Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes

Transcription

1 Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes Jacqueline Mandelbaum 1 ' 2 ' 5, Joelle Belai'sch-Allart 3, Anne- Marie Junca 4, Jean-Marie Antoine 2, Michelle Plachot 1 ' 3, Sylvia Alvarez 2, Marie-Odile Alnot 1, and Jacques Salat-Baroux 2 'Laboratoire de Recherche en FIV et Biologie de la Reproduction, Hopital Necker, 149 rue de Sevres, Paris, 2 Service du Pr Dadoune, Hopital Tenon, Paris, 3 CHI Jean Rostand, Sevres, and 4 Laboratoire d'eylau, Paris, Hopital Tenon, Paris, Hopital Necker, Paris, France 5 To whom correspondence should be addressed at: Laboratoire de Recherche en FIV et Biologie de la Reproduction, Hopital Necker, 149 rue de Sevres, Paris, France Human embryo cryopreservation represents an indispensable extension of in-vitro fertilization (IVF) programmes as long as they are based upon the recovery of a large number of oocytes. The most widely used procedures include the cryopreservation of human zygotes or embryos in early cleavage, using 1,2-propanediol and sucrose as cryoprotectants. Our results over a 10 year period ( ) on 5032 thawed cycles involving stored embryos make it possible to appraise the results and the contribution of embryo freezing to assisted reproduction. Embryos survived the freeze-thaw process in 73% of cases leading to 4590 transfers of 2.2 embryos (91% of thawed cycles). The clinical pregnancy rate per transfer was 16%, the live birth rate 12%, and the rate of babies born alive per transferred embryo was 6%. Embryo freezing monitored 10 years later produced an average of 8% of additional births. By then, 86% of stored embryos had been thawed for transfer to patients. Destruction or donation were required for only 8% of all frozen embryos and there was no news from the parental couple in relation to almost 6% of embryos. The fate of the vast majority of embryos was decided during the first 5 years of storage. Blastocyst cryopreservation is making new strides, thanks to co-culture systems and embryo selection. Micromanipulation procedures seem to have little impact on the outcome of embryo freezing. Human oocyte freezing is again clinically applied. Indeed, much of the concern about injuries to the oocyte structures through the freeze-thaw process do not seem to be justified, and the problems with frozen-thawed oocyte fertilization has been overcome using intracytoplasmic sperm injection (ICSI). As long as oocyte in-vitro maturation is not well controlled, better results will probably be obtained with mature oocyte Human Reproduction Volume 13 Supplement European Society for Human Reproduction and Embryology 161

2 J.Mandelbaum et al. cryopreservation. Emerging methods include the freezing of immature oocytes, follicles and ovarian tissue. Key words: cryopreservation/embryo/human/oocyte Introduction Cryopreservation of mammalian embryos has been available since 1972, when Whittingham et al. obtained live mice after the transfer of frozen-thawed morulae. Cryotechnology derived from rodents was applied to cattle where embryo transfers have been done each year since In humans, the first births from frozen embryos involved twins (Zeilmaker et al, 1984), followed by a singleton in 1984 (Trounson and Mohr, 1983). Embryo freezing spread rapidly, with the help of optimized and simplified biological procedures such as the use of propanediol and sucrose as cryoprotectants (Lassalle et al., 1985), and it became an indispensable extension of in-vitro fertilization (IVF). It is now possible, after >10 years, to appraise the contribution of embryo cryopreservation to assisted reproduction and to ask ourselves if this 'routine' procedure can be improved. This period of time was also necessary to obtain the successful cryopreservation of human oocytes (Chen, 1986), after the first births of mice from unfertilized oocytes frozen and stored at -196 C, (Whittingham, 1977). However, even by 1997, very few successful pregnancies have arisen from cryopreserved human oocytes, and only a handful of live births have been reported in the literature (Chen, 1986; Van Uem, 1987) despite considerable interest in this technique. The main negative points include low survival rates, poor fertilizability and a high incidence of polyploidy in human oocytes frozen and thawed according to the techniques used in other species and especially in rodents (Al Hasani, 1987; Mandelbaum et al, 1988a; Hunter et al, 1991). Animal studies raised concerns about the possibility of aneuploidy caused by injury to the meiotic spindle after exposure to cryoprotectants or by the freeze-thaw procedure (Bernard and Fuller, 1996). Lately, fundamental and experimental studies have been carried out which give a more optimistic outlook with regard to the preservation of cell structures after freeze-thaw procedures on mature oocytes (Imoedemhe, 1992; Gook et al, 1994; Van Blerkom and Davis, 1994). These results have been recently followed by new sets of clinical trials (Tucker et al, 1996), and the last led to the first new birth after 10 years in the domain of human oocyte cryopreservation (Porcu, 1997). Freezing immature oocytes does not appear to be more efficient, and the cryopreservation of in-vitro matured human oocytes seems to be unlikely because maturation in vitro still remains difficult (Mandelbaum, 1988b; Toth, 1994a,b; Son, 1996). The technique does not seem to be safe enough for routine, reliable oocyte freezing in IVF programmes. 162

