Department of Obstetrics and Gynecology, NYU Fertility Center, NYU School of Medicine, New York 10016, USA; 2

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1 RBMOnline - Vol 18 No Reproductive BioMedicine Online; on web 8 April 2009 Article Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies Dr Noyes has worked full time in infertility since She received an undergraduate and medical degree from the University of Vermont and then completed residency in obstetrics and gynaecology and fellowship in reproductive endocrinology at the New York Hospital Cornell Medical Center in New York City. She is board-certified in obstetrics and gynecology as well as in reproductive endocrinology. Dr Noyes has been involved in the treatment of over 16,000 infertility patients using assisted reproductive technologies. In 2004, she and her partner, Dr Jamie Grifo, established a successful fertility preservation programme at the NYU Fertility Center of NYU School of Medicine. Dr Nicole Noyes N Noyes 1,4, E Porcu 2, A Borini 3 1 Department of Obstetrics and Gynecology, NYU Fertility Center, NYU School of Medicine, New York 10016, USA; 2 Infertility/Reproductive Unit, University of Bologna, Bologna 40138; 3 Technobios Procreazione, Centre for Reproductive Health, Bologna 40125, Italy 4 Correspondence: nnoyes01@gmail.com Abstract Over the past decade, the number of reported live births resulting from oocyte cryopreservation has rapidly increased. To appreciate the true number of children born, verified live births were tabulated and assessed. A literature search was performed; authors were then contacted to verify birth outcomes and provide updates. A database including all verified live born infants was constructed. A total of 58 reports ( ) were reviewed, which included 609 live born babies (308 from slow freezing, 289 from vitrification and 12 from both methods). Additionally, 327 other live births were verified. Of the total 936 live borns, 1.3% (12) were noted to have birth anomalies: three ventricular septal defects, one choanal and one biliary atresia, one Rubinstein Taybi syndrome, one Arnold Chiari syndrome, one cleft palate, three clubfoot and one skin haemangioma. Compared with congenital anomalies occurring in naturally conceived infants, no difference was noted. With more live born data accumulating, this procedure may become mainstream as a fertility preservation option, particularly for women diagnosed with malignancy requiring cytotoxic therapy. A registry would help to assure the safest, most expeditious development of this technology. Keywords: live born, oocyte cryopreservation, pregnancy outcome, slow freezing, vitrification Introduction Over the past decade, and particularly in the last 2 years, the number of reported live births resulting from the transfer of cryopreserved, then thawed or warmed, fertilized oocytes has rapidly increased (Borini et al., 2007, Chian et al., 2008). Running in parallel has been an exponential rise in the number of oocyte cryopreservation cycles being performed around the world. The twenty-first century is marked by significant scrutiny of all new technologies, equipment and medications. Because of its relatively slow start and early lack of reproducibility, oocyte cryopreservation has endured more medical scepticism than prior novel assisted reproduction procedures; in 2006, the American Society of Reproductive Medicine labelled it experimental (Practice Committee ASRM, 2006). The first reported live birth resulting from the transfer of frozen thawed, fertilized oocytes occurred 23 years ago (Chen, 1986), only 2 years after that of frozen thawed embryos (Zeilmaker et al., 1984). However, it was not until 1998, over a decade later, that the first two series of oocyte cryopreservation births were reported (Borini et al., 1998; Porcu et al., 1998b). Fortunately, around this hiatus and continuing to the present day, extensive biological assessment of oocyte cryopreservation, including chromosome and meiotic spindle integrity (Pickering et al., 1990; Gook et al., 1994; Van Blerkom and Davis, 1994; Cobo et al., 2001; Boiso et al., 2002; Coticchio et al., 2006; Noyes et al., 2006; DeSantis et al., 2007b), premature cortical granule release with resultant zona pellucida hardening Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 770 (Johnson, 1989; Schalkoff et al., 1989; Bernard et al., 1992; Gook et al., 1995; Nottola et al., 2007) and oolemma membrane permeability alteration (Mazur, 1963; Meryman, 1971; Knox et al., 1980; Dowgert and Steponkus, 1984; Takahashi et al., 1985; Paynter et al., 2005; Gardner et al., 2007) has been undertaken, all the while, live birth reports have continued to appear from around the world. A comprehensive overview of oocyte cryopreservation technology and safety issues can be found in a recently published article by Gook and Edgar (2007). As oocyte cryopreservation usage expands, both in volume and application (despite a relative paucity of available outcome data), the authors felt it imperative to attempt a tabulation of the actual current