Implantation Potential and Clinical Impact of Cryopreservation A Review

Transcription

1 Placenta (2003), 24, S27 S33 doi: /s (03) Implantation Potential and Clinical Impact of Cryopreservation A Review E. M. Kolibianakis a, K. Zikopoulos and P. Devroey Centre for Reproductive Medicine, Dutch-Speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium Paper accepted 21 May 2003 The ability of human embryos to survive the freezing and thawing process is reflected in their implantation potential. Although cryopreservation appears to affect adversely the capacity of human embryos to implant, it has also been shown to enhance consistently IVF outcome. Moreover, despite the reduced implantation potential of cryopreserved embryos as compared to fresh embryos, multiple pregnancies are frequent in frozen embryo transfer (FRET) cycles. There is no conclusive evidence that the stage of development at the time of freezing provides a clear advantage for the outcome of a FRET cycle. On the other hand, a decreased chance for pregnancy achievement appears to exist with advanced maternal age. Neither the mode of endometrium preparation nor the length of cryostorage appears to affect the outcome of FRET cycles which is positively associated to the achievement of pregnancy in the corresponding fresh cycle. Available evidence suggests that there are no adverse consequences in the babies born after embryo cryopreservation, although larger studies are necessary to allow solid conclusions. Placenta (2003), 24, S27 S Elsevier Ltd. All rights reserved. INTRODUCTION Cryopreservation, available for mammalian embryos since 1972 (Whittingham, Leibo and Mazur, 1972), was applied for the first time in the human in the late 1970s (Edwards and Steptoe, 1980). Refinement of cryopreservation protocols has made feasible the achievement of pregnancy from frozen thawed human oocytes, two pronuclei (2-pn) zygotes, cleavage stage embryos and blastocysts, establishing cryopreservation as an essential part of in-vitro fertilization (IVF). The current review addresses several issues related to the success of cryopreservation and its role in assisted reproductive technologies (ART) twenty years after the first pregnancy reported from frozen thawed human embryos (Trounson and Mohr, 1983). Does cryopreservation affect adversely the implantation potential of human embryos? In order to assess reliably the above question, a comparison between embryos with similar quality that are transferred either fresh or after freezing and thawing, needs to be performed. Moreover, the uterine environment in both the fresh and the frozen transfer should be of similar quality. Consequently, comparison between a fresh embryo transfer performed in a stimulated endometrium for IVF and a frozen transfer performed in endometrium prepared with hormonal substitution is probably misleading. Stimulation for IVF has a To whom correspondence should be addressed. Fax: ; stratis@easynet.be /03/$ see front matter consistently been related to abnormal endometrium histology both before (Marchini et al., 1991) and at oocyte retrieval (Ubaldi et al., 1997; Kolibianakis et al., 2002). Up to date, few studies devoid of the above confounding factors have been performed to assess the impact of cryopreservation on the implantation potential of human embryos. Levran et al. (1990) showed that cryopreservation at the 2 4 cell stage significantly reduces the capacity of human embryos to implant. In an oocyte-sharing programme aspirated donor eggs were distributed to the donor herself and recipients. Recipients were prepared by a standard protocol of hormone replacement and were assigned at random to the transfer of either fresh or frozen thawed embryos. Eggs were randomly allocated for donation, no selection of embryos was performed before freezing and all frozen embryos were thawed and transferred, provided that they were not degenerating. Twenty-four percent of the fresh embryos transferred to the recipients were successfully implanted as compared with only 7.7 per cent of the frozen thawed embryos (P<0.01). A significant adverse effect of cryopreservation at the 2-pn stage on embryo quality and a tendency for a lower implantation rate compared to fresh cycles was in addition observed by Selick et al. (1995). Oocytes from the same donor were randomly allocated to two or more ovum recipients resulting in at least one fresh and one frozen embryo transfer. An identical hormonal preparation of the endometrium was used in the transfer cycle, whether fresh or frozen embryos were used. The power of the study to detect a difference in implantation rate (12.6 per cent vs 8.1 per cent, respectively), however, was limited Elsevier Ltd. All rights reserved.

