrasound with 3D, color, and spectral Doppler modality has become a widely accepted “one-stop shop” fertility scan [1–3]. In case a physician is not able to provide optimal imaging service to subfertile and infertile patients, he/she should refer them to the centers where such services are available. The gynecologist and reproductive endocrinologist (REI) have a range of treatment options, including IUI, IVF-ET, and third-party reproduction opportunities (e.g., oocyte donation, sperm donation, gestational surrogacy). Conscientious objection in medicine is the notion that a healthcare provider can abstain from offering certain types of medical care with which he/she does not personally agree. This includes care that would otherwise be considered medically appropriate (e.g., a physician may not agree with oocyte donation treatment option or PGA). A potential solution for conscientious objection, in this case, is that the patient can be referred to the provider who is trained and willing to honor a patient’s request for this type of service [4, 5]. Evidence-based medicine includes three components: research-based evidence, clinical expertise, and the patient’s values and preferences [6]. An honest dialogue between the patients and the provider improves transparency and quality of care, leading to better outcomes.

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Ethical Issues in Ultrasound Imaging Practice Recently “one-stop shop” pelvic ultrasound of an infertile patient was proposed as a rational, noninvasive, and cost-beneficial investigation that can shorten the assessment allowing earlier access to appropriate treatment [1, 3, 6, 7]. Debate continues about the timing of the scan. The majority of the studies on ovarian reserve assessment reported best results if pelvic ultrasound is performed on days 2–3 of the menstrual cycle, while some authors prefer the scan on days 10–12 to determine follicular development and endometrial morphology and thickness associated with ovulatory changes [3, 7]. This type of pelvic ultrasound exam consists of native or contrast-enhanced two-­ dimensional (2D) and 3D ultrasound assessment of the uterus and cervix, fallopian tubes, and ovaries.

Uterine and Cervical Assessment The uterine scan aims to determine the size, position, mobility, and relationship of the uterus to surrounding organs. The endometrium is assessed for thickness, texture, and the presence of focal lesions or fluid in a midline sagittal plane [1]. 3D volume acquisition of the uterus is also performed in the midsagittal plane, allowing the visualization of the entire length of the uterine cavity with the cervix on one end and the fundus on the other. Upon acquisition of the 3D uterine volume, the standardized multiplanar view allows simultaneous assessment of the sagittal, transverse, and coronal planes of the uterus, which enables visualization of diffuse and focal myometrial and endometrial thickening and lesions. Surface rendering and volume contrast imaging (VCI) enable improved assessment of the junctional zone (JZ) and evaluation of the external serosal surface contour of the uterus [1, 6]. 3D ultrasound facilitates visualization of adenomyosis based on detection of JZ irregularities (e.g., thickening, disruption, and protrusion of the endometrium into the myometrium) and visualization of small cystic inclusions within the myometrium [8]. Timely recognition of sonographic signs of adenomyosis is of paramount importance since some centers reported decreased clinical pregnancy rate with IVF-ET compared to those without adenomyosis [9]. Careful evaluation of the morphologic and vascular changes of the endometrium in the early postmenstrual period, mid-cycle, and in the late luteal phase in spontaneous menstrual cycle is highly recommended prior to fertility treatment [10]. 2D and 3D color Doppler assessment of the endometrial receptivity by estimation of the uterine and endometrial blood flow may give further insight into endometrial receptivity and may be recommended prior to ET. The uterine cavity is carefully assessed before ART because acquired uterine cavity abnormalities may be expected in 11–40% of the sub- or infertile patients

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[11]. The most common intracavitary lesions are endometrial polyps, visualized as focal endometrial thickening with a feeder vessel easily detected by color and power Doppler ultrasound. The most symptomatic subtype of uterine fibroids, commonly associated with abnormal uterine bleeding, infertility, recurrent pregnancy loss, preterm delivery, and fetal malpresentation, is submucosal fibroids. They are visualized as broad-based well-defined solid lesions, distorting the interface between the endometrium and myometrium and showing acoustic attenuation. Transvaginal 2D ultrasound has sensitivity and specificity of 87% and 80%, for the detection of endometrial polyps, and 95% and 96% for the detection of submucosal fibroids, respectively [12]. Following 3D saline infusion sonography (SIS), these values are improved to almost 100% and 93% for endometrial polyps and 99% and 98% for submucosal fibroids comparable to hysteroscopy (gold standard) [12]. Improved spatial orientation with 3D SIS has increased its sensitivity and specificity in detecting intrauterine lesions [13, 14]. With high accuracy of 3D SIS in diagnosing uterine cavity abnormalities, it is considered as an alternative to diagnostic hysteroscopy. Müllerian defects are not uncommon in women seeking ART; the arcuate and septate uterus is the most common müllerian anomalies [15]. A recent study using 3D ultrasound reported that the rate of congenital uterine anomalies in the subfertile female population is estimated to 13.3% [16]. The most common obstetrical complications occurring in patients with congenital uterine anomalies are second trimester miscarriages, prematurity, intrauterine growth restriction, antepartum and postpartum hemorrhage, gestational hypertension and preeclampsia, cervical incompetence, premature delivery, malpresentation, and operative delivery [17, 18]. Following hysteroscopic metroplasty term delivery rates increase from approximately 5 to 75% [17], live birth rates increase from approximately 5 to 85% [19], and the rate of miscarriages decreases from 57.7 to 11.9% [20]. In the past uterine cavity, anomalies were detected by X-ray hysterosalpingography (HSG). Combined hysteroscopy (direct inspection of the uterine cavity) and laparoscopy (evaluation of the external uterine contour) was considered the gold standard for the assessment of suspected müllerian anomalies. MRI provides high accuracy by simultaneous visualization of the uterine cavity, fundus, vagina, and renal anomalies, but its use is limited by high cost and availability. Similar to MRI, 3D ultrasound allows simultaneous assessment of the uterine cavity, external fundal contour, and cervix, with comparable diagnostic accuracy to MRI müllerian anomalies [21, 22]. 3D ultrasound can also be efficiently used for follow-up of the hysteroscopic metroplasty and detection of the residual septum [17]. Recent advances made by 3D ultrasound and operative hysteroscopy should change the “old school of thoughts” that surgical correction is required only for patients with a history of recurrent (≥3) miscarriages. In patients with history ≥1 unexplained miscarriages but with a septate uterus on 3D ultrasound, hysteroscopic metroplasty should be considered [22]. However, a septal incision is not always necessary and should be based on the patient’s reproductive history and desires, the size of the septum, and consultation with specialists in the field of infertility and hysteroscopy. Decisions on when and whom to treat should be discussed with the

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patient and done on an individual basis due to the lack of randomized controlled trials. In patients with recurrent pregnancy loss (RPL), it is important to rule out other potential causes of infertility, such as genetic, endocrine, and metabolic abnormalities, before proceeding with surgical treatment. In patients with septate uterus and primary infertility, the role of metroplasty continues to be controversial, and although some patients have been treated solely, for this reason, the pregnancy rate in this subpopulation has been much lower than when the indication for hysteroscopic metroplasty is pregnancy loss or other obstetrical complications. Another ethical concern is when a physician who was performing ultrasound self-refers to endoscopy facility in which he has a financial interest or if the same physician is performing both imaging and hysteroscopy.

Fallopian Tube Data from the literature indicate that sonographically visible hydrosalpinges affect the outcome of IVF-ET procedures. 3D inverse mode ultrasound is superior to 2D ultrasound in demonstration of the morphological characteristics of the hydrosalpinx such as sausage or retort-like shape, incomplete septations, pseudopapillomatous projections, cogwheel sign, mural nodules, and clear distinction from the ipsilateral ovary. During the last three decades, the influence of the presence of hydrosalpinx on IVF-ET success rate was thoroughly studied, and improved clinical pregnancy rates after surgical removal of hydrosalpinx either by proximal tubal closure, salpingectomy, or aspiration were reported [23]. An astute clinician should always perform a detailed 3D pelvic ultrasound exam and discuss the negative effect of hydrosalpinx fluid on pregnancy rates and discuss surgical options. The benefits of a salpingectomy before embryo transfer should be discussed with patients undergoing IVF-ET. There are some controversial reports on the potential decrease in uterine perfusion with salpingectomy, which may negatively affect receptivity. For patients requiring evaluation of tubal patency, 3D hystero-contrast-­ salpingography (Hy-Co-Sy) should be recommended. Being as accurate as X-ray HSG in assessing tubal patency and having the advantage of simultaneous evaluation of the coronal plane of the uterine cavity and adnexal pathology, Hy-Co-Sy has become the first-line diagnostic test for tubal assessment [24].

Ovaries and Cul-de-Sac Antral follicle count (AFC) and ovarian volume measurements have been reported as accurate measures for the assessment of the ovarian reserve and are complementary to anti-müllerian hormone (AMH) testing [25]. Measurements of the ovarian volume, AFC, and increased ovarian stromal flow assessed by color/power Doppler

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are associated with better ovarian response to controlled ovarian hyperstimulation (COH), a higher number of retrieved oocytes and pregnancy rates. Similarly, these ovarian parameters may be used for prediction of the ovarian hyperstimulation syndrome (OHSS). The size of the follicles correlates with a competency of retrieved eggs for fertilization; hence, REI should be proficient in the follicular monitoring and considerate in determining the best timing for oocyte retrieval [26].

Ethical Issues in Clinical Practice Important ethical issues in the clinical practice of reproductive medicine include respect for patient autonomy, privacy, informed consent, discrimination, and broader societal consequences [27]. To ensure appropriate service delivery, clinical practice, and patient safety in reproductive medicine and ART, each society and institution must provide clear and specific regulations for this type of service. Here we will review the most common ethical dilemmas facing practicing clinicians in reproductive medicine.

Oocyte and Embryo Donation and Surrogacy While fertility preservation and reproduction in patients facing gonadotoxic therapies is not controversial, there are many ethical issues on the use of donor gametes and embryos, especially related to donors’ compensation, confidentiality, and protection of donors’ identity, as well as the increased risk of the future incestuous relationships [28–30]. Another ethical concern is the use of donor gametes without the consent of the partner, especially after divorce or the partner’s death [31]. Challenging dynamics of a partnership sometimes result in withholding sensitive information related to a third-party reproduction from each other, which may open a Pandora box of ethical issues. Another ethical dilemma is regarding the disposition of abandoned embryos. It is estimated that about 600,000 cryopreserved embryos are stored in fertility centers/ clinics across the United States [32, 33]. Many couples are not appropriately counseled or educated to make a decision about what to do with their embryos once their families are complete. Also, there are many questions about the use of donor gametes in fertility treatment by gays, lesbians, and unmarried persons [34]. A paucity of research exists about the relationship and experience between children and their lesbian, gay, or bisexual (LGB) parents with known and unknown donors [35]. There is ethical concern regarding the provision of this type of service for donors and surrogates at increased risk of complications during fertility treatment or pregnancy, respectively [36]. Another important issue is that many donors and surrogates are not appropriately screened, evaluated, educated, and consented. Sometimes

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they are not informed about the health risks, short- and long-term complications associated with gamete donation, pregnancy risks, psychological and emotional issues associated with pregnancy, and the postpartum period [37]. There is a special concern for using family members as gamete donors or gestational carriers, and ART programs are encouraged to develop policies and procedures for dealing with the request of intrafamilial gamete donors and gestational surrogates [38]. Currently, we are missing a systematic follow-up of the patients who conceived and families who were built through a third-party reproduction, nor reproductive outcomes of the donors and surrogates [36, 38]. Many oocytes and sperm donors are not informed about the outcomes and success of the collaborative ART procedures in which they participated [36]. This is concerning, because there may be a mutual benefit of sharing the biologic material or information in case medical need arises from either side (donor or recipient family), and a comprehensive national and international third reproduction party registry should be created to track the donors, surrogates, and the families built through collaborative reproduction to protect the interest of donors [39]. More recently, infertility centers start offering couples the “compassionate transfer” of cryopreserved surplus embryos [40]. This procedure consists of the transfer of the thawed embryos into the vagina, during the period when conception is not possible. Sometimes couples may decide to stop paying storage fees, which transfers responsibility for the destiny of the stored embryos to the infertility center/ institution, while other couples or patients decide to donate their gametes and embryos for research. Ethicists generally agree that creating extra embryos for research purposes or as a means of generating embryonic stem cells should not be encouraged and legalized [36, 41]. At this moment, the NIH Guidelines on Human Stem Cell Research permit federal funding for research on stem cell lines created from embryos donated by couples who have completed their infertility treatment, but they rule out funding for research using lines created from embryos produced purely for research [42]. Although human rights, technically, cannot be applied to an embryo, an embryo should be perceived as having cumulative rights. However, in most cultures these rights can only be accessible if born alive [43].

Preimplantation Genetic Analysis (PGA) Sex selection is a sensitive and complex subject with different implications in different societies. PGA requires strict regulation and education to avoid misuse; the treatment of patients using this technique for sex selection and identification of abnormal genetic traits such as sickle cell genes for the purposes of avoiding transmittable diseases is a serious ethical dilemma since a child born with sickle cell disorder could still add value to the society.

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Conclusions ART has emerged as one of the most widely accepted and successful medical technologies in the last decades. Ethical challenges in this dynamic and ever-changing field are emerging, challenging REI specialists to continuously monitor, offer, and deliver the best and up-to-date care with full social and moral responsibility. Due to recent scientific and technological advances, we may need to reassess our clinical and imaging practices to ensure that the infertility management and ART are performed, documented, and followed up in ethical and socially responsible manner.

References 1. Kupesic Plavsic S, Arya S. Future of imaging in human reproduction. In: Schenker JG, Sciarra J, Mettler L, editors. Reproductive medicine for clinical practice: medical and surgical aspects: Springer; 2019. p. 93–103. ISBN 978-3-319-78008-5. 2. Kupesic S. Editorial: the present and future role of three-dimensional ultrasound in assisted conception. Ultrasound Obstet Gynecol. 2001;18(3):191–4. 3. Kelly SM, Sladkevicius P, Campbell S, Nargung G. Investigation of the infertile couple: a one stop ultrasound-based approach. Hum Reprod. 2001;16(12):2481–4. 4. Savulescu J. Conscientious objection in medicine. BMJ. 2006;332:294–7. 5. Adams KE. Moral diversity among physicians and conscientious refusal of care in the provision of abortion services. J Am Med Womens Assoc. 2003;58:223–6. 6. Chervenak FA, Kupesic Plavsic S, McCulough LB.  Professionally responsible referral for assisted reproduction. J Reprod Med. 2019;64(2):87–9. 7. Kupesic S, Kurjak A.  Predictors of IVF outcome by three-dimensional ultrasound. Hum Reprod. 2002;17(4):950–5. 8. Luciano DE, Exacoustos C, Albrecht L, et al. Three-dimensional ultrasound in the diagnosis of adenomyosis: histologic correlation with ultrasound targeted biopsies of the uterus. J Minim Invasive Gynecol. 2013;20(6):803–10. 9. Vercellini P, Consonni D, Dridi D, Bracco B, Frattaruolo MP, Somigliana E. Uterine adenomyosis and in vitro fertilization outcome: a systematic review and meta-analysis. Hum Reprod. 2014;29(5):964–77. 10. El-Mazny A, Abou-Salem N, El-Sherbiny W, Saber W.  Outpatient hysteroscopy: a routine investigation before assisted reproductive techniques? Fertil Steril. 2011;95(1):272–6. 11. Kupesic S, Kurjak A. Uterine and ovarian perfusion during the periovulatory period assessed by transvaginal color Doppler. Fertil Steril. 1993;60(3):439–43. 12. Bingol B, Gunenc Z, Gedikbasi A, Guner H, Tasdemir S, Tiras B. Comparison of diagnostic accuracy of saline infusion sonohysterography, transvaginal sonography, and hysteroscopy. J Obstet Gynaecol. 2011;31:54–8. 13. Sylvestre C, Child TJ, Tulandi T. A prospective study to evaluate the efficacy of 2 and 3D SHG in women with intrauterine lesions. Fertil Steril. 2003;79:1222–5. 14. La Sala GB, Blasi I, Gallinelli A, Debbi C, Lopopolo G, Vinci V, Villani MT, Iannotti F. Diagnostic accuracy of sonohysterography and transvaginal sonography as compared with hysteroscopy and endometrial biopsy: a prospective study. Minerva Ginecol. 2011;63:421–7. 15. Bermejo C, Martinez TP, Cantarero R, Diaz D, Pedregosa P, Barrón E, Labrador E, Ruiz Lopez L.  Three-dimensional ultrasound in the diagnosis of Mullerian duct anomalies and concordance with magnetic resonance imaging. Ultrasound Obstet Gynecol. 2010;35:593–601. 16. Jayaprakasan J, Chan YY, Sur S, et al. Prevalence of uterine anomalies and their impact on early pregnancy in women conceiving after assisted reproduction treatment. Ultrasound Obstet Gynecol. 2011;37:727–32.

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17. Woelfer B, Salim R, Banerjee S, Elson J, Regan L, Jurkovic D.  Reproductive outcomes in women with congenital uterine anomalies detected by three-dimensional ultrasound screening. Obstet Gynecol. 2001;98(6):1099–103. 18. Chan YY, Jayaprakasan K, Tan A, Thornton JG, Coomarasamy A, Raine-Fenning NJ. Reproductive outcomes in women with congenital uterine anomalies: a systematic review. Ultrasound Obstet Gynecol. 2011;38(4):371–82. 19. Grimbizis GF, Camus M, Tarlatzis BC, et al. Clinical implications of uterine malformations and hysteroscopic treatment results. Hum Reprod Update. 2001;7:161. 20. Kupesic S, Kurjak A, Skenderovic S, Bjelos D. Screening for uterine abnormalities by three-­ dimensional ultrasound improves perinatal outcome. J Perinat Med. 2002;30(1):9–17. 21. Jurkovic D, Geipel A, Gruboeck K, et al. Three-dimensional ultrasound for the assessment of uterine anatomy and detection of congenital anomalies: a comparison with hysterosalpingography and two-dimensional sonography. Ultrasound Obstet Gynecol. 1995;5(4):233–7. 22. Kupesic S. Clinical implications of sonographic detection of uterine anomalies for reproductive outcome. Ultrasound Obstet Gynecol. 2001;18:387–400. 23. Tsiami A, Chaimani A, Mavridis D, Siskou M, Assimakopoulos E, Sotiriadis A. Surgical treatment for hydrosalpinx prior to in-vitro fertilization embryo transfer: a network meta-analysis. Ultrasound Obstet Gynecol. 2016;48(4):434–45. 24. Kupesic S, Plavsic BM. 2D and 3D hysterosalpingo-contrast-sonography in the assessment of uterine cavity and tubal patency. Eur J Obstet Gynecol Reprod Biol. 2007;133(1):64–9. 25. Kupesic S, Kurjak A, Bjelos D, Vujisic S. Three-dimensional ultrasonographic ovarian measurements and in vitro fertilization outcome are related to age. Fertil Steril. 2003;79(1):190–7. 26. Rosen MP, Shen S, Dobson AT, Rinaudo PF, McCulloch CE, Cedars MI. A quantitative assessment of follicle size on oocyte developmental competence. Fertil Steril. 2008;90(3):684–90. 27. Schweikart S. AMA code of medical ethics’ opinions related to global reproductive health. AMA J Ethics. 2018;20(3):247–52. 28. Goldman RH, Racowsky C, Farland LV, et al. Predicting the likelihood of live birth for elective oocyte cryopreservation: a counseling tool for physicians and patients. Hum Reprod. 2017;32(4):853–9. 29. Druckenmiller S, Goldman KN, Labella PA, et  al. Successful oocyte cryopreservation in reproductive-­aged cancer survivors. Obstet Gynecol. 2016;127(3):474–80. 30. Cobo A, García-Velasco JA, Coello A, et al. Oocyte vitrification as an efficient option for elective fertility preservation. Fertil Steril. 2016;105(3):755–764; e8. 31. Bahm SM, Karkazis K, Magnus D.  A content analysis of posthumous sperm procure ment protocols with considerations for developing an institutional policy. Fertil Steril. 2013;100(3):839–843; e6. 32. Ethics Committee of the American Society for Reproductive Medicine. Disposition of abandoned embryos: a committee opinion. Fertil Steril. 2013;99(7):1848–9. 33. Boys KS, Walsh JS. The dilemma of spare embryos after IVF success: social workers’ role in helping clients consider disposition options. Adv Soc Work. 2017;18(2):583–94. 34. Goldberg AE, Allen KR. Donor, dad, or? Young adults with lesbian parents’ experiences with known donors. Fam Process. 2013;52:338–50. 35. Bos H, van Rijn-van Gelderen L, Gartrell N. Self-esteem and problem behavior in Dutch adolescents conceived through sperm donation in planned lesbian parent families. J Lesbian Stud. 2020;24(1):41–55. https://doi.org/10.1080/10894160.2019.1625671. 36. Levens ED, DeCherney AH. Human oocyte research: the ethics of donation and donor protection. J Am Med Assoc. 2008;300(18):2174–6. 37. The Ethics Committee of the American Society for Reproductive Medicine. Financial compensation of oocyte donors: an Ethics Committee opinion. Fertil Steril. 2017;107:1136–42. 38. Marshall LA.  Intergenerational gamete donation: ethical and societal implications. Am J Obstet Gynecol. 1998;178:1171–6. 39. Jadva V, Imrie S. Children of surrogate mothers: psychological well-being, family relationships, and experiences of surrogacy. Hum Reprod. 2014;29:90–6.

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40. Riggan KA, Allyse M. “Compassionate transfer”: an alternative option for surplus embryo disposition. Hum Reprod. 2019;34(5):791–4. 41. Dresser R. Stem cell research as innovation: expanding the ethical and policy conversation. J Law Med Ethics. 2010;38(2):332–41. https://doi.org/10.1111/j.1748-­720X.2010.00492.x. 42. Guidelines for research involving hESCs in the Federal Register for public comment. 74 Fed. Reg. 18578 (April 23, 2009). https://stemcells.nih.gov/policy/2009-­guidelines.htm. 43. Solter D. Politically correct human embryonic stem cells? N Engl J Med. 2006;354(11):1209.

Reproduction at Advanced Parental Age Eran Altman and Yoel Shufaro

Reproduction at Advanced Parental Age Women above 45 years can conceive and deliver following the transfer of cryopreserved self embryos or from the transfer of embryos originating from young donor oocytes. When taken to the extreme, even cases of deliveries in women way over 60 have been reported in peer-reviewed literature and also in the popular media [1–4]. As the aging of the human uterus occurs (in average) more than a decade after the human ovaries, the transfer and successful implantation of embryos into the uterus of women far beyond their natural reproductive years is readily accomplished [5, 6]. As a consequence, women beyond the extended fecundity period enabled by assisted reproduction, and even up to the seventh decade of life, are now able to conceive, carry pregnancies, and deliver live-borns. In industrialized rich societies, the maternal age is constantly rising in favor of the fulfillment of personal achievements and inspirations. Thus the number of women contemplating and achieving pregnancies at an age previously considered adequate for grandparenthood is constantly rising. Contrary to the ovarian age, it appears that embryo implantation is less affected by the endometrial age; therefore oocyte donation cycles in elderly women are as successful as assisted reproduction in the donors’ age group [7]. The uterus retains its receptivity to embryo implantation for a substantial period of time after the ovarian germ cell reserve diminishes, as long as adequate endogenous or exogenous hormonal support exists or is provided. On the other hand, the apparently good pregestational maternal health can be misleading, and pregnancies at an advanced maternal age (AMA) are associated with increased maternal and fetal and neonatal morbidities [8, 9]. Although the implantation rate of embryos from donated oocytes is almost

E. Altman · Y. Shufaro (*) Infertility and IVF Unit, Beilinson Hospital RMC, Petah Tikva, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel e-mail: [email protected] © International Academy of Human Reproduction 2021 J. G. Schenker et al. (eds.), Clinical Management of Infertility, Reproductive Medicine for Clinicians 2, https://doi.org/10.1007/978-3-030-71838-1_3

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unaffected by the recipients’ age [8], the chance for a successful final outcome is significantly affected by the occurrence of complications associated with AMA. Pregnancies resulting from assisted reproduction at AMA (contrary to the rare ones occurring spontaneously) raise major medical and ethical concerns regarding the maternal, fetal, and neonatal well-being. Therefore it is important to publicly debate these issues in order to facilitate a discussion toward determining the positions of the healthcare systems and society regarding expediting pregnancies and childbearing beyond the natural fecundity. This discussion should be held in accordance to local parenthood values and available resources to bear the financial, social, and personal risk costs. The adverse effect of AMA on offspring health has been studied extensively and is currently well recognized [10–12]. In contrast, the association between advanced paternal age (APA) and poor reproductive outcome has been less defined and studied, and its exact effect remained somewhat uncertain. In this chapter this issue will also be reviewed including the relationship between APA and changes in testicular function [13], reproductive hormones [14], sperm parameters [15, 16], sperm DNA fragmentation [17], telomere length [18], de novo autosomal dominant mutations [19] and other genetic and epigenetic factors risks, childhood cancer, congenital anomalies, neurodevelopmental outcomes, and obstetrical complications [20]. Naturally some association exists between the maternal and paternal age, so the net effect of the paternal age is quite difficult to establish. The objective of this chapter is to raise the medical and ethical concerns associated with parenthood at advanced maternal and paternal ages and to try to answer some of them from our perspective which represents our specific social and medical setup.

Reproduction at Advanced Maternal Age Pregestational Considerations Starting at age 35 in average, a woman’s fertility and fecundity decrease in parallel to her aging [21]. The leading cause for this age-related decline in fecundity is the deterioration of the quantitative ovarian reserve and the incline in the rate of oocyte chromosomal aberrations [22] resulting in embryo aneuploidy, failed implantation, and high miscarriage rate (20–40% at age 45). The endometrial receptivity and the chance of implantation also decrease with aging [23], but in a significantly lower rate. In most cases endometrial receptivity is preserved by hormonal replacement or additive therapy [5]. When oocytes from young donors are used instead of those originating from the aging patients, after adequate endometrial preparation with estrogens and progesterone, the pregnancy and miscarriage rates correspond to those obtained with assisted reproduction in the donor’s age group [7]. Therefore as long as the uterine factor is normal or even acceptable, pregnancy with embryos originating from young (donor or self) oocytes can be readily achieved even in the seventh decade of life. The principal issue is the events that follow this success.

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The hemodynamic, respiratory, renal, and endocrinologic changes induced by pregnancy pose a considerable stress even for young women. The cardiac output gradually increases during pregnancy reaching its maximum at the first weeks of the third trimester—140% of the baseline value. During labor, transient increases above this level occur, especially in the second stage. The ability of the cardiovascular system to adapt to these changes at advanced age is not obvious, even in patients with an apparently normal baseline health and good cardiovascular exercise capacity. The pulmonary respiratory volumes and effort, as well as the renal glomerular filtration rate, are also significantly increased during pregnancy [24]. The prevalence of hypertension, heart diseases, diabetes, chronic lung diseases, renal diseases, and essential hypertension (and resulting placental complications) are directly associated with the maternal age [25, 26], as well as the mortality associated with neoplasms and heart diseases [2]. At the fifth and sixth decades, such diseases might exist at a subclinical level and be unveiled by the extraordinary prolonged strain of pregnancy, jeopardizing the health of the mother and fetus, sometimes necessitating the premature termination of the pregnancy. In addition, gestational trophoblastic diseases, uterine fibroids, and urinary tract infections are also more prevalent at advanced age and might also complicate pregnancies in these patients [27–29]. Thus, it is well established that pregnancy in the older population potentially constitutes a major maternal health risk. Our main dilemmas as healthcare and fertility care providers are (a) setting up an age limit for conception attempts and (b) to determine in which modalities and to what extent to screen the candidates in order to avoid serious maternal morbidity on the one hand but allow pregnancy to those who can go through the gestation relatively safely on the other hand. Dynamic tests designed to unmask occult diseases like stress ergometry and echocardiography, thallium scans, spirometry, or even maximal oxygen consumption are of short duration and do not constitute a reliable model of the changes occurring in advanced pregnancy. No clinical diagnostic test actually and reliably simulates the conditions of pregnancy, neither in length nor in amplitude. For example, it is unclear if a pregestational normal baseline and stress-­ induced heart function at echocardiography and a negative thallium scan actually predict a pregnancy free from cardiac complications. The same applies for pulmonary and renal tests, and the negative predictive value of normal basic and functional heart, lung, or renal tests performed before pregnancy is uncertain.

Gestational and Obstetrical Considerations Advanced maternal age is the risk factor for chronic hypertension, pregnancy-­ induced or exacerbated hypertension, and preeclampsia [25]. The prevalence of impaired glucose tolerance and frank diabetes are also in correlation with age. Therefore it is not surprising that diabetes diagnosed during pregnancy is more prevalent at advanced maternal age [25]. These two complications were reported to be of high prevalence in the advanced age group in almost all the published reports

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on this issue and to be in direct correlation with advanced age [30]. Moreover, in women over 50, the occurrence of cardiac pathologies, hypertension, diabetes, and preeclampsia were all elevated in comparison to women in the 40–49 age group [26]. These observations were confirmed by other studies [8, 9, 30]. The risk for any complication requiring hospitalization was substantially higher over 50 than under 50 years of age and reached 63%. These risks are higher in elderly women conceiving through oocyte donation than in the rare ones who conceive spontaneously. In a historical Israeli cohort including 405 singleton live births of women above 45, the risk of preeclampsia was compared between natural conceptions and assisted reproduction conceptions with donor eggs. The absolute risk of preeclampsia in oocyte donation recipients over 45 was 12.6% compared to 1.1% in natural conceptions. Contrary to natural conceptions, the preeclampsia risk in oocyte donation recipients over 45 was constant and was unaffected by parity or increasing age [31]. Age is also independently correlated with the occurrence of placental abruption and placenta previa, even when after correction for confounding factors such as smoking or hypertension [28]. After stratification, the prevalence of both these complications was found to be in direct correlation with maternal age, even within the oocyte donation recipient group [30]. Changes in the uterine microvasculature occurring with aging might be the explanation [32]. In the case of multi-fetal gestations, a common result of assisted reproduction, such hypertensive and placental complications are markedly increased [8]. Therefore women of advanced age are more likely to experience preterm labor or complications necessitating premature delivery [2, 8, 9, 26, 30, 33, 34]. As a consequence, the prevalence of preterm deliveries and low birth weight is significantly increased over 50 for multiple as well as for singleton gestations [8]. The occurrence of various patterns of labor and instrumental deliveries in older primiparas varies between reports even after correction for confounding factors [35, 36]. For obvious reasons, infertility and especially oocyte donation pregnancies are strongly associated with delivery by cesarean section [33], and cesarean section rate has been shown to rise in correlation with maternal age [37]. It is reasonably safe to state that currently most of primiparas over 50, who deliver in a setup of suitable updated obstetrical care, are delivered by cesarean section, even in the absence of complications or other obstetrical indications. The implications of this mode of delivery are mainly short termed, since these women are unlikely to have multiple repeated cesarean sections in most cases. Although cesarean section is associated with longer hospitalizations and increased febrile morbidity in comparison to vaginal deliveries, the utilization of intensive care unit hospitalizations and blood transfusion were not significantly increased in older patient group [30]. Since most of the partuitants of advanced age conceiving by assisted reproduction will not deliver again, more serious long-term sequelae such as scar placenta accreta or uterine rupture are unlikely. Although the maternal morbidity is definitely increased parallel to aging, the overall neonatal outcome does not appear to be affected by the maternal age if comparing the same gestational ages and birth weights [33, 36].