number of live births. These enumerated oocyte cryopreservation data were then compared with live births resulting after natural conception. Finally, a worldwide or alternatively, country-specific, registry of egg freezing births is strongly encouraged to ensure that technology progresses in the safest, most expeditious, unbiased direction. Materials and methods A literature search using all available internet and university library resources, including a formal NYU Medical Centre library services query, MEDCat, Medline (Ovid), PubMed and Google, was conducted. Search words included oocyte (egg), cryopreservation, freezing, slow freeze, vitrification, birth, birth outcomes, live birth, congenital (birth) defects or anomalies, complications and safety. In addition, a review of major infertility and oocyte cryopreservation meeting abstracts from the last decade was performed. Each publication was reviewed, recording all valid pregnancy outcome data into a cumulative working spreadsheet. Pregnancy was defined as the presence of at least a single gestational sac visualized on ultrasound. Live birth was defined as any pregnancy where one viable infant was delivered beyond 25 weeks gestation. Any reported birth defect was recorded and classified as major or minor. Any author reporting ongoing pregnancies or whose publication contained incomplete or confusing birth data was contacted for updated information. Any author group with more than one existing publication or meeting presentation was also contacted to assure no duplicate accounting of delivered infants occurred. If the same live births were included in more than one publication, they were usually counted in the manuscript containing the most births. All but five contacted authors responded to , in-person or telephone outcome inquiries. Questionable data from unreachable authors were omitted from the data pool. The resulting database was then compared with United States national statistics for birth outcome (Centre for Disease Control, 1997). Results A total of 58 reports (43 using slow freeze, 12 vitrification and three both methods) from 1986 to 2008 were reviewed. Fortyfive were peer-reviewed journal articles and 13 were meeting abstracts (Table 1). The time line of reported births resulting from transfer of thawed/warmed and fertilized, previously slowfrozen and vitrified oocytes is shown in Figure 1. Cumulatively, there were 609 live born babies (308 from slow freezing, 289 from vitrification and 12 from a combination of slow freezing and vitrification). Slow-freezing reports span from 1986 to 2008 while vitrification birth reports began in Although 13 years later than the first slow-freeze birth, the number of reported babies born as a result of vitrified oocytes is now approaching that of slow-frozen oocytes (Figure 1). Table 2 shows birth outcome data from oocyte cryopreservation case reports (n = 23) while Table 3 shows cycle and birth data from series reports (n = 35), all listed in Table 1. More pregnancy-specific information was available in the case reports, whereas more cycle data could be tabulated in the series reports. Gender information was available for a total of 395 babies; there were 209 female and 186 male infants born. Multiple-pregnancy status was available in 452 pregnancies; 81% were single while 19% were multiple gestations. To adequately assess birth weight, gestational age and prematurity, one would need to know these parameters for the single versus multiple births; this information was not clearly delineated in many of the series manuscripts. The majority of babies reported had a mean gestational age greater than 36 weeks. For the 30 live born babies reported in case reports, the mean gestational age and weight at birth was 36.9 ± 0.6 weeks and 2720 ± 116 g in the slow-freezing group and 39 ± 1 weeks and 3318 ± 201 g in the vitrification group. Review of all case and series oocyte cryopreservation reports revealed a total of eight birth anomalies: three ventricular septal cardiac defects, one choanal atresia, one biliary atresia, one Rubinstein Taybi syndrome, one clubfoot and one skin haemangioma. Three of these anomalies were reported in the 308 slow-freeze births, and five in the 289 vitrification births. Additionally, there are another 327 live births from seven fertility centres around the world known to have occurred at the writing of this manuscript (Table 4); 224 were the result of transferring embryos created from slow-frozen oocytes and 103 from vitrified oocytes. Four birth defects were found among these babies; one was diagnosed with Arnold Chiari syndrome (slow freezing), one with cleft palate (slow freezing) and two with clubfoot (one slow freezing and one vitrification). In total, there were four minor and eight major birth anomalies. The minor anomalies included three cases of clubfoot and one skin haemangioma; all other anomalies were major. There were no epigenetic malformations reported. The overall anomaly rate was 1.3%. The incidence in slow freeze births was 6/532 (1.1%) and in vitrification births 6/392 (1.5%). Outcome comparison of live born babies resulting from oocyte cryopreservation and natural conception was then performed (Table 5). According to the Center for Disease Control (CDC), major structural or genetic birth defects occur in approximately 3% of live births in the United States (Hoyert et al., 2006). In the present study population, the overall incidence of reported birth defects was 1.3% (12 in 936 live borns). The most common birth anomalies that occur in naturally conceived newborn infants are cardiac malformations, neural tube defects and clefts of the lip and palate. Of the 12 birth defects found in babies born following transfer of embryos derived from previously cryopreserved oocytes, three were ventricular septal defects; this is the most common cardiac anomaly occurring in up to one per 100 naturally conceived newborns. One baby had a cleft palate; the incidence in naturally conceived infants is one per 700 births (Bellis, 1999). Three babies born were found to have clubfoot; the incidence in natural conceptions is approximately

3 Table 1. Case and series birth reports for oocyte slow freezing and vitrification by year of publication. Case reports were those that only included one to three deliveries, while series reports had greater than three deliveries or included data on cycles not achieving pregnancy. Report type Cryopreservation method Slow-freeze Vitrification Both Case reports Chen (1986) Kuleshova et al. (1999) (n = 23) Van Uem et al. (1987) Katayama et al. (2003) Chen (1988) Kyono et al. (2005) Porcu et al. (1997) Polak de Fried et al. (1998) Porcu et al. (1998a) Tucker et al. (1998) Porcu et al. (1999a) Wurfel et al. (1999) Kyono et al. (2001) Chen et al. (2002) Huttelova et al. (2003) Miller et al. (2004) Notrica et al. (2004) Tjer et al. (2005) Montag et al. (2006) Barritt et al. (2007) Gook et al. (2007) Yang et al. (2007) Porcu et al. (2008) Series reports Borini et al. (1998) Yoon et al. (2003) Grifo et al. (2006) (n = 35) Porcu et al. (1998b) Chian et al. (2005) Smith et al. (2006) Porcu et al. (1999b) Kim et al. (2005) Noyes et al. (2008) Porcu et al. (2000) Okimura et al. (2005) Winslow et al. l (2001) Kuwayama et al. (2005) Yang et al. (2002) Ruvalcaba et al. (2005) Quintans et al. (2002) Lucena et al. (2006) Boldt et al. (2003) Antinori et al. (2007) Fosas et al. (2003) Cobo et al. (2008) Borini et al. (2004) Azambuja et al. (2005) Chen et al. (2005) Li et al. (2005) Porcu (2005) Boldt et al. (2006) Borini et al. (2006) Chamayou et al. (2006) LaSala et al. (2006) Levi-Setti et al. (2006) Petracco et al. (2006) Bianchi et al. (2007) DeSantis et al. l (2007) Konc et al. (2007) one per 735 births (Center for Disease Control, 1997). One infant had a skin haemangioma; the incidence of this disorder in natural births is one per 50 to 225 births (Simpson, 1959). One baby had an Arnold Chiari malformation (displacement of the cerebellar tonsils, medulla and the fourth ventricle into the spinal canal); the natural incidence of this anomaly is approximately one per 1200 newborns (Tasdemir et al., 2004). One infant was noted to have a gastrointestinal atresia, notably biliary atresia (obliteration or discontinuity of the extrahepatic biliary system causing obstruction of bile flow). This disorder represents the most common surgically treatable cause of cholestasis encountered during the newborn period. The reported frequency of this disorder is one per 10,000 15,000 births (Schwartz, 2007). One child had choanal atresia, the most common nasal abnormality seen in newborn infants, affecting about one in every 7000 naturally conceived live births (Goverman, 2005). One child was diagnosed with Rubinstein Taybi syndrome (a genetic disease involving chromosome 16, characterized by broad thumbs and toes, short stature, distinctive facial features, and varying degrees of mental retardation). This syndrome 771

4 No. of liveborn babies Slow freeze (n = 308) Vitrification (n = 289) Both methods (n = 12) 9 years n = Year Figure 1. Number of live births from reports of slow-frozen and vitrified, then thawed warmed, fertilized and transferred oocytes from 1986 to Table 2. Data from oocyte cryopreservation birth case reports reported from listed in Table 1 (n = 23 reports: 20 slow-freeze, three vitrification). All oocyte were fertilized by intracytoplasmic sperm injection other than in the first three slow-freeze reports ( ; n = 4 babies). No birth deformities were reported in any of these cases. Parameter Cryopreservation method Slow-freeze Vitrification Patient (n) Oocyte age (years) 30.1 ± 1? Day of zygote/embryo transfer 1 to 5 2 to 5 No. of pregnancies 22 4 No. of deliveries 20 4 No. of live born babies 26 4 Single/multiple gestation 14 single, 6 multiple 4 single Gestational age (weeks) 36.9 ± 0.6 (range: 29 40) 39 ± 1 (range: 37 40) Baby gender 14 female, 8 male, 4 unknown 2 female, 2 male Birth weight (g) 2720 ± 116 (range: ) 3318 ± 201 (range: ) Prematurity (n) <1500 g: 1, g: 5 <1500 g: 0, g: 0 Birth defects None None Table 3. Data from oocyte cryopreservation birth series reports