Cryopreservation of supernumerary oocytes in IVF/ICSI cycles

Transcription

1 Human Reproduction Vol.21, No.2 pp , 2006 Advance Access publication October 20, doi: /humrep/dei347 Cryopreservation of supernumerary oocytes in IVF/ICSI cycles P.E.Levi Setti 1,2, E.Albani 1, P.V.Novara 1, A.Cesana 1 and G.Morreale 1 1 UO di Medicina della Riproduzione, IRCCS Istituto Clinico Humanitas, Rozzano (Milano), Italy 2 To whom correspondence should be addressed. paolo.levi_setti@humanitas.it BACKGROUND: The aim of the present study is to investigate cryopreservation of oocytes in patients refusing embryo cryopreservation for ethical reasons, patients from whom no sperm could be retrieved and patients with enough oocytes to yield a number of fresh and cryopreserved embryos to transfer. METHODS: A total of 2900 oocytes out of 6216 retrieved were cryopreserved in 286 patients undergoing 303 cycles. The reasons for cryopreservation were because no sperm was found in 16 cycles, for ethical or personal reasons in 80, and in 207 only supernumerary oocytes were frozen. In 159 cycles, the oocytes were thawed and the surviving metaphase II oocytes microinjected. RESULTS: A toal of 1087 oocytes were thawed, 760 (69.9%) survived and 687 were microinjected. We obtained 368 (53.5%) normally cleaved embryos, 331 were transferred and 37 were cryopreserved. One hundred and forty-five transfers (range 1 3 embryos/patient) were performed and 18 (12.4%) pregnancies were obtained. Twelve patients delivered 13 healthy children, and six first trimester abortions were observed (33.3%). CONCLUSION: Although a low implantation rate was observed and a higher abortion rate than in fresh cycles, our results show that in sibling oocytes, the process of cryopreservation apparently does not affect the fertilization and cleavage rate. In this group of patients, producing a large number of mature gametes, oocyte cryopreservation gives the couple extra chances to achieve a pregnancy within a single retrieval and is a good effort towards reducing the number of embryos cryopreserved and enhancing our experience in this new technology. Key words: assisted reproduction/cleavage/embryo transfer/fertilization/oocyte cryopreservation Introduction Since the birth of the first human through assisted reproduction in 1978, the number of couples seeking infertility treatment has increased dramatically. In 2001, > cycles of clinical infertility treatments occurred in Europe alone (ESHRE, 2005). This is due in part to the increase in patient awareness and acceptability of fertility treatments. During routine clinical treatment, ovarian stimulation is carried out to increase oocyte production. Increasing success rates have led to more concerns regarding multiple gestation pregnancies. This has caused most clinicians to limit the number of embryos transferred during a cycle and to cryopreserve the supernumerary embryos. This policy raised, in many countries, the problem of longterm storage of embryos and stimulated research trials on oocyte cryopreservation. In humans, a possible application of oocytecryopreservation techniques could be fertility preservation in women at risk of premature menopause, which can have several causes: recurrent or severe ovarian diseases such as cysts, benign tumours and endometriomas; ovary removal to treat endometriosis or genital cancer; and chemotherapy or radiotherapy to treat cancer or other systemic diseases. In addition, oocytecryopreservation has been seen as a successful alternative for storing the excess oocytes during assisted reproduction therapies, thus avoiding ethical, moral and religious dilemmas and reducing the number of embryos stored for future use. The first report of a pregnancy from a frozen egg was described by Chen (1986). A few other births were achieved shortly afterwards (Al-Hasani et al., 1987; Van Uem et al., 1987), but for many years the experiences conducted on oocyte freezing remained sporadic. Gook et al (1995) suggested for the first time that ICSI could improve fertilization rates in frozen oocytes, thereby bypassing possible fertilization failures derived from premature cortical granule release and zona hardening. However, in spite of several successes being reported (Porcu et al., 1997, 2000; Kuleshova et al., 1999; Levi Setti et al., 2004; Borini et al., 2004), there are still technical problems associated with oocyte freezing. Studies have shown that oocyte survival rates after