3 Human embryos and oocyte cryopreservation Embryo freezing: clinical and technical requirements Embryo freezing enables IVF patients to reduce their risk of multiple pregnancies without destroying excess embryos, helps to avert risky transfers in cases of hyperstimulation and allows embryo quarantine for oocyte donation. Nevertheless, a huge amount of literature has focussed on the problems raised especially by the fate of frozen embryos after long-term storage. An alternative to routine IVF is possible, namely IVF performed in spontaneous cycles after ovulation has been triggered by exogenous human chorionic gonadotrophin (HCG). In studies involving 80 normal ovulatory patients, Foulot et al. (1990) obtained 17.5% developing pregnancies per oocyte retrieval but after high rates of cancellation and premature luteinzing hormone (LH) discharges (45%). Cooke and Lenton (1997) utilized 675 spontaneous cycles but obtained only 7% births. Lindheim et al. (1996) reported higher fertilization rates (89 versus 48%) and lower pregnancy rates (14 versus 7%) per recovery in poor responders in a study including 35 spontaneous cycles which were compared with 27 stimulated cycles in which three or fewer oocytes were recovered. The average number of inseminated oocytes was 1.1 and 2.3 respectively. IVF in spontaneous cycles can occasionally produce interesting results, but it remains useless in routine clinical practice. The technical requirements for human embryo freezing are well documented. In IVF laboratories, there has been a tendency for a particular cryoprotectant to be applied to each embryonic stage: 1,2-propanediol (PROH) for zygotes and early cleaved embryos (Lassalle et al., 1985), and glycerol for blastocysts (Cohen et al, 1985) with a slow freeze to -40 C (0.3 C/min) and a rapid thaw to 37 C or room temperature. Dimethyl sulphoxide (DMSO) is less used now for multicellular embryo freezing since it has not proved to be any more effective. It lengthens the procedure since it involves a slow freeze to -80 C and either a slow thaw (Trounson and Mohr, 1983) or rapid freezing, on crushed ice (Van der Elst, 1995). The efficiency of the ice-seeding technique is of major importance (Blefco, 1996), and seeding may be impaired in some freezers with automatic systems, thus leading to a failure of the whole freezing programme. Careful storage is also essential. Recent work by Tyler et al. (1996) has stressed again the importance of a rapid handling of vials, and especially straws, containing frozen embryos. Exposure times of <40 s at room temperature may lead to potentially detrimental temperature changes, since a 0.25 ml straw has reached -7 C, the eutectic point, in 1 min. Cryopreservation of cleaving human embryos: 10 years experience Overall results Collaborative work since 1986 with three IVF clinical teams at Tenon Hospital, Sevres Hospital and La Muette Clinic, involved the freezing of supernumerary 163

4 J.Mandelbaum et al. Table I. Cryopreservation of cleaving human embryos Data from four centres (Necker, Tenon, Sevres and La Muette) Thawing cycles Transfers Embryos thawed Mean number of embryos transferred Clinical pregnancies Live births Implantation rate per transferred embryo Babies born per transferred embryo Number / Percentage /transfer 12/transfer Table II. Follow-up of 727 fully-documented pregnancies after the cryopreservation of cleaving human embryos ( ). Data from four centres (Necker, Tenon, Sevres, La Muette) Total no. Result Number Percentage Pregnancies Multiple pregnancies Still births Live births Anomalies Sex ratio Girls Boys Delivered Abortions Ectopic Twins Triplets Single Sets of twins Sets of triplets Babies born At birth Therapeutic abortions early cleaved embryos at day 2 or 3 post-insemination using PROH and sucrose as cryoprotectants, as described by Mandelbaum et al. (1987). Over this 10 year period ( ), 5032 thawing cycles were carried out, leading to a transfer of 2.2 embryos on average in 91% of the cases (Table I). Among the stored embryos that were thawed, 73% had at least one half of their initial blastomeres still intact, which represents the survival rate. The clinical pregnancy rate per transfer was 16% (754/4590) and the implantation rate per transferred embryo reached 8.4% (864/10 333). The percentages of live births per transfer was 12% and of babies born alive per transferred embryo was 6%. Amongst the pregnancies achieved by means of frozen-thawed embryo transfer, 727 pregnancies were monitored, i.e. 96.4% of all pregnancies (Table II). Spontaneous abortions occurred in 23% of them and ectopic implantation in 4%, leading to 73% of births per initiated pregnancy as also found in transfers without embryo storage. Despite the low mean number of embryos per transfer (2.2), multiple pregnancies still occurred (13%), 86% of them resulting in twin 164