reported from listed in Table 1 (n = 35 reports: 23 slow-freeze, nine vitrification, three both cryopreservation methods). All oocytes were fertilized by intracytoplasmic sperm injection. Parameter Cryopreservation method Slow-freeze Vitrification Both 772 No. of embryo transfers No. of oocytes thawed/warmed 11, No. of oocytes survived % oocyte survival 68 (range: 22 90) 81 (range: 69 99) 90 % 2-pronucleate fertilization 73 (range: 50 86) 79 (range: 59 93) 81 No. of live born babies Baby gender (gender information 99 female, 69 male 86 female, 103 male 8 female, available for 168 slow-freeze, male vitrification and 12 both methods) Birth defects 1 ventricular septal defect, 1 2 ventricular septal defect, None choanal atresia, 1 Rubinstein 1 biliary atresia, 1 clubfoot, Taybi syndrome 1 skin haemangioma

5 Table 4. Unpublished births resulting from cryopreserved oocytes a. Cryopreservation No. of Birth anomalies method live born babies Slow-freeze cleft palate, 1 clubfoot, 1 Arnold Chiari syndrome Vitrification clubfoot a Data were obtained from centres that have previously published live birth outcomes following oocyte cryopreservation. Table 5. Birth anomalies in natural conception versus oocyte cryopreservation, listed most common to most rare. Birth anomaly Approximate incidence in Incidence in total of 936 natural conception births oocyte cryopreservation births (n) All One in (one in 78) Skin haemangioma One in Cardiac defects One in Neural tube defects One in Cleft lip and palate One in Clubfoot One in Arnold Chiari syndrome One in Choanal atresia One in Biliary atresia One in 10,000 15,000 1 Rubinstein Taybi syndrome One in 100, ,000 1 is rare, affecting 1 in 100, ,000 newborns (Genetics Home Reference, 2007). Most cases are sporadic and are likely due to a de-novo mutation; some are inherited as autosomal dominant. It is not known whether there was any history in the affected baby s family. Discussion From this extensive literature and meeting review, it is clear that many more births have resulted from the transfer of cryopreserved, then thawed/warmed, fertilized oocytes than was previously considered. It appears that there are at least 532 live born children that have resulted from slow-frozen oocytes, 392 from vitrified oocytes and 12 from a combination of both methods, totalling 936 children at the time this manuscript was written. For obvious reasons, but unlike that which occurred following the introduction of previous innovative assisted reproduction procedures (e.g. transfer of frozen thawed embryos in the 1980s or intracytoplasmic sperm injection in the 1990s), oocyte cryopreservation is considered experimental. Clearly, the most important consideration in all IVF procedures is patient safety, both for the women undergoing the procedures and any potential resulting offspring. Although preliminary, the incidence of all birth anomalies found to date in babies born from cryopreserved oocytes does not appear different from that of infants born through natural conception. The most common anomaly noted in the oocyte cryopreservation cycles was a ventricular septal defect, also one of the most common major anomalies noted in naturally conceived newborns. The incidence of such is 1/125 (0.8%) in naturally conceived newborns and was 0.3% in the oocyte cryopreservation population. Several syndromes involving epigenetic alterations have been associated with assisted reproductive technologies, especially where intracytoplasmic sperm injection was used to achieve fertilization (Cox et al., 2002; DeBuan et al., 2003; Gicquel et al., 2003; Moll et al., 2003). These include Beckwith Wiedemann syndrome, Angelman syndrome and retinoblastomas. No epigenetic disorders were reported in the present series of oocyte cryopreservation births. Continued monitoring of birth outcomes will help to assess any increased risks associated with oocyte cryopreservation. Unfortunately, the current conservative attitude towards oocyte cryopreservation has presented a double-edged sword for the technology; while the number of centres offering oocyte freezing, and the number of cycles and indications for the technology are rapidly expanding despite an experimental label, the reporting and scientific advancement appears somewhat stifled by fear of criticism. Certainly, for most young cancer patients in need of potentially life-saving, but cytotoxic cancer therapy, oocyte preservation appears to be the best means of preserving fertility potential. In addition, as success improves, donor oocyte banking (in countries that allow use of donor gametes) will afford quarantine and more equitable distribution of retrieved oocytes. Lastly, as pregnancy rates approach or surpass those achieved using frozen thawed embryos, oocyte cryopreservation will afford IVF centres to shift away from the practice of cryopreserving supernumerary embryos in favour of unfertilized, supernumerary oocytes, a trend that will reduce the current worldwide surplus of frozen embryos. 773

