Uterus Transplantation [1st ed. 2020] 978-3-319-94161-5, 978-3-319-94162-2

Table of contents :
Front Matter ....Pages i-xi
Introduction: Uterus Transplantation (Mats Brännström)....Pages 1-10
The Ethics of Uterus Transplantation: Moral Challenges and Recommendations for Progress (Arthur L. Caplan, Brendan Parent, Pasquale Patrizio)....Pages 11-23
Patients with Uterine Factor Infertility: General (Cesar Diaz Garcia)....Pages 25-32
MRKH Patients and Their Preparations for Uterus Transplantation (Dorit Schöller, Sara Brucker)....Pages 33-37
Rodent Animal Research in UTx (Randa Akouri, Min Jong Song, Cesar Diaz Garcia)....Pages 39-50
Domestic Species Research in Uterus Transplantation (Mats Brännström)....Pages 51-55
Nonhuman Primate Research in Uterus Transplantation (Iori Kisu, Yusuke Matoba, Kouji Banno, Daisuke Aoki)....Pages 57-67
Human Preclinical Research in Uterus Transplantation (Mats Brännström)....Pages 69-72
Medical Work-Up of the Recipient (Jana Pittman, Rebecca Deans, Mats Brännström)....Pages 73-78
Live or Deceased Uterus Donor (Michael Olausson)....Pages 79-82
Medical Work-Up of the Live Donor (Mats Brännström, Pernilla Dahm-Kähler)....Pages 83-87
Medical Work-Up of the Deceased Donor (Anne C. Davis, Rebecca Flyckt, Tommaso Falcone)....Pages 89-93
Psychological Evaluations Before Uterus Transplantation (Stina Järvholm)....Pages 95-101
Assisted Reproduction Before and After Uterus Transplantation (Lars B. Nilsson, Jan I. Olofsson)....Pages 103-110
Surgical Technique of Live Donor in Uterus Transplantation (Mats Brännström, Pernilla Dahm-Kähler)....Pages 111-117
Surgical Technique of Deceased Donor in Uterus Transplantation (Andreas Tzakis, Michael Olausson, Tommaso Falcone)....Pages 119-127
Surgical Technique in Preparation of Recipient (Janusz Marcickiewicz, Mats Brännström)....Pages 129-133
Back-Table Preparation and Flushing of the Uterus (Niclas Kvarnström, Mats Brännström)....Pages 135-138
Surgical Technique for Vascular Anastomosis of the Uterine Graft (Michael Olausson, Niclas Kvarnström)....Pages 139-145
Fixation of the Uterine Graft After Uterus Transplantation (Mats Brännström, Pernilla Dahm-Kähler)....Pages 147-149
Immunosuppression and Treatment of Rejection in Uterus Transplantation: Current Practice and Future Potential (Matthew H. H. Young, Dawn Truong, Jana Ekberg, Stefan G. Tullius)....Pages 151-166
Evaluation of Graft Function After Uterus Transplantation (Milan Milenkovic, Mats Brännström)....Pages 167-170
Rejection Diagnosis After Uterus Transplantation (Johan Mölne, Verena Bröcker)....Pages 171-176
Psychological Aspects After Uterus Transplantation (Stina Järvholm)....Pages 177-182
Obstetrical and Pediatric Follow-Up After Uterus Transplantation (Hans Bokström, Mats Brännström, Henrik Hagberg)....Pages 183-188
Infections After Uterus Transplantation (Steven Van Laecke, Steven Weyers)....Pages 189-207
Indications and Surgical Technique for Hysterectomy After Uterus Transplantation (Pernilla Dahm-Kähler, Mats Brännström, Niclas Kvarnström)....Pages 209-214
The Future Expansion of Patient Groups for Uterus Transplantation (Steven Weyers, Petra De Sutter)....Pages 215-218
The Bioengineered Uterus: A Possible Future (Mats Hellström, Mats Brännström)....Pages 219-230

Citation preview

Uterus Transplantation Mats Brännström Editor

123

Uterus Transplantation

Mats Brännström Editor

Uterus Transplantation

Editor Mats Brännström University of Gothenburg Gothenburg Sweden

ISBN 978-3-319-94161-5    ISBN 978-3-319-94162-2 (eBook) https://doi.org/10.1007/978-3-319-94162-2 © Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

This first book about uterus transplantation is dedicated to those nine brave women who participated as recipients in the first uterus transplantation trial and their nine unselfish relatives and friends who donated their uteri. Thanks to these Swedish women and the results of the scientific trial they participated in; uterus transplantation evolved as the first treatment of uterine factor infertility. The book is also dedicated to gynaecologist and associate professor, Jane Thorburn Olsson and her family, who through Jane and Dan Olsson Foundation for Science supported important preclinical research on uterus transplantation and the Swedish clinical trials on human uterus transplantation.

Preface

Absolute uterine factor infertility, because of lack of a uterus or presence of a non-­ functional uterus, has for long been regarded as untreatable. The proof-of-concept of uterus transplantation as a treatment of absolute uterine factor fertility came with the birth of the first baby after uterus transplantation. This birth of baby Vincent in Sweden in 2014 has been followed by several births, both after live donor and deceased donor uterus transplantation. Importantly, the births have occurred in four continents so the technique has spread around the globe. This book is a comprehensive review of the field of uterus transplantation. World experts have come together to share knowledge and experience concerning their specific expertise in the broad field of uterus transplantation. The book covers the important animal-based research that paved the way for the clinical trials of uterus transplantation. All essential steps of clinical uterus transplantation are also included, from screening of recipients/donors through surgical techniques, and also covering follow-up after transplantation and of pregnancy. It is my hope that this book will be of great interest and help not only for all health professionals that are in the process of starting up programs in clinical uterus transplantation but also for those that want to broaden their knowledge in this new field of gynaecology, reproductive medicine, and transplantation surgery. Gothenburg, Sweden

Mats Brännström

vii

Contents

1 Introduction: Uterus Transplantation������������������������������������������������������   1 Mats Brännström 2 The Ethics of Uterus Transplantation: Moral Challenges and Recommendations for Progress��������������������������������������������������������  11 Arthur L. Caplan, Brendan Parent, and Pasquale Patrizio 3 Patients with Uterine Factor Infertility: General ����������������������������������  25 Cesar Diaz Garcia 4 MRKH Patients and Their Preparations for Uterus Transplantation������������������������������������������������������������������������  33 Dorit Schöller and Sara Brucker 5 Rodent Animal Research in UTx��������������������������������������������������������������  39 Randa Akouri, Min Jong Song, and Cesar Diaz Garcia 6 Domestic Species Research in Uterus Transplantation��������������������������  51 Mats Brännström 7 Nonhuman Primate Research in Uterus Transplantation����������������������  57 Iori Kisu, Yusuke Matoba, Kouji Banno, and Daisuke Aoki 8 Human Preclinical Research in Uterus Transplantation������������������������  69 Mats Brännström 9 Medical Work-Up of the Recipient����������������������������������������������������������  73 Jana Pittman, Rebecca Deans, and Mats Brännström 10 Live or Deceased Uterus Donor����������������������������������������������������������������  79 Michael Olausson 11 Medical Work-Up of the Live Donor��������������������������������������������������������  83 Mats Brännström and Pernilla Dahm-Kähler 12 Medical Work-Up of the Deceased Donor ����������������������������������������������  89 Anne C. Davis, Rebecca Flyckt, and Tommaso Falcone

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Contents

13 Psychological Evaluations Before Uterus Transplantation��������������������  95 Stina Järvholm 14 Assisted Reproduction Before and After Uterus Transplantation�������� 103 Lars B. Nilsson and Jan I. Olofsson 15 Surgical Technique of Live Donor in Uterus Transplantation�������������� 111 Mats Brännström and Pernilla Dahm-Kähler 16 Surgical Technique of Deceased Donor in Uterus Transplantation������ 119 Andreas Tzakis, Michael Olausson, and Tommaso Falcone 17 Surgical Technique in Preparation of Recipient ������������������������������������ 129 Janusz Marcickiewicz and Mats Brännström 18 Back-Table Preparation and Flushing of the Uterus������������������������������ 135 Niclas Kvarnström and Mats Brännström 19 Surgical Technique for Vascular Anastomosis of the Uterine Graft ���������������������������������������������������������������������������������� 139 Michael Olausson and Niclas Kvarnström 20 Fixation of the Uterine Graft After Uterus Transplantation ���������������� 147 Mats Brännström and Pernilla Dahm-Kähler 21 Immunosuppression and Treatment of Rejection in Uterus Transplantation: Current Practice and Future Potential���������������������� 151 Matthew H. H. Young, Dawn Truong, Jana Ekberg, and Stefan G. Tullius 22 Evaluation of Graft Function After Uterus Transplantation���������������� 167 Milan Milenkovic and Mats Brännström 23 Rejection Diagnosis After Uterus Transplantation �������������������������������� 171 Johan Mölne and Verena Bröcker 24 Psychological Aspects After Uterus Transplantation������������������������������ 177 Stina Järvholm 25 Obstetrical and Pediatric Follow-Up After Uterus Transplantation������������������������������������������������������������������������������������������ 183 Hans Bokström, Mats Brännström, and Henrik Hagberg 26 Infections After Uterus Transplantation�������������������������������������������������� 189 Steven Van Laecke and Steven Weyers 27 Indications and Surgical Technique for Hysterectomy After Uterus Transplantation������������������������������������������������������������������� 209 Pernilla Dahm-Kähler, Mats Brännström, and Niclas Kvarnström

Contents

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28 The Future Expansion of Patient Groups for Uterus Transplantation������������������������������������������������������������������������ 215 Steven Weyers and Petra De Sutter 29 The Bioengineered Uterus: A Possible Future���������������������������������������� 219 Mats Hellström and Mats Brännström

1

Introduction: Uterus Transplantation Mats Brännström

1.1

Introduction

Research in the area of uterus transplantation (UTx) was initiated in the 1960s and then mostly conducted in dogs (Eraslan et al. 1966; Barzilai et al. 1973). The aim was to develop a method to cure tubal factor infertility, one of the largest subgroups of female infertility. This was in an era before introduction of effective immunosuppression agents. Although this historical UTx research, which included transplantation of the uterus together with the oviducts, was continued into the 1970s, the results were poor in terms of successful transplants. The major breakthrough in infertility treatment of the previous century was the introduction of in vitro fertilization (IVF) in the late 1970s after the paper by Edwards and Steptoe in 1978, reporting the first IVF baby (Steptoe and Edwards 1978). By means of introduction of IVF and the rapid spread of this technique around the world, the research on transplantation of the utero-tubal compartment to circumvent infertility because of blocked tubes promptly disappeared. Nevertheless the small group of absolute uterine factor infertile (AUFI) women, because of lack of a functional or anatomical uterus, remained as a significant group of women that remained untreatable. The research in UTx was reinitiated in the late 1990s, when the calcineurin inhibitor cyclosporine was introduced widely as an effective immunosuppressant in solid organ transplantation. Early research on UTx utilized mostly rodent species, and pregnancy was soon demonstrated in the syngeneic UTx model of the mouse (Racho El-Akouri et al. 2002). Around the same time, the first human UTx attempt was made public (Fageeh et al. 2012). That live donor (LD) UTx attempt, M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_1

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conducted in Saudi Arabia in 2000, was not prepared by any structured research activities and it ended with uterine removal 3 months after UTx. Nevertheless, the attempt undoubtedly stimulated several groups around the world to initiate research programs on the topic of UTx. Thus research developed in multiple animal models, also including domestic species and non-human primate species (Díaz-García et al. 2012). In 2011, the world’s second human UTx attempt was performed, and this was a deceased donor (DD) UTx procedure (Ozkan et al. 2013). The first clinical trial of UTx, involving a series of nine LD UTx cases, was performed in Sweden in 2013 (Brännström et al. 2014). The world’s first live birth after UTx was from a woman of that series with UTx conducted in February 2013 and with delivery of a healthy boy in September 2014 (Brännström et  al. 2015). The boy, Vincent, is now almost 5  years old and is in good health (The road to Vincent n.d.). That birth was followed by seven more births from the Swedish trial (Brännström et  al. 2016; Mölne et  al. 2017) and then in December 2017 births in USA after LD UTx (Testa et al. 2018) and in Brazil after DD UTx (Ejzenberg et al. 2019). The UTx procedure is still at its experimental stage (Brännström et al. 2010), even though a total of almost 20 have taken place up until mid-2019 and also the fact that the births have occurred with a geographical spread between Europe, North America, Latin America, and Asia. All current efforts of human UTx should be performed in well-prepared and systematic ways, as outlined in many chapters of this book, with results (positive and negative) published as proper, peer-reviewed scientific papers.

1.2

 terus Transplantation Research: The Historical U Background and Modern Attempts

The first experimental study with relation UTx is from 1966, when Eraslan and coworkers published a study on “replantation” of the utero-tubal-ovarian compartment in the dog (Eraslan et al. 1966). Vascular dissection was conducted both on the venous and arterial side, and this was followed by division of the common iliac artery, flushing of the uterus in situ, and then reanastomosis of the arterial vessels. The vagina was clamped, but never transected, during flushing and anastomosis. Accordingly, the major surgical interventions were dissection, division of arteries, flushing, and reconnection of arteries. A factual autologous UTx was never performed. In the early 1970s, the technique in the dog was developed further, by including also transection of the vagina (Barzilai et  al. 1973), but the organ was still flushed in its normal anatomical position and the veins were never dissected. Allogeneic UTx in the dog was reported around the same time, with vascular pedicles including the common iliacs (Paldi et al. 1975) or lower aorta/vena cava (Wingate et al. 1970), thus mimicking a DD UTx situation. Rejection occurred after various times due to the ineffectiveness of the immunosuppression agents used at that time. The abovementioned experiments in the dog did not produce any remarkable results, but they represent the historical backbone of the subsequent decades of structured UTx research in animal species.

1  Introduction: Uterus Transplantation

3

During the time from the first human UTx case in 2000 (Fageeh et al. 2012), research on UTx has been conducted in an organized way with use of numerous animal models to examine UTx-specific facets such as surgery, ischemia, rejection, immunosuppression, and fertility (Díaz-García et al. 2012; Brännström et al. 2010). Investigations were done with autologous and syngeneic transplantation models to mainly test the results of surgery. Additional effects of immunosuppression and rejection were tested in allogeneic UTx models. The key experiments concerning fertility in animal models are summarized below. Separate chapters will go through these and other animal experiments in detail. The first demonstration of pregnancy, but not going to term, in a true UTx setting was in the mouse, with a heterotopically positioned uterus after syngeneic transplantation and use of caval-caval and aortic-aortic anastomoses (Racho El-Akouri et  al. 2002). After surgical modifications to include a cervical-cutaneous stoma, instead of the original methodology with abdominally positioned cervix (Racho El-Akouri et  al. 2002), pregnancies were allowed to go to term, with the resultant pregnancy rate and offspring growth trajectory/fertility being normal (Racho El-Akouri et al. 2003). In the rat, an orthotopic UTx model with end-to-side anastomoses between the common iliacs vessels of the graft and of the recipient was used (Wranning et  al. 2011). Pregnancy rate, after natural mating, in a syngeneic transplantation model, was similar in UTx animals as compared to controls (Wranning et al. 2011). Noteworthy is that fertility after allogeneic UTx was for the first time ever reported (Díaz-García et  al. 2010) in this species. Birth weights and growth trajectory of offspring from UTx were normal as compared to controls (Díaz-García et al. 2014). Live births in a large domestic animal, the sheep, was first demonstrated in 2010, by use of an autologous UTx model, with end-to-side vascular anastomoses of the uterine artery, the utero-ovarian vein, and the ovarian artery to the external iliacs (Wranning et al. 2010). The year after, studies with an allogeneic sheep UTx model, with cyclosporine immunosuppression, demonstrated pregnancies and one live birth (Ramirez et al. 2011). This was the first live birth from a large animal undergoing allogeneic UTx (Ramirez et al. 2011). The original, and so far solitary, offspring reported in a non-human primate species was after autologous UTx in the cynomolgus macaque (Mihara et al. 2012). Pregnancy was after natural mating and delivery was by cesarean section.

1.3

 he Live Donor Human Uterus Transplantation T Attempts and Live Births

The first UTx attempt ever in modern history was performed in Saudi Arabia in 2000, when an unrelated peri-menopausal woman, planned for elective ovarian surgery on benign indication, donated her uterus to a woman that had experienced emergency peripartum hysterectomy (Fageeh et  al. 2012). A necrotic uterus was removed 99 days post-UTx. The general view was that this case was not preceded by proper research studies and team preparations.

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In 2013, the Swedish team initiated a clinical LD UTx trial (Brännström et al. 2014), after systematic UTx research for more than a decade (Díaz-García et  al. 2012). The trial included eight MRKH recipients and one recipient with previously diagnosed cervical cancer, treated successfully with radical hysterectomy and pelvic lymph node dissection more than 5 years prior to UTx. Seven of the nine donors were related (five mothers, maternal aunt, sister) to the recipient and one was a family friend and one a mother-in-law. Five donors were postmenopausal. Donor surgery (Brännström et al. 2014) included retrieval of deep uterine veins, including patches/segments of the internal iliac veins, and arterial supply included uterine arteries and internal iliac arteries. Duration of retrieval surgery was 10–13 h. Recipient surgery, with duration of 4–5 h, included dissection of vessels and vaginal vault, bilateral anastomosis to the external iliac vessels, vaginal anastomosis, and structural fixation of the uterus. The 6-month outcome of these LD UTx cases was that seven out of nine uteri showed regular menstruations and were still viable (Brännström et al. 2014). Bilateral thrombotic uterine vessel occlusion and persistent intrauterine infection were the causes of the two uterine removals. Embryo transfer (ET) was performed from 1 year after UTx, a time span after transplantation, which is in accordance with international recommendations for solid organ transplantations. Two patients became pregnant and delivered healthy babies as a result of their first ETs (Brännström et  al. 2015, 2016), and altogether six of the seven women who underwent ET later delivered healthy babies, with two of them being pregnant twice and getting two babies (Mölne et al. 2017). The accumulated take-home baby rate among the seven patients that went through the complete IVF-UTx-ET procedure was 86% and the pregnancy rate was 100%. One patient had repeated miscarriages, but with no delivery. Live donor UTx case number 11 in the world was done in China in November 2015, when a 42-year-old mother donated her uterus to her daughter with MRKH (Wei et al. 2017). The procedure was a full robotic-assisted laparoscopic retrieval surgery with the utero-ovarian veins being the exclusive venous outflow. The organ was extracted through the vagina. Recipient surgery was conventional, as in the Swedish trial (Brännström et al. 2014). Menstruation occurred after 2 months. In January 2019, a live birth from this case was reported in the media. The group around Jiri Fronek in Prague started in early 2016 a clinical trial involving up to ten cases of LD UTx, with the early results of the first five LD cases recently published (Chmel et al. 2018). The recipients were all MRKH women (18– 25 years old). The donors (49–58 years of age) were mothers in four cases and a maternal aunt in one case. Traditional laparotomy surgery was used in both donor and recipient. In a majority of cases, ovarian veins were used as sole venous outflows. One uterus was removed 2 weeks post-UTx due to vascular thrombosis. One live birth has been reported in the media. The group around Sara Brucker in Tubingen, Germany, reported three LD UTx attempts, with one terminated after organ retrieval prior to transplantation (Brucker et al. 2018). In the two cases where actual transplantation occurred, spontaneous and regular menstrual bleedings resumed within 2  months post-UTx. Two live births have been reported in the media.

1  Introduction: Uterus Transplantation

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In the initial trial of LD UTx in the USA (Dallas), the first three recipients lost their grafts within the initial 2 weeks, due to vascular complications (Testa et al. 2017). This initial negative outcome was surprising, in light of that two of the co-­ surgeons were from the Swedish team and that the team leader is an experienced transplant surgeon. Moreover, the transplantation procedures were performed in one of the largest transplantation units in the USA, and the three altruistic donors were of relatively young (42–55  years), non-smokers, and with low BMI.  These early setbacks, in spite of the preparatory factors listed above, highlight the importance of team training, including both gynecologists and transplant surgeons, in a large animal model to be prepared for any new activity concerning human UTx. The surgeries of the subsequent two cases were uneventful and spontaneous menstruations occurred (Testa et al. 2017). In December 2017, the fourth case (uterus from altruistic donor of age around 33 years at donation) delivered a baby by elective cesarean section already at week 33 + 1 after achieving pregnancy at her first ET, which was performed as soon as 6 months after UTx (Testa et al. 2018). There have also been media reports concerning three additional births. Our group performed, in collaboration with the Serbian team of Milan Milenkovic and the Harvard team of Stefan Tullius, one LD UTx procedure between identical twins (donor with three children, recipient with MRKH) in March 2017, in Belgrade, Serbia. No immunosuppression was needed since there were total tissuetype match and no donor-specific antibodies. The recipient delivered a healthy baby boy in June 2018, as reported in the media. An Indian team embarked in May 2017 on a partial conventional laparoscopic approach for LD UTx (Puntambekar et al. 2018). Although the team was experienced in laparoscopy, there had been no preparations for this human UTx endeavor in the research setting, before the two cases. One recipient had the MRKH and one was born with a uterus but had severe intrauterine adhesions after a previous pregnancy that resulted in neonatal death and endometritis. The donors were mothers of ages 42 and 45. Surgery, which at later stages was through a large midline incision, went uneventful in both cases, and follow-up has shown graft viability during the initial months. One birth (2018) has been reported in the media.

1.4

 he Deceased Donor Human Uterus Transplantation T Attempts and Live Birth

To date there are seven published cases of deceased donor (DD) UTx. The first case, which was also globally the second UTx attempt, was performed in Antalya, Turkey, in 2011 (Ozkan et al. 2013). A woman with MRKH was transplanted with a uterus from a 22-year-old nulliparous multi-organ donor. The uterus was the prioritized organ and was retrieved prior to retrieval of the traditional transplantation organs, which is a sequence of organ procurement that is not realistic in a standard clinical situation. The retrieval surgery lasted for 2 h and the transplantation, with bilateral end-to-side anastomosis of the internal iliac vessels of the graft to the external iliac vessels, took 5 h. No live birth has so far been reported from this case although a

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very early pregnancy was reported some years ago (Erman Akar et al. 2013). There exists no information whether the uterus is still in place in the recipient. The second DD UTx in the world was performed in Cleveland, USA, in February 2016. The case was initially reported to be successful, but 2 weeks after transplantation, an acute fungal infection of the graft caused vessel aneurysms, and hysterectomy had to be performed (Flyckt et al. 2016). The largest series of DD UTx attempts is from the Czech Republic, where Jiri Fronek and his team performed four procedures, starting with the initial case in the first half of 2016 (Chmel et  al. 2018). The four recipients (17–21  years old) had MRKH and the DDs were in two cases postmenopausal (age 56 and 57) and with previous normal vaginal births. The other two DDs were of young age (20 and 24 years) and nulliparous, thus with no indication whether the uteri would be functional, in regard to pregnancy. Surgical time of the recipient was 6–8 h, with two uterine veins being used in all cases and additionally two ovarian veins in two cases (Chmel et al. 2018). One uterus was removed after 1 week because of thrombosis and another after 7 months because of endometrial degeneration, secondary to herpes simplex virus infection. No pregnancy has yet been reported. The first successful DD UTx case in the world was performed in Brazil in September 2016, when a 32-year-old MRKH woman received a uterus from a brain-­dead (subarachnoid hemorrhage) 45-year-old, 3-parous DD (Ejzenberg et al. 2019). The cold ischemic time was over 6 h and subsequent recipient surgery (10 h) included bilateral end-to-side anastomosis of the uterine/internal iliac vessels to the external vessels. The recipient became pregnant at her first ET, as early as 6 months after UTx, and elective cesarean section was performed at the recommended time of gestational week 35+. A healthy baby girl was delivered and hysterectomy was performed at the same occasion. Notably, prominent intimal fibrous hyperplasia of the uterine arteries was observed, although the uterus was of relatively young age (Ejzenberg et al. 2019).

1.5

 he Importance of the International Society of Uterus T Transplantation (ISUTx) and Its Registry

The International Society of Uterus Transplantation (ISUTx, www.isutx.org) was founded at a meeting in Gothenburg, Sweden, in January 2016. Around 70 clinicians and scientists with activity in the field of UTx participated in a 2-day meeting, where they drew up the general lines of the society, as well as discussed activities, by-laws, and formation of an international quality registry. The missions of the society were decided to be (1) to facilitate networking between scientists, clinicians, and paramedics worldwide, (2) to advocate patient rights, (3) to educate the public and medical professionals, (4) to share current knowledge and new discoveries through the ISUTx website and the congresses of ISUTx, (5) to promote multidisciplinary collaborative research, (6) to develop consensus and guidelines on uterus transplantation, and (7) to establish and maintain an international registry of uterus transplantation cases with follow-up of patients, children, and donors. The ISUTx had its first international congress in September 2017 (Gothenburg, Sweden) and

1  Introduction: Uterus Transplantation

7

its first state-­of-­the-art meeting in October 2018 (Ghent, Belgium). These meetings were followed by the second international congress of ISUTx in Cleveland, USA, in September 2019. There have been an increasing number of attendees at the formal meetings with participation of most of the leading groups in the field of human UTx. The meetings have been important arenas for exchange of technical details regarding the transplantations and also providing a general update of the global results, through short presentations of unpublished details of the cases, confidentially among attendees. At the meeting in Cleveland, in September 2019, more than 70 UTx procedures had been performed, with the majority being LD UTx procedures and involving transplantation to women with MRKH. Live births had been demonstrated both after LD and DD UTx, as described above. One important agenda for ISUTx is to launch an international UTx quality registry. Through activities by board members of the society and with input from attendees of the first two formal meetings of ISUTx, a web-based registry has been developed. The intention is that data should be entered after each step of the UTx process for any patient undergoing UTx. The data sets relate to recipient and donor characteristics and medical facts; surgery details including complications, immunosuppression, and rejection; pregnancy outcome; and hysterectomy. Similar registries exist for other vascularized composite allograft (VCA) transplantations, such as hand and face transplantation (Petruzzo and Dubernard 2011).

1.6

The Future of Uterus Transplantation

Uterus transplantation is currently spreading globally, but appreciatively mostly in an organized fashion and within clinical trials. The ISUTx registry will be important to keep track of all the cases and for quality control during these coming years. One aspect of the LD UTx procedure that most likely will develop further is use of minimal invasive surgery of the donor. There have been cases using traditional laparoscopic technique (Puntambekar et al. 2018) and robotic-assisted laparoscopy (Wei et  al. 2017) of the donor, but with no attempts to use deep uterine veins and instead using ovarian veins as sole venous outflow, necessitating bilateral oophorectomy. Our group has initiated a trial of LD surgery by robotic-assisted laparoscopy, but also accomplishing harvesting of the deep uterine veins. So far eight procedures have been performed and the first live birth is occurred in April 2019. It is my prediction that robotic-assisted laparoscopy will gradually be used more and more in LD UTx (Brännström et al. 2018), initially for LD surgery but later also for recipient surgery. This development is in line with that of LD kidney transplantation, which at many centers is performed as a fully robotic-assisted laparoscopy procedure in both LD and recipient. Through use of this minimal invasive surgery, we can anticipate a much shorter hospital stay for patients and a short time until they are back into normal activities of daily life. This minimal invasive surgery will of course also open up for nondirected altruistic uterine donors, as it is a reality in LD kidney transplantation (Matas et al. 2000).

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One important aspect of both DD and LD UTx is to include appropriate screening of donors to ensure a good surgical outcome but also live birth after transplantation. Several of the graft failures that have been reported worldwide are most likely due to unfortunate selection of the donors, most concerning quality of uterine arteries. Lifestyle factors, genetics, and chronological age will affect both the arterial and venous vascular systems. Concerning uterine vascularity, it is evident that uterine age is not the major determining factor of success since the first live birth occurred from a uterus that was 63 years at child birth (Brännström et al. 2015) and had been in a postmenopausal state for more than 5 years prior to UTx. Moreover, one of the youngest uteri that have been transplanted in a surgically successful UTx procedure was 22 years of age at donation, but this uterus has not been able to hold a viable pregnancy, despite several years of ET attempts (Ozkan et al. 2013; Erman Akar et al. 2013). We, and others, have found that older uteri may have vessels that are compromised by atherosclerosis and intimal hyperplasia and that UTx procedures on such uteri often have ended in hysterectomy because of uterine necrosis, thrombosis, or non-functionality. Different imaging modalities have been used to visualize the size of the vascular lumen of the uterine arteries, and results from usage of these modalities have to be compared with clinical outcome both in DD and LD UTx. Moreover, other non-/less invasive techniques to pre-donation evaluation of microcirculatory perfusion, implantation ability, and placentation capacity should be developed and included in donor screening. Results from all of the abovementioned screening modalities should be available on short notice, in order to have enough time, also in a DD situation, to base any inclusion/exclusion decision on as many medical facts as possible. Repeated rejection episodes have occurred after a majority of UTx procedures. The rejection episodes have been reversible in a vast majority (>95%) of the episodes after treatment by intensified immunosuppression. The diagnosis of rejection has during the last years solely been based on evaluation of protocol biopsies, taken from the ectocervix. The basis for this is a preliminary scoring system, developed from experience of the initial Swedish trial (Mölne et al. 2017). However, further developments of rejection diagnosis are needed. This may include other less invasive methods for detection of rejection, which could be detected by molecular biology methods of substances/profiles in cytology smears, cervical washings, and other fluids. Another important factor to take into account concerning rejection diagnosis is that normal fluctuations of rejection indicators, such as leukocyte density of the ectocervix, may occur and that we may over-treat some episodes, which actually constitute normal physiological fluctuations. The type of recipients of uterine grafts may also change in the future. Up until today, UTx recipients have been MRKH women in the vast majority but also included single cases of women with hysterectomy post-cervical cancer and hysterectomy post-postpartum bleeding and one woman with Asherman’s syndrome. All women have been in stable relationships or married to male partners, and IVF with the gametes of the couple has been done prior to UTx. It is likely that the UTx procedure in the future will also be available to single women, utilizing IVF treatments with donor sperms. Another group that has been discussed for UTx is transgenders

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of type male-to-female, who naturally would need donor oocytes in IVF treatment with male partner. Similarly, women with androgen insensitivity syndrome (AIS), who genetically also are XY but phenotypically female, would be candidates for UTx but also then with a need for donor oocytes.

1.7

Conclusion

Uterus transplantation is a rapidly evolving field with a geographical spread to most continents. An important factor for a safe and effective transition of this procedure, from an experimental method into a clinical infertility/transplantation treatment, is to share scientific data and to perform modifications of the procedure in a systematic way. This book will be helpful for all groups active in the area of uterus transplantation, or those planning to start clinical activities within the field. It is my wish that the book will be updated only after a few years, to accumulate all new knowledge that surely will be gathered within this period.

References Barzilai A, Paldi E, Gal D, Hampel N. Autotransplantation of the uterus and ovaries in dogs. Isr J Med Sci. 1973;9:49–52. Brännström M, Wranning CA, Altchek A.  Experimental uterus transplantation. Hum Reprod Update. 2010;16:329–45. Brännström M, Johannesson L, Dahm-Kähler P, et al. First clinical uterus transplantation trial: a six-month report. Fertil Steril. 2014;101:1228–36. Brännström M, Johannesson L, Bokström H, et al. Livebirth after uterus transplantation. Lancet. 2015;14:607–16. Brännström M, Bokström H, Dahm-Kähler P, et al. One uterus bridging three generations: first live birth after mother-to-daughter uterus transplantation. Fertil Steril. 2016;106:261–6. Brännström M, Dahm-Kähler P, Kvarnström N.  Robotic-assisted surgery in live-donor uterus transplantation. Fertil Steril. 2018;109:256–7. Brucker SY, Brännström M, Taran FA, et  al. 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. Chmel R, Novackova M, Janousek L, et al. 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. 2018;19:855–64. https://doi.org/10.1111/ajt.15096. Díaz-García C, Akhi SN, Wallin A, et al. First report on fertility after allogeneic uterus transplantation. Acta Obstet Gynecol Scand. 2010;89:1491–4. Díaz-García C, Johannesson L, Enskog A, et al. Uterine transplantation research: laboratory protocols for clinical application. Mol Hum Reprod. 2012;18:68–78. Díaz-García C, Johannesson L, Shao R, et al. Pregnancy after allogeneic uterus transplantation in the rat: perinatal outcome and growth trajectory. Fertil Steril. 2014;102:1545–52. Ejzenberg D, Andraus W, Baratelli Carelli Mendes LR, et al. Livebirth after uterus transplantation from a deceased donor in a recipient with uterine infertility. Lancet. 2019;392:2697–704. Eraslan S, Hamernik RJ, Hardy JD. Replantation of uterus and ovaries in dogs, with successful pregnancy. Arch Surg. 1966;92:9–12. Erman Akar M, Ozkan O, Aydinuraz B, et al. Clinical pregnancy after uterus transplantation. Fertil Steril. 2013;100:1358–63.

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Fageeh W, Raffa H, Jabbad H, Marzouki A.  Transplantation of the human uterus. Mol Hum Reprod. 2012;18:68–78. Flyckt RL, Farrell RM, Perni UC, et al. Deceased donor uterine transplantation: innovation and adaption. Obstet Gynecol. 2016;128:837–42. Matas AJ, Garvey CA, Jacobs CL, Kahn JP. Nondirected donation of kidneys from living donors. N Engl J Med. 2000;343:433–6. Mihara M, Kisu I, Hara H, et al. Uterine autotransplantation in cynomolgus macaques: the first case of pregnancy and delivery. Hum Reprod. 2012;27:2332–40. Mölne J, Broecker V, Ekberg J, et al. Monitoring of human uterus transplantation with cervical biopsies: a provisional scoring system for rejection. Am J Transplant. 2017;17:1628–36. Ozkan O, Akar ME, Ozkan O, et al. Preliminary results of the first human uterus transplantation from a multiorgan donor. Fertil Steril. 2013;99:470–6. Paldi E, Gal D, Barzilai A, et al. Genital organs. Auto and homotransplantation in forty dogs. Int J Fertil. 1975;20:5–12. Petruzzo P, Dubernard JM. The international registry on hand and composite tissue allotransplantation. Clin Transpl. 2011:247–53. Puntambekar S, Telang M, Kulkarni P, et  al. Laparoscopic-assisted uterus retrieval from live organ donors of uterine transplant; our experience of two patients. J Minim Invasive Gynecol. 2018;25:622–31. Racho El-Akouri R, Kurlberg G, Dindelegan G, et al. Heterotopic uterine transplantation by vascular anastomosis in the mouse. J Endocrinol. 2002;174:157–66. Racho El-Akouri R, Kurlberg G, Brännström M. Successful uterine transplantation in the mouse: pregnancy and post-natal development of offspring. Hum Reprod. 2003;18:2018–23. Ramirez ER, Ramirez Nessetti DK, Nessetti MB, et al. Pregnancy and outcome of uterine allotransplantation and assisted reproduction in sheep. J Minim Invasive Gynecol. 2011;18:238–45. Steptoe PC, Edwards RG. Birth after the reimplantation of a human embryo. Lancet. 1978;2:366. Testa G, Koon EC, Johannesson L, et  al. Living donor uterus transplantation: a single center’s observations and lessons learned from early setbacks to technical success. Am J Transplant. 2017;17:2901–10. Testa G, McKenna GJ, Gunby RT Jr, et al. First live birth after uterus transplantation in the United States. Am J Transplant. 2018;18:1270–4. The road to Vincent. (n.d.). https://www.salomonssonagency.se/books/vagen-till-vincent. Wei L, Xue T, Tao KS, et al. 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. Wingate MB, Karasewich E, Wingate L, et al. Experimental uterotubovarian homotransplantation in the dog. Am J Obstet Gynecol. 1970;106:1171–6. Wranning CA, Marcickiewicz J, Enskog A, et al. Fertility after autologous ovine uterine-tubal-­ ovarian transplantation by vascular anastomosis to the external iliac vessels. Hum Reprod. 2010;25:1973–9. Wranning CA, Akhi SN, Diaz-Garcia C, Brännström M. Pregnancy after syngeneic uterus transplantation and spontaneous mating in the rat. Hum Reprod. 2011;26:553–8.

2

The Ethics of Uterus Transplantation: Moral Challenges and Recommendations for Progress Arthur L. Caplan, Brendan Parent, and Pasquale Patrizio

Abbreviations AUFI IRB ISUTx IVF UTx VCA

2.1

Absolute uterine factor infertility Institutional review board International Society of Uterus Transplantation In vitro fertilization Uterus transplantation Vascularized composite allograft

Introduction

A Swedish team at the Sahlgrenska Academy, University of Gothenburg, has achieved a series of successful live births after the transplantation of uteri from living related donors (Brännström et al. 2015;Brännström 2017). From its first description and up to the time of this report (January 2019), more than 50 women have received uterine transplants from in a majority living donors. The countries that have performed human uterus transplantation (UTx) procedures are, in a chronological order of the first case of the nation, Saudi Arabia, Turkey, Sweden, China, the USA, the Czech Republic, Brazil, Germany, Serbia, India, and Lebanon. A total of 14 healthy children born from uterus transplant recipients have been reported in A. L. Caplan · B. Parent Division of Medical Ethics, NYU Langone Medical Center, New York, NY, USA e-mail: [email protected]; [email protected] P. Patrizio (*) Yale School of Medicine, Yale Fertility Center and Yale Center for Bioethics, New Haven, CT, USA e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_2

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the scientific literature and in the media (Brännström, personal communication). Concerning the first eight babies that were born after UTx, all emerged from the Swedish trial. The ages of the children are, as of January 2019, between 4.5 and 1.5 years and all are healthy and mothers and live donors are also with good health (Brännström, personal communication). This remarkable achievement continues a string of innovations in vascularized composite allotransplantation (VCA) including the hands, face, penis, and larynx. The proof of concept demonstrated by the University of Gothenburg group has led to other efforts at UTx. In the USA, the Cleveland Clinic performed an operation with a deceased donor, which ended in failure requiring the removal of the organ (Tribune News Service 2016). At Baylor University in Dallas, the initial four were performed with living donors in the span of a week, and three were removed within 3  weeks due to low blood flow (Baylor Scott and White Health 2016). This is a reminder that there remain a host of under-examined issues accompanying this novel form of transplantation that require careful consideration before this treatment of absolute uterine factor infertility (AUFI) attains widespread clinical use. In this paper, we expand on the initial literature that characterized ethical issues with UTx before trials in humans began by considering the results of the first attempts. Because UTx is underway, we shall address concerns that go beyond the potential physical risks of untested research incurred by the transplant recipients and any resulting fetuses. We aim to characterize requirements that should be met by any team considering UTx (Table 2.1). These include the need for a study design to guide this highly experimental procedure, criteria to be used for subject selection, criteria for selecting living or cadaver donors, the need to define success with respect to this form of transplantation, a strategy for managing failure, and understanding the challenge of access for those seeking to reproduce in terms of cost, options, and legal requirements.

2.2

Should Uterus Transplantation Be Attempted at All?

Uterus transplantation, like other forms of VCA, is not life-saving. It is life-creating by enabling women with AUFI to gestate. But that admirable goal still raises hard questions. The procedure involves exposure, albeit limited, to risky drugs for both mother and fetus; surgery, which takes approximately 4 h, to put in the uterus (Petrini et al. 2017; Falcone et al.2017) and remove it (after live birth is achieved); a C-section delivery that has some degree of risk for both mother and baby; and, in the case of living donation, yet another surgery to obtain a uterus (about 8–12 h). These many steps involve real risks and significant financial costs. Since there are often other options available, these must be weighed against transplantation. Women without a functioning uterus may choose to adopt. They may, in some parts of the world, hire a surrogate. Or, they may have a relative or friend willing

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Table 2.1  Necessary considerations prior to undertaking uterus transplantation Necessary considerations prior to uterus transplantation Related issues Patient selection Psychological screening, comorbidities, family/social support, economic resources, tolerance for publicity Donor selection Living or deceased, consent to specific transplant, family support; if living donor, psychological screening, comorbidities, social support, relationship to patient (lack of indication of coercion), tolerance for publicity Informed consent For patient, patient’s family, living donor, living donor’s family, confirm understanding of the following: length and nature of all associated procedures, potential complications of procedures (hemorrhages, infections, IVF complications, etc.), alternatives, risks/benefits, that successful transplant does not guarantee successful childbirth, costs, long-term care, need for future operations, difficulty ensuring anonymity, intent to publish results IRB/ethics board Review by IRB/ethics board members familiar with VCA (vascularized approval composite allograft); study design including all other elements in this table for review Cost assignment Transplant; transport; long-term physical care; medications; IVF; long-term mental, emotional, and social support; unforeseen outcomes for donor, patient, resulting child Long-term care Immunosuppression, medication, removal of uterus, physical care, counseling/therapy Publishing System for collecting and recording data, compliance with anonymity policies, policy regarding sharing data between multiple institutions System for periodic check-ins with patient, donor, and resulting children Monitoring long-term health/ success Strategy for Disposition of uterus; plan to protect well-being of patient, resulting managing failure fetus, and live donor if used; protocol for allowing/disallowing second attempts at transplant same patient; protocol for allowing/disallowing second attempt at pregnancy

to carry their child to term as an act of altruistic love. Each of these options should be given significant consideration and their benefits and burdens weighed against those of UTx. The US Organ Procurement and Transplantation Network (OPTN) recommends that before a vascularized composite allograft can be given consideration as a treatment option, all other forms of treatment must be explored and found unsatisfactory (Organ Procurement Transplantation Network 2015). For example, although face transplants show great promise in terms of restoring form and function, the OPTN recommendation recognizes that face transplant is still experimental research and subjects the potential recipient to the uncertainty of waiting for a donor and, if matched, to the significant risks of immunosuppression and transplant rejection. Not enough data exists to demonstrate long-term safety, and the consequences of a failed face transplant would be grave.

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Similarly, the alternative “treatments” for AUFI—adoption and surrogacy—do not pose physical risks to the intended parents and only pose the standard risks of pregnancy on the individual giving birth, whereas UTx poses greater risks to the donor (in living donor UTx), the recipient, and potentially the child. Ethically, to justify experimenting on a mother, a fetus, and possibly a donor, it is incumbent on the medical team to show that all other options, if available and permissible, were thoroughly discussed and thoughtfully considered. In offering UTx it may be necessary to initially restrict availability to those women who cannot, for financial, legal, religious, or availability reasons, pursue a less risky option. As the procedure becomes well-characterized and the risks reduced, this restriction can be eliminated.

2.3

Patient Selection

There are a variety of patients with AUFI, who might serve as subjects in studies of UTx. Some are women born without uteri; others may have severely malformed or irreparably damaged organs, or have had their uteri removed due to medical problems. In the USA alone it is estimated that there are some 5000 hysterectomies performed annually in women younger than 24 years old (Nair et al. 2008). Therefore, it is likely that there are many women in the USA, as well as all over the world, who have not yet had the chance to start or complete their families at a time when hysterectomy is performed. Some in the USA, despite the permissibility of gestational surrogacy, may wish to consider experimental UTx particularly considering the high price tag (about $80,000) for alternatives such as gestational surrogacy. In one clinical trial, 500 women signed up to be experimental participants before UTx had been successful in humans (Woessner et al. 2015). This demonstrates a deep desire to not only have biologically related children but also to gestate one’s own children. Because the procedure is currently considered research, the performing institution will cover most of operation costs. However, this will change if UTx becomes an established option for the treatment of AUFI. Individuals potentially interested in UTx may be young or relatively older; have other children or not; have had a child and lost that child; be single, married or remarried, or transgendered; and/or have other comorbid conditions. Age, motivations, child-rearing capacity, and amount of infertility treatment required have been proposed as uterus allocation criteria (Bayefsky and Berkman 2016), but performing institutions will need to determine the extent to which these and other demographic factors should be considered in transplant eligibility. The process of determining eligibility criteria should focus foremost on predicting thresholds for objective medical success. Criteria should be scientifically linked to the likely success of UTx and the capacity to bear children. Judgments regarding the reasons for having a child or the capacity to be a good parent should be reserved.

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It is the healthcare professional’s duty to provide optimal care, not to decide who is worthy. For example, UTx teams will need to consider eligibility of unpartnered women. While IVF is generally available to women without partners, UTx adds several additional risks, for which a dependable support network will likely be necessary. Other family members will likely be able to fill this role as long as they live locally and demonstrate that they will be available to provide necessary support. The state of a given candidate’s support network should be assessed before recipient selection and methods for supplementing support implemented. This could include integrating a budget into long-term costs for infant care service, social worker consultations, and/or helping nonlocal family members temporarily relocate to assist the mother and child. The amount of risk the medical team is willing to bear and impose on a potential recipient will have to be mediated by the fact that the procedure is intended to enable a woman to gestate for creating a new life and to improve her quality of life, but not save it. Potential recipients might also face significant pressure to undergo UTx from partners or family, especially those that come from pronatalistic cultures in which creating an offspring or large family size is expected and not achieving the “right” number of children can carry serious social and physical consequences (Mumtaz 2012). It is important to involve the potential recipient’s partner, if one exists, in the consent process for this procedure, because it implies the desire to procreate. The partner must be made equally aware of the risks and limitations of the transplant, including potential health complications both foreseeable and unforeseeable, the fact that it will not solve other potential infertility conditions, the involvement of a donor (either living or deceased) who will know or whose family may know the recipient’s identity, and that it does not guarantee children. Involving both parties in the consent process, learning their motivations and expectations, and confirming their understanding will be crucial to promoting positive psychosocial outcomes. Strict and transparent selection criteria must be promulgated with an eye toward generating useful data on safety and efficacy and toward promoting fair access with rules as existent for other organs. Paying someone to “donate” a uterus (live donation) is illegal. This requires screening donors to ensure they are not sellers. Donation from a family member or stranger should be allowed with appropriate counseling and consent. Policies must be formulated for allowing non-residents of the USA to be UTx patients or donors, and guidance should be crafted for physicians asked to advise those considering going abroad for the procedure. Because UTx research is still in its infancy, not enough data exists to confirm safety or success for the recipient, living donor, or resulting children. The recipient must undergo at least two major abdominal surgeries—one to implant the uterus and one to remove it (after child birth so the subject will not be permanently on immunosuppressant therapy). The living donor must undergo a major abdominal surgery lasting 8–12 h, resulting in some amount of pain, scarring, and recovery time (Brännström 2017). In a study using a minimally invasive robotic retrieval technique, the procurement operation only took 6 h and likely benefitted

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from less operator fatigue and less blood loss, but resulted in premature surgical menopause (Wei et al.2017;Falcone et al.2017). The initial Cleveland Clinic uterus transplant, with an organ from a deceased donor, was removed due to a yeast infection, which severely constrained necessary blood supply. If the organism originated from donor tissue, the recipient would not have been infected if the transplant had not taken place. If the organism originated in the recipient, it would have had negligible consequences if not for the transplant and immunosuppressant medication (Grady 2016). If a yeast infection spreads to the bloodstream, it can be fatal. These case studies draw attention to the need for detailed publicly available data from each research study, so that teams can share information toward developing best practices. Although the children born from transplanted uteri appear healthy in their first years of life, the risks of immunosuppressant medications on the growing fetuses are not definitive (Orentlicher 2012). Some numbers of pregnancies have been complicated by transplantation of other organs, which poses the question as to whether pregnancy complications are made more likely by transplanting the organ bearing the direct stress of pregnancy (Catsanos et al. 2013). Furthermore, the risks of organ rejection can never be fully eliminated, and they implicate not only the recipient but also a potential fetus, which might be older than 24 weeks. Catsanos et al. expressed an additional concern that these risky and expensive transplants would be for nothing because of low likelihood of establishing a pregnancy in a transplanted uterus (Catsanos et al. 2013), but this seems to have been proven wrong by the eight recent Swedish births. Because of the risks, prospective candidates must be informed of all alternatives including gestational surrogacy, when available, and adoption. After selected recipients and their partners are informed about potential risks, the medical team should come to prior written agreement with the recipients on several possible outcomes regarding the need for early removal of the uterus, or termination of the pregnancy at various stages. Although the performing institution might bear most of operation cost, several peripheral costs remain, including transportation, IVF, maintenance of the surplus cryopreserved embryos, follow-up physical care, medication, and psychosocial support. Research subjects who cannot afford these services face additional and unnecessary risks if the services are not otherwise provided. Fair access requires that efforts be made to determine which costs will be covered by the institution, insurance, and/or government programs and then to assist initial subjects who require help with uncovered costs.

2.4

Who Ought to Be a Donor?

