Evaluation of Z-VAD-FMK as an anti-apoptotic drug to prevent granulosa cell apoptosis and follicular death after human ovarian tissue transplantation

Purpose 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.
Methods In vitro, granulosa cells were exposed to hypoxic conditions, reproducing early ischemia after ovarian tissue trans- plantation, 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. Results 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. Conclusions In situ administration of Z-VAD-FMK slightly improves primary follicular preservation and reduces global apo- ptosis 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.