RBMOnline - Vol 14. No Reproductive BioMedicine Online; on web 14 November 2006

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1 RBMOnline - Vol 14. No Reproductive BioMedicine Online; on web 14 November 2006 Novel protocols have increased survival and fertilization rates of cryopreserved oocytes. Nevertheless, in most cases clinical experiences have been disappointing or contradictory. Human oocytes of 141 patients were cryopreserved using a modified slow-cooling protocol involving 1.5 mol/l propane-1,2-diol (PrOH) and 0.2 mol/l sucrose during dehydration, while rehydration was conducted applying decreasing concentrations of PrOH and 0.3 mol/l sucrose. One thousand and eighty-three oocytes were frozen and 403 were thawed, with a survival rate of 75.9%. Among the 306 surviving oocytes, 252 were microinjected and 192 (76.2%) showed two pronuclei. One hundred and eighty zygotes (93.8%) cleaved. The proportion of good quality embryos (grade I and II) was 86.2%. All embryos were transferred and 17 clinical pregnancies were obtained. Pregnancy rates were 21.3% per transfer, 21.8% per patient, and 18.9% per thawing cycle. The implantation rate was 13.5% while the miscarriage rate was 11.8%. To date, four babies have been delivered, while the remaining pregnancies are ongoing. Increased oocyte survival rates can be achieved by moderately high sucrose concentrations in the freezing and thawing solutions. This also ensures elevated success rates in terms of fertilization, embryo development and clinical outcome. Keywords: cryopreservation, fertility preservation, oocyte, pregnancy rate, slow cooling, sucrose concentration Oocyte cryopreservation could make possible the perspective of preserving fertility in women who intend to postpone pregnancy to later in life or in those at risk of prematurely losing their gonadal function. It could also represent a valid alternative to embryo cryopreservation, which often raises ethical and religious concerns, as well as problems related to legal ownership (Gook and Edgar, 1999). Furthermore, restrictive legislations may impose a need for oocyte cryopreservation. In particular, the Italian IVF law prescribes that only a maximum of three oocytes may be inseminated at one time. Therefore, it would be advantageous to freeze for later use supernumerary oocytes obtained from a single stimulation cycle, in order to spare patients additional stimulations (Borini et al., 2006a). The first pregnancies resulting from frozen oocytes were obtained about 20 years ago (Chen, 1986; Al-Hasani et al., 1987), but such attempts remained sporadic. Oocyte cryopreservation was proposed again after about a decade, with the publication of studies (Porcu et al., 1997; Borini et al., 1998; Tucker et al., 1998) performed using the original slow-cooling technique developed for embryo freezing (Lassalle et al., 1985). These initial results showed that conventional cryopreservation techniques were not able to guarantee the successful storage of oocytes, especially as a consequence of low post-thaw survival rates Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 It is believed that the main biophysical factor limiting human oocyte survival following slow freezing is the low surface-tovolume ratio of this cell. Such a condition impedes adequate cryoprotectant (CPA)-mediated dehydration, with the consequent formation of intracellular ice crystals, a factor believed to be a major source of cell injury. During freezing procedures, cell dehydration may be modulated by CPA concentration and exposure time, as well as freezing and thawing rates (Jackowski et al., 1980; Leibo, 1980). The original slow-cooling protocols included exposure first to 1.5 mol/l of an intracellular CPA, propane-1,2-diol (PrOH) or dimethyl sulphoxide, and subsequently to a mixture also containing 0.1 mol/l sucrose, an extracellular CPA. Following the disappointing outcome of the initial attempts at cryopreserving oocytes, many modifications in the slow freezing protocol have been proposed. Such changes have included increasing the concentration of sucrose in the freezing solution (Fabbri et al., 2001), replacing sodium with choline in the freezing medium (Stachecki et al., 1998, 2004; Quintans et al., 2002) or microinjecting intracellular trehalose (Eroglu et al., 2002; Wright et al., 2004). However, despite the growing number of pregnancies obtained with such alternative protocols (Porcu et al., 2000; Boldt et al., 2003; Fosas et al., 2003; Chen et al., 2005; Borini et al., 2006b; Levi Setti et al., 2006), implantation rates have remained low or difficult to reproduce. Nevertheless, oocyte freezing is emerging as a valid alternative procedure for the preservation of reproductive potential, although it still appears unable to compete with embryo freezing. Recently, it has been suggested that oocyte storage may be successfully achieved with vitrification. This methodology has been reported to