P-075 A new clinically relevant classification of seminiferous tubules in Klinefelter Syndrome patients based on the karyotype of the Sertoli cells
Authors: Gül S , Vloeberghs V , Gies I , Goossens E 
Published: July 2024

Abstract

Study question
Can a new seminiferous tubule classification be made that indicates the chance of sperm retrieval by TESE in Klinefelter Syndrome (KS) patients?

Summary answer
Spermatogonia are only present in tubules with euploid Sertoli cells. Classification of tubules based on Sertoli cell ploidy may predict sperm retrieval success with TESE.

What is known already
KS patients can have children thanks to focal spermatogenesis. However, this can only happen if sperm is produced. Histological classification of seminiferous tubules in these patients has been made based on the maturity of the Sertoli cells (type A and type B seminiferous tubules). However, so far there is no classification predicting the presence of germ cells.
In case two X chromosomes are present, one is inactivated. The histone-3-lysine-27-tri-methylation (H3K27me3) protein is involved in X chromosome inactivation and its immunohistochemical staining may be used for a new seminiferous tubule classification based on the karyotype of Sertoli cells.

Study design, size, duration
Inactive X (Xi) chromosomes were immunohistochemically stained with H3K27me3 and seminiferous tubules were classified as Xi+ tubules (presence of inactive X in Sertoli cells) or Xi- tubules (absence of inactive X in Sertoli cells). Testicular biopsies were used from 17 non-mosaic KS adult patients.

Participants/materials, setting, methods
After tissue fixation, processing, and paraffin embedding, sections were cut at 5 µm. Immunohistochemical staining was applied with antibodies H3K27me3 (for inactive X chromosome), MAGE-A4 (for spermatogonia), and SOX9 (for Sertoli cells). Seminiferous tubules in testicular sections were classified as Xi+ or Xi- tubules and analyzed for the presence of spermatogonia or focal spermatogenesis. X chromosome number in spermatogonia and Sertoli cells was confirmed by the FISH method using chromosome 18, Y, and X probes.

Main results and the role of chance
A total of 311 (18±16) seminiferous tubule sections were analyzed in testicular tissue of 17 KS men. Of the 311 tubules analyzed, 253 (14±16) of the tubules were Xi- tubules and 58 (3±4) were Xi+ tubules. Spermatogonia were present in 41 of the Xi- tubules. As an exception, only one patient had spermatogonia in two Xi+ tubules, but these tubules were immature and spermatogenesis was not observed. Ongoing spermatogenesis was observed in two of Xi- tubules in one of the patients. Sixteen of the patients had both Xi- tubules and Xi+ tubules in their testicular sections. In 13 of these 16 patients with Xi- tubules, sperm was either obtained as a result of TESE or spermatogonia were observed in the Xi- tubules.

Limitations, reasons for caution
In Klinefelter patients, there are few seminiferous tubules due to widespread testicular fibrosis and degeneration of seminiferous tubules. Studies including more patient samples are needed for more definitive and comprehensive conclusions.

Wider implications of the findings
Spermatogonia are only present in tubules with euploid Sertoli cells. Therefore, we hypothesize that focal spermatogenesis only can occur in tubules with euploid Sertoli cells. Xi+ and Xi- seminiferous tubule classification based on H3K27me3 staining may thus be used to predict sperm production in KS patients.

Trial registration number
Not applicable