Effect of coasting on the implantation potential of embryos transferred after cryopreservation and thawing

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1 IN VITRO FERTILIZATION Effect of coasting on the implantation potential of embryos transferred after cryopreservation and thawing Murat Arslan, M.D., a,b Silvina Bocca, M.D., Ph.D., a Estella Jones, M.S., a Jacob Mayer, Ph.D., a Laurel Stadtmauer, M.D., Ph.D., a and Sergio Oehninger, M.D., Ph.D. a a Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia; and b Department of Obstetrics and Gynecology, Mersin University, Mersin, Turkey Objective: To examine the effect of withholding gonadotropins on the outcome of embryos after cryopreservation and thawing. Design: Retrospective clinical evaluation of patients having cryopreserved thawed ET trials with coasting during the corresponding ovarian stimulation cycle. Setting: Academic tertiary clinical care unit. Patient(s): Patients with cryopreserved embryos having coasting in their fresh IVF cycle and age-matched controls without coasting, both groups receiving the same stimulation protocol (long GnRH agonist plus recombinant FSH). Intervention(s): All patients had a cycle in which embryos were transferred fresh and a cycle of thawing of cryopreserved embryos with the aim of transferring in a steroid-supplemented cycle. Main Outcome Measure(s): Embryo survival, implantation, and clinical pregnancy rates. Result(s): Post-thawing embryo survival (66.4% vs. 73%), implantation (12.3% vs. 13.0%), and clinical pregnancy rates (31.5% vs. 38.0%) were similar in study and control groups, respectively. Patients with coasting for 3 days had significantly lower post-thawing embryo survival rates compared with patients having shorter duration of coasting ( 3 days) and controls. Implantation and pregnancy rates, however, were similar in the three groups. Conclusion(s): Coasting did not seem to have a detrimental effect on oocyte and embryo quality because the implantation competence of transferred concepti after cryopreservation and thawing was similar to that of controls. However, prolonged coasting ( 3 days) had a subtle negative impact on the post-thaw survival rate. (Fertil Steril 2005;84: by American Society for Reproductive Medicine.) Key Words: Cryopreservation thawing, coasting, embryo quality, estradiol, survival rate Received January 14, 2005; revised and accepted March 31, Reprint requests: Sergio Oehninger, M.D., Ph.D., The Jones Institute for Reproductive Medicine, 601 Colley Avenue, Norfolk, Virginia (FAX: ; OehninSC@EVMS.edu). Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic and potentially dramatic clinical condition initially characterized by increased capillary permeability and shift of intravascular fluid to extravascular spaces. Elevated E 2 and cytokine levels have been implicated in its pathogenesis (1). Women prone to developing OHSS are commonly younger ( 35 years) with lean body mass and/or with polycystic ovary syndrome (2). Despite the well-established predictive value of such markers, some OHSS cases cannot be anticipated before the initiation of ovarian stimulation (1). Serum E 2 levels at the time of hcg administration have been found to be closely correlated with OHSS (1). Critical values of E 2 that trigger the syndrome are under debate and differ for assisted reproductive techniques ( 4,000 pg/ml) and conventional ovulation induction methods ( 1,700 pg/ml), possibly as a reflection of the patient s physiological background and pretreatment ovarian suppression (2). Because hcg is the only known triggering factor for the development of OHSS, withdrawing gonadotropins and postponing hcg administration ( coasting ) has been a commonly proposed method for reducing the risk of OHSS in those patients with E 2 above certain critical values (3, 4). The concept of gonadotropin withdrawal during the late follicular phase was originally introduced by Jones et al. (5) as a means of allowing for follicle and oocyte maturation before ovulation triggering according to the interval between FSH/LH and hcg administration. Substitution of hcg by shorter-acting compounds like GnRH agonists (6), use of the recently introduced recombinant LH (7), and cryopreservation of all embryos, thus avoiding transfer in the fresh cycle (8, 9), have also been proposed as alternatives to decrease or prevent the occurrence of the syndrome /05/$30.00 Fertility and Sterility Vol. 84, No. 4, October 2005 doi: /j.fertnstert Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. 867

