Paternal MTHFR deficiency leads to reproductive decline across generations in association with hypomethylation of young retrotransposons. Paternal MTHFR deficiency leads to reproductive decline across generations in association with hypomethylation of young retrotransposons

5, 10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in the folate metabolic pathway with a key role in generating methyl groups. As MTHFR deficiency impacts male fertility and sperm DNA methylation, there is the potential for epimutations to be passed to the next generation. Here, we assessed whether the impact of MTHFR deficiency on testis morphology and sperm DNA methylation is exacerbated across generations. While MTHFR deficiency in F1 fathers has only minor effects on sperm counts and testis weights and histology, F2 generation sons show further deterioration in reproductive parameters. Extensive loss of DNA methylation is observed in both F1 and F2 sperm, with >80% of sites shared between generations, suggestive of regions consistently susceptible to MTHFR deficiency. These regions are generally methylated during late prenatal germ cell development and are enriched in young retrotransposons. As retrotransposons are resistant to reprogramming of DNA methylation in prenatal germ cells, their hypomethylated state in the sperm of F1 males could contribute to the worsening reproductive phenotype observed in F2 MTHFR- deficient males, findings compatible with the intergenerational passage of epimutations. Overall design: In order to study the reproductive health of Mthfr mice across generations, Mthfr +/- mice were mated in order to obtain an F1 generation of both Mthfr +/+ and Mthfr -/- male mice. Upon sexual maturity, male mice of the F1 generation were mated with Mthfr +/- mice to obtain the F2 generation. An external cohort of mice (Mat_Def_colony) was also examined, where maternal- and paternal-deficient Mthfr mice were mated to obtain Mthfr -/- male mice.