Disruption of folate metabolism causes poor alignment and spacing of mouse conceptuses for multiple generations

ABSTRACT Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with an Mtrrgt hypomorphic mutation. MTRR is necessary for methyl group utilisation from the folate cycle, and the Mtrrgt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrrgt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes (e.g., eccentric chorioallantoic attachment). Notably, we report misalignment of some Mtrrgt conceptuses within their implantation sites from E6.5. The degree of skewed growth occurs across a continuum, with eccentric chorioallantoic attachment now re-characterised as a severe form of conceptus misalignment. Additionally, some Mtrrgt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Embryo development is influenced by skewed growth since developmental delay and heart malformations (but not neural tube defects) associate with severe misalignment of Mtrrgt/gt conceptuses. Patterning of trophoblast lineage markers is largely unaffected in skewed Mtrrgt/gt conceptuses at E8.5 indicating trophoblast differentiation was normal when misaligned. Typically, the uterus guides conceptus orientation. Accordingly, we manipulate the maternal Mtrr genotype and assess conceptus alignment. Mtrr+/gt, and Mtrrgt/gt mothers, plus Mtrr+/+ mothers, exhibit misaligned conceptuses at E6.5. While progesterone and/or BMP2 signalling required for decidualisation might be disrupted, normal gross decidual morphology, patterning, and blood perfusion is evident regardless of conceptus alignment, arguing against a uterine defect. Given the important finding that Mtrr+/+ mothers also display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrrgt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe skewing associates with the Mtrr genotype of either maternal grandparent. Moreover, misalignment is independent of the uterus and instead is attributed to an embryonic mechanism based on blastocyst transfer experiments. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. Our study casts light on the complex role of folate metabolism during development beyond a direct maternal effect.