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

doi:10.21203/rs.3.rs-1745658/v2

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

https://doi.org/10.21203/rs.3.rs-1745658/v2

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Background: Klinefelter syndrome (KS; 47,XXY) affects 1-2 in 1000 males. Most KS men suffer from an early germ cell loss and testicular fibrosis from puberty onwards. Mechanisms responsible for these processes remain unknown. Previous genomics studies on KS tissue focused on germ cell loss, while a transcriptomic analysis focused on testicular fibrosis has not yet been performed.

This study aimed to identify factors involved in the fibrotic remodelling of KS testes by analysing the transcriptome of (non-)fibrotic testicular tissue.

Material and methods: RNA sequencing was performed to compare the genetic profile of testicular samples with (KS and testis atrophy) and without (Sertoli cell-only syndrome and fertile controls) fibrosis (n=5, each). Additionally, differentially expressed genes (DEGs) between KS and testis atrophy samples were studied to reveal KS-specific fibrotic genes. DEGs were considered significant when p<0.01 and log2FC>2. Next, downstream analyses (GO and KEGG) were performed. Lastly, RNA scope was performed to validate the results.

Results: The first analysis (fibrotic vs non-fibrotic) resulted in 734 significant DEGs (167 up- and 567 down-regulated). Genes involved in the extracellular structure organization (e.g. VCAM1) were found upregulated. KEGG analysis showed an up-regulation of genes involved in the TGF-β pathway.

The KS vs TA analysis resulted in 539 significant DEGs (59 up- and 480 down-regulated). Chronic inflammatory response genes were found upregulated. The overlap of X-linked DEGs from the two analyses revealed three genes: matrix-remodelling associated 5 (MXRA5), doublecortin (DCX) and variable charge X-Linked 3B (VCX3B). RNA in situ hybridization showed an overexpression of VCAM1, MXRA5 and DCX within the fibrotic group compared with the non-fibrotic group.

Conclusion: In this study, DEGs between fibrotic and non-fibrotic tissue were first compared, revealing fibrotic genes, including VCAM1. Secondly, DEGs between KS and TA samples were studied and the analysis revealed KS-specific, fibrotic genes. When comparing X-linked genes of the first analysis (fibrotic versus non-fibrotic) with those of the second analysis (KS versus TA), KS-related X-linked fibrotic genes were revealed, i.e. MXRA5, DCX and VCX3B. Their potential role in KS-related testicular fibrosis needs further study.

Transcriptomics

Klinefelter syndrome

testicular fibrosis

testis biopsy

Klinefelter syndrome (KS) was first documented in 1942 and remains the most common sex-chromosome related disorder to this day with KS being diagnosed in approximately 1 to 2 in 1000 new-born males (1, 2). Men diagnosed with KS show at least one supplementary X-chromosome (47,XXY) and may present typical phenotypical features including gynaecomastia and hypogonadism. Due to the absence of any significant symptoms or features in many patients, this disorder remains highly underdiagnosed, with only a presumable 25% of all patients actually being diagnosed (3, 4). Diagnosis mainly occurs during adulthood when KS men discover they are unable to conceive naturally, since azoospermia can be seen in more than 90% of these patients. Germ cell loss is already noticeable during childhood (5) and has been suggested to be a consequence of either the chromosomally aberrant germ cells present in KS testes, a disturbed testicular environment or the altered gene dosage (6). Fertility strategies in adult KS patients include a testicular sperm extraction (TESE) procedure followed by an intracytoplasmatic sperm injection. However, the TESE procedure only appears to be successful in about 48% of adult KS patients (7). The success-rate of finding spermatozoa/spermatogonia is hampered because of testicular fibrosis, which occurs from puberty onwards, and which eventually leads to the degeneration of the seminiferous tubules within the KS testicular tissue (5, 8). Unfortunately, the exact mechanisms responsible for the initiation of testicular fibrosis in KS men are still unknown.

