Ovarian pieces were collected from women < 35 yr of age who were undergoing surgery because of clinical conditions other than ovarian pathology. Human tissue collection was approved by the Institutional Review Board. Ovary samples were rinsed with Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 100 U/ml penicillin, 10 ^g/ml streptomycin, 50 ^g/ml amphotericin B, and 0.5% BSA and dissected free of any broken antral follicle and luteal remnant. Ovarian pieces from each woman were processed separately for follicle isolation, and class 1 and 2 follicles were dissociated and collected as described previously. birth control yasmin
Class 1 follicles were < 90 ^m in diameter with 1-2 layers of granulosa cells, and class 2 follicles were < 220 but > 90 ^m in diameter with > 3-4 layers of granulosa cells. Moreover, class 2 follicles were without an antral cavity and any definitive thecal layer. Previously, we have shown that these classes of human follicles can produce steroid hormones in response to FSH. Preantral follicles from each ovary were rinsed twice with DMEM supplemented with ITS+; insulin, 6.25 ^g/ml; transferrin, 6.25 ^g/ml; selenium, 6.25 ^g/ml; BSA, 1.25 mg/ml; and linoleic acid, 5.35 ^g/ml; Collaborative Research, Bedford, MA).