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Maternal Mortality The reported direct and indirect maternal mortality is in correlation with age. The primary reasons are mainly preexisting conditions exacerbated by pregnancy or the occurrence of pregnancy-associated complications such as preeclampsia, placental abruption, postpartum hemorrhage, and thromboembolic phenomena [38]. The almost universal performance of cesarean sections for delivering women of advanced age who conceived through oocyte donation does not appear to significantly contribute to the maternal mortality [2]. Sporadic maternal deaths following ovum donation in advanced age have been published [38], but it can be assumed that sub-reporting of such cases in the literature exists in order to avoid litigation. However, the prevalence of maternal mortality, even at advanced age, is extremely low in developed countries with an up-to-date prenatal care system, from which most of these women originate. Even if the relative risk of maternal mortality at advanced age is increased in comparison to the general population, the absolute mortality risk with proper pregestational screening and adequate antenatal care is still very low. In a well-screened population specifically determined to be healthy and free of chronic diseases, the maternal morbidity and mortality is considered low enough to permit the undertaking of oocyte donation and pregnancy despite the age factor [39]. When careful obstetrical management is provided, the maternal and neonatal outcomes are reasonably good. The basic strategy should involve caution and active expectation for complications. Pregnancies at advanced age should be regarded as ones with a high maternal and neonatal risk, even if the mother is found healthy at the pregestational screen and there is no apparent problem during pregnancy. In a case of meticulous preceding health screen and prenatal care, even if complications do occur, the outcome will be generally favorable. As applies for any age, only medical conditions which might be seriously exacerbated during the gestation and endanger the life of the mother or fetus should be contraindications to pregnancy. In addition to the routine tests performed in the general IVF patient population, most centers providing oocyte donation for women over 45 perform the following workup: a complete medical history and physical exam; a cardiac workup consisting an ECG; glucose tolerance testing; a complete hematological, hepatic, renal, and lipid profile; and imaging studies to rule out occult breast cancer. Nevertheless, pregnancy complications may arise despite a comprehensive negative pregestational evaluation; therefore it should be anticipated by the healthcare professional who provides the antenatal care.

The Neonate The prevalence of low birth weight and stillbirths is reported to be increased in neonates born to mothers of advanced age [2, 7, 26, 33, 40, 41], resulting from an increased risk of preterm labor, complications necessitating preterm delivery, and abnormal placental function. On the other hand, the rate of low Apgar scores,

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asphyxia, and metabolic complications in live births is not increased in women of advanced age [30, 33]. The prevalence of chromosomal and congenital anomalies is increased in children of older premenopausal women conceiving from self oocytes, but not when oocytes from young donors are used [7]. The long-term psychological and social impact of being the child of an elderly mother, father, or parents varies greatly between countries, populations, and societies.

Donor Egg-Based Fertility Treatments The availability of oocytes from young donors is the basis for postmenopausal pregnancies and deliveries. Spontaneous pregnancies after age 45 continuing to term are quite rare, and the number of women utilizing own embryos cryopreserved more than a decade before is also not significant. Donor oocytes are currently used in cases of advanced age, timely and premature ovarian insufficiency, and low ovarian response to exogenous gonadotropins and replace autologous oocytes in the cases of carriership of disorders inherited through the mitochondrial DNA or other maternally inherited conditions in which pregestational diagnosis is not feasible. The oocytes are obtained either from IVF patients donating surplus oocytes or from financially compensated volunteers—not very accurately termed as “donors.” If fresh embryo transfer is contemplated, then the uterus must be prepared and synchronized. Alternatively, oocytes or embryos can be cryopreserved and transferred to the recipient in an independent cycle [42]. In menopausal recipients, artificial endometrial preparation is required and achieved by administration of exogenous estrogens followed by addition of progesterone agents, in a manner similar to artificial cycle for frozen-thawed embryo transfer [5]. After pregnancy is confirmed, the endometrial support administration should be continued until placental steroid hormone production autonomy occurs. In the case that ovarian activity still exists, embryos can be transferred based on the endogenous ovarian steroid secretion without artificial endometrial preparation. The average reported global ongoing pregnancy and delivery rate is currently approximately 50% per transfer [43]. The increasing effectiveness of embryo and oocyte cryopreservation and its central role in current IVF practice has decreased patient willingness to donate oocytes, since most patients will prefer to cryopreserve any surplus of oocytes or embryos for their future use [44]. Therefore in most cases, oocytes for the recipients were aspirated from paid volunteers who are “compensated” for their “donation.” Candidates must be under 35, in good current and past health and free of diseases transmitted through body fluids. Similar to sperm donors, screening for common hereditary disorders, fragile X permutation, and structural chromosomal rearrangements are performed by most programs. The volunteers to donate oocytes undergo moderate ovarian stimulation and oocyte recruitment under ultrasonographic guidance. Excessive ovarian stimulation is hazardous to the donor and is also detrimental to the quality of the obtained oocytes. A treatment protocol using a GnRH antagonist for protection against spontaneous LH surge and a GnRH analog instead of hCG for triggering oocyte maturation [45] effectively protects the donor from the

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ovarian hyper-stimulation syndrome and its metabolic complications, without reducing the chance of implantation in the endometrium of the separately prepared recipient. In countries in which oocyte donation from compensated volunteers is performed, relevant legislation or directives exist, usually as a part of the human reproduction regulations. In most cases such regulation is set to protect the health, rights, and anonymity of the donor [46]. Presently the entire stimulation and pick-up procedure bear little risk for the donors allowing oocyte donation to peri- and postmenopausal women to be performed in many centers worldwide. Women of advanced age with low or absent ovarian reserve are the major patient group in need of oocyte donations, surpassing young low responders and genetic cases by far. However, on the recipients’ side, there is a paucity of regulations regarding the medical work-up required to test their suitability, despite the significant hazards that pregnancy might impose on them. A large share of oocyte donation treatments is performed across borders, with no actual supervision on age and health status of the recipients.

Advanced Paternal Age Definition and Incidence of APA There is no consensus on the definition of APA. Different studies in the subject have used different age cutoffs, a fact that complicates our ability to evaluate systematically the exact effect of paternal age of the reproductive outcome. Having said that, a frequently used criterion for APA is any man 40 years or older at the time of conception. Accordingly, in its “Statement on guidance for genetic counseling in advanced paternal age” in 2008, the American College of Medical Genetics (ACMG) has defined APA as 40 years of age or older at conception [47]. Unfortunately, other societies including the American College of Obstetrics and Gynecology (ACOG) have not formed guidelines on this issue. The American Society for Reproductive Medicine (ASRM) did refer to APA in its guidelines for sperm donation and determined that “The donor should be younger than 40 years of age so that potential hazards related to aging are diminished.” The trend of delayed childbearing, which is AMA maternal age, has not skipped men. This trend is a result of multiple factors including increased life expectancy, advanced age of marriage, significant change in role of women in society, and the accessibility of ART. In the United States, for example, the birth rate among men in the ages of 35–49 during the year of 2015 was 69.1 per 1000. These rates constitute a rise of more than 60% in comparison to the birth rate in 1980 which was reported to be 42.8 per thousand [48]. Interestingly, the World Health Organization report concerning aging and health issued in 2015 suggested that the trend of increased paternal age was not limited to the first world countries, but was seen even in the low- and middle-income countries.

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Fertility and Sperm Parameters at APA Fertility has been reported to decline with increasing paternal age. Ford et al. found that men above 40 years of age were 30% less likely to spontaneously conceive with their female partners during a 1-year period when compared to men below the age of 30 [49]. Similarly, Hassan et al. eliminated maternal age factor and reported that only 52.9% of couples consisted of men older than 45 years of age conceived spontaneously after a 6-month period, compared to 76.8% of couples where males were aged less than 25 [50]. Semen analysis is a fundamental step of the evaluation of the male fertility potential. The guidelines formed by the WHO in 2010 regarding the examination of human semen which refer to the fifth percentile as the lower limit of normal sperm parameters have become the consensus. Most studies, but not all, reported changes in semen parameters in aging men which starts around the age of 34 [51]. A significant decline in semen volume and in sperm forward motility was shown by many researches to parallel aging [16, 51–54]. The percentage of normal morphological spermatozoa was reported to decrease with age by Brahem et al. [16] and Kidd et al. [51], but not by Eskenazi et al. [52] or Hossain et al. [53]. In most studies a significant change in sperm concentration was not shown [51–53]. Kidd et al. suggested that the mechanism responsible for the decline in semen volume and for sperm morphology was the age-related seminal vesicle inadequacy and prostate atrophy [51]. Stone et al. studied the sperm samples and the spontaneous pregnancy rate of 5081 men aged between 16 and 72 years [55]. They found a correlation between the deterioration in sperm quality and quantity after the age of 34 and a decline in spontaneous birth rate, after controlling for the age of the female partner factor. Abortion rates in spontaneous pregnancies as well as in pregnancies achieved by intrauterine insemination of washed sperm have been reported to increase with advanced paternal age. Slama et al. studied the influence of paternal age on 5121 spontaneous pregnancies and found that the relative risk for a miscarriage in female partners of men older than 35 years was 1.26 in comparison to men younger than 35 years of age [55]. Similarly, Belloc et al. studied the effect of paternal age on miscarriage rate in 17,000 intrauterine insemination cycles and reported a high 32.4% abortion rate when the male partner was older than 35, while only 13.7% rate when less than 35 [56]. Sperm DNA fragmentation (SDF) is single- or double-strand breaks in the sperm’s DNA, which is caused mainly by oxidative stress. The latter occurs when there is an increased production of reactive oxygen species or reduced antioxidant reserves [57–60]. Abnormal protamination or abnormal protamines compaction can also contribute to sperm DNA fragmentation by the resulting detrimental presence of histones which is not converted to protamine [61, 62]. Many studies have reported an increment in sperm DNA damage with age. Moskovtsev et al. studied the DNA fragmentation index (DFI) of the sperm of different age groups. They found a consistent increase in DFI with age, with an average DFI levels for men younger than 30 years of age, 30–35, 35–40, and above 45 years of 15.2, 19.4, 20.1, 26.4, and 32% [17]. Singh et al. found similar results in their study [63]. Another study that

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included 215 couples in Denmark reported on a doubling of sperm DNA damage from the age of 25–55 years [64]. Finally, a comprehensive meta-analysis that consisted of 26 studies including 10,220 men showed similar consistent increase of DNA fragmentation rates with increased parental age. SDF was found to be an independent predictor of male fertility status [65, 66], as well as of the likelihood of achieving natural pregnancy [67]. Moreover, high SDF was associated with greater incidence of abortions of both natural pregnancies and ART-induced pregnancies [68–73] and with modest decrease in IVF pregnancy rates [73–75]. Due to that age-related incline in SDF and its negative impact on fertility, Humm et al. recommended to perform SDF tests to APA patients before beginning ART [76]. Increasing amount of retrospective data about the negative effect of APA on ART results is being accumulated. Frattarelli et al. [77] found that male age negatively impacts embryo development in donor oocyte ART cycles. Luna et al. [78] reported similar results, as well as reduction of fertilization, implantation, pregnancy, and life birth rates. Poorer pregnancy and life birth rates were reported by Ferreira et al. [79] and Klonoff-Cohen et al. [80] as well. Interestingly, Ferreira et al. [79] reported that the poorer outcome in ART for APA patients was limited to men with oligozoospermia, while those with normal sperm counts have not been negatively affected. Regarding the miscarriage rate in ART pregnancies among APA couples, there is a conflicting data. While de la Rochebrochard et al. [81] have shown increased miscarriage rates, others have not demonstrated such an incline in association with paternal age [80, 82, 83].

The Genetic Risks of Advanced Paternal Age  e Novo Autosomal Dominant Mutations D Unlike the female ovum, male spermatogonia divide during embryogenesis and resume mitotic divisions at puberty, which then continues throughout the male lifespan. At the age of 20, spermatogonial cells have already undergone approximately 150 mitoses, and by the age of 50, the number of cell divisions has surpassed 800 [19, 84, 85]. This ongoing process increases the probability of germline replication errors and therefore increases the risk for de novo point mutations [86]. The reduced activity of antioxidant enzymes in seminal plasma, and the lack of DNA repair mechanisms in male germ cells, contributes to the risk as well [48, 87, 88]. The phenomenon is intensified with age, due to age-related compromise in DNA replication and repair [19]. Kong et al. [86] estimated that the risk for paternal de novo mutation is increased by 4% per year. Unfortunately, due to selfish spermatogonial selection, the mutated cells are positively selected resulting in their clonal expansion [89]. Those de novo autosomal dominant mutations cause a small number of rare syndromes termed paternal age effect (PAE) disorders which characterized with a triad of features: paternal origin of mutations, strong paternal age effect, and high germline mutation rate [89]. Most of the PAE disorders are caused by mutations in the fibroblastic growth factor receptor (FGFR) and in the RET gene [90].

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Mutations in the FGFR2 gene are associated with the Apert, Crouzon, and Pfeiffer syndromes, while mutations in the FGFR3 gene are strongly associated with achondroplasia [89, 90]. Due to this PAE, the risk for an offspring of a 50-year-old father to be born with achondroplasia is as high as 1:1250, compared to the general population risk of 1:15,000 [47]. Mutation in the RET gene increases the risk for multiple endocrine neoplasia [89]. The overall incidence of the PAE syndromes is low and is estimated as less than 0.5% above paternal age of 40 years [91].

Chromosomal Abnormalities The maternal age-related increase in chromosomal aneuploidies due to non-equal division of the chromosomes in daughter cells during miosis as a result of nondisjunction is a well-known phenomenon. However, most studies suggest that the risk for aneuploidies does not increase with paternal age [92–94]. Having said that, approximately 10% of the cases of trisomy 21—Down syndrome—originate from the fathers, and several studies did show an association between paternal age and the risk for Down syndrome in the offsprings [95–97]. The strong confounder of maternal age in studies of aneuploidies obligates us to take those results with a grain of salt. In conclusion, the evidence for any paternal age relation to aneuploidies is weak and should be further studied while better dealing with the maternal age confounder.  ther Genetic Risks O Structural chromosomal abnormalities such as deletions, duplications, and translocations are more likely to be of paternal origin. Thomas et al. showed that balanced translocations are predominantly paternal in origin and associated with significant increase with paternal age [98]. The odds was observed to double every 10 years after age 25. There is also some evidence that advanced paternal age increases the risk for specific x-linked disorders in such as hemophilia, Duchenne muscular dystrophy, Hunter syndrome, and Lesch-Nyhan disease [99, 100]. Congenital Anomalies Advanced paternal age appears to constitute a small risk for several birth defects. Many inconsistent reports regarding this subject have been published over the years, which raises the suspicion of positive finding publication bias among others. Polednak et al. [101] reported back in 1976 a higher incidence of syndactyly, club foot, and oral cleft. Lian et al. [102] showed higher risk for situs inversus and chondrodystrophy with APA, and McIntosh et al. [96] showed higher incidence of neural tube defects, cataract, and upper limb reduction defects, in addition to the increased risk for Down syndrome reported earlier. Bille et al. [103] used the comprehensive Danish Facial Cleft Register and reported an increased risk for cleft palate, which has no maternal age influence. This data was reinforced by a later meta-analysis published by Herkrath [104]. However, Su et al. [105] used the registry of all birth in Denmark and failed to show a relation between APA and elevated risk for overall heart defects. They did find increased risk of patent ductus arteriosus in that population. Interestingly, Archer et al. [106] did not find any correlation between the risk

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of congenital anomalies and APA using the Texan Birth Defect Registry 1996–2002. In contrast, the well-designed US national birth defect prevention study, which was adjusted for multiple confounders and included all birth defects categories with more than 100 cases for each category, revealed that APA was associated with statistically significant increased risk of cleft lip (OR 1.02), diaphragmatic hernia (OR 1.04), right ventricular outflow tract obstruction (OR 1.03), and pulmonary stenosis (OR 1.02) with each year of paternal age.

Childhood Cancers While several types of childhood cancers such as leukemia [107, 108], non-­ Hodgkin’s lymphoma, central nervous system tumors, and breast cancer [109] have been suspected to be associated with APA, the only type that has been consistently shown to be associated with APA was ALL (acute lymphoblastic leukemia). A weaker correlation has been shown between childhood retinoblastoma and advanced paternal age as well [110–112]. Larfors et al. [113] used the Swedish population-­ based register data in order to study the association between different types of leukemias and APA and found a higher risk of childhood ALL and AML (acute myeloblastic leukemia). However, a recent meta-analysis which investigated this subject found only correlation between APA and ALL, but not AML. The reported risk for ALL was increased by 4% every 5 years of paternal age [114]. An English case-control study also showed increased risk of ALL with advanced paternal age [110], and a Danish analysis consisted of nearly two million children born between 1978 and 2010 reported an increase in hazard ratio of 1.13 for every 5  years of paternal age, without an increase in any other childhood cancer [115]. There are several hypothesized mechanisms which can contribute to this increased risk in childhood cancers. First, unlike other cells wherein the telomere becomes shorter with age, the sperm’s telomere tends to increase in length with age [116], and offspring of older fathers has longer leukocyte telomere length [116, 117]. Interestingly, telomere length has been linked to longevity and decreased risk for atherosclerosis, but on the other hand to increased risk of malignancy [117]. Second plausible mechanism to be responsible for the higher risk for cancer connected to APA is the tendency of those fathers to have fewer children [108]. Consequently, those children are less exposed to childhood infections and may develop a weaker immune system, which makes them prone to immune-related diseases and cancers [118, 119].

Neurodevelopmental Outcomes Schizophrenia The vast majority of studies support that the risk of schizophrenia is increased with advanced paternal age [120–122]. Schizophrenia has a strong genetic influence, and a quarter of all cases was estimated to be associated with APA [123, 124]. A Swedish population-based cohort study [122] showed a 1.47 increase in the risk of an offspring to have schizophrenia for every 10-year increase in paternal age. Similar

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results were reported in the Japanese population [125]. Wohl et al. [126] showed that the risk of schizophrenia increased exponentially with parental age, from odds ratio of 1.16  in the age group of 25–34  years to 5.92 above 55  years of age. Interestingly, Lee et al. [127] described paternal age-related schizophrenia as a variant of schizophrenia, which is characterized by verbal and performance intelligence. There is no consensus regarding the mechanism behind that age-related increase. One proposed mechanism is the accumulation of de novo mutations in sperm [122, 124]. Another possible mechanism is that the age-related dysregulation of epigenetics occurring in the sperm is responsible for the increased risk of schizophrenia [128].

 utism Spectrum Disorders (ASDs) A Among the neurodevelopmental disorders, ASDs probably have the strongest correlation with APA [120, 129–132]. Buizer-Voskamp et al. [120] found that paternal age above 45 constituted a 3.3 times higher risk of offspring with ASD than paternal age younger than 20. Similarly, Sandin et al. [132] united data from the registry of Denmark, Sweden, Norway, Australia, and Israel and reported increased risk of ASD in children of older fathers. Hultman et  al. [130] controlled in their meta-­ analysis for confounders such as maternal age and found that fathers older than 50 years of age had twice the chance to conceive offspring with ASD than fathers younger than 29  years. Interestingly, Frans et  al. [133] showed association even between advanced grandfather’s age (either maternal or paternal) at the time the parent was born and the risk of ASD in his grandchild. This paternal age-related increased risk of ASD is consistent with the findings showing that de novo mutations, including copy number variants, contribute to the development of ASD [134, 135].  ther Psychiatric Disorders and Neurodevelopmental Outcomes O The association between other psychiatric disorders such as bipolar, eating, and attention-deficit disorders and APA is less robust although some evidence does exist. While McGrath et al. [136] showed increased risk of all groups of psychiatric disorders except eating disorders beyond paternal age of 45, by studying the total Danish population born between 1955 and 2006, Mikkelsen et  al. reported no increased risk of attention-deficit disorders although using the same database. Frans et  al. [133] investigated the Swedish registry and found that offspring of fathers older than 55 years of age had 1.37 times higher risk of developing bipolar disorders, compared to fathers aged 20–24 after controlling for maternal age and family history. An association between paternal age and poor neurocognitive outcomes was reported as well. Malaspina et al. [124] investigated data derived from exams performed by 44,175 soldiers in the Israeli army and reported a U-shaped relationship between intelligence quotient and paternal age. Similarly, Saha et al. [137] analyzed data from multiple neurocognitive assessments performed by more than 30,000 children and found that APA was significantly associated with poorer performance.

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 bstetrical Complications Associated with APA O The risk of spontaneous abortions was reported to be associated with APA in several studies [78, 81, 138], but not in all [78, 139]. Rochebrochard et al. [81] performed a multicenter European study and compared the risk for abortion in different paternal age groups [20–64] while all the mothers were between 20 and 29 years of age. The calculated odds ratios for the different paternal groups were 1, 1.06, 1.31, and 1.8, respectively. Belloc et al. [56] reported an increase in risk for miscarriage as well. They compared couples where fathers were above 45 years of age to those younger than 30 years and found a significant increase in the older group (32.4% vs 13.7%). The data regarding the risk for preterm birth is inconsistent as well. Zhu et al. [140] compared the risk of preterm birth in paternal age groups 25–29, 30–34, 35–39, 40–44, and 45–49 to the risk in the reference age group of 20–24 and reported increased odds ratio of 1.3, 1.4, 1.7, 1.6, and 2.1, respectively. Similarly, in a population-based retrospective cohort study between 1989 and 2005, advanced paternal age (older than 45 years) was associated with a 13% increased risk of preterm birth (OR 1.13; 95% CI, 1.05–1.22) [141]. Having said that, other studies did not find any significant association between the two [142–144]. It seems that APA is linked to increased risk or low birth weight. Alio et al. [141] reported in their previously mentioned cohort study, a 19% increased risk of conceiving low birth weight offspring in fathers older than 45 years (OR 1.19; 95% CI, 1.09–1.29). Similarly, Reichman et al. [145] found a 1.9 times increased risk of low birth weight when father’s age was above 35 years, compared to 20–34-year group. APA constitutes a risk factor to stillbirth as well. Alio et al. [141] reported an increased risk of 48% (OR 1.48; 95% CI, 1.04–2.10) for fathers older than 45 years, and Nybo et al. [146] found an increase of 40% risk in the 45–49-year age group, after adjustment for maternal age. APA was linked not only to increased risk for the fetus, but for the mother as well by increasing the risk for preeclampsia during the pregnancy [141, 147]. Harlap et al. reported an increase in the odds ratio in 35–39-year, 45–49-year, and 50–54-­ year paternal age groups by 1.3, 1.89, and 1.54, respectively, compared to the 25–29-year group and after adjustment for maternal age [147].

Ethical Considerations Egg donation neutralizes the age element in fertility treatments, and its success rate at advanced ages is high [43]. Any reasonably healthy woman with a uterus is a potential treatment candidate. The results of the pregnancy, on the other hand, might not be as favorable. The first issue is that of putting an age limitation to this reproductive technology that will protect the mother and her child. When taking into account the welfare of the patient, offspring, family, and society, limitations of the reproductive choice should be seriously considered against the right to personal freedom in making reproductive choices. The attitude toward enabling elderly women to conceive using advanced technology and donor oocytes should be decided by each society individually. In addition, in places where the financial and egg

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donation resources are limited, a priority system for their allocation should also be established, reflecting the local values and laws.

 he Issue of Choice T The will to reproduce has been evolutionarily strong in most human societies that ever existed. From the mid-twentieth century, modern liberal societies cherish and value individual rights for autonomous choices, especially in the issue of reproduction. Assisted reproduction has opened new options and choices that did not exist before [5]. Oocyte donation from a young woman to an older woman is such a reproductive choice [148]. In an era of prolonged life expectancy and improving quality of life, a woman may not feel unsuitable to deliver and raise a child even if she is menopausal, at an age considered adequate for grandparenthood a few decades earlier. In developed countries with a good health system, a woman who has reached 50  in good health has an average additional life expectancy of at least another 25 years [149], long enough to raise a child to adulthood. Many women decide to postpone childbearing to the age after the achievement of career and financial goals, so the age that a woman (or couple) decides to conceive might easily be quite advanced. Childbearing in younger women with significant medical conditions or with inherited disorders is not prohibited in open societies and even encouraged and supported by the public health system. Only in the most extreme conditions, in which the risk of maternal mortality is high, a young woman would be prohibited from conceiving and fertility treatments (if needed) denied. Refusing the presently feasible option of oocyte donation to women of advanced age, for this reason solely without any other actual risk, is a denial of a basic right for reproductive choice and even gender-based discrimination [150]. When it comes to age and reproduction, men and women are not equally viewed; while older women are considered unable to conceive naturally, older men are considered suitable for parenting with younger female partners. In the natural fecundity setup, this cannot be regarded as discriminatory, since women bear children, deliver them, nurse them, and bear the medical risks of pregnancy. But when the relatively safe age of biological motherhood is extended, it is only fair to modestly correct this long-standing gender-based difference in parenting opportunities [150]. It can be argued against this that enabling pregnancy at an age in which it is naturally impossible might be a hazardous action against our biological limitations [148]. Adversely, this argument can be extended to other “unnatural” situations resolved by assisted reproduction such as severe male factor and mechanical infertility. Currently, it seems totally unacceptable to deny childbearing from young couples with severe oligoteratoasthenospermia, blocked fallopian tubes, premature ovarian failure, or anovulation on the grounds that assisted reproduction is “unnatural.” It is obvious to all that assisted reproductive technologies should be made available exactly in these very cases. Then why should women be denied conception on the grounds of age alone? The current trends in liberal societies emphasize women’s right for career achievements, equal contribution, and opportunities. However other circumstances can be the reason for a woman or couple to seek oocyte donation [151]. Some

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women find themselves in fifth and sixth decades in a new relationship and desire a child with their new partner. Others tragically lose their grown-up children and would like to deliver again as rehabilitation from their tragedy. In other situations, repeated artificial reproduction attempts fail and time passes [151]. Denying access to oocyte donation in such circumstances, based solely on age, is obviously unfair and even cruel.

The Welfare of the Child Opponents to pregnancies in woman/parents of advanced age reason their position based on the interest and welfare of the future offspring, thus implying that older individuals are less capable of coping with parenthood, physically and mentally [152]. The main argument is that having parents of advanced age may cause children to endure a greater generation gap, grow up without grandparents, cope with parental geriatric diseases prematurely, and finally become orphans at a young age [148]. On the other hand, older parents, singles or couples, in general are more mature and experienced than younger ones. In average they have more free time and emotional and material resources to nurture children and cope with their raising. The long-lasting desire for a child in cases of prolonged infertility might be of more benefit than harm to the child [151]. Taking all these into consideration, it is reasonable to assume that the welfare of children born to mothers or parents of more advanced age is not negatively affected by this factor. Nevertheless, creating a supporting family and social backup system in case of parental impairment or death is a wise step in the case of parents of advanced age.  he Law and Religion T Oocyte donation is not entirely arranged by law in many countries. Legislation is important to guarantee the rights of the donors, regulate the relationship between the oocyte donor and the recipient, and guard the rights of the offspring and the method of performance and documentation. Gamete donation, sperm and oocytes, is prohibited because of religious reasons in several Catholic European countries, in South America, and in Sunni Muslim countries. According to the Judaism, oocyte donation is allowed only from unmarried donors. In Christianity, the Roman Catholic, Eastern Orthodox, and the Protestant faiths prohibit oocyte donation. In Sunni Islam oocyte donation parallels adultery and is therefore impermissible. Hinduism and Buddhism do not address the issue of oocyte donation [153]. Oocytes for donation are available in limited numbers, and the issue of their allocation to the potential recipients is an ethical dilemma. Unfortunately, allocation is most often based on the free market (i.e., financial resources) rather than the recipients’ urgency and strength of need. Though often theoretical, an ideal society should aspire that women and couples that have been deprived of children as a result of a biomedical condition are given priority before those who purposely delayed childbearing. It might seem ethically acceptable to prioritize young women with premature ovarian insufficiency or ovarian dysgenesis, rather than to menopausal woman with or without children. Ideally oocyte donation should be voluntary and

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altruistic, but this is a naive expectation in our time. Currently donor payments are defined as financial “compensation” for time and effort. However other forms of donor compensation, like free oocyte vitrification for the donors’ elective fertility preservation, should be considered and encouraged instead of direct payment. Any restrictions on providing fertility treatments to women of advanced age must be based on medical evidence, or psychological/social grounds, but not on fears, personal beliefs, or prejudice. Reproduction is a basic right in our society, and withholding donor oocytes or other treatments from those who need them essentially negates this right. However life expectancy and quality is not unlimited, and an age limit to assisted reproduction should exist and be determined by society and the medical community in an open lucid discussion. Some suggest that this limit should be set to 60, based on the average life expectation, minus 20 years—the approximate age of adulthood. In the popular media, those who provide oocyte donation to this age group are depicted as greedy, ruthless, acting irresponsibly, or playing God. On the other hand, the opponents are portrayed as paternalistic and deniers of human rights. The public opinion in most developed countries where late motherhood using donor oocytes is feasible is often split. The questions of where and how to draw the line and who should decide are the issue of ongoing public debate. Our opinion is that when setting an age limit to recipients of oocyte donation, medical considerations must be taken into account, together with social and ethical positions. Therefore any age limit to maternal age in assisted reproduction should be set in accordance to local medical circumstances and social values, by doctors, experts in bioethics, sociologists, prominent public figures, and politicians. Local life expectancy; public health conditions; the quality, level, and accessibility of medical services; the availability of adequate fertility treatments; and the value of childbearing in the local culture should all be considered when an age limit is set. In Israel a law arranging all aspects of oocyte donation treatments was passed in 2010. The maximal age of the recipients of embryos originating from oocytes donated by Israeli donors was set to 54 years. This limit was established after years of public debates on oocyte donation from designated paid volunteers. This recipients’ age limit set up by this law reflects the life expectancy in Israel, the high level and good availability of the public health system, and the significance of biological parenthood in the Israeli society. Although this age limit was narrowly set to be the upper limit for Israeli recipients of eggs donated by Israeli women, it was quickly adopted by the fertility care providers in Israel as an upper age limit to other types of assisted reproduction (transfers of self frozen-thawed embryos and off-shore oocyte donations). This decision-making process can result in a different age limit elsewhere or in another time.