5 Human embryos and oocyte cryopreservation Table III. Benefit of embryo freezing during in-vitro fertilization (IVF) (1986, 1987, 1988). Data on 1117 cycles with embryos transferred and frozen in four centres (Necker, Tenon, Sevres and La Muette) IVF attempt Births Babies born Birth rate (%) Transfers with fresh embryos Transfers with frozen-thawed embryos Total *8% additional births for women with frozen embryos. implantations (83/97) and 14% in triplets (14/97). The stillbirth rate per pregnancy was 1.1%. The percentage of anomalies, including therapeutic abortions and major abnormalities at birth (2.2%), was similar to those gained after transfers of fresh embryos. Cryopreservation appears to be a quite efficient and safe procedure. Similar results had already been reported by Wada et al. (1994) and by FIVNAT (1994), comparing the birth characteristics and perinatal outcome of babies conceived from fresh and cryopreserved embryos. Moreover, two studies focussing on anomalies and development in children aged 1-9 years old (254 cases) arising from cryopreserved embryos again showed no difference from normally conceived children (Sutcliffe et al, 1995; Olivennes et al, 1996). Contribution of embryo freezing to IVF programmes The main purpose of embryo cryopreservation was to provide further possibilities for conception in addition to those obtained through the initial cycle using fresh embryos. By analysing results from three consecutive years (1986, 1987, 1988), it is now possible to calculate the cumulative birth rate (Table III). It is clear that women who had transfers of fresh and frozen embryos obtained 8% additional births, by using their stored embryos. Similar results were reported by other authors in long-term studies (Toner, 1991; Kahn et al, 1993; Wang et al, 1994; Jones et al, 1997). The transfer of frozen-thawed embryos from a given IVF procedure were mainly carried out after the failure of the initial transfer or after a miscarriage. These cases comprised 80% of the births (n = 74), while 20% (n = 18) were initiated to obtain a second birth in the couple. Extended storage of embryos cryopreserved in 1986, 1987 or 1988 did not affect the outcome of thawed cycles (Mandelbaum et al, 1994). The clinical pregnancy rate per transfer was 16% after 1-4 years of cryopreservation and 26% for 5-10 years of storage, with implantation rates of 7 and 13% respectively. The differences were not statistically significant. However a slight, but significant, decrease in survival rates occurred after the fourth year of storage, to 51% compared with 67% for shorter periods. Other authors also found no impairment in the viability and even survival of frozen embryos whatever the storage length (Lin et al, 1995; Avery et al, 1995). 165

6 J.Mandelbaum et al. Table IV. Outcome of long-term storage four centres (Necker, Tenon, Sevres and and human cryopreserved embryos*, using data from La Muette) Outcome (1997) Still in storage Destroyed1 Donated No news Legal problems No. couples No. embryos Percentage of all stored embryos *1986, 1987, 1988: data on 2873 cryopreserved embryos were analysed. The fate of frozen embryos During 1986, 1987 and 1988, 2873 embryos had been frozen in the course of our IVF programme. In 1997, it was possible to obtain insights into the outcome of embryo storage (Table IV). Only one couple have asked to keep their remaining embryo in store for possible transfer, although the wife is now aged 42. In 4.7% of cases, embryos had been destroyed, according to the patients' wishes. Although embryo research is not allowed in France, one-half of the couples were in favour of donating their spare embryos for research purposes. Donation to a sterile couple was accepted for 3.4% of the stored embryos but embryo donation is still not regulated in France, since the necessary procedures have yet to be established. For 5.4% of the embryos (n = 156), no instructions were received from the parents, with no replies to circular letters, and no available recent address in one third of the cases. Finally, 0.2% of all stored embryos (n = 6) gave rise to legal problems after the death of one partner in a couple, or after a divorce. Thus, 86.2% of all cryopreserved embryos had been thawed for transfer to the patients. In 1983, a similar survey of the same period evaluated data obtained after the fifth year of storage. Until 1997, only 1.1% of frozen embryos (32 embryos for 10 couples) had been additionally thawed for transfer, and 1.2% (35 embryos) were destroyed or donated. Parents of the embryos with an unknown issue were still undecided about their decision. Therefore, 5 years would appear to be a reasonable limit for embryo storage, provided that an extended delay can be allowed if necessary. Cryopreserved embryos and micromanipulation Freezing of ICSI embryos Cryopreservation of intracytoplasmic sperm injection (ICSI)-derived zygotes or embryos gives, for the majority of IVF teams, similar results to the freezing of classical IVF embryos (Van Steirteghem et al, 1994; Al-Hasani et al, 1996; Hoover et al, 1997; Kim et al, 1997), and our results are also similar (Table V). However, Lejeune et al (1997) recently reported that implantation rates for ICSI frozen-thawed embryos (4%) were reduced in comparison to IVF frozen-thawed 166