2 S28 Placenta (2003), Vol. 24 Check et al. (2001) analysed pregnancy and implantation rates in oocyte recipients who had their first embryo transfer performed either during a fresh or a FRET cycle (where no synchronization with the donor was feasible). A significantly higher pregnancy rate and implantation rate was observed in recipients who had their first embryo transfer performed during a fresh cycle as compared to those who had their first embryo transfer performed during a FRET cycle (63.4 per cent vs 43.6 per cent; 31.8 per cent vs 15.3 per cent, respectively). It therefore appears that cryopreservation adversely affects the implantation potential of human embryos. Following thawing, a proportion of embryos will be fully intact, some will survive partially and some will degenerate. This has been related to their morphological quality at the time of freezing (Mandelbaum et al., 1987; Karlstrom et al., 1997), although it could also occur randomly (Burns et al., 1999). The ability of embryos to survive the freezing and thawing process is reflected in their implantation potential. Van den Abbeel et al. (1997) retrospectively analysed the implantation and in-vivo development of embryos which were either fully intact or had lost a proportion of their blastomeres after cryopreservation. A significantly higher implantation rate was obtained after transfer of fully intact embryos than after transfer of partially damaged embryos (11.4 per cent vs 3.5 per cent, respectively). Burns et al. (1999) in a retrospective analysis showed a significantly higher clinical pregnancy rate when frozen transfers included at least one embryo with intact blastomeres as compared to transfers of embryos in which not all their blastomeres were intact. Moreover, Guerif et al. (2002) reported that a significantly higher implantation rate per transferred embryo was achieved when all transferred embryos had intact blastomeres (100 per cent blastomere survival) as compared with damaged embryos (50 or 75 per cent blastomere survival). Similar results have been reported by Edgar et al. (2000). The decrease in developmental potential of embryos that have lost part of their blastomeres could be due to diminished intrinsic embryonic viability and/or to inhibitory effects of the damaged blastomeres. The probability, however, that transfer of cryopreserved embryos can lead to multiple pregnancies should not be underestimated. How frequent are multiple pregnancies after transfer of frozen thawed embryos? Mandelbaum et al. (1998) analysed 5032 thawed cycles leading to 4590 embryo transfers. Despite the low mean number of embryos transferred per cycle (2.2), a 13 per cent (97/727) multiple pregnancy rate was observed (86 per cent twins 84/97 and 14 per cent triplets 13/97). A 13 per cent multiple pregnancy rate was in addition reported by the Australian and New Zealand registry (Hurst, Shafir and Lancaster, 1999). Moreover, in a retrospective analysis of 3570 frozen embryo transfers by Wang, Yap and Matthews (2001) a 17.7 per cent multiple pregnancy rate occurred in women <40 years of age by transferring 1.9 embryos per cycle. It therefore appears that despite the reduced implantation potential of cryopreserved embryos as compared to fresh embryos, multiple pregnancy occurrence can be a source of concern in FRET cycles. This might be a reflection of improved endometrial receptivity in FRET cycles as compared with fresh IVF cycles (Check et al., 1995, 1999; de Ziegler et al., 1998), compensating for the decreased quality of the frozen thawed embryos. Although in FRET cycles a lower capacity of human embryos to implant probably exists as compared with fresh embryos, the clinical question of interest is to what extent cryopreservation can enhance IVF pregnancy rates. A simple evaluatory formula for reporting pregnancy rates involving cryopreserved material, however, has so far remained elusive (Jones, Veeck and Muasher, 1995). What is the benefit from cryopreservation for IVF results? An increase of 5.2 per cent in the baby take-home rate for 485 women entering an IVF programme was reported by Kahn et al. (1993), when embryos cryopreserved at the 2 8 cell stage were subsequently transferred in 124 initiated thawed cycles. Similarly, Wang et al. (1994) observed a 4 per cent increase in ongoing pregnancy rate in 2707 couples entering their IVF programme, as a result of cryopreservation at the cleavage stage. This figure increased to 11 per cent for those couples who returned for a FRET cycle (886 couples out of 1497 with frozen embryos after a fresh cycle). Bergh et al. (1995) followed for a period of 3 years 398 couples