目的 探讨精浆外泌体miR-26a-5p及其靶基因在特发性畸形精子症中的作用机制，揭示其调控精子形态异常的通路，为临床诊断和靶向干预提供依据。 方法 采用病例对照研究设计，纳入154例男性受试者（畸形精子症组TZS 84例，正常对照组NC 70例）。通过精浆外泌体分离，筛选差异表达miRNA，分析预测靶基因及信号通路。利用双荧光素酶报告基因实验验证miR-26a-5p与PTEN的直接结合作用，通过实时荧光定量PCR、Western blot检测miRNA及蛋白表达水平。采用Spearman相关性分析评估miR-26a-5p、PTEN与精子形态参数的关系，并通过ROC曲线分析诊断效能。 结果 精浆外泌体miRNA测序共鉴定出11种差异表达miRNA，其中miR-26a-5p在TZS组显著上调（P < 0.05），且与TZS组的正常形态精子占比呈负相关（r = -0.762，P < 0.05）。PTEN为miR-26a-5p的直接靶基因，其表达在TZS组显著降低（P < 0.05），并与TZS组的精子正常形态率呈正相关（r = 0.821，P < 0.05）。miR-26a-5p与PTEN呈负相关（r = -0.878，P < 0.05）。双荧光素酶实验证实miR-26a-5p可特异性抑制PTEN 3'UTR活性（荧光素酶活性降低45%，P < 0.05）。功能富集分析显示，miR-26a-5p-PTEN轴通过调控PI3K/AKT/mTOR、p53、Wnt/β-catenin及NF?κB通路，加剧氧化应激、DNA损伤及精子细胞分裂异常。ROC曲线分析表明，miR-26a-5p与PTEN对畸形精子症的诊断效能分别为AUC^ROC = 0.785（灵敏度78.9%，特异度71.3%）和AUC^ROC = 0.754（灵敏度73.3%，特异度75.1%）。 结论 miR-26a-5p通过靶向抑制PTEN，激活PI3K/AKT/mTOR等通路并抑制DNA修复功能，导致氧化应激积累及精子形态异常。精浆外泌体miR-26a-5p高表达与PTEN低表达的组合模式显示出对特发性畸形精子症的诊断潜力，其分子机制为标志物开发提供了理论依据。靶向沉默miR-26a-5p或联合AKT/mTOR抑制剂及抗氧化治疗，可能成为改善精子形态的新策略。

Objective This study aims to elucidate the functional mechanism through which seminal plasma exosomal miR-26a-5p and its target genes contribute to idiopathic teratozoospermia， with emphasis on their regulatory pathways in sperm morphogenesis defects， thereby establishing a theoretical foundation for novel diagnostic markers and targeted therapies. Methods A case-control study design was adopted， enrolling 154 male subjects （84 in the teratozoospermia group ［TZS］ and 70 in the normal control group ［NC］）. Seminal plasma exosomes were isolated to screen differentially expressed miRNAs， followed by prediction and analysis of target genes and signaling pathways. Dual-luciferase reporter gene assays were employed to validate the direct binding of miR-26a-5p to PTEN. Quantitative real-time PCR and Western blot were used to measure miRNA and protein expression levels. Spearman correlation analysis evaluated relationships between miR-26a-5p， PTEN， and sperm morphological parameters， while ROC curve analysis assessed diagnostic efficacy. Results Seminal plasma exosomal miRNA sequencing identified 11 differentially expressed miRNAs， with miR-26a-5p significantly upregulated in the TZS group （P < 0.05） and negatively correlated with the percentage of normal sperm morphology in TZS （r = -0.762， P < 0.05）. PTEN was confirmed as a direct target of miR-26a-5p， with its expression significantly reduced in the TZS group （P < 0.05） and positively correlated with normal sperm morphology （r = 0.821， P < 0.05）. A negative correlation was observed between miR-26a-5p and PTEN （r = -0.878， P < 0.05）. Dual-luciferase assays demonstrated that miR-26a-5p specifically inhibited PTEN 3'UTR activity （45% reduction in luciferase activity， P < 0.05）. Functional enrichment analysis revealed that the miR-26a-5p-PTEN axis exacerbates oxidative stress， DNA damage， and abnormal spermatocyte division by regulating the PI3K/AKT/mTOR， p53， Wnt/β-catenin， and NF?κB pathways. ROC analysis showed diagnostic efficacy for teratozoospermia with AUC^ROC values of 0.785 （sensitivity 78.9%， specificity 71.3%） for miR-26a-5p and 0.754 （sensitivity 73.3%， specificity 75.1%） for PTEN. Conclusions miR-26a-5p suppresses PTEN through targeted inhibition， activating PI3K/AKT/mTOR signaling pathways while impairing DNA repair functions. This cascade leads to accumulated oxidative stress and sperm morphological abnormalities. The combined pattern of elevated seminal plasma exosomal miR-26a-5p expression and reduced PTEN levels demonstrates diagnostic potential for idiopathic teratozoospermia， with its molecular mechanism providing a theoretical foundation for biomarker development. Targeted silencing of miR-26a-5p， either alone or in combination with AKT/mTOR inhibitors and antioxidant therapy， may represent a novel strategy for improving sperm morphology.