Freeze-all: enhanced outcomes with cryopreservation at the blastocyst stage versus pronuclear stage using slow-freeze techniques

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1 Reproductive BioMedicine Online (2010) 21, ARTICLE Freeze-all: enhanced outcomes with cryopreservation at the blastocyst stage versus pronuclear stage using slow-freeze techniques Eric Surrey a, *, Jennifer Keller b, John Stevens c, Robert Gustofson a, Debra Minjarez a, William Schoolcraft a a Colorado Center for Reproductive Medicine, RidgeGate Circle, Lone Tree, CO 80124, USA; b Dept. of Obstetrics-Gynecology, Exempla Saint Joseph Hospital, Denver, CO, USA; c Fertility Laboratories of Colorado, Lone Tree, CO, USA * Corresponding author. addresses: esurrey@colocrm.com, esurrey@earthlink.net (E Surrey). Eric Surrey, MD, is Medical Director of the Colorado Center of Reproductive Medicine in Lone Tree, CO. He is a board certified reproductive endocrinologist who completed residency and fellowship training in the Department of Obstetrics and Gynecology at the UCLA School of Medicine. Subsequently, he was an Associate Clinical Professor of Ob-Gyn at UCLA and acting director of the Division of Reproductive Medicine at Cedars- Sinai Medical Center. He is Past President of the Society for Assisted Reproductive Technology, Society of Reproductive Surgeons and Pacific Coast Reproductive Society and past member of the Board of Directors of the American Association of Gynecologic Laparoscopists. Abstract This retrospective cohort study compared outcomes from transfer of embryos cryopreserved at the pronuclear versus blastocyst stage following freeze-all IVF cycles without fresh transfer for 87 consecutive IVF patients <40 years, who underwent cryopreservation of all viable embryos followed by at least one subsequent frozen embryo transfer (FET) between January 2003 and July Cryopreservation of all embryos from one oocyte retrieval was performed at either the pronuclear (1.5 mol/l propanediol and 0.1 mol/l sucrose) (group A) or blastocyst (10% glycerol) (group B) stage. Main outcome measures included survival, live birth and implantation rates. A total of 110 FET cycles were analysed. Live birth and implantation rates observed after the first FET were significantly higher (P = and P = 0.002) in group B (67.7% and 40.8%) than in group A (41.1% and 21.5%) despite a higher survival rate in group A. After two FET cycles, 32.1% of group A had not conceived despite thaw of all available embryos, compared with 6.5% of group B. When freeze-all is necessary, blastocyst cryopreservation leads to higher implantation and live birth rates compared with pronuclear-stage cryopreservation despite lower survival rates. Prolonged embryo culture may allow for more optimal embryo selection. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: blastocyst, cryopreservation, in-vitro fertilization, ovarian hyperstimulation syndrome /$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 412 E Surrey et al. Introduction Successful transfer of cryopreserved and thawed human embryos was first reported in 1983, and has since become an integral component of the assisted reproductive technologies (Trounson and Mohr, 1983). Cryopreservation of supernumerary embryos provides not only the means to reduce the number of embryos transferred per cycle, thereby diminishing the multiple pregnancy rate, but also to maximize the cumulative pregnancy rate per oocyte retrieval while reducing overall treatment costs (Kjellberg et al., 2006; Tiitenen et al., 2001; Veeck et al., 1993). Cryopreservation of all embryos in an IVF cycle ( freeze-all ) offers a valuable alternative to cycle cancellation in a variety of unforeseen circumstances in which a fresh transfer would not be advantageous, such as the case of ovarian hyperstimulation syndrome, premature elevation of serum progesterone concentrations and previously undiagnosed hydrosalpinges (Ferraretti et al., 1999; Queenan et al., 1997; Schoolcraft et al., 1991; Shapiro et al., 2008; Yovel et al., 1995). Additionally, cryopreservation of all embryos allows for planned embryo banking for fertility preservation. Successful outcomes have been reported after transfer