cryopreservation could be affected by morphological and biophysical factors. Morphological characteristics of oocytes such as maturity and size, and biophysical factors such as cryoprotectant compositions are particularly important. For oocytecryopreservation with the slow freezing method, cryoprotectant solution usually consists of 1.5 mol/l membrane-permeating cryoprotectant (i.e. propanediol) and mol/l sucrose. It has been reported that an increase in sucrose concentrations could improve the survival 370 The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 No effect of oocyte cryopreservation on fertilization rate of frozen thawed human oocytes (Fabbri et al., 2001; Chen et al., 2004). Some recent reports have shown improvements in success when compared with those from the past two decades (Quintans et al., 2002; Boldt et al., 2003; Fosas et al., 2003), but the success rates remain suboptimal, with highly variable fertilization rates [e.g. from 17 to 100% (Quintans et al., 2002) and from 0 to 100% (Boldt et al., 2003)]. An overview of the literature shows that most studies use a similar freezing and thawing procedure (slow freezing, rapid thawing), and similar seeding points and cryoprotectants (1,2-propanediol and sucrose) (Fabbri et al., 2001). Several babies have been born by using oocytecryopreservation, according to recent reports (Fosas et al., 2003; Borini et al., 2004; Levi Setti et al., 2005a). Our team has been involved in oocyte cryopreservation since 1998, after our local ethical vommittee stimulated studies in order to offer a possible option to young patients before chemotherapy, to reduce the number of cryopreserved embryos and to provide a possible option for couples refusing embryo cryopreservation for personal reasons. The aim of the present study is to report our experience in oocyte cryopreservation with a slow freezing 1,2 propanediol mol/l sucrose protocol, before Italian Law regulated the practice of assisted reproduction. Materials and methods Study population During the period October 1999 to December 2003 in 286 patients undergoiing 303 IVF or ICSI cycles, 6216 oocytes were retrieved and 2900 were cryopreserved. This group of cycles represents 11.6% of the inductions for IVF/ICSI performed during the same period (303/2600). All patients signed an informed consent describing the experimental nature of the study. By a decision of our ethical committee, all the costs of the procedure of cryopreservation and storage of the oocytes were covered by the hospital and nothing was charged to the patient. In 16 cycles, oocyte cryopreservation was performed because no sperm was found the day of the retrieval and the patients desire was not to lose all the gametes after the stress of the procedure and the costs of the induction therapy, offered by the National Health System. In 80 cycles, the couple signed up, for ethical or other personal reasons, to use a limited number of oocyte in the fresh cycle or to have no embryos cryopreserved, even though they understood the possible reduction in pregnancy rate. In one of these couples, all the oocytes were cryopreserved due to a high risk of hyperstimulation. In 207 cycles when >12 good quality oocytes, or 15 in cases of extremely severe male factor, were retrieved and the couple consented to cryopreserve the supernumerary oocytes (OSO cycles) that would have not been used, in order to reduce the number of embryos stored. Our policy was to obtain embryos for only one or two postcryopreservation attempts after the analysis showed that >60% of the couples with cryopreserved embryos had delivered at least one child as a result of the fresh cycle. Cycles with all the oocytes cryopreserved, or in which a limited number of fresh oocytes were used, were considered separately as non-oso cycles, to test possible differences in the pregnancy rate in this subgroup of patients. Female age in the 286 couples was 34.1 ± 3.5 (mean ± SD) years, 55% of the couples were undergoing their first attempt, 26% were at their second attempt and 11% at their third attempt. Eight percent of the couples had undergone >3 previous attempts. Pituitary desensitization was induced by the administration of a GnRH agonist (Decapeptyl 3.75 mg, depot, 4 amp. IH Ibsen SPA, Italy). Multiovulation was induced with daily s.c. doses of recombinant FSH (Gonal F, Serono Pharma, Italy), ranging from 150 to 450 IU, and final maturation was induced by 5000 or IU of