Oocyte vitrification technology has made egg-sharing donation easier in China

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Reproductive BioMedicine Online (2012) 24, 186 190 www.sciencedirect.com www.rbmonline.

De-Chun Chai, Ri-Cheng Chian, Jia-Yin Liu * The State Key Laboratory of Reproductive Medicine and The Center for Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University,

Dr Ling-Bo Cai was awarded his Master of Science at Nanjing Agricultural University in 2002.

1 Reproductive BioMedicine Online (2012) 24, ARTICLE Oocyte vitrification technology has made egg-sharing donation easier in China Ling-Bo Cai 1, Xiao-Qiao Qian 1, Wei Wang, Yun-Dong Mao, Zheng-Jie Yan, Cui-Zhen Liu, Wei Ding, Jie Huang, De-Chun Chai, Ri-Cheng Chian, Jia-Yin Liu * The State Key Laboratory of Reproductive Medicine and The Center for Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing , PR China * Corresponding author. address: jyliu_nj@126.com (J.-Y. Liu). 1 The first author with equal contribution. Dr Ling-Bo Cai was awarded his Master of Science at Nanjing Agricultural University in From August 2002 to date, he has worked as a clinical embryologist at the Center for Reproductive Medicine of The First Affiliated Hospital of Nanjing Medical University. He has been pursuing his PhD studies on female fertility preservation from 2007 at the State Key Laboratory of Reproductive Medicine, Nanjing Medical University. Abstract When infertile women undergoing IVF or intracytoplasmic sperm injection (ICSI) have more than 20 mature oocytes retrieved, at least 15 oocytes are inseminated by their husband s spermatozoa. The extra oocytes are cryopreserved by vitrification. If the patients became pregnant and have healthy live births, the patients are encouraged to donate their remaining cryopreserved oocytes. Forty-seven egg-sharing donors were recruited after having normal deliveries and they donated their remaining oocytes, totalling 395 cryopreserved oocytes, to 75 recipients. The survival rate of vitrified warmed oocytes was 83.0%. Following insemination by ICSI, the fertilization and cleavage rates were 83.8% and 89.8%, respectively. Out of 75 recipients, 71 recipients completed the treatment cycles and 30 of them became pregnant with clinical pregnancy and implantation rates of 42.3% and 25.5%, respectively. The birthweight of the new-born infants (22 from singleton and two from one set of twins) were ± g and ± g, respectively. No birth defects were observed for the live births. These results indicate that oocyte vitrification is an effective methodology for an egg-sharing donation programme, with acceptable pregnancy and implantation rates. RBMOnline ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: implantation, live birth, oocyte, pregnancy, sharing donation, vitrification Introduction Oocyte donations began in the 1990s and have developed since. It has become one of the conventional techniques used in reproductive medicine. The laws and regulations about oocyte donations vary in different countries based on culture, religion and other local factors. The shortage of oocytes is a global problem (Kramer et al., 2009). Elsewhere, oocyte /$ - see front matter ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 Egg-sharing donation programme with oocyte vitrification 187 donors are recruited by an agency or by IVF centres as volunteers or from relatives and friends. To meet the demands in the USA, it is estimated that more than 100,000 young women have sold or donated their oocytes (Schneider, 2008). In recent years, the concept of egg sharing has been accepted in some countries, including the UK (Blyth et al., 2004), Belgium (Pennings and Devroey, 2006) and the People s Republic of China (Heng and Zhang, 2007). In China, the regulations controlling oocyte donation were issued in 2003, with a supplement in According to Decree no. 44 (2006), the donor oocytes can only be produced from patients who undergo IVF/intracytoplasmic sperm injection (ICSI) and oocyte donation is permitted only when the patient has 20 or more mature oocytes retrieved and at least 15 oocytes being retained for the patient s own use. The remaining oocytes can be donated; the embryos produced from the donor oocytes must be cryopreserved and they cannot be transferred in a fresh cycle. Six months