O-139 Testicular mosaicism in non-mosaic Klinefelter Syndrome patients with focal spermatogenesis

Abstract Study question Do testis-specific cells have a normal karyotype in regions with spermatogonia (SPG) and ongoing spermatogenesis in non-mosaic Klinefelter Syndrome (KS) patients? Summary answer While SPG and Sertoli cells (SC) have 46,XY karyotype in regions with spermatogenesis, most Leydig cells (LC) and peritubular myoid cells (PTMC) have 47,XXY karyotype. What is known already Although the majority of non-mosaic KS patients is azoospermic, some patients with focal spermatogenesis (FS) can have children with artificial reproductive technologies. However, the genetic origin of FS is unknown. Independent of FS, dimorphism in sex chromatin has been reported amongst the seminiferous tubules of KS men. However, the karyotype of testis-specific cells in FS regions and other regions has not been clearly revealed so far. The histone-3-lysine-27-tri-methylation (H3K27me3) modification is involved in suppression of the extra X chromosome, and its immunohistochemical staining allows the analysis of the X chromosome aneuploidy. Study design, size, duration Testicular biopsies were taken from 7 patients in whom spermatozoa were found by testicular sperm extraction (TESE). Muscle cells of female colon samples were used as positive control. Participants/materials, setting, methods After tissue processing and paraffine embedding, sections were cut at 5 µm. Immunohistochemical staining was performed with antibodies for H3K27me3 (inactive X chromosome), and MAGE-A4 (SPG), SOX9 (SC) and CYP17A1 (LC). PMTC was evaluated according to their position around the tubules and elongated shape. Inactive X (Xi) positive (Xi+) and Xi negative (Xi-) cells were counted and, Xi+ cells were evaluated as having the 47,XXY karyotype; Xi- cells were evaluated as having the 46,XY karyotype. Main results and the role of chance A total of 25 SPG+ tubule sections were detected in 4 of 7 samples. Both SPG and SC were Xi-, i.e. 46,XY karyotype, in all of these tubules. In some of the Sertoli cell only (SCO) tubules, SCs were Xi+ (i.e. 47,XXY), while in others they were Xi- (i.e. 46,XY). The rate of Xi+ SC in SCO tubules containing Xi+ SCs was 31.6%. Similarly, the Xi+ ratio in muscle cells in the positive control was 31.0%. PTMCs surrounding tubules with SPG present, or ongoing FS were Xi+ (i.e. 47,XXY). LCs were Xi+ (i.e. 47,XXY) around all tubules. However, the percentage of Xi+ PTMCs (21.4%) and LC (24.6%) was low compared to the female control (31.0%), suggesting mosaicism for these cells as well. Limitations, reasons for caution The fact that not all of the cell nuclei coincide with the section plane and the cells are in the mitotic stage limit the detection of Xi with H3K27me3. To overcome this limitation, X chromosome analysis could be performed by different techniques on intact cells isolated from fresh tissue. Wider implications of the findings FS occurs only in tubules with 46,XY SPGs and 46,XY SCs. Non-mosaic 47,XXY (diagnosed on blood cells) KS patients may present with testicular mosaicism, increasing their chances for sperm retrieval. Trial registration number Not applicable