Summary and Conclusions Presently it is more common and socially acceptable for peri- and postmenopausal women to conceive and deliver children. Advances in assisted reproduction and the availability of donor oocytes have made pregnancies in women beyond the

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biological barrier of the human ovary possible. The impact of advanced age on the maternal well-being and the outcome of the pregnancy remains controversial. Apparently pregnancy at advanced age might be hazardous to the mother and fetus, but with appropriate preconception screening and meticulous prenatal and intrapartum care, a reasonably successful pregnancy outcome can be achieved in most cases. Patients should undergo a meticulous medical and psychological preparation before contemplating pregnancy at an advanced age in order to exclude those who will not be able to cope physically or psychologically. This concern raises a debate whether or not to limit the age of the recipients and on what ground. Categorically denying older women a biologically available option to reproduce contradicts their rights and personal freedom. On the other hand, it is our duty to protect these women from harm via sound and transparent guidelines which will assure that pregnancy will be achieved safely and result in a good outcome. Even when all screening tests turn out to be negative, we believe that an absolute line should eventually be drawn. The precise position of this line should be in accordance to specific social and medical factors such as culture, religion, life expectancy, quality of medical care, and antenatal management. Such a limitation should be periodically revised and updated in accordance to life expectancy, the level of the available medical support, and social trends. Advanced paternal age is a risk factor of varying degrees for several neonatal adverse outcomes including de novo autosomal dominant mutations, specific types of congenital anomalies, childhood cancers, neurodevelopmental disorders, and obstetrical complications. Unfortunately, the molecular mechanisms responsible for these findings are still poorly understood. It is important to note that the reported correlation between APA and reproductive risks does not necessarily prove causality. Age is a biological process by all means but is a social construct as well. Possibly, men with health or mental disabilities become fathers later in life. Similarly, men with subfertility tend to succeed in conceiving at older age, and subfertility is a known risk factor for poorer reproductive outcomes. Nevertheless, regardless of causality, the current data supports the adequate counselling for couples with APA regarding the reproductive risks associated with paternal aging. The current body of evidence on the adverse outcomes associated with APA certainly does not justify any solid recommendation against parenthood at an advanced paternal age.

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28. Naeye RL.  Maternal age, obstetric complications, and the outcome of pregnancy. Obstet Gynecol. 1983;61(2):210–6. 29. Tuck SM, Yudkin PL, Turnbull AC. Pregnancy outcome in elderly primigravidae with and without a history of infertility. Br J Obstet Gynaecol. 1988;95(3):230–7. 30. Yogev Y, Melamed N, Bardin R, Tenenbaum-Gavish K, Ben-Shitrit G, Ben-Haroush A.  Pregnancy outcome at extremely advanced maternal age. Am J Obstet Gynecol. 2010;203(6):558.e1–7. 31. Dior UP, Laufer N, Chill HH, Granovsky-Grisaru S, Yagel S, Yaffe H, et al. Increased incidence of preeclampsia in mothers of advanced age conceiving by oocyte donation. Arch Gynecol Obstet. 2018;297(5):1293–9. 32. Zalud I, Shaha S. Three-dimensional sonography of the placental and uterine spiral vasculature: influence of maternal age and parity. J Clin Ultrasound. 2008;36(7):391–6. 33. Dulitzki M, Soriano D, Schiff E, Chetrit A, Mashiach S, Seidman DS.  Effect of very advanced maternal age on pregnancy outcome and rate of cesarean delivery. Obstet Gynecol. 1998;92(6):935–9. 34. Sheffer-Mimouni G, Mashiach S, Dor J, Levran D, Seidman DS.  Factors influencing the obstetric and perinatal outcome after oocyte donation. Hum Reprod. 2002;17(10):2636–40. 35. Kirz DS, Dorchester W, Freeman RK.  Advanced maternal age: the mature gravida. Am J Obstet Gynecol. 1985;152(1):7–12. 36. Ziadeh S, Yahaya A.  Pregnancy outcome at age 40 and older. Arch Gynecol Obstet. 2001;265(1):30–3. 37. Smith GC, Cordeaux Y, White IR, Pasupathy D, Missfelder-Lobos H, Pell JP, et al. The effect of delaying childbirth on primary cesarean section rates. PLoS Med. 2008;5(7):e144. 38. Bewley S, Wright JT. Maternal death associated with ovum donation twin pregnancy. Hum Reprod. 1991;6(6):898–9. 39. Edwards RG.  Pregnancies are acceptable in post-menopausal women. Hum Reprod. 1993;8(10):1542–4. 40. Schenker JG.  The therapeutic approach to infertility in cases of ovarian failure. Ann N Y Acad Sci. 1991;626:414–30. 41. Kenny LC, Lavender T, McNamee R, O’Neill SM, Mills T, Khashan AS. Advanced maternal age and adverse pregnancy outcome: evidence from a large contemporary cohort. PLoS One. 2013;8(2):e56583. 42. Cobo A, Bellver J, Domingo J, Perez S, Crespo J, Pellicer A, et al. New options in assisted reproduction technology: the Cryotop method of oocyte vitrification. Reprod Biomed Online. 2008;17(1):68–72. 43. Noyes N, Hampton BS, Berkeley A, Licciardi F, Grifo J, Krey L. Factors useful in predicting the success of oocyte donation: a 3-year retrospective analysis. Fertil Steril. 2001;76(1):92–7. 44. Shufaro Y, Schenker JG. Cryopreservation of human genetic material. Ann N Y Acad Sci. 2010;1205:220–4. 45. Humaidan P, Kol S, Papanikolaou E. GnRH agonist for triggering of final oocyte maturation: time for a change of practice? Hum Reprod Update. 2011;17(4):510–24. 46. Schenker JG.  Sperm, oocyte, and pre-embryo donation. J Assist Reprod Genet. 1995;12(8):499–508. 47. Toriello HV, Meck JM, Professional P, Guidelines C.  Statement on guidance for genetic counseling in advanced paternal age. Genet Med. 2008;10(6):457–60. 48. Martin RH. Genetics of human sperm. J Assist Reprod Genet. 1998;15(5):240–5. 49. Ford WC, North K, Taylor H, Farrow A, Hull MG, Golding J. Increasing paternal age is associated with delayed conception in a large population of fertile couples: evidence for declining fecundity in older men. The ALSPAC Study Team (Avon Longitudinal Study of Pregnancy and Childhood). Hum Reprod. 2000;15(8):1703–8. 50. Hassan MA, Killick SR. Effect of male age on fertility: evidence for the decline in male fertility with increasing age. Fertil Steril. 2003;79(Suppl 3):1520–7. 51. Kidd SA, Eskenazi B, Wyrobek AJ.  Effects of male age on semen quality and fertility: a review of the literature. Fertil Steril. 2001;75(2):237–48.

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52. Eskenazi B, Wyrobek AJ, Sloter E, Kidd SA, Moore L, Young S, et al. The association of age and semen quality in healthy men. Hum Reprod. 2003;18(2):447–54. 53. Hossain MM, Fatima P, Rahman D, Hossain HB. Semen parameters at different age groups of male partners of infertile couples. Mymensingh Med J. 2012;21(2):306–15. 54. Mukhopadhyay D, Varghese AC, Pal M, Banerjee SK, Bhattacharyya AK, Sharma RK, et al. Semen quality and age-specific changes: a study between two decades on 3,729 male partners of couples with normal sperm count and attending an andrology laboratory for infertility-­ related problems in an Indian city. Fertil Steril. 2010;93(7):2247–54. 55. Slama R, Bouyer J, Windham G, Fenster L, Werwatz A, Swan SH. Influence of paternal age on the risk of spontaneous abortion. Am J Epidemiol. 2005;161(9):816–23. 56. Belloc S, Cohen-Bacrie P, Benkhalifa M, Cohen-Bacrie M, De Mouzon J, Hazout A, et al. Effect of maternal and paternal age on pregnancy and miscarriage rates after intrauterine insemination. Reprod Biomed Online. 2008;17(3):392–7. 57. Agarwal A, Said TM.  Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update. 2003;9(4):331–45. 58. Alshahrani S, Agarwal A, Assidi M, Abuzenadah AM, Durairajanayagam D, Ayaz A, et al. Infertile men older than 40 years are at higher risk of sperm DNA damage. Reprod Biol Endocrinol. 2014;12:103. 59. Moustafa MH, Sharma RK, Thornton J, Mascha E, Abdel-Hafez MA, Thomas AJ Jr, et al. Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility. Hum Reprod. 2004;19(1):129–38. 60. Sharma RK, Said T, Agarwal A. Sperm DNA damage and its clinical relevance in assessing reproductive outcome. Asian J Androl. 2004;6(2):139–48. 61. Erenpreiss J, Spano M, Erenpreisa J, Bungum M, Giwercman A. Sperm chromatin structure and male fertility: biological and clinical aspects. Asian J Androl. 2006;8(1):11–29. 62. Zhang X, San Gabriel M, Zini A.  Sperm nuclear histone to protamine ratio in fertile and infertile men: evidence of heterogeneous subpopulations of spermatozoa in the ejaculate. J Androl. 2006;27(3):414–20. 63. Singh NP, Muller CH, Berger RE. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertil Steril. 2003;80(6):1420–30. 64. Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril. 2000;73(1):43–50. 65. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, et al. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod. 2007;22(1):174–9. 66. Oleszczuk K, Augustinsson L, Bayat N, Giwercman A, Bungum M.  Prevalence of high DNA fragmentation index in male partners of unexplained infertile couples. Andrology. 2013;1(3):357–60. 67. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod. 1999;14(4):1039–49. 68. Absalan F, Ghannadi A, Kazerooni M, Parifar R, Jamalzadeh F, Amiri S.  Value of sperm chromatin dispersion test in couples with unexplained recurrent abortion. J Assist Reprod Genet. 2012;29(1):11–4. 69. Ford HB, Schust DJ. Recurrent pregnancy loss: etiology, diagnosis, and therapy. Rev Obstet Gynecol. 2009;2(2):76–83. 70. Khadem N, Poorhoseyni A, Jalali M, Akbary A, Heydari ST. Sperm DNA fragmentation in couples with unexplained recurrent spontaneous abortions. Andrologia. 2014;46(2):126–30. 71. Simon L, Brunborg G, Stevenson M, Lutton D, McManus J, Lewis SE. Clinical significance of sperm DNA damage in assisted reproduction outcome. Hum Reprod. 2010;25(7):1594–608. 72. Zini A. Are sperm chromatin and DNA defects relevant in the clinic? Syst Biol Reprod Med. 2011;57(1–2):78–85.

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96. McIntosh GC, Olshan AF, Baird PA. Paternal age and the risk of birth defects in offspring. Epidemiology. 1995;6(3):282–8. 97. Zhu JL, Madsen KM, Vestergaard M, Olesen AV, Basso O, Olsen J. Paternal age and congenital malformations. Hum Reprod. 2005;20(11):3173–7. 98. Thomas NS, Durkie M, Van Zyl B, Sanford R, Potts G, Youings S, et al. Parental and chromosomal origin of unbalanced de novo structural chromosome abnormalities in man. Hum Genet. 2006;119(4):444–50. 99. Francke U, Felsenstein J, Gartler SM, Migeon BR, Dancis J, Seegmiller JE, et al. The occurrence of new mutants in the X-linked recessive Lesch-Nyhan disease. Am J Hum Genet. 1976;28(2):123–37. 100. Sommer SS, Scaringe WA, Hill KA. Human germline mutation in the factor IX gene. Mutat Res. 2001;487(1–2):1–17. 101. Polednak AP. Paternal age in relation to selected birth defects. Hum Biol. 1976;48(4):727–39. 102. Lian ZH, Zack MM, Erickson JD. Paternal age and the occurrence of birth defects. Am J Hum Genet. 1986;39(5):648–60. 103. Bille C, Skytthe A, Vach W, Knudsen LB, Andersen AM, Murray JC, et al. Parent’s age and the risk of oral clefts. Epidemiology. 2005;16(3):311–6. 104. Herkrath AP, Herkrath FJ, Rebelo MA, Vettore MV. Parental age as a risk factor for non-­ syndromic oral clefts: a meta-analysis. J Dent. 2012;40(1):3–14. 105. Su XJ, Yuan W, Huang GY, Olsen J, Li J. Paternal age and offspring congenital heart defects: a national cohort study. PLoS One. 2015;10(3):e0121030. 106. Archer NP, Langlois PH, Suarez L, Brender J, Shanmugam R.  Association of paternal age with prevalence of selected birth defects. Birth Defects Res A Clin Mol Teratol. 2007;79(1):27–34. 107. Murray L, McCarron P, Bailie K, Middleton R, Davey Smith G, Dempsey S, et al. Association of early life factors and acute lymphoblastic leukaemia in childhood: historical cohort study. Br J Cancer. 2002;86(3):356–61. 108. Teras LR, Gaudet MM, Blase JL, Gapstur SM. Parental age at birth and risk of hematological malignancies in older adults. Am J Epidemiol. 2015;182(1):41–8. 109. Yip BH, Pawitan Y, Czene K. Parental age and risk of childhood cancers: a population-based cohort study from Sweden. Int J Epidemiol. 2006;35(6):1495–503. 110. Dockerty JD, Draper G, Vincent T, Rowan SD, Bunch KJ.  Case-control study of parental age, parity and socioeconomic level in relation to childhood cancers. Int J Epidemiol. 2001;30(6):1428–37. 111. Heck JE, Lombardi CA, Meyers TJ, Cockburn M, Wilhelm M, Ritz B. Perinatal characteristics and retinoblastoma. Cancer Causes Control. 2012;23(9):1567–75. 112. Moll AC, Imhof SM, Kuik DJ, Bouter LM, Den Otter W, Bezemer PD, et al. High parental age is associated with sporadic hereditary retinoblastoma: the Dutch retinoblastoma register 1862-1994. Hum Genet. 1996;98(1):109–12. 113. Larfors G, Hallbook H, Simonsson B. Parental age, family size, and offspring’s risk of childhood and adult acute leukemia. Cancer Epidemiol Biomark Prev. 2012;21(7):1185–90. 114. Sergentanis TN, Thomopoulos TP, Gialamas SP, Karalexi MA, Biniaris-Georgallis SI, Kontogeorgi E, et al. Risk for childhood leukemia associated with maternal and paternal age. Eur J Epidemiol. 2015;30(12):1229–61. 115. Nybo Andersen AM, Urhoj SK. Is advanced paternal age a health risk for the offspring? Fertil Steril. 2017;107(2):312–8. 116. Kimura M, Cherkas LF, Kato BS, Demissie S, Hjelmborg JB, Brimacombe M, et  al. Offspring’s leukocyte telomere length, paternal age, and telomere elongation in sperm. PLoS Genet. 2008;4(2):e37. 117. Aviv A, Susser E. Leukocyte telomere length and the father’s age enigma: implications for population health and for life course. Int J Epidemiol. 2013;42(2):457–62. 118. Printz C.  Father’s age at birth of child may increase child’s blood cancer risk. Cancer. 2015;121(17):2863.

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Cross-Border Reproductive Care: Current State of the Art Tomer Singer, Liron Bar-El, Laurence B. McCullough, and Frank A. Chervenak

Introduction It is estimated that worldwide more than 70 million people of childbearing age are infertile and that around 40.5 million (56%) of them seek infertility treatments [1]. Changing laws in some countries allow groups previously denied access to assisted reproductive technology (ART), such as single women and men, same-sex couples, and older women, in their fifth and sixth decades of life, the same right to access ART as heterosexual, married, or young couples. These, along the internet, that allowed for better communication platforms and many other factors, result in an increase in the use of ART in general and the rise in cross-border reproductive care in particular. Historically, local regulations regarding termination of pregnancy (TOP) were the main cause for reproductive migration. While the legalization of abortion in many countries has resulted in a steady decline of TOP-motivated travels, a new migration trend, related to varied accessibility to ART, has increased. The legal restrictions vary immensely from country to country, often reflecting different cultural and religious values. Thus, in some countries, certain technologies or treatments may be restricted for specific categories of intended parents (i.e., single individuals or same-sex couples) or entirely forbidden to all by law (i.e., social sex selection, surrogacy). Many obstacles arise even in countries with access to treatments: the technology might not be sufficiently advanced (i.e., lack of preimplantation genetic diagnosis); the privacy protections might be inadequate; the T. Singer (*) Department of Obstetrics and Gynecology, Zucker School of Medicine of Hofstra/Northwell, Lenox Hill Hospital, New York, NY, USA Shady Grove Fertility, New York, NY, USA L. Bar-El · L. B. McCullough · F. A. Chervenak Department of Obstetrics and Gynecology, Zucker School of Medicine of Hofstra/Northwell, Lenox Hill Hospital, New York, NY, USA © International Academy of Human Reproduction 2021 J. G. Schenker et al. (eds.), Clinical Management of Infertility, Reproductive Medicine for Clinicians 2, https://doi.org/10.1007/978-3-030-71838-1_4

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waiting lists may be too long in one’s home country (i.e., oocyte donation); and the out-of-pocket costs may be too high to utilize. Inevitably, the crossing of national borders to have children has gained momentum and became a rapidly growing phenomenon, as part of the wider trend of “medical tourism” [2]. Many terms had been used to describe this phenomenon. The terms “procreative tourism,” “reproductive tourism,” and “fertility tourism” were suggested and used in early publications [3–8] as a variant of the wider trend of “medical tourism.” However, the word “tourism” implies choice, pleasure, and leisure, a representation that might not be appropriate to describe the physical and emotional challenges of fertility treatments [9, 10]. The term “reproductive exile” was suggested in some publications, in order to emphasize the fact that the patients are compelled into leaving their home countries on a journey abroad in order to access the treatments they need [9, 11]. The use of a negative term in this context drew many objections and was largely abandoned as it invokes the idea of law evasion. Other terms were suggested as “reproductive travel” or “ART travel” that suggest a more neutral attitude, though they have not achieved wide acceptance thus far. Although the term “reproductive tourism” has already been accepted by the media and the public opinion, a terminology change was recently applied, and the most current, widely used term is “cross-border reproductive care” (CBRC). The term CBRC has been used by several committees, including the European Society of Human Reproduction and Embryology (ESHRE) [10] and the American Society for Reproductive Medicine (ASRM) [12] with the acknowledgment that for some, “care” may not necessarily be an appropriate term. The purpose of this chapter is to describe major aspects of cross-border reproductive care from a global perspective.

Global Perspective on Cross-Border Reproductive Care It is a major challenge to accurately study the flow of patients seeking reproductive care overseas. Concerns of law evasion, medical confidentiality, and the personal and emotional issues related to infertility may preclude the display of that sensitive data. The main method in which data on the extent of CBRC was collected is by voluntary questionnaires handed to the patients during, before, or after their treatments. Only a small number of centers and patients agree to participate in these studies; thus the results should be taken with necessary caution. In 2008, the European IVF Monitoring (EIM) and the Task Force on Ethics and Law of the “European Society of Human Reproduction and Embryology” (ESHRE) started a study to collect data on CBRC.  The Task Force on Cross-Border Reproductive Care, coordinated by Françoise Shenfield, designed two questionnaires that were distributed in centers of six European countries: Belgium, Czech Republic, Denmark, Switzerland, Slovenia, and Spain. The data were collected from 46 ART centers, with the main goal of revealing the quantity and motivations behind seeking cross-border reproductive care. Among the cross-border patients participating, almost two-thirds came from four countries, Italy (31.8%), Germany

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(14.4%), the Netherlands (12.1%), and France (8.7%), followed by Norway (5.5%), the UK (4.3%), and Sweden (4.3%). The mean age of the participants was 37.3 years for all countries (range 21–51 years), 69.9% were married, and 90% were heterosexual. Their reasons for crossing international borders for treatment varied by countries of origin: legal reasons were predominant for patients traveling from Italy (70.6%), Germany (80.2%), France (64.5%), Norway (71.6%), and Sweden (56.6%). Better access to treatment compared to the country of origin was more often noted for UK patients (34.0%) than for other nationalities. Quality of care was an important factor for patients from most countries [13]. In 2010 Hughes and Dejean [14] undertook a questionnaire survey of Canadian and US clinics and clinicians. Although partially based on estimations, their survey shows some patterns of movement of Canadian patients to the USA and a smaller number of US patients seeking IVF in India, Asia, Europe, and Canada. The most commonly reported cross-border treatment sought by Canadians was anonymous donor-oocyte in  vitro fertilization. For patients entering Canada and the USA to receive fertility treatment, the largest demand was for IVF.  The main origin of patients received by US clinics was Europe (25%) and Latin America (39%) [14]. Interestingly, other regions of the world are joining the reproductive globalization. According to a study by Marcia Inhorn, the United Arab Emirates has also become a popular destination for patients, particularly those who have familial, cultural, or economic affinities with this region [15]. Furthermore, in an ethnographic study, not directly focused on CBRC, Thailand was highlighted as a destination for couples seeking fertility treatment away from home, again, particularly for those with familial links to the country [16]. The organization and coordination of CBRC may be carried out in many different forms, from independent travel and self-referrals at one end of the spectrum to intricately connected shared-care arrangements on the other. There is no reliable information regarding the prevalence and market proportion of these different types, nor any systematic comparison of outcomes, experiences, and patient satisfaction. Although business interests undoubtedly constitute a crucial aspect of the global assisted reproduction market [17], there is very little transparency about how this business functions and manifests in CBRC arrangements [18]. According to estimates, which might be conservative, the business of surrogacy in India alone is close to $445 million a year [19, 20], and the World Bank anticipates that it will be a US$2.5 billion industry by the year 2020 [21].

In Vitro Fertilization In vitro fertilization (IVF) has evolved tremendously in the last four decades. Since the first successful IVF in 1978 (bypassing tubal factor infertility) resulting in the birth of Louise Brown, most of the IVF steps have improved resulting in very high pregnancy rates per cycle. It is estimated that over 25 million IVF cycles have been performed worldwide and about 8 million babies have been born using this

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technology. In the USA alone, every year over 250,000 IVF and frozen embryo transfer (FET) cycles are completed and with more than 120,000 oocyte retrievals. The steps of IVF include stimulation period, lasting usually 8–12 days, during which the female patient will usually administer different types of gonadotropins to stimulate her ovaries to produce more than one mature follicle, followed by a final maturation injection (or spray) with GnRH agonist or human chorionic gonadotropins, mimicking the natural LH surge. Following the stimulation portion, an oocyte retrieval is performed, using a transvaginal approach and ultrasound guidance. The patient is sedated for the procedure that usually lasts less than 30 min. Within 2–4 h of the retrieval, the oocytes are inseminated with sperm (partner’s or donor sperm, fresh or frozen) either with insemination or using a technique known as intracytoplasmic sperm injection (ICSI). The fertilized oocytes are then cultured for 2–7 days (no live birth was reported with embryos cultured beyond day 7) in special media, aimed to mimic the fallopian tube environment, allowing for initial screening of abnormal embryos and for the selection of the finest embryo for transfer or for cryopreservation. Lastly an embryo or embryos are transferred to the patient’s (or a surrogate) uterus using a soft catheter (and usually under trans-abdominal ultrasound guidance), and the patient is given progesterone supplementation for luteal phase support. In the past four decades, many improvements have been implemented for each step of the IVF process, from medication protocols to developing new medications (short and long acting) while focusing on lowering cost and improving success and safety (reduction of ovarian hyperstimulation and multiple gestation pregnancies). The oocyte retrieval technique and the retrieval needles used for the aspiration of the follicular fluid have improved as well. In the USA (Society for Assisted Reproductive Technology—SART), Canada, some European countries, Australia and New Zealand (ANZARD), and few other countries, IVF and its outcome is highly monitored by national agencies. These countries usually report high success rates and, as such, host CBRC from countries with less transparent process and usually less resources and experience. Cost is thought to play the most significant driver, as many countries do not cover IVF treatments for their citizens as part of their national healthcare budget, so infertile patients without the means to cover fertility treatment choose to travel to countries which offer low-cost IVF, despite lower success rate and minimal data concerning safety and outcome. Legal aspects are also important; in Asia, many countries forbid single woman or same-sex couple from undergoing in vitro fertilization or even egg freezing. When the need to seek CBRC arises, there are two successful models: 1. Travel the country where all the treatment is provided, and remain there until a pregnancy or treatment goal has been achieved. 2. Collaborate with doctors and clinics in the country of origin, and only travel for specific treatments, to undergo an egg retrieval and/or for embryo (Lower key) transfer. In some cases the process is divided into two separate events, in which embryos are created at an earlier time or shipped from a different clinic.

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Current data suggest that in embryo transfer on a blastocyst stage, day 5 or day 6 postfertilization is the preferred time both for selection of the preferred embryo and to yield the highest implantation rates. However, extended culture also means higher IVF cost (due to extended cultures of the embryos) as well as longer stay in the country in which the medical treatment is provided (added cost and the burden). Therefore, many clinics would offer the patient to transfer embryos at a much earlier stage of development in order to lower the cost. They might also offer to transfer more than one embryo in order to compensate for the lack of good embryo selection and to improve their pregnancy rate. Once an oocyte retrieval is performed and the oocytes are inseminated or injected with sperm, there are a few options to complete the IVF process: 1 . To perform fresh embryo(s) transfer on days 1–3 postfertilization 2. To perform fresh embryo(s) transfer at the blastocyst stage (supported by international guidelines: ASRM, ESHRE, and others) 3. To Freeze all embryos and perform a frozen-thawed embryo transfer at the following month or at a later stage—a “freeze-all” strategy. Cases where no fresh embryos are transferred are usually done to prevent ovarian hyperstimulation syndrome (OHSS) which occurs in up to 5–10% of cycles (based on Cochrane database) or to allow for an embryo biopsy and preimplantation genetic screening or testing to be completed. In the last two decades, nearly every step in the IVF process has been revised and improved. Many improvements in embryo culture techniques have been developed, with several commercial culture media now available, improving both the quality and safety of the procedures. As a result of these enhancements, pregnancy rates have increased significantly reaching over 50% per cycle in patients younger than 35 years of age. New incubators including the EmbryoScope™ (time-lapse microscopy) are now used in many IVF centers, monitoring every minute, hour, and day in the embryo development providing clinical information to the embryologist, the reproductive endocrinologist, and the patients. Both transfer techniques and freezing techniques (different freezing protocols and cryopreservation devices) have been perfected with dozens of different options that are now available for physicians to tailor to their IVF patients’ needs. Many countries in Africa, Asia, South America, and Europe have rules or laws limiting who can undergo fertility treatment. In some countries, same-sex couples and single woman are prohibited from using IVF treatments, whereas in some it is permitted as long as sperm donation or egg donation is not being used. Specific countries will not allow fertility treatment to couple, until they established stable heterosexual relationship for at least a year, while other countries will allow access to IVF only to legally married couples. The rules and regulation are very dynamic and vary from country to country. In general, most prohibitory rules are derived from concerns of creating nontraditional families and ‘unnatural relationships’ between children and their genetic or biological parents. In countries, where religion is more involved, certain fertility treatments

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are banned. For example, in Italy, Germany, and France, where the Roman Catholic Church is more intertwined, IVF and especially transferring, freezing, and discarding embryos have strict guidelines. The Church also raises concerns about artificial insemination as it deviates from the natural conception achieved through a procreation between a man and a woman. Israel, the USA, and Mexico are some of the leading destinations for IVF treatment. The main reasons are liberal laws, higher pregnancy rates, and reduced cost. Most women traveling to the USA for IVF treatment come from India, Canada, China, and other Asian countries. In Asia, the two leading countries to travel to, for the purpose of IVF treatment, are India and Thailand where the cost of IVF is significantly lower than in the USA.

