Use of trypan blue staining to assess the quality of ovarian cryopreservation

Objective: To check the efficiency of the cryopreservation procedure by using trypan blue staining of ovarian test fragments to assess the quality of frozen-thawed ovarian tissue. Design: Prospective study. Setting: University hospital. Patient(s): Patients with polycystic ovary syndrome undergoing laparoscopic ovarian drilling. Intervention(s): Ovarian cortical biopsies obtained from polycystic ovary syndrome patients were frozen using a slow freezing-rapid thawing protocol. Main Outcome Measure(s): Primordial and primary follicle viability was assessed with trypan blue staining. Unstained isolated follicles were studied by transmission electron microscopy. Histologic and immunohistochemical analysis of apoptosis was performed on tissue sections. Result(s): The percentage of unstained follicles considered live was lower (P=.015) after freezing/thawing (71.9%) than before cryopreservation (87.3%). Transmission electron microscopy on follicles considered live confirmed the lack of ultrastructural damage. After freezing/thawing, tissue morphology was preserved, but immunohistochemical analysis shows a significant increase in the apoptosis process. Conclusion(s): Ovarian cortical test fragments combined with trypan blue staining on enzymatically isolated follicles is a useful and quick method of assessing the initial quality and viability of follicles in cryopreserved ovarian tissue. This type of test should be used routinely as quality control in ovarian cryopreservation procedures.