Background  Non-obstructive azoospermia (NOA) is the most serious condition in male infertility patients, and its pathophysiology remains unclear.

  Objective  To preliminary investigate the candidate biomarkers of NOA and analyze seminal plasma of NOA by liquid chromatography with tandem mass spectrometry (LC-MS/MS) quantitative proteomics.

  Methods  Totally 21 NOA patients who underwent semen routine analysis in the First Medical Center of Chinese PLA General Hospital from October 2020 to January 2022 were recruited, and they were divided into primary hypogonadism group (PH group, n=12), secondary hypogonadism group (SH group, n=6) and normal sex hormone group (NH group, n=3). Identification and relative quantification of proteins using label-free proteomics via LC-MS/MS was carried out on seminal plasma from NOA patients. MS data files were imported to Proteome Discoverer software to identify the proteins. Then bioinformatics analysis of differentially expressed proteins (DEPs) was performed.

  Results  A total of 1 144 proteins were detected. Compared with group NH, there were 32 up-regulated DEPs and 8 down-regulated DEPs in PH group, 14 up-regulated DEPs and 99 down-regulated DEPs in SH group. Compared with SH group, 146 up-regulated DEPs and 21 down-regulated DEPs were found in PH group. The functions of DEPs mainly included hydrolase activity, enzyme catalytic activity, metabolism, peptide endonuclease regulation activity and receptor binding GO terms, and the pathways of DEPs mainly enriched into lysosome, complement and coagulation cascade, glucose degradation and amino acid metabolism pathways. Protein-protein interaction network by STRING revealed that Malate dehydrogenase (MDH2), Peptidyl-prolyl cis-trans isomerase (PPIA), Inter-alpha-trypsin inhibitor heavy chain (ITIH4), Heat shock protein 90 kDa (HSP90B1), Alpha-2-HS-glycoprotein (AHSG), CD44 antigen (CD44) and Complement component (C9) might play important roles in the spermatogenesis regulation network in NOA, and we focused on two proteins, MDH2 and HSP90B1, which were associated with spermatogenesis. Compared with SH group, MDH2 was down-regulated and HSP90B1 was up-regulated in PH group.

  Conclusion  In conclude, MDH2, PPIA, ITIH4, HSP90B1, AHSG, CD44 and C9 may play important roles in the spermatogenesis regulation network in NOA. Moreover, low expression of MDH2 is caused by testicular dysfunction leading to NOA, while the low expression of HSP90B1 is caused by regulating lipid metabolism affecting sex hormone levels. The functional enrichment analysis of DEPs suggests that the abnormal amino acid metabolism and lysosome digestion may contribute to disorder of hypothalamic-pituitary-gonad axis negative and positive feedback regulation. Abnormal metabolism pathway, disrupted glucose degradation, and disrupted complement and coagulation cascade may lead to testicular dysfunction.