Rhfsh gonal f

48 hours after transfection COS-7 cells were incubated in modified Hank's buffer (5.36 mM KCl; 0.44 mM KH₂PO₄; 0.41 mM MgSO₄; 0.33 mM Na₂HPO₄; 5.55 mM Glucose) supplemented with 1.3 mM CaCl₂; 280 mM Sucrose; 0.2% BSA and 2.5% milk powder in the presence of 80.000 cpm/ml of ¹²⁵I-bTSH (Thermo Fisher Scientific, B.R.A.H.M.S), or 10.000–20.000 cpm/ml ¹²⁵I-rhFSH (Perkin Elmer) with increasing concentrations of unlabeled ligand (0–100 mU/ml bTSH – NIDDK National Hormone and Pituitary Program; 0–1000 ng rhFSH – Gonal-F, Merck Serono) at 4°C for four hours. Afterwards cells were washed with the same ice-cold buffer, solubilized with 1N NaOH, and radioactivity was measured in a gamma-counter. Specific binding was determined by subtracting the amount of radioligand unspecific bound to cells transfected with the empty pcDNA3.1/Zeo⁽⁻⁾ vector from each data set of the characterized receptor variants. The parameter maximal binding capacity was determined by nonlinear regression of competition binding curves using Graph Pad Prism 4.0 for Windows assuming a one-site binding model. The maximal binding capacity of wild type receptor was set at 100%, and the maximal binding of all mutants was calculated according to this.

All participants underwent controlled ovarian stimulation with gonadotrophin‐releasing hormone (GnRH) agonist long protocol. Briefly, patients received 0.1 mg/day of GnRH agonist (Decapeptyl, Ferring, Germany) from day 20 of a spontaneous menstrual cycle until the day of human chorionic gonadotrophin (hCG) injection. When pituitary down‐regulation was achieved (usually after 14 days of GnRH agonist injection), ovarian stimulation was initiated with a minimum of 150 IU/day of recombinant human FSH (rhFSH, GonalF, Merck Serono). The dosages of rhFSH were adjusted according to serum E2 levels and follicle growth of the patients. 250 µg of recombinant hCG (Ovidrel, Merck Serono) was administered, when at least three follicles were >17 mm in diameter. Transvaginal aspiration was performed 34‐36 hours later to retrieve oocytes and follicular fluids. The follicular fluids from follicles >14 mm in diameter and without obvious blood contamination were collected. The follicular fluids were centrifuged at 340 g for 8 minutes to pellet the GCs. The cell pellets were resuspended in 1× PBS solution (Gibco), overlaid on 40%/80% gradient solution (PureCeption, SAGE) and centrifuged at 320 g for 20 minutes. GCs in the interface were collected and washed with 1× PBS solution.