BackgroundNon-obstructive azoospermia (NOA) is the most severe type that leads to 1% of male infertility. Wnt signaling governs normal sperm maturation. However, the role of Wnt signaling in spermatogonia in NOA has incompletely been uncovered, and upstream molecules regulating Wnt signaling remain unclear.MethodsBulk RNA sequencing (RNA-seq) of NOA was used to identify the hub gene module in NOA utilizing weighted gene co-expression network analyses (WGCNAs). Single-cell RNA sequencing (scRNA-seq) of NOA was employed to explore dysfunctional signaling pathways in the specific cell type with gene sets of signaling pathways. Single-cell regulatory network inference and clustering (pySCENIC) for Python analysis was applied to speculate putative transcription factors in spermatogonia. Moreover, single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) determined the regulated genes of these transcription factors. Finally, spatial transcriptomic data were used to analyze cell type and Wnt signaling spatial distribution.ResultsThe Wnt signaling pathway was demonstrated to be enriched in the hub gene module of NOA by bulk RNA-seq. Then, scRNA-seq data revealed the downregulated activity and dysfunction of Wnt signaling of spermatogonia in NOA samples. Conjoint analyses of the pySCENIC algorithm and scATAC-seq data indicated that three transcription factors (CTCF,AR, andARNTL) were related to the activities of Wnt signaling in NOA. Eventually, spatial expression localization of Wnt signaling was identified to be in accordance with the distribution patterns of spermatogonia, Sertoli cells, and Leydig cells.ConclusionIn conclusion, we identified that downregulated Wnt signaling of spermatogonia in NOA and three transcription factors (CTCF,AR, andARNTL) may be involved in this dysfunctional Wnt signaling. These findings provide new mechanisms for NOA and new therapeutic targets for NOA patients.