HCG (Profasi, Serono Pharma, Italy). The luteal phase was supported by 50 mg i.m. daily progesterone in oil (Prontogest, Amsa SRL, Italy). In the period September 2000 to December 2003 in 159 cycles in 120 patients, the oocytes were thawed and the normally fertilized and cleaved embryos were transferred. Mean female age in the thawing cycles was 35.0 ± 3.4 years. Transfer of the embryos obtained from cryopreserved oocytes was performed after endometrial preparation. The administration of GnRH agonist (Decapeptyl 3.75 mg depot) induced pituitary desensitization, and estradiol 6 mg a day per os (Progynova 2 mg, Shering, Italy) in divided doses was administered for 9 15 days. When an endometrial thickness of >7 mm was observed, 100mg of progesterone in oil was started (Prontogest, Amsa SRL) and the oocytes were thawed the same day. Our policy was to transfer no more than two embryos in patients aged 36 years and three embryos in women >36 years old. Embryos from fresh and cryopreserved oocytes were transferred 72 h after ICSI or IVF into the mid-cavity of the uterus under transabdominal ultrasound guidance. A clinical pregnancy was considered if the HCG level reached a value >1000 miu/ml or a gestational sac was observed by ultrasound. The implantation rate was considered as the number of gestational sacs with a fetal heart beat/transferred embryos. In all pregnancies, a karyotype was performed and we waited to present our data after a 6-month follow-up of babies. Oocyte cryopreservation and thawing Cumulus oocyte complexes were transferred to IVF medium (B2 INRA Medium, Laboratoire CCD, Paris, France) at 37 C in an atmosphere of 5% CO 2 in air. Complete removal of the cumulus and corona cells was performed using hyaluronidase (80 IU/ml; Sigma, St Louis, MO) and mechanical disruption with fine bore glass pipettes. All oocytes were examined for the presence of the polar body. Only metaphase II oocytes were included in this study. Oocytes were frozen 3 4 h after retrieval using the slow freeze rapid thaw propanediol method (Fabbri et al., 2001). Eggs were equilibrated in stepwise additions of the cryoprotectant to a final concentration of 1.5 mol/l 1.2- propanediol (Sigma Aldrich Srl, Milan Italy) plus 0.3 mol/l sucrose (Sigma Aldrich Srl) and a serum protein supplement (Pacific Andrology, CGA/Diasint, Florence, Italy). The oocytes were loaded into plastic straws (Paillettes Cristal, 133 mm; Cryo Bio System, Paris, France) and transferred into an automated Kryo 10 series III biological vertical freezer (Planer Kryo 10/1.7 GB; PLANER plc, Middlesex, UK). The initial chamber temperature was 23 C. Then the temperature was slowly reduced to 8 C at a rate of 2 C/min. Ice nucleation was induced manually at 8 C. Then the straws were cooled slowly to 30 C at a rate of 0.3 C/min and then rapidly to 150 C at a rate of 50 C/min. After 5 min of temperature stabilization, the straws were transferred into liquid nitrogen. Oocytes were thawed by plunging straws into warm water (30 C) followed by the expulsion of oocytes and the stepwise dilution of the cryoprotectants. Thawed eggs were transferred to IVF medium at 37 C in an atmosphere of 5% CO 2 in air. ICSI After at least 1 h, thawed eggs were transferred to individual microdrops in an injection dish maintained at 37 C. A single morphologically normal, motile spermatozoon was isolated, immobilized and injected into the oocyte. Injected eggs were returned to culture in B2 medium at 37 C in an atmosphere of 5% CO 2. Fertilization and cleavage Oocytes were examined h post-icsi. Fertilized oocytes were transferred to fresh medium. Oocyte survival, fertilization and embryo 371

3 P.E.Levi Setti et al. development were examined using a Nikon inverted microscope with Hoffman optics. Embryos were scored as grade 1 5. Grade 1 was assigned to the best quality embryos containing equally sized, symmetrical blastomeres with no fragmentation, grade 2 was assigned if they had blastomeres of equal size with minor cytoplasmic fragmentation occupying <10%, grade 3 if the blastomeres were of distinctly unequal size and variable fragmentation, grade 4 if the blastomeres were of equal or unequal size and cytoplasmic fragmentation >10%, and grade 5 were embryos with few blastomeres and fragmentation of >50% of their cytoplasm (Veeck, 1999). Statistical analysis Data were extracted from our general dedicated database (certified in 2001 by the Joint Commission International) and