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Vertebrate reproductive science and technology
RESEARCH ARTICLE

Effect of different vitrification protocols for human ovarian tissue on reactive oxygen species and apoptosis

Gohar Rahimi A C , Eugenia Isachenko A , Heinrich Sauer B , Vladimir Isachenko A , Maria Wartenberg B , Jürgen Hescheler B , Peter Mallmann A and Frank Nawroth A
+ Author Affiliations
- Author Affiliations

A Department of Obstetrics and Gynaecology, University of Cologne, Cologne, Germany.

B Institute of Neurophysiology, University of Cologne, Cologne, Germany.

C To whom correspondence should be addressed. email: shiva7de@yahoo.com

Reproduction, Fertility and Development 15(6) 343-349 https://doi.org/10.1071/RD02063
Submitted: 31 July 2002  Accepted: 29 August 2003   Published: 29 August 2003

Abstract

The aim of the present study was to evaluate the effect of different vitrification protocols on reactive oxygen species (ROS) and apoptosis in human ovarian tissue. Human ovarian tissue pieces were exposed to different vitrification solutions. The intracellular redox state level was measured using the fluorescent dye dichlorodihydrofluorescein diacetate. Imaging of apoptotic cells was monitored by anti-caspase-3 immunolabelling after vitrification and warming. Following equilibration in either 40% ethylene glycol (EG) (v/v), 0.35 M sucrose + 10% egg yolk extract (v/v) or 40% EG (v/v), 18% Ficoll-70 (w/v) + 0.35 M sucrose for 6 min, ovarian pieces were cooled to −196°C using four different protocols. Tissue that was cooled very rapidly (plunged directly into liquid nitrogen in straws or on grids or plunged directly into metal filings precooled to −196°C) showed no statistically significant increase in either tissue ROS levels or the number of apoptotic cells after warming. In contrast, cooling using a less rapid method (nitrogen vapour at −120°C) resulted in significantly elevated ROS levels and apoptosis after warming. There were no significant differences between the two vitrification solutions. This indicates that human ovarian tissue pieces should be vitrified using very rapid cooling rates.


Acknowledgment

The study was supported by Koeln Fortune Program/Faculty of Medicine, University of Cologne.


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