Successful pregnancy occurred from slowly freezing human oocytes using the regime of 1.5 mol/l 1,2-propanediol with 0.3 mol/l sucrose

Shee-Uan Chen1, Yih-Ron Lien1, Yi-Yi Tsai1, Li-Jung Chang1, Hong-Nerng Ho1 and Yu-Shih Yang,1

1 Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan


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Dear Sir,

We read the article by Fabbri et al. with interest (Fabbri et al., 2001Go). They found that the survival rate was increased when the sucrose concentration was tripled in conjunction with a longer exposure time of 15 min before lowering the temperature. Recently, we used this protocol to cryopreserve eight oocytes with removal of cumuli from a 33-year-old woman with tubal infertility who had 22 oocytes retrieved for an IVF cycle. The remaining 14 fresh oocytes were inseminated and seven fertilized normally. However, no pregnancy was achieved after transfer of four 4-cell embryos to the uterus. The other three embryos were cryopreserved.

During the next cycle, the couple decided to use the frozen oocytes. At the day of ovulation of the woman's natural cycle, the eight cryopreserved oocytes were thawed, and all of them (100%) were morphologically intact. One oocyte was in the metaphase I stage. Seven oocytes were in metaphase II and were micro-fertilized with ICSI as recommended (Fabbri et al., 2001Go). The timing for ICSI was 3 h after completion of thawing. Four normal zygotes (57%) were obtained, and the four cleaving embryos (100%) were placed into the uterus at the 4-cell stage. A successful twin pregnancy was achieved and was ongoing at 9 weeks gestation.

Two major problems that had been associated with oocyte cryopreservation were poor survival and injury of meiotic spindle (Eroglu et al., 1998Go; Fabbri et al., 2001Go). Fabbri et al. increased the sucrose concentration to 0.3 mol/l which was considered to dehydrate the oocyte more adequately, and accomplished a survival rate of 82% (183/224). However, they used only the oocytes frozen in 0.2 mol/l sucrose solution for their clinical trial, and did not specify the timing of ICSI in the article.

Another study (Eroglu et al., 1998Go) found that insemination of slowly cryopreserved mouse oocytes, immediately after thawing, caused a delay in the spindle rotation, second polar body formation, pronuclear formation, migration, and formation of the mitotic spindle. In contrast, insemination after 1 h of incubation resulted in fertilization dynamics similar to those of controls. We examined the patterns of meiotic spindles of vitrified mouse oocytes immediately after dilution, and at 1, 2, and 3 h intervals after dilution and found that they recuperated in a time-dependent process (Chen et al., 2000Go, 2001Go). After 2 or 3 h of incubation, the spindle patterns and fertilization outcomes were significantly improved.

The effects of cryopreservation on the meiotic spindles of human oocytes and their recovery after thawing are not clear and may merit further research. Sufficient restoration of microtubules for the injured spindle after incubation is critical. Early insemination may lead to abnormal fertilization and development. In another aspect, late insemination would result in compromised embryos from aging of oocytes. The timing of insemination of cryopreserved human oocytes varied in reports in the literature, ranging from 1–4 h post-thawing (Chen et al., 2001Go), and may deserve further studies. With reference to our mouse experiments (Chen et al., 2001Go) and in consideration of the possible sophistication of human oocytes, we performed ICSI for human frozen oocytes at 3 h post-thawing.

Our successful initial experience of oocyte cryopreservation encourages further clinical application of this programme. At present, embryo cryopreservation is commonly used in IVF centres for excessive oocytes. However, considering the wider implications of oocyte cryopreservation (Wennerholm, 2000Go), more studies should be conducted to investigate the survival, fertilization, cleavage, and pregnancy rates.


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1 To whom correspondence should be addressed: E-mail: ysyang{at}ha.mc.ntu.edu.tw Back


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Chen, S.U., Lien, Y.L., Chen, H.F., Chao, K-H., Ho, H-N. and Yang, Y-S. (2000) Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum. Reprod., 15, 2598–2603.[Abstract/Free Full Text]

Chen, S.U., Lien, Y.L., Cheng, Y.Y., Chen H-F., Ho, H-N. and Yang, Y-S. (2001). Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids. Hum. Reprod., 16, 2350–2356.[Abstract/Free Full Text]

Eroglu, A., Toth, T.L. and Toner, M. (1998) Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes. Fertil. Steril., 69, 944–957.[ISI][Medline]

Fabbri, R., Porcu, E., Marsella, T., Rocchetta, G., Venturoli, S. and Flamigni, C. (2001) Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum. Reprod., 16, 411–416.[Abstract/Free Full Text]

Wennerholm, W.B. (2000) Cryopreservation of embryos and oocytes: obstetric outcome and health in children. Hum. Reprod., 15, (Suppl. 5), 18–25.





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