starting their first IVF treatment. The use of frozen thawed embryos at the cleavage stage in 228 cycles added an extra 19 per cent of deliveries to those achieved in 826 fresh IVF cycles. Van Voorhis et al. (1995) by analyzing 610 patients, who performed 1000 fresh cycles and 373 frozen transfers of embryos cryopreserved at the 2-pn stage, suggested that FRET cycles could enhance ongoing pregnancy rate by 6.6 per cent. Mandelbaum et al. (1998) reported an 8 per cent of additional births by analyzing 5032 FRET cycles, from embryos frozen at the early cleavage stage. The percentage of live births per transfer was 12 per cent and per transferred embryo 6 per cent. It can be concluded that there is a consistent enhancement of IVF outcome attributed to cryopreservation. However, results fluctuate among centers, probably due to the several factors influencing the success of cryopreservation, among which is the stage at which embryos are cryopreserved. Does stage of embryo development at freezing affect the results of cryopreservation? In a retrospective study, Demoulin et al. (1991) compared implantation rates from cryopreserved 2-pn oocytes (112

3 Kolibianakis et al.: Cryopreservation of Human Embryos FRET cycles) and multicellular embryos (110 FRET cycles). A significantly higher implantation rate and ongoing pregnancy rate and was observed in the 2-pn group as compared to the cleavage stage group (10.7 per cent and 17.9 per cent vs 4.7 per cent and 5.5 per cent, respectively). In a semi-randomized study including 382 patients, Senn et al., (2000) compared cumulative live birth rates obtained after cryopreservation of either pronucleate zygotes or early cleavage embryos. The best-cleaved embryos were not selected for fresh transfer, but rather all cleaved embryos were graded and allocated in groups of equal quality between those to be transferred or cryopreserved. Significantly higher implantation rates and pregnancy rates after cryopreserved embryo transfers were obtained in the 2-pn group as compared with the cleavage stage group, leading to higher cumulative pregnancy rates and live birth rates. However, the outcome of cycles in the early cleavage stage that did not lead to embryo cryopreservation was not taken into consideration for the calculation of the cumulative pregnancy rate. Moreover, significantly more cumulus oocyte complexes were retrieved in the 2-pn group, which might have influenced the results obtained by putting the 2-pn group at an advantage, before any embryo freezing took place. In a randomized controlled trial including 364 patients, cryopreservation at the 2-pn stage was compared with that at the cleavage stage in a two embryo transfer programme (Horne et al., 1997). A lower pregnancy rate in fresh embryo transfers in the group with 2-pn freezing as compared with the group with early cleavage freezing was observed, while the reverse was true for frozen embryo transfers, resulting in similar cumulative rates between the two groups. Up to date limited data are available considering blastocyst cryopreservation (Kolibianakis and Devroey, 2002). Behr et al. (2002) suggested that blastocyst cryopreservation results in a 16 per cent implantation rate for embryos frozen either on day 5 or day 6 of culture, while implantation rates of 21.9 per cent (Yokota et al., 2001) and 23.7 per cent (Choi et al., 2000) have been reported following vitrification and subsequent thawing of human blastocysts. In summary, there is no conclusive evidence that the stage of development at the time of freezing provides a clear advantage for the outcome of a FRET cycle. However, the method used for oocyte fertilization may be a confounding factor. It has been reported that embryos with a cracked zona, particularly those with holes in the zona, have higher rates of degeneration after thawing (Cohen et al., 1986), although the breach in zona pellucida during ICSI is known to disappear after withdrawal of the pipette used (Schwartz et al., 1996). On the other hand, despite evidence that pronuclear formation occurs faster with ICSI than with standard insemination (Nagy et al., 1994), it has been shown that human embryos obtained after ICSI survive the cryopreservation procedure equally well as do embryos resulting from IVF, while their capacity to cleave further is not altered (Van den Abbeel and Van Steirteghem, 2000). Is the insemination method related to the outcome of cryopreservation? S29 Obasaju et al. (1994) in a retrospective study compared the survival of 28 embryos with perforated zonae from subzonal insemination (SUZI) with that of 140 embryos with intact zone frozen at the 2 8 cell