Article Highly efficient vitrification method for cryopreservation of human oocytes

RBMOnline - Vol 11. No 3. 2005 300-308 Reproductive BioMedicine Online; www.rbmonline.com/article/1827 on web 15 July 2005 Article Highly efficient vitrification method for cryopreservation of human oocytes Dr Masashige Kuwayama Masashige Kuwayama (PhD) is currently the Scientific Director of Kato Ladies Clinic (Tokyo, Japan), the world s largest IVF unit. In 1986, he began work in the field of embryology with Dr Hanada. They developed assisted reproduction techniques (IVM, IVF, vitrification, embryo culture, ES cell) and established a bovine embryo mass production system as the leader of a National Project in Japan in 1990. He obtained the first calves after oocyte vitrification, IVF, invitro culture and blastocyst transfer in 1992. He moved to human IVF in 1999, developed the Cryotop vitrification method for human oocytes and established the first human oocyte bank in 2001. The first babies following oocyte vitrification in USA and Japan were obtained by his group using the Cryotop method. He is also interested in rejuvenescence of old defective oocytes, and obtained the first calf from old infertile cattle with germinal vesicle transfer in 2002. Masashige Kuwayama 1,4, Gábor Vajta 2, Osamu Kato 1, Stanley P Leibo 3 1 Kato Ladies Clinic, Tokyo 160 0023, Japan; 2 Population Genetics and Embryology, Department of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark; 3 Audubon Centre for Research of Endangered Species, Department of Biological Sciences, University of New Orleans, New Orleans, LA 70131, USA 4 Correspondence: Tel: +81 3 33663777; Fax: +81 3 53327373; e-mail: masaabc@bekkoame.ne.jp Abstract Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.1 μl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation. Keywords: birth, bovine, Cryotop, human, pregnancy, vitrifi cation 300 Introduction The capability to cryopreserve oocytes could be a valuable tool in human assisted reproductive techniques (Oktay et al., 1998; Porcu et al., 2000; Pool and Leibo, 2004; Paynter, 2005). It would permit patients suffering from various types of malignant diseases to have their oocytes collected prior to the beginning of chemo- or radiotherapy, with the expectation of having their oocytes fertilized after recovery from treatment (Meirow, 2000). As the ability of the oocytes to be fertilized and develop to term dramatically decreases in parallel with age, oocyte cryopreservation would permit women to delay maternity because of career demands. If oocyte cryopreservation were reliable and efficient, a woman s oocytes could be collected when she is young and preserved until she wished to have a child. Women whose husbands need treatment for sterility could avail themselves of this technology. Recent advances in cryobiology have made it possible to preserve various types of reproductive cells with relatively little loss of viability (reviewed in Karow and Critser, 1997; Fuller and Paynter, 2004). However, other types of reproductive cells, especially oocytes, are sensitive to chilling injury and are damaged when cooled slowly by standard equilibrium methods (Vincent and Johnson, 1990; Bernard and Fuller, 1996; Leibo et al., 1996). Vitrification is an alternative method to cryopreserve those biological specimens that are sensitive to chilling injury. Since the first report of vitrification of mammalian embryos by Rall and Fahy (1985), successful vitrification of reproductive cells of more than 11 species, including humans, have been reported; among the most studied are oocytes and embryos of cattle (reviewed by Rall, 2001). First births of normal calves derived from vitrified blastocysts that had been produced by IVF were reported 13

years ag go (Kuwayamaa et al., 1992). Calves have also been producedd by transfer of embryos obtained by IVF and in-vitro culture (IVC) of bovine oocytes that had been vitrified after in- vitro maturation (IVM) to the metaphase II stage (Hamano and Kuwayama, 1992). Similar procedures have also been used to vitrify po orcine blastocysts (Kuwayamaa et al., 1997; Kobayashi et al.,, 1998) and oocytes (Nagashimaa et al., 1999), both of which are considered to be especially difficult to cryopreserve because of their extreme sensitivity to chilling injury (Leibo et al., 1996). Several methods have been described for vitrification of bovine and porcine embryos. Among these are: in-straw dilution (ISD; Kuwayama, 1994), open-pulled straw (OPS; Vajta a et al., 1998, Isachenko et al., 2003); and the Cryotop method, a