Comparison of open and a novel closed vitrification system with slow freezing for human ovarian tissue
Yodo Sugishita1,2,3, Enes Taylan1, Tai Kawahara1,2
1Laboratory of Molecular Reproduction and Fertility Preservation, Department of Obstetrics, Gynecology & Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, FMB 224, New Haven, CT, 06520, USA.
Purpose:
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.
Methods:
A total of 50 pieces of 5 × 5 × 1 mm ovarian cortical pieces were harvested (5 donor ovaries; mean age 31 ± 6.62 years). 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 8-10 weeks of cryostorage, tissues were evaluated 4 h 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.
Results:
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.
Conclusion:
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.
