A genome-wide association study identifies GNPNAT1 as a candidate risk locus for endometriosis in the Tehran Cardio-metabolic genetic study

Background Population studies elucidating the genetic architecture of endometriosis have predominantly focused on individuals of European ancestry, thereby leaving a gap in understanding the genetic influences within non- European populations. This present study aims to identify potential genetic variants associated with endometriosis in Iranian women. Research design and methods We conducted a genome-wide association study on endometriosis involving a discovery group of 1978 women, comprising 305 women with endometriosis and 1673 unaffected individuals. An independent confirmation cohort comprising 829 women (101 cases and 728 controls) was selected from the TCGS cohort. Over 9 million genetic variants were analyzed using the Genome-wide Complex Trait Analysis framework, followed by integrative bioinformatics and functional annotation analyses.

In the discovery phase, we identified rs12886544 at the GNPNAT1 locus as genome-wide significant for endometriosis susceptibility (OR = 1.66, p-value = 5.89 × 10⁻⁸). In the confirmation cohort, the direction of effect was consistent with the discovery phase, although the association did not remain statistically significant after correction for multiple testing (OR = 1.4, 95% [CI 0.89–2.11], p = 0.14). A pooled analysis of the discovery and confirmation cohorts demonstrated a statistically significant association between rs12886544 and endometriosis risk (combined p-value = 0.001, OR = 1.59, 95% CI 1.19–1.89). Epigenomics results suggest that rs12886544 may affect a regulatory motif associated with the Bobby Sox homolog (BBX) transcription factor in women, implicating a potential role in modulating gene expression. Page 2 of 13 Najd-Hassan-Bonab et al. Middle East Fertility Society Journal (2026) 31:37

Endometriosis is a chronic, estrogen-dependent inflam - matory disorder characterized by the ectopic presence of endometrial-like tissue outside the uterine cavity, the pel- vic peritoneum, and the ovaries. Affecting approximately 5–10% of women of reproductive age and up to 20–50% of those with infertility, it presents with significant clini - cal heterogeneity. It imposes a substantial burden on reproductive health, quality of life, and healthcare systems worldwide [ 1, 2]. Although primarily benign, endome - triosis can, in rare cases, progress to become particularly severe within reproductive organs, such as the ovaries. Despite its high prevalence, diagnosis remains chal - lenging due to nonspecific symptoms and the need for surgical confirmation, often leading to delays of several years [3]. The etiology of endometriosis is multifactorial, involving complex interactions between genetic predis - positions and environmental influences such as age, body mass index (BMI), and reproductive history. The genetic factors are estimated to account for 50% of disease sus - ceptibility. Family and twin studies provide strong evi - dence for heritability: first-degree relatives of affected women have up to a 7-fold increased risk, and monozy - gotic twins show significantly higher concordance than dizygotic twins, with heritability quantified at approxi - mately 51% [ 4– 7]. These findings highlight the central role of gene architecture in disease development. Early genetic studies of endometriosis focused on candidate genes, particularly those involved in estrogen signaling (ESR1, PGR) and inflammation (TNF, IL-1), reflecting the hypothesis that hormonal and immune dysregulation drive the disease. Later, genetic associa - tion shifted to genome-wide association studies (GWAS). The first robust GWAS signal emerged from a Japanese cohort (Uno et al., 2010), identifying rs10965235 in CDKN2BAS on chromosome 9p21, a genomic region implicated in cell cycle regulation [ 8– 11]. Soon after, Painter et al. [ 12] reported a significant association with

Our findings provide supportive evidence that rs12886544 at the GNPNAT1 locus is associated with endometriosis susceptibility in Iranian women. The association was supported by a consistent direction of effect in an independent cohort and a significant pooled analysis, although further validation in larger independent populations is required. Additionally, observed differences in allele frequencies between Iranian and European populations highlight the importance of trans-ethnic studies for understanding population-specific genetic architecture in endometriosis.