continuous variables were analysed with Statistica release 5.1 (Statsoft, Tulsa, OK). The results are reported as mean ± SD. The Mann Whitney U-test was applied to test differences between groups for continuous variables, and Yates corrected χ 2 as required. Variables considered in the study were: fertilization and cleavage rate; pregnancy rate; and implantation rate in fresh (OSO and non-oso) and cryopreserved oocyte cycles. Cycles with cryopreserved embryos were not considered in the study because >40% of the embryos are still frozen and most of them belong to couples who have at least one child. The general cumulative pregnancy rate will be elaborated to reduce the many biases involved when all the oocyte and embryos would be thawed. Patients with a live birth after fresh transfer coming for a frozen transfer were not considered in the study. P < 0.05 was considered statistically significant. Results During the period October 1999 to December 2003, 303 oocyte retrievals were performed. In 16 cycles, no sperm was found on the day of the retrieval and in one cycle all the retrieved oocytes were cryopreserved due to a high risk of hyperstimulation; therefore, IVF or ICSI was performed in 286 cycles. A total of 2445 oocytes were inseminated or microinjected (8.1 ± 3.6 per patient), according to semen characteristics, 1629 (66.6%) were fertilized, 1590 (97.6%) cleaved embryos were obtained and 719 were transferred (Table I). A mean of 2.5 embryos per patient were transferred (range 1 4). A toal of 263 transfers were performed and 126 (47.9% per transfer) pregnancies were obtained. The implantation rate was 23.2% (167 out of 719) and the abortion rate was 19.0% (24 out of 126). In OSO fresh cycles (207), the mean female age was 34.2 ± 3.8 years. A total of 2033 oocytes were inseminated or microinjected (9.8 ± 2.6 per patient), 1335 (66.6%) were fertilized, 1303 (97.6%) cleaved embryos were obtained and 516 were transferred. One hundred and eighty-seven transfers were performed and 91 (48.7% per transfer) pregnancies were obtained. The implantation rate was 23.2% (120 out of 516). In non-oso fresh cycles (79), the mean female age was 33.7 ± 4.7 years. A total of 412 oocytes were inseminated or microinjected (5.2 ± 1.8 per patient), 294 (71.4%) were fertilized, 287 (97.6%) cleaved embryos were obtained and 203 were transferred. Seventy-six transfers were performed and 35 (46.1% per transfer) pregnancies were obtained. The implantation rate was 23.1% (47 out of 203). No statistically significant difference between OSO and non-oso groups was found. In the 159 thawing cycles (120 patients), 1087 oocytes were thawed, 760 (69.9%) survived and 687 were microinjected 372 Table I. Results of fresh (286 patients) and thawing cycles (120 patients) *Yates corrected χ 2. Not significantly different. **P <0.05. Fresh cycles Thawing cycles No. of cycles Thawed oocytes 1087 Survived oocytes 760 Survival rate (%) 69. Inseminated/injected oocytes Inseminated/injected oocytes/ 8.1 ± ± 2.1 patients (mean ± SD) Fertilized oocytes Fertilization rate (%)* Cleaved embryos Cleavage rate (%)* Normal cleaved embryos Normal cleavage rate (%)* Cryopreserved embryos Transferred embryos No pregnancy Pregnancies Abortion rate (%)* Suspended cycles 17 4 Non-fertilization 3 6 Transfers suspended 20 4 Transfers Performed Pregnancy rate per cycle (%)** Pregnancy rate per transfer (%)** Implantation rate (%) ** (4.3 ± 2.1 per patient). A total of 464 (66.6%) fertilized oocytes and 413 (89.0%) cleaved embryos were obtained. Of these embryos, 368 (89.1%) were considered eligible for embryo transfer or cryopreservation; for this reason, 331 were transferred, 37 cryopreserved and 45 anomalous embryos (grade 5 embryos or arrested division) were discarded. No statistically significant differences were found in the fertilization rate and cleavage rate between fresh and thawed oocytes (65.7 versus 67.5% and 97.6 versus 89.0%, respectively). Comparing the rate of abnormal cleavage or poor quality embryos between fresh (1427 out of 1590) and thawed cycles (368 out of 413), no statistically significant difference was found. A total of 145 transfers were performed and a mean of 2.2 embryos per patient were transferred (range 1 3). Eighteen (12.4%) pregnancies were obtained and the implantation rate was 5.7% (19 out of 331). Twelve patients delivered 13 healthy children, and six first trimester abortions were observed (33.3%). No