The differences in genetic background between 46,XY males and 47,XXY KS males has been the subject of multiple studies, reporting an altered gene expression pattern in KS men. The degree of differential gene expression is believed to be associated with the variability in clinical phenotype, although it is not certain whether the differential expression does translate into significant changes at protein level.
In this study, the differential gene expression analysis was performed on KS and control testicular tissue to reveal a number of genes which may be responsible for the KS-related testicular fibrosis. First, a gene expression analysis of fibrotic versus non-fibrotic testis tissue was performed. In aging men, testicular fibrosis occurs naturally (22), however, a statistical analysis showed no significant differences between the two patient groups in their respective age periods. Even though samples with and without ongoing spermatogenesis were included within both the fibrotic and non-fibrotic group, the most down-regulated biological functions remained associated with fertilization and spermatogenesis. For the up-regulated biological functions, both processes involving hormonal changes and processes of extracellular structure organization were found. Testicular fibrosis is characterized by excessive ECM synthesis and it has been shown that the tubular wall is remodelled in men with impaired spermatogenesis (23). A recent study of our group revealed significant changes in the ECM of KS compared to SCO and FC, i.e. a loss of alpha smooth muscle actin 2 and an altered expression of collagen I and IV (24). Several genes are involved within the ECM organization, including some collagen genes, VCAM1 and small leucine-rich proteins such as decorin and lumican. Decorin has already been shown to be highly expressed in KS tissue, especially in hyalinized tubules (25). Loss of lumican has been shown to ameliorate cardiac fibrosis (26). VCAM1 is expressed in the interstitium of the testis and is known to play a role in the testicular immunoregulation (27, 28). Recently, VCAM1 has been shown to be crucial for the reconstruction of testicular interstitial tissue in vitro (29). In addition, VCAM1 is a key element of the inflammation pathway, contributing to several immunological disorders as well as cancer. Furthermore, VCAM1 has already been associated with pulmonary fibrosis (30, 31). Our results also showed that DEGs involved in the TGF-β1 pathway were significantly up-regulated. It is known that testicular fibrosis is mediated by TGF-β1 and that its expression is increased in men with impaired spermatogenesis (32, 33). Moreover, TGF-β1 production is regulated by decorin and vice versa (34). In addition, it has been shown that VCAM1 is a TGF-β1 inducible gene (30). In accordance, our research also revealed a significantly higher expression of TGF-β1 in KS biopsies compared with fertile controls. The interaction between VCAM1, decorin and the TGF-β pathway is displayed in supplementary Fig. 8. Recently, a study from Mahyari et al. revealed different up-regulated genes related to fibrosis, specifically expressed by Leydig cells, through a single-cell analysis of KS biopsies (18). Three of these profibrotic genes (IGFBP5/WFIKKN2/ INHBA) were also found up-regulated in our data.
Winge et al. recently compared all published studies concerning differentially expressed analyses and revealed an overlap of only eight transcripts which were up-regulated in at least two studies, while no down-regulated genes overlapped between the studies (35). Of these eight genes, only one was found differentially expressed in our analysis of fibrotic versus non-fibrotic tissue, e.g. DLK1. This marker for immature Leydig cells has already been shown to be up-regulated in adult men with testicular pathologies, including KS males (36).
Secondly, a gene expression analysis between the two fibrotic groups, KS and TA, was performed. This analysis, in combination with the first analysis, revealed an overlap of three genes: MXRA5, DCX and VCX3B. MXRA5 is a proteoglycan which has a function in cell adhesion and ECM remodelling. MXRA5 has been associated with several diseases and conditions, including preeclampsia and colorectal cancer (37, 38). A few years ago, MXRA5 was identified as an anti-inflammatory and anti-fibrotic molecule, regulated by TGF-β1 (39). However, no exact function of MXRA5 within the testis has been elucidated. DCX has been elaborately studied within the brain, as it is a microtubule-associated protein required for cerebral cortex development. In addition, it has been correlated with several disorders of the neuronal structures, including lissencephaly, epilepsy and developmental dyslexia (40). A novel human DCX domain containing gene which is expressed mainly in the adult testis was discovered several years ago, however, the role of this protein within the testis is unknown (41). VCX3B is a member of the VCX/Y gene family, of which all the members are exclusively expressed in male germ cells. The exact function of these genes is currently unknown, although one of the family members, VCX-A, has already been associated with mental retardation, a condition also found in some KS patients (42, 43). Moreover, the KEGG analysis revealed genes involved in steroidogenesis pathogenesis to be up-regulated in the KS samples, which is in accordance with research showing an increased intratesticular testosterone level in KS men (44). Although no changes in the blood vessel density were found in KS testicular biopsies compared to controls, as reported in our previous research (25), the above finding may contribute to the hypothesis that a disturbed vascularization is present within the KS testis (45, 46). In KS men, as in females, one of the two X-chromosomes is silenced to compensate for the dosage of X-linked genes. However, it is known that about 15% of these genes escape X-inactivation, resulting in an overexpression of several X-linked genes. This overexpression could be related to the clinical features seen in KS men, including the testicular fibrosis (47–49). In this study, the only known escapee X-linked DEG found was MXRA5 (50), substantiating its role in KS-related testicular fibrosis.
In this study, snap-frozen testicular biopsies were used to perform the gene expression analysis, in contrast to most other studies using blood samples or extracted RNA from paraffin-embedded tissue. This allowed us to focus on testicular fibrosis. Nevertheless, this study has a few limitations. Only a small number of samples could be included, as KS samples for research are rather scarce. Since ideal control samples with a fibrotic score of zero were not available, we included samples with fibrotic scores one and two in the non-fibrotic group. A last limitation includes the use of bulk RNA sequencing, which is sensitive to differences in cell-type proportions.