Hum. Reprod. Advance Access originally published online on February 27, 2004
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Human Reproduction, Vol. 19, No. 4, 988-990,
April 2004
© 2004 European Society of Human Reproduction and Embryology
Case report: successful pregnancy after vitrification of a human blastocyst that had completely escaped from the zona pellucida on day 6
1 Kinutani Women’s Clinic, 2-1-4-3F, Ohtemachi, Naka-ku, Hiroshima 730-0051, Japan
2 To whom correspondence should be sent. e-mail: mkinu0826{at}aol.com
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Abstract |
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This case report describes a successful pregnancy after vitrification of a human hatched blastocyst. A 31-year-old woman, after failed stimulated and thaw cycles, underwent short-treatment protocol stimulation, and oocytes were recovered transvaginally with ultrasound guidance. Eight mature oocytes were obtained and six were fertilized with conventional IVF. Consecutive embryo transfer was performed, in which two cleaved embryos were transferred on day 3 and a single blastocyst was transferred on day 5, but no implantation occurred. On day 6, one of the non-transferred embryos developed into a blastocyst that had completely escaped from the zona pellucida. The zona-free hatched blastocyst was vitrified using a cryotop procedure after artificial shrinkage, which in our clinical experience has proved to be effective for zona-intact blastocysts. Six months after the previous retrieval cycle, the cryopreserved hatched blastocyst survived the warming process and was transferred to the patient’s uterus. Implantation resulted in a healthy pregnancy; the pregnancy is ongoing at 33 weeks. This is the first report of a pregnancy after vitrification of a human blastocyst that had completely escaped from the zona pellucida.
Key words: artificial shrinkage/cryotop/hatched blastocyst/human/vitrification
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Introduction |
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Since the first pregnancy after vitrification of a human blastocyst was reported using cryostraws (Yokota et al., 2000
), more attention has been focused on an ultra-rapid vitrification that involves direct contact between a reduced volume of cryoprotectant and liquid nitrogen. By greatly increasing the cooling and thawing rate, the formation of ice crystals and chilling injuries are reduced. Many human pregnancies that originate from an ultra-rapid vitrification technique are achieved after cryopreservation of blastocysts using the cryotop (Kuwayama and Kato, 2000), the cryoloop (Mukaida et al., 2001, 2003; Reed et al., 2002), electron microscope grids (Choi et al., 2000; Son et al., 2003) or the hemi-straw (Vanderzwalmen et al., 2003). In addition, the survival rate of expanded blastocysts after vitrification increases when, to reduce ice crystal formation, the blastocoele is artificially shrunk with pipetting (Motoishi, 2000), or the use of a glass micro-needle (Vanderzwalmen et al., 2002) or two 29-gauge needles (Son et al., 2003). However, to the best of our knowledge, there are no reports of pregnancies following human hatched blastocyst vitrification. We did find two reports in which hatched blastocysts are frozen by a slow-cooling method (Khorram et al., 2000; Quintans et al., 2003). In this case report, we present a successful pregnancy after the transfer of a vitrified human blastocyst at the hatched stage of development using a cryotop procedure after artificial shrinkage of the blastocoele. |
Case report |
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A 31-year-old woman and her 31-year-old husband consulted our clinic because of secondary infertility. The woman had tubal factor infertility. The husband’s semen characteristics were within normal parameters according to World Health Organization criteria (World Health Organization, 1999
).
After the first unsuccessful IVF cycle (day 2 transfer) and one unsuccessful thaw (day 2 embryo) cycle, the patient was treated with the GnRH analogues buserelin acetate (MOCHIDA, Tokyo, Japan) and HMG (Nikken, Tokyo, Japan) using a short-treatment protocol. On cycle day 11, 10 000 IU of HCG (TEIZO, Tokyo, Japan) was administered; ovum pick-up was performed 35 h later. Following recovery of eight mature oocytes, six were fertilized following conventional IVF and were cultured in vitro until day 5 in order to perform a consecutive embryo transfer (Goto et al., 2003). Briefly, two cleaved embryos were transferred on day 3 and a single blastocyst was transferred on day 5, but no implantation occurred. The remaining three embryos continued to be cultured in vitro until day 6. One of these grew to a hatched stage blastocyst that had completely escaped from the zona (Figure 1D).
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Since July 2002, we have been cryopreserving expanded zona-intact blastocysts clinically by the method developed by Kuwayama (2001
) using a cryotop, which consists of polyethylene laminate film (20 mm x 0.7 mm x 0.1 mm, L x W x T; Kitazato Supply, Inc., Shizuoka, Japan). In our blastocyst vitrification system, we used aspiration in and out (pipetting) of a glass pipette to shrink the blastocoele (Motoishi, 2000) (Figure 1B). Figure 1A and C shows the morphology of human expanded zona-intact blastocysts before cryopreservation. From October 2002 to October 2003, a total of 19 vitrified expanded blastocysts from 11 patients have been warmed. Eighteen blastocysts (95%) re-expanded 3 h after warming and were transferred into 10 patients. The implantation rate was 44% (8/18) and the pregnancy rate was 60% (6/10). One of the women delivered a healthy boy (2810 g) at 39 weeks of gestation on November 8, 2003. No obvious anomalies were detected. All of the other five pregnancies are proceeding normally. To date, there have been no spontaneous abortions. Based on the above clinical results, we applied the same vitrification protocol to the hatched blastocyst.
