Effects of Cryopreservation on the Molecular Integrity of Ovarian Grafts

Abstract

INTRODUCTION: The objective of this study was to compare the effects of slow freezing and vitrification on the molecular integrity of ovarian grafts. METHODS: Fresh bovine ovaries were processed and divided into three groups (fresh control, slow freezing, and vitrification) before transplantation. For slow freezing and vitrification, 1.5 M dimethylsulfoxide-based cryoprotectant and a two-step vitrification method were used, respectively. Two pieces of ovarian tissue were transplanted onto the back muscle of the severe combined immunodeficient mouse (six animals for each group). Ovarian grafts were recovered on day 7 and day 28 after transplantation to assess DNA damage, apoptosis, and autophagy by histology, immunohistochemistry, and Western blot analysis. RESULTS: The slow freezing group showed significantly higher normal follicle count compared with the vitrification group. There was no significant difference in apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, caspase 3) among three groups; however, autophagy (LC3B) was decreased in both slow freezing and vitrification groups. On day 28, a significant decrease in LC3B in the vitrification group was noted compared with the slow freezing group. There was no difference in expression of DNA damage and repair markers (γH2AX, Rad51, PCNA) and vascular density makers (CD31). CONCLUSION: The expression pattern of biochemical markers for DNA damage and repair and apoptosis in ovarian grafts was similar between three groups. There is not yet strong evidence that vitrification is better than slow freezing. Therefore, further improved vitrification protocols are needed.