Cellular and molecular impact of vitrification versus slow freezing on ovarian tissue.

OBJECTIVE: To evaluate a vitrification protocol from histology to gene expression to slow freezing. METHODS: Ovaries from twelve pre-pubertal ewes. The same ovary was cut into fragments studied fresh, frozen and vitrified. Follicle morphology by HES staining, vitality by trypan blue and apoptosis by marking cleaved caspase-3 were studied. Expression of gene: AMH, CYP11A, and STAR (granulosa cells); GDF9 and ZP3 (oocytes); and CCND2 and CDKN1A (cell cycle regulation), was evaluated by rt-qPCR. RESULTS: The slow freezing protocol had a significant negative impact on intact primordial follicles compared to fresh tissue (37.6% vs. 62.5%, p = 0.003). More intact follicles after vitrification were observed compared to slow freezing (p = 0.037). The apoptotic primordial follicles were similar after slow freezing and vitrification (12.6% vs 13.9%). Concerning granulosa cells genes, slow freezing led to a trend towards overexpression of AMH mRNA (p = 0.07); while vitrification led to a significant overexpression of CYP11A mRNA (p = 0.003), and a trend towards an overexpression of STAR mRNA (p = 0.06). Concerning oocytes genes, both techniques did not lead to a difference of GDF9 and ZP3 mRNA. Concerning cell cycle genes, slow freezing led to a significant underexpression of CCND2 (p = 0.04); while vitrification did not lead to a difference for CCND2 and CDKN1A mRNA. CONCLUSION: Vitrification preserved follicular morphology better than slow freezing and led to gene overexpressed, while slow freezing led to gene underexpressed.