Impact of oocyte cryopreservation on embryo development

Impact of oocyte cryopreservation on embryo development M. Cristina Magli, M.Sc., Michela Lappi, B.Sc., Anna P. Ferraretti, M.D., Alessandra Capoti, B.Sc., Alessandra Ruberti, B.Sc., and Luca Gianaroli, M.D. Reproductive Medicine Unit, Societa Italiana Studi Medicina della Riproduzione, Bologna, Italy Objective: To verify whether the morphologic evaluation of zygotes and embryos derived from thawed oocytes could provide some relevant information regarding their developmental performance. Design: Fertilization, zygote, and embryo morphology from sibling fresh and frozen oocytes was compared. Setting: Reproductive Medicine Unit, Societa Italiana Studi Medicina della Riproduzione, Bologna, Italy. Patient(s): Two hundred thirty-four patients underwent intracytoplasmic sperm injection cycles from which 1,101 spare metaphase II oocytes were cryopreserved. Subsequently, 256 thawing cycles were performed, and 997 oocytes were thawed. Intervention(s): Intracytoplasmic sperm injection was performed on both fresh and frozen oocytes. Main Outcome Measure(s): Fertilization rates, pronuclear zygote morphology, and embryo cleavage rates. Result(s): Thawed oocytes had lower chances of being fertilized and developing into top-quality zygotes and regularly cleaving embryos when compared with sibling fresh oocytes irrespective of female age. As a result, the percentage of transferred cycles was significantly lower in frozen cycles compared with fresh cycles (79% and 93%, respectively); the proportion of transferred top-quality embryos followed the same trend. Conclusion(s): Reduced fertilization and cleavage rates in frozen cycles when compared with sibling fresh oocytes suggest that, even if surviving thawing, the process of slow freezing has a negative impact on the potential of further growth that is evident as early as the first cleavage divisions. (Fertil Steril Ò 2010;93:510 6. Ó2010 by American Society for Reproductive Medicine.) Key Words: Fertilization rate, cleavage rate, oocyte cryopreservation, pronuclear zygote morphology, propanediol, slow-freezing, top-quality embryos, vitrification Although the theoretical advantages of human oocyte cryopreservation as an alternative to embryo freezing are undeniable, the corresponding clinical outcome appears to be critically inferior to that reported after zygote and embryo thawing (1, 2). The first pregnancies resulting from frozen oocytes were reported in the 1980s (3, 4), but for a long time only sporadic reports followed these initial results, leading to the conclusion that the technique of oocyte cryopreservation was quite far from being efficient and clinically applicable. In the following years, some important technical modifications have been proposed including an increased concentration of sucrose in the freezing and thawing solutions with propanediol as cryoprotectant (5, 6) and the use of freezing media in which choline replaced sodium (7, 8). More recently, vitrification has been reported to be a very promising technique (9 13), and several studies are ongoing to confirm its clinical validation. To date, an increasing number of studies and clinical pregnancies have been reported confirming the growing interest in Received November 22, 2008; revised January 16, 2009; accepted January 27, 2009; published online April 1, 2009. M.C.M. has nothing to disclose. M.L. has nothing to disclose. A.P.F. has nothing to disclose. A.C. has nothing to disclose. A.R. has nothing to disclose. L.G. has nothing to disclose. Reprint requests: Luca Gianaroli, M.D., S.I.S.M.E.R., Via Mazzini, 12, 40138 Bologna, Italy (FAX: 39-051-302933; E-mail: luca.gianaroli@ sismer.it). oocyte cryobanking (14 18). This is true not only in those countries where it represents the only option for IVF patients, zygote and embryo freezing being forbidden by local regulations (19), but also in consideration of the several applications that could be of wider interest. These include the preservation of fertility in women at risk of premature ovarian failure because of hormonal dysfunction or the use of gonadotoxic therapies for the treatment of cancer, the impossibility of having sperm available at the time of the partner s oocyte insemination, and cases of ethical or religious concerns, as