The Mechanism Of Cyclophosphamide-induced Endoplasmic Reticulum Stress To Damage Ovarian Function

Objective:To clarify the value of AMH in evaluating ovarian reserve function;to clarify the damage of CTX on ovarian function,to explore the effect of CTX on the secretion of AMH from ovarian granulosa cells,and to further clarify that CTX-induced endoplasmic reticulum stress inhibits AMH secretion and damages ovarian functionMethods:①144 premenopausal breast cancer patients who received adjuvant cyclophosphamide-based chemotherapy were collected.The serum AMH levels of the patients were measured before and 6 months after chemotherapy,and their menstrual changes were followed up② In animal experiments,single-dose CTX(200 mg/kg)was used to treat female C57BL/6J mice in different month-old groups(3 and 6 months old)Before and one week after CTX treatment,the serum AMH level and the number of ovarian follicles of the mice were measured.In addition,the mice model with breast cancer were constructed and randomly divided into Control group,CTX group and GnRHa group,and intraperitoneally inject the same volume of NS,CTX and CTX+GnRHa,respectively.Serum AMH levels were detected before chemotherapy and 1,3 and 5 weeks after treatment.The ovaries of mice were collected and the number of primordial follicles and growing follicles were counted.③KGN cells were treated with CTX alone and the levels of AMH and ER stress-related markers in KGN cells were detected.Results:①The menstrual state of breast cancer patients receiving CTX-based chemotherapy was significantly related to the level of serum AMH before chemotherapy(P<0.05).After CTX-based chemotherapy,the≥35-year-old group had a higher amenorrhea rate and lower menstrual recovery rate than the<35-year-old group.The levels of serum AMH and E2 decreased more significantly(P<0.05),and the increase of FSH levels was greater than<35 year-old-group,but the difference was not statistically significant(P>0.05).There was no significant decrease in serum AMH level in patients treated with CTX combined with GnRHa(P=0.315).② Before and after CTX treatment in mice of different month-old groups,serum AMH levels decreased significantly,and the number of ovarian primordial follicles and growing follicles was significantly less.The differences were statistically significant(P<0.05),and the decrease was more significant in the 6-month-old group(P<0.05).After CTX treatment of tumor-bearing mice,the serum AMH levels,the number of ovarian primordial follicles and the number of growing follicles decreased significantly with the time of chemotherapy.However,the decrease was not significant in the CTX+GnRHa group.③After KGN cells were treated with CTX,the intracellular AMH protein expression was significantly increased and the extracellular AMH secretion was reduced.CTX combined with GnRHa to treat KGN cells alleviated the effect of CTX in inhibiting AMH secretion in KGN cells.After treatment of KGN cells with CTX,the expression of endoplasmic reticulum stress-related proteins increased significantly.Immunofluorescence staining indicated that AMH accumulated on the endoplasmic reticulum.Conclusion:Serum AMH level is an effective indicator to evaluate ovarian reserve function;CTX-based chemotherapy obviously damages ovarian function,and the older the patients are,the more serious the damage is;co-treatment with GnRHa during chemotherapy can relieve the damage of CTX to ovaries;CTX-induced endoplasmic reticulum stress inhibits AMH secretion,reduces the level of AMH in serum,promotes the development of primordial follicles into growing follicles and damages ovarian reserve function.