Solid-surface vitrification is an appropriate and convenient method for cryopreservation of isolated rat follicles

Weijie Xing,Yubin Li,Canquan Zhou,Tao Li,Markus Montag,Jiang Bian,Yanwen Xu

doi:10.1186/1477-7827-8-42

Abstract

BackgroundCryopreservation of isolated follicles may be a potential option to restore fertility in young women with cancer, because it can prevent the risks of cancer transmission. Several freezing protocols are available, including slow-rate freezing, open-pulled straws vitrification (OPS) and solid-surface vitrification (SSV, a new freezing technique). The purpose of our study was to investigate the effects of these freezing procedures on viability, ultrastructure and developmental capacity of isolated rat follicles.MethodsIsolated follicles from female Sprague-Dawley rats were randomly assigned to SSV, OPS and slow-rate freezing groups for cryopreservation. Follicle viability assessment and ultrastructural examination were performed after thawing. In order to study the developmental capacity of thawed follicles, we performed in vitro culture with a three-dimensional (3D) system by alginate hydrogels.ResultsOur results showed that the totally viable rate of follicles vitrified by SSV (64.76%) was slightly higher than that of the OPS group (62.38%) and significantly higher than that of the slow-rate freezing group (52.65%; P < 0.05). The ultrastructural examination revealed that morphological alterations were relatively low in the SSV group compared to the OPS and slow-rate freezing groups. After in vitro culture within a 3D system using alginate hydrogels, we found the highest increase (28.90 ± 2.21 μm) in follicle diameter in follicles from the SSV group. The estradiol level in the SSV group was significantly higher than those in the OPS and slow-rate freezing groups at the end of a 72-hr culture period (P < 0.05).ConclusionsOur results suggest that the SSV method is an appropriate and convenient method for cryopreservation of isolated rat follicles compared with the conventional slow-rate freezing method and the OPS method.

Highlights

Cryopreservation of isolated follicles may be a potential option to restore fertility in young women with cancer, because it can prevent the risks of cancer transmission
The aim of our study was to investigate the effects of the three freezing methods (SSV, open-pulled straws vitrification (OPS) and slow-rate freezing) on viability, ultrastructure and developmental capacity of isolated rat follicles
Digestion was terminated by adding an equal volume of phosphate-buffered saline (PBS; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) at 4°C

Summary

Introduction

Cryopreservation of isolated follicles may be a potential option to restore fertility in young women with cancer, because it can prevent the risks of cancer transmission. Ovarian cortical tissue contains high numbers of primordial and primary follicles. Cryopreservation of ovarian cortex has become a potential option to restore fertility in young women with cancer [1]. SSV, a new freezing technique, has been successfully applied to preserve oocytes [5,6,7] and ovarian tissues [8]. SSV uses a metal surface, which is precooled to -180°C by partial immersion into liquid nitrogen (LN2) and serves as a template to cool microdrops of vitrification solution containing embryos or oocytes [9]. SSV provides enough space for tissue, maximizes cooling rates, and avoids the generation of gas phase of LN2 bubbles [10]

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