Study design

For the current study ITT from six patients was thawed as previously described (39) and allocated to prepubertal (ages 1, 2 and 3) and peripubertal (ages 9, 11 and 12) groups ( Table 1 ). We excluded any patients who had previous gonadotoxic therapy, patients with potential malignant cells in their ITT (e.g., testicular cancer, leukemia) and patients with differences in sexual development ( Table 1 ). Selected cryopreserved ITT samples were removed from liquid nitrogen dewars and thawed rapidly in a 37°C water bath, as described previously (39) ( Figures 1C, D ). Thawed testicular tissue for each patient was divided into five small fragments (~1 mm³) when possible (see explanation for missing data in Supplementary Table 1 ). One ITT fragment was fixed in 4% paraformaldehyde (PFA) overnight and labeled as post-thaw sample and four fragments were placed in organotypic culture at the air-liquid interface as previously described by De Michele and colleagues (32). Briefly, ITTs were placed in 12 mm diameter/0.4 µm polycarbonate membrane Transwell^® inserts (Corning^® Incorporated) and cultured in a 5% CO2 humidified incubator at 34°C for 7, 16 or 32 days. The culture medium, CTS™ KnockOut™ DMEM/F-12 culture medium (Cat. No. A1370801, Thermofisher Scientific) supplemented with 10% CTS Knockout SR xenofree medium (KSR, Cat. No. 12618012, Thermofisher Scientific) and 1% penicillin-streptomycin (Cat. No. 15140122, Thermofisher, Gibco), was refreshed every 48 hours. ITT fragments of each patient were cultured under four conditions: 1) no gonadotropins, 2) 5 IU/L FSH (Gonal-F 75 IU, Merck Serono), 3) 1 IU/L hCG (Cat. No. CG5-1VL, lyophilized powder, vial of ~5,000 IU, Sigma-Aldrich) or 4) 5 IU/L FSH + 1 IU/L hCG, starting from day 2 ( Figures 1D, E ). Tissues from an individual patient were divided among the four gonadotropin treatment groups (no gonadotropins, FSH, hCG, FSH + hCG). There was not enough tissue from individual patients to also spread across the three culture timepoints. Therefore, one prepubertal sample (1 y/o) and one peripubertal sample (9 y/o) were analyzed on culture day 7; a second prepubertal (2 y/o) and second peripubertal (11 y/o) sample were analyzed on culture day 16 and a third prepubertal (3 y/o) and third peripubertal (12 y/o) sample were analyzed on culture day 32. Tissues from both pre- and peripubertal groups and each gonadotropin treatment group were collected on days 7, 16 and 32 of culture; fixed in 4% PFA overnight and paraffin embedded for histology and immunofluorescence staining ( Figures 1F, H, I ). Spent media was collected on days 4, 8 and 12 of culture and assayed for testosterone ( Figure 1G ). The testosterone experiment required additional samples because the d8 and d12 timepoints could not be collected from the cultures that were terminated on day 7 ( Supplementary Table 2 ).

Patient groups for histology and IHC studies.