Mendelian Randomization to Examine the Causal Effects of Cystatin on Ovarian Lesions

Background: Cystatin, a superfamily of cysteine protease inhibitors, are implicated in extracellular matrix remodeling, immune modulation, and tumor progression. Observational studies have reported associations between specific cystatin and gynecological pathologies, including ovarian cancer. However, whether these associations reflect causality remains uncertain due to potential confounding and reverse causation. Methods: We performed a two-sample Mendelian randomization (MR) study to investigate the causal relationships between genetically predicted levels of seven cystatin subtypes (Cystatin B, C, D, F, M, S, and Cystatin 8) and the risk of various ovarian lesions. Genetic instruments (single nucleotide polymorphisms, SNPs) for cystatin were selected from genome-wide association studies (GWAS) at a significance threshold of P < 5× 10 − 6 , with clumping for linkage disequilibrium (LD R 2 < 0.001, window = 10,000 kb). Summary-level data for outcomes including ovarian cysts, primary ovarian failure, ovarian torsion, and major histologic subtypes of ovarian cancer (high/low-grade serous, mucinous, clear cell, endometrioid) were obtained from the FinnGen, UK Biobank and Ovarian Cancer Association Consortium (OCAC). The primary analysis used the inverse-variance weighted (IVW) method, complemented by Weighted Median, MR-Egger, Mode-based methods, and sensitivity analyses for pleiotropy and heterogeneity. Results: Genetically predicted Cystatin-8 was significantly associated with a reduced risk of endometrioid ovarian cancer (OR = 0.898, 95% CI: 0.836– 0.965, P =0.003). Cystatin-C showed a suggestive protective effect against high-grade serous ovarian cancer (OR = 0.908, 95% CI: 0.828– 0.994, P =0.038). Cystatin-F was associated with a reduced risk of polycystic ovarian syndrome (OR = 0.852, 95% CI: 0.725– 0.999, P = 0.049). No significant causal relationships were observed for the other cystatin subtypes with the studied ovarian lesions. Sensitivity analyses were conducted to validate the MR assumptions. Cochran’s Q test was used to assess heterogeneity across the genetic instruments. The MR-Egger intercept test was applied to detect and adjust for directional horizontal pleiotropy. Additionally, a leave-one-out analysis was performed to ensure that no single SNP disproportionately drove the causal estimates. Conclusion: This MR study provides genetic evidence supporting a potential causal, protective role for Cystatin-8 against endometrioid ovarian cancer, and suggestive protective roles for Cystatin-C in high-grade serous ovarian cancer and Cystatin-F in polycystic ovarian syndrome. These findings highlight specific cystatin as potential biomarkers or etiological factors warranting further mechanistic and clinical investigation in ovarian pathophysiology. Keywords: cysteine protease inhibitors, ovarian cancer, ovarian lesions, Mendelian randomization, causal inference