7 Human embryos and oocyte cryopreservation Table V. Cryopreservation of embryos derived from in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) ( ), using data from three centres (Necker, Tenon and Sevres) Embryo freezing No. thawed cycles No. transfers (%) No. thawed embryos No. transferred embryos (%) Best-quality embryos (%) Clinical pregnancies per transfer (%) Classical IVF (95) a (78) b ICSI (96) a (78) b y} test = not significant. a Percentage of thawed embryos. b Percentage of thawed embryos corresponding to the survival rate. embryos (13%). More information is needed to exclude the possibility of deleterious effects of micromanipulation on the outcome of the freeze-thaw procedure. Schwartz et al. (1996) showed by scanning electron microscopy that the breach in the zona pellucida rapidly disappears after withdrawal of the ICSI pipette. Assisted hatching of cryopreserved embryos Assisted hatching, either by partial zona dissection (PZD) (Tucker et al, 1991), zona drilling (Check et al, 1996), or laser (Germond, 1996) might enhance the implantation rate of frozen-thawed zygotes or cleaved embryos. The only randomized and significantly different results were reported by Check et al. (1996). They obtained 14% implantation rate per transferred frozen-thawed embryo in 79 cycles with assisted hatching compared with a 5% implantation rate in 79 control cycles. If the freeze-thaw procedure leads to zona hardening which is far from being obvious (Matson et al., 1997), then assisted hatching may improve the results of human embryo cryopreservation. Such studies, as well as large randomized series, are needed to evaluate the benefit of assisted hatching after cryopreservation. Cryopreservation of other embryonic stages Pronucleate eggs According to published data, zygotes can be successfully frozen using propanediol and sucrose, results being similar to those gained with multicellular embryos at 2 or 3 days post-fertilization. The best time to cryopreserve zygotes seems to be h after insemination, when pronuclei are in close contact, thus avoiding the risks of cooling during pronuclear migration or DNA synthesis (Wright etal, 1990). A study by Demoulin et al. (1991) obtained better pregnancy rates after the 167

8 J.Mandelbaum et al. transfer of frozen-thawed zygotes when compared with multicellular embryos. Generally, either zygote or cleaved embryo freezing is used within a clinic, so that detailed comparisons cannot be made. Blastocyst freezing Some controversy has arisen over the benefits of cryopreservation of embryos at early or late developmental stages. Blastocyst cyropreservation using glycerol as the cryoprotectant was abandoned, despite initial good results (Cohen et al, 1985), since suboptimal culture conditions allowed only 25% of human zygotes to develop to the expanded blastocyst stage in vitro. Co-culture on feeder cells improved this percentage to 50-60%, and after freezing and thawing, blastocysts were able to implant at rates between 9-18 % (Menezo et al, 1992; Hazout et al, 1995). With a simplified protocol, comprising glycerol (9%) and sucrose (0.2 M) for freezing and sucrose (0.5 and 0.2 M) only in 2-step thawing, Menezo and Veiga (1997) again reported high results on 816 thawing cycles. When 100 blastocysts were frozen and thawed, 10 babies were obtained. When comparing zygote, multicellular and blastocyst freezing, it is difficult to provide conclusive data on the eventual superiority of one of the protocols. Indeed, when all supernumerary zygotes and 97% of excess transferrable cleaved embryos are cultured in vitro, only 50-60% result in blastocysts. A two-fold increase in implantation rate should therefore be expected per frozen blastocyst owing to embryo selection. This is exactly what is obtained: 13% live births for transferred blastocysts (Menezo et al, 1997) in comparison with 6% for cleaving embryos in our series. Moreover, the live birth rate reaches 12% when single embryos are transferred after being frozen at the 8-cell stage at 3 days postinsemination (unpublished data). Early cleaved embryos with the best morphology and developmental rate have the same implantation potential as blastocysts. Therefore, the advantages of blastocyst freezing seems to depend on embryo selection, and both procedures are equally efficient and complementary rather than antagonistic. Recent improvements in culture media, making it possible to obtain high rates of blastocysts without co-culture systems, will certainly lead to an increasing use of this technique. Whatever the developmental stage of frozen embryos, many of the failures in implantation are due to poor uterine receptivity. Human oocyte freezing Oocyte cryopreservation represents one of the tools still missing in the range of IVF-derived technologies. It would solve some difficult situations arising when the husband is unable to produce a viable sperm sample or when no spermatozoa can be found in the testis at a given moment in cases of non-obstructive azoospermia. It could enable young women to store their gametes before undergoing therapies which lead to infertility, such as chemotherapy or 168