well as possession of property related to embryo cryopreservation. In addition, the oocyte cryobank could be extremely convenient in the organization and management of an oocyte donation program (20). All these possible applications promoted an increasing interest in oocyte cryopreservation and a great deal of research on factors and strategies that could improve the overall outcome of oocyte freezing as a valid alternative to a new fresh cycle. Concomitantly, novel information on oocyte physiology has been obtained, especially on the structures and processes that, being affected by the freezing-thawing procedures, could be responsible for the decreased competence of embryos derived from cryopreserved oocytes (21 23). The aim of this study was to verify whether the morphologic evaluation of embryos derived from thawed oocytes could provide some relevant information regarding their developmental performance. For this reason, the morphology 510 Fertility and Sterility â Vol. 93, No. 2, January 15, 2010 0015-0282/10/$36.00 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2009.01.148

of two-pronuclear oocytes and embryos generated from thawed oocytes was compared with that resulting from sibling fresh oocytes. The analysis was conducted in relation to maternal age. MATERIALS AND METHODS Patients Between March 2004 and March 2008, 234 patients (aged 35.2 6.61 years) underwent an intracytoplasmic sperm injection (ICSI) cycle from which 1,101 spare metaphase II oocytes were cryopreserved. Subsequently, these patients returned for a thawing cycle because of [1] no pregnancy achieved in the fresh cycle (n ¼ 221) and [2] spontaneous abortion (n ¼ 13). Two hundred fifty-six thawing cycles were performed, and 997 oocytes were thawed. Induction of multiple follicular growth was obtained by administering exogenous gonadotropins after a long desensitization protocol with long-acting GnRH analogues (24, 25). Oocytes were retrieved transvaginally via ultrasound guidance at 34 to 36 hours after hcg administration and cultured in human tubal fluid (HTF) (SAGE CooperSurgical Inc., Pasadena, CA) supplemented with 5% human serum albumin (HSA, SAGE) at 37 C in a 5% CO 2 humidified gas atmosphere. Insemination (see later in this article) was performed at 4 to 5 hours after retrieval, and spare oocytes were cryopreserved at 41.2 2.2 hours after hcg. Thawing cycles were performed by administering a hormonal replacement therapy consisting of E 2 valerate (Progynova; Schering, Milan, Italy) at 2 mg/d from day 1 to day 5 of the menstrual cycle, 4 mg/d from day 6 to day 10, 6 mg/ d from day 11 to day 13, and 4 mg/d from day 14. On day 14, 50 mg/d of P was given, and the dose was increased to 100 mg/d from day 15 (26). The performance of cycles was evaluated in relation to maternal age by subdividing patients with an age %35 years versus those with an age R36 years. Freezing Procedure Oocyte cryopreservation was performed after a slow freezing protocol having propanediol and sucrose as cryoprotectants (5). In brief, freezing solutions were based on Dulbecco s phosphate-buffered saline (PBS) solution (GIBCO, Life Technologies Ltd., Paisley, Scotland) and plasma protein supplement (Plasmanate; Medicult, Jyllinge, Denmark) to prepare equilibration (1.5 mol/l propanediol þ 30% Plasmanate in PBS) and loading solutions (1.5 mol/l propanediol þ 0.3 mol/l sucrose þ 30% Plasmanate in PBS). Cumulus-free oocytes were left 10 minutes at room temperature in the equilibration solution and then transferred to the loading solution. In 1 minute, the oocytes were loaded individually in plastic straws (Paillettes Crystal 133 mm; Cryo Bio System, Paris, France) and cooled in a programmable freezer (Kryo 10 Series; Planer Products, Sunbury Thames, United Kingdom) at a rate of 2 C/min from 20 Cto 7 C. At this point, seeding was performed manually, and the cooling was continued at a rate of 0.3 C/min to 30 C, and 50 C/min to 150 C. Finally, the straws were plunged into liquid nitrogen and stored. Only metaphase II oocytes with a normal ooplasmic morphology were cryopreserved. Thawing Procedure The thawing solutions (TS) contained decreasing concentrations of propanediol in a constant concentration of 0.3 mol/l sucrose: TS1 (1.0 mol/l propanediol þ 0.3 mol/l sucrose þ 30% Plasmanate), TS2 (0.5 