Oocyte Donation In this unique in vitro fertilization treatment, a donated oocyte is being used, rather than the autologous oocyte. The most common indications for oocyte donation are advanced maternal age (typically age 42–55), diminished ovarian reserve (high serum follicle-stimulating hormone [FSH] levels, low anti-Mullerian hormones [AMH], and/or low antral follicle count seen on ultrasound), poor oocyte quality, premature ovarian failure, maternal genetic disease, or failure of other fertility treatments using ones’ autologous oocytes. The process of oocyte donation involves retrieving oocytes from young (typically 21–32  years of age), healthy women that are willing to undergo medical, genetic, and psychological screening and donate their oocytes to a third party. The process includes self-administration of daily hormonal injections to stimulate follicular growth, followed by the surgical procedure of harvesting the oocytes from both ovaries. The oocyte retrieval is performed via an ultrasound-guided transvaginal approach, while the patient is sedated. The hormonal stimulation process usually lasts 8–14 days during which the patient may experiences side effects, the most common being pain, erythema at the site of injection, and bloating. The risks are rare but include infection, bleeding, ovarian hyperstimulation (if many follicles grow of a harsh response to stimulation), ovarian cyst, ovarian torsion, and anesthesia or sedation complication. There are three oocyte donation options available for the recipients: • Known oocyte donor—typically a sister, a cousin, a female partner (in same-sex relationship), or other relative or friend. • Using frozen oocyte from commercially available “egg banks” or the clinic’s own “egg bank.” • Fresh anonymous oocyte donation—the most common route. This option is also divided into using the clinic’s own oocyte donor pool or using donor agency that specializes in recruiting donors with certain specific features, such as ethnicity, religion, education, etc.

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Oocyte donation is illegal in many parts of the world. In other countries, oocyte donation might be legal, but the compensation for the donors is limited, resulting in low supply. Both scenarios contribute to the popularity and demand of CBRC to other countries who have a large pool of readily available donor oocytes. Many western European citizens seek oocyte donation in Eastern European countries like, Ukraine, and Czechoslovakia. Similarly, British and Canadian citizens usually seek oocyte donation in the USA, and Australian/New Zealand citizens often seek oocyte donation in South American countries like Argentina and Chile. The preferred oocyte donors are young, fertile proven donors, who underwent prior successful cycles (with proven pregnancies) and who have good ovarian reserve (assessed by serum AMH, FSH, and antral follicle count). New stimulation protocols and the implementation of GnRh antagonist protocols for stimulation and GnRh agonist injection for final egg maturation allow clinicians to retreive more oocytes than previously possible, sometimes more than 20–30, allowing them to share that cohort among 2–3 recipients (each usually receives a minimum of 6 mature oocytes). The ASRM recently published very clear guidelines regarding the practice of oocyte donation. The guidelines include recommendations of the appropriate tests and examinations for the donors prior to the donation, recommended stimulation protocols, standardized compensation for the donors, and a limitation of maximum six oocyte retrievals per donor. In addition, ASRM guidelines recommend that the clinics should obtain a mental health evaluation of both the recipients and oocyte donors (either by a psychologist or a social worker) prior to any procedure. The risk of pregnancy for women over age 40 remains very high and includes complications for both the mother and the fetus. Reports have shown increased risk for hypertensive disorders (gestational hypertension, preeclampsia, eclampsia, HELLP syndrome), gestational diabetes, prematurity, intrauterine growth restriction (IUGR), malpresentation, operative delivery, and cesarean sections. Current ASRM guidelines recommend against transferring embryos to any recipient beyond 55 years of age. Patients of advanced maternal age who undergo embryo transfer, resulting from the use of an oocyte donor, are advised to consult with maternal-fetal medicine specialist, a cardiologist, and any other relevant specialist, in addition to the routine screening test (mammogram, colonoscopy, pap smear, CBC, cholesterol, and TSH) performed in nonpregnant woman at the fourth and fifth decades of life.

Embryo Donation SART defines embryo donation as “a procedure that enables embryos that were created by couples undergoing fertility treatment or created from donor sperm and oocyte donation to be transferred to a different infertile patient”. By conservative assessment, based on the SART data, it is estimated that over million embryos are cryopreserved in fertility centers across the USA alone. Even when assuming a very low implantation rate of 25%, much lower rate than seen in good prognosis patients, the potential for live birth—if these embryos were to be

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transferred—is immense. In addition, the high demand for oocyte donation among women of advanced reproductive age, the increase in life expectancy, coupled with the high divorce rate in western countries and the steep single women/men ratio in some states, have created a demand for embryo donation as a low-cost fertility treatment option. Instead of the long, expensive, and challenging process of child adoption, embryo adoption became a preferred option for many couples and single woman, who failed other fertility treatment. We now know that even beyond the age of menopause and well into the sixth decade of life, fertility rates, using oocyte donation and embryo donation, are very high if an intact uterus is medically prepared for implantation and pregnancy. Embryos are mostly donated by families that had used REI for conception and were left with excess of embryos after completing their family planning. Therefore, one might consider using donated embryos as a more successful method than using donated oocytes/sperm since most of the donated embryos are sibling embryos from successful cycles with proven pregnancies and deliveries. Furthermore, the cost for embryo donation is much lower than oocyte donation, given the fact that most of the cost for creating the embryos, in the setting of autologous IVF, as well as the screening and freezing processes, have been accounted for by the donors. The clinics also have an interest in using these embryos for transfer rather than assuming the liability of cryopreserving the embryos for decades and eventually discarding them. Unfortunately, embryo donation is illegal in many countries. In the USA, however, very few states have specific legislation concerning embryo donation, and it is legally accepted in most states. This makes the USA a popular country to travel to for patients seeking embryo donation.

Sperm Donation At first, sperm donation was used in couples where the husband was assumed to be sterile, and fresh sperm was used to inseminate the female partner around the time of ovulation. With the improvement in techniques allowing cryopreservation of sperm and since the first sperm bank was opened in 1976 in California, a steady rise in the use of frozen sperm donation was seen. Hundreds of sperm banks are now commercially available throughout the USA and many more across the globe, offering thousands of donor sperm vials every year. With the social changes and legal changes seen in the past 30–40 years, single woman in most western countries (unlike in most countries in Asia) can now conceive using sperm donation, and the same goes for same-sex female couples. The testing for sperm donors, which is monitored by the FDA, ranges from basic infectious disease screening (testing for hepatitis B, hepatitis C, HIV, syphilis, and CMV), basic genetic screening (based on the donors ethnicity), and blood type to extensive genetic screening, psychological screening, drug use screening, and other additional testing which each sperm bank chooses to implement for marketing and other reasons.

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Woman from Asia and other part of the world where sperm donation is illegal have traveled for decades to receive care using sperm donation in western countries. With the improvement in safety and shipment of cryopreserved human tissue, many sperm banks are now offering shipping vials of sperm across the ocean to clinics for the purpose of intrauterine insemination (IUI), intracervical insemination (ICI), and in vitro fertilization (IVF). Many sperm banks are also offering discrete online purchasing and shipping to private residence for “home insemination.” Sperm donation has evolved significantly. In the past it was reserved for couples with male factor infertility; however, the development of ICSI in 1992, which proved that injecting single sperm into an oocyte can yield a life birth (even in severe male factor infertility and azoospermia), had reduced the need for donor sperm for male factor infertility. Therefore, currently, donor sperm is mostly used by single women and same-sex female couples. Given the ease of recruiting sperm donors, the low cost in freezing and cryopreserving sperm, and the simplicity of shipping sperm, it is a main aspect of CBRC. It is not uncommon for an Asian patient to use a South American or Eastern European donor sperm for a treatment conducted in the USA.

Preimplantation Genetic Testing Preimplantation genetic testing (PGT) is used as part of IVF treatment when the patient or the physician wishes to screen or test the embryos for chromosomal abnormalities and genetic abnormalities, identifying embryos affected by balanced translocation, and for gender selection. The first reported live birth following PGT was in 1990 [22]. Since then the field has evolved dramatically and many changes were implemented, including the embryological techniques, the phase in which the biopsy is performed, and the platforms that are used for DNA analysis. The most common process incorporating PGT into the IVF treatment involves growing embryo to the blastocyst stage (5–7 days) where there is a clear distinction between the inner cell mass (the embryo) and the trophectoderm (the future membranes and placenta). At that point, using laser, three to five cells are removed from the trophectoderm and sent to a reference lab, while the embryo is cryopreserved (using a technique called vitrification, or fast freeze) for future use. There are three types of analysis available: • PGT-A = screening for aneuploidy (mainly trisomies 13, 15, 16, 18, 21, 22, X, and Y as well as monosomy x) • PGT-M = diagnosing embryos affected by a specific mutant gene that is responsible for a significant morbidity and mortality in the offspring • PGT-SR = diagnosing embryos affected by unbalanced translocation If the embryo biopsy reveals a euploid (when PGT-A is performed), carrier- or disease-free (when PGT-M is performed), or free from balanced translocation (when PGT-SR is performed), a single (or rarely more than one) embryo can be thawed and

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transferred or frozen for use in a later phase, usually within the following 2 months. If all embryos are found to be aneuploid or affected, they are either discarded, donated to research, or kept cryopreserved until gene therapy or other future treatments become available. The technology used to analyze the biopsied cells has radically changed from the first case in 1990 to 2020. For many years, fluorescence in situ hybridization (FISH) was used as the leading technology to analyze chromosomal abnormalities. In recent years comparative genome hybridization (CGH) and array CGH were introduced as well as polymerase chain reaction (PCR), allowing us to analyze all chromosomal monosomies and trisomies. Interestingly, the most recently developed technology, next-generation sequencing (NGS) became the gold standard, with the global understanding that a day will come when whole genome amplification (WGA) will be the common practice. The newest technologies can assess all 23 sets of chromosomes, including the small base pairs areas, and can detect copy number variants (CNV). This provides us with significantly more information but also introduces a great challenge in the clinical interpretation, due to the unknown importance of these small variations in the human genome. The indication for performing preimplantation genetic screening for healthy couples, aimed to select the best embryo to transfer, has been the topic of many studies as well as committee opinions and practice bulletins. Whether or not this screening technique increases live birth or improve pregnancy rates, most authors agree that the use of PGT for detection of a mutation (PGT-M) caused by autosomal recessive gene (i.e., cystic fibrosis, sickle cell disease), X-linked condition (i.e., Duchenne muscular dystrophy, hemophilia), or autosomal dominant (Huntington, neurofibromatosis) is recommended. The same is true for screening embryos for couples where one of the parents is affected by balanced translocation. The main concerns are that evidence-based data is still lacking regarding: • • • •

False-negative results False-positive results Theoretical risk to the embryo(s) from the biopsy itself Loss of euploid embryo between cleavage stage (day 3 of embryo development) and the blastocyst stage • Effect of both the cryopreservation and the thawing on the embryo The American Society for Reproductive Medicine (ASRM) guidelines recommend that the use of preimplantation genetic screening for aneuploidy (PGT-A) in healthy individuals should be assessed on a case-by-case basis and may be helpful in reducing pregnancy losses which result from aneuploidy (either having an extra chromosome or missing a chromosome). The authors concluded that the value of PGT-A as a universal screening test for all IVF patients has yet to be determined [23]. Since the completion of the human genome project in April 2003 and the identification of over 22,300 protein-coding genes, some have raised concerns that the PGT technology will be used to screen for obscure genes and genes that have no impact on morbidity and mortality. This slippery slope in testing and “eliminating”

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certain genes has been the focus of ethics committees at ASRM and European Society of Human Reproduction and Embryology (ESHRE) and others. Another concern is that these advanced technology and skills are limited to certain countries that have access to both the lab equipment necessary and the sophisticated genetic analysis platforms, therefore providing access to this innovative treatment only to patients with financial means who can afford it.

Surrogacy Surrogacy is a method of assisted reproduction whereby a woman agrees to become pregnant for the purpose of gestating and giving birth to a child for others to raise. In this section, we seek to identify the key ethical themes within the surrogacy component of CBRC.

Surrogacy Around the World Laws differ widely from one country to another. The USA has no federal surrogacy or parentage law, leaving each state to make up its own laws. There are a substantial number of states with relatively liberal laws and policies surrounding the assisted reproduction treatments and gestational surrogacy. A growing number of states, by statute or court decision, authorize pre-birth or post-birth orders for intended parents, at least for married couples with a genetic tie to the child (embryo, spermatozoa, or eggs), thereby establishing a legal relationship between intended parents and the child upon birth [24]. This can make a post-birth adoption unnecessary and protects the intended parents’ legal status, their gestational surrogate, and one another (the latter can be important if donor eggs or spermatozoa were used). When there is no genetic connection to the child, establishing legal parentage can be less predictable and is much more variable from state to state [25]. In Canada, the Assisted Human Reproduction Act permits only altruistic surrogacy; surrogate mothers may be reimbursed for approved expenses, but payment of any other consideration or fee is illegal [26, 27]. Quebec law, however, renders all surrogacy contracts, whether commercial or altruistic, unacceptable. There is no consensus in Europe regarding surrogacy; the pendulum moves from complete restrictions and illegal status for any involvement with surrogacy to complete acceptance and encouragement of commercial surrogacy. In England, commercial surrogacy arrangements are not legal and are prohibited by the Surrogacy Arrangements Act 1985. A surrogate mother still maintains the legal right for the child, even if they are genetically unrelated. Unless a parental order or adoption order is made, the surrogate mother remains the legal mother of the child [28]. In Ireland, Denmark, Belgium, and the Netherlands, altruistic surrogacy is legal, while commercial surrogacy is illegal. Italy, Spain, Norway, Switzerland, France, Austria, and Germany ban all forms of surrogacy.

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Examples of countries where surrogacy is completely legal are Ukraine, Russia, and Georgia. In Ukraine, only married couples can legally go through gestational surrogacy [29]. In Russia, while there must be a certain medical indication for surrogacy, foreigners have the same rights as Russian citizens [30, 31]. In Georgia, surrogate mother cannot exercise any parental rights over the child [32]. In Sweden, surrogacy is not clearly regulated. The legal procedure most equivalent would be an adoption of the child from the surrogate mother. It is illegal for Swedish fertility clinics to make surrogate arrangements [29]. As it can be seen, laws on surrogacy in Europe are varied and for some countries, vague and nonexistent. Up until recently surrogacy in India was legal and unregulated, turning India into one of the busiest locations for surrogacy. New restrictions require all commissioning couples to meet certain criteria including being married for a minimum of 2 years. Gay marriage is not recognized in India, precluding surrogacy for single intended parents and gay couples, a change of policy that is significant and being met with strong resistance. In Australia, all states, except Tasmania which bans all surrogacy, recognize altruistic surrogacy as legal; however, arranging commercial surrogacy is a criminal offense [33]. The Science Council of Japan proposed a ban on surrogacy. Doctors, agents, and clients will be punished for commercial surrogacy arrangements. Surrogacy is neither forbidden nor expressly permitted by law in China. The Ministry of Health has established “departmental rules” which prohibit medical professionals from performing surrogacy procedures, with violations punished by fines (but not criminal liabilities). In practice, surrogacy arrangements are common in China, with an underground market for commercial surrogacy estimated to encompass between 400 and 500 agencies in 2012 [34]. Costa Rica is the only country to ban IVF entirely, and the repercussions and impact of the most recent ruling are still unfolding [35]. In 1999, Iran issued a verdict electively permitting surrogacy. This ruling achieves acceptance in parts of the Shi’ite population, e.g., Iran, Lebanon, and part of Saudi Arabia, Bahrain, Iraq, Syria, Afghanistan, India, and Pakistan [36]. All surrogacy arrangements (both commercial and altruistic) are legal and popular in Iran. In March 1996, the Israeli government legalized gestational surrogacy under the “Embryo Carrying Agreements Law.” This law made Israel the first country in the world to implement a form of state-controlled surrogacy in which each and every contract must be approved directly by the state [37]. A state-appointed committee permits surrogacy arrangements to be filed only by Israeli citizens who share the same religion [38]. The numerous restrictions on surrogacy under Israeli law have prompted some intended parents to turn to surrogates outside of the country. Same-­ sex couples can enter a surrogacy arrangement outside of Israel and have their legal parenthood recognized within Israel.

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In South Africa, altruistic surrogacy is allowed including traditional and gestational surrogacy. The South Africa Children’s Act of 2005 enabled the “commissioning parents” and the surrogate to have their surrogacy agreement validated by the High Court before fertilization. This allows the commissioning parents to be recognized as legal parents from the outset of the process and helps prevent uncertainty [39]. The law permits single people and gay couples to be commissioning parents [40]. If the surrogate is the genetic mother, she has until 60 days after the birth of the child to change her mind. The surrogate also has the right to unilaterally terminate the pregnancy, but she must consult with and inform the commissioning parents, and if she is terminating for a nonmedical reason, she may be obliged to refund any medical reimbursements she had received. In Nigeria, Ghana, and Kenya, commercial surrogacy is popular, even though there is no formal legal framework in place. The United Arab Emirates is one of the few Middle Eastern countries with readily available ART clinics; however it also enacts ART legislation. It does not permit any form of third-party reproductive assistance (i.e., sperm and egg donation, embryo donation, or surrogacy) [15].

Traditional vs. Gestational Surrogacy The term surrogacy incorporates two significantly different modes of reproduction. While some providers and countries continue to offer “traditional” surrogacy options (artificial insemination of the surrogate with either the intended father’s or a sperm donor’s spermatozoa, which results in the surrogate being the genetic mother of any offspring), the majority of intended parents seek, and professionals offer, “gestational” surrogacy (IVF using the intended mother’s or an egg donor’s eggs, but not those of the surrogate). Although gestational surrogacy is undeniably more expensive as it requires IVF as opposed to artificial insemination, it avoids any genetic connection between the child and the gestational surrogate and allows both parents to share their genetic load in creating the offspring. This approach reduces the legal risk that is involved in surrogacy as in some jurisdictions around the world, the surrogate will be considered the legal mother as they recognize motherhood based on genetics and/or intention rather than gestation in the context of surrogacy [24]. Cases in which the surrogate mother is also genetically related to the child present a problem for the courts in particular, in that not only the legitimacy of surrogacy contracts has been called into question, but the issue has also been raised of whose right to the child is greater: the genetic and biological mother or the genetic father and his partner, the social parent.

Multiple Embryo Transfers and Abortions Surrogates are commonly encouraged to accept multiple embryos in order to maximize the probability of a successful implantation and thus reduce costs to the client.

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A multiple pregnancy is a dangerous medical condition that is avoided by fertility doctors in the West [41]. Yet, it is attractive as a cost-saving measure, since more transferred embryos raise the chance of a pregnancy and reduces the number of attempts needed to become pregnant [42, 43]. This trend is a factor influencing both the autonomy of the surrogate and the nature of her informed consent, since it increases the risk for the need of a fetal reduction or even abortion that has to be done for several medical reasons. It remains unknown whether surrogates from conservative cultures are aware of this likelihood since they might not be culturally/ religiously comfortable with these procedures. Efforts must be made to ensure that the surrogate has a full understanding of all long- and short-term risks involved in a multiple pregnancy and the possibility of essential embryo reduction or termination of pregnancy, as well as the liberty to choose to avoid it.

Summary Cross-border reproductive care has evolved in parallel to the milestones achieved in the field of reproductive medicine. Since the inception of the first successful IVF in London in 1978, through the first successful oocyte donation in the early 1980s and the development of ICSI and PGT in the 1990s, fertility treatments achieved improved outcomes and became more affordable and readily available. Although basic IVF is now available in most developing countries, its overall success in these countries remains low due to poor technology, lack of professional personnel, and limiting legislation and religious beliefs. These, along with other limiting factors, prohibit the use of donated gametes, surrogacy, embryo biopsy, and access to care for single intended parents and same sex couples in many countries. Until these limitations and obstacles are resolved, we foresee a rise in cross-border reproductive care in the next few decades. This is especially true for third-party reproduction care (oocyte, sperm, and embryo donation, as well as surrogacy) with citizens of countries in Asia and Africa traveling to Europe and the USA to receive care.

References 1. Boivin J, et al. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum Reprod. 2007;22(6):1506–12. 2. Gray HH, Poland SC. Medical tourism: crossing borders to access health care. Kennedy Inst Ethics J. 2008;18(2):193–201. 3. Cohen J.  Procreative tourism and reproductive freedom. Reprod Biomed Online. 2006;13(1):145–6. 4. Afshar Y, Tabsh K. Pregnancy and subsequent uterine rupture in a 72-year-old gravida: medical tourism versus procreative freedom. J Obstet Gynaecol. 2018;38(5):716–8. 5. Knoppers BM, LeBris S. Recent advances in medically assisted conception: legal, ethical and social issues. Am J Law Med. 1991;17(4):329–61. 6. Pennings G.  Reproductive tourism as moral pluralism in motion. J Med Ethics. 2002;28(6):337–41.

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7. Blyth E, Farrand A. Reproductive tourism-a price worth paying for reproductive autonomy? Crit Soc Policy. 2005;25(1):91–114. 8. McKelvey A, et al. The impact of cross-border reproductive care or ‘fertility tourism’ on NHS maternity services. BJOG. 2009;116(11):1520–3. 9. Inhorn MC, Patrizio P. Rethinking reproductive “tourism” as reproductive “exile”. Fertil Steril. 2009;92(3):904–6. 10. Pennings G, et al. ESHRE task force on ethics and law 15: cross-border reproductive care. Hum Reprod. 2008;23(10):2182–4. 11. Matorras R. Reproductive exile versus reproductive tourism. Hum Reprod. 2005;20(12):3571; author reply 3571–2. 12. Cross-border reproductive care: an Ethics Committee opinion. Fertil Steril. 2016;106(7):1627–33. 13. Shenfield F, et  al. Cross border reproductive care in six European countries. Hum Reprod. 2010;25(6):1361–8. 14. Hughes EG, Dejean D. Cross-border fertility services in North America: a survey of Canadian and American providers. Fertil Steril. 2010;94(1):e16–9. 15. Inhorn MC, Shrivastav P. Globalization and reproductive tourism in the United Arab Emirates. Asia Pac J Public Health. 2010;22(3 Suppl):68S–74S. 16. Whittaker A. Global technologies and transnational reproduction in Thailand. Asian Stud Rev. 2009;33(3):319–32. 17. Spar DL. The baby business: how money, science, and politics drive the commerce of conception. Boston: Harvard Business School Press; 2006. TURAL. 18. Inhorn MC, Gurtin ZB.  Cross-border reproductive care: a future research agenda. Reprod Biomed Online. 2011;23(5):665–76. 19. Van den Akker OB. Psychological trait and state characteristics, social support and attitudes to the surrogate pregnancy and baby. Hum Reprod. 2007;22(8):2287–95. 20. Edelmann RJ. Surrogacy: the psychological issues. J Reprod Infant Psychol. 2004;22(2):123–36. 21. Hyder N. India debates new surrogacy laws. Bio News. 2011;594:117–31. 22. Handyside AH, et  al. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature. 1990;344(6268):768–70. 23. The use of preimplantation genetic testing for aneuploidy (PGT-A): a committee opinion. Fertil Steril. 2018;109(3):429–36. 24. Crockin SL, Altman AB. Statutory and case law governing the practice of third-party reproduction. In: Principles of oocyte and embryo donation: Springer; 2013. p. 351–68. 25. Crockin SL.  Growing families in a shrinking world: legal and ethical challenges in cross-­ border surrogacy. Reprod Biomed Online. 2013;27(6):733–41. 26. Brahams D.  The hasty British ban on commercial surrogacy. Hastings Cent Rep. 1987;17(1):16–9. 27. Lawrence DE. Surrogacy in California: genetic and gestational rights. Golden Gate Univ Law Rev. 1991;21:525. 28. Serratelli A. Surrogate motherhood contracts: should the British or Canadian model fill the US legislative vacuum. George Washington J Int Law Econ. 1992;26:633. 29. Pennings G.  Legal harmonization and reproductive tourism in Europe. Hum Reprod. 2004;19(12):2689–94. 30. Svitnev K.  Legal regulation of assisted reproduction treatment in Russia. Reprod Biomed Online. 2010;20(7):892–4. 31. Svitnev K. 196 surrogacy and its legal regulation in Russia. Reprod Biomed Online. 2010;20:S90. 32. Pennings G.  Reply: reproductive exile versus reproductive tourism. Hum Reprod. 2005;20(12):3571–2. 33. Australia W.  Legislative council, standing committee on uniform legislation and statutes review, report 3. Administrative Practices and Procedures and Parliamentary Processes involving Treaties entered into, or proposed to be entered into, by the Commonwealth. 2007. p. 20.

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34. De Wert G, et al. ESHRE Task Force on Ethics and Law 23: medically assisted reproduction in singles, lesbian and gay couples, and transsexual people. Hum Reprod. 2014;29(9):1859–65. 35. Crockin SL, et al. Costa Rica’s absolute ban on in vitro fertilization deemed a human rights violation: implications for US assisted reproductive technology policy and “personhood” initiatives. Fertil Steril. 2013;100(2):330–3. 36. Inhorn MC. Local babies, global science: gender, religion and in vitro fertilization in Egypt. New York: Routledge; 2012. 37. Teman E. Birthing a mother: the surrogate body and the pregnant self. Berkeley: University of California Press; 2010. 38. Schenker JG. ‘Surrogate Pregnancies: Ethical and Legal Aspects’. Ethical Dilemmas in Perinatal Medicine, Jaypee Brothers Medical Publishers (P) Ltd. 2010. https://doi.org/10.5005/ jp/books/11363_17. 39. Kriari-Catranis I. Human assisted procreation and human rights-The Greek response to the felt necessities of the time. Eur J Health Law. 2003;10:271. 40. Schmidt L, et al. Demographic and medical consequences of the postponement of parenthood. Hum Reprod Update. 2011;18(1):29–43. 41. Schieve LA, et al. Low and very low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med. 2002;346(10):731–7. 42. Hurst T, Shafir E, Lancaster P.  Assisted conception, Australia and New Zealand. Sydney: AIHW National Perinatal Statistics Unit; 1996. 43. Deonandan R, Green S, Van Beinum A. Ethical concerns for maternal surrogacy and reproductive tourism. J Med Ethics. 2012;38(12):742–5.

Uterine Transplantation Victor Gomel

Introduction Successful human uterine transplantation (UTx) with viable births was achieved after several years of preliminary animal experimentation using a systematic and scientific approach by a large multidisciplinary research and medical team headed by Mats Brännström. The teams included transplant and gynecologic surgeons, reproductive endocrinologist, high-risk obstetrician, anesthesiologist, transplant psychiatrist, infectious disease specialist, social worker, patient advocate, and research and coordinator nurses [1]. This success initiated great interest internationally and stimulated the development of similar programs in several countries, including the USA, China, India, Japan, Brazil, Czech Republic, France, and the UK. The multiple causes aligned with “uterine factor infertility” [1] may be condensed under four headings: congenital absence of the uterus, prior surgical removal (hysterectomy), uterine malformation, and nonfunctional uterus. Fallopian tube and uterine transplantations have been attempted since the 1960s largely with animal experiments. In 1966, Eraslan and coworkers reported an autologous transplantation in a dog, which led to a subsequent pregnancy [2]; however, in human trials, autologous transplants of fallopian tubes failed. It was speculated at the time that the failure was due to inadequate immunosuppressants [3]. In the early 2000s, syngeneic uterine transplantation on rats demonstrated a pregnancy rate, number of pups, and growth trajectory similar to controls. However, the numbers of resorbed and arrested pregnancies were more common in those with UTx° [4, 5]. Not until 2010 was a positive pregnancy outcome reported following allogeneic The content of this chapter was published as an article in the journal Climacteric on “2019; 22(2): 117–121” and is republished here with permission of the journal. V. Gomel (*) Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, BC, Canada © International Academy of Human Reproduction 2021 J. G. Schenker et al. (eds.), Clinical Management of Infertility, Reproductive Medicine for Clinicians 2, https://doi.org/10.1007/978-3-030-71838-1_5

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uterine transplantation in rats [6]. The procedure was also attempted in rabbits [7], and swine, without any attempt at pregnancy [8]. Ovine models produced the first live birth from a large animal [9, 10]. The first ever pregnancy in a nonhuman primate following autologous uterine transplantation was in a cynomolgus macaque. Subsequent to an abruptio placenta, a live birth was obtained [11]. Brännström’s team experimented on 15 nonhuman primates before attempting human transplantation in the process developing surgical techniques for uterus retrieval and transplantation. In addition, they studied subsequent ovarian function [12]. They tested immunosuppression on 18 female baboons submitted to allogenic uterine transplantation. In four untreated baboons, there was severe rejection. In another four treated with monotherapy immunosuppression, all uteri became necrotic. The outcome on postoperative days 32–35 of the remaining ten treated with multi-therapy immunosuppression was as follows: Two baboons developed pulmonary edema and died; in six the uteri became necrotic, one of which was in the vagina; in one the uterus was grossly normal; the tenth had a normal uterus and demonstrated hormonal cyclicity but no menses [13]. In another experiment of uterine autotransplantation on 16 baboons, in 8 cases where uterine vessels were anastomosed end to end, vascular thrombosis resulted in all. In the other eight animals, end-to-side anastomosis of uterine vessels to the internal iliac vessels was performed resulting in 90% of patency. The authors did not provide information about the functionality of the uteri [14].

Human Uterine Transplantation Before the successful first delivery of a child from a transplanted uterus, there were two prior attempts at uterine transplantation. The first of these was in 2000 in Saudi Arabia. The recipient was a 26-year-old woman who had a prior hysterectomy for postpartum hemorrhage, and the donor was a 46-year-old woman who was submitted to an enlarged hysterectomy to preserve tissue and vascular integrity. The recipient experienced an episode of acute rejection on postoperative day 9, which was treated. Acute vascular thrombosis occurred on postoperative day 9, which appeared to be caused by inadequate uterine support and for which a hysterectomy was undertaken [14]. The second attempt was in Turkey in 2011. The recipient was a 23-year-old woman, with complete Müllerian agenesis for which a prior vaginal reconstruction had been performed. The donor was 22 years old, a deceased multi-organ donor. Eighteen months after the transplantation, embryo transfer was attempted twice; one day 3 thawed embryo was transferred under real-time ultrasound guidance, 3 days after the initial Progesterone application in both ET cycles. Eleven days after the first transfer, serum hCG levels were consistent with a biochemical pregnancy (hCG, 35.7  IU/L) suggesting a biochemical gestation, but dropped 4  days later. Following the second embryo transfer, transvaginal ultrasound confirmed an intrauterine pregnancy of 5 gestational weeks; but the gestation failed to develop further [15, 16].