9 Human embryos and oocyte cryopreservation radiotherapy. It would also lead the way to oocyte banking, which could offer assistance to anonymous oocyte donation. Mature oocyte freezing: first clinical applications The initial report by Chen (1986) of the first births (one singleton and one twin) following human mature oocyte cryopreservation was highly encouraging. Using chilled DMSO in a one-step procedure, with slow freezing and rapid thawing, he obtained a survival rate of 76%, after classical insemination and a fertilization rate of 71% from 50 frozen and thawed oocytes. Two births, of three normal babies, occurred after seven embryo transfers. Van Uem et al. (1987) obtained the third birth and the fourth baby reported in the literature. Until 1994, three other groups reported data on human oocyte freezing for clinical purposes using either vitrification (Feichtinger et al, 1987) or propanediol and sucrose as cryoprotectants (Al-Hasani et al, 1987; Serafini, 1994). They obtained only short-lived pregnancies. The combined data indicate that 383 mature oocytes had been thawed, resulting in the birth of four babies, i.e. 1% of living babies per thawed oocyte. There were various reasons for this lack of success: low survival rates (25^40%), low fertilization rates after classical insemination, a high incidence of polyploidy and poor developmental ability of embryos. All these concerns, together with the success of embryo freezing, persuaded IVF clinics to move away from the clinical application of oocyte cryopreservation. Simultaneously, an extensive evaluation was made of the effects of various steps in the freeze-thaw protocol on oocyte structures involved in fertilization and embryo growth (for reviews see Van Blerkom and Davis, 1994; Bernard and Fuller, 1996). Studies focussed on several of the cytoplasmic inclusions in developing oocytes: the meiotic spindle and its loosely bound chromosomes; microfilaments essential for polar body extrusion, pronuclear migration and cytokinesis; cytoplasmic organelles such as mitochondria; damage to the zona pellucida such as breaches and hardening, and a premature cortical reaction. Numerous anomalies were reported, especially in mice. These concerns over mature oocyte cryopreservation prompted some teams to turn to immature oocyte freezing, which was thought to be less sensitive to cryo-injury. Immature human oocyte freezing Prophase I oocytes, collected in stimulated (Mandelbaum et al, 1988a,b; Toth et al, 1994a) or spontaneous cycles (Toth et al, 1994b; Son et al, 1996), have been frozen and thawed with propanediol and sucrose. Surviving oocytes were fertilized in vitro and the embryos allowed to develop into blastocysts. Pooled data of the literature on 504 thawed immature oocytes showed how 37% of them collected in stimulated cycles were able to reach metaphase 2 as compared with only 17% achieving meiosis after being recovered in spontaneous cycles. In both groups, mature and intact oocytes were fertilized in 43% of the 169

10 J.Mandelbaum et al. Table VI. Cryopreservation of immature (prophase I) oocytes during intracytoplasmic sperm injection (ICSI). Data from three centres (Necker, Sevres and Tenon) Study No. of oocytes Survival rate Metaphase II Injected oocytes with pronuclei two Experimental Clinical Total /7* *Two transfers made with a single embryo gave no resulting pregnancies. cases. No advantage of immature oocyte freezing accrued from these results, despite the ability of 3-17% of resulting embryos to develop to blastocysts. Our small series on the cryopreservation of immature prophase I oocytes was gained during ICSI, whether carried out for experimental or therapeutic purpose (Table VI). High survival rates (78%) were achieved with the protocol used for embryo freezing, and one-half of the intact oocytes extruded the first polar body during the first 24 h of culture. Seven oocytes were microinjected by ICSI, resulting in two transfers of a single normal embryo. No pregnancies arose. Return to mature oocyte freezing Two main developments led to the revival of mature oocyte freezing. First, reappraisals of the effects of freezing on oocyte structures gave a more optimistic outlook (Gook et al, 1993, 1994; Van Blerkom and Davis, 1994). At least 60% of surviving oocytes had normal spindles and chromosome configurations, and there was no evidence of an increased frequency of freezing-associated aneuploidy as assessed by fluorescence or cytogenetics. Breaks in individual tubules could result in stray chromosomes that were never observed in 103 cryopreserved pronucleate and in 34 lysed and failed-fertilized oocytes (Gook et al, 1994). Cortical granule distribution was similar in cryopreserved and non-cryopreserved human oocytes, using a lectin stain, excluding spontaneous cortical reactions resulting from cryo-injury (Gook et al, 1993). A high rate of parthenogenetic activation (27%) was observed (Gook et al, 1995a), but these oocytes had a single pronucleus and would have been discarded. The second major change was the introduction of ICSI to fertilize in-vitro cryopreserved oocytes, thus by-passing the eventual problems related to cortical granules or zona pellucida defects (Gook et al, 1995b; Kazem et al, 1995). ICSI brought fertilization rates of cryopreserved oocytes to the same levels as those obtained with control oocytes. The way was open for new clinical studies following the procedure used in embryo freezing, and again using propanediol and sucrose as cryoprotectants. Tucker et al. (1996) obtained 16% of cleaved embryos per thawed oocytes during studies on oocyte donation and performed five transfers with unfortunately three miscarriages, two of them with normal karyotypes. Porcu et al (1997) focussed on IVF cycles for tubal sterility in young volunteer women (aged 30 ± 3 years) 170