Donors may be living or deceased, fertile but finished with their procreative activity, post-menopausal, and of varying degrees of health. Here again it is necessary to develop eligibility criteria (Table 2.1) that promote the greatest likelihood of success

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while minimizing risks and respecting altruism. It is also important to respect a living donor’s right to refuse, or change their mind, and to provide a medical exemption to those who wish one. For cadaveric donors, protections must be in place to prevent reproductive organs from being utilized without their express prior consent as well as the consent of their immediate family. Privacy concerns must be attended since a family might not realize that a cadaver donation might not be possible due to the lifestyle of the donor or the detection of a transmissible disease that might be stigmatizing. The first US uterus transplant, performed at the Cleveland Clinic in February 2016 and removed in March due to complications, used a deceased donor to avoid the risks to a living donor (Tribune News Service 2016) likely at the behest of the IRB. It is unclear at this point whether the benefits of using a live donor justify the concomitant risks. Transplants using both living and deceased donors should proceed to determine the relative technical merits, while paying adequate attention to the ethical issues related to each. The nine UTx procedures completed in Sweden in 2013 were made possible by living donors, many of whom were biologically related to the recipients (Brännström et al. 2015). Using living related donors might help overcome issues of histocompatibility and, because of the shorter period of ischemia, the quality of the organ could be superior to that obtained from a deceased donor. It is suggested that living donor uteri be selected from mothers who have demonstrated the functionality of their uteri to increase the chances of success in a new recipient. (Brännström et al. 2016) However, as discussed earlier, using living donors requires careful surgery and protection of key blood vessels by skilled surgeons, which can take up to 11 h, whereas procuring a uterus from a deceased donor is simpler and faster and does not put the donor at risk (Grady 2015). At least one team used a novel minimally invasive approach with robotic assistance, which resulted in shorter surgical time but introduced a new risk of premature surgical menopause (Falcone et al.2017). It has yet to be demonstrated whether the benefits of this procedure outweigh the additional risks, or whether the retrieval method has any direct effect on implantation in the recipient or pregnancy. Potential donors and recipients must be informed of the potential risks of this procedure and how they might compare to other retrieval methods. A potential living donor and recipient should also discuss whether the potential donor might carry the child herself (if surrogacy is allowed), in consultation with a social worker or patient advocate. This discussion is best implemented as a requirement prior to consenting to UTx. There are those who believe that potential donors should be allowed to decide for themselves what risks they are willing to incur, even for an experimental procedure. Because of the lack of data, it is impossible for living donor research participants to understand the nature or extent of risks. Informed consent should include the most current data on the physical and psychological health of uterine transplant living donors, recipients, and resulting children, with the caveat that the limited information is anecdotal. Donors must be prepared for the possibility that the donation will not lead to successful child birth and, while perhaps obvious to most, should also

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understand that they will not be getting their uteri back. Just as potential transplant recipients might be subject to undue pressure, so too might living donors. Women with AUFI and/or their partners might pressure their relatives who have already had children to donate. A donor advocate, completely separated from recipient advocates, should be assigned to the living donor (Woessner et al. 2015) to ensure free choice. During eligibility determination, the motivations of the potential donor should be assessed regarding her relationship with the recipient. It should be confirmed that the potential donor does not have any expectations for the recipient’s actions, or for any resulting children, in exchange for donation. After transplant, the relationship between donor and recipient should continue to be assessed during psychosocial support, and anonymous data comparing pre-transplant relationship assessment and post-transplant relationship outcomes should be included in publication. International guidelines are required with respect to mediating donor eligibility and, for selected donors, ensuring voluntariness, understanding risks, and calibrating expectations. For donations from deceased, it is likely that few individuals who are registered as cadaver organ donors are aware that their uteri are among donatable parts. Family members might deny procurement from deceased individuals who would have approved of donating their uterus but were unaware of the possibility during their lives. On the other hand, family members might allow procurement from those who would have disapproved of the practice. Awareness of UTx (as well as other VCA transplants) should be promoted through donor registries and education campaigns to ensure meaningful donor consent (Parent 2014).

2.5

Experimental Protocol

Petrini and co-authors suggest a risk of “therapeutic misconception,” which implies the need to ensure that potential recipients view the procedure strictly as research and that they must not be misled into believing they will receive any therapeutic benefit (Petrini et al.2017). Since UTx is still in its infancy, it is true that it must still be treated as a research activity and not strictly as a therapy. However, it is unlikely that any recipient would agree to the procedure strictly for research purposes. Accordingly, UTx might be better viewed as research with an innovative therapy component. The informed consent process should acknowledge the intended clinical outcome (the capacity to bear children), but should disclose information to potential participants that emphasizes the lack of existing safety data. Hope might blind potential recipients to the risks, which should inform how the discussion is framed by providers. Thorough informed consent must be obtained from potential recipients and, if used, living donors, ensuring a full description and comprehension of risks

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and unknowns. The current state of the procedure in terms of outcomes should be made known. Discussion must occur with subjects, properly documented, about what will happen to embryos or fetuses if the mother’s health is in jeopardy. Strategies must be in place and fully disclosed to patients for managing any and all decisions to prematurely end a pregnancy. Costs for the procedure and follow-up care must be transparent and assigned ahead of time. All cases ought to be followed post-surgery, published, and included in a registry using standardized information to permit assessment and improvement in this form of surgery. The International Society of Uterus Transplantation (ISUTx) has just launched a web-based registry including details of donors, recipients, surgery, immunosuppression, and pregnancy outcome. The registration of all cases, including those with early negative outcomes, should be mandatory for all centers involved with activities in human UTx.

2.6

Proceed Collaboratively and with Care

The early successes and failures of UTx will affect the speed with which the field can advance. Early setbacks, whether avoidable or not, are highly publicized and have the capacity to turn away potential donors, recipients, transplanting teams, sponsoring hospitals, and most importantly insurers. To move from research to standard of care for AUFI and for insurers to cover the significant associated costs, safety and efficacy relative to alternatives will need to be demonstrated across large study samples. This means that universally accepted metrics for success must be developed that reflect patient values and account for the long-term health of potential living donors, recipients, and resulting children.

2.7

Weighing Merits

All VCA transplants are intended to be life-improving, and none are life-saving procedures. They also carry significant risks, including complications with surgery, potential susceptibility to disease due to prolonged immunosuppressant therapies and other uncertain health effects, psychological distress of the recipient and family members, and potential undue pressure placed on the recipient and on living donors. A successful face transplant (of which there have been approximately 30) can restore the recipient’s ability to breathe, blink, eat, sleep, smile, and re-enter society. A successful penis transplant not only restores the recipient’s capacity to procreate but can also restore basic voiding function and appearance and can significantly reduce pain. Uterus transplants provide recipients with the ability to carry a child to term, which might play an important role in identity for some women (Catsanos

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et al. 2013) and might restore self-esteem and their sense of femininity (Petrini et al. 2017). Many women might find gestation as important to their qualities of life as breathing. On the other hand, it has been argued that society too deeply associates childbearing with femininity, placing unfair expectations and pressure on women to bear children and thus devaluing them if they are unable or choose not to do so (Catsanos et  al. 2013). It is possible that developing this medical procedure, the sole purpose of which is to create childbearing opportunities for women without functioning uteri, exacerbates the incorrect notion that such women are “broken” and should be “fixed.” Uterus transplants are not the only means of providing a woman with a child. At least two studies indicate that pregnancy confers no physical or psychological benefits for the mother, the child, or their relationship as compared with using a surrogate or adopting (Golombok et al. 2006; Söderström-Anttila et al. 2015). On the other hand, there is some correlation between the development of mother-child bond and the mother’s oxytocin levels while the fetus is in utero (Allen-West 2007), which might support the benefits of UTx. It is also possible that physical sensation plays an important role in the development of this bond, but because a transplanted uterus will not be innervated as a native uterus and any resulting babies will be delivered via elective cesarean sections, it has been speculated that the mother with a transplanted uterus will not “feel” the pregnancy in the traditional sense (Mumtaz 2012; Catsanos et al. 2013). These facts must be considered along with examples of strong bonds between mothers and adopted children and between mothers and children born from surrogacy. However, adoption and surrogacy are not without their own challenges. When adopting from foster care—the least expensive route—it is very difficult to get a child younger than 3 years old, and many of these children carry histories of emotional and physical abuse (Newman 2008). Adopting from agencies domestic or abroad can cost up to $40,000 (Newman 2008). Surrogacy on the other hand is not legal in many states (Lewin 2014) and is forbidden in several European countries including Austria, Denmark, Finland, France, Germany, Hungary, Italy, Portugal, Spain, and Sweden. These economic constraints and legal prohibitions leave scores of women unable to fulfill their desire to create a family. Even where legal in the USA, surrogacy is not covered by insurance and is very expensive (about $80,000) (Covington and Patrizio 2013). Furthermore, surrogacy court cases demonstrate the myriad associated technical and ethical complications, including the possibility of coercion of the surrogate (Saul 2009), determining the disposition of the child when intended parents change their minds (In re Marriage of Buzzanca 1998) or when the surrogate becomes attached to the fetus (In re Baby M 1988), and liability if something happens to the baby or the baby is not “as expected” (Glionna 2001). There are also cultural barriers to surrogacy and adoption. For example, the Islamic faith holds that the only true mother of the child is the birth mother, and according to some, adopted children cannot “carry on the patriline, or inherit the family property” (Mumtaz 2012).

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Bayefsky and Berkman accept that UTx bears a significant and ethical value proposition and believe the most relevant moral consideration is fair allocation (Bayefsky and Berkman 2016). The four allocation criteria they lay out—motivation to seek treatment, age, child-rearing capacity, and amount of infertility treatment required—are logical and promote fairness. While we agree that UTx has the potential to reach a point where allocation is the most valid concern, we must also consider when the benefits outweigh the risks for each potential recipient and how to ensure that transplant teams are adequately prepared to protect the health and rights of recipients, family members, donors, and resulting children. The primary concerns regarding the physical and psychosocial risks inherent in using either a deceased unaware donor or a live related donor for a transient quality-­of-­life transplant have not changed since Caplan et  al. sketched out the potential risks in 2007 (Caplan et al.2007). Protocol must first be established to protect against and monitor several risks unrelated to access, including health consequences for recipients and fetuses in utero; complications in the development of resulting children; exacerbation of illegal organ trade, particularly in pronatalistic countries that already have significant black markets for organs; how long a recipient should be allowed to continue facing associated risks including immunosuppression if a live birth has not been achievable; undue pressure on recipients or living donors; and the procedure’s impact on relationships between living donors, recipients, and partners before, during, and after transplant. Fourteen births are very promising, but few more are necessary to establish evidence of safety and efficacy. In conclusion, UTx procedures are likely to become an important treatment option for women with AUFI who feel that carrying a child is an essential part of their lives, adding to their quality-of-life paradigm, or for those unable to resort to alternative means of creating families for legal, financial, or religious prohibitions. Initial data on safety and success is promising. Still, the practice must be informed by the accumulation of evidence. It is possible that the physical and psychosocial risks that weigh against UTx and in favor of surrogacy or adoption can be alleviated after carefully planned and ethically vetted research, but this research endeavor should not proceed until protocol is developed with attention to the considerations detailed in this paper. It is critical that the health and well-being of the mothers, living donors, and resulting children in live births, as well as the parties involved in ongoing activities, are carefully monitored and that the data is published. Published data should include negative outcomes and should be shared, comparing how different methods of retrieval and implantation affect outcomes toward establishing standard practice. The ISUTx registry has an important role here with planned annual reports as well as possibility to extract data for research studies, which include data from an international perspective. Given the potential for failures and the risky consequences, it is mandatory for any surgical team wanting to offer UTx to acknowledge and comply with the list of ethical considerations.

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References Allen-West C. Level of oxytocin in pregnant women predicts mother-child bond. Association for Psychological Science; 2007. http://www.psychologicalscience.org/index.php/news/releases/ level-of-oxytocin-in-pregnant-women-predicts-mother-child-bond.html. Bayefsky MJ, Berkman BE. The ethics of allocating uterine transplants. Camb Q Healthc Ethics. 2016;25(3):350–65. Baylor Scott & White Health. Uterine transplant clinical trial update—October 5, 2016. Baylor Scott & White Health; 2016. http://news.bswhealth.com/releases/ uterine-transplant-clinical-trial-oct-5-2016. Brännström M. Womb transplants with live births: an update and the future. Expert Opin Biol Ther. 2017;17(9):1105–12. Brännström M, Johannesson L, Bokström H, Kvarnstromet N, Mölne J, Dahm-Kähler P, et  al. Livebirth after uterus transplantation. Lancet. 2015;385:607–16. Brännström M, Bokström H, Dham-Khaler P, Diaz-Garcia C, Ekberg J, Enskog A, et al. One uterus bridging three generations: first live birth after mother-to-daughter uterus transplantation. Fertil Steril. 2016;106(2):261–6. Caplan A, Perry C, Plante LA, Saloma J, Batzer FR.  Moving the womb. Hastings Cent Rep. 2007;37:18–20. Catsanos R, Rogers W, Lotz M. The ethics of uterus transplantation. Bioethics. 2013;27(2):65–73. Covington SN, Patrizio P. Gestational carriers and surrogacy. In: Sauer MV, editor. Principles of oocyte and embryo donation. London: Springer-Verlag; 2013. p. 277–88. Falcone T, Farrell R, Flyckt R. The future of human uterine transplantation: can minimally invasive techniques provide a uterus suitable for transplant? Fertil Steril. 2017;108(2):243–4. Glionna J.  Twins rejected, surrogate birth mother sues. 2001. http://articles.latimes.com/2001/ aug/11/local/me-33076. Golombok S, Murray C, Jadva V, Lycett E, MacCallum F, Rust J. Non-genetic and non-gestational parenthood: consequences for parent–child relationships and the psychological well-being of mothers, fathers and children at age 3. Hum Reprod. 2006;21:1918–24. Grady D. Uterus transplants may soon help some infertile women in the US become pregnant. NY Times. 2015. http://www.nytimes.com/2015/11/13/health/uterus-transplants-may-soon-helpsome-infertile-women-in-the-us-become-pregnant.html?_r=1. Grady D.  Yeast infection led to removal of transplanted uterus. NY Times. 2016. http://www. nytimes.com/2016/04/09/health/yeast-infectionled-to-removal-of-transplanted-uterus. html?_r=2. In re Baby M, 537 A.2d 1227, 109 N.J. 396 (N.J. 1988). In re Marriage of Buzzanca, 61 Cal. App. 4th 1410 (Cal. App. 4th Dist. 1998). Lewin T.  Surrogates and couples face a maze of laws, state by state. NY Times. 2014. http:// www.nytimes.com/2014/09/18/us/surrogates-and-couples-face-a-maze-of-laws-state-bystate. html?_r=0. Mumtaz Z. Ethics criteria for uterine transplants: relevance for low-income, pronatalistic societies? J Clin Res Bioethics. 2012;S:1–5. Nair A, Stega J, Richard Smith J, Del Priore G. Uterus transplant; evidence and ethics. Ann N Y Acad Sci. 2008;1127:83–91. Newman S. Why more people don’t adopt. Psychol Today. 2008. https://www.psychologytoday. com/blog/singletons/200810/why-more-people-don-t-adopt. Orentlicher D. Toward acceptance of uterus transplants. Hastings Cent Rep. 2012;42(6):12–3. Organ Procurement and Transplantation Network. VCAs from living donors. 2015. http://optn.transplant.hrsa.gov/resources/by-organ/vascular-composite-allograft/vcas-from-living-donors/. Parent B. Informing donors about hand and face transplants: time to update the uniform anatomical gift act. J Health Biomed Law. 2014;10:309–26. Petrini C, Gainotti S, Morresi A, Nanni Costa A. Ethical issues in uterine transplantation: psychological implications and informed consent. Transplant Proc. 2017;49:707–10.

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Saul S.  New Jersey judge calls surrogate legal mother of twins. NY Times. 2009. http://www. nytimes.com/2009/12/31/us/31surrogate.html. Söderström-Anttila V, Wennerholm UB, Loft A, Pinborg A, Aittomäki K, Romundstad LB, et al. Surrogacy: outcomes for surrogate mothers, children and the resulting families—a systematic review. Hum Reprod Update. 2015;22(2):260–76. Tribune News Service. Cleveland clinic says first uterus transplant in U.S. fails. Chicago Tribune. 2016. http://www.chicagotribune.com/lifestyles/health/ct-uterus-transplant-fails20160314-story.html. Wei L, Xue T, Tao K-S, Zhang G, Zhao G-Y, Yu S-Q. 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(2):346–56. Woessner J, Blake V, Arora K. Ethical considerations in uterus transplantation. Theor Med Bioeth. 2015;5:81–8.

3

Patients with Uterine Factor Infertility: General Cesar Diaz Garcia

3.1

Introduction

Absolute uterine factor infertility (AUFI) refers to a type of infertility that is 100% attributable to the absence of a normal uterus, either by anatomical or functional causes, which prevents the implantation of an embryo or the ability to carry a term pregnancy. The cause of AUFI can be either congenital or acquired. There are also some uterine abnormalities, whose presence can cause variable degrees of infertility or subfertility, although it is difficult to prove that such uterine abnormality is the major cause of infertility in each specific case. The group is referred to as relative uterine factor infertility (RUFI). Patients belonging to the RUFI group may often benefit from other established medical or surgical treatments, and uterus transplantation (UTx) should only be considered as the last resort when all other therapeutic options have failed. Because of the abovementioned issues, we can describe a cause specificity when it comes to uterine infertility. The prevalence of uterine infertility among patients of childbearing age is not exactly known, but it is likely to be significant, with a recent estimation of 12,000–15,000 uterine-infertile patients in the UK (Sieunarine et al. 2005). This estimation would indicate the presence of more than 150,000 uterine-­ infertile patients in Europe, although obviously only a portion of these would have the desire to obtain a pregnancy through UTx. As a general rule, the more serious the cause of uterine infertility is, the less prevalent the condition (Table 3.1). Women lacking an anatomical uterus will naturally belong to the AUFI group. Women with an anatomical uterus are considered to have RUFI.

C. D. Garcia (*) IVI-London, IVI-RMA Global, London, UK e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_3

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Table 3.1  Main causes of infertility of uterine origin susceptible of being treated by uterus transplantation (UTx) Related infertility/ Prevalence (%) Cause sterility (%) Absolute infertility (only treatable by adoption, surrogacy, or UTx)  Uterine agenesis 0.0002 100  Leiomyomas requiring hysterectomy 1 100  Post-partum hysterectomy 0.04–1.25 100  Hysterectomy for cervical neoplasia 0.00004–0.0001 100  Uterine hypoplasia 0.038 100a Relative infertility (patients in whom UTx should only be considered as a last-line treatment)  Intrauterine adhesions 25% and cytomegalovirus (CMV) viremia in three animals. Menstruation did not occur after surgery in any animals. Basic experiments on autologous UTx in 16 baboons were performed in Saudi Arabia prior to the first clinical application of UTx (Fageeh et al. 2002). In early experiments, end-to-end anastomosis of the uterine vessel was used, and vascular thrombosis and graft failure occurred in 75% of anatomized vessels, resulting in anastomotic occlusion and pelvic abscesses. In subsequent experiments, procedures for end-to-side anastomosis of the internal iliac and uterine vessels were improved and vascular patency was confirmed in 90% of vessels, indicating a greater success rate of anastomosis. Detailed outcomes, including individual births and deaths, recovery of menstruation, and pregnancy, were not described. Uterine perfusion has also been examined in baboons as background research for UTx. In baboon models in which uterine veins or uterine arteries and veins were ligated, uterine and ovarian viability was not affected and cyclical sexual skin turgescence and menstruation were observed in all animals (Shockley et  al. 2017). This study proved that perfusion and drainage of the baboon uterus can be achieved through the utero-ovarian vessels. In a subsequent study by the same group, an angiosome model using microsurgically anastomosed utero-ovarian vessels and lacking uterine arteries and veins was shown to support gestation to live birth (Beran et al. 2017). Pregnancy occurred in all animals and live birth in two of four gestations was achieved, with stillbirth in two of four gestations and fetal growth restriction in one of out of four. These studies show that utero-ovarian vessels alone may support pregnancy and delivery and allow living uterine donors to undergo less-­ invasive and shorter donor hysterectomy procedures.

7.3

Studies in Rhesus Macaque

A group in the USA (New York) has conducted research on preservation and restoration of fertility by transplantation of reproductive organs and preliminary experiments on UTx in 27 rhesus monkeys and systematic UTx in five of the animals (Del Priore et al. 2008). Allogeneic UTx was performed with cyclosporine and the surgical procedure was described in detail, but no detailed outcomes were reported in the article.

7.4

Studies in Cynomolgus Macaque

Cynomolgus macaques are commonly used for experiments because of their anatomic and physiologic similarity to humans, but their small body weight, ranging from 3 to 4 kg, is a significant disadvantage. Our group in Japan has conducted most basic UTx research with cynomolgus macaques, including studies of autologous and allogeneic UTX, uterine blood flow, organ perfusion, and ischemia.

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Indocyanine green (ICG) fluorescence imaging was used to examine uterine hemodynamics and vessels associated with uterine blood flow (Kisu et al. 2012a). Observation of intraoperative uterine hemodynamics using ICG fluorescence imaging with selection of various nutrient vessels by clamping of blood vessels suggested that uterine arteries contribute to the blood supply significantly more than ovarian arteries, and that blood flow from the uterine artery perfuses mainly ovarian veins, rather than superficial uterine veins. However, deep uterine veins were not evaluated in this study. Therefore, it was difficult to conclude exactly which veins (ovarian vessels, uterine vessels, and deep uterine veins) are the main contributors to uterine drainage. In a pilot study of whether childbearing is supported by a unilateral uterine artery and vein (Kisu et al. 2013b), the subject was observed while blood flow to the uterus was maintained by the right uterine artery and vein alone. After resumption of regular menstruation, natural pregnancy and full-term delivery were achieved with collateral vessels. These results indicated that pregnancy and delivery can be supported by a unilateral uterine artery and vein, but with the major limitation of the study being performed in only one animal. We first conducted initial autologous orthotopic UTx in six cynomolgus macaques (Kisu et al. 2012b; Mihara et al. 2011, 2012). In the first four cases, death occurred in three animals during surgery due to hemorrhage from microvascular anastomosis and inadequate anesthetic management. This occurred because fine vascular dissection in the pelvic floor and microvascular anastomosis with deep uterine veins were required. Recovery of menstruation occurred in only one case out of four. From case 5 onward, autologous blood was prepared before surgery and ovarian veins, instead of deep uterine veins, were mainly used for vascular anastomosis. Using this approach, cases 5 and 6 both survived and one had recovery of menstruation. The average times required for vascular anastomosis and the surgical procedure in all cases were ~4.5 h and ~14 h, respectively. The sixth monkey became pregnant spontaneously after surgery and the first delivery worldwide in a nonhuman primate was achieved (Mihara et al. 2012). A group in China (Guangzhou) also performed six autologous UTx procedures in cynomolgus macaques (Wang et al. 2014), with five surviving after surgery and two resuming cyclicity. The average vascular anastomotic time and total operative times of ~2 h and 6 h, respectively, were much shorter than in our procedures. Detailed information is limited because the article was published in Chinese, with only the abstract in English. After the first delivery in autologous UTx, we examined allogeneic UTx in cynomolgus macaque (Kisu et al. 2014). In a preliminary study, uteri were interchanged between two animals and then transplanted orthotopically. Immunosuppressive protocols included use of three agents (tacrolimus, mycophenolate mofetil, and methylprednisolone) in case 1 and two agents (tacrolimus and methylprednisolone) in case 2, all by oral administration. The surgical time was ~13.5 h and the vascular anastomosis time was ~3.5 h in both cases. Both animals had long-term survival (survival rate, 100%). Rejection occurred in both cases after surgery due to poor control of immunosuppressant levels, but then resolved, and menstruation occurred transiently in case 1. Ischemia in the uterus was associated with rejection, and uterine atrophy was found in case 2. Due to the need for complicated vascular

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anastomosis and dissection of uterine vessels, longer and thicker vessels including common iliac vessels (n = 2) or abdominal aorta/inferior vena cava (n = 2) were utilized in a subsequent study, assuming a deceased donor model (Obara et  al. 2016). Prior to these studies, perfusion via the femoral artery and/or external iliac artery, for organ perfusion in the abdomen, were examined and this procedure was found to be useful in procurement of a uterus (Kisu et al. 2016). In the allogeneic UTx study, the vascular anastomosis times were shortened to ~2.5 h for anastomosis of the common iliac vessels and ~1 h for the abdominal aorta/inferior vena cava, and the respective operative times in recipients were ~9 h and ~6 h, with long-term survival of all animals. Moreover, we succeeded first pregnancy after allogeneic UTx in nonhuman primate models (unpublished data). Therefore, a stable surgical procedure for allogeneic UTx in cynomolgus macaque was established by performing vascular anastomosis with major blood vessels. Immunological findings are not described in the published article. In other studies in cynomolgus macaque, a surgical technique using ovarian veins, assuming potential living donor surgery for UTx, was shown to have a decreased donor surgical time, indicating that this donor surgery is less invasive for UTx compared with use of uterine veins (Kisu et al. 2015). A study of the allowable warm ischemic time and morphological and biochemical changes in uterine ischemia/ reperfusion injury showed that warm ischemia in the uterus in cynomolgus macaque is tolerated for up to 4 h and that reperfusion after uterine ischemia causes no further morphological and biochemical changes (Adachi et al. 2016; Kisu et al. 2017).

7.5

 roblems and Limitations in Nonhuman Primate P Models for Uterus Transplantation

Validation of UTx procedures in nonhuman primate models, have many advantages for extrapolation to humans, but there are certain problems and limitations with experimental use of nonhuman primates for UTx (Tzakis 2013; Kisu et al. 2013c). The size of female macaques is approximately 3–4 kg, which is very small compared to humans, and complex vascular dissection of the pelvic floor is required in UTx. Therefore, fine surgical techniques are required in the macaque model. Postoperative management also has some difficulties. Control of the blood concentration of immunosuppressive agents is important because of effects on graft survival, but it is difficult to administer these agents orally in nonhuman primates as scheduled, and delivery through enteral feeding is problematic because of marked anorexia after invasive surgery and side effects of immunosuppressive agents. Intravenous administration is simple in humans, but difficult to perform in nonhuman primate models. These difficulties also cause marked postoperative weight loss that cannot be recovered by supplementation with oral nutrients alone, and excessive weight loss leads to an endpoint of the experiment in most settings. Moreover, postoperative examinations including echography and biopsy cannot be easily performed because sedation is required. Blood test items also are limited in nonhuman primate, in contrast to humans.

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Reproductive outcomes are very important components of UTx experiments. A further problem is that assisted reproductive technology (ART) is essential after UTx in humans, but this aspect of the procedure is not well-established in nonhuman primates. Sperm counts in male baboons are extremely low and there is little experience with ART in this species. In cynomolgus macaque, technology to conduct transtubal embryo transfer has gradually been developed, but intra-abdominal adhesion is severe after UTx and the fallopian tube is often obstructed. Transvaginal embryo transfer could be an alternative procedure, as in humans, but bending and tortuosity of the cervical canal in cynomolgus macaque make this impossible. The cost of experiments using nonhuman primates is also a major problem and this limits the number of animals that can be used. The small number of animals in each study limits the margin of error and the opportunity for corrective action. There are also limitations from the perspective of animal welfare and regulatory restrictions, in comparison with small animals. Therefore, the number of animal centers that can keep nonhuman primates is limited in each country. Consequently, researchers often have to visit a primate center from afar, and sometimes from overseas, which causes difficulties with frequent visits, observations, examinations, and prompt treatment after the procedures.

7.6

Conclusion

Data from animal studies, including studies in nonhuman primates, have provided the basis for clinical application of UTx as a medical technology in humans, despite the procedure still being in an experimental stage. However, there are few outcomes of UTx in nonhuman primates and delivery after allogeneic UTx has not been achieved due to difficulties with postoperative control by immunosuppressive agents, limited ART, surgical procedures in small animals, and costs. Therefore, further validation in nonhuman primate models is needed for resolution of these issues for UTx in humans. Data accumulation in basic experiments in nonhuman primates is essential for establishment of the safety and efficacy of the procedure. Conflict of Interest  None declared.

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Brännström M, Johannesson L, Bokström H, Kvarnström N, Mölne J, Dahm-Kähler P, Enskog A, Milenkovic M, Ekberg J, Diaz-Garcia C, Gäbel M, Hanafy A, Hagberg H, Olausson M, Nilsson L. Livebirth after uterus transplantation. Lancet. 2015;385:607–16. Del Priore G, Schlatt S, Malanowska-Stega J. Uterus transplant techniques in primates: 10 years’ experience. Exp Clin Transplant. 2008;6(1):87–94. 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(11):1491–4. Enskog A, Johannesson L, Chai DC, Dahm-Kähler P, Marcickiewicz J, Nyachieo A, Mwenda JM, Brännström M.  Uterus transplantation in the baboon: methodology and long-term function after auto-transplantation. Hum Reprod. 2010;25(8):1980–7. Fageeh W, Raffa H, Jabbad H, Marzouki A. Transplantation of the human uterus. Int J Gynaecol Obstet. 2002;76(3):245–51. 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(6):1640–8. Johannesson L, Enskog A, Mölne J, Diaz-Garcia C, Hanafy A, Dahm-Kähler P, Tekin A, Tryphonopoulos P, Morales P, Rivas K, Ruiz P, Tzakis A, Olausson M, Brännström M.  Preclinical report on allogeneic uterus transplantation in non-human primates. Hum Reprod. 2013;28(1):189–98. Kisu I, Banno K, Mihara M, Lin LY, Tsuji K, Yanokura M, Hara H, Araki J, Iida T, Abe T, Kouyama K, Suganuma N, Aoki D.  Indocyanine green fluorescence imaging for evaluation of uterine blood flow in cynomolgus macaque. PLoS One. 2012a;7(4):e35124. Kisu I, Mihara M, Banno K, Hara H, Yamamoto T, Araki J, Iida T, Hayashi Y, Moriguchi H, Aoki D. A new surgical technique of uterine auto-transplantation in cynomolgus monkey: preliminary report about two cases. Arch Gynecol Obstet. 2012b;285(1):129–37. Kisu I, Banno K, Mihara M, Suganuma N, Aoki D. Current status of uterus transplantation in primates and issues for clinical application. Fertil Steril. 2013a;100(1):280–94. Kisu I, Banno K, Yanokura M, Nogami Y, Umene K, Tsuji K, Masuda K, Ueki A, Kobayashi Y, Aoki D.  Indocyanine green fluorescence imaging in the pregnant cynomolgus macaque: childbearing is supported by a unilateral uterine artery and vein alone? Arch Gynecol Obstet. 2013b;288(6):1309–15. Kisu I, Banno K, Mihara M, Aoki D. Uterus transplantation in nonhuman primates. Fertil Steril. 2013c;100(1):e3. Kisu I, Mihara M, Banno K, Hara H, Masugi Y, Araki J, Iida T, Yamada Y, Kato Y, Shiina T, Suganuma N, Aoki D. Uterus allotransplantation in cynomolgus macaque: a preliminary experience with non-human primate models. J Obstet Gynaecol Res. 2014;40(4):907–18. Kisu I, Banno K, Mihara M, Hara H, Umene K, Adachi M, Nogami Y, Aoki D. A surgical technique using the ovarian vein in non-human primate models of potential living-donor surgery of uterus transplantation. Acta Obstet Gynecol Scand. 2015;94(9):942–8. Kisu I, Kato Y, Yamada Y, Matsubara K, Obara H, Emoto K, Adachi M, Umene K, Nogami Y, Banno K, Kitagawa Y, Aoki D.  Organ perfusion for uterus transplantation in non-human primates with assumed procurement of a uterus from a brain-dead donor. Transplant Proc. 2016;48(4):1266–9. Kisu I, Umene K, Adachi M, Emoto K, Nogami Y, Banno K, Itagaki I, Kawamoto I, Nakagawa T, Narita H, Yoshida A, Tsuchiya H, Ogasawara K, Aoki D. Allowable warm ischemic time and morphological and biochemical changes in uterine ischemia/reperfusion injury in cynomolgus macaque: a basic study for uterus transplantation. Hum Reprod. 2017;32(10):2026–35. Mihara M, Kisu I, Hara H, Iida T, Yamamoto T, Araki J, Hayashi Y, Moriguchi H, Narushima M, Banno K, Suganuma N, Aoki D, Koshima I.  Uterus autotransplantation in cynomolgus macaques: intraoperative evaluation of uterine blood flow using indocyanine green. Hum Reprod. 2011;26(11):3019–27. 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

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I. Uterine autotransplantation in cynomolgus macaques: the first case of pregnancy and delivery. Hum Reprod. 2012;27(8):2332–40. Milliez J. Uterine transplantation FIGO committee for the ethical aspects of human reproduction and women’s health. Int J Gynaecol Obstet. 2009;106(3):270. Obara H, Kisu I, Kato Y, Yamada Y, Matsubara K, Emoto K, Adachi M, Matoba Y, Umene K, Nogami Y, Banno K, Tsuchiya H, Itagaki I, Kawamoto I, Nakagawa T, Ishigaki H, Itoh Y, Ogasawara K, Saiki Y, Sato SI, Nakagawa K, Shiina T, Aoki D, Kitagawa Y. Surgical technique for allogeneic uterus transplantation in macaques. Sci Rep. 2016;6:35989. 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(2):238–45. Shockley M, Arnolds K, Beran B, Rivas K, Escobar P, Tzakis A, Falcone T, Sprague ML, Zimberg S. Uterine viability in the baboon after ligation of uterine vasculature: a pilot study to assess alternative perfusion and venous return for uterine transplantation. Fertil Steril. 2017;107(4):1078–82. Tryphonopoulos P, Tzakis AG, Tekin A, Johannesson L, Rivas K, Morales PR, Wagner J, Mölne J, Enskog A, Diaz-Garcia C, Dahm-Kähler P, Berho M, Zimberg S, Falcone T, Ruiz P, Olausson M, Brännström M. Allogeneic uterus transplantation in baboons: surgical technique and challenges to long-term graft survival. Transplantation. 2014;98(5):e51–6. Tzakis AG.  Nonhuman primates as models for transplantation of the uterus. Fertil Steril. 2013;100(1):61. Wang Y, Zhu Y, Yu P, Chen G, Cai B, Zhang Z, Liu N, Lü X, Xiong J, Zhong L, Rao J. Uterine autologous transplantation in cynomolgus monkeys: a preliminary report of 6 case. Zhonghua Yi Xue Za Zhi. 2014;94(47):3774–7. in Chinese.

8

Human Preclinical Research in Uterus Transplantation Mats Brännström

There exist a small number of preclinical research studies, with use of human material, concerning uterus transplantation (UTx). An initial preclinical human study evaluated the effect of cold ischemia on small tissue pieces of human myometrium that were excised directly after benign hysterectomy, performed during the peri-menopausal period (Wranning et al. 2005). The tissue pieces of about 15 × 25 mm size were from the myometrium of the fundal region, and they were brought to the laboratory to be submerged into either of two preservation solutions (the intracellular-like University of Wisconsin solution (UW) or the extracellular-like Perfadex solution (PER)) or in Ringer’s acetate (RA) for 6 h or 24 h. They were evaluated concerning spontaneous contractility, prostaglandin-­induced contractility, and levels of the energy source adenosine triphosphate (ATP) and by transmission electron microscopy. The tissue that had been in proper preservation solutions (UW, PER) showed normal ultra-morphology, as evaluated by electron microscopy, but degenerative changes were seen in tissue that had been kept for 24 h in RA (Wranning et al. 2005). The levels of ATP were also higher after preservation for 24 h in UW and PER as compared to storage in RA. Functionality of the tissue was examined by evaluating contractile patterns. It was shown that both spontaneous and prostaglandin-induced contractions after 6 h storage in UW, but less in PER, were relatively similar to fresh control tissue. However, after 24 h of storage, the contractile patterns were clearly negatively affected. Taken together, the results showed that human myometrial tissue is resistant toward cold ischemia for at least 6 h if stored in proper preservation solution, with an advantage of intracellular-like preservation solutions, such as UW, over extracellular-like solutions, such as PER. However, possible damage during reperfusion was not assessed in these incubation experiments. M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_8

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Another study looked at the tolerance of the human uterine tissue after preservation in Celsior preservation solution in a transplantation situation (Sieunarine et al. 2008). The reason to test the Celsior solution is that that is frequently used in cardiac transplantation and that the heart, like the uterus, is essentially a muscular tissue. This study evaluated morphology on the light and electron microscopy levels with findings indicating acceptable morphology after 24-h preservation in Celsior, with unchanged structural integrity of the uterine myo-endometrium. Major structural damage was seen after preservation for 48 h. Thus, the results suggest that a long (up to 24 h) cold-ischemia period in a human UTx situation is acceptable, but in line with other transplantation settings, the rule of “less ischemic time is better” should also apply for UTx. In human live donor UTx, most cases have so far been performed with the live donor (LD) concept, and that was also the technique our group had explored in domestic species, in primates, and lastly in the initial human clinical UTx trial (Brännström et al. 2014). Before that trial, only one LD human UTx case had been performed and that was in year 2000 (Fageeh et al. 2002). In the latter case, the uterine arteries and veins on both sides were too short to reach the anastomosis sites on the external iliac vessels, and both uterine arteries and veins were elongated with segments of the saphenous veins. The failure of that case (Fageeh et al. 2002) may have been related to these extensions, with several anastomosis lines that would be potential origins of thrombosis formation. We conducted a study in patients undergoing open radical hysterectomy for cervical/uterine cancer with the aim to separately dissect uterine arteries and veins (Johannesson et al. 2012). This was done to explore how long vascular pedicles could be retrieved and if they were lengthy enough to make LD-UTx feasible without interposing with another vascular graft. The accomplished lengths of the free vascular pedicles of uterine arteries and veins were around 6 cm. In the same patients, we measured the inter-iliac external distance to around 10 cm, so the acquired pedicle lengths on each side of the centrally positioned cervix (diameter of around 4 cm) would permit direct, bilateral anastomosis on the external iliac vessels. This was an important finding to reassure that LD human UTx would be feasible, by a technique that excluded additional vascular grafts. Dissection of the deep uterine veins toward the internal iliac vein is the most difficult part in uterine retrieval from LD donor, but it is also important to be well acquainted with the anatomy of the internal iliac vessels in a DD situation. In an important study (Beran et al. 2018), including literature review and laparoscopic dissection of cadaveric pelvic vasculature, it was established that there are minimal anatomical variations in the anatomy of the internal iliac artery but with variations in the anatomy of the internal iliac vein. Relative to the internal iliac artery, the vein can lie medially or laterally. Around 80% of women have normal venous anatomy, as defined by bilateral common iliac veins, formed by ipsilateral external and internal veins. Three studies have evaluated the attainability of graft recovery from deceased donor (DD), when performing the uterine retrieval, after cross clamping of the aorta and vena cava with retrieval of vital abdominal organs. This order of organ retrieval would ensure effective and safe procurement of the vital organs. The initial uterus

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retrieval study from 2007 was performed in New York City, and unexpectedly the rate of acceptance to exercise the uterine procurement was less than 10% (Del Priore et al. 2007). The low acceptance rate from relatives of the DD participants may have been related to that UTx had not proved to be a feasible infertility treatment at that time. Procurement experiments were performed in nine patients, and the plan was to harvest the uterus with the feeding and draining uterine vessels, including internal iliac vessels up to their branching from the common iliacs. However, this was only achieved bilaterally in two out of nine cases, with some also having unilateral loss of complete uterine vessels. The study pointed out the complex pelvic vascular anatomy and indicates that time should be spent on dissection of vessels in situ in a procurement situation, also from DD. A similar study (Gauthier et al. 2014) conducted in Limoges, France, in 2014 showed a very high acceptance rate of relatives for participation by the DD. The difference in acceptance rate to participate in comparison to the low of 2007 (Del Priore et al. 2007) is possibly related to that the knowledge of UTx as a possible novel infertility treatment was now widespread. In this study (Gauthier et al. 2014) they developed a technique for flushing of the uterus through femoral artery catheters, with complete bilateral internal iliac arteries and veins harvested in >85% of cases and a retrieval time of less than 1 h. A detailed step-by-step guide for the retrieval of a viable uterus from a DD was recently presented, also with inclusion of video footage (Richards et al. 2018). It has also been suggested that the uterine retrieval could occur prior to the procurement of other organs in the DD setting (Testa et al. 2018), although there would be a risk of vaginal bacterial contamination to other organs in that situation. In conclusion, the preclinical human studies on UTx have been instrumental in the development of several aspects of human UTx specifically to find limits for cold ischemic tolerance and surgical techniques to isolate the uterine vasculature both in the LD and DD setting.

References Beran BD, Shockley M, Arnolds K, et al. Anatomy of the internal iliac vein: implications for uterine transplant. J Minim Invasive Gynecol. 2018;25:329. Brännström M, Johannesson L, Dahm-Kähler P, et al. The first clinical uterus transplantation trial: a six months report. Fertil Steril. 2014;101:1228–36. Del Priore G, Stega J, Sieunarine K, et al. Human uterus retrieval from a multi-organ donor. Obstet Gynecol. 2007;109:101–4. Fageeh W, Raffa H, Jabbada A, Marzouki A. Transplantation of the human uterus. Int J Gynecol Obstet. 2002;76:245–51. Gauthier T, Piver P, Pichon N, et al. Uterus retrieval process from brain dead donor. Fertil Steril. 2014;102:476–82. Johannesson L, Diaz-Garcia C, Leonhardt H, et al. Vascular pedicle lengths after hysterectomy: toward future human uterus transplantation. Obstet Gynecol. 2012;119:1219–25. Richards EG, Flyckt R, Tzakis A, et al. Uterus transplantation: organ procurement in a deceased donor model. Fertil Steril. 2018;110:183.

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Sieunarine K, Lindsay I, Ungar L, et al. Cold ischaemic preservation of human uterine tissue. Int Surg. 2008;93:366–72. Testa G, Anthony T, McKenna GJ, et  al. Deceased donor uterus retrieval: novel technique and workflow. Am J Transplant. 2018;18:679–83. Wranning CA, Mölne J, El-Akouri RR, et  al. Short-term ischaemic storage of human uterine myometrium-­basic studies towards uterine transplantation. Hum Reprod. 2005;20:2736–44.

9

Medical Work-Up of the Recipient Jana Pittman, Rebecca Deans, and Mats Brännström

9.1

Introduction

Uterus transplantation (UTx) has been successfully performed in numerous centres worldwide, using both live and deceased donors. The factors associated with graft failure have predominantly been related to donor characteristics. However, the selection of recipients’ eligibility also is likely to influence success, particularly post-operative recovery, tolerability to immunosuppression and risk factors for obstetric complications, such as preeclampsia, that can affect both the offspring and the UTx recipient. Accumulated data from trials out of Sweden (Brännström et al. 2014), the USA (Testa et al. 2017), the Czech Republic (Chmel et al. 2019), Germany (Brucker et  al. 2018) and India (Puntambekar et  al. 2018, 2019) have identified several variations in recipient characteristics that may influence procedural outcomes. Factors and investigations that are important for the optimal selection of recipients are discussed below.

J. Pittman Blacktown Hospital, Sydney, NSW, Australia e-mail: [email protected] R. Deans University of New South Wales, Sydney, NSW, Australia Royal Hospital for Women, Sydney, NSW, Australia Sydney Children’s Hospital, Sydney, NSW, Australia Genea Ltd., Sydney, NSW, Australia e-mail: [email protected] M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_9

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Age and Lifestyle Factors

Minimal differences are seen across published trials concerning recipients for both age (range 28–36 years) and body mass index (BMI, range 20.9–23.9 kg/m2). Both the lower and upper age limits are important in selection of recipients. Current recommendations based on existing data are that the recipients be a minimum of 20 years and an upper limit of 38 years (Brännström et al. 2014; Testa et al. 2017; Brucker et  al. 2018). This span should give a high likelihood of optimum health status, in order to reduce post-operative complications. Furthermore, this ensures a greater chance of adequate ovarian reserve with possibility to get a large number of high-grade embryos. However, it should be kept in mind that if a patient at the upper age limit around 38 years undergoes UTx, a possible second pregnancy could be around age 42–43 years, and this is an age with caution for age-related pregnancy-­ associated complications. The upper BMI limit for recipients has in general been 28 (kg/m2) prior to surgery. In some cases, potential recipients may be required to lose weight prior to proceeding with investigations in order to be eligible. The recipient is preferably a lifelong nonsmoker and should be free from current or previous abuse of alcohol or other illicit drugs. Aside from profoundly increased complication rates, smoking greatly affects the quality of the arterial vasculature and by that it increases the risk of graft failure, as shown in kidney transplantation (Khalil et al. 2017). There is also a well-established association between smoking and poor neonatal outcomes (Dennis et al. 2010).

9.3

Anatomical Variations

Currently UTx has only been performed in genotypical females with strict absolute uterine factor infertility. This condition may be due to a complete absence of the uterus as result of hysterectomy due to conditions such as benign or malignant gynaecologic disease or after obstetric complications. Absolute uterine factor infertility may also be present in women with an anatomically present uterus, but with non-­ functionality due to conditions such as severe intrauterine adhesions or major malformation. Currently more than 90% of UTx cases in the literature have been performed in women with Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome (Brännström et al. 2014; Ejzenberg et al. 2019; Testa et al. 2017; Chmel et al. 2019; Brucker et al. 2018; Puntambekar et al. 2018, 2019). The non-MRKH cases have, after the intial case in 2000, have been one case with hysterectomy after cervical cancer (Brännström et  al. 2014) and one case with severe and non-treatable intrauterine adhesions (Puntambekar et al. 2018). The MRKH syndrome is defined as a congenital aplasia of the vagina and the uterus with normal female karyotype and ovarian function (Chan et  al. 2011). Importantly, 30–50% of these cases are also associated with kidney malformations, such as unilateral kidney agenesis, pelvic kidney, or supernumerary ureter. In the original Swedish trial (Brännström et  al. 2014), the three recipients, who

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experienced preeclampsia, all had MRKH with a single kidney. In the US trial, no recipients were included with kidney malformations and thus far preeclampsia has not been reported (Testa et al. 2017). Kidney malformations do not currently exclude women from inclusion into UTx trials providing they have a normal glomerular filtration rate (GFR) >90 and that uro-magnetic resonance imaging (uro-MRI) shows a normal iliac anatomy. Women with kidney agenesis will need heightened monitoring and preparation during pregnancy (Mishra et al. 2017). Another surgical consideration is the minimum vaginal length (and stability of length) required for optimal attachment of the vaginal cuff of the graft to the vaginal vault. To optimize anastomosis of the transplant to the vaginal cuff, a minimum vaginal length of 7–8 cm is required to avoid wound dissidence and traction on the newly anastomosed vessels. The average vaginal length in women is between 6.5 and 12.5 cm (Lloyd et al. 2005). Many women with MRKH have vaginal agenesis and require the creation of a neovagina. Options available for vaginal lengthening can be either surgical or non-surgical depending on the decision of the patient and availability of procedures in their country. Non-surgical options include self-­ dilators, where therapy uses graduated dilators (Frank 1938; Lee 2006), or dilation via regular sexual intercourse. If surgical vaginal lengthening is required, techniques, such as the Vecchietti procedure (Vecchietti 1965) preferably by the laparoscopic approach (Brucker et al. 2008), can be used. Other augmenting neovagina procedures including large or small bowel segments are not recommended in the recipient of UTx, as this may lead to an increased risk of transplant failure due to uterine infection in the immunosuppressed recipient and possible implantation failure, related to the rich secretion of mucus and with an intestinal bacterial flora. After neovagina creation, the average length is 7.0 cm with a range from 5.5 to 9.0 cm (Pastor et al. 2017). To a lesser degree, women post-hysterectomy may also experience a decrease in vaginal length and may require monitoring and dilation. Therefore, it is important to educate participants on maintaining vaginal length, and assessment of this should be repeated so that it is carried out prior UTx.

9.4

Medical Examination and Laboratory Tests

Recipients should undergo several pre-clinical medical screening exams prior to inclusion. These should proceed in a stepwise manner from minimally invasive tests initiated prior to embryo creation via assisted reproductive technologies. The first step needs to be a standard women’s health check involving extensive blood chemistry, including tests for liver function, kidney function, coagulation studies and haematological status. Electrocardiography (ECG), exercise ECG and chest-X ray should also be included. Following this, a full assessment using both serology and ultrasound to ensure ono-infectious state and sufficient ovarian reserve, with awareness of current pelvic anatomy is beneficial. At a minimum these blood tests should include anti-Müllerian hormone and serology for HIV, hepatitis B, hepatitis C, HTLV 2 and syphilis. These serology tests should also be done in the

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recipient’s partner, once in vitro fertilization is planned (see below). The ultrasound examination should be concentrated on investigations of size and position of uterine rudiments and on the existing uterus, in the few cases where a uterus is still in situ. If the uterus is present, the endometrial lining should be investigated. These ultrasound investigations are most often carried out through transvaginal ultrasound, even when there is a short vaginal length. The most important part of ultrasound is assessment of the ovaries. In non-MRKH cases, this investigation can be done by the transvaginal approach, but in MRKH patients, a combination of transvaginal and abdominal ultrasound may have to be used. In these patients the vagina is often shorter than normal, and the ovaries may be located in a more cranial and lateral (lateral to iliac vessels) position than the normal, making them more difficult to examine. When assessing the ovaries, the size of the ovaries and the antral follicle count (AFC) should also be evaluated. Further serology for infectious diseases screening is paramount, due to the subsequent requirement for future immunosuppression. Several serology tests are already included in the initial step as a requirement for assisted reproduction, but additional tests should include serology for rubella and varicella status, toxoplasmosis, cytomegalovirus (CMV) and Epstein-Barr virus (EBV). A gynaecological examination with sampling for sexually transmitted diseases (gonorrhoea, chlamydia, mycoplasma) and cytology including high-risk human papilloma virus (HPV) should be performed. A glucose tolerance test, or at a minimum ensuring adequate fasting glucose levels and HbA1c, is recommended. This should be done in order to reduce the risk of gestational diabetes during pregnancy. Vaccination status should also be recorded. Finally, there are some MRKH-specific tests. As mentioned above, kidney function must also be established at baseline for women with MRKH and kidney malformations. This should be done by assessing true glomerular filtration rate (GFR) and by acquiring data on electrolytes/urea/creatinine (EUC). The only further imaging that may be required is the uro-MRI, previously mentioned in relation to kidney malformations in some women with MRKH.

