Retrospective analysis of outcomes following transfer of previously cryopreserved oocytes, pronuclear zygotes and supernumerary blastocysts

Reproductive BioMedicine Online (2011) 23, 118 123 www.sciencedirect.com www.rbmonline.com ARTICLE Retrospective analysis of outcomes following transfer of previously cryopreserved oocytes, pronuclear zygotes and supernumerary blastocysts Brooke Hodes-Wertz *, Nicole Noyes, Christine Mullin, Caroline McCaffrey, Jamie A Grifo NYU Fertility Center, New York University School of Medicine, 660 First Avenue, New York, NY 10016, USA * Corresponding author. E-mail address: hodesb01@med.nyu.edu (B Hodes-Wertz). Dr. Brooke Hodes-Wertz graduated from Vanderbilt University with a degree in neuroscience and subsequently received both her Medical Doctorate and Masters in public health from the University of Medicine and Dentistry of New Jersey. Currently, she is completing her residency in obstetrics and gynaecology at New York University where she will remain as a Fellow in reproductive endocrinology and infertility. Since beginning residency, she has participated in research focusing on cryopreservation as well as female and male infertility. Abstract Oocyte cryopreservation still bears the experimental label. Remarkable innovation in this field has led to immense improvement in clinical outcomes and has even resulted in outcomes comparable to those achieved following fresh embryo transfers. Such success has prompted this centre to investigate outcomes of cryopreservation options (oocyte versus pronuclear zygote versus supernumerary day-5 blastocyst after fresh embryo transfer). This study retrospectively analysed 200 cryopreservation cycles which were divided into three groups according to cryopreservation option, which were all cultured to blastocyst-stage post thaw/warming from January 2005 to December 2008, and compared them with 400 fresh embryo transfer cycles from the same time period. When compared with fresh embryo transfer, frozen embryo transfers originating from previously cryopreserved oocytes or pronuclear zygotes resulted in similar implantation, pregnancy and live-birth rates; however, frozen embryo transfers originating from supernumerary day-5 blastocysts resulted in lower outcomes. Thus, oocyte and/or pronuclear zygote cryopreservation appear to be the most viable options for women desiring fertility preservation. Cryopreservation of supernumerary blastocysts may lead to a slightly lower live-birth rate since the best-quality blastocysts are generally transferred during the fresh embryo transfer attempt. RBMOnline ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: blastocyst, cryopreservation, oocyte, zygote Introduction The first successful human oocyte freeze thaw pregnancy was reported by Chen (1986). However, despite this early success, subsequent cryopreservation studies including treatments such as ovarian tissue cryopreservation with subsequent autologous transplantation have resulted in substandard outcomes (Andersen et al., 2007; Demeestre et al., 1472-6483/$ - see front matter ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2011.03.020

Cryopreserved oocytes and zygotes lead to outcomes similar to fresh blastocysts 119 2007; Donnez et al., 2004; Meirow et al., 2005; Roberts and Oktay, 2005). Improvements in oocyte freezing and thawing methods over the past 10 years have led to much improved oocyte survival and, hence, pregnancy rates (Bianchi et al., 2007; Fabbri et al., 2001; Kuwayama et al., 2005; Schoolcraft et al., 2009; Yoon et al., 2000). Several studies have even shown pregnancy rates following oocyte cryopreservation that are comparable to those achieved following fresh embryo transfer cycles (Cobo et al.,2010; Grifo and Noyes, 2010; Nagy et al., 2009). Notably, Nagy et al. (2009) reported achieving pregnancies from gametes frozen twice, first as oocytes and then subsequently as resultant embryos. To date, there is no apparent increased risk in congenital anomalies in women who use their frozen thawed oocytes. A recent review of 936 infants born after oocyte cryopreservation demonstrated a 1.2% incidence of major congenital anomalies, which is lower than the 3% incidence found in the general population (Noyes et al., 2009a). Such crucial clinical