mol/l propanediol þ 0.3 mol/l sucrose þ 30% Plasmanate), and TS3 (0.3 mol/l sucrose þ 30% Plasmanate). The procedure was carried out at room temperature. Each straw was held in air for 30 seconds and then left for 40 seconds in a water bath at 30 C. The oocyte was released and transferred sequentially to TS1 and TS2 for 5 minutes respectively, to TS3 and to PBS þ 30% Plasmanate for 10 minutes respectively, followed by 10 additional minutes in PBS þ 30% Plasmanate at 37 C. Thawed oocytes were considered intact when showing zona pellucida integrity, cytoplasmic clearness, and absence of shrinkage or swelling. Intact oocytes were incubated in 100 ml drop of cleavage medium (SAGE) supplemented with 5% HSA (SAGE) under warm mineral oil (SAGE) at 37 C in a humidified 5% CO 2 atmosphere. Insemination and Embryo Culture According to the national regulation on IVF prohibiting the formation of more than three embryos per cycle, up to three vital oocytes per fresh and thawing cycle were inseminated (19). The selection of the oocytes to be inseminated was performed according to an accurate analysis of their morphology. Insemination was performed by ICSI at 4 to 5 hours after retrieval for the fresh cycles and at approximately 4 hours after thawing for the thawed cycles. Fertilization was assessed 16 hours later for the presence of two pronuclei and two polar bodies, and fertilized oocytes were evaluated for their morphology as previously described (27). In brief, five patterns of pronuclear shape and location were described: A juxtaposed and centralized, B juxtaposed and peripheral, C centralized and separated, D different in size, and E fragmented. The position and size of nucleoli within pronuclei was classified as 1 large size and aligned; 2 large size, scattered; 3 large size, aligned in one pronucleus and scattered in the other; 4 small size, scattered; and 5 any other configuration in which the two pronuclei totally differ between them. Finally, the position of polar bodies was related to the longitudinal axis of pronuclei: a in the longitudinal axis 30 degrees, b perpendicular to the longitudinal axis 30 degrees, and g in different angles with a rotation >30 degrees off the longitudinal or the perpendicular axis. The configurations A1a,A2a,A1b, and A2b that, according to previous studies, had been associated with the highest chances of chromosome euploidy, embryo development, Fertility and Sterility â 511

FIGURE 1 Two pronuclear configurations, A1a, A2a, A1b, and A2b, defining top-quality zygotes. and implantation (27) were defined as top-quality zygotes (Fig. 1). Fertilized oocytes were transferred to fresh cleavage medium supplemented with 10% HSA and cultured individually until the day of transfer that was normally performed on day 2 or 3. The culture period was further extended when three embryos developed regularly with the aim of favoring their selection in culture. This was done to minimize the occurrence of three-embryo transfers, keeping in mind the obligation by law to transfer all the generated viable embryos (19). In this case, day 3 embryos were transferred to blastocyst medium (SAGE) supplemented with 10% HSA. Embryo scoring was performed on day 2 at 40 hours after insemination, and then regularly at 24-hour time intervals. Embryos were evaluated for cell number and blastomere appearance (presence of vacuoles, multinucleation, cytoplasmic darkness or inclusions, unevenness size of blastomeres) and consequently graded as 1 to 4 (28). Top-quality embryos on day 2 were those grade 1 with four regular blastomeres and no fragments at the observation performed at 40 hours after insemination; top-quality embryos on day 3 had eight regular cells, grade 1, without fragmentation at the observation performed at 64 hours after insemination; top-quality embryos on day 4 were compacted morulas at the observation performed at 88 hours after insemination; top-quality embryos on day 5 were morphologically normal blastocysts (grade 3 4, A or B for inner cell mass and trophectoderm according to Gardner and Schoolcraft [29]) at the observation performed at 112 hours after insemination. No viable embryos were identified as those that arrested in culture for at least 48 hours with clear signs of degeneration. Statistical Analysis Data were analyzed by