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 ( C9orf72 ) [area under the curve (AUC) =0.861] and cartilage-associated protein ( CRTAP ) (AUC =0.917) were the hub genes of NOA, and the RT-qPCR analysis revealed the critical expression of both genes in NOA. 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 C9orf72 and CRTAP play critical roles in NOA due to their high expression in macrophages.

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