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 ... -  《-》

When does germ cell loss and fibrosis occur in patients with Klinefelter syndrome?

Van Saen D ，Vloeberghs V ，Gies I ，Mateizel I ，Sermon K ，De Schepper J ，Tournaye H ，Goossens E ... -  《-》

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 ... -  《-》

Long-term culture of human Sertoli cells from adult Klinefelter patients as a first step to develop new tools for unravelling the testicular physiopathology.

Are Sertoli cells (SCs) from adult Klinefelter men (47,XXY) capable of proliferating in vitro and maintaining their main phenotypical and functional characteristics as do SCs from adult 46,XY patients? Isolated SCs from patients with Klinefelter syndrome (KS) can be expanded in vitro while maintaining their characteristics and a stable karyotype, similar to SCs from 46,XY patients. The mechanism leading to testicular tissue degeneration in KS is still unknown. A few recent studies highlight the main role played by SCs in the physiopathology of the disease, but new study models based on co-culture or testicular organoids are needed to further understand the SC's involvement in the mechanism of testicular degeneration and fibrosis, and to find therapeutical targets. KS SC expansion could be the first step towards developing such in vitro study models. SCs have been isolated from 46,XY men and expanded in vitro while maintaining the expression of phenotypical and functional markers, but propagation of SCs from KS men has not been achieved yet. Testicular tissue was obtained during a testicular sperm extraction procedure for infertility treatment between 2019 and 2021 from three azoospermic adult KS (47,XXY) men (33±3.6 years old) and from three control patients (46,XY) (36±2 years old) presenting with obstructive azoospermia. SCs isolated from frozen-thawed tissue of KS and 46,XY patients were cultured for 60 days and compared. All patients signed an informed consent according to the ethical board approval of the study protocol. Testicular biopsies obtained from KS (n = 3) and 46,XY (n = 3) adult patients were slow-frozen. After tissue thawing SCs were isolated using a double-step enzymatic digestion and differential plating, and cultured for 60 days in DMEM medium containing FBS. Analyses were performed at different culture times (passages 5 (P5) and 10 (P10)). Quantification of cells using immunofluorescence (IF) for cell type-specific markers (Sox9, GATA4, ACTA2, INSL3, MAGEA4), SCs characterization using both IF and quantitative real-time PCR for GDNF, BMP4, AR and CLDN11 and cells karyotyping were performed. We demonstrate for the first time that a small population of human SCs isolated from frozen-thawed testis of adult KS patients can be expanded in vitro while retaining expression of characteristic markers of SCs and the 47,XXY karyotype, and exhibiting cell-specific functional proteins and gene expression (GDNF, BMP4, AR, and CLDN11) after 60 days in culture. At P10, 83.39 ± 4.2% of cultured cells from KS men and 85.34 ± 4.1% from 46,XY men expressed Sox9, and 88.8 ± 3.9% of KS cells versus 82.9 ± 3.2% of the control cells were positive for GATA4 without any differences between two groups; both Sox9 and GATA4 are typical SC markers. No differences were found between KS and 46,XY SCs in vitro in terms of cells expansion (exponential growth between P1 and P10 with an average cell count of 2.8±1.5×107 versus 3.8±1.2×107 respectively for the KS and control groups at P10). There was no significant statistical difference for functional proteins and genes expressions (GDNF, BMP4, AR, and CLDN11) neither between KS SCs and control SCs nor between P5 and P10. The small number of donor samples is a limitation but it is due to limited availability of tissue for research in KS populations. Although no differences were observed in SCs function in the culture of isolated SCs after 60 days, the possibility of a SCs dysfunction needs to be investigated in more complex 3-dimensional models allowing the establishment of a proper cell organization and further analyses of cell functions and interactions during longer culture periods. The demonstration of the possibility to propagate KS SCs in vitro could be useful to build new in vitro models for deciphering testicular cell interactions, determining deficient signalling pathways involved in impaired spermatogenesis, and identifying targets for infertility treatment in KS. As the cell numbers achieved in this study are higher than cell numbers used to develop testicular organoids, we may expect to be able to understand the behaviour and physiopathology of SCs in the disease during the long-term culture of these organoids. Such models could be further applied to understand other causes of deficiencies in seminiferous tubules. M.G.G is funded by a grant from the Cliniques Universitaires Saint-Luc (FRC) for the research project on Klinefelter Syndrome Physiopathology. The authors declare no conflicts of interest. NCT05997706.

Giudice MG ，Kanbar M ，Poels J ，Duquenne A ，Wyns C ... -  《-》

Irradiation affects germ and somatic cells in prepubertal monkey testis xenografts.

