ESPE Abstracts

The most common genetic abnormality found among men is the presence of an additional X-chromosome which causes Klinefelter syndrome (KS). KS has an estimated prevalence of 1.5/1000, but only ~40% receive the diagnosis, probably because the phenotype varies considerably. Men with KS often show eunuchoid body proportions, cognitive and psycho-social difficulties, and have an increased risk of developing cardiometabolic diseases. But the most persistent symptoms are small testes, non-obstructive azoospermia, and hypergonadotropic hypogonadism. The additional X chromosome arises due to a non-disjunction during paternal or maternal meiosis and is thought to be subjected to dosage compensation by X-inactivation and expression of the long non-coding RNA, XIST. In adult men with KS, the testes appear degenerated and typically only contain Sertoli cell-only (SCO) tubules without germ cells, degenerated “ghost” tubules, and Leydig cell hyperplasia. Occasionally, focal spermatogenesis can be observed and is crucial for the ability to father a child by in vitro fertilisation. However, what biologically determines whether focal spermatogenesis can occur and what causes testicular degeneration remains unknown. With the emergence of bulk RNA- and later single-cell RNA-sequencing, several studies have indicated that the Sertoli cells play a central role in testicular degeneration. Interestingly, earlier morphological studies have described that two different types of SCO tubules exist, in which, the Sertoli cells show differences in the presence of the Barr body. The Barr body represents a dense inactivated X chromosome and was observed in the type B SCO tubules containing immature-like Sertoli cells but never in the type A SCO tubules containing adult mature-like Sertoli cells. This could indicate a relationship between X-inactivation and the maturity of the Sertoli cells. Using ultra-sensitive single-molecule RNA and DNA in situ hybridisation with probes targeting XIST and the X chromosome, we show that the undifferentiated type B Sertoli cells highly express XIST and have two X-chromosomes, while the differentiated type A have lost XIST expression and the additional X-chromosome. Hence, the maturity of the Sertoli cells seems related to the loss of the additional X-chromosome. In focal tubules with spermatogenesis, both Sertoli cells and spermatogonia were also found to have lost XIST expression and the additional X-chromosome. The additional X-chromosome hence seems incompatible with the maturation of Sertoli cells and spermatogenesis, while focal rescue of spermatogenesis can occur in conjunction with a micro-mosaic loss of the additional X-chromosome both in Sertoli cells and spermatogonia.