Gnrh antagonist

Flexible GnRHant protocol was used in this study. When the leading follicle was observed to be ≥14 mm in diameter or estradiol concentration reached ≥400pg/ml, GnRH-antagonist (0.25mg/day, Merck Serono, Coinsins, Switzerland) injection was started until the trigger day. When three dominant follicles reached 17 mm in diameter, the final maturation of oocytes was induced by recombinant hCG (250ug; Merck Serono, Coinsins, Switzerland). The oocyte aspiration was performed 35.5~36.5 hours after triggering.

Normal hGCs were obtained from patients (ages < 35) with tubal occlusion. The exclusion criteria is that the women with previous radiotherapy, ovarian surgery, known abnormal karyotype or autoimmune diseases. Ethics approval information and informed consent from patients were obtained (3 (link)). Recombinant FSH (Puregon; Schering Plough, New Jersey, USA) and GnRH antagonist (Merck, Frosst, Montreal, Canada) were employed to treat all subjects. Follicular development was monitored by vaginal ultrasound examinations. Ten thousand International Unit of human chorionic gonadotropin (hCG) (Pregnyl, Merck) was used to induce maturation of follicle. During the process of oocyte retrieval, follicular fluid was collected. Purified hGCs were acquired by density centrifugation method as previously described (3 (link)). Culture medium for primary hGCs was formed, that included DMEM/F12 media (Thermo, USA), 1% penicillin/streptomycin, 100 mg/ml streptomycin sulfate (Thermo, USA), 1X GlutaMAX (Thermo, USA), and 10% fetal bovine serum (complete medium). CTX (Sigma, USA) was used at different doses (20, 40, and 60 μg/ml) for treating hGCs, CTX with 60 μg/ml was used at different time point, respectively (0, 3, 6, 9, and 12 days). In all of the experiment, the culture medium was changed every other day, the information of patients were listed in Supplemental Table 2.