Generation of spermatid-like cells from testicular biopsies obtained from pre-pubertal boys

OBJECTIVE: To study whether functional human spermatids can be generated from spermatogonial stem cells obtained from testicular biopsies of prepubertal boys newly diagnosed with cancer using a three-dimensional human testicular organoid system. DESIGN: Experimental laboratory study using a three-dimensional human testicular organoid model incorporating Sertoli cells and endothelial cells to recreate the testes microenvironment. SUBJECTS: Testicular biopsy specimens obtained from prepubertal boys newly diagnosed with cancer prior to initiation of gonadotoxic cancer therapy for fertility preservation. EXPOSURE: Culture of prepubertal testicular tissue in a three-dimensional human testicular organoid system with growth factors to support germ cell differentiation. MAIN OUTCOME MEASURES: Generation of differentiated spermatids followed by verification of postmeiotic marker expression by immunofluorescence, and assessment of functional competence through fertilization assays. RESULTS: Germ cells differentiated into round spermatid-like cells within our testicular organoid system. Immunofluorescence confirmed expression of markers consistent with postmeiotic germ cell differentiation. Functional competence was demonstrated by successful fertilization assays using mouse oocytes, indicating the ability of the spermatid-like cells generated ex vivo to activate oocytes. CONCLUSION: Spermatid-like cells can be generated from prepubertal testicular tissue in a three-dimensional organoid culture system, supporting the potential feasibility of fertility preservation for boys undergoing gonadotoxic cancer treatments.