11 Human embryos and oocyte cryopreservation with normal partners and a fair response to stimulation (i.e. 18 oocytes on average). Survival rates reached 57%, and fertilization rates after ICSI were 64-77%. An average of 3.1 embryos were transferred either in the same cycle or some months later. Pregnancy rates per transfer averaged 17%, but the percentage of ongoing implantations still remained at 1% per thawed oocyte. Nevertheless, this first birth for 10 years, and some following pregnancies, are reassuring given that this technique had fallen into disuse for almost a decade. Other interesting approaches are in progress in the field of cryopreservation including freezing of follicles and ovarian tissue (Gosden, 1997). Autografting procedures or follicle storage and culture in humans could revolutionise the means of preserving female reproductive potential. Conclusions Human embryo cryopreservation offers an efficient solution to the problem of supernumerary embryos whatever their developmental stage: zygote, cleaved embryos, blastocyst. Thanks to this flexibility, clinics could be able to choose the time to cryopreserve their embryos and the various strategies of transfer. Improvements will be difficult, even though 20-30% of cryopreserved embryos still fail to survive thawing. Changes may arise from cryotechnology with new macromolecules or antifreeze proteins, new procedures such as vitrification, or better strategies in embryo culture and selection. Better knowledge is accumulating about uterine receptivity. The long-term storage of human embryos raises problems that could be solved by a clear and carefully constructed law. Human oocyte cryopreservation has reappeared but in-vitro maturation remains poorly controlled. Better results should be obtained with the freezing of mature oocytes aspirated from their follicles. Success is still elusive and births are rare (e.g. Tucker etal, 1996; Porcu etal, 1997). The risks, e.g. spindle disorganization, seem to have been overestimated, while ICSI has increased the chances of fertilization. References Al-Hasani, S., Diedrich, K., van der Ven, H. et al. (1987) Cryopreservation of human oocytes. Hum. Reprod., 2, Al-Hasani, S., Ludwig, M., Gagsteiger, F. et al. (1996) Comparison of cryopreservation of supernumerary pronuclear human oocytes obtained after intra-cytoplasmic sperm injection (ICSI) and after conventional in-vitro fertilization. Hum. Reprod., 11, Avery, S., Marcus, S., Spillane, S. et al. (1995) Does the length of storage time affect the outcome of frozen embryo replacement? J. Assist. Reprod. Genet., 12, 67S. Bernard, A. and Fuller, BJ. (1996) Cryopreservation of human oocytes: a review of current problems and perspectives. Hum. Reprod. Update, 2, BLEFCO, Denis, I., Mandelbaum, J.and Testart, J. (1996) Congelation des embryons humains: enquete sur la situation en France ( ). Contracept. Fertil. Sex., 24,

