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Hum. Reprod. Advance Access originally published online on January 31, 2008
Human Reproduction 2008 23(4):819-826; doi:10.1093/humrep/den002
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© The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Integrity rate of pronuclei after cryopreservation of pronuclear-zygotes as a criteria for subsequent embryo development and pregnancy

Vladimir Isachenko1,2,3, Plamen Todorov1,2, Yosif Dimitrov2 and Evgenia Isachenko1,2

1 Institute of Biology and Immunology of Reproduction, Sofia, Bulgaria 2 Medical Center of Reproduction, Sofia, Bulgaria

3 Correspondence address. Department of Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Tel: +49-228-28714379; Fax: +49-228-2874651; E-mail: v.isachenko{at}yahoo.com

BACKGROUND: The aim of this study was to examine whether the integrity rate of pronuclei, after cryopreservation of pronuclear zygotes, could be a predictor of future embryo development and implantation.

METHODS: Two-pronuclei stage zygotes (n = 862) were cryopreserved by aseptic rapid freezing in 15% ethylene glycol + 15% DMSO + 0.2 M sucrose with a 4-step exposure in 12% (v:v), 25, 50 and 100% rapid freezing solution for 2, 1, 1 min and 30–50 s, respectively, at room temperature, and then plunged into liquid nitrogen. Zygotes were rapidly warmed at a speed of 30 000°C/min and subsequently expelled into a graded series of sucrose solutions (1.0, 0.5, 0.25, 0.12 and 0.06 M) at 2.5 min intervals and in vitro cultured for 5 days. If embryos had developed to blastocysts on the 5th day, they were transferred to the recipients. The rest of the embryos were fixed for evaluation of chromatin.

RESULTS: Zygote development, up to the expanded blastocyst stage, after in vitro culture, was 40%, if the integrity rate of pronuclei was high, and 4% if the integrity rate was low (P < 0.05). The pregnancy rate after transfer of the 5-day blastocysts depended on the pronuclear integrity rate: 43% (125 pregnancies aftertransfer of 291 blastocysts) for those with a high integrity, and only 1 in 5 (20%) for the few blastocysts which had shown a pronuclear low integrity rate (P < 0.05).

CONCLUSIONS: Integrity rate of pronuclei after cryopreservation of pronuclear zygotes was a predictor of future embryo development and implantation: high integrity rate resulted in high pregnancy rate, while zygotes with low integrity rate of pronuclei after cryopreservation had low developmental potential.

Key words: zygotes/cryopreservation/pronuclei/morphology/integrity rate

Submitted on August 27, 2007; resubmitted on December 14, 2007; accepted on December 31, 2007.


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