Student s t-test and c 2 analysis applying the Yates correction, 2 2 contingency tables. RESULTS A total of 2,298 oocytes were collected from the 234 ICSI cycles, of which 702 were inseminated and 1,101 were cryopreserved. After thawing 997 oocytes, the survival rate was 72.8% (726/997) and the fertilization rate was 73% (450/ 619). As represented in Table 1, this last figure was significantly different from that observed in the fresh cycles (83%, 585/702, P<.001). The analysis of pronuclear zygote morphology was aimed at determining the frequency of those configurations, A1a, A2a, A1b, and A2b, defined as top quality. The proportion of zygotes having one of these four patterns was significantly higher in fresh cycles than in frozen cycles (61% vs. 50%, P<.001). Embryo development was similar in fresh and frozen cycles (91% and 88%, respectively), whereas a significant difference was found in the proportion of day 2 top-quality embryos (37% vs. 19% respectively, P<.001) and day 3 top-quality embryos (23% vs. 8%, respectively, P<.001) (Fig. 2). Embryo culture was extended to day 4 in 96 cycles and to day 5 in 56 cycles. As shown in Figure 2, the development to morula on day 4 and to blastocyst on day 5 demonstrated the same trend as for day 2 and day 3 although no statistical significance was achieved. TABLE 1 Overall outcome of fresh and frozen cycles. Fresh Frozen No. cycles 234 256 No. inseminated oocytes 702 619 No. fertilized oocytes (%) 585 (83) 450 (73) a No. zygotes with the configurations A1a, A2a, 354 (61) 225 (50) a A1b, and A2b (%) No. embryos (%) 534 (91) 396 (88) No. transferred cycles (%) 218 (93) 203 (79) a 52 (24) 65 (32) No. cycles with one transferred embryo (%) a P<.001 compared with fresh. 512 Magli et al. Embryo development from thawed oocytes Vol. 93, No. 2, January 15, 2010

FIGURE 2 Top-quality embryo development after culture to day 2, day 3, day 4, and day 5. Values with same superscripts are significantly different. differences were detected for the cleavage rate (92% vs. 90%) and embryo development on day 4 and 5. Also in this group of patients, the percentage of transferred cycles was superior in fresh cycles (94% vs. 81% in thawed cycles, P<.005) and, although the mean number of transferred embryos was similar between fresh and frozen cycles (2.0 0.8 vs. 1.8 0.9, respectively), the proportion of transferred top-quality embryos was significantly higher in fresh cycles compared with frozen cycles (0.7 0.8 vs. 0.3 0.7, P<.001). As a result of fertilization and embryo development, the proportion of transferred cycles was superior in fresh cycles (93%) in comparison with frozen cycles (79%, P<.001), and the proportion of cycles that had only one embryo available for transfer was not significantly different between the two groups (24% vs. 32%, respectively). The analysis of the data divided according to maternal age is presented in Table 2. In the group of younger patients (%35 years), the fertilization rate was significantly higher in fresh cycles (82% vs. 72% in frozen cycles, P<.01) with the frequency of top-quality zygotes following the same trend (57% in fresh cycles vs. 46% in frozen cycles, P<.025). Although the cleavage rate did not differ between fresh and frozen cycles (90% and 86%, respectively), the number of top-quality embryos on day 2 was significantly higher in fresh cycles when compared with frozen cycles (35% and 17%, respectively, P<.001). A similar trend was observed for top-quality embryos on day 3 with proportions of 23% and 10%, respectively (P<.01), whereas no differences were detected on day 4 and day 5. Finally, the percentage of transferred cycles was significantly higher in fresh cycles compared with frozen cycles (92% and 78%, respectively, P<.005). Although the mean number of transferred embryos did not differ between fresh and frozen cycles (1.8 0.80 vs. 1.9 0.93, respectively), the proportion of transferred top-quality embryos was significantly higher in fresh cycles compared with frozen cycles (0.7 0.8 vs. 0.4 0.7, P<.005). Similarly, in the group of patients aged R36 years, the following parameters were significantly higher in fresh cycles than in frozen cycles: the fertilization rate was 85% versus 73%, P<.001; the proportion of top-quality zygotes was 64% versus 54%, P<.025; the proportion