1 Kinutani Womens 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
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: artificial shrinkage/cryotop/hatched blastocyst/human/vitrification
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Case report |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
The hatched blastocyst was vitrified using a two-step protocol (Kuwayama, 2001). As the base medium, Dulbeccos 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 23 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 patients 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.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cervera RP and Garcia-Ximénez F (2003) Vitrification of zona-free rabbit expanded or hatching blastocysts: a possible model for human blastocysts. Hum Reprod 18,21512156.
Choi DH, Chung HM, Lim JM, Ko JJ, Yoon TK and Cha KY (2000) Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVF-ET program. Fertil Steril 74,838839.[CrossRef][Medline]
Goto S, Takebayashi K, Shiotani M, Fujiwara M, Hirose M and Noda Y (2003) Effectiveness of 2-step (consecutive) embryo transfer. Comparison with cleavage-stage transfer. J Reprod Med 48,370374.[Medline]
Khorram O, Shapiro SS and Jones JM (2000) Transfer of nonassisted hatched and hatching human balstocysts after in vitro fertilization. Fertil Steril 74,163165.[CrossRef][Medline]
Kuwayama M (2001) Vitrification of human oocytes and embryos. In Suzuki S (ed.) IVF Update [in Japanese]. Medical View Co., Tokyo, Japan, pp. 230234.
Kuwayama M and Kato O (2000) All round vitrification of human oocytes and embryos [abstract]. J Assist Reprod Genet 17,477.
Motoishi M (2000) Cryopreservation of human blastocyst [Japanese]. J Clin Embryol 5,614.
Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M and Takahashi K (2001) Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 76,618620.[CrossRef][Medline]
Mukaida T, Nakamura S, Tomiyama T, Wada S, Oka C, Kasai M and Takahashi K (2003) Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 18,384391.
Quintans CJ, Donaldson MJ, Bertolino MV and Pasqualini RS (2003) Birth resulting from transfer of blastocysts cryopreserved with propanediol after spontaneous hatching. Reprod Biomed. Online 6,6668.[Medline]
Reed ML, Lane M, Gardner DK, Jensen NL and Thompson J (2002) Vitrification of human blastocysts using the cryoloop method: successful clinical application and birth of offspring. J Assist Reprod Genet 19,304306.[CrossRef][Medline]
Son WY, Yoon SH, Yoon HJ, Lee SM and Lim JH (2003) Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod 18,137139.
Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, van Roosendaal E, Vandervorst M, Bollen N, Zech H, Mukaida T, Takahashi K et al. (2002) Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 17,744751.
Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, Bollen N, van Roosendaal E, Vandervorst M, Schoysman R and Zech N (2003) Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum Reprod 18,15041511.
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction, 4th edn, Cambridge University Press, Cambridge, pp. 433.
Yokota Y, Sato S, Yokota M, Ishikawa Y, Makita M, Asada T and Araki Y (2000) Successful pregnancy following blastocyst vitrification. Hum Reprod 15,18021803.
Submitted on September 1, 2003; resubmitted on November 14, 2003; accepted on January 26, 2004.