Several studies on the transcriptomic analysis of KS samples have been reported. In five of these studies, RNA was isolated from whole blood to compare the transcriptome of KS patients to controls (9–13). Vawter et al. specifically studied the genes associated with verbal cognition whereas the research of Huang et al. was more focussed on the relation between the gene expression and the regulation of metabolism in KS patients compared to controls (12, 13). Other transcriptomic analyses were performed to unravel which genes are responsible for the typical KS phenotype, revealing differentially expressed genes (DEGs) involved in multiple pathways, including, among others, insulin resistance, male infertility, azoospermia and obesity (9–11). D’Aurora et al. performed microarray transcriptome analyses on fresh testicular biopsies from azoospermic KS patients (14) and testicular biopsies from KS patients with ‘hypospermatogenesis’ (15) to uncover DEGs related to germ cell loss. These analyses revealed DEGs involved in steroidogenesis, spermatogenesis failure, germ cell function and morphology, cell death and blood-testis barrier maintenance. Winge et al. performed a transcriptome analysis on fetal KS and age-matched controls to find an answer on how the supplemental X-chromosome affects adult spermatogenesis. The results from this research showed changes in the germ cell differentiation pattern and DEGs involved in somatic cell maturation, hormone response, cell death and testis development (16). In addition, transcriptomic analysis on the adult human KS paraffin-embedded testis was performed to uncover DEGs involved in germ cell loss and the destruction of seminiferous tubules. This analysis showed the disturbed differentiation of the somatic cell populations in the KS testes and revealed that transcriptomes of testes from different developmental ages show limited overlap (17).

Since most of these studies were focussed on the DEGs involved in germ cell loss, the role of genes which may be involved in the testicular fibrotic process were not specifically investigated. Nevertheless, the inflammation pathway was uncovered as one of the associated processes in three of the studies (9, 14, 15). In addition, a recent study performed single cell RNA sequencing on KS testis tissue and revealed a couple of genes related to testicular fibrosis (18).

The aim of the current study was to reveal DEGs which are involved in the fibrotic process in KS. Unravelling the mechanisms behind the testicular fibrosis in KS patients could uncover how to delay or even stop the fibrotic process, leading to a higher chance of sperm retrieval in KS patients through a TESE procedure. Therefore, RNA sequencing was performed on fresh testicular biopsies from adult KS patients and patients with mixed testis atrophy (TA), who show testicular fibrosis but no germ cell loss (fibrotic group), Sertoli cell only syndrome (SCO) patients, who are infertile but do not show any fibrosis in their testes and fertile men (non-fibrotic group).

Patient samples

Testicular biopsy samples were obtained from adult KS (n = 5), SCO (n = 5) and TA patients (n = 5) during a TESE procedure in function of their fertility treatment. In addition, samples from men who underwent a vasectomy reversal (n = 5) were included as fertile controls (FC). Written informed consent was obtained from all patients included in this study. Patient samples were divided over four categories: non-fibrotic samples containing spermatozoa (FC), non-fibrotic samples lacking germ cells (SCO), samples with a normal karyotype showing testicular fibrosis (TA) and samples with the KS karyotype lacking germ cells and showing fibrosis (KS). More information about the patients included in this study can be found in table I.

Immediately after the biopsy was retrieved, samples were snap frozen in liquid nitrogen after which they were stored at -80°C for next generation sequencing. In addition, biopsy samples were fixed in alcohol-formalin acetic acid mixture (AFA; Q02022; international medical products, Oudergem, Belgium) for at least one hour and further dehydrated and fixed with TISSUE TEK-VIP (5990; Sakura, Berchem, Belgium) overnight after which the tissue was embedded in paraffin. For validation by RNA in situ hybridization, sections of 5 mm thick were cut at three different depths using a microtome (SM2010R; Leica, Brussels, Belgium).

Histological examination

The fibrotic score of each testicular tissue sample included in this study was determined as previously described (5). In brief, paraffin-embedded tissue was used to perform a haematoxylin-periodic acid Schiff’s reagent-haematoxylin staining. The histology of the tissue was scored according to the degree of fibrosis. A mean score of five evaluated image fields was given to each tissue sample: (: (0) ideal, normal histology; (1) normal tubules but some interstitial fibrosis; (2) degenerated tubules and interstitial fibrosis; (3) hyalinized tubules and severe fibrosis; (4) severe fibrosis, complete loss of tubules. Only samples with a fibrotic score < 2 were included in the non-fibrotic groups while for the fibrotic groups, only samples with a score of 3 or 4 were included.

