Single-cell and spatial transcriptomics identify SPP1 + trophoblast necroptosis as a potential contributor to...

Late-onset preeclampsia (LOPE) is a complex placental disorder characterized by inflammation and placental dysfunction, yet the underlying molecular mechanisms remain incompletely understood. Necroptosis, a regulated inflammatory form of cell death, has been implicated in various inflammation-associated diseases, but its role in LOPE has not been fully elucidated.

We performed an integrated analysis of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) in placental tissues from LOPE and healthy pregnancies. Necroptosis activity was evaluated using gene-set scoring and validated by Western blotting and immunohistochemistry in human placentas. Pseudotime trajectory analysis, CellChat, and enrichment analysis were applied to characterize pathological cellular states and intercellular communication. Spatial deconvolution using RCTD was employed to map the localization and colocalization of specific cellular subtypes within placental niches. Functional validation was conducted in vivo using an L-NAME-induced preeclampsia-like mouse model in Mlkl knockout mice, together with multicolor immunofluorescence staining of human placental samples.

Single-cell transcriptomic profiling revealed marked cellular heterogeneity between LOPE and healthy placentas and identified activation of necroptosis-related pathways in trophoblast populations in LOPE. A distinct SPP1 + extravillous trophoblast (EVT) subtype associated with necroptosis signatures was identified. Trajectory analysis indicated that this SPP1 + EVT subset represented a pathological state enriched for cell-death and inflammatory signatures. Spatial transcriptomics demonstrated specific localization of SPP1 + EVTs within the placental microenvironment and their spatial proximity to macrophages. Cell-cell communication analysis further highlighted a prominent SPP1-CD44 signaling axis mediating interactions between SPP1 + EVTs and macrophages. In vivo, Mlkl knockout attenuated L-NAME-induced preeclampsia-like phenotypes and reduced placental SPP1 expression. Consistently, multicolor immunofluorescence confirmed increased abundance of SPP1 + EVTs and spatially adjacent macrophages in LOPE placentas.

Necroptosis is a predominant trophoblast cell death pathway in LOPE. The SPP1-CD44 axis may mediate EVT-macrophage crosstalk, contributing to placental inflammation and dysfunction. These findings provide mechanistic insight into LOPE pathogenesis, nominate necroptosis as a potential therapeutic target, and highlight SPP1 as a promising biomarker candidate.