Timing matters: Letrozole-induced hyperandrogenism and its effects on pregnancy outcomes in mice

Miscarriage (pregnancy loss before 20 weeks) is the most common complication of human pregnancies, affecting one in five worldwide. The risk of miscarriage is elevated even further in women with polyendocrine metabolic ovarian syndrome – PMOS (formerly known as polycystic ovarian syndrome – PCOS). Hyperandrogenism is a hallmark of PMOS, causing disruption to the hormonal environment. The aim of this study was to investigate whether transient increases in androgens at different time points in early gestation cause adverse pregnancy outcomes in C57BL/6 mice using letrozole in drinking water. Firstly, to find a suitable mouse model, non-pregnant mice were treated with letrozole (0, 0.5 or 1 mg/kg/day) for 14 days. On day 14, plasma testosterone was significantly elevated by 0.5 mg/kg/day (p<0.01) and 1 mg/kg/day (p<0.05). This then had to be tested in a pregnancy context where a separate cohort of mice were mated then treated with letrozole (0.5 or 1 mg/kg/day) from embryonic day 0.5 (E0.5) to E13.5 alongside controls. All controls delivered pups, alongside one 0.5 mg/kg/day mouse and one 1 mg/kg/day mouse. Plasma testosterone at E13.5 for 1 mg/kg/day was significantly elevated (p<0.01), so that dosage was continued for further experiments. The teratogenic effects observed following chronic letrozole exposure through gestation prompted a shift in experimental design, with subsequent experiments restricting letrozole exposure to one of three gestational periods. To determine at which timeframe letrozole was having the most effects on pregnancy, another cohort of mated mice were treated with letrozole (1 mg/kg/day) at three separate embryonic time periods E0.5-E6.5 (T1), E6.5-E13.5 (T2), E13.5-E18.5 (T3). Bloods were taken from all groups (including controls) at E6.5, E13.5 and E18.5. T1 had significantly fewer pups per litter compared to controls (p<0.01), and significantly higher levels of testosterone (p<0.01). As T1 was the only group to have significantly lower number of pups compared to the controls, further investigation into this timeframe was conducted. Blastocysts were collected from letrozole and control mice on E3.5 to determine if letrozole was affecting pregnancy pre-implantation. There was no significant difference in the number of blastocysts between the two groups. Plasma testosterone and LH levels were also measured and found to be significantly elevated in the letrozole mice (p<0.001; p<0.01, respectively). Since high levels of LH could indicate depletion of oestradiol (E2), uterine weights (as an E2 proxy) were taken from non-mated mice after three days letrozole (1 mg/kg/day) treatment. The letrozole uterine weights were significantly smaller in the letrozole mice compared to the controls (p<0.05). In letrozole-induced hyperandrogenic mice, the process of implantation is disrupted leading to adverse pregnancy outcomes. From this study, further questions such as whether this is due to a depletion in E2 levels, direct effects of letrozole or higher levels of androgens remains to be determined, therefore refining of the hyperandrogenic mouse model is warranted. By identifying the window in which letrozole-induced hyperandrogenism had detrimental effects, this could encourage further research into potential treatments to help reduce the risk of miscarriage in PMOS women.