RNAseq – Wetlab protocol

Total RNA was extracted from the testicular tissue using the RNeasy Micro kit (74004; Qiagen, Antwerp, Belgium) following the manufacturer’s guidelines. RNA concentration was determined with a Nanodrop spectrophotometer (Isogen Life Science, De Meern, the Netherlands). The subsequent steps of the RNAseq procedure were carried out at the BrightCore facility (UZ Brussel). Extracted RNA from KS, SCO, TA and FC biopsies was analysed on the Agilent 2100 Bioanalyzer system (G2939BA; Agilent technologies), leading to an RNA integrity number (RIN) score. Samples with RIN scores above 4.7 were considered pure enough to continue with the experiments. Subsequently, an input of 150 ng RNA per sample was processed using the KAPA RNA HyperPrep with RiboErase (08098131702; Roche, Vilvoorde, Belgium) library preparation kit with the following steps: oligo hybridization and RNA depletion, 2.2X bead-based RNA depletion clean-up, DNase digestion, 2.2X bead-based DNase digestion clean-up, RNA elution, fragmentation and priming, first strand synthesis, second strand synthesis and A-tailing, adaptor ligation, first 0.63X bead-based post-ligation clean-up, second 0.7X bead-based post-ligation clean-up, library amplification and library amplification clean-up. These steps were carried out according to the manufacturer’s protocol. Library validation was performed through the measurement of cDNA fragment concentrations, using the Victor Nivo Multimode Microplate Reader (HH35000500; PerkinElmer, Zaventem, Belgium), and the length of the different fragments was measured by the Fragment-analyzer (M5311AA; Agilent technologies). These steps were carried out according to the manufacturer’s protocol in order to obtain a library insert size of 300-400bp.

Libraries were pooled according to the two different patient groups (fibrotic vs non-fibrotic tissue) as well as pooled within the fibrotic group (KS vs TA). The pooled libraries were sequenced with the Illumina NovaSeq 6000 (Illumina, Eindhoven, the Netherlands).

RNAseq – Differential expression and downstream analysis

The reads were mapped to the human genome (hg19) using the tool STAR v2.5.0c (19). The mapped reads were then translated into a quantitative measure of gene expression, counting the number of reads that map to each exon/gene using HTSeq v0.11.0 (20).

Following quantification of expression levels, the differential expression between the different groups was determined. Focus was put on the protein coding genes (19265 genes, downloaded from Ensemble biomart in May 2019). In a first step, a PCA analysis was run to check for outliers. Next, the differential expression analysis was run after which the genes that were significantly up- or down- regulated with an absolute log2 fold change > 2 after shrinkage (type apeglm) and an adjusted p-value < 0.01 were extracted. This was done with the DESeq2 package v1.24.0 in R v3.6.1 (21).

Then, two gene ontology (GO) analyses were run using i) the set of up-regulated genes and ii) the set of down-regulated genes using the enrichGO function of the Bioconductor package clusterProfiler. On these two gene sets, a kyoto encyclopedia of genes and genomes (KEGG) analysis was run using the enrichKEGG function of the same package.

RNA in situ hybridization

RNA in situ hybridization was used as validation method to confirm the overexpression of VCAM1, DCX and MXRA5 in the fibrotic tissue. This was performed by the ‘extended RNA analysis’ facility at VUB (https://lmmo.research.vub.be/en/era-facility). The RNAscope 2.5 HD Duplex Assay (322430; Advanced Cell Diagnostics, Newark, CA, USA) was used according to the manufacturer's instructions with adapted pretreatment conditions (15 minutes target retrieval and 15 minutes protease plus). Probes used were B. subtilis duplex negative control (320751; Advanced Cell Diagnostics), H. sapiens duplex positive control (321641; Advanced Cell Diagnostics) and H. sapiens MXRA5 (419691; Advanced Cell Diagnostics), VCAM1 (440371-C2; Advanced Cell Diagnostics) and DCX (489551; Advanced Cell Diagnostics). Imaging was performed with the EVOS™ M7000 Imaging System (AMF7000; Invitrogen, Merelbeke, Belgium).

Statistical analysis

The age of the patients in each group was compared by an unpaired t-test (GraphPad Prism8 software). The difference was considered statistically significant if p < 0.05.

