Sertoli cell function and the dose of denosumab is critical for the response to RANKL inhibition in human testis cultures

Currently, no treatment options exist to improve semen quality for the majority of infertile men, but it is well known that the interaction between Sertoli- and germ cells is essential to ensure complete spermatogenesis. A recent study suggested that denosumab, an inhibitor of receptor activator of nuclear factor kappa-B ligand (RANKL) signaling, may decrease germ cell apoptosis in human ex vivo testis cultures and stimulate sperm production in some infertile men. Here, we propose that the level of osteoprotegerin (OPG) expression in Sertoli cells may be indicative of dysgenesis in human testis tissue cultured ex vivo and that the expression of the RANKL signaling system, in combination with the dose, is predictive for the testicular response to denosumab. Human testis from orchiectomies was cultured ex vivo for 48 hours (34°C, 5% CO2) and treated with 1, 30, or 100 ng/ml denosumab or vehicle. Samples positive for D2-40, a marker of germ cell neoplasia in situ (the precursor cell for testicular cancer), were excluded. Immunohistochemical staining for OPG was used to assess dysgenesis in Sertoli cells in each seminiferous tubule of testis cultures. Proliferation and apoptosis were assessed by quantification of poly(ADP-ribose) polymerase cleavage (cPARP) and Bromdeoxyuridin (BrdU) positive germ cells, respectively. Treatment with 100 ng/ml of denosumab increased germ cell proliferation and decreased apoptosis in testis cultures with low or moderate OPG expression in Sertoli cells, while lower doses of denosumab had no effect. In contrast, 100 ng/ml denosumab increased germ cell apoptosis in testis tissue with high OPG expression in Sertoli cells but did not affect germ cell proliferation. Lower doses of denosumab did not affect germ cell proliferation or apoptosis in testis tissue with high OPG expression. This study suggests that the effect of denosumab depends on the function of Sertoli cells, with high OPG expression being a marker of dysgenesis. In addition, the testicular effect of denosumab is determined by the dose, and future clinical trials in infertile men should aim for a dose resulting in an intratesticular concentration close to 100 ng/ml.