ensure very high survival rates (90 95%) (Katayamaa et al., 2003; Kuwayama a et al., 2005; Lucena et al., 2006), although its clinical validation necessitates confirmation through more extensive studies. To date, one of the most successful modifications in the original slow-cooling protocol has been the increase in sucrose concentration in the freezing solution, from 0.1 to mol/l, with the aim of achieving a higher oocyte dehydration (Fabbri et al., 2001). Protocols involving high sucrose concentration have been applied with success in terms of survival, fertilization and cleavage rate, while having generated conflicting clinical outcomes (Porcu et al., 2000; Boldt et al., 2003; Fosas et al., 2003; Chen et al., 2005; Borini et al., 2006b; Levi-Setti et al., 2006). The aim of the present work was to test whether a modified version of the conventional slow-cooling protocol could result in high implantation rate. Elevated survival and fertilization rates were recently reported with another protocol (Fabbri et al., 2001), but experience suggests that this was not sufficient for achieving an improved clinical outcome (Borini et al., 2006b). Cryopreservation mixtures and exposure times were established with the objective of minimizing osmotic stress and increasing standardization. Oocytes were frozen in a solution containing 0.2 mol/l sucrose as an extracellular CPA, while rehydration after thawing was in solutions containing a higher (0.3 mol/l) concentration of the same CPA. The increase in the sucrose concentration in the freezing solutions was aimed at augmenting the dehydration rate, while the maintenance of a higher sucrose concentration in the thawing solutions was adopted to achieve a more controlled rehydration. The hypothesis behind this strategy was that by improving dehydration and rehydration conditions, while maintaining the sucrose concentration difference in the freezing and thawing solutions, it is possible to achieve a better preservation of oocyte viability. These measures had been already attempted successfully by Jericho et al. (2003) for the cryopreservation of cleavage-stage biopsied embryos, which are especially sensitive to freezing. However, on the basis of previous work on the osmotic response to CPA exposure (Paynter et al., 2005) this protocol was adapted in an attempt to establish, at least during dehydration, concentration and exposure time to the extracellular CPA specifically suited to the unfertilized oocyte. Institutional approval for the present study was obtained by the Institutional Review Board (IRB). From April 2004, date of introduction of the Italian IVF law, to December 2005, 141 patients (mean age ± SD, 35.4 ± 4.3 years) requiring an IVF treatment were included in the study by offering them the opportunity to have their surplus oocytes cryopreserved, as the insemination of more than three gametes at one time is prohibited by the new legislation. Only oocytes with an extruded polar body and presumably at the metaphase II stage were cryopreserved. Ovarian hormonal stimulation, oocyte collection, cumulus cell removal (carried out about 2 h following retrieval), culture conditions and time of microinjection were described previously (Dal Prato et al., 2001; Borini et al., 2006b). The fresh treatments data are relevant to a cohort of patients comparable to the frozen oocyte group by age, indications for treatment and ovarian response ( 10 retrieved oocytes). All the freezing solutions were prepared using Dulbecco s phosphate-buffered saline (PBS) solution (Gibco, Life Technologies Ltd, Paisley, Scotland) and plasma protein supplement (PPS) (Baxter AG, Vienna, Austria). The equilibration solution contained 1.5 mol/l PrOH + 20% PPS in PBS and the loading solution consisted of 1.5 mol/l PrOH mol/l sucrose + 20% PPS in PBS, as described by Porcu et al. (1997). The thawing solutions contained a gradually decreasing concentration of PrOH and a constant 0.3 mol/l sucrose concentration. They were prepared as follows: (i) 1.0 mol/l PrOH mol/l sucrose + 20% PPS; (ii) 0.5 mol/l PrOH mol/l sucrose + 20% PPS; (iii) 0.3 mol/l sucrose + 20% PPS. Cumulus-free oocytes were incubated in the equilibration solution for 10 min at room temperature (RT) and then transferred to the loading solution for 5 min. The oocytes were finally loaded in plastic straws (Paillettes Crystal 133 mm; Cryo Bio System, France) individually or in small groups (maximum three oocytes per straw). The temperature was lowered through an automated Kryo 10 series III biological freezer (Planer Kryo 10/1,7 GB) from 20 C to 8 C at a rate of 2 C/min. Manual seeding was performed at 8 C and this temperature was maintained for 10 min in order to allow uniform ice propagation. The temperature was then decreased to 30 C at a rate of 0.3 C/min and finally rapidly brought to 150 C at a rate of 50 C/min. The straws were then directly plunged into liquid nitrogen at 196 C and stored for later use.