of both supernumerary and freeze-all embryos cryopreserved at the 2 pronuclear (2PN), cleavage and blastocyst stages, but the optimal stage of embryo cryopreservation has not been established (Ferraretti et al., 1999; Griesinger et al., 2007; Salumets et al., 2003; Shapiro et al., 2010; Testart et al., 1987; Tummon et al., 2004; Veeck, 2003). Success at the pronuclear stage has been attributed to the lack of a spindle apparatus in the single-celled pronucleate embryo which may provide greater protection from the freezing process. Comparative trials have demonstrated higher survival and cumulative delivery rates after transfer of cryopreserved pronucleate embryos than cleavage-stage embryos (Demoulin et al., 1991; Horne et al., 1997; Senn et al., 2000). However, a disadvantage of cryopreservation at these earlier stages of embryonic development is the limited amount of available information regarding embryo morphology and developmental potential. The development of sequential media and enhanced culture techniques has resulted in the achievement of high implantation rates with fresh blastocyst-stage embryo transfer (Gardner et al., 1998, 2004; Karaki et al., 2002; Papanikolaou et al., 2006). If culture conditions are optimal, cryopreservation of embryos at the blastocyst stage would allow for enhanced embryo selection due to a greater ability to assess embryo morphology after activation of the embryonic genome. This could result in enhanced outcomes while allowing the clinician to delay the decision to freeze-all if the severity of the clinical condition is not completely established. High implantation rates following transfer of cryopreserved and thawed blastocyst-stage embryos have been described (Anderson et al., 2003; Veeck et al., 2004). However, investigations comparing cryopreservation at the blastocyst with other developmental stages have typically only evaluated outcomes with supernumerary embryos, a circumstance in which the highest quality embryos have already been transferred (Anderson et al., 2003; Veeck et al., 2004). The outcomes from cryopreservation and transfer of supernumerary embryos do not account for factors contributing to the development and quality of embryos obtained in the fresh cycle. Few reports have been published detailing the comparative efficacy between pronuclear, cleavage and blastocyst frozen embryo transfer (FET) following freeze-all. The aim of this study, therefore, was to compare outcomes from FET following freeze-all IVF cycles performed at the pronuclear versus blastocyst stage. Materials and methods All patients who underwent freeze-all at the Colorado Centre for Reproductive Medicine between January 2003 and July 2007 met inclusion criteria and had completed at least one subsequent FET were evaluated in this investigation. Patients were included if they were <40 years of age at transfer, had a normal endometrial cavity documented by ultrasound examination and office hysteroscopy performed within 6 months of the FET and had an endometrial lining 8 18 mm in thickness with an adequate pattern documented sonographically prior to transfer. Exclusion criteria included cycles involving oocyte donation, preimplantation genetic diagnostic testing and those in which embryos were cryopreserved by vitrification. A total of 110 FET cycles were analysed in 87 patients. Patients were divided into two groups based on the stage of embryo development at cryopreservation. Group A consisted of 56 patients with all embryos cryopreserved at the pronuclear stage and group B consisted of 31 patients with all embryos cryopreserved at the blastocyst stage. All embryos from a given oocyte aspiration procedure were cryopreserved at a single developmental stage. The time period during which the cycles were performed was the same for both groups. Pronuclear-stage cryopreservation was performed by employing a slow-freeze protocol using 1.5 mol/l propanediol and 0.1 mol/l sucrose as ultimate cryoprotectant concentrations (Testart et al., 1987). All 2PN embryos were cryopreserved in cryovials on the day that normal fertilization was identified. Blastocyst cryopreservation was performed employing a slow-freeze protocol using 