2 The introduction of embryo cryopreservation has been instrumental in the advancement of assisted reproductive technology. Embryo cryopreservation offers multiple advantages, including [1] allowing the possibility of inseminating all retrieved mature oocytes without the need to discard any embryos, [2] limiting the number of embryos transferred to reduce the incidence of multiple pregnancy, [3] enhancing couples chances of pregnancy by allowing multiple transfers originating from a single stimulated cycle (thereby optimizing their total reproductive potential), [4] aiding in the clinical management of OHSS, and [5] providing a valid ethical means for evaluation of research alternative protocols (10). Although there are many factors affecting embryo survival and clinical outcome after cryopreservation (10, 11), embryo quality seems to be the most significant determinant of viability after cryopreservation and thawing (12). The potential impact of coasting on cycle outcome in patients at imminent risk of ovarian hyperstimulation has been analyzed in several studies. There are conflicting reports regarding a potential detrimental effect of gonadotropin withdrawal on pregnancy outcome in the fresh cycle (4, 13 17). Ulug et al. (18) investigated the effect of the duration of the coasting period on conception and reported that although embryo quality was not affected, reduced implantation and pregnancy rates were observed in patients who required coasting for 3 days. It was speculated that the poor implantation rates observed in such patients were due to disruption of synchronization between endometrial and embryonic development. A similar outcome was demonstrated in oocyte donation cycles, with lower implantation and pregnancy rates in the recipients after fresh transfers with 4 days of coasting (19). The investigators speculated that this result was due to detrimental effects of prolonged coasting on oocyte and embryo quality not detected by current oocyte and embryo grading systems. We hypothesized that if coasting has a negative impact on oocyte and embryo quality, then further evidence of this effect could be sought by evaluating the outcome of cryopreserved thawed embryos originating from such stimulation cycles. Moreover, the effects of variable coasting periods could be examined. Cryopreserved thawed embryos are typically transferred in hormonally (estrogen/progesterone)-supplemented cycles in which the endometrium is not exposed to the hyperestrogenic milieu that characterizes the stimulated cycles. Hence, the primary objective of the present study was to determine the effect of coasting on the outcome of embryos after cryopreservation and thawing. The primary endpoint was the examination of post-thawing embryo survival and implantation competence. MATERIALS AND METHODS Subjects: Study and Control Groups We retrospectively evaluated all IVF cycles performed during a 5-year period in our program between September 1999 and September The institutional review board of Eastern Virginia Medical School approved this study. From the computerized database we identified patients who were prepared to undergo a transfer cycle with cryopreserved thawed embryos and who had coasting in their corresponding ovarian stimulation ( fresh ) cycle. Coasting was defined as withholding gonadotropin administration for at least 24 hours before triggering ovulation with hcg. The decision to coast was based on the presence of E 2 levels 4,000 pg/ml associated with 20 follicles, each 12 mm in diameter and some of them in the range of mm. Cryopreservation thawing cycles resulting from the use of a different ovarian stimulation protocol other than the one described below, or in which intracytoplasmic sperm injection (ICSI) or assisted hatching had been performed, were excluded from the analysis. Thirty-nine cryopreserved thawed ET cycles performed in 33 patients who had undergone coasting during ovarian stimulation fulfilled the inclusion criteria and constituted the study group. Thirty-three IVF patients with 42 cryopreserved thawed ET cycles were used as the controls. These patients were identified from the database as the consecutive IVF patient to each study subject who received the same stimulation protocol, achieved a peak serum E 2 level of 3,000 4,000 pg/ml at the time of hcg administration, did not undergo coasting, and had excess embryos that were cryopreserved. In addition, groups were matched by woman s age at the time of their fresh IVF cycle and basal cycle serum day-3 FSH levels. IVF Protocol In the fresh IVF cycle, all patients were down-regulated according to a long protocol with a GnRH agonist and stimulated with recombinant FSH. The GnRH agonist leuprolide acetate was initiated on day 21 of the preceding luteal phase (0.5 mg/d SC) until menses and dropped to 0.25 mg/d until triggering ovulation. Ovarian stimulation was started with a fixed regimen for the first 4 days of recombinant FSH (225 IU/d SC), and thereafter the gonadotropin dose was continued according to the individual s ovarian response with a step-down