The paternal preconception environment has been implicated as a modulator of phenotypic traits and disease risk in F1offspring. 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. Usingin vitrofertilisation (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 lineage regulators in blastocysts, particularly affecting extra-embryonic tissues. Conversely, paternal low-protein high-sugar diet associates with subtle preimplantation developmental delay. We further identify gene expression variability as a paternally-induced F1phenotype, and highlight confounding issues for IEI such as batch-effects and under-sampling. Finally, paternal genetic background and age exert a dominant influence over the inherited environmental signature. This study systematically characterises how paternal conditioning programmes subtle but detectable molecular responses in early embryos, and suggests guiding principles to dissect intergenerational phenomenology.