10% glycerol as the ultimate cryoprotectant concentration (Horne et al., 1997). All blastocysts of grade 2/3 or more were cryopreserved in cryovials on day 5 or 6 after oocyte aspiration. Patients included in group A were primarily those whose indications for freeze-all was clearly identified prior to oocyte aspiration, e.g. high risk for ovarian hyperstimulation syndrome (OHSS), previously undiagnosed hydrosalpinges suspected sonographically during ovarian hyperstimulation and premature elevation of serum progesterone concentrations. In this group, the decision to perform freeze-all was made prior to oocyte aspiration. Patients included in group B were primarily those who were being monitored after oocyte aspiration for suspected OHSS or evidence of persistent fluid accumulation in the endometrial cavity. Failure of these issues to resolve or progression of symptoms by the day of embryo transfer would necessitate a delayed freeze-all. Thus, group B included patients for whom the decision to freeze-all was not made until after oocyte aspiration had been performed. Previously undiagnosed tubal

3 Freeze-all: cleavage vs. blastocyst stage 413 or uterine abnormalities which had necessitated freeze-all were corrected prior to initiation of the frozen embryo transfer cycle. Patients underwent endometrial preparation for FET with transdermal oestradiol patches 0.1 mg (Vivelle Dot; Novartis Pharmaceuticals, East Hanover, NJ) self-administered at progressively increasing doses to achieve an endometrial thickness of 8 18 mm documented sonographically. Gonadotrophin-releasing hormone agonist (Lupron; Abbott Laboratories, Chicago, IL) down-regulation was administered at physician discretion to patients with regular menses and to all patients with irregular menses. Luteal support consisted of progesterone in oil 50 mg i.m. daily (Watson Pharmaceuticals, Corona, CA) or micronized progesterone 200 mg vaginally three times daily (Prometrium; Solvay Pharmaceuticals, Marietta, GA) in conjunction with continuation of oestradiol supplementation. Pronuclear embryos were thawed on the second day of progesterone administration and transferred on the subsequent day after documentation of cleavage. Blastocysts were thawed and transferred on the sixth day of progesterone administration. When patients had blastocyst-stage embryos cryopreserved on both day 5 and 6, day-5 embryos were preferentially thawed and transferred. Embryo transfers were performed under ultrasound guidance as has been previously described (Gardner et al., 1998). Serum human chorionic gonadotrophin concentrations were measured 14 days after supplemental progesterone initiation and ultrasound examinations to document pregnancy viability were performed on at least two occasions 2 4 weeks after evidence of normally rising human chorionic gonadotrophin concentrations was obtained. Oestradiol and progesterone supplementation was maintained until evidence of a viable intrauterine pregnancy on ultrasound examination was noted after which doses were progressively tapered. Survival rate was defined as the number of embryos which appeared to survive morphologically and, in the case of pronuclear-stage embryos cleaved prior to transfer, per number of embryos thawed. Implantation rate was defined as the number of intrauterine gestational sacs with cardiac activity visible on at least two ultrasound examinations performed at least 4 weeks after documentation of a positive pregnancy test per the total number of embryos transferred. Live birth rate was defined as the number of deliveries resulting in live births per number of embryo transfer procedures. Delivery of a multiple pregnancy was considered to be a single live birth. Biochemical pregnancy rate was defined as the number of positive pregnancy tests which subsequently declined prior to performance of a first ultrasound examination per number of embryo transfer procedures. Spontaneous abortion rate was defined as the number of nonviable intrauterine pregnancies documented by ultrasound evaluation per number of embryo transfer procedures. Data were analysed by unpaired t-tests, chi-squared analyses and Fisher s exact tests as appropriate. P-values <0.05 were