3 The thawing procedure was carried out at RT. Plastic straws were held in air at RT for 30 s and then transferred into a water bath at 30 C for 40 s. Stepwise CPA dilution was performed by transferring the oocytes in thawing solution (i) for 5 min, then in solution (ii) for additional 5 min and finally in solution (iii) for 10 min before final dilution in PBS + 20% PPS for 20 min (10 min at RT and 10 min at 37 C). Oocytes were immediately checked for survival evaluating zona pellucida integrity, cytoplasmic clearness, absence of shrinkage or swelling, and then transferred to a 20 μl drop of cleavage medium (Cook IVF, Brisbane, Australia) under warm mineral oil (Cook IVF) at 37 C in an atmosphere of 5% CO2. A further check for confirming survival was made 3 h later, just before intracytoplasmic sperm injection (ICSI) according to previous work (Borini et al., 2006b). In adherence with the Italian IVF law that in practice prohibits the injection of more than three oocytes during each IVF attempt, only a proportion (n = 252, 82.4%) of the 306 survived oocytes were employed. In a few cases, it was possible to select the oocytes to be destined for microinjection on the basis of minor cytoplasmic characteristics, but more generally the oocytes that were not used for treatment were indistinguishable from those that were microinjected. Sixteen to 18 h after microinjection, normal fertilization was checked by the presence of two pronuclei and two polar bodies. All the fertilized oocytes were transferred into fresh cleavage medium and cultured until transfer. On day 2, at h post-insemination, embryos were evaluated for cell number and rate of fragmentation and consequently graded as I IV (best worst) (Borini et al., 2006b). Embryo transfers were all performed on day 2. Endometrial development was supported using micronized 17β-oestradiol in patches (Esclima; Schering, Milan, Italy) with increasing dosage, μg. The length of administration varied from 10 to 18 days, depending on the patient (Borini et al., 1996). Progesterone supplementation either as injections of 100 mg in oil (Prontogest; Amsa, Rome, Italy) or 180 mg micronized doses in gel (Crinone 8; Serono, Rome, Italy) via vaginal route was started on the day of oocyte thawing (day 0). Embryo transfers were performed on day 3 of progesterone administration (day 2). Endometrial thickness was checked on day 0 and the cycle suspended if the line was thinner than 8 mm or thicker than 12 mm. In cases of pregnancy, endometrial support treatment was continued for 60 days after transfer. Clinical pregnancy was defined as the presence of a gestational sac at ultrasound examination. One thousand and eighty-three oocytes from 141 patients were frozen. To date, 90 thawing cycles involving 78 patients have been carried out, for a total of 403 supernumerary oocytes thawed. Three hundred and six oocytes were recovered as viable after thawing, with a survival rate of 75.9% estimated immediately after thawing. No change in survival rate was observed 3 h after thawing. During each thawing cycle of a given patient, only a fraction of the oocytes, which had been previously frozen individually or in groups of two to three, were recovered from storage. This was aimed at obtaining in as many cases as possible three viable oocytes suitable for microinjection to comply with the already mentioned Italian law, while maximizing the overall chances of achieving a pregnancy in each cycle of stimulation. Two hundred and fifty-two oocytes were microinjected and 192 of them fertilized normally, with a rate of 76.2%. Abnormal fertilization (one or three pronuclei) was observed in 16 cases (6.3%), while 11 oocytes (4.4%) degenerated after ICSI. Embryo evaluation was performed shortly before transfer, showing that 180 of the 192 zygotes had cleaved, with a rate of 93.8%. Embryo grading was assessed taking into account both cell division and rate of fragmentation as described previously (Borini et al., 2005). Embryos at the 2-, 3- and 4-cell stage were 65 (36.1%), 25 (13.9%) and 76 (42.2%) respectively. The remaining 14 embryos showed more than four cells (7.8%). The large majority of embryos displayed a degree of fragmentation classified as grade I or II (i.e. less than 25% of fragments). The