Letrozole Reduces Ovulatory Responsiveness in In Vitro-Grown Mouse Follicles

Purpose: This study aimed to determine whether continuous aromatase inhibition with letrozole reduces peri-ovulatory responsiveness to ovulatory stimulation in in vitro-grown mouse follicles. Methods: Early antral follicles were isolated from 21 to 24-day-old C57BL/6jjcl mice and cultured individually for 5 days with letrozole (0.01, 0.1, or 1 μM) or vehicle. Follicle survival and growth were assessed by measuring follicle diameter. Ovulatory responsiveness was evaluated by hCG/EGF stimulation. Expression of ovulation-related genes (Lhcgr, Ptgs2, and Runx1) and a luteinization-related gene (Foxo1) was analyzed using RT-qPCR. The effect of estradiol supplementation was also examined. Results: Letrozole did not affect follicle survival or growth. However, ovulation rates were reduced in a dose-dependent manner (p < 0.0001). At 0.1 μM, letrozole significantly decreased mRNA levels of Lhcgr, Runx1, and Ptgs2 compared with vehicle-treated follicles. Estradiol supplementation restored the expression of these genes and partially rescued ovulatory capacity. In contrast, Foxo1 expression increased in letrozole-treated follicles and was attenuated by estradiol. Conclusions: Continuous letrozole exposure reduces ovulatory responsiveness in in vitro-grown mouse follicles and is associated with decreased Lhcgr expression after hCG stimulation, likely due to estrogen deficiency. These findings suggest that prolonged aromatase inhibition during follicular growth may impair acquisition of peri-ovulatory competence.