N6-methyladenosine methylation regulators can serve as potential biomarkers for endometriosis related infertility

Abstract

Endometriosis (EMS) is a chronic inflammatory disease frequently associated with infertility. N6-methyladenosine (m6A) methylation, the most common form of methylation in eukaryotic mRNAs, has gained attention in the study of female reproductive diseases, including EMS and infertility. This study aimed to investigate the role of m6A regulators in EMS-related infertility. To begin, specific m6A regulators were identified by analyzing the GSE120103 dataset, followed by receiver operating characteristic (ROC) curve analysis. A nomogram model was then constructed, and unsupervised clustering of m6A regulators was performed to identify distinct m6A molecular clusters. Functional enrichment analysis of differentially expressed genes (DEGs) between these clusters, along with immune cell infiltration analysis, was subsequently conducted. In addition, the single-cell dataset GSE214411 was analyzed to explore the role of m6A regulators in various cell types. Finally, clinical samples were collected, and immunohistochemistry analysis was performed. The study identified seven key m6A regulators with significant diagnostic value for EMS-related infertility and two distinct m6A molecular clusters. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEGs between the clusters revealed that m6A clustering was strongly associated with immune pathways. Immune cell infiltration analysis further demonstrated that the expression levels of m6A regulators had a notable impact on immune cell infiltration. Single-cell analysis revealed that HNRNPA2B1 and HNRNPC were significantly elevated in endometrial immune cells from infertile EMS patients but notably decreased in stromal cells. Immunohistochemical staining confirmed that HNRNPA2B1 and HNRNPC expression levels were significantly higher in the eutopic endometrium of fertile women compared to ovarian EMS patients. These findings suggest that m6A regulators play critical roles in the development and progression of EMS-related infertility. Notably, HNRNPA2B1 and HNRNPC may serve as potential biomarkers for this condition.

Figure 1.

Expression features of m6A regulators in eutopic endometrium from infertile patients with EMS and identification of key diagnostic candidate regulators. (A) Differences in the expression of m6A regulators in endometrial tissue between normal fertile women and infertile patients with EMS; (B) Differences in the expression of m6A regulators in endometrial tissue between fertile and infertile patients with EMS; (C) Differences in the expression of m6A regulators in endometrial tissue between normal fertile women and fertile EMS patients; (D) Correlation analysis of differentially expressed m6A regulators in the endometrial tissue of normal fertile women, fertile EMS patients, and infertile EMS patients; (E–K) ROC curve analysis of intersecting m6A regulators. m6a: N6-methyladenosine; EMS: Endometriosis; ROC: Receiver operating characteristic.

Figure 2.

Construction of the nomogram model for key candidate m6A regulators and identification of molecular clusters via unsupervised clustering. (A) Nomogram model based on key candidate m6A regulators; (B) DCA curve of the nomogram model; (C) The clinical impact curve of the nomogram model; (D) Calibration curve of the nomogram model; (E) m6A molecular clusters based on candidate regulators with K ═ 2; (F) The CDF curve (K ═ 2–9); (G) The variation in the area under CDF curve (K ═ 2–9). m6a: N6-methyladenosine; DCA: Decision curve analysis.

Figure 3.

Cluster grouping of key candidate m6A regulators and GO and KEGG analyses of DEGs. (A) PCA of two m6A clusters; (B) PCA of the adjusted two clusters; (C) Clustering heatmap of key candidate m6A regulators in the adjusted two clusters; (D) Differences in the expression levels of key candidate m6A regulators in the adjusted two clusters; (E) Statistical plot of DEGs in the adjusted two clusters; (F) GO analysis of DEGs in the adjusted two clusters (top 30 GO terms); (G) KEGG analysis of DEGs in the adjusted two clusters (top 20 pathways). m6a: N6-methyladenosine; GO: Gene Ontology; DEG: Differentially expressed gene; KEGG: Kyoto Encyclopedia of Genes and Genomes; PCA: Principal component analysis.

Figure 4.

Relationships among key candidate m6A regulators, cluster grouping and immune cell infiltration. (A) The correlation between the two m6A clusters and immune cell infiltration; (B) Correlations between key candidate m6A regulators and immune cell infiltration; (C) Differences in immune cell infiltration between groups with low and high HNRNPC expression; (D) Differences in immune cell infiltration between groups with low and high HNRNPA2B1 expression; (E) Differences in immune cell infiltration between groups with low and high LRPPRC expression; (F) Differences in immune cell infiltration between groups with low and high IGF2BP1 expression; (G) Differences in immune cell infiltration between groups with low and high FTO expression; (H) Differences in immune cell infiltration between groups with low and high IGFBP3 expression; (I) Differences in immune cell infiltration between groups with low and high YTHDF2 expression. m6a: N6-methyladenosine.

Figure 5.

Analysis of the expression levels of key candidate m6A regulators in various cell types. (A) The characteristically expressed genes of different cell types; (B) Distribution of the expression levels of various cell types; (C) The expression levels of each key candidate m6A regulator in various cell types of eutopic endometrium; (D) The expression levels of HNRNPA2B1, HNRNPC, YTHDF2, and LRPPRC in various cell types within eutopic endometrium of normal fertile women and infertile EMS patients. m6a: N6-methyladenosine.

Figure 6.

HNRNPA2B1 and HNRNPC can serve as potential biomarkers for EMS-related infertility. (A) Sierra figures of the expression levels of HNRNPA2B1, HNRNPC, YTHDF2, and LRPPRC in various cell types within eutopic endometrium of normal fertile women and infertile patients with EMS; (B) Analysis of the expression levels of HNRNPA2B1 and HNRNPC in various cell types in the eutopic endometrium of normal fertile women and infertile patients with EMS. The immune cells are marked by blue arrows, and the stromal cells are marked by red arrows; (C) Immunohistochemical staining analysis of HNRNPA2B1 in eutopic endometrium from normal fertile women and infertile patients with EMS; (D) Immunohistochemical staining analysis of HNRNPC in eutopic endometrium from normal fertile women and infertile patients with EMS. EMS: Endometriosis; NK: Natural killer.