considered to be statistically significant. Results are expressed as mean ± SD unless otherwise indicated. Results Patient characteristics and indications for freeze-all are displayed in Table 1. The mean age of patients in group A was slightly greater than that of group B, a difference which was statistically significant (P < 0.01) but unlikely to be of clinical significance given the relatively young ages of both groups. There were no significant differences in ovarian reserve testing or characteristics of ovarian stimulation between the groups with the exception of the administration of significantly higher gonadotrophin doses in group A (P < 0.01). As expected, the distribution of indications for Table 1 Characteristic Patient characteristics. Group A Group B No. of patients Stage of cryopreservation 2PN Blastocyst Age (years) 33.8 ± ± 2.7 a Day-3 FSH (miu/ml) 7.4 ± ± 2.5 Gonadotrophin dose (units) ± ± a Gonadotrophin duration (days) 11.3 ± ± 3 Peak oestradiol (pg/ml) ± ± No. of oocytes retrieved 20.9 ± ± 7.8 a No. of mature oocytes/oocytes retrieved 734/940 (78.1) 505/626 (80.7) No. of embryos cryopreserved 13.1 ± ± 3.9 a Reason for freeze-all High-risk OHSS 23/56 (41.1) 28/31 (90.3) a Premature progesterone elevation 15/56 (26.8) 1/31 (3.2) a Ultrasound evidence of hydrosalpinx 7/56 (12.5) 0/31 (0) a Endometrial abnormalities 7/56 (12.5) 1/31 (3.2) a Fertility preservation 3/56 (5.4) 0/31 (0) Other 1/56 (1.8) 1/31 (3.2) Values are mean ± SD or number/total (%). OHSS = ovarian hyperstimulation syndrome. a P < 0.01 versus group A.

4 414 E Surrey et al. freeze-all was different between the two groups. The indications for the 56 patients in group A included incipient OHSS (41.1%), premature elevation of serum progesterone concentrations (26.8%), evidence of previously undiagnosed hydrosalpinx suggested by ultrasound evaluation during ovarian hyperstimulation (12.5%), endometrial cavity abnormalities (12.5%) and fertility preservation (5.4%), whereas the indication for freeze-all in group B was primarily due to incipient OHSS (90.3%). There were no patients whose embryos were cryopreserved on day 3, 151 blastocysts from 26 patients were cryopreserved on day 5 and 122 blastocysts from 29 patients were cryopreserved on day 6. Therefore, the majority of patients had embryos cryopreserved on both days (24 patients, 231 embryos). Two patients (18 embryos) only had blastocysts cryopreserved on day 5 and five patients (24 embryos) only had blastocysts cryopreserved on day 6. All patients who initiated a frozen embryo transfer cycle subsequently underwent a transfer in that cycle. No patients who were scheduled to undergo blastocyst freeze-all failed to have embryos available for cryopreservation. Details of the first FET after freeze-all are presented in Table 2. The survival rate as well as mean number of embryos thawed and transferred was significantly higher in group A than in group B (P = 0.003, P = and P = 0.008, respectively). However, significantly higher implantation rates (40.8% versus 21.5%, P < 0.002) and live birth rates (67.7% versus 41.1%, P = 0.025) were observed for group B in comparison with group A. The differences in biochemical pregnancy and spontaneous abortion rates between the groups were not statistically significant. The implantation rate per total number of embryos thawed was significantly lower in group A than B (11.3% versus 23.8%, P < 0.01). As previously stated, the laboratory s policy is to preferentially thaw and transfer blastocysts which have been cryopreserved on day 5. Therefore, only five patients who had no day-5 embryos cryopreserved during the freeze-all cycle underwent transfer of day-6 embryos during their first FET cycle. Of these patients, four experienced a live birth. The implantation rate in this subgroup was 45.5% (5/11), which was not significantly different from those undergoing transfer of cryopreserved and thawed day-5 blastocysts (40.0%, 26/65). Analysis of cumulative FET cycle outcomes again demonstrated a higher mean total number of embryos thawed (8.2 versus 5.0) and transferred (4.2 versus 3.0) for group A than group B, yet significantly higher live birth (80.6% versus 57.1%; P = 0.026) and