Evaluation of Z-VAD-FMK as an anti-apoptotic drug to prevent granulosa cell apoptosis and follicular death after human ovarian tissue transplantation
To evaluate the efficiency of ovarian tissue treatment with Z-VAD-FMK, a broad-spectrum caspase inhibitor, to prevent follicle loss induced by ischemia/reperfusion injury after transplantation.
In vitro, granulosa cells were exposed to hypoxic conditions, reproducing early ischemia after ovarian tissue transplantation, and treated with Z-VAD-FMK (50 μM). In vivo, cryopreserved human ovarian fragments (n = 39) were embedded in a collagen matrix containing or not Z-VAD-FMK (50 μM) and xenotransplanted on SCID mice ovaries for 3 days or 3 weeks.
In vitro, Z-VAD-FMK maintained the metabolic activity of granulosa cells, reduced HGL5 cell death, and decreased PARP cleavage. In vivo, no improvement of follicular pool and global tissue preservation was observed with Z-VAD-FMK in ovarian tissue recovered 3-days post-grafting. Conversely, after 3 weeks of transplantation, the primary follicular density was higher in fragments treated with Z-VAD-FMK. This improvement was associated with a decreased percentage of apoptosis in the tissue.
In situ administration of Z-VAD-FMK slightly improves primary follicular preservation and reduces global apoptosis after 3 weeks of transplantation. Data presented herein will help to guide further researches towards a combined approach targeting multiple cell death pathways, angiogenesis stimulation, and follicular recruitment inhibition.
KeywordsFertility preservation Apoptosis Granulosa cells Ovarian tissue transplantation Z-VAD-FMK
The authors thank the CPMA-CHR Citadelle Liège for their contribution to follicular fluids collection. The authors are very grateful to M. C. Masereel and S. Schoenen for their help for follicular quantification. The authors acknowledge M. Dehuy, E. Konradowski, E. Feyereisen, and I. Dasoul for their excellent technical assistance.
The authors thank the Fonds de la Recherche Scientifique-FNRS (F.R.S.-FNRS, Télévie, Belgium; 7.4597.12-7.4622.14-7.4590.16), the Fondation contre le Cancer (foundation of public interest, Belgium), the Fonds spéciaux de la Recherche (University of Liège), the Centre Anticancéreux près l’Université de Liège, the Fonds Léon Fredericq (University of Liège), the Direction Générale Opérationnelle de l’Economie, de l’Emploi et de la Recherche from the Service Public de Wallonie (SPW, Belgium), the Interuniversity Attraction Poles Programme-Belgian Science Policy (Brussels, Belgium), the Plan National Cancer (Service Public Fédéral), and the Actions de Recherche Concertées (University of Liege, Belgium). The authors also thank the GIGA (Groupe Interdisciplinaire de Génoprotéomique Appliquée, University of Liege, Belgium) for the access to the GIGA-Imaging and Flow Cytometry platform and Dr. S. Ormenese and R. Stephan for their support with FACS analyses.
MF performed experiments, interpreted data, and wrote the manuscript. LN contributed to in vitro experiments. LH and SL interpreted data and revised the manuscript. SB performed the computer images analysis. MN conceived and designed the study and corrected the manuscript. CM conceived and designed the study, interpreted data, corrected the manuscript, and substantially contributed to critical revisions. All authors read and approved the final manuscript.
Compliance with ethical standards
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
The use of human follicular fluid and ovarian tissue was approved by the Ethics Committee of the CHR Citadelle, University of Liège (CE412/1508 and CE412/1448, respectively). The use of the xenograft model was approved by the Animal Ethics Committee of the University of Liège (Reference 1304, October 2012).
Informed consent was obtained from all individual participants included in the study.
Conflict of interest
The authors declare that they have no conflict of interest.
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