9.5

Test of Established Ovarian Function

Prior to inclusion in any UTx clinical trial, fertility must be assured through the creation of viable embryos or that a large number of unfertilized oocytes can be stored. So far only embryo cryopreservation has been done in the published papers on UTx, but it is of course also possible to cryopreserve unfertilized oocytes. The required number and developmental stage of embryo cryopreservation prior to initiation of UTx has been variable in different countries. In the original Swedish study (Brännström et al. 2014), the minimum required number of embryos was ten high-grade embryos, which could be of cleavage stage, blastocyst stage or a mix of these (Brännström et al. 2014). In the trial out of the Czech Republic, ten embryos, of unspecified stage, were the requirement (Chmel et al. 2019). In the US trial, four

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blastocysts (Testa et al. 2017) were the set prerequisite, but all of these blastocysts had undergone genetic testing for aneuploidy and were found to be euploid. Live births have been reported from the trials of Sweden (Brännström et al. 2015) and the USA (Testa et al. 2018). Based on the accumulated knowledge, our current recommendation now is cryopreservation of at least eight to ten high-grade embryos to have a good chance for birth of two children from this cohort of embryos that are created before UTx. In the event of cryopreservation of only unfertilized oocytes, the recommendation would be that the woman should have at least 20 cryopreserved oocytes.

References Brännström M, Johannesson L, Dahm-Kähler P, et al. The first clinical uterus transplantation trial: a six months report. Fertil Steril. 2014;101:1228–36. Brännström M, Johannesson L, Bokström H, et al. Live birth after uterus transplantation. Lancet. 2015;385:607–16. Brucker SY, Gegusch M, Zubke W, et al. Neovagina creation in vaginal agenesis: development of a new laparoscopic Vecchietti-based procedure and optimized instruments in a prospective comparative interventional study in 101 patients. FertilSteril. 2008;90(5):1940–52. Brucker SY, Brännström M, Taran FA, et  al. 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. Chan Y, Jayaprakasan K, Zamora J, et  al. The prevalence of congenital uterine anomalies in unselected and high-risk populations: a systematic review. Hum Reprod Update. 2011;17(6):761–71. Chmel R, Novackova M, Janousek L, et al. Revaluation and lessons learned from the first 9 cases of a Czech uterus transplantation trial: four deceased and five living donor uterus transplantations. Am J Transplant. 2019;19:855–64. Dennis O, Mook-Kanamori M, Steegers P, et al. Risk factors and outcomes associated with first-­ trimester fetal growth restriction. JAMA. 2010;303(6):527–34. Ejzenberg D, Andraus W, BaratelliCarelli Mendes LR, et al. Livebirth after uterus transplantation from a deceased donor in a recipient with uterine infertility. Lancet. 2019;392:2697–704. Frank RT.  The formation of an artificial vagina without operation. Am J Obstet Gynecol. 1938;35:1053–5. Khalil M, Tan J, Khamis S, Khalil M, et al. Cigarette smoking and its hazards in kidney transplantation. Adv Med. 2017;2017:6213814. Lee MH.  Non-surgical treatment of vaginal agenesis using a simplified version of Ingram’s method. Yonsei Med J. 2006;47(6):892–5. Lloyd J, Crouch N, Minto C, et  al. Female genital appearance: normality unfolds. BJOG. 2005;112(5):643–6. Mishra VV, Mistry KM, Nanda SS, et al. Pregnancy outcome in patients with solitary kidney. J Obstet Gynaecol India. 2017;67(3):168–72. Pastor Z, Fronêk J, Nováčková M, et  al. Sexual life of women with Mayer-Rokitansky-Küster-­ Hauser syndrome after laparoscopic Vecchietti vaginoplasty. Sex Med. 2017;5(2):106–13. Puntambekar S, Telang M, Kulkarni P, et  al. Laparoscopic-assisted uterus retrieval from live organ donors of uterine transplant; our experience of two patients. J Minim Invasive Gynecol. 2018;25:622–31. Puntambekar S, Puntambekar S, Telang M, et al. Novel anastomotic technique for uterine transplant using utero-ovarian veins for venous drainage and internal iliac arteries for perfusion in two laparoscopically harvested uteri. J Minim Invasive Gynecol. 2019;4:628–35.

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Testa G, Koon EC, Johannesson L, et  al. Living donor uterus transplantation: a single center’s observations and lessons learned from early setbacks to technical success. Am J Transplant. 2017;17:2901–10. Testa G, McKenna GJ, Gunby RT Jr, et al. First live birth after uterus transplantation in the United States. Am J Transplant. 2018;18:1270–4. Vecchietti G. Creation of an artificial vagina in Rokitansky-Kuster-Hauser syndrome. Attual Ostet Ginecol. 1965;11:131–47.

Live or Deceased Uterus Donor

10

Michael Olausson

10.1 Introduction The objective of this chapter is to describe the two modalities of organ donation available in uterus transplantation (UTx) as well as pros and cons for choosing one or the other. The ethical questions for live donation are covered in a separate chapter, discussing ethics in UTx, and thus only mentioned briefly here. As one will understand, the basic concept of using live donors is to a large extent an ethical question (Olausson et al. 2014), partially dependent on local traditions and regulations. In many countries with a long tradition of organ transplantation, live organ donation is a natural part of the therapeutic arsenal mainly offered to patients in need of a kidney or liver transplant. Pancreas, small bowel, and lung transplantations are also offered as live donor procedures, but in much fewer centers. In contrast to the abovementioned procedures, UTx is not a lifesaving procedure but rather performed as quality-of life-enhancing procedure, and it is also the first ephemeral type of transplantation.

10.2 Live Donor Donation Live organ donors have been used since the first successful kidney transplantation by Joseph Murray in 1954. In many countries, like in the Scandinavian countries, between 25 and 50% of all donated kidneys originate from a live donor. This was one of the reasons for the Gothenburg group to consider this option in the first trial of UTx in the world.

M. Olausson (*) Department of Transplantation, Sahlgrenska Academy at Gothenburg University, Sahlgrenska University Hospital, Göteborg, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_10

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Some advantages of live uterus donation are as follows: 1. The extensive experience in performing hysterectomies in women. According to statistics from the USA, hysterectomy is the second most common procedure performed in women after cesarean section. Many of the steps of the surgical procedure and the complications specific to the surgery are well known (Centers for Disease Control and Prevention Website 2015). 2. The possibility for evaluating the donor before transplantation, theoretically increasing the success of pregnancy and a healthy baby, is obvious. 3. The numbers of potential donors are larger than in deceased donation since the vast majority of deaths occur outside the hospital and these women will therefore never be considered for donation. 4. Furthermore, elderly donors may be considered as donors, since there will be time to perform more elaborate studies of the vessels of the donors, which is usually not possible in a deceased donor. 5. In postmenopausal women the function of the uterus can be tested by reinsertion of hormones to resume monthly periods before donation. 6. So far the vast majority (20) of the 23 healthy babies born worldwide are after live donation—proving that live donation works with a relatively high efficiency. Three births have occurred after UTx from a deceased donor, and there are further ongoing pregnancies both after live and deceased uterus donations. Some disadvantages of live uterus donation are as follows: 1. The procedure is performed in a healthy individual who does not need this procedure for medical reasons. The risk therefore has to be low to be justifiable. Clearly, we do not know the risks and therefore have to rely on calculations. In the Gothenburg trial, the procedure was compared to live donor nephrectomy before kidney transplantation. The mortality of live donor nephrectomy is reported to be 0.02% (Matas et al. 2003). In comparison, nephrectomy for renal carcinoma carries a 2.8% mortality risk (Thoroddsen et al. 2003), indicating that selection of healthy individuals certainly decreases the risk of dying from the procedure. Hysterectomy is considered a less dangerous procedure with a mortality of 0.1– 1% (Wingo et al. 1985), despite the fact that this includes pregnant and elderly women as well as cases with neoplasm. The estimate is therefore a fair assumption. 2. The time for the surgical procedure in the donor was significantly longer than anticipated for the first cases. Lately, the same procedure now takes 4–5 h compared to 11–12 h initially. The reasons for this may be several, but most importantly the surgical field was found to be new and more difficult than in non-human primates and other large animals tested. The present time for the procedure is more closely related to the time for a radical hysterectomy, but more than five times longer than for a total hysterectomy on benign indication.

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3. The complexity of surgery in the deep pelvis is another matter of concern. Clearly, experience of vascular surgery will be helpful to overcome the learning curve.

10.3 Deceased Donor Donation Organs from deceased donors are the most common source in organ transplantation globally. There is a discrepancy between the demand and the supply, with many patients never receiving an organ despite that they are on a waiting list. In general, live donation can help bridge the gap of demand and supply, but not completely. The wish to donate seems to be larger for the uterus than for other organs, judging from early experiences, but it is still too early to say with certainty until more patients are transplanted. Some advantages of deceased uterus donation are as follows: 1 . The donor takes no risk at all, eliminating one ethical issue with the procedure. 2. The vessels to and from the uterus can be kept longer than in a live donor procedure. This is certainly an advantage making the recipient operation easier. Both the donor uterine veins and the donor uterine arteries are sometimes complex regarding dissection in the donor. Theoretically the retrieval of the uterus from a deceased donor will simplify the dissection in the deep pelvis, thus reducing the total surgical operation time. Some disadvantages of deceased uterus donation are as follows: 1. The time for evaluating the organ is shorter than in a live donor. This may be a problem especially in the elderly donor close to or after menopause. In these cases the donor vessels have to be carefully evaluated, preferably with an angiography before the retrieval. This is not always possible. An alternative would then be an angiography on the back-table. A functional test of postmenopausal uterus will not be possible. 2. Nulliparous donors are not recommended, especially not in the deceased donors. Vessels are more likely to be smaller in diameter, and the ultimate test whether a pregnancy has been feasible does not exist. 3. The original thought that the duration of organ retrieval would be substantially reduced in a deceased donor uterine retrieval, as compared to live donor uterine retrieval, has proven to be of less significance. Thus, experience has shown that more dissection in the deceased donor is necessary to avoid damages to the small-diameter vessels to and from the uterus. The main reason for this is that small structures are difficult to identify after perfusion of preservation fluids in the multiorgan donor. Still, it is true that longer vessels can be utilized than in a live donor.

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References Centers for Disease Control and Prevention Website. Key statistics from the national survey of family growth. Atlanta: Centers for Disease Control and Prevention; 2015. http://www.cdc. gov/nchs/nsfg/key_statistics/h.htm#hysterectomy. Accessed 23 June 2015. Matas AJ, Bartlett ST, Leichtman AB, Delmonico FL. Morbidity and mortality after living kidney donation, 1999–2001: survey of United States Transplant Centers. Am J Transplant. 2003;3(7):830–4. https://doi.org/10.1046/j.1038-5282.2001.00400.x-i1. Olausson M, Johannesson L, Brattgård D, Diaz-Garcia C, Lundmark C, Groth K, Marcickiewizc J, Enskog A, Akouri R, Tzakis A, Rogiers X, Janson PO, Brännström M.  Ethics of uterus transplantation with live donors. Fertil Steril. 2014;102(1):40–4. https://doi.org/10.1016/j. fertnstert.2014.03.048. Thoroddsen A, Gudbjartsson T, Jonsson E, Gislason T, Einarsson GV. Operative mortality after nephrectomy for renal cell carcinoma. Scand J Urol Nephrol. 2003;37(6):507–11. https://doi. org/10.1080/00365590310015732. Wingo PA, Huezo CM, Rubin GL, Ory HW, Peterson HB.  The mortality risk associated with hysterectomy. Am J Obstet Gynecol. 1985;152(7):803–8. https://doi.org/10.1016/ S0002-9378(85)80067-3.

Medical Work-Up of the Live Donor

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Mats Brännström and Pernilla Dahm-Kähler

11.1 Introduction Uterus transplantation (UTx) has been performed successfully with a uterine graft both from live donor (Brännström et al. 2015) and from deceased donor (Ejzenberg et al. 2019). The accumulated knowledge about UTx from live donor is from published results of case series out of Sweden (Brännström et al. 2014), the USA (Testa et al. 2017), the Czech Republic (Chmel et al. 2019), Germany (Brucker et al. 2018) and India (Puntambekar et al. 2018, 2019). In addition, there are reports of single cases of live donor UTx in Saudi Arabia (Fageeh et al. 2002) and China (Wei et al. 2017). There are variations in the age and status of the donors in the trials, with some morbidity and graft failures reported. These may in some cases be related to improper selection of donors. Below we discuss some of the factors and investigations, which are important in the proper selection of donors.

11.2 Age Both the lower and upper age limits of a donor are important in selection of live donors. Concerning the lower age limit, a live donor should in principle be past her childbearing years. This is in general around 45  years of age. However, if there is a clear decision on unwillingness to have more children, patients younger

M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] P. Dahm-Kähler Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_11

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than 45  years may be accepted. The reluctance can be confirmed by that the woman has undergone sterilization, but in such a case and also among non-sterilized potential donors of age below 45 years, proper counselling at several occasions should be performed so that the donating woman understands that uterus donation leads to irreversible infertility. In the live donor trial in the USA, three of their first five donors were below 45 years, with ages 34, 26 and 42 years (Testa et al. 2017). These were altruistic donors that had no contact with the potential recipients, and they had all given birth to at least two children. In our Swedish trial (Brännström et al. 2014), one of the donors was below 45 years at donation. That was a 37-year-old woman, with four children, who donated her uterus to her 4  years younger sister, who was born with no uterus. Thorough psychological counselling preceded the donation, and she has, since the donor surgery in 2013, not expressed any regrets but has been very happy that she helped her younger sister to acquire motherhood by this means. There are no live donors below the age of 45 years who yet have been included in the trials of the Czech Republic (Chmel et al.2019) or Germany (Brucker et al. 2018). In the original UTx case in Saudi Arabia (Fageeh et  al. 2002), the donor was 46  years old. The age of the donating mother in the live donor UTx case in China was 42  years (Wei et  al. 2017), and this was also the age of one of the four donating mothers in the Indian trial, with the rest of them being between 45 and 50 years of age (Puntambekar et al. 2018, 2019). Our current recommendation on upper age limit is menopausal age plus 5 years, but this can be extended for some years if the potential donor has been on hormone replacement therapy since menopause, since the treatment may have protected her uterine vessels from the age-related intimal hyperplasia and constriction. Noteworthy is that two of the three oldest donors in the original Swedish UTx study (Brännström et al. 2014) donated organs that resulted in early graft failures. These donors were 58 and 62 years at donation and in one case (58 years) clear atherosclerotic plaques were seen at organ procurement. This uterus showed a low flow after reperfusion, and after UTx, the recipient acquired an intrauterine infection, possibly related to endometrial hypoperfusion. The uterus was removed by hysterectomy 3.5 months after UTx. In the other case (donor 62 years) there was no flow at the back-table, but the procedure was finalized with UTx, and some but restricted flow was seen after unclamping. Thrombosis developed in the vasculature of the graft within some days, and a partly necrotic uterus was removed on the third postoperative day (Brännström et al. 2014). A similar case was experienced in the live donor UTx trial in Germany, with the donating mother being a previous smoker, somewhat overweight and at age 61  years (Brucker et  al. 2018). Atherosclerosis was seen during organ procurement, and flow through the uterus could not be established on the back-table. The case was aborted and surgery in the daughter was not done. On the other hand, it is of importance to note that the donor of the first successful UTx procedure in the world was 61 years when donating, albeit she was in excellent health, with a BMI of 20, and was a non-smoker (Brännström et al. 2015).

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11.3 Lifestyle Factors Since the surgery of the donor is demanding, with long duration, it is important that the donor is in good shape to reduce risk for postoperative complications and to shorten the hospital stay and the time of return to daily activities. Thus, we have an upper limit of body mass index (BMI) of the donor of 28 (kg/m2), and in some cases of potential donors, we have asked them to lose weight before we would start to do more investigations. Smoking will affect the quality of the arterial vasculature, but it also has profound effects on complication rates. The donor should be a non-smoker for at least 3 months prior to surgery, but it is preferable that she has been a non-smoker for many years. One donor in the German trial had been a heavy smoker for many years, but stopped smoking before the trial (Brucker et al. 2018). The donor surgery was performed but the case was cancelled for further transplantation due to no flow at back-table flushing, as described above. Naturally, the donor should be free from current or previous abuse of alcohol or drugs.

11.4 Obstetric History We require all live donors to have at least one normal pregnancy with delivery at term. There should be no history of preterm delivery, preeclampsia or other obstetric complications. The mode of previous delivery should preferably be vaginal, but we would also accept donors with up to two caesarean sections, if ultrasound does not indicate any thin inferior segment of the uterus. Moreover, uteri with a history of multiple implantation failures or repeated miscarriages should be excluded, due to the possibility of internal uterine factor that may have negative influences on pregnancy potential.

11.5 Medical Data and Laboratory Tests The donors should be screened by extensive blood chemistry, including tests for liver function, kidney function, coagulation as well as haematology status. Moreover, serology for cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV) and syphilis should be obtained. Electrocardiography (ECG), exercise ECG and chest-X ray should also be included. Gynaecological examination with sampling for sexually transmitted diseases (gonorrhoea, chlamydia, mycoplasma) and cytology including high-risk human papilloma virus should be performed.

11.6 Imaging of the Uterus and Vasculature In the original Swedish study, vaginal ultrasound and contrast-enhanced magnetic resonance imaging (MRI) were the means for evaluation of the uterus and its vasculature (Brännström et al. 2014). This would exclude uterine pathology that could

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affect functionality, such as myoma, adenomyosis and polyps. We used contrast-­ enhanced MRI to evaluate the arteries to the uterus. However, it was only possible to see that there was a flow out through the proximal part of the uterine artery on each side and any detailed evaluation of flow or arterial dimensions could not be performed. Thus, the vascular evaluation only by MRI was insufficient to acquire information of the low quality of the arteries in the two failed attempts of older donors of the Swedish study (Brännström et al. 2014) and also in the aborted case in the German study (Brucker et al. 2018). In addition, computed tomography (CT), as used in the study in the USA, was not able to exclude one donor with intimal hyperplasia and constricted lumen (Testa et al. 2017). In the Czech trial, contrast-­ enhanced CT angiography (CTA) was used, and no donor uterus with low patency was included, in spite of the age range of the five donors being 47–58 years (Chmel et al. 2019). We are presently conducting a prospective study within the ongoing Swedish trial of robotic-assisted UTx in order to compare pelvic vessels, particularly uterine arteries, by the methods of contrast-enhanced MR angiography (MRA), CTA in the arterial contrast phase and conventional digital subtraction angiography (DSA). Preliminary results show that, if MRA shows good flow, it would be sufficient as a single imaging modality to include for uterus donation. If MRA is inconclusive, the next investigative procedure would be CTA, which provides some more details on the uterine artery. If both of these investigations are inconclusive, DSA will give the superior image of the uterine arteries with fairly accurate measurements of the arterial lumen. We have a cutoff of 2 mm lumen (at three standardized locations), but in selected cases of younger donors, we have accepted a lumen on one side down to 1.5 mm. We hope that this investigation will shed some light on the issue of imaging of uterine arteries and cutoff values of vascular dimensions for proceeding with donor investigations towards a UTx procedure with a great chance of live birth(s).

References Brännström M, Johannesson L, Dahm-Kähler P, et al. The first clinical uterus transplantation trial: a six months report. Fertil Steril. 2014;101:1228–36. Brännström M, Johannesson L, Bokström H, et al. Live birth after uterus transplantation. Lancet. 2015;385:607–16. Brucker SY, Brännström M, Taran FA, et  al. 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. Chmel R, Novackova M, Janousek L, et al. Revaluation and lessons learned from the first 9 cases of a Czech uterus transplantation trial: four deceased and five living donor uterus transplantations. Am J Transplant. 2019;19:855–64. Ejzenberg D, Andraus W, BaratelliCarelli Mendes LR, et al. Livebirth after uterus transplantation from a deceased donor in a recipient with uterine infertility. Lancet. 2019;392:2697–704. Fageeh W, Raffa H, Jabbad H, Marzouki A. Transplantation of the human uterus. Int J Gynaecol Obstet. 2002;76:245–51.

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Puntambekar S, Telang M, Kulkarni P, et  al. Laparoscopic-assisted uterus retrieval from live organ donors of uterine transplant; our experience of two patients. J Minim Invasive Gynecol. 2018;25:622–31. Puntambekar S, Puntambekar S, Telang M, et al. Novel anastomotic technique for uterine transplant using utero-ovarian veins for venous drainage and internal iliac arteries for perfusion in two laparoscopically harvested uteri. J Minim Invasive Gynecol. 2019;4:628–35. Testa G, Koon EC, Johannesson L, et  al. Living donor uterus transplantation: a single center’s observations and lessons learned from early setbacks to technical success. Am J Transplant. 2017;17:2901–10. Wei L, Xue T, Tao KS, et al. 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.

Medical Work-Up of the Deceased Donor

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Anne C. Davis, Rebecca Flyckt, and Tommaso Falcone

12.1 Introduction Thorough medical evaluation of a deceased donor for uterus transplantation (UTx) is critical to the success of the procedure. Although this assessment can be logistically challenging, it is balanced by the proposed benefits of using a deceased donor: the elimination of risk to the donor, the quality of premenopausal tissue available for the graft, the ability to obtain wider and longer vascular pedicles, and the speed of recovering the uterus (Flyckt et al. 2018). In this chapter, we will discuss briefly the history of deceased donor uterine transplant and explore the major components of the medical evaluation of a deceased uterine donor, including obtaining the medical and surgical history, serological and microbiological evaluation, and preoperative imaging. We will also briefly discuss the challenges associated with obtaining consent from a deceased donor’s family.

12.2 History of Deceased Uterine Donor Transplantation The first deceased donor human uterus transplantation (UTx) took place in Turkey in 2011. In this case, the recipient was a 21-year-old patient with MRKH who received a uterus from a brain-dead multi-organ donor who was of similar age (Ozkan et al. 2013). As of the time of this publication, this transplanted uterus remains in situ and has shown evidence of menstrual function. However, despite several early failed pregnancies, no successful live births have resulted from the graft to date (Erman et al. 2013). The first

A. C. Davis IVI America, Basking Ridge, NJ, USA R. Flyckt · T. Falcone (*) Cleveland Clinic, Women’s Health Institute, Cleveland, OH, USA e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_12

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attempted deceased donor UTx in the United States took place in 2015 at the Cleveland Clinic as part of a clinical trial of human UTx (Flyckt et al. 2017). While perfusion of the graft was demonstrated postoperatively, a severe fungal infection developed which caused an anastomotic failure of one of the vascular connections shortly after transplant. This led to the recipient undergoing hysterectomy 12 days after the transplant. Following careful review of this first attempt, revisions to the protocol and consent were made, and additional deceased donor UTx procedures are forthcoming to complete the clinical trial. Other US-based and international transplant research groups are also exploring the use of a deceased donor for human uterine transplantation (Testa et al. 2018; Fronek et al. 2016).

12.3 Obtaining the Medical History One of the most challenging aspects of using a deceased donor uterus is obtaining an accurate and complete medical history of the donor. As much information as possible must be found through detailed examination of medical records as well as from thorough interview with the donor’s family. While medical records of the current admission as well as the cause of death are likely readily available, prior records especially those pertinent to the determination of graft quality and suitability for potential transplant may be more difficult to obtain. A medical history of any condition that could affect the vascular quality or vascular anatomy of the graft is a major concern. Donors with chronic hypertension, diabetes, and/or severe hyperlipidemia should be excluded. Obese donors should not be included as this makes access to the deep pelvis for adequate dissection extremely difficult. In addition, an attempt to obtain detailed obstetric and gynecologic records is essential, especially to explore any history of infertility or obstetrical complications. If available, medical records should be queried for the donor’s gravidity and parity, mode of delivery for any live births, mode of evacuation for any abortions (spontaneous or otherwise), history of uterine instrumentation, and history of uterine anomalies. In addition, any history of sexually transmitted infections, endometriosis, fibroids, or prior pelvic surgeries should be determined as significant pelvic adhesions may present a challenge in recovery and dissection of the necessary vessels. A history of any genito-urinary infections should be identified prior to transplant; however, most donor families will not have knowledge of such infections, and documentation may be scarce or not present at all. The process by which these records are obtained and reviewed should be carried out with the same protocols that are currently used in solid organ transplant programs.

12.4 Serologic and Microbiologic Testing Serologic testing has been standardized under other solid organ transplant protocols and typically includes HIV, hepatitis B and C, HTLV 1 and HTLV 2, HSV, EBV, and CMV status. Major infectious conditions that would compromise graft survival and recipient outcome can be determined from this panel of serologic testing, as well as from the review of existing medical records and thorough family interviews. In

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addition, the matching of ABO group as well as CMV compatibility is strongly recommended and may pose a significant limitation for some patients, as some blood groups are quite rare. In this respect, deceased donor grafts do not differ significantly from other types of grafts. With respect to microbiological testing, uterine grafts pose a unique challenge not previously faced in solid organ transplantation. Specifically, the continuity of the uterus with the cervix and vagina and potential for contamination with normal and abnormal microbes that may be present is concerning. For this reason, in addition to traditional microbial prophylaxis, we recommend obtaining vaginal swabs of the deceased donor to detect pathogens such as candida, gonorrhea, chlamydia, and trichomonas. Antibiotic and antifungal agents should be considered during the recovery of the uterus and should be incorporated into the recipient protocol during and after organ transplantation.

12.5 Imaging With respect to imaging of the potential donor, we recommend pelvic ultrasound and/or CT or MRI of the pelvis prior to organ recovery in order to exclude potential donors with unknown uterine or vascular anomalies. Some donors may have recent imaging which can be used; others may require imaging to be ordered preoperatively if no imaging is available for review. These studies will allow for preoperative planning of vascular dissection, as well as determining the presence of any anatomic abnormalities that may require special attention at the time of surgical recovery or may even exclude the donor as a candidate. Hysteroscopy can be also considered for additional preoperative visualization of the endometrial cavity; however, this does mean additional instrumentation of the uterus along with increased potential for introducing microbes from the vagina into the graft.

12.6 Graft Availability and Consent While there are many benefits in the deceased donor model (Table12.1), significant logistic limitations present unique challenges. The unpredictable timing of deceased donor recoveries may pose difficulty for both surgeons and recipients; however, this is not unlike other solid organ transplants. In addition, the availability of Table 12.1  Advantages and disadvantages of deceased uterine donors Advantages –  No medical/surgical risk to donor –  Younger grafts available – Wider/longer vascular pedicles and greater range of vessels available – Elimination of “donor guilt” if the graft is not used or fails to produce a pregnancy

Disadvantages – Unverified medical history and more limited preoperative assessment – Consent from next of kin may be difficult to obtain –  Greater distance from recipient – Less convenient scheduling of surgical teams –  Scarcity of appropriate donors

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reproductive-­aged female deceased donors that are not “high-risk” donors may be severely limited. A significant factor contributing to the scarcity of available donors may be willingness of next of kin to consent for UTx (Flyckt et al. 2016). In an early US study, only 6% of families agreed to procurement of the uterus for research purposes, even with the understanding that it would never be transplanted (Del Priore et al. 2007). However these attitudes may be changing. More recent publications from France and the UK demonstrate that, with improved education and familiarity, there is increased support for uterus donation for transplant (Rodrigue et al. 2017; Saso et al. 2015; Jones et al. 2016; Gautheir et al. 2014). In addition, a recently published US study evaluating attitudes about vascular composite allografts (VCA) demonstrated that 87.6% of respondents “supported” or “strongly supported” VCA transplantation of the uterus and 74.4% of women would be willing to donate their own uterus (Rodrigue et al. 2017). Because donor uteri for transplantation are considered vascularized composite allografts (VCAs), which are life-enhancing rather than life-saving, their transplantation process is typically performed under a research protocol. VCAs are regulated in a fashion similar to other solid organ transplants and require special consent. Not only must the donor family specifically authorize donation of the uterus, but they must also consent to the use of the organ specifically for research purposes.

12.7 Ischemia Time Ischemia time is a factor in both living donor and deceased donor models (Flyckt et  al. 2018). In a deceased donor, the uterus is typically recovered following the removal of all other life-saving organs after cross clamp; this extends the cold ischemia time of the organ. In addition, the graft must be transported to the recipient, who may be located in a different geographic location than the donor. While it is not currently known what the optimal cold ischemia time is for uterine grafts, initial studies suggested that the myometrium is resistant to ischemia for at least 6  h (Wranning et al. 2005). A recent study from France found that the uterus was resistant to cold ischemia for up to 24  h based on histology and apoptosis assays (Gautheir et al. 2014). However, because of the still relatively unknown effects of cold ischemia time on donor uteri, geographic consideration may limit the availability of suitable organ donors (Flyckt et al. 2016). Because the deceased donor may not be in the same facility as the recipient, even with expedited transport, the cold ischemia time will likely be longer in a deceased donor versus a living donor model (Flyckt et al. 2018). This longer ischemia time may be associated with diminished graft function and possibly increased risk of rejection (Flyckt et al. 2016).

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12.8 Conclusions When considering the benefits of deceased donor UTx, careful attention must be paid to the medical evaluation so as to minimize potential risk to the recipient. Collection of medical, gynecologic, and surgical history based on all available records as well as thorough family interview will decrease risk. However, augmentation of the evaluation with serological, microbiological, and radiological assessments is essential, as well as careful consideration of challenges in obtaining consent for transplantation.

References Del Priore G, Stega J, Sieunarine K, et al. Human uterus retrieval from a multi-organ donor. Obstet Gynecol. 2007;109:101–4. Erman A, Ozkan O, Aydinuraz B, Dirican K, Cincik M, Mendilcioglu I, et al. Clinical pregnancy after uterus transplantation. Fertil Steril. 2013;100:1358–63. Flyckt R, Falcone T, Eghtesad B, Fung J, Tzakis A. Uterus transplantation: medical considerations. Curr Transplant Rep. 2016;3:380–4. Flyckt R, Kotylar A, Arian S, Eghtesad B, Falcone T, Tzakis A. Deceased donor uterine transplantation. Fertil Steril. 2017;107:e13. Flyckt R, Davis A, Farrell R, Zimberg S, Tzakis A, Falcone T. Uterine transplantation: surgical innovation in the treatment of uterine factor infertility. J Obstet Gynaecol Can. 2018;40(1):86–93. Fronek J, Janousek L, Chmel R.  Deceased donor uterus retrieval—the first Czech experience. Rozhl Chir. 2016;95:312–6. Gautheir T, Piver P, Pichon N, et al. Uterus retrieval process from brain dead donors. Fertil Steril. 2014;102:476–82. Jones B, Saso S, Yazbek J, Smith J.  Uterine transplantation: past, present and future. BJOG. 2016;123:1434–8. Ozkan O, Akar ME, Erdogan O, Ozkan O, Hadimioglu N. Uterus transplantation from a deceased donor. Fertil Steril. 2013;100:e41. Rodrigue J, Tomich D, Fleishman A, Glazier A.  Vascularized composite allograft donation and transplantation: a survey of public attitudes in the United States. Am J Transplant. 2017;17:2687–95. Saso S, Clarke A, Bracewell-Milnes T, Al-Memar M, Hamed A, Thum M, et al. Survey of perceptions of health care professionals in the United Kingdom toward uterine transplant. Prog Transplant. 2015;25:56–63. Testa G, Anthony T, McKenna G, Koon E, Wallis K, Klintmalm G, et al. Deceased donor uterus retrieval: a novel technique and workflow. Am J Transplant. 2018;18(3):679–83. Wranning C, Molne J, El-Akouri R, Kurlberg G, Brannstrom M.  Short-term ischaemic storage of human uterine myometrium—basic studies towards uterine transplantation. Hum Reprod. 2005;20:2736–44.

Psychological Evaluations Before Uterus Transplantation

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Stina Järvholm

The way of handling psychological aspects of planning for uterus transplantation (UTx) will differ between countries and depends on legislation and if the team doing UTx is mainly coming from gynecology/reproductive medicine or transplantation surgery/medicine. Regardless of where the UTx team is physically and organizationally positioned, it is important to keep in mind the unique psychological aspects of UTx, placing itself somewhere in the borderline of these two disciplines. The psychological evaluation also differs from most other evaluations since you have to address several individuals in a shared project both on individual and relational level. The psychological evaluation prior to UTx has at least three goals: • Select individuals who are suitable for UTx. • Screen out individuals with behaviors that put them at risk, and if these behaviors not are changeable, exclude them from UTx. • Establish a relationship that promotes counseling that will later on encourage the recipient, partner, and possible donor to seek for help when needed. Themes that need to be covered are the same with other types of transplantation such as general psychological health, cognition, social support, mental illness, previous compliance, and substance abuse. In addition there are also specific domains for UTx to evaluate, such as being able to adjust to a temporary life as transplanted, to move from “healthy to sick,” and aspects regarding childlessness (Järvholm et al. 2018). This chapter focuses mainly on the evaluations concerning latter UTx-­ specific parts. S. Järvholm (*) Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, Sahlgrenska Academy, University of Gothenburg, Institute of Clinical Sciences, Gothenburg, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_13

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13.1 General Psychological Well-Being To evaluate if a recipient, her partner, and a living donor are potential candidates for UTx, it is a good way to start with their strengths. How have they managed strains before, as individuals and as couples? Have they experienced previous adverse life events and which coping skills were they able to use to adhere to the situation? To gain knowledge of how an individual have dealt with difficulties in the past is a way to understand the concept of resilience. Rutter (1987) published the framework of the theory about resilience. He states that resilience is a way to understand how people adapt to adversity. Key components in this theory are risk and protective factors and a focus on strengths rather than deficits. At some point in people’s lives, they experience a period of anxiety and/or depression which is common (Ahrnsbrak et al. 2017; Johansson et al. 2013), and there is no reason to be excluded for UTx, based solely on this fact. When anxiety and depression are present in the history of a patient, it is important to know if the condition was treated properly and if help-seeking behavior was present. More severe psychopathology such as personality disorders and/or psychotic symptoms should be a cause for exclusion in this early stage of UTx. This viewpoint is based both on concerns regarding the individual’s own health and, in the case of the recipient and her partner, from the perspective of a future child, a perspective in accordance with that existing in general in reproductive medicine.

13.2 Coping with Childlessness The question about childlessness and how the woman and her partner previously have handled their wish for children is to be discussed. If they do not have children before UTx, the following questions could be addressed: Is UTx their first choice or are they “forced” to opt for this course of action due to legislation, economical, or cultural settings? Have they gone through other attempts such as adoption or gestational surrogacy or are they restricted from these options in some way? And if they have previous children, how will the children be affected when the mother/parents enter a process to undergo UTx? Does the couple have previous possibly traumatic experience in their reproductive history, such as traumatic births, large bleedings, or emergency peripartum hysterectomy? Another important angle is the relatively long time perspective in UTx, and if the couple enters this road, they may put other options on hold and not be able to choose these afterward, for example, passing the age limit for adoption. Regarding the questions about other options, it is also important to inform the couple of what possibilities they have due to legislation in association to UTx. For example, is it possible to later on transfer frozen embryos obtained before UTx to gestational surrogacy if there is a graft failure or failure of pregnancy?

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13.3 Knowledge About the Procedure The psychological evaluation is aiming to understand if the participants have sufficient knowledge about the procedure and sufficient skills to adhere to the extensive medical follow-up after transplantation. Moreover, they should be able to understand the extent of medical and psychological issues that may arise after donation/transplantation. Themes to discuss during evaluation could be cognitive limitations or an unrealistic view of the own effort and contribution to the procedure. In cases where the former is present, this is disadvantageous for compliance (Jin et al. 2008).

13.4 Social Situation Both the couple and the donor need to be in a stable social situation that enhances them to handle UTx, both at time for surgery and for the couple over several years thereafter. Concerning the couple, questions about relationship stability and consistency regarding the decision about UTx should be assessed. Having sufficient social support seems in general to favor good psychological health (Ahnquist et al. 2012; Lindström and Eriksson 2005), and this has in heart transplantation been shown to be related to better quality of life (White-Williams et al. 2013). The socioeconomic situation should be stable, and the state should also have tolerance for that the recipient and donor have time off from work for extended periods during recovery and for the recipient also at the frequent follow-up visits. The social situation is influenced by cultural setting, and therefore general recommendations on this area are hard to establish.

13.5 Donor Questions A living donor can be both a directed donor (DD) who is then chosen by the recipient and her partner or a non-directed (altruistic) donor (ND). In both cases, an alternate psychologist as to the one who does the evaluation of the recipient and her partner should perform the evaluation of the donor. This is for avoidance of confusion concerning perspectives and to secure that any hesitation or unsuitability to donate is being noticed. Of course there is the risk in close relationships to feel obligated to donate, and that should be addressed in the evaluation and is facilitated when using two psychologists, one for the recipient and one for the donor. Nevertheless if the DD is the recipient’s mother or another close relative, it has been shown in other living-donor programs (Forsberg et al. 2004; Perez-SanGregorio et  al. 2017) that a related (parental) donor in general seems to adjust well to this experience in a psychological perspective. There could be a risk of

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psychological distress for the related DD if the pregnancy attempt fails in that she would feel, at least partly, guilty of the negative outcome (Lavoué et al. 2017). This risk for strain for DD could favor the use of ND living donor. In case of using an ND donor, it is important to cover motives for donation and that the donor has sufficient social support in relation to the decision to donate and support during recovery (Warren et al. 2018). In the evaluation concerning donor-specific psychological aspects, the donor’s own continued motherhood should be brought up for discussion. Is the potential donor in menopause? This question is restricted to responsibility for small/young children and how they can be affected by surgery, postoperative period and recovery, loss of income, etc. Is the donor still in her reproductive age? This adds a more complex question even though the donor may state that she has finished her wish for children. For example, what will happen if changes occur in the donor’s romantic relationship and then she wants to start a second family formation? Even if the general chance of pregnancy at an age above 40 years is as low as below 5%, mostly because of oocyte-specific factors, there are other options up to around 45 years of age. The older woman can undergo IVF with donor oocytes or her own, if cryopreserved at younger age. Nevertheless, it has been shown in several studies (Benzing et al. 2015; Gross et al. 2013) that there is none or a positive impact of quality of life among living donors as compared with the norm population. This fact probably overlaps with traits prior to transplantation such as being an individual willing to donate, showing suitability, and passing the evaluation procedure before donation. There is no reason to expect that this will be different for women who would donate their uterus (Kvarnström et al. 2017); if differences would exist, even less strain is predicted since the organ only will be donated when it no longer can be used by the donor as its only physiological purpose is to carry a pregnancy.

13.5.1 Deceased Donor When the UTx is performed with a uterus from a deceased donor, there is the question of consent from the relatives or from the donor during her life. The regulation around this issue differs between countries. Concerning the recipient and her partner, this setup can be a psychological strain. The couple that is on this pathway for UTx would have to cope with the means of living with an unknown waiting time and unpredictability concerning the availability of eligible donors (Dickens 2016). On the other hand, an issue that can be of less psychological strain is the knowledge that there is no risk for complications for the donor. Moreover, possible burden of guilt or gratitude toward the donor after donation is less likely to occur.

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13.6 Assessment Tools To collect the information needed to evaluate whether UTx is the right choice to achieve parenthood for the recipient and her partner as well as a secure choice for the possible donor, both questionnaires and clinical interview are useful tools. The questionnaires have the advantage in that each individual can be compared with norm populations. This kind of assessment also gives the individuals a possibility to give their answers on potentially sensitive questions not upfront to the caregiver. However, the disadvantage is the tendency to answer in a socially expected way to be accepted for the treatment. Prior to UTx, a suggestion is that the questionnaires would cover the key elements mood (anxiety and depression), health-related quality of life, marital relationship, and body image. Concerning living donors, also donor-specific questionnaires could be used. For the recipient and her partner, also a questionnaire regarding infertility can be added. The clinical interview has the advantage in that it is possible to achieve more complex knowledge about the individual’s perspectives, possible strengths, and vulnerability before UTx. If the interview is carried out in a non-judgmental and confidential relationship, individuals tend to speak freely and also term questions that are more complex and not always in favor of the desired treatment, in this context the UTx. Domains that could be covered for the woman and her partner are shown in Table 13.1. The interview provides knowledge about important areas in UTx. The Table 13.1  Semi-structured interview guide used before inclusion in uterus transplantation (Johannesson and Järvholm 2016) Domains Psychological well-being

Relationship

Managing childlessness (CL)

Constructs Identity

Interview questions How do you feel about yourself in present/past?

Skills Coping strategies Empowerment Support Consistency

Psychological/psychiatric burden? Experiences of help, from whom? How have you managed prior difficulties? Duration of the relationship? Have they learned about the project together? Aspects that they think differently from each other? How have they as a couple managed burdens in the past? How have they managed the CL up until now?

Coping strategies Goals Commitment

Alternative plans except UTx for becoming a parent? Thoughts/feelings about the risk of permanent CL? How are they affected by CL in their daily life? Openness about CL? (continued)

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Table 13.1 (continued) Constructs Domains Knowledge about the Outcome UTx expectance Contact with the team Procedural knowledge Risk

Relation with the donor

Perceived risk/ threat The choice Support Complications

Interview questions How did they get to know about the UTx? How have you perceived the contact with the team? What information have they got so far? Voluntariness? Thoughts/feelings about risks? How have you managed to go from being independent to being dependent of health care? How/to whom did they ask the question? Feelings? Has the relation with the donor been affected by the decision? In what way? Worries for the donor?

guide can also be used for living donor with adjustments, excluding questions about childlessness and adding questions regarding feelings of the decision to be a donor and support/lack of backing of this decision. If possible, it is advisable to perform both individual and couple interviews at inclusion. In the future other groups will enter the UTx program, for example, single women and the transgender group, and this will of course lead to a need for modification of the themes evaluated. But to conclude, the frame for psychological evaluation in UTx is already well stated in general by Collins and Labott (2007) “that the setup of the evaluation before transplantation is that it is not mainly performed to exclude participators more to identify potential risks and when needed offer support.”

References Ahnquist J, Wamala SP, Lindstrom M. Social determinants of health—a question of social or economic capital? Interaction effects of socioeconomic factors on health outcomes. Soc Sci Med. 2012;74(6):930–9. https://doi.org/10.1016/j.socscimed.2011.11.026. Ahrnsbrak R, Bose J, Hedden S, Lipari R, Park-Lee E. Key substance use and mental health indicators in the United States: results from the 2016 National Survey on Drug Use and Health. Rockville: Center for Behavioral Health Statistics and Quality, Substance Abuse and Mental Health Services Administration; 2017. Benzing C, Hau H-M, Kurtz G, Schmelzle M, Tautenhahn H-M, Morgül MH, et al. Long-term health-related quality of life of living kidney donors: a single-center experience. Qual Life Res. 2015;24(12):2833–42. Collins CA, Labott SM. Psychological assessment of candidates for solid organ transplantation. Prof Psychol Res Pract. 2007;38(2):150. Dickens BM.  Legal and ethical issues of uterus transplantation. Int J Gynecol Obstet. 2016;133(1):125–8.

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Forsberg A, Nilsson M, Krantz M, Olausson M. The essence of living parental liver donation– donors’ lived experiences of donation to their children. Pediatr Transplant. 2004;8(4):372–80. Gross C, Messersmith EE, Hong BA, Jowsey SG, Jacobs C, Gillespie BW, et al. Health-related quality of life in kidney donors from the last five decades: results from the RELIVE study. Am J Transplant. 2013;13(11):2924–34. Jin J, Sklar GE, Min Sen Oh V, Chuen Li S. Factors affecting therapeutic compliance: a review from the patient’s perspective. Ther Clin Risk Manag. 2008;4(1):269–86. Johannesson L, Järvholm S. Uterus transplantation: current progress and future prospects. Int J Womens Health. 2016;8:43. Johansson R, Carlbring P, Heedman Å, Paxling B, Andersson G.  Depression, anxiety and their comorbidity in the Swedish general population: point prevalence and the effect on health-­ related quality of life. Peer J. 2013;1:e98. Järvholm S, Warren AM, Jalmbrant M, Kvarnström N, Testa G, Johannesson L. Preoperative psychological evaluation of uterus transplant recipients, partners, and living donors: suggested framework. Am J Transplant. 2018;18(11):2641–6. Kvarnström N, Järvholm S, Johannesson L, Dahm-Kähler P, Olausson M, Brännström M. Live donors of the initial observational study of uterus transplantation—psychological and medical follow-up until 1 year after surgery in the 9 cases. Transplantation. 2017;101(3):664–70. Lavoué V, Vigneau C, Duros S, Boudjema K, Levêque J, Piver P, et al. Which donor for uterus transplants: brain-dead donor or living donor? A systematic review. Transplantation. 2017;101(2):267–73. Lindström B, Eriksson M. Salutogenesis. J Epidemiol Commun Health. 2005;59(6):440–2. Perez-San-Gregorio M, Martín-Rodríguez A, Luque-Budia A, Conrad R. Concerns, mental health, and quality of life in living kidney donation–parent donor candidates worry less about themselves. Front Psychol. 2017;8:564. Rutter M.  Psychosocial resilience and protective mechanisms. Am J Orthopsychiatry. 1987;57(3):316. Warren A, Testa G, Anthony T, McKenna G, Klintmalm G, Wallis K, et  al. Live nondirected uterus donors: psychological characteristics and motivation for donation. Am J Transplant. 2018;18(5):1122–8. White-Williams C, Grady KL, Naftel DC, Myers S, Wang E, Rybarczyk B. The relationship of socio-demographic factors and satisfaction with social support at five and 10 yr after heart transplantation. Clin Transplant. 2013;27(2):267–73.

Assisted Reproduction Before and After Uterus Transplantation

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Lars B. Nilsson and Jan I. Olofsson

14.1 Assisted Reproduction Pretreatment Assessments As an integral part of uterus transplantation (UTx), it is advised that the initial evaluations and treatment planning will take place in an accredited sizeable reproductive medicine unit which has in house capacity, or close access to, of all the testing’s and IVF procedures. Importantly, a dedicated electronic medical record and patient management system for all individuals constituting the future parent couples where detailed documentation of all assisted reproduction laboratory and clinical procedures is essential. The unit will also need to feature a secured cryopreservation facility. Details of such setups can be found in text book literature (Bento et  al. 2013) describing total quality management strategies in assisted reproduction (Olofsson et al. 2013). Prior to embarking on clinical treatments using IVF most countries regulatory authorities require mandatory screening of all patients to be screened for human immunodeficiency virus (HIV), hepatitis B and C, syphilis and for the woman, rubella immunity. While screening both partners for Chlamydia trachomatis and other sexually transmitted agents has not reached widespread usage in many fertility clinics, it can well be considered for patients in a UTx program. The male partner is suggested to have andrology screening including a complete semen evaluation as per WHO’s guideline (WHO 2010). It is universally agreed that all couples should L. B. Nilsson Reproductive Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden Department of Obstetrics and Gynecology, Institute of Clinical Sciences, Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden e-mail: [email protected] J. I. Olofsson (*) Reproductive Medicine, Karolinska University Hospital, Stockholm, Sweden Department of Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_14

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be offered thorough clinical and psychological counseling, patient education and finalization of consent forms prior to embarking on assisted reproduction with IVF. In addition to careful consideration of an age limit for the recipient (recommended below 35 years, with upper limit of 38 years), given that chronological age remains the most reliable predictor of IVF treatment outcome, we also consider it of special importance to obtain an estimate of the ovarian reserve (Amato 2017). This will enable tailor-made treatment planning and dosing of the ovarian stimulation gonadotropins. Available tests include biochemical markers, i.e., serum levels of FSH and LH, estradiol, anti-Mullerian hormone (AMH), and possibly inhibin B in addition to ultrasound imaging, including e.g., ovarian antral follicle count (AFC) and ovarian volume.

14.2 O  varian Stimulation, Oocyte Retrieval, and Embryo Transfer in the Swedish Study Controlled ovarian stimulation, or hyperstimulation, is a mix of medications designed to stimulate multiple ovarian follicle development, aiming at the generation of a large (typically 10–15) pool of mature and fertilizable oocytes and constitutes a fundamental step of IVF that has been in practice since its initial practice in the 1970s (Beall and DeCherney 2012). Urinary derived preparations such as menopausal human gonadotropin (hMG) and later recombinant follicle stimulating hormone (rFSH) are routinely used in clinical practice for ovarian stimulation as part of IVF (Fig. 14.1). These treatments are used in combination with either long-acting gonadotropin-releasing hormone (GnRH) agonist protocols or with short-acting GnRH antagonist protocols to inhibit premature LH surges during stimulation that may result in premature ovulation and cycle cancellation. Since the discovery of GnRH analogues, different types thereof have offered multiple options for ovarian stimulation regimens. While there remains some controversy regarding the optimal protocol to choose, an updated 2011 Cochrane systematic review reported no statistically significant differences in live birth rates in women undergoing IVF cycles using rFSH in comparison with urinary gonadotropins for ovarian stimulation, irrespective of the LH downregulation protocol used (Van Wely et al. 2011). In a recent large retrospective database study real-life evidence show that ovarian stimulation with hMG and rFSH in either a GnRH agonist or antagonist protocol are equally successful, with no significant advantage by important covariates such as age, oocyte yield, total dose of FSH administered, treatment history, or embryo score (Karlström et al. 2018). In the first series of patients in the Swedish UTx study, a majority of women were stimulated by either hMG alone or mixed in combination with rFSH and went through oocyte retrieval with cryopreservation of suitable pre-embryos at least on two occasions before UTx was performed. This was done with the aim to ascertain an acceptable pool of good quality embryos being cryopreserved for each couple. As the study was designed already in the early years of this millennium’s second decade, initially only cleavage stage embryos and slow freezing was utilized with

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GnRH Agonist Long Protocol

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GnRH agonist

GnRH antagonist

FSH

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LH

hCG

hCG/ GnRH ag

E2 E2

21...