regimen. Human chorionic gonadotropin (10,000 IU) was administered IM to trigger ovulation when at least two follicles were 17 mm in diameter. Transvaginal follicular aspiration was performed under ultrasound guidance, hours after hcg injection. Gamete processing and embryo culture procedures and transfer techniques were previously described (20). Embryo quality (cleavage and morphology) was assessed according to the criteria of Veeck (21). Transfer of embryos was performed on day 3. All stimulation cycles underwent a fresh cycle. The luteal phase was supported with micronized P vaginally (600 mg/d). Clinical pregnancy was defined as visualization of a gestational sac with a heart beat by transvaginal ultrasound in patients with normally rising serum -hcg levels. Implantation rate was defined as the number of gestational sacs visualized divided by the number of embryos transferred. 868 Arslan et al. Effect of coasting on embryo quality Vol. 84, No. 4, October 2005

3 Cryopreservation Thawing Protocol Cryopreservation of embryos was performed at the pronuclear or cleaving (day-3) stage according to a slow-freeze, slow-thaw protocol (10, 22) with a programmed cell freezer (Planer Kryo ; T.S. Scientific, Perkasie, PA) and 1.5 mol/l 1,2 propanediol as the cryoprotectant. Briefly, the zygote freezing protocol started at room temperature and continued at 1 C/min to 6 C; this was followed by 5 minutes holding, manual seeding with forceps (to avoid ice crystal formation), holding again for 5 minutes, and freezing to 80 C at a rate of 0.5 C/min (slow cooling). Specimens were kept in cryovials containing 0.3 ml of cryoprotective medium and maintained at 196 C under liquid nitrogen in storage tanks (35 VHC liquid nitrogen storage tank; Taylor-Warton Cryogenic Equipment, Indianapolis, IN). Slow thawing of the embryos to room temperature was followed by dilution of the cryoprotectant and transfer of the specimen to equilibrated culture medium and incubation at 37 C under 5% CO 2 in air until cleavage was established. The freezing protocol for cleavage-stage embryos was very similar to that described above except for the addition of sucrose (0.2 mol/l) to the freezing medium and with a post-seeding cooling rate of 0.3 C/min. Cleavage-stage embryos were plunged into liquid nitrogen at 30 C. Thawing of day-3 embryos was performed with a 37 C water bath for 1 minute, followed by 5 minutes at room temperature. Assessment of Embryo Survival Post-thaw survival of pronuclear-stage embryos was defined as the ability of the zygotes to enter syngamy and proceed to at least the first cleavage division; one-cell specimens that appeared viable but failed to meet these criteria were not considered survivors. Thawed day-3 embryos were defined as surviving morphologically after identification of zona pellucida intactness and 50% intact blastomeres (22). Transfer of Cryopreserved Thawed Embryos Patients were prepared for ET in all cycles as follows. Transdermal 17 -E 2 patches were used (Vivelle Dot; Novogyne Pharmaceuticals, Miami, FL; each patch delivering 0.1 mg/day of E 2 ) and replaced every other day. On day 1, two patches were applied, and then E 2 administration was gradually increased on cycle days 7 (to three patches every other day) and on cycle day 11 (to four patches every other day). In addition, from cycle day 12 and every other day, 1 mg 17 -E 2 (Estrace; Warner Chilcot, Rockaway, NJ) was started vaginally and continued with the same dose every other alternate day to the estrogen patch. From cycle day 15, the transdermal E 2 dose was decreased (to two patches every other day) alternating with the vaginal Estrace pills (at the same dose). From cycle day 15 on, vaginal micronized P (Prometrium; Solvay Pharmaceutical, Baudette, MN) was added to the regimen (600 mg/d). Pronuclear stage concepti were thawed on day 16 and transferred on day 17, whereas day-3 embryos were thawed on day 17 and transferred on day 18. Statistical Analysis Data are presented as mean SE. Comparisons between groups of continuous outcomes were performed by Student s t-test, Mann-Whitney U test, analysis of variance with least significant difference post hoc test, or the Kruskal-Wallis rank sum test, as appropriate, after testing for normal distribution by the Kolmogorov-Smirnov test. Nominal data were analyzed by 2 test. These analyses were performed with commercial software (SPSS for Windows, version 9.05; SPSS, Chicago, IL). RESULTS The mean age and serum FSH levels on basal cycle day 3 for patients in the study and controls were years vs years and miu/ml vs miu/ml, respectively. There was no difference between study ( miu/ml) and control ( miu/ml) groups in mean serum LH levels on basal cycle day 3. The etiology of infertility was similar: tubal factor, 35% and 31%; unexplained, 25% and 