Enzymatic isolation of human primordial and primary ovarian follicles with Liberase DH: protocol for application in a clinical setting

Abstract

Objective: To set up a protocol to isolate human preantral follicles with an enzyme produced in good manufacturing practice conditions for use in a clinical setting.

Design: For follicle isolation, ovarian biopsies were divided into two halves: one was treated with collagenase IA and the other with Liberase DH (Dispase High) Research Grade.

Setting: Academic research unit.

Patient(s): Twelve women undergoing laparoscopy for benign gynecological disease.

Intervention(s): Follicle isolation.

Main outcome measure(s): Follicles were counted, their morphology was analyzed, and follicular viability was evaluated by live/dead assays before and after 7 days of in vitro culture. Their structural preservation was assessed after isolation by electron microscopy.

Result(s): A total of 1,030 follicles were obtained after isolation: 566 with Liberase DH and 464 with collagenase IA. The percentage of viable follicles (with <10% of dead granulosa cells [GC]) was not found to be statistically different before or after culture in either group (Liberase DH: 95% and 81%, respectively; collagenase: 96% and 87%, respectively). A significant increase in follicle size was observed in both groups after culture. Liberase DH did not affect the ultrastructure of isolated follicles.

Conclusion(s): Liberase DH allows isolation of a high number of preantral follicles, maintaining their viability, even after in vitro culture, and their ultrastructure. In addition, Liberase DH can be produced in good manufacturing practice conditions, allowing use of isolated preantral follicles for future clinical applications.