Short-term oral supplementation with nicotinamide mononucleotide restores oocyte quality in chemotherapy-induced ovarian damage mouse models

Previous studies suggest that nicotinamide mononucleotide (NMN) restores fertility in aged mice, improving oocyte mitochondrial activity and redox status. However, these positive effects against aging should be validated in the worst ovarian scenario of cancer treatments. High-dose chemotherapy with alkylating drugs induces deleterious changes on the ovaries, leading to follicle depletion and impaired oocyte quality. Thus, we aim to investigate if a short NMN administration is able to enhance the oocyte quality of growing follicles in chemotherapy-induced mouse models of diminished ovarian reserve (DOR) and primary ovarian insufficiency (POI). Twenty-six 7-week-old C57BL/6 female mice were randomly allocated to young-healthy (n=8), DOR (n=8), and POI (n=10) groups. DOR and POI were induced by a single intraperitoneal injection of a reduced or standard dose of cyclophosphamide and busulfan, respectively. One week later (day 0), half of the animals from each model were supplemented with 2g/L NMN in drinking water for 14 days whereas the remaining mice received standard drinking water and were used as controls for each ovarian condition. On day 11, mice underwent ovarian hyperstimulation, collecting metaphase-II oocytes on day 14. Part of the recovered MII-oocytes was subjected to in vitro fertilization (IVF) and embryo culture while NAD+ levels and reactive oxygen species (ROS) were measured by a FRET-microscopy for live cell imaging in the remaining ones. Mann-Whitney U-tests were used to compare results between NMN and Control mice while chi-square tests were applied to analyze IVF outcomes per oocyte. NMN supplementation reduced the percentage of morphologically abnormal MII-oocytes in all models (DOR-NMN: 1.1±2.3% vs. DOR-Control: 2.3±3.1%; POI-NMN: 1.6±2.1% vs. POI-Control: 2.6±4.5%; p=N.S.) although did not increase the MII-oocyte yield in any ovarian condition. After IVF, NMN supplementation enhanced fertilization (NMN vs. Control; DOR: 82% vs. 68%; POI: 68% vs. 59%), blastocyst formation (DOR: 73% vs. 52%; POI: 38% vs. 27%) and hatching (DOR: 68% vs. 43%; POI: 26% vs. 18%) rates in both ovarian damage mouse models, reaching statistically significant differences in DOR (p=0.062, p=0.016 and p=0.007, respectively). By contrast, these outcomes were slightly impaired in NMN-treated young-healthy mice. In the DOR model, NMN treatment increased NAD+ levels in MII-oocytes (NMN: 0.4±0.0 vs. Control: 0.3±0.0; p<0.001), while in POI mice a decrease in ROS was detected (NMN: 0.9±0.2 vs. Control: 1.4±1.4; p=0.040). NMN oral supplementation during the late stages of folliculogenesis increases NAD+ levels in ovulated oocytes from chemotherapy-induced DOR mice, improving their ability to be fertilized and reach the blastocyst stage. In the POI condition, this treatment also showed positive effects on oocyte quality, by reducing ROS levels, increasing fertilization and embryo development.