The hatched blastocyst was vitrified using a two-step protocol (Kuwayama, 2001). As the base medium, Dulbecco’s phosphate-buffered saline solution (PBS 1x; Irvine Scientific, CA) plus 20% (v/v) serum substitute supplement (SSS; Irvine) was used. The equilibration solution contained 7.5% (v/v) ethylene glycol (EG) (Sigma Chemical Co., MO) and 7.5% (v/v) dimethylsulfoxide (DMSO) (Kanto Chemical Co., Tokyo, Japan). The vitrification solution was composed of 15% (v/v) EG, 15% (v/v) DMSO and 0.5 mol/l sucrose (Nacalai Tesque, Inc., Kyoto, Japan). Both cryoprotectant solutions were warmed briefly in an incubator at 37°C, and the hatched blastocyst was handled on the stage warmer of a dissecting microscope at 38°C.
Before starting the vitrification procedure, artificial shrinkage of the hatched blastocyst was performed in the equilibration solution. First, pipetting of the blastocyst was conducted with a fine hand-drawn glass pipette slightly smaller in diameter than the hatched blastocyst (Figure 1E). After confirmation of slight shrinkage of the blastocoele, pipetting was performed with a pipette slightly smaller in diameter than the first one. This procedure was repeated 2–3 times until the blastocoele completely collapsed (Figure 1F).
After contraction of the blastocoele, the hatched blastocyst was equilibrated in the equilibration solution for another 2 min before exposure to the vitrification solution. The hatched blastocyst was then incubated in the vitrification solution and loaded on to the tip of the cryotop with 
1 µl of cryoprotectant solution for 45 s. Then the cryotop was immediately plunged into liquid nitrogen.
Before warming the hatched blastocyst, 1.0 mol/l sucrose solution, 0.5 mol/l sucrose solution and the base medium were warmed briefly in an incubator at 37°C. The warming procedure was done as follows. The tip of the cryotop with the hatched blastocyst was plunged directly into 1.0 mol/l sucrose solution for 1 min. The hatched blastocyst was then transferred to 0.5 mol/l sucrose solution for 3 min and washed twice in the base medium for 5 min. All the steps were completed on the stage warmer of a dissecting microscope at 38°C. The warmed hatched blastocyst was cultured for 2 h until embryo transfer (Figure 1G and H).
The warmed hatched blastocyst was transferred to the patient’s uterus during a natural cycle, 6 months after the previous retrieval cycle. On day 9 after the embryo transfer, pregnancy was confirmed with a positive HCG; 6 weeks later, an ultrasound showed a healthy beating fetal heart inside a clear and distinct gestational sac. At the time of writing, the pregnancy has progressed to 33 weeks.
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Discussion |
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TopAbstractIntroductionCase reportDiscussionReferences |
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Our results demonstrated that a blastocyst that had completely escaped from the zona pellucida could be cryopreserved by an ultra-rapid vitrification and, following embryo transfer, result in a successful pregnancy. Two possible explanations could account for this success. First, the blastocoele was artificially shrunk during equilibration. Secondly, the hatched blastocyst was transferred 2 h after warming.
In human zona-intact expanded blastocysts, results improve when the blastocoelic cavity is shrunk artificially (Motoishi, 2000; Vanderzwalmen et al., 2002; Son et al., 2003). In addition, rabbit zona-free blastocysts are satisfactorily vitrified by a two-step cryoprotectant addition procedure in which blastocoelic cavity reduction is observed (Cervera and Garcia-Ximénez, 2003). In our case, the blastocoele was artificially shrunk during equilibration, which could be why no cryoinjury by ice crystal formation was observed.
In an in vitro model for studying human embryo implantation into the endometrial stroma, hatched human blastocysts placed on the stromal cell layer remain expanded but unattached for a number of hours. After attaching to the stroma, they appear to undergo contraction and become less expanded before entering the invasive stage (Carver et al., 2003). After the blastocyst has completely escaped from the zona pellucida, the hatched blastocyst may attach to the endometrium as the hatched blastocyst fully expands. Warmed expanded zona-intact blastocysts artificially shrunk by pipetting re-expanded with approximately the same degree of expansion as before cryopreservation 3 h after warming (our personal observation). This was the reason the warmed hatched blastocyst was transferred before becoming fully re-expanded, i.e. 2 h after warming.
As was studied by Khorram et al. (2000), hatching of human blastocysts by day 6 is a favourable prognostic factor for IVF outcome. Embryos that hatch by day 6 may have a higher implantation potential. In conclusion, the zona-hatched blastocyst cryopreserved by an ultra-rapid vitrification could contribute to preventing wastage of higher quality supernumerary embryos and to extending the strategy of blastocyst vitrification in human assisted reproductive technology.
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References |
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Submitted on September 1, 2003; resubmitted on November 14, 2003; accepted on January 26, 2004.
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