1 Yokota Ob/Gyn Clinic, 1-16-5, Shimokoide, Maebashi, Gunma 371-0031, and 2 The Institute for ARMT, 2-39-3, Kamikoide, Maebashi, Gunma 371-0037, Japan
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Abstract |
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Key words: blastocyst/cryopreservation/IVF/vitrification
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Introduction |
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On the other hand, recent studies have indicated the possibility of producing viable human blastocysts in a sequential culture medium (Gardner and Lane, 1997; Gardner et al., 1998
). In IVF, the transfer of blastocysts facilitates high pregnancy rates, thereby reducing the number of embryos needing to be transferred. Thus, this procedure minimizes the risk of multiple gestation (Gardner et al., 1998
). A simple procedure for the cryopreservation of supernumerary blastocysts for infertility treatment is needed.
While the slow cooling method of cryopreservation is now widely used, there has been some recent experimentation with ultra rapid freezing (vitrification) of human embryos. The technique of vitrification may be improved with regard to both the laborious nature of the technique itself and the survival of embryos upon recovery. Previously reported attempts to vitrify human embryos have been unsuccessful in achieving pregnancy (Quinn and Kerin, 1986; Trounson et al., 1988
). In the 1990s, there were reports of vitrification of human cleavage stage embryos and some successful deliveries following thaw and transfer using several cryosolutions (Barg et al., 1990
; Gordts et al., 1990
; Feichtinger et al., 1991
; Ohta et al., 1996
; Mukaida et al., 1998
; Kosuge et al., 1999
). However, to the best of our knowledge, there have been no reports of pregnancies following human blastocyst vitrification.
In the present case, human blastocysts were successfully vitrified using modified VSED cryosolution (Ishimori et al., 1993), containing modified-human tubal fluid (m-HTF) with 20% serum, ethylene glycol and dimethyl sulphoxide (DMSO) at a 2:1:1 ratio. After thawing, viable blastocysts were transferred in utero, resulting in one healthy pregnancy, now in its 25th week.
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Case report |
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Although the patient had regular ovulatory cycles, a bilateral hydrosalpinx was also demonstrated by hysterosalpingography. Ovarian stimulation was achieved via a combination of gonadotrophin-releasing hormone (GnRH), human menopausal gonadotrophin (HMG; Nikken, Tokyo, Japan) and 10000 IU human chorionic gonadotrophin (HCG, Pregnyl; Organon, Oss, The Netherlands). Both were administered when the leading follicle had reached a mean diameter of 1820 mm.
Following recovery of 12 mature oocytes, nine were fertilized following conventional IVF. Three days after insemination, three fresh embryos were transferred to the uterus, but all failed to result in pregnancy. The remaining six embryos continued to be cultured in vitro until day 5. Two grew to the blastocyst stage, and the resulting blastocysts were cryopreserved in VSED solution. Blastocysts were exposed to 10% ethylene glycol for 5 min, then placed into 50% VSED for 1 min, and finally (within 30 s), loaded into straws containing VSED at room temperature. The straws were placed in liquid nitrogen vapour for 2 min, and then plunged immediately into liquid nitrogen.
The thawing procedure involved warming the straws in a 25°C water bath, after a one-step dilution of the cryoprotectant was carried out using 0.5 mol/l sucrose solution; the blastocysts were the cultured in vitro overnight. Informed consent was obtained from the couple before the use of VSED.
The two frozenthawed blastocysts were transferred to the patient's uterus during a natural cycle, 3 months after the previous retrieval cycle. Pregnancy was achieved, and a HCG concentration of 50 mIU/ml in the urine was detected 2 weeks after the blastocysts had been transferred. A singleton pregnancy with a visible heartbeat was seen at 6 weeks gestation and is now ongoing in its 25th week.
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Discussion |
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Recently, a case involving a unique combination of high survival and meiotic normality together with good preservation using egg yolk was described (Isachenko and Nayudu, 1999). Another author (Park et al., 1999
) demonstrated that higher survival of vitrifiedthawed bovine blastocysts can be obtained using electron microscope grids rather than plastic straws as embryo containers during freezing. Furthermore, vitrification has another advantage in that it may improve survival if the procedure is further optimized for human blastocysts, since all of the physical and chemical injuries caused by extracellular ice are eliminated in vitrification.
The present case report demonstrated the effectiveness of a simple vitrification method using VSED solution for the cryopreservation of human blastocysts. This is the first report of a pregnancy resulting from vitrification of a human blastocyst in Japan.
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Note added in proof |
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Notes |
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References |
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Gardner, D.K., Vella, P., Lane, M. et al. (1998) Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil. Steril., 69, 8488.[ISI][Medline]
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Park, S.P., Kim, E.Y., Kim, D.I. et al. (1999) Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids. Hum. Reprod., 14, 28382843.
Quinn, P. and Kerin, J.F.P. (1986) Experience with the cryopreservation of human embryos using the mouse as a model to establish successful techniques. J. In Vitro Fertil. Embryo Transfer, 3, 4045.[Medline]
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World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. 4th edn. Cambridge University Press, Cambridge, UK, pp. 433.
Submitted on January 4, 2000; accepted on May 2, 2000.