Embryonic signatures of intergenerational epigenetic inheritance across paternal environments and genetic backgrounds.

The paternal preconception environment has been implicated as a modulator of phenotypic traits and disease risk in F1 offspring. However, the prevalence and mechanisms of such intergenerational epigenetic inheritance (IEI) in mammals remain poorly defined. Moreover, the interplay between paternal exposure, genetics, and age on emergent offspring features is unexplored. Here, we measure the quantitative impact of three paternal environments on early embryogenesis across genetic backgrounds. Using in vitro fertilisation (IVF) at scale, we capture batch-robust transcriptomic signatures of IEI with single-blastocyst resolution. Amongst these, paternal gut microbiota dysbiosis is linked with aberrant expression of (extra-)embryonic lineage regulators in blastocysts. In contrast, a paternal low-protein high-sugar diet associates with subtle preimplantation growth effects. We further identify gene expression variability as a paternally induced F1 phenotype, and highlight confounding issues for IEI, such as batch effects and under-sampling. Finally, while genetic background dominantly modifies the inherited signature of paternal environment, aged fathers universally impact F1 expression programmes across genetic contexts. This study systematically characterises how paternal conditioning programs subtle but detectable molecular responses in early embryos, and proposes guiding principles to dissect intergenerational phenomenology.