Exploration of Diagnostic Biomarkers in Seminal Plasma and Metabolic Mechanisms of Non-Obstructive Azoospermia Based on Metabolomics and Transcriptomics

Background Non-obstructive azoospermia (NOA) is a severe form of male infertility for which diagnosis may involve invasive testicular biopsy. This study aimed to identify candidate non-invasive biomarkers in seminal plasma and explore metabolic pathways associated with NOA. Methods We conducted a cross-sectional study of seminal plasma from 20 men with NOA and 20 normospermic controls recruited at the Second Hospital of Shanxi Medical University between December 2022 and November 2023. Targeted liquid chromatography–mass spectrometry quantified 414 metabolites. Differential metabolites were identified using orthogonal partial least-squares discriminant analysis (variable importance in projection > 1) and t tests (P < 0.05), and their diagnostic performance was assessed by receiver operating characteristic analysis. Pathway enrichment was integrated with publicly available testicular transcriptomic data (GSE4797; 16 NOA and 12 control samples). Results The NOA and control groups separated in orthogonal partial least-squares discriminant analysis (Q² = 0.72; R²Y = 0.887; both P < 0.05). Seventy-three metabolites met the differential-abundance criteria (variable importance in projection > 1 and P < 0.05), including 42 with higher and 31 with lower abundance in NOA. Five metabolites had reported receiver-operating-characteristic areas under the curve of 0.900 for L-pipecolic acid, 0.998 for phenylpyruvic acid, 0.930 for thymidine, 0.905 for thiamine pyrophosphate, and 0.887 for L-methionine sulfone. Integration with transcriptomic data implicated purine, amino-acid, and energy-metabolism pathways and suggested an association with oxidative-stress-related metabolic dysregulation. Conclusions The findings identify candidate seminal-plasma biomarkers and metabolic pathways associated with NOA. The biomarkers require evaluation in larger, independent and multicenter cohorts, including comparison with obstructive azoospermia, before clinical use.