details are illustrated in Table 1. Eighty of the 90 thawing cycles progressed to the embryo transfer stage. Failure to achieve embryos suitable for transfer involved mainly patients with very few frozen oocytes and was due to complete degeneration of thawed oocytes (one cycle), or failure of survived oocytes to fertilize (seven cycles) or cleave (two cycles). Seventeen pregnancies were confirmed by ultrasound assessment, with 24 gestational sacs and 19 fetal heart beats (FHB). The pregnancy rates were 21.3, 18.9 and 21.8% per embryo transfer, thawing cycle, and patient, respectively, and the implantation rate was 13.5%. The multiple pregnancy rate was 29.4% (5/17), with three twin and two triplet gestations. To date, two patients miscarried with a miscarriage rate of 11.8% (Table 2). Four healthy babies have been delivered, while the remaining pregnancies have so far progressed beyond weeks 7 (n = 3), 17 (n = 3), 27 (n = 2), and 39 (n = 3) of gestation. Because it is well established that patient age has an impact on success rate, the results were also analysed by taking into consideration only those obtained from patients of relatively younger age ( 38 years). In such a group (Table 3) (n = 62) the mean age was 33.7 ± 3.4 (mean ± SD), 942 oocytes were cryopreserved and 325 thawed. Two hundred and forty-four oocytes were successfully thawed, with a survival rate of 75.1%. Two hundred and three oocytes were microinjected and 157 (77.3%) of them showed two normal pronuclei h after ICSI. The rate of abnormal fertilization (one or three pronuclei) was 7.4%, which does not differ from previous results obtained using a previously tested protocol (Borini et al., 2006b). Only seven oocytes (3.4%) showed signs of degeneration after microinjection. A total of 146 normally fertilized oocytes underwent one or more cell divisions, with a cleavage rate of 93.0%. Most of the embryos were classified as grade I (73/146 with a rate of 50.0%) and grade II (53/146, 36.3%). In this group of younger patients, 72 thawing cycles were performed, with 144 embryos transferred in 65 transfers. Pregnancy rates were 26.2, 23.6 and 27.4%, per transfer, thawing cycle, and patient respectively. The implantation rate was 16.7% (Table 4). During the same period, the results achieved in 139 patients undergoing fresh transfers are reported in Table 5. This group was similar to the frozen oocyte treatments, by age, type of infertility and number of retrieved oocytes ( 10).

4 Table 1. Survival, fertilization and cleavage of oocytes frozen using a modified oocyte cryopreservation protocol based on the use of 0.2 and 0.3 mol/l sucrose in the freezing and thawing solutions respectively. No. of patients 141 No. of patients undergoing 78 at least one thawing cycle No. of thawing cycles 90 No. of oocytes Frozen 1083 Thawed 403 Survived (%) 306 (75.9) Microinjected 252 Fertilization 2PN (%) 192 (76.2) 1PN/3PN (%) 16 (6.3) Degenerated (%) 11 (4.4) No. of embryos Cleaved (%) 180 (93.8) G1 (%) 83 (46.2) G2 (%) 72 (40.0) G3 (%) 12 (6.6) G4 (%) 13 (7.2) 2-cell (%) 65 (36.1) 3-cell (%) 25 (13.9) 4-cell (%) 76 (42.2) >4-cell (%) 14 (7.8) Table 2. Clinical outcome of patients after transfer of embryos derived from frozen oocytes. No. of patients undergoing 78 at least one thawing cycle No. of thawing cycles 90 No. of embryo transfers 80 No. of embryos transferred 178 No. pregnancies 17 Pregnancy rate/embryo transfer % 21.3 (17/80) Pregnancy rate/thawing cycle % 18.9 (17/90) Pregnancy rate/patient % 21.8 (17/78) Multiple pregnancy rate % 29.4 (5/17) No. of gestational sacs 24 No. of FHB 19 Implantation rate % 13.5 (24/178) Abortion rate % 11.8 (2/17) Values in parentheses are numbers. FHB = fetal heart beat. PN = pronucleate. Table 3. Survival, fertilization and cleavage of frozen oocytes from patients 38 years using the modified cryopreservation protocol. No. of patients 118 No. of patients undergoing 62 at least one thawing cycle No. of thawing cycles 72 No. of oocytes Frozen 942 Thawed 325 Survived (%) 244 (75.1) Microinjected 203 Fertilization 2PN (%) 157 (77.3) 1PN/3PN (%) 15 (7.4) Degenerated (%) 7 (3.4) No. of embryos Cleaved (%) 146 (93.0) G1 (%) 73 (50.0) G2 (%) 53 (36.3) G3 (%) 11 (7.5) G4 (%) 9 (6.2) 2-cell (%) 54 (37.0) 3-cell (%) 20 (13.7) 4-cell (%) 63 (43.2) >4-cell (%) 9 (6.2) Table 4. Clinical outcome of patients 38 years after transfer of embryos derived from frozen oocytes. No. of patients undergoing 62 at least one thawing cycle No. of thawing cycles 72 No. of embryo transfers 65 No. of embryos transferred 144 No. of pregnancies 17 Pregnancy rate/embryo transfer % 26.2 (17/65) Pregnancy rate/thawing cycle % 23.6 (17/72) Pregnancy rate/patient % 27.4 (17/62) Multiple pregnancy rate % 29.4 (5/17) No. of gestational sacs 24 No. of FHB 19 Implantation rate % 16.7 (24/144) Abortion rate % 11.8 (2/17) Values in parentheses are numbers. FHB = fetal heart beat.

