Differential Effects of Folic Acid and 5‐Methyltetrahydrofolate on Mice Oocytes and Embryo Development In Vitro and In Vivo: Implications for Reproductive Health

Folic acid (FA) is widely used to support one-carbon metabolism; however, at high doses, it can circulate predominantly as unmodified FA (UMFA), potentially leading to metabolic imbalances and adverse effects on reproductive performance. This study aimed to compare the effects of high doses (20X) of FA and 5-methyltetrahydrofolate (5mTHF) on the developmental competence of mouse oocytes, both in vitro and in vivo, using standard dose FA as a control. For the in vitro experiment, female mice were hormonally stimulated and the collected cumulus-oocyte-complexes (COCs) were matured in vitro with the add-on of variable concentrations of FA or 5mTHF, and the reproductive outcomes were recorded. In vivo, 3-week female mice received high-dose FA or 5mTHF supplementation during the weaning period (5 weeks), after which they were either mated with fertile males or hormonally stimulated for COCs collection. Results indicated that high FA concentrations impaired oocyte maturation, spindle assembly, fertilization, and blastocyst development, whereas similar high concentrations of 5mTHF did not induce such deterioration and even improved some outcomes. Post-mating, high-dose FA increased embryo reabsorption rates, while 5mTHF reduced them. Blood analyses showed comparable folate levels across groups, but high-dose FA significantly raised formaldehyde levels-a genotoxic metabolite-whereas 5mTHF markedly decreased formaldehyde. In vitro, 5mTHF supported better oocyte maturation compared to FA, and in vivo, it provided reproductive benefits without inducing formaldehyde toxicity. These findings suggest 5mTHF as a safer, more effective folate form for reproductive health, emphasizing concerns over high-dose FA's genotoxic potential and advocating for cautious clinical use.