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3

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Fig. 14.1  Schematic view of controlled ovarian stimulation. Stimulation procedures for long GnRH agonist (Panel a) and short GnRH antagonist (Panel b) and minimal stimulation protocols for IVF. Typically, a GnRH long agonist protocol starts with daily administration of sc 0.1 mg GnRH agonist (e.g., triptorelin) or nasal spray (e.g., 0.3 mg buserelin or 0.8 mg nafarelin) 7 days after verification of midcycle ovulation as monitored by urinary LH dip sticks and/or daily serial serum progesterone, followed 2 weeks later by s.c. self-injection rFSH/hMG at 150–300 international units (IU) daily. The adjustment of gonadotropin dose is based on daily transvaginal sonography (TVS) assessment of follicular development which can be commenced on day 5 or 6 of FSH stimulation. Continual administration of GnRH agonist and gonadotropin lasts until the start of human chorionic gonadotropin (HCG) injection to induce final oocyte maturation, which is approximately 10–12 days post FSH start or follicles reached from 18 to 20 millimeters (mm) in size. For the GnRH antagonist protocol (Panel b), administration of gonadotropin at 150–300 IU daily is initiated after monitoring of patients’ follicles size on menstrual cycle, day 2/3, or alternatively, as in patients without a uterus, after 2–3 weeks of oral pretreatment using an estrogen/progestin combined or progestin only contraceptive pill. Gonadotropin dosage varies according to the follicular response and on approximately the fifth to sixth day of gonadotropin injection, or when leading follicle size reaches more than or equal to 14  mm, subcutaneous administration of the GnRH antagonist (e.g., 0.5 mg ganirelix) is initiated. In both protocols, there is routine monitoring of patients via TVS and hormonal profiling of FSH, LH, estrogen, and progesterone levels of patients. After 35–37 h of HCG injection mature oocytes are retrieved by ultrasound guided ovarian follicular puncture. It is important to note that in excessive response in terms of follicular recruitment the cycles should be discontinued and/or HCG injection be deferred as there is added risk of ovarian hyper stimulation syndrome (OHSS). The benefit of using a GnRH antagonist cycle is that the final oocyte maturation injection can then be replaced by administration of 0.1  mg GnRH agonist where after oocyte pickup (OPU) can be performed at the same time interval as after conventional HCG priming, with a diminutive risk of OHSS. As all embryos will be cryopreserved for later usage, there is no need to use luteal phase support regimens

the aim of generating ten or more embryos (Brännström et al. 2014), but this was later changed to extended culture to obtain expanded blastocysts (aiming eight embryos) which were subsequently subjected to cryopreservation using vitrification (Brännström et al. 2016). Since it was decided to perform segmentation of all IVF treatments and to freeze all embryos before moving forward with UTx, gonadotropin stimulation and oocyte retrieval had to be performed in patients lacking a uterus. The only previous experience was from similar treatments of MRKH women, where the

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fertilized oocytes were transferred into surrogate mothers (Ben-Rafael et al. 1998; Raziel et al. 2012). Given the lack of information of menstruation pattern cyclicity, it was decided to use the long GnRH agonist protocol and a mixed protocol with rFSH and hMG in combination to avoid stimulation failure in case of low LH (WHO type one, hypo-­hypo anovulation). Final ovulatory follicle development and oocyte maturation were induced using hCG injection. Since none of the study patients had experienced menstrual bleedings, and because the ovaries in many of the patients were positioned lateral to the external iliac vessels and in a more cranial position than their normal, anatomical position, the non-stimulated ovaries were often not readily visible (especially in the early follicular phase) neither using vaginal nor abdominal ultrasound scanning. Consequently, the patients typically went through one or more cyclic assessments of LH (urinary as well as serum), FSH, estradiol, and progesterone. All showed reasonably regular ovulatory cycles with identifiable LH peaks. Treatment with GnRH agonist as commenced 8–9 days after an LH peak, when an elevated and luteal phase affirming progesterone value was demonstrated. Since follicular measurements were occasionally uncertain, especially in some of the patients with latero-cranially placed ovaries, monitoring was based mainly on estradiol levels and developmental curves of this steroid in the first 8–12 days, until follicles could be measured either by abdominal or vaginal ultrasound scanning. Mature oocytes in the largest follicles can be expected after a sustained increment in serum estradiol levels over baseline for 7–9 days, according to our experience. Oocytes were aspirated 36–37 h after the injection of hCG, using routine transvaginal technique in most cases. In some MRKH patients, with extra pelvic location of ovaries (Fedele et  al. 2007), the mature follicles were not easily reachable by transvaginal approach thereby necessitating abdominal percutaneous aspiration, using a modified method based on our original method for transvesical aspiration (Wikland et  al. 1983, 1987; Raziel et  al. 2006). In all cases, moderate sedation/ analgesia (“conscious sedation”) using alfentanil and local anesthesia was used. Sperm preparation and incubation procedures were standard, and fertilization accomplished by routine IVF or by intracytoplasmic single sperm injection (ICSI), as judged by the actual sperm sample. After normal fertilization was confirmed, embryos were normally cultured for ordinary slow freezing of four cell stage embryos on day 2 or vitrification of blastocysts on day 5/6. Embryos were individually cryopreserved in liquid nitrogen for different time periods, until used for frozen/thawed embryo transfer (FET). In most patients, the stimulation-aspiration-freezing procedures had to be repeated one or several times to accumulate a certain number of embryos. Our later experience has shown that, if needed, there is a possibility to perform additional stimulated cycles with oocyte harvest after the transplantation procedure. Hence, three of this first series of seven patients have undergone one or more oocyte retrieval cycles after UTx, in two of these patients resulting in pregnancy and live birth of a second child. At least 1 year after the UTx, patients returned for FET of one single embryo. To identify the period of optimal endometrial receptivity most transfers were performed in the natural cycle, 3–6 days after the LH peak, depending on the intended

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transfer embryo developmental stage. As transplantation surgery could possibly have negatively impacted ovarian blood supply, it can be considered valuable to supplement the luteal phase for 2–3 weeks following FET with micronized vaginal progesterone or as postulated for patients with skin graft neovaginas, who could have a decreased uptake of vaginal progesterone, supplementary luteal phase support can be given by daily i.m. progesterone (25–50  mg) for 2–3  weeks or oral dydrogesterone. As an alternative, endometrial stimulation by sequential estrogen/ progesterone medication can be recommended, although there are some concerns of increased number of miscarriages in addition to a longer medication period should a pregnancy occur (Groenewoud et  al. 2013). Routine pregnancy testing using repeated serial serum-hCG was used for all patients measured no earlier than 12 days after embryo transfer. Two out of seven patients that underwent the complete set of IVF, UTx, and FET became pregnant with resulting live birth at the first ET attempt but some patients delivered on FET number five to seven.

14.3 R  uminations Based on the First Series of Swedish Patients 14.3.1 Ovarian Reserve Testing All the patients in the Swedish study were evaluated for inclusion during 2010– 2014. Recent data suggests that MRKH women with the atypical form (type B) (Oppelt et  al. 2006) have lower AMH levels and AFC compared to age-matched controls and the Israeli group (Raziel et al. 2012) also reported that women with the atypical MRKH form had decreased ovarian response to gonadotropins and lower fertilization rates compared with women with the type A MRHK form. Since MRKH patients with a single kidney could also constitute a risk factor for preeclampsia, when taken together, we propose cautious evaluation of MRHK type B patients prior to consideration of inclusion in UTx programs.

14.3.2 Alternatives for Anesthesia/Analgesia During Oocyte Retrievals Since women devoid of a uterus and also after UTx do not experience sensory neural triggered pain from the lower uterine segment, it can be considered using a preovarian block as per our earlier experience (Cerne et al. 2006) in uterus intact patients. In the first series, we did not find it necessary to recommend deep sedation/analgesia using propofol, although recognizing there may be cases in which this is recommendable (Matsota et  al. 2015). Notably, acceptable anesthesia for abdominal percutaneous follicular aspirations may also be obtained by transversus abdominis plane (TAP) block (Tsai et al. 2017).

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14.3.3 Embryo Selection and Transfer, Potential for Further Developments Given the inherent risks of multiple pregnancies being closely associated with preterm birth and other obstetric risks, in a program for UTx, it is strongly advised to recommend single embryo transfer (SET). This strategy has predominantly been advocated in all assisted reproduction programs throughout in Sweden following national recommendations from 2003, and is demonstrated to have a clear benefit in reducing multiple births and perinatal health risks (Thurin et al. 2004; Tobias et al. 2016). This has also become the standard treatment regimen for conventional IVF in many fertility clinics worldwide (Kushnir et al. 2017). Additional rationale for using SET can be found in recent data (De Vos et al. 2016) showcasing superior outcome with shorter time to pregnancy using blastocyst transfer compared to cleavage stage embryos. While the search for a marker enabling selection of the single best embryo for transfer continues to be the major challenge, recent developments is the embryology laboratory technology has indicated that embryo viability can be assessed by time-lapse microscopy (Pribenszky et  al. 2017), alone or in combination with preimplantation genetic testing for aneuploidy (PGT-AS) which is widely used in IVF and aims to improve treatment outcomes by avoiding selection and transfer of aneuploid embryo transfers (Dahdouh et al. 2015). While these and other methods, may represent very promising as markers of embryo viability, it is still not entirely clear which methods, or combinations thereof, constitutes the optimal strategy of embryo selection, why additional prospective randomized studies will provide additional insights before precise guidelines can be established. As noted above with regards to possible alterations of ovarian/endometrial vascularization and effects on ongoing immunosuppression in the UTx situation, there may still be room for optimization in selection of day of FET, which may differ from routine management in most ART programs. Hence, while recent evidence suggests that each woman may have a personal window of implantation, a test—the endometrial receptivity test (ERA), was postulated to increase implantation rate in those transplanted uteri where repeated implantation failure had occurred, but at the time of writing there is no clear supportive evidence (Tan et al. 2018) that such testing should be included in routine evaluation of UTx cases prior to first ET. However, the ERA test was used in some patients of the initial UTx study and in two cases the day of ET was adjusted according to ERA result. Subsequent pregnancies with live births were seen in those cases. During the ET procedure, a standard method with a full urinary bladder and abdominal ultrasound scanning was used to visualize the transfer catheter tip when the embryo was injected into the uterine cavity in the middle or low portion of the endometrium. All transfers hitherto have been uneventful, although a few have been technically difficult due to poor visualization of the external os of the cervix, due to an altered position of the uterus so that the cervix will enter the vagina in a nearly right angle and to partial stenosis of the vaginal-vaginal anastomosis.

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14.4 Conclusions The lack of a functional uterus renders the woman absolutely infertile which, as defined by WHO, generates disability (an impairment of function), where access to health care falls under the Convention on the Rights of Persons with Disability (United Nations 2006). In addition, the possibility to give the woman a child birth, adds reason as an additional ethical justification for UTx, particularly in countries where gestational surrogacy is not available to women. Earlier reports on MRHK patients have indicated lower than expected outcomes in surrogacy gestations (Friedler et al. 2016). Contrasting to this, ART outcomes in the Swedish series of UTx were excellent. This success rate with a cumulative live birth rate of 86% per initially successful UTx in the women that underwent IVF-­ UTx-­ET, illustrates the potential development of this method to reach equal outcome results to that of gestational surrogacy. Given that any pregnancy carries with it an incumbent risk of obstetric complication, and recent evaluations in large registry data series, have clearly established that such a risk is even higher in recipient patients following oocyte or embryo donation ART, such combinations in a UTx setting will need even more careful medico-ethical scrutinizing before approval even in a clinical research setting. In closing, many societies have recognized and addressed ethically challenging issues not only in ART procedures involving gamete procurement and embryo cryopreservation, but also which patients can be accepted as recipients. Hence, the importance of facilitating international experience sharing and collaboration is underscored, also in areas of ethical governance and regulations, since in this emerging field of reproductive medicine inevitably attracts opportunistic and competitive groups.

References Amato P. Ovarian reserve testing. In: Falcone T, Hurd W, editors. Clinical reproductive medicine and surgery. Cham: Springer; 2017. Beall SA, DeCherney A. History and challenges surrounding ovarian stimulation in the treatment of infertility. Fertil Steril. 2012;97:795–801. Ben-Rafael Z, Bar-Hava I, Levy T, Orvieto R. Simplifying ovulation induction for surrogacy in women with Mayer-Rokitansky-Kuster-Hauser syndrome. Hum Reprod. 1998;13:1470–1. Bento F, Esteves SC, Agarwal A, editors. Quality management in ART clinics: a practical guide. New York: Springer; 2013. isbn:978-1-4419-7139-5. Brännström M, Johannesson L, Bokström H, Kvarnström N, Mölne J, et al. Livebirth after uterus transplantation. Lancet. 2014;385:607–16. Brännström M, Bokström H, Dahm-Kähler P, Diaz-Garcia C, Ekberg J, et al. One uterus bridging three generations: first live birth after mother-to-daughter uterus transplantation. Fertil Steril. 2016;106:261–6. Cerne A, Bergh C, Borg K, Ek I, Gejervall AL, et al. Pre-ovarian block versus paracervical block for oocyte retrieval. Hum Reprod. 2006;21:2916–21. Dahdouh EM, Balayla J, García-Velasco JA. Impact of blastocyst biopsy and comprehensive chromosome screening technology on preimplantation genetic screening: a systematic review of randomized controlled trials. Reprod Biomed Online. 2015;30:281–9.

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De Vos A, Van Landuyt L, Santos-Ribeiro S, Camus M, Van de Velde H, et al. Cumulative live birth rates after fresh and vitrified cleavage-stage versus blastocyst-stage embryo transfer in the first treatment cycle. Hum Reprod. 2016;31:2442–9. Fedele L, Bianchi S, Frontino G, Ciappina N, Fontana E, Borruto F. Laparoscopic findings and pelvic anatomy in Mayer-Rokitansky-Kuster-Hauser syndrome. Obstet Gynecol. 2007;109:1111–5. Friedler S, Grin L, Liberti G, Saar-Ryss B, Rabinson Y, et al. The reproductive potential of patients with Mayer–Rokitansky–Küster–Hauser syndrome using gestational surrogacy: a systematic review. Reprod Biomed Online. 2016;32:54–61. Groenewoud ER, Cantineau AEP, Kollen BJ, et al. What is the optimal means of preparing the endometrium in frozen–thawed embryo transfer cycles? A systematic review and meta-­ analysis. Hum Reprod Update. 2013;19:458–70. Karlström PO, Holte J, Hadziosmanovic N, Rodriguez-Wallberg KA, Olofsson JI. Does ovarian stimulation regimen affect IVF outcome? a two-centre, real-world retrospective study using predominantly cleavage-stage, single embryo transfer. Reprod Biomed Online. 2018;36:59–66. Kushnir VA, Barad DH, Albertini DF, Darmon SK, Gleicher N. Systematic review of worldwide trends in assisted reproductive technology 2004–2013. Reprod Biol Endocrinol. 2017;15:6. Matsota P, Kaminioti E, Kostopanagiotou G. Anesthesia related toxic effects on in vitro fertilization outcome: burden of proof. Biomed Res Int. 2015;2015:475362. Olofsson JI, Banker MR, Sjoblom LP. Quality management systems for your in vitro fertilization clinic’s laboratory: why bother? J Hum Reprod Sci. 2013;6:3–8. Oppelt P, Renner SP, Kellermann A, Brucker S, Hauser GA, et  al. Clinical aspects of Mayer-­ Rokitansky-­Kuester-Hauser syndrome: recommendations for clinical diagnosis and staging. Hum Reprod. 2006;21:792–7. Pribenszky C, Nilselid AM, Montag M. Time-lapse culture with morphokinetic embryo selection improves pregnancy and live birth chances and reduces early pregnancy loss: a meta-analysis. Reprod Biomed Online. 2017;35:511–20. Raziel A, Vaknin Z, Schachter M, Strassburger D, Herman A, et al. Ultrasonographic-guided percutaneous transabdominal puncture for oocyte retrieval in a rare patient with Rokitansky syndrome in an in vitro fertilization surrogacy program. Fertil Steril. 2006;86:1760–3. Raziel A, Friedler S, Gidoni Y, Ben Ami I, Strassburger D, Ron-El R. Surrogate in vitro fertilization outcome in typical and atypical forms of Mayer–Rokitansky Küster–Hauser syndrome. Hum Reprod. 2012;27:126–30. Tan J, Kan A, Hitkari J, Taylor B, Tallon N, et al. The role of the endometrial receptivity array (ERA) in patients who have failed euploid embryo transfers. J Assist Reprod Genet. 2018;35(4):683– 92. https://doi.org/10.1007/s10815-017-1112-2. Thurin A, Hausken J, Hillensjö T, Jablonowska B, Pinborg A, et al. Elective single-embryo transfer versus double-embryo transfer in in vitro fertilization. N Engl J Med. 2004;351:2392–402. Tobias T, Sharara FI, Franasiak JM, Heiser PW, Pinckney-Clark E. Promoting the use of elective single embryo transfer in clinical practice. Fertil Res Pract. 2016;2:1. Tsai HC, Yoshida T, Chuang TY, Yang SF, Chang CC, et al. Transversus abdominis plane block: an updated review of anatomy and techniques. Biomed Res Int. 2017;2017:8284363. United Nations. Convention on the Rights of Persons with Disabilities (CRPD). 2006. https:// www.un.org/development/desa/disabilities/convention-on-the-rights-of-persons-with-disabilities.html. Accessed 4 Feb 2018. Van Wely M, Kwan I, Burt AL, Thomas J, Vail A, Van der Veen F, Al-Inany HG. Recombinant versus urinary gonadotrophin for ovarian stimulation in assisted reproductive technology cycles. Cochrane Database Syst Rev. 2011;(2):CD005354. https://doi.org/10.1002/14651858. CD005354.pub2. WHO laboratory manual for the examination and processing of human semen. 5th ed. Cambridge: WHO Cambridge University Press; 2010. Wikland M, Nilsson L, Hansson R, Hamberger L, Janson PO. Collection of human oocytes by the use of sonography. Fertil Steril. 1983;39:603–8. Wikland M, Enk L, Hammarberg K, Nilsson L. Use of a vaginal transducer for oocyte retrieval in an IVF/ET program. J Clin Ultrasound. 1987;15:245–51.

Surgical Technique of Live Donor in Uterus Transplantation

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Mats Brännström and Pernilla Dahm-Kähler

15.1 Introduction The first uterus transplantation (UTx) case in the world was performed already in year 2000, and this was a live donor case (Fageeh et al. 2002). The technique of this donor surgery is unfortunately not well specified in the article and there is no mention of surgical duration. The surgery included harvesting of the uterus with attached oviducts, to rely on natural conception. Only short vascular pedicles were retrieved and at back-table preparation, both uterine veins and arteries had to be elongated by saphenous grafts. The donor surgery was uneventful, apart from occurrence of a laceration of one of the ureters, and this injury was repaired perioperatively. More than a decade later, the first clinical trial of live donor UTx was performed and our series included nine live donor UTx cases (Brännström et al. 2014). Our team had for many years performed extensive preparations by surgery (Díaz-García et al. 2012) in three large animal species (pig, sheep, and baboon). Allogeneic live donor UTx was performed in the baboon, with surgical duration for donor surgery of around 200  min (Johannesson et  al. 2013). Thus, our assumption before the human trial was that the surgical duration of a uterus donating woman would be maximum 360 min, based on that essentially an identical technique would be used as in the baboon (Johannesson et al. 2013) and that the anatomical structures would be larger. However, the surgery proved to be more demanding than expected and the range of duration for retrieval surgery of the nine donors was around 700 min. We M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] P. Dahm-Kähler Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_15

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have now performed well over 20 live donor surgical procedures, both by laparotomy technique and robotic-assisted laparoscopy. Our accumulated experience and other results from published reports, concerning surgical technique by open surgery and minimal invasive surgery of live donor are described below.

15.2 L  aparotomy in Live Donor Uterus Transplantation Surgery The traditional surgical approach of organ retrieval in live donor UTx is a vertical midline incision, beginning below the umbilicus and extending to the upper part of the pubic bone. The incision could be expanded 1–3  cm above the umbilicus in patients with a short sub-umbilical distance. The large incision is required to ensure adequate exposure of pelvic structures up to the level of the common iliac arteries and veins. The type of retractor is essential for good exposure. Preferably, a retractor of the type with a field post attached to the operating table, with a one-piece support arm and a frame with a possibility to use several small retractors that can be placed in optimal positions should be used. Good packing of intestines in the upper abdomen is essential for superior exposure of the pelvis. Most of the surgery is done by diathermy, monopolar or bipolar, to keep an operating field essentially free of blood. Surgery starts with division of the round ligaments at lateral positions and placement of marking sutures in the ligaments. Then, by monopolar diathermy, a large flap of the bladder peritoneum (nearly to the level of the bladder dome) with the uterus is acquired and recommended. The peritoneal flap will later be part of fixation of the grafted uterus in the recipient to cover the vesicouterine fossa, which will minimize the risk of intestinal herniation. Thereafter, the identification of the ureters just distal to their passage over the iliac vessels is done. The ureters are then dissected toward the bladder. During ureteric dissection we prefer to use bipolar, rather than monopolar, diathermy to coagulate overriding vessels, in order to avoid widespread thermic injury from monopolar diathermy. In our second live donor UTx case, the donor acquired a ureteric-vaginal fistula 2 weeks after surgery (Brännström et al. 2014). An assumption is that this injury was due to extensive use of monopolar diathermy close to the ureter, which leads to thermal injury of the ureter wall and subsequent fistulation. The critical part of the ureteric dissection is the dissection of the ureteric tunnel, under the overriding uterine artery, and adjacent uterine veins. During dissection of the ureteric tunnel, care must be taken to not cause any injury to the large deep uterine veins, which in a majority of cases go under the ureter. We try to do most of the dissection of the ureteric tunnel from the cranial angle, so that a plane of the tunnels is reached that go beyond the crossing of the vessels. Of note is that from the uterine artery, there will be branching of two small ureteric arteries, one in a cranial direction and one caudal, and often also branching of two additional caudal branches to the upper and lateral part of the bladder. These small arterial branches have to be properly secured with ligations or appropriate coagulation before transection, in order to avoid leakage after reperfusion of the organ in the

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recipient. When the ureter is relatively free from the vessels and the paracervical tissue, for a distance of the major extent of the ureteric tunnel, the ureter can be identified anteriorly to the uterine artery and dissection should proceed all the way to the bladder. Several vein plexuses will be firmly attached to the ureters and they have to be gently lifted and dissected away from the ureter. Care should be taken to avoid injury to major uterine veins that may go up toward the bladder in and then arch down to the internal iliac veins. All vascular dissections, as described above and further below, should be performed with a non-touch technique, since these vessels will be the sole inflow and outflow parts of blood after transplantation. Thus, the vessels should not be subjected to any injury or tissue stress, which could increase the risk of constriction and thrombosis. The dissection is then directed toward that of the arterial supply to the uterus. Noteworthy is that in a normal anatomical situation the uterus is supplied by six major arteries (bilateral vaginal, uterine and ovarian arteries) but after transplantation the blood flow to the entire uterus will be solely through the bilateral uterine arteries. The uterine artery has during the ureteric dissection been identified and partly freed at a distal part, where it overrides the ureter. The arterial vasculature is then dissected distally from the interior iliac artery toward the uterine artery. The arterial segment between the bifurcation of the posterior branch (gluteal artery) of the iliac artery and cervix are kept in the graft, while all the other branches, which may contain distinct branches of iliolumbar artery, lateral sacral artery, pudendal artery, middle rectal artery, vaginal artery, obturator artery, and umbilical artery, are ligated and transected at their divisions from this segment. Thus, the goal is to achieve one major trunk of the arterial supply through the major anterior branch of the internal iliac artery. Depending on the venous anatomy, the divisions of these arterial branches may be done during this initial artery dissection, during the subsequent venous dissection, or during the final step of clamping of vessels and removal of graft. Noteworthy is that the major posterior branch of the internal iliac artery, also named the gluteal artery, is kept in the donor to avoid gluteal ischemia. In some patients, this artery has been included in the graft vasculature out of anatomical reasons. The contralateral gluteal artery should in these cases be left intact. One patient experienced ischemic symptoms at walking during the initial postoperative months. However, subsequently the donor has had no problems at physical activity or in daily life, most likely due to development of new collateral circulation. There are several alternatives for venous outflow from a uterine graft. Our recommendation is that optimal harvesting of the uterine venous outflow should on each side include one deep uterine vein with segment of the internal iliac vein, as well as the proximal part of the utero-ovarian vein. As discussed below, this will not necessitate oophorectomy and it will provide the surgeons with several options for venous outflow in case of that some of the veins are considered inadequate or suboptimal for creation of anastomosis in the recipient. Dissection of the deep uterine veins begins on the pelvic sidewall, in order to identify the internal iliac vein, and then progresses toward the uterus. These veins and plexuses, toward the uterus, have very many variations and are difficult to properly assess preoperatively. The

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objective of venous dissection is to create an optimal outflow conduit, with a large lumen, on each side of the uterus. The venous vascular pedicle should, at its end, comprise a segment of the internal iliac vein, in order to get a vein with well-defined walls that will make anastomosis surgery in the recipient easier. During isolation of this segment, several major venous branches have to be divided and closed, preferably by suturing. Positions and sizes of the two to three deep uterine veins determine whether one dominating uterine vein is chosen or if two uterine veins are used for venous drainage. Dissection of the deep uterine veins is exceptionally complex due to the close position and adherence of these veins to the ureters and the paravaginal/cervical tissue, as well as presence of several branches and plexuses between veins. Noteworthy is that in cases where two modest sized uterine veins, instead of one larger vein, are chosen on one side for outlet and if they ride on either side of the ureter, one of the veins has to be divided before the graft is removed and then sutured end-to-end during back-table preparation. This maneuver is done since the ureter will ride in between the two uterine veins, which converge into the internal iliac vein. A salvage procedure, which may be used, is to utilize the proximal part of the utero-ovarian vein for later anastomosis to the internal iliac segment or to the deep uterine vein, to increase venous drainage. These vessels are dissected and isolated as a late procedure of the donor surgery, when the oviducts are removed (see below). When only this proximal part of the utero-ovarian vein is utilized, the blood flow to and from an ovary, that is left in situ, will not be compromised. There are also reports on laparotomy procedures of live donor surgery that has only used the proximal part of the utero-ovarian vein for venous outflow (Testa et al. 2018) and also reports of usage of only the entire utero-ovarian veins (Wei et al. 2017; Puntambekar et al. 2018). The ovarian artery is riding on top of this vein and is firmly attached to the common utero-ovarian vein and at the distal site of the artery, before entering the ovarian medulla, rides on top of the ovarian branch of this utero-ovarian. This vascular anatomy is important, in the understanding that oophorectomy has to be performed when any part of the common utero-ovarian vein is harvested with the graft. The rectovaginal space is then opened and the sacrouterine ligaments are identified and divided, preferably around 20  mm from the uterine cervix, to simplify subsequent uterine fixation in the recipient. At this stage we divide the ligament between the ovary and the uterus and excise the oviducts, with care taken not to disturb the proximal part of the utero-ovarian vein, which is located just under the oviduct. This vein is dissected all the way to the inlet of the ovarian branch, in order to isolate this as a salvage maneuver. Paravaginal tissue is then dissected together with the vaginal arteries and veins, which are firmly attached to the lateral portions of the upper vagina and are divided. The vagina is divided with a cuff of with a length of around 20 mm on the side of the graft. The division of the vagina should preferably be performed by alternate use of bipolar diathermy and scissors. Diathermy methods with more widespread thermal coagulation should be avoided since such a procedure may be related to the risk of developing vaginal stenosis in the recipient.

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At this point of the surgery, the dissection is almost completed, and the uterus is only attached by the bilateral arterial and venous vascular pedicles. Vascular clamps are then placed, first on the internal iliac arteries just distal to the branching of the gluteal arteries, and on the internal iliac veins to obtain a segments of this vein on each side. The vessels are divided just distal to the clamps, and the uterus is quickly taken to the back-table for chilling and flushing. The division sites on the major arteries and veins are then closed, by sutures that are firmly and thoroughly placed over the four transection locations. Ovaries are usually sutured and fixed to the pelvic sidewalls, lateral to the iliac vessels, to avoid future torsion or displacement into the rectovaginal fossa. Surgery is then ended by standard technique including hemostatic control, mass suture, subcutaneous suture, and skin suture. A wound drain is usually not applied. The durations of laparotomy retrievals in the initial live donor UTx study in Sweden was between 600 and 780 min (Brännström et al. 2014) and in subsequent trials decreased surgical durations are reported. Thus, in the Czech trial the surgical duration was 320–430 min (Chmel et al. 2019) and surgical times between 480 and 540 min were reported in the live donor UTx trial in the USA (Testa et al. 2017). The estimated blood loss was between 300 and 2400  mL in the Swedish trial (Brännström et al. 2014) and 100–1000 mL in the Czech trial (Chmel et al. 2019). Length of hospital stay (LOS) for the donors in open UTx procedures was 6 days for all nine donors in our Swedish trial (Brännström et  al. 2014), while LOS was 5–7 days and 6–11 days in the USA (Testa et al. 2017) and Czech (Chmel et al. 2019) trials, respectively. One major concern in the live donor UTx setting is the surgical risk of the donor. Among the published laparotomy UTx cases, some major intra- and postoperative complications of the donors were reported. Intraoperative unilateral ureteric laceration occurred in the initial UTx case (Fageeh et al. 2002) and in one of the five live donors of the Czech trial (Chmel et al. 2019) As mentioned above, a ureteric-vaginal fistula was diagnosed 2 weeks postoperatively in the second patient in our initial Swedish trial (Brännström et al. 2014). Less severe and easily treatable/reversible reported complications include urinary tract infection, fecal impaction, temporary gluteal pain, minor depression, and reversible bladder hypotonia.

15.3 L  aparoscopy in Live Donor Uterus Transplantation Surgery Minimal invasive surgery is slowly moving into the arena of live donor surgery at UTx. The first attempt to use minimal invasive surgery in UTx was in China (Wei et al. 2017) with a fully robotic-assisted laparoscopy procedure. The surgery followed essentially the same steps used in open surgery, as described above. The surgery team used two 8-mm ancillary robotic trocars and two accessory trocars of 10- and 5-mm for use by the assistant surgeon. One modification of surgery from the principles of laparotomy was that the uterus, with the oviducts and attached ovaries, was removed through the vagina instead of through an abdominal incision.

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The graft was not protected inside a sterile bag. The ovaries and oviducts were removed at the back-table. Another, aberration from the standard procedure was that merely the utero-ovarian veins were used as venous outflow, in order to simplify the procedure. The surgery lasted for 360 min and with minimal (100 mL) bleeding. Length of hospital stay was 5 days. There are also four cases of donor surgery performed by partly laparoscopic procedure (Puntambekar et al. 2018, 2019). The last parts of the procedures were by laparotomy through midline incision in two cases and transverse incision in two cases. The uterus was retrieved with utero-ovarian veins as venous outflow parts and the uterus was lifted out through the abdominal incision. Oophorectomy was done in all donors. The surgical durations were only 144–240 min and blood loss was 100 mL in all cases. The length of hospital stay was 6–7 days. No complications were reported. Other centers, including our Swedish center, are developing and performing robotic-assisted laparoscopic retrieval of the uterus for transplantation and more details concerning these cases will be presented in the future.

15.4 Conclusion There will be a continuous development in live donor surgery for uterus transplantation. Minimal invasive surgery will become more common in the future and will most likely have positive impacts on surgical duration, tissue trauma, hospital stay, postoperative pain, time to return to common daily activities, and on rates of complications.

References Brännström M, Johannesson L, Dahm-Kähler P, et al. The first clinical uterus transplantation trial: a six months report. Fertil Steril. 2014;101:1228–36. Chmel R, Novackova M, Janousek L, et al. Revaluation and lessons learned from the first 9 cases of a Czech uterus transplantation trial: four deceased and five living donor uterus transplantations. Am J Transplant. 2019;3:855–64. Díaz-García C, Johannesson L, Enskog A, et al. Uterine transplantation research: laboratory protocols for clinical application. Mol Hum Reprod. 2012;18:68–78. Fageeh W, Raffa H, Jabbad H, Marzouki A. Transplantation of the human uterus. Int J Gynaecol Obstet. 2002;76:245–51. Johannesson L, Enskog A, Mölne J, et al. Preclinical report on allogeneic uterus transplantation in non-human primates. Hum Reprod. 2013;28:189–98. Puntambekar S, Telang M, Kulkarni P, et  al. Laparoscopic-assisted uterus retrieval from live organ donors of uterine transplant; our experience of two patients. J Minim Invasive Gynecol. 2018;25:622–31. Puntambekar S, Puntambekar S, Telang M, et al. Novel anastomotic technique for uterine transplant using utero-ovarian veins for venous drainage and internal iliac arteries for perfusion in two laparoscopically harvested uteri. J Minim Invasive Gynecol. 2019;4:628–35.

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Testa G, Koon EC, Johannesson L, et  al. Living donor uterus transplantation: a single center’s observations and lessons learned from early setbacks to technical success. Am J Transplant. 2017;17:2901–10. Testa G, McKenna GJ, Gunby RT Jr, et al. First live birth after uterus transplantation in the United States. Am J Transplant. 2018;18:1270–4. Wei L, Xue T, Tao KS, et al. 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.

Surgical Technique of Deceased Donor in Uterus Transplantation

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Andreas Tzakis, Michael Olausson, and Tommaso Falcone

16.1 Introduction The objective of this chapter is to describe the technique for graft recovery in a deceased donor (DD). The medical screening and general approach is covered in a separate chapter. The sole final uterine evaluation is with a hysteroscopy if no other pelvic imaging was performed. However pre-procedural imaging of the uterus is preferred. Uterine retrieval from a DD provides several advantages over retrieval from a live donor (LD; Flyckt et al. 2016, 2017a, b). First, dissection of the uterine artery and vein from the ureter and the para-cervical tissue, which is the most time-­ consuming part of LD surgery, is avoided. Second, in many cases of LD dissection of the uterine veins, this is such a complex surgical procedure that many centers do not use the uterine veins and use the utero-ovarian veins that have a very small caliber and potentially limited length (Flyckt et al. 2017b). This is never an issue with DD. Third, in LD minimal vaginal length is removed that can lead to vaginal stenosis in the recipient. This again is not a concern with DD. There are several concepts that should be explored before procurement and discussed with the other surgical teams. The recovery of the uterus from a DD is part of multiorgan recovery. Extreme care is taken to prevent any damage to the vital organs which are recovered contemporaneously. The a priori decision is at what stage is the A. Tzakis Cleveland Clinic, Weston, FL, USA e-mail: [email protected] M. Olausson Department of Transplantation, Sahlgrenska Academy at Gothenburg University, Sahlgrenska University Hospital, Göteborg, Sweden e-mail: [email protected] T. Falcone (*) Cleveland Clinic, Cleveland, OH, USA e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_16

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surgical dissection of the uterus started and how much is it completed before core flushing. In our original experience, all other procurement teams proceeded first. The thoracic, abdominal, and pelvic cavities were filled with ice. Vital organ retrievals proceeded in the usual fashion with the heart, liver, pancreas, and kidney. The uterus was removed only after all teams were complete but still under cold ischemia. The main issue with this approach is that multiple sites of bleeding from veins are not identified until after transplantation into the recipient and thereby leading to significant blood loss and unnecessary extension of the duration of recipient surgery. Subsequently, we modified our technique to perform maximal dissection and ligation of blood vessels to the uterus before cross clamping and flushing. When all other teams were complete, we would return and complete the procurement by ligating dissected vessels and clamping and opening the vagina. The other participating teams are warned that the uterine part of the recovery will add 2–3 h to the operation before core flushing. Furthermore extreme care needs to be observed as hemorrhage can occur resulting in more hemodynamic instability. Finally, in the third approach as published by the Turkish (Ozkan et al. 2012) and Dallas group (Testa et al. 2018), the uterus is removed before any of the other procurement teams have started. The flushing of the organ was performed on the back table rather than in vivo. This approach is appealing but requires more thought. The most important is the fact that the vagina is opened and contamination of the operative field has occurred as a hysterectomy is a “clean-contaminated” case. Therefore for the moment, we perform maximal dissection without opening the vagina requiring approximately 2 h before flushing and then return to finish the procedure.

16.2 Procedural Steps Upon acceptance of the donor, a vaginal prep is ordered including a betadine wash followed by a miconazole suppository. This is repeated in the operating room (OR). Intravenous micafungin is given on call to the OR.  The patient does not require lithotomy position for the uterine removal. Exposure of the pelvic structures is facilitated through a complete midline abdominal with bilateral inguinal incisions which are extended laterally to the 12th rib (Fig.  16.1, Tzakis incision). This incision, combined with a sternotomy for the recovery of the thoracic organs, offers the best possible exposure for recovery of not only the uterus but all abdominal organs.

16.2.1 Step 1: Dissection of the Ureters from the Pelvic Brim (Fig. 16.2) The purpose of this step is to give adequate length of ureters for the kidneys. The dissection starts by identifying the ureters at the pelvic brim as they cross over the iliac vessels and is typically medial to the ovarian vessels. The dissection is carried downward toward the uterine arteries but keeping at least 2 cm away from the parametrium. The ureters are ligated, tagged distally as they serve as a marker of the uterine artery location, and identified proximally and moved out of the way for the kidney transplant surgeons.

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Fig. 16.1  Incision for adequate exposure

16.2.2 Step 2: Ligation of the Round Ligaments and Dissection of the Bladder (Fig. 16.3) The round ligaments are ligated and retroperitoneal space completely opened and the loose areolar tissue identified. The round ligament on the uterus is tagged for orientation. It is very important that the parametrium with the uterine vessels is never opened but kept attached to the uterus. The dissection is carried anteriorly where the bladder peritoneum is dissected extensively downward to identify the anterior vagina.

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Fig. 16.2  Ureters have been dissected and transected distally

Fig. 16.3 Round ligaments have been ligated and ovarian vessels ligated

16.2.3 Step 3: Dissection of the Utero-Ovarian and Ovarian Vessels (Fig. 16.3) The dissection of the loose areolar tissue is carried cranially from the ligated round ligaments in such a way that the entire vascular supply of the ovary is left attached to the uterus. This pedicle can serve as an additional venous return if needed. The ovary is removed and left within the body cavity.

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16.2.4 Step 4: Identification and Ligation of the Obliterated Umbilical Arteries (Fig. 16.4) The lowermost identifiable structure of the specimen will be the obliterated umbilical arteries. These are ligated distally at the level of the bladder. They are dissected proximally toward the parametria. This vessel will lead to the branching of the uterine artery from the anterior trunk of the internal iliac artery. However the uterine arteries should not be dissected into the parametria but become part of the parametrial tissue.

16.2.5 Step 5: Dissection of the Internal Iliac Vessels (Fig. 16.5) The dissection is started at the bifurcation of the common iliac vessels. The internal iliac artery and vein will be harvested from just distal to the bifurcation. The dissection of the artery proceeds caudad. The gluteal, obturator, and inferior rectal branches of the internal iliac artery are ligated or oversewn on the side of the specimen to avoid bleeding after reperfusion. The identification of these vessels is relatively easy. Lateral are the branches of the internal iliac vein. These are more easily injured so dissection is slower. Meticulous identification and ligation is required. These vessels are dissected toward the parametrial mass, but it is important to keep the parametrial mass intact because of the extensive branching. The vascular pedicle is ready for ligation but not performed until after cross clamping.

Fig. 16.4 Obliterated umbilical artery has been ligated

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Fig. 16.5  Internal iliac artery and vein dissection

16.2.6 Step 6: Dissection of the Recto-Vaginal Space (Fig. 16.6) Dissection of this space is started in the midline posteriorly and proceeds caudally until well below the cervix. It is important that the sacrouterine ligaments are preserved from some lengths on the uterus. They are tagged and form the lowermost posterior margin. This ligament will be used for support at transplantation. An umbilical tape is placed around the vagina. At this time the other teams return and finish their procurement.

16.3 Completing the Procurement The donor is heparinized, and arterial cannula is placed in one external iliac artery near the inguinal ligament. The aorta is clamped for core cooling above its bifurcation so that pelvic flush is effective. The contralateral iliac artery is clamped in order to vigorously cool the uterus. After completion of the other teams and with the uterus still under cold ischemia, the vagina is transected and the internal iliac vessels are clamped and ligated. If the graft needs to be transported, it is sealed with a surgical stapler or running suture. The graft will include the uterus with the vault of the vagina, the bilateral internal iliac arteries and veins, as well as the superior uterine/ovarian vessels, tagged round ligaments, tagged ureters, tagged utero-sacral ligaments, and internal iliac vessels (Figs.  16.7 and 16.8). An angiogram of the specimen reveals excellent vascular supply (Fig. 16.9a, b).

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Fig. 16.6  Dissection of the recto-vaginal space

Fig. 16.7  The final specimen includes transected ureter, tagged round ligament, tagged obliterated umbilical artery, and internal artery and veins. Notice the ovaries were removed from the final specimen. The bubble area is a zone that should not be dissected so as not to damage small veins and arteries

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Fig. 16.8 Completed specimen

Fig. 16.9 (a, b) Angiogram of completed specimen showing intact parametrial vessels

a

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References Flyckt RL, Farrell RM, Perni UC, Tzakis AG, Falcone T. Deceased donor uterine transplantation: innovation and adaptation. Obstet Gynecol. 2016;128(4):837–42. Flyckt R, Kotlyar A, Arian S, Eghtesad B, Falcone T, Tzakis A. Deceased donor uterine transplantation. Fertil Steril. 2017a;107(3):e13. Flyckt R, Davis A, Farrell R, Zimberg S, Tzakis A, Falcone T.  Uterine transplantation: surgical innovation in the treatment of uterine factor infertility. J Obstet Gynaecol Can. 2017b;15:1701–2163. Ozkan O, ErmanAkar M, Ozkan O, Erdogan O, Hadimioglu N, Yilmaz M, et al. Preliminary results of the first human uterus transplantation from a multiorgan donor. Fertil Steril. 2012;99:470–6. Testa G, Anthony T, McKenna GJ, et al. Deceased donor uterus retrieval: a novel technique and workflow. Am J Transplant. 2018;18:679–83.

Surgical Technique in Preparation of Recipient

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Janusz Marcickiewicz and Mats Brännström

17.1 Timing of Surgery and Incision In both deceased donor (DD) and live donor (LD) uterus transplantation (UTx), the uterus recipient should be surgically prepared so that the uterus can be brought into the pelvis of the recipient, as soon as the uterine graft is properly flushed and prepared during the back-table procedure. This will minimize the cold ischemic time of the organ, which in a typical LD situation is around 1 h and in a DD situation may be up to 6–8 h. The uterus is at a low temperature when brought into the pelvis of the recipient, and this cooling of the organ has been accomplished by uterine flushing with chilled preservation solution and then submersion of the uterus into ice slush at back-table. The uterus should be brought into the recipient’s pelvis with the uterine fundus wrapped inside a gauze, which have been soaked with cold preservation solution. This exercise will slow the gradual warming of the uterus during anastomosis surgery, which usually takes about 1–1.5 h. This period is referred to as the warm ischemic period in the recipient, or second warm ischemic period, and this is less harmful for the tissue as compared to the first warm ischemic period in the donor, when the organ has the body temperature, with a high metabolic rate, from the initiation of ischemia. The surgical plan, after entrance of the abdominal cavity, will vary between patients with no uterus from birth, as the Mayer-Rokitansky-Küster-­Hauser (MRKH) J. Marcickiewicz Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden e-mail: [email protected] M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_17

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syndrome, and those with existing uterus or previous hysterectomy. A great majority of the around 60 UTx procedures that up until mid-­2019 have been performed, are with MRKH women as recipients. In order to synchronize LD and recipient surgeries, and to avoid a long cold ischemic period of the grafted uterus, the surgery of the recipient should be initiated in a separate operating room, located close to the operating room for LD surgery. By our experience, the preparation of the recipient takes about 1 h, from first skin incision until all tissues are dissected, cleared, and with fixation sutures placed at the key locations for uterine fixation. However, to minimize cold ischemic time it would be advisable to start the recipient surgery around 0.5 h before predicted vascular clamping with subsequent retrieval of the uterus. In a LD situation, where presurgery assessment of the quality and diameters of the uterine arteries have not been totally convincing concerning acceptable uterine artery quality, it would be advisable to keep the recipient under anesthesia but not to start the surgery of the recipient until it is evident, by the results of back-table flushing, that the perfusion of the uterine graft is acceptable. In a DD situation, with much more uncertainty of the quality of the uterus and its vascularity, the decision to start with recipient surgery has to await results of flow at back-table flushing but also results of the specific investigations that may take place on the back-table to exclude pathological conditions of the uterine cervix and the endometrium. These investigations may include colposcopy of the portio, hysteroscopy and histological analysis of any tissue biopsy. The laparotomy surgery of the recipient can best be performed through a sub-­ umbilical midline incision. An indwelling urinary catheter has been placed before. The skin incision should go all the way down to the pubic bone in order to provide good access for the subsequent vaginal-vaginal anastomosis. Care should be taken to minimize bleeding during incision through the layers of the abdominal wall, since any bleeding may disturb the surgical field for the following complex surgery. The peritoneal cavity should be carefully inspected and any adhesions dissected and cleared. A retractor system, with a field post attached to the operating table and a frame with possibility to attach multiple and flexible retractors, should be used to allow maximum exposure during the critical elements of the UTx procedure, such as vascular anastomoses and vaginal attachment.

17.2 Surgical Technique in the Mayer-Rokitansky-Küster-­ Hauser Patient The MRKH patients that will undergo UTx may, some years prior to UTx, have undergone diagnostic laparoscopy as the only surgical procedure of the pelvic region and none or only minor adhesions will be present. Preoperative imaging has given information about positions of kidneys and if ureters are present unilaterally or bilaterally. There is no need for stenting ureters in order to visualize them during surgery, since all dissections will be far away from their anatomical position. The ureters can be easily palpated in case the surgeon has to ascertain their positions.

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A typical anatomic situation of a MRKH patient is presence of an elongated uterine rudiment just cranial and posterior to the top of the bladder. There will also be uterine-like tissue on the pelvic sidewalls and these are usually connected to the round ligaments. Typically, fairly large arteries, most likely remnants of the uterine arteries, enter this uterine-like tissue buds on each pelvic sidewall. The ovaries and the connected oviducts are typically situated at a more cranial position than the normal anatomical position and in around half of MRKH women they are positioned laterally to the external/iliac arteries. In a typical MRKH pelvis, the sacrouterine ligaments are easily identified by upward traction of the uterine rudiment. The first step of the surgery is to identify the vaginal vault and to dissect this free from the bladder, which characteristically covers the top of the vagina. We use a sacrocolpopexy probe, constructed of a metal shaft and a silicon sphere at the distal end, in order to move the vaginal vault upward and to indicate the position of the vaginal top to the surgeon and the assisting surgeon. Ideally, a second assisting-­ surgeon, positioned between the legs of the patient, holds the vaginal probe. In many MRKH-women, the longitudinal elasticity of the vagina is less than that of a non-MRKH patient and the functional length of the vagina is relatively short. It is also important that the assisting surgeon ascertains that the probe is placed inside the vagina and not mistakenly inside the rectum, which we have experienced at recipient surgery. The confirmation of a correct position of the probe can be done by rectal palpation, just after placement of the vaginal probe. Pressing of the probe cranially allows identification of the direction for dissection of the vaginal vault, even if the vault typically is fully covered by the bladder. The edge between the frontal aspect of the uterine rudiment and the bladder can typically be found after incision of the peritoneum and fine dissection. The bladder visualization can be aided by filling the bladder with 100–150 mL saline. The bladder is then gently dissected free from the frontal aspect of the vaginal vault using mainly monopolar electrocautery. For full exposure of the vaginal vault, the rudiment uterine tissue has to be cleaved in the midline. The cleavage can be done by monopolar electrocautery and the distance for cleavage varies between 2 and 4 cm, and in some cases also a small rudimentary uterine cavity, that may contain fluid, will be reached. Although monopolar diathermy is used for cleavage, there will be some additional bleeding from the medial aspects of the cleaved rudiment and hemostasis with bipolar diathermy or sutures may be needed. Usually restricted dissection is also needed on the posterior aspect of this rudiment and then care has to be taken to avoid injuries to the rectum, which may be attached fairly high on the posterior aspect of the vaginal vault. An area of about 50 mm in longitudinal direction and about 40  mm in transverse direction should be freed from bladder and rectum and only the fascia covering the vaginal vault should remain. The vagina is then considered fully prepared for later opening and immediate vaginal-vaginal end-to-end anastomosis. We prefer to delay the opening of the vagina until accomplishments of vascular anastomoses with the organ well reperfused. Then the time of open communication between the sterile milieu inside the abdomen and the vagina, with its bacterial content, will be minimal. To allow easy orientation and identification of the vaginal vault at later opening of the vagina and also in order to

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make the anastomosis surgery easier, two stay sutures can be placed in fascia of the anterior and posterior aspect of the vaginal wall clearing. The next step is to identify and place fixation sutures in the structures that will support the anatomical position of the uterus and also prevent prolapse or torsion of the uterus after transplantation. We place non-absorbable monofilament sutures (1-0 polypropylene) in the sacrouterine ligaments, the round ligaments, and in the bisected uterine rudiments for structural support. The sutures on the round and sacrouterine ligaments should be placed as distal as possible in these ligamentous structured, in order to enable effortless fixation when the uterus will take a considerable space in the pelvis. The sutures in the bisected midline, uterine rudiment will, at transplantation, be linked to the lateral aspects of the lower cervix, and will by that anatomical position be similar to the cardinal ligaments. The needles of the polypropylene fixation sutures should remain on the threads since stitches will be placed on the corresponding parts of the uterine graft. The sacrouterine sutures are pulled over the cranial aspect of abdominal incision and the other sutures are pulled over the caudal aspect of the incision, to enable uncomplicated fixation after anastomoses of the blood vessels and vagina have been performed. The surgery is then directed toward preparation of the external iliac vessels for subsequent anastomosis. The iliac artery and vein should be cleared separately for a distance of around 5–7 cm. Rubber slings should be placed around each vessel, as aids for vascular clamping and anastomosis surgery. The bilateral uterine rudiments (buds) on the pelvic sidewalls, typically attached to the round ligaments, in many MRKH cases, may be in an anatomical place so that they will disrupt the natural, slightly curved direction of the vascular pedicle from the graft to the external iliacs. This unobstructed passage is especially important for the veins, with their thin vessel walls and with a vascular low-pressure system to reach the external iliac veins low in the pelvis. Accordingly, the round ligaments are generally cleaved immediately lateral to the pelvic lateral rudiments, which are then dissected free and repositioned to a more cranial and lateral position.