27%; male infertility, 21% and 24%; endometriosis, 14% and 16%; and other causes, 5% and 2%, respectively, for study and control groups. The results of the fresh cycles for the study and control groups are presented in Table 1. By study design, all patients reached the transfer stage. The groups were similar in serum E 2 levels at the time of hcg administration, number of metaphase II oocytes harvested per retrieval, fertilization rate, and mean number of embryos transferred per cycle. As expected, the day of hcg administration was significantly delayed in the study group (P.01). There were no significant differences between groups regarding the quality of the embryos transferred: proportion of embryos grade 1 (best quality), 25.1% and 26.1%; grade 2, 39.7% and 35.2%; grade 3, 35.2% and 38.6%, respectively, for study and control groups. No grade 4 to 5 embryos were transferred in fresh cycles in any group. Patients with coasting (study group) had significantly lower implantation and pregnancy rates compared with controls: 7.7% vs. 26.9% and 18.1% vs. 48.4%, respectively (P.01 for all comparisons). Table 2 presents the characteristics of the transfer cycles after embryo cryopreservation and thawing. The mean duration of freezing was comparable in both groups (8.1 and 8.7 months in study and control groups, respectively, P.05). There was no significant difference between groups regarding the stage at cryopreservation: 36% pronuclear and 64% cleaving, and 45% pronuclear and 55% cleaving, for study and control groups, respectively. In the study group (with coasting) there were 39 thawing cycles, and 38 of them reached the transfer stage. There were 42 thawing cycles in Fertility and Sterility 869

4 TABLE 1 Results of the fresh IVF cycles in the study (with coasting) and control (without coasting) groups. Variable Study group (coasting) Control group (no coasting) No. of patients/transfers Total dose of rfsh (IU) 1, , Day of hcg a E 2 at the beginning of coasting (pg/ml) 5, E 2 at hcg administration (pg/ml) 3, , Duration of coasting (d) No. of metaphase II oocytes retrieved Fertilization rate (%) Mean no. of embryos transferred Implantation rate, n (%) 7/91 (7.7) a 24/89 (26.9) Clinical pregnancy rate, n (%) 6/33 (18.1) a 16/33 (48.4) Note: Data are presented as mean SE, unless otherwise noted. a P.01. Arslan. Effect of coasting on embryo quality. Fertil Steril the control group. Embryo viability (survival rate) was 66.4% and 73.0% in study and control groups, respectively, and the difference was not statistically significant. In the study group, one cycle was canceled because there was no viable embryo available after thaw. The mean number of embryos transferred, implantation rates, and pregnancy rates were not significantly different between groups. There were no significant differences between groups regarding the quality of the embryos at the time of cryopreservation (not shown). Cycles in the study group (with coasting) were further divided into two subgroups according to the duration of coasting ( 3 days or 3 days). Results of the fresh IVF cycles are presented in Table 3. As expected, serum E 2 levels at the time of coasting were significantly higher in patients with 3 days of coasting (P.05). Also, the day of hcg administration was significantly delayed in patients with 3 days of coasting compared with patients with 3 days of coasting or controls (P.01). Although the fertilization rates and the number of embryos transferred were similar in the groups, both subgroups of patients receiving coasting had significantly lower implantation and pregnancy rates than the control group (P.05 for all comparisons). Table 4 presents results of the same three subgroups of patients after embryo cryopreservation and thawing. There were no differences between groups in the quality of the cryopreserved embryos (not shown). The survival rate of embryos in patients with 3 days of coasting was significantly lower compared with controls and compared with the subgroup of patients having coasting 3 days (P.05). The numbers of embryos transferred, the implantation rates, and pregnancy rates were similar in all groups. TABLE 2 Results of cycles of cryopreserved thawed embryos in patients with and without coasting in their fresh IVF cycle. Variable Study group (coasting) Control group (no coasting) No. of cycles with thaw No. of transfers Mean ( SE) duration of freezing (mo) Embryo survival (%) Mean ( SE) no. of embryos transferred Implantation rate, n (%) 14/113 (12.3) 15/115 (13.0) Clinical pregnancy rate, n (%) 12/38 (31.5) 16/42 (38.0) Arslan. Effect of coasting on embryo quality. Fertil Steril Arslan et al. Effect of coasting on embryo quality Vol. 84, No. 4, October 2005

