DIA-based analysis of the menstrual blood proteome identifies association between CXCL5 and IL1RN and endometriosis

Endometriosis is a gynecological disease related to menstruation that affects nearly 10% of reproductive-age women. However, so far, there are no reliable diagnostic biomarkers for endometriosis, causing a delay in diagnosis of 6.7 ± 6.2 years. Menstrual blood is a non-invasive source of endometrial tissue that can be analyzed for biomarkers of endometriosis. In this study, menstrual blood samples were collected from women with (n = 8) and without (n = 8) endometriosis. Data Independent Acquisition (DIA)-based mass spectrometry and bioinformatic analysis were used to quantify and identify differentially expressed proteins (DEPs) using the thresholds of fold change >1.5 and P value <0.05. A total of 95 DEPs were identified in menstrual blood from women with endometriosis compared to women without endometriosis, of which 64 were up-regulated and 31 were down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to functionally annotate DEPs. Protein-protein interaction (PPI) network analysis was then conducted to identify hub genes and the MCODE plugin placed CXCL1, CXCL3, CXCL5, CCL18, and IL1RN in the most significant cluster network. The expression of the above candidate proteins was confirmed by enzyme-linked immunosorbent assay (ELISA), among which CXCL5 and IL1RN protein expression was increased in patients with endometriosis, indicating that CXCL5 and IL1RN in menstrual blood may be useful biomarkers to diagnose endometriosis from non-invasive samples. SIGNIFICANCE: Endometriosis is a common gynecological disease that causes discomfort in many women. Unfortunately, the diagnosis of endometriosis is frequently delayed due to a lack of reliable non-invasive biomarkers. To our knowledge, this is the first time that DIA-MS was used to characterize the proteome and identify the differentially expressed proteins in menstrual blood from women with endometriosis. The results, as confirmed by ELISA, showed that CXCL5 and IL1RN protein expression is significantly increased in patients with endometriosis, indicating that these proteins can be used as biomarkers for endometriosis. This study contributes to the identification of putative endometriosis biomarkers from non-invasive samples and lays the groundwork for future research into the roles of CXCL5 and IL1RN in the pathogenesis of endometriosis.