modification of the minimum volume cooling (MVC) procedure (Hamawaki et al., 1999). This paper reports the results of two experiments designed to derive an efficient method of cryopreserving human oocytes by vitrification. In the first experiment, the three methods are compared for vitrification of bovine oocytes. In the second experiment, using the method that yielded the best results in experiment 1, the effect of two concentrations of ethylene glycol and the presence or absence of cumulus cells on survival of human oocytes after vitrification are compared. Another group of oocytes was cryopreserved by standard equilibration cooling. The normality of those human embryos derived from vitrified oocytes was examined by fluorescence in-situ hybridization (FISH). Day 2 and day 5 embryos derived from intracytoplasmic sperm injection (ICSI) after vitrification of human oocytes were transferred to recipients to obtain data regarding their full developmental competence. Materials and methods Bovine oocyte recovery and maturation Bovine ovaries were obtained from a local slaughterhouse and transported to the laboratory at 33 35 C in 0.9% saline containing 0.1 mg/ml kanamycin. All visible follicles (2 5 mm in diameter) on the ovarian surface were aspirated with an 18-gauge needle. Cumulus oocyte complexes were cultured in groups of 20 in 100 μl droplets covered with oil of maturation medium in a culture dish (35 mm 15 mm; Nunclon #153066; Roskilde, Denmark) in 5% CO 2 in air with maximum humidity at 38.5 C for 22 h The maturation medium was TCM199 medium with Earle s salts, HEPES and bicarbonate (Gibco, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (Sigma Chemical Co., St Louis, MO, USA) and 250 μg/ml gentamycin sulphate. Bovine IVF After maturation, bovine oocytes were subjected to IVF and IVC as previously described (Kuwayama a et al., 1992). In brief, capacitation was induced by incubating frozen thawed and washed bovine spermatozoa in 5 mmol/l theophylline and 10 μg/ml heparin. Insemination was performed by introducing the oocytes into a sperm suspension at a concentration of 2 10 7 spermatozoa/ml. After co-culture with spermatozoa for 5 h, oocytes were transferred to a 100 μl droplet of Synthetic Oviductal Fluid Medium (SOFM; Fukui et al., 1996)) containing essential and non-essential amino acids and BSA (A-2153; Sigma Chemical Co.; 5 mg/ml), then cultured in a humidified atmosphere of 5% O 2, 5% CO 2 and 90% N 2 at 38.5 C for 7 days. Patients Experiments were conducted with patients who gave informed consent and with IRB approval. They were without spermatozoa at IVF because of the husband s personal reasons, or were testicular sperm extraction cases. Sixty-seven patients (32.3 ± 6.1 years old; mean ± SD) underwent vitrification experiments. The ovarian stimulation was performed by the clomiphene cycle with human menopausal gonadotrophin (HMG). Clomiphene (Clomid; Shionogi Co., Ltd, Osaka, Japan) was initiated on day 3 of the cycle by 50 mg/day and continued until the flare-up of LH, caused by spraying 300 μg of gonadotrophin-releasing hormone agonist. HMG (Humegon; Organon Co., Ltd, Oss, The Netherlands) was initiated on day 8 by 150 IU and continued every other day until the day before LH flare-up. ICSI and IVC of human oocytes after vitrification After recovery of the oocytes, they were cultured for 2 h in the basic medium, i.e. TCM199 medium buffered with 11 mmol/l HEPES, 9 mmol/l Na HEPES, 5 mmol/l NaHCO 3 and supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific, Santa Ana, CA, USA) in 5% CO 2 in air at 37 C and maximum humidity. Those oocytes considered to have survived, as judged by morphological criteria (Figure 2, see below), were inseminated by ICSI, as described by Palermo et al. (1992). For ICSI, semen specimens, either fresh or frozen thawed from an anonymous donor or from the patient s husband with >90% motility, were used for insemination. After insemination, oocytes with 2 pronuclei and a second polar body were transferred into 0.5 ml droplets of P1 medium (Irvine Scientific) containing 10% SSS. Embryos that developed beyond the 4-cell stage were then transferred into 100 μl droplets of G2.2 medium (IVF Science, Göteborg, Sweden) and cultured up for 6 days after ICSI. Vitrification of bovine oocytes Cumulus cells were partially removed from cumulus oocyte complexes by gentle pipetting until only a few layers remained. All oocytes were then transferred into 1.6 mol/l ethylene glycol diluted in TCM199 medium and kept for 5 15 min at ~22 C until the oocytes had completely recovered