Comparison of open and a novel closed vitrification system with slow freezing for human ovarian tissue cryopreservation.

Abstract

To investigate the differences concerning post-thawing/warming follicle survival, DNA damage and apoptosis in human ovarian tissues cryopreserved by slow freezing, open, or closed vitrification methods. A total of 50 pieces of 5 × 5 × 1mm ovarian cortical pieces were harvested (5 donor ovaries; mean age 31 ± 6.62years). From each donor, one cortical piece was used as baseline; the remaining were randomly assigned to slow freezing (SF), vitrification using open device (VF-open), or closed device (VF-closed) groups. After 8-10weeks of cryostorage, tissues were evaluated 4h after thawing/warming. Histological analysis was evaluated for follicle survival (primordial and primary follicle densities) by H&E staining. The percentages of primordial and primary follicles with DNA double-strand breaks (γH2AX) and apoptotic cell death pathway activation (AC3) were immunohistochemically assessed. Data were analysed using one-way ANOVA and LSD post hoc comparison. Compared to the baseline, primordial follicle (pdf) densities significantly declined in all cryopreserved groups (SF, VF-open, and VF-closed, P < 0.05). However, the total and non-apoptotic pdf densities were similar among SF, VF-open, and VF-closed. SF and VF with either open or closed devices did not increase the percentages of primordial or primary follicles with DNA double-strand breaks (DSBs) or apoptosis compared to the baseline or among the freezing methods in the present study. Based on the intact primordial follicle survival, DNA damage, and apoptosis rates after thawing/warming, SF vs VF with either open or newly developed closed devices appear to be comparable.