The maternal microbiome influence on pregnancy success: breeding comparison of germ-free and conventionalized mice.

Germ-free (GF) animals, which are entirely devoid of all microorganisms, are one of the most powerful tools for studying the role of the microbiome in a phenotype, moving the microbiome field from association to causation. They allow the introduction of specific microbes or microbial communities to interrogate the causality of microbiomes in protecting against or contributing to a phenotype. Here, we report critical and underappreciated challenges in using GF models to investigate the intergenerational effects of maternal diet and microbiota on offspring health. Using 57 GF and littermate conventionalized GF dams, we observed unexpectedly high maternal (odds ratio 11.5, p < 0.0001) and offspring (odds ratio 4.12, p < 0.0001) mortality in GF animals. Remarkably, GF dams had an extremely low pregnancy and parturition (pmicrobiome < 0.0001) and a high incidence of cecal torsion (18.2%) compared to the conventionalized group, underscoring the indispensable role of the maternal microbiome in reproductive success and early development. Notably, even conventionalized GF mothers on high-fiber diets exhibited poor fertility, suggesting that microbial colonization timing and maternal microbial capacity to metabolize fiber are crucial. These findings not only reveal significant limitations in GF breeding protocols but also indicate that the maternal microbiota might influence offspring health far earlier than previously recognized, with implications for the developmental origins of health and disease research.