significant difference was found (33.3 versus 19%) in the abortion rate between fresh and post-thawing pregnancies. The mean gestational age at delivery was 37.1 weeks and the mean weight was 2807 g. The karyotypes of the nine female and four male children and their 6-month follow-up examinations were normal. In the OSO cycles (94), 678 oocytes were thawed and 467 survived (68.9%), 418 were injected (4.5 ± 2.2 per patient), 286 (68.4%) were fertilized and 259 (90.6%) were cleaved. Among these, 199 were transferred, 28 cryopreserved and 32 discarded. No statistically significant difference was found in the fertilization rate between fresh and post-cryopreservation oocytes in the OSO patients (65.7 versus 68.4%). Eighty-six transfers were performed and 12 pregnancies were obtained (14.0% per transfer). The implantation rate was 6.0% (12 out

4 No effect of oocyte cryopreservation on fertilization rate of 199). In the non-oso cycles (65), 409 oocytes were thawed and 293 survived (71.6%), 266 were injected (4.3 ± 1.9 per patient), 178 (66.9%) were fertilized and 154 (86.5%) were cleaved. Among these, 132 were transferred, nine cryopreserved and 13 discarded. Fifty-nine transfers were performed and six pregnancies were obtained (10.1% per transfer). The implantation rate was 5.3% (seven out of 132). No statistically significant difference was found between OSO and non-oso cycles in the thawed cycles. In 12 patients, 37 embryos were frozen after oocyte cryopreservation. Twenty-eight embryos survived after thawing and were transferred. One pregnancy was obtained with the delivery of a normal baby (Levi Setti et al., 2005a). An implantation rate of 3.5% was obtained with a pregnancy rate of 8.3%. Discussion The development of efficient methods of oocyte cryopreservation would bring about a major breakthrough in human IVF. In fact, egg storage has the potential to circumvent several ethical and legal problems associated with embryo freezing, as well as to preserve female fertility in patients at risk of premature ovarian failure, or in women who intend to postpone motherhood for various reasons. More recently, the application of ICSI has coincided with the achievement of several pregnancies (Porcu et al., 1997, 2000; Borini et al., 1998; Yang et al., 2002; Levi Setti et al., 2004). These studies were conducted using a slow cooling/rapid thawing method based on propanediol and sucrose as cryoprotectants, according to criteria originally optimized for embryo freezing (Lassalle et al., 1985). These protocols have proved to be suboptimal in terms of survival rate (35 55%). Our results on a large sample of patients showed, however, a high survival rate (69.9%) with 0.3 mol/l sucrose. Replacement of sodium with choline in the cryoprotectant solution appears to protect mouse (Stachecki et al., 1998) and human (Quintans et al., 2002) oocytes frozen with slow cooling, by preventing damage caused by increased salt concentration during the liquid to solid phase transition. This modification has been tested clinically (Boldt et al., 2003), producing five live births and four pregnancies in 11 women. However, from this evidence, it is not clear whether improved survival results were derived from replacement of sodium with choline in the freezing solution, higher seeding temperature ( 6 C) or increased sucrose concentration (0.5 mol/l) in the thawing solution. An improvement in survival has also been associated with the application of higher subzero temperatures at which ice formation (ice seeding) is promoted. Temperatures of 6 to 4.5ºC instead of 8ºC are able to improve survival rates of immature human oocytes and those that failed to fertilize (Trad et al., 1998), but it remains unclear to what extent these oocytes offer a reliable experimental model for fully mature fresh oocytes. The intra-oocyte injection of a sugar (trehalose), which has the ability to protect cell membranes from cooling, has been described to improve survival without compromising normal fertilization and development to term in a murine model (Eroglu et al., 2003). Post-thaw survival is no guarantee of unaltered developmental ability, and sperm microinjection may resolve fertilization failure originating from zona hardening, but poor fertilization may be caused by a myriad of other types of cell injury due to freezing. In association with reduced survival, a suboptimal fertilization rate (45%) has often been observed (Borini et al., 