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Experience of the Swedish Team As part of their research program, the Swedish team carried out nine human uterine transplantations with live donors. Eight of the recipients were women with congenital absence of the uterus, and one had a prior hysterectomy for cervical cancer. Ovarian stimulation, oocyte aspiration, and embryo cryopreservation were performed prior to the transplantation [1]. The majority of the donors were relatives of the recipients and had themselves more than one pregnancy and live birth. Five of them were postmenopausal. Both donors and recipients were submitted to rigorous medical evaluation by the multidisciplinary team of specialists. Before removal of the uterus, the postmenopausal donors were given cyclical hormone therapy for a few months until a regular menstrual pattern was achieved. In these series the duration of surgery for the donors varied from 10 to 13 h and for the recipients from 4 to 6 h. In seven of the nine recipients, the uterus was viable and regular menses commenced at 6 months. Mild signs of rejection were noted in four of these seven women; they were successfully treated with corticosteroids. Two of the nine recipients required removal of the graft due to complications; one experienced thrombosis of both uterine arteries and veins on the third postoperative day. The other suffered a long episode of uterine infection, uterine abscess, and septicemia requiring prolonged treatment (postoperative days 33–105) [17, 18]. In the pregnancy, which resulted in the first live birth after uterine transplantation, there were three episodes of mild rejection, all successfully treated with corticosteroids. Fetal growth parameters and uterine and umbilical cord blood flow remained normal until the patient’s admission to the hospital with preeclampsia at 31 weeks and 5 days. Due to abnormal fetal heart rate, cesarean section was performed 16 h later, and a male infant weighing 1775 g with normal Apgar scores was delivered. A hysterectomy was performed 3.5 months later. The surgery was complicated due to adhesions around the uterine fundus and extensive paracervical adhesions requiring double-J ureteric stents to aid in the dissection. The anastomosis sites were fully patent, and Doppler-estimated blood flow of the uterine arteries was 50 and 35 mL/min on the left and right side, respectively. The duration of surgery was 4 h and 35 min, with a blood loss of 600 mL [17, 18]. Surgical technique of the donor  To enter the abdominal cavity, a midline incision from the pubis to the umbilicus is made. The need to obtain long vascular pedicles requires dissection of the uterine veins and arteries from the ureters and mobilization of the internal iliac arteries starting from their bifurcations. The dissection includes severance of multiple major vascular branches. Bilateral salpingectomy is also performed. The vagina is transected 10–15 cm below the vaginal fornix, the major feeding arteries and veins are then clamped and cut, and the uterus removed from the pelvis [1, 17, 18]. The donor graft is then perfused first with heparinized saline and then flushed with an approved organ-preservation solution, instead of physiologic saline.

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Table 1  Current results of the Swedish program # pat Diagnosis 1 MRKH single kidney 2 Cervical cancer 2b Cervical cancer 3 MRKH 4 MRKH single kidney 5 MRKH single kidney 5b MRKH single kidney 6 MRKH

Gest. week at birth 31 + 6

Pregnancy complication Pre-eclampsia

Sex Male

Apgar score 9-10-10

Infant outcome (Sept 2017) Healthy

35 + 0

–

Male

9-10-10

Healthy

37 + 0

–

Female

9-10-10

Healthy

34 + 4 34 + 4

Male Male

8-8-8 3-7-10

Healthy Healthy

35 + 3

Cholestasis Pre-eclampsia cholestasis Pre-eclampsia

Female

9-10-10

Healthy

35 + 6

–

Male

9-9-10

Healthy

37 + 1

–

Female

9-10-10

Healthy

Surgical technique of the recipient  A second team of surgeons prepares the recipient in an adjacent OR. A midline incision from the pubis to umbilicus permits adequate entry to the abdomen. The vaginal vault is dissected free from the bladder and rectum. For the subsequent organ fixation, 1-0 polypropylene sutures are applied to the uterine rudiment. The external iliac artery and vein are bilaterally separated from each other and from adjacent tissue to a distance of 60 mm. The uterine vessels are placed in their normal position in the pelvis and bilateral end-to-­ side vascular anastomoses performed between the graft vessels and the external iliac vessels with the use of continuous 7-0 polypropylene sutures for arterial anastomosis and 8-0 for venous anastomosis. The vaginal rim of the graft is anastomosed to the top of the recipient’s vagina with continuous absorbable 2-0 suture [1, 17, 18]. Results  In 2017 the Swedish team reported on live births in four patients one of whom had two pregnancies resulting in viable births [19]. Their most recent results have been generously provided to the author and are shown in Table 1. Six patients have had live births and among them two women had two successive live births.

Technical Modifications Success with uterine transplantation has created universal interest resulting in the creation of such programs in many countries. This has resulted in a number of technical variations and also increased use of uteri from deceased donors. Three recent publications reported robotic-assisted procurement of the uterus from a living donor and subsequent transplantation. Wei and coworkers who

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commenced UTx studies on the sheep model [20] reported recently use of the utero-­ ovarian veins for venous drainage to simplify the procedure. The duration of surgery on the donor was 6 h and on the recipient 8 h and 50 min. The uterus has remained in situ for a year, and the recipient has had menstrual cycles [21]. Another center found the robotic approach for deep pelvic dissection to be superior to laparotomy [22] although operating times remain long on both donor and recipient. The Swedish team has also started a robotic-assisted live donor UTx trial of ten cases using the same criteria as before. Ethical approval was obtained in the spring of 2016, and the study commenced in 2017. The aim of the study is to reduce the surgical time for donors, with the same safety and outcome [23, 24]. The first birth of a healthy child following uterus transplantation in the USA, reported in 2018, was achieved with a uterus procured from a living donor. The two major modifications used in this case were having only the utero-ovarian veins as venous outflow, which significantly simplified live donor surgery, and to greatly shorten the time from transplantation to embryo transfer with the aim to decrease exposure to immunosuppression by the recipient and lower the risk for potential adverse effects from these medications [25] (Table 2). A recent publication from the Czech Republic program reported the main characteristics, perioperative and postoperative courses of both recipients and donors after four deceased donor and five living donor uterus transplantations. Except for one live and one deceased donor recipient that relied on two uterine veins for venous Table 2  Details of the Dallas program

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outflow, in all other recipients, two uterine and two ovarian veins were used. Other than one transplant using uterine veins for outflow, all others were surgically successful, and follow-up posttransplant ultrasound examinations revealed normal uterine blood supply and outflow. To date no successful pregnancy has been reported [26, 27].

Discussion Success with UTx and delivery of live children is a great achievement, and the Swedish team from Gothenburg must be congratulated for the systematic and scientific way with which they proceeded. Nonetheless uterine transplantation remains an experimental procedure. Live donors carry a significant risk in order to potentially benefit the recipient [1, 28]. Surgery on a live donor is a highly invasive, complex, hazardous procedure that presents a full range of irreducible levels of risks that may decrease her quality of life and induce negative influences on her psychological well-being. The dedication of women to subject themselves to such risks, so that others might bear children, appears to exceed commonplace altruism. Another ethical concern is that, in tight-knit personal relationships, individuals could feel under familial or social pressure to act against their own interests or preferences for the benefit of others close to them. In the UK, the Human Tissue Authority regulates all living organ donations. It insists that the consent provided by the living donor is fully informed and that there is no evidence of coercion, duress, or reward. For the recipient in addition to the risks of extensive surgery are risks of immunosuppression and pregnancy. In all three reports of living donor transplants, from Sweden, Saudi Arabia, and Texas, there have been major complications reported [1, 21]. This procedure has many medical, ethical, and social issues that require discussion. The Japan Society created in 2014 for this purpose suggests to proceed with caution and stated “the safety and efficacy remain unclear, despite several clinical applications” [29]. The American Society for Reproductive Medicine (ASRM) recognizes uterus transplantation as the first successful medical treatment of absolute uterus factor infertility, while cautioning health professionals, patient advocacy groups, and the public about its highly experimental nature. They recently published an extensive position statement on uterine transplantation [30], and in an earlier one, they reminded: “As with all our patients seeking to build their families, it is important to understand the full array of options available to them, including adoption, gestational carriers, and child-free living” [31]. The choice to donate must be fully informed and free of any coercion, because the transplantation procedure puts the donor not only to risks of extensive surgery, duration of anesthesia, and postsurgical sequela but also being left with short vaginal length, premature menopause with younger donors, and potential risks associated with giving estrogens to postmenopausal ones. These may lead to a decreased quality of life and negative influences on psychological well-being.

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The recipient must also be equally prepared to make informed decisions about receiving the organ, including the risks of surgery, ongoing immunosuppression, and the possibility of organ rejection. Given its complexity, the cost of the transplantation procedures and downstream care for the donor may be quite high. Study design should include a clear and ethically justifiable method of defining the thresholds for acceptable levels of risk for all of the involved parties [32, 33]. The use of a deceased donor would partly simplify the issue. With regard to deceased donor uteri, initial studies looked into uterus retrieval techniques in brain-­ dead multi-organ donors [34]. In a study by Gauthier and coworkers, in situ perfusion of the organs was performed with catheters placed in the femoral arteries. Retrieval of the uterus from a deceased donor allows a wide dissection of the vascular pedicles, which permits wider anastomosis reducing the risk of thrombosis. In this study the internal iliac arteries and veins were successfully preserved bilaterally in six of seven cases. In addition it avoids the dissection of the ureteral tunnel, which decreases the surgical time [35, 36]. This strategy also provides an alternative to having a gestational surrogate, which is not practiced in many countries because of ethical, religious, or legal reasons. A recent video article describes the advantages of a deceased donor of a live donor model: eliminates the risk of surgical complications of a living donor, eliminates the ethical issues inherent in live donation, provides easier access to generous vascular pedicles for anastomosis, and faster procurement time [37]. As the article was going to print, we were delighted to learn of a live birth after uterus transplantation from a deceased donor. The donor was a 45-year-old woman who died as a result of a subarachnoid hemorrhage. Major organs having been retrieved first for other transplant operations, it took 8  h before the uterus was removed. The recipient was a 32-year-old woman who was born without a uterus. The duration of the surgery on the recipient was 10.5 h. She was given immunosuppressive therapy until birth. As the recipient was having regular menstruations, a single embryo was transferred after 7 months. A baby girl was born by cesarean section at 35 weeks and 3 days, weighing 2550 g. The uterus was removed during the same intervention and the immunosuppressive drugs stopped [38]. Both types of uterus donation pose unique regulatory challenges, including how to allocate donated organs; whether the donor and/or the donor’s family has any rights to the uterus and resulting child; how to manage contact between the donor, donor’s family, recipient, and resulting child; and how to track outcomes moving forward [39]. The ASRM Ethics Committee recommends 55 years as upper age for transfer of donor oocytes and embryos in healthy women. In Swedish team’s initial trial of nine UTx with live donors, six were under 55 years of age (Table 3). Would it not be more practical and reasonable for these donors to carry the pregnancy instead of submitting themselves and the recipient to extensive, expensive, and lengthy surgical procedures? Such approach would greatly simplify the process and avoid major surgical operations in two individuals, avoid harmful effects from medications used to prevent rejection of the graft, and very likely improve the outcomes.

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Table 3  The status and ages of the donors and recipients of the Swedish program

In summary, successful outcomes with UTx have ignited interest in the procedure in several countries that have initiated programs, and few of these have achieved to have live births. In recent count there have been five live births internationally other than those obtained in the Swedish program. Nonetheless, uterine transplantation remains an experimental procedure that requires the study and resolution of ethical, technical, financial, social issues, all very important. Technical feasibility is not a sufficient reason to perform a procedure. There must be a real indication, and the procedure must yield a real benefit to the patient [40].

References 1. Brännström M, Johannesson L, Dahm-Kahler P, Enskog A, Molne J, Kvarnström N, Diaz-­ Garcia C, Hanafy A, Lundmark C, Marcickiewicz J, Gabel M, Groth K, Akouri R, Eklind S, Holgersson J, Tzakis A, Olausson M.  First clinical uterus transplantation trial: a six-month report. Fertil Steril. 2014;101:1228–36. https://doi.org/10.1016/j.fertnstert.2014.02.024. [PubMed] [CrossRef]. 2. Eraslan S, Hamernik RJ, Hardy JD. Replantation of uterus and ovaries in dogs, with successful pregnancy. Arch Surg. 1966;92:9–12. [PubMed]. 3. O’Leary JA, Feldman M, Gaensslen DM.  Uterine and tubal transplantation. Fertil Steril. 1969;20:757–60. 4. El-Akouri RR, Kurlberg G, Brännström M. Successful uterine transplantation in the mouse: pregnancy and post-natal development of offspring. Hum Reprod. 2003;18:2018–23.

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5. Akhi SN, Diaz-Garcia C, El-Akouri RR, Wranning CA, Molne J, Brännström M.  Uterine rejection after allogeneic uterus transplantation in the rat is effectively suppressed by tacrolimus. Fertil Steril. 2013;99:862–70. https://doi.org/10.1016/j.fertnstert.2012.11.002. [PubMed] [CrossRef]. 6. Díaz-García C, Akhi SN, Wallin A, Pellicer A, Brännström M. First report on fertility after allogeneic uterus transplantation. Acta Obstet Gynecol Scand. 2010;89:1491–4. https://doi. org/10.3109/00016349.2010.520688. 7. Saso S, Hurst S, Chatterjee J, Kuzmin E, Thum Y, David AL, Hakim N, Corless DJ, Boyd M, Noakes DE, Lindsay I, Ghaem-Maghami S, Del Priore G, Smith JR. Test of long-term uterine survival after allogeneic transplantation in rabbits. J Obstet Gynaecol Res. 2014;40:754–62. https://doi.org/10.1111/jog.12256. [PubMed] [CrossRef]. 8. Avison DL, DeFaria W, Tryphonopoulos P, Tekin A, Attia GR, Takahashi H, Jin Y, Palaios E, Pararas N, Carreno MR, Santiago S, Bazer F, Ruiz P, Tzakis A. Heterotopic uterus transplantation in a swine model. Transplantation. 2009;88:465–9. https://doi.org/10.1097/ TP.0b013e3181b07666. [PubMed] [CrossRef]. 9. Wranning CA, Marcickiewicz J, Enskog A, Dahm-Kahler P, Hanafy A, Brännström M. Fertility after autologous ovine uterine-tubal-ovarian transplantation by vascular anastomosis to the external iliac vessels. Hum Reprod. 2010;25:1973–9. https://doi.org/10.1093/humrep/deq130. 10. Ramirez ER, Ramirez Nessetti DK, Nessetti MB, Khatamee M, Wolfson MR, Shaffer TH, Ramirez VZ, Ramirez HA. Pregnancy and outcome of uterine allotransplantation and assisted reproduction in sheep. J Minim Invasive Gynecol. 2011;18:238–45. https://doi.org/10.1016/j. jmig.2010.11.006. 11. Mihara M, Kisu I, Hara H, Iida T, Araki J, Shim T, Narushima M, Yamamoto T, Moriguchi H, Kato Y, Tonsho M, Banno K, Aoki D, Suganuma N, Kagawa N, Takehara Y, Kato O, Koshima I. Uterine autotransplantation in cynomolgus macaques: the first case of pregnancy and delivery. Hum Reprod. 2012;27:2332–40. https://doi.org/10.1093/humrep/des169. [PubMed] [CrossRef]. 12. Enskog A, Johannesson L, Chai DC, Dahm-Kahler P, Marcickiewicz J, Nyachi Brännström M.  Uterus transplantation in the baboon: methodology and long-term function after auto-­ transplantation. Hum Reprod. 2010;25:1980–7. https://doi.org/10.1093/humrep/deq109. [PubMed] [CrossRef]. 13. Johannesson L, Enskog A, Dahm-Kähler P, Hanafy A, Chai DC, Mwenda JM, Díaz-García C, Olausson M, Brännström M. Uterus transplantation in a non-human primate: long-term follow­up after autologous transplantation. Hum Reprod. 2012;27:1640–8. https://doi.org/10.1093/ humrep/des093. Epub 2012 March. 14. Fageeh W, Raffa H, Jabbad H, Marzouki A. Transplantation of the human uterus. Int J Gynecol Obstet. 2002;76:245–51. https://doi.org/10.1016/S0020-­7292(01)00597-­5. [PubMed] [CrossRef]. 15. Ozkan O, Akar ME, Ozkan O, Erdogan O, Hadimioglu N, Yilmaz M, Gunseren F, Cincik M, Pestereli E, Kocak H, Mutlu D, Dinckan A, Gecici O, Bektas G, Suleymanlar G. Preliminary results of the first human uterus transplantation from a multiorgan donor. Fertil Steril. 2013;99:470–6. https://doi.org/10.1016/j.fertnstert.2012.09.035. [PubMed] [CrossRef]. 16. Erman Akar M, Ozkan O, Aydinuraz B, Dirican K, Cincik M, Mendilcioglu I, Simsek M, Gunseren F, Kocak H, Ciftcioglu A, Gecici O, Ozkan O. Clinical pregnancy after uterus transplantation. Fertil Steril. 2013;100:1358–63. https://doi.org/10.1016/j.fertnstert.2013.06.027. [PubMed] [CrossRef]. 17. Johannesson L, Kvarnström N, Molne J, Dahm-Kahler P, Enskog A, Diaz-Garcia C, Olausson M, Brännström M.  Uterus transplantation trial: 1-year outcome. Fertil Steril. 2015;103:199–204. https://doi.org/10.1016/j.fertnstert.2014.09.024. [PubMed] [CrossRef]. 18. Brännström M, Johannesson L, Bokstrom H, Kvarnström N, Molne J, Dahm-Kahler P, Enskog A, Milenkovic M, Ekberg J, Diaz-Garcia C, Gabel M, Hanafy A, Hagberg H, Olausson M, Nilsson L.  Live birth after uterus transplantation. Lancet. 2015;385:607–16. https://doi. org/10.1016/S0140-­6736(14)617281. [PubMed] [CrossRef].

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19. Castellón LAR, Amador MIG, González RED, Eduardo MSJ, Díaz-García C, Kvarnström N, Brännström M. The history behind successful uterine transplantation in humans. JBRA Assist Reprod. 2017;21:126–34. 20. Wei L, Xue T, Yang H, Zhao GY, Zhang G, Lu ZH, Huang YH, Ma XD, Liu HX, Liang SR, Yang F, Chen BL. Modified uterine allotransplantation and immunosuppression procedure in the sheep model. PLoS One. 2013;8:e81300. https://doi.org/10.1371/journal.pone.0081300. 21. Wei L, Xue T, Tao KS, Zhang G, Zhao GY, Yu SQ, Cheng L, Yang ZX, Zheng MJ, Li F, Wang Q, Han Y, Shi YQ, Dong HL, Lu ZH, Wang Y, Yang H, Ma XD, Liu SJ, Liu HX, Xiong LZ, Chen BL. Modified human uterus transplantation using ovarian veins for venous drainage: the first report of surgically successful robotic-assisted uterus procurement and follow-up for 12 months. Fertil Steril. 2017;108:346–56. https://doi.org/10.1016/j.fertnstert.2017.05.039. 22. Fornalik H, Fornalik N.  Uterus transplantation: robotic surgeon perspective. Fertil Steril. 2018;109:365. https://doi.org/10.1016/j.fertnstert.2017.10.038. Epub 2017 Dec 13. PMID: 29246556. 23. Brännström M, Dahm-Kähler P, Kvarnström N. Robotic-assisted surgery in live-donor uterus transplantation. Fertil Steril. 2018;109:256–7. PMID: 29395094. 24. Brucker SY, Brännström M, Taran FA, Nadalin S, Königsrainer A, Rall K, Schöller D, Henes M, Bösmüller H, Fend F, Nikolaou K, Notohamiprodjo M, Rosenberger P, Grasshoff C, Heim E, Krämer B, Reisenauer C, Hoopmann M, Kagan KO, Dahm-Kähler P, Kvarnström N, Wallwiener D. Selecting living donors for uterus transplantation: lessons learned from two transplantations resulting in menstrual functionality and another attempt, aborted after organ retrieval. Arch Gynecol Obstet. 2018;297:675–84. https://doi.org/10.1007/s00404-­017-­4626-­z. Epub 2017 Dec 2. 25. Testa G, McKenna GJ, Gunby RT Jr, Anthony T, Koon EC, Warren AM, Putman JM, Zhang L, de Prisco G, Mitchell JM, Wallis K, Klintmalm GB, Olausson M, Johannesson L. First live birth after uterus transplantation in the United States. Am J Transplant. 2018;18:1270–4. 26. Nováčková M, Pastor Z, Matěcha J, Čekal M, Froněk J, Chmel R. Pregnancy in women with solid-organ transplants. Ceska Gynekol. 2018;83:62–8. 27. Chmel R, Novackova M, Janousek L, Matecha J, Pastor Z, Maluskova J, Cekal M, Kristek J, Olausson M, Fronek J. Revaluation and lessons learned from the first 9 cases of a Czech uterus transplantation trial: four deceased donor and 5 living donor uterus transplantations. Am J Transplant. 2019;19(3):855–64. https://doi.org/10.1111/ajt.15096. PMID: 30151893. 28. Testa G, Johannesson L.  The ethical challenges of uterus transplantation. Curr Opin Organ Transplant. 2017;22(6):593–7. https://doi.org/10.1097/MOT.0000000000000467. Review. 29. Kisu I, Banno K, Matoba Y, Adachi M, Aoki D. Current status of uterus transplantation and approaches for future clinical application in Japan. Transplant Proc. 2018;50:2783–8. https:// doi.org/10.1016/j.transproceed.2018.02.198. Epub 2018 Mar 19. 30. Practice Committee of the American Society for Reproductive Medicine. American Society for Reproductive Medicine Position statement on uterus transplantation: a committee opinion. Fertil Steril. 2018;110:605–10. 31. ASRM Bulletin, Volume 19, Number 31 December 4, 2017. 32. Farrell RM, Falcone T.  Uterine transplant: new medical and ethical considerations. Lancet. 2014;385:581–2. 33. Farrell RM, Falcone T. Uterine transplantation. Fertil Steril. 2014;101:1244–5. 34. Del Priore G, Stega J, Sieunarine K, Ungar L, Smith JR. Human uterus retrieval from a multi-­ organ donor. Obstet Gynecol. 2007;109:101–4. PMID: 17197594. 35. Gauthier T, Piver P, Pichon N, Bibes R, Guillaudeau A, Piccardo A, Pesteil F, Tricard J, Gardet E, Laskar M, Lalloué F, Marquet P, Aubard Y. Uterus retrieval process from brain dead donors. Fertil Steril. 2014;102:476–82. https://doi.org/10.1016/j.fertnstert.2014.04.016. Epub 2014 May 13. 36. Tricard J, Ponsonnard S, Tholance Y, Mesturoux L, Lachatre D, Couquet C, Terro F, Yardin C, Marquet P, Piccardo A, Gauthier T. Uterus tolerance to extended cold ischemic storage after auto-transplantation in ewes. Eur J Obstet Gynecol Reprod Biol. 2017;214:162–7. https://doi. org/10.1016/j.ejogrb.2017.05.013. Epub 2017 May 16.

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37. Richards EG, Flyckt R, Tzakis A, Falcone T.  Uterus transplantation: organ procurement in a deceased donor model. Fertil Steril. 2018;110(1):183. https://doi.org/10.1016/j.fertnstert.2018.04.014. PMID: 28535402. 38. Ejzenberg D, Andraus W, Mendes L, et  al. Livebirth after uterus transplantation from a deceased donor in a recipient with uterine infertility. Lancet. 2019;392(10165):2697–704. https://doi.org/10.1016/S0140-­6736(18)31766-­5. 39. Bruno B, Arora KS. Uterus transplantation: the ethics of using deceased versus living donors. Am J Bioeth. 2018;18(7):6–15. https://doi.org/10.1080/15265161.2018.1478018. 40. Gomel V. Operative laparoscopy: time for acceptance. Fertil Steril. 1989;52:1–11.

Part II Clinical Challenges: Patients and Therapies

Pediatric and Adolescent Gynecology Physiology and Pathology Dvora Bauman

Introduction Pediatric and adolescent gynecology is a subspecialty of gynecology, dealing with gynecological issues in girls from neonatal period till the completion of adolescence (up to age of 20–24 years). This period of life is characterized by dynamic changes in anatomy and physiology of the reproductive system. The changes are unique and may be an important hallmark for later reproductive health and fertility. Any deviation from the “normal” may result in unfavorable and irreversible consequences, as, for example, short stature in case of untreated precocious puberty. The fluctuation in hypothalamic-pituitary-gonad axis activity, characterized by “on-off-on-neuroendocrine switch” of the gonadotropin-releasing hormone (GnRH) secretion, is essential for health and reproduction. Understanding the hormonal and metabolic homeostasis peculiar to this period enables correct approach to many disorders, as, for example, polycystic ovary syndrome (PCOS). The hormonal disturbances of PCOS resemble very much normal changes of puberty; thus implication of adult diagnostic criteria of PCOS in young adolescents may lead to overdiagnosis and unfortunately to unnecessary treatment. Another example is irregularity of menstrual cycle that may be physiologically infrequent once in 80 days at the first menstrual year. Many peculiar disorders may appear for the first time during adolescence as bleeding disorders, in girls with heavy menstrual bleeding or obstructive müllerian anomalies in girls with primary amenorrhea. This chapter will focus on normal anatomy and physiology of pediatric and adolescent reproductive organs and on abnormalities, resulting in pathologic disorders.

D. Bauman (*) Obstetrics and Gynecology, Hadassah University Medical Center, Jerusalem, Israel e-mail: [email protected] © International Academy of Human Reproduction 2021 J. G. Schenker et al. (eds.), Clinical Management of Infertility, Reproductive Medicine for Clinicians 2, https://doi.org/10.1007/978-3-030-71838-1_6

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Anatomy and Physiology The external and internal genitalia are generated at the 6–12 weeks of intrauterine life with completion of the development about 22  weeks. During childhood and adolescence, the system goes through cyclic changes of shrinkage and hypertrophy (Table 1). Those changes correlate with “on-off-on-neuroendocrine switch” of the GnRH secretion and fluctuating levels of estrogen. The clinical expression consists of mini-puberty at postneonatal period, then the “physiologic hibernation” of childhood, and finally pubertal revival [1]. Newborn Infant: During the first few weeks after birth, process known as “mini-­ puberty” takes place. Follicle-stimulating hormone (FSH) rises and may reach preovulatory levels. The hyperestrogenic state manifested by clinical signs as breast budding, hypertrophy of labia majora (Fig. 1), and bloody vaginal discharge occurring in 5% of neonates. This phenomenon is accepted to be physiologic in the first few weeks of life; however some researches link it to higher incidence of endometriosis [2]. Prepubertal Child: Few months after birth, the hypothalamic-pituitary-ovarian axis activity declines gradually, achieving full suppression known as “childhood hibernation” by 2  years. The restraint on GnRH pulse generator is obtained by Table 1  Normal volume of the ovaries and uterus Age Neonate 3 months–1 year 1–2 years 2–8 years 8–16 years

Ovarian volume (mL) 1–3.6 1–2.7 99.9% 99.9% 99.9%

PPV

82%

37%

49%

Future While there is no doubt that noninvasive testing by cell-free DNA will be the preferred choice for a growing number of pregnant women for detecting genetic conditions prenatally in the future, invasive tests will remain the gold standard for a conclusive diagnosis for the foreseeable future. Professional counseling should be based on facts, should be up to date, should be comprehensive, should be empathic, and should be non-directional. The many options now available for screening pose a challenge for the counselors. It always has to be kept in mind that trisomy 21 is associated with only 8% of the anomalies at birth; therefore NIPT should not be associated with the concept of just detecting fetal trisomy 21. It should not be offered without ultrasound screening in pregnancy care. It has always to be kept in mind that every pregnant woman and couple have the right not wanting to know, and this right has to be protected by the healthcare providers and society at large.

References 1. Schmorl G.  Pathologisch-anatomische Untersuchungen über Puerperal-Eklampsie. Leipzig: FCW Vogel; 1893. 2. Lapaire O, Holzgreve W, Oosterwijk JC, Brinkhaus R, Bianchi DW. Placenta.: on trophoblasts in the maternal circulation. Placenta. 2007;28(1):1–5. Epub 2006 Apr 18. 3. Holzgreve W, Hahn S, Zhong XZ, Lapaire O, Hösli I, Tercanli S, Miny P. Genetic communication between fetus and mother: short- and long-time consequences. Am J Obstet Gynecol. 2007:372–82. 4. Skrzypek H, Hui L. Noninvasive prenatal testing for fetal aneuploidy and single gene disorders. Best Pract Res Clin Obstet Gynaecol. 2017;42:26–38. https://doi.org/10.1016/j.bpobgyn.2017.02.007. Epub 2017 Feb 28.

