Identification of Differentially Expressed Genes in Human Testis Biopsies with Defective Spermatogenesis

The study was designed to identify differences in the expressed transcriptome of the two pathological human testis sample groups compared to normal spermatogenesis samples, with the objective of discovering genes related to the pathological condition. Transcriptional differences in human testis biopsies collected from men with normal spermatogenesis (NSP, n=3; intact germ cells, clinical score - 10), spermatid arrest (SDA, n=2; contains somatic cells and germ cells up to round spermatids, clinical score – 4-5) and Sertoli cell-only (SCO, n=2; complete absence of germ cells, clinical score - 0) phenotypes were assessed by RNA sequencing. A total of over 49 million reads were generated in NSP samples, over 24 million reads in SDA samples, and over 13 million reads in SCO samples. Differentially expressed genes (DEG) were identified based on a mean minimum count => 5, FDR <= 0.05 and log2FC fold change of >0.585 or <-0.585. Genes differentially expressed in pathological groups SDA and SCO compared to NSP, and genes differentially expressed in between the two pathological groups SDA and SCO were filtered. The study discovered a number of significantly differentially expressed genes (DEG) between groups as follows: NSP vs SDA n=1,873; SDA vs SCO n= 4,017; and NSP vs SCO n=10,253. A number of genes (n=10) were selected for further analysis by qRT-PCR and the detection of the protein (n=2) localization in testis by immunohistochemistry. Overall design: Examination of differentially expressed genes and its transcript levels in normal and abnormal spermatogenesis conditions in human

本研究旨在鉴定两类病理性人类睾丸样本组相较于正常精子发生样本的表达转录组差异，以期发掘与该病理状态相关的基因。本研究通过RNA测序，对来自正常精子发生（Normal Spermatogenesis, NSP，n=3；生殖细胞完整，临床评分10）、精子发生阻滞（Spermatid Arrest, SDA，n=2；包含体细胞及发育至圆形精子细胞阶段的生殖细胞，临床评分4~5）以及唯支持细胞综合征（Sertoli Cell-Only, SCO，n=2；完全缺失生殖细胞，临床评分0）的男性睾丸活检组织的转录差异进行了评估。NSP样本共生成超过4900万条reads，SDA样本超过2400万条reads，SCO样本超过1300万条reads。差异表达基因（Differentially Expressed Genes, DEG）的筛选基于以下标准：平均最小计数≥5、错误发现率（False Discovery Rate, FDR）≤0.05、log2倍变化（log2 Fold Change, log2FC）>0.585或<-0.585。本研究分别筛选了病理性组SDA、SCO与NSP之间的差异表达基因，以及两类病理性组SDA与SCO之间的差异表达基因。最终鉴定得到多组具有显著统计学差异的表达基因：NSP与SDA组间共1873个，SDA与SCO组间共4017个，NSP与SCO组间共10253个。本研究选取了10个基因进行定量实时荧光PCR（qRT-PCR）进一步分析，并通过免疫组织化学（Immunohistochemistry）检测其中2个基因在睾丸组织中的蛋白定位情况。总体实验设计：探究人类正常与异常精子发生状态下的差异表达基因及其转录水平。