stage within the same period. Similar survival rates after thawing were present between the two groups. Similar survival rates after ICSI and IVF have also been reported by Van Steirteghem et al. (1994) who compared retrospectively 1171 ICSI embryos with 2495 IVF embryos frozen at the 2 16 cell stage. A tendency, however, for a higher incidence of preclinical abortions following transfer of human embryos cryopreserved after ICSI as compared to IVF was observed (40.9 per cent vs 27.0 per cent respectively). In agreement with the previous study, Kowalik et al. (1998) reported similar results between IVF and ICSI for post-thaw survival rates and pregnancy rates in a retrospective study in which embryos were cryopreserved at the 2-pn stage or at the cleavage stage. In addition, a tendency for a higher preclinical loss in thawed embryos derived from ICSI as compared to those derived from IVF was observed (23.8 per cent VS 16.7 per cent, respectively). Macas et al. (1998) in a retrospective study analysed a cohort of 408 ICSI zygotes and 299 IVF zygotes frozen after selection of the best four to five 2-pn oocytes for further culture. Similar post-thaw survival rates after IVF (24 cycles) and ICSI (44 cycles) were reported. However, a significantly lower implantation rate was observed in the ICSI as compared to the IVF group (10.9 per cent vs 25.0 per cent) while no difference was noted in implantation rates between embryos developed from fresh ICSI and IVF sibling zygotes (21.0 per cent vs 22.5 per cent, respectively). In addition, a tendency for a higher preclinical loss in the ICSI group as compared with the IVF group was again observed (4/14 vs 1/17, respectively). On the other hand, in a prospective non-randomized study comparing the outcome of 67 patients (356 frozen thawed embryos) who underwent a FRET cycle, a significantly higher post-thaw survival rate and a tendency for a higher implantation rate were observed in the ICSI group as compared to the IVF group, with either 2-pn or cleavage-stage embryo freezing (Hu et al., 1999). Furthermore, in an observational study Damario et al. (1999) compared the clinical outcome of frozen embryo transfers using cryopreserved pronuclear stage oocytes that had undergone either ICSI (67 cycles) or conventional IVF (72 cycles). Survival rates post thawing as well as implantation rates and delivery rates were similar between the two groups compared. In addition, no increase was observed in the rate of preclinical pregnancy loss in the ICSI group after a FRET cycle. Hoover et al. (1997) in a comparative observational study analysed 99 patients who performed 153 oocyte retrievals. In these cycles, no fresh ET was carried out and all embryos were cryopreserved at the pronuclear stage, after fertilization with either ICSI or IVF. Similar results between ICSI and IVF for post-thaw survival, implantation rates, clinical pregnancy rates and preclinical pregnancy loss rates

4 S30 Placenta (2003), Vol. 24 were reported. In addition, Al-Hasani et al. (1996) in a retrospective study observed similar post-thaw survival rates, clinical pregnancy rates and spontaneous abortion rates after transfer of supernumerary cryopreserved 2-pn oocytes derived either from ICSI (n=744) or IVF (n=333). In summary, it is not clear from the existing retrospective studies if ICSI as compared to IVF is associated with a higher preclinical pregnancy loss in FRET cycles. A valid answer to this question can only be provided from prospective studies that will take into account additional factors such as the age of the patients analysed, known to be related to the quality of the oocytes retrieved and cryopreserved. Does age of the female affect cryopreservation outcome? A trend toward a decline in delivery rates per frozen embryo transfer in line with an increase in the female age was reported by Damario et al. (2000) by analysing 103 frozen transfers when embryos were cryopreserved at the 2-pn stage. In a larger series, Karlstrom et al. (1997) analysed 660 FRET cycles (551 couples) in which freezing took place at the early cleavage stage and showed that women >40 years of age had a significantly lower birth rate than women <40 years of age. Similarly, a reduced pregnancy rate following frozen embryo transfer was recorded with increasing female age, with a significant reduction occurring after 40 years of age in a retrospective analysis of 3570 FRET cycles (1438 couples), where embryos were frozen at the early cleavage stage (Wang, Yap and Matthews, 2001). Therefore, a decreased chance for achievement of pregnancy appears to exist with advanced maternal age in FRET cycles. This