Tehran cardiometabolic genetic study (TCGS), GWAS, Endometriosis, GNPNAT1 Graphical abstract Page 3 of 13 Najd-Hassan-Bonab et al. Middle East Fertility Society Journal (2026) 31:37 the intergenic SNP rs12700667 on chromosome 7p15.2 in women of European ancestry, a locus harboring genes involved in uterine development. Subsequent studies in European populations identified additional loci, includ - ing HOXA10, which is a key transcriptional regulator of uterine development and endometrial receptivity, and NFE2L3, a transcription factor linked to cellular dif - ferentiation and stress-responsive regulation that may influence endometrial remodeling [12– 15]. Variants near VEZT further implicate cell-cell adhesion and epithelial integrity, processes relevant to endometrial implantation- like behavior and lesion establishment, while GREB1, an estrogen-responsive gene, supports the central role of hormone-driven signaling in endometriosis pathogen - esis, with primary effects expected in endometrium and ectopic pelvic lesions [ 16– 18]. Moreover, associations involving IL1A and STAT3 genes underscore the con - tribution of pro-inflammatory cytokine signaling and downstream transcriptional programs, consistent with an inflammatory microenvironment in endometrium, peri - toneal cavity, and lesion-associated immune cells [ 12]. An Icelandic study identified signals near KDR (encod - ing VEGFR2), TTC39B, and RTN4RL1 genes, pointing toward angiogenesis (KDR/VEGFR2) that may support lesion vascularization in ovarian/peritoneal implants, alongside loci potentially related to metabolic regulation (TTC39B) and neurobiological processes (RTN4RL1), which may contribute to symptom heterogeneity, includ- ing pain [ 19]. In contrast, a Polish cohort was unable to confirm 22 previously reported SNPs [20]. These discrep- ancies underscore the importance of ethnic diversity in genetic studies, indicating that some risk variants may be population-specific. Despite recent advances, the genetic landscape of endometriosis in Middle Eastern and Iranian populations remains largely unexplored. While large-scale GWAS in European and East Asian cohorts have identified numer - ous risk loci, the generalizability of these findings and the potential for population-specific genetic risk factors in underrepresented groups are unclear. This gap hinders the development of precision medicine approaches in underrepresented populations. To address this gap in genetic data from Middle East - ern populations and to investigate the potential for ancestry-specific risk factors, we conducted a GWAS in a large Iranian cohort with the following objectives: (1) to discover candiadate and population-specific genetic variants associated with endometriosis; (2) evaluate the transferability of established risk loci from prior large- scale studies in our cohort ancestries; and (3) to perform functional annotation of identified variants to elucidate their potential roles in disease pathogenesis. Our study aims to expand the global understanding of endome - triosis genetics and contribute to the development of targeted diagnostics and therapies tailored to the Iranian population. Patients and methods Study population We utilised data from the Tehran Lipid and Glucose Study (TLGS), an ongoing cohort study initiated in 1998 to investigate risk factors for non-communicable diseases among urban residents [ 21, 22]. Within this framework, the Tehran Cardio-metabolic Genetic Study (TCGS) was established to identify genetic determinants of key car - diometabolic traits using high-throughput genotyping and sequencing technologies [23]. The TLGS initially recruited 15,005 participants, all of whom were aged three years or older. Trained personnel collected detailed information regarding demographics, reproductive health, lifestyle factors, and medical history through standardized interviews. Skilled general practi - tioners conducted physical and anthropometric measure- ments at baseline, with follow-ups occurring every three years across seven phases. For this analysis, we included non-menopausal women aged 18–50 from the 6th follow-up visit, which included an expanded reproductive questionnaire [24]. Participants were asked about a prior diagnosis of endometriosis, and all self-reported cases were verified through a review of their medical records. Medical records were also exam - ined for women reporting moderate to severe chronic pel- vic pain, dysmenorrhea, or dyspareunia. An experienced sonographer performed a Transabdominal (3.5 MHz) or transvaginal (5 MHz) ultrasound. For women without a prior diagnosis, suspected endometriosis was assessed using the standardized protocol of the International Deep Endometriosis Analysis (IDEA) group [25]. A total of 3,450 eligible female participants were ran - domly assigned into two groups: 70% ( n = 2,415) for the discovery phase and 30% ( n = 1,035) for the confirmation phase. The study’s flowchart is presented in Fig. 1. Definition of endometriosis In the current study, participants were designated as endometriosis cases if they self-reported a diagnosis that was subsequently confirmed through medical records. Additionally, women with a positive ultrasound diag - nosis of endometriosis, regardless of the presence of symptoms such as chronic pelvic pain, dysmenorrhea, or dyspareunia, were included in the endometriosis group. Participants exhibiting symptoms like chronic pelvic pain or moderate to severe dysmenorrhea or dyspareunia but with negative ultrasound findings were excluded, as a negative ultrasound does not definitively exclude endo - metriosis, particularly superficial peritoneal disease. This classification follows the 2022 ESHRE guide - lines, which recommend reserving laparoscopy for Page 4 of 13 Najd-Hassan-Bonab et al. Middle East Fertility Society Journal (2026) 31:37 