Highlights d Four methods, PCA, MDS, t-SNE, and UMAP, are evaluated on 71 bulk transcriptomic datasets d UMAP is overall superior to PCA and MDS and shows some advantages over t-SNE d UMAP can efficiently and effectively reveal clusters in twodimensional space d Clusters revealed by UMAP are associated with biological features and clinical traits
The immune privilege of the testes is necessary to prevent immune attacks to gamete-specific antigens and paternal major histocompatibility complex (MHC) antigens, allowing for normal spermatogenesis. However, infection and inflammation of the male genital tract can break the immune tolerance and represent a significant cause of male infertility. Different T cell subsets have been identified in mammalian testes, which may be involved in the maintenance of immune tolerance and pathogenic immune responses in testicular infection and inflammation. We reviewed the evidence in the published literature on different T subtypes (regulatory T cells, helper T cells, cytotoxic T cells, γδ T cells, and natural killer T cells) in human and animal testes that support their regulatory roles in infertility and the orchitis pathology. While many in vitro studies have indicated the regulation potential of functional T cell subsets and their possible interaction with Sertoli cells, Leydig cells, and spermatogenesis, both under physiological and pathological processes, there have been no in situ studies to date. Nevertheless, the normal distribution and function of T cell subsets are essential for the immune privilege of the testes and intact spermatogenesis, and T cell-mediated immune response drives testicular inflammation. The distinct function of different T cell subsets in testicular homeostasis and the orchitis pathology suggests a considerable potential of targeting specific T cell subsets for therapies targeting chronic orchitis and immune infertility.
Transcriptome profiling and differential gene expression constitute a ubiquitous tool in biomedical research and clinical application. Linear dimensionality reduction methods especially principal component analysis (PCA) are widely used in detecting sample-to-sample heterogeneity in bulk transcriptomic datasets so that appropriate analytic methods can be used to correct batch effects, remove outliers and distinguish subgroups. In response to the challenge in analysing transcriptomic datasets with large sample size such as single-cell RNA-sequencing (scRNA-seq), non-linear dimensionality reduction methods were developed. t-distributed stochastic neighbour embedding (t-SNE) and uniform manifold approximation and projection (UMAP) show the advantage of preserving local information among samples and enable effective identification of heterogeneity and efficient organisation of clusters in scRNA-seq analysis. However, the utility of t-SNE and UMAP in bulk transcriptomic analysis has not been carefully examined. Therefore, we compared major dimensionality reduction methods (linear: PCA; nonlinear: multidimensional scaling (MDS), t-SNE, and UMAP) in analysing 71 bulk transcriptomic datasets with large sample sizes. UMAP was found superior in preserving sample level neighbourhood information and maintaining clustering accuracy, thus conspicuously differentiating batch effects, identifying pre-defined biological groups and revealing in-depth clustering structures. We further verified that new clustering structures visualised by UMAP were associated with biological features and clinical meaning. Therefore, we recommend the adoption of UMAP in visualising and analysing of sizable bulk transcriptomic datasets.
Chronic epididymitis (CE) refers to a long-lasting inflammatory condition of the epididymis, which is considered the most common site of intrascrotal inflammation and an important aetiological factor of male infertility. Recent studies demonstrate that small RNAs secreted from epididymal epithelium modulate embryo development and offspring phenotypes via sperm transmission, and the resulting modifications may lead to transgenerational inheritance. However, to date, the genome-wide analysis of small RNA together with the transcriptomic expression profiles of human epididymis and CE is still lacking. In this study, we facilitated next-generation sequencing and bioinformatics to comprehensively analyze the small RNA and mRNA in an integrative way and identified signatures associated with CE. Both of the small RNA and mRNA expression data demonstrated relatively larger molecular differences among the segmental region of the epididymides, including caput, corpus, and cauda, than that of the inflammatory conditions. By comparing the inflamed caputs to the controls, a total of 1727 genes (1220 upregulated and 507 downregulated; 42 most significant genes, adjusted P <0.05) and 34 miRNAs (23 upregulated and 11 downregulated) were identified as differentially expressed. In silico functional enrichment analysis showed their roles in regulating different biological activities, including leukocyte chemotaxis, extracellular milieu reconstruction, ion channel and transporter-related processes, and nervous system development. Integrative analysis of miRNA and mRNA identified a regulatory network consisting of 22 miRNAs and 31 genes (miRNA-mRNA) which are strong candidates for CE. In addition, analysis about other species of small RNA, including (miRNA), piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), Y RNA, and rsRNA identified the distinct expression pattern of tsRNA in CE. In summary, our study performed small RNA and miRNA profiling and integrative analysis in human CE. The findings will help to understand the role of miRNA-mRNA in the pathogenesis of CE and provide molecular candidates for the development of potential biomarkers for human CE.
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