Transcriptomic analysis - fibrotic vs non-fibrotic tissue

No significant difference (p = 0.568) was found between the fibrotic and non-fibrotic group concerning age at biopsy (Supplementary Fig. 1).

Transcriptomic analysis resulted in 17493 genes which were differentially expressed between the fibrotic and non-fibrotic group. After using an adjusted p-value < 0.001 and an absolute log2 fold change of ≥ 2, excluding non-significant DEGs, a total of 734 genes were found, of which 167 were up-regulated and 567 were down-regulated (Fig. 1A-B). Results showed that all FC samples clustered together, as did the KS samples, while the gene expression in SCO and TA samples was more diverse. Nevertheless, the TA samples with a fibrotic score of 3 clustered together with the KS samples (Fig. 1C). GO analysis showed diverse biological functions for the up-regulated DEGs. The top ten of the most up-regulated functions is shown in Fig. 2A. The most interesting function found through this GO analysis included the extracellular structure organization, since testicular fibrosis can be attributed to an excessive deposition of ECM components. The genes involved the extracellular structure organization, were, among others, decorin, lumican, vascular cell adhesion molecule 1 (VCAM1) and several collagen-related genes. The network of genes involved in this biological function is displayed in Fig. 2B. GO analysis for the down-regulated genes resulted in biological functions which are mainly related to spermatogenesis and fertilization, such as spermatid development and differentiation (Supplementary Fig. 2A). KEGG analysis showed, among others, an up-regulation of genes involved in the transforming growth factor beta-1 (TGF-β) pathway (Fig. 2C). The upregulation of TGF-β in KS patients compared with SCO patients and fertile controls has been validated by RT-qPCR analysis and was found significant (Supplemental experimental procedure and supplementary Fig. 3). Moreover, a downregulation of genes involved in the extracellular matrix (ECM)-receptor interaction pathway was seen (Supplementary Fig. 2B). A total of 15 downregulated and 11 upregulated DEGs were located on the X-chromosome (Fig. 2D).

Transcriptomic analysis - KS vs TA

Next, a transcriptomic analysis to find DEGs between the two fibrotic groups, KS and TA samples, was performed. A total of 482 significant DEGs were found, of which 57 were up-regulated and 425 were down-regulated (Fig. 3A-B). Enrichment analysis revealed the most significant biological functions for both the up- and down-regulated genes. Among the up-regulated biological functions, the chronic inflammatory response (PTGES/GJA1/CYP19A1) was found (Fig. 3C). Processes involved in cilium movement, meiotic cell cycle and the cellular process involved in reproduction in multicellular organisms were found as most down-regulated biological functions (Supplementary Fig. 4). KEGG analysis for the up-regulated genes showed the steroid hormone biosynthesis as most up-regulated pathway. For the down-regulated genes, several pathways were found, including ABC transporters, tight junction and glycosaminoglycan degradation (Supplementary Fig. 5).

Twenty-one downregulated genes were located on the X-chromosome, while three upregulated DEGs could be found on the X-chromosome (Fig. 3D). Concerning the X-linked genes, three genes were found in both the first analysis, comparing fibrotic and non-fibrotic tissue, and the second analysis, comparing fibrotic tissue with and without a normal karyotype: up-regulated matrix-remodelling associated 5 (MXRA5), up-regulated doublecortin (DCX) and down-regulated variable charge X-Linked 3B (VCX3B).

Validation

RNAscope was performed to visualize three selected genes from our two analyses, namely VCAM1, MXRA5 and DCX. VCAM1 was selected due to its role in the inflammation pathway, while MXRA5 and DCX were chosen as the two up-regulated X linked genes which overlapped in the two analyses of this study. The duplex array combined VCAM1 with MXRA5 and with DCX. In both combinations, an overexpression of VCAM1 was clearly observed in KS samples compared with FC samples. VCAM1 RNA molecules were mainly present in the interstitial tissue in FC and SCO samples, while in KS samples, VCAM1 was mostly present within the fibrotic patches. MXRA5 expression was barely seen in FC and SCO samples, while visibly more expression was detected in KS and TA samples. A lot of DCX RNA molecules could be distinguished in KS and TA samples, in contrast to SCO and FC samples. In FC samples, some positive patches were seen in the center of the seminiferous tubules, however, this was also visible in the negative control of all FC samples included, confirming these patches did not correspond with real DCX expression (Fig. 4 and supplementary Figs. 6–7).