advances have allowed women more viable options for ovarian preservation, both for medical and elective intentions. Cryopreservation alleviates the stress associated with the urgency to select donor spermatozoa to inseminate eggs, in order to create embryos for cryopreservation for women without a male partner. Such circumstances can occur when women face acute infertility from treatment of a newly diagnosed cancer or sub-acute infertility associated with the natural ageing process. Other potential benefits of oocyte cryopreservation include: reducing infectious diseases by quarantining oocytes, as is done with donor spermatozoa (Noyes et al., 2010), providing women who are uncomfortable with freezing embryos, for ethical or religious reasons, the opportunity to freeze gametes and preventing severe ovarian hyperstimulation syndrome in high-risk women. The aim of this paper was to assess the success of oocyte and embryo cryopreservation performed at various stages to age-matched fresh IVF cycles performed at this institution over a 4-year period. Materials and methods Patient selection A total of 200 cryopreservation thaw/warming and IVF embryo transfer cycles performed at New York University (NYU) Fertility Centre from January 2005 to December 2008 in women less than 37 years of age were reviewed retrospectively. Cycles included both autologous and anonymous donor oocytes in which all patients underwent a day-5/6 blastocyst embryo transfer using female gametes harvested before the age of 37. The 200 cycles were divided into three cryopreservation groups: oocyte, pronuclear zygote and day-5 supernumerary blastocyst after fresh embryo transfer. The control group consisted of 400 fresh embryo transfer cycles performed during the same time period that were individually matched within 1 year of oocyte age. There were 11 patients in the pronuclear group and seven patients in the day-5 supernumerary blastocyst group that underwent more than one treatment cycle. None of the women in the oocyte cryopreservation or IVF control group were treated more than once. Follicular-phase/IVF stimulation A patient s ovarian stimulation protocol was individualized to achieve adequate numbers of mature oocytes at retrieval. Most patients underwent either down-regulation with a gonadotrophin-releasing hormone (GnRH) agonist followed by treatment with combinations of recombinant human FSH (rfsh) and/or human menopausal gonadotrophin, or patients were started on gonadotrophins on day 2 with a GnRH antagonist either when lead follicles reached a mean diameter of 13 mm or when oestradiol concentration reached 1000 pg/ml. Patients were followed with periodic ultrasound and oestradiol assessment. When lead follicles reached a mean diameter of 17 18 mm, human chorionic gonadotrophin (HCG) was given. Oocyte retrieval Approximately 34 h after injection of HCG, oocytes were collected by ultrasound-guided transvaginal aspiration, isolated from follicular fluid and immediately placed in 75-ll droplets of human tubal fluid (Irvine Scientific, Irvine, CA, USA) supplemented with 6% plasmanate (plasma protein fraction (human) 5%, USP; Bayer, Elkhart, IN, USA) overlaid with Sage mineral oil (Cooper Surgical, Trumbull, CT, USA). Oocyte cryopreservation, thawing and fertilization Ninety minutes after oocyte retrieval, enzymatic removal of the corona cumulus complex was achieved using hyaluronidase (Sigma Aldrich, St. Louis, MO, USA) or cumulase (Halozyme Therapeutics, San Diego, CA, USA). Oocytes were then examined for nuclear maturity. Only mature oocytes (metaphase II; MII) were cryopreserved; those with a germinal vesicle were discarded. Oocytes were then cryopreserved by either the slow cooling method or vitrification according to the methods described by Grifo and Noyes (2010). Cryopreservation was performed by designated embryologists. All available frozen/vitrified oocytes were thawed/warmed in the thaw/warming cycle as described by Grifo and Noyes (2010). On the day of oocyte thaw/warming, a fresh or thawed semen sample was obtained from the male partners or donors and specimens were processed via isolate (Irvine Scientific) or swim-up techniques as described elsewhere (Carrell et al., 1998). Based on prior published data (Gook et al., 1995; Porcu et al., 