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Hum. Reprod. Advance Access published online on January 29, 2004
Human Reproduction, doi:10.1093/humrep/deh151
© 2004 by European Society of Human Reproduction and Embryology
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Received August 26, 2003
Revised November 4, 2003
Accepted December 8, 2003

Article

Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration

Vladimir Isachenko 1*, Markus Montag 2, Eugenia Isachenko 3, Frank Nawroth 3, Salvatore Dessole 4, and Hans van der Ven 2

1 Department of Obstetrics and Gynaecology, University of Cologne, Cologne, Germany; Department of Gynaecological Endocrinology and Reproductive Medicine, University of Bonn, Bonn, Germany
2 Department of Gynaecological Endocrinology and Reproductive Medicine, University of Bonn, Bonn, Germany
3 Department of Obstetrics and Gynaecology, University of Cologne, Cologne, Germany
4 Department of Obstetrics and Gynaecology, Sassari University, Sassari, Italy

* To whom correspondence should be addressed. E-mail: VovaIsachenko{at}yahoo.com.


  Abstract

BACKGROUND: This study compared the viability of human pronuclear oocytes subjected to vitrification followed by post-thaw step-wise removal of cryoprotectants versus direct rehydration, in terms of their subsequent in vitro survival and ultrastructural features. METHODS: A total of 115 three-pronuclei stage oocytes were cryopreserved in super-open-pulled straws by vitrification in 40% ethylene glycol + 0.75 mol/l sucrose for either 1 min or 10 s at 38°C, followed by direct plunging into liquid nitrogen. After thawing, oocytes vitrified for 1 min (group 1) or 10 s (group 2) were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) for removal of the cryoprotectant in five 2.5 min steps. A second batch of oocytes vitrified for either 1 min (group 3) or 10 s (group 4) were directly expelled into culture medium at 38°C after thawing. Finally, the ultrastructural changes occurring in oocytes in each of the treatment groups were evaluated. RESULTS: Oocyte development (division to two-blastomere stage) rates after in vitro culture were 82, 83, 0 and 0% for groups 1, 2, 3 and 4, respectively. The harsh osmotic process involved in direct rehydration provoked ultrastructural changes, including the disruption of cytoplasmic and pronuclear membranes as well as intracellular organelles. CONCLUSION: The direct post-thaw rehydration of human pronuclear oocytes has lethal osmotic effects, such that protocols for vitrifying human pronuclear oocytes should include the step-wise removal of the cryoprotectant.

Key words: Key words: damage/direct rehydration/human oocytes/ultrastructure/vitrification


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