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.

When comparing DEGs between fibrotic and non-fibrotic tissue, this study revealed genes which may play a role in testicular fibrosis. One of these genes is VCAM1. In addition, this analysis revealed a pertinent role for genes involved in the TGF-β1 pathway. Secondly, a differential gene expression analysis between the fibrotic groups, KS and TA samples, was performed. GO analysis revealed genes involved in the chronic inflammatory response. When comparing the X-linked genes of the first analysis (fibrotic versus non-fibrotic) with those of the second analysis (KS versus TA), fibrotic genes on the X-chromosome were revealed, i.e. MXRA5, DCX and VCX3B. These genes may play a role in the KS-related testicular fibrosis.

AFA Alcohol-formalin acetic acid mixture

DCX Doublecortin

DEGs Differentially expressed genes

ECM Extracellular matrix

FC Fertile controls

GO Gene ontology

KEGG Kyoto encyclopedia of genes and genomes

KS Klinefelter syndrome

MXRA5 Matrix-remodelling associated 5

RIN RNA integrity number

SCO Sertoli cell only syndrome

TA Mixed testis atrophy

TESE Testicular sperm extraction

TGF-β Transforming growth factor beta-1

VCAM1 Vascular cell adhesion molecule 1

VCX3B Variable charge X-Linked 3B

Ethics approval

The study was approved by the ethical committee of the UZ Brussel (2015/121). All methods were performed in accordance with the relevant guidelines and regulations.

Availability of data and materials

The datasets used and analysed during the current study have been deposited in the GEO public database (GSE200680).

Consent for publication

Not applicable

Competing interests

No conflict of interest is declared by any of the authors towards the contents of this manuscript.

Funding

This study was supported by the scientific Fund Willy Gepts (D.V.S and E.G.) from the UZ Brussel and grants from the Vrije Universiteit Brussel (E.G.) as well as a grant from the Fonds Wetenschappelijk Onderzoek (12M2819N, D.V.S.).

Author’s contributions

M.W. performed the experiments, acquired and analysed the data, and drafted and approved the final manuscript. C.O. acquired and analysed the RNA seq data and was involved in the critical revision and approval of the final manuscript. C.B. was involved in the acquisition of the RNA seq data, revised and approved the final manuscript. V.V. was involved in the acquisition of adult patient samples and data, critical revision and approval of the final manuscript. J.D.S and H.T. were involved in the conception and design of the study, critical revision and approval of the final manuscript. D.V.S. and E.G. were involved in the conception and design of the study, critical revision and approval of the final manuscript.

Acknowledgements

The authors are thankful to Yves Heremans, Pierre Hilven, Annick Callaerts and Sarah Peeters for their technical assistance. The authors are also thankful to Céline Devriendt for her help with the RNA scope analysis. The authors would also like to thank the pathology department for providing the testicular tissue samples.

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Table I: Individual information on patients included

