Characterization of the stem cell niche components within the seminiferous tubules in testicular biopsies of Klinefelter patients.
To characterize the tubular environment in testicular biopsy tissues from patients with Klinefelter syndrome (KS).
Observational immunohistochemical study.
Academic research unit.
Males with KS and controls at different developmental time points: fetal, prepubertal, peripubertal, and adult.
Immunohistochemical analysis of testicular biopsies samples to characterize maturation of Sertoli cells and tubular wall components-peritubular myoid cells (PTMC) and extracellular matrix (ECM) proteins.
Intensity of antimüllerian hormone staining; proportion of Sertoli cells expressing androgen receptor (AR); and expression of tubular wall markers as characterized by identifying abnormal staining patterns.
Decreased expression for alpha smooth muscle actin 2 (ACTA2) was observed in peripubertal and adult KS as well as in Sertoli cell only (SCO) patients. Altered expression patterns for all ECM proteins were observed in SCO and KS biopsy tissues compared with controls. Only for collagen I and IV were altered expression patterns observed between KS and SCO patients. In peripubertal samples, no statistically significant differences were observed in the maturation markers, but altered ECM patterns were already present in some samples.
The role of loss of ACTA2 expression in PTMC in the disintegration of tubules in KS patients should be further investigated. Future research is necessary to identify the causes of testicular fibrosis in KS patients. If the mechanism behind this fibrotic process could be identified, this process might be altered toward increasing the chances of fertility in KS patients.
Van Saen D
，Vloeberghs V
，Gies I
，De Schepper J
，Tournaye H
，Goossens E
... -
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Testicular immune cells and vasculature in Klinefelter syndrome from childhood up to adulthood.
Is the distribution of immune cells and the testicular vasculature altered in testicular biopsies from patients with Klinefelter syndrome (KS)?
Increased numbers of macrophages and mast cells, an increased expression of decorin and an increased blood vessel density were found in KS samples compared to controls.
Most KS patients are infertile due to an early germ cell loss. From puberty onwards, testicular fibrosis can be detected. How this fibrotic process is initiated remains unknown.
In this study, the number of macrophages, mast cells and their secretory products were evaluated in KS, Sertoli cell only (SCO) and control patient samples. The association between immune cell numbers and level of fibrosis in KS tissue was examined. In addition, the vascularization within these testicular tissue biopsies was studied. For immunohistochemical evaluation, KS patients at different stages of testicular development were included: prepubertal (aged 4-7 years; n = 4), peripubertal (aged 11-17 years; n = 21) and adult (aged >18 years; n = 37) patients. In addition, testicular tissue biopsies of adult SCO (n = 33) and control samples for the three KS age groups (prepubertal n = 9; peripubertal n = 5; adult n = 25) were analysed. Gene expression analysis was performed on adult testicular tissue from KS (n = 5), SCO (n = 5) and control (n = 5) patients.
Adult (>18 years) KS, SCO and control testicular tissue biopsies were obtained during a testicular sperm extraction procedure. KS peripubertal (11-18 years), prepubertal (<11 years) and age-matched control biopsies were obtained from the biobank of the university hospital. Immunohistochemistry was used to determine the tubular structure (H/PAS), the number of spermatogonia (MAGE-A4), macrophages (CD68) and mast cells (tryptase) and the blood vessel density (Von Willebrand factor). In addition, quantitative real-time polymerase chain reaction was used to determine the expression of secretory products of macrophages and mast cells (tryptase, tumour necrosis factor alpha and decorin).
A significant increase in the number of macrophages (P < 0.0001) and mast cells (P = 0.0008) was found in the peritubular compartment of testes of adult KS patients compared to control samples. However, no association between the number of immune cells and the degree of fibrosis was observed. In adult SCO samples, a significant increase was seen for peritubular macrophage (P < 0.0001) and mast cell (P < 0.0001) numbers compared to control samples. In the interstitial compartment, a significant increase in mast cell number was found in adult SCO samples compared to KS (P < 0.0001) and control (P < 0.0001) tissue. A significant difference (P = 0.0431) in decorin expression could be detected in adult KS compared to control patients. Decorin expression was mostly seen in the walls of the seminiferous tubules. When comparing the vascularization between KS patients and age-matched controls, a significant increase (P = 0.0081) in blood vessel density could be observed only in prepubertal KS testicular tissue.
N/A.
As controls for this study, testicular tissue biopsies of men who underwent a vasectomy reversal or orchiectomy were used, but these men may not represent fertile controls. In addition, a high variability in immune cell numbers, secretory products expression and number of blood vessels could be observed amongst all patient samples.