Does irradiation evoke adverse effects in germ and somatic cells in testis xenografts from prepubertal monkeys? In addition to the expected depletion of germ cells, a dose-dependent effect of irradiation was observed at the mRNA and protein level in Sertoli and peritubular myoid cells. Testicular irradiation studies in monkeys have focused on the dose-dependent effects on germ cells. Previous studies using intact animals or xenografts reported that germ cells are highly sensitive to irradiation. Their depletion was demonstrated by morphometric and histological analyses. The effect of irradiation on expression of Sertoli and peritubular myoid cell markers, however, has not yet been described. The testes of two prepubertal macaques (Macaca fascicularis) were dissected into testicular fragments. Fragments were randomly exposed in vitro to one of the following three doses of irradiation: 0 Gy, n = 60; 1 Gy, n = 54; 4 Gy, n = 72. Non-irradiated control fragments (0 Gy) were placed into the Faxitron for 6.6 min without irradiation. For 1 Gy and 4 Gy irradiation was applied for 1.7 and 6.6 min, respectively. Grafts were then either immediately analyzed or subcutaneously implanted under the back skin of 39 nude mice and analyzed after 6.5 months. Post grafting, 133 testicular xenografts were retrieved. The body weight, serum testosterone level and seminal vesical weight of the host mice as well as the number and weight of retrieved grafts were determined. Larger grafts were used to evaluate both mRNA expression profiles and protein expression patterns. In total, 71 testicular fragments were used for morphometric and histological analysis while 68 fragments were analyzed for gene expression. For PCR arrays, M. fascicularis-specific primer sequences were employed. Irradiation-induced changes in the transcript levels of 34 marker genes were determined for each testicular graft. The effects of irradiation on peritubular myoid cells and Sertoli cells were confirmed by immunohistochemical analysis of chemokine (C-X-C motif) ligand type 11 (CXCL11), alpha smooth muscle actin (SMA) and chemokine (C-X-C motif) ligand type 12 (CXCL12). The four testes gave rise to 106 xenografts, which were individually analyzed, limiting the role of chance despite using only two monkeys in the study. Prior to grafting, the two donors displayed spermatogonia as the most advanced germ cell type in 95% and 70% of seminiferous tubules, respectively, while remaining tubules contained SCO. No spermatocytes were encountered prior to grafting in either monkey. After 6.5 months, non-irradiated grafts displayed spermatocytes in 15.4% and 1.8% of seminiferous tubules indicating an induction of meiosis. Irradiation resulted in a complete absence of spermatocytes. The percentage of seminiferous tubules containing spermatogonia declined in a dose-dependent manner. In non-irradiated xenografts, ~40% of tubules contained spermatogonia. This proportion was reduced to 3.4% and 4.3% in the 1 Gy treated group and to 1.3% and 0.2% in 4 Gy irradiated grafts. A dose-dependent decline in mRNA levels of selected germ cell marker genes supported the morphologically detected loss of germ cells. Irradiation had no effect on CXCL12 transcript levels. At the protein level, CXCL12-positive Sertoli cells were most abundant in the 1 Gy group compared to the 4 Gy group (P < 0.05), indicating a potential role of CXCL12 during recovery of primate spermatogenesis. The most prominent radiation-evoked changes were for CXCL11, which was localized to smooth muscle cells of blood vessels and seminiferous tubules. Transcript levels declined in a dose-dependent manner in grafts from both monkeys (MM687: P < 0.01 (0 Gy versus 4 Gy), MM627: P < 0.05 (0 Gy versus 4 Gy), P < 0.001 (1 Gy versus 4 Gy)). CXCL11 patterns of protein expression revealed irradiation-dependent changes as well. That peritubular cells are affected by X-irradiation was substantiated by changes at the transcript level between 1 and 4 Gy exposed groups (P < 0.01) and at the protein level of SMA (P < 0.05, 0 Gy versus 4 Gy). n/a. The spermatogonial stem cell system in primates is remarkably different from rodents. Therefore, data from a non-human primate may be more relevant to man. However, species-specific differences amongst primates cannot be fully excluded and the use of only two donors may raise concerns toward the generalization of the findings. There may also be important differences across the prepubertal period (e.g. infancy, early childhood) that are not represented by the ages included in the present study. This study is the first to indicate relevant testicular somatic cell responses following irradiation of prepubertal primate tissue. In addition to the well-known depletion of germ cells, the changes in Sertoli, and in particular peritubular myoid, cells may have important consequences for spermatogenic recovery. These novel findings should be taken into consideration when irradiation effects are assessed in tumor survivors. Interdisciplinary Center for Clinical Research (IZKF) Münster (Schl2/001/13) and the Excellence Cluster 'Cells in Motion' at the University Münster. There are no conflicts of interest to declare.

Tröndle I ，Westernströer B ，Wistuba J ，Terwort N ，Schlatt S ，Neuhaus N ... -  《-》