Poster Presentation The Annual Scientific Meeting of the Endocrine Society of Australia and the Society for Reproductive Biology 2014

Viability assessment using in vitro culture or xenografts of prepubretal ovine ovarian cortex cryopreserved by slow freezing or vitrification (#325)

Michael J Bertoldo 1 , Lucie Grecias 1 , Nicolas Duffard 1 , Pascal Mermillod 1 , Yann Locatelli 1
  1. Physiologie de la Reproduction et des Comportements, INRA, Nouzilly, France

Ovarian tissue cryopreservation and transplantation is a fertility preservation technology offered to females at risk of losing ovarian function following cancer treatment.While births have occurred following cryopreservation and transplantation, folliculogenesis is significantly perturbed following thawing and the procedure is still considered experimental.The aim of the present study was to validate two methods of cryopreservation (slow freezing/vitrification) using lamb ovarian material by in vitro culture or xenografts.To assess cryopreservation method,ovarian fragments were either slow frozen or vitrified and subsequently xenografted to the ovarian bursa or cultured. Slow freezing was performed using 30min exposure at 4°C in culture medium supplemented with 10% FCS, 10%DMSO and 0.1M sucrose and exposure to a conventional slow freezing curve.Non-equilibrium vitrification was performed after 5min exposure to culture medium supplemented with 10% FCS+1X[CP;cryoprotectant](CP=5% DMSO,5% Ethylene Glycol and 0.125M sucrose),5min exposure to culture medium supplemented with 10% FCS+2X[CP] and 2min in medium supplemented with 10%FCS+4X[CP] before being plunged into liquid nitrogen. Following 2 and 10 weeks,xenografted tissue was removed, assessed for the development of antral follicles,and oocytes recovered and placed in culture. Histological and PCNA analysis was carried out on cryopreserved fragments following thawing/warming and then at 2,6,10 and 14 days of culture.We observed greater activation of primordial follicles in vitrified/warmed tissue; indicated by the greater numbers of primary and secondary follicles when compared to freshly cultured tissue and slow frozen/thawed tissue.However, a greater proportion of follicles within slow-frozen tissue were positive for PCNA staining.At 2 and 10 weeks following xenotransplantation,there were no differences between cryopreservation treatments in follicle distribution.However, there was a tendency for more primordial follicles in cryopreserved than fresh tissue.We were able to recover a single GVBD oocyte from fresh xenografted tissue after 10 weeks.The results of this preliminary study demonstrate that the 2 cryopreservation methods did not significantly alter folliculogenesis following culture or xenotransplantation.