with an Identity by Descent (IBD) value of ≥ 18.5%, and (4) extreme heterozygosity levels > ± 3SD. Statistical analysis For our power calculations, we used the Genetic Asso - ciation Study (GAS) Power Calculator [ 29]. The follow - ing parameters were specified based on our available data and published estimates: Risk allele frequency: approxi - mately 0.49; Genotype relative risk: 1.5; Endometriosis prevalence: 20% and Genome-wide significance thresh - old of 5 × 10 − 8 for the discovery stage and 0.05 for the confirmation stage.Under these assumptions and our available cases (305 cases in discovery and 101 cases in confirmation), the estimated power was 96% and 92% for the discovery and confirmation phases, respectively. The normality of continuous variables was assessed using the Kolmogorov–Smirnov test. Variables follow - ing a normal distribution are expressed as mean ± stan- dard deviation (SD) and compared between groups using Student’s t-test. Categorical variables are presented as percentages and compared with the chi-squared (χ²) test. The statistical method was conducted separately for the discovery and confirmation cohorts. In the dis - covery phase, genome-wide association testing was performed for autosomal variants using the FastGWA

implemented in Genome-Wide Complex Trait Analysis (GCTA; version 1.93 beta). FastGWA applies a mixed linear model under an additive genetic framework symptomatic patients with negative imaging or ineffec - tive treatment and advice against routine laparoscopic screening in asymptomatic individuals with negative imaging [26]. As a result, the final analysis included 406 women diagnosed with endometriosis and 2401 control participants. Genotyping quality control Genotyping for TCGS participants was performed using the Illumina Human OmniExpress-24-v1-0 bead chip, which comprises 652,919 SNP loci, at deCODE genet - ics/Amgen in Iceland, following the manufacturer’s protocols (Illumina Inc., San Diego, CA, USA) [ 23]. A subset of 1,462 samples was selected for whole-genome sequencing. Variant imputation was performed using the IMPUTE hidden Markov model (HMM), where chip- genotyped individuals were imputed based on shared haplotypes with the training set. Before association anal - ysis, quality control procedures were applied to ensure the reliability of genotype data at both the individual and SNP levels [27, 28]. Genetic variants were excluded based on the following criteria: (1) genotyping rate below 95%, (2) minor allele frequency (MAF) less than 0.05, and (3) deviation from Hardy-Weinberg Equilibrium ( p-value < 1 × 10 − 6 ). Participants were excluded for the follow - ing reasons: (1) sex discrepancies identified via PLINK’s “sex check, ” (2) missing data exceeding 5%, (3) relatedness Fig. 1 Overview of study design and analysis workflow used in the current GWAS on endometriosis. N indicates the sample size Page 5 of 13 Najd-Hassan-Bonab et al. Middle East Fertility Society Journal (2026) 31:37 and accounts for relatedness and population structure through a genetic relationship matrix (GRM). Case–con - trol status for endometriosis was modeled as the out - come variable, and all tests were two-sided. Analyses were adjusted for age at menarche, body mass index (BMI), smoking, with and without dysmenorrhea, and 10 principal components (PCs) capturing popula - tion structure. Although the Iranian population is rela - tively homogeneous, subtle genetic substructure arising from regional or ethnic diversity (Persian, Turkic, Kurd - ish, and Baloch) may exist. To address this, PCs were derived from an LD-pruned subset of autosomal SNPs using PLINK, and the genetic relationship matrix (GRM) was estimated with GCTA. Genomic inflation was evaluated using quantile–quantile (Q–Q) plots and the genomic control factor (λ). Manhattan and Q–Q plots were generated using the qqman package in R (version 4.0.2). Genome-wide statistical significance was defined as P-values < 5 × 10⁻⁸ to account for multiple testing. Regional association plots for significant loci were gener - ated using LocusZoom. In the confirmation phase, association testing was per - formed using logistic regression under an additive genetic model implemented in PLINK. Resampling permutation testing with 100,000 iterations was applied to evaluate the robustness of association signals. Odds ratios (ORs) and corresponding two-sided P-values were reported, with statistical significance defined as P-values < 0.05. Functional annotation of GWAS results Functional interpretation of GWAS results was carried out using HaploReg, which helps identify tagging SNPs in high linkage disequilibrium and provides insights into chromatin states, conserved regions, and transcription factor motif alterations [30]. To further contextualize the findings, we integrated epigenomic mapping data from ENCODE and the Roadmap Epigenomics Mapping Con - sortium, enabling interpretation at both the variant and gene levels. We also leveraged additional resources, including GWAS Atlas, FinnGen, PheWeb, and Open Targets, to explore phenotypic annotations and previously reported trait associations [ 31– 34]. These open-access databases facilitate the identification and prioritization of poten - tially causal variants and genes, providing a comprehen - sive functional understanding of GWAS results [35].

