Endocrine Abstracts

Background: Variations in sex development represent a natural condition that provides insights into fertility and gonadal development. Research on pathogenic variants associated with DSD not only improves the diagnosis and prognosis, but can also identify previously unrecognized concomitant diseases. Myelin Regulatory Factor (MYRF) is a critical transcriptional regulator essential for CNS myelination and is also expressed in other tissues. Disruptive variants in MYRF lead to a recently described syndrome involving cardiac and urogenital syndrome (CUGS) and DSD.

Methods: We analyzed archived FFPE gonadal tissue from a patient with DSD carrying a MYRF mutation (p.Q838*) and compared it with control gonadal tissue. We performed IHC, IF, RNAscope in situ Hybridization and VisiumHD spatial transcriptomics using the WTA probes. These approaches were used to explore defects in somatic and germ cell differentiation through gene and protein expression analysis at near single-cell resolution.

Results: The patient’s testicular tissue is characteristic of Sertoli cell-only Syndrome. The peritubular structure showed a thick lamina around the seminiferous tubules. ACTA2 IF staining revealed two distinct layers of peritubular myoid cells, suggesting an abnormal organization of the tubular basal lamina. Sertoli cells (SCs) have lost their polarity and immunostaining for the tight junction protein CLDN11 contributing to the blood-testis-barrier (BTB) revealed an irregular and disrupted pattern along the seminiferous tubules. While some SCs expressed CLDN11 protein others did not. This finding was also confirmed by RNA expression, indicating a disruption of the BTB. VisiumHD data showed a high number of Leydig cells (LCs) positive for INSL3 and DLK1. In the patient VisiumHD sample we analyzed 116,148 spatial barcodes representing segmented cells. Most of these LCs (23.9%, 27612 barcodes) express only the immature LCs marker DLK1, while 6.5% (7370 barcodes) express only the mature LC marker INSL3, and 8.8% (10114 barcodes) express both genes.

Conclusions: Our findings revealed immature features of testicular architecture in a patient with a MYRF variant, characterized by Sertoli cell-only syndrome with immature Sertoli cell characteristics and disruption of the BTB. We also observed an abnormal peritubular organization. Transcriptomic analysis suggests altered LCs maturation, with a prevalence of cells expressing the immature marker DLK1.