later, oocyte donors must be tested again for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and other relevant communicable diseases. With such explicit regulation, it is impossible to recruit donors for fresh oocyte donation and because potential donor patients expect their own pregnancy first, patients would hardly donate their oocytes to another couple before confirming their pregnancy. Recently, it has been proven that another option for female fertility preservation can be oocyte cryopreservation using vitrification technology (Huang et al., 2006) and it has also been established that oocyte vitrification can safely be applied for an egg-donation programme (Chian et al., 2008). With the strict regulation in China, the current study explores an alternative strategy for an egg-sharing donation programme with oocyte vitrification technology, in which live births for donors were confirmed before donating the remaining eggs to recipients. Materials and methods Based on strict regulation in China, the egg-sharing donation programme was established at the study centre in The programme was approved by the hospital s ethical committee and the local and central governments. Institutional Review Board approval was granted. Potential egg-sharing donors An egg-sharing donation programme has been carried out in the study centre from The patients underwent IVF treatment using either the gonadotrophin-releasing hormone (GnRH)-agonist long protocol or the flare-up protocol. The potential donors were given human menopausal gonadotrophin or recombinant FSH ( IU/day) for ovarian stimulation. When more than 20 mature oocytes were retrieved, the patients were informed that 15 oocytes would be inseminated with their partner s spermatozoa and the remaining oocytes would be cryopreserved in order to be potentially donated with the condition that the patients first had a healthy live birth. After the procedure was explained, patients signed a consent form for oocyte cryopreservation. Table 1 Demographic characteristics of potential donors with fresh or frozen embryo transfer. Patients (n = 398) Age (years) 28.2 ± 3.3 Duration of infertility (years) 3.7 ± 2.0 Cause of infertility Male factor 136 Tubal factor 204 Endometriosis 7 PCOS 26 Unexplained 25 Oocytes retrieved 9652 (24.3 ± 6.3) Oocytes vitrified 3018 (7.6 ± 3.3) Patients with healthy live births 168 (42.2) from fresh transfer cycle Patients with healthy live births 71 (17.8) from frozen thawed transfer cycle Patients with healthy live births from 239 (60.1) fresh and frozen thawed embryos Values are mean ± SD, n, n (mean ± SD) or n (%). PCOS = polycystic ovary syndrome. Three months after the healthy live birth with fresh-embryo transfer or frozen thawed-embryo transfer (produced from fresh oocytes), the patients were contacted to discuss cryopreserved egg donation. Those with no live birth, or who had HIV, HBV, HCV and other relevant communicable diseases as well as any genetic disease in family members, were excluded as potential donors. Potential donors were selected on the screening results of the patient s family history, karyotyping and blood type, as well as by the prenatal screening in the third trimester of pregnancy for antibodies for HIV, HBV, HCV and syphilis. An individual donor was permitted to donate oocytes resulting in pregnancies in up to five recipients. By the end of 2010, a total of 398 infertile women who underwent IVF/ICSI treatment had 3018 extra oocytes vitrified (7.6 oocytes/cycle; Table 1). Of the potential eggsharing donors, 47 patients agreed and signed a consent form to donate their remaining vitrified oocytes after a healthy live birth (Table 2). Oocyte vitrification, warming and fertilization procedures The mature oocytes were vitrified using a vitrification kit (Jieying Laboratory, Longueuil, Quebec, Canada). Briefly, the mature oocytes were suspended in vitrification medium 1 (7.5% ethylene glycol (EG) + 7.5% 1,2-propanediol (PROH), v/v) for equilibration for 5 min at room temperature, then transferred to vitrification medium 2 (15% EG + 15% PROH mol/l sucrose) for 1 min at room temperature, loaded into JY straws (Jieying Laboratory) and then plunged immediately into liquid nitrogen for storage. The vitrified oocytes were warmed with a thawing kit (Jieying Laboratory). Briefly, a JY straw was directly

