Can cumulative pregnancy rates be increased by freezing and thawing single embryos?

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1 Can cumulative pregnancy rates be increased by freezing and thawing single embryos? Marie Prades, Pharm.D., a Jean-Louis Golmard, Ph.D., b Daniele Vauthier, M.D., c Gilles Lefebvre, M.D., c and Catherine Poirot, Ph.D. a a Reproductive Biology Unit, b Statistical Unit, and c Department of Obstetrics and Gynecology, University Hospital, H^opital Pitie-Salp^etriere, Paris, France Objective: To evaluate the extent to which transfers of frozen single embryos increase cumulative pregnancy rates. Design: Retrospective analysis. Setting: IVF unit of a university hospital. Patient(s): Patients undergoing IVF cycles that were carried out from 2001 to 2005 (n ¼ 1758). Patients were assigned to three groups according to the number of embryos frozen: group A, no cryopreservation; group B, a single embryo frozen; group C, several embryos frozen. Intervention(s): Analysis of fresh ETs as a function of the number of embryos frozen and comparison outcomes for the thawing of a single embryo between subgroups B* (only one embryo frozen and thawed) and C* (last embryo of the cohort thawed). Main Outcome Measure(s): Implantation and pregnancy rates after fresh ETs and embryo survival and pregnancy rates after the transfer of a single thawed embryo. Result(s): The pregnancy rate per fresh ET increased significantly with the number of embryos frozen: 16.2% in group A, 21.4% in group B, and 26.5% in group C. For single thawed embryos, survival was higher in group C* (91.7%) than in group B* (72.6%). The pregnancy rate was also significantly higher in group C* (19.4% vs. 0%). Conclusion(s): The freezing of single embryos is of no benefit in cumulative pregnancy rates. ET strategies should therefore be reviewed. (Fertil Steril Ò 2009;91: Ó2009 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, single frozen embryo, implantation, cumulative pregnancy rate, embryo survival The freezing of embryos is a complementary procedure offered to couples undergoing treatment with assisted reproductive technologies. The main aim of this process is to facilitate the storage of nontransferred embryos to decrease the risk of multiple pregnancies by reducing the number of fresh embryos transferred (1). It also increases the cumulative pregnancy rate for a given treatment cycle (2, 3). Frozen ET (FET) cycles result in about 30% 40% fewer implantations than fresh IVF/intracytoplasmic sperm injection (ICSI) cycles (4, 5) owing to the detrimental effects of freezing and thawing on embryo viability (6, 7). For this reason, cryopreservation is only effective for embryos of sufficiently high morphological quality as assessed by fragmentation rate or cleavage stage before freezing (4, 8, 9). Survival rates vary between centers but are currently between 45% and 90% (10, 11). Studies focusing on the number of embryos transferred in FET have shown that pregnancy rates increase with the number of embryos transferred (12 14). Cryopreservation allows pregnancy rates of 11% 32% per transfer (12, 15). By producing an extra 8% of births on average (16), this technique allows a few teams to achieve cumulative pregnancy Received June 28, 2007; revised and accepted November 20, Reprint requests: Marie Prades, Unite fonctionnelle de Biologie de la Reproduction du Professeur Catherine Poirot, Batiment 85, Avenue de la Nouvelle Pitie, 83 Boulevard de l H^opital, Paris cedex 13, France (FAX: ; marie.prades@psl.aphp.fr). rates of 74% per cycle (17). Embryo freezing has thus become a routine procedure in IVF laboratories. Cumulative pregnancy rates tend to increase with the number of embryos frozen after fresh ET. We evaluated the extent to which the transfer of single frozen and thawed embryos increased cumulative pregnancy rates. We first considered the outcome after fresh ET as a function of the number of embryos frozen. For each attempt, the main outcomes analyzed were implantation and pregnancy rates. We then investigated the data for single frozen and thawed embryos. We compared single embryo thawing from cycles in which only one embryo