implantation (40.9% versus 23.6%; P = 0.003) rates for group B versus group A (Table 3). Furthermore, the percentage of patients who had not yet conceived despite the thaw of all available embryos was higher for group A (32.1%) than for group B (6.5%; P = 0.007). Discussion The results of the current investigation demonstrate that when freeze-all is necessary, cryopreservation of embryos at the blastocyst stage leads to higher implantation and live Table 2 Outcomes of first frozen embryo transfer. Group A Group B RR (95% CI) P-value No. of frozen embryo transfers No. of embryos thawed 6.0 ± ± No. of embryos transferred 3.2 ± ± Survival rate 286/336 (85.1) 91/130 (70.0) ( ) Live birth rate 23/56 (41.1) 21/31 (67.7) ( ) Implantation rate 38/177 (21.5) 31/76 (40.8) ( ) Biochemical pregnancy 7/56 (12.5) 4/31 (12.9) ( ) NS Spontaneous abortion rate 3/56 (5.4) 2/31 (6.5) ( ) NS Values are mean ± SD or number/total (%). NS = not statistically significant. Table 3 Cumulative outcomes of frozen embryo transfer. Group A Group B RR (95% CI) P-value No. of patients Total no. of frozen embryo transfers No. of embryos thawed 8.2 ± ± No. of embryos transferred 4.2 ± ± Cumulative live birth rate 32/73 (43.8) 25/37 (67.6) ( ) Cumulative implantation rate 56/237 (23.6) 38/93 (40.9) ( ) No conception/patient 24/56 (42.9) 6/31(19.4) ( ) No conception, no embryos remaining 18/56 (32.1) 2/31 (6.5) ( ) Values are mean ± SD or number/total (%).

5 Freeze-all: cleavage vs. blastocyst stage 415 birth rates than cryopreservation at the pronuclear stage. Notably, after two FET cycles, a higher percentage of group A patients had not conceived after thaw of all available embryos despite having more embryos available for cryopreservation and a higher post-thaw survival rate. There are several possible explanations for this phenomenon. It is theoretically possible, but unlikely, that the pronuclear-stage cryopreservation techniques employed may result in a higher degree of immediate survival, but may impair subsequent embryo development after transfer. As far as is known, there are no data which would support this contention. In contrast, it is more likely that allowing embryos to develop to the blastocyst stage prior to cryopreservation allows for freezing of a higher percentage, but lower number, of developmentally competent embryos which are subsequently replaced into a more receptive prepared uterine environment. Prolonged embryo culture, therefore, may allow for more optimal embryo selection if optimal culture conditions exist, thus increasing the likelihood of pregnancy as has been demonstrated with fresh embryo transfers in which implantation rates have been shown to be significantly higher with transfer of blastocystversus cleavage-stage embryos (Gardner et al., 1998; Papanikolaou et al., 2006). Cryopreservation at the blastocyst stage may also provide further benefit in cases of incipient OHSS. Various strategies have been proposed to prevent severe OHSS, including cycle cancellation, albumin administration, coasting and administration of dopamine agonists (Al-Shawaf et al., 2001; Alboulghar et al., 2002; Alvarez et al., 2007; Sher et al., 1993). Freeze-all provides an adjunctive and alternative approach that decreases the risk of severe OHSS while preserving the chance of pregnancy from embryos derived from the immediate cycle (Ferraretti et al., 1999; Griesinger et al., 2007; Queenan et al., 1997). Because not all patients with high serum oestradiol concentrations and a large cohort of follicles develop clinically significant OHSS, the ability to successfully cryopreserve all embryos at the blastocyst stage, as demonstrated in this trial, allows the clinician time to more accurately identify those high-risk patients who do not develop symptoms by the time of embryo transfer. Thus, the ultimate decision to freeze-all can be deferred with no compromise in cycle outcome. Existing data regarding cryopreservation of embryos largely results from trials which evaluated outcomes with supernumerary embryos (Veeck et al., 2003; Anderson et al., 2003). Anderson et al. reported upon results from 224 cleavage-stage and 29 blastocyst-stage frozen embryo transfers (Anderson