their original isotonic volume, as judged by microscopic observation. Liquid nitrogen, used for cooling oocytes, was filtered through a Teflon membrane filter (0.22 μm pore diameter; Millipore Corp., Bedford, MA, USA). To prepare straws for the ISD method, 25 μl of a vitrification solution (6.8 mol/l ethylene glycol + 1.0 mol/l sucrose in TCM199 medium) and 150 μl of a diluent solution (0.5 mol/l sucrose in TCM199) were aspirated with syringes into plastic artificial insemination straws (0.25 ml; IMV, l Aigle, France) without any air bubbles between the two columns of media. After being equilibrated in 1.6 mol/l ethylene glycol, oocytes were washed in 4.5 ml of vitrification solution in a culture dish for 30 s, picked up by pipette and introduced into the vitrification solution in the straw. Each straw was sealed and plunged directly into liquid nitrogen. The time between introducing oocytes into the 301

302 vitrification solution and plunging the straw into liquid nitrogen was approximately 1 min. For warming of vitrified samples, the straw was first held at room temperature air for 5 s and was then submerged and gently shaken in a 37 C water bath for 8 s. The two solutions were mixed by holding the straw with its plug-end up for 1 min and then horizontally for 5 min at room temperature. The entire contents of the straw were expelled into a dish, the oocytes retrieved and washed twice for 5 min in TCM199 medium containing 20% calf serum, and were cultured in vitro. In the OPS method, 0.25 mm standard insemination plastic mini-straws were heat-softened over a hot plate and pulled manually, as originally described by Vajta a et al. (1998). The inner diameter and the wall thickness of the pulled part of the straw were approximately 0.8 and 0.07 mm respectively. Equilibration and loading methods were slightly modified compared with the original description. After being equilibrated in 1.6 mol/l ethylene glycol, oocytes were transferred sequentially between three 8 μl droplets of vitrification solution for 10 s each. Oocytes were loaded into the pulled straws by placing the narrow end of the pulled straw in the third droplet and aspirating oocytes within a 2 3 mm long liquid column (1 1.5 μl) using capillarity. The straws were then cooled by being plunged directly into liquid nitrogen and stored briefly. For warming, the open end of the straw was immersed vertically into 4.5 ml of 1.0 mol/l sucrose in TCM199 on a warm stage at 37 C. The solidified vitrification solution became liquid within 1 2 s, whereupon the sucrose solution entered the straw, and the oocytes immediately flowed out of the straw into the culture dish. One minute after warming, oocytes were transferred into 0.5 mol/l sucrose for 3 min, washed twice in TCM199 + calf serum for 5 min each, and then cultured as described above. In the Cryotop method, after being equilibrated in 1.6 mol/l ethylene glycol, oocytes were washed in 4.5 ml of vitrification solution in a culture dish for 30 s. Individual oocytes were then picked up in an extremely small volume (<0.1 μl) of vitrification solution and placed on top of a very fine polypropylene strip (0.4 mm wide 20 mm long 0.1 mm thick) attached to a hard plastic handle specially constructed according to specifications by Kitazato Supply Co, Fujinomiya, Japan (Figure 1). The droplet volume was estimated from the length of the fluid column within the pipette tip. As soon as the oocyte was placed onto the thin polypropylene strip of the Cryotop, it was immediately submerged vertically into filtered liquid nitrogen. Then, the thin strip was covered with a hard plastic cover (3 cm long) on top of the Cryotop sheet to protect it during storage in liquid nitrogen containers. For warming, the protective cover was removed from the Cryotop while it was still submerged in liquid nitrogen, and the polypropylene strip of the Cryotop was immersed directly into ~3 ml of 1.0 mol/l sucrose prepared in TCM199 at 37 C for 1 min. Oocytes were retrieved and transferred into 4.5 ml of diluent for 3 min, and then washed twice in TCM199 + serum for 5 min each, and then cultured. Standard equilibrium cooling For comparison with standard methods of cryopreservation, a small number of human oocytes were frozen in 1.5 mol/l propylene glycol (PG) + 0.3 mol/l sucrose by the method of Lassalle et al. (1985), which was designed for embryo freezing and is commonly applied. After cumulus cells had been removed, oocytes were exposed to the cryoprotectant solution, and then aspirated into 0.25 ml straws; these were cooled to 6 C, seeded, and