3 188 L-B Cai et al. Table 2 Demographic and cycle characteristics of eggsharing donors. Donors (n = 47) Age (years) 28.1 ± 3.2 Duration of infertility (years) 3.6 ± 1.9 Cause of infertility Male factor 21 Tubal factor 21 Unexplained 3 PCOS 2 Oocytes retrieved 1238 (26.3 ± 6.6) Oocytes vitrified 395 (5.1 ± 0.9) Fresh oocytes inseminated 809 Fresh oocytes fertilized 607 (75.0) Embryos transferred 89 (1.5 ± 0.3) Embryos implanted 47 (52.8) Live births with fresh transfer 45 Live births with frozen thawed transfer 2 Values are mean ± SD, n, n (mean ± SD) or n (%). PCOS = polycystic ovary syndrome. inserted into thawing medium 1 (1.0 mol/l sucrose) for 1 3 min. The warmed oocytes were transferred to thawing medium 2 (0.50 mol/l sucrose) and thawing medium 3 (0.25 mol/l sucrose), each for 3 min, and then washed in thawing medium 4 (no sucrose) for 3 min. All these steps were performed at room temperature. After washing, the oocytes were transferred to an incubator for culture for at least 2 h. Oocyte survival was evaluated on the basis of the integrity of the oocyte membrane and the zona pellucida after warming and 2 h in culture. Viable oocytes were inseminated by ICSI with the recipient s husband s spermatozoa and fertilization was confirmed h post ICSI by observing 2 pronuclei (PN) in the cytoplasm. The fertilized 2PN were cultured for 2 days before embryo transfer. Recipients, endometrial preparation and embryo transfer The recipients fell into the following four groups: (i) women without ovarian function or women afflicted with premature ovarian failure; (ii) older women who either had a late marriage in life or deliberately delayed childbearing after marriage; (iii) women who had lost their children; and (iv) women with recurrent failures for IVF/ICSI with poor-quality embryos. The recipients were also screened for antibodies to HIV, HBV, HCV and syphilis. The blood type of the egg-sharing donor and the recipient were matched. The recipient s endometrium was prepared in a natural cycle with oral oestrogen (Progynova; Schering), based on the endometrial thickness. When the endometrial thickness reached at least 8 mm, the oocytes were warmed, and on the same day the recipient was given 60 mg progesterone (Xianju Pharma) by injection plus 20 mg oral dydrogesterone (Solvay Pharmaceuticals) and 6 mg oral oestrogen. The embryo transfer was performed on day 3 after insemination with ultrasound guidance. Pregnancy was confirmed by serum b-human chorionic gonadotrophin on day 14 after the embryo transfer and clinical pregnancy was confirmed by the observation of a gestational sac with a fetal heartbeat on ultrasound evaluation on day 45 of gestation. Results As shown in Table 3, of the 75 recipients who started treatment cycles, only 71 recipients completed the treatment using oocytes from 47 egg-sharing donors. The four failed cycles were due to no embryo available for transfer (fertilization and cleavage failure) in each cycle (3/5, 3/5, 4/6 and 3/5 oocytes survived following warming). The mean age of the recipients was 39.7 ± 6.9 years. Each recipient received 5.1 ± 0.9 warmed eggs and the survival rate was 83.0% (328/395) after warming. The fertilization rate was 83.8% (275/328) following ICSI and the cleavage rate of fertilized zygotes was 89.8% (247/275). Following the transfer of 1.9 ± 0.4 embryos/recipient, 42.3% (30/71) recipients had a clinical pregnancy, with implantation and live-birth rates of 25.5% (36/141) and 32.4% (23/71), respectively. There were 23 healthy live births with 24 babies. The mean birthweight was ± g and ± g for singleton and twin babies, respectively. No birth defects were observed in these infants. Discussion This study demonstrates that oocyte vitrification is an effective tool for an egg-sharing donation programme with acceptable pregnancy rates. These results also highlight another important issue: oocyte vitrification technology Table 3 Cycle characteristics and pregnancy outcomes for recipients of cryopreserved oocytes derived from egg-sharing donors. Recipients (n = 75) Transfer cycles 71 Age (years) 39.7 ± 6.9 Eggs warmed 395 (5.1 ± 0.9) Eggs survived 328 (83.0) Eggs fertilized 275 (83.8) Zygotes cleaved 247 (89.8) Embryos transferred 141 (1.9 ± 0.4) Embryos implanted 36 (25.5) Biochemical pregnancies a 4 (5.6) Clinical pregnancies a 30 (42.3) Singleton pregnancies a 29 (40.8) Multiple pregnancies a 1 (1.4) Live births a,b 23 (32.4) Miscarriages c 7 (23.3) Ectopic pregnancies c 2 (6.3) Values are n, mean ± SD, n (mean ± SD) or n (%). a Percentages are per patient. b Twin pregnancy counted as one live birth. c Percentages are per pregnancy.

