Causes for miscarriages and congenital malformations can be genetic, environmental, or a combination of both. Factors such as genetic variants, hypoxia, or undernutrition individually may not affect embryo development but collectively may do so. Previously, we showed that genetic disruption of nicotinamide adenine dinucleotide (NAD) synthesis via homozygous loss-of-function mutations in HAAO or KYNU, two genes of the kynurenine pathway, caused NAD deficiency and multiple congenital malformations in humans and mice. NAD is synthesized de novo from tryptophan and from vitamin B3.
Here, we show that dietary restriction of vitamin B3 and tryptophan during pregnancy can induce NAD deficiency and cause congenital malformations in C57BL/6J wildtype mice. The types of malformations match those seen in mouse embryos with homozygous loss-of-function in Haao. When the dietary NAD-precursor restriction was combined with a heterozygous mutation in Haao, which by itself does not cause NAD deficiency or malformations, the incidence of embryo loss and multiple malformations was significantly increased. Accordingly, NAD levels in the mother and embryos were even more reduced. Similarly, mild hypoxia exacerbated the embryo outcome when combined with one or both other factors. The maternal NAD levels during pregnancy are the main determinant for the embryo outcome.
Together, our data show that NAD deficiency as a cause of embryo loss and congenital malformation is not restricted to the rare cases of homozygous mutations in NAD synthesis pathway genes. Instead, heterozygous genetic variants and environmental factors can result in similar outcomes when occurring in combination. The results expand our understanding of the causes of congenital malformations and the importance of enough supply of NAD precursors during pregnancy.