1 Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash University, Wright Street, Clayton, Victoria, Australia and 2 SISMER srl, Bologna, Italy
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Abstract |
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Key words: birth after freezing/cryopreservation/oocyte/oocyte cryosurvival/vitrification
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Introduction |
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Case report |
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Oocytes were recovered after conventional ovarian stimulation for IVF as previously described (Ferraretti et al., 1996). Two to seven of the mature metaphase II oocytes from the four patients were randomly allocated to the vitrification study. Within 4 h of aspiration from follicles, the oocytes were denuded of cumulus cells by a brief exposure to hyaluronidase enzyme (Hyase®; Scandinavian IVF Sciences, Göteborg, Sweden). Cumulus denuded mature metaphase II oocytes were transferred to a 10% (v/v) ethylene glycol (Sigma, St Louis, MO, USA) solution in phosphate buffered saline (PBS) (Gibco BRL, Paisley, Renfrewshire, UK) containing 10 mg/ml human serum albumin (HSA) (Advanced Reproductive Technologies Inc., San Clemente, CA, USA) for 40 s. Oocytes were then transferred to 20% ethylene glycol (v/v) in PBS + 10 mg/ml HSA for 30 s and finally to 40% ethylene glycol (v/v) and 20.54% (w/v) (0.6 mol/l) sucrose (Analar®; BDH Laboratory Supplies, Poole, Dorset, UK) in PBS + 10 mg/ml HSA for a further 60 s. All the procedures were carried out on a warm stage at 37°C. The oocytes were then drawn into a finely drawn plastic straw by capillary action (Vajta et al., 1998
) and the open pulled straw (OPS) rapidly cooled to 196°C (at about 20 000°C/min) by direct transfer to liquid nitrogen. The OPS straws were drawn from heated plastic 0.25 ml insemination straws as described by Vajta et al. (1998). Oocytes were drawn into the OPS in 12 µl medium and transferred immediately to liquid nitrogen.
Oocytes were warmed by transfer of the vitrified OPS contents into pre-warmed sucrose solutions maintained on a warm stage at 37°C. The oocytes were expelled into 13.69% sucrose (0.4 mol/l) in PBS + 10 mg/ml HSA for 23 min, then to 8.56% sucrose (0.25 mol/l) in PBS + 10 mg/ml HSA for 23 min and finally 4.28% sucrose (0.125 mol/l) in PBS + 10 mg/ml HSA for 36 min. Oocytes were washed in IVF50 medium (Scandinavian IVF Sciences) for 4 h before ICSI and returned to culture in IVF50 medium. Ejaculated spermatozoa were used to fertilize six oocytes for three patients, and for the other patient frozenthawed testicular spermatozoa obtained by testicular sperm extraction (TESE) were used for ICSI with five oocytes.
Two pronuclear oocytes were cultured to the 6- to 8-cell stage (5868 h after insemination) and then either transferred to the patients or subjected to preimplantation genetic diagnosis (PGD) for aneuploidy determination by the biopsy of a single cell and fluorescent in-situ hybridization (FISH) for chromosomes X,Y, 13, 14, 15, 16, 18, 21 and 22, as previously described (Magli et al., 1998). Embryos suitable for transfer were transferred 6472 h after insemination using a Wallace catheter (Smith Industries Medical Systems, Hythe, Kent, UK).
Of 17 vitrified oocytes, 11 (65%) survived intact and were injected with spermatozoa for ICSI. Four of the six oocytes injected with ejaculated spermatozoa had two pronuclei and one of the five oocytes injected with testicular spermatozoa had two pronuclei. Three of the pronucleate zygotes developed to apparently normal 7- or 8-cell embryos. Two of these embryos were transferred to the two original patients from whom the oocytes were collected, together with one or two non-frozen embryos but pregnancy did not occur. One embryo was biopsied and was identified as disomic for chromosomes X, 13, 14, 15, 16, 18, 21 and 22. The embryo was transferred at 93 h after insemination to a 47 year old nulliparous recipient who had failed on four previous attempts to become pregnant by donation of non-frozen oocytes after embryo transfer. Pregnancy was confirmed by ultrasound and a normal female karyotype confirmed by chorionic villus sampling at 12 weeks gestation. A healthy 3500 g baby girl was delivered by Caesarian section at 37 weeks gestation on June 20, 1999.