12 J.Mandelbaum et al. Check, J.H., Hoover, L., Nazari, A. et al. (1996) The effect of assisted hatching on pregnancy rates after frozen embryo transfer. Fertil. Steril., 65, Chen, C. (1986) Pregnancy after human oocyte cryoprevervaton. Lancet, i, Cohen, J., Simons, R.F., Edwards, R.G. et al. (1985) Pregnancies following the frozen storage of expanding human blastocysts. J. In Vitro Fertil. Embryo Transfer, 2, Cooke, I.D.and Lenton, E.A. (1997) IVF in the natural cycle. J. Assist. Reprod. Genet, 14, Demoulin, A., Jouan, C, Gerday, C. et al. (1991) Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages. Hum. Reprod., 6, Feichtinger W., Benko I. and Kemeter P. (1987) Freezing human oocytes using rapid techniques In Feichtinger, W. and Kemeter, P. (eds), Future Aspects in Human In Vitro Fertilization. Springer-Verlag, Berlin, Heidelberg, Germany, pp FIVNAT (1994) Pregnancy outcome after replacement of frozen-thawed embryos and after transfer of fresh embryos in French IVF registry. Contracept. Fertil. Sex., 22, Foulot, H., Ranoux, C, Dubuisson, J.B. et al. (1989) In vitro fertilization without ovarian stimulation: a simplified protocol applied in 80 cycles. Fertil. Steril., 52, Germond, M, Nocera, D., Senn, A. et al. (1995) Microdissection of mouse and human zona pellucida using a im diode laser beam: efficacy and safety of the procedure. Fertil. Steril, 64, Gook, D.A., Osborn, S.M., Bourne, H. et al. (1994) Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Hum. Reprod., 9, Gook, D.A., Osborn, S.M. and Johnston, W.I.H. (1993) Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum. Reprod., 8, Gook, D.A., Osborn, S.M. and Johnston, W.I.H. (1995a) Parthenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol. Hum. Reprod., 10, Gook, D.A., Schiewe, M.C., Osborn, S.M. et al. (1995b) Intra-cytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol. Hum. Reprod., 10, Gosden, R.G. (1997) Cryopreservation of ovarian tissue. In 10 th World Congress on In Vitro Fertilization and Assisted Reproduction. Monduzzi Eds, Bologna, Italy, pp Hazout, A., Nathan-Abbou, C, Cohen-Bacrie, P. et al. (1995) Results of replacement cycles in patients with frozen cocultured supernumerary blastocysts. J. Assist. Reprod. Genet., 12, 67-68S. Hoover, L., Baker, A., Check, J.H. et al. (1997) Clinical outcome of cryopreserved human pronuclear stage embryos resulting from intracytoplasmic sperm injection. Fertil. Steril, 67, Hunter, J.E., Bernard, A., Fuller, B. et al (1991) Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperatures and cryopreservation: a comparison of two techniques. Hum. Reprod., 6, Imoedemhe, D.G. and Sigue, A.B. (1992) Survival of human oocytes cryopreserved with or without the cumulus in 1,2-propanediol. J. Assist. Reprod. Genet., 9, Jones, H.W., Jones, D. and Kolm, P. (1997) Cryopreservation: a simplified method of evaluation. Hum. Reprod., 12, Kahn, J.A., von During, V., Sunde, A. et al. (1993) The efficacy and efficiency of an in-vitro fertilization programme including embryo cryopreservation: a cohort study. Hum. Reprod., 8, Kazem, R., Thompson, L.A., Srikantharajah, A. et al. (1995) Cryopreservation of human oocytes and fertilization by two techniques: in-vitro fertilization and intra-cytoplasmic sperm injection. Hum. Reprod, 10, Kim, J.W., Han, M.H., Byun, H.K. et al. (1997) Comparison of transfer of cryopreserved supernumerary embryos obtained after conventional IVF and ICSI. In 10 th World Congress on In-Vitro Fertilization and Assisted Reproduction. Monduzzi Eds, Bologna, Italy, pp Lassalle, B., Testart, J. and Renard, J.P. (1985) Human embryo features that influence the success of cryopreservation with the use of 1 2 propanediol. Fertil. Steril, 44,

