Multiomics and Systematic Analyses Reveal the Roles of Hemoglobin and the HIF-1 Pathway in Polycystic Ovary Syndrome

Abstract

Polycystic ovary syndrome (PCOS) affects reproductive and cardiometabolic health, yet its pathogenesis remains unclear. Emerging evidence links hemoglobin levels to metabolic disorders, suggesting a potential role in PCOS development. Here, we integrated a large-scale cohort study, Mendelian randomization (A genetic tool to infer causal relationships), bioinformatics analyses, and in vitro experiments to investigate the relationship between hemoglobin levels and PCOS. In a cohort of 20 602 women, each 10 g L^-1 elevation in hemoglobin levels is associated with 22% higher odds of PCOS (adjusted odds ratio: 1.22, 95% confidence interval: 1.15-1.29, P < 0.001) and PCOS manifestations, particularly hyperandrogenism. Mendelian randomization analysis confirms that higher hemoglobin levels are associated with increased PCOS risk and elevated testosterone levels. The hypoxia-inducible factor 1 (HIF-1) pathway is enriched, identifying three testosterone-associated genes (nuclear factor kappa B (NFKB1), insulin receptor (INSR), protein kinase C alpha. Colocalization and druggability analysis supports shared genetic regions and confirmed these genes as druggable targets. Upregulation of NFKB1 and INSR are confirmed in both blood and ovarian granulosa cells of PCOS patients. The findings demonstrate that higher-end normal hemoglobin levels are associated with increased PCOS risk, potentially through a mechanism of elevating testosterone levels involving the HIF-1 pathway.

Keywords: causality; hemoglobin; hyperandrogenism; hypoxia‐inducible factor; polycystic ovary syndrome.

Figure 1

Central illustration of this study. Systematic analyses of the association between hemoglobin and polycystic ovary syndrome identify the potential effect of HIF‐1 pathway. Abbreviations: DEGs, differentially expressed genes; eQTL, expression quantitative trait loci; HIF, hypoxia‐inducible factor pathway; INSR, insulin receptor; NFKB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; PCOS, polycystic ovary syndrome; PRKCA, protein kinase C alpha; SD, standard deviation; SNP, single nucleotide polymorphism.

Figure 2

The associations between Hb levels and PCOS and its manifestations. A) Univariate and multivariate logistic regression model analysis to characterize the associations between Hb levels and PCOS, PCOS phenotypes, and separate manifestations. Model 1 was adjusted for intervals of appointment date, age, body mass index, and education; Model 2 was additionally adjusted for systolic blood pressure, endometriosis status, history of ovary surgery, LH/FSH ratio, fasting blood glucose, total cholesterol, triglycerides, and high‐ and low‐density lipoprotein levels. B) Nonlinear associations between Hb levels and PCOS, PCOS phenotypes, and separate manifestations, adjusted for confounders in Model 2. Abbreviations: PCOS, polycystic ovary syndrome; LH, luteinizing hormone; FSH, follicle‐stimulating hormone.

Figure 3

MR results and pathway enrichment analysis. A) Associations of Hb levels with PCOS and related traits according to the Mendelian randomization analysis. B) Significant results for KEGG pathway enrichment analysis of candidate genes from Hb IVs used in MR analysis. C) Significant tissue‐specific eQTL MR results in whole blood and ovary. D) eQTL and pQTL MR results for INSR, NFKB1, and PRKCA on testosterone levels. E) LocusCompare plots comparing genetic signals for NFKB1 levels and TT levels in colocalization analysis. Abbreviations: BT, bioavailable testosterone; TT, total testosterone; SHBG, sex hormone binding globulin; PH4, posterior probability for shared causal variants.

Figure 4

In vitro validation experiments in peripheral blood cells and granulosa cells. Hb levels, qPCR analyses of INSR, NFKB1 and PRKCA expression in A) peripheral blood cells and B) granulosa cells. The blue dots indicate the mRNA levels in the control group, and the red dots indicate the mRNA levels in the PCOS patients. P values, two‐tailed Student's t‐test or Mann‐Whitney test. C) Representative Western blot analysis of INSR and NF‐κB in the granulosa cells of the control and PCOS groups in three independent experiments, with the GAPDH level used as a control. D) Linear regression analysis of hemoglobin levels and INSR and NFKB1 gene expression in peripheral blood cells and granulosa cells after adjusting for BMI. E) Linear regression analysis of INSR and NFKB1 gene expression and testosterone level in peripheral blood cells before and after adjusting for BMI.