4 Egg-sharing donation programme with oocyte vitrification 189 can make an egg-sharing donation programme workable even with the restrictive laws and tight regulation. Women who have premature ovarian failure, who have married late in life, deliberately delayed childbearing after marriage or lost their children by accident or disease or who have had recurrent failures of infertility treatment need egg donation. This has led to a large increase in demand for oocyte donation by infertility services worldwide. In China, oocyte donation from fertile woman and commercial transactions of human oocytes are strictly prohibited by law. Only patients who have undergone successful IVF/ICSI treatment and who can share their remaining oocytes with recipients can qualify as donors. In addition, the embryos produced from egg-sharing donors cannot be transferred to the recipients immediately, so the embryos must be cryopreserved at least for 6 months with quarantine for communicable diseases. Such regulations have led to a severe shortage of egg donors. In fact, in China, patient donors prefer to keep their eggs or embryos until they have had a live birth. The particular value of egg cryopreservation is cultural. The very idea of obtaining eggs from an infertile woman is cultural anathema. Postponing treatment of the recipient until 3 months after the delivery of a healthy baby makes egg donation culturally acceptable and emphasizes the flexibility of egg sharing. Furthermore, when the donor receives nothing in cash or kind, the delay in treatment conforms to Chinese cultural norms. Nevertheless, although the patient donors had a live birth, most of them still wanted to keep their eggs without donation. As shown in Table 1, of a total of 239 patients who had a healthy live birth, only 47 patient donors were recruited (Table 2). Recently it has been reported that the use of vitrified warmed oocytes has resulted in acceptable pregnancy and live-birth rates (Chian et al., 2008; Cobo et al., 2008; Lucena et al., 2006; Nagy et al., 2009; Rienzi et al., 2010, 2008; Selman et al., 2006; Yoon et al., 2007), indicating that oocyte cryopreservation with vitrification can be applied efficiently for female fertility preservation (Chian et al., 2009). As shown in Table 2, the majority of those 47 patient donors had male or tubal factors. These patients donated their oocytes to 75 recipients (each recipient received 5.1 ± 0.9 frozen thawed oocytes) in the study centre s egg-sharing donation programme. Of 75 recipients, 71 of them completed the donation cycles and subsequently 30 became pregnant (42.3% clinical-pregnancy rate and 32.4% live-birth rate; Table 3), comparing favourably with fresh egg-donation programmes (de Ziegler et al., 2011; Trokoudes et al., 2011). Although the 23.3% miscarriage rate (including two ectopic pregnancies) seems higher than the original cohort of 398 fresh-embryo transfers (6.7%, 12/180), it is comparable with the 20% miscarriage rate reported in an earlier study (Cobo et al., 2008). Studies including a larger number of patients are needed to draw firm conclusions regarding the miscarriage and ectopic-pregnancy rates. The live-birth results from the current egg-sharing donation programme also indicate that there were no abnormalities detected in terms of birth defects from the vitrified oocytes derived from the patient donors and the birthweights were in the normal range. Concerning the egg-sharing donation programme, there are some aspects that need to be explored because there is a potential risk that the requirement that prospective donors must have 20 or more retrieved mature oocytes may encourage the injudicious use of unnecessarily high dosages of gonadotrophins on good-prognosis patients, such as infertile patients with male factor or tubal factor aetiologies (Heng, 2008). It has been proven that high gonadotrophin dosage stimulations are associated with an increased risk of ovarian hyperstimulation syndrome (OHSS) in those patients with a good response (Budev et al., 2005). Therefore, it would be unethical to subject good-prognosis women to stimulation with high dosages of gonadotrophins in order to maximize the number of retrievable oocytes available for potential egg-sharing donation (Heng, 2009). In the study centre, less than 1% of patients develop minor or severe OHSS, and in less than 10% of treatment cycles were more than 15 oocytes obtained. The priority for patients undergoing oocyte cryopreservation was to preserve the oocytes primarily for their own use. For patients who fail to achieve a pregnancy, these cryopreserved oocytes would be used for the patient s own purpose. Interestingly, although there were 26 patients with polycystic ovary syndrome who had more than 20 oocytes retrieved, none of them had any severe OHSS symptoms; therefore, no one underwent any treatment after oocyte retrieval. Only egg-sharing donors who had a healthy live birth were permitted to donate their extra cryopreserved oocytes. In fact, in the present study, 15 fresh mature oocytes were inseminated when more than 20 mature oocytes were retrieved and the patient was encouraged to have the remaining oocytes cryopreserved. After analysis of these results, it was evident that 10 mature oocytes retrieved from each cycle gave optimal pregnancy and live-birth rates (unpublished data). Therefore, it is enough to use those 10 fresh mature oocytes for the primary treatment cycle to maximize the pregnancy potential. It may be necessary to suggest to the central Government of China a revision of the regulation concerning oocyte donation so that the required number of oocytes retrieved from a potential donor is reduced from 20 to 15. In conclusion, the results of the present study demonstrated that the strategy of egg-sharing donation with oocyte vitrification is a practical and efficient programme in China, especially given the strict regulations. This egg-sharing donation programme may provide another option for egg donation to resolve the shortage of oocyte resources in the world. References Blyth, E., Crawshaw, M., Daniels, K., Policy formation in gamete donation and egg sharing in the UK a critical appraisal. Soc. Sci. Med. 59, Budev, M.M., Arroliga, A.C., Falcone, T., Ovarian hyperstimulation syndrome. Crit. Care Med. 33 (Suppl. 10), S301 S306. Chian, R.C., Huang, J.Y.J., Tan, S.L., Lucena, E., Saa, A., Rojas, A., Ruvalcaba Castellon, L.A., Garcia Amador, M.I., Montoya Sarmiento, J.E., Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod. BioMed. Online 16, Chian, R.C., Gilbert, L., Huang, J.Y.J., Demirtas, E., Holzer, H., Benjamin, A., Buckett, W.M., Tulandi, T., Tan, S.L., Live birth after vitrification of in vitro matured human oocytes. Fertil. Steril. 91,

