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 12 prepubertal ewes. The same ovary was cut into fragments, studied fresh, frozen, and vitrified. Follicle morphology by hematoxylin-eosin-safran staining, vitality by Trypan Blue, and apoptosis by marking cleaved caspase-3 were studied. The expression of gene: anti-Müllerian hormone (AMH), cytochrome p450 family 11 subfamily A member 1 (CYP11A), and steroidogenic acute regulatory protein (STAR; granulosa cells); growth differentiation factor 9 (GDF9) and zona pellucida glycoprotein 3 (ZP3; oocytes); and cyclin D2 (CCND2) and cyclin-dependent kinase inhibitor 1A (CDKN1A; cell cycle regulation), was evaluated by reverse transcription quantitative polymerase chain reaction. Results: The slow freezing protocol had a significant negative impact on intact primordial follicles compared with fresh tissue (37.6% vs. 62.5%, p = 0.003). More intact follicles after vitrification were observed compared with slow freezing ( p = 0.037). The apoptotic primordial follicles were similar after slow freezing and vitrification (12.6% vs. 13.9%). Concerning granulosa cell genes, slow freezing led to a trend toward overexpression of AMH messenger RNA (mRNA; p = 0.07); while vitrification led to a significant overexpression of CYP11A mRNA ( p = 0.003), and a trend toward an overexpression of STAR mRNA ( p = 0.06). Concerning oocyte 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.