then cooled at 0.3 C/min to 35 C before being placed directly into liquid nitrogen. Frozen straws were thawed rapidly, and the PG removed by stepwise dilution. These oocytes were assayed by the same criteria used for the other treatments. Biopsy and FISH of blastocysts Biopsy and FISH were performed according to the procedures previously described by Monk et al. (1988) and Munné et al. (1998). Several trophectodermal cells were removed by biopsy using suction and excision with glass microneedles from the abembryonic pole of embryos that were at the expanding blastocyst stage. The biopsied cells were placed in a hypotonic solution (0.075 mol/l) of KCl for 5 min at room temperature and then were transferred in a small volume of hypotonic solution onto glass slides. Under a stereomicroscope, fixative (1:3 mixture of acetic acid and ethanol respectively) was dropped onto the biopsied piece. The fixative was spread by continuous and gentle blowing until the cytoplasm dissolved. The position of the cells on the slides was marked with a thin marker pen. Hybridization was carried out according to the manufacturer s directions using probes of CEP18, CEPX, and CEPY (Vysis Inc., Downers Grove, IL, USA). In brief, the hybridization mixture was added, then slides were covered with coverslips and sealed with rubber cement. Hybridization was performed at 75 C for 2 min and then incubated for 6 h or overnight at 37 C. Coverslips were then removed, and slides were washed with buffer. The DNA was counter-stained with DAPI, and the specimens were examined using an ECLIPSE E800 fluorescence microscope (Nikon, Tokyo, Japan) using a Quad band-pass filter set (Vysis Inc.). Statistical analysis Data were analysed by the chi-squared test. P-values lower than or equal to 0.05 were regarded as significant Measurements of cooling and warming rates The volumes of solutions in which the oocytes were cooled were very different in the three vitrification methods used in the present experiments, with supposedly considerable effect on the cooling and warming rates. For exact determination of these parameters, an ultra-fine thermocouple probe (0.1 mm diameter; Chino Ltd, Tokyo, Japan) was introduced into the liquid column (for the ISD and OPS methods) or into the small droplet (Cryotop method). Subsequently, the standard straw, the open-pulled straw or the polypropylene strip was submerged directly into liquid nitrogen, held briefly inside, then warmed in a 37 C water bath. Temperature changes were measured with an electronic thermometer (Model EB22005;

Chino Ltd). For each method, the time required for the temperat ture to drop from 20 C to 100 C (or the reverse) was measured during cooling and during warming of five replicates each, and the average cooling and warming rates were calculated. Experiment 1 The survival of bovine oocytes cryopreserved by the three vitrification methods was assessed, and those results compared with the development of control oocytes that were not subjected to any cooling. A total of 606 IVM bovine oocytes were randomly distributed into four groups. In all, 152, 149 and 153 oocytes were vitrified with the ISD, OPS or the Cryotop method respectively, as described above. A total of 152 oocytes were used as controls. After vitrification and warming, the morphological survival was evaluated under a stereomicroscope, then the oocytes were fertilized and cultured in vitro. Cleavage and blastocyst rates were determined on days 2 and 7 after fertilization respectively. Experiment 2 In experiment 2, human oocytes were cryopreserved either by standard equilibrium cooling or by vitrification. On the basis of results of experiment 1, the Cryotop method was selected for further vitrification work. Two concentrations of ethylene glycol were compared for vitrification of human oocytes either surrounded or free of cumulus cells respectively. Cryopreserved and thawed oocytes were subjected to ICSI and embryo culture as described above. Cleavage of the embryos and blastocyst formation (Figure 2) were observed on days 2 and 6 respectively. Five day 6 blastocysts were subjected to biopsy and the obtained cells were investigated by FISH. Twenty-nine embryo transfers (mean number of the embryos per embryo transfer was 2.2) were performed on days 2 and 5. All the embryo transfers were performed non-surgically under ultrasound guidance. Pregnancy was diagnosed by the formation of the gestational sacs by ultrasound observations on 6 weeks after their last menstrual period. Results Measurement of cooling and warming rates As shown