Total oxidative status of mouse vitrified pre-antral follicles with pre-treatment of alpha lipoic acid

Abstract

Background: Cryopreservation of pre-antral follicles is a hopeful technique to preserve female fertility. The aim of the present study was to evaluate reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of mouse vitrified pre-antral follicles in the presence of alpha lipoic acid (ALA).

Methods: Isolated pre-antral follicles (140-150 µm in diameter) were divided into vitrified-warmed and fresh groups. Each group was subjected to in vitro maturation with or without ALA for 12 days, followed by adding human chronic gonadotropin to induce ovulation. In vitro fertilization was performed to evaluate their developmental competence. In parallel, the amount of ROS and TAC were assessed after 0, 24, 48, 72, and 96 h of culture by 2',7'-dichlorofluorescin assay and ferric reducing/antioxidant power assay, respectively.

Results: The respective rates of survival, antrum formation, and metaphase II oocytes were significantly higher in ALA-supplemented groups compared to the groups not treated with ALA. TAC and ROS levels were significantly decreased and increased, respectively during the culture period up to 96 h in the absence of ALA in both vitrified and non-vitrified samples. However, with pretreatment of ALA, TAC levels were increased significantly and remained constant up to 96 h in vitrified-warmed pre-antral follicles, while ROS levels completely returned to the level of starting point after 96 h of culture in the presence of ALA.

Conclusion: Pretreatment of ALA positively influences development of pre-antral follicles in vitrified and non-vitrified samples through increasing follicular TAC level and decreasing ROS levels.

Keywords: Vitrification; Pre-antral follicle; Alpha lipoic acid; Reactive oxygen species; Total antioxidant capacity.

Fig. 1

Cultured pre-antral follicle: initial day (a), day 2 (b), day 4 (c), day 6 (d), day 8 (e), day 10 (f) day 12 (g), and ovulated MII oocyte after hCG stimulus (h).

Fig. 2

Total antioxidant capacity (TAC) levels in cultured vitrified-warmed and fresh pre-antral follicles with or without pretreatment of alpha lipoic acid (ALA). In all cases 4 experimental replicates were performed. Different superscripts (A, B, C) reflect different levels of significant differences at same times of culture among the different groups (P<0.05). Different superscripts (a, b, c) reflect different levels of significant differences at different times of cultivation period within the same group (P<0.05).

Fig. 3

Reactive oxygen species (ROS) concentrations in cultured vitrified-warmed and fresh pre-antral follicles with or without pretreatment of alpha lipoic acid (ALA). In all cases 4 experimental replicates were performed. * indicates significant difference with initial time in the same group (P<0.05); # indicates significant difference at same times of cultivation period between respective groups with or without pretreatment of ALA (P<0.05). ROS, reactive oxygen species; ALA, Alpha lipoic acid