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Hum. Reprod. Advance Access published online on September 30, 2005
Human Reproduction, doi:10.1093/humrep/dei320
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© The Author 2005. 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
Received May 26, 2005
Revised August 22, 2005
Accepted August 25, 2005

Article

Evaluation of Liberase, a purified enzyme blend, for the isolation of human primordial and primary ovarian follicles

Marie-Madeleine Dolmans 1, Nathalie Michaux 1, Alessandra Camboni 2, Belen Martinez-Madrid 1, Anne Van Langendonckt 1, Stefania Annarita Nottola 3, and Jacques Donnez 1*

1 Department of Gynecology, Université Catholique de Louvain, Avenue Hippocrate 10, 1200 Brussels, Belgium
2 Department of Gynecology, Université Catholique de Louvain, Avenue Hippocrate 10, 1200 Brussels, Belgium; Department of Anatomy, University of Rome ‘La Sapienza’, Rome 00161, Italy
3 Department of Anatomy, University of Rome ‘La Sapienza’, Rome 00161, Italy

* To whom correspondence should be addressed.
Jacques Donnez, E-mail: Donnez{at}gyne.ucl.ac.be


  Abstract

BACKGROUND: The purpose of this study is to evaluate the effectiveness of a standardized mixture of purified enzymes (Liberase), for the isolation of human ovarian follicles. METHODS: This is an experimental prospective study. Ovarian biopsies were obtained from eight young women undergoing laparoscopy for benign gynaecological disease. Follicles were isolated by Liberase or collagenase enzymatic digestion. Follicle quality was assessed by evaluating their general morphology and viability after fluorescent staining, and the ultrastructure by electron microscopy. RESULTS: The number of fully isolated follicles recovered from the Liberase-treated group was lower than from the collagenase group (156 versus 263) despite equal-sized biopsies being taken. A high proportion of follicles (98.6%, 70/71) were viable after Liberase isolation and most follicles were of good morphology with a complete granulosa cell layer (70.4%, 31/44). Ultrastructural studies indicated that Liberase-isolated follicles showed signs of atresia only occasionally and that the oolemma-follicular cell interface was well preserved. CONCLUSIONS: Liberase treatment allows the isolation of highly viable follicles from human ovarian tissue, with an unaltered morphology and ultrastructure. This purified endotoxin-free enzyme preparation is a promising alternative to impure collagenase preparations for the reproducible isolation of intact primordial and primary follicles for culture and grafting purposes.

Keywords: collagenase/fertility preservation/human follicles/isolation/Liberase.
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