17.3 Surgical Technique in Non-MRKH Patient There are two typical scenarios with non-MRKH patients that may become uterus recipients. There are recipients with previous hysterectomy and those with a present uterus that will be removed at the same surgical session as the UTx procedure. The most common causes of previous hysterectomy are cervical cancer, postpartum bleeding, and large leiomyoma. In cervical cancer, hysterectomy has been radical to also include the upper vagina and the accompanying lymph node dissection may give rise to lymphocele and extensive adhesions. In the benign causes of hysterectomy, and typically in the emergency postpartum hysterectomy, a subtotal hysterectomy, with preservation of the uterine cervix, is usually performed.

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In the totally hysterectomized uterus recipient, the dissection will essentially follow that of the MRKH patient as described above, but with minor modifications. Adhesions and abnormal positions of ovaries, after previous surgery, may present difficulties when the pelvic region is surgically exposed. The dissection of any uterine rudiment, as in a MRKH patient, is not needed and this would make it easier to accomplish a satisfactory exposure of the vaginal vault. The low and lateral fixation sutures that in a MRKH patient is typically placed in the medial aspects of the cleaved uterine rudiment, should in the hysterectomized patient be placed through the fibrous tissue lateral to the top of the vagina. In this step, it is important to assure that these sutures are not placed in close proximity to the ureters. The vagina should also in these recipients be opened after satisfactory uterine reperfusion has been established. In a patient that has undergone subtotal hysterectomy, here will most likely not be any useful vessels in the cervical stump for subsequent arterial or venous anastomoses to a uterine graft. Moreover, anastomosis of a graft cranial to a present cervical stump will most likely lead to problems with cervical stenosis, which can render problems at embryo transfers. Thus, the cervical stump should be removed as part of the surgery. We have no personal experience in this but suggest that the cervical stump can be cleaved like the uterine rudiment in a MRKH-patient and then later be used in placement of the low and lateral fixation sutures. Care should be taken not to enter the vagina during this bisection since the vagina should be opened after accomplishment of anastomoses and organ reperfusion. If the cervix is bulky, and possibly can make anastomosis surgery more difficult or prevent flow in the vascular pedicles after anastomosis, the cranial and medial parts of the cervix could be resected during the preparatory surgery. In a patient that has a full nonfunctional uterus, such as a patient with severe intracavitary adhesions (Asherman’s syndrome), the hysterectomy would be performed as part of the preparatory surgery of the recipient. We have no personal experience in this but a suggestion is that the uterine arteries and deep uterine veins, at a level under the ureter should be dissected and identified as possible vascular connections during the subsequent UTx procedure. These vascular ends can be clamped with bulldog clamps and the hysterectomy could proceed to perform a subtotal hysterectomy and not open the vagina. Steps will then follow what is reported above for the subtotally hysterectomized women.

Back-Table Preparation and Flushing of the Uterus

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Niclas Kvarnström and Mats Brännström

18.1 Introduction Back-table preparation in the context of uterus transplantation (UTx) is defined as the procedure when the uterus is prepared ex vivo, following procurement and prior to transplantation. The primary focus of the back-table procedure is flushing and cooling of the uterus to change the state of warm ischemia of the organ into cold ischemia. After the organ has been properly flushed and cooled, the uterus has to be prepared for transplantation and reperfusion in the recipient. This preparation can include reconstruction of the vascular tree that will be used at transplantation, sealing of open vessels, and removal of excess donor tissue. Although the uterus is an organ that tolerates several hours of cold ischemia, the back-table procedure, after proper flushing and cooling of the uterus, should be kept as short as possible. Moreover, in order to minimize the cold ischemic time on the back-table and warm-­ ischemia at transplantation, meticulous and precise dissection when harvesting the uterus should be performed, since excessive tissue, especially adjacent to the vascular pedicles, may impede and slow down the back-table preparation as well as the anastomosis surgery.

N. Kvarnström Department of Transplantation, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden e-mail: [email protected] M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_18

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18.2 Preparations of the Back-Table Before the Procedure It is important to have the back-table well prepared before the uterus comes out from the donor, in order to minimize initial warm ischemia. The sterile back-table should be located in the same operating theater as of organ procurement for fast delivery of the uterus from the pelvis of the donor to the back-table. The table should have a large container with ice slush of frozen saline, solutions for flushing, several flushing cannulas of different dimensions, ties, sutures (4-0 to 8-0 polypropylene), clips, bulldog clamps, as well as instruments for small vessel surgery. In our experience, the back-table preparation is by preference performed by two surgeons (one transplant surgeon and one gynecology surgeon) and one experienced operating nurse. The surgeons should be those that have been responsible for major parts of the vessel dissection in the donor, so that they are familiar with the specific vascular anatomy of the graft. It is important to have good illumination, if possible both by an operating lamp and a headlight, in order to be able to fully identify and dissect the small vessels. The two surgeons should work with the aid of magnifying loupes.

18.3 Back-Table Procedures The procured uterus, wrapped in a soaked operating gauze and in a kidney dish with ice slush, should be brought to the back-table immediately after clamping of vessels and transection of the major vessels (most often the anterior divisions of the internal iliac arteries, the proximal parts of the utero-ovarian veins, and bilateral segments of the internal iliac veins) in the donor. The uterus is then either kept in the kidney dish or placed in another basin with ice slush, with the anterior surface facing upward so that the internal iliac arterial ends and the uterine arteries are well exposed. The anterior surface of the uterus can easily be identified by the large, frontal peritoneal flap of the bladder. Care should be taken to avoid direct contact between the organ and ice. The free pedicles of the bilateral arterial tree should be fully visualized, and it is important to make sure that the pedicle is not twisted. We have usually placed long (2–3 cm) ligations in the umbilical artery, so that these vessels can easily be identified to aid in the orientation of the vessel. We first flush through the arterial side with a solution of saline plus lidocaine and with added heparin through the arteries on both sides. This can be done simultaneously with one surgeon doing each side. This pre-­flushing solution will help to dilate the vessels. This pre-flushing is done by two handheld syringes (10 mL) and flushing cannulas, which should fit the open ends of internal iliac parts of the arteries. If this is not possible, the uterine artery must be cannulated with the risk of damaging the intima. The venous ends are observed to see that blood flows out on both sides. The chilled preservation solution is then administered through an infusion-set coupled to two cannulas, with possibility to apply pressure on the bag. In the

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initial Swedish UTx trial (Brännström et al. 2014) and in the Brazilian deceased donor case (Ejzenberg et al. 2019), histidine-tryptophan-ketoglutarate preservation solution was used. However, any organ perfusion solution approved for kidneys may be used for uterus flushing, although solutions with high viscosity could be more difficult to flush with since the resistance to flow of the uterus is great. We usually do not apply pressure to the bag with preservation solution, which is instead elevated around 1 m above the back table. The flushing rate will initially be low but will increase by flushing time. External pressure on the bag may be needed initially, but the organ should be appropriately perfused without this extra pressure after a while. In order to open up the vascular tree, flushing of around 200 mL on each side is needed. Concerning the arteries used for anastomosis, the adjacent parts of the internal iliac artery may be shortened but preferably not cut down to a cuff. Remaining branches of the internal iliac artery should each be secured by a suture ligature, placed over any ligations performed in situ. Failure to achieve good perfusion may also indicate need for arterial reconstruction, and this may be successful in case only the first part of the uterine artery is damaged or if there is insufficient internal diameter in proximity to the origin of the artery. There is one published case, where minimal flow through the uterus was seen, although very high pressure was applied (Brucker et al. 2018). This latter case was aborted on the back-table, and the organ was never transplanted. Histopathological analysis showed massive intimal hyperplasia and a minimal lumen of the uterine artery on both sides. When the uterus is cooled down and it is full cold ischemia, the next step is to decide which veins that should be used on each side. It is critical to keep several options for vein anastomosis open if possible. This decision is based on the qualities, lengths, and perfusion flow rates on back-table. For evaluation and identification of the dominant vein outflow on each side, bulldog clamps may be used in sequence on different veins during the perfusion. One or two veins on each side are preferably used for anastomosis. Two veins on one side may on the back-table be connected into one venous outflow part if it is technically possible. This will reduce the numbers of anastomosis sites in the recipient. Back-table cleaning of the utero-­ ovarian vein and proximal part of the uterine vein is hazardous as the vessels may be branched and are very thin walled. The parts of the adjacent internal iliac veins, with thicker vessel wall, may be cleaned. The time for back-table preparation was specified to 1.5  h in the Brazilian deceased donor UTx case (Ejzenberg et  al. 2019). In the original Swedish study (Brännström et al. 2014), including nine patients with organs from live donors, the mean cold ischemic time was 78  min (range 54–180  min), and in the Brazilian deceased donor case, the cold ischemic time was 400 min (Ejzenberg et al. 2019). Times for cold-ischemia and back-table preparation are not given in other relevant studies.

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References Brännström M, Johannesson L, Dahm-Kähler P, et al. The first clinical uterus transplantation trial: a six months report. Fertil Steril. 2014;101:1228–36. Brucker SY, Brännström M, Taran FA, et  al. 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. Ejzenberg D, Andraus W, Baratelli Carelli Mendes LR, et al. Livebirth after uterus transplantation from a deceased donor in a recipient with uterine infertility. Lancet. 2019;392:2697–704.

Surgical Technique for Vascular Anastomosis of the Uterine Graft

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Michael Olausson and Niclas Kvarnström

19.1 Introduction The objective of this chapter is to describe the surgical technique for the vascular anastomoses in uterus transplantation (UTx) and some of the complications that may occur. Solid organ transplantation is to a large part vascular surgery, based on techniques described during the early 1900. Alexis Carrel (1907) developed many of the organ transplantation modalities still used until this day, an achievement that resulted in the Nobel Prize in Physiology or Medicine in 1912. The surgical technique includes choosing a relevant site for the placement of the donor organ. This will largely determine how one will design the anastomoses and what vessel of the recipient to use.

19.2 Site of Uterus Transplantation A transplanted organ can either be placed heterotopically or orthotopically. A heterotopically placed organ is transplanted in a non-anatomic position, while an orthotopic site is the anatomic location. An example of the former is kidney transplantation, which in most cases are transplanted to the pelvic region instead of the normal anatomical position. This makes the recipient operation much easier and carries less risk for the patient than trying to position the graft close to the aorta and vena cava. This has no negative bearing on the graft or recipient survival. In some cases, the kidney can be placed orthotopically—like in small children receiving an adult kidney, in which case the M. Olausson (*) · N. Kvarnström Department of Transplantation, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_19

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graft has to be transplanted in the abdominal cavity instead of retroperitoneally as the standard location. An example of an orthotopically placed organ transplant is a standard liver transplantation. Even in cases where only part of the liver is transplanted, the most common placement is an orthotopic position. As in the case of kidney transplantation, there are circumstances where a heterotopic position has been chosen, but is rare. Another example is multivisceral organ transplantation, where sometimes the kidney is kept together with a multi-organ graft. Uterus transplantation in the experimental situation has been carried out using both techniques, but in nonhuman primates, an orthotopic position was chosen (Johannesson et al. 2013). In humans the decision concerning where to place the organ was based on anatomical studies in nonhuman primates and humans (Enskog et al. 2010; Johannesson et al. 2012). To be exact, the position is a mix between the methods. For practical reasons, the uterus is positioned in the pelvis with anastomosis to the vagina to facilitate future embryo transfer, while the vascular anastomoses are performed to the external iliac vessels (Fig. 19.1) instead of the technically more demanding internal iliac vessels. The template for the vascular anastomoses mainly follows that of kidney transplantation.

19.3 The Venous Anastomoses The veins in uterus transplantation (UTx) are connected to the external iliac veins on both sides. Depending on the number of veins and their quality and size, this may include advanced reconstruction options on the back-table before the implant can proceed. Ideally, good quality uterine veins on a cuff from the internal iliac veins are present. These then can be sutured end-to-side to the external iliac veins (Fig. 19.2). This is the standard technique in renal transplantation. The anastomoses are

Fig. 19.1  External iliac vein and artery exposed before starting the anastomoses. (Photo Jiri Fronek, IKEM Prague)

19  Surgical Technique for Vascular Anastomosis of the Uterine Graft Fig. 19.2 (a) End-to-side venous anastomosis between the uterine vein of the donor and the external iliac vein of the recipient. (Photo Lennart Wiman, Gothenburg). (b) End-to-side venous anastomosis between the uterine vein and the ovario-uterine vein of the donor and the external iliac vein of the recipient. Both veins used on each side. (Photo Jiri Fronek, IKEM Prague)

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typically sutured with a running suture, using 6-0 or 7-0 monofilament, followed by opening the iliac veins after clamping with a bulldog on the organ side. In some instances, the uterine veins are multiple, in which case a longer patch or a segment of the internal vein must be retrieved. This part can be quite demanding and difficult, as pointed out in another chapter. Not seldomly, the resulting veins also have poor quality, being thin-walled. Great caution must be taken not to tear the veins during the venous anastomoses. Use of fine quality sutures 7-0, or sometimes 8-0, may be required. The presence of valves in the internal iliac as described by Caggiati (2013) must be explored when using whole segments of internal iliac branches. Another surprise may be small diameter uterine veins. In this case, you can use the proximal part of the ovarian-uterine vein—either after performing a back-table reconstruction, or together with the uterine vein using two separate anastomoses (Fig. 19.3) or, in case of an unusable uterine vein, alone as a substitute for the uterine vein. Although the proximal part of ovarian-uterine vein,

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Fig. 19.3  Uterus graft from a deceased donor including upper ovarian veins as well as caval vein and aortic artery conduits. (Photo Lennart Wiman, Gothenburg)

before the inlet from the ovary usually is short, it may be long enough to make a decent anastomosis without tension. Consent for removing the ovary on one or both sides may facilitate the dissection and help achieving appropriate length of the vein. Great care must be taken not to dissect this vein from surrounding tissue, since it often divides into “micro” branches of collateral veins with small holes on the intimal side as a result. In nulliparous women the ovarian-uterine veins can be of very small diameter and not suitable for vascular anastomosis. Having four vessels to choose from—two on each side—is the preferred way, although it may be tempting to avoid dissecting the deep uterine veins. Even if this has been successfully performed, it will not work in all cases. As in all solid organ transplantation, venous outflow is paramount for a successful transplantation. Obstruction will inevitably lead to vascular thrombosis. In deceased donor transplantation, you may use the entire ovarian-uterine veins, theoretically with patches on their inlets into the cava on the right side and renal vein on the left side. This is usually not an advantage, since the resulting vessels will be too long, thus susceptible to twisting and compression from the outside. Furthermore, the upper part of the ovarian veins are very often multiple and thin walled. For the deep uterine veins, a deceased donor allows for a much better segment or patch of the internal iliac vein than otherwise possible to retrieve from a live donor.

19.4 The Arterial Anastomoses The uterine artery branches off from the internal iliac artery and the obliterated umbilical artery. As pointed out in the chapters concerning surgery of donors, this junction is a major landmark in the dissection of the donor. Before the implant, you

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have to carefully assess the uterine artery, since preoperative imaging is not 100% sufficient to rule out that the uterus may be unsuitable for transplantation. Several cases have been aborted before transplantation due to this fact. It is common that the initial flush of preservation solution on the back-table is slow, but after 10–15 min, the flow usually improves. If this is not the case, there may be a problem with the artery of the graft. Angiogram on the back-table might further diagnose this, but usually the color of the uterus (should be whitish) and the flow from the veins are sufficient signs to determine on an adequate arterial system. A problem that may occur is a thick intima at the junction of the uterine and the internal iliac artery. The partial obliteration of the artery may cause this thickening. An obstructed lumen of the uterine artery may be reconstructed on the back-table, although this sometimes can be avoided by good quality preoperative imaging, in case of a live donor. As spare parts, you can use small branches from the internal iliac artery from either side, or an allogeneic vascular graft from another donor. Such grafts are usually present in any large size transplant center and may prove critical to the outcome of the transplantation. Ideally and in most cases, the uterine artery will be present on a cuff or a segment of the internal iliac artery. The size of this cuff is comparable with a renal artery from a live kidney donor. The anastomoses are typically sutured with a running suture, using 6-0 or 7-0 monofilament (Fig. 19.4) and clamped bilaterally with a bulldog on the organ side.

19.5 Reperfusion Depending on the timing of the donor and recipient operation, the vascular procedure usually takes 1–2 h. During the procedure, the uterus should be kept as cool as possible in order to avoid warm ischemia. When all four anastomoses are completed, the uterus is re-perfused after removal of the bulldog clamps on both artery and vein on one side first, followed by the other Fig. 19.4 End-to-side arterial anastomosis between internal iliac artery of the donor and the external iliac artery of the recipient. (Photo Lennart Wiman, Gothenburg)

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Fig. 19.5 Completed anastomoses before reperfusion. (Photo Jiri Fronek, IKEM Prague)

side without delay (Fig. 19.5). The pale, whitish uterus should immediately return to its normal color, and there should be observed a small amount of bleeding from the vaginal cuff. The blood flow should be assessed early by using devices such as a flowmeter, a power Doppler, a fluorescence camera (with ICG), or a heat camera.

19.6 Complications The most frequent complication in UTx so far is the vascular thrombosis of the artery and the vein (Brännström et al. 2014; Testa et al. 2017). The three large centers so far (Gothenburg, Prague and Dallas) all report either arterial or venous thromboses. Possible reasons for arterial thromboses are arteriosclerosis or otherwise vascular lumina of small diameter lumen. To avoid performing UTx with a donor organ with arteriosclerosis, you can perform preoperative imaging using either computer tomography angiography (CTA), magnet resonance angiography (MRA), or conventional selective angiography (DSA). Ongoing studies from the Gothenburg group will shed light on what modality is best and in what sequence the methods should be used. The small diameter of the uterine artery makes the imaging a challenge. Another reason for arterial thrombosis is a narrow anastomosis. By using fine sutures in combination with magnification loupes or surgical microscope, you can perfect the technique to minimize iatrogenic reasons for failure. Venous thromboses are another cause for early failure. The reasons are often poor quality of the veins due to varices, but outflow obstruction due to small diameter or rotation may occur. As mentioned above, this part of the procedure can prove to be very challenging. Reconstructions of the veins add to complexity and the risk of thromboses, emphasizing the need of proper anticoagulants post-surgery. Furthermore, like in all solid organ transplantations, one should put particular emphasis to the venous outflow since any obstruction in a low flow system like the uterus transplantation would increase the risk of thromboses.

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Apart from thromboses, hemorrhages are well-known complications in any solid organ transplantation. This was also reported in one of the cases performed in the USA. Early bleedings are easily discovered and usually do not result in any major problem, while late bleedings are devious and difficult to recognize. In one patient, bleeding was caused by a fungus infection, which is a dreaded complication in kidney, pancreas, and liver transplantation. To prevent this from reappearance, all programs now include fungus prophylaxis at the time of UTx.

References Brännström M, Johannesson L, Dahm-Kähler P, Enskog A, Mölne J, Kvarnström N, Diaz-Garcia C, Hanafy A, Lundmark C, Marcickiewicz J, Gäbel 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. PMID: 24582522. Caggiati A. The venous valves of the lower limbs. Phlebolymphology. 2013;20(2):87–95. Carrel A. The surgery of blood vessels. Johns Hopkins Hosp Bull. 1907;18(190):18–28. Enskog A, Johannesson L, Chai DC, Dahm-Kähler P, Marcickiewicz J, Nyachieo A, Mwenda JM, 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. PMID: 20519250. Johannesson L, Diaz-Garcia C, Leonhardt H, Dahm-Kähler P, Marcickiewicz J, Olausson M, Brännström M.  Vascular pedicle lengths after hysterectomy: toward future human uterus transplantation. Obstet Gynecol. 2012;119:1219–25. https://doi.org/10.1097/ AOG.0b013e318255006f. PMID: 22617587. Johannesson L, Enskog A, Mölne J, Diaz-Garcia C, Hanafy A, Dahm-Kähler P, Tekin A, Tryphonopoulos P, Morales P, Rivas K, Ruiz P, Tzakis A, Olausson M, Brännström M. Preclinical report on allogeneic uterus transplantation in non-human primates. Hum Reprod. 2013;28:189–98. https://doi.org/10.1093/humrep/des381. PMID: 23108346. Testa G, Koon EC, Johannesson L, McKenna GJ, Anthony T, Klintmalm GB, Gunby RT, Warren AM, Putman JM, dePrisco G, Mitchell JM, Wallis K, Olausson M. Living donor uterus transplantation: a single center’s observations and lessons learned from early setbacks to technical success. Am J Transplant. 2017;17(11):2901–10. https://doi.org/10.1111/AJT.14326.

Fixation of the Uterine Graft After Uterus Transplantation

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Mats Brännström and Pernilla Dahm-Kähler

20.1 Introduction The uterus is held in its normal anatomical position by several ligamentous structures. These structures and the muscle layer of the pelvic floor will prevent the uterus from descending into the vagina and eventually from protruding from the introitus of the vagina. In the first case of human uterus transplantation (UTx) that was performed in year 2000 (Fageeh et al. 2002), the recipient experienced uterine prolapse after around 3 months post UTx. The uterus was then partly necrotic and with thrombotic uterine arteries and veins. The authors and surgeons speculated that the prolapse of the uterus had caused bending of the uterine vessels and secondary to that impaired blood flow that caused thrombus formations. The surgical technique used was with no fixation of the uterus, apart from the connections between blood vessels and between the cervix and the vaginal vault. They suggested fixation of the uterus to the inside of the abdominal wall in future cases to prevent this complication that led to graft failure (Fageeh et al. 2002). In our initial series of live donor UTx attempt in Sweden (Brännström et  al. 2014), performed in 2013, we used a more extensive fixation method as compared to the original case in Saudi Arabia (Fageeh et al. 2002). Accordingly, we did not experience any tendency to uterine prolapse in our series of seven cases that were followed for 2–5 years. The fixation method we used in 2013 and are still using in our ongoing robotic UTx trial is described in more details below. M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] P. Dahm-Kähler Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_20

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20.2 Normal Structural Support of the Uterus The uterus is held in its normal position by several ligamentous structures. The broad ligament is a sheet of peritoneum that covers the uterus on all aspects but most prominent on the latter aspect. This mesometrial part of the broad ligament wraps the iliac vessels, the uterine vessels, the ureters, and the round ligaments. The round ligament, a remnant of the embryonic gubernaculum, originates from the uterine horn and attaches to the labia majora, after passage through the inguinal canal. It is an important anatomical landmark in most types of uterine surgery by laparotomy or minimal invasive surgical techniques. The round ligaments are also present and easily identified in patients with the Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, who have been the most common patients as UTx recipients. After previous hysterectomy, these ligaments can also be found at the lateral abdominal wall, close to the inguinal ligament. The cardinal ligaments are also known as the lateral, transverse cervical ligaments. They are not prominent as distinct ligaments but are rather an area with dense collagen-rich extracellular matrix between the lateral part of the cervix/vaginal fornix and the pelvic sidewall. The sacrouterine ligaments are also named the sacro-cervical ligaments. They are two distinct ligaments that ride on each side of the rectum, to connect the lateral, dorsal aspect of the cervix to the frontal, lateral aspect of the sacrum. These ligaments are present also in patients with MRKH, but may be a little more difficult to identify in previously hysterectomized patients.

20.3 Fixation Technique Already at donor surgery, it is of importance to plan for acceptable fixation locations of the graft in the recipient. Thus, an extensive leaf of the bladder peritoneum should be harvested along with the uterine graft. Moreover, long (3–4 cm) segments of the round and sacrouterine ligaments should be preserved on the graft. The endings of these ligaments should be marked by sutures in order to have the ligaments easily identified at transplantation. The pelvic cavity of the recipient should also be prepared by placing fixation sutures (non-resorbable) at the round ligaments, in the sacrouterine ligaments, and in the cardinal ligaments/cleaved uterine rudiment. The fixation sutures of the sacrouterine ligament in the recipients should be placed cranially in the abdominal incision and the other four sutures caudally over the pubic bone, when the uterine graft is placed into position in the pelvic cavity and surgery for vascular anastomosis is prepared. The first procedure at transplantation is to accomplish the vascular anastomoses, usually minimum four end-to-side connections on the external iliac vessels. When

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appropriate reperfusion of the uterus has been secured, the larger vaginal-vaginal anastomosis will be sutured and the vagina closed early to minimize bacterial contamination. At this stage the uterus is prevented from moving or twisting, but is still very sensitive to movements that can be harmful to the delicate anastomosis lines on the vasculature. Thereafter, the fixation procedure can start with the first part to attach the sacrouterine ligaments of the recipient and graft to each other by usage of the non-­ resorbable sutures that have been previously placed in the ligaments of the recipient. During this procedure, the uterus has to be gently tipped forward to get satisfactory exposure to the rectovaginal fossa. Secondly, the cardinal ligament sutures are adapted and placed. The non-­ resorbable sutures attachment sutures may have been placed in the paracervical tissue of a patient with previous hysterectomy or in the cleaved uterine rudiment in a patient with MRKH. We attach these sutures to the graft’s vaginal rim, just above the anastomosis line of the vagina, rather than to the cervix, in order not to disturb the blood flow to the cervix. Thirdly, the round ligaments are attached to each other. This surgery is easily accomplished and can be done without any movement of the uterus. Lastly, we suture the leaf of the bladder peritoneum of the graft on top of the bladder of the recipient. The free bladder peritoneum will be about 4–5 cm broad when stretched and should be received all the way from the round ligament to the contralateral round ligament. We adapt the bladder peritoneal flap with sutures at 3–4 points close to the cervix, with sutures placed only through the superficial bladder peritoneum of the recipient. Thereafter, we suture the edge of the graft’s bladder peritoneum flap on to the frontal upper part of the bladder, so that the leaf is overlaying the top of the bladder. We believe that this peritoneal attachment is not only important for fixation of the uterus but also to prevent intestinal herniation into the cavity between the frontal aspect of the grafted uterus and the bladder of the recipient. We are aware that future modifications and simplification of this rather cumbersome fixation methodology will occur.

References Brännström M, Johannesson L, Dahm-Kahler P, et al. First clinical uterus transplantation trial: a six-month report. Fertil Steril. 2014;101:1228–36. Fageeh W, Raffa H, Jabbad H, Marzouki A. Transplantation of the human uterus. Int J Gynaecol Obstet. 2002;76:245–51.

Immunosuppression and Treatment of Rejection in Uterus Transplantation: Current Practice and Future Potential

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Matthew H. H. Young, Dawn Truong, Jana Ekberg, and Stefan G. Tullius

21.1 Introduction Although principles of immunosuppression in uterus transplantation (UTx) are largely based on the experience in solid organ transplantation (SOT), a unique set of challenges and potential opportunities require consideration (McKay and Josephson 2006; Webster et al. 2017). Some experience has been gained through UTx animal models (Castellón et al. 2017). Here we review (1), the immunosuppressive armamentarium at our current disposal, (2), examine recent immunosuppression protocols employed as induction, maintenance, and rejection treatment for UTx and (3), discuss specific aspects of immunosuppression during pregnancy. Immunosuppression is generally applied as maintenance immunosuppression usually consisting of double or triple immunosuppression after a perioperative induction treatment. Rejection treatment is usually short term and based on clinical or histological evidence.

M. H. H. Young Christiana Care Health System, Newark, DE, USA e-mail: [email protected] D. Truong Harvard University, Cambridge, MA, USA e-mail: [email protected] J. Ekberg Department of Transplantation, Sahlgrenska Academy at the University of Gothenburg, Göteborg, Sweden e-mail: [email protected] S. G. Tullius (*) Division of Transplant Surgery and Transplantation Surgery, Research Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_21

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21.2 Induction Immunosuppression Induction therapy is administered before, during, and, for a short-term period, after transplantation to ameliorate the alloimmune responses initiated after the recognition of the allograft. Immunosuppressive induction usually consists of mono- or polyclonal antibodies and an augmented dose of steroids (McKay and Josephson 2006). Induction agents are used in the majority of SOT immunosuppression protocols and offer the opportunity to withdraw corticosteroids and to lower the maintenance immunosuppression, thereby minimizing the potential adverse effects associated with these agents (McKeage and McCormack 2010). Anti-thymocyte globulin (ATG) is composed of purified polyclonal immune globulins isolated from the sera of rabbits or horses that have been immunized with human thymocytes or T-cell lines (Mohty 2007). This ATG targets a wide variety of proteins on the surface of lymphocytes (including markers such as CD2, CD3, CD4, CD8, CD11a, CD18, CD25, CD44, CD45, HLA-DR, HLA Class I heavy chains, and beta 2 microglobulin) and on other cell types (granulocytes, platelets, bone marrow cells). It may also ameliorate the response to B cells, natural killer cells, and dendritic cells. T-cell depletion is caused by complement-dependent lysis or apoptosis from T-cell activation. Although basic principles on how ATG exerts its immunosuppressive effects have been characterized, many mechanistic aspects still need to be defined (Gaber et al. 2010). The usual dosage of ATG administered prior to reperfusion of solid organ transplants (SOT) is 1.5 mg/kg iv once daily for 4–7 days in either a high flow or peripheral vein. There are, however, broad variations in dosing and timing in the UTx studies that have been published so far (Table 21.1). Patients require monitoring for cytokine release syndrome with associated clinical symptoms such as hypotension, tachycardia, and pulmonary edema. White blood cell counts (WBC) and platelets may drop rapidly and are usually monitored daily during treatment. Rarely, patients may develop serum sickness presenting up to 1–3  weeks following the last dose with symptoms including intermittent fevers, polyarthralgia, and acute renal failure. Basiliximab is a humanized and chimeric IgG monoclonal antibody that selectively binds to the α subunit of interleukin 2 receptor (IL-2R; CD25) on the surface of activated T cells, thereby inhibiting IL-2-mediated activation and proliferation of T lymphocytes (Chapman and Keating 2003). The usual dosage of basiliximab for adults is 20 mg administered perioperatively, with a second dose applied by day 4 after transplant (McKeage and McCormack 2010). While basiliximab has a comparable immunosuppressive efficacy to ATG in standard risk patients (Thomusch et al. 2016), effects appear less potent in immunological high-risk patients. Basiliximab is currently used in the Swedish robotic-assisted live donor UTx study, with six completed surgeries in 2017–2018. Compared to an induction with alemtuzumab (see below), basiliximab is associated with more frequent episodes of biopsy-confirmed acute rejections. Basiliximab was found to be as tolerable as placebo and appeared to have a favorable side effect profile compared to ATG.

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Table 21.1  Available data on immunosuppressive protocols used in uterus transplantation Immunosuppression protocols Maintenance Tac 0.2 mg/kg, MMF 2 g/day, and 20 mg prednisolone for first 12 months. MMF was discontinued and replaced with AZA at postoperative month 12. At ET: prednisolone 10 mg/day, AZA 50 mg/day, Tac 3 mg/ day – Tac twice daily Brännström – ATG 2.5 mg/kg (from day 0, average et al. 2014 (day 0, and day 1) trough level of – MMF 1 g 10 ng/mL first preoperatively 6 months and 8.5 – MP 500 mg just until pregnancy); before uterine – MMF twice daily reperfusion (from day 0; MPA AUC (target 40–60 mg/L∗h); withdrawal between month 7 and 9 or replacement with AZA (2 mg/kg per day) if multiple rejections – Prednisolone 5 mg once day 0, and then twice daily for the initial 4 postoperative days During pregnancy: – Tac average trough level 6 ng/mL – If AZA (1–2 mg/kg; depending on side effects) – Prednisolone 5 mg once daily

Reference Akar et al. 2013

Induction – ATG 2.0 mg/dL daily for 10 days, and 1 mg prednisolone

Rejection treatment – Adjustment of prednisolone and AZA dosages

Laboratory monitoring Complete blood count, liver enzymes, creatinine, blood urea nitrogen

– Borderline episode treated with 4-week taper of oral prednisolone – Mild rejection episode treated with MP 500 mg iv for 3 days, followed by oral prednisolone starting at 10 mg twice daily and tapered over 4–5 weeks. Then both AZA plus prednisolone were added (if no rejection, then TAC monotherapy was continued). If repeated or not fully resolved rejection Tac was increased by 30% and prednisolone maintained at 5 mg daily – Steroid-­resistant rejection: ATG 6 mg/kg (cumulative dose)

Tac trough levels, MPA AUC, complete blood count, creatinine, liver enzymes, glucose, c-reactive protein, urine albumin/ creatinine ratio

(continued)

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Table 21.1 (continued) Immunosuppression protocols Reference Swedish ongoing roboticassisted trial

Induction Prior reperfusion MP 500 mg and BSX 20 mg (day 0); BSX 20 mg (day 4)

Maintenance Tac twice daily, start day 0, target trough level 10 ng/mL (month 1), 8 ng/mL (month 2–3), 5–7 ng/mL onward. Month 2 switch to once daily formula AZA once daily 2 mg/ kg, start day 0 prednisolone taper from 80 mg day 1, withdrawal day 6

Testa et al. 2017

ATC 1.5 mg/kg iv every 2 days × 3

Tac 0.2 mg/kg orally daily (initial trough 7–12 ng/mL); MMF (720 mg orally twice a day) Steroid taper over 49 days

Rejection Laboratory treatment monitoring – Borderline: no As above treatment – Repeated borderline/mild rejection: prednisolone 10 mg twice daily for 2 weeks, if response taper and withdrawal (2 months) – Moderate rejection: hydrocortisone iv. 3 day course (500-250250 mg) – Severe/ steroid-resistant rejection: ATG 6 mg/kg (total dose) – Repeated rejection: addition of oral prednisolone 5 mg onward

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Table 21.1 (continued) Immunosuppression protocols Reference Flyckt, in correspondence with Cleveland Clinic

Induction Maintenance – ATG 1.5 mg/kg iv – Tac orally with a starting dose of on day 0, 2 and 4; 1–2 mg twice a day. doses adjusted for Levels adjusted with thrombocytopenia target trough level of – MP1000 mg iv 7–11 ng/mL perioperatively – Steroid taper based on plan for maintenance on steroid therapy (3 weeks–6 months) – MMF will be started orally postoperatively at 1000 mg q12h Replacement with mTOR inhibitors, if CNIs are not tolerated: sirolimus (loading dose of 5 mg × 1 and maintenance dose of 1–2 mg daily), or everolimus (1–2 mg twice daily). Medications adjusted to maintain a trough level of 7–10

Rejection treatment – Mild to moderate rejection – Steroid bolus and augmented Tac – Severe rejection—ATG and steroid bolus; and if necessary a second course of ATG

Laboratory monitoring Complete blood count with differential, Tac levels blood chemistry profile, magnesium and phosphorus levels, liver enzymes, CMV-DNA

ATG anti-thymocyte globulin, Tac tacrolimus, MMF mycophenolate mofetil, AZA azathioprine, MP methylprednisolone, AUC area under the curve

Alemtuzumab is a humanized anti-CD52 monoclonal antibody that binds to CD52, triggering an antibody-dependent lysis of T and B lymphocytes. Although there is no consensus regarding a standard dosage regimen of alemtuzumab, doses of 20–30 mg on the day of transplant surgery, with a second dose on postoperative days 1 or 4 have proven effective (Gabardi and Grafals 2011). While a meta-­analysis found no statistically significant difference in the efficacy or safety of alemtuzumab versus ATG (Zheng and Song 2017), individual studies have suggested that alemtuzumab lowers the risk of biopsy-confirmed acute rejections when compared to IL-2R antibodies. The immunosuppressive efficacy has been comparable to ATG therapy (Morgan et al. 2012), especially in low risk patients (Zhang et al. 2012). At this time, there is no clinical experience with alemtuzumab in UTx.

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21.3 Maintenance Immunosuppression 21.3.1 Calcineurin Inhibitors Cyclosporine (CyA) and tacrolimus (Tac) are the two predominant calcineurin inhibitors used in transplant immunosuppression. More recently, Tac has been used preferably in SOT, based on clinical studies suggesting a slightly more potent immunosuppressive potential (European FK506 Multicentre Liver Study Group 1994; Johnson et al. 2000; The U.S. Multicenter FK506 Liver Study Group 1994; Ahsan et al. 2001; Ekberg et al. 2007). Tac has also been the preferred maintenance immunosuppression in UTx (Table 21.1). Tac is a macrolide derived from soil fungus (Kino et al. 1987) that inhibits T-lymphocyte activation and proliferation as well as the T-helper-cell-dependent B-cell response. Patients should be dosed to achieve whole blood trough concentrations, obtained 12 h after their last ingestion of the drug, with a target range between 8 and 12 ng/mL. Side effects of Tac include nephrotoxicity, tremor, central nervous system disturbances, hair loss, and hyperglycemia. It is not known whether in utero exposure to Tac interferes with fetal development, but the agent has been deemed safe in pregnancy and breastfeeding (Webster et al. 2017). Most recently, prolonged-release Tac and novel-prolonged-release Tac have become available allowing for once-daily dosing. Several studies have demonstrated improved adherence (Gerken et  al. 2011; Doesch et  al. 2013) and less intra-subject variability in drug levels enhancing patient and graft survival. While extended-­release and twice-daily Tac have similar absorption, distribution, metabolism, and excretion characteristics, the extended-release formulations were associated with decreased maximum concentrations and delayed time to maximum concentration. Hence, on the basis of the stricter criteria for narrow-therapeutic-index drugs, the immediate-release and prolonged-release formulations are not bioequivalent [in terms of the area under the concentration–time curve from 0 to 24 h (AUC0–24) or the minimum concentration (Cmin)] and patients require an appropriate dosage modification if converted from twice-daily to once-daily regimen (Staatz and Tett 2015). Of note is that Tac has been used as a monotherapy, by some patients included in studies of UTx, in the presence of “clean” protocol biopsies. Notably, the Gothenburg group switched to Tac monotherapy prior to embryo transfer (ET) if cervical biopsies showed no rejection and in some cases with frequent rejection episodes, mycophenolate mofetil (MMF) was shifted to azathioprine (AZA) (Table 21.1). Similarly, in the Chinese robotic-assisted living donor UTx case, maintenance immunosuppression that initially started with Tac, MMF, and steroids was to be reduced to Tac monotherapy prior to ET to avoid potential fetal malformations caused by MMF (Wei et al. 2017).

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21.3.2 Antiproliferative Agents Mycophenolic acid (MPA) selectively inhibits the nucleotide synthesis pathways of T and B lymphocytes and has been most frequently used in UTx recipients prior to ET.  This antiproliferative agent comes in two variants—mycophenolate mofetil (MMF; marketed as CellCept) and enteric-coated mycophenolate sodium (EC-MPS; marketed as Myfortic). Ideally, MPA is initiated prior to transplantation (Table 21.1). MMF is administered as 1 g orally and EC-MPS as 720 mg orally twice a day (the first dose ideally given in the evening prior to transplantation, and an additional 720 mg on the morning of transplant). MMF is hydrolyzed in the liver to become MPA, the active metabolite, whereas EC-MPS has an enteric coat that dissolves at pH  >  5, supporting the absorption in the small intestine. The predominant side effects of MPA include gastrointestinal disturbances and myelosuppression. The daily dosing may be applied three times/day to reduce gastrointestinal side effects. The FDA has issued a black box warning for increased risk of first-trimester pregnancy loss and congenital malformations. Prior to embryo transfer, MPA has to be switched to azathioprine (AZA) at least 6 weeks before conception to avoid possible birth defects (Webster et al. 2017). Azathioprine (AZA) is the antimetabolite of choice after discontinuation of MPA. Mechanisms of action include the inhibition of purine nucleotide synthesis. As the fetus lacks the enzyme necessary to convert AZA into its active metabolite, there is reduced concern regarding its use in pregnancy (Le Ray et al. 2004; Webster et  al. 2017). It is important to note, that the field of UTx is rapidly changing. Immunosuppression is expected to be impacted by shorter times between transplantation and ET and the traditional 12 months period between transplantation and first ET has, by some groups, been reduced to 6 months. Most recently, AZA, instead of MMF, in combination with Tac has been as part of a dual maintenance immunosuppression after UTx. This combination allows reduced exposure to nephrotoxic Tac, as compared to Tac monotherapy, and opens earlier a window for first ET.

21.3.3 mTOR Inhibitors Mammalian target of rapamycin (mTOR) inhibitors, such as sirolimus and everolimus, are antifungal agents with immunosuppressive and antiproliferative properties. These mTOR inhibitors have been used to treat tuberous sclerosis, psoriasis, and malignancies. They are also hypothesized to have anti-ageing properties and inhibitory effects on neurodegenerative, lung, and metabolic diseases (Li et  al. 2014). Multiple trials have evaluated the use of mTOR inhibitors specifically for organ transplant recipients. When calcineurin inhibitors are replaced with mTOR

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inhibitors, there is increased risk for bone marrow suppression, dyslipidemia, infection, and acute rejection within 1 year post-transplant (MacDonald 2001; Webster et al. 2006; Lim et al. 2014). As a result of its teratogenicity and contraindication in pregnancy, sirolimus should be discontinued at least 12  weeks prior to ET and should be switched to Tac (EBPG Expert Group on Renal Transplantation 2002).

21.3.4 Co-stimulatory Blockade Belatacept, a co-stimulatory blockade agent, has been shown by some as being as effective as a CyA-based immunosuppression at preventing acute rejection after SOT (Vincenti et  al. 2017). Moreover, given that belatacept is a calcineurin-free immunosuppressive, it holds promise for sustaining long-term graft function (>10  years) in the absence of nephrotoxic side effects (Vincenti et  al. 2005). However, belatacept has also been shown to increase risks of acute rejection, graft loss, and death in SOT recipients, compared to Tac and MMF (Figs. 21.1 and 21.2). The effect of belatacept use in pregnant SOT recipients, however, is unknown (Kirk et al. 2014; Vincenti et al. 2017).

Alemtuzumab

Corticosteroids

CD 3, 2, 4, 8, 11 a

2

CD5

FK-506

ATG

CD18 25, 44, etc.

X

R

2 IL-

mTOR

FKB

PA

RA

P

CaN

BS

MMF Inflammatory Genes

IL-2 Cytokine Production

NFAT

NFxB

Cell Cycle

Nucleotide Synthesis

AZA

Fig. 21.1  Immunosuppression at the cellular level. FK-506 tacrolimus, ATG anti-thymocyte globulin, MMF mycophenolate mofetil, FKBP FK506 binding protein, RAPA rapamycin, mTOR mechanistic target of rapamycin, Aza azathioprine, BSX basiliximab, CaN calcineurin, IL-2 interleukin-­2, NFAT nuclear factor of activated T-cells, NFxB NFxB transcriptional regulator (image credit: Michelle Long, Harvard University)

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Uterine Transplant With Implanted Embryo

Uterine Transplant

Embryo Implantation

Induction: MP and ATG or BSX

Maintenance:

Maintenance:

Tac ± Aza ± pred

Tac ± MMF or Aza ± pred

Rejection:

Rejection:

MP or ATG

MP or ATG

Fig. 21.2  Immunosuppressants before and after embryo transfer, current experience. FK-506 tacrolimus, ATG anti-thymocyte globulin, MMF mycophenolate mofetil, FKBP FK506 binding protein, RAPA rapamycin, mTOR mechanistic target of rapamycin, Aza azathioprine, BSX basiliximab, CaN calcineurin, IL-2 interleukin-2, NFAT nuclear factor of activated T-cells, NFxB NFxB transcriptional regulator (image credit: Michelle Long, Harvard University)

21.3.5 Corticosteroids As part of the induction treatment in UTx, high dose of steroids is applied perioperatively. Steroids are ideally tapered rapidly posttransplantation once calcineurin inhibitor levels are in a therapeutic range. Short and long-term side effects of steroids include hyperglycemia, psychological disorders (anxiety, depression, hallucinations, delusions), hypertension, hypercholesterolemia, and increased bone metabolism leading to osteopenia and osteoporosis. Adverse effects to the fetus are rare since the drug is mostly metabolized by the placenta, although there are reports of adrenal suppression in newborns (Webster et al. 2017). Hence steroids are still the first choice treatment of rejection, either in oral or iv form, depending on rejections severity.

21.4 Dosage Monitoring and Adjustment As a result of volume expansion and placental metabolism, calcineurin inhibitor levels can fluctuate and therefore must be monitored closely to maintain adequate blood concentrations (Webster et al. 2017). Two recent studies on the use of Tac in pregnancy recommended that calcineurin inhibitor dosing be substantially increased to maintain target trough levels during gestation (Aktürk et  al. 2015; Kim et  al. 2015). However, other studies also recommend administering a consistent dose of calcineurin inhibitor before and during pregnancy, regardless of any decrease in calcineurin inhibitor trough levels (Kainz et al. 2000; Normand et al. 2017).

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21.5 Infection Prophylaxis Prophylactic antibiotic selection should take “the resident flora of the transplanted site, the prevalent bacterial flora known to cause wound infections, and the antibiotic susceptibilities at a particular institution” into account (Soave 2001). Postoperatively, P. carinii pneumonia (PCP), cytomegalovirus (CMV), and fungal infections, especially Candida albicans, are of particular concern and prophylaxis for these infections should be considered. Prophylactic trimethoprim-­ sulfamethoxazole (TMP-SMX), which has nearly eliminated PCP, is recommended for all SOT recipients for 6  months posttransplantation (Soave 2001). Prophylaxis for CMV can be achieved with aciclovir, ganciclovir, or valaciclovir, all of which significantly reduced the risk of CMV disease compared to placebo or no treatment. In direct comparisons, ganciclovir was more effective than aciclovir, while valganciclovir and iv ganciclovir were as effective as oral ganciclovir across CMV-high risk constellations (CMV-positive recipient, CMV-negative recipient/CMV-positive donor) (Hodson et al. 2005). In the Gothenburg series, valganciclovir at a 450 mg daily oral dose was given for 3 months when recipient was CMV positive, and for 6 months in a donor positive/recipient negative constellation, but no prophylaxis was given if donor/recipient were both negative (Brännström et al. 2014). Immunosuppression predisposes to certain infections. High-risk human papilloma virus (HPV) should be tested for in donors. Additionally, the Gothenburg team obtained cervical bacterial swabs in recipients at regular intervals and treated colonization by non-vaginal bacteria with short courses of vaginal antibiotics (Brännström 2015). Considering the high incidence of posttransplant fungal infections and the associated morbidity, antifungal prophylaxis is reasonable. The first deceased donor human UTx in the United States experienced a severe Candida infection that infiltrated the vasculature of the uterus and disrupted an arterial anastomosis requiring a transplant hysterectomy on around 2 weeks after UTx (Flyckt et al. 2016). In SOT patients, echinocandins (micafungin) at 50 mg/day or fluconazole 400 mg/day have been proposed as a perioperative Candida prophylaxis (Bow et al. 2010; Silveira and Kusne 2013).

21.6 Risk to Fetus All immunosuppressants cross the maternal-placental-fetal interface (Webster et al. 2017) and have been categorized based on their teratogenic potential. The categories for safety of immunosuppressants in pregnancy, as outlined by Food and Drug Administration (FDA) is outlined in Tables 21.2 and 21.3.

