Apoptosis in autotransplanted cryopreserved rat ovarian tissues

Abstract

OBJECTIVE: To evaluate apoptosis in autoransplanted rat ovarian tissues after Vitrification (V) or Slow Freezing (SF) cryopreservation. DESIGN: Prospective laboratory study. MATERIALS AND METHODS: Mature rat (n=14) ovaries were placed in phosphate buffered saline (PBS) and randomly assigned to one of two treatments: V plus autotransplantation (VA) or SF plus autotransplantion (SFA). The V (n=8) was done using 1M dimethylsulfoxide(DMSO) followed by 2M DMSO + 1M Acetamide + 1M propylene glycol in PBS. Ovaries were thawed at 25°C for 30 sec in PBS containing 0.25M sucrose. SF (n=6) was done in 1.5 M DMSO and 0.1 M Sucrose. Ovaries were initially cooled to -6°C at -2°C/min, ice nucleated, cooled at -0.3°C/min to -40°C then at -10°C/min to -80°C then plunged in LN. Thawing was done at 25°C using 0.75M DMSO, and 0.25M sucrose. Ovarian autotransplantation (n=5) was performed within 1 h of thawing. Pre-freeze, post-freeze and post-transplant sections were microscopically analyzed for apoptosis with DNA staining using DAPI and TUNEL. Results were analyzed by ANOVA with Tukey post-hoc testing and paired- t-test. RESULTS: Follicular apoptosis was lower in pre-frozen ovarian tissue as compared to post-frozen tissue in SF and V methods (p=0.0478 and p=0.0009 respectively). No difference was found in the number of total follicles between SF and V methods but the amount of damage to living follicular cells was increased in V (p=0.0071). After transplantation, the number of apoptotic cells was lower in pre-frozen tissue than in post-transplanted tissue (p=0.0001). No significant difference was seen in the amount of apoptotic cells between post-frozen and post-transplanted ovarian tissue, suggesting that more damage to the follicles occurred during the freezing/thawing process than by autotransplantation. CONCLUSIONS: As indicated by apoptosis, SF may be less traumatic, post thaw, than V and the freezing/thawing process contributes more to the follicular loss than autotransplantation.