Probes for TGF-β

Study question Which genes are differentially expressed between patients with and without testicular fibrosis? Summary answer This study revealed three X-related genes MXRA5, DCX and VC3BX, which may be involved in Klinefelter-related testicular fibrosis. What is known already Klinefelter syndrome (KS; 47,XXY) affects 1-2 in 1000 males. Most KS men suffer from azoospermia due to a loss of spermatogonial stem cells. Additionally, testicular fibrosis is detected from puberty onwards. However, mechanisms responsible for fibrosis and germ cell loss remain unknown. Previous genomics studies on KS tissue focused on germ cell loss, however, differential gene expression analyses focused on testicular fibrosis have not been performed before. This study aimed to identify factors involved in the fibrotic remodeling of KS testes by analyzing the transcriptome of (non-)fibrotic testicular tissue. Study design, size, duration Transcriptome analysis on extracted RNA from testicular biopsies was performed. RNA scope analysis and immunohistochemistry were performed as validation for the findings of the transcriptomics study. Participants/materials, setting, methods RNA sequencing was performed to compare the genetic profile of testicular biopsies from patients with (KS and testis atrophy) and without (Sertoli cell-only syndrome and fertile controls) testicular fibrosis (n = 5, each). Next, differentially expressed genes (DEGs) between KS and testis atrophy samples were compared. To gain insight in potential functions of DEGs (significant when p < 0.01 and log2FC > 2), gene-ontology and KEGG analyses were performed. To validate the gene expression results, immunohistochemistry and RNA scope were performed. Main results and the role of chance A first transcriptomic analysis of fibrotic versus non-fibrotic testis tissue resulted in 734 significant DEGs (167 up- and 567 downregulated), of which 26 were X-linked. In the top upregulated biological functions, DEGs involved in the extracellular structure organization were found, including vascular cell adhesion molecule 1 (VCAM1). KEGG analysis showed an upregulation of genes involved in the TGF-β pathway. The second analysis of KS versus testis atrophy samples resulted in 539 significant DEGs (59 up- and 480 downregulated). One of the biological functions found though gene ontology analysis was the chronic inflammatory response. When looking at the overlap of DEGs on the X-chromosome from the first and second analysis, three genes were found: matrix-remodeling associated 5 (MXRA5), doublecortin (DCX) and variable charge X-Linked 3B (VCX3B). Through validation by immunohistochemistry and RNA scope, an overexpression of VCAM1, MXRA5 and DCX was found within the fibrotic group compared to the non-fibrotic group. Limitations, reasons for caution The study included fresh testis tissue from adult KS patients, however these are quite scarce, resulting in a low number of included patients per group (n = 5). Wider implications of the findings This study revealed genes which may play a role in testicular fibrosis, including VCAM1. In addition, fibrotic genes on the X-chromosome were revealed: MXRA5, DCX and VCX3B. Up- or downregulation of these genes may prevent testicular fibrosis and thus enhance the chances at retrieving spermatozoa from KS patients. Trial registration number NA