1999), intracytoplasmic sperm injection (ICSI) was performed on all surviving oocytes within 3 h post thaw/warming, regardless of semen parameters. Fertilization was accessed 18 h post ICSI by visualization of two pronuclei. Following this stage, the pronuclear zygotes were cultured similarly to fresh embryos for transfer on day 5/6. Pronuclear zygote cryopreservation and thawing Approximately 4 6 h post retrieval, oocytes were fertilized with routine insemination or ICSI when indicated for male factor or a prior history of poor fertilization. Oocytes inseminated by ICSI were transferred to Quinn s Advantage Cleavage Media (Q1; Cooper Surgical) supplemented by 10%

120 B Hodes-Wertz et al. plasmanate. Fertilization was assessed 18 h post insemination/icsi by visualization of two pronuclei. On day 2, pronuclear zygote embryos were cryopreserved using propanediol and sucrose. In the thaw cycles, a designated number of pronuclear zygotes were rapidly thawed in a 31 C waterbath with the cryoprotectants removed using a three-step dilution procedure (Testart et al., 1986; Veeck et al., 1993). Following this stage, pronuclear zygotes were cultured similarly to fresh embryos for transfer on day 5. Blastocyst cryopreservation and thawing after fresh embryo transfer Supernumerary good-quality blastocysts, graded 3BB or better (Gardner et al., 2001) were cryopreserved using a variation of Ménézo s standard slow freezing, two-step process (Ménézo et al., 1992) using glycerol and sucrose as cryoprotectants following a fresh day-5 blastocyst transfer. For the thaw cycle, blastocysts were rapidly thawed in a 31 C waterbath with a three-step dilution/removal of cryoprotectants 1 3 h prior to transfer. Blastocysts were observed at 200 magnification immediately following thaw and the percentage of cell survival as well as the degree of re-expansion were observed and recorded following thaw and again prior to transfer that same day. Blastocysts needed to have >50% survival and demonstrate evidence of re-expansion 2 6 h post thaw to meet criteria for embryo transfer (Noyes et al., 2009b). Blastocyst transfer The decision on the number of embryos to transfer was in collaboration with the embryologist, physician and patient in accordance to the guidelines of the American Society for Reproductive Medicine (ASRM, 2009). Transcervical blastocyst transfer was performed using a soft-tipped Wallace (Portex, Hythe, UK) catheter under ultrasound guidance. Immediately following embryo transfer, catheters were flushed and the media inspected microscopically to confirm absence of blastocysts. Management of luteal phase and early pregnancy in cryopreservation cycles The patient s uterus was either prepared with a 2-week course of graduated oral oestradiol (Bristol-Meyers Squibb, Hamilton, NJ, USA) beginning on cycle day 2 or a patient s natural cycle was used. Oestrogen treatment was then supplemented daily with intramuscular progesterone injections (50 mg in oil) beginning 4 6 days prior to thaw depending on the stage of cryopreservation. Daily intramuscular progesterone injections were continued until 10 weeks of gestation or until a nonviable or failure of pregnancy was diagnosed. Management of luteal phase and early pregnancy in fresh embryo transfer cycles Luteal support consisted of intramuscular progesterone injections (50 mg in oil) daily. The patient continued daily injections of progesterone until a viable intrauterine pregnancy was diagnosed by ultrasound. Statistical analysis Using the Statistical Package for Social Sciences (SPSS, Chicago, IL, USA), variables in the study groups were compared by chi-squared analyses and Fisher s exact test as appropriate. Kruskal Wallis ANOVA was used for comparison of continuous variables in more than two study groups. P-values <0.05 were considered to be of statistical significance. Cycle outcomes were compared with each other and to an age-matched control group of fresh blastocyst transfers. Spontaneous abortion, implantation, clinical pregnancy and live-birth rates were evaluated. The implantation rate was calculated as the number of intrauterine gestational sacs visualized on ultrasound per total number of embryos transferred. Clinical pregnancy was defined as the presence of an intrauterine gestational