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5. Ganshirt-Ahlert D, Borjesson-Stoll R, Burschyk M, Dohr A, Garritsen HS, Helmer E, Miny P, Velasco M, Walde C, Patterson D, et al. Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting. Am J Reprod Immunol. 1993;30:194–201. 6. Troeger C, Zhong XY, Burgemeister R, Minderer S, Tercanli S, Holzgreve W, Hahn S.  Approximately half of the erythroblasts in maternal blood are of fetal origin. Mol Hum Reprod. 1999;5:1162–5. 7. Bianchi DW, Flint AF, Pizzimenti MF, Knoll JH, Latt SA. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc Natl Acad Sci U S A. 1990;87:3279–83. 8. Simpson JL, Elias S. Isolating fetal cells from maternal blood. Advances in prenatal diagnosis through molecular technology. JAMA. 1993;270:2357–61. 9. Bianchi DW, Simpson JL, Jackson LG, Elias S, Holzgreve W, Evans MI, Dukes KA, Sullivan LM, Klinger KW, Bischoff FZ, Hahn S, Johnson KL, Lewis D, Wapner RJ, de la Cruz F. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diagn. 2002;22:609–15. 10. Zimmermann B, Holzgreve W, Zhong XY, Hahn S. Inability to clonally expand fetal progenitors from maternal blood. Fetal Diagn Ther. 2002;17:97–100. 11. Babochkina T, Mergenthaler S, De Napoli G, Hristoskova S, Tercanli S, Holzgreve W, Hahn S. Numerous erythroblasts in maternal blood are impervious to fluorescent in situ hybridization analysis, a feature related to a dense compact nucleus with apoptotic character. Haematologica. 2005;90:740–5. 12. Anker P, Mulcahy H, Chen XQ, Stroun M. Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients. Cancer Metastasis Rev. 1999;18:65–73. 13. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997;350:485–7. 14. Fan HC, Gu W, Wang J, et al. Non-invasive prenatal measurement of the fetal genome. Nature. 2012;487:320–4. 15. Kitzman JO, Snyder MW, Ventura M, et  al. Noninvasive whole-genome sequencing of a human fetus. Sci Transl Med. 2012;4:137ra76. 16. Costa JM, Benachi A, Gautier E, Jouannic JM, Ernault P, Dumez Y. First-trimester fetal sex determination in maternal serum using real-time PCR. Prenat Diagn. 2001;21:1070–4. 17. Faas BH, Beuling EA, Christiaens GC, von dem Borne AE, van der Schoot CE. Detection of fetal RHD-specific sequences in maternal plasma. Lancet. 1998;352:1196. 18. Gautier E, Benachi A, Giovangrandi Y, Ernault P, Olivi M, Gaillon T, Costa JM. Fetal RhD genotyping by maternal serum analysis: a two-year experience. Am J Obstet Gynecol. 2005;192:666–9. 19. Lo YM, Hjelm NM, Fidler C, Sargent IL, Murphy MF, Chamberlain PF, Poon PM, Redman CW, Wainscoat JS. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med. 1998;339:1734–8. 20. Zhong XY, Holzgreve W, Hahn S. Detection of fetal Rhesus D and sex using fetal DNA from maternal plasma by multiplex polymerase chain reaction. BJOG. 2000;107:766–9. 21. Van der Schoot CE, Soussan AA, Koelewijn J, Bonsel G, Paget-Christiaens LG, de Haas M. Non-invasive antenatal RHD typing. Transus Clin Biol. 2006;13(1–2):53–7. 22. George SM. Millions of missing girls: from fetal sexing to high technology sex selection in India. Prenat Diagn. 2006;26:604–9. 23. Kaiser J. Prenatal diagnosis: an earlier look at baby’s genes. Science. 2005;309:1476–8. 24. Smith RP, Lombaard H, Soothill PW. The obstetrician’s view: ethical and societal implications of non-invasive prenatal diagnosis. Prenat Diagn. 2006;26:631–4. 25. Zhong XY, Hahn S, Holzgreve W.  Prenatal identification of fetal genetic traits. Lancet. 2001;357:310–1. 26. Santacroce R, Vecchione G, Tomaiyolo M, Sessa F, Sarno M, Colaizzo D, Grandone E, Margaglione M. Identification of fetal gender in maternal blood is a helpful tool in the prenatal diagnosis of haemophilia. Haemophilia. 2006;12:417–22. 27. Miny P, Filges L, Tercanli S, Holzgreve W. Fetal diagnosis in eLS. Chichester: Wiley; 2018.

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28. Bell CJ, Dinwiddie DL, Miller NA, et al. Carrier testing for severe childhood recessive diseases by next-generation sequencing. Sci Transl Med. 2011;3(65):65ra4. 29. Stevens B, Krstic N, Jones M, et al. Finding middle ground in constructing a clinically useful expanded carrier screening panel. Obstet Gynecol. 2017;130:279–84. 30. Dan S, Yuan Y, Wang Y, et al. Non-invasive prenatal diagnosis of lethal skeletal dysplasia by targeted capture sequencing of maternal plasma. PLoS One. 2016;11:e0159355. 31. Wax JR, Chard R, Cartin A, et al. Noninvasive prenatal testing: the importance of pretest trisomy risk and posttest predictive values. Am J Obstet Gynecol. 2014;212:548–9. 32. Grace MR, Hardisty E, Green NS, et al. Cell free DNA testing-interpretation of results using an online calculator. Am J Obstet Gynecol. 2015;213:30–2. 33. Alberry M, Maddocks D, Jones M, et al. Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast. Prenat Diagn. 2007;27:415–8. 34. Dar P, Curnow KJ, Gross SJ, et al. Clinical experience and follow-up with large scale single-­ nucleotide polymorphism-based noninvasive prenatal aneuploidy testing. Am J Obstet Gynecol. 2016;211:527.e1–17. 35. Hartwig TS, Ambye L, Sørensen S, Jørgensen FS. Discordant non-invasive prenatal testing (NIPT)—a systematic review. Prenat Diagn. 2017;37:527–39. 36. Mackie FL, Hemming K, Allen S, et  al. The accuracy of cell-free fetal DNA-based non-­ invasive prenatal testing in singleton pregnancies: a systematic review and bivariate meta-­ analysis. BJOG. 2017;124:32–46. 37. Curnow KJ, Wilkins-Haug L, Ryan A, et al. 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.

Kurjak Antenatal Neurodevelopmental Test (KANET): A Useful Tool for Fetal Neurodevelopmental Assessment Asim Kurjak, Milan Stanojevć, Lara Spalldi Barišić, and Erden Radončić

Introduction Fetal functional studies by four-dimensional ultrasound (4D US) and in particular Kurjak Antenatal Neurodevelopmental Test (KANET) are useful and valuable in the assessment of fetal behavior and fetal facial expressions during pregnancy [1–8]. This evaluation is particularly important in latter stages of the gestation, from the third trimester of pregnancy onward, in order to verify fetal well-being and central nervous system (CNS) maturation and development. Additionally, it is possible to determine several functional aspects and separate fetuses with normal, borderline, and abnormal neurobehavior at the high risk for neurological impairments [5, 7–9]. Recently it was also speculated how KANET can help in the evaluation of “fetal psychiatry” and increasing the fetal and the mother’s resilience [10, 11]. The 4D US assessment of fetal behavior including the differential diagnosis between normal and abnormal features is reviewed in our previous works [8–15].

A. Kurjak Medical School, University of Zagreb, Zagreb, Croatia Medical School, University of Sarajevo, Sarajevo, Bosnia and Herzegovina University Sarajevo School of Science and Technology, Sarajevo, Bosnia and Herzegovina e-mail: [email protected] M. Stanojević (*) Department of Obstetrics and Gynecology, Medical School, University of Zagreb, “Sveti Duh” Hospital, Zagreb, Croatia e-mail: [email protected] L. S. Barišić Polyclinic “Veritas”, Zagreb, Croatia E. Radončić Polyclinic for Gynecology and Reproductive Medicine “Repromed”, Zagreb, Croatia © International Academy of Human Reproduction 2021 J. G. Schenker et al. (eds.), Clinical Management of Infertility, Reproductive Medicine for Clinicians 2, https://doi.org/10.1007/978-3-030-71838-1_19

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Growing biological and epidemiological data combined with the evidence from molecular studies suggest that there is prenatal origin of most neurological impairments. An attempt to solve this problem in the past by increasing cesarean section (CS) rates resulted in the same prevalence and incidence of cerebral palsy (CP), while maternal short- and long-term morbidity and mortality due to higher rates of CS are increasing [13].

About the KANET The KANET is the first prenatal test evaluating neurological integrity of the fetus by applying 4D US. In 2005, Kurjak et al. studied fetal behavior through all three trimesters of normal and abnormal pregnancy, confirmed behavioral pattern continuity from prenatal to postnatal life, and set the normal standards for fetal neurobehavioral development [2, 5]. In 2008, Kurjak et al. [8] developed the new scoring system for fetal neurobehavior by 4D US. KANET is structured, integrated, and well developed based on the concept that fetal behavioral patterns are reflecting development and maturation of fetal CNS particularly the brain [9, 14]. It is combining parameters from prenatal evaluation (such as general movement assessment) and postnatal Amiel-Tison Neurological Assessment at Term (ATNAT). Over the several years, KANET was modified and passed the first and second consensus statement in 2010 in Osaka and in 2015 in Bucharest [15, 16]. In Osaka the test was simplified (instead of ten, new test has eight parameters) and standardized to increase the reproducibility, which was followed by many studies from different centers [15, 17–39]. At the second consensus on KANET held in Bucharest [16] in 2015, based on the results of the studies, KANET was recommended as a valuable diagnostic tool to be introduced into everyday clinical practice, becoming even more powerful with introduction of the new 4D US high-definition (HD) image quality [16].

How to Perform KANET? Each of the conducted studies had baseline methods with the regard to a KANET performance. The test is standardized and clear instructions are well known (Box 1) [15, 16]. In the presented studies KANET was performed according to the standards, informed consent of the patient is taken, and ethical committee approval was acquired. Box 1 Instructions for Performing the KANET [15, 16] Instructions for performing KANET test: Gestational age to perform KANET: 3rd trimester of pregnancy, from 28 weeks of gestation onwards The duration of KANET should be between 15 and 20 min Preferably performed in a slightly (15%) right (or left) lateral-tilt position to reduce inferior vena cava compression by the gravid uterus

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Instructions for performing KANET test: At periods that the fetus is awake, if not awake—repeat the test in 30 min or the next day If score is abnormal or borderline, repeat the test every 2 weeks until delivery + do additional Doppler assessment The learning curve for KANET test is very reasonable, cca. 80 test performed Clinicians who apply KANET should have adequate training and experience in low- and high-risk pregnancies Inter-observer and intra-observer variability has to be documented Recommendation regarding the ultrasound machines is to have good resolution and quality for 4D scanning with the frame rate of at least 24 volumes/s 2-year postnatal follow-up should be available and documented for all fetuses in order to draw safe conclusions

The best gestational age at which KANET should be performed is the third trimester of pregnancy, from 28 weeks of gestation onward. The duration of KANET should be from 15 to 20 min (maximum up to 30 min) and preferably performed in a slightly (up to 15%) right (or left) lateral-tilt position to reduce inferior vena cava compression by the gravid uterus. The fetus should be assessed while awake. If this is not the case, and the fetus is quiet for a longer period of time, KANET should be repeated within 30 min or postponed till the following day, at a minimum interval of 14–16 h. If KANET score is abnormal or borderline, it is advisable to repeat it every 2 weeks until delivery and to perform additional Doppler assessment. The number of movements should be documented accordingly [15, 16, 40]. There are in total eight parameters for scoring in the KANET, correlating with the different types of fetal movements (isolated head ante-flexion, cranial sutures and head circumference, isolated eye blinking, facial alteration or mouth opening, isolated leg movement, isolated hand movement or hand-to-face movements, finger movements, and gestalt perception of general movements (Table 1) [15, 16]. The results of KANET are divided into three groups: abnormal, when the score is 0–5, borderline for a score 6–9, and normal for a score 10–16 (Table 1). Ideally postnatal at least 2-year follow-up should be available and documented for all fetuses that KANET has been applied, in order to draw safe conclusions.

The Results of KANET from the Multicentric Studies The results from the previous published studies [41] till the year 2016 on the clinical value on KANET are listed in Table 2. The interpretation and the significance of the studies are published, explained, and well known and could be found in the literature and will not be discussed here in detail. We want to share with you the results from the recent studies of KANET conducted in ten collaborative centers from Croatia, Greece, Japan, India, Brazil, United Arab Emirates, Poland, Bosnia and Herzegovina, Romania, and Turkey, which are presented in the Table 3, as some new insights of KANET have emerged,

Cranial sutures and head circumference

Isolated head anteflection

Sign

Table 1  KANET scoring table [15, 16] 1 Small range (0–3 times of movements)

OVERLAPPING Normal cranial sutures with CRANIAL measurement of HC SUTURES below or above the normal limit (−2 SD) according to GANot fluent (1–5 blinking)

Score 0 ABRUPT

Normal cranial sutures with normal measurement of HC according to GA

2 Variable in full range, many alternation (>3 times of movements)

Sign score

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Facial alteration (grimace or tongue expulsion)

Isolated eye blinking

Not present

Not fluent (1–5 Fluency (>5 times times of alteration) of alteration)

(continued)

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Score Not present

Cramped

Sign Or mouth opening (yawning or mouthing)

Isolated leg movement

Table 1 (continued)

Poor repertoire or Variable in full small in range (0–5 range, many times of movement) alternation (>5 times of movements)

Not fluent (1–5 Fluency (> 5 times times of alteration) of alteration)

Sign score

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Or hand to face movement

Isolated hand movement

Cramped or abrupt

Not fluent (1–5 Fluency (>5 times times of alteration) of alteration)

Poor repertoire or Variable in full small in range (0–5 range, many times of movement) alternation (>5 times of movements)

(continued)

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Total score 0–5 6–9 10–16

Interpretation Abnormal Borderline Normal

Normal Total score

Definitely abnormal

Gestalt perception of GMs Borderline

Score Cramped invariable Smooth and Unilateral or bilateral clenched finger movements complex, variable finger movements fist, (neurological thumb)

Sign Fingers movements

Table 1 (continued) Sign score

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Multi-­ center cohort

Cohort

Talic et al. [38] 2011

2011

Honemeyer et al. [44]

Cohort

2010

Miskovic et al. [43]

Multi-­ center

2010

Kurjak et al. [9]

Study Cohort

Year 2005

Authors Kurjak et al. [42]

Unselected

High risk

Prospective

Prospective

High risk

High risk

Study population High risk

Prospective

Prospective

Study design Retrospective

Unselected

Multiple

Multiple

Multiple

Indication Multiple

100

620

226

288

No. 220

28–38

26–38

20–36

20–38

GA (weeks) 20–36

N/A

15–20

30

30

Time (minutes) 30

Table 2  Previously published studies that applied KANET for the detection of neurological impairment [41]

Positive

Positive

Positive

Positive

Result Positive

(continued)

Summary Introduction of scoring system for antenatal assessment of fetal neurobehavior First proof of prognostic value of KANET for detection of neurological impairment in fetuses. Correlated severe fetal anatomical anomalies, with neurological damage Comparison of prenatal (KANET) and postnatal (ATNAT) findings. KANET differences when applied to high-and low-risk populations KANET’s significance was proved in distinguishing normal from borderline and abnormal cases. Abnormal KANET was predictive of both intrauterine and neonatal death KANET showed a very good negative predictive value, reassuring of a good neurological outcome

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Cohort

Cohort

Lebit et al. [22] 2011

2012

2012

Abo-Yaqoub et al. [45]

Vladareanu et al. [24]

Cohort

Study Multi-­ center cohort

Year Authors Talic et al. [39] 2011

Table 2 (continued)

Prospective

Prospective

Prospective

Study design Prospective

High risk

High risk

Low risk

Study population High risk

Multiple

Multiple

Normal 2D examination

Indication Ventri-­ culomegaly

196

80

144

No. 240

24–38

20–38

7–38

GA (weeks) 32–36

N/A

15–20

15–20

Time (minutes) 10–15

Positive

Positive

Positive

Result Positive

Summary KANET was applied to cases with ventrkulomegaly and compared to low-risk cases. KANET score was worse as the degree of ventrkulomegaly increased, particularly when combined with other anomalies Specific neurobehavioral patterns were described for each stage of pregnancy Differences in KANET scores between high and low-risk cases were shown. All abnormal KANET scores had postnatal confirmation with an abnormal neurological assessment Most fetuses with normal KANET → low risk, those with borderline → IUGR fetuses with increased MCA RI and most fetuses with abnormal KANET → threatened PTD with PPROM. Differences m fetal movements were identified between the two groups. For normal pregnancies → 93.4% of fetuses had normal score, for high-risk pregnancies → 78.5% of fetuses had a normal score

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Cohort

Case study

2013

2014

Kurjak et al. [32]

Predojevic et al. [30]

Study Cohort

Year 2013

Authors Honemeyer et al. [25]

Prospective

Prospective

Study design Prospective

High risk

High and low risk

Study population High and low risk

IUGR

Multiple

Indication Multiple

5

869

No. 56

31–39

28–38

GA (weeks) 28–38

30

20

Time (minutes) 30 maximum

Positive

Positive

Result Positive

(continued)

Summary Introduction of the average KANET icon (combination of the mean value of KANET scores throughout pregnancy). Relationship of fetal diurnal rhythm with the KANET score Statistical differences regarding the distribution of normal, abnormal, and borderline results of KANET between low-risk and high-risk groups found fetal behavior was significantly different between the normal group and the high-risk subgroups KANET could recognize pathologic and borderline behavior in IUGR fetuses with or without blood flow redistribution. Combined assessment of hemodynamic and motoric parameters could enable in better diagnosis and consultation

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Study Cohort

Cohort

Cohort

Cohort

Neto et al. [17] 2015

2016

2016

Hanaok et al. [46]

Hata et al. [47]

Propective

Prospective

Prospective

Study design Prospective

Mixed (male vs female)

Mixed (Asian and Caucasian)

High and low risk

Study population Unselected (high and low risk)

51

167

112

Multiple

Muiltiple

No. 152

Multiple

Indication Multiple (IUGR, PET, and GDM)

3rd trimester

3rd trimester

3rd trimester

GA (weeks) 2nd and 3rd trimesters

20

N/A

20

Time (minutes) N/A

No

Positive

Positive

Result Positive

Summary The neurodevelopmental trimester score was higher m the low risk in comparison to that of high-risk group (p 16 3 2 5 5 18 2 0 35

of 141 patients, 69% had normal KANET score, 27% had borderline KANET score, and only 4% had abnormal KANET score [12]. Abnormal KANET score was present in 5 out of 12 fetuses from high-risk pregnancies (severe IUGR with oligohydramnios and type I diabetes mellitus), borderline scores were present in another 5 out 12, while in 2 out of 12 high-risk fetuses, KANET scores were normal. Out of 129 patients in the low-risk pregnancies, 96 (74.4%) had a normal KANET score, and 33 (25.6%) had a borderline KANET score, while abnormal KANET scores were not found [12]. The data of the postnatal follow-up are not available yet. Panchal et al. in India conducted prospective study on the value of KANET in high-risk pregnancies [12]. There were 135 high-risk patients with singleton pregnancy included in the study during 2 and a half years. This cohort included 13 patients with diabetes mellitus (DM), 18 patients with gestational diabetes (GDM), 31 with pregnancy-induced hypertension (PIH), 16 with thyroid gland pathology, 8 with infections during pregnancy, and 1 with cardiac disease (Table 8). Forty-eight gravid women were older than 35 years of age. KANET was performed twice: between 28 and 32 weeks of gestation and between 34 and 36 weeks of gestation. There were 134 fetuses in the range of normal KANET scores and one with the borderline score. At birth neonates were assessed routinely and with postnatal follow-­up by the pediatrician at the age of 24 h and 1, 3, 6, 9, 12, 18, and 24 months [12]. The neonates and infants till now are on follow-up and now aging from 10 to 18 months. Four infants showed delayed milestones at 16 and 17 months of age (KANET score 11 to 13, mothers had GDM and PIH), respectively [12]. Delay in the milestones was also

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found in patients in whom the KANET score decreased significantly in two assessments of KANET scores. Fetuses with score above 14 were considered to be at a lower risk for any post birth neurological deficit than ones with the lower scores between 11 and 14 [12]. Those who had a decreasing score at the second assessment were also considered at a higher risk than those who maintained the score on both scans. Authors of this Indian study concluded that KANET has a good potential for detecting the fetuses that are at risk to develop neurodevelopmental delay. Antsaklis et al. in Greece included 40 pregnant women with preexisting insulin-­ dependent diabetes mellitus (DM) or gestational insulin-dependent diabetes mellitus (GDM) and compared KANET scores with 40 low-risk pregnant women without diabetes mellitus or GDM [12]. There were no statistically significant differences regarding maternal age (30.5 ± 5.1 years for DM and GDM group vs. 29.8 ± 6.2 years for nondiabetic group) and gestational age (32 ± 1.6 weeks for the DM and GDM group compared to 33 ± 1.2 weeks for the nondiabetic group) [12]. The KANET score was higher in the nondiabetic group of fetuses. It is apparent that there are differences in fetal behavior between diabetic (DM and GDM) and nondiabetic fetuses. They identified movements which were different between diabetic and nondiabetic group of fetuses [48]. Specifically there were no abnormal scores identified in the nondiabetic group, while the borderline scores were found in 15 (11%) and the normal scores in 120 (89%) fetuses. In the diabetic group, the following KANET scores were noted: 2 abnormal (1.3%), 23 borderline (14.7%), and 131 (84%) normal. Further analysis of the parameters that are used for the KANET score showed that the biggest difference between the two groups was for isolated eye blinking, facial expressions, and finger movements [48]. Esin et al. in Turkey started a prospective study of fetal assessment with the KANET in high-risk pregnancies complicated by congenital heart disease (CHD) [12]. Until present they evaluated eight pregnancies all prenatally diagnosed with the following CHD: four fetuses with the d-transposition of the great arteries (d-TGA), one fetus with tetralogy of Fallot, and three fetuses with the interruption of the aortal arch, of whom one was postnatally diagnosed with trisomy 18 and excluded from the study [12]. All KANET scores were normal in fetuses diagnosed prenatally with significant structural CHD.  This study has very low number of cases, but if KANET score is abnormal than other conditions accompanied with CHD, it should be searched for like chromosomopathy. Honemeyer et  al. from Dubai, United Arab Emirates (UAE), studied two cases of fetal akinesia deformation sequence (FADS) [12]. The relevance of KANET scoring system becomes more than obvious in the clinical scenario of FADS, where the examiner is “stuck” by total immobility of a fetus which may otherwise exhibit only minor structural or postural abnormalities [12]. Here, the hint at a fatal neuropathology of the fetus comes essentially from the abnormal result of fetal motor assessment [12]. In this case study, KANET was applied in two cases of polyhydramnios with absent fetal movements/abnormal Gestalt perception and led to the diagnosis of FADS. In both cases KANET score was abnormal (respectively, 3 and 2). One fetus was in breech position with extended legs as shown in Fig. 2 and postnatally in Fig. 3 [12].

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Fig. 2  3D HD live US technique showing hyperflexed hand in the forced position

Fig. 3  Postpartum image of the same baby, notice similarities with the prenatal findings (hyperflexed hand, etc.)

We will show the ultrasound figures of fetuses important for KANET assessment. Figure 4 is showing sequence of images from 4D surface ultrasound imaging technique. Fetus at 29 gestational weeks bothered by umbilical cord around the neck, trying to pull up the umbilical cord and to remove it. Notice targeted, goal-­ oriented movements and fine movements of the fingers which are assessed during KANET. Figure 5 is showing 3D surface US image of the tongue protrusion from a slightly gaping mouth. Figure 6 depicts 3D HD live image of fetal face with low set of ears and face dysmorphology which may affect KANET results. Figure 7 of 3D surface ultrasound imaging showed clenched hand and overlapping toes, which are important during fetal neurological assessment.

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Fig. 4  Sequence of images from the 4D surface ultrasound imaging technique. Fetus at 29 gestational weeks bothered by umbilical cord around the neck, trying to pull up the umbilical cord and to remove it. Notice targeted, goal-oriented movements and fine movements of the fingers

Fig. 5  3D surface US imaging showing tongue protruded from a slightly gaping mouth

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Fig. 6  3D HD live technique showing low set of ears and face dysmorphology which may affect KANET test results Fig. 7  3D surface US imaging showing clenched hand and overlapping toes

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Discussion In all mentioned studies, the clinical features and value of KANET were assessed and clinical outcomes evaluated postnatally. KANET was used among random pregnancies, in specific patient groups such as low- and high-risk pregnancies and in pregnancies complicated by diabetes mellitus (DM) or gestational diabetes mellitus. In one study the fetal acoustic stimulation was applied during KANET to evaluate its advantages. In one case study, KANET was used to evaluate its diagnostic efficiency in detecting abnormal fetal behavior and neuromorbidity. When KANET was used in high- and low-risk pregnancies, it is shown that more abnormal and borderline cases come from the high-risk group. Apart from abnormal cases, with the respect to the scoring, it was clear that all fetuses with the borderline scores had to be followed postnatally closely, because in this group more false negatives and false positives can be found, respectively. We found that in the specific high-risk pregnancies such as diabetic patients, there appear to be differences in the fetal behavior between diabetic and nondiabetic fetuses. We identified specific KANET parameters, fetal movements that were different between the two groups [51]. In the study where acoustic stimulation during KANET testing was performed, it was concluded that it does not evoke more fetal activity and it does not seem to be useful for KANET testing. Possible explanations to this include the low number of studied fetuses and choice of acoustic stimulation. Further, fetal behavioral patterns are reflecting development and maturation of fetal CNS particularly the brain. At the moment, follow-up of most studies is still ongoing, and complete data will be available soon. The main limitations of some studies could be the fact that number of patients was small and that the postnatal follow-up is not always standardized or even not available in the more rural parts of the countries. However, we do believe that this multicenter study offers a complete approach for the assessment of fetal behavior, by using the KANET score and by simply observing not only the quantity but also the quality of fetal movements with respect to increasing fetal age in relation to maturation of CNS.  Some of the studies are still ongoing, and complete postnatal follow-up data are still not available at present. Understanding and placing the evolution of fetal movements in the timeline by 4D US throughout pregnancy and how these movements reflect the development and integrity of fetal nervous system were and still are a great challenge [40]. It was found that during the first trimester of pregnancy, the development of the frequency and the complexity of fetal movements are more important, while during the second trimester, the variation of fetal movements develops, with more detailed movements (facial expressions and eye blinking) appearing at the end of this trimester [37]. Finally, at the end of third trimester, the number of fetal movements declines as a result of the increase of fetal rest periods, due to fetal brain maturation, which is experienced by the most pregnant women near-term [40]. It is important to perform the postnatal follow-up of infants after KANET testing in pregnancy. Postnatally both minor and major neurological disability can be detected, and the most important is not to miss cerebral palsy (CP), diagnosis of which is very demanding. By now it is evident that CP is not one condition that

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presents uniformly. CP shares frequent clinical features of nonprogressive disability of motor control (movement) and posture [13]. CP is a very heterogeneous condition, with different clinical types, multiple causes, and different associated comorbidities [13]. More proper name in the future would be cerebral palsy spectrum of disorders (CPS). Even more difficult circumstance in making the diagnosis is the fact that the diagnosis of CP can be rejected or made only after 2 years of age or even later, due to still ongoing neurological development which further continues during childhood [21, 23, 36]. The common belief that CP is exclusively reserved for fetal hypoxia and ischemia during and around the delivery was overthrown since there is clear evidence from epidemiological studies that the origins of CPs are mainly prior to labor (more than 90%) [13]. This is apparent from the data presented on the prevalence of CP compared with cesarean birth rates over the last 50 years [10]. Regardless of the sixfold increase in cesarean section birth, worldwide CP rates did not change at all; prevalence stayed stable at about 2–2.5/1000 births [13]. This elucidates once again that CP has almost nothing to do with the mode of delivery. Recent genetic studies link sporadic cases of CP with the genetic causes [13]. Many studies elucidated the fact that genetic risk for autism can influence multiple aspects of behavior, but alone it is often not enough to provoke the full spectrum of behavior needed for a diagnosis of autism spectrum disorder (ASD) [40, 52–63]. Another aspect of CP is that it is a lifelong disability, mostly starting in the childhood, but it is not a condition that affects exclusively children [63]. Even though the initial injury that induces the brain damage by definition does not aggravate through the lifetime, the effects of CP manifest differently throughout the life span [50, 51, 53, 61–66]. High prevalence of CP along with the high percentage of children surviving into adulthood, no cure, and not known course of CP over the lifetime make this condition extremely relevant for clinical practice. An attempt to detect the fetuses, by using the KANET, as early as possible in prenatal period which could be at higher risk for development of CP, ASD, or other neurological impairments becomes highly relevant as well. Latest state of the art 4D US imaging is advanced so far, giving clinicians the power to be more precise and efficient in their diagnosis. Now more than ever before, we need to find the way to detect these fetuses at risk for neurodevelopmental impairments as early as possible. Data from the studies done in the past 10 years are summarized in the Table 1, and all relevant details and conclusions are discussed in our previous works [40, 41].

Are There Any Obstacles for the Use of KANET? One of the obstacles is the high prize of the 4D US machines. This is still reality in the less-developed countries; unfortunately these are also the places that could have the highest profit out of the KANET due to higher incidence of CP and other

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neurodevelopmental impairments. Luckily, technology sector is growing really fast, and the cost of the ultrasound machines and equipment are becoming more affordable by each day. Education and skills training on how to perform KANET and good-quality fetal ultrasound should not be an issue since there are many trained and licensed doctors who could help to teach others.

Conclusion Over the last 10 years, KANET has become a valuable functional test for assessing the fetal neurological status and identifying fetuses at high risk for various neurological impairments as we could see from the many conducted studies so far that revealed the value of the test in daily clinical work. Taking everything into consideration, as we look into the future, identifying the factors that influence the neurodevelopment prenatally and detecting altered fetal behavior early is clinically very relevant. Collaborative data from multicenter studies, with the multi-ethnic population, allows generalizability to wider population and underpins breakthrough in knowledge about the fetal brain function. In the future there may be also possibility for additional screening for the CP cases in high-risk fetuses or newborns with a panel of known targeted genes. Personalized approach to each fetus at high risk for CP is needed as well as holistic approach to management and treatment. Ultimately, we all strive to prevent the cerebral palsy spectrum (CPS) disorders, ASD, and other neurological impairments, and we are convinced that KANET is helpful to realize this important aim. This is expected from both patients and healthcare professionals.

