Single-cell Spatial Landscape Reconstruction Of Human Extravillous Trophoblast Invasion Trajectory In Early Pregnancy

Objectives:The maternal-fetal interface is mainly composed of the placenta and decidua,and plays a vital role in successful reproduction.Defective placentation and abnormal maternal-fetal microenvironment underpin common pregnancy disorders.Extravillous trophoblasts(EVTs)derive from placenta villi and invade through the decidua and superficial myometrium of the uterus,which is essential to the maternal arterial remodeling and placenta development.The objective of this study is to dissect the regulatory network accompanies early process of placentation and determine the role of maternal-fetal interface in EVT invasive behavior.Methods:In this study,a total of 4 normal pregnancies at 6+0～6+6 gestational weeks and 3 cases of invasive hydatidiform mole(HHM)were included,and samples of chorionic villi and decidua were collected,respectively.In the first part of the study,single-cell RNA sequencing was used to generate the cell atlas of the maternal-fetal interface,which was further validated by identifying the cell origin via integrating the single-nucleotide polymorphism information.EVT differentiation trajectory was then reconstructed using the RNA splicing velocity.Furthermore,differences in gene expression and KEGG pathway enrichment between normal pregnancies and IHM cases were analyzed to identify signature genes,imprinting genes,and differentiation-driving genes closely related to EVT invasive behavior.In the second part of the study,spatial transcriptome sequencing was used to construct the spatial transcriptome landscape of the maternal-fetal interface,which was further validated by high-resolution deconvolution via integrating the singlecell atlas.The co-localization of EVT with specific cells was analyzed using non-negative matrix factorization,and the maternal-fetal communication network was then reconstructed using CellPhoneDB.Key cells and signaling pathways involved in the regulation of EVT invasion were then verified by means of cell to cell interaction analysis.Results:1.In early pregnancy,the chorionic villi is mainly composed of EVT,villous cytotrophoblasts(VCT),syncytiotrophoblasts(SCT),fetal fibroblasts(fFB),Hofbauer cells(HB),and fetal vascular endothelial cells[Endo(f)].The decidua mainly contains decidual stromal cells(dS),endometrial glandular epithelial cells(Epi),perivascular cells(dP),decidual natural killer cells(dNK),T lymphocytes(Tcells),dendritic cells(DC1),decidual macrophages(dM),and maternal vascular endothelial cells[Endo(m)].Chorionic villi and decidua share certain cell types including VCT,SCT,dNK,Tcells,dM,and Endo(m).2.EVT signature genes are enriched in KEGG pathways related to cell proliferation and differentiation,cell adhesion,migration,directed growth,and tumor microenvironment.In comparison with normal pregnancy,the expression of proto-oncogenes and tumor-invasion genes in IHM-EVT is significantly upregulated.3.There is a remarkable difference in the genomic imprinting of EVT between normal pregnancies and IHM cases.The former exhibits an upregulation of maternal allele expression,mainly playing a role in inhibiting tumor proliferation,while the latter shows an upregulation of paternal allele expression,mainly promoting tumor development.4.During the differentiation,EVT acquires tumor-like biological characteristics,including cell migration and invasion ability,while exhibits self-protective mechanisms including anti-inflammatory and anti-phagocytic capabilities.In contrast,IHM-EVT significantly enhances the proliferative and invasive abilities during its differentiation process,which may be related to the disturbed expression of differentiation-driving genes.5.In the chorionic villi,EVT co-localizes with fFB and VCT1,while in the decidua,EVT shows co-localization with dS3,Endo(m),Endo L,dP2,DC 1,dMl,dM3,Epi1,Epi2,dCD8,dNK1,and dNK2.6.fFB in chorionic villi promotes the proliferation and invasion of EVT through AREBEGFR and other pathways,while decidua plays a bidirectional regulating role in EVT proliferation and invasion through the TGFB,WNT and NOTCH-related pathways.7.The antigen-presenting system centered on DC1 activates the maternal-fetal interface immune response.The microenvironment of the maternal-fetal interface has two mechanisms in regulating EVT invasion:negative regulation and immune tolerance.8.EVT may establish a self-protective mechanism by interacting with immune cells through the immune checkpoint pathway,inhibiting innate and adaptive immune responses,while inducing immune tolerance.Conclusions:The invasive behavior of EVT involves multiple pathways,including cell cycle,cytoskeleton,cell migration,and immune response.The delicate balance between maternal and paternal imprinting genes is crucial for the normal function maintenance of EVT.EVT acquires tumor-like biological characteristics during differentiation,and the disruption of driving gene expression may be involved in abnormal EVT invasion.EVT interacts with decidual cells along its invasion trajectory,regulating its own and target cell growth.The antigen-presenting system centered on DC1 activates the immune response at the maternal-fetal interface and participates in the negative regulation and immune tolerance of EVT invasion.EVT acts on immune cells through the immune checkpoint pathways,inhibiting immune responses and establishing self-protection mechanisms.These findings indicate that the invasive behavior of EVT in early human pregnancy is regulated by the intrinsic epigenetic control and the external maternal-fetal interface microenvironment.Our findings provide a new perspective for the etiological research and early prediction of placenta-related pathological pregnancies.