Background: Obstetric antiphospholipid syndrome (OAPS) is a subset of APS manifestated as placenta-derived complications, such as pre-eclampsia, fetal growth restriction (FGR) and premature delivery before 34 weeks of gestation, but the pathogenesis was not uncovered completely. Adequate trophoblast differentiations from cytotrophoblast (CTB) to extra-villous trophoblast (EVT) and syncytiotrophoblast (SCT) are essential for placental formation and function. Although previous in-vitro and in-vivo studies confirmed the adverse effects of antiphospholipid antibodies (aPLs) on EVT proliferation, invasion and migration, the differentiational status of trophoblast and underlying mechanisms in OAPS was not identified.

Objectives: This study was to explore the differentiational status of placental trophoblast in OAPS patients at single-cell and spatial resolution, and to investigate the potential mechanisms of trophoblast differentiational disorders in OAPS.

Methods: Seven decidua basalis and placental villous samples from OAPS patients with severe pre-eclampsia and/or FGR, 5 samples from OAPS patients without any pregnancy complications after effective treatments and 3 samples from healthy controls (HCs) were collected for cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and spatial transcriptomics (ST). Pseudo-time analysis and RNA velocity analysis were used for analyzing the potential differentiational states of identified trophoblast subtypes. Differentially expressed genes (DEGs) and enrichment analysis were used for describing the functional differences of trophoblast. CellPhone DB was used to explore the intercellular communications of placental cells. Immunofluorescence (IF) and trophoblast organoids were used to validate the findings of CITE-seq and ST.

Results: An integrated single-cell multi-omic atlas, and a spatial transcriptomic map, of placental villous and decidua basalis for OAPS patients and HCs was drawn. By using CITE-seq, three clusters of CTB (CTB, CTB proliferation and CTB fusion), three types of STB (SCT nascent, SCT pre-mature and STB mature) and two types of EVT (intestinal EVT and vascular EVT) were identified, as well as various stromal cells, fibroblasts, myeloid cells, NK cells and T cells clusters were defined. We found two main differentiational trajectories of trophoblasts, the one is from CTB fusion, SCT nascent, SCT pre-mature to SCT mature, and the other is from CTB to intestinal EVT and vascular EVT. In OAPS patients with severe pre-eclampsia and/or FGR, the EVT differentiation were inhibited, and STB differentiation were enhanced compared to OAPS patients received effective treatments and HCs. The significant DEGs in trophoblasts from OAPS patients with severe pre-eclampsia and/or FGR were significantly enriched in the terms of wound healing, response to hypoxia and inflammation and multi-multicellular organism process, and the significant DEGs in which from OAPS received effective treatments and HCs were enriched in the terms of cell invasion, cell migration and blood vessels formation. The core transcriptomic factors (TFs) of EVT differentiation (DLX5, DLX6, ZNF 439) were reduced, and TFs of trophoblast fusion (GATA3, GATA2, TFAP2A) were increased in trophoblasts of OAPS patients with severe pre-eclampsia and/or FGR. Enhanced intercellular communications between CCR2 ⁺ macrophages, CD16 ^bright natural killer (NK) cells, cytotoxic T cells and EVT via TNF-TLRs and other inflammatory cytokines ligand-receptor pairs were identified by CellPhone DB, and the closer sites between EVTs and these above pro-inflammatory immune cells were confirmed by ST and IF, in OAPS patients with severe pre-eclampsia and/or FGR. By EVT differentiation organoids and co-culture system, we validated that primary decidual CCR2 ⁺ macrophages, CD16 ^bright NK cells and cytotoxic T cells also inhibited EVT differentiations in vitro. However, we did not observe the direct effect of the above-mentioned immune cells on trophoblast fusion. The enhanced STB differentiation in OAPS patients may be related to the compensation caused by hypoxia.

Conclusions: There was significant but different abnormality in trophoblast differentiation from CTB to EVT or from CTB to STB, among placenta of OAPS patients with severe pre-eclampsia and/or FGR compared to OAPS patients received effective treatments and HCs. Increased pro-inflammatory immune cells (etc. CCR2 ⁺ macrophages, CD16 ^bright NK cells and cytotoxic T cells) and cytokines may be triggers for inhibited EVT differentiation in OAPS, but enhanced STB differentiation may be the compensation for hypoxia. Promoting trophoblast differentiation and inhibiting inflammatory effects may be a new strategy for treating OAPS in the future.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.