sac(s) with fetal cardiac activity as documented by ultrasound. Reporting of this data was approved by the New York University School of Medicine Institutional Review Board. The oocyte cryopreservation data was collected and is reported under NYU Langone Medical Centre IRB R# 05 463. Results Cycle demographics for patients who underwent a thaw/warming and embryo transfer cycle from January 2005 to December 2008 of previously cryopreserved oocytes (n = 19), pronuclear zygotes (n = 28) and supernumerary blastocysts (n = 153) in women less than 37 years of age are shown in Table 1. All cryopreservation groups used significantly less gonadotrophins (P = 0.0001) and had significantly more eggs retrieved (P = 0.001 0.038). In the oocyte group, 81% (269/331) cryopreserved oocytes were mature (metaphase; MII) at time of freezing/vitrification. Thaw/warming survival was judged by the appearance and translucency of the cytoplasm and the integrity of the oolema and failure to survive was characterized by a dark or shrunken appearance. Of the mature cryopreserved oocytes, 45% (121/269) had been slow frozen whereas 55% (148/269) had undergone vitrification. Four vitrified oocytes were not found from the cryopreservation device at the time of warming, leaving a total of 265 MII oocytes for evaluation. The survival rate of recovered oocytes was 89% (237/265). There was no statistically significant difference between slow freezing versus vitrification groups in terms of survival (85% versus 93%, respectively) and fertilization (81% versus 76%, respectively). Within the same time period, 28 cycles resulted in patients opting to freeze all embryos at the pronuclear zygote stage. Cryopreservation occurred for various reasons including: medical (i.e. patients recently diagnosed with cancer; n = 9), personal scheduling issues (n = 8), ovarian hyperstimulation syndrome (n = 7) and uterine factor discovered during the follicular phase (i.e. uterine polyp or fluid within the uterine cavity; n = 5).

Cryopreserved oocytes and zygotes lead to outcomes similar to fresh blastocysts 121 Table 1 Cryopreservation cycle demographics as compared with fresh embryo transfer. Oocytes (n = 19) Pronuclear zygotes (n = 28) Supernumerary blastocysts (n = 153) Fresh blastocysts (n = 400) Oocyte age at retrieval 30.6 ± 4.4 28.6 ± 4.5 a 30.8 ± 4.4 30.8 ± 4.0 (years) Donor cycles (%) 32 39 33 33 Serum oestradiol on day of 2646 ± 1322 b 2900 ± 1945 2231 ± 1401 2063 ± 1054 c HCG (pg/ml) Gonadotrophins used (IU) 1981 ± 744 1773 ± 782 2151 ± 897 2649 ± 1344 d Oocytes retrieved 24 ± 9 22 ± 14 22 ± 9 16 ± 8 e Two-pronuclear fertilization (%) 78% (186/237 mature oocytes) 61% (495/809 oocytes retrieved) 66% (2481/3743 oocytes retrieved) 56% (3652/6480 oocytes retrieved) Embryos transferred 2.2 ± 0.6 1.9 ± 0.8 2.1 ± 0.6 2.0 ± 0.4 Values are mean ± standard deviation unless stated. All other comparisons are not significant. a The pronuclear group was significantly younger than the supernumerary and fresh blastocyst group (P = 0.0001 0.029). b,c P = 0.021. d All three cryopreservation groups used significantly less gonadotrophins (P = 0.0001). e All three cryopreservation groups had significantly more eggs retrieved (P = 0.001 0.038). Table 2 Cryopreservation cycle clinical outcome data per embryo transfer as compared with fresh embryo transfer. Oocytes (n = 19) Pronuclear zygotes (n = 28) Supernumerary blastocysts (n = 153) Fresh blastocysts (n = 400) Spontaneous 0 (0) 2 (7) 12 (8) 30 (8) abortions Implantations 19/42 (45) 23/54 (43) 105/321 (33) a 375/788 (48) b Clinical pregnancies 11 (58) 14 (50) 70 (46) c 246 (62) d Live births 11 (58) 13 (46) 70 (46) e 240 (60) f Values are n (%). All other comparisons are not statistically significant. a,b P = 0.0001. c,d P = 0.0008. e,f P = 0.003. Within the same time period, 153 cycles where supernumerary blastocysts were cryopreserved on day 5 (grade 3BB embryo or better, Gardner criteria; Gardner et al., 2001) and subsequently thawed and transferred were evaluated. In this group, 493 blastocysts were frozen following 181 fresh embryo transfers (average 2.7 ± 3.6 per cycle). A statistical comparison of live-birth outcome for all transferred cryopreservation cycles was compared with consecutive oocyte age-matched fresh embryo transfer cycles (n = 400) completed from the same time period at the study centre. This group had a significantly lower number of oocytes retrieved (P = 0.001 0.038) compared with the three cryopreservation groups. Clinical outcomes for all groups are summarized in Table 2. When compared with fresh embryo transfer cycles, embryo transfers performed using previously cryopreserved oocytes or pronuclear zygotes resulted in similar implantation, clinical pregnancy and live-birth rates whereas frozen embryo transfer using supernumerary blastocysts resulted in lower implantation, clinical pregnancy and live-birth rates as compared with fresh cycles; all other outcome comparisons were similar between the groups. Discussion The development of cryopreservation of both gametes and embryos has allowed for novel reproductive possibilities. Recently, there has been an increased interest in cryopreservation options for both medical and elective indications. Creating an embryo when a patient is single and/or unmarried, i.e. forcing a woman to choose between possible offspring versus no reproductive future, can be quite distressing and some might even say unethical. Allowing women to freeze gametes helps to avoid pressure to create embryos in order to achieve better outcomes. While oocyte cryopreservation is still considered experimental, the current data confirm previous work (Cobo et al., 2010; Grifo and Noyes, 2010; Nagy et al., 2009; Noyes et al., 2011) that has shown that outcomes from cryopreserved oocytes are comparable to that of fresh embryo transfer. Such results give women broader cryopreservation options and the confidence that these options will lead to the same pregnancy outcomes as a fresh IVF cycle. Likewise, women that start an IVF cycle but are confronted with disruptions to the treatment such as timing issues with a donor, medical problems, personal emergencies

122 B Hodes-Wertz et al. or hyperstimulation can feel secure that their embryos have the same chance of a successful pregnancy as a fresh cycle. However, patients that are transferring supernumerary embryos from a previous IVF cycle should be aware that their pregnancy rates may be slightly lower, as their best-quality embryos were transferred in their fresh IVF cycle. One limitation of this study is that it is a retrospective analysis, with the attendant possible selection bias and variables not appreciated by review. In addition, between the four study groups, there are many variables in each cycle such as type of stimulation and type of fertilization used making it difficult to control all of them. The oocyte cryopreservation group consisted of oocytes cryopreserved by two different methods, of which the more superior method was not clearly discerned at the time that this study was performed. Currently, the study centre is performing a randomized controlled trial comparing slow freeze versus vitrification and there are pregnancies in both groups. More data is needed to determine which, if either, method is superior. This data also represents a single fertility centre with extensive oocyte cryopreservation experience and the results may not be reproducible at other centres. In summary, oocyte, pronuclear zygote and/or supernumerary blastocyst cryopreservation appear to be viable options for women desiring fertility preservation. Of note, such clinical outcomes may only be achieved at centres with extensive oocyte cryopreservation experience. These results should aid physicians in counselling patients about the available and evidence-based options for their current reproductive condition and future ambitions. Acknowledgements The authors wish to thank the embryology staff, nurses and physicians at the NYU Fertility Centre who have contributed to the care of the patients. 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Cryopreserved oocytes and zygotes lead to outcomes similar to fresh blastocysts 123 and thaw of pronuclear stage oocytes. Fertil. Steril. 59, 1202 1207. Yoon, T.K., Chung, H.M., Lim, J.M., Han, S.Y., Ko, J.J., Cha, E.Y., 2000. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil. Steril. 734, 180 181. Declaration: The author reports no financial or commercial conflicts of interest. Received 9 October 2010; refereed 22 March 2011; accepted 22 March 2011.