研究目的：母胎界面主要由胎盘与蜕膜构成，在成功生殖中扮演关键角色，其形成缺陷与多种病理妊娠密切相关。绒毛外滋养细胞（extravillous trophoblast，EVT）通过入侵蜕膜及子宫肌层，参与血管重铸，在胎盘形成与发育中发挥重要作用。本研究使用单细胞RNA测序，绘制人类妊娠早期母胎界面细胞图谱，寻找与EVT侵袭行为相关的驱动基因以及印记基因，然后借助空间转录组平台，对母胎界面细胞图谱进行验证，同时构建母胎微环境细胞通讯网络，寻找与EVT侵袭功能相关的关键细胞及信号转导通路，揭示母胎界面微环境对EVT侵袭行为的调控机制。

方法：本研究一共纳入4例6⁺⁰ ~ 6⁺⁶周正常妊娠，以及3例侵袭性葡萄胎病例（invasive hydatidiform mole，IHM），分别采集绒毛及底蜕膜样本。研究第一部分使用单细胞RNA测序绘制母胎界面单细胞转录组图谱，引入单核苷酸多态性信息鉴定细胞母胎来源，解析母胎界面细胞构成，然后使用RNA剪接速率重建EVT分化轨迹，通过比较正常妊娠与IHM组间基因表达差异，寻找与EVT侵袭行为相关的特征性基因、印记基因、以及分化驱动基因。研究第二部分使用空间转录组测序，绘制母胎界面空间转录组图谱并进行高分辨率去卷积，对单细胞RNA数据进行验证，通过非负矩阵分解观察EVT与特定细胞共定位现象，然后使用CellPhoneDB重建母胎界面通讯网络，最后借助空间近场通讯对目标受体-配体作用进行验证，鉴定参与EVT侵袭行为调控的关键细胞及信号转导通路。

结果：1.妊娠早期绒毛组织主要由EVT、细胞滋养细胞（VCT）、合体滋养细胞（SCT）、胎儿成纤维细胞（fFB）、霍夫鲍尔巨噬细胞（HB）、胎儿血管内皮细胞[Endo (f)]构成，蜕膜组织主要由蜕膜间质细胞（dS）、子宫内膜腺体上皮细胞（Epi）、血管周细胞（dP）、蜕膜自然杀伤细胞（dNK）、T淋巴细胞（Tcells）、树突状细胞（DC1）、蜕膜巨噬细胞（dM）、血管内皮细胞[Endo (m)]构成，两种组织共享VCT、SCT、dNK、Tcells、dM、Endo (m)等细胞类型。

2.EVT特征基因在细胞增殖与分化、细胞黏附、迁移、定向生长、肿瘤微环境等KEGG通路中出现富集。与正常妊娠相比，IHM-EVT原癌基因以及肿瘤侵袭基因表达出现显著上调。

3.正常妊娠EVT与IHM-EVT基因组印记存在显著差异，前者父源印记基因表达上调，主要发挥抑制肿瘤增殖作用，后者母源印记基因表达上调，主要发挥促进肿瘤发生作用。

4. EVT在分化过程中逐渐获得肿瘤生物学特性，包括细胞运动与侵袭能力，同时表现出抗炎与抗吞噬等自我保护机制，IHM-EVT在分化过程中增殖及侵袭能力显著增强，出现失控倾向，可能与分化驱动基因表达紊乱相关。

5.绒毛组织中，EVT与fFB、VCT1存在共定位现象，蜕膜组织中，EVT与dS3、Endo (m)、Endo L、dP2、DC1、dM1、dM3、Epi1、Epi2、dCD8、dNK1、dNK2存在共定位现象。

6.绒毛组织中fFB通过AREB-EGFR等通路促进EVT增殖与侵袭，蜕膜组织通过TGFB、WNT、NOCTH相关通路对EVT增殖与侵袭发挥以正向调节为主的双向调控作用。

7.以DC1为中心的抗原呈递系统激活母胎界面免疫应答，针对EVT侵袭行为，母胎界面免疫微环境同时存在负向调控与免疫耐受两种机制。

8. EVT可能通过免疫检查点通路作用于T淋巴细胞等免疫细胞，抑制针对自身的固有及获得性免疫应答，并介导免疫耐受，从而建立自我保护机制。

结论：EVT侵袭行为涉及细胞周期、细胞骨架、细胞迁移、免疫应答等多种功能通路，母源印记及父源印记的精确平衡对于维持EVT正常功能具有重要意义，EVT在分化过程中逐渐获得类肿瘤生物学特性，分化驱动基因紊乱与EVT侵袭行为异常相关。EVT在侵入母胎蜕膜的过程中，与蜕膜中多种实质细胞发生广泛的细胞间通讯，调节自身及靶细胞生长；以DC1为中心的抗原呈递系统激活母胎界面免疫应答，参与对EVT侵袭行为的负向调控与免疫耐受；EVT通过免疫检查点相关通路作用于T淋巴细胞等免疫细胞，抑制针对自身的免疫应答，建立自我保护机制。以上结果说明，在人类妊娠早期，EVT侵袭行为受内在表观遗传调控及外部母胎界面微环境的双重调节。

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 (IHM) 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 single-cell 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, DC1, dM1, dM3, Epi1, Epi2, dCD8, dNK1, and dNK2.

6. fFB in chorionic villi promotes the proliferation and invasion of EVT through AREB-EGFR 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.