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48. Antsaklis P, Porovic S, Daskalakis G, Kurjak A. 4D assessment of fetal brain function in diabetic patients. J Perinat Med. 2017;45(6):711–5. https://doi.org/10.1515/jpm-­2016-­0394. 49. Hata T, Kanenishi K, Mori N, AboEllail MAM, Hanaoka U, Koyano K, Kato I, Kusaka T. Prediction of postnatal developmental disabilities using the antenatal fetal neurodevelopmental test: KANET assessment. J Perinat Med. 2018;47(1):77–81. https://doi.org/10.1515/ jpm-­2018-­0169. 50. Tan KH, Smyth RMD, Wei X. Fetal vibroacoustic stimulation for facilitation of tests of fetal wellbeing. Cochrane Database Syst Rev 2013;(12):CD002963. https://doi.org/10.1002/14651858. CD002963.pub2. 51. Barker DJ.  The developmental origins of chronic adult disease. Acta Paediatr Suppl. 2004;93(446):26–33. 52. Rudra A, Belmonte MK, Soni PK, Banerjee S, Mukerji S, Chakrabarti B. Prevalence of autism spectrum disorder and autistic symptoms in a school-based cohort of children in Kolkata, India. Autism Res. 2017;10:1597–605. 53. Robinson EB, St Pourcain B, Anttila V, Kosmicki JA, Bulik-Sullivan B, Grove J, et al. Genetic risk for autism spectrum disorders and neuropsychiatric variation in the general population. Nat Genet. 2016;48:552–5. https://doi.org/10.1038/ng.3529. 54. O’Callaghan ME, MacLennan AH, Gibson CS, McMichael GL, Haan EA, Broadbent JL, Goldwater PN, Dekker GA, Australian Collaborative Cerebral Palsy Research Group. Epidemiologic associations with cerebral palsy. Obstet Gynecol. 2011;118(3):576–82. https:// doi.org/10.1097/AOG.0b013e31822ad2dc. 55. Kelly, et al. Impaired development of the cerebral cortex in infants with congenital heart disease is correlated to reduced cerebral oxygen delivery. Nat Sci Rep. 2017;7:15088. https://doi. org/10.1038/s41598-­017-­14939-­z. 56. Ronan L, Fletcher PC.  From genes to folds: a review of cortical gyrification theory. Brain Struct Funct. 2015;220(5):2475–83. 57. Armstrong E, Schleicher A, Omran H, Curtis M, Zilles K. The ontogeny of human gyrification. Cereb Cortex. 1995;5(1):56–63. 58. Diallo FB, Fombonne E, Kisely S, Rochette L, Vasiliadis HM, Vanasse A, et al. Prevalence and correlates of autism spectrum disorders in Quebec. Can J Psychiatry. 2018;63:231–9. 59. Raina SK, Chander V, Bhardwaj AK, Kumar D, Sharma S, Kashyap V, et al. Prevalence of autism spectrum disorder among rural, urban, and tribal children. J Neurosci Rural Pract. 2017;8:368–74. 60. Zablotsky B, Black LI, Maenner MJ, Schieve LA, Blumberg SJ.  Estimated prevalence of autism and other developmental disabilities following questionnaire changes in the 2014 national health interview survey. Natl Health Stat Rep. 2015;13:1–20. 61. Delobel-Ayoub M, Klapouszczak D, van Bakel MME, Horridge K, Sigurdardottir S, Himmelmann K, et al. Prevalence and characteristics of autism spectrum disorders in children with cerebral palsy. Dev Med Child Neurol. 2017;59:738–42. 62. Nardone S, Elliott E. The interaction between the immune system and epigenetics in the etiology of autism spectrum disorders. Front Neurosci. 2016;10:329. https://doi.org/10.3389/ fnins.2016.00329. 63. Haak P, Lenski M, Hidecker MJ, Li M, Paneth N. Cerebral palsy and aging. Dev Med Child Neurol. 2009;51(Suppl 4):16–23. https://doi.org/10.1111/j.1469-­8749.2009.03428.x. 64. Kunze A, Achilles A, Keiner S, Witte OW, Redecker C.  Two distinct populations of doublecortin-­positive cells in the perilesional zone of cortical infarcts. BMC Neurosci. 2015;16:20. https://doi.org/10.1186/s12868-­015-­0160-­8. Published 2015 Apr 15. 65. Kunze A, Grass S, Witte OW, Yamaguchi M, Kempermann G, Redecker C.  Proliferative response of distinct hippocampal progenitor cell populations after cortical infarcts in the adult brain. Neurobiol Dis. 2006;21(2):324–32. https://doi.org/10.1016/j.nbd.2005.07.013. 66. Spalldi Barišić L, Kurjak A, Kimata Pooh R.  Three-dimensional sonography in fetal syndromes. In: Merz E, Kurjak A, editors. Donald School textbook current status of clinical use of 3d/4d ultrasound in obstetrics and gynecology. 2nd ed: Jaypee Brothers Medical Pub; 2019.

Part V Recurrent Implantation Failure and Pregnancy Loss

Recurrent Implantation Failure Natali Schachter-Safrai, Alex Simon, and Neri Laufer

Introduction Recurrent implantation failure (RIF) is a term used in the in vitro fertilization (IVF) field when transferred embryos fail to implant following several treatment cycles. In the absence of a formal criterion, various definitions for RIF are currently to be found in the literature. Some practitioners recommend defining RIF as a failure of at least three consecutive IVF-ET attempts in which good-quality embryos are transferred [1], while others take into consideration maternal age and define RIF as the failure to achieve clinical pregnancy after four transfers of good-quality embryos or following at least three fresh or frozen IVF cycles in women younger than 40 [2]. Implantation success involves two main components: a good-quality embryo that has the capability to implant and a receptive endometrium. The interaction between the two, involving many mediators including the maternal immune system, is mandatory for successful implantation and subsequent normal placentation. Therefore, the etiologies attributed to RIF concern either factors associated with the embryo or the mother. The aim of this chapter is to review the putative etiologies and possible treatments for RIF and to offer a clinical approach to the management of patients suffering from it.

N. Schachter-Safrai · A. Simon · N. Laufer (*) Department of Obstetrics and Gynecology, Hadassah Hebrew University Hospital, Jerusalem, Israel e-mail: [email protected]; [email protected]; [email protected] © International Academy of Human Reproduction 2021 J. G. Schenker et al. (eds.), Clinical Management of Infertility, Reproductive Medicine for Clinicians 2, https://doi.org/10.1007/978-3-030-71838-1_20

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Maternal Factors Uterine Anatomy Anatomical malformations of the uterus, including Müllerian (septate uterus and bicornuate uterus) or acquired abnormalities (uterine fibroids, endometrial polyps, intrauterine synechiae, and hydrosalpinges), might interfere with normal implantation. Patients diagnosed with RIF should have their uterine cavity assessed by hysteroscopy, with the use of three-dimensional ultrasound (US) and hysterosalpingogram (HSG) as additional tools. If uterine abnormality is diagnosed, treatment options to be considered include septectomy, polypectomy, adhesiolysis, and myomectomy—particularly of submucosal fibroids [3–5]. Although not considered an integral part of the uterine cavity, several mechanisms by which hydrosalpinx can interfere with implantation have been suggested, including impairment of embryo development [6], a negative impact on the endometrial receptivity [7], and mechanical flushing of the embryo from the uterine cavity [4]. Therefore, if diagnosed in a patient with RIF, it is recommended that hydrosalpinx be removed [4].

Thrombophilia Similar to recurrent pregnancy loss, the possible pathophysiology by which thrombophilia results in RIF is by reducing blood flow to the endometrium, thereby reducing endometrial receptivity. Yet, the association between RIF and thrombophilia, either acquired or inherited, is the subject of some debate. Some investigators have reported higher rates of inherited thrombophilia in women with RIF [8], while others have not demonstrated such an association [9]. As a consequence, some controversy exists as to the yield of using antithrombotic agents in women with RIF. Some investigators now advocate the use of low-molecular-weight heparin (LMWH) [10], while other studies demonstrated no advantage when compared to controls [11].

Infection Chronic endometritis is associated with RIF [12]. Chronic endometritis is a condition that has traditionally been diagnosed on visualization of characteristic findings with hysteroscopy, histological examination, and bacterial culture, with newer and more promising molecular methods recently described [13]. The presence of bacteria causes inflammation, which results in disrupted endometrial receptivity. A trend toward higher implantation rates in patients cured of

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their infection was demonstrated, with statistically significant higher pregnancy and live birth rates in the subsequent IVF cycle [12]. The varieties of endometrial bacterial flora have been suggested to affect implantation. A recent study showed that women with lactobacillus-dominant endometrium had higher implantation and live birth rates as compared to women with non-lactobacillus-dominant endometrium [14].

Immunological Factors Several components of the immune system have been implicated in the pathophysiology of RIF. A role has been suggested for natural killer (NK) cells, with the demonstration of higher NK cell levels in the periphery in woman with RIF [15]. Higher levels of TH1 cells expressed by their cytokine, TNFα, and elevated TH1\ TH2 ratio have also been shown to be associated with RIF.  Moreover, increased mean ratios of TNFα\IL4 and TNFα\IL10 were measured in a woman with RIF [16]. The use of Tacrolimus, an immunosuppressive drug, in patients with elevated TH1\TH2 ratio, has had encouraging results, demonstrating improved implantation and live birth rates compared to controls [17]. The HLA compatibility system is one of the routes by which immunomodulation occurs during implantation and pregnancy. An inadequate response of the maternal immune system has been implicated due to HLA allele sharing of the couple and might result in RIF. If similarity is found after HLA crossmatch, then two courses of high-dose IVIG should be offered: the first before ET, followed by a second course after visualization of a heartbeat [18].

The Endometrium During the menstrual cycle, the endometrium exhibits morphologic and histologic changes that enable successful implantation. Only after the completion of these changes, and only within a certain time interval defined as the “window of implantation,” is the embryo able to attach, invade, and implant in the endometrium. For the implantation window to occur, the endometrium must first proliferate, and following ovulation, it must undergo molecular changes in order to become receptive. Endometrial thickness measured by US is used to assess these morphological changes and their adequacy during the follicular phase. The minimal endometrial thickness required for successful implantation varies among studies, ranging between 6 and 8 mm. Some controversy surrounds the association between endometrial thickness and successful implantation, with some teams reporting a strong association [19–21], while others fail to establish such association [22, 23]. Thin endometrium is hard to manage. Limited treatment options have been described including GCSF [24, 25], sildenafil citrate [26], and vitamin E [27]. There are studies describing increased implantation, pregnancy, and live birth rates in a woman with RIF who underwent local endometrial injury induced by endometrial

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biopsy catheter and sampling [28, 29]. The possible mechanism might be attributed to increased local cytokines such as leukemia inhibitory factor (LIF), which is also required for implantation [30]. In order to become receptive, a hormonally mediated, coordinated development of the endometrial glands and stroma must proceed, with certain markers, such as leukemia inhibitory factor (LIF), mouse ascites Golgi (MAG), and cyclin E, expressed during the appropriate time [31–33]. Not only is the window of implantation delayed when uncoupling of the stromal and glandular development occurs [34], but also when a timed development takes place, but the pattern of certain markers is disrupted [32, 33]. This explains why endometrial dating based on histology alone is not a sufficient method to detect endometrial developmental abnormalities, since it does not relate to the molecular changes exhibited by the endometrium. Based on this understanding, the endometrial receptivity array (ERA) test was developed in order to date the endometrium based on molecular transformation and subsequently synchronize ET according to it. However, results so far have been ambiguous [35, 36].

The Embryo Genetics Abnormal karyotype of the embryo is one of the major reasons for failure of implantation and miscarriage. Balanced translocations are the most common chromosomal abnormalities found in couples with RIF [37, 38]. Therefore, karyotype analysis is advised in patients with RIF and their partners, since PGD (preimplantation genetic diagnosis) can be offered to select only embryos with a normal chromosomal excluding those with balanced translocations. Mosaicism, inversions, and deletions have also been described in increased incidences of patients with RIF [38]. Analysis of the genetic material obtained from embryos from RIF patients has revealed not only more than twice as many chromosomal abnormalities [39] but also more complex genetic abnormalities [40]. Although preimplantation genetic screening (PGS) has been suggested to be efficient in diagnosing chromosomal abnormalities in RIF patients with normal karyotype, large controlled studies have failed to confirm its advantage [41]. The absence of a clinical effect of PGS might be attributed either to the presence of mosaicism in abnormal embryos [42] or to the capability of abnormal early cleavage embryos to correct themselves throughout cell divisions [43]. Array comparative genomic hybridization (aCGH) is a high-resolution, more sophisticated method to detect deletion or excess in the DNA content that may reduce the implantation potential. Similar to standard CGH, it cannot detect balanced translocation, as the total amount of DNA tested is the same as in the control sample. Further drawbacks include its cost and availability and the fact that it usually takes more than 24  h. Thus, if preformed on a blastocyst, this approach

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necessitates abandoning the option of fresh ET. Finally, the test might detect abnormalities of unknown significance. To date, the data from studies performing PGS via aCGH are limited.

Embryonic Factors The preference to transfer blastocysts rather than cleavage stage embryos is becoming increasingly popular in many IVF units. Culturing an embryo to the blastocyst stage serves two goals: the first is to improve the selection of embryos for transfer and the second is to enable a better synchronization between the embryo and the endometrium. These advantages enable the transfer of fewer embryos, thus decreasing the risk of multiple pregnancies. Regarding the RIF population, several reports have demonstrated the superiority of blastocyst compared to cleavage stage embryo transfer, manifested by higher implantation, pregnancy, and live birth rates [44–46], though with a higher cancellation rate [46]. Sequential ET has been proposed to overcome this disadvantage. This method increases the odds of striking the “window of implantation” and subsequently improving pregnancy rates. Indeed, some reports have demonstrated increased pregnancy rates for sequential ET compared to a control group in which a similar mean number of embryos was transferred [47, 48]. Hatching of the blastocyst through the zona pellucida is an essential step in promoting implantation. Abnormalities in the hatching process have been suggested as part of the pathophysiology responsible for implantation failure. Hence, assisted hatching is another treatment option available for patients with RIF, which has demonstrated increased implantation rate [49–51].

Paternal Factor Low Sperm Quality Increased incidence of chromosomal abnormalities has been described in male partners of women with RIF [52]. There are reports demonstrating a correlation between poor sperm morphology and a high rate of DNA fragmentation [53], which in turn is associated with decreased fertilization and implantation rates and low embryo quality [54–57]. The effect on embryo quality might be expressed only on day 3 after fertilization, when the paternal genome is activated. Consequently, a high-­ quality cleavage stage embryo might not properly reflect its actual developmental potential. Therefore, advanced morphological analysis of sperm may be included as part of the assessment of patients with RIF. Intracytoplasmic morphologically selected sperm injection (IMSI) is a technique by which motile, morphologically normal sperms are selected under high

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magnification (*6000) in order to detect minor morphological changes not identified in conventional ICSI.  The assumption underlying this method is that these subtle morphological differences might reflect defects in the DNA content. The benefits of IMSI over ICSI have been a matter of debate, with some investigators demonstrating higher implantation, pregnancy, and live birth rates [58] while others failed to show any effect [59].

Conclusion Recurrent implantation failure is a broad classification incorporating several etiologies and possible treatments. The clinical approach to this problem must be a systematic one, taking into consideration both the mother and the embryo (Table 1), followed by a personalized treatment plan developed according to the findings of clinical evaluation. Table 1  Evaluation of repeated implantation failure Etiology Anatomical

Diagnosis Hysteroscopy 3DUS HSG

Impaired endometrial receptivity

Endometrial thickness on US ERA test

Infection

Immunological factors

Hysteroscopy Tissue biopsy for histological examination and culture HLA crossmatch

Thrombophilia

Tests for thrombophilia

Male factor

Motile sperm organelle morphology examination (MSOME) under high magnification Karyotype

Genetics Embryo development

Treatment Salpingectomy Myomectomy Polypectomy Salpingectomy Adhesiolysis GCSF Sildenafil citrate Vitamin E LIF Endometrial injury HD estrogen treatment Aspirin Surrogacy Antibiotics

IVIG Tacrolimus LMWH Aspirin IMSI PGD Blastocyst transfer Sequential transfer Assisted hatching

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Evolving methods, including continuous image monitoring of the cultured embryo and “omics” technologies, will shed more light on embryonal development and the processes involved in the endometrium during implantation. These technologies might help to better synchronize the crosstalk between the embryo and endometrium, resulting in improved implantation rates.

References 1. Simon A, Laufer N. Assessment and treatment of repeated implantation failure (RIF). J Assist Reprod Genet. 2012;29:1227–39. 2. Coughlan C, et al. Recurrent implantation failure: definition and management. Reprod Biomed Online. 2014;28:14–38. 3. Margalioth EJ, Ben-Chetrit A, Gal M, Eldar-Geva T. Investigation and treatment of repeated implantation failure following IVF-ET. Hum Reprod. 2006;21:3036–43. 4. Practice Committee of American Society for Reproductive Medicine, Society of Reproductive Surgeons. Salpingectomy for hydrosalpinx prior to in vitro fertilization. Fertil Steril. 2008;90(5 Suppl):S66–8. 5. Demirol A, Gurgan T. Effect of treatment of intrauterine pathologies with office hysteroscopy in patients with recurrent IVF failure. Reprod Biomed Online. 2004;8:590–4. 6. Kodaman PH, Arici A, Seli E. Evidence-based diagnosis and management of tubal factor infertility. Curr Opin Obstet Gynecol. 2004;16:221–9. 7. Bildirici I, Bukulmez O, Ensari A, Yarali H, Gurgan T. A prospective evaluation of the effect of salpingectomy on endometrial receptivity in cases of women with communicating hydrosalpinges. Hum Reprod. 2001;16:2422–6. 8. Azem F, et al. Increased rates of thrombophilia in women with repeated IVF failures. Hum Reprod. 2004;19:368–70. 9. Martinelli I, et al. Embryo implantation after assisted reproductive procedures and maternal thrombophilia. Haematologica. 2003;88:789–93. 10. Qublan H, et al. Low-molecular-weight heparin in the treatment of recurrent IVF-ET failure and thrombophilia: a prospective randomized placebo-controlled trial. Hum Fertil (Camb). 2008;11:246–53. 11. Berker B, Taşkin S, Kahraman K, Taşkin EA, Atabekoğlu C, Sönmezer M. The role of low-­ molecular-­weight heparin in recurrent implantation failure: a prospective, quasi-randomized, controlled study. Fertil Steril. 2011;95:2499–502. 12. Cicinelli E, et  al. Prevalence of chronic endometritis in repeated unexplained implantation failure and the IVF success rate after antibiotic therapy. Hum Reprod. 2015;30:323–30. 13. Moreno I, et  al. The diagnosis of chronic endometritis in infertile asymptomatic women: a comparative study of histology, microbial cultures, hysteroscopy, and molecular microbiology. Am J Obstet Gynecol. 2018;218:602.e1–602.e16. 14. Moreno I, et al. Evidence that the endometrial microbiota has an effect on implantation success or failure. Am J Obstet Gynecol. 2016;215:684–703. 15. Sacks G, Yang Y, Gowen E, Smith S, Fay L, Chapman M.  Detailed analysis of peripheral blood natural killer cells in women with repeated IVF failure. Am J Reprod Immunol. 2012;67:434–42. 16. Kwak-Kim JYH, et al. Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF. Hum Reprod. 2003;18(4):767–73. 17. Nakagawa K, et al. Immunosuppression with tacrolimus improved reproductive outcome of women with repeated implantation failure and elevated peripheral blood TH1/TH2 cell ratios. Am J Reprod Immunol. 2015;73(4):353–61.

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18. Elram T, Simon A, Israel S, Revel A, Shveiky D, Laufer N. Treatment of recurrent IVF failure and human leukocyte antigen similarity by intravenous immunoglobulin. Reprod Biomed Online. 2005;11:745–9. 19. Bergh C, Hillensjo T, Nilsson L. Sonographic evaluation of the endometrium in in vitro fertilization IVF cycles. Away to predict pregnancy? Acta Obstet Gynecol Scand. 1992;71:624–8. 20. Check JH, Nowroozi K, Choe J, Dietterich C. Influence of endometrial thickness and echo patterns on pregnancy rates during in vitro fertilization. Fertil Steril. 1991;56:1173–5. 21. Dickey RP, Olar TT, Curole DN, Taylor SN, Rye PH.  Endometrial pattern and thickness associated with pregnancy outcome after assisted reproduction technologies. Hum Reprod. 1992;7:418–21. 22. Khalifa E, Brzyski RG, Oehninger S, Acosta AA, Muasher SJ. Sonographic appearance of the endometrium: the predictive value for the outcome of in vitro fertilization in stimulated cycles. Hum Reprod. 1992;7:677–780. 23. Oliveira JB, Baruffi RL, Mauri AL, Petersen CG, Campos MS, Franco JG Jr. Endometrial ultrasonography as a predictor of pregnancy in an in  vitro fertilization programme. Hum Reprod. 1993;8:1312–5. 24. Gleicher N, et al. A pilot cohort study of granulocyte colony-stimulating factor in the treatment of unresponsive thin endometrium resistant to standard therapies. Hum Reprod. 2013;28:172–7. 25. Li J, Mo S, Chen Y.  The effect of G-CSF on infertile women undergoing IVF treatment: a meta-analysis. Syst Biol Reprod Med. 2017;63:239–47. 26. Sher G, Fisch JD.  Effect of vaginal sildenafil on the outcome of in  vitro fertilization (IVF) after multiple IVF failures attributed to poor endometrial development. Fertil Steril. 2002;78:1073–6. 27. Miwa I, Tamura H, Takasaki A, Yamagata Y, Shimamura K, Sugino N. Pathophysiologic features of “thin” endometrium. Fertil Steril. 2009;91:998–1004. 28. Barash A, Dekel N, Fieldust S, Segal I, Schechtman E, Granot I. Local injury to the endometrium doubles the incidence of successful pregnancies in patients undergoing in vitro fertilization. Fertil Steril. 2003;79:1317–22. 29. Siristatidis C, Kreatsa M, Koutlaki N, Galazios G, Pergialiotis V, Papantoniou N. Endometrial injury for RIF patients undergoing IVF/ICSI: a prospective nonrandomized controlled trial. Gynecol Endocrinol. 2017;33:297–300. 30. Zeyneloglu HB, Onalan G. Remedies for recurrent implantation failure. Semin Reprod Med. 2014;32:297–305. 31. Nachtigall MJ, Kliman HJ, Feinberg RF, Olive DL, Engin O, Arici A. The effect of leukemia inhibitory factor (LIF) on trophoblast differentiation: a potential role in human implantation. J Clin Endocrinol Metab. 1996;81:801–6. 32. Catalanotti JS, Spandorfer SD, Barmat LI, Rosenwaks Z, McSweet JC, Kliman HJ. Mouse ascites Golgi mucin expression abnormalities in natural cycle endometrial biopsies predict subsequent in  vitro fertilization-embryo transfer (IVF-ET) failure in patients with previous IVF-ET failures. Fertil Steril. 2006;85:255–8. 33. Dubowy RL, et  al. Improved endometrial assessment using cyclin E and p27. Fertil Steril. 2003;80:146–56. 34. Benadiva CA, Metzger DA. Superovulation with human menopausal gonadotropins is associated with endometrial gland-stroma dyssynchrony. Fertil Steril. 1994;61:700–4. 35. Ruiz-Alonso M, et  al. The endometrial receptivity array for diagnosis and personalized embryo transfer as a treatment for patients with repeated implantation failure. Fertil Steril. 2013;100(3):818–24. 36. Hashimoto T, et  al. Efficacy of the endometrial receptivity array for repeated implantation failure in Japan: a retrospective, two-centers study. Reprod Med Biol. 2017;16:290–6. 37. De Sutter P, Stadhouders R, Dutré M, Gerris J, Dhont M. Prevalence of chromosomal abnormalities and timing of karyotype analysis in patients with recurrent implantation failure (RIF) following assisted reproduction. Facts Views Vis Obgyn. 2012;4:59–65.

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38. Raziel A, Friedler S, Schachter M, Kasterstein E, Strassburger D, Ron-El R. Increased frequency of female partner chromosomal abnormalities in patients with high-order implantation failure after in vitro fertilization. Fertil Steril. 2002;78:515–9. 39. Pehlivan T, et al. Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients. Reprod Biomed Online. 2003;6:232–7. 40. Voullaire L, Collins V, Callaghan T, McBain J, Williamson R, Wilton L. High incidence of complex chromosome abnormality in cleavage embryos from patients with repeated implantation failure. Fertil Steril. 2007;87:1053–8. 41. Harper JC, Sengupta SB.  Preimplantation genetic diagnosis: state of the ART 2011. Hum Genet. 2012;131:175–86. 42. Hatirnaz S, et  al. Pre-implantation genetic screening among women experiencing recurrent failure of in vitro fertilization. Int J Gynaecol Obstet. 2017;137:314–8. 43. Greco E, Minasi MG, Fiorentino F. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts. N Engl J Med. 2015;19(373):2089–90. 44. Cruz JR, Dubey AK, Patel J, Peak D, Hartog B, Gindoff PR. Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril. 1999;72:218–20. 45. Guerif F, et  al. Efficacy of blastocyst transfer after implantation failure. Reprod Biomed Online. 2004;9:630–6. 46. Levitas E, et al. Blastocyst stage embryo transfer in patients who failed to conceive in three or more day 2–3 embryo transfer cycles: a prospective randomized study. Fertil Steril. 2004;81:567–71. 47. Almog B, et al. Interval double transfer improves treatment success in patients with repeated IVF/ET failures. J Assist Reprod Genet. 2008;25:353–7. 48. Loutradis D, et al. A double embryo transfer on days 2 and 4 or 5 improves pregnancy outcome in patients with good embryos but repeated failures in IVF or ICSI. Clin Exp Obstet Gynecol. 2004;31:63–6. 49. Cohen J, Alikani M, Reing M, Troubridge J, Tucker M. Selective assisted hatching of human embryos. Ann Acad Med Singap. 1992;21:565–70. 50. Cohen J, Alikni M, Troubridge J, Rozenwaks Z.  Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod. 1992;7:685–91. 51. Stein A, Rufas O, Amit S, Fisch B, Avrech O.  Assisted hatching by partial zona pellucida dissection of human pre-embryos in patients with recurrent implantation failure. Fertil Steril. 1995;63:838–41. 52. Saleh RA, et al. Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril. 2002;78:313–8. 53. Skowronek F, et al. DNA sperm damage correlates with nuclear ultrastructural sperm defects in teratozoospermic men. Andrologia. 2012;44:59–65. 54. Bungum M, Humaidan P, Spano M, Jepson K, Bungum L, Giwercman A. The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod. 2004;19:1401–8. 55. Jiang H, He RB, Wang CL, Zhu J. The relationship of sperm DNA fragmentation index with the outcomes of in-vitro fertilisation-embryo transfer and intracytoplasmic sperm injection. J Obstet Gynaecol. 2011;31:636–9. 56. Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aamold ET, Evenson DP.  Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril. 2003;80:895–902. 57. Virro MR, Larson-Cook KL, Evenson DP. Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in  vitro fertilization and intracytoplasmic sperm injection cycles. Fertil Steril. 2004;81:1289–95.

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58. Shalom-Paz E, et al. Can intra cytoplasmatic morphologically selected sperm injection (IMSI) technique improve outcome in patients with repeated IVF-ICSI failure? A comparative study. Gynecol Endocrinol. 2015;31:247–51. 59. El Khattabi L, et  al. Is intracytoplasmic morphologically selected sperm injection effective in patients with infertility related to teratozoospermia or repeated implantation failure? Fertil Steril. 2013;100:62–8.

Recurrent Pregnancy Loss Howard Carp

Introduction The term recurrent pregnancy loss (RPL) refers to the loss of pregnancy irrespective of the stage of gestation. The term recurrent has been defined as either three or more consecutive spontaneous pregnancy losses [1] or two or more consecutive losses [2, 3]. It has been estimated that 1–2% of reproducing couples suffer from recurrent pregnancy loss if the three or more definition is used [1] and 5% if the two or more definition is used. Miscarriage is defined in North America as pregnancy loss prior to 20 weeks. In Europe, the term miscarriage includes all pregnancy losses from the time of implantation until 24 weeks of gestation, if recurrent the term recurrent miscarriage (RM) is used. Investigation has a number of objectives: to reach an accurate diagnosis of cause, to allow an accurate prognosis for future pregnancies, and to offer specific treatment in order to prevent a recurrence. However, even after exhaustive investigation, almost 50% of patients will be unexplained. In the past, the specific cause for recurrent miscarriage could seldom be diagnosed. Newer diagnostic techniques in genetics have revolutionized diagnosis in that many cases of genetic anomalies which could not previously be diagnosed are now diagnosed. In recent years, a specific prognosis has become possible based on the patient’s age, number of miscarriage, and genetic assessment of past miscarriages. Additionally, new treatment options have become available.

H. Carp (*) Department of Obstetrics and Gynecology, Sheba Medical Center, Tel Hashomer, Israel Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel e-mail: [email protected] © International Academy of Human Reproduction 2021 J. G. Schenker et al. (eds.), Clinical Management of Infertility, Reproductive Medicine for Clinicians 2, https://doi.org/10.1007/978-3-030-71838-1_21

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Table 1  Relative prognoses according to clinical features No. of miscarriages Age Embryo aneuploidy 1° or 2° aborter Early or late losses Infertility NK cells Family history

Good prognosis 2 3 20s Aneuploid 2° Early Normal fertility Normal None

Medium prognsis 4 30s Euploid 1° or 3° Early

Poor prognosis 5 6 7 8 9 40s Euploid 1° or 3° Late Infertility High

Close relatives

Prognosis The prognosis for a subsequent live birth is dependent upon the following: 1. Number of previous pregnancy losses. As the number of previous losses increases, the chance of a live birth decreases. The chance of a third pregnancy loss after two miscarriages is approximately 20%, and the chance of a sixth miscarriage after five previous miscarriages is approximately 75%. 2. Maternal age. The incidence of miscarriage increases with maternal age, particularly after the age of 40, possibly due to the increased risk of foetal aneuploidy in the older age groups. 3. Primary or secondary aborter status. The secondary aborter has a better prognosis than the primary aborter. 4. Karyotype of previous miscarriage. The patient with an aneuploid abortion has a better chance of a live birth. Embryonic aneuploidy may be a sporadic event. However, repeat aneuploidy does occur. 5. Early or late pregnancy losses, as the patient with late losses has a worse prognosis. The recognition that RPL exhibits a high degree of heritability implies that susceptibility genes for RPL may be inherited by genetic linkage in families with several siblings experiencing RPL [4]. Table 1 summarizes the factors affecting prognosis and should be taken into account when explaining the prognosis to the patient. If an 80% live birth rate is assumed after two losses, the two or more definition is problematic as in any research trial of treatment effect, there will be an 80% live birth rate in the control group. Therefore, inclusion of patients with two miscarriages in a trial will preclude any positive result.