et al., 2003). Cryopreserved blastocysts demonstrated higher post-thaw survival, clinical pregnancy and implantation rates (81%, 69% and 43%, respectively) than cleavage-stage embryos (73%, 42% and 18%, respectively). Veeck and colleagues (2003) described similar survival rates, but significantly higher clinical pregnancy and implantation rates for frozen thawed blastocysts (77.2%, 64.2% and 38.5%, respectively) compared with pronuclear-stage (76.4%, 42.1% and 17.1%, respectively) and cleavage-stage (78.6%, 37.4% and 15.2%, respectively) embryos. These investigators, however, evaluated excess embryos cryopreserved after fresh transfer of those of higher or at least equivalent quality. In the present analysis, only patients who had cryopreservation of all embryos were included which eliminates this confounding variable. The decision to cryopreserve at the pronuclear or blastocyst stage in the current investigation was not dependent on embryo quality or number, but rather on timing of the decision for freeze-all. This study was not designed to evaluate comparative outcomes from transfer of blastocysts cryopreserved on day 5 versus day 6 and, as a result, combined data are presented. As previously mentioned, the laboratory s protocol is to preferentially transfer blastocysts which have been cryopreserved on day 5. Therefore, the vast majority of first cycle frozen embryo transfers were of day-5 blastocysts. The small sample size of patients who underwent transfer of only cryopreserved and thawed day-6 blastocysts cannot yield meaningful data. However, it is encouraging to note that the outcomes in this subgroup were no different than in the blastocyst transfer group as a whole. This confirms the findings of Shapiro et al. (2010) who reported similar pregnancy rates with transfer of day-5 versus day-6 blastocysts in FET cycles employing a prepared endometrium, but much poorer outcomes with fresh cycle transfer of day-6 blastocysts, suggesting the predominant role of endometrial receptivity if embryo quality is otherwise equal. Multiple components play roles in the success of frozen embryo transfer, including laboratory factors, the quality of embryos transferred, the age of the patient and the aetiology of infertility (Salumets et al., 2006; Wang et al., 2001). The retrospective nature of this analysis allowed for a large number of freeze-all cycles to be compared. Nevertheless, this retrospective study cannot control for other confounding variables. The mean age of the patients in the present study was significantly higher in group A than in group B. However, given the fact that the mean ages of both patient populations are below 35 years, the clinical impact of this difference is unlikely to be significant. Additionally, the differing indications for freeze-all could potentially confound the results, although the indications for freeze-all such as hydrosalpinges, endometrial cavity abnormalities, OHSS and aberrant progesterone rises were resolved prior to the frozen embryo transfer cycle. Patients in group A were more likely to be those in whom a decision to cryopreserve all embryos was made prior to oocyte aspiration, whereas in group B, the decision to freeze-all was more likely to be made after oocyte aspiration, thus explaining the preponderance of patients with incipient OHSS in the latter group. Nevertheless, it is hard to imagine that these different indications for freeze-all, once resolved, would have an impact on the ultimate outcome of a frozen embryo transfer into a uterus with a prepared endometrium. A prospective, randomized trial assessing outcomes for patients with equally distributed ages, aetiology of infertility and indications for freeze-all would be beneficial to control for these factors. Vitrification, or the rapid-freezing of embryos, has been implemented in many IVF programmes and has been shown in comparative trials to be associated with a higher post-thaw survival rate than slow-freezing techniques (Loutradi et al., 2008). Although cycles in which vitrification of embryos was utilized were excluded in this study, the outcomes seen here would likely be enhanced with routine implementation of blastocyst vitrification.

