1) Researchers vitrified 17 mature human oocytes from 4 patients using a solution of 40% ethylene glycol and 0.6 mol/l sucrose. 11 oocytes survived vitrification. 2) After ICSI, 5 oocytes were fertilized forming pronuclear zygotes, of which 3 developed into embryos. One embryo was transferred to a 47 year old recipient. 3) This resulted in the successful delivery of a healthy baby girl, demonstrating the potential of oocyte vitrification for human fertility preservation and treatment.

1. Human Reproduction vol.14 no.12 pp.3077–3079, 1999
CASE REPORT
Birth following vitrification of a small number of human
oocytes
Lilia Kuleshova1, Luca Gianaroli2, Cristina Magli2, been encouraging but the methods were the same as the earlier,
larger study (Tucker et al., 1998). We have been exploring theAnna Ferraretti2 and Alan Trounson1,3
use of vitrification using very rapid cooling for cryopreservation1Centre for Early Human Development, Monash Institute of
of human oocytes and report the birth of a normal childReproduction and Development, Monash University, Wright Street,
resulting from the vitrification and transfer of a small numberClayton, Victoria, Australia and 2SISMER srl, Bologna, Italy
of human oocytes.3To whom correspondence should be addressed
We report the birth of a healthy baby girl at 37 weeks
Case report
gestation to a 47 year old recipient, after vitrification of
Four patients of mean age 32.5 years (range 27–35) attendingmature oocytes from four in-vitro fertilization (IVF)
the SISMER in-vitro fertilization (IVF) clinic (Bologna, Italy)patients. A total of 17 oocytes was vitrified in 1–2 µl of
agreed to have their surplus oocytes vitrified with the intentionethylene glycol (40%) and 0.6 mol/l sucrose (20.54%) in
of reducing the number of surplus zygotes and embryos thatopen pulled straws. Eleven oocytes survived after vitrifica-
needed to be frozen in the particular cycle of treatment. Ation and five pronuclear zygotes were obtained after intra-
total of 17 oocytes from the four patients was vitrified, 13cytoplasmic sperm injection (ICSI). Three embryos were
were intended to be used by the patients to form embryos fortransferred to three patients, two of whom were the original
their own use and four were donated to recipients seekingoocyte donors and pregnancy was not established. The
donor oocytes.third embryo was donated to a 47 year old infertile woman
Oocytes were recovered after conventional ovarian stimula-after preimplantation diagnosis had confirmed euploidy
tion for IVF as previously described (Ferraretti et al., 1996).for chromosomes X, 13, 14, 15, 16, 18, 21 and 22. The
Two to seven of the mature metaphase II oocytes from thesuccessfully completed pregnancy is encouraging for fur-
four patients were randomly allocated to the vitrification study.ther research to explore the potential benefits of vitrification
Within 4 h of aspiration from follicles, the oocytes werefor the cryopreservation of human oocytes, given the
denuded of cumulus cells by a brief exposure to hyaluronidaserelatively low success of conventional freezing of human
enzyme (Hyase®; Scandinavian IVF Sciences, Go¨teborg,oocytes by slow cooling methods.
