Direct comparative analysis of conventional and directional freezing for the cryopreservation of whole ovaries

Abstract

Objective: To compare conventional slow equilibrium cooling and directional freezing for cryopreservation of whole ovaries.

Design: Experimental animal study.

Setting: Academic research environment.

Animal(s): Adult ewes.

Intervention(s): Eighty-one ovaries were randomly assigned to fresh control, conventional freezing (CF), and directional freezing (DF) group. Ovaries of CF and DF groups were perfused via the ovarian artery with Leibovitz L-15 medium, 10% fetal bovine serum, and 1.5 M dimethyl sulfoxide for 5 minutes. Each ovary was inserted into a glass test tube containing 10 mL of the same solution and cooled to -100°C or -70°C, respectively. Ovaries were stored in liquid nitrogen for a minimum of 2 weeks.

Main outcome measure(s): Structural integrity of cortical and medulla regions, vascular integrity, follicle in vitro development, cell proliferation, and DNA damage and repair.

Result(s): All examined parameters indicate that the structure of DF ovaries remains largely intact and comparable to fresh controls, whereas significant damages were observed in CF ovaries.

Conclusion(s): Directional freezing allows good preservation of whole ovaries, with most of the parameters taken into consideration almost identical to those recorded in fresh control samples. This encourages a reconsideration of the possible use of whole-ovary cryopreservation as a viable alternative to cortical fragments.

Keywords: Whole ovary; cryopreservation; directional freezing; sheep.