6 416 E Surrey et al. The cryopreservation of embryos provides value in many situations in an IVF programme. Freeze-all cycles are beneficial to preserve fertility, to reduce the risks associated with OHSS and to improve cycle outcome in cases of premature luteinization or other unforeseen complications. The ultimate goal in any IVF cycle is the establishment of a healthy singleton pregnancy. The results of the current retrospective analysis would suggest that even if the decision to cryopreserve all embryos is made prior to oocyte aspiration, extended embryo culture with blastocyst-stage cryopreservation may allow for selection of higher quality embryos and ultimately provide the opportunity to optimize pregnancy outcomes. Acknowledgements The authors would like to gratefully acknowledge the superb care provided by the embryology and nursing teams at the Colorado Centre for Reproductive Medicine, without whom, this work would not have been possible. References Alboulghar, M., Evers, J.H., Al-INany, H., Intravenous albumin for preventing severe ovarian hyperstimulation syndrome. Cochrane Database Syst. Rev. 2, CD Al-Shawaf, T., Zosmer, A., Hussain, S., et al., Prevention of severe ovarian hyperstimulation syndrome in IVF with or without ICSI and embryo transfer: a modified coasting strategy based on ultrasound for identification of high-risk patients. Hum. Reprod. 16, Alvarez, C., Marti-Bonmati, L., Norella-Maestre, E., et al., Dopamine agonist cabergoline reduces hemo-concentration and ascites in hyperstimulated women undergoing assisted reproduction. J. Clin. Endocrinol. Metab. 92, Anderson, A.R., Weiker, W.L., Crain, J.L., Determining the most optimal stage for embryo cryopreservation. Reprod. Biomed. Online 8, Demoulin, A., Jouan, C., Gerday, C., Dubois, M., Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages. Hum. Reprod. 6, Ferraretti, A.P., Gianaroli, L., Magli, C., Fortini, D., Selman, H.A., Feliciani, E., Elective cryopreservation of all pronucleate embryos in women at risk of ovarian hyperstimulation syndrome: efficiency and safety. Hum. Reprod. 14, Gardner, D.K., Schoolcraft, W.B., Wagley, L., Schlenker, T., Stevens, J., Hesla, J., A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum. Reprod. 13, Gardner, D.K., Surrey, E., Minjarez, D., Leitz, A., Stevens, J., Schoolcraft, W.B., Single blastocyst transfer: a prospective randomized trial. Fertil. Steril. 81, Griesinger, G., von Otte, S., Schroer, A., Ludwig, A., Diedrich, K., Al-Hasani, S., Elective cryopreservation of all pronuclear stage oocytes after GnRH agonist triggering of final oocyte maturation in patients at risk of developing OHSS: a prospective, observational proof of concept study. Hum. Reprod. 22, Horne, G., Critchlow, J.D., Newman, M.C., Edozien, L., Matson, P.L., Lieberman, B.A., A prospective evaluation of cryopreservation strategies in a two-embryo transfer programme. Hum. Reprod. 12, Karaki, R.Z., Samarraie, S.S., Younis, N.A., Lahloub, T.M., Ibrahim, M.H., Blastocyst culture and transfer: a step toward improved in-vitro fertilization outcome. Fertil. Steril. 77, Kjellberg, A.T., Carlsson, P., Bergh, C., Randomized single versus double embryo transfer: obstetric and paediatric outcome and a cost-effectiveness analysis. Hum. Reprod. 21, Loutradi, K.E., Kolibianakis, E.M., Venetis, C.A., et al., Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil. Steril. 90, Papanikolaou, E.G., D haeseleer, E., Verheyen, G., et al., Live birth rate is significantly higher after blastocyst transfer than after cleavage-stage embryo transfer when at least four embryos are available on day 3 of embryo culture. A randomized prospective study. Hum. Reprod. 