Identification of biomarkers associated with macrophage infiltration in non-obstructive azoospermia using single-cell transcriptomic and microarray data.

Background: Non-obstructive azoospermia (NOA) is a common clinical cause of male infertility. Research suggests that macrophages are linked to testicular function; however, their involvement in NOA remains unknown. Methods: To evaluate the importance of macrophages infiltration in NOA and identify the macrophage-related biomarkers, the gene-expression microarray data GSE45885 and the single-cell transcriptomic data GSE149512 were utilized from the Gene Expression Omnibus (GEO). A single-sample gene set enrichment analysis (ssGSEA) was conducted to investigate immune cell proliferation. The Seurat package was used for the single-cell data analysis, and the limma package was used to identify the differentially expressed genes between the NOA and normal samples. Moreover, we conducted a weighted gene co-expression network analysis (WGCNA) to identify the macrophage-related key modules and genes, and conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses for the functional exploration. To identify the macrophage-related biomarkers, we conducted least absolute shrinkage and selection operator (LASSO) and support vector machine-recursive feature elimination (SVM-RFE) analyses. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to verify the marker genes present in NOA. Results: We confirmed that open reading frame 72 gene on chromosome 9 ( Conclusions: Through the combination of tissue transcriptomic and single-cell RNA-sequencing analyses, we concluded that macrophage infiltration is significant in different subtypes of NOA, and we hypothesized that