13 Human embryos and oocyte cryopreservation Lejeune, B., Vanderzwalmen, P., Vandamme, B. et al. (1997) Reduced implantation rate after the transfer of frozen-thawed embryos obtained by ICSI. Hum. Reprod., 12 (Abstr. Book 1), 3. Lin, Y.P., Cassidenti, D.L., Chacon, R.R. et al. (1995) Successful implantation of frozen sibling embryos is influenced by the outcome of the cycle from which they were derived. Fertil. Sterii, 63, Lindheim, S.R., Vidali, A., Ditkoff, E. et al. (1996) Poor responders to ovarian hyperstimulation may benefit from an attempt at natural cycle oocyte retrieval. J. Assist. Reprod. Genet., 14, Mandelbaum, J., Junca, A.M., Plachot, M. et al. (1987) Human embryo cryopreservation, intrinsic and extrinsic parameters of success. Hum. Reprod., 2, Mandelbaum, J., Junca, A.M. and Tibi, C. (1988a) Cryopreservation of immature and mature hamster and human oocytes. Ann. N.Y. Acad. Sci., 541, Mandelbaum, J., Junca, A.M. and Plachot, M. (1988b) Cryopreservation of human embryos and oocytes. Hum. Reprod., 3, Mandelbaum, J., Plachot, M., Junca, A.M. et al. (1994) Human embryo cryopreservation in an IVF program. Limits, facts, prospects. In Mori, T., Aono, T., Tominaga, T., and Hiroi, M. (eds), Frontiers in Endocrinology. Ares Serono Symposia Publications, no. 4, pp Matson PL., Graefling J., Junk S.M. et al. (1997) Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme. Hum. Reprod., 12, Menezo, Y., Nicollet, B., Herbaut, N. et al. (1992) Freezing cocultured human blastocysts. Fertil. Sterii., 58, Menezo, Y. and Veiga, A. (1997) Cryopreservation of blastocysts. In 10 th World Congress on In Vitro Fertilization and Assisted Reproduction. Monduzzi Eds, Bologna, Italy, pp Olivennes, F., Schneider, Z., Remy, V. et al. (1996) Perinatal outcome and follow-up of 82 children aged 1-9 years old conceived from cryopreserved embryos. Hum. Reprod., 11, Porcu, E., Fabbri, R., Seracchioli, R. et al. (1997) Intra-cytoplasmic sperm injection of cryopreserved human oocytes. In 10 th World Congress on In Vitro Fertilization and Assisted Reproduction. Monduzzi Eds, Bologna, Italy, pp Schwartz, P., Magerkurth, C. and Michelmann, H.W. (1996) Scanning electron microscopy of the zona pellucida of human oocytes during intracytoplasmic sperm injection (ICSI). Hum. Reprod., 11, Serafini, P., Tran, C. and Tan, T. (1995) Cryopreservation of human oocytes. A clinical trial. J. Assist. Reprod. Genet., 12, 6S. Son, W.Y., Park, S.E., Lee, K.A. et al. (1996) Effects of 1,2-propanediol and freezing on the in vitro developmental capacity of human immature oocytes. Fertil. Sterii., 66, Sutcliffe, A.G., D'Souza, S.W., Cadman, J. et al. (1995) Minor congenital anomalies, major congenital malformations and development in children conceived from cryopreserved embryos. Hum. Reprod., 10, Toner, J.P., Brzyski, R.G., Oehninger, S. et al. (1991) Combined impact of the number of preovulatory oocytes and cryopreservation on IVF outcome. Hum. Reprod., 6, Toth, T.L., Baka, S. and Veeck, L.L. (1994b) Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil. Sterii, 61, Toth, T.L., Lanzendorf, S.E., Sandow, B.A. et al. (1994a) Cryopreservation of human prophase I oocytes collected from unstimulated follicles. Fertil. Sterii., 61, Trounson, A. and Mohr, L. (1983) Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature, 305, Tucker, M.J., Cohen, J., Massey, J.B. et al. (1991) Partial dissection of the zona pellucida of frozen-thawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates. Am. J. Obstet. Gynecol, 165, Tucker, M.J., Wright, G., Morton, P. et al. (1996) Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose. Hum. Reprod., 11, Tyler, J.P.P., Kime, L., Cooke, S. et al. (1996) Temperature change in cryo-container during short exposure to ambient temperatures. Hum. Reprod., 11, Van Blerkom, J.and Davis, PW. (1994) Cytogenetic, cellular, and developmental consequences of 173

14 J.Mandelbaum et al. cryopreservation of immature and mature mouse and human oocytes. Microscopy Res. TecnoL, 27, Van der Elst, J., Camus, M., Van den Abbeel, E. et al. (1995) Propective randomized study on the cryopreservation of human embryos with dimethylsulfoxide or 1,2-propanediol protocols. Fertil. Steril, 63, Van Steirteghem, A.C., Van der Filst, J., Van den Abbeel, E. et al. (1994) Cryopreservation of supernumerary multicellular human embryos obtained after intracytoplasmic sperm injection. Fertil. Steril, 62, Van Uem, J.F., Siebzehnruebl, E.R., Schuh, B. et al. (1987) Birth after cryopreservation of unfertilized oocytes. Lancet, ii, Wada, I., Macnamee, M.C., Wick, K. et al. (1994) Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum. Reprod., 9, Wang, X.J., Ledger, W, Payne, D. et al. (1994) The contribution of embryo cryopreservation to in-vitro fertilization/gamete intra-fallopian transfer: 8 years' experience. Hum. Reprod., 9, Whittingham, D.G. (1977) Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at -196 C. J. Reprod. Fertil., 49, Whittingham, D.G., Leibo, S.P. and Mazur, P. (1972) Survival of mouse embryos, frozen to C and -289 C. Science, 178, 411^14. Wright, G., Wiker, S., Eisner, C. et al. (1990) Observations on the morphology of pronuclei and nucleoli in human zygotes and implications for cryopreservation. Hum. Reprod., 5, Zeilmaker, G.H., Alberda, A.T., Van Geest, L. et al. (1984) Two pregnancies following transfer of intact frozen-thawed embryos. Fertil. Steril., 42,