5 Table 5. Clinical outcome of patients after transfer of embryos derived from fresh treatments with mean age and number of retrieved oocytes ( 10) comparable to the frozen oocyte cycles. Age Overall 38 years No. of patients (fresh cycles) Age (years, mean ± SD) 34.9 ± ± 3.4 Retrieved oocytes Inseminated oocytes Fertilized oocytes (%) 310 (79.7) 250 (79.6) Embryos (%) 302 (97.4) 244 (97.6) Transferred embryos No. of transfers Pregnancies FHB Pregnancy rate per ET 22.7 (29/128) 27.2 (28/103) Implantation rate 14.2 (41/289) 17.3 (40/231) Abortion rate 10.3 (3/29) 10.7 (3/28) Values in parentheses are numbers unless otherwise stated. ET = embryo transfer; FHB = fetal heart beat. In particular, in 112 patients of younger age (33.4 ± 3.4, mean ± SD), fertilization, cleavage and implantation rates were 79.6, 97.6 and 17.3% respectively. This gave raise to a pregnancy rate per transfer of 27.2%. Oocyte cryopreservation has undoubtedly been improved in terms of survival rate (Fabbri et al., 2001; Boldt et al., 2003), a progress that in the last few years has resulted in several pregnancies. It is still debated, however, whether high oocyte survival rate at thawing is necessarily associated to a high clinical outcome obtained after fertilization and embryo transfer. This uncertainty has prevented oocyte freezing from being accepted as a well-established IVF procedure. The difficulty in cryopreserving the human oocyte originates from its unique characteristics. The particular cryo-sensitivity of this cell makes unfeasible the application of standard protocols of cryopreservation. For this reason, several factors that affect freezing, such as cooling rate, seeding, role of CPA and permeability kinetics, need thorough investigation in order to develop improved protocols (Fuller and Paynter, 2004). Empiricism has in some cases generated interesting results, but it appears obvious that a rational approach to oocyte cryopreservation could give a more fundamental contribution to the development of improved protocols. Theoretical models have been developed to explain and, to some extent, predict optimum cooling rates (Leibo et al., 1974, 1978; Mazur, 1984). The principal limiting factor in slow cooling is probably the flow of water and intracellular CPA through the cell membrane, from which dehydration and, as a consequence, prevention of intracellular ice formation depends. More specifically, dehydration and rehydration are influenced by: (i) the composition and the permeability characteristics of the cell membrane; (ii) the surface-to-volume ratio of the cell; (iii) the temperature; (iv) the difference in osmotic pressure between the intra-cellular and extracellular environment. These factors must be taken into account when dehydrating and rehydrating cells (Ashwood-Smith, 1980; Jackowski et al., 1980; Leibo, 1980). The presence of CPA (both intracellular and extracellular) in the freezing solutions minimizes cell damage during the freezing and thawing processes. In standard oocyte cryopreservation procedures, intracellular CPA concentrations are usually applied at around 1.5 mol/l, a concentration several times higher than that of any other component in the medium. Intracellular CPA enters the cell, replacing the intracellular water drawn out by the osmotic gradient (Hunter et al., 1992; Shaw et al., 2000). The protective action of extracellular agents, including monosaccharides, disaccharides and trisaccharides was reported by Friedler (1988). These agents do not enter the cell but exert their beneficial effects by causing further cellular dehydration by establishing an osmotic gradient. However, exposure