Isolation and long-term culture of human preantral follicles

Abstract

To isolate intact preantral follicles from the human ovary for in vitro studies. Premenopausal human ovary was digested by a mixture of collagenase and deoxyribonuclease (DNase) for 1 hour at 37 degrees C and for 36 hour at 4 degrees C. Intact preantral follicles at classes 1 and 2 were isolated and cultured for up to 120 hours in a serum-free Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) with ITS+ (insulin, transferrin, selenium, linoleic acid, and bovine serum albumin; Collaborative Research, Bedford, MA) with or without FSH. Follicular DNA synthesis was measured by [3H]thymidine incorporation, whereas steroidogenic capacity was assessed by P, androstenedione (A), and 17 beta-E2 RIA. The morphology of long-term cultured follicles was studied by routine histologic procedure. A large number of intact, preantral follicles was efficiently dissociated from a portion of ovary. Morphologically, follicles were healthy and did not have any thecal layer. Follicle-stimulating hormone, in vitro, induced follicular DNA synthesis by 24 hours and antrum formation in class 2 follicles after 120 hours. Both class 1 and 2 follicles were able to produce a small basal amount of P and A, but they did not produce any measurable E2. However, both classes synthesized increasing amounts of P, A, and E2 after FSH exposure. This is a first report of a successful enzymatic isolation of human preantral follicles and their long-term culture in vitro. The growth and differentiation of preantral follicles by FSH clearly indicate the importance of FSH in human follicular development. These results provide an opportunity for quantitative studies on factors regulating folliculogenesis in the human and a novel future direction for human IVF.