Patient ID	Reason for TESE procedure	Diagnosis/Anamnesis	Testicular histology	Age at biopsy	Karyotype	Fibrotic score
KS1	Non-obstructive azoospermia	Klinefelter syndrome	Severe tubular atrophy, Leydig cell hyperplasia, fibrosis	36	47, XXY	4
KS2	Non-obstructive azoospermia	Klinefelter syndrome	Sertoli cell only image, hyalinized tubules, patches of fibrosis	29	47, XXY	3
KS3	Non-obstructive azoospermia	Klinefelter syndrome	Tubular atrophy, Leydig cell hyperplasia	47	47, XXY	3
KS4	Non-obstructive azoospermia	Klinefelter syndrome	Tubular atrophy, Leydig cell hyperplasia	30	47, XXY	3
KS5	Non-obstructive azoospermia	Klinefelter syndrome	Total fibrotic tissue	43	47, XXY	4
TA1	Non-obstructive azoospermia	Ideopathic testicular atrophy	Tubular atrophy	37	46, XY	2
TA2	Non-obstructive azoospermia	Ideopathic testicular atrophy / Cryptorchidy	Mixed tubular atrophy and Sertoli cell only syndrome, sclerotic blood vessels	35	46, XY	2
TA3	Non-obstructive azoospermia	Ideopathic testicular atrophy	Hyalinized tubules, a lot of fibrotic patches	38	46, XY	3
TA4	Non-obstructive azoospermia	Ideopathic testicular atrophy	Hyalinized and atrophic tubules	29	46, XY	3
TA5	Non-obstructive azoospermia	Ideopathic testicular atrophy	Hyalinized tubules, a lot of fibrotic patches	49	46, XY	3
SCO1	Non-obstructive azoospermia	Sertoli Cell Only syndrome	Normal histology, tubules with Sertoli cell only image	31	46, XY	1
SCO2	Non-obstructive azoospermia	Sertoli Cell Only syndrome	Normal histology, tubules with Sertoli cell only image and some hyalinized tubules	29	46, XY	2
SCO3	Non-obstructive azoospermia	Sertoli Cell Only syndrome	Normal histology, tubules with Sertoli cell only image and some atrophic tubules	35	46, XY	2
SCO4	Non-obstructive azoospermia	Sertoli Cell Only syndrome	Normal histology, tubules with Sertoli cell only image and some hyalinized tubules	34	46, XY	2
SCO5	Non-obstructive azoospermia	Sertoli Cell Only syndrome	Normal histology, tubules with Sertoli cell only image	37	46, XY	1
FC1	Vasectomy reversal	Vasectomy	Normal histology	52	46, XY	1
FC2	Vasectomy reversal	Vasectomy	Normal histology	49	46, XY	1
FC3	Vasectomy reversal	Vasectomy	Normal histology with few hyalinized tubules	50	46, XY	2
FC4	Vasectomy reversal	Vasectomy	Normal histology	39	46, XY	1
FC5	Vasectomy reversal	Vasectomy	Normal histology	37	46, XY	1

No competing interests reported.

Suppfigure1.jpg
Supplementary figure 1: Age of patients included in the fibrotic group compared to those of the non-fibrotic group. No statistical difference was found.
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Supplementary figure 2: Enrichment analysis of the fibrotic versus non-fibrotic analysis. A) Gene ontology analysis of the biological functions of the downregulated genes. B) Downregulated genes involved in the ECM/receptor interaction (data on KEGG graph, rendered by Pathview).
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Supplementary figure 3: RT-qPCR analysis for TGF-β1 in KS, SCO and FC testicular samples. A significant difference was found between KS and FC samples. *p=0.0016
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Supplementary figure 4: Gene ontology analysis of the biological functions of the down-regulated genes found in the analysis comparing KS and TA samples.
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Supplementary figure 5: KEGG analysis found in the analysis comparing KS and TA samples. A) Up-regulated DEGs. B) Down-regulated DEGs.
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Supplementary figure 6: Validation of the gene expression results by RNA scope for KS and FC samples, including positive and negative controls, as well as overview pictures. A) Duplex analysis showing a higher expression for both VCAM1 and MXRA5 in KS samples compared with FC samples B) Duplex analysis showing a higher expression for both VCAM1 and DCX in KS samples compared with FC samples.
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Supplementary figure 7: Validation of the gene expression results by RNA scope. A) Duplex analysis showing a higher expression for both VCAM1 and MXRA5 in TA samples compared with SCO samples B) Duplex analysis showing a higher expression for both VCAM1 and DCX in TA samples compared with SCO samples.
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Supplementary figure 8: Interaction and downstream pathways of KS upregulated genes TGF-β, decorin and VCAM1, leading to fibrosis.
Supplementalexperimentalprocedure.docx

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Editorial decision: Major revision
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You are reading this latest preprint version

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

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Abstract

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Background

Methods

Patient samples

Histological examination

RNAseq – Wetlab protocol

RNAseq – Differential expression and downstream analysis

Statistical analysis

Results

Transcriptomic analysis - fibrotic vs non-fibrotic tissue

Transcriptomic analysis - KS vs TA

Validation

Discussion

Conclusions

Abbreviations

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References

Tables

Additional Declarations

Supplementary Files

Status:

Version 2