in Table 1, the sample volumes had dramatic effect on the cooling and warming rates of solutions. The differences between the cooling and warming rates achievable in ISD and Cryotop methods were approximately 5- and 30-fold respectively, while rates obtained with the OPS technique were between values obtained with the two other methods. Experiment 1 Results of experiment 1 (Table 2) showed that 78% of 152 control oocytes cleaved after IVF and 45% of the total developed into blastocysts. Although equally high percentages of oocytes subjected to the three cryopreservation methods appeared to have survived based on morphological criteria, there were significant differences in developmental rates among the treatments after IVF and culture (P P < 0.05). Significantly more oocytes cryopreserved by the Cryotop method cleaved (60%; P < 0.05) and 23% of the total that had been treated developed into blastocysts, a survival rate about 3- to 4-fold higher than for oocytes cryopreserved by the ISD or OPS method. Experiment 2 As shown by the results in Table 3, significantly more human oocytes exhibited normal morphology after vitrification in 5.0 mol/l ethylene glycol than in 6.8 mol/l (P P < 0.05). When vitrified in 6.8 mol/l ethylene glycol, more oocytes appeared to survive when enclosed within cumulus cells, although only a small number of cumulus-free oocytes were vitrified in that concentration of ethylene glycol. The best results were achieved with cumulus-intact oocytes suspended and vitrified in 5.0 mol/l ethylene glycol. Of 64 human oocytes vitrified by the Cryotop method, 91% of them appeared morphologically normal after warming and recovery. After ICSI and IVC, 81% developed to the 4-cell stage, and 32 (50% of the original number treated) of them reached the blastocyst stage in culture. Of a small number of oocytes cryopreserved by standard equilibrium cooling, only two appeared morphologically normal and neither was fertilized by ICSI. All the cells (five of five) removed by biopsy from blastocysts derived from vitrified oocytes were diploid, showing X and Y chromosomes, and two no. 18 chromosomes. As shown in the Table 4, in total 29 embryo transfers (mean number of embryos per embryo transfer was 2.2) were performed on days 2 and 5. Twelve pregnancies were obtained (pregnancy rate = 41.3%). Seven healthy singletons were born and, and three pregnancies are ongoing. Table 1. Comparison of sample volumes and cooling/warming rates measured between 20º and 100ºC of specimens treated by various vitrification methods. The values shown are the means of five replicate measurements for each method. Vitrifi cation method Volume of Cooling rate Warming rate solution (μl) (ºC/min) (ºC/min) In-straw dilution 25.0 4460 1300 Open-pulled straw 1.5 16,340 13,900 Minimum volume cooling <0.1 22,800 42,100 303

c a b d Figure 1. The Cryotop vitrification tool. The polypropylene strip (a) is attached to a hard plastic handle (b). After vitrification, a hard plastic cover (c) is attached to protect strip during storage in liquid nitrogen (d). a b c d 304 Figure 2. Cumulus-enclosed (a) and cumulus-free human oocytes before (b) and after (c) Cryotop vitrification. A day 6 expanded blastocyst t (d) developed from the vitrified oocytes. Bar represents 100 mm.

Table 2. In-vitro development of bovine oocytes after vitrification by three methods. Details of the methods are described in the text. Vitrifi cation method No. oocytes No. (%) No. (%) Developed into treated with normal cleaved blastocysts morphology In-straw dilution 152 144 (94.7) 60 (39.5) a 8 (5.3) a Open-pulled straw 149 143 (96.0) 71 (47.7) a 11 (7.4) a Cryotop 153 148 (96.7) 91 (59.5) b 35 (22.9) b Fresh control 152 118 (77.6) c 68 (44.7) c a,b,c Values with different superscripts within the same columns are significantly different (P < 0.05). Table 3. In-vitro survival of cumulus-enclosed or cumulus-free human oocytes after vitrification by the Cryotop method using two different cryoprotectant concentrations compared with freezing by standard equilibrium cooling. CPA = cryoprotective additive. Cryopreservation CPA Cumulus No. of oocytes No. (%) No. No. (%) method concentration layer cryopreserved with normal fertilized of (mol/l) morphology blastocysts Cryotop vitrification 6.8 d + 33 25 (75.8) b 23 8 (24.2) b 6.8 d 10 3 (30.0) c 2 0 5.0 d + 64 58 (90.6) a 52 32 (50.0) a Standard equilibrium 1.5 e 9 2 (22.2) c 0 0 cooling a,b,c Values with different superscripts within the same columns are significantly different (P < 0.05). d,e Cryoprotectants ethylene glycol and propylene glycol respectively. Table 4. In-vivo development of the vitrified human oocytes after embryo transfer on day 2 and day 5. ET = embryo transfer. Day of ET (no. No. of ET No. (%) of No. of No. of embryos/et) (no. embryos) pregnancies deliveries a ongoing pregnancies 2 (2) 1 (2) 1 (100) 0 0 2 (3) 17 (51) 6 (35.3) 4 1 5 (1) 11 (11) 5 (45.5) 3 2 Total 29 (64) 12 (41.3) 7 3 a Take-home babies. 305

