Protein N-glycosylation supports extravillous trophoblast cell lineage development in hypoxia microenvironment

Extravillous trophoblasts (EVT) are essential for placental invasion into the maternal decidua and spiral artery remodeling, ensuring a successful pregnancy. It is well-established that early placental development occurs under physiologically hypoxic conditions (~2% O₂), while the later stages proceed under increased physiological oxygen levels (~8% O₂). However, the heterogeneity of EVT across these distinct developmental conditions remains poorly characterized. Moreover, the molecular mechanisms governing EVT lineage development and function under low oxygen conditions remain largely elusive. Herein, we systematically characterize the transcriptome dynamics underlying the differentiation of cytotrophoblasts (VCT) and human trophoblast stem cells (hTSC) into EVT at single-cell resolution. Our analysis reveals pronounced functional and phenotypic heterogeneity among EVT from early versus late gestational stages, as well as between in vivo and in vitro models. We demonstrate that oxygen tension is a pivotal factor driving EVT heterogeneity, giving rise to distinct subtypes with invasive/migratory and secretory potentials. Furthermore, we elucidate that hypoxia impairs EVT lineage development from hTSC in vitro. Hypoxia reduces intracellular UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) level, thereby possibly disrupting protein N-glycosylation. We further demonstrate that N-glycosylation pathway activity is significantly elevated during the differentiation of hTSC into EVT, as pharmacological inhibition with tunicamycin (TM) completely blocks this process. UDP-GlcNAc supplementation rescues the EVT differentiation defect under hypoxia, demonstrating that protein N-glycosylation is essential for EVT lineage commitment under low oxygen conditions. Our findings uncover a hypoxia-sensitive glycosylation in EVT development and provide new insights into early placental development.