means probably that the negative association of age and oocyte/embryo quality is also present after thawing. If the quality of embryos in the fresh cohort is reflected in the frozen thawed cohort, then achievement of pregnancy in a fresh cycle, known to be positively related to embryo quality, might predict a successful outcome in a subsequent FRET cycle. Is pregnancy achievement in a fresh cycle associated with an increased chance to obtain a further pregnancy in a subsequent FRET cycle? In a retrospective analysis of 375 consecutive frozen ETs according to the outcome of the corresponding fresh cycle, Lin et al. (1995) suggested that sibling embryos from a prior successful ART cycle, cryopreserved at the pronucleate or the early cleavage stage, have a significantly higher chance to initiate a pregnancy in a FRET cycle. In a retrospective study by Toner et al. (1991), 367 couples who had their cryopreserved pre-embryos subsequently thawed were analysed. Pregnancy achievement in the fresh IVF cycle predicted higher implantation rates in the FRET cycle. Similarly, Wang, Yap and Matthews (2001) by analysing retrospectively the outcome of 3570 FRET cycles, in which embryos were cryopreserved in the early cleavage stage, reported a significantly higher pregnancy rate in FRET cycles, if pregnancy had been achieved in the corresponding fresh cycle. In addition, Karlstrom et al. (1997) analysed 598 FRET cycles in which embryos were frozen at the early cleavage stage according to the outcome of the corresponding fresh cycle and observed similar results. It therefore appears that establishment of pregnancy in a fresh cycle affects positively the chance of pregnancy achievement in a subsequent FRET cycle. Besides age and outcome of the corresponding fresh cycle, additional factors may exert an influence on the outcome of a FRET cycle, including the type of stimulation during the fresh cycle, the mode of endometrium preparation in the FRET cycle, the aetiology of infertility and the length of cryostorage. Does the type of endometrium preparation affect the outcome of a FRET cycle? Al-Shawaf et al. (1993) analysed retrospectively 77 FRET cycles in which endometrium was prepared during a natural cycle, in women with regular cycles and 72 cycles in which hormone replacement therapy was used following downregulation with GnRH agonists, in women with anovulation or irregular cycles. No difference was observed in implantation and pregnancy rates between the two groups compared. Similar results have been reported by Sathanandan et al. (1991) in a prospective series of FRET cycles, in patients who had their frozen embryos thawed and replaced after downregulation with GnRH agonist and hormonal substitution (n=84) or during a natural cycle (n=78). It therefore appears that there is no benefit from using a natural or a hormone replacement scheme in order to increase the chance of pregnancy in a FRET cycle. Consequently the natural cycle should probably be the first choice for endometrium preparation in women with regular cycles. Is ovarian stimulation related to the outcome of cryopreservation? Van der Elst et al. (1996) performed a retrospective study of patients entering an IVF programme in which a triple embryo transfer was performed after stimulation with CC/HMG (106 cycles) or GnRH agonists/hmg (80 cycles). Supernumerary embryos were frozen at the cleavage stage and transferred in 42 FRET cycles after CC/HMG stimulation and in 30 FRET cycles after GnRH agonists/hmg stimulation. Significantly lower implantation and delivery rates were observed in the GnRH agonist/hmg group as compared to the CC/HMG group. Seelig et al. (2002) by retrospectively analysing 342 couples who underwent a FRET cycle, showed that the implantation potential of frozen 2-pn embryos is independent

5 Kolibianakis et al.: Cryopreservation of Human Embryos of the gonadotropin-releasing hormone analogue and gonadotropin chosen for the collection cycle. Similar results have been reported by Nikolettos et al. (2000) in a smaller series. Do infertility type and length of cryostorage affect cryopreservation outcome? In a retrospective analysis Wang, Yap and Matthews (2001) suggested that patients with tubal blockage had a significantly lower implantation rate than patients with non-tubal factor aetiology of infertility. In addition, although an early study by Testart et al. (1987) suggested that survival rate of frozen thawed embryos is negatively associated to the length of embryo cryostorage, this was not confirmed in several subsequent studies (Schalkoff et al., 1993; Lin et al., 1995; Wang, Yap and Matthews, 2001). Safety of