of day 2 top-quality embryos was 39% versus 20%, P<.001; the proportion of day 3 top-quality embryos was 24% versus 6%, P<.001, and no DISCUSSION The present study compares development and morphology of embryos generated in fresh and frozen cycles from the same patients with the aim of detecting whether the performance after thawing was similar to that observed in the corresponding fresh cycle. The first notable difference was a decreased fertilization rate in thawed oocytes, denoting their reduced viability irrespective of the apparent survival to the cryopreservation procedure. As represented in Figure 2, this observation was consistent with the analysis of embryo development and morphology, revealing that after thawing the morphologic and developmental characteristics of the resulting embryos were of lower grade when compared with the performance in the corresponding fresh cycles. This was evident immediately after fertilization, as in thawed oocytes the proportion of pronuclear zygotes presenting morphologic patterns that are more predictive of euploidy and implantation was significantly reduced compared with sibling fresh oocytes. In agreement with these observations, zygotes from thawed oocytes tended to develop lower proportions of top-quality embryos, on both day 2 and day 3. As only a few cycles were transferred on day 4 and day 5, the development to morula and blastocyst did not provide significant information. Although the evaluation of the clinical performance was beyond the purpose of this study, it is clear that a positive clinical outcome in the fresh cycle was the logical expectation because of the high proportion of top-quality zygotes and embryos. Nevertheless, the great majority of the patients included in this study were not pregnant in the fresh cycle (94%) or underwent spontaneous abortion (6%). Failure in the fresh cycle could insinuate the suspicion that these couples could have characteristics of poor prognosis for pregnancy, being not representative of the general ICSI population. This possibility was not confirmed by their clinical parameters and history, and by the fact that the overall performance of their thawed cycles was within the normal range of the results that are obtained normally by thawing supernumerary oocytes (18% clinical pregnancy rate per transferred cycle). This clinical outcome in thawed cycles is inferior only to that observed in the category of patients with the best prognosis, namely those that underwent elective cryopreservation of all their oocytes because of the high risk of development of hyperstimulation syndrome (24% clinical pregnancy rate per transfer, 32% per patient [30]). Fertility and Sterility â 513

TABLE 2 Fertilization and embryo development in fresh and frozen cycles according to maternal age. %35 y R36 y Fresh cycles Frozen cycles Fresh cycles Frozen cycles No. cycles 117 120 117 136 Age (mean SD) (y) 31.9 4.6 31.7 4.1 38.5 4.6 38.5 4.2 No. inseminated oocytes 351 303 351 316 No. fertilized oocytes (%) 287 (82) a 219 (72) a 298 (85) b 231 (73) b No. zygotes with the configurations 164 (57) c 101 (46) c 190 (64) d 124 (54) d A1a, A2a, A1b, and A2b (%) No. embryos (%) 259 (90) 189 (86) 275 (92) 207 (90) Day 2 top-quality embryos (%) 91 (35) e 33 (17) e 106 (39) f 41 (20) f Day 3 top-quality embryos (%) 58/255 (23) g 10/102 (10) g 57/240 (24) h 5/87 (6) h Day 4 top-quality embryos (%) 43/128 (34) 4/16 (25) 44/117 (38) 1/8 (13) Day 5 top-quality embryos (%) 22/78 (28) 1/4 (25) 25/77 (32) 0/2 No. transferred cycles (%) 108 (92) i 93 (78) i 110 (94) j 110 (81) j a P<.01, fresh vs. frozen. b P<.001, fresh vs. frozen. c P<.025, fresh vs. frozen. d P<.025, fresh vs. frozen. e P<.001, fresh vs. frozen. f P<.001, fresh vs. frozen. g P<.01, fresh vs. frozen. h P<.001, fresh vs. frozen. i P<.005, fresh vs. frozen. j P<.005, fresh vs. frozen. In light of these observations, it can be affirmed reasonably that the patients included in the present study are not likely to belong to a poor prognosis category, but they just represent a negatively selected group because of the precise scope of comparing the development in culture of sibling oocytes in both fresh and thawed cycles. It is logical indeed that the great majority of patients entering the study were not pregnant in the fresh cycle, as those