Effects of PD-1 inhibitors on ovarian function-based on two-sample mendelian randomization and visualization experimental validation

Abstract

Although immune checkpoint inhibitors (ICIs) have transformed cancer treatment by improving survival, their ovarian safety remains uncertain. This study combined Mendelian randomization (MR) and experimental validation to assess the impact of PD-1 inhibitors on ovarian function. MR analysis used summary statistics from large European-ancestry genome-wide association studies (GWAS), applying the inverse-variance weighted (IVW) method, supported by sensitivity analyses. For in vitro experiments, mouse follicles were cultured with 10 µg/ml PD-1 inhibitor ch15mt (clinically relevant concentration), 200 nM doxorubicin (DOX), or PBS control. Follicular morphology was evaluated via diameter measurements; endocrine function by estradiol (E2) quantification using ELISA. Real-time cytoplasmic Ca²⁺ dynamics were monitored using FRET-based Cyto-Ca2 + probes for high-resolution stress assessment. MR results showed no significant association between genetically predicted PD-1 levels and risks of premature ovarian insufficiency, infertility, or alterations in ovarian hormones including AMH and E2. Sensitivity analyses confirmed MR robustness. In vitro, PD-1 inhibition did not affect follicular size or E2 secretion. Notably, DOX induced rapid Ca²⁺ elevation, while PD-1 inhibitor treatment had no detectable effect on Ca²⁺ fluctuations. This first integrative MR and experimental study demonstrates that PD-1 inhibitors at clinically relevant concentrations lack acute ovarian toxicity. While further work is needed to assess long-term effects, these findings demonstrate that standard anti-PD-1 immunotherapy regimens do not compromise follicular viability or endocrine function, strongly supporting their safety in fertility-sparing oncology protocols.