Effects of urinary and recombinant gonadotropins on in vitro maturation outcomes of mouse preantral follicles

Abstract

Gonadotropins including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play a crucial role in human-assisted reproduction techniques. Despite wide use of recombinant gonadotropins in clinical practice, the efficacy of urinary gonadotropins and the dosage of LH component have not yet been elucidated. This study was designed to investigate the difference of follicle culture outcomes according to various compositions of gonadotropins during in vitro culture of mouse preantral follicles. Ovaries were obtained from the 14-day-old C57BL/6 mice, and preantral follicles were isolated and cultured in culture media supplemented with human menopausal gonadotropin (hMG) 200 mIU/mL (group 1), recombinant FSH and LH (rFSH + rLH) 200 mIU/mL each (group 2), rFSH 200 mIU/mL + rLH 100 mIU/mL (group 3), or rFSH 200 mIU/mL + rLH 20 mIU/mL (group 4). Follicle survival rate was significantly lower in group 4. Antral follicles in lower doses of LH (groups 3, 4) showed a statistically significant larger diameter and tended to have a higher antral formation rate. However, follicles in group 1 tended to have a higher oocyte maturation rate. Estradiol concentration from conditioned media from 2:1 FSH/LH (group 3) was significantly higher than those from 1:1 FSH/LH (group 2) or 10:1 FSH/LH (group 4). Half dose of rLH to rFSH facilitated upregulation of growth differentiation factor 9 (Gdf9) expression in granulosa cells when compared to 1:1 FSH/LH or 10:1 FSH/LH. Conclusively, recombinant gonadotropins provided a comparable condition to hMG, and half dose of rLH to rFSH seems to be more suitable for follicular development during in vitro culture.

Keywords: in vitro maturation; ovarian stimulation; preantral follicles; recombinant gonadotropins; urinary gonadotropin.

Figure 1.

Development of preantral follicles during in vitro culture. Follicles had been cultured with hMG 200 mIU/mL (A-C), recombinant FSH 200 mIU/mL plus recombinant LH 200 mIU/mL (D-F), recombinant FSH 200 mIU/mL plus recombinant LH 100 mIU/mL (G-I), and recombinant FSH 200 mIU/mL plus recombinant LH 20 mIU/mL (J-L). Typical follicles of each group at days 1, 6, and 10 during in vitro maturation are shown. Original magnification was 60× and scale bars represent 100 μm. In the hMG group, granulosa cells were denser than other groups on day 10 (C). FSH indicates follicle stimulating hormone; hMG, human menopausal gonadotropin; LH, luteinizing hormone.

Figure 2.

A, Typical antral follicles are shown. Original magnification was 60× and scale bars represent 100 μm, applied to all images. B, Mean diameters of antral follicles cultured with 4 different gonadotropin components on day 12 during in vitro maturation. Data represent diameters (mean ± standard error [SE]). (A-1) hMG 200 mIU/mL, (A-2) rFSH: rLH 200 mIU/mL: 200 mIU/mL, (A-3) rFSH: rLH 200 mIU/mL: 100 mIU/mL, and (A-4) rFSH: rLH 200 mIU/mL: 20 mIU/mL. *Follicles in group 3 or 4 showed a statistically significant larger diameter compared to group 1 or 2 (P < .001). No significant difference was observed between groups 3 and 4. rFSH indicates recombinant follicle stimulating hormone; hMG, human menopausal gonadotropin; rLH, recombinant luteinizing hormone.

Figure 3.

Comparison of estradiol concentrations from conditioned media. Data present estradiol level (ng/mL, mean ± standard error [SE]) measured from conditioned media on day 12 of in vitro culture. Different letters indicate a significant difference (P < .05).

Figure 4.

Relative expression of oocyte development-related genes (Oct4, Bmp15, and Gdf9) in oocytes (A) granulosa cells (B). The expression of each gene was calculated relative to that of group 1. A, All measures in oocytes have no significant difference with regard to relative expression of all 3 genes. B, *Relative expression of Oct4 in group 4 was lower than that in group 1 (P < .001) and 3 (P < .01) and relative expression of Bmp15 in group 2 was lower than that in group 1 (P < .001). Relative expression of Gdf9 in group 3 was higher than that in group 2 (P < .05) and 4 (P = .001). A statistical difference was also observed between group 1 and group 4 in terms of Gdf9 expression (P < .001).