306 Discussion In the first part of the present study, bovine oocytes were used as a model to try to establish a better method to cryopreserve human oocytes. The highest survival rate of vitrified bovine oocytes was obtained with the use of the Cryotop method. Probably the most remarkable difference among the three tested methods that might influence the results was the cooling and warming rate. These findings confirm earlier data suggesting that survival of vitrified oocytes, especially their viability after IVF and IVC, seems to be dependent on the rate at which they are cooled and/or warmed. Using a small electron microscope grid as a support for bovine oocytes, Martino et al. (1996) were able to improve significantly functional survival and development after IVF and culture. Healthy calves were also obtained after vitrification of both matured oocytes and blastocyst derived from them after IVF and IVC by using the OPS method (Vajta et al., 1998). Analogous methods have been used by other investigators to minimize the volume of the droplets of solution in which oocytes were cooled to increase the cooling and warming rates during cryopreservation (Arav and Zeron, 1997; Chung et al., 2000; Papis et al., 2000), resulting in higher survival of mammalian oocytes after vitrification. The oocyte is one of the largest cells in the mammalian body and, because of its spherical shape, has the lowest surface area-to-volume ratio, compared with other cell types with irregular shapes (Smith and Silva, 2004; Wright et al., 2004). Moreover, as was first shown many years ago for mice, oocytes have lower permeability to cryoprotective additives (CPA) compared with zygotes and embryos (Jackowski et al., 1980). One major obstacle of vitrification is the deleterious effect of high CPA concentration that is usually regarded as indispensable to induce the glass-like solidification. Oocytes at MII stage are not only difficult to equilibrate with CPA, but they are also quite sensitive to physical/chemical insults because of their vulnerable spindle apparatus (Zenzes et al., 2001; Stachecki et al., 2004). Such damage can be caused to oocytes that are only exposed to vitrification solutions before cooling (Fuku et al., 1995). Consequently, one of the most promising approaches to improve their functional survival after vitrification might be to minimize the amount of CPA added to vitrification solutions, so as to reduce possible chemical toxicity and osmotic injury. It is well known, however, that the tendency for water to vitrify depends not only on the concentration of CPA, but also the cooling rate. If pure water is cooled very rapidly, it is even possible to vitrify it without the addition of CPA. For example, hot steam cooled by direct contact on a copper sheet held in liquid nitrogen solidified with no ice crystal formation in an amorphous state (Sakai, 1987). Evidently, a decrease of the volume of solution containing oocytes at plunging into liquid nitrogen increases cooling rates and may allow lower CPA concentration required for vitrification Reducing the CPA concentration of vitrification solutions may be one reason for the improved survival after the vitrification procedure used in this study. Higher survival of human oocytes was obtained when the ethylene glycol concentration in the vitrification solution was 5.0 mol/l rather than 6.8 mol/l. This suggests that it may be possible to improve the results even more by reducing the ethylene glycol and/or sucrose concentration of vitrification solutions. It should be mentioned that according to some earlier experiments, successful cryopreservation can also be performed with rapid cooling and by using surprisingly low CPA concentrations that are insufficient to induce complete vitrification. For example, in studies with mice, embryos vitrified in a solution referred to as 40EFS, which contains 40% ethylene glycol plus Ficoll plus sucrose, exhibited very high survival rates (Kasai et al., 1990), yet equally high survival rates were also obtained for embryos vitrified in 18% ethylene glycol. Similarly, 8-cell mouse embryos cooled rapidly in a solution of 2 mol/l ethylene glycol