Transcriptomic differences between fibrotic and non-fibrotic testicular tissue reveal possible key players in Klinefelter syndrome-related testicular fibrosis

Introduction & Objective: Klinefelter syndrome (KS; 47,XXY) affects 1-2 in 1000 males. Most (95%) KS men suffer from azoospermia due to the loss of spermatogonial stem cells. Additionally, testicular fibrosis is detected from puberty onwards. However, mechanisms responsible for fibrosis and germ cell loss remain unknown. This study aimed to identify factors which may be involved in the fibrotic remodeling of KS testes by analyzing the transcriptome of (non-)fibrotic testicular tissue. 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). In addition, differentially expressed genes (DEGs) between the KS and testis atrophy samples were studied to reveal KS-specific fibrotic genes. DEGs were considered significant when p < 0.01 and log2FC > 2. To gain insight in the potential functions of the DEGs, gene-ontology and KEGG analyses were performed. To validate the gene expression results, immunohistochemistry and RNA scope were performed. Results: 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 analysis, three genes were found: matrix-remodeling associated 5 (MXRA5), doublecortin (DCX) and variable charge X-Linked 3B (VCX3B). As validation, an overexpression of VCAM1, MXRA5 and DCX was found within the fibrotic group compared to the non-fibrotic group through immunohistochemistry and RNA scope. Conclusion: Comparing DEGs between fibrotic and non-fibrotic tissue resulted in genes which may play a role in testicular fibrosis, including VCAM1. When comparing the X-linked genes of the first analysis with those of the second analysis (KS versus testis atrophy) were compared, fibrotic genes on the X-chromosome were revealed. MXRA5, DCX and VCX3B may play a role in KS-related testicular fibrosis.