cryopreservation Wada et al. (1994) by comparing birth characteristics of 283 babies born after FRET cycles to those of 961 babies born after fresh IVF cycles, observed no difference in the perinatal mortality rate and described a significantly lower major malformation rate in the cryopreservation group. Sutcliffe et al. (1995) compared the outcome of 91 children born after cryopreservation to that of 83 normally conceived control children of a similar age, sex and social class. The incidence of minor congenital anomalies and major congenital malformation rates were similar in both groups compared. Similar results have been reported by Olivennes et al. (1996) and Bonduelle et al. (1998). Although available evidence suggests that there are no adverse consequences of embryo cryopreservation in humans (Wennerholm, 2000), the limited power of the existing studies to detect potential differences between babies born after fresh or frozen embryo transfers should be kept in mind. Large registry studies will be required to reach concrete conclusions on the safety of cryopreservation. The role of cryopreservation in ART Cryopreservation of human embryos is related to several clinical benefits, among which the most important is probably the increase of IVF pregnancy rates in a cost effective way (Van Voorhis et al., 1995). Cryopreservation can in addition facilitate synchronization between donors and recipients or allow storage of cryopreserved donated embryos. Alternatively, oocyte cryopreservation could be used in a donation programme or an IVF programme (Porcu, 2001). Although oocyte freezing is still not widely available, it can be valuable for cancer patients about to undergo chemotherapy and can assist in overcoming religious or ethical objections to embryo freezing (Lockwood, 2002). On the other hand, cryopreservation should probably not be considered as a method to decrease multiple pregnancy rates, as frequently stated in the literature. Cryopreservation is a way to handle supernumerary embryos, a consequence of current IVF technology and ovarian hyperstimulation practice. Its aim is to enhance pregnancy rates after oocyte retrieval. A decrease in multiple pregnancy rates is intimately related to the decision to replace fewer embryos per cycle. This is based on the awareness of the problems associated with the occurrence of multiple pregnancies in ART and is and should be independent from the availability for cryopreservation. Moreover, cryopreservation is not a method to decrease the occurrence of ovarian hyperstimulation syndrome (OHSS), as has been suggested. This can be achieved by coasting stimulation, by withholding hcg or by using GnRH agonist as an ovulation triggering signal in high risk cases. If the risk of OHSS becomes apparent after oocyte retrieval, freezing all embryos is certainly an option but should not be considered as a way to avoid OHSS, when it is in fact simply compensating our lack of knowledge on how the ovary responds to gonadotrophin stimulation, introducing at the same time various ethical problems associated with cryopreservation (Shenfield et al., 2001). Perhaps the adoption of alternative strategies for ovarian stimulation in ART, aiming at fewer oocytes and in turn in the production of fewer embryos is worth considering. This would probably decrease the chance of developing OHSS but might also increase the chance of delivery following fresh transfer of a single embryo in a less compromised by ovarian hyperstimulation endometrium, eliminating at the same time the risk of multiple pregnancies. Cryopreservation under this setting can still play an important role. Conclusions S31 Cryopreservation appears to affect adversely the implantation potential of human embryos. The ability of embryos to survive the freezing and thawing process is reflected in their ability to implant. Despite the reduced implantation potential of cryopreserved embryos as compared to fresh embryos, multiple pregnancies are frequent in FRET cycles. A consistent enhancement of IVF outcome appears to result through cryopreservation programmes. There is no conclusive evidence that the stage of development at the time of freezing provides a clear advantage for the outcome of a FRET cycle. In FRET cycles, a decreased chance for achievement of pregnancy appears to exist with advanced maternal age. Establishment of pregnancy in a fresh cycle affects positively the chance of pregnancy achievement in a subsequent FRET cycle. Neither the mode of endometrium preparation nor the length of cryostorage appears to affect the outcome of FRET cycles.

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