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Discussion |
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Mature oocytes and early cleavage stage embryos of many species are sensitive to cryopreservation (Leibo et al., 1996). Vitrification of the extremely sensitive early cleavage stage bovine embryo in a 12 µl mixture of ethylene glycol (16.5%), dimethyl sulphoxide (16.5%) and sucrose (0.5 mol/l), in narrow bore plastic straws (OPS) has been shown to be very successful (Vajta et al., 1998
). Furthermore, in the same report it was shown that mature unfertilized bovine oocytes could be successfully vitrified in 20% ethylene glycol + 20% dimethyl sulphoxide and 0.5 mol/l sucrose using the same OPS rapid cooling system, if cumulus cells were removed during maturation in vitro. When warmed after vitrification, 25% of oocytes fertilized and developed to blastocysts, compared with 48% of non-vitrified (fresh) oocytes. The blastocysts were revitrified and they retained the capacity to develop to normal calves at term (Vajta et al., 1998
). The mixture of ethylene glycol (40%) and 0.6 mol/l sucrose is a stable vitrification solution (Kuleshova et al., 1999
) with relatively low toxicity to embryos. Survival of 65% of the mature human oocytes after vitrification is higher than most reports for cryosurvival of human oocytes (Porcu et al., 1998a
). Survival rates after freezing of large numbers of human oocytes by conventional slow cooling or equilibrium cooling methods was 56%, with 63% fertilization after ICSI and a cleavage rate of 90%. Six implanted embryos developed to term from the 709 thawed embryos (Porcu et al., 1998b
). Very similar data were reported (Tucker et al., 1998
) using the same freezing methods for cryopreservation of human oocytes in 1,2-propanediol. Numbers for fertilization (4/6 oocytes fertilized after ICSI with ejaculated spermatozoa), development to 8-cell stage (3/5 pronuclear oocytes) and development to term (1/3 transferred embryos) in the present study, are encouraging and worth following up because they are in the upper range for rates of survival and development to term of other published cryopreservation methods. The birth rate of 1/17 (6%) vitrified oocytes may indicate that substantial improvements can be achieved to the present developmental success rates of around 1% births for conventional freezingthawing of human oocytes (Porcu et al., 1998b
; Tucker et al., 1998
).
Vitrification has been used to cryopreserve human 4- and 8-cell embryos (Mukaida et al., 1998): 40% ethylene glycol together with 18% Ficoll and 0.3 mol/l sucrose were used as a low toxicity solution with stable vitrification properties. Of 52 vitrified embryos, 42 (81%) were considered suitable for transfer and two implanted and developed to term (5% implantation/birth rate). It remains to be determined whether the relatively low implantation rate can be improved for human embryo vitrification or if this represents chromosomal damage as observed for some rapid freezing methods (Shaw et al., 1991b
). However, it should be noted that this chromosomal cryodamage was caused by inadequate concentrations of permeating cryoprotectant when rapid cooling and this would not be anticipated where cryoprotectant concentrations are very high for stable vitrification.
PGD was used to assess aneuploidy for X, Y, 13, 14, 15, 16, 18, 21 and 22 (Magli et al., 1998) in the one donated embryo. This embryo was euploid for these chromosomes. The application of PGD to embryos derived from cryopreserved oocytes could be an important quality assurance procedure because of the reported low implantation rate. This may enable an improved selection of euploid embryos and could raise implantation rates by discarding those diagnosed as aneuploid. This might be important for donation of cryopreserved oocytes to provide the recipient with some assurance of a reasonable implantation rate. It is very important to derive reliable data on the potential increase in embryonic aneuploidy resulting from oocyte freezing and vitrification.
This case report identifies the potential use of the vitrification of oocytes for IVF patients, for oocyte donation and the storage of oocytes for patients who are at risk of sterility because of radio- and/or chemotherapy, and those wishing to delay conception for other reasons. The combination of vitrification in a low toxicity solution, rapid cooling in 12 µl volumes in OPS, ICSI for fertilization and PGD to avoid aneuploidy for some chromosomes, enabled the birth of a healthy baby girl for a 47 year old recipient after oocyte donation.
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Acknowledgments |
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Notes |
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References |
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Submitted on July 5, 1999; accepted on September 24, 1999.