Study population characteristics A total of 2,807 women were included in the analysis, comprising 406 women with endometriosis and 2,401 controls. They were randomly assigned to two phases: the discovery phase (305 cases and 1,673 controls) and the confirmation phase (101 cases and 728 controls). Baseline characteristics stratified by endometriosis status and study phase are presented in Table  1. As expected, women with endometriosis reported higher rates of dys - menorrhea and chronic pelvic pain compared to controls. Discovery phase After quality control, approximately 9  million imputed variants were analyzed using FastGWA, adjusting for age at menarche, BMI, smoking status, and 10 principal components in the primary GWAS model. We then rein- troduced dysmenorrhea as an additional covariate in a secondary analysis to assess the stability of our findings. The genomic inflation factor (λ = 1.08) indicated minimal test-statistic inflation, confirming adequate control for population stratification, suggesting the absence of false- positive association signals (Fig. 2A). Table 1 Baseline characteristics of the study population stratified by study phase and endometriosis status Characteristic Discovery Phase Confirmation Phase Endometriosis (n = 305) Non-Endometriosis (n = 1673) P-value Endometriosis (n = 101) Non-Endometriosis (n = 728) P-value Age (years) 34.0 ± 12 34.8 ± 12 0.21 32.1 ± 12.5 32.3 ± 11.7 0.11 Age at menarche (years) 13 ± 1.5 13 ± 1.50 0.046 13 ± 1.2 13 ± 1.3 0.35 Ever Smoke (n%) 11(3.7%) 56 (4.4%) 0.0001 4 (4.0%) 26 (3.6%) 0.0001 Number of parities 2.2 ± 1.0 2.3 ± 1.2 0.11 2.3 ± 1.1 2.2 ± 1.1 0.11 Number of gravidities 2.6 ± 1.2 2.7 ± 1.4 0.13 2.7 ± 1.3 2.6 ± 1.3 0.12 Number of abortions 1.4 ± 0.6 1.4 ± 0.7 0.29 1.3 ± 0.5 1.3 ± 0.7 0.23 SBP (mm Hg), mean SD 126 ± 20.2 120 ± 20.5 0.04 109 ± 12.3 111 ± 13.5 0.02 DBP (mm Hg), mean SD 80 ± 10.8 78 ± 11.02 0.07 75 ± 12.3 75 ± 9.2 0.1 BMI (kg/m2) 27 ± 5.1 27 ± 5.03 0.53 27 ± 4.6 27 ± 5.2 0.51 Waist Circumferences(cm) 93 ± 12.3 89 ± 12.3 0.02 83 ± 10.9 85 ± 11.08 0.2 Hip Circumferences (cm) 105 ± 9.9 104 ± 9.5 0.2 104 ± 8.9 103 ± 8.8 0.15 Wrist Circumferences(cm) 16.45 ± 1.09 16.1 ± 1.01 0.32 15.21 ± 0.9 15.86 ± 0.9 0.23 Data are presented as mean ± standard deviation, except for smoking status, which is presented as number (%) BMI body mass index, SBP Systolic Blood Pressure, DBP Diastolic Blood Pressure P-values were calculated using the independent samples t-test for continuous variables and the chi-square test for categorical variables Page 6 of 13 Najd-Hassan-Bonab et al. Middle East Fertility Society Journal (2026) 31:37 We identified one genome-wide significant locus at chromosome 14q22.1 (rs12886544; OR = 1.66, 95% CI: 1.35–2.04; p-value = 5.89 × 10⁻⁸) as shown in Manhattan plot in Fig.  2(B). This intronic variant is located within GNPNAT1 (Glucosamine-Phosphate N-Acetyltrans - ferase 1), a gene encoding a key enzyme in the hexos - amine biosynthesis pathway. Notably, the risk allele (C) shows marked population differentiation; minor allele frequency (MAF) = 0.495 in the TCGS cohort versus MAF = 0.001 in European populations, suggesting this locus may contribute to population-specific endometrio - sis susceptibility. Regional association plotting confirmed a distinct peak centered on GNPNAT1 without evidence of multiple independent signals (Fig. 3). The results of this sensitivity analysis demonstrated high consistency with our original findings (primary GWAS model). The key genome-wide significant association, represented by rs12886544 (OR = 1.66; p = 4.99 × 10⁻⁸), remained robust Fig. 3 Regional association (LocusZoom) plot of the GNPNAT1 locus on chromosome 14. SNPs are plotted according to genomic position (x-axis) and −log10 ( P) value (y-axis), reflecting association results from the genome-wide association analysis. The lead SNP is indicated, and surrounding variants are shown based on their local linkage disequilibrium structure Fig. 2 A Quantile–quantile (Q–Q) plot of observed versus expected −log10 ( P) values from the genome-wide association analysis of endometriosis. The genomic inflation factor (λ) was 1.08. B Manhattan plot showing genome-wide association results for endometriosis. Each point represents a single SNP plotted according to its chromosomal position (x-axis) and −log10 ( P) value (y-axis). The horizontal red line denotes the genome-wide significance threshold (P ≤ 5 × 10⁻⁸) Page 7 of 13 Najd-Hassan-Bonab et al. Middle East Fertility Society Journal (2026) 31:37 after additional adjustment for dysmenorrhea and other covariates. Furthermore, we observed a modest increase in the number of suggestive loci, from 13 SNPs in the dys- menorrhea unadjusted model (Supplementary Table 1 A) to 17 SNPs in the dysmenorrhea-adjusted model (Sup - plementary Table 1B). These results collectively indicate that the inclusion or exclusion of dysmenorrhea does not materially alter the detected genetic associations, thereby reinforcing the robustness and interpretabil - ity of our findings. To our knowledge, this locus has not been reported in prior endometriosis GWAS analyses. Three independent signals mapped to the MICAL3 locus (22q12.1), suggesting a potential regulatory hotspot. To assess whether established genetic risk factors for endometriosis are shared in the Iranian population, we examined previously reported loci and found that six variants (near WNT4, FN1, and VEZT genes) reached nominal significance, each demonstrating effect direc - tions consistent with those reported in large prior meta-analyses(Supplementary Table S2). Confirmation phase The top SNP (rs12886544) identified in the discovery phase was further evaluated in an independent confirma - tion cohort consisting of 101 women with endometriosis and 728 controls. Logistic regression analyses were per - formed in PLINK, adjusting for the covariates used in the discovery phase. Following correction for multiple testing using the false discovery rate (FDR), none of the tested variants reached statistical significance in the con - firmation phase. For the lead variant rs12886544, the direction of effect size in the confirmation phase was consistent with that observed in the discovery phase. However, the asso - ciation did not remain statistically significant after mul - tiple testing correction (OR = 1.4, 95% [CI 0.89–2.11], p-value = 0.14). A pooled analysis combining the dis - covery and confirmation phase was subsequently con - ducted using logistic regression in PLINK, applying the same covariates and analytical framework as used in the confirmation phase. This combined analysis dem - onstrated a statistically significant association between rs12886544 and endometriosis susceptibility (combined p-value = 0.001, OR = 1.59, 95% CI 1.19–1.89; Table  2). Although the association of rs12886544 did not reach statistical significance after multiple testing correction in the confirmation cohort, the consistent direction of effect and the statistically significant pooled analysis provide supportive evidence for its role in endometriosis suscep - tibility, warranting further validation in larger indepen - dent populations. Functional annotation analysis results of endometriosis- associated SNPs To characterize the potential biological function of rs12886544, we performed integrative functional annota - tion of rs12886544 using HaploReg, PheWeb, Open Tar - gets, ENCODE, Roadmap Epigenomics, GWAS Atlas, and FinnGen (Supplementary Table S3-S6). No prior GWAS has linked this variant to endometriosis. It is not in significant linkage disequilibrium (r² > 0.1) with known coding variants in the 1000 Genomes Project, suggesting an independent signal. Variant-level annotation At the variant level, rs12886544 is predicted to reside within genomic regions annotated with histone modifica- tions characteristic of enhancer and promoter elements, according to HaploReg. The variant also overlaps DNase I hypersensitivity sites, indicative of open chromatin across multiple cell types and tissues, notably in adipose and ovarian tissues. These tissues are critically involved in the pathophysiology of endometriosis, highlighting the potential functional relevance of rs12886544 in disease mechanisms through epigenetic modulation [36]. Annotations from the Roadmap Epigenomics and ENCODE projects further indicate that rs12886544 is a functionally significant variant within the reproductive system. This SNP is predicted to modify a regulatory motif and to interact with the transcription factor Bobby Sox homolog (BBX). Subsequent analysis has revealed epigenetic signals associated with rs12886544, spe - cifically the trimethylation of histone H3 at lysine 4 (H3K4me3) in reproductive tissues, including the ovary, placenta, and cervix. These findings underscore the pre - dicted regulatory role of the reproductive system, provid- ing a basis for hypothesis generation regarding potential mechanisms in endometriosis. Gene-level annotation To further investigate at the gene level, associations by using the PheWeb identified the GNPNAT1 gene as being correlated with non-inflammatory disorders of Table 2 Result for the top SNP in the pooled (combined) GWAS and confirmation study Chr: Position SNP Risk allele Nearest Gene Consequence Phase Control/case P-value OR chr14:52837402 rs12886544 T GNPNAT1 intron variant GWAS 1673/305 5.89E-08 1.66 Confirmation 728/101 0.143 1.39 Combined 2401/406 0.001 1.59 Chr chromosome, OR odds ratio Page 8 of 13 Najd-Hassan-Bonab et al. Middle East Fertility Society Journal (2026) 31:37 the cervix, conditions related to the amniotic cavity and membranes, and Leukemia in UK Biobank data. Moreover, experimental data from the Open Target platform revealed that mouse knockout studies impli - cate the GNPNAT1 gene as essential for multiple devel - opmental phenotypes, including the absence of amnion, allantois, and chorion, reduced cellular proliferation, abnormal morphology of extraembryonic tissue, and embryonic lethality occurring between implantation and somite formation, with complete penetrance.

derived from the GWAS Atlas further impli - cated GNPNAT1 in reproductive traits, such as age at menarche, menstrual quality of life, and gestational weight gain, as well as metabolic traits including waist- hip ratio, BMI, and fat mass distribution in the legs and arms. Consistent with these findings, data from the Finn - Gen cohort corroborated associations between GNP - NAT1 and diverse gynecological conditions, including adenomyosis (endometriosis of the uterus), deep endo - metriosis, endometriosis involving the fallopian tube, rectovaginal septum, and vagina, in addition to female infertility, pain syndromes, and other disorders related to the female genital organs and menstrual cycle (Supple - mentary Table S6). So, gene-level evidence indicates that GNPNAT1 is associated with reproductive and metabolic traits, highlighting the potential involvement of GNP - NAT1 in reproductive and related traits. Pathway for the GNPNAT1 Gene We utilized the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases to identify key biochemical pathways associated with endometriosis. The result revealed that the GNPNAT1 gene is involved in the biosynthesis of UDP-N-acetylglucosamine, a vital donor molecule in the initial two steps of the N-glycan precursor biosynthesis pathway. Subsequently, UDP- N-acetylglucosamine serves as a substrate for further enzymatic modifications following the attachment of the glycan precursor to the target protein. In addition, at the epigenetic level, it influences transcription factor binding activity, notably that of the Bobby Sox homolog (BBX), which is critically involved in developmental processes and the regulation of Wnt signaling pathways. The signif- icantly enriched pathways identified in this analysis were subsequently compared with those previously inves - tigated in the literature through candidate gene asso - ciation studies to evaluate consistency [ 37]. These gene regulatory pathway links are predicted from functional genomic and association databases. Further mechanistic studies can clarify the functional roles of rs12886544 and GNPNAT1 in endometriosis pathogenesis.

