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See detailSupplementation of transport and freezing media with anti-apoptotic drugs improves ovarian cortex survival
HENRY, Laurie ULg; Fransolet, Maïté ULg; LABIED, Soraya ULg et al

in Journal of Ovarian Research (2016)

Background: Ovarian tissue preservation is proposed to patients at risk of premature ovarian failure, but this procedure still needs to be optimized. To limit injury during ovarian tissue cryopreservation ... [more ▼]

Background: Ovarian tissue preservation is proposed to patients at risk of premature ovarian failure, but this procedure still needs to be optimized. To limit injury during ovarian tissue cryopreservation, anti-apoptotic drugs were added to the transport and freezing media of ovarian cortex tissue. Methods: Sheep ovaries were transported, prepared and frozen in solutions containing vehicle or anti-apoptotic drugs (Z-VAD-FMK, a pan-caspase inhibitor, or sphingosine-1-phosphate (S1P), a bioactive lipid). After the tissue was thawed, the ovarian cortex was cultured for 2 or 6 days. Follicular quantification and morphological and proliferation analyses were performed on histological sections. Results: After 2 days of culture, S1P improved the quality of primordial follicles; higher densities of morphologically normal and proliferative primordial follicles were found. Z-VAD-FMK displayed similar effects by preserving global primordial follicular density, but this effect was evident after 6 days of culture. This drug also improved cell proliferation after 2 and 6 days of culture. Conclusions: Our results showed that the addition of S1P or Z-VAD-FMK to the transport and freezing media prior to ovarian tissue cryopreservation improves primordial follicular quality and therefore improves global tissue survival. This should ultimately lead to improved fertility restoration after auto-transplantation. [less ▲]

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See detailIn vitro evaluation of an anti-apoptotic drug, Z-VAD-FMK, for further use in ovarian tissue transplantation
Fransolet, Maïté ULg; HENRY, Laurie ULg; LABIED, Soraya ULg et al

Poster (2015, June 14)

Study question: In a model reproducing early ischemia after ovarian tissue transplantation, does the pan-caspase inhibitor Z-VAD-FMK could prevent granulosa cell apoptosis? Summary answer: Results ... [more ▼]

Study question: In a model reproducing early ischemia after ovarian tissue transplantation, does the pan-caspase inhibitor Z-VAD-FMK could prevent granulosa cell apoptosis? Summary answer: Results obtained with HGL5 granulosa cell line suggest that Z-VAD-FMK is efficient to protect granulosa cells from etoposide or CoCl2 induced apoptosis. What is known already: Removal, cryopreservation and subsequent graft of ovarian strips after cancer treatment have been successfully used to re-establish female fertility. However, the pregnancy rate after autografting of cryopreserved tissue is about 30%. Indeed, the major problem after transplantation is follicular loss due to ischemic reperfusion injury. Study design, size, duration: Three human granulosa cell lines (GC1a, HGL5 and COV434) were cultured during 48h with Z-VAD-FMK with or without etoposide to induce apoptosis. To reproduce the ischemic phase of the graft, cells were cultured without serum under reduced O2 (1%) or with CoCl2 (chemical hypoxia). Participants/materials, setting, methods: Granulosa cells were used as a model since they are essential for oocyte survival. Metabolic cell activity was evaluated by the WST-1 assay. Cell apoptosis was analyzed by flow cytometry after annexin V-FITC and propidium iodide double staining. The mRNA levels and protein expression of apoptotic markers were evaluated using RT-qPCR and western blot analysis. Main results and the role of chance: Flow cytometry showed that cells co-treated with etoposide and Z-VAD-FMK displayed a higher percentage of viable cells as compared to etoposide alone. When in vivo ischemic stage was mimicked (1% O2), no beneficial effect of the Z-VAD-FMK was detected. However, a significant decrease of the number of early apoptotic cells was evidenced by flow cytometry for HGL5 cells treated with Z-VAD-FMK. RT-qPCR and western blot analysis revealed that apoptotic molecules were not modulated. Metabolic activity of the 3 cell lines was reduced by CoCl2. For HGL5 cells, this decrease was partially reversed by Z-VAD-FMK. The number of viable cells was reduced by CoCl2 in HGL5 cells but Z-VAD-FMK allowed to preserve a similar number of viable and apoptotic cells than in control condition. Limitations, reasons for caution: In this study we used 3 different cell lines but granulosa cells represent only a part of the cell types present in ovarian tissue biopsies. Experiences on the effect of Z-VAD-FMK on primary culture of granulosa cells have not yet been realized. Wider implications of the findings: This study suggests that the use of an antiapoptotic drug could be efficient to improve ovarian tissue transplantation outcomes. Ovarian tissue grafting studies using our xenograft murine model will be performed to test the potential efficacy of this drug to improve tissue viability and primordial follicles preservation after transplantation. [less ▲]

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See detailIsoform 165 of vascular endothelial growth factor in collagen matrix improves ovine cryopreserved ovarian tissue revascularisation after xenotransplantation in mice.
Henry, Laurie ULg; LABIED, Soraya ULg; Fransolet, Maïté ULg et al

in Reproductive biology and endocrinology (2015), 13(1), 15

BACKGROUND: Aggressive anti-cancer treatments can result in ovarian failure. Ovarian cryopreservation has been developed to preserve the fertility of young women, but early graft revascularisation still ... [more ▼]

BACKGROUND: Aggressive anti-cancer treatments can result in ovarian failure. Ovarian cryopreservation has been developed to preserve the fertility of young women, but early graft revascularisation still requires improvement. METHODS: Frozen/thawed sheep ovarian cortical biopsies were embedded in collagen matrix with or without isoform 165 of vascular endothelial growth factor (VEGF165) and transplanted into ovaries of immunodeficient mice. Ovaries were chosen as transplantation sites to more closely resemble clinical conditions in which orthotopic transplantation has previously allowed several spontaneous pregnancies. RESULTS: We found that VEGF165 significantly increased the number of Dextran-FITC positive functional vessels 3 days after grafting. Dextran- fluorescein isothiocyanate (FITC) positive vessels were detectable in 53% and 29% of the mice in the VEGF-treated and control groups, respectively. Among these positive fragments, 50% in the treated group displayed mature smooth-muscle-actin-alpha (alpha-SMA) positive functional vessels compared with 0% in the control group. CD31 positive murine blood vessels were observed in 40% of the VEGF165 transplants compared with 21% of the controls. After 3 weeks, the density of murine vessels was significantly higher in the VEGF165 group. CONCLUSION: The encapsulation of ovarian tissue in collagen matrix in the presence of VEGF165 before grafting has a positive effect on functional blood vessel recruitment. It can be considered as a useful technique to be improved and further developed before human clinical applications in female cancer patients in the context of fertility preservation. [less ▲]

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See detailCancer et droit à la maternité
Henry, Laurie ULg; Labied, Soraya ULg; Chiaradia, Fanny et al

in Revue Médicale de Liège (2014), 69(4), 188-193

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