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Table 21.2  FDA category definitions Pregnancy category A B C

D

X

Meaning – “Adequate and well-controlled studies in pregnant women have failed to demonstrate a risk to the fetus in the first trimester of pregnancy (and there is no evidence of a risk in later trimesters)” –  “Animal reproduction studies have failed to demonstrate a risk to the fetus,” and –  “There are no adequate and well-controlled studies in pregnant women” – “Animal reproduction studies have shown an adverse effect on the fetus” – “There are no adequate and well-controlled studies in humans,” and – “The benefits from the use of the drug in pregnant women may be acceptable despite its potential risks” – “There is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans,” but – “The potential benefits from the use of the drug in pregnant women may be acceptable despite its potential risks (for example, if the drug is needed in a life-threatening situation or serious disease for which safer drugs cannot be used or are ineffective)” – “Studies in animals or humans have demonstrated fetal abnormalities,” or – “There is positive evidence of fetal risk based on adverse reaction reports from investigational or marketing experience,” and – “The risk of the use of the drug in a pregnant woman clearly outweighs any possible benefit (for example, safer drugs or other forms of therapy are available)”

Table 21.3  FDA categories for safety of immunosuppressants in pregnancy Immunosuppressant drug Calcineurin inhibitors (CNI) – Tacrolimus (Prograf) – Cyclosporine (Neoral, Sandimmune, Gengraf) Antiproliferative agents – Mycophenolate mofetil (CellCept, Myfortic) – Azathioprine (Imuran) – Sirolimus (Rapamune) – Leflunomide (Arava) Corticosteroids – Prednisone Antirejection agents – Methylprednisolone – Muromonab-CD3 (Orthoclone OKT3) – Anti-thymocyte globulin (Thymoglobulin, ATGAM) – Anti-thymocyte globulin, antilymphocyte globulin (ATGAM, ATG) – Alemtuzumab (Campath-1H) – Basiliximab (Simulect)

FDA category C C D D C X D C C C C C B

The FDA formerly provided a risk assessment for drug use in pregnancy (https://www.gpo.gov/ fdsys/pkg/CFR-2004-title21-vol4/pdf/CFR-2004-title21-vol4-sec201-57.pdf) (as of June 2015, the FDA no longer uses letter categories and has required their removal from all product labeling (https://s3.amazonaws.com/public-inspection.federalregister.gov/2014-28241.pdf))

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21.7 Challenges in Managing and Treating Rejection The Gothenburg series relied on ecto-cervical biopsies to detect early rejection and a grading system based on the histological appearance (Mölne et al. 2017). Protocol biopsies were obtained by 1, 2, and 4  weeks, and monthly thereafter with additional for cause biopsies. Most patients in the Gothenburg first series experienced some level of rejection. Borderline rejections resolved without requiring the addition of steroids. Acute rejections (≥ grade 1/mild) were seen in all but one patient, and these episodes were treated with a short-term steroid bolus or increased oral prednisolone course. Two patients experienced acute rejection during the pregnancy and these resolved with steroids. One steroidresistant rejection was successfully treated with a short-term ATG treatment. With respect to cumulative incidence of histological rejection a majority of patients (5/7) were kept on augmented maintenance, low-­dose triple treatment (Tac + AZA + steroid). However, the clinical relevance of histological rejection diagnosis remains to be explored by future studies. Of note, no graft was lost due to rejection and all babies born thus far have been healthy. Antibody mediated rejection (ABMR) remains a barrier to long-term allograft function. Acute and chronic ABMR are characterized by tissue injury, antibody, and vascular endothelium interactions, and serologic donor HLA molecules. Both are challenging to treat and associated with poorer outcomes. There are no consensus guidelines for the treatment of ABMR. For cases of acute ABMR, a combination of monoclonal antibodies, plasmapheresis, MMF, Tac, and steroids have been shown to improve therapeutic response. ATG induction therapy has been shown to reduce de-novo donor-specific antibodies, inhibit B-cell activation, and improve 1-year allograft survival. Ideally, ABMR therapies would remove existing antibodies and inhibit their redevelopment. Several uncontrolled or controlled nonrandomized studies support the use of rituximab, bortezomib, plasmapheresis, and IVIG. Eculizumab has also been used to treat ABMR, but there are no randomized controlled studies that demonstrate the efficacy of this drug. Without strong evidence to support a specific therapy, the Kidney Disease: Improving Global Outcomes Transplant Work Group recommends using corticosteroids, plasmapheresis, IVIG, anti-CD20 antibodies, and lymphocyte-depleting antibodies, either alone or in combination to treat ABMR (Djamali et  al. 2014). Notably, there may be contraindications to the usage of rituximab, especially in the second and third trimesters, due to rituximab-induced B-cell depletion in the fetus (Østensen et al. 2008). However, it has also been suggested that rituximab and eculizumab administration during pregnancy is safe, even though both agents can cross the placenta (Ton et  al. 2011). Although potentially less relevant for UTx, chronic ABMR is a leading cause of late allograft failure while standard immunosuppression therapy (CNIs, MMF, and prednisone) combined with rituximab have been shown to improve graft survival. However, given the severity and advancement of tissue damage upon the diagnosis of ABMR, immunosuppressive treatment often fails.

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21.8 Discontinuing Immunosuppression In contrast to SOT and other composite tissue transplants, UTx is the first and only type of transplant that is intended to be transient, thereby limiting immunosuppressant exposure to only a few years and decreasing the risk of long-term adverse effects, such as nephrotoxicity, diabetes, hypertension, and malignancy, as compared to other transplant populations (Brännström 2017). Conceptually, side-effects of immunosuppressive therapies are linked to the overall immunosuppressive load. Given the increase in life expectancy after SOT, the side effects of long-term immunosuppression and the possibility of discontinuation must be considered. Long-term immunosuppression in SOT has attempted substitution and reduction of immunosuppressive agents to curb metabolic disease, cardiovascular disease, renal damage, and other long-term complications. In SOT, low-dose CNI combined with MMF regimen has been shown to reduce side effects related to MMF therapy and CNI-induced nephrotoxicity. There is also evidence to demonstrate that a gradual decrease of CNIs at least 6  months post SOT, with simultaneous introduction of MMF, are effective in lowering the risk of graft rejection. CNI free immunosuppressive protocols or, alternatively, withdrawing CNIs as early as 1 months are, at least in theory, an option. However, complete withdrawal of CNI is not recommended, due to increased risk of acute and chronic rejection. At this time, there is no sufficient experience in the reduction of immunosuppression in UTx. While combination therapies can be beneficial, drug interactions need to be monitored particularly between CNIs and aminoglycosides, nonsteroidal anti-­ inflammatory drugs, angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, and/or amphotericin. Simultaneous administration of CNIs and mTOR inhibitors may potentiate nephrotoxicity; at the same time, the combination of Tac and sirolimus, may allow for the reduction of CNIs (Undre 2003; Shipkova et al. 2005; Wilkinson and Pham 2005; Creput et  al. 2007; Karie-Guigues et  al. 2009; Beckebaum and Cicinnati 2011).

21.9 Future Directions Much research has been devoted to understanding the complex interplay between fetal trophoblasts, maternal decidual immune cells, and the vast panoply of implicated immune protagonists—from T regulatory cells to uterine dendritic cells, uterine natural killer (uNK) cells, macrophages, and more (PrabhuDas et al. 2015). For example, it has been demonstrated that maternal decidual macrophages help preventing uNK cells from attacking cytotrophoblasts that facilitate the implantation of the fertilized egg into the uterine lining (Co et al. 2013). Mechanisms understanding “reproductive immune tolerance” have distinguished “local” factors including uterine sequestration of antigen-presenting cells, and chemokine-mediated gene silencing by decidual stromal cells from “systemic” factors with enhanced by

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progesterone-driven immunomodulation, and the liberation of “tolerogenic placental debris” into the maternal circulation (PrabhuDas et  al. 2015). Relevant tissue engineering efforts have also constructed uterine patches, which have been successfully grafted in mice and even supported pregnancy (Hellstrom et  al. 2016). The clinical potential of those efforts needs to be determined.

References Ahsan N, Johnson C, Gonwa T, et al. Randomized trial of tacrolimus plus mycophenolate mofetil or azathioprine versus cyclosporine oral solution (modified) plus mycophenolate mofetil after cadaveric kidney transplantation: results at 2 years. Transplantation. 2001;72(2):245–50. Akar M, Ozkan O, Aydinuraz B, Dirican K, Cincik M, Mendilcioglu I, et al. Clinical pregnancy after uterus transplantation. Fertil Steril. 2013;100(5):1358–63. Aktürk S, Çelebi ZK, Erdogmus S, et al. Pregnancy after kidney transplantation: outcomes, tacrolimus doses, and trough levels. Transplant Proc. 2015;47:1442–4. Beckebaum S, Cicinnati VR. Conversion to combined mycophenolate mofetil and low-dose calcineurin inhibitor therapy for renal dysfunction in liver transplant patients: never too late? Dig Dis Sci. 2011;56(1):4–6. Print. Bow EJ, et al. Canadian clinical practice guidelines for invasive candidiasis in adults. Can J Infect Dis Med Microbiol. 2010;21(4):e122–50. Print. Brännström M. Uterus transplantation. Curr Opin Organ Transplant. 2015;20:621–8. Brännström M. Womb transplants with live births: an update and the future. Expert Opin Biol Ther. 2017;17(9):1105–12. Brännström M, Johannesson L, Dahm-Kähler P, Enskog A, Mölne J, Kvarnström N, et al. First clinical uterus transplantation trial: a six-month report. Fertil Steril. 2014;101(5):1228–36. Brännström M, Johannesson L, Bokström H, Kvarnström N, Mölne J, Dahm-Kähler P, et  al. Livebirth after uterus transplantation. Lancet. 2015;385(9968):607–16. Castellón L, Amador A, González R, Eduardo M, Díaz-García C, Kvarnström N, Brännström M.  The history behind successful uterin transplantation in humans. JBRA Assist Reprod. 2017;21(2):126–34. Chapman T, Keating G. Basiliximab: a review of its use as induction therapy in renal transplantation. Drugs. 2003;63(24):2803–35. Co EC, et al. Maternal decidual macrophages inhibit NK cell killing of invasive cytotrophoblasts during human pregnancy. Biol Reprod. 2013;88:155. Creput C, et al. Long-term effects of calcineurin inhibitor conversion to mycophenolate mofetil on renal function after liver transplantation. Liver Transpl. 2007;13(7):1004–10. Print. Djamali A, Kaufman DB, Ellis TM, Zhong W, Matas A, Samaniego M. Diagnosis and management of antibody-mediated rejection: current status and novel approaches. Am J Transplant. 2014;14(2):255–71. Doesch A, Mueller S, Katus H, et al. Increased adherence eight months after switch from twice daily calcineurin inhibitor based treatment to once daily modified released tacrolimus in heart transplantation. Drug Des Devel Ther. 2013;7:1253–8. EBPG Expert Group on Renal Transplantation. Section IV: long-term management of the transplant recipient. IV.10. Pregnancy in renal transplant recipients. Nephrol Dial Transplant. 2002;17(Suppl 4):50–5. Ekberg H, Tedesco-Silva H, Demirbas A, et al. Reduced exposure to calcineurin inhibitors in renal transplantation. N Engl J Med. 2007;357(25):2562–75. European FK506 Multicentre Liver Study Group. Randomised trial comparing tacrolimus (FK506) and cyclosporin in prevention of liver allograft rejection. Lancet. 1994;344(8920):423–8. Flyckt RL, Farrell RM, Perni UC, Tzakis AG, Falcone T. Deceased donor uterine transplantation: innovation and adaptation. Obstet Gynecol. 2016;128(4):837–42.

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Gabardi S, Grafals M.  Induction immunosuppressive therapies in renal transplantation. Am J Health Syst Pharm. 2011;68(3):211–8. Gaber AO, Monaco AP, Russell JA, et al. Rabbit antithymocyte globulin (thymoglobulin): 25 years and new Frontiers in solid organ transplantation and haematology. Drugs. 2010;70(6):691. Gerken G, Beckebaum S, de Geest S, et al. Efficacy, safety, and immunosuppressant adherence in stable liver transplant patients converted from a twice-daily tacrolimus-based regimen to once-­ daily tacrolimus extended-release formulation. Transpl Int. 2011;24(7):666–75. Hellstrom M, et al. Bioengineered uterine tissue supports pregnancy in a rat model. Fertil Steril. 2016;106(2):487–96.e1. Print. Hodson E, Jones C, Craig J, et al. Antiviral medications to prevent cytomegalovirus disease and early death in recipients of solid-organ transplants: a systematic review of randomised controlled trials. Lancet. 2005;365(9477):2105–15. Johnson C, Ahsan N, Gonwa T, et al. Randomized trial of tacrolimus (Prograf) in combination with azathioprine or mycophenolate mofetil versus cyclosporine (Neoral) with mycophenolate mofetil after cadaveric kidney transplantation. Transplantation. 2000;69(5):834–41. Kainz A, et al. Analysis of 100 pregnancy outcomes in women treated systemically with tacrolimus. Transpl Int. 2000;13(Suppl 1):S299–300. Print. Karie-Guigues S, et al. Long-term renal function in liver transplant recipients and impact of immunosuppressive regimens (calcineurin inhibitors alone or in combination with mycophenolate mofetil): the try study. Liver Transpl. 2009;15(9):1083–91. Print. Kim H, Jeong JC, Yang J, et al. The optimal therapy of calcineurin inhibitors for pregnancy in kidney transplantation. Clin Transpl. 2015;29:142–8. Kino T, Hatanaka H, Hashimoto M, et al. FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics. J Antibiot. 1987;40:1249–55. Kirk AD, et al. Renal transplantation using belatacept without maintenance steroids or calcineurin inhibitors. Am J Transplant. 2014;14(5):1142–51. Le Ray C, et al. Mycophenolate mofetil in pregnancy after renal transplantation: a case of major fetal malformations. Obstet Gynecol. 2004;103(5 Pt 2):1091–4. Li J, Kim SG, Blenis J. Rapamycin: one drug, many effects. Cell Metab. 2014;19(3):373–9. Lim WH, Eris J, Kanellis J, Pussell B, Wiid Z, Witcombe D, Russ GR. A systematic review of conversion from calcineurin inhibitor to mammalian target of rapamycin inhibitors for maintenance immunosuppression in kidney transplant recipients. Am J Transplant. 2014;14(9):2106– 19. https://doi.org/10.1111/ajt.12795. MacDonald AS. A worldwide, phase III, randomized, controlled, safety and efficacy study of a sirolimus/cyclosporine regimen for prevention of acute rejection in recipients of primary mismatched renal allografts. Transplantation. 2001;71(2):271–80. McKay D, Josephson M. Pregnancy in recipients of solid organs—effects on mother and child. NEJM. 2006;354:1281–93. McKeage K, McCormack P. Basiliximab: a review of its use as induction therapy in renal transplantation. BioDrugs. 2010;24(1):55–76. Mohty M.  Mechanisms of action of antithymocyte globulin: T-cell depletion and beyond. Leukemia. 2007;21(7):1387–94. Mölne J, Broecker V, Ekberg J, Nilsson O, Dahm-Kähler P, Brännström M. Monitoring of human uterus transplantation with cervical biopsies: a provisional scoring system for rejection. Am J Transplant. 2017;17:1628–36. Morgan R, O’Callaghan J, Knight S, Morris P. Alemtuzumab induction therapy in kidney transplantation: a systematic review and meta-analysis. Transplantation. 2012;94(10):972. Normand G, et al. Pregnancy outcomes in simultaneous pancreas and kidney transplant recipients: a National French Survey Study. Transpl Int. 2017;30(9):893–902. Print. Østensen M, et al. Update on safety during pregnancy of biological agents and some immunosuppressive anti-rheumatic drugs. Rheumatology. 2008;47(Suppl_3):iii28–31. Print. PrabhuDas M, Bonney E, Caron K, et  al. Immune mechanisms at the maternal-fetal interface: perspectives and challenges. Nat Immunol. 2015;16(4):328–34.

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Shipkova M, et al. Mycophenolate mofetil in organ transplantation: focus on metabolism, safety and tolerability. Expert Opin Drug Metab Toxicol. 2005;1(3):505–26. Print. Silveira F, Kusne S.  Candida infections in solid organ transplantation. Am J Transpl. 2013;13(4):220–7. Soave R. Prophylaxis strategies for solid-organ transplantation. Clin Infect Dis. 2001;33(1):S26–31. Staatz CE, Tett SE. Clinical pharmacokinetics of once-daily tacrolimus in solid-organ transplant patients. Clin Pharmacokinet. 2015;54(10):993–1025. Testa G, Koon EC, Johannesson L, McKenna GJ, Anthony T, Klintmalm GB, Gunby RT, Warren AM, Putman JM, dePrisco G, Mitchell JM, Wallis K, Olausson M. Living donor uterus transplantation: a single center’s observations and lessons learned from early setback to technical success. Am J Transplant. 2017;17:2901–10. The U.S. Multicenter FK506 Liver Study Group. A comparison of tacrolimus (FK 506) and cyclosporine for immunosuppression in liver transplantation. N Engl J Med. 1994;331(17):1110–5. Thomusch O, et al. Rabbit-ATG or basiliximab induction for rapid steroid withdrawal after renal transplantation (harmony trial). Lancet. 2016;388(10063):3006–16. Ton E, et  al. Safety of rituximab therapy during twins’ pregnancy. Rheumatology (Oxford). 2011;50(4):806–8. Print. Undre NA. Pharmacokinetics of tacrolimus-based combination therapies. Nephrol Dial Transplant. 2003;18(Suppl 1):i12–5. Print. Vincenti F, et al. Costimulation blockade with belatacept in renal transplantation. N Engl J Med. 2005;353(8):770–81. Print. Vincenti F, et al. Ten-year outcomes in a randomized phase Ii study of kidney transplant recipients administered belatacept 4-weekly or 8-weekly. Am J Transplant. 2017;17(12):3219–27. Print. Webster AC, Lee VW, Chapman JR, Craig JC. Target of rapamycin inhibitors (TOR-I; sirolimus and everolimus) for primary immunosuppression in kidney transplant recipients. Cochrane Database Syst Rev. 2006;(2):CD004290. https://doi.org/10.1002/14651858.CD004290.pub2. Webster P, Lightstone L, McKay D, Josephson M. Pregnancy in chronic kidney disease and kidney transplantation. Kidney Int. 2017;91:1047–56. Wei L, et al. 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(2):346–56.e1. Print. Wilkinson A, Pham PT. Kidney dysfunction in the recipients of liver transplants. Liver Transpl. 2005;11(Suppl 2):S47–51. Print. Zhang X, Han S, Fu S, Wang L, Huang H. Alemtuzumab induction in renal transplantation: a meta-­ analysis and systemic review. Transpl Immunol. 2012;27(2–3):63–8. Zheng J, Song W.  Alemtuzumab versus antithymocyte globulin induction therapies in kidney transplantation patients a systematic review and meta-analysis of randomized controlled trials. Medicine. 2017;96(28):1–8.

Evaluation of Graft Function After Uterus Transplantation

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Milan Milenkovic and Mats Brännström

22.1 Monitoring of Graft During In-Patient Stay The goal of close monitoring of the graft is to find pathological conditions at an early stage when they are treatable by surgical or pharmacological intervention. The typical recipient, after a standard surgical procedure by laparotomy, would stay in the hospital for around 1  week. During the hospital stay, standard postoperative controls, including laboratory tests, should be performed daily. Early mobilization from the first postoperative day is important to decrease the risk of thrombosis formation. The blood level of calcineurin inhibitor is checked daily and the dose adjusted accordingly. Blood flow distal to the anastomosis lines of the internal iliac/ uterine arteries is controlled on a daily basis. This can easily be accomplished by Doppler scanning through an abdominal probe, placed just cranial to the inguinal ligament. We perform the first gynaecological examinations on the second postoperative day, and this investigation includes visual inspection, vaginal swab for bacterial culture and attainment of cervical biopsy for rejection grading. The vaginal examination is repeated every second day, and no further biopsy is taken during this first postoperative week, if initial biopsy is normal. Transvaginal ultrasound examination will not add any information during this initial postoperative week.

M. Milenkovic CLINTEC, Karolinska Institute, Stockholm, Sweden BeoGyn, Belgrade, Serbia M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_22

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22.2 Interval of Outpatient Monitoring After the recipient leaves the hospital, we see the patient for blood tests and gynaecological examination as outpatient procedures. During the second postoperative week, we recommend gynaecological examination, including ultrasound with measurement of blood flow indices, and blood test twice. An ectocervical biopsy, for rejection monitoring, is taken at the second occasion during this postoperative week two. The examinations, including attainment of ectocervical biopsy and blood tests, are then spaced out to be once weekly during postoperative week three and four. The blood tests will after that become more frequent than the gynaecological and ultrasound examinations. Furthermore, we generally then exclude observation of blood flow parameters (see below), since the timeframe for great risk of thrombosis formation is over, and even if low blood flow would be seen, it would not lead to any acute surgical intervention. During the second and third postoperative month, we advise gynaecological examinations, with transvaginal ultrasound and biopsies, forth-nightly and then monthly up to and including months 6. After that and until pregnancy, we evaluate the patient every second month with gynaecological examination, including biopsy from the ectocervix and vaginal swab for bacterial culture. If repeated pathology, such as rejection and infection, has been seen during the initial period, we will see the patient more frequently than every second month, following an individualized scheme.

22.3 Self-Evaluation The patient is asked to alert us if change of vaginal discharge or irregular bleeding. Menstrual bleeding is one parameter that is fully evaluated and monitored by the patient. The first spontaneous menstrual bleeding in the original Swedish uterus transplantation trial appeared within 2 months after surgery in the seven patients with graft success (Brännström et al. 2014). During the first year, the bleedings were more or less regular, with intervals of 27–32 days (Johannesson et al. 2015).

22.4 Gynecological Examination of Cervix and Vagina The gynaecological examination starts with sampling with vaginal swab for bacterial culture. Generally, we only perform ordinary bacterial culture, but in some cases it has been expanded to also include culture for group B streptococcus, actinomyces and fungi. This expansion of examination for vaginal/cervical pathogens could also include PCR detection of chlamydia and gonorrhoea, if suspicion by signs of un-normal discharge or mucosal inflammation. The vaginal anastomosis line should be inspected at every clinical visit. There are not yet any reports on leakage/opening of this anastomosis line, but UTx centres have reported progressive stenosis over the site for vaginal end-to-end anastomosis

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(Chmel et al. 2019). We did not observe this in any of the seven patients with graft success of the initial Swedish trial (Brännström et al. 2014). In our ongoing robotic UTx trial, we initially modified the technique of vaginal transection from bipolar diathermy and scissors in the original study (Brännström et al. 2014) to use of only the LigaSure® device (unpublished data). In the first three cases of this robotic series, with use of LigaSure® device, vaginal stenosis occurred. There was a progressive decline of the vaginal lumen over the anastomosis, and in all cases vaginal surgery had to be performed during the first 6 months post UTx, in order to widen the stenosis. The surgery was a combination of forced dilation and several small incisions to perpendicularly cut through the fibrotic ring. In the last two cases of the robotic UTx trial, we used the traditional vaginal transection method (bipolar diathermy plus scissors), and we have not observed any vaginal stricture during their observation period for over 6  months. We speculate that extensive use of the LigaSure® device will permanently impair microcirculation for a relatively large distance on the vaginal cuff of the graft, and this will lead to ischemia with scarring, by invasion of fibroblast and production of collagen-rich extracellular matrix. The examination also includes macroscopic inspection of the uterine cervix that is usually of a diameter of around 3 cm and pale in colour. Signs of discoloration or ulcerations would be suspicious for severe rejection, serious infection or impaired blood flow. Any of these signs should lead to further investigative procedures including colposcopy and attainment of multiple cervical biopsies. In the situation with a macroscopically normal uterine cervix, protocol cervical biopsies should be taken, for rejection diagnosis and grading (Mölne et al. 2017).

22.5 Ultrasound Examinations Ultrasound examinations of the uterine graft include both transvaginal ultrasound and abdominal ultrasound with Doppler measurements. The transvaginal ultrasound examination will give a coarse view of the status of the graft, by evaluation of the myometrial thickness and the echogenicity as well as width of the endometrium. Moreover, blood flow within the outer parts of the myometrium can be seen by colour Doppler, but it is difficult to quantitate intramural blood flow. During the time after the first menstruation, transvaginal ultrasound can be performed for some months more frequently to assure that the growth and echogenicity of the endometrium follow the expected pattern, with development of a thick endometrium and with triple line during the late stage of the follicular phase followed by hyper-echogenicity during the luteal phase. If normal patterns are seen, there is no reason to perform transvaginal ultrasound at each visit, but this can rather be performed at demand. Endometrial thickness varies depending on hormonal status, and in the original Swedish UTx trial, we saw an increase from a median of 7.3 (3.2– 10.4) mm in proliferative phase to 14.4 (11.2–18.7) mm in luteal phase (Johannesson et al. 2015). An abdominal ultrasound probe is used for assessment of blood flow of the uterine arteries. The abdominal probe should typically be placed perpendicular to the

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inguinal ligament, just cranial to the middle part. We evaluated the blood flow indices in seven patients of the original Swedish study (Johannesson et  al. 2015). Repetitive Doppler measurement of uterine arteries of the transplanted uterus showed minimal variations during the first postoperative year. Median (ranges) of pulsatility index (PI), resistance index (RI) and peak systolic velocity (PSV) were 1.9 (0.5–5.4), 0.8 (0.3–1.4) and 25.4 cm/s (9.2–59.4), respectively. In another UTx study, from Czech Republic, including seven surgically successful grafts from four live donor cases and three deceased donor cases, the median PIs and PSV were 1.2 and 25.4 cm/s (Chmel et al. 2019). Taken together, these figures for blood flow indices indicate a solid blood flow that does not change over time during the initial post-UTx year.

22.6 Evaluation Before Embryo Transfer The first attempt for pregnancy after UTx is recommended by us to be 10–12 months after UTx, although some groups have recommended earlier start from 6 months post UTx. Before the first embryo transfer (ET), it is important to ascertain that the ET can be achieved easily in the patient that would come in some days after ovulation in a natural cycle ET or after sequential estradiol-progesterone in a hormone replacement therapy (HRT) cycle. We advise monitoring of the endometrium and mock-ET in a cycle before the cycle of the sharp transfer. Transvaginal ultrasound should be used to see that the normal growth of the endometrium to more than 7 mm is achieved during the follicular phase of the natural cycle or during oestrogen-only stimulation in a HRT cycle. At the correct point of the cycle, a mock-ET procedure, with an empty transfer catheter, is recommended. The patient should have a full bladder so that transabdominal ultrasound could be used to visualize the ET catheter and a little of content of air that could be flushed for better imagining. We have experienced in some patients that the external os of the cervix can be in an atypical position and that needs to be described before a sharp ET. Moreover, some transplanted patients will for unknown reasons have elongated cervices with distances between external and internal os of 7–8 cm. That is also valuable information. Moreover, a relative vaginal stenosis may make the transfer more difficult, and a try out transfer will aid.

References Brännström M, Johannesson L, Dahm-Kähler P, et al. The first clinical uterus transplantation trial: a six months report. Fertil Steril. 2014;101:1228–36. Chmel R, Novackova M, Janousek L, et al. Revaluation and lessons learned from the first 9 cases of a Czech uterus transplantation trial: four deceased and five living donor uterus transplantations. Am J Transplant. 2019;19(3):855–64. Johannesson L, Kvarnström N, Mölne J, et al. Uterus transplantation trial: 1-year outcome. Fertil Steril. 2015;103:199–204. Mölne J, Broecker V, Ekberg J, et al. Monitoring of human uterus transplantation with cervical biopsies: a provisional scoring system for rejection. Am J Transplant. 2017;17:1628–36.

Rejection Diagnosis After Uterus Transplantation

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Johan Mölne and Verena Bröcker

23.1 Introduction Human uterus transplantation has become reality mainly, due to the successful uterus transplantation trial, with preceding solid research, in Gothenburg, Sweden. Today, several centers worldwide have started their own transplantation trials. This emphasizes the need for a robust and standardized histopathological grading scheme to monitor rejections and treatment. Here, we report our histological experience gained on ectocervical biopsies from human uterus transplants.

23.2 Immunological Mechanisms Rejection of solid organ allografts is a form of inflammation aimed at foreign antigens, mainly HLA- or ABO-antigens. Foreign epitopes on cells in organ transplants activate the recipient’s immune system directly or indirectly via antigen presentation in lymph nodes. Antigen-presenting cells activate CD4+ T-helper cells, which subsequently activate CD8+ T-suppressor cells, macrophages, and B-cells. This leads to a T-cell-dominated response, in which mainly CD8+ cells attack the graft of the recipient, as well as an antibody-mediated response, in which B-cells become antibody-producing plasma cells. Most histopathological rejection schemes today divide the immunological response into a cellular and a humoral (antibody-­ mediated) component. Likely, both mechanisms occur simultaneously but one of them might dominate. Both cells and antibodies attack vascular endothelial cells, whereas T-cells also attack the parenchyma. Antibodies initiate a complement-­ mediated attack that can be visualized in biopsies using immunohistochemistry J. Mölne (*) · V. Bröcker Department of Pathology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_23

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against the complement split product C4d as the latter binds covalently to the endothelium. In addition, donor-specific anti HLA-antibodies (DSA) can be measured in the peripheral blood to diagnose humoral rejection using an ELISA-assay (Luminex®).

23.3 Animal Models To prepare for human uterus transplantation (UTx), Mats Brännström established a research group in 1999, and one of the authors (JM) was at an early stage recruited as a central part of the research team. We started to investigate transplantation solutions for mouse (Racho El-Akouri et al. 2003a, b) and rat (Wranning et al. 2008) uteri and studied ischemic changes after cold (Wranning et  al. 2005) and warm (Diaz-Garcia et  al. 2013) storage. Surgical techniques were developed and later transplantation trials commenced. The mouse vessels are very small, and a substantial number of transplants were lost due to initial technical problems but otherwise the methods worked. The research was also extended to large domestic species (Dahm-Kahler et  al. 2008; Wranning et  al. 2006). Importantly, we also studied rejection morphology in rodent models (El-Akouri et al. 2006; Groth et al. 2009) in order to gain knowledge that later could be used in the non-human primate (Johannesson et  al. 2012, 2013). We developed immunosuppressive protocols, attained functional rodent uteri transplants also in allogeneic settings and finally achieved pregnancies. The offspring was followed and has developed normally and achieved successful mating and offspring themselves. The basis for the rejection grading system of human UTx today (Mölne et al. 2017), as discussed further below, is our results from the non-human primate studies of allogeneic UTx.

23.4 Human Experience The initial Swedish human UTx trial was performed with surgeries mainly in 2013. The aim was to transplant ten women with live donor uteri. Finally, nine patients were included in the study. In short, seven transplants were successful, and so far eight healthy babies have been born in six women. The seventh woman has had multiple early miscarriages but no childbirth so far. Two uteri failed early after transplantation due to thrombosis and infection, respectively. A detailed description of the biopsy results and a preliminary grading system for rejection in UTx (Fig. 23.1) using ectocervical biopsies covered by squamous epithelium was published in 2017 (Mölne et al. 2017). At the beginning of the trial, a protocol biopsy schedule was established according to which weekly biopsies during the first month and monthly thereafter until pregnancy were performed. Only two biopsies were planned during pregnancy, at around gestational weeks 20 and 30, as a safety measure. Based on our histological experience and following re-­ evaluation of all the biopsies after 3 years of the trial, a rejection scheme was developed. Biopsies from ectocervical tissue were graded as normal, borderline changes or rejection grade 1–3 (Table  23.1). A borderline category, which designated

23  Rejection Diagnosis After Uterus Transplantation Fig. 23.1 Schematic illustrations of rejection patterns and grading system in cervical biopsies. (a) Normal cervical biopsy with few inflammatory cells. (b) Borderline changes. Small nests of inflammatory cells, dominated by lymphocytes, can be seen at the stromal-epithelial interface. (c) Grade 1 rejection. A mixed inflammatory infiltrate at the stromal-epithelial interface is dominated by lymphocytes. There is low grade stromal inflammation and edema. (d) Grade 2 rejection. A moderate interface inflammatory infiltrate with intraepidermal influx of inflammatory cells is dominated by lymphocytes and has some neutrophils. There is often a reduced surface epithelial thickness and a marked mixed stromal inflammation with edema. (e) Grade 3 rejection. There is a significant diffuse, mixed inflammatory cell infiltrate dominated by lymphocytes, with presence of neutrophils and eosinophils. Apoptotic bodies, epithelial erosions/ ulcerations, and focal necrosis can be seen. The stromal infiltrate (mixed) is intense and continuous

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biopsies as suspicious but not definite for rejection, was added to avoid overdiagnosis in slightly inflamed biopsies. A similar category is found in rejection grading schemes for other solid organs, especially kidney, in order to allow for inflammation of uncertain significance at the low end of the spectrum (Fig. 23.2). Further research may help to shed light on these uncertain inflammatory changes.

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Table 23.1  Grading system for uterus transplantation, cervical biopsies Grade Borderline changes Grade 1 rejection Grade 2 rejection Grade 3 rejection

Morphology At least two, small, nested foci of interphase inflammation in the basal cell layers, dominated by lymphocytes. Focal intercellular edema Minimal stromal inflammation, particularly in the papillary stroma Mild mixed inflammatory cell infiltrate in the basal epidermal layer, dominated by lymphocytes. Single epithelial apoptotic bodies Low-grade stromal inflammation and edema Moderate inflammatory cell infiltrate with intraepidermal influx (exocytosis), dominated by lymphocytes and with some neutrophils. May show reduced surface epithelial thickness, some epithelial apoptotic bodies. Marked, mixed stromal inflammation and edema Significant diffuse, mixed inflammatory cell infiltrate dominated by lymphocytes and presence of neutrophils and eosinophils. Apoptotic bodies. Epithelial erosions/ulcerations, focal to complete. Focal necrosis can be seen Dense and continuous stromal infiltrate (mixed)

From Mölne et al. AJT 2017

Fig. 23.2  Ectocervical biopsies with rejection grade 1 (a) and after treatment with steroids (b). (a) There is a dense lymphocyte-predominant infiltration of inflammatory cells in the upper stroma and the basal epidermal layer (arrows) and some intracellular edema (arrowhead). (b) After treatment there are very few infiltrating inflammatory cells remaining at the interface between stroma and squamous epithelium (arrow)

In total, 163 biopsies from the seven functioning transplants were taken during the first 36 months of the trial (Mölne et al. 2017). Only protocol and follow-up biopsies were obtained. No biopsies for cause were taken, as there were no clinical signs to suggest rejection. Thirteen biopsies (8%) showed rejection and another 15 (9%) were classified as borderline changes, while 135 (83%) were normal. All follow-­up biopsies after rejection episodes showed normalization of findings following initial treatment, except in one patient. She had a 3-month period of repeated or on-going rejection episodes and finally a grade 3 rejection, which was treated with anti-thymocyte globulin (ATG). There was no evidence of humoral rejection in any of the biopsies as all biopsies were negative for C4d and microvascular inflammation was absent. The DSA were negative during the study period, but one patient developed DSA 8 months post transplantectomy.

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Our findings show that rejections are relatively infrequent in UTx using a kidney-­ based immunosuppression protocol. However, the findings also demonstrate that rejections in UTx are clinically silent, underscoring the necessity for a protocol biopsy program. At present the specificity of any inflammation in the cervix for rejection is uncertain and subject to further research studies. Mild and unspecific inflammation can even be found in non-transplant cervical biopsies, especially at the squamocolumnar junction and in the endocervix. It therefore needs to be emphasized that our proposed grading system for UTx is based on biopsies from the ectocervix, which is covered by squamous epithelium and usually shows much less inflammation. We do not have enough experience of other types of tissue yet to give any suggestions how to determine or grade rejection in the endocervix. However, the successful outcome of the trial with eight healthy babies born argues for that the grading scheme is feasible to monitor UTx and to guide treatment until the research community accumulates further data that may lead to substantial changes in this preliminary scheme. Of note, none of the five uteri that have been removed after childbirth so far showed any signs of severe acute or chronic rejection (unpublished observations).

References Dahm-Kahler P, et  al. Transplantation of the uterus in sheep: methodology and early reperfusion events. J Obstet Gynaecol Res. 2008;34:784–93. https://doi. org/10.1111/j.1447-0756.2008.00854.x. Diaz-Garcia C, Akhi SN, Martinez-Varea A, Brannstrom M.  The effect of warm ischemia at uterus transplantation in a rat model. Acta Obstet Gynecol Scand. 2013;92:152–9. https://doi. org/10.1111/aogs.12027. El-Akouri RR, Molne J, Groth K, Kurlberg G, Brannstrom M. Rejection patterns in allogeneic uterus transplantation in the mouse. Hum Reprod. 2006;21:436–42. https://doi.org/10.1093/ humrep/dei349. Groth K, Akouri R, Wranning CA, Molne J, Brannstrom M. Rejection of allogenic uterus transplant in the mouse: time-dependent and site-specific infiltration of leukocyte subtypes. Hum Reprod. 2009;24:2746–54. https://doi.org/10.1093/humrep/dep248. Johannesson L, et al. Uterus transplantation in a non-human primate: long-term follow-up after autologous transplantation. Hum Reprod. 2012;27:1640–8. https://doi.org/10.1093/humrep/ des093. Johannesson L, et  al. Preclinical report on allogeneic uterus transplantation in non-human primates. Hum Reprod. 2013;28:189–98. https://doi.org/10.1093/humrep/des381. Mölne J, Broecker V, Ekberg J, Nilsson O, Dahm-Kähler P, Brännström M. Monitoring of human uterus transplantation with cervical biopsies: a provisional scoring system for rejection. Am J Transplant. 2017;17(6):1628–36. Racho El-Akouri R, Wranning CA, Molne J, Kurlberg G, Brannstrom M. Pregnancy in transplanted mouse uterus after long-term cold ischaemic preservation. Hum Reprod. 2003a;18:2024–30. Racho El-Akouri R, Kurlberg G, Brannstrom M. Successful uterine transplantation in the mouse: pregnancy and post-natal development of offspring. Hum Reprod. 2003b;18:2018–23.

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Wranning CA, Molne J, El-Akouri RR, Kurlberg G, Brannstrom M. Short-term ischaemic storage of human uterine myometrium—basic studies towards uterine transplantation. Hum Reprod. 2005;20:2736–44. https://doi.org/10.1093/humrep/dei125. Wranning CA, et al. Auto-transplantation of the uterus in the domestic pig (Sus scrofa): surgical technique and early reperfusion events. J Obstet Gynaecol Res. 2006;32:358–67. https://doi. org/10.1111/j.1447-0756.2006.00426.x. Wranning CA, Akhi SN, Kurlberg G, Brannstrom M.  Uterus transplantation in the rat: model development, surgical learning and morphological evaluation of healing. Acta Obstet Gynecol Scand. 2008;87:1239–47. https://doi.org/10.1080/00016340802484966.

Psychological Aspects After Uterus Transplantation

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Stina Järvholm

To be able to provide support during the long time of follow-up, from inclusion through, IVF, transplantation, pregnancy attempts, pregnancy and to hysterectomy the psychologist needs to be a part of the multidisciplinary team that would handle all aspects of UTx. Differences will exist between countries and settings concerning the provision for different kind of support but in this early stage of establishment of UTx toward the full clinical stage, psychological follow-up is recommended to be offered at least once a year for recipient, partner, and the donor. Comparable with when ordinary in vitro fertilization treatment (IVF) started up in the 1980s, careful psychological follow-up of the children born after UTx will be crucial but this will not be discussed in this chapter.

24.1 Recovery The first time period after transplantation is associated with a psychological strain in several ways also when the outcome is successful. The recipient and her partner need to adjust to both recovery and to live a life much closer to healthcare facilities, with medications, and changes in everyday life. The recipient has to cope with the fact that she has moved from “healthy to sick.” The “healthy to sick” concept have been found useful to discuss both prior to and past transplantation in order to prepare the recipient for this practical and psychological change (Järvholm et al. 2015a, b). Concerning both recipients and donors, the approximate first 3  months after transplantation is a time period when support from social network, medical team, and psychologist is beneficial (Järvholm et al. 2015a, b; Kvarnström et al. 2017).

S. Järvholm (*) Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, Sahlgrenska Academy, University of Gothenburg, Institute of Clinical Sciences, Gothenburg, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_24

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24.2 The Renewed Body The recipient has a body after transplantation that needs much more attention from healthcare than she is used to. Just the fact that you are experiencing your first menstruation when you are an adult woman in your 20–30s or to resume the menstruation after several years without such is cause to attention. Notable is that after experiencing menstruation for some period most recipients speak about the menstrual bleeding as an unwanted necessity. Feelings of relief were expressed when they no longer where having their menstruation after the hysterectomy was performed. After UTx, both recipients and donors express difficulties adjusting to the scars after surgery and that the scars limit them in situations where they are exposing themselves, for example on the beach. Furthermore, questions about sexuality after surgery should be brought up, regarding both recipient and donor. Medical aspects of sexuality and body image that can be brought up are pain or lack of arousal as well as psychological aspects, for example lack of confidence with the renewed body.

24.3 Rejection and Graft Failure Those uterine recipients that experience a nonfunctioning organ and/or rejection and a possible graft failure will be subjected to several psychological issues. The anxiety that take place when facing rejection is expected and well known in other types of organ transplantation (Dabbs et al. 2004; Nilsson et al. 2011). Concerning the recipient and her partner after UTx, there are both resemblances and differences from other transplanted patients in the case of rejection. The uterus is life enhancing, not lifesaving, and therefore it is possible to remove it without severe risk of impairment or death. But in a psychological way the threat with rejection has resemblance with other organs since the uterus represent the desired way of how they want to live their life after transplantation. Some will need psychological support during times of rejection and/or in the period of grief after early graft removal. Psychological counseling can also be needed for reorientation toward other pathways to parenthood such as adoption or gestational surrogacy or adjust to a life without becoming a parent. When using a directed living donor (DD) graft failure can also affect the donor negatively (Lentine et al. 2012). With graft failure after UTx there is also the question of not becoming a grandparent if the donor is the mother of the recipient. Therefore psychological support should be offered to the donor as well in the case of graft failure.

24.4 Trying to Achieve Pregnancy and Parenthood When starting up the pregnancy attempts, the participants in UTx will share a well-­ known situation with other patients that undergo embryo transfer after IVF.  This period is associated with increased levels of anxiety and this increase manifests more for the women than for her partner (Chachamovich et al. 2010; El Kissi et al. 2013).

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If the donor is the woman’s mother or another close relative, such as sister or mother-in-law, these will also be affected of the pregnancy attempts since the child and parenthood is the real goal for the transplantation and the final proof of a functioning organ. To be in “trying situation” together can both be a possibility for mutual support but can also add strain if significant other burdens exist at the same time. The women that achieve pregnancy after UTx, all face the well-known and common psychological strains that are associated with pregnancy and parenthood. These are increased risk for anxiety/depression and decline in relationship satisfaction (McKenzie and Carter 2013; Philpott et al. 2017) and also the added strains after IVF treatment (Gourounti 2016). In addition, the pregnant woman with a uterine graft and her partner can also experience that the tightly managed pregnancy after UTx, does not fully belong to the couple but rather to the healthcare system/ transplantation team. In order to succeed to become parent after UTx, there is a requirement of a balance between the need for medical monitoring and the task to psychologically facilitate the to-be parents feelings of efficacy and possibilities to start the process of attachment to the prospective child. The influence of parenthood on satisfaction with marital quality is affected by the presence of factors related to protection and risk. A long-term relationship prior to parenthood, good economic standard, high education, and good health are all protective factors while lack of consensus regarding child upbringing or division of household labor increase risk of relationship termination (Loft 2011). Lawrence et al. (2008) found that parenthood hastens marital decline even for those couples that select themselves to this transition and that a planned for pregnancy seems to protect from these declines. The general protective factors, such as long-term relationship as well as having extremely well planned pregnancies would be in favor for marital satisfaction during the transition to parenthood in couples undergoing UTx.

24.5 Psychological Challenges Recipient, partners, and donors will be affected by the UTx procedure over a long time-period over several years. Critical and/or stressful medical and/or situations of everyday life will for sure occur during the follow-up. In order to support and accomplish compliance to the extensive care, a supportive nonjudgmental relationship with the team is needed so that challenging events are brought up and can be discussed at any time during these years. There are several medical issues that can be raised throughout the period from UTx but also for several years after childbirth(s) and hysterectomy. When is time to give up if the UTx does not lead to pregnancy? In cases when a transplanted woman do not agree with the medical advice regarding time for hysterectomy, who have the final say? There also exist questions of everyday life. What happens if the couple decides to get a divorce? Do legalization accept that the woman continue in a UTx procedure as a single mother? Has she got enough support to go through with UTx on her own? It is the responsibility of the team to handle that any participant’s determination to be a part of this innovative procedure, that may lead to parenthood, could build

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up a facade showing that they are extraordinarily strong in psychological terms. They may then avoid to share expected and common difficulties with the team. This viewpoint is contra-productive when it comes to compliance and undermines the possibility to offer psychological support when required.

24.6 Relationship with Donor When the donor is a directed living donor, the relationship might be affected by the situation. In our experience, the relation between donor and recipient turns back to as it was before the donation or may be experienced as strengthened for the UTx participants and this is comparable to other transplantations (Benzing et al. 2015; Gross et al. 2013). However, for some a guiltiness of gratitude may occur for recipient and partner toward the donor and this may be in the reversed direction in cases of a nonfunctioning uterine graft. In these situations psychological counseling is of great value in order to bring these subjects and feelings up in order to normalize and to prevent strains in the relationship.

24.7 Hysterectomy The recipients and partners that have experienced parenthood after UTx, has described the subsequent hysterectomy as psychologically uncomplicated and transition to “back to normal.” The women define themselves as back to the person they were before they entered UTx and that their body feels like their own again. Some will express the feeling of secondary childlessness when they will not be able to have as many children as they intended with UTx due to medical safety reasons, legislation, or financial aspects. Concerning the recipients and partners who did not experience parenthood after UTx, the time of the exit from the project is more ambivalent and psychologically complicated. Nevertheless these women also, after a period of mourning, described relief with the fact that their body were “back to normal” again. All women, regardless of outcome, have so far described the uterus as a temporal mean to achieve parenthood and not as an essential part of their body, self-esteem or a wish for the uterus in itself. Of course other groups to come for UTx in the future may feel differently about this aspect, for example the transgender group may see the uterus itself as an important part of their self-image.

24.8 Long-term Psychological Aspects Since the UTx procedure is novel, with only a few years since the first successful attempt, there is no accumulated knowledge concerning the long-term psychological consequence for women, partners, donors, and future children of those

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participating in UTx. The full knowledge of this is yet to come during upcoming decades. What we already know today is that psychological consequences will differ between individuals due to traits and experiences before UTx. Different psychological consequences will also be caused by the experiences during UTx and perhaps also differ due to cultural settings. So far the psychological strains after UTx is considered to be manageable and several aspects have been found to be psychologically favorable. For the group of women with infertility related to absolute uterine factor it is apparent that the evolving field of UTx is promising but it will never be an option for all. In order to achieve sound long-term psychological consequences for all participating stakeholders in UTx it is of importance to recruit those who are right for the procedure, to offer those who go through with UTx support during the process, and to help those who will not succeed with an acceptable exit process.

References Benzing C, Hau H-M, Kurtz G, Schmelzle M, Tautenhahn H-M, Morgül MH, et al. Long-term health-related quality of life of living kidney donors: a single-center experience. Qual Life Res. 2015;24(12):2833–42. Chachamovich JR, Chachamovich E, Ezer H, Fleck MP, Knauth D, Passos EP. Investigating quality of life and health-related quality of life in infertility: a systematic review. J Psychosom Obstet Gynecol. 2010;31(2):101–10. https://doi.org/10.3109/0167482X.2010.481337. Dabbs ADV, Hoffman LA, Swigart V, Happ MB, Dauber JH, McCurry KR, Iacono A. Striving for normalcy: symptoms and the threat of rejection after lung transplantation. Soc Sci Med. 2004;59(7):1473–84. El Kissi Y, Romdhane AB, Hidar S, Bannour S, Idrissi KA, Khairi H, Ali BBH.  General psychopathology, anxiety, depression and self-esteem in couples undergoing infertility treatment: a comparative study between men and women. Eur J Obstet Gynecol Reprod Biol. 2013;167(2):185–9. Gourounti K.  Psychological stress and adjustment in pregnancy following assisted reproductive technology and spontaneous conception: a systematic review. Women Health. 2016;56(1):98–118. Gross C, Messersmith EE, Hong BA, Jowsey SG, Jacobs C, Gillespie BW, et al. Health-related quality of life in kidney donors from the last five decades: results from the RELIVE study. Am J Transplant. 2013;13(11):2924–34. Järvholm S, Johannesson L, Brännström M. Psychological aspects in pre-transplantation assessments of patients prior to entering the first uterus transplantation trial. Acta Obstet Gynecol Scand. 2015a;94(10):1035–8. https://doi.org/10.1111/aogs.12696. Järvholm S, Johannesson L, Clarke A, Brännström M.  Uterus transplantation trial: psychological evaluation of recipients and partners during the post-transplantation year. Fertil Steril. 2015b;104(4):1010. https://doi.org/10.1016/j.fertnstert.2015.06.038. Kvarnström N, Järvholm S, Johannesson L, Dahm-Kähler P, Olausson M, Brännström M. Live donors of the initial observational study of uterus transplantation—psychological and medical follow-up until 1 year after surgery in the 9 cases. Transplantation. 2017;101(3):664–70. Lawrence E, Rothman AD, Cobb RJ, Rothman MT, Bradbury TN. Marital satisfaction across the transition to parenthood. J Fam Psychol. 2008;22(1):41. Lentine KL, Schnitzler MA, Xiao H, Axelrod D, Davis CL, McCabe M, et al. Depression diagnoses after living kidney donation: linking United States registry data and administrative claims. Transplantation. 2012;94(1):77.

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Loft LTG.  Child health and parental relationships. Int J Sociol. 2011;41(1):27–47. https://doi. org/10.2753/IJS0020-7659410102. McKenzie SK, Carter K.  Does transition into parenthood lead to changes in mental health? Findings from three waves of a population based panel study. J Epidemiol Community Health. 2013;67(4):339–45. https://doi.org/10.1136/jech-2012-201765. Nilsson M, Forsberg A, Bäckman L, Lennerling A, Persson LO. The perceived threat of the risk for graft rejection and health-related quality of life among organ transplant recipients. J Clin Nurs. 2011;20(1–2):274–82. Philpott LF, Fitzgerald S, Leahy-Warren P, Savage E. Stress in fathers in the perinatal period: a systematic review. Midwifery. 2017;55:113–27.