Sweden). Cumulus denuded mature metaphase II oocytes wereKey words: birth after freezing/cryopreservation/oocyte/oocyte
transferred to a 10% (v/v) ethylene glycol (Sigma, St Louis,cryosurvival/vitrification
MO, USA) solution in phosphate buffered saline (PBS) (Gibco
BRL, Paisley, Renfrewshire, UK) containing 10 mg/ml human
serum albumin (HSA) (Advanced Reproductive Technologies
Introduction Inc., San Clemente, CA, USA) for 40 s. Oocytes were then
transferred to 20% ethylene glycol (v/v) in PBS ϩ 10 mg/mlThe cryopreservation of human oocytes has remained a difficult
and inefficient procedure despite considerable effort to improve HSA for 30 s and finally to 40% ethylene glycol (v/v) and
20.54% (w/v) (0.6 mol/l) sucrose (Analar®; BDH Laboratorythe original methods described by Trounson (1986). The
introduction of 1,2 propanediol as an alternative to dimethyl Supplies, Poole, Dorset, UK) in PBS ϩ 10 mg/ml HSA for a
further 60 s. All the procedures were carried out on a warmsulphoxide as the penetrating cryoprotectant and intracyto-
plasmic sperm injection (ICSI) to overcome fertilization failure stage at 37°C. The oocytes were then drawn into a finely
drawn plastic straw by capillary action (Vajta et al., 1998) andafter cryopreservation has had little effect on the overall
success rates (Porcu et al., 1997). Of more than 709 oocytes the open pulled straw (OPS) rapidly cooled to –196°C (at
about 20 000°C/min) by direct transfer to liquid nitrogen.thawed, six babies were reported (Porcu et al., 1998b), which
is less than 1% of all thawed oocytes. Similarly, Tucker et al. The OPS straws were drawn from heated plastic 0.25 ml
insemination straws as described by Vajta et al. (1998). Oocytes(1998) reported around 1–2% of more than 400 thawed oocytes
developing to term using a very similar technique. These data were drawn into the OPS in 1–2 µl medium and transferred
immediately to liquid nitrogen.are little different from those which resulted in the first
pregnancies from frozen oocytes (Chen, 1986; Van Uem et al., Oocytes were warmed by transfer of the vitrified OPS
contents into pre-warmed sucrose solutions maintained on a1987). Smaller studies, such as that of 10 donor oocytes frozen
(de Fried et al., 1998), which resulted in a single birth, have warm stage at 37°C. The oocytes were expelled into 13.69%
© European Society of Human Reproduction and Embryology 3077

2. L.Kuleshova et al.
sucrose (0.4 mol/l) in PBS ϩ 10 mg/ml HSA for 2–3 min, for human oocyte cryopreservation. The vitrification solution
chosen was a mixture of 40% ethylene glycol and 0.6 mol/lthen to 8.56% sucrose (0.25 mol/l) in PBS ϩ 10 mg/ml HSA
for 2–3 min and finally 4.28% sucrose (0.125 mol/l) in PBS sucrose. These concentrations enable a stable vitrified solution
to form during rapid cooling and rewarming but can be quiteϩ 10 mg/ml HSA for 3–6 min. Oocytes were washed in IVF50
medium (Scandinavian IVF Sciences) for 4 h before ICSI and toxic to embryonic cells if they are exposed for 2 min or
longer at temperatures of 25°C or higher (Kasai et al., 1992).returned to culture in IVF50 medium. Ejaculated spermatozoa
were used to fertilize six oocytes for three patients, and for Consequently the brief times of exposure to the vitrification
solutions are probably also critical for survival of humanthe other patient frozen–thawed testicular spermatozoa obtained
by testicular sperm extraction (TESE) were used for ICSI with oocytes.
Mature oocytes and early cleavage stage embryos of manyfive oocytes.
Two pronuclear oocytes were cultured to the 6- to 8-cell species are sensitive to cryopreservation (Leibo et al., 1996).