was frozen and the last remaining embryo of cycles in which several embryos were frozen. Embryo survival and pregnancy rates after thawing for these two groups of embryos were analyzed. MATERIALS AND METHODS Patients Studied In this retrospective study, cycles involving classical IVF or ICSI carried out between January 2001 and December 2005 at the Pitie-Salp^etriere Reproductive Biology Unit (Paris, France) were investigated. During this period of time, 1758 IVF/ICSI cycles were carried out, resulting in 1601 ETs. These cycles were assigned to three groups: group A (no embryo cryopreservation; n ¼ 1192), group B (only one embryo frozen; n ¼ 133), and group C (several embryos frozen; n ¼ 433) /09/$36.00 Fertility and Sterility â Vol. 91, No. 2, February doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 In the second part of the study, only IVF attempts in which a single embryo was thawed were considered. Two subgroups were compared: group B* (cycles from group B in which the single embryo was thawed; n ¼ 106) and group C* (cycles from group C in which the embryo thawed was the last of a cohort of cryopreserved embryos; n ¼ 36). Ovarian Stimulation and Oocyte Retrieval The procedures used remained essentially unchanged between 2001 and Ovulation was stimulated by two protocols: [1] a long agonist protocol including a phase of pituitary down-regulation with a GnRH agonist (Decapeptyl; Ipsen Biotech, Paris, France) for days, followed by a phase of recombinant FSH stimulation (Gonal F; Serono Merck, Boulogne, France; or Puregon; Organon, Eragny, France), or [2] a short protocol in which administration of the GnRH agonist began with gonadotropin administration. Ovulation was induced by injecting 10,000 units of hcg (Organon). Ultrasound-guided transvaginal oocyte retrieval was carried out 36 hours later. IVF and ICSI Procedures Ovarian follicular fluids were transferred from the operating theater to the laboratory in a thermostatically controlled case at 37 C. Oocyte-granulosa cell complexes were identified, rinsed, and then incubated in Universal IVF Medium (Medi- Cult; Limonest, France) in microdrops under oil (CryoBio- System; l Aigle, France). Insemination or microinjection was carried out 4 6 hours after oocyte retrieval. The oocytes were checked for the presence of pronuclei and polar bodies hours after insemination or ICSI. Zygotes were transferred to fresh Universal IVF Medium and were cultured for the next 24 hours. Embryos were transferred hours after insemination or ICSI, and their quality was assessed based on the number of blastomeres and the degree of fragmentation. Degree of fragmentation was scored as follows: grade A, no fragmentation; grade B, 1% 20% fragmentation; grade Bc, 21% 30% fragmentation; grade C, 31% 50% fragmentation; grade D, more than 50% fragmentation. Embryo Freezing Embryos with less than 31% cytoplasmic fragmentation (morphology grades A, B, and Bc) were considered suitable for freezing. Cryopreservation was carried out using automated LC 40 (Air Liquide; Marne-la-vallee, France) until 2003 and Planer Kryo 560 (CryoBioSystem) thereafter. A slow-freezing protocol with 1,2-propandiol (PROH; Sigma- Aldrich; Saint Quentin Fallavier, France) and sucrose (Sigma-Aldrich) as cryoprotectants was used (18). The freezing solutions were supplemented with 20% human serum. Embryos were first incubated in 1.5 M PROH freezing solution at room temperature for 15 minutes and then in 1.5 M PROH and 0.1 M sucrose freezing solution for 5 minutes. Embryos were loaded into plastic straws (CryoBioSystem) and frozen as follows: embryos were placed in the freezing machine at 20 C, cooled at 2 C/minute to 7 C, held at 7 C for manual seeding, cooled at 0.3 C/minute to 35 C and then at 35 C/minute to 150 C, before being plunged into liquid nitrogen. The number of embryos frozen per straw depended of the number of fresh embryos transferred. In any case, at least half of the total number of straws contained one embryo, allowing the adjustment of the number of embryos transferred in case of damage of another one. Embryo Thawing and Transfer Frozen ET was performed in a stimulated or substituted cycle. The straw