The objective of the present study was to investigate the differences in the proteomic profiles of sperm from infertile males with severe oligoasthenoteratozoospermia requiring intracytoplasmic sperm injection (ICSI) and normal control sperm from fertile males. Isobaric tag for relative and absolute quantitation labeling and liquid chromatography–tandem mass spectrometry was performed for identifying proteins in the sperm of infertile and fertile males. Differentially expressed proteins were analyzed via the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases through the Database for Annotation, Visualization, and Integrated Discovery, and protein–protein networks were produced using the Search Tool for Retrieval of Interacting Genes. Immunofluorescence and western blotting verified the differential expression of Y-box-binding protein 1(YBX1), adenylate kinase 1 (AK1), and aconitase 2, mitochondrial (ACO2) proteins. Altogether, 3444 proteins were identified in the sperm of infertile and fertile males, and 938 were differentially expressed between the two groups. Pairwise comparisons revealed that 226 and 712 proteins were significantly upregulated and downregulated in infertile males, respectively. These proteins were significantly enriched in metabolic pathways as per KEGG enrichment analysis. YBX1 expression was upregulated in the sperm heads of patients requiring ICSI treatment, whereas AK1 and ACO2, which are critical enzymes involved in energy metabolism, were downregulated in the sperm tails of the same patients. This result indicates that metabolism may have a crucial role in maintaining normal sperm function. Overall, our results provide insights that will further help in investigating the pathogenic mechanisms of infertility and possible therapeutic strategies.

本研究的目的是调查来自需要胞浆内单精子注射 (ICSI) 的严重少弱畸形精子症的不育男性和来自可育男性的正常对照精子的蛋白质组学特征的差异。用于相对和绝对定量标记的等压标签和液相色谱-串联质谱法用于鉴定不育和可育男性精子中的蛋白质。通过基因本体论和京都基因和基因组百科全书 (KEGG) 数据库通过注释、可视化和集成发现数据库分析差异表达的蛋白质，并使用检索相互作用基因的搜索工具生成蛋白质-蛋白质网络。免疫荧光和蛋白质印迹验证了 Y-box 结合蛋白 1 (YBX1)、腺苷酸激酶 1 (AK1) 和乌头酸酶 2、线粒体 (ACO2) 蛋白的差异表达。总共在不育和可育男性的精子中鉴定出 3444 种蛋白质，其中 938 种在两组之间差异表达。成对比较显示，226 和 712 种蛋白质在不育男性中分别显着上调和下调。根据 KEGG 富集分析，这些蛋白质在代谢途径中显着富集。YBX1 在需要 ICSI 治疗的患者的精子头部中表达上调，而作为参与能量代谢的关键酶 AK1 和 ACO2 在相同患者的精子尾部中表达下调。这一结果表明新陈代谢可能在维持正常的精子功能方面发挥着至关重要的作用。总体而言，我们的结果提供的见解将进一步有助于研究不孕症的致病机制和可能的治疗策略。