Vitrification of the extremely sensitive early cleavage stagestage (58–68 h after insemination) and then either transferred
to the patients or subjected to preimplantation genetic diagnosis bovine embryo in a 1–2 µl mixture of ethylene glycol (16.5%),
dimethyl sulphoxide (16.5%) and sucrose (0.5 mol/l), in narrow(PGD) for aneuploidy determination by the biopsy of a
single cell and fluorescent in-situ hybridization (FISH) for bore plastic straws (OPS) has been shown to be very successful
(Vajta et al., 1998). Furthermore, in the same report it waschromosomes X,Y, 13, 14, 15, 16, 18, 21 and 22, as previously
described (Magli et al., 1998). Embryos suitable for transfer shown that mature unfertilized bovine oocytes could be suc-
cessfully vitrified in 20% ethylene glycol ϩ 20% dimethylwere transferred 64–72 h after insemination using a Wallace
catheter (Smith Industries Medical Systems, Hythe, Kent, UK). sulphoxide and 0.5 mol/l sucrose using the same OPS rapid
cooling system, if cumulus cells were removed during matura-Of 17 vitrified oocytes, 11 (65%) survived intact and were
injected with spermatozoa for ICSI. Four of the six oocytes tion in vitro. When warmed after vitrification, 25% of oocytes
fertilized and developed to blastocysts, compared with 48%injected with ejaculated spermatozoa had two pronuclei and
one of the five oocytes injected with testicular spermatozoa of non-vitrified (fresh) oocytes. The blastocysts were revitrified
and they retained the capacity to develop to normal calves athad two pronuclei. Three of the pronucleate zygotes developed
to apparently normal 7- or 8-cell embryos. Two of these term (Vajta et al., 1998). The mixture of ethylene glycol
(40%) and 0.6 mol/l sucrose is a stable vitrification solutionembryos were transferred to the two original patients from
whom the oocytes were collected, together with one or two (Kuleshova et al., 1999) with relatively low toxicity to embryos.
Survival of 65% of the mature human oocytes after vitrificationnon-frozen embryos but pregnancy did not occur. One embryo
was biopsied and was identified as disomic for chromosomes is higher than most reports for cryosurvival of human oocytes
(Porcu et al., 1998a). Survival rates after freezing of largeX, 13, 14, 15, 16, 18, 21 and 22. The embryo was transferred
at 93 h after insemination to a 47 year old nulliparous recipient numbers of human oocytes by conventional slow cooling or
equilibrium cooling methods was 56%, with 63% fertilizationwho had failed on four previous attempts to become pregnant
by donation of non-frozen oocytes after embryo transfer. after ICSI and a cleavage rate of 90%. Six implanted embryos
developed to term from the 709 thawed embryos (Porcu et al.,Pregnancy was confirmed by ultrasound and a normal female
karyotype confirmed by chorionic villus sampling at 12 weeks 1998b). Very similar data were reported (Tucker et al., 1998)
using the same freezing methods for cryopreservation of humangestation. A healthy 3500 g baby girl was delivered by
Caesarian section at 37 weeks gestation on June 20, 1999. 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
Discussion development to term (1/3 transferred embryos) in the present
study, are encouraging and worth following up because theyVitrification of cells is a potentially less damaging procedure
than freezing because it avoids the formation of intracellular are in the upper range for rates of survival and development
to term of other published cryopreservation methods. The birthice and damaging osmotic effects that occur as a result of ice
formation during equilibrium cooling and warming. Rapid rate of 1/17 (6%) vitrified oocytes may indicate that substantial
improvements can be achieved to the present developmentalcooling in high concentrations of penetrating cryoprotectants
enable high rates of survival of mouse embryos of all develop- success rates of around 1% births for conventional freezing–
thawing of human oocytes (Porcu et al., 1998b; Tuckermental stages (Shaw et al., 1991a). Penetrating cryoprotectant
can be replaced with sugars and polymers (Kasai et al., 1992; et al., 1998).
