Effects Of Two Freezing Methods On Cryopreservation And Autotransplantation In Mouse Ovarian Tissue

Objective: Two different methods were used to freeze mice ovarian tissues and the effects of ovarian tissues cryopreservation and autotransplantation were evaluated by detecting estrous cycle,serum AMH level and follicle apoptosis rate to provide experimental evidence for the fertility preservation of women with cancer in clinic.Method: 90 mice were randomly divided into three groups,and there were 30 mice in each group.Group A was the control group,and group B and group C were the experimental groups.In group A,autotransplantation was taken directly after ovariectomy.In group B,autotransplantation was performed 21 days after the ovarian tissues cryopreservation by slow programmed freezing method immediately after ovariectomy.In group C,autotransplantation was performed 21 days after the ovarian tissues cryopreservation by vitrification method immediately after ovariectomy.Blood samples were obtained at the day ovariectomied,the day ovarian transplantated and 21 days after ovarian transplantated to detect the level of serum AMH.The follicle apoptosis rates were detected in the fresh ovarian tissues,thawing ovarian tissues and transplanted ovarian tissues by TUNEL.The estrous cycles of the mice were observed daily during the experiment.Result: The estrous cycle disappeared in mice of the two experimental groups during the time from the ovariectomy to transplantation,and the estrous cycles in group C were significantly longer than those in group B(P<0.05).The estrous cycle days after transplantation in mice of the three groups were significantly longer than those before ovariectomy(P<0.05).The serum AMH levels of two experimental groups mice were significantly lower at the day ovariectomy than those before the transplantation(P<0.05).21 days after the transplantation,the serum AMHlevels increased significantly than before(P<0.05),and the AMH levels in group B were significantly higher than those in group C(P<0.05).21 days after the transplantation,the serum AMH levels of the three groups were significantly lower than those before ovariectomy(P<0.05).After the transplantation,the apoptosis rates of primordial follicle and antral follicle in two experimental groups were significantly higher than those in control group(P<0.05),and the apoptosis rates of the preantral follicle in group C were significantly higher than those in group A and group B(P<0.05).In group A,the apoptosis rates of primordial follicle and preantral follicle in transplanted ovarian tissues were significantly lower than those in fresh ovarian tissues(P<0.05).In group B and group C,the apoptosis rates of primordial follicle,preantral follicle and antral follicle in ovarian tissues after thawing and tansplantation were significantly lower than those in ovarian tissues before the cryopreservation(P<0.05).Conclusion:1.Both the programmed freezing and vitrification could damage the follicles of mice to prolong the estrous cycle.2.Both the programmed freezing and vitrification could cause the apoptosis of antral follicles in mice to affect the secretion of AMH.But this influence by slow programmed freezing was less than that by vitrification.3.The effects of cryopreservation on the structure and function of the mice ovary could be recovered after transplantation,but it could not reach the level before ovariectomy.4.Compared between the two cryopreservation methods,slow programmed freezing was more advantageous in freezing mice ovarian tissues.