with frozen embryos was removed from liquid nitrogen and placed at room temperature. Embryos were first incubated in a series of PROH solutions of decreasing concentration (1, 0.5, 0.25, and 0 M) in Universal IVF Medium supplemented with 0.2 M sucrose and 20% human serum for 5 minutes at each concentration. They were then incubated twice, for 5 minutes each, in Universal IVF Medium supplemented with 20% human serum, before transfer to M3 culture medium (MediCult) for incubation overnight at 37 C. Embryos were transferred on the day of thawing or after 24 hours of culture. Embryo survival was defined as the retention of at least one intact blastomere after thawing. Pregnancy and Implantation of Transferred Embryos Progesterone (100 mg twice a day; Utrogestan; Besins- Iscovesco, Paris, France) was administered to women after transfers. Serum b-hcg concentrations were determined on days 9 and 11 after ET. Once b-hcg was detected, subsequent determinations and an ultrasound scan were prescribed for the fifth week. Clinical pregnancy was defined as a concentration of b-hcg exceeding 1000 IU/mL and was confirmed by the observation of a gestational sac with a fetal heartbeat on ultrasound evaluation. Statistics Groups A, B, and C were compared using nonparametric analysis of variance, with Kruskal-Wallis tests, followed by pair-wise comparisons by two-sample Wilcoxon tests for quantitative variables and c 2 -tests or Fischer s exact tests for qualitative ones. Univariate logistic regression analysis was carried out to assess the relationship between pregnancy rates per transfer and the number of embryos frozen for group C. Finally, two stepwise multivariate logistic regression analyses were performed to identify independent prognostic factors for pregnancy rates. The candidate factors included in the stepwise regression were those significant at the 5% level in an initial univariate analysis. The factors retained in the final model were significant at the 5% level in Wald s test. Groups B* and C* were compared using two-sample Wilcoxon tests for quantitative variables and c 2 -tests or Fisher s exact tests for qualitative variables. All the tests were two-sided. P<.05 was considered statistically 396 Prades et al. Single embryo freezing Vol. 91, No. 2, February 2009

3 significant. SAS version 8 was used for statistical analysis (SAS Institute, Cary, NC). RESULTS The overall freezing rate, which was defined as the proportion of cycles with embryos cryopreserved, was 32% for all groups together. Outcomes after Fresh ET as a Function of the Number of Embryos Frozen (Table 1) Group A (no cryopreserved embryo) differed significantly from the other two groups (B, single embryo frozen; and C, several embryos frozen) in terms of maternal age, number of IVF attempts, fertilization rate, and number of embryos transferred. The women in group A were older (35.1 years old vs and 33.6 years old in groups B and C, respectively) and had undergone a larger number of IVF attempts (2.4 vs. 2.0 in groups B and C, respectively). The fertilization rate was lower in group A (65.2% vs. 76.6% and 81.1%, for groups B and C, respectively), and fewer embryos were transferred (1.7 vs. 2.0 in groups B and C). Groups B and C were statistically similar for all these variables. The number of oocytes retrieved differed significantly between the groups, with the lowest number in group A (7.5), the intermediate number in group B (10.8), and the highest number in group C (14.5). Similar observations were also made for mature oocytes (5.9 in group A, 8.5 in group B, and 11.9 in group C), pronuclear stage embryos (3.7 in group A, 6.3 in group B, and 9.5 in group C), and cleavage diploid embryos (3.6 in group A, 6.0 in group B, and 9.3 in group C). Cleavage rates were similar in the three groups. Pregnancy rates (PRs) differed between groups. The PR expressed per retrieval was lower in group A (14.2%) than in groups B (21.1%) and C (26.1%), with these two groups being statistically similar. The PR expressed per transfer was significantly lower in group A (16.2%) than in group C (26.5%), with no significant difference between groups A and B. The implantation