achieving a term pregnancy tended to delay or discard the search for a second child. Moreover, it should be kept in mind that the evaluation of the clinical performance of the cycles presented here must take into consideration the restrictive conditions imposed by the national law on IVF that prohibits the generation of more than three embryos per cycle, as well as any form of embryo selection or cryopreservation (19). The diverse oocyte development and growth after cryopreservation in comparison with the corresponding sibling fresh oocytes give an estimation of the effects caused by freezing and thawing and, indirectly, of the expectations related to oocyte cryobanking. The difference in terms of oocyte fertilization and development was independent from age, suggesting that the mechanisms regulating oocyte activation and the first cleavage divisions are especially sensitive to low temperatures. Accordingly, the proportion of top-quality embryos, on both day 2 and day 3, was lower in thawed cycles, and this is in agreement with the studies suggesting a high sensitivity of meiotic and mitotic spindles to temperature variations (31 33). It has been reported that spindle errors may result in lagging or broken chromosomes that are expelled into fragments, being the percentage of fragmentation closely related to mosaicism (34, 35). As a result, a decreased chance of transferring embryos in thawed cycles was observed, and particularly top-quality embryos. As already mentioned, because of the prohibition by law to perform embryo selection, only a few embryos were excluded from transfer even in case of poor morphology, and this happened just when, after repeated visual analysis, they clearly had started a degeneration process. For this reason, the proportion of transferred top-quality embryos gives better information on the cycle performance, provided that morphology is an objective indicator of viability (27, 28). In agreement with these considerations, the mean number of top-quality embryos that were transferred was significantly lower in thawed cycles than in fresh cycles, in both young and older patients. These findings suggest that if the first cleavage division takes place, the probability of developing top-quality embryos depends on the state of the oocyte, fresh or frozen, but not on the woman s age. Cleavage defects after oocyte cryopreservation are a consequence of a cryogenic damage that ultimately affects 514 Magli et al. Embryo development from thawed oocytes Vol. 93, No. 2, January 15, 2010

implantation. The recent improvements in the technique of oocyte cryopreservation by slow-freezing have resulted in several pregnancies (6, 18). Nevertheless, the implantation rate per thawed oocyte remains extremely low implying that the efficiency of oocyte cryopreservation by slow-freezing methods is still far from being optimal (17). The complexity in freezing human oocytes is due to their high temperature sensitivity that makes the standard protocols of cryopreservation inapplicable to these cells. For this reason, the critical factors that affect the process have been studied thoroughly, and the most relevant achievements regard the survival rate that is now >70% when high sucrose concentration is used in freezing solutions (5, 6). Having overcome the obstacle of losing cell integrity during freezing and thawing does not mean necessarily that the deriving embryos are viable, and recent reports demonstrate that high survival is not synonymous of improved clinical outcome (1, 17, 36, 37). Therefore, the next step in this research area is aimed first at identifying the reasons for which these embryos generally have a reduced competence that severely affects their developmental potential, and second at defining the conditions that may preserve their viability (23, 38 42). The results reported here are in agreement with previous data generated on a small number of cases (43, 44) reporting on a slower cleavage rate in frozen cycles when compared with the sibling fresh oocytes. This behavior suggests that even if surviving thawing, the process of oocyte cryopreservation by slow freezing has a negative effect on the potential of further growth that is evident as early as the first cleavage divisions. 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