5 TABLE 3 Evaluation of fresh IVF cycles subgrouped according to duration of coasting. Variable Coasting <3 days (n 22) Coasting 3 days (n 11) Controls (no coasting) (n 33) Age (y) Basal FSH (miu/ml) E 2 at the beginning of coasting (pg/ml) 5, a 6, E 2 at hcg administration (pg/ml) 3, , , Day of hcg administration b c Total dose of FSH (IU) 1, , , No. of metaphase II oocytes retrieved Fertilization rate (%) Mean no. of embryos transferred Implantation rate, n (%) 5/62 (8.0) d 2/29 (6.9) d 24/89 (26.9) Clinical pregnancy rate, n (%) 4/22 (18.1) d 2/11 (18.1) d 16/33 (48) Note: Data are presented as mean SE, unless otherwise noted. a P.05 vs. the coasting 3 days group. b P.01 vs. the coasting 3 days group. c P.01 vs. controls. d P.05 vs. controls. Arslan. Effect of coasting on embryo quality. Fertil Steril DISCUSSION To test the hypothesis that coasting during ovarian stimulation has a detrimental effect on oocyte/embryo quality, we examined the effect of withholding gonadotropins on the outcome of embryos after cryopreservation and thawing. To the best of our knowledge, this is the first study to evaluate the effect of coasting on the performance of cryopreserved thawed embryos. We compared patients with coasting having excess cryopreserved embryos with a well-matched control group of patients without coasting. Although our own previous results have demonstrated that ICSI does not have an impact on embryo quality after cryopreservation and thawing (23), others have reported a negative impact of oocyte and zona pellucida micromanipulation techniques on implantation potential after thawing (11). Therefore, we excluded from the analysis all cases in which ICSI or assisted hatching had been performed. Results of this study showed that coasting did not seem to have a detrimental effect on oocyte and embryo quality because the implantation competence of transferred concepti after cryopreservation thawing was similar to that of con- TABLE 4 Performance of cryopreserved thawed embryos subgrouped according to duration of coasting. Variable Coasting <3 days (n 22) Coasting 3 days (n 11) Controls (no coasting) (n 33) No. of cycles with thaw No. of transfers Embryo survival (%) a b Mean ( SE) no. of embryos transferred Implantation rate, n (%) 8/67 (11.9) 6/46 (13.0) 15/115 (13.0) Clinical pregnancy rate, n (%) 7/23 (30.4) 5/15 (33.3) 16/42 (38) a P.05 vs. coasting 3 days group. P.05 vs. controls. Arslan. Effect of coasting on embryo quality. Fertil Steril Fertility and Sterility 871

6 trols. However, prolonged coasting ( 3 days) had a negative impact on the post-thaw survival rate. There are many studies in the literature evaluating the effects of coasting on fresh IVF cycle outcomes (3, 13 17, 24 27). As detailed in a review by Delvigne and Rozenberg (28), fertilization rate (36.7% 71%), implantation rate (9.5% 31%), and pregnancy rate (20% 63%) have varied, mainly because of differences in patient selection criteria and design of the studies. Of all such studies, only a few focused directly on the effects of coasting on oocyte and embryo quality. Aboulgar et al. (27) found a lower percentage of high-quality oocytes and a decreased fertilization rate in a group of patients who developed OHSS and had coasting in their stimulation protocol. However, because the number of oocytes was large enough, this impaired percentage of high-quality oocytes affected neither the quality nor the number of embryos transferred nor the pregnancy rate. Delvigne et al. (16) investigated oocyte quality and IVF outcomes of fresh cycles after different periods of coasting. These investigators could not demonstrate any difference in oocyte quality and mean embryo quality scores compared with controls. Isaza et al. (19) also evaluated oocyte and embryo quality after coasting and their effects on implantation and pregnancy rates after fresh transfer in oocyte donation cycles. Because of the study design, they were able to eliminate the possible detrimental effect of high serum E 2 levels on endometrium and implantation. Similar to our experiment, the study and control donor cycles had comparable serum E 2 levels on the day of hcg administration, but there were higher serum peak E 2 levels in coasted patients. There were no differences between groups in terms of embryo quality or implantation rates after transfer in the recipients. However, when donors were further subdivided into two groups according to the length of coasting ( 4 days and 4 days), both implantation and pregnancy rates in recipients were lower with longer duration of coasting. This finding was coincident with no difference between groups in the quality of the embryos transferred. The investigators concluded that prolonged coasting had possible deleterious effects on oocyte and embryo quality that were unable to be determined by currently used oocyte and embryo grading systems. Our results demonstrated, overall, that