rate was significantly higher in group C (15.4%) than in TABLE 1 Comparison of clinical and biological results after oocyte retrieval and fresh transfer as a function of the number of embryos frozen. Group A: no cryopreservation (n [ 1192) Group B: single embryo frozen (n [ 133) Group C: several embryos frozen (n [ 433) P Age, years , a,b , a , b 33.0 <.0001 a,b [32.0, 39.0] [31.0, 37.0] [30.0, 37.0] No. of IVF attempts , a,b , a , b 1.0 <.05 a, <.0001 b [1.0, 3.0] [1.0, 3.0] [1.0, 2.0] Oocytes retrieved , a,b , a,c , b,c 13.0 <.0001 a,b,c [3.0, 10 0] [7.0, 14.0] [10.0, 19.0] Mature oocytes , a,b , a,c , b,c 11.0 <.0001 a,b,c [2.0, 8.0] [5.0, 11.0] [8.0, 15.0] Pronuclear stage , a,b , a,c , b,c 8.0 <.0001 a,b,c embryos [1.0, 5.0] [4.0, 7.0] [6.0, 12.0] Fertilization rate, % 65.2 a,b 76.6 a 81.1 b <.0001 a,b Cleavage diploid , a,b , a,c , b,c 8.0 <.0001 a,b,c embryos [1.0, 5.0] [4.0, 7.0] [6.0, 12.0] Cleavage rate, % NS Embryos transferred , a,b , a , b 2.0 <.0001 a,b [1.0, 2.0] [2.0, 2.0] [2.0, 2.0] Clinical PR per 14.2 a,b 21.1 a 26.1 b <.05 a, <.0001 b retrieval, % Clinical PR per 16.2 a a <.0001 a transfer, % Implantation rate, % 7.4, a (148/2005) 9.4, b (25/267) 15.4, a,b (132/860) <.0001 a, <.05 b Clinical abortion rate, % NS Note: Quantitative variables are expressed as mean SD, median [lower quartile Q1, upper quartile Q3] unless otherwise specified. Values with the same superscript are significantly different. NS ¼ not statistically significant; PR ¼ pregnancy rate. Prades. Single embryo freezing. Fertil Steril Fertility and Sterility â 397

4 the other two groups (7.4% in group A and 9.4% in group B). Clinical abortion rates did not differ significantly among the three groups. In cases in which more than one embryo was frozen, the logistic regression procedure established no relationship between PRs per transfer and the number of embryos frozen. The results of multivariate analysis confirmed those of univariate analysis, with significant differences in PR among the three groups. This analysis also made it possible to identify two other independent factors affecting PR: maternal age and number of embryos transferred (Table 2). Thawing of Single Embryos: Comparison between Groups B* (n [ 106) and C* (n [ 36) (Table 3) In group C*, a mean of 3.3 embryos had been frozen per attempt. The patients of groups B* and C* were similar in terms of age (33.8 years old in groups B* vs years old in group C*) and number of IVF attempts (2.0 vs. 1.8, respectively). The numbers of oocytes, mature oocytes, pronuclear stage embryos, and early cleavage diploid embryos recovered were significantly lower in group B* than in group C*. Fertilization and cleavage rates were similar in the two groups (75.9% and 97.1%, respectively, in group B*, vs. 75.8% and 96.9%, respectively, in group C*). Pregnancy and implantation rates did not differ after the transfer of fresh embryos (15.4% and 4.4%, respectively, in group B*, vs. 13.9% and 9.3%, respectively in group C*). The survival rate of frozen embryos after thawing was significantly lower in group B* (72.6%) than in group C* (91.7%): the observed percentages of fully intact embryos (42.5% vs. 58.3%) or embryos presenting the loss of at least one blastomere (30.2% vs. 33.3%) were lower in group B*, but these differences were not statistically significant. The proportion of completely damaged embryos was significantly higher in group B* than in group C* (27.4% vs. 8.3%). PRs per thawing and per FET were much higher in group C* (19.4% and 21.2%, respectively) than in group B* (0%). IVF and ICSI procedures did not differ between groups B (IVF, 45%; ICSI, 55%) and C (IVF, 48%; ICSI, 52%; P¼.69) or between groups B* (IVF, 44%; ICSI, 56%) and C* (IVF, 46%; ICSI, 54%; P¼.84). The proportion of IVF procedures was statistically increased in group A (IVF, 55%; ICSI, 45%) compared with groups B and C (P<.05). DISCUSSION The aim of this study was to evaluate the benefit of using single frozen and thawed ETs in terms of cumulative PRs. We initially explored implantation and PRs after fresh ET as a function of the number of embryos frozen. Outcomes after the thawing of a single embryo were then compared