Ficoll density gradient method for recovery of isolated human ovarian primordial follicles.

OBJECTIVE: To develop a simple and efficient technique to allow rapid recovery of a maximum number of good quality isolated follicles. DESIGN: Prospective experimental study. SETTING: Academic research unit of the department of gynecology in a university hospital. PATIENT(S): Biopsies were obtained from five women (between 26 and 31 years of age). INTERVENTION(S): Biopsies were cut with a tissue sectioner. Enzymatic digestion was performed in a collagenase solution for 90 min at 37 degrees C. The follicles were recovered using a discontinuous Ficoll density gradient method. MAIN OUTCOME MEASURE(S): The number of follicles present in the interface layers of Ficoll gradient was quantified. Follicular viability of these recovered follicles was assessed with live-dead stains, using calcein-AM and ethidium homodimer-I. RESULT(S): Out of a total of 6,811 recovered follicles, we found 63% (n = 4,201) at the medium-1.06 Ficoll interface and 36.9% (n = 2,590) at the 1.06-1.09 Ficoll interface, which represents 99.9% of total recovered follicles. Analysis by vital fluorescent staining showed that 95.8% of the follicles treated with Ficoll were totally viable. CONCLUSION(S): The Ficoll density gradient method allows us to maximize the recovery of isolated human ovarian follicles and minimize the manipulation time while maintaining high follicular viability.