2004) with a protocol of 0.1 mol/l sucrose. In our hands, according to a 0.3 mol/ l sucrose protocol, we obtained a high fertilization rate not different from that observed with fresh oocytes. On the other hand, protocols whose use coincides with improved survival rates (Yang et al., 2002; Fosas et al., 2003; Yoon et al., 2003) also appear to ensure high fertilization rates (70 80%), suggesting that perhaps the incidence of survival somehow also reflects the oocyte fertilization ability. Despite the fact that with some protocols survival and fertilization rates are reduced, cleavage rates are not necessarily compromised (Borini et al., 2004), approaching the value normally achieved with embryos from fresh oocytes. Other reports confirm this finding, describing similar cleavage rates (Porcu et al., 2000; Yoon et al., 2000; Boldt et al., 2003). Data on the quality of embryos developed from frozen eggs are scant, but at least in one case (Borini et al., 2004) oocyte freezing did not appear to compromise early cleavage, with 97% of day 2 embryos being considered suitable for transfer. In most cases, pregnancies have been achieved with the application of slow freezing protocols. As previously discussed in the case of slow freezing, data need to be collected on a larger scale before drawing unambiguous conclusions. In terms of clinical outcome, in the study of Borini et al. (2004), pregnancy (per cycle) and implantation rates were 22 and 16%, respectively. Similar pregnancy rates were described by Porcu et al. (2000) and Yoon et al. (2000) (18 and 21%, respectively), although in the former study the implantation rate was not reported, while in the latter this value was relatively low (6.4%). Yang et al. (2002) were able to achieve extraordinarily high pregnancy and implantation rates (45 and 25%, respectively). However, the oocytes frozen in this study were presumably of particularly good quality, since they were derived from selected donors. Although a high survival rate and fertilization rate were obtained, our results show a lower pregnancy rate. Our implantation rate (5.7%) after thawing is impressively lower than our implantation rate after the transfer of fresh embryos (23.2%). In the light of these data, it appears rather obvious that comprehensive evaluation of the clinical efficiency of diverse protocols is unattainable at present, due to the fact that patient populations in the various reports differ substantially. In the final analysis, evaluation of the efficiency of different protocols will require the comparison of implantation rates derived from the treatment of homogeneous groups of patients and estimated on the basis of the number of frozen thawed oocytes. Perhaps more importantly, the possibility that the oocyte can become a valid option for preservation of fertility is inevitably dependent on the development of methods able to give success rates similar to embryo freezing. In this respect, despite the lack of controlled studies, it has been suggested that the efficiency of egg freezing is 50% compared with that guaranteed by cryopreserved embryos (Borini et al., 2004). This conclusion has not been supported by the ESHRE European Register. In 2001, after embryo cryopreservation, the pregnancy rate for transfer in Italy in the 2416 reported cycles 373

5 P.E.Levi Setti et al. was 11.7% for thawing and 14.4% for transfer, not very different from the pregnancy rate (12.4%) obtained in our oocyte freezing programme (ESHRE, 2005). The safety of oocyte freezing has always been a cause for major concern, especially in relation to the susceptibility of the meiotic spindle to low temperatures. Pregnancy losses are sometimes reported, but appear insufficient in terms of sample size (Quintans et al., 2002), while it is not known whether these failures were caused by either chromosomal anomalies or other factors. Our data show a very high abortion rate (33.3%), despite not being statistically different from the abortion rate observed with fresh cycles (19%). This result probably indicates that even with a high survival and fertilization rate, a reduced number of embryos are really competent, leading to lower implantation and higher abortion rates. These data are confirmed by the very low implantation rate obtained with embryos frozen after oocyte thawing. Moreover, irrespective of its overall efficiency, slow freezing does not affect the rate of aneuploidy in human oocytes (VanBlerkom et