between two different populations. The number of high-quality embryos in a cohort, as indicated by the number of frozen embryos, has been shown to be of strong predictive value for PRs after fresh ETs. In cases in which no high-quality embryos remained after transfer (group A), an overall PR per transfer of 16.2% was obtained. If only one embryo was of suitable quality for freezing (group B), an overall PR of 21.4% was achieved, and when several embryos were frozen (group C) the PR reached 26.5%. Implantation rates were clearly higher in the group with several frozen embryos. Our data also suggest that implantation potential may differ between patients, depending on the number of high-quality embryos remaining after fresh transfer. Implantation rates of 7.4% and 9.4% were obtained for groups A and B, whereas implantation rates reached 15.4% in group C. These results are consistent with those obtained by Volpes et al. in 2004: PR increases with the number of high-quality embryos available on the morning of day 3 (defined as embryos reaching the 8-cell stage with less than 20% fragmentation) (19). No data have yet been published concerning pregnancies after the freezing and thawing of a single embryo. As explained above, the freezing of an embryo indicates that it was considered to be of high quality. Embryo quality before freezing is known to be a major factor determining the success of FET (8, 10, 20). However, the morphological features of the embryos before freezing were similar in groups B* and C*. Nonetheless, none of the 106 thawed single embryos in group B* resulted in a pregnancy. Other studies in which single FETs were analyzed did not specify whether the embryo transferred was the only embryo frozen, the only embryo surviving the freezing and thawing process, or the last in TABLE 2 PRs and multivariate logistic regression results. Variable PR per retrieval P PR per transfer P Group: A/C [0.423, 0.732] [0.436, 0.755] B/C [0.462, 1.184] [0.466, 1.195] Maternal age [0.927, 0.983] [0.934, 0.991].0109 Embryos transferred [1.377, 2.044] < [1.106, 1.726].0044 Note: Data are OR [95% CI, Wald confidence limits]. Prades. Single embryo freezing. Fertil Steril Prades et al. Single embryo freezing Vol. 91, No. 2, February 2009

5 TABLE 3 Comparison of clinical and biological results for the thawing of a single embryo in groups B* and C*. Group B* (n [106) Group C* (n [ 36) P Age , 34.0 [31.0, 37.0] , 32.0 [30.0, 34.5] NS No. of IVF attempts , 2.0 [1.0, 3.0] , 1.0 [1.0, 2.0] NS Oocytes recovered , 10.0 [6.0, 14.0] , 15.0 [10.5, 20.0] <.0001 Mature oocytes , 8.0 [5.0, 11.0] , 12.0 [9.0, 16.5] <.0001 Pronuclear stage embryos , 5.0 [4.0, 8.0] , 8.5 [7.0, 11.5] <.0001 Fertilization rate, % NS Cleavage diploid embryos , 5.0 [4.0, 7.0] , 8.5 [7.0, 11.0] <.0001 Cleavage rate, % NS Embryos transferred , 2.0 [2.0, 2.0] , 2.0 [2.0, 2.0] NS Clinical PR per retrieval, % 15.1 (16/106) 13.9 (5/36) NS Clinical PR per transfer, % 15.4 (16/104) 13.9 (5/36) NS Implantation rate, % NS Clinical abortion rate, % NS Thawed embryo quality: Intact embryos, % 42.5 (45/106) 58.3 (21/36) NS Completely damaged 27.4 (29/106) 8.3 (3/36) <.05 embryos, % Embryos that had lost at 30.2 (32/106) 33.3 (12/36) NS least one blastomere, % Survival rate, % 72.6 (77/106) 91.7 (33/36) <.05 PR per thawing, % 0 (0/106) 19.4 (7/36) <.0001 PR per transfer, % 0 (0/77) 21.2 (7/33) <.0001 Note: Quantitative variables are expressed as mean SD, median [lower quartile Q1, upper quartile Q3] unless otherwise specified. NS ¼ not statistically significant. Prades. Single embryo freezing. Fertil Steril a cohort of several frozen embryos. Reported PRs for single FET vary from 0% (13) to 40.6% (21). Group C*, in which several embryos were candidates for cryopreservation, was used as a control population for group B*. We checked that groups B* and C* were similar, to eliminate possible skew due to differences in maternal age, number of IVF attempts, IVF or ICSI procedures, or PRs for fresh ET. However, children were born after single FET for the cycles included in group C*: a clinical PR per FET of 21.2% was obtained. Thus, the transfer of a single thawed embryo is not in itself