exhibited the same high rate of development into blastocysts as embryos that were cooled rapidly in 6 mol/l ethylene glycol, despite the fact that the former solution crystallized during cooling, whereas the latter solution vitrified (Leibo and Oda, 1992). In the second experiment, human oocytes were successfully preserved with the method selected on the basis of bovine results. Post-thaw survival rates of the cumulus-enclosed oocytes were significantly higher than those in the nontreated group. It has been suggested that establishment of the technology to vitrify oocytes at the GV stage and other stages of maturation will also be very important for clinical and experimental use of the technology. As immature oocytes require the cumulus cells connected to oocytes through gap junctions for their maturation, the vitrification protocol might be helpful for the purpose. Mammalian oocytes are known to be activated by exposure to a low temperature and to develop as parthenogenetic embryos. However, in this study, all the biopsied cells from blastocysts derived from vitrified oocytes were judged to be normal diploid cells. Therefore, these results indicate that the embryos were not parthenogenetically activated by low temperatures during vitrification. Rather, they developed from the normal process of fertilization. Although case reports in available publications are low, births of healthy babies derived from oocytes cryopreserved both by conventional slow freezing and by vitrification have already been reported (van Uem et al., 1987; Kuleshova et al., 1999; Wurfel et al., 1999). These results suggest that the safety of this approach might be similar to that of embryo cryopreservation, which is now routinely used in fertility clinics world-wide. The 29 embryo transfers performed by us on days 2 and 5 after ICSI and IVC of vitrified thawed oocytes resulted in 12 initial pregnancies (41% pregnancy rate), seven healthy babies and three ongoing pregnancies. So far as is known, the efficiency of the present technique regarding both in-vitro and in-vivo development is higher than that of other methods published so far. Work is continuing to increase the biopsy numbers and hopefully the pregnancy data. In conclusion, the Cryotop method is a highly efficient

vitrification procedure that may open a new way to resolve the various aspects of the problem of human oocyte cryopreservation. Acknowledgements The authors thank Dr T Nagai for the critical reading of the manuscript, and also Ms K Hata, M Aoki and K Hayashi for their excellent technical assistance. References Arav A, Zeron Y 1997 Vitrification of bovine oocytes using modified minimum drop size technique (MDS) is affected by the composition and concentration of the vitrification solution and by the cooling conditions. Theriogenology 47, 341. Bernard A, Fuller BJ 1996 Cryopreservation of human oocytes: a review of current problems and perspectives. Human Reproduction Update 2, 193 207. Chung HM, Hong SW, Lim JM et al. 2000 In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages. Fertility and Sterility 73, 545 551. Fuku E, Xia L, Downey BR 1995 Ultrastructural changes in bovine oocytes cryopreserved by vitrification. Cryobiology 32, 139 156. Fukui Y, Lee ES, Araki N 1996 Effect of medium renewal during culture in two different culture systems on development to blastocysts from in vitro produced early bovine embryos. Journal of Animal Sciences 74, 2752 2758. Fuller B, Paynter S 2004 Fundamentals of cryobiology in reproductive medicine. Reproductive BioMedicine Online 9, 680 691. Hamano S, Kuwayama M 1992 Development of in vitro matured bovine oocytes after cryopreservation by vitrification and in vitro fertilization. Proceedings of the 12th International Congress for Animal Reproduction, The Hague, The Netherlands, pp. 1421 1423. Hamawaki A, Kuwayama M, Hamano S 1999 Minimum volume cooling method for bovine blastocyst vitrification. Theriogenology 51, 165. Isachenko V, Selman H, Isachenko E et al. 2003 Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure. Reproductive BioMedicine Online 7, 211 216. Jackowski S, Leibo SP, Mazur P 1980 Glycerol permeabilities of fertilized and unfertilized mouse ova. Journal of Experimental Zoology 212, 329 341. Karow A, Critser JC (eds) (1997) Reproductive Tissue Banking. Academic Press, San Diego, CA, USA. Kasai M, Komi JH, Takakamo A 1990 A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. Journal of Reproduction and Fertility 89, 91 97. Kobayashi S, Takei M, Kano M et al. 1998 Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants. Cryobiology 