The Study Of Freezen-thawed Human Ovarian Tissue Isolation And Preantral Follicle In Vitro Culture

ObjectiveIn this study, we adopted direct cover vitrification (DCV) to freezen- thawed and preserve human ovarian tissue. Follicles were isolated after ovarian tissue was thawed, we isolated follicles with enzymatic digestion-mechanical isolation and mechanical isolation after follicles grown within human ovarian cortical strip for 6 days in vitro. Integrity survival follicles were selected, then cultured in vitro, using calcium alginate hydrogels culture system to maintain the cell-cell and cell-matrix connections. We cultured follicles in serum and serum-free medium respectively. The objective of this study was to explore easy and effective isolation methods and appropriate culture medium of human preantral follicles growth and development though comparing before and after follicle diameter and estradiol levels.MethodsOvarian cortical tissue was obtained by biopsy from twenty -three patients who were treated by ovarian tumorectomy in the First Affiliated Hospital of Zhengzhou University after obtaining written informed consent. Ovarian cortical tissue was cutted approximate size 0.5 mm x 0.5 mm x 1 mm under stereoscopic microscope and with DCV.After thawing, we isolated follicles with enzymatic digestion-mechanical isolation and mechanical isolation after follicles grown within human ovarian cortical strip for 6 days in vitro. Follicles which were embedded in Calcium alginate matrix were cultured in 10% fetal calf serum (FBS) and 0.3% bovine serum albumin (BSA) medium respectively. Follicle diameters were measured using a dissecting microscope with a crossed micrometer and on Day 2, half the culture medium was removed and replaced with fresh medium.The collected culture medium was stored in refrigerator at -20℃. The level of estradiol was measured with the method of electrochemiluminescence immunoassay (ECLIA). Statistical analysis on the experimental data was done by SPSS 17.0 software package.Result1. We isolated follicles with enzymatic digestion-mechanical isolation. The survival rate of follicles in different developmental stages was not statistically significant before and after cryopreservation(P>0.05). The survival rate of the primordial follicle was highest. It showed Cryopreservation did not effect the survival rate of the follicles, but the primordial follicles were most tolerant in Cryopreservation damage.2. Adopt serum-free culture system for follicular culture. Six days after ovarian tissue culture in vitro, the follicles which we isolated with mechanical method were less than follicles which isolated with enzymatic digestion-mechanical isolation, but the survival rate of the secondary follicles was higher and there were statistical significance between two groups (P<0.05).3. We isolated follicles in serum-free and serum medium with enzymatic digestion-mechanical isolation. We obtained more follicles in BSA group, but the survival rate of follicles in different developmental stages were not Statistically significant between two groups (P<0.05).4. The diameter of follicles which were isolated with mechanical isolation at the end of the 6 day culture period and were cultured for 6 or 10 days in serum-free medium was significantly increased compared other three groups(P<0.05).5. The estradiol release of follicles which were isolated with mechanical isolation at the end of the 6 day culture period and were cultured for 6,8 or 10 days in serum-free medium was significantly increased compared other three groups(P< 0.05).Conclusions1. Direct cover vitrification cryoapplication does not effect the survival rate of human primordial, primary and secondary follicles;2. The end of the 6 day culture period in serum-free medium, follicles which were isolated with mechanical isolation develop quickly;3. Follicles which were isolated in serum-free medium were more than in serum medium, the survival rate of follicle at all levels;4. Serum-free medium is conducive to promote the growth of preantral follicles.