Cross-species single-cell transcriptomics of placental development highlights conserved and divergent programs during gestation

Supporting healthy pregnancy outcomes requires a comprehensive understanding of the cellular hierarchy and underlying molecular mechanisms in the ruminant placenta during gestation. Here, we constructed a single-nucleus transcriptomic atlas of 304,726 placental nuclei spanning 13 developmental stages in cows, identifying 13 major cell types and 14 trophoblast subtypes. Bioinformatics analyses and multiple validation experiments suggested that placental trophoblast cells exhibited stage-specific differences across gestation, and identified HAND1and DLX5as novel key regulators of maternal recognition of pregnancy. We found that binucleate (BNC) cells arisefrom specific uninucleate (UNC) subpopulations at E24, with their differentiation trajectories co-regulated by imprinted genes and metabolic reprogramming. Integrated genome-wide association study (GWAS) analysis identified early trophoblast subtypes significantly associated with gestation length, along with key pathways and risk genes (e.g., CYCS, HMGA1, VDAC1), with these signals under strong evolutionary constraint. Furthermore, we found that placental macrophages originate at E30 in cow and are significantly associated with pregnancy loss in both cattle and humans, sharing common risk pathways. Collectively, this study provides a cross-stage cellular and molecular map of the ruminant placenta, delineates the key regulatory networks at the maternal–fetal interface, and offers a theoretical framework and potential molecular targets for understanding pregnancy maintenance and improving reproductive traits in ruminants.