This study presents the first genome-wide association scan for endometriosis in an Iranian population, provid - ing critical insights into the disease’s genetic architec - ture within this underrepresented ancestry. Our analysis identified a candidate susceptibility locus rs12886544, an intronic variant within the GNPNAT1 gene on chromosome 14, that demonstrates a significant asso - ciation with increased endometriosis risk(OR = 1.66, p-value = 5.89 × 10⁻⁸). Beyond being the first GWAS of endometriosis in an Iranian population, our study con - tributes by evaluating genetic risk in an understudied ancestry, where differences in allele frequencies and LD structure may reveal signals not captured in predomi - nantly European and East Asian cohorts. This finding is further supported by the consistency observed across our independent discovery and confirmation cohorts (com - bined p-value = 0.001, OR = 1.59). To contextualize our findings, we assessed previously reported loci and observed that six variants (near WNT4, FN1, and VEZT genes) showed nominal significance with effect directions consistent with prior large meta-analy - ses, providing supportive internal validation and indicat - ing that aspects of endometriosis genetic architecture are shared across.We also highlight the emerging role of GNPNAT1 in reproductive biology, while noting that molecular mechanisms remain to be clarified. Impor - tantly, observed differences in allele frequencies between Iranian and European populations, along with variability in sample sizes across existing GWASs, likely contribute to the heterogeneous results reported in the literature, highlighting the value of studying diverse ancestries [38]. Although the functional impact of rs12886544 on endometriosis pathogenesis has not been experimentally validated, genomic annotation reinforces the potential involvement of this non-coding variant near the GNP - NAT1 gene in reproductive disorders. UK Biobank data demonstrate significant associations between the GNP - NAT1 gene and a spectrum of genital tract conditions, supporting the gene’s involvement in relevant patho - physiology. GNPNAT1 encodes a critical enzyme in the hexosamine biosynthesis pathway (HBP), responsible for producing UDP-GlcNAc, a central metabolite inte - grating glucose, amino acid, nucleotide, and fatty acid metabolism. UDP-GlcNAc serves as an essential sub - strate for both N-linked and O-linked glycosylation pro - cesses, which are pivotal for protein folding, stability, and function. Proper N-glycosylation in the Golgi apparatus ensures the correct maturation of membrane and secre - tory proteins, while defects in this pathway lead to pro - tein misfolding, endoplasmic reticulum stress, altered cell surface proteomes, and subsequent disruptions in cellular signaling and fate determination [ 39– 41]. Based on genomic and regulatory annotations of rs12886544, Page 9 of 13 Najd-Hassan-Bonab et al. Middle East Fertility Society Journal (2026) 31:37 suggesting a potential role in gene regulation, we hypoth- esize that this variant may influence endometrial cell behavior [ 42] by modulating GNPNAT1-dependent processes such as cell adhesion, migration, and signal transduction. We emphasize that although GNPNAT1’s biological role in glycosylation pathways is well estab - lished, direct experimental evidence linking rs12886544 to altered GNPNAT1 expression or function in endome - trial tissue has yet to be demonstrated. Furthermore, O-GlcNAcylation, a dynamic post- translational modification mediated by HBP metabo - lites, is frequently upregulated in tumor cells, promoting mechanisms like proliferation. Inhibiting the HBP has induced growth arrest and apoptosis in tumor models. Consistently, in vitro studies demonstrate that GNP - NAT1 knockdown reduces proliferation and invasive - ness in breast cancer cell lines, supporting a functional role for this gene in cell growth and migration [ 43, 44]. Collectively, these data highlight GNPNAT1 as a biologi - cally plausible candidate locus influencing endometriosis susceptibility through its involvement in critical glycosyl- ation-mediated cellular processes [39, 43]. Recent studies have linked UDP-GlcNAc to the regu - lation of Wnt signaling. Neitzel et al. demonstrated that UDP-GlcNAc modulates Wnt pathway activity via protein glycosylation, a modification critical for proper receptor function and the downstream signaling cascade [ 45]. In addition to its role in Wnt signaling, UDP-GlcNAc influ - ences hyaluronan-CD44 interactions and immune modu- lation, processes regulated in part by Leukemia inhibitory factor (LIF), a cytokine essential for endometrial receptiv- ity and embryo implantation. Disruption of LIF-mediated signaling, as observed in infertile women, impairs down - stream effectors, including STAT3, MAPK, and Protein kinase C (PKC)pathways, which are integral to blastocyst adhesion [ 46– 48]. Notably, elevated STAT3 expression has been reported in both endometriosis and endometrial cancer, implicating STAT3 as a potential risk factor for these disorders [49, 50]. Glycosylation plays a pivotal role in endometrial func - tion, with the FERM Domain Containing Kindlin 2 (FERMT2) gene, located adjacent to GNPNAT1, regu - lating the glycosylation of highly glycosylated proteins, such as MUC1, which are essential for integrin-mediated adhesion and implantation. In parallel, the O-GlcNAc modification supports endometrial cell proliferation and invasion during the secretory phase, underscoring the contribution of glycosylation-dependent pathways to endometrial receptivity and fertility [ 51]. The FERMT2, a member of the FERMT adaptor protein family, is highly expressed in reproductive tissues, particularly the endometrium and uterus, and has been shown to facili - tate trophoblast adhesion and invasion throughout ges - tation [ 52]. Notably, gene-based association analyses reveal that FERMT2 and GNPNAT1 exhibit comparable significance levels and a strong correlation, suggesting a potential co-regulatory relationship that influences glycosylation-mediated adhesion and implantation mechanisms processes that may also contribute to endo - metriosis susceptibility [53]. Our findings are consistent with prior studies. Based on PheWeb data, several of our suggestive variants showed associations with reproductive and metabolic traits. The variants rs8143037 and rs401910 (intron variants of the MICAL3 gene) were associated with irregular