Vitrification has been used to cryopreserve human 4- andShaw et al., 1997; Kuleshova et al., 1999). It is also possible
to cryopreserve mature bovine oocytes successfully by using 8-cell embryos (Mukaida et al., 1998): 40% ethylene glycol
together with 18% Ficoll and 0.3 mol/l sucrose were used asvery rapid cooling methods and brief exposure to vitrification
solutions (Martino et al., 1996; Vajta et al., 1998). These a low toxicity solution with stable vitrification properties. Of
52 vitrified embryos, 42 (81%) were considered suitablevitrification techniques continue to be revised and optimized
and are now in widespread use for embryo cryopreservation for transfer and two implanted and developed to term (5%
implantation/birth rate). It remains to be determined whetherfor a number of species. The opportunity to explore the
response of human oocytes to vitrification in the present case the relatively low implantation rate can be improved for human
embryo vitrification or if this represents chromosomal damagereport suggests that further research should be undertaken to
determine the likely benefits for infertile patients of the method as observed for some rapid freezing methods (Shaw et al.,
3078

3. Birth after vitrification of human oocytes
Porcu, E., Fabbri, R., Savelli, L. et al. (1998a) Cryopreservation of human1991b). However, it should be noted that this chromosomal
oocytes: state of the art. In Kempers, R.D., Cohen, J., Haney, A.F. and
cryodamage was caused by inadequate concentrations of per- Younger, J.B. (eds) Fertility and reproductive medicine. Elsevier, New
York, pp. 599–613.meating cryoprotectant when rapid cooling and this would not
Porcu, E., Fabbri, R., Seracchioli, R. et al. (1998b) Birth of six healthybe anticipated where cryoprotectant concentrations are very
children after intracytoplasmic sperm injection of cryopreserved human
high for stable vitrification. oocytes. Hum. Reprod., 13, 124.
PGD was used to assess aneuploidy for X, Y, 13, 14, 15, Shaw, J.M., Diotallevi, L. and Trounson, A.O. (1991a) A simple rapid 4.5M
dimethyl sulphoxide freezing technique for the cryopreservation of one-cell16, 18, 21 and 22 (Magli et al., 1998) in the one donated
to blastocyst stage preimplantation mouse embryos. Reprod. Fertil. Develop.,embryo. This embryo was euploid for these chromosomes. 3, 621–626.
The application of PGD to embryos derived from cryopreserved Shaw, J.M., Kola, I., MacFarlane, D.R. et al. (1991b) An association between
chromosomal abnormalities in rapidly frozen two-cell mouse embryos andoocytes could be an important quality assurance procedure
the ice forming properties of the cryoprotective solution. J. Reprod. Fertil.,because of the reported low implantation rate. This may enable
91, 9–18.
an improved selection of euploid embryos and could raise Shaw, J.M., Kuleshova, L.L., MacFarlane, D.R. et al. (1997) Vitrification
implantation rates by discarding those diagnosed as aneuploid. properties of solutions of ethylene glycol in saline containing PVP, Ficoll,
or Dextran. Cryobiology, 35, 219–229.This might be important for donation of cryopreserved oocytes
Trounson, A. (1986) Preservation of human eggs and embryos. Fertil. Steril.,
to provide the recipient with some assurance of a reasonable
46, 1–12.
implantation rate. It is very important to derive reliable data Tucker, M.J., Morton, P.C., Wright, G. et al. (1998) Clinical application of
human egg cryopreservation. Hum. Reprod., 13, 3156–3159.on the potential increase in embryonic aneuploidy resulting
Vajta, G., Holm, P., Kuwayami, M. et al. (1998) Open pulled straw (OPS)from oocyte freezing and vitrification.
vitrification: a new way to reduce cryoinjuries in bovine ova and embryos.
This case report identifies the potential use of the vitrification Mol. Reprod. Develop., 51, 53–58.
of oocytes for IVF patients, for oocyte donation and the storage Van Uem, J.F.H.M., Siebzehnrubl, E.R., Schun, E.R. et al. (1987) Birth after
cryopreservation of unfertilized oocytes. Lancet, i, 752–753.of oocytes for patients who are at risk of sterility because
of radio- and/or chemotherapy, and those wishing to delay Received on July 5, 1999; accepted on September 24, 1999
conception for other reasons. The combination of vitrification
in a low toxicity solution, rapid cooling in 1–2 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.
Acknowledgements
This research was supported by Monash IVF Pty Ltd as a grant to
two of us (Lilia Kuleshova and Alan Trounson).
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