5 190 L-B Cai et al. Cobo, A., Kuwayama, M., Perez, S., Ruiz, A., Pellicer, A., Remohí, J., Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil. Steril. 89, de Ziegler, D., de Mouzon, J., Fauque, P., Zanette, M., Marszałek, A., Blanchet, V., Boissonas, C.C., Wolf, J.P., Chapron, C., Multiplying recipients paired with oocyte donors optimizes the use of donated oocytes. Fertil. Steril. 95, Health Ministry of China. Decree on the Regulation of Human Clinical Assisted Reproduction Technologies and Accreditation of Human Sperm Banks (Decree No. 44 of 2006). Promulgated on 7th February Full document in Chinese Language. (12 September 2008, data last accessed). Heng, B.C., Egg sharing in return for subsidized fertility treatment ethical challenges and pitfalls. J. Assist. Reprod. Genet. 25, Heng, B.C., Stringent regulation of oocyte donation in China. Hum. Reprod. 24, Heng, B.C., Zhang, X., Perspectives on compensated egg-sharing in the People s Republic of China. Reprod. BioMed. Online 14, Huang, J., Tan, S.L., Chian, R.C., Fertility preservation for female. J. Reprod. Contracept. 17, Kramer, W., Schneider, J., Schultz, N., US oocyte donors: a retrospective study of medical and psychosocial issues. Hum. Reprod. 24, Lucena, E., Bernal, D.P., Lucena, C., Rojas, A., Moran, A., Lucena, A., Successful ongoing pregnancies after vitrification of oocytes. Fertil. Steril. 85, Nagy, Z.P., Chang, C.C., Shapiro, D.B., Bernal, D.P., Elsner, C.W., Mitchell-Leef, D., Toledo, A.A., Kort, H.I., Clinical evaluation of the efficacy of an oocyte donation program using egg cryo-banking. Fertil. Steril. 92, Pennings, G., Devroey, P., Subsidized in-vitro fertilization treatment and the effect on the number of egg sharers. Reprod. BioMed. Online 13, Rienzi, L., Ubaldi, F.M., Iacobelli, M., Minasi, M.G., Romano, S., Ferrero, S., Sapienza, F., Baroni, E., Litwicka, K., Greco, E., Significance of metaphase II human oocyte morphology on ICSI outcome. Fertil. Steril. 90, Rienzi, L., Romano, S., Albricci, L., Maggiulli, R., Capalbo, A., Baroni, E., Colamaria, S., Sapienza, F., Ubaldi, F., Embryo development of fresh versus vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study. Hum. Reprod. 25, Schneider, J., Fatal colon cancer in a young egg donor: a physician mother s call for follow-up and research on the long-term risk of ovarian stimulation. Fertil. Steril. 90, 2016.e e5. Selman, H., Angelini, A., Barnocchi, N., Brusco, G.F., Pacchiarotti, A., Aragona, C., Ongoing pregnancies after vitrification of human oocytes using a combined solution of ethylene glycol and dimethyl sulfoxide. Fertil. Steril. 86, Trokoudes, K.M., Pavlides, C., Zhang, X., Comparison outcome of fresh and vitrified donor oocytes in an egg-sharing donation program. Fertil. Steril. 95, Yoon, T.K., Lee, D.R., Cha, S.K., Chung, H.M., Lee, W.S., Cha, K.Y., Survival rate of human oocytes and pregnancy outcome after vitrification using slush nitrogen in assisted reproductive technologies. Fertil. Steril. 88, Declaration: The authors report no financial or commercial conflicts of interest. Received 29 May 2011; refereed 14 October 2011; accepted 1 November 2011.

Synchronization between embryo development and endometrium is a contributing factor for rescue ICSI outcome

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