to CPA may be source of osmotic stress and/or chemical toxicity. Measurements of cell volume changes during CPA exposure have the potential to provide crucial information to minimize osmotic stress, from which oocyte viability may depend (Paynter, 2005). The low oocyte surfaceto-volume ratio certainly plays a negative role in the exchange of water and CPA between the extra-cellular and intracellular compartments, enhancing osmotic stress. Interestingly, the traditional slow-cooling protocol (Lassalle and Testart, 1988) that is unable to ensure high oocyte survival rates (Borini et al., 2004), on the contrary produces high survival rates with pronuclear stage eggs (Senn et al., 2000), whose surfaceto-volume ratio is essentially identical in comparison to unfertilized mature oocytes. This apparent paradox may be explained by the fact that, in rats, various types of aquaporins, proteins that regulate the flux of water and polar molecules through the plasmalemma, are differentially expressed during

6 oocyte maturation and early development (Edashige et al., 2000; Ford et al., 2000). On the basis of previous work (Paynter et al., 2005), it was aimed to test a modified cryopreservation protocol involving a freezing solution containing 1.5 mol/l PrOH and 0.2 mol/l sucrose in order to better control the impact of shrinkage, known to be a possible cause of cell injury, during dehydration caused by the extracellular CPA. It was observed, in fact, that after around 3 min of exposure to 1.5 mol/l PrOH in the presence of 0.3 mol/l sucrose, oocyte volume shrinks drastically exceeding rapidly the 30% threshold excursion believed to be detrimental to cell viability. Upon exposure to a solution with a sucrose concentration of 0.2 mol/l, the 30% volume change is not reached until after 10 min of exposure, dehydration being achieved more gradually and presumably less traumatically. The use of 0.2 mol/l instead of 0.3 mol/l sucrose in the freezing solution has the additional advantage of providing a wider temporal window for oocyte loading within the straws while dehydration rate changes only moderately. This makes the procedure less prone to variations introduced by the time of incubation in the freezing mixture, which in the day-to-day routine is influenced by the number oocytes to be frozen. However, the sole adoption of 0.2 mol/l sucrose in the freezing solution may not be sufficient to preserve post-thaw oocyte viability, as shown by the fact that in the absence of other protocol modifications success rates remain inadequate in terms of survival, fertilization and implantation (Porcu et al., 2000; Fabbri et al., 2001). As a further measure, a higher sucrose concentration (0.3 mol/l) was employed during thawing, applying a principle previously suggested by the original work of Lassalle et al. (1985). The rationale for fine tuning differential sucrose concentrations in both freezing and thawing solutions is not a novel concept, having also been tested by Jericho et al. (2003) for the cryopreservation of biopsied cleavage stage embryos. These authors, in fact, applied 0.2 and 0.3 mol/l sucrose concentrations in freezing and thawing solutions respectively. This allowed a dramatic improvement in the survival rate and implantation ability of biopsied embryos. To date, implantation rates per thawed oocyte have been very low following slow cooling rapid thawing when compared with embryo storage. In previous studies, in effect a poor outcome was observed, irrespective of the use of either low (0.1 mol/l) (Borini et al., 2004) or high (0.3 mol/l) (Borini et al., 2006b) sucrose concentration in the freezing mixtures (2.3 and 2.6% implantation rates per thawed oocyte respectively). Recently, it has been suggested that vitrification could be a better answer to the challenge of oocyte storage. Isolated births were described by Kuleshova et al. (1999) and Katayama et al. (2003) following vitrification of a few oocytes. Yoon et al. (2003) reported the birth of several babies, but in this study the overall implantation rate per thawed oocyte (1.7%) did not exceed the one achieved with slow-cooling protocols. After vitrification and the use of cryotop as a storage device, Kyono et al. (2005) obtained a viable pregnancy by transferring