Protective effect of N-Acetyl-L-Cysteine on cryopreserved-thawed human ovarian tissue
Objective To investigate the protective effect of antioxidant N-Acetyl-L-Cysteine(NAC)on human ovarian tissue by human ovarian tissue xenotransplantation based on the current human ovarian tissue cryopreservation and thawing protocol in Department of Gynecological Endocrinology,Beijing Obstetrics and Gynecology Hospital,Capital Medical University.Methods The ovarian tissues from 4 patients were cryopreserved and thawed according to the currently-used protocol or the modified protocol(adding with 5 mm NAC).the fresh ovarian tissues underwent Calcein acetoxymethyl ester(Calcein-AM)staining and hematoxylin-eosin(HE)staining to evaluate follicle activity and follicle counting.Thirty-six female nude mice who underwent ovariectomy were randomly divided into 4 groups.The thawing ovarian tissues were transplanted under the bilateral renal capsules of the nude mice.According to the different freezing-thawing protocol,the four group were divided as current freezing-thawing protocol group(control group),the modified protocol group(NAC group),ovariectomy group and normal group(no surgery).The nude mice were killed on day 3,day 7 and day 21 respectively,and serum and ovarian tissue grafts were taken.Serum samples were detected by enzyme-linked immunosorbent assay(ELISA)for estradiol(E2),follicle stimulating hormone(FSH)and anti-mullerian hormone(AMH).One ovarian graft was taken for HE staining to observe follicle development and one graft for total antioxidant capability(TAC).Results There was no significant difference in follicle development stages between the two transplantation groups at each time point after transplantation(P>0.05).The TAC level of NAC group was significantly higher than that of control group(P<0.05),but no difference was found between NAC group and fresh ovarian tissue(P>0.05).The level of serum E2 in control group was significantly higher than that in ovariectomy group on the 3rd day of transplantation(P<0.05),and there was no significant difference between the two transplantation groups and ovariectomy group at other time points(P>0.05).The level of FSH at day 3 and 21 after transplantation was lower than that in ovariectomy group(P<0.05),and had no significant difference from that in normal group(P>0.05).On the 3rd day after transplantation,AMH in the control group was significantly higher than that in the ovarian ovariectomy group(P<0.05),but no significant difference was found between the control group and the other groups(P>0.05).On 7th day after transplantation,AMH level in the two transplantation groups decreased,and was significantly lower than that in the normal group(P<0.05).On the 21st day after transplantation,AMH levels in both transplantation groups were significantly higher than those in ovariectomy group(P<0.05),but had no significant difference with normal group(P>0.05).Conclusions Both the currently-use protocol and the modified protocol can effectively restore the endocrine function of ovarian tissue.Compared with the currently-use cryopreservation regimen,the modified regimen can improve the antioxidant capacity of transplanted ovarian tissue,reduce the primordial follicle activation at the initial stage of transplantation,and preserve more primordial follicles.
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