6 774 Perhaps one hesitation regarding oocyte cryopreservation partially lies in the commercial interests of companies promoting premature elective fertility preservation. In addition, competing interests exist for those who manufacture oocyte storage containers and/or oocyte cryopreservation culture media. Clearly, clinicians and embryologists need to be cognizant of these latter conflicts as more publications appear in the literature. One shortcoming of this publication is that not all reported births within the manuscript have been subjected to the scrutiny of peer-review literature. Because of this, care was taken to contact authors in order to clarify any confusing or conflicting birth information. A second shortcoming is that not all desired outcome data were available for every reported pregnancy and birth. This is in large part because no currently acceptable registry exists for oocyte cryopreservation cycles. The authors made their best efforts to obtain as much information as possible. A third shortfall is that the accumulated data does not lend itself to evaluation of the efficiency of oocyte cryopreservation (albeit, this was not the intent of the manuscript). Because established cryopreservation techniques are just now beginning to spread rapidly across the globe, the authors feel pregnancy rates would best be assessed in 1 2 years. The authors also feel it would be premature to judge the relative efficiency of slow-freeze versus vitrification cryopreservation methods. This is because the data that currently exist are not comparable. At present, the majority of slow freezing information has been generated from cycles performed in Italy where there are national restrictions on the number of oocytes that can be thawed, fertilized and transferred (Fineschi et al., 2005); thus, the majority of these cases have been performed because of the three-egg fertilization constraint with supernumerary oocytes being cryopreserved for a subsequent IVF attempt. The majority of vitrification births have been achieved in South America, Spain and Asia; these cycles are being performed for a variety of indications, including oocyte donation, which clearly has the best chance for a positive pregnancy outcome. A comparison of the two major cryopreservation techniques is only possible through a large, randomized controlled trial performed at centres with established successes using both methods. Noyes and Grifo at NYU Fertility Centre and Smith at the University of Michigan Health System are currently conducting IRB-approved randomized trials comparing slow freeze to vitrification success; accrued data at this time are too preliminary for judgment. Regardless, a working knowledge of the actual number of babies born as a result of transferred cryopreserved, thawed warmed fertilized oocytes, including fetal wellbeing, is an important step towards adequately judging the merits of this highly sought after technology. The most efficient way to achieve this goal is to organize a real-time worldwide oocyte cryopreservation registry, ideally supervized by one of the larger infertility organizations. Alternatively, each country could collect their respective egg freezing birth data; the latter strategy would be easier to manage but would make it more difficult to quickly amass large comparative numbers. To date, at least 936 live born infants have been delivered as a result of this technology and birth defects in these cases do not appear more common than what has been reported in naturally conceived newborns. Although this is still a small number of children born and the comparisons are somewhat limited, no indication has been found that there is a worse outcome for newborns conceived through this technology. It is necessary to continue scrutiny and review of oocyte cryopreservation technology in a way that closely monitors birth outcomes; this would best be achieved through a systematic outcome reporting system. Acknowledgements All those who attended and contributed to the informal Oocyte Cryopreservation Safety Forum held during the 24th annual meeting of the European Society of Human Reproduction and Embryology in Barcelona, Spain in July This included the authors, as well as Ricardo Azambuja, Alejandro Chavez- Badiola, Patrizia Maria Ciotti, Anna Cobo, Jacques Cohen, Giovanni Coticchio, Lucia DeSantis, David Edgar, Debra Gook, Karl Gosta-Nygren, Koichi Kyono, Paolo Emanuele Levi Setti, Elkin Lucena, Zsolt Peter Nagy, Ester Polak de Fried, Luis Ruvalcaba, Antonio Sanoja, Giulia Scaravelli, Gary Smith, Maria Teresa Vilani, Ulla-Brit Wennerholm, and Maureen Wood, representing 12 countries worldwide. The authors would also like to thank the organizers of the annual ESHRE meeting for providing a venue for this discussion. References Antinori M, Licata E, Dani G et al Cryotop vitrification of human oocytes results in high survival rate and healthy deliveries. 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Fertility and Sterility 42, Declaration: The authors report no financial or commercial conflicts of interest. Received 29 September 2008; refereed 17 November 2008; accepted 23 February 2009.