21, Queenan, J.T., Veeck, L.L., Toner, S.O., Muasher, S.J., Cryopreservation of all prezygotes in patients at risk of severe hyperstimulation does not eliminate the syndrome, but the chances of pregnancy are excellent with subsequent frozen-thaw transfers. Hum. Reprod. 12, Salumets, A., Tuuri, T., Makinen, S., et al., Effect of developmental stage of embryo at freezing on pregnancy outcome of frozen-thawed embryo transfer. Hum. Reprod. 18, Salumets, A., Suikkari, A.M., Makinen, S., Karro, H., Roos, A., Tuuri, T., Frozen-embryo transfers: implications of clinical and embryological factors on pregnancy outcome. Hum. Reprod. 21, Schoolcraft, W., Sinton, E., Schlenker, T., Huynh, D., Hamilton, F., Meldrum, D., Lower pregnancy rate with premature luteinization during pituitary suppression with leuprolide acetate. Fertil. Steril. 55, Senn, A., Vozzi, C., Chanson, A., De Grandi, P., Germond, M., Prospective randomized study of two cryopreservation policies avoiding embryo selection: the pronucleate stage leads to higher cumulative delivery rate than the early cleavage stage. Fertil. Steril. 74, Shapiro, B., Daneshmand, S., Garner, F., Aguirre, M., Ross, R., Contrasting patterns in in vitro fertilization pregnancy rates among fresh autologous, fresh oocyte donor and cryopreserved cycles with the use of day 5 or day 6 blastocysts may reflect differences in embryo-endometrium synchrony. Fertil. Steril. 89, Shapiro, B.S., Daneshmand, S.T., Garner, F.C., Aguirre, M., Hudson, C., Thomas, S., Embryo cryopreservation rescues cycles with premature luteinization. Fertil. Steril. 93, Sher, G., Salem, R., Feinman, M., Dodge, S., Zouves, C., Knutzen, V., Eliminating the risk of life-endangering complications following overstimulation with menotropin fertility agents: a report on women undergoing in vitro fertilization and embryo transfer. Obstet. Gynecol. 81, Testart, J., Lassalle, B., Forman, R., Gazengel, A., Belaisch-Allart, J., Hazout, A., et al., Factors influencing the success rate of human embryo freezing in an in vitro fertilization and embryo transfer program. Fertil. Steril. 48, Tiitenen, A., Halttunen, M., Harkki, P., Vuoristo, P., Hyden-Granskog, C., Elective single embryo transfer: the value of cryopreservation. Hum. Reprod. 16, Trounson, A., Mohr, L., Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305, Tummon, I.S., Contag, S.A., Thornhill, A.R., Session, D.R., Dumesic, D.A., Damario, M.A., Cumulative first live birth after elective cryopreservation of all embryos due to ovarian hyperresponsiveness. Fertil. Steril. 81, Veeck, L.L., Does the developmental stage at freeze impact on clinical results post-thaw? Reprod. Biomed. Online 6,

7 Freeze-all: cleavage vs. blastocyst stage 417 Veeck, L.L., Amundson, C.H., Brothman, L.J., DeScisiolo, C., Maloney, M.K., Muasher, S.J., et al., Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertil. Steril. 59, Veeck, L.L., Bodine, R., Clarke, R.N., Berrios, R., Libraro, J., Moschini, R.M., et al., High pregnancy rates can be achieved after freezing and thawing human blastocysts. Fertil. Steril. 82, Wang, J.X., Yap, Y.Y., Matthew, C.D., Frozen-thawed embryo transfer: influence of clinical factors on implantation rate and risk of multiple conception. Hum. Reprod. 16, Yovel, I., Yaron, Y., Amit, A., Reuben Peyser, M., David, M.P., Kogosowski, A., et al., High progesterone levels adversely affect embryo quality and pregnancy rates in in vitro fertilization and oocyte donation programs. Fertil. Steril. 64, Declaration: The authors report no financial or commercial conflicts of interest. Presented in part at the 64th Annual Meeting of the American Society for Reproductive Medicine, San Francisco, CA, 8 12 November Received 28 November 2009; refereed 30 March 2010; accepted 7 April 2010.