embryos transferred after cryopreservation and thawing in hormonally supplemented cycles had similar implantation competence when compared with controls without coasting (Table 2). Because our study was designed as a model to exclude an endometrial effect due to the hyperestrogenism of ovarian hyperstimulation cycles, we might therefore conclude that coasting seems not to have a detrimental effect on oocyte and embryo quality. Our results only point out a subtle effect of prolonged coasting, manifested by a lower survival rate in cryopreserved thawed embryos with 3 days of coasting in their stimulation cycle (Table 4). Nevertheless, although the embryo survival rate was diminished in the group with prolonged coasting, implantation and pregnancy rates were not. This can be explained by the fact that the number of embryos surviving thawing was large enough to still select good-quality embryos for transfer. We cannot, therefore, entirely rule out that high serum E 2 levels or an extended exposure in the group with prolonged coasting might have affected the quality of oocytes, resulting in decreased resistance to freezing and thawing. If present, this effect could be due to either the absolute increase in serum concentration of the steroid or to increased time of exposure to a high estrogenic milieu. Owing to the high risk of development of OHSS and ethical concerns, it was not acceptable to examine a group of patients having peak serum E 2 levels similar to those of the coasted patients but in whom coasting was not performed. Ng et al. (29) examined oocyte and embryo quality in patients having an excessive ovarian response and reported no difference in the number of blastomeres per embryo comparing groups of patients with different levels of E 2 levels on the day of hcg administration. It has also been shown that high serum E 2 concentrations in fresh IVF cycles do not impair implantation and pregnancy rates in subsequent cryopreserved thawed ET cycles (30). However, in an in vitro study, Valbuena et al. (31) found direct evidence of a detrimental effect of E 2 on embryo developmental competence that might lead to lower embryo adhesion capability. It is obvious that the effect of high levels of serum E 2 during ovarian stimulation on embryo quality is still under debate and is open to new studies. Although our main objective was to determine the effect of coasting on the outcome of embryos transferred after cryopreservation and thawing, results of the study and control groups in their fresh cycles were quite different and worth analyzing. We observed lower implantation and pregnancy rates in the group of patients with coasting, irrespective of its duration (short or prolonged) (Tables 1 and 3). This could be due to the associated hyperestrogenism leading to either an oocyte or an endometrial effect. The fact that the results of cryopreserved thawed embryos showed no impact on implantation suggests an endometrial effect. The relationship between decreased endometrial receptivity and ovarian hyperstimulation in fresh IVF cycles has been studied for more than a decade (32). It has been hypothesized that ovarian hyperstimulation, by leading to supraphysiologic E 2 levels, prematurely induces P receptors in the endometrium in the follicular phase (33, 34). This might result in extremely advanced endometrium that negatively affects implantation (35). Simon et al. (36) reported lower implantation rates when serum levels of E 2 were 2,500 pg/ml on the day of hcg administration. These investigators presented further and strong evidence in favor of a detrimental effect of high E 2 levels on uterine receptivity (37, 38). Conversely, other studies have shown no detrimental effects (39 41). The study of Toner et al. (39) did not reveal 872 Arslan et al. Effect of coasting on embryo quality Vol. 84, No. 4, October 2005

7 impaired implantation rates in high responders, except for the infrequent group of patients with peak serum E 2 levels 5,000 pg/ml in whom there was a remarkable absence (down-regulation) of P4 receptors. We are presently conducting studies that might shed some light on this issue through the comparative analysis of gene expression profiles in stimulated and natural cycles (42). In conclusion, our studies showed that, overall, coasting does not seem to have a detrimental effect on oocyte/embryo quality, because the implantation competence of transferred concepti after cryopreservation and thawing was similar to that of controls. Prolonged coasting ( 3 days), on the other hand, had a subtle and negative impact on post-thaw survival rates, but because of the adequate number of embryos available to transfer demonstrating implantation competence, pregnancy rates were not affected. 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