menstrual bleeding, anatomical abnormalities of the ureters, and dysmenorrhea. The variants rs706042 (intron variant of the CHIC1 gene) and rs6593654 (nearest gene: RWDD3) were linked to disorders of the cervix, prolapse of vagi - nal walls and vaginal vault after hysterectomy, premeno - pausal menorrhagia, and ovarian cysts. The rs12981001 variant (nearest gene: IL4I1) was associated with endo - metrial hyperplasia, disorders of menstruation, abnormal bleeding from the female genital tract, irregular men - strual cycle, and absent or infrequent menstruation. The rs396717 variant (intron variant of the MICAL3 gene) was also associated with endometrial hyperplasia. The rs17058669 variant (nearest gene: ZNF516) was associ - ated with menopause and placenta previa. The variants rs111741344 (intron variant of the B3GALT1 gene) and rs6593654 (nearest gene: RWDD3) were associated with ovarian cysts. Additionally, rs35913552 (nearest gene: APOB) and rs13011615 (nearest gene: NCKAP1L) were associated with ectopic pregnancy. Notably, variants rs706042 (CHIC1), rs35913552 (APOB), and rs13011615 (NCKAP1L) were also associated with type 2 diabetes, disturbances in lipid metabolism, and hypercholester - olemia, conditions that may be more prevalent among women with endometriosis. The biological functions of these genes, including roles in actin cytoskeleton regula - tion (MICAL3), immune modulation (IL4I1, NCKAP1L), lipid metabolism (APOB), and protein glycosylation (B3GALT1), may provide mechanistic insights into the pathophysiology of endometriosis [54]. Although evidence of epigenetic contributors to endo - metriosis has historically been limited, recent in vitro and in vivo studies have increasingly elucidated a signifi - cant epigenetic role in the pathogenesis of this condition. Complementary data from the HaploReg database pro - vide evidence supporting the involvement of rs12886544 in epigenetic regulation, specifically through histone modification marked by H3K4me1 in ovarian tissue. The risk allele of this variant may influence transcription fac - tor binding activity, notably that of BBX. BBX harbors a high-mobility group (HMG) box domain, which is criti - cally involved in developmental processes and the regu - lation of Wnt signaling pathways [ 55]. Interestingly, the findings indicate that Wnt signaling is involved in all Page 10 of 13 Najd-Hassan-Bonab et al. Middle East Fertility Society Journal (2026) 31:37 forms of endometriosis, as demonstrated by evidence from both single-SNP analyses and pathway-based asso - ciation studies of Wnt genes [12– 57]. A recent survey by Marquardt et al. demonstrated that epigenetic dysregulation, particularly of histone modifications such as H3K4me1, plays a crucial role in altering gene expression, contributing to ectopic endo - metrial growth and infertility. H3K4me1 is differen - tially enriched in endometriotic stromal cells, regulating genes involved in proliferation, invasion, and immune response [ 58]. Dysregulated H3K4me1 at this enhancer may impact pathways such as the Wnt/β-catenin or TGF- β pathways, which are known to drive the formation of endometriotic lesions and promote inflammation, as previously explained. Additionally, experimental data from Timofeeva and colleagues suggest that the absence of specific proteins and their corresponding RNAs in the oocyte affects the development of the preimplantation embryo. These key proteins include transcription fac - tors such as BBX and zinc finger protein 646, along with histone modifier Ankyrin repeat domain 12 (ANKRD12) [59]. Collectively, these findings underscore the critical importance of the epigenetic signature associated with this variant, suggesting it may have a substantial influ - ence on the molecular mechanisms underpinning the pathophysiology of endometriosis. This warrants further investigation into its functional impact on the regulation of endometrial genes. The observed epigenetic modifica - tions suggest potential effects on chromatin accessibility, transcription factor recruitment, and gene expression regulation within endometrial tissue [45]. Importantly, we observed notable differences in allele frequencies between our Iranian cohort and European- ancestry reference populations (Ensembl), suggesting potential heterogeneity in genetic architecture across ancestries. In addition to variability in sample sizes across prior GWAS (ranging from 171 to 17,045 participants) [60], differences in ethnic and genetic backgrounds likely contribute to the inconsistent results reported in the lit - erature [61]. Therefore, our identification of rs12886544 as a candidate genetic locus associated with endome - triosis may be partly attributable to these underlying population-specific genetic variations and the diverse demographic characteristics of the study cohorts. For example, SNPs such as rs706042, rs12981001, rs251151, rs13011615, rs111741344, and rs35913552 exhibit mark - edly divergent effect allele frequencies, with higher fre - quencies observed in European populations (0.816, 0.792, 0.75, 0.83, 0.82, and 0.835) compared to Iranian popula - tions (0.14, 0.21, 0.15, 0.19, 0.23, and 0.19), respectively. In contrast, the allele frequencies of most endometrio - sis-associated variants were broadly comparable between Iranian and European populations, with the exception of rs868941587 and rs17058669 (Supplementary Table 1). The markedly low frequency of these two variants in our Iranian cohort may contribute to the significant associa - tion observed exclusively among Iranian women, poten - tially explaining why these signals remained undetected in previous Euro-centric studies where their effects may have been masked.This pattern aligns with prior research demonstrating that ancestry-specific analyses can reveal distinct genetic contributors to endometriosis. For exam- ple, studies focused solely on individuals of East Asian descent have identified several variants associated with endometriosis that were not detected in predominantly European cohorts, including five SNPs reported by two independent studies [ 9, 62] and 14 SNPs identified by Wang et al. [ 63]. In contrast, studies incorporating more ethnically diverse populations, such as those by Sapkota et al., [ 64] and Sobalska-Kwapis et al., have reported broader but non-overlapping sets of associated SNPs, further highlighting substantial cross-population het - erogeneity. Collectively, these observations underscore the importance