al., 1994). This is also inferred by the evidence that comparable aneuploidy frequencies were observed in embryos obtained from fresh or frozen oocytes (28 and 26%, respectively), by performing a fluorescence in situ hybridization analysis and employing specific probes for chromosomes 13, 18, 21, X and Y (Cobo et al., 2001). In the absence of comprehensive data on clinical efficiency and safety, oocyte cryopreservation at the moment cannot be considered a routine form of treatment. Investigation of potential adverse effects of cryopreservation on oocyte viability remains a priority and should not be restricted to the cytoskeletal apparatus and associated chromosomes, but rather expanded to other cellular attributes, such as mitochondrial and calcium stores. Multicentre studies should also be conducted to facilitate the collection of clinical data and test the reproducibility of alternative methods. Despite these uncertainties, it appears more than likely that oocyte cryopreservation will soon become a valid option for IVF patients. The establishment of oocyte banks could improve the safety of fertility treatments for women using oocyte donors by allowing improved screening of donors for potential transmittable diseases. Finally, patients who find gamete cryopreservation more acceptable than embryo cryopreservation could cryopreserve their oocytes, reducing the number of supernumerary embryos generated (Karow, 1997; Levi Setti et al., 2004). Oocyte cryopreservation is an option for this group of patients, but we strongly agree that it should still be considered an experimental technique (ASRM Practice Committee, 2004) and not offered as a means to defer reproductive ageing. We report one of the largest experiences in oocyte cryopreservation in a group of good prognosis patients, producing large numbers of gametes, in which we could use many thawed oocytes, select the best embryos to transfer and even freeze embryos for future attempts. A possible bias of our study is probably that the high pregnancy rate in this group of patients does not allow us to present the cumulative pregnancy rate after fresh, embryo and oocyte cryopreserved embryo transfer. This was not the aim of the present study, but we hope to be able to report these data in the near future. 374 These results were obtained before the passing of the Italian law which allows the fertilization of only three fresh or frozen eggs, and time is needed to assess the real effects of this restrictive policy on pregnancy and implantation rates. Although not originally the aim of this study, our results show that, even using a limited number of oocytes, the same pregnancy rate could be obtained (non-oso patients) in fresh and post-oocyte thawing cycles, compared with patients in whom a large number of oocytes are injected. All our efforts now need to move from embryo to oocyte quality in order to maintain good results and reduce the need for reproductive tourism that is exploding in Italy. More attention should be paid to the general results of assisted reproduction, when embryo cryopreservation in Europe led in 2001 to a 16.6% pregnancy rate per transfer (ESHRE, 2004) and to the effects of the restrictive Italian law, because it remains unethical even if, in selected centres, the clinical pregnancy rate is confirmed as not significantly different (Levi Setti et al., 2005b; Ragni et al., 2005). Acknowledgements We wish to thank Professor Giorgio Pardi for his support in performing this investigation and all the Medical and Biological staff of the IRCCS Istituto Clinico Humanitas who contributed to the results of this study. The authors wish also to thank Ms Rosalind Roberts for revising the manuscript. These data were presented in part in Berlin at the ESHRE Annual Meeting in June References ASRM Practice Committee (2004) Ovarian tissue and oocyte cyropreservation. Fertis Steril 82, Al-Hasani S, Diedrich K, Van der Ven H, Reineke A, Hartje M and Krebs D (1987) Cryopreservation of human oocytes. Hum Reprod 2, ESHRE (2005) Assisted reproductive technology in Europe, Results generated from European Registers by ESHRE. Hum Reprod 20, Boldt J, Cline D and McLaughlin D (2003) Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles. Hum Reprod 18, Borini A, Bafaro MG, Bonu MA, Di Stratis V, Serini E, Sciaino R and Serrao L (1998) Pregnancies after oocyte freezing and thawing: preliminary data. Hum Reprod 13 (Abstract Book 1, ). Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M and Flamigni C (2004) Pregnancies and births after oocyte cryopreservation. Fertil Steril 82, Chen C (1986) Pregnancy after human oocyte cryopreservation. Lancet 1, Chen ZJ, Li M, Li Y, Zhao LX et al. (2004) Effects of sucrose concentration on the development potential of human frozen thawed oocytes at different stages of maturity. Hum Reprod 19, Cobo A, Rubio C, Gerli S, Ruiz A, Pelliceri A and Remohi J (2001) Use of fluorescence in situ hybridization to assess the chromosomal status of embryos obtained from cryopreserved oocytes. Fertil Steril 75, Eroglu A, Lawitts JA, Toner M and Toth TL (2003)Quantitative microinjection of trehalose into mouse oocytes and zygotes, and its effect on development. Cryobiology 46, Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S and Flamigni C (2001) Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 16, Fosas N, Marina F, Torres PJ, Jove I, Martin P, Perez N, Arnedo N and Marina S (2003) The births of five Spanish babies from cryopreserved donated oocytes. Hum Reprod 18, Gook, DA, Osborn, SM, Bourne H and Johnston WI (1994) Fertilization of human oocytes following cryopreservation: normal karyotypes and absence of stray chromosomes. Hum Reprod 9, Gook DA, Schiewe MC, Osborn SM, Asch RH, Jansen RPS and Johnston WIH (1995) Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol. Hum Reprod 10, Karow AM (1997) Implications of tissue banking for human reproductive medicine. In Karow AM and Critser JK (eds), Reproductive Tissue Banking, Scientific Principles. Academic Press, San Diego, pp

6 No effect of oocyte cryopreservation on fertilization rate Kuleshova L, Gianaroli L, Magli C, Ferraretti A and Trounson A (1999) Birth following vitrification of a small number of human oocytes: case report. Hum Reprod 14, Lassalle B, Testart J and Renard JP (1985) Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril 44, Levi Setti PE, Albani E, Morreale G, Bianchi S, Novara P, Cesana A, Parini V, Marras A, Zannoni E, Baggiani AM, Colombo GV and Alagna F (2004) Supernumerary oocyte cryopreservation in 303 IVF/ICSI cycles. Hum Reprod 19 Suppl 1,i Levi Setti PE, Albani E, Novara PV, Cesana A, Bianchi S and Negri L (2005a) Normal birth after transfer of cryopreserved human embryos generated by microinjection of cryopreserved testicular spermatozoa into cryopreserved human embryos. Fertil Steril 83, e9 e10. Levi Setti PE, Albani E, Novara PV, Morreale G, Marras A and Parini V (2005b) Results of IVF/ICSI cycles after the new Italian law. Hum Reprod 20,i65. Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O and Flamigni C (1997) Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 68, Porcu E, Fabbri R, Damiano G, Giunchi S, Fratto R, Ciotti PM, Venturoli S and Flamigni C (2000) Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 169, Quintans CJ, Donaldson MJ, Bertolino MV and Pasqualini RS (2002) Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium. Hum Reprod 17, Ragni G, Alegra A, Anserini P, Causio F, Ferraretti AP, Greco E, Palermo R and Somigliana E (2005) The 2004 Italian legislation regulating assisted reproduction technology: a multicentre survey on the results of IVF cycles. Hum Reprod 20, Stachecki JJ, Cohen J and Willadsen SM (1998) Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology 37, Trad FS, Toner M and Biggers JD (1998) Effects of cryoprotectants and iceseeding temperature on intracellular freezing and survival of human oocytes. Hum Reprod 14, Van Uem JFHM, Siebzehrubl ER, Schuh B, Koch R, Trotnow S and Lang N (1987) Birth after cryopreservation of unfertilized oocytes. Lancet i, VanBlerkom J and Davis PW (1994) Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Tech 27, Veeck LL (1999) Preembryo grading and degree of cytoplasmic fragmentation. In Veeck LL (ed) An Atlas of Human Gametes and Conceptus. Parthenon, New York, pp Yang D, Winslow KL, Blohm PL, Brown SE, Nguyen K and Brubaker C (2002) Oocyte donation using cryopreserved donor oocytes. Fertil Steril 78 (Suppl.), S14. Yoon TK, Chung HM, Lim JM, Han SY, Ko JJ and Cha KY (2000) Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program [letter]. Fertil Steril 74, Yoon TK, Kim TJ, Park SE, Hong SW, Ko JJ, Chung HM and Cha KY (2003) Live births after vitrification of oocytes in a stimulated in vitro fertilizationembryo transfer program. Fertil Steril 79, Submitted on June 9, 2005; resubmitted on September 12, 2005; accepted on September 20,