Increased numbers of macrophages and mast cells have previously been described in non-KS infertile men. Our results show that these increased numbers can also be detected in KS testicular tissue. However, no association between the number of macrophages or mast cells and the degree of fibrosis in KS samples could be detected. Decorin has previously been described in relation to fibrosis, but it has not yet been associated with testicular fibrosis in KS. Our results suggest a role for this proteoglycan in the fibrotic process since an increased expression was observed in adult KS tissue compared to controls. Impaired vascularization in KS men was suggested to be responsible for the KS-related disturbed hormone levels. Our results show a significant difference in blood vessel density, especially for the smallest blood vessels, between prepubertal KS samples and age-matched controls. This is the first study to report differences between KS and control testicular tissue at prepubertal age.
The project was funded by grants from the Vrije Universiteit Brussel (E.G.) and the scientific Fund Willy Gepts from the UZ Brussel (D.V.S.). D.V.S. is a post-doctoral fellow of the Fonds voor Wetenschappelijk Onderzoek (FWO; 12M2819N). No conflict of interest is declared for this research project.
Willems M
，Vloeberghs V
，Gies I
，De Schepper J
，Tournaye H
，Goossens E
，Van Saen D
... -
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Testicular mosaicism in non-mosaic postpubertal Klinefelter patients with focal spermatogenesis and in non-mosaic prepubertal Klinefelter boys.
Do testis-specific cells have a normal karyotype in non-mosaic postpubertal Klinefelter syndrome (KS) patients with focal spermatogenesis and in non-mosaic prepubertal KS boys?
Spermatogonia have a 46, XY karyotype, and Sertoli cells surrounding these spermatogonia in postpubertal patients also have a 46, XY karyotype, whereas, in prepubertal KS boys, Sertoli cells surrounding the spermatogonia still have a 47, XXY karyotype.
A significant proportion of patients with non-mosaic KS can have children by using assisted reproductive techniques thanks to focal spermatogenesis. However, the karyotype of the cells that are able to support focal spermatogenesis has not been revealed.
Testicular biopsy samples from non-mosaic KS patients were included in the study. Karyotyping for sex chromosomes in testis-specific cells was performed by immunohistochemical analysis of inactive X (Xi) chromosome and/or fluorescent in situ hybridization (FISH) analysis of chromosomes 18, X, and Y.
A total of 22 KS patients (17 postpubertal and 5 prepubertal) who were non-mosaic according to lymphocyte karyotype analysis, were included in the study. After tissue processing, paraffin embedding, and sectioning, the following primary antibodies were used for cell-specific analysis and Xi detection; one section was stained with MAGE A4 for spermatogonia, SOX9 for Sertoli cells, and H3K27me3 for Xi; the other one was stained with CYP17A1 for Leydig cells, ACTA2 for peritubular myoid cells, and H3K27me3 for Xi. Xi negative (Xi-) somatic cells (i.e. Sertoli cells, Leydig cells, and peritubular myoid cells) were evaluated as having the 46, XY karyotype; Xi positive (Xi+) somatic cells were evaluated as having the 47, XXY. FISH stain for chromosomes 18, X, and Y was performed on the same sections to investigate the karyotype of spermatogonia and to validate the immunohistochemistry results for somatic cells.
According to our data, all spermatogonia in both postpubertal and prepubertal non-mosaic KS patients seem to have 46, XY karyotype. However, while the Sertoli cells surrounding spermatogonia in postpubertal samples also had a 46, XY karyotype, the Sertoli cells surrounding spermatogonia in prepubertal samples had a 47, XXY karyotype. In addition, while the Sertoli cells in some of the Sertoli cell-only tubules had 46, XY karyotype, the Sertoli cells in some of the other Sertoli cell-only tubules had 47, XXY karyotype in postpubertal samples. In contrast to the postpubertal samples, Sertoli cells in all tubules in the prepubertal samples had the 47, XXY karyotype. Our data also suggest that germ cells lose the extra X chromosome during embryonic, fetal, or neonatal life, while Sertoli cells lose it around puberty. Peritubular myoid cells and Leydig cells may also be mosaic in both postpubertal patients and prepubertal boys, but it requires further investigation.
The number of prepubertal testicle samples containing spermatogonia is limited, so more samples are needed for a definitive conclusion. The fact that not all the cell nuclei coincide with the section plane limits the accurate detection of X chromosomes by immunohistochemistry and FISH in some cells. To overcome this limitation, X chromosome analysis could be performed by different techniques on intact cells isolated from fresh tissue. Additionally, there is no evidence that X chromosome inactivation reoccurs after activation of the Xi during germ cell migration during embryogenesis, limiting the prediction of X chromosome content in germ cells by H3K27me3.
Our findings will lay the groundwork for new clinically important studies on exactly when and by which mechanism an extra X chromosome is lost in spermatogonia and Sertoli cells.
This study was funded by The Scientific and Technological Research Council of Türkiye (TUBITAK) (2219 - International Postdoctoral Research Fellowship Program for Turkish Citizens) and the Strategic Research Program (SRP89) from the Vrije Universiteit Brussel. The authors declare no competing interests.
N/A.
Gül S
，Vloeberghs V
，Gies I
，Goossens E
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