of considering population genetic struc - ture and ancestral background when interpreting genetic association results. The principal strengths of the present study are embed- ded within its robust methodological framework, which is distinguished by a large, population-based design. This approach enhances the generalizability and statis - tical power of the findings by minimizing selection bias and ensuring a representative sample. Furthermore, the comprehensive nature of the study design enables a more accurate assessment of genetic associations within diverse demographic groups, thereby enhancing the reli - ability and validity of the results. It is essential to acknowledge the limitations of our study. The primary limitation is the absence of a com - prehensive evaluation of all endometriosis cases via laparoscopy, which remains the definitive diagnostic standard for this condition. Additionally, our dataset lacks sufficient reliability for the precise identification of endometriosis severity. Moreover, individuals with moderate to severe dysmenorrhea but without a con - firmed diagnosis of endometriosis were excluded if their ultrasound imaging did not indicate the presence of endometriosis. However, because a negative ultrasound

cannot definitively exclude endometriosis, espe - cially in cases of superficial peritoneal involvement, our

may not entirely reflect the population with less severe manifestations of the condition. Furthermore, our study was conducted with a relatively modest sam - ple size compared with contemporary GWAS, which may limit precision in effect estimation. Power calcula - tions performed based on assumptions of a risk allele frequency of 0.49, genotype relative risk of 1.5, disease prevalence of 20%, and standard genome-wide signifi - cance thresholds, indicated adequate statistical power. Page 11 of 13 Najd-Hassan-Bonab et al. Middle East Fertility Society Journal (2026) 31:37 However, these estimates reflect optimistic assumptions and should be interpreted accordingly. We also recog - nize the potential impact of the winner’s curse, whereby effect sizes observed in initial discovery analyses may be inflated due to statistical selection bias [ 65]. Although this bias affects studies across populations, the identi - fication of this variant specifically in our cohort raises the possibility of ancestry-related heterogeneity or a genuinely larger effect in this population. Nonetheless, independent replication in a larger cohort of the same ancestry will be essential to confirm this association and obtain an unbiased estimate of effect size. Another key limitation of this study is the absence of an independent external cohort for replication. Because large-scale genomic datasets from Middle Eastern or Ira - nian populations are currently scarce, we were unable to validate our lead association in an ancestrally matched cohort. Establishing this locus will require replication in independent datasets, ideally including samples from ancestrally relevant populations or large multi-ancestry biobanks such as All of Us or similar resources. Such external validation is essential to confirm the robustness, generalizability, and true biological significance of the candidate locus. Our research concentrated exclusively on genetic vari - ants situated on the autosomal chromosomes, thereby omitting analysis of the X chromosome. Subsequent studies incorporating sex chromosome data could yield further valuable insights. Furthermore, our analysis was limited to single-variant association testing and did not incorporate haplotype-based analyses or cross-popula - tion comparisons of linkage disequilibrium (LD) struc - ture surrounding the GNPNAT1 locus. Additionally, LD Score Regression (LDSC) could not be performed due to the absence of publicly available Iranian LD reference panels; applying European LD reference panels to our cohort would introduce bias, given documented popula - tion-specific differences in LD architecture.

In conclusion, our GWAS identification of rs12886544 is presented as a candidate population-specific locus that may arise from these ancestry differences. In sum, our study adds (i) the largest Iranian GWAS dataset to date, (ii) evidence of concordance with established loci (mod - est internal validation), and (iii) population-specific allele-frequency that can inform future trans-ancestry meta-analyses and functional follow-up. Independent replication cohorts and inclusion in meta-analyses will be required validate this association and elucidate its clinical relevance for risk stratification and therapeutic development. Supplementary Information The online version contains supplementary material available at h t t p s : / / d o i . o r g / 1 0 . 1 1 8 6 / s 4 3 0 4 3 - 0 2 6 - 0 0 3 2 4 - 2 . Supplementary Material 1: Supplementary Tables. Authors’ contributions Leila Najd-Hassan-Bonab contributed to conceptualization, methodology, software, and writing the original draft. Farzaneh Motafeghi contributed to data curation and investigation. Samaneh Chegeni contributed to data curation, review, and editing. Fereidoon Azizi contributed to the review and editing. Maryam S. Daneshpour contributed to methodology, validation, review, and editing. Fahimeh Ramezani Tehrani contributed to methodology, validation, and review editing. All authors saw and approved the final version, and no other person made a substantial contribution to the paper. Funding Research reported in this publication was supported by Elite Researcher Grant Committee under award number [IR.NIMAD.REC.4040334] from the National Institute for Medical Research Development (NIMAD), Tehran, Iran Data availability Data Availability On Request. Declarations Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki and institutional guidelines. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1Cellular and Molecular Endocrine Research Center, Research Institute for Endocrine Molecular Biology, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2Reproductive Endocrinology Research Center, Research Institute for Endocrine Molecular Biology, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 4Endocrine Research Center, Research Institute for Endocrine Disorders, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran 5Foundation for Research & Education Excellence, Vestavia Hills, AL, USA Received: 1 December 2025 / Accepted: 23 April 2026

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