responsible for a failure to achieve pregnancy. Other specific elements must be involved. Our findings show that survival after thawing is significantly higher if more than one embryo was frozen for the cycle concerned. Embryo survival rates were 72.6% for group B* and 91.7% for group C* (P<.05). Moreover, 27.4% of the embryos in group B* were completely damaged, versus 8.3% in group C* (P<.05). It is difficult to compare survival results with those obtained by other teams, as different definitions of embryo survival have been used. Most teams consider an embryo to have survived if at least 50% of its blastomeres remain intact (9, 22). In this study, we defined survival as the retention of at least one intact blastomere. The survival rate of 91.7% in the control population (group C*) is, nevertheless, at least as good as the 57% and 90% obtained in other studies using the same criteria (10, 23). If we had applied the more widely used definition of cryosurvival, we would have obtained survival rates of 70% and 78% in groups B* and C*, respectively. Salumets and Mandelbaum and coworkers, in independent studies, reported survival rates of 79.3% and 73%, respectively (14, 16). The PR after FET of 21.2 % in group C* is also comparable to the 13% 14.7% obtained by other teams after the transfer of a single thawed embryo (11, 24). The freezing-thawing techniques used in the laboratory are therefore acceptable. Two different freezing machines were employed during the study period the LC 40 until 2003 and then the Planer Kryo 560. The proportions of embryos frozen with each of the machines were similar between groups B* and C*. Moreover, embryo survival did not seem to depend on the freezing machine used. PRs after fresh ETs, embryo survival, and PRs after FET suggest that these differences between groups are probably due to intrinsic differences in embryo cohort quality. This hypothesis is supported by the notion of cohort homogeneity (25), potentially accounting for the development potential of a cohort of embryos. Embryos from the same cohort are thought to have a similar implantation potential (26). Embryo survival and PR data suggest that the results obtained for frozen embryos are similar to those for fresh embryos from the same cohort (15, 27). Consistent with this theory of cohort Fertility and Sterility â 399

6 homogeneity, many studies have reported better pregnancy results after FET of embryos resulting from cohorts for which a pregnancy was achieved with fresh embryos (28, 29). This concept of cohort homogeneity could also account for our results and suggests that the existence of only one embryo suitable for freezing after fresh transfer may be strongly predictive of the quality of the embryo cohort. Toner et al. evaluated the impact of the number of retrieved oocytes and cryopreservation on IVF outcome (30). Taking into account the observation that PRs after fresh ETs were statistically similar regardless of the number of oocytes retrieved, they concluded that the chances of achieving pregnancy from one cycle increased with the number of oocytes retrieved. Toner et al. confirmed the benefit of FET for increasing cumulative PRs. Our results add to these findings, showing that the increase in cumulative PRs is also clearly due to the higher PR obtained after fresh ET when several embryos are frozen. This study shows that the PR after fresh ET increases with the number of embryos frozen after that transfer. Survival and PRs after the thawing of single embryos showed that frozen embryos from cohorts of several frozen embryos had a greater development potential than single embryos frozen in isolation. Thus, in our center, the freezing of single embryos is of no benefit for increasing cumulative PRs. As embryo freezing is time-consuming and requires human and financial resources, the freezing of single embryos as part of an ET strategy should be reviewed. REFERENCES 1. Tiitinen A, Halttunen M, Harkki P, Vuoristo P, Hyden-Granskog C. Elective single embryo transfer: the value of cryopreservation. Hum Reprod 2001;16: Bergh C, Josefsson B, Nilsson L, Hamberger L. 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