Methods
Fresh and cryopreserved-thawed testis fragments (1-2 mm3) were cultured at a gas-liquid interphase (34°C, 5% CO2) in Minimum Essential Medium alpha + 10% knock-out serum replacement medium containing 10-7 M melatonin and 10-6 M retinoic acid, with or without 3 IU/L FSH/LH supplementation. The effect of culture conditions on testicular fragments was weekly assessed by histological evaluation of germ cell development and immunohistochemical identification of spermatogonia (using MAGEA4), proliferative status of spermatogonia and Sertoli cells (using proliferating cell nuclear antigen [PCNA]), intratubular cell apoptosis (by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) and Sertoli cells maturation (using Anti-Müllerian Hormone [AMH] versus Androgen Receptor [AR]). Additionally, Leydig cells' functionality was determined by measuring testosterone concentration in the culture media supernatants. Main