Puregon

GC were cultured in DMEM/F12 medium supplemented with 10 % FCS and antibiotics for 72 h to regain a more native reaction pattern before hormonal stimulation [24 (link)]. After three days in culture, GC were either untreated (control) or treated for 48 h with 30 ng/ml of r-FSH (Puregon®, Schering Plough, Courbevoie, France) or 30 ng/ml of hCG (Ovitrelle ®, Merck Serono, Lyon, France), individually or in combination. One μl of Androstenedione (Sigma, Poole, UK) was added to the culture medium to provide specific substrate to GC, for oestrogen synthesis. Therefore, 17 β-estradiol quantification by immuno-chemiluminescence using a commercially kit (ADVIA® Centaur, Bayer Diagnostics, Tarrytown, NY, USA) allowed to check GC viability (Additional file 3: Figure S1). From day 3, FCS was replaced in culture medium by Nutridoma-CS (Roche Applied Science, Germany) in order to avoid lipoproteins contamination. After 5 days of culture, culture medium and GC samples were retrieved and stored at –80 °C. Each culture’s experiment was performed in triplicate and 10 times.

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.