Seminal extracellular vesicle sncRNA profiles reveal miRNAs/isomiRs as predictive biomarkers of testicular sperm retrieval outcome in azoospermic patients

Non-invasive molecular biomarkers for differential diagnosis of the origin of azoospermia into obstructive azoospermia (OA) or secretory azoospermia (SA), as well as for prediction of the presence of residual spermatogenesis in testicular tissue of patients presenting with SA, are of great interest for TESE outcome prediction in assisted reproduction. Our previous study of several hundreds of miRNAs suggested the use of some sEV-miRNAs from seminal plasma as biomarkers for the origin of azoospermia. In this regard, studying more in depth sncRNAs in EVs from semen of azoospermic individuals will be useful to select additional non-invasive biomarkers with diagnostic/prognostic purposes which could help in the identification of those individuals with real chances of positive sperm recovery on the testicular biopsy prior to assisted reproduction technology (ART) treatment. A high-throughput sncRNA profiling analysis using paired-end small RNA-seq was performed in seminal sEVs from azoospermia and normozoospermic individuals. A case and control prospective study was performed. In this pilot study we aimed at extensively characterizing seminal sEV sncRNA profiles of azoospermic individuals with different testicular sperm recovery outcome. For that, we have profiled and quantified the expression of annotated sncRNAs by paired-end small RNA-seq and determined the expression pattern of not only miRNAs (analyzed at the isomiR level), but also piRNAs, and tsRNAs within the human seminal sEVs in a small number of normozoospermic (n=4) and azoospermic [OA-N n=4; SA(Sp+) n=5; SA(Sp-) n=4] individuals. RT-qPCR validation analysis of selected miRNAs was additionally performed in a larger number of individuals.

用于鉴别诊断无精子症为梗阻性无精子症（OA）或分泌性无精子症（SA），同时可预测分泌性无精子症（SA）患者睾丸组织中残余精子发生状态的无创分子生物标志物，对于辅助生殖领域睾丸精子提取术（TESE）的结局预测具有重要临床价值。我们此前针对数百例临床样本的微小RNA（miRNAs）研究表明，精浆来源的部分小细胞外囊泡微小RNA（sEV-miRNAs）可作为无精子症病因分型的生物标志物。鉴于此，对无精子症患者精液来源细胞外囊泡（EVs）中的小非编码RNA（sncRNAs）开展更深入的研究，将有助于筛选出更多具备诊断与预后价值的无创生物标志物，从而辅助识别那些在接受辅助生殖技术（ART）治疗前，通过睾丸活检即可获得成功精子回收的患者。本研究采用双端小RNA测序（paired-end small RNA-seq），对无精子症及正常精子症个体的精浆小细胞外囊泡开展高通量小非编码RNA谱分析，并实施了前瞻性病例对照研究。在本次预实验中，我们旨在全面解析不同睾丸精子回收结局的无精子症患者的精浆小细胞外囊泡小非编码RNA表达谱。为此，我们通过双端小RNA测序对注释过的小非编码RNA进行了表达谱分析与定量，不仅在异构体（isomiR）水平上分析了微小RNA的表达模式，还同时检测了人精浆小细胞外囊泡中的Piwi互作RNA（piRNAs）与tRNA来源小RNA（tsRNAs），共纳入少量正常精子症个体（n=4）及无精子症个体[OA-N n=4; SA(Sp+) n=5; SA(Sp-) n=4]。此外，我们在更大样本量的队列中对筛选出的微小RNA进行了实时定量聚合酶链反应（RT-qPCR）验证分析。