Obstetrical and Pediatric Follow-Up After Uterus Transplantation

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Hans Bokström, Mats Brännström, and Henrik Hagberg

25.1 Antenatal Care In the original Swedish uterus transplantation (UTx) trial (Brännström et al. 2014), with so far (mid-2019) eight live births and one ongoing pregnancy, the patients were followed closely during their entire pregnancies. All recipients were monitored by regular clinical visits and laboratory tests that started from gestational week (gw) 8. The frequency of these controls was initially every second week, and after gw 34, they were weekly. The patients were seen by an obstetrician (feto-­ maternal-­medicine specialist) and a midwife. They worked in close collaboration with a nephrologist, having expertise in strategies for immunosuppression medication. The clinical examination comprised macroscopic inspection of the transplanted uterine cervix and vaginal rim as well as cervical cultures for general bacteria, including group B streptococci. Cervical biopsies were obtained at two predetermined time points (gw 12–16 and 28–30). These biopsies were taken by the same gynecologist, who had followed the patient during the interval between UTx and embryo transfer (ET). In the event of verified rejection in analysis of cervical biopsy, the immunosuppression was temporarily elevated, and biopsies were taken every second week until a biopsy verified non-rejection.

H. Bokström · H. Hagberg Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden e-mail: [email protected]; [email protected] M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_25

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At each clinical visit, transvaginal ultrasound was performed to assess the cervical length, and by abdominal ultrasound, fetal biometry was achieved, with measurement of biparietal diameter, abdominal diameter, and femur length. Doppler ultrasound was used to evaluate flow velocity patterns of uterine arteries. Waveform characteristics and measurement of blood flow velocity at peak systole and peak diastole were obtained to calculate the pulsatile index. Routine blood tests were performed to assess blood status, liver function, and kidney function and to detect infection. Tacrolimus levels were measured. In addition to pharmacological treatment by immunosuppressants, the patients were treated with oral folic acid (250 μg twice daily) from 2 weeks before ET. In the event of frozen ET, the woman received oral estradiol (2 mg three times daily) plus vaginal progesterone (100 mg three times daily) until around gw 8. The patient was on oral acetylsalicylic acid (75 mg daily) from the time of transplantation, and the original recommendation by us was to stay on this dose during pregnancy. However, in later and ongoing pregnancies, the dosage was increased to 160 mg daily, based on indications that this dose may decrease the risk for development of preeclampsia (Rolnik et al. 2017). The dose of tacrolimus was elevated during pregnancy to keep acceptable trough levels, in line with accumulated knowledge from pregnant solid organ transplantation patients. Psychological support, to the pregnant woman and her partner, during pregnancy was by the midwife and obstetrician, and on demand by the psychologist of the team.

25.2 Delivery The recommended mode of delivery is elective cesarean section. This advice is because of special anatomical conditions of a transplanted uterus, with non-­ physiological fixations and sensitive vascular connections. Moreover, the vagina may be less predisposed to the normal dilatation during vaginal birth. There will always exist a fibrous ring, at the site of the vaginal anastomosis line, and in some reported cases this has developed into true vaginal stenosis, that has necessitated surgical opening (Chmel et al. 2019). It is unlikely that this fibrotic ring will dilate further than a couple of cm during a trial of vaginal birth. Moreover, the vast majority of uterus recipients in the ongoing clinical trials have the Mayer-Rokitansky-­ Kuster-Hauser (MRKH) syndrome, and some years prior to UTx, they have undergone vaginal dilatation or surgery to create a vagina of functional length for UTx. This non-physiological vagina will not have the elasticity of a normal vagina. The time of optimal delivery after UTx remains to be defined. Initially, we aimed for delivery by cesarean section at gw 35 + 0. Eventually, after gaining more experience, this recommendation was changed to gw 37 + 0 if no signs of complication had occurred. The postponement from gw 35 to 37 was in order to achieve optimal fetal lung maturation. According to local principles iv, corticosteroid administration for lung maturation was performed if delivery occurred before gw 34 + 0.

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The elective cesarean section is preferably done in spinal anesthesia, which can be combined with epidural anesthesia if difficult dissection is anticipated. The surgeons performing the cesarean section could if possible be one obstetrician and two of the gynecologists that have been involved in the specific UTx surgery, in order to be familiar with any alterations from the normal anatomy. A transplant surgeon is preferably standby in case that injury to any of the uterine arteries or veins would occur during the cesarean section. We have performed the section through a midline skin incision, following the scar after UTx. Typically, the bladder peritoneum will be attached a little higher than normal, and adhesions may be present from the omentum to the uterus. After dividing any adhesions that may disturb the operating field, the bladder peritoneum should be divided high to remove the top of the bladder from the site of uterine incision. Before the transverse incision on the lower uterine segment, the surgeons should make sure that the uterine arteries or veins are not overriding the lateral aspects of the planned transverse uterine incision. In one of our nine cesarean sections after UTx, the uterine arteries rolled up on the front side of the uterus, and by mistake one of the uterine arteries was cut. The ends were clamped and re-anastomosed end-to-end by the transplant surgeon, at the end of the cesarean section. Cesarean section is then by a low transverse uterine incision and further by standard technique. After the baby and placenta have been lifted out, we suture the uterine incision in two layers and do not use any suture on the bladder peritoneum. There have been no problems with uterine atony after any of the cesarean sections after UTx that we have performed. There are some special considerations of the cesarean section that should be taken into account. In cases where preoperative fetal heart monitoring has shown abnormalities, fetal heart monitoring, with the probe inside a sterile bag and applied to the surface of the uterus, can be used. In cases planning for hysterectomy in conjunction with the cesarean section, general anesthesia can be applied after the delivery of the child. After delivery by cesarean section, the uterus will in most cases stay for delayed (1–3  months after delivery) hysterectomy or for attempt for a second pregnancy. The reason to delay hysterectomy for some months after childbirth, and not to do this in conjunction with cesarean section, is to have some observation time to assure the health of the newborn. Postoperative care can be done according to local routines. Concerning immunosuppression, the dose of tacrolimus can after delivery typically be reduced to half of the dose prior to delivery. During the first week(s), daily measurements of tacrolimus levels should be done until acceptable levels are reached, and then measurements can be done weekly. In case of prematurity, the child should be admitted to a neonatal unit. Breastfeeding should be allowed, but in the case of prematurity, after control of immune status of the newborn. The typical discharge of mother and non-­ premature child would be on day 3–5 after delivery. Postoperative control is recommended after 6  weeks and will include clinical examination and ultrasound.

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25.3 Postnatal Follow-Up The children of the initial UTx trial in Sweden (Brännström et al. 2014) were followed by the Swedish routine medical pediatric control program with measurement of length, weight, and head circumference together with physical and neurological health development evaluation at set intervals. This is then continued in school until the age of 18 years. We have also added a battery of other tests/questionnaires to examine neuropsychiatric development as well as immune function. There are no indications that these parameters should not be normal in children from organ transplant patients with immunosuppression medication, but thorough investigations should be done after UTx, since these children have fetal development in a grafted uterus and under the influence of immunosuppression. The neuropsychological and neuropsychiatric tests that we use are Bailey, M-CHAT, Essence-Q-rev, and SDQ at 3 years of age. At the age of 6 years, the tests that we use are WPPSI-IW/WISC, SDQ, 5–15, SNAP IV, and SCQ. All tests are performed and evaluated by a clinical neuropsychologist together with a pediatric psychiatrist. Immune status is checked when the children are 5  years old, and this is by FACS analysis of whole blood concerning subpopulations of immune cells, with additional functional test when pathological conditions are seen concerning any immune cell distribution.

25.4 Results of Published Live Births So far detailed results of four pregnancies after UTx have been published. The facts from these four pregnancies are reviewed in detail below. The first live birth after UTx occurred in September 2014 from patient number five of the original Swedish study (Brännström et al. 2014). The pregnancy and birth have been described in detail (Brännström et al. 2015). The 35-year-old recipient, with MRKH syndrome, was transplanted 1  year before pregnancy, which was achieved by the first ET. The donor was a 61-year-old family friend, who had two normal vaginal deliveries and was 7 years post menopause. The recipient was on triple immunosuppression with tacrolimus, azathioprine, and corticosteroids. The pregnancy was complicated by anemia with hemoglobin of 8.4 g/dL in gw 8, despite oral iron supplementation. One dose of 500 mg iv ferric carboxymaltose was then administered, and treatment was then with 60 μg darbepoetin alfa, given sc once a week. The hemoglobin value thereafter stabilized around 10.0–10.7  g/dL during remaining pregnancy (Brännström et al. 2015). One asymptomatic mild rejection episode was diagnosed on cervical biopsy at gw 18 + 2, and this was treated with iv methylprednisolone for 3 days (250 mg day 1; 125 mg day 2 and 3). Control biopsy 2 weeks later and protocol biopsy at gw 30 were normal. The pregnancy was followed with ultrasound. Fetal growth parameters were all normal until delivery. Umbilical blood flow velocity index was normal, and uterine blood flow pulsatility index was in the low to normal range during pregnancy. Cervical length varied between 43 and 50  mm. In gw 31  +  5, the patient was admitted for severe preeclampsia, and a cesarean section was performed due to variable decelerations on

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fetal heart monitoring. The baby boy weighed 1775 g (−9%) and had normal Apgar score as well as normal blood gas/acid base status (Brännström et al. 2015). The weight-gain of the mother during pregnancy was 8 kg. The infant was treated for respiratory distress syndrome and required phototherapy for neonatal icterus. The reason for development of preeclampsia is not known, but it is likely that underlying factors can be a combination of that the patient had a single kidney and the effects of immunosuppression. The boy was discharged 16 days after delivery, and breastfeeding was initiated after 2 weeks and continued for 6 weeks. The baby underwent elective surgery for unilateral inguinal hernia, when aged 6 months. The child has developed normally and is today (mid 2019) almost 5 years old. The second pregnancy from the Swedish trial (Brännström et al. 2016) was in a 28-year-old woman with MRKH syndrome. The donor was the 50-year-old mother, with a history of three normal vaginal deliveries. Twelve months after UTx, the first ET attempt was done, and pregnancy was achieved on this blastocyst transfer in the natural cycle. The recipient was on triple immunosuppression with tacrolimus, azathioprine and prednisolone. The pregnancy was without histological or clinical signs of rejection. A creatinine elevation up to 137 μmol/L was noted from gw 18, and this elevation, together with moderate hydronephrosis, lasted for the remainder of the pregnancy. Hemoglobin count declined to 7.9 g/dL in gw 18, and this anemia was treated in the same way as the first pregnant UTx patient (see above). Hemoglobin levels were subsequently around 9.5 g/dL. Blood pressure, blood glucose levels, and cervical length were in the typical span during pregnancy. On abdominal ultrasound, fetal growth parameters were in the normal range, and Doppler showed normal blood flow indices in umbilical and uterine arteries. The pregnancy advanced routinely until gw 33, when pruritus developed. This developed in gw 34 to intense pruritus, and elevated levels of bile acids indicated intrahepatic cholestasis of pregnancy. The elective cesarean section, which initially was planned for gw 35 + 0, was now brought forward some days, and at gw 34 + 4 a boy of normal size 2335 g (−7%), with normal Apgar score and normal umbilical artery blood gas/acid base balance, was born. The baby showed signs of mild respiratory distress, which was treated for 2 days with continuous positive airway pressure and surfactant. The mother and child were discharged 8 days after delivery. Breastfeeding was initiated after 2 weeks and continued for 3 months. The boy has developed fully normally and is now (mid 2019) 4.5 years old. These two initial births of the Swedish UTx study (Brännström et al. 2014) were followed by six more births from that study (Mölne et al. 2017), but details of those pregnancies have not been published. Taken together, these were the set of first live births after UTx in the world, and they were all from live donor UTx attempts. The ninth UTx birth in the world, which was the first outside Sweden, occurred in Dallas, USA (Testa et  al. 2018), in late November 2017. This was more than 3  years after the initial UTx birth (Brännström et  al. 2015). The recipient was a 29-year-old MRKH woman, who received a uterus from a 32-year-old altruistic donor that had delivered twice. Pregnancy was achieved at the first ET. A vaginal bleeding occurred at gw 13 and a subchorionic hematoma developed, which was fully resolved at gw 22. Elective cesarean section was performed already at gw

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33 + 1. The decision of this early delivery was according to the authors “driven by the risk-benefit ratio of the baby having reached a gestational age and growth associated with the highest chance of uneventful birth and protection of the mother’s kidney function.” The weight of the boy was 1995  g and Apgar score was 8/9. Hysterectomy was done as the last procedure of cesarean section surgery. The mother was discharged after 4 days and the infant stayed at hospital for 4 weeks. The article does not mention any details concerning the postnatal care of the infant or if breastfeeding was attempted. The tenth UTx baby in the world occurred in Sao Paolo, Brazil, and this was the first delivery after UTx from a deceased donor (Ejzenberg et al. 2019). This pregnancy actually started almost the same time as the USA pregnancy (see above), but the birth in December 2017 took place in gw 35 + 3, as compared to elective cesarean section in gw 33 + 1 in late November 2017. The recipient was a 32-year-old woman with MRKH, and the deceased donor was a 45-year-old, 3-parous woman, with brain death caused by subarachnoid bleeding. The patient became pregnant at the first ET, and the pregnancy developed normally with normal blood pressure and a weight gain of the patient of 15 kg. In gw 32, the woman was treated for pyelonephritis. Elective cesarean section was scheduled according to recommendations of the initial Swedish protocol, and in gw 35 + 3 a girl was delivered. The weight was 2550 g and the girl presented with good Apgar (9/9/10). The transplanted uterus was removed at cesarean section, with histopathology showing intimal hyperplasia of uterine arteries.

References Brännström M, Johannesson L, Dahm-Kähler P, et al. The first clinical uterus transplantation trial: a six months report. Fertil Steril. 2014;101:1228–36. Brännström M, Johannesson L, Bokström H, et al. Livebirth after uterus transplantation. Lancet. 2015;385:607–16. Brännström M, Bokström H, Dahm-Kähler P, et al. One uterus bridging three generations: first live birth after mother-to-daughter uterus transplantation. Fertil Steril. 2016;106:261–6. Chmel R, Novackova M, Janousek L, et al. Revaluation and lessons learned from the first 9 cases of a Czech uterus transplantation trial: four deceased and five living donor uterus transplantations. Am J Transplant. 2019;19:855–64. Ejzenberg D, Andraus W, Baratelli Carelli Mendes LR, et al. Livebirth after uterus transplantation from a deceased donor in a recipient with uterine infertility. Lancet. 2019;392:2697–704. Mölne J, Broecker V, Ekberg J, et al. Monitoring of human uterus transplantation with cervical biopsies: a provisional scoring system for rejection. Am J Transplant. 2017;17:1628–36. Rolnik D, Wright D, Poon L, et al. Aspirin versus placebo in pregnancies at high risk for preterm preeclampsia. N Engl J Med. 2017;377:613–22. Testa G, McKenna GJ, Gunby RT Jr, et al. First live birth after uterus transplantation in the United States. Am J Transplant. 2018;18:1270–4.

Infections After Uterus Transplantation

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Steven Van Laecke and Steven Weyers

26.1 Introduction Uterus transplantation (UTx) has dramatically changed the scope of female infertility. The ethical framework of this innovative technique is partially based upon the temporary exposure to immunosuppressive drugs. Since in UTx, the graft is planned for removal after the accomplishment of one or two successful pregnancies, this exposure is restricted to a limited amount of years. This should theoretically, and in the absence of carry-over effects, eliminate an additional risk of infections and cancer after withdrawal of immunosuppressive drugs shortly after graft removal. Much attention must be paid to the ongoing increased infection risk during exposure to immunosuppressive drugs. The benefit/harm ratio should be optimized as these young women, mostly without meaningful comorbidity, undergo a non-life-saving intervention. As the number of transplantations so far performed is rather limited and the duration of follow-up is short, data on posttransplant complications and especially infections are scarce (Brannstrom 2018; Brannstrom et al. 2018; Favre-­ Inhofer et al. 2018; Kisu et al. 2018). Considering the defensible prime focus on ethical, psychosocial, and technical surgical aspects, this topic has also been somewhat underemphasized in the published reports and review papers (American Society for Reproductive Medicine position statement on uterus transplantation: a committee opinion 2018; Brannstrom 2018; Brannstrom et al. 2015; Favre-Inhofer et al. 2018; Flyckt et al. 2016b; Testa et al. 2018b). The first year graft survival is steadily increasing but remains affected by a relatively high risk of immediate posttransplant thrombotic complications, especially in recipients of cadaveric UTx

S. Van Laecke (*) Renal Division, Ghent University Hospital, Ghent, Belgium e-mail: [email protected] S. Weyers Women’s Clinic, Ghent University Hospita, Ghent, Belgium e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_26

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grafts (Chmel et al. 2019; Fageeh et al. 2002; Johannesson et al. 2015; Kisu et al. 2018; Testa et al. 2017). This should not defer us from considering the optimization of immunosuppressive treatment to control rejection rate with the lowest possible incidence of attributable infections as critical for a successful UTx program. In this chapter, we describe the epidemiology of infections after transplantation and especially in the context of UTx. The technical procedure, the specific properties of this organ including the presence of commensal microbial flora, and the prospect of future pregnancy shift the attention particularly toward fungal infections, adapted prophylactic strategies, and transfection of microorganisms and especially cytomegalovirus (CMV). We discuss the conditions affecting infection rate in solid organ transplant recipients and other immunocompromised patients and will separately consider bacterial, viral, and fungal infections with particular focus on UTx recipients. Finally, we will include the recently published data on pre-­transplant donor and acceptor screening, post-transplant prophylaxis and on infections reported so far across active UTx programs worldwide.

26.2 General Concepts 26.2.1 Immunosuppression and Infections The net state of immunosuppression is the most crucial factor, which determines the infection risk in solid organ transplant recipients next to environmental factors (Fishman 2017). This environmental exposure includes not only interaction with resident or commensal microbial organisms but also surgery, traveling abroad, contact with domestic animals, or residing in spaces with high risk of exposure to molds or fungi such as barns and attics (Fig. 26.1). The net state of immunosuppression is especially determined by the degree or level of immunosuppression and is not quantifiable (Fishman 2017). It also varies upon the duration of exposure to immunosuppressive drugs and translates into inhibitory effects on both humoral and cellular immunity (Fishman 2017) (Fig.  26.1). Hypogammaglobinemia and (especially CD4+ T cell) lymphopenia are respective exponents and potentially prognostic biomarkers (Fernandez-Ruiz et  al. 2014; Florescu 2014). Hypogammaglobinemia is especially associated with the development of fungal and viral infections including CMV (Florescu 2014). Other contributing factors are the presence of other immune-­ modulating viruses next to CMV such as HIV, zika virus (ZKV), and hepatitis C, disrupted muco-cutaneous barriers by catheters or drains or technical complications including wounds and fluid collections (Fishman 2017; Foo et al. 2017). Additional contributing pretransplant factors such as autoimmune disease, uremia, diabetes, liver cirrhosis, and malnutrition supposedly are not relevant for UTx recipients as these conditions preclude listing for transplantation. This theoretically implies that the infection risk of UTx recipients is lower than other solid organ transplant recipients, who are generally older and often have complicated medical histories and substantial comorbidity. Importantly, this theoretical advantage could be offset easily if immunosuppressive drug regimens incorporate polyclonal induction, advocate high

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Net state of immunosuppression Surgery Disruption of mucocutaneous barrier (catheters, lines, drains)

Epidemiological exposure Prophylactic drugs Bacteria

Viruses

Yeasts (fungi)

Immunosuppressive Drugs

Age Genetic background

Lymphopenia Neutropenia Hypogammaglobulinemia

Commensal flora (Candida) Potential donor transmission (CMV, bacteria, fungi) Environmental exposure (all micro-organisms) Reactivation (CMV) Immunomodulation (CMV,EBV, ZIKV,HCV)

Fig. 26.1  Etiopathogenesis of the increased infection risk in uterus transplant recipients. The net state of immunosuppression is determined by the surgery itself, preexisting characteristics but especially by immunosuppressive drugs of which the exposure is declining gradually after transplantation and which can cause predisposing immunological disturbances such as lymphopenia. Environmental exposure includes exposure to commensal and nosocomial microorganisms starting from the day of transplantation. Arrows in green depict inhibitory effects on these microorganisms, while arrows in red represent stimulatory effects

tacrolimus drug concentrations the first months after transplantation and include overtly aggressive anti-rejection treatment for borderline or mild rejections. Exposure of relatively immunocompetent people to immunosuppressive drugs generally translates into an increased infection risk and especially of severe infections. This was consistently observed for instance in people with psoriasis or rheumatoid arthritis according to registry data and meta-analyses of randomized controlled trials (RCT) (Ai et al. 2015; Downey 2016; Mabille et al. 2017; Minozzi et al. 2016). According to a systematic review of placebo-controlled RCT not only the risk of infection (OR 1.19; 95%CI of 1.10–1.29) but especially of opportunistic infections (OR 1.90; 95%CI of 1.21–3.01) was increased in patients with inflammatory bowel disease treated with biological agents (Bonovas et al. 2016). According to registry data from 6273 patients with Crohn disease, both prednisone therapy (HR = 1.57; 95% CI of 1.17, 2.10) and infliximab treatment (HR = 1.43; 95% CI of 1.11–1.84) were associated with an increased risk of serious infections (Lichtenstein et al. 2012). In a recent RCT in 262 patients with IgA nephropathy with a mean age of 39 years and mildly compromised kidney function, allocation to a high-dose corticosteroid regimen versus placebo resulted in renal protection but at the expense of serious infections in 8% leading to death in two subjects in the intervention group (Lv et al. 2017). This led to premature termination of the study despite beneficial renal effects (Lv et al. 2017). The occurrence of three cases of Pneumocystis jirovecii pneumonia (PJP) in the study group underpins the need for microbial prophylaxis upon installation of immunosuppressive drugs even in apparent immunocompetent subjects.

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The highest exposure to immunosuppressive drugs culminates shortly after induction therapy with concomitant exposure to boluses of corticosteroids and relatively high drug levels of the calcineurin inhibitors cyclosporine and especially tacrolimus the first weeks after transplantation. This archetypical timing of drug administration explains the increased risk of infection especially during the first weeks and months after transplantation. Peaks of especially respiratory and urinary tract infections (UTI) occur the first posttransplantation month with a major contribution of nosocomial and procedure-related infections due to leaks, postoperative wounds, indwelling (bladder) catheters, and mechanical ventilation, rather than donor-derived infections which are much more uncommon (Fishman 2017; Karuthu and Blumberg 2012; Kutinova et al. 2006). According to the classical timeline of transplantation-­related infections, opportunistic infections are not generally contracted during this time period but more typically appear from the second month on (Fishman 2017). They are more difficult to diagnose and treat and sometimes need prolonged courses of treatment even up to 12 months, with examples being Nocardia and Cryptococcal infections (Clark and Reid 2013; Henao-Martinez and Beckham 2015). Opportunistic infections, which are virtually absent in immunocompetent persons, are much more common in solid organ transplant recipients than in the normal population. According to registry data from kidney transplant recipients, opportunistic pathogens are the cause of infection-related deaths in 5% of all cases (Kinnunen et al. 2018). After 1 year posttransplantation, most infections are community-acquired in parallel with the general population albeit with a higher frequency and severity although opportunistic infections may appear even many years after transplantation (Fishman 2017; Karuthu and Blumberg 2012). In light of this, UTx recipients might still encounter opportunistic infections as long as they are exposed to immunosuppressive drugs.

26.2.2 Infections During Pregnancy Pregnant women, irrespective of intake of immunosuppressive drugs, are already more susceptible to infections at least for some particular viruses such as influenza and herpes simplex virus (HSV) the latter of which has the potential to disseminate even in immunocompetent women (Kourtis et al. 2014). Also the risk of invasive listeria infection especially in the third trimester is excessively higher than that in the general population (Kourtis et al. 2014). This increased infection risk is allegedly due to hormonal and immunological changes including a gradual decrease in especially T-cell and NK-cell activity during pregnancy (Kourtis et al. 2014). The concept of pregnancy as a state of systemic immunosuppression however seems outdated also considering the increased innate immunity and regulatory T cells during the last months of pregnancy (Pazos et al. 2012). Rather, pregnancy should be considered as a modulated immunological condition (Kourtis et al. 2014). Pregnancy-associated infections are of particular concern. Next to potential transfection due to some viral infections (such as CMV and

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Toxoplasma), hemodynamic alterations in the case of septicemia are also possibly harmful to the unborn child. Urinary tract infections are common during pregnancy partially because of urinary stasis due to an enlarging uterus, and may provoke preterm labor (Grio et al. 1994). According to registry studies, the risk of serious infections during pregnancy in women exposed to auto-immunosuppressive drugs including corticosteroids, anti-tumor necrosis factor, or biological agents for a diversity of autoimmune diseases including systemic lupus erythematosus (SLE) was 0.2% with a crude incidence rate between one and four per 100 patient years (Desai et al. 2017). Of note, high-dose corticosteroid was an independent risk factor for serious infections in this analysis (Desai et al. 2017). This explains why early embryonic transfer shortly after UTx, and especially when complicated by graft rejection, should be avoided not only for immunological reasons (Table 26.1).

Table 26.1  Strategies to decrease infection risk in uterus transplant recipients

Strategies General measures Avoidance of early embryo transfer (before 12 months posttransplantation) Avoid CMV-positive cadaveric uterus donors for CMV-negative acceptors Avoid living uterus donors residing in ZIKV endemic regions 6 months before donation Consider an urological assessment before transplantation (wait-listing) Culture preservation fluid especially with special attention for fungi Avoid cadaveric donors with meningitis of uncertain origin as cause of death Remove catheters (for instance, bladder catheter) as soon as possible Microbial prophylaxis Consider miconazole ointment to the cadaveric donor uterus PJP prophylaxis (6 m of TMP-SMX) Herpes simplex prophylaxis acyclovir three times 800 mg daily for 30 days (CMV donor and acceptor negative) CMV prophylaxis valganciclovir 900 mg daily for 100 days (CMV acceptor positive) CMV prophylaxis valganciclovir 900 mg for 200 days (CMV donor positive and acceptor negative) IV Piperacillin-tazobactam 4 g four times daily (3–5 days), antibacterial prophylaxis from the time of transplantation

Very early (pretransplant until first month)

Early (month 1–6)

Late (after 6 months)

NA

X

X

X

NA

NA

X

NA

NA

X

NA

NA

X

NA

NA

X

NA

NA

X

X

X

X

NA

NA

X X

X NA

NA NA

X

X

NA

X

X

X

X

NA

NA

(continued)

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Table 26.1 (continued)

Strategies Po Fluconazole 200 mg 1–2 times daily (5–10 days), antifungal prophylaxis from the time of transplantation Immunosuppression Avoid tacrolimus through levels >15 ng/mL Avoid tacrolimus through levels >10 ng/mL Avoid induction with polyclonal antibodies unless necessary for immunological reasons (immunized receptor) Avoid polyclonal antibodies as antirejection treatment as much as possible Avoid boluses of corticosteroids for borderline rejection Consider dose reduction of MMF/ azathioprine in case of leukopenia or severe lymphopenia

Very early (pretransplant until first month) X

Early (month 1–6) NA

Late (after 6 months) NA

X NA X

X X NA

X X NA

X

X

X

X

X

X

X

X

X

NA not applicable, CMV cytomegalovirus, ZIKV zika virus, PJP Pneumocystis jirovecii, TMP-­ SMX trimethoprim-sulfamethoxazole, po peroral, IV intravenous, MMF mycophenolate mofetil

26.2.3 Antimicrobial Prophylaxis Antimicrobial prophylaxis is warranted in solid organ transplant recipients including UTx recipients (Table  26.1). Thus, PJP prophylaxis by trimethoprim-­ sulfamethoxazole (TMP-SMX) is generally recommended for 6  months after transplantation and offers additional protection. It protects against the development of Toxoplasma gondii, Nocardia, and Listeria and possibly against other infections such as UTI (Giullian et al. 2010; Singh et al. 2015). Breakthrough Nocardia infections have been reported while on TMP-SMX prophylaxis despite proven sensitivity to this antibiotic, which could raise concerns about non-adherence or suboptimal dosing (Coussement et  al. 2016). The use of TMP-SMX is prohibited during pregnancy. In solid organ transplant recipients, it remains yet uncertain whether the preemptive strategy which advocates regular measurement of CMV viremia is non-inferior to universal CMV prophylaxis in those without donor positivity and acceptor negativity, where prophylaxis is mandatory (Caskurlu et al. 2019; Mumtaz et al. 2015). After implementation of a preemptive strategy, CMV infections occurring beyond 6 months after kidney transplantation were associated with a more aggressive course when compared to early infections (Ono et al. 2019). The situation for UTx recipients seems rather straightforward. CMV viremia without clinical symptoms should absolutely be avoided considering the prospect of early posttransplant conception with inherent risk of transfection and ensuing birth defects in the child. Therefore,

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most UTx centers will advocate CMV prophylaxis with valganciclovir for 100 days in the intermediate risk group (acceptor CMV positivity with or without donor positivity). In the high-risk group, 200 days of prophylaxis is associated with a lower infection risk of CMV infection the first 2 years after kidney transplantation (Humar et al. 2010). This time period falls within the time frame of possible conception in UTx recipients. CMV prophylaxis in solid organ transplant recipients also decreases the risk of not only other herpes but also of bacterial and protozoal infections (Hodson et al. 2013). Some UTx centers (Cleveland Clinic, Ghent) have adapted their allocation policy to avoid the CMV donor-positive/acceptor-negative constellation in cadaveric UTx recipients (Flyckt et al. 2016a). In CMV seronegative solid organ transplant recipients with a seronegative donor, many transplant centers advocate the use of acyclovir prophylaxis the first 4 weeks after transplantation which could be a valid strategy as well in UTx recipients considering the pathogenic role of herpes simplex in the development of cervicitis. The optimal short-term antibacterial prophylaxis on the contrary remains uncertain. Prophylactic antibiotics decrease the risk of postoperative infection including pelvic infections (RR 0.28; 95% CI 0.20–0.39) after both vaginal and abdominal hysterectomy (Ayeleke et  al. 2017). There is an ongoing controversy concerning which antibiotic should be administered before hysterectomy. The current evidence is mostly based on low to moderate quality studies from decades ago, and they cannot address this question properly (Ayeleke et al. 2017). As the infection rate after vaginal hysterectomy in theoretically immunocompetent women without prophylaxis is about 20–50% (Ayeleke et al. 2017; Mittendorf et al. 1993), prophylaxis in UTx recipients seems valid and should be well-targeted. As the lower genital tract is an area abundant with normal resident flora including anaerobic microorganisms (Larsen and Monif 2001), not only gram-positive aerobic bacteria should be targeted. The use of antibiotic prophylaxis in uncomplicated trans-cervical, intrauterine procedure remains of unproven benefit as there are currently no RCTs assessing the effects of antimicrobial prophylaxis on the infection risk (Minas et  al. 2014; Thinkhamrop et al. 2013). In immune-depressed UTx recipients, taking into account the long duration of surgery, the associated trauma, and the potential exposure to both aerobic and anaerobic commensal flora with contamination of uterine cavity with adjacent vaginal or cervical flora, a strategy encompassing broad-spectrum antibiotic prophylaxis seems warranted. Most UTx centers propagate short courses (1–5  days) of broad-spectrum antibiotics with anaerobic coverage mostly with piperacillin-tazobactam (Brannstrom et al. 2014; Chmel et al. 2019; Ejzenberg et al. 2019; Ozkan et al. 2013) or alternatively ceftriaxone together with ornidazole (Wei et al. 2017). Invasive fungal infections are other important causes of morbidity and even mortality in solid organ transplant recipients and especially in lung and small bowel transplant recipients, while they are less frequent in liver, heart, and especially kidney transplant recipients (Giannella et al. 2018; Pappas et al. 2010). This difference can largely be explained by the potential environmental exposure to fungi or molds as commensal pathogens, which can become direct pathogens in people with

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abolished host defense. Concerning UTx recipients, antifungal prophylaxis, especially directed against the commensal pathogen Candida albicans, could theoretically decrease the risk of early-onset uterine and/or systemic infections. A Candida infection of the graft led to early removal of the first UTx ever performed in the United States (Flyckt et al. 2016b). Invasive candidiasis occurs earlier than other invasive mycoses, mostly within the first 3 months and is being classified as nosocomial infection (Giannella et  al. 2018). Arteritis is a potential threat caused by fungal infection and this can develop into of a mycotic aneurysm with potential to rupture and become life-threatening (Tang et al. 2017). In UTx recipients, also the theoretical concern for graft transmission through the donor or by contamination of the preservation fluid seems genuine. In liver transplant recipients, prophylaxis for 3 or 4 weeks not only reduces colonization but also fungal infection and its related mortality according to current guidelines (Pappas et al. 2009). Taking into account the role of colonization with Aspergillus, almost all lung transplant centers have adopted an antifungal prophylaxis strategy which, according to a meta-analysis of observational studies, decreases the incidence of invasive aspergillosis, although the duration of prophylaxis remains controversial and is often extended (3–6 months) (Pilarczyk et al. 2016). In small bowel transplant recipients, antifungal prophylaxis is universal and fluconazole has been the agent of choice, for the duration of at least 4 weeks, until the anastomosis has completely healed and in the absence of rejection (Giannella et al. 2018; Silveira and Kusne 2013). In non-neutropenic kidney and pancreas transplant recipients, antifungal prophylaxis is not recommended. Some open questions concerning antimicrobial prophylaxis remain unresolved for UTx recipients. It is unclear how immunosuppression affects the normal bacterial vaginal flora and more particularly the potential regulatory role of lactobacillus species or other commensal pathogens. The duration of prophylaxis in UTx recipients remains elusive as reflected by the variable duration across transplant centers (Table 26.1). Also, the role of echinocandins should possibly be explored considering the efficacy of these drugs in prophylaxis according to RCT (Giannella et al. 2018). Echinocandins lack clinically significant drug interactions and harmful effects on endogenous adrenal production both of which are associated with the use of azoles (Giannella et al. 2018). Widespread fluconazole use can drive the emergence of azole-resistant non-albicans strains, which are still covered by echinocandins (Giannella et  al. 2018; Singh 2000). The currently applied antifungal prophylaxis in UTx recipients is mostly 5–10  days of fluconazole which is well below the evidence-based treatment duration (3–4 months) considered in lung or small bowel transplant recipients, whereas in other solid organ transplant recipients, the duration of prophylaxis is also less outlined (Patterson et al. 2016).

26.3 Bacterial Infections The “classical” respiratory infections and especially UTI are the mainstay of all bacterial infections after organ transplantation (Kutinova et al. 2006). In the preliminary published data, UTI were rather frequently reported in UTx recipients

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(without further grading of the severity of disease) albeit without healthy controls and thus without a clear estimate of the attributable risk in UTx recipients (Chmel et al. 2019; Ozkan et al. 2013; Testa et al. 2018b). We have previously observed that a high proportion (29%) of 14 screened potential recipients with Mayer-Rokitansky-­ Küster-Hauser (MRKH) syndrome listed for UTx in our center had a bladder residual volume of >100 mL on uroflowmetry (unpublished observation). This is not in line with what could be expected in healthy asymptomatic women of the same age group selected from the general population (N = 308) with a mean post-void residual volume of 2.92 ± 3.69 mL (Barapatre et al. 2009). Bladder dysfunction is not a recognized trait of MRKH, which is associated with various development abnormalities of the genitourinary tract, including renal agenesis and dysplasia (Ledig and Wieacker 2018). Our observation however mandates further investigation. Other more rare infections are expected to occur more often as well after transplantation. One important consideration is chronic endometritis, a persistent inflammation of the endometrial mucosa caused by both gram-positive and gram-negative bacterial pathogens with potential negative effects on fertility, implantation, and miscarriage frequency (Moreno et al. 2018). In line with this, CMV endometritis, diagnosed in the context of abdominal pain, was described in liver transplant recipients (Sayage et al. 1990). Overall, the incidence of endometritis in solid organ transplant recipients is not well specified. Molecular microbiology might be helpful in the detection of nonculturable microorganisms and hence become potentially relevant in UTx recipients (Moreno et al. 2018). Histological abnormalities include the infiltration of plasma cells in the presence of lymphoid cells, possibly mimicking rejection of the UTx graft, the latter being disclosed by serial surveillance biopsies of the cervix (Molne et al. 2017). In animal models, the infiltration of lymphocytes into the uterus endometrium is an early sign of rejection (El-Akouri et al. 2006). A failure to discriminate both problems after UTx could provoke unnecessary treatment of assumed rejection inflicting a deterioration of the infectious problem with possible implantation failure. Also bacterial cervicitis might complicate UTx. The Swedish UTx group have described an UTx complicated by cervicitis/uterine infection 1 month posttransplantation due to Enterococcus faecalis with the development of uterine abscess despite prolonged antibiotic treatment and repeated surgical drainages, and finally necessitating the removal of the graft at month 3 (Johannesson et al. 2015). This underpins that in UTx recipients and in contrast with other solid organ transplant recipients, infections might directly contribute to graft loss. Opportunistic infections after transplantation compose a minority of all bacterial infections, and there is no reason to believe this does not hold true as well for UTx recipients. Their diagnosis however can be challenging, and they often needs prolonged treatment upon diagnosis. A potential threat to the organ transplant recipient also includes mycobacterial infection. According to a meta-analysis of RCTs, mycobacterial infection is much more common in patients with rheumatoid arthritis exposed to biological therapies with an OR of 3.73 (95% CI of 1.7–8.13) versus placebo (Kourbeti et al. 2014). In transplant recipients, and especially in countries with a low endemic prevalence, the absolute risk of contracting mycobacterial infection remains rather low with a comparable increase in relative risk (Lopez de Castilla and Schluger 2010).

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26.4 Viral Infections In line especially with decreased cellular immunity, viral infections occur more frequently in organ transplant recipients. This holds especially true for herpes viruses of which CMV is the most renowned. Infection by CMV in immunocompromised subjects can cause a generalized disease with not only fatigue but also fever and tissue-invasive disease including hepatitis, central neurological involvement, retinal disease, esophagitis, colitis, myocarditis, and more rarely vasculitis or renal involvement (Fishman 2017). Also, CMV endometritis and cervicitis have been described in immunocompromised patients, and even in immunecompetent subjects (Abou and Dallenbach 2013; Frank et al. 1992; Giraldo-Isaza et al. 2011; Lusk and Konecny 2008). Next to direct effects, indirect effects of CMV should also be taken into account, and these are largely driven by the immunomodulatory properties of this virus which might explain the increased incidence of opportunistic infections, including invasive fungal disease, in organ transplant recipients (De Keyzer et al. 2011; Fishman 2017; Yong et al. 2018). Immunomodulatory effects, mediated by decreased T cell and phagocytic function and cytokine dysregulation, also include an increased risk of rejection, accelerated immune-senescence, and possibly higher risk of cardiovascular disease (Haidar and Singh 2017; La Rosa and Diamond 2012; Meijers et al. 2015). Reactivation or primo-infection during pregnancy in UTx recipients is challenging, considering the risk of transfection and the impossibility to treat CMV infection by ganciclovir, which has teratogenic effects. This drug should generally be administered intravenously for at least 2 weeks or longer until the patient has two negative CMV quantitative nucleic acids tests. Novel antiviral agents such as maribavir and brincidofovir are currently being tested as alternatives especially for ganciclovir-resistant strains (Haidar and Singh 2017). These teratogenic drugs however are no future options for the treatment of CMV infection during pregnancy. The alternative and currently available but less efficacious treatment is intravenous CMV hyperimmune immunoglobulins, of which the evidence to support its use is doubtful (Hodson et al. 2008). Induction with polyclonal antibodies in organ transplant recipients is associated with less rejection but at the cost of more CMV infection (Hill et al. 2017; Webster et al. 2010). Polyclonal induction induces impairs T-cell proliferation the first 2 years after transplantation and is associated with long-lasting CD4+ T cell lymphopenia for at least 5 years after transplantation (Weimer et  al. 2014). In the context of UTx, these findings seem important. CMV infection is a relevant and patient-centered outcome in UTx recipients. Following this line of reasoning, the constellation of donor-positive and acceptor-negative cadaveric UTx recipients should ideally be avoided, and the use of polyclonal antibodies should be restricted as much as possible. Other herpes viruses are also relevant in UTx recipients. Epstein-Bar virus (EBV), of which 90–95% of adults have protective immunity, can escape immune-­ surveillance upon immunosuppression and reactivate. Uncontrolled EBV viral replication due to decreased immune-surveillance can modify lymphocytic differentiation and proliferation, promoting the development of posttransplant lymphoproliferative disorder (PTLD), which ranges from lymphoid proliferation to

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overt lymphoma in which the monomorphic forms carry the worst prognosis (Dierickx and Habermann 2018; Green and Michaels 2013). The highest risk to develop PTLD is observed in donor-positive and acceptor-negative subjects, a rather rare but avoidable constellation (Dierickx and Habermann 2018). In case of EBV viremia, a preemptive approach consists of decreasing the overall immunosuppression, also considering that both acyclovir and ganciclovir have no activity against latent EBV (Haidar and Singh 2017). Also, herpes simplex is relevant for the UTx recipient. This virus very rarely causes disseminated disease in organ transplant recipients including hepatitis or esophagitis (Basse et al. 2008). Moreover, this sexually transmittable virus can cause cervicitis and local inflammation in the graft, possibly leading to graft loss as reported in one UTx recipient due to HSV-2 (Chmel et al. 2019). Of note, CMV prophylaxis with valganciclovir will also protect against herpes simplex infections the first months after UTx (Wilck and Zuckerman 2013). A theoretical concern in organ transplant recipients is the increased incidence of HPV viral infections, which are often of high-risk serotypes (Hinten et al. 2017; Meeuwis et al. 2015). In the Swedish UTx trial, one receptor had CIN2 lesions on a cervical biopsy 8  months posttransplantation with the presence of the high-risk HPV31 serotype (Johannesson et al. 2015). Closely related, the incidence of genital warts is almost five times higher in kidney transplant recipients than in the general population according to registry data (Larsen et  al. 2019). It is clear that HPV-­ induced lesions in both donor and acceptor should preclude transplantation (Favre-­ Inhofer et al. 2018). Meticulous surveillance of HPV-associated pathology is needed in organ transplant recipients including UTx recipients as lesions theoretically can develop more rapidly into (pre) malignant disease. It is clear that pretransplant HPV vaccination of the acceptor should be adopted in each UTx program (Johannesson et al. 2014). There is an ongoing doubt concerning the immunogenicity of this vaccine and whether acquired immunological protection does not decrease after transplantation. In kidney transplant recipients, the seropositivity of quadrivalent recombinant HPV vaccine was between 50 and 75% depending on the genotype, while it was 100% in control subjects with chronic kidney disease (Nelson et al. 2016). The best serological response in kidney transplant recipients was in those with the lowest through levels of tacrolimus, demonstrating the negative role of immunosuppression on the humoral response to vaccination (Nelson et al. 2016). The mosquito-borne zika virus (ZIKV) is another potential threat in pregnant women, with a history of traveling to endemic areas, considering its close association with microcephaly in particular in the newborn (Nogueira et al. 2017). It is also transmitted through blood and sexual contact (especially from men to women) raising awareness about this problem in the public domain (Mead et al. 2018). In immunocompetent people, the clinical picture, apart from the neurological symptoms, is mostly asymptomatic (80%) or mild and nonspecific with mild fever, rash, arthralgia, and conjunctivitis, mimicking other arboviruses such as dengue and chikungunya (Levi 2017). The course of a contracted infection with this immunomodulatory virus in immunocompromised people is less outlined. In a recently published case series, ZIKV infection in liver and kidney transplant recipients was not associated with neurological symptoms such as meningoencephalitis and Guillain–Barré

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syndrome but rather with worsening liver or kidney function, thrombocytopenia, and bacterial superinfection (Nogueira et al. 2017). The treatment of ZIKV infection is purely symptomatic, and awareness should exist to potential threats concomitant bacterial infections. Transmission through organ transplantation so far has not been reported (Haidar and Singh 2017). There is some controversy whether to accept solid organ donors with previous trips to endemic areas. Donor screening by real-time polymerase chain reaction (RT-PCR) has not been approved by the Food and Drug Administration (FDA), and wide-scale screening for ZIKV in blood donors was recently demonstrated to have a very low yield and to be very costly (Haidar and Singh 2017; Saa et al. 2018). The Dallas group has advocated avoidance of potential living donors of uterus grafts residing in endemic regions 6 months before donation (Testa et  al. 2018a). Given the potential of viral persistence in organs in the absence of viremia, there is certainly a need for the development of guidelines for deceased and living donor screening in UTx recipients (Levi 2017).

26.5 Fungal Infections The risk of developing fungal infections in organ transplant recipients is significantly higher than in immunocompetent people (Pappas et  al. 2010). The organ-­ specific risk is related to the degree of immunosuppression, including cumulative exposure to corticosteroids next to the occurrence of CMV infection but also to the particular exposure to commensals in the body or in the material environment (Fishman 2017; Lionakis and Kontoyiannis 2003; Yong et al. 2018). In line with this, especially lung and small bowel and to a lesser degree liver transplant recipients are more prone to fungal infections than kidney and pancreas recipients in which antifungal prophylaxis is not advocated (Pappas et al. 2010). Also, in UTx recipients in particular the yeast, Candida albicans is a potential threat, which urges the need for prophylactic treatment. In most UTx programs including the Swedish and the Lebanese, prophylaxis with fluconazole is administered for 5–10 days after transplantation (Chmel et al. 2019; Ejzenberg et al. 2019; Erman Akar et al. 2013). The potential dismal outcome due to yeast infections was illustrated by the early removal of an infected uterus graft by a Candida albicans strain (Flyckt et al. 2016b) and the development of a fungal cervicitis, with a small lesion of necrosis, at week 3  in the Lebanese program (unpublished data). Invasive candidiasis remains the most common invasive fungal infection following organ transplantation, and emerging non-albicans strains with variable sensitivity to antifungal therapy have the potential to alter current guidelines on prevention and treatment. One other concern in solid organ transplant recipients is the rare but potentially dangerous complication of fungal arteritis, which has been described in case series of affected kidney transplant recipients (Tang et  al. 2017). Fungal contamination seems to play an important role and can occur during organ retrieval, preservation, and/or transplantation (Tang et al. 2017). In UTx, attention should be paid toward this complication considering the presence of yeasts in the commensal vaginal flora. This also provides an argument not only in favor of antifungal prophylaxis but also of systematic

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cultures of preservation fluid in UTx recipients. Finally, the differential diagnosis with colonization is often difficult to achieve which especially holds true in lung transplant recipients (Herrera and Husain 2018). In this regard, the development of diagnostic and prognostic immunological markers may facilitate the therapeutic approach of fungal infections after transplantation (Herrera and Husain 2018). The usage of PJP prophylaxis significantly decreases the risk of PJP infection in the highest period at risk (the first 6  months after transplantation). Nevertheless, observational data indicate that infections often occur late (on average 6 years posttransplantation) and especially in older recipients with lymphopenia (Werbel et al. 2018). Considering the relative short time of exposure to immunosuppressive drugs in the younger UTx recipients, the risk of PJP infection should be estimated accordingly.

26.6 Donor Transmission Donor-derived infections are uncommon in solid organ transplant recipients but can never be excluded. They mostly occur early and especially the first 6 weeks after transplantation (Fischer 2019). Especially cadaveric donors with prolonged stay in the intensive care unit with ventilator-associated pneumonia and indwelling catheters at risk of colonization with fungi or multiresistant gram-negative bacteria (Fischer 2019). All but especially potential donors at risk of recently contracting transmittable viruses such as HIV and hepatitis B and C should be meticulously screened, although negative testing considering the lag time of seroconversion and the possibility of viral loads below the detection limit cannot guarantee completely safe organ transfer (Fishman 2017). Donor-derived infections are more likely to occur while using organs of cadaveric donors with unidentified meningoencephalitis (Trotter et al. 2016). Also, epidemiological exposure of the potential donor should be taken into account. Previous residence in countries with high prevalence of Strongyloides stercoralis should urge for serological screening of the potential donor and prophylactic treatment of the UTx receptor by ivermectin at the time of transplantation. Obviously, environmental risk assessment should incorporate local epidemiological data that can affect pretransplant screening and prophylaxis worldwide. Cadaveric donors of UTx in many centers are treated with vaginal miconazole ointments to promote the containment of C. albicans in particular.

26.7 Conclusions UTx recipients have increased risk of infections during the time frame in which they are exposed to immunosuppressive drugs. In parallel with other solid organ transplant recipients, there is an increased risk of bacterial, viral, and fungal infections. The latter is related to the exposure to commensal flora and especially Candida, which justifies antifungal prophylaxis. In UTx recipients, although aggregated

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safety data are still limited, it seems that infections per se can lead to graft loss, which is a rather unique constellation in organ transplantation. The role of microbial exposure on allo-immunization and rejection risk remains undetermined. Particular attention should be attributed to CMV, considering the potential deleterious role of transfection. This could urge the avoidance of CMV-positive donor grafts into CMV-negative acceptors considering the estimated risk despite prolonged prophylaxis for 200 days the first 2 years after transplantation, coinciding with embryonic transfer. However, the International Society of Uterus Transplantation (ISUTx) should strive to develop recommendations and guidelines taking into account the current knowledge in the field of infectious diseases after transplantation while integrating outcome data, which are generated by a universal and compulsory registry which is in progress.