a single blastocyst developed from a cryopreserved oocyte. More importantly, with a specially developed vitrification method, Kuwayama et al. (2005) vitrified 64 human oocytes, obtaining a very high survival rate (91%). Fertilization and blastocyst rates were also elevated (90 and 62% respectively). After the transfer of 29 embryos, the pregnancy rate was 43%. If confirmed, these results would mark a major breakthrough in oocyte cryopreservation. This should encourage more extensive studies on such a protocol. Recently, oocyte cryopreservation with the cryotop vitrification technique has been described to generate high survival (87.2%), fertilization (89.2%), and pregnancy rates (56%), although it should be noted that the majority of pregnancies (9/13) were obtained from oocyte donation cycles (Lucena et al., 2006). This study provides evidence that by more carefully adjusting the sucrose concentration during dehydration and rehydration, the implantation rate per thawed oocyte remarkably increases to 5.9% (24/403). In fact, in more practical terms this value approaches 7%, considering that, in compliance with the Italian IVF law, only a proportion (n = 252, 82.4%) of the 306 survived oocytes were used for treatment. Such a success rate appears even higher than the one normally achieved with embryo freezing, in which case the implantation rate per inseminated oocyte has been estimated to correspond to about 4% (Gook et al., 1999; Borini et al., 2006b). The results also suggest that, in comparison to fresh oocytes, the protocol tested in this study does not seem to affect the ability of frozen thawed oocytes to fertilize, develop and implant. It is reasonably hopeful that these preliminary data may be reproduced on a larger scale, on the basis that the most significant parameters measured in this work (survival, fertilization, and implantation) did not vary appreciably throughout the entire period of study (data not shown). From a retrospective comparison of all the major biological and clinical parameters generated by this study and previous work (Borini et al., 2004, 2006b), it appears that the protocol applied in this study gives rise to survival, fertilization and cleavage rates that are significantly improved when compared with the protocol using 0.1 mol/l sucrose, while in this respect no major differences are observed in comparison to the use of 0.3 mol/ l sucrose protocol. However, the most noticeable difference may be in terms of implantation rates, which are clearly improved with the 0.2 mol/l protocol, as discussed above. It is most likely that the 0.1 mol/l sucrose concentration is not sufficient to ensure an adequate dehydration before cooling, as consistently demonstrated by poor survival rate described in previous studies. On the other hand, it can be speculated that the higher shrinkage induced by 0.3 mol/l sucrose concentration may lead to cell injury compatible with postthaw survival and yet responsible for compromising overall viability, as suggested by the poor implantation rate (Coticchio et al., 2005). It was recently established that the disappointing performance of the 0.3 mol/l sucrose protocol is not attributable to disruption of the chromosome segregation apparatus, as the frequency of oocytes with normal spindle and chromosome configurations were very similar in fresh and frozen thawed groups (Coticchio et al., 2006). Stachecki et al. (2004) also confirmed that the spindle might be safely preserved in stored oocytes. However, it has been argued that depolymerization and repolymerization of microtubules, immediately after thawing and during rehydration, may compromize spindle function (Rienzi et al., 2005). So far, comparative clinical studies to investigate the relative importance of sucrose concentration have not been performed. However, from the work of Porcu et al. (2000) as well the authors own experiences, it seems that the 0.2 mol/l protocol described in this manuscript could represent an appropriate compromise, especially if associated with a higher concentration (0.3 mol/l) of the same CPA in the thawing solutions to prevent excessive osmotic stress. The protocol could be further improved by other changes, such as

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