Aim: Can anti-Mullerian hormone (AMH) protect human ovarian follicles and stroma from 4-hydroxycyclophosphamide (4-HC)-induced DNA damage and apoptosis?
Methods: Fresh ovarian cortical biopsies from healthy women (aged 25–35) were cultured with vehicle (DMSO), 2µM 4-HC ± 200ng/mL AMH, or AMH only. Cultures were maintained for 4 hours or 7 days following 24-hour exposure (n=5-7 patients) to assess immediate and prolonged effects. Analyses included histology (follicle stage and morphology), and immunostaining for DNA damage (TUNEL and γH2AX), apoptosis (cleaved-PARP) and proliferation (Ki67).
Results: At 4 hours, exposure to 4-HC compromised histological health of non-growing follicles (vehicle: 24±4% unhealthy follicles vs 4-HC: 46±8%, p<0.0001) and increased stromal cell TUNEL staining intensity 2.5-fold compared to vehicle (p=0.04). AMH attenuated morphological damage (30±6% unhealthy follicles, p<0.0001 vs 4-HC) and reduced stromal TUNEL staining intensity 2.3-fold compared to 4-HC exposed tissue (p=0.01).
At 7 days following 24h 4-HC exposure, non-growing follicle health remained compromised (vehicle: 32±8% unhealthy follicles vs 4-HC: 47±3%, p<0.001), with evidence of increased DNA damage measured by both γH2AX (vehicle: 26±7% γH2AX+ oocytes vs 4-HC: 61±14%, p<0.0001) and TUNEL (vehicle: 6±3% TUNEL+ oocytes vs 4HC: 23±12%, p<0.0001). This corresponded with a 4-fold increase in apoptotic follicles identified by cleaved-PARP (vehicle: 8±3% cPARP+ oocytes vs 4-HC: 35±9%, p<0.0001). Co-culture with AMH reduced morphological damage (27± 8% unhealthy follicles, p<0.05), DNA damage (30±10% γH2AX+ oocytes, p<0.001, and 10±4% TUNEL+ oocytes, p<0.001) and follicle apoptosis (4±2% cPARP+ oocytes, p<0.0001).
While the proportion of growing follicles doubled in tissue analysed at 7 days versus 4 hours, there were no significant differences in follicle stage distribution (both timepoints) or Ki67 staining (7 days only) between different treatment groups following 4-HC exposure.
Conclusions: AMH confers both immediate and sustained protection against chemotherapy-induced damage in human ovarian follicles and stroma, supporting its potential as a targeted ovarian chemoprotective agent.