36, 20 31. Kuleshova L, Gianaroli L, Magli C, Trounson A 1999 Birth following vitrification of a small number of human oocytes. Human Reproduction 14, 3077 3079. Kuwayama M 1994 In straw dilution of bovine IVF-blastocysts cryopreserved by vitrification. Theriogenology 41, 231. Kuwayama M, Holm P, Jacobsen H et al. 1997 Successful cryopreservation of porcine embryos by vitrification. Veterinary Record 141, 365. Kuwayama M, Hamano S, Nagai T 1992 Vitrification of bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro. Journal of Reproduction and Fertility 96, 187 193. Lassalle B, Testart J, Renard J-P 1985 Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertility and Sterility 44, 645 651. Leibo SP, Oda K 1992 High survival of mouse zygotes and embryos cooled rapidly or slowly in ethylene glycol plus polyvinylpyrrolidone. Cryo-Letters 14, 133 144. Leibo SP, Martino A, Kobayashi S, Pollard JW 1996 Stagedependent sensitivity of oocytes and embryos to low temperatures. Animal Reproduction Science 42, 45 53. Martino A, Songsasen N, Leibo SP 1996 Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biology of Reproduction 54, 1059 1069. Meirow D 2000 Reproduction post-chemotherapy in young cancer patients. Molecular and Cellular Endocrinology 169, 123 131. Monk M, Muggleton-Harris AL, Rawlings E et al. 1988 Preimplantation diagnosis of HPRT-deficient male and carrier female mouse embryos by trophectoderm biopsy. Human Reproduction 3, 377 381. Munné S, Marquez C, Magli C et al. 1998 Scoring criteria for preimplantation genetic diagnosis of numerical abnormalities for chromosomes X, Y, 13, 16, 18 and 21. Molecular Human Reproduction 4, 863 870. Nagashima H, Cameron R, Kuwayama M 1999 Survival of porcine delipated oocytes and embryos after cryopreservation by freezing or vitrification. Journal of Reproduction and Development 45, 167 176. Oktay K, Newton H, Aubard Y et al. 1998 Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology? Fertility and Sterility 69, 1 7. Palermo G, Joris H, Devroey P, Van Steirteghem AC 1992 Pregnancies after intracytoplasmic sperm injection of single spermatozoa. Lancet 340 (8810), 17 18. Papis K, Shimizu M, Izaike Y 1999 The effect of gentle preequilibration on survival and development rates of bovine in vitro matured oocytes vitrified in droplets. Theriogenology 51, 173. Paynter SJ 2005 A rational approach to oocyte cryopreservation. Reproductive BioMedicine Online 10, 578 586. Pool TB, Leibo SP 2004 Introduction. Reproductive BioMedicine Online 9, 132 133. Porcu E, Fabbri R, Damiano G et al. 2000 Clinical experience and applications of oocyte cryopreservation. Molecular and Cellular Endocrinology 169, 33 37. Rall WF 2001 Cryopreservation of mammalian embryos, gametes, and ovarian tissues. In: Wolf DP, Zelinski-Wooten M (eds) Contemporary Endocrinology: Assisted Fertilization and Nuclear Transfer in Mammals. Humana Press, Towata, NJ, pp. 173 187. Rall WF, Fahy GM 1985 Ice-free cryopreservation of mouse embryos by vitrification. Nature 313, 573 575. Sakai A 1989 Freezing and vitrification of aqueous solutions. In: Cryopreservation. Asakurashoten Co. Ltd, Tokyo, Japan, pp. 46 54 (Japanese). Smith GD, Silva CASE 2004 Developmental consequences of cryopreservation of mammalian oocytes and embryos. Reproductive BioMedicine Online 9, 171 178. Stachecki JJ, Munné S, Cohen J 2004 Spindle organization after cryopreservation of mouse, human and bovine oocytes. Reproductive BioMedicine Online 8, 664 672. Vajta G, Kuwayama M, Holm P et al. 1998 Open pulled straw vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Molecular Reproduction and Development 51, 33 58. van Uem JF, Siebzehnrübl ER, Schuh B et al. 1987 Birth after cryopreservation of unfertilized oocytes. Lancet 1 (8535), 1:752 753. Vincent C, Johnson JH 1992 Cooling cryoprotectants and the cytoskeleton of the mammalian oocyte. Oxford Review in Reproductive Biology 14, 73 100. Wright DL, Eroglu A, Toner M, Toth TL 2004 Use of sugars in cryopreserving human oocytes. Reproductive BioMedicine Online 9, 179 186. Wurfel W, Schleyer M, Krusmann G et al. 1999 Fertilization 307

of cryopreserved and thawed human oocytes by injection of spermatozoa (ICSI) medical management of sterility and case report of a twin pregnancy. Zentralblatt für Gynakologie 121, 444-448 (German). Zenzes MT, Bielecki R, Casper RF, Leibo SP (2001) Effects of chilling to 0 C on the morphology of meiotic spindles in human metaphase II oocytes. Fertility and Sterility 75, 769 777. Received 18 April 2005; 25 May 2005; accepted 15 June 2005. 308