Assessment of RPL The hitherto accepted method for assessing RPL was to subject the patient to a series of investigations to diagnose a single cause of RPL.  If a cause was found, it was assumed that “the” cause had been diagnosed. However, the list of investigations

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varied from protocol to protocol and according to which investigations were thought relevant at any particular time. For example, tissue typing for HLA antigens was thought to be relevant in the 1980s but is not thought relevant today. Treatment was performed on an entire RPL population, and the results of treatment were compared to non-treatment or placebo treatment. Efficacy was assumed if the benefit was statistically significant. Statistical significance is still the gold standard of evidencebased medicine and the standard by which recommendations are made in standard guidelines. More recently, a personalized approach has become more evident in clinical medicine. In oncology in particular, genomic diagnosis is often performed in order to detect genetic mutations which may affect the tumour occurrence or behaviour, e.g. BRCA mutations and breast or ovarian cancer. In addition, sensitivity assays can be used to determine the most effective drug regimen. For example, gefitinib is effective in non-small cell lung tumours harbouring the EGFR mutation [5], but not in patients with non-small cell lung tumours not harbouring the EGFR mutation. Personalized medicine is nothing more than accurate diagnosis. In RPL there are still no diagnoses which have the same degree of specificity, but the principle is the same, and treatment should be specific for specific patients, not based on one “basket” of RPL according to two or three or more pregnancy losses.

Problems with the Evidence-Based Approach Confounding Factors Embryonic genetic aberrations account for approximately 54% of RPL using the two or more definition and CGH microarrays [6]. Few trials of therapy for a maternal factor exclude the confounding effect of embryonic genetic aberrations. The effect of confounding factors can be seen in the case of vaginal micronized progesterone and RM, where two studies have produced conflicting results. Stephenson and colleagues [7] studied the role of vaginal progesterone in women with two or more unexplained miscarriages under 10 weeks and miscarriages with chromosome errors excluded. Pregnancy was only allowed after vaginal micronized progesterone increased cyclin E (a marker of endometrial maturation [8]) levels. The ongoing pregnancy rate increased from 6% (16/255) to 69% (OR, 2.1 CI 1.0–4.4) with progesterone [7]. The PROMISE study [9], however (a randomized, double-blind, placebo-controlled international multicentre trial of micronized vaginal progesterone), took no account of embryonic aneuploidy or state of the endometrium. Using these nonselective criteria, there was no evidence of progesterone leading to a significant difference between the groups for any of the outcomes.  he Question Asked of a Meta-Analysis T Paternal leucocyte immunization, although always controversial, became the treatment of choice for RM in the 1980s and 1990s. However, leucocyte immunization fell out of favour after Ober et al.’s trial [10] claimed that the prognosis was worse after leucocyte immunization. However, Ober et al.’s [10] trial used immunizations prepared from old cells which were stored in the refrigerator overnight. All previous trials had used fresh cells. It has been reported that refrigeration renders

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immunizations ineffective [11]. There has since been a Cochrane database systematic review which asked the question whether paternal leucocyte immunization improved the live birth rate in patients with RPL [12]. There were 12 trials included comprising 641 patients. The summary odds ratio of the effect of treatment was 1.23 (95% confidence interval 0.89 to 1.70, p = 0.21, nonsignificant). There was significant heterogeneity of the effect of treatment across all trials. However, if the question were to be “Does immunization with fresh cells improve the live birth rate?”, Ober et al.’s trial [10] would have to be excluded. Daya [13] has calculated that the remaining 11 trials would show a summary odds ratio of 1.63 (95% confidence interval 1.13–2.36, p = 0.009) indicating a statistically significant effect in favour of immunotherapy [13].

Diagnosis RPL may be due to an abnormal embryo with either abnormal chromosomes or severe structural malformations which are incompatible with life or due to maternal factors causing miscarriage of a normal embryo. Eighteen percent of miscarriages are due to structural malformations with no chromosomal aberrations [14]. If the embryonic causes of pregnancy loss are not investigated, it is impossible to arrive at an accurate diagnosis. A presumptive diagnosis is usually made after investigations for maternal causes only. The presence of a maternal cause of pregnancy loss does not guarantee a normal embryonic chromosome complement. Even in antiphospholipid syndrome (APS), 30% of aborted embryos have a chromosomal aberration [15, 16]. Carp et al. [17] have found four chromosomal aberrations in the embryos of 16 patients with hereditary thrombophilia (de novo balanced translocation, 16 trisomy, 13 trisomy, and 45XO). The investigation of maternal factors causing repeated pregnancy loss requires a comprehensive history of the type of presentation and investigation of hormonal, anatomical, genetic, thrombotic, and immunological factors. Table  2 shows our investigation protocol.

Genetic Aberrations The genetic assessment of the abortus allows an accurate diagnosis, prognosis, and direct treatment to foetal factors (PGT-A) or maternal factors. Additionally, if treatment has been provided for a maternal cause of RPL, it allows audit of the failure of treatment. Table 3 shows how embryonic genetic assessment affects the results of treatment. Hence, foetal genetic assessment is recommended in the RCOG guideline [1]. In recent years complete chromosomal analysis (CCA) (using comparative genomic hybridization) has replaced the older karyotype binding techniques, and allows diagnosis at a much higher resolution. The majority of aberrations are trisomies, of which 16 trisomy is the most common. CGH

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Table 2  Investigation of recurrent pregnancy loss •  Genetic assessment of the embryo at the time of curettage • If genetic assessment has not been performed on the products of conception, paraffin block analysis of previous miscarriages • Accurate history of past losses, missed abortions, blighted ova, abortions of live embryos, mixed pattern of losses, and primary or secondary aborters •  Treatment given in past pregnancies •  Cycle length • History of associated conditions, diabetes, thyroid disease, infertility, or autoimmune disease • Karyotype of both parents, whole exome sequencing if embryonic anomalies have been found •  Hysteroscopy, sonohysteroscopy, or 3-D ultrasound •  Autoantibody screen for anticardiolipin antibody and lupus anticoagulant • Screening for thrombophilias, including proteins C and S and antithrombin, factor V Leiden, and prothrombin gene mutation •  Follicular phase FSH, LH, and ultrasound of ovaries to exclude polycystic ovaries Table 3  Cause of disparate results according to embryonic aneuploidy Progestogen supplementation

Filgrastim (G-CSF)

Genetic aberrations not excluded PROMISE (2015) (RR, 1.04; 95% CI, 0.94–1.15) NS (>3 miscarriages, aneuploidy ignored) Zafardous et al. (2017) (OR, 0.90 95% CI, 0.28–2.89) NS (>2 miscarriages, aneuploidy ignored)

Genetic aberrations excluded Luteal start (Stephenson et al. 2017) (OR, 2.1; 95% CI, 1.0–4.4) (>2 miscarriages, ≥1 euploid embryo) Scarpellini and Sbracia (2009) (OR, 5.1; 95% CI, 1.5–18.4). (>5 miscarriages, ≥1 euploid embryo)

analyses can only detect excess or lack of DNA. CGH cannot detect sequencing disorders. In the near future whole exome sequencing may replace CGH as the optimal method of testing [18]. Most often, the patient presents in the interval between pregnancies, and no results are available for genetic assessment of the abortus. In these cases, if curettage has been performed, CGH can be undertaken on either paraffin blocks or histological slides. Paraffin block testing is routinely used for genomic analysis of tumours [19, 20] and is being increasingly used for RPL. As embryonic aneuploidy may be a chance occurrence and not recurrent, two publications have reported the subsequent live birth rate after embryonic aneuploidy [15, 21]. The prognosis is good (65%), compared to 38% for patients losing euploid embryos. There are numerous other genetic defects which may lead to RM, in addition to aneuploidy. These include epigenetic changes which may cause misreads of the

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DNA sequence, skewed X inactivation which allows recessive genes to be represented in phenotype, uniparental disomy, isolated gene defects, immunity-related genes, angiogenesis-related, and apoptosis-related genes.

Parental Genetic Aberrations Parental karyotyping is often performed in order to identify structural chromosomal rearrangements which may be transmitted to the embryo in an unbalanced form. The most common types of aberration are balanced translocations, either reciprocal or Robertsonian, or inversions. Opinions are divided as to whether parental chromosomal aberrations should be examined. Testing is not recommended by ESHRE [2] or the RCOG [1]. Lack of support for parental testing is based on the low yield for a balanced parental rearrangement (2–4%) and that most carriers of parental translocation will succeed in having successful pregnancies without intervention [22, 23]. CGH arrays are not used on the parents as there is no excess or deficiency of DNA. However, whole exome sequencing (WES) may soon supersede karyotyping as the procedure of choice. In cases of malformations, WES is increasingly performed on the trio of foetus and both parents.

Biomarkers The personalized approach requires a biomarker to suggest which patients will benefit from treatment, e.g. the presence of hormone receptors in breast cancer to indicate the need for hormone or anti-hormone therapy. Unfortunately, most trials in RPL have only used the evidence-based approach and have not sought biomarkers or a personalized approach. This “one-size-fits-all approach” has created an illusion of futility and that no treatment modality has any effect. However, there are some hints as to which biomarkers may be effective in directing treatment. Biomarkers are discussed in the next section where the evidence is presented for various treatment modalities.

Maternal Causes of RPL Uterine Anomalies Uterine assessment is considered to be an important part of the evaluation of patients with RPL.  Diagnosis is usually made by hysteroscopy, hydrosonography, or 3-D ultrasound. A review of several studies has found that congenital uterine anomalies are present in 4.3% of the general population of fertile women and in 12.6% of patients with RPL (defined as two or more losses) [24]. A high rate of miscarriage has been reported in the presence of septate, bicornuate, and arcuate uteri.

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Uterine septa may be corrected by hysteroscopic surgery. It could be said that the septum is a biomarker for hysteroscopic resection. However, there is a dearth of information about the efficacy of treatment. Sugiura-Ogasawara [25] published a comparative cohort study on 109 women with two or more miscarriages who underwent septotomy (hysteroscopic or by open surgery) and compared the live birth rates to 15 women who did not undergo surgery. Although the study was underpowered to show a statistically significant effect, there was a 20% benefit from surgery (81% live births after surgery compared to 61.5% without surgery). However, hysteroscopic metroplasty is associated with a substantial and as yet non-­quantified, increased risk of uterine rupture during subsequent pregnancies [26, 27]. In the bicornuate uterus, metroplasty was not associated with any beneficial effect in Sugiura-Ogasawara et  al.’s [25] study. In the case of acquired uterine anomalies such as polyps, adhesions, etc., there is some evidence favouring surgery in terms of increased fertility, but none regarding RPL.

Antiphospholipid Syndrome (APS) In APS pathological antiphospholipid antibodies (aPL; lupus anticoagulant, anticardiolipin, and β2 microprotein antibodies) are thought to have detrimental effects on the developing trophoblast, including inhibition of villous cytotrophoblast differentiation and extravillous cytotrophoblast invasion into the decidua [28] and induction of syncytiotrophoblast apoptosis [29]. aPL significantly reduce hCG release and early embryonic development [28, 30]. These effects can be seen histologically by decreased vasculosyncytial membranes, increased synctial knots, substantially more fibrosis, hypovascular villi, and infarcts than women without APS [31]. These actions may then cause vasoconstriction, or subsequent blood clotting in the small vessels leading to the placenta. Therefore the foetus suffers from growth retardation and may die in utero. The syndrome may be associated with first trimester losses, but there is a heavy preponderance of later losses. Fewer than 5% of women with RM and no other autoimmune or thrombotic disease have aPL [32]. A significant proportion of otherwise healthy subjects have positive aPL results [32, 33]. However, positive tests for aPL (aCL or aβ2-GP-I antibodies) have been found in 9.6% of foetal deaths (≥20 weeks of gestation), and positive results for aCL antibodies were associated with a fivefold odds of stillbirth, while aβ2-GP-I antibodies were associated with a threefold odds of stillbirth [34]. Although there are numerous reports of patients with aPL having successful pregnancies, there is a general consensus that APS should be treated. Standard treatment consists of an anticoagulant to prevent excess coagulation and aspirin to prevent the platelet aggregation predisposing to subsequent coagulation. However, the aspirin and anticoagulant regimen has never been tested in a randomized controlled trial. Anticoagulants have been assessed in addition to aspirin in a meta-analysis by Ziakas et al. [35] and have shown an 18% benefit compared to aspirin alone (OR = 0.39, CI 0.24–0.65). Therefore, the role of

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aspirin could be questioned. Two meta-analyses have assessed aspirin compared to placebo [36, 37]. Each meta-analysis assessed three papers (five in total). There was no improvement in the live birth rate.

 he Refractory Patient T Despite treatment with anticoagulants and aspirin, there are always patients who continue to lose pregnancies. For example, the PREGNANTS study [38] consisted of 750 women with aPL antibodies and at least one clinical feature of APS. The authors found that women who were triple positive for aPL antibodies had only a 30% rate of successful pregnancy in spite of treatment with a heparin agent and aspirin. If the subsequent pregnancy loss is in the first trimester, the abortus should be genetically assessed. If the embryo was aneuploid, the same treatment regimen can be repeated, as neither anticoagulants nor aspirin can correct genetic aberrations. However, if the pregnancy loss was a late loss (where aneuploidy is uncommon), or the embryo euploid, there is treatment failure, and an alternative treatment regimen may be required. However, there is, as yet, little evidence of efficacy of alternative treatment regimens. Low-dose prednisolone [39] and hydroxychloroquine (HCQ) [40] have been added to heparin LMWH and LDA. A retrospective, international multicentre study [41] concluded that the addition of HCQ treatment was associated with a significantly higher live birth rate in women with a history of one or more pregnancies refractory to conventional therapy although this report did not take account of embryonic aneuploidy as a cause of miscarriage. In the case of subsequent pregnancy losses being due to late obstetric complications, intravenous immunoglobulin (IVIg) may be helpful. IVIg has not been shown to increase the live birth rate when given as an adjunct to heparin and aspirin [42– 45], but is associated with a lower incidence of intrauterine growth restriction, pregnancy-­ induced hypertension, gestational diabetes, and preterm labour [42, 43, 46]. Varying degrees of successful pregnancy outcomes have also been reported in high-risk or refractory obstetric APS using plasmapheresis [47, 48].

Hereditary Thrombophilias Hereditary thrombophilias may cause pregnancy loss due to thrombosis in the small vessels of the placenta as in APS. However, the hereditary thrombophilias do not have the anti-trophoblast effects of APS. As well as pregnancy loss, thrombophilias may predispose to foetal growth retardation, preeclampsia, and deep vein thromboses in the mother. The association between hereditary thrombophilias and RPL has been assumed due to studies showing a higher prevalence of thrombophilias in patients with pregnancy losses [49, 50]. However in RPL or RM, the results have been inconsistent. There does, however, seem to be an increased prevalence in

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women with late pregnancy losses [49, 51, 52]. It is also controversial whether the presence of a thrombophilia increases the chance of a subsequent pregnancy loss. Carp et al. [44] found the live birth rate to be similar to that expected in recurrent miscarriage, whether the patient had factor V Leiden, G20210A, MTHFR, protein C or S, or antithrombin deficiencies. Due to these controversies, major organizations do not advise thrombophilia testing in their guidelines [1–3] and do not advise thromboprophylaxis. Isolated reports of thromboprophylaxis, have been summarized by Inbal and Carp [45]. When the results are taken from four studies, there was a statistically significant 15% improvement in the live birth rate. Therefore, this author does recommend thromboprophylaxis in RPL with hereditary thrombophilias.

Alloimmune Pregnancy Loss Despite intense research into reproductive immunology, no concept of immune pregnancy loss has stood the test of time. Current concepts of alloimmune pregnancy loss involve an inbalance of cytokines causing the mother to lose tolerance to the paternal antigens expressed by the trophoblast. Th-1 (proinflammatory) cytokines are thought to promote breakdown of tolerance, whereas Th-2 (anti-­ inflammatory) cytokines are thought to enhance tolerance. Preponderance of Th-2 cells is enhanced by increased production of Treg cells, whereas Th-17 responses enhance the Th-1 effect. The inappropriate cytokine response is thought to activate natural killer cells (NK) in the decidua to attack the trophoblast. The problem is that decidual NK cells have never been shown to kill trophoblast in vivo. Their role is in remodelling decidual blood vessels and possibly immunosurveillance. Based on this and other models, various forms of testing have been devised, and various forms of immunotherapy have been used. However, all forms of immunotherapy have been tried on an unselected group of RPL patients, without assessing any subgroup or excluding embryonic aneuploidy. Some of the results are discussed below.

Intravenous Immunoglobulin When all patients with three or more miscarriages are treated as one homogeneous group with immunoglobulin (IVIg), there is no beneficial effect, as a systematic review in the Cochrane database has shown [12]. Again as with other maternal causes of RPL, IVIg cannot correct aneuploid pregnancies. There have been attempts to classify patients on a clinical basis. Hutton et al. [53] analysed the series which were assessed in the Cochrane database meta-analysis and found that IVIg had a statistically significant effect when secondary aborters were analysed as a separate subgroup. Similarly, in Christiansen et al.’s [54] placebo controlled trial, in secondary aborters, the live birth rate was 50% in treated women compared to 23% in placebo treated women.

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Additionally, the timing of IVIg administration is all important. Coulam [55] has assessed nine trials in which IVIg was administered, based on obstetric history alone or obstetric history and immunologic test results. Five trials gave IVIg before conception and four of the five showed significant benefit in enhancing live birth rates. Five trials delayed treatment until pregnancy was established and none demonstrated benefit from treatment (P = 0.04, Fisher’s exact test). Hutton et al. [53] have published similar results. When IVIg was administered prior to pregnancy, there was a statistically significant benefit (OR = 2.02 CI 1.04–3.92). Some of the trials administering IVIg administered the medication up to 8 weeks of pregnancy, with no ultrasound control as to foetal viability. Hence, IVIg may have been administered after foetal demise, too late to have any effect. The author uses IVIg in women with five or more miscarriages. In these cases there was a statistically significant 20% benefit. The author tends to reserve IVIG for the most resistant cases [56].

Intralipid Intralipid, a 20% intravenous fat emulsion containing soybean oil, egg yolk phospholipids, gylcerine, and water, is probably the most widely used immunomodulatory agent. It is used instead of IVIg as it is cheaper and not a blood product. Fatty acids have been shown to affect NK activity through peroxisome proliferator-­ activated receptors (PPARs) [57], G-protein-coupled receptors [58], and CD1 receptors [59]. Coulam and Accacio [60] have reported the results of 200 women with reproductive failure and elevated NK cell cytotoxicity, treated with intralipid, and 242 age- and indication-matched women treated with IVIg. The overall live birth or ongoing pregnancy rate per cycle of treatment was 61% for women treated with intralipid and 56% with IVIg. However, to date, there is no randomized trial of intralipid compared to controls in RM. Filgrastim Filgrastrim is a cytokine growth factor (G-CSF). The use of filgrastim in RPL is supported by a randomized controlled study [61]. The live birth rate in treated women was 82.8%, compared to 48.5% in the control group (p = 0.0061, NNT = 2.9). The strength of Scarpellini and Sbracia’s [61] study lay in the inclusion criteria. Only women with more than four previous miscarriages, failure of previous therapy for RPL, negative results for other known causes of RPL, and loss of a euploid embryo in the previous miscarriage were included. As in other trials of immunotherapy, when the selection criteria were widened to include all patients with RPL irrespective of embryonic chromosomal analysis and a good prognosis (two or more pregnancy losses) [62], the results became insignificant. The above two papers show how including a nonselected group of patients can confound the results of a trial which is designed to test treatment in appropriate patients.

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Empirical Treatment Regimens Hormone Supplementation Progestogens Since the first use of progestogen preparations in the 1950s, the therapeutic benefit has been disputed. A relative or absolute progesterone deficiency could contribute to RPL by delaying endometrial development. Low progesterone levels have been found in recurrent miscarriage [63–65] and have even been used to make predictions about subsequent pregnancy success. However, the relevance of using serum progesterone levels has been disputed. Ogasawara et  al. [66] have reported that serum progesterone concentrations are not reflective of endometrial levels of progesterone and are not predictive of pregnancy outcome. Additionally, progesterone is secreted in a pulsatile fashion [67], and blood may be drawn at a pulse peak or pulse nadir. There have been several meta-analyses evaluating the use of progestogens for the prevention of subsequent miscarriage after RM. A meta-analysis of six trials in the Cochrane database concluded that there was a slight benefit in women receiving progestogen in terms of live births (RR 1.07, 95% CI 1.00–1.13) [68]. Saccone et al. [69] published a meta-analysis of ten trials including 1586 women The miscarriage risk was lower (RR 0.72, 95% CI 0.53–0.97), and live birth rate was higher (RR 1.07, 95% CI 1.02–1.15) after progesterone supplementation. However, the papers included in the meta-analyses did not account for embryonic aneuploidy, nor did they stratify for the number of miscarriages, the particular progestogen used, maternal age, or primary or secondary aborter status, all of which have impact on the outcome. The role of micronized vaginal progesterone is doubtful. Coomarasamy et al. [9] performed a multicentre, randomized, placebo-controlled study (PROMISE) to investigate whether treatment with micronized progesterone would increase the live birth rate among women with unexplained recurrent miscarriage. The live birth rate was 65.8% in the progesterone group which was comparable to 63.3% in the placebo group (NS). However, as other studies described above, the PROMISE study took no account of embryonic aneuploidy or state of the endometrium. Stephenson and colleagues [7] studied the role of vaginal progesterone in women with two or more unexplained miscarriages under 10 weeks and miscarriages with chromosome errors excluded. After correction of cyclin E levels, the ongoing pregnancy rate increased from 6% (16/255) to 69% (OR, 2.1 CI 1.0–4.4) with vaginal micronized progesterone. In Saccone et al.’s [69] meta-analysis, which includes multiple types of progestogen, the effect of dydrogesterone stands out. The author of this chapter has published a meta-analysis of three trials of dydrogesterone [70]. There was a 10.5% (29/275) miscarriage rate after dydrogesterone administration compared to 23.5% in control women (OR 0.29 CI 0.13–0.65). The effect of dydrogesterone may be due

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to the higher bioavailability than progesterone itself and higher receptor binding selectivity [71]. On summarizing the above literature and trials, it seems that there is an advantage to using progestogens in recurrent miscarriage. However, it remains to define a population who can respond and the appropriate diagnostic tests to determine who can benefit.

 uman Chorionic Gonadotropin (hCG) H The actions of hCG in feto-placental tissues are paracrine, autocrine, and immune in nature. Regular hCG stimulates the corpus luteum to produce progesterone, promotes angiogenesis, causes trophoblast differentiation, and prepares the endometrium for the implanting embryo. The hyperglycosylated isoform of hCG (H-hCG) is autocrine enhancing implantation by promoting the growth and invasion of the trophoblast [72]. The immune effects are due to lymphocytes from pregnant women expressing the hCG receptor gene [73]. Uzumcu et al. [74] have shown that increasing doses of hCG have been found to cause a dose-dependent increase in TNFα and IL-6 secretion. hCG has also been reported to stimulate secretion of IL-1β and inhibit IL-2 expression by human monocyte cells in culture [74]. A number of trials have assessed whether hCG supplementation has a beneficial effect in women with recurrent miscarriage. These have been assessed in a Cochrane database meta-analysis by Morley et al. in 2013 [75]. The initial analysis performed on five studies of 302 patients showed a statistically significant benefit of hCG treatment in reducing subsequent miscarriages in women with RPL (RR  =  0.51 (CI, 0.32–0.81). hCG supplementation is the only treatment modality shown to have a beneficial effect in the Cochrane database. Carp [76] observed a statistically significant benefit of 15% by using hCG (OR, 1.88, 95% CI, 1.16–3.04) [76]. When the analysis involved only the subgroup of women with five or more miscarriages— those with a “poor” prognosis—the beneficial effect of hCG was more evident with an absolute benefit of 34% (OR, 4.33, CI, 1.7–11.3). Again, there was no accounting for foetal aneuploidy, but it is important to note that no detrimental effects of hCG were observed.

Empirical Anticoagulants Heparins Heparins have anti-inflammatory actions by inhibiting TNFα production and increasing TNF binding protein. Heparin has also been reported to enhance trophoblast invasion in APS and increase hCG production. Multiple, prospective studies have looked at the effect of anticoagulants on successful pregnancy outcome in women with RPL and no inherited thrombophilia. The results have been summarized by Merriam and Paidas [77]. There were three positive trials and nine negative trials. However, the trials are too heterogeneous to be combined in a meta-analysis. Similarly, a Cochrane review of nine studies by de Jong et al. [78] did not find an improvement in pregnancy outcomes in women with

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RPL and no history of thrombophilia when anticoagulants were used during pregnancy. Given these findings the use of anticoagulants during pregnancy in women with RPL but without a history of a thrombophilia is not recommended by multiple societies including ACOG [79], the Royal College of Obstetricians and Gynaecologists [1], and the American College of Chest Physicians [80].

Aspirin Aspirin has been reported to inhibit the proinflammatory cytokines TNFα and IL-8 in stroke. TNFα induces thrombin generation. IL-8 causes polymorph accumulation. Polymorphs react with fibrin and damaged tissues to form clots. Hence aspirin may also modify cytokine-mediated thrombosis. The trials assessing aspirin in RPL have been summarized by Merriam and Paidas [77]. Four reports were summarized, comprising 713 treated patients and 608 non-treated or placebo-treated patients. None of the studies showed any benefit; however, the trial designs were inconsistent with various control groups. Hence, ACOG currently does not recommend the use of aspirin alone for the prevention of early miscarriage in women with RPL or for prevention of later pregnancy foetal demise, unless the later occurs with a diagnosis of preeclampsia [79].

Assisted Reproductive Technology (ART) Many centres offer empirical in vitro fertilization (IVF) for RM. The rationale is that IVF shortens the time to conceive and that sperm selection techniques improve sperm quality, embryo quality by selecting the best embryos for transfer either by miscoscopy before transfer or by time lapse embryoscopy, and synchrony between embryo and endometrium. All of these concepts have been discounted by Kirshenbaum and Orvieto [81], who suggest that IVF is not an appropriate technique especially as IVF is a technique used in order to make a patient conceive and subfertility is not a problem in most couples with RPL. IVF is only indicated if there is associated secondary infertility.

 regestational Testing for Aneuploidy (PGT-A) P The concept of PGT is attractive, in that a large number of RMs are due to embryonic aneuploidy. Therefore, PGT can be used to exclude aneuploidy. However, since 2015, the whole concept of PGT-A has come under increasing scrutiny. There are reports of patients who experienced miscarriages after PGT-A, in which chromosomal reassessment was found to be aneuploid, raising the spectre of false-negative results [82]. False-positive results were shown after replacement of embryos reported to be aneuploid led to the birth of normal infants [83, 84]. Additionally a certain amount of aneuploid mosaicism is normal in human embryos [85, 86]. However, the question is really about whether PGT-A can increase live birth rates in RPL. Murugappan et al. [87] reported similar pregnancy, live birth, and clinical miscarriage rates after PGT-A or expectant management, with a shorter time to pregnancy (3.0 vs 6.5  months) in the patients managed expectantly. Moreover,

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Murugappan et al. (45) did not find PGT-A a cost-effective strategy for increasing live births. Murugappan et al.’s [87] study is usually quoted to show that PGT-A is not warranted. However, closer inspection of the results reveals a different picture. The results were broken down into age groups. In the age group of 20–35, the patients managed expectantly did better; however in the 35–45 age group, the PGT patients had higher live birth rates, significantly so in the age group 40–45. The effect of PGT in the advanced age groups is entirely logical as the older age groups have increased rates of embryonic aneuploidy. The older age groups may therefore be a targeted population for treatment. Indeed PGT has been said to cancel the effect of age [88]. The author has published a list of indications for PGT-A [89]. These include advanced maternal age (if enough ova are available for biopsy), repeat foetal aneuploidy, and foetal aneuploidy in the presence of parental karyotypic aberrations.

Gamete Donation In some cases of PGT-A, there may be no embryos for transfer or the biopsy for PGT may damage the embryos leaving it unable to implant. Additionally in the older age groups, there may be insufficient embryos for biopsy, or no eggs can be retrieved. In these cases ovum donation may be required to overcome RPL. The author has seen 75 women who attended the RM clinic after ovum donation. Ovum donation does not invariably overcome RM. Remohi et al. [90] reported on 12 ovum donation cycles in eight RM couples. The pregnancy rate was 75%, delivery rate 66.6%, and miscarriage rate per cycle 11.1%, which is lower than expected in RM.  estational Carrier Surrogacy G Gestational carrier surrogacy may be indicated for patients who lose euploid embryos and are refractory to simpler forms of treatment such as IVIG. Severe APS may be another indication in whom pregnancy is contraindicated due to the internal medical complications of the syndrome. However, there are few reports of surrogacy in RM. Raziel et al. [91] reported a normal live birth in a patient with 24 prior pregnancy losses. The author has advised surrogacy in a secondary aborter with 12 miscarriages. The surrogate delivered normal twins. The logic of surrogacy in patients with large numbers of miscarriages is due to the poor prognosis and low incidence of chromosomal aberrations.

Late Pregnancy Losses Patients with recurrent second trimester foetal deaths have a poorer prognosis than after first trimester losses [92]. Detailed ultrasound or embryoscopy may diagnose foetal structural anomalies. Diabetes should be excluded as diabetes predisposes to foetal anomalies.

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Thrombotic mechanisms, either due to APS or hereditary thrombophilias, are more likely to cause foetal demise than first trimester miscarriages [92]. If either of these is found, in the presence of recurrent second trimester foetal deaths, treatment by anticoagulants is warranted. Another condition which has been identified is chronic histiocytic intervillositis [93, 94]. The aetiology remains unclear, but the aberrant recruitment of maternal immune cells to the maternal-foetal interface suggests an anomalous maternal immunological response to foetal tissue. Immunosuppression by steroids has been reported to be useful and superior to anticoagulants and aspirin [95].

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