Preservation of the fertile potential of cryptorchid boys by generating in vitro Leydig-like cells starting from gubernaculum testis biopsies

Cryptorchidism is a common condition in paediatric andrology and is associated with infertility, hypogonadism, and impaired testicular function, primarily due to a deficiency of Leydig cells. The development of regenerative strategies capable of restoring testosterone production represents a clinically relevant goal of great interest. Since the gubernaculum testis shares developmental aspects and molecular features with testicular tissue and is easily accessible during orchiopexy, the surgical operation that relocates the undescended testis into the scrotum, this thesis investigates its potential as an autologous source for generating Leydig-like cells in vitro. Gubernacular cells obtained from two cryptorchid patients were cultured and subjected to a differentiation protocol based on three key molecules: SAG, LiCl, and T3 hormone. Following treatment, the cells acquired phenotypic and functional characteristics typical of Leydig cells, including the accumulation of lipid droplets rich in cholesteryl esters, increased total and accessible cholesterol, and the transfer of cholesterol to the mitochondria, where steroidogenesis begins. Activation of the steroidogenic machinery was further confirmed by an increase in interaction between key proteins of the process, detected through proximity ligation assay (PLA). Analyses of autophagy, lysosomal markers, and electron microscopy also revealed an enhancement of lipophagy, a vital process for mobilizing stored cholesterol from lipid droplets toward steroid hormone synthesis. Overall, this work demonstrates that gubernacular cells can be directed toward a steroidogenic Leydig-like phenotype, opening up promising perspectives for future clinical application, such as autologous reimplantation in conditions of hypogonadism or the generation of testicular organoids to support in vitro spermatogenesis.