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Larsen B, Monif GR. Understanding the bacterial flora of the female genital tract. Clin Infect Dis. 2001;32:e69–77. https://doi.org/10.1086/318710. Larsen HK, Thomsen LT, Haedersdal M, Dehlendorff C, Schwartz Sorensen S, Kjaer SK. Risk of genital warts in renal transplant recipients-a registry-based, prospective cohort study. Am J Transplant. 2019;19(1):156–65. https://doi.org/10.1111/ajt.15056. Ledig S, Wieacker P. Clinical and genetic aspects of Mayer-Rokitansky-Kuster-Hauser syndrome. Med Genet. 2018;30:3–11. https://doi.org/10.1007/s11825-018-0173-7. Levi ME. Zika virus: a cause of concern in transplantation? Curr Opin Infect Dis. 2017;30:340–5. https://doi.org/10.1097/qco.0000000000000384. Lichtenstein GR, et al. Serious infection and mortality in patients with Crohn’s disease: more than 5 years of follow-up in the TREAT registry. Am J Gastroenterol. 2012;107:1409–22. https:// doi.org/10.1038/ajg.2012.218. Lionakis MS, Kontoyiannis DP.  Glucocorticoids and invasive fungal infections. Lancet. 2003;362:1828–38. https://doi.org/10.1016/s0140-6736(03)14904-5. Lopez de Castilla D, Schluger NW. Tuberculosis following solid organ transplantation. Transpl Infect Dis. 2010;12:106–12. https://doi.org/10.1111/j.1399-3062.2009.00475.x. Lusk MJ, Konecny P.  Cervicitis: a review. Curr Opin Infect Dis. 2008;21:49–55. https://doi. org/10.1097/QCO.0b013e3282f3d988. Lv J, et al. Effect of oral methylprednisolone on clinical outcomes in patients with IgA nephropathy: the TESTING randomized clinical trial. JAMA. 2017;318:432–42. https://doi.org/10.1001/ jama.2017.9362. Mabille C, Degboe Y, Constantin A, Barnetche T, Cantagrel A, Ruyssen-Witrand A. Infectious risk associated to orthopaedic surgery for rheumatoid arthritis patients treated by anti-TNFalpha. Joint Bone Spine. 2017;84:441–5. https://doi.org/10.1016/j.jbspin.2016.06.011. Mead PS, Hills SL, Brooks JT. Zika virus as a sexually transmitted pathogen. Curr Opin Infect Dis. 2018;31:39–44. https://doi.org/10.1097/qco.0000000000000414. Meeuwis KA, et al. Cervicovaginal HPV infection in female renal transplant recipients: an observational, self-sampling based, cohort study. Am J Transplant. 2015;15:723–33. https://doi. org/10.1111/ajt.13053. Meijers RW, Litjens NH, Hesselink DA, Langerak AW, Baan CC, Betjes MG. Primary cytomegalovirus infection significantly impacts circulating T cells in kidney transplant recipients. Am J Transplant. 2015;15:3143–56. https://doi.org/10.1111/ajt.13396. Minas V, Gul N, Rowlands DJGS.  Role of prophylactic antibiotics in endoscopic gynaecological surgery; a consensus proposal. Gynecol Surg. 2014;11:153–6. https://doi.org/10.1007/ s10397-014-0841-9. Minozzi S, et al. Risk of infections using anti-TNF agents in rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis: a systematic review and meta-analysis. Expert Opin Drug Saf. 2016;15:11–34. https://doi.org/10.1080/14740338.2016.1240783. Mittendorf R, et al. Avoiding serious infections associated with abdominal hysterectomy: a meta-­ analysis of antibiotic prophylaxis. Am J Obstet Gynecol. 1993;169:1119–24. Molne J, Broecker V, Ekberg J, Nilsson O, Dahm-Kahler P, Brannstrom M. Monitoring of human uterus transplantation with cervical biopsies: a provisional scoring system for rejection. Am J Transplant. 2017;17:1628–36. https://doi.org/10.1111/ajt.14135. 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. https://doi.org/10.1016/j.ajog.2018.02.012. Mumtaz K, Faisal N, Husain S, Morillo A, Renner EL, Shah PS. Universal prophylaxis or preemptive strategy for cytomegalovirus disease after liver transplantation: a systematic review and meta-analysis. Am J Transplant. 2015;15:472–81. https://doi.org/10.1111/ajt.13044. Nelson DR, Neu AM, Abraham A, Amaral S, Batisky D, Fadrowski JJ.  Immunogenicity of human papillomavirus recombinant vaccine in children with CKD. Clin J Am Soc Nephrol. 2016;11:776–84. https://doi.org/10.2215/cjn.09690915. Nogueira ML, et al. Zika virus infection and solid organ transplantation: a new challenge. Am J Transplant. 2017;17:791–5. https://doi.org/10.1111/ajt.14047.

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Ono G, Medina Pestana JO, Camargo LFA. Late CMV infections after kidney transplantation under the preemptive strategy: risk factors and clinical aspects. Transpl Infect Dis. 2019;21:e13035. https://doi.org/10.1111/tid.13035. Ozkan O, et  al. 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. Pappas PG, et  al. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis. 2009;48:503–35. https://doi. org/10.1086/596757. Pappas PG, et  al. Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Network (TRANSNET). Clin Infect Dis. 2010;50:1101–11. https://doi.org/10.1086/651262. Patterson TF, et  al. Executive summary: practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis. 2016;63:433–42. https://doi.org/10.1093/cid/ciw444. Pazos M, Sperling RS, Moran TM, Kraus TA. The influence of pregnancy on systemic immunity. Immunol Res. 2012;54:254–61. https://doi.org/10.1007/s12026-012-8303-9. Pilarczyk K, et al. Is universal antifungal prophylaxis mandatory in adults after lung transplantation? A review and meta-analysis of observational studies. Clin Transplant. 2016;30:1522–31. https://doi.org/10.1111/ctr.12854. Saa P, et  al. Investigational testing for zika virus among U.S. blood donors. N Engl J Med. 2018;378:1778–88. https://doi.org/10.1056/NEJMoa1714977. Sayage L, Gunby R, Gonwa T, Husberg B, Goldstein R, Klintmalm G. Cytomegalovirus endometritis after liver transplantation. Transplantation. 1990;49:815–7. Silveira FP, Kusne S.  Candida infections in solid organ transplantation. Am J Transplant. 2013;13(Suppl 4):220–7. https://doi.org/10.1111/ajt.12114. Singh N. Antifungal prophylaxis for solid organ transplant recipients: seeking clarity amidst controversy. Clin Infect Dis. 2000;31:545–53. https://doi.org/10.1086/313943. Singh R, Geerlings SE, Bemelman FJ.  Asymptomatic bacteriuria and urinary tract infections among renal allograft recipients. Curr Opin Infect Dis. 2015;28:112–6. https://doi.org/10.1097/ qco.0000000000000120. Tang M, et al. Fifty-one cases of fungal arteritis after kidney transplantation: a case report and review of the literature. Transpl Infect Dis. 2017;19. https://doi.org/10.1111/tid.12781. Testa G, et  al. Living donor uterus transplantation: a single center’s observations and lessons learned from early setbacks to technical success. Am J Transplant. 2017;17:2901–10. https:// doi.org/10.1111/ajt.14326. Testa G, et al. Deceased donor uterus retrieval: a novel technique and workflow. Am J Transplant. 2018a;18:679–83. https://doi.org/10.1111/ajt.14476. Testa G, et al. First live birth after uterus transplantation in the United States. Am J Transplant. 2018b;18:1270–4. https://doi.org/10.1111/ajt.14737. Thinkhamrop J, Laopaiboon M, Lumbiganon P.  Prophylactic antibiotics for transcervical intrauterine procedures. Cochrane Database Syst Rev. 2013;(5):CD005637. https://doi. org/10.1002/14651858.CD005637.pub3. Trotter PB, Robb M, Hulme W, Summers DM, Watson CJ, Bradley JA, Neuberger J. Transplantation of organs from deceased donors with meningitis and encephalitis: a UK registry analysis. Transpl Infect Dis. 2016;18:862–71. https://doi.org/10.1111/tid.12621. Webster AC, et al. Interleukin 2 receptor antagonists for kidney transplant recipients. Cochrane Database Syst Rev. 2010;(1):CD003897. https://doi.org/10.1002/14651858.CD003897.pub3. Wei L, et al. 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;108:346–356.e341. https://doi.org/10.1016/j.fertnstert.2017.05.039. Weimer R, et al. ATG induction in renal transplant recipients: long-term hazard of severe infection is associated with long-term functional T cell impairment but not the ATG-induced CD4 cell decline. Hum Immunol. 2014;75:561–9. https://doi.org/10.1016/j.humimm.2014.02.015.

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Werbel WA, Ison MG, Angarone MP, Yang A, Stosor V.  Lymphopenia is associated with late onset Pneumocystis jirovecii pneumonia in solid organ transplantation. Transpl Infect Dis. 2018;20:e12876. https://doi.org/10.1111/tid.12876. Wilck MB, Zuckerman RA. Herpes simplex virus in solid organ transplantation. Am J Transplant. 2013;13(Suppl 4):121–7. https://doi.org/10.1111/ajt.12105. Yong MK, Slavin MA, Kontoyiannis DP.  Invasive fungal disease and cytomegalovirus infection: is there an association? Curr Opin Infect Dis. 2018;31:481–9. https://doi.org/10.1097/ qco.0000000000000502.

Indications and Surgical Technique for Hysterectomy After Uterus Transplantation

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Pernilla Dahm-Kähler, Mats Brännström, and Niclas Kvarnström

27.1 Introduction Uterus transplantation (UTx) is a novel infertility treatment and type of transplantation, which has proved to be successful in several settings worldwide, both after live donor and deceased donor UTx. There are a number of distinctive features with UTx in comparison to the traditional solid organ transplantations. Thus, UTx is a type of transplantation that is performed to create new life within the recipient, rather than to save the life of the recipient. Moreover, all traditional solid organ transplants are intended for life-long use, but UTx is instead an ephemeral type of transplantation. Thus, the uterine graft should be removed by hysterectomy, after the recipient has delivered one or more children. This type of hysterectomy is what is planned prior to UTx, but there can be several other situations that necessitate a hysterectomy (Brannstrom et  al. 2014; Testa et  al. 2017) as also outlined below. Each specific situation of hysterectomy will come with its medical and psychological issues for the patient.

P. Dahm-Kähler Department of Obstetrics and Gynecology, University of Gothenburg, Gothenburg, Sweden e-mail: [email protected] M. Brännström (*) Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] N. Kvarnström Department of Transplantation, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_27

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Hysterectomy is the most common gynaecological surgical procedure performed worldwide (Committee on Gynecologic Practice 2017), and approximately 400,000 hysterectomies are performed annually in the USA (Topsoee et  al. 2016; Wright et al. 2013). Nevertheless, the hysterectomy performed after UTx is different from an ordinary hysterectomy for various reasons such as different location of the uterine arteries and veins and the requirement of complete removal of the uterine graft vessels in order to minimize future immunization of the recipient. Moreover, dense adhesions will in some cases be present, and the ureters are usually dislocated to an atypical anatomical location.

27.2 General Principles and Indications of Hysterectomy There are some special features with UTx in relation to graft removal. The graft is intended for use for a limited time, but the true functionality of the graft is not shown until several months after transplantation. Although regular menstruations and ultrasound-verified growth of the endometrium during the ovarian cycle are indications of a functional uterus, the true functionality is not shown until attempts for achieving pregnancy take place. Traditionally, we have delayed embryo transfer (ET) attempts until 12 months after UTx, in order to avoid pregnancy at a time of an increased risk for acute rejection episodes. Some groups, with reported successful UTx procedures, in terms of live births, initiated ET after 6 months, and accordingly we have changed our conservative approach to performance of ET 10 months after UTx. The full potential of the transplanted uterus will only be proven when a pregnancy has gone to term and a baby has been delivered. The time it takes for pregnancy and delivery of one or more babies have to be balanced so that the time of exposition of the recipient to immunosuppression is minimized. There exist several indications to perform a hysterectomy after UTx, and those are listed and discussed below. One obvious reason for hysterectomy is an early post-transplantation complication, most typically a vascular complication especially thrombosis of the vessels of the graft. Attempts can be made to perform thrombectomy including potential corrections of anastomotic sites. A bilateral persistent thrombosis will later be evident by a necrotic appearance of the cervix at gynaecological examination although the uterus may be viable after a unilateral thrombosis or an immediate successful thrombectomy. This type of complication typically occurs during the initial 2 weeks. Hysterectomy is often easily performed at this stage since there has been no firm adhesion or regrowth of tissue between graft and recipient. Another cause for hysterectomy before attempts of pregnancy is that there is clear evidence of non-functionality of the uterine graft. An early sign may be that the blood flow to the uterus is decreased, indicating a hypoperfusion. The lowered uterine blood flow will primarily affect the central part of the uterus, since the blood vessels, with the spiral arteries, are penetrating the uterus from the serosal aspect, through the myometrium and with an extensive network of arterioles and capillaries in the endometrial stroma. This low blood flow will in most cases be harmful to the

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endometrial lining of the uterus. Thus, menstruations will not occur, and there will be no signs of endometrial growth on trans-vaginal ultrasound scan. Attempts should be made to induce endometrial growth by exogenous estradiol if no spontaneous endometrial growth is seen. Hysteroscopy, with inspection and resection of small areas of the wall of the endometrial cavity, should also be considered and performed. If there are no visual signs of viable endometrium and if histopathological findings of biopsies show absence of endometrium, a final and definite decision to perform elective hysterectomy may be performed. One cause to perform hysterectomy before or after pregnancy is a severe endometritis. In our second attempt of the original Swedish UTx trial, the recipient had signs of endometritis some months post-transplantation (Brannstrom et al. 2014). The endometritis could not be treated successfully despite intense i.v. antibiotic therapy and repeated attempts of surgical draining of the developing abscess. The infectious agent was a faecal bacterium. Signs of septicaemia developed and the uterus was removed 3.5 months after transplantation. The uterus had not shown any spontaneous menstruations after UTx. Another cause of untimely hysterectomy is an acute or chronic rejection, which is resistant to therapy resulting in irreversible damage to the uterus. Rejection episodes are most frequent during the initial 8 months after UTx but may occur also later and during pregnancy. In our hands all (>30) rejection episodes that we have experienced have been reversible upon either interval steroid treatment, increased levels of the calcineurin inhibitor used or in one case necessitating thymoglobulin treatment. A progressively rejecting uterus will first show severe inflammation and then tissue necrosis. This can be verified on tissue biopsies from the ectocervix in combination with core biopsies from the myometrium that can be obtained through ultrasound-guided trans-abdominal approach or vaginally. One reason for early hysterectomy can also be severe somatic disease, which is incompatible with future pregnancy or continued immunosuppression. Examples of these somatic diseases are malignancy, severe heart disease, and severe, chronic lung disease. The immunosuppression given to avoid rejection of the uterine graft has specific side effects. In exceptional cases, these side effects may lead to that a premature hysterectomy is recommended. The serious side effects are mainly related to decreased kidney function, susceptibility for serious infections, development of diabetes, and malignancy. A predictable cause of premature hysterectomy is when the uterus has not achieved a pregnancy with delivery of a healthy baby in an optimally defined time frame after UTx. This can be due to multiple implantation failures. In our personal experience, we had to wait until almost ten ET attempts in some patients to achieve a viable pregnancy. It is known from accumulated data in the usual IVF setting that the cumulative pregnancy rate increases with each ET attempt well over 20 attempts. Thus, more than 20 attempts with ET of good quality embryos should be performed before a transplanted uterus is considered to have an inherent implantation failure. There will also be patients that experience repeated miscarriages. These patients should be encouraged to go on with repeated pregnancy attempts, but out of physiological and medical

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issues, hysterectomy would be recommended after an excessive number (>8–10) of miscarriages, in cases where hysteroscopy and other miscarriage-related investigations have been performed to find any treatable pathology. To rule out embryo-specific causes of multiple implantation failures or repeated miscarriages, pre-genetic testing of aneuploidy could be considered on blastocysts, and there is also the possibility to use donor oocytes. The most obvious cause to perform hysterectomy is when the uterine graft has delivered the number of desired babies. So far, some patients have delivered twice from a transplanted uterus, but in the future it is likely that three and four deliveries will take place from a transplanted uterus. We have chosen to do hysterectomy in direct conjunction with the C-section and birth of a second child, but have waited for around 3 months with hysterectomy after initial childbirth, in cases that the woman only desires one child or if medical issues have made us to recommend that the graft should be removed. This waiting period for some months will of course expose the woman to an additional surgery, but we believe it is important to have some observation period of the baby to ascertain good health. The limiting factor for number of childbirths before hysterectomy is the side effects by the immunosuppression medication including the risk for a progressive decline in kidney function, as well as increased risk for serious infections, hypertension, diabetes, and certain malignancies. There is a delicate balance between these risks and the desire of the woman to have several children. In our hands we have generally recommended that the graft should be removed after 5–6 years, in order to avoid serious long-term side effects of immunosuppressants. One should be aware that a transplanted woman may want to have removal of the graft at any time, as has been seen in hand transplantation. Hysterectomy in these cases should only be performed after meticulous counselling of doctors and psychologist, in order to ascertain that the patient has understood the impact of a hysterectomy and that the decision is on solid grounds.

27.3 T  echnique of Hysterectomy Surgery After Uterus Transplantation As mentioned above, hysterectomy of a uterine grafts can be performed as an acute or elective procedure and in some cases also immediately after a caesarean section. Preoperatively, insertion of ureter stents is recommended to facilitate the identification of the ureters in order to avoid demanding dissection and accidental injury to the ureters. This is preferably performed during anaesthesia just prior the hysterectomy surgery. The uterine vessels should be completely and safely removed. This is up until today simplest achieved by a laparotomy approach. The surgical procedure starts with the preferred skin incision, most often a subumbilical midline incision in the same incision line as the incision at UTx. After opening the abdomen, a thorough anatomical investigation of the uterus including the cervix and vaginal cuff, uterine vessels, bladder, and ureters should be performed.

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The round ligaments should be divided bilaterally, and opening of the bladder peritoneum will follow, with dissection of the bladder from the uterus and the ventral part of the cervix. The uterine vessels will in most cases have an abnormal anatomical location and are typically located ventrally, since the anastomoses have been made to the external iliac vessels, with a shorter distance from their origin to their attachmernts above the ureters. We have experienced uterine arteries that are attached to the ventral surface of the uterus, rather that entering the uterus from a lateral angle. All sites of vessel anastomosis to the recipient should be identified by careful dissection. The arterial anastomosis is preferably first dissected and the complete uterine artery removed from the inlet from the external iliac artery. A small cuff of the graft artery adjacent to the anastomoses may remain in order to safely close the vessel without narrowing of the lumen of the external artery. Thereafter, in most cases the vein anastomosis is easier to visualize and access in the deep pelvic cavity. The vein is gently dissected to the inlet of the external vein and removed in a similar fashion to the described principle of the artery. The procedure is performed with repetitive exact identification of the ureter and thereafter repeated on the other side. The next step is to follow the vessels to the uterus and detaching the uterus and parametrium including the uterine vessels down to the cervix and to identify the vagina. The vagina is opened and the uterus should then be removed after which the vagina is closed by a running suture. Before closing the abdomen, the cavity is again inspected and haemostasis is established and performed. There is normally not an indication for a surgical drain.

27.4 Complications In any surgery or hysterectomy performed, complications may occur (Makinen et al. 2001; Wallace et al. 2016), and the severity of the complications are due to the surgical procedure and complexity performed as well as the patient’s risk factors (Bohlin et al. 2016; Mehta et al. 2017). The rate and type of complications at hysterectomy post-UTx is still unknown since very few cases have been published. Our personal experience of eight cases is that one patient acquired a vaginal cuff hematoma and another patient got an incision hernia more than 1 year after hysterectomy. One may consider the risks of post-UTx hysterectomy at least as large as after hysterectomy performed on benign indication, which is around 7–8% (Wallace et al. 2016). The recipient, although perfectly healthy when transplanted, has typically been immunosuppressed for some years and thus will be more susceptible for infections and may also have impaired wound healing. The surgery should therefore be performed by experienced surgeons with detailed knowledge of the anatomy of the transplanted uterus and also understanding of the complications related to the immunosuppression. The registry from the International Society for Uterus Transplantation (ISUTx) will in the future reveal specific complications and rates to be considered.

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References Bohlin KS, Ankardal M, Stjerndahl JH, Lindkvist H, Milsom I. Influence of the modifiable life-­ style factors body mass index and smoking on the outcome of hysterectomy. Acta Obstet Gynecol Scand. 2016;95:65–73. Brannstrom M, Johannesson L, Dahm-Kahler P, Enskog A, Molne J, Kvarnstrom 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. Committee on Gynecologic Practice. Committee opinion no 701: choosing the route of hysterectomy for benign disease. Obstet Gynecol. 2017;129:e155–e59. Makinen J, Johansson J, Tomas C, Tomas E, Heinonen PK, Laatikainen T, Kauko M, Heikkinen AM, Sjoberg J.  Morbidity of 10 110 hysterectomies by type of approach. Hum Reprod. 2001;16:1473–8. Mehta A, Xu T, Hutfless S, Makary MA, Sinno AK, Tanner EJ 3rd, Stone RL, Wang K, Fader AN. Patient, surgeon, and hospital disparities associated with benign hysterectomy approach and perioperative complications. Am J Obstet Gynecol. 2017;216:497.e1–97.e10. Testa G, Koon EC, Johannesson L, McKenna GJ, Anthony T, Klintmalm GB, Gunby RT, Warren AM, Putman JM, dePrisco G, Mitchell JM, Wallis K, Olausson M. Living donor uterus transplantation: a single center’s observations and lessons learned from early setbacks to technical success. Am J Transplant. 2017;17:2901–10. Topsoee MF, Ibfelt EH, Settnes A.  The Danish hysterectomy and hysteroscopy database. Clin Epidemiol. 2016;8:515–20. Wallace SK, Fazzari MJ, Chen H, Cliby WA, Chalas E. Outcomes and postoperative complications after hysterectomies performed for benign compared with malignant indications. Obstet Gynecol. 2016;128:467–75. Wright JD, Herzog TJ, Tsui J, Ananth CV, Lewin SN, Lu YS, Neugut AI, Hershman DL. Nationwide trends in the performance of inpatient hysterectomy in the United States. Obstet Gynecol. 2013;122:233–41.

The Future Expansion of Patient Groups for Uterus Transplantation

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Steven Weyers and Petra De Sutter

It is difficult to discuss about new indications for uterus transplantation (UTx), while we have not yet figured out what the current indications today are. Do we already agree on which causes of uterine infertility qualify for UTX? Up till now, worldwide, more than 50 UTx procedures have been performed, the indications on which there is more or less a consensus at present being congenital absence of the uterus and post-hysterectomy. It is, however, clear that there is still a lot of controversies around what are good or acceptable indications for UTx. Uterine factor infertility (UFI) stems from the anatomical or physiological inability of a uterus to sustain gestation; it is a condition that affects about 5% of all infertile couples. Often the term absolute uterine infertility (AUFI) is used, thus indicating that the chance of pregnancy is zero. The condition of UFI can have anatomical or physiological causes. Typical anatomical causes are the congenital absence of the uterus or the presence of a congenital abnormal uterus (primary UFI), but also the absence of the uterus as a result of a hysterectomy and the presence of a defective uterus as a result of Asherman syndrome or multiple fibroids (secondary UFI) are common anatomical causes. Examples of physiological UFI causes are conditions such as endometrial non-­ receptivity (primary or secondary), adenomyosis, or other uterine diseases (secondary). Only in case of an absent uterus we can truly talk of AUFI. All of the above groups are relatively small. However, if we take them together, they make up a large group. That will be discussed below in this chapter. In 2012, after the first two cases of UTx in human, the Montreal criteria for ethical feasibility of uterine transplantation were drawn up (Lefkowitz et  al. 2012). Concerning the recipient the following recommendations were made: it should be a genetic female of reproductive age with no medical contraindication for S. Weyers (*) · P. De Sutter Department Obstetrics, Gynaecology and Reproductive Medicine, Ghent University Hospital, Ghent, Belgium e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_28

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transplantation, who has documented congenital or acquired UFI which has failed conservative therapy, has a personal or legal contraindication for surrogacy or adoption and desires a child or seeks UTx solely as a measure to experience gestation with an understanding of its limitations. Furthermore, the candidate should be psychologically stable and suited for motherhood and responsible enough to consent, informed enough to make a responsible decision and not under coercion. Approximately, a year later these guidelines were slightly updated, adding a phrase about the compliancy to take immunosuppressive medication and stay under the follow-up of the transplantation team (Lefkowitz et  al. 2013). Moreover, ‘other populations’ were also discussed, namely, transgender patients, people with body image identity disorders and people from low-income pronatalistic countries/societies. Important questions, however, still remain: should we give recommendations about the removal of the uterus after one or two successful pregnancies? Should we only treat women who have their own oocytes, or can donor oocytes be used? What about women who already have children? What about 46XY women with complete androgen insensitivity syndrome (AIS) and what about other XY women? When addressing these questions, we must keep in mind that there is indeed an ethical tension between the principles of ‘non-maleficence’ and ‘autonomy of the individual’ (Olausson et al. 2014). In our role as medical professionals, the principle of ‘non maleficence’ is of utmost importance. Thus, we have to keep in mind that UTx is a potentially life-­ threatening experimental treatment without the aim of improving the physical health of the individual, let stand save its life. That individual, however, has the fundamental right to reproduce even if there are risks involved. Nowadays, we do treat women with pre-existing medical conditions (congenital heart disease, cystic fibrosis, solid organ transplant recipients, etc.) that pose danger to themselves and/or their prospective foetuses/children. As physicians we always have to weigh the potential benefits (even if psychosocial) of an intervention against its biophysical risks. Of course, UTx will only be introduced as a routine treatment option of UFI after research finds it reasonably safe. Up until today UTx has been performed in women with the Mayer-Rokitansky-­ Kuster-Hauser (MRKH) syndrome (congenital) and women who have had hysterectomy (acquired). The other causes of acquired UFI are quite a difficult group in relation to indication for UTx. First of all, in most cases, the uterine infertility which results is not absolute but relative, such as women with Asherman syndrome, adenomyosis and fibroids. Women with these conditions do get pregnant and often have successful pregnancies. Moreover, if we would keep to the Montreal criteria, conservative treatments have to be unsuccessful. But what is the definition of failed conservative treatment? How many years of failed conception should they undergo before UTx can be brought up as an alternative. How many documented pregnancy losses should they have? As for other non-life-saving transplantations (face, hand, larynx, penis), the indication for this group of relative UFI will always be debatable. Even more difficult is the question of UTx in other types of congenital absence of the uterus, namely, the persons with disorder of sexual differentiation (DSD) and

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transgender individuals. Several arguments against performing this procedure in this group of patients can be put forward, and some of them are mentioned in the updated version of the Montreal criteria for the ethical feasibility of uterine transplantation (Lefkowitz et al. 2013). Up until today no experiments (in animal nor in human) involving UTx to non-­ genetic female individuals have been performed. What makes a trans-woman anatomically different from, for example, a biological woman born without a uterus? A trans-woman lacks the presence of a uterine vascularisation. This is also the case in women with MRKH, where the vascular anastomoses of the uterine vessels are done at the level of the external iliac vessels. Trans-women (like some DSD-women) do not have ovaries making them non-capable of carrying their own genetic offspring. But this is also the case in many other less conventional reproductive situations such as biological women getting pregnant through oocyte donation, lesbian couples where one partner carries the child originating from the oocytes of the other or gay men using an oocyte donor and surrogate mother. Trans-women will have the necessity for appropriate hormonal replacement therapy (HRT) throughout pregnancy. However, this is also the case in pregnancies in women without ovaries. Moreover trans-women are continuously on HRT. What about the placement of a uterus in a male type of pelvis? About 20% of biological women also present with a so-called android pelvis, with the only reproductive consequence for them being that they have a higher risk of getting a caesarean section. Probably of most importance is the fact that in transgender patients the vagina is skin-lined or made from intestinal mucosa. This could possibly increase the risk of intrauterine infections both before and during pregnancy. What other arguments can be found in favour of UTx in this group? Well, at first sight no ethical reason really counts why DSD and transgender individuals should be deprived of having the possibility to carry their own child. Both DSD women and trans-woman are legally female and unequivocally suffer from AUFI. Moreover, the principle of autonomy is not sex-specific. We already mentioned the right to procreate and this right indeed is not absolute, but this equally counts for MRKH or post-­ hysterectomy women. And the medical and surgical risks for trans- and DSD women are the same as in biological women, and so are the risks for their future child. Maybe even more controversial would be a person who, for example, after hysterectomy, requires a UTx procedure to feel completely as a woman without having a child wish. On one hand, we have to realise that the ethicality of many medical interventions does not solely depend on the purpose and/or motivation of the patient. On the other hand, when weighing the ‘non-maleficence’ principle against ‘autonomy of individual’, there is one major argument against doing performing UTx in this group, namely, the risk of life-long immunosuppressive therapy. To exclude this group the Montreal criteria state that the only motivation should be to gestate a pregnancy. There is one group, without any doubt the most controversial one, concerning indication of UTx, and that is UTx in men. Some authors state that the possibility of carrying offspring should not be limited to women (born or not born female) and that men could also claim this right. In a recent paper from Timothy Murphy from

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Chicago he states that ‘pregnancies in men cannot be totally excluded’, but also that ‘only a focused line of research could answer that question, after the evidence becomes clearer about what UTx can do for non-transgender women with absolute uterine infertility’ (Murphy 2015). In conclusion, it is really important to realise that UTx up to date still is merely an experimental technique, and a lot of future research is necessary to confirm its success and safety. Moreover, since no ‘typical’ groups have been defined, ‘expansion to other groups’ is in fact no real issue at this moment. Indications for its use will have to be defined once UTx is accepted as ‘common practice’ (and covered by health insurance systems) and could very well differ from country to country. For the time being, while still in an experimental phase, restricting its use to genetic women with AUFI would indeed probably be wise. However, it could very well be that within one or two decades, UTx will be a possibility for all women with UFI and also men wishing to gestate a pregnancy.

References Lefkowitz A, Edwards M, Balayla J. The Montreal criteria for the feasibility of uterine transplantation. Transpl Int. 2012;25:439–47. Lefkowitz A, Edwards M, Balayla J. Ethical considerations in the era of the uterine transplant: an update of the Montreal criteria for the ethical feasibility of uterine transplantation. Fertil Steril. 2013;100:924–6. Murphy T. Assisted gestation and transgender women. Bioethics. 2015;29:389–97. Olausson M, Johannesson L, Brattgård D, et  al. Ethics of UTx with live donors. Fertil Steril. 2014;102:40–3.

The Bioengineered Uterus: A Possible Future

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Mats Hellström and Mats Brännström

Abbreviations 3D Three-dimensional ECM Extracellular matrix ES Embryonic stem (cells) GFP Green fluorescent protein HHP High hydrostatic pressure iPS Induced pluripotent stem (cells) MSCs Mesenchymal stem cells SDS Sodium dodecyl sulfate STAT3 Signal transducer and activator of transcription 3 UTx Uterus transplantation M. Hellström (*) Laboratory for Transplantation and Regenerative Medicine, Institute of Clinical Science, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Department of Obstetrics and Gynecology, Institute of Clinical Science, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden e-mail: [email protected] M. Brännström Laboratory for Transplantation and Regenerative Medicine, Institute of Clinical Science, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Department of Obstetrics and Gynecology, Institute of Clinical Science, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden Stockholm IVF-EUGIN, Stockholm, Sweden e-mail: [email protected] © Springer Nature Switzerland AG 2020 M. Brännström (ed.), Uterus Transplantation, https://doi.org/10.1007/978-3-319-94162-2_29

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29.1 Introduction The successful births of healthy babies from recipients with a uterine allograft are exceptional outcomes after the world’s first controlled human clinical trial (Brännström et al. 2014, 2015). The initial results from the global uterus transplantation (UTx) society are both positive and negative with much focus on the choice and quality of the donor source (old/young, live/deceased donor) and modifications to the immunosuppressive treatment regime following engraftment. Donor source and immunosuppression are central issues that play essential parts concerning success or failure following UTx. The live donor approach has multiple advantages compared to a deceased donor model since it facilitates logistical challenges and consequently minimizes the cold ischemia injury and allows specialized physicians to conduct the surgery. Furthermore, the medical history of the donor and proof-of-­ function of the donated uterus can be considered. However, the complicated and risky live donor surgery is a major disadvantage. Therefore, several groups are currently investigating the relevance of using deceased multi-organ donors for UTx instead of using live donors (Testa et al. 2018) and has thus far resulted in one birth of a healthy baby (Ejzenberg et al. 2019). Independent of choice, the recipient will have to remain under strict observation for possible organ rejection events and must follow the arranged immunosuppressive treatment protocols. These drugs can protect the graft from rejection and ultimately deterioration, but have side effects such as the induction of atherosclerosis, nephrotoxicity, hypertension, diabetes, and reduced fertility in the recipient (Azimzadeh et al. 2011). Novel ideas using bioengineering and a uterus graft derived from the patient’s own cells may overcome problematic issues such as donor source and immunosuppression. Significant progress concerning organ reconstruction, stem cells, and tissue engineering have been made in experimental animal studies during the last decade (Badylak et al. 2011; Crapo et al. 2011), including on uterine tissue (Hellström et al. 2017; Campo et al. 2017b). Thus, this donor option may become a future clinical reality.

29.2 C  ombining Stem Cells and Biomaterials for Tissue Reconstruction Tissue engineering, or bioengineering, is a multidisciplinary research area in regenerative medicine that combines knowledge from stem cells, biomaterials, transplantation, and many other areas with the aim to produce customized autologous implants/ grafts to improve or restore tissue function. To date, this field is dominated by various bone- and skin regeneration applications where significant clinical progress has been made. Constructs for artificial heart valves and blood vessels have also been broadly examined and a wide range of scientists and clinicians now explore bioengineering applications as a utility to stimulate tissue repair, or as an allograft substitute for various transplantation procedures (Peloso et al. 2015; Feinberg 2012).

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29.2.1 Stem Cells The most popular types of stem cells used for tissue engineering include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and mesenchymal stem cells (MSCs). All these cell types have the ability to proliferate into large amount of cells, but have their unique advantages and disadvantages that should be considered. For example, pluripotent ES cells are an attractive cell source since they can differentiate into any type of adult tissue. However, to obtain a specific homogenous cell population using ES cells is challenging since it requires a precise cocktail of growth- and differentiation factors to avoid the formation of cancer-like teratomas that include a range of different cell phenotypes. Furthermore, these cells are not autologous since they are derived from a donated aborted embryo and may therefore induce an immune rejection after cell transfer. The use of ES cells may also cause some ethical concerns regarding their origin. Therefore, many scientists now prefer using iPS cells. These cells can be obtained via a blood sample or through a skin biopsy and are thus autologous. The isolated cells can then be reprogramed in vitro to become a pluripotent stem cell by, e.g., the introduction of the Yamanaka factors (by inducing an overexpression of Oct3/4, Sox2, cMyc, and Klf4) (Takahashi and Yamanaka 2006) and thus turning somatic cells into an astonishing similarity to ES cells. For these reasons, iPS cells are interesting for various tissue engineering applications and have been used to create a functional human liver-like tissue structure in a mouse model (Takebe et al. 2013). However, overexpression of the transcription factors and the redifferentiation process performed in vitro still need to be proven safe for clinical use since it may induce potentially detrimental phenotypic- or epigenomic changes. The MSCs are possibly the most comprehensively studied stem cell type. MSCs are multipotent and thus slightly easier to guide into the correct phenotype but are less versatile compared to ES cells or iPS cells. However, they can still be differentiated into both soft and hard tissues and can be harvested from bone marrow, cartilage, adipose- or fibrous connective tissue. Furthermore, MSCs can be isolated directly from the patient, expanded and/or differentiated in vitro, and then be transplanted back to the patient for the therapeutic purpose without causing a negative immune response. They are also popular due to their unique beneficial immune modulating effect and have shown to reduce the host–foreign–body response to biomaterials and to be involved in tissue repair and regeneration (Anasiz et al. 2017; Borger et al. 2017; Li and Hua 2017). Uterine stem cells have been identified in several mammals, including the mouse, rat, sheep, and human (Cervello et al. 2015a, b; Ono et al. 2007; Ulrich et al. 2013; Emmerson and Gargett 2016; Masuda et al. 2010). These MSCs-like uterus stem cells can be harvested by an endometrial biopsy and be expanded to high clonogenic numbers. Hence, this cell source may be suitable for the development of transplantable autologous uterine constructs derived from decellularized uterus tissue.

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29.2.2 Biomaterials For decades, various biomaterials made of metals such as stainless steel, titanium, or cobalt alloys have successfully been used in the clinic to improve recovery after trauma. Several examples include dental or hip implants, stents, meshes, heart valves, and bone replacements (Prakasam et al. 2017). For soft tissues however, various polymers may be more suitable for tissue engineering applications since they have lower density than metallic products and can be made biodegradable. Furthermore, both synthetic and natural polymers can be used to create hydrogels in specific shapes that are able to absorb and retain large quantities of water similar to normal tissues. Hydrogel is thus a promising supporting material for wound healing, drug delivery systems, muscle, bladder, and ovary reconstruction (Vedadghavami et al. 2017; Atala et al. 2006; Shea et al. 2014) and can be used as ink for novel three-dimensional (3D) bioprinting applications (Datta et al. 2017). However, hydrogels created from natural polymers (e.g., alginate, fibrin, collagen, or hyaluronic acid) have a tendency to lack mechanical strength while synthetic polymers (e.g., polyethylene glycol or polyacrylamide) need to be modified to become bioactive (Vedadghavami et al. 2017). Thus, hydrogels generally lack the complex composition of interwoven extracellular matrix (ECM) macromolecules that provide both mechanical and biochemical support to surrounding cells. Therefore, generating organ-specific biomaterials through a process called decellularization has become popular (Hellström et  al. 2017). This process involves a procedure that starts with a donor tissue/organ that is exposed to various detergents, ionic solutions, enzymes, and/or mechanical forces followed by extensive washing protocols to remove all immunogenic cellular components. The remaining structure after such treatment comprises the organ-specific ECM with the 3D-appearance of the original structure that contains patent conduits for blood vessels that enable anastomoses for in  vivo downstream applications (Padma et  al. 2018; Hellström et  al. 2014; Miyazaki and Maruyama 2014). Importantly, the remaining structure can be remodeled by repopulated cells and, depending on the decellularization procedure, has also shown to be immune compatible after transplantation (Wong et al. 2016). Some groundbreaking experiments on whole-organ decellularization and recellularization was published in 2008–2010 using the rat model. In these studies, decellularized heart, liver, lung, and kidney were repopulated with assorted cells and then transplanted in vivo (Ott et al. 2008, 2010; Song et al. 2013; Uygun et al. 2010; Petersen et al. 2010). Although the grafts were only assessed during the initial hours after transplantation and only rudimentary organ-like features were observed, these experiments created a positive hype in regenerative medicine using decellularized organs as scaffolds. Today, most tissues have successfully been decellularized for various applications. However, most published papers discuss the decellularization process and the remaining scaffold composition, and only few studies show convincing unbiased results on successful recellularization of the decellularized tissues. Even fewer studies have truly evaluated the efficacy of recellularized tissues and whole organs in vivo. Nevertheless, the current status of using decellularized scaffolds for organ/tissue reconstruction is promising, in particular for uterine tissue reconstruction (Fig. 29.1) (Campo et al. 2017b; Hellström et al. 2017).

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Fig. 29.1  Different synthetic or biological materials can be utilized as starting tissues for tissue engineering applications. The aim is to develop a biocompatible scaffold material that can be recellularized by the patient’s own cells, thus customizing a grafting material that will benefit the patient without the need for immunosuppressive drugs

29.3 Uterine Bioengineering 29.3.1 In Vitro Applications Several reports on uterine tissue reconstruction have been published using semisynthetic and biologically derived scaffolds. Most studies focus on fertility issues such as decidual differentiation and implantation studies. However, tissue engineering may also provide a good in vitro platform to study endometrial cancer cell mechanisms and epithelial and stromal cell communication (Meng et al. 2009; Kim et al. 2005; Sengupta et al. 2008; Park et al. 2003; Benbrook et al. 2008; Arnold et al. 2001). Many uterine-related biomaterials/hydrogels were created from an agar/ matrigel/collagen mix and were assessed in vitro using conventional 2D-cell culturing techniques together with mouse , rabbit or human myometrial, endometrial, and/or epithelial cells (Lu et al. 2009; Schutte and Taylor 2012; Wang et al. 2010). Silk-collagen scaffolds with isolated cervical mid-canal stromal cells for cervix reconstruction have also been evaluated for an extended time period in vitro (House et al. 2010). In this study, spinner flasks significantly improved the bioengineered constructs, indicating that a more advanced cell culturing system may be required to allow better diffusion into the deeper tissue layers of the uterine tissue for the cellular reconstruction (House et al. 2010). Decellularized uterine tissue has also been evaluated as biomaterial for in vitro applications. Pregnant rat and human myometrial tissue were decellularized with a trypsin/ethanol/water combination and was then repopulated with human or rat myocyte cell lines. After 51 days in vitro, these constructs showed a stratified multicellular superficial structure with cell clusters aggregating deeper in the scaffolds. Evidenced by organ bath experiments, these bioengineered constructs obtained some elementary muscular function (Young and Goloman 2013). Recently, 500-μm-thick sections of premenopausal human endometrium was decellularized

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and then reconstructed with primary endometrium cells. A stepwise hormone protocol was included in the media to mimic a 28-day menstrual cycle and the reconstructed tissue responded to the hormonal stimuli (Olalekan et al. 2017). When the same recellularized constructs were cultured in a more sophisticated microfluidic culture system under the same 28-day hormone regime, cells were still proliferating and expressed estrogen and progesterone receptors indicating an active endometrial stroma at the end of the cycle (Xiao et al. 2017).

29.3.2 Bioengineered Patches for Partial Uterus Repair In Vivo Using tissue engineering to reconstruct transplantable patches for partial uterine repair could potentially become an effective treatment regimen to cure infertility or fetal morbidity caused by severe uterus scarring/malformations as a result from repeated uterine surgeries (e.g., due to myomectomy, resection of adenomyoma, resection of placental tumors, or from repeated caesarean deliveries). Several studies therefore started to evaluate the in vivo biocompatibility and usefulness of these bioengineered constructs in rodent models to cover/replace a full-thickness uterine wall injury. For example, a collagen-derived uterine tissue scaffold was repopulated with MSCs and was successfully implanted to repair a full-thickness uterine wall injury (Ding et al. 2014). The construct improved healing and recruited endogenous endometrial and myometrial cells in the graft up to 90 days post transplantation. Embryo development took place in the grafted area, thus indicating that the bioengineered constructs were able to support plasticity during pregnancy (Ding et al. 2014). Comparable results were also obtained in an earlier study when autologous tubular-shaped myofibroblastic tissue was used to repair a similar injury (Campbell et al. 2008). We, and others, have successfully assessed decellularized uterus segments for the same application (Hellström et  al. 2016; Miyazaki and Maruyama 2014; Hellström et  al. 2014; Santoso et  al. 2014; Miki et  al. 2019). For example, Santoso et al. (2014) decellularized segments of full-thickness rat uterus by using the common decellularization reagent sodium dodecyl sulfate (SDS) and compared it to a novel protocol using high hydrostatic pressure (HHP) (Santoso et al. 2014). Both these cell-free constructs were shown to support uterus repair and local recruitment of host uterine cells. Similarly to the previous patch studies, these biomaterials gave support strength during fetal development. Later, the same people used the mouse model to show that the spontaneous repopulations by host cells of the decellularized uterine segments, were signal transducer and activator of transcription 3 (STAT3) -dependent and estrogen- and progesterone independent (Hiraoka et al. 2016). These are promising and scientifically valuable results. However, in a UTx setting, these studies have the limitations of not being able to replace a donor since the scaffolds are too small and cannot be anastomosed to the vascular system of the host.

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29.3.3 Whole-Uterus Bioengineering Thus, several efficient techniques for whole-uterus decellularization was developed for the rat uterus (Hellström et al. 2014; Miyazaki and Maruyama 2014) and recently also for the pig uterus (Campo et al. 2017a). These whole-uterus scaffolds maintain patent blood vessel conduits and can thus be anastomosed to the host vasculature at the time of transplantation. Depending on the decellularization procedure used, the remaining composition of the ECM varies between protocols (e.g., levels of fibronectin, elastin, collagen, and other important cell-matrix biomolecules). Consequently, this affects the recellularization efficiency and in vivo functionality and should be considered for future applications (Hellström et al. 2016). The biocompatibility and functionality of these constructs were assessed using the partial uterus patch repair as described above. Both Miyazaki and Maruyama (2014) and our own constructs supported uterine repair and showed normal uterine-like morphology after engraftment (Hellström et al. 2014, 2016; Miyazaki and Maruyama 2014). The constructs for these studies had been repopulated with MSCs and uterine primary cells prior to transplantation and showed to improve fertility rates compared to untreated rats. It is difficult to assess and compare results between studies since there are many technical and protocol variations between groups (e.g., cell source and cell numbers used in the recellularization process, size of bioengineered construct, and the time point when pregnancy was induced post transplantation). Naturally, these variables can influence fertility outcomes in the affected uterus. However, based on the literature and our in house experiences, we believe that a mild decellularization protocol improve the downstream applications (Simsa et al. 2018). After the transplantation of our most successful construct derived from a mild decellularization protocol (perfusion of Triton-x100, DMSO and dH2O repeatedly for a total of 5 days), the grafts supported uterus repair to such degree so that fertility was not significantly reduced compared to non-operated uterus horn (Hellström et al. 2016). However, follow-up studies made on larger animal organs in our lab have shown that this decellularization protocol is not effective on the sheep uterus (unpublished observations). Likewise, Campo et al. (2017a, b) required the more aggressive detergent SDS to effectively decellularize the large pig uterus, yet convincingly showed that the remaining ECM supported a good growth environment for human uterus side population cells (Campo et al. 2017a). Hence, species-­ dependent protocols will need to be developed during the translational progress and must also be matched for the associated cell types that will be used in the reconstruction phase. For the reconstruction of the decellularized uterine tissue, we generally use green fluorescent protein (GFP)-labeled MSCs. To permanently pre-label the cells before use is a major advantage for analyzing the recellularization efficiency since they cannot be mistaken for lingering non-labeled donor cells in the decellularized tissue. It also allows locating grafted cells after transplantation. In our patch study, we noticed that all the MSCs used in the recellularization had been replaced by host

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cells 9 weeks post transplantation. However, they were crucial for graft survival, since cell-free scaffolds completely degraded 3  months after transplantation (Hellström et  al. 2016). This is somewhat contrary to the results published by Santoso et al. (2014). However, their scaffold production is different to ours and this may explain the dissimilarity (Santoso et al. 2014). Recently, it was also shown that the spatial orientation of the decellularized tissue graft is important to consider for preventing topology-related tissue defects during uterine regeneration (Miki et al. 2019). Nevertheless, the above mentioned progress show great potential using uterine tissue engineering for partial uterine repair. In our opinion, the field is ready to evaluate bioengineered constructs in larger animal models with a partial uterine damage to assess the clinical value of using tissue engineered patches for repair or to reduce uterine scaring. However, to replace a donor uterus in a UTx setting, additional animal studies will be required to further optimize methods that support the construction of transplantable whole-uterus bioengineered constructs, including pedicles for vascular anastomosis.

29.4 Conclusions and Future Challenges Recent progress show that the uterus has a good ability to regenerate if a biocompatible grafting material is used to repair a full-thickness wall injury of the uterus. Results also suggest that MSCs can potentiate such repair. Furthermore, uterine stem cells have been isolated from many different species, and collectively, these findings suggest that we should move forward and assess bioengineered uterus patches for partial uterine repair in large animal models. Such effort would also give clues how we may improve our protocols further. This is necessary knowledge so that larger engineered constructs effectively can be developed to also include constructs that can be perfused and anastomosed. It is still a major challenge to succeed with the recellularization process of large scaffolds. Thus, future research need to address: (a) what appropriate stem cell sources should be used for each specific tissue type, (b) how stem cells effectively should be applied to the scaffold and in what sequence, and (c) how these large and complex bioengineered constructs should be cultured/maintained in  vitro to obtain efficient recellularization prior to transplantation. For whole organ reengineering, the in vitro conditions most likely have to include novel and sophisticated 3D-cell culturing techniques using specialized growth media compositions and various types of sophisticated perfusion bioreactors to regulate perfusion pressure, oxygen supplement, and other important parameters. All these factors are variables that possibly will need to be optimized for each species, type of uterus scaffold, and cell type used during the reengineering process. Success in such endeavors will improve the grafting materials and could potentially lead to the development of an autologous bioengineered donor uterus construct suitable for a human UTx.

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Acknowledgements  The authors report no conflict of interest. The work was supported by Wilhelm and Martina Lundgren research foundation, Hjalmar Svensson research foundation, Adlerbertska research foundation, the Swedish Government LUA grant, Wallenberg Foundation and the Swedish Science Research Council (Vetenskapsrådet; Grant No. 116008).

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