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Problem: In the cell column of anchoring villi, the cytotrophoblast differentiates into extravillous trophoblast (EVT) and invades the endometrium in contact with maternal immune cells. Recently, chemokines were proposed to regulate the decidual immune response. To investigate the roles of chemokines around the anchoring villi, we examined the expression profiles of chemokines in the first‐trimester trophoblast‐derived Swan71 cells using a three‐dimensional culture model. Method of Study: The gene expressions in the spheroid‐formed Swan71 cells were examined by microarray and qPCR analyses. The protein expressions were examined by immunochemical staining. The chemoattractant effects of spheroid‐formed Swan71 cells were examined by migration assay using monocyte‐derived THP‐1 cells. Results: The expressions of an EVT marker, laeverin, and matrix metalloproteases, MMP2 and MMP9, were increased in the spheroid‐cultured Swan71 cells. Microarray and qPCR analysis revealed that mRNA expressions of various chemokines, CCL2, CCL7, CCL20, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, and CXCL10, in the spheroid‐cultured Swan71 cells were up‐regulated as compared with those in the monolayer‐cultured Swan71 cells. These expressions were significantly suppressed by hypoxia. Migration assay showed that culture media derived from the spheroid‐formed Swan71 cells promoted THP‐1 cell migration. Conclusion: This study indicated that chemokine expressions in Swan71 cells increase under a spheroid‐forming culture and the culture media have chemoattractant effects. Since three‐dimensional cell assembling in the spheroid resembles the structure of the cell column, this study also suggests that chemokines play important roles in the interaction between EVT and immune cells in their early differentiation stage. [ABSTRACT FROM AUTHOR]

Purpose: Extravillous trophoblasts (EVTs) invade the endometrium to establish a fetomaternal interaction during pregnancy. Epidermal growth factor (EGF) and heparin‐binding EGF‐like growth factor (HB‐EGF) stimulate EVT invasion by binding to the EGF receptor (EGFR). We examined the role of the small GTP‐binding protein Rap1 in EGF‐ and HB‐EGF‐stimulated EVT invasion. Methods: Expression of Rap1 in the first‐trimester placenta was examined by immunohistochemistry. Effect of EGF or HB‐EGF on Rap1 activation (GTP‐Rap1) and Rap1 knockdown on invasion was assessed in EVT cell line (HTR‐8/SVneo). In addition, effect of Rap1 knockdown and Rap1GAP (a Rap1 inactivator) overexpression on the activation of EGF signaling and EGFR expression were examined. Results: Rap1 was expressed by EVTs, villous cytotrophoblasts, and syncytiotrophoblasts in the placenta. EGF and HB‐EGF activated Rap1 and promoted invasion of HTR‐8/SVneo, and these effects were inhibited by Rap1 knockdown. The EGF‐ and HB‐EGF‐induced phosphorylation of AKT, ERK1/2, p38MAPK, and Src was inhibited by Rap1 knockdown. Furthermore, the knockdown of Rap1 reduced the EGFR protein level. Overexpression of Rap1GAP repressed EGF‐ and HB‐EGF‐induced Rap1 activation and reduced EGFR expression. Conclusion: Rap1 may function as a mediator of EGF and HB‐EGF signaling pathways and can modulate EGFR expression in EVTs during placental development. [ABSTRACT FROM AUTHOR]

Preeclampsia is one of the most severe obstetric complications, yet its pathogenesis remains unclear. Decidual natural killer (dNK) cells, the most abundant immune cells at the maternal-fetal interface, are closely associated with preeclampsia due to abnormalities in their quantity, phenotype, and function. This review summarizes the molecular mechanisms by which dNK cells regulate extravillous trophoblast (EVT) invasion, promote uterine spiral artery remodeling, and maintain immune tolerance. Furthermore, it explores how disruptions in these mechanisms and changes in the decidual microenvironment alter dNK cell properties, driving the progression of preeclampsia. Understanding the mechanisms of dNK cells and identifying potential therapeutic targets may provide new insights for clinical intervention., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Trophoblast cell differentiation is of paramount importance for successful pregnancy. Krüppel-like factor 6 (KLF6), a transcription factor with diverse roles in cell physiology and tumor biology, is required for trophoblast differentiation through the syncytial pathway. Herein, we demonstrate that extravillous trophoblast (EVT) cell migration and mesenchymal phenotype are increased upon KLF6 downregulation or the expression of a deletion mutant lacking its transcriptional regulatory domain (KDac). Raman spectroscopy revealed molecular modifications compatible with increased differentiation in cells stably expressing the KDac mutant. Moreover, abnormally invasive placenta showed lower KLF6 immunostaining compared with the normal placenta. Thus, impaired KLF6 expression or function stimulates EVT migration and differentiation in vitro and may contribute to the physiopathology of the abnormally invasive placenta. [ABSTRACT FROM AUTHOR]

Aim: To determine which genes are important in placenta by network analysis. Methods: Placenta expressing genes were screened from RNA‐Seq data. Protein–protein interaction data were downloaded from STRING (v11.0) database. Google PageRank (PR) algorithm was used to identify important placental genes from protein interaction network. Six placental disease‐related datasets were downloaded from NCBI GEO database, and the differential expression of the 99 genes was identified. Results: We calculated PR for each placenta expressing gene and defined the top 99 genes with high PR as important genes. GAPDH has the highest PR. The 99 genes had different expression pattern in placental cell types. FN1 is up‐regulated in 8 w EVT compared to 8 w CTB and 24 w EVT compared to 8 w EVT. HSPA4 is down‐regulated in 8 w EVT compared to 8 w CTB and 24 w EVT compared to 8 w EVT. MIB2, TLR4, and UBB are consistently changed in preeclampsia (PE). UBB and ACTG1 were identified to be down‐regulated in fetal growth restriction (FGR). SOD1 is down‐regulated in preterm birth placenta. Conclusion: Our findings confirmed that the importance of these genes in placenta‐related diseases, and provide new candidates (MIB2, UBB, ACTG1, and SOD1) for placenta‐related disease diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Purpose: Extracellular matrix remodeling is essential for extravillous trophoblast (EVT) cell migration and invasion during placental development and regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteases (TIMPs). Sphingosine kinases (SPHK1 and SPHK2) synthesize sphingosine‐1‐phosphate (S1P), which works either intracellularly or extracellularly via its receptors S1PR1‐5 in an autocrine or paracrine manner. The role of SPHKs/S1P in regulating the expression of MMPs and TIMPs in EVT is mostly unknown and forms the primary objective of the study. Methods: HTR‐8/SVneo cells were used as a model of EVT. To inhibit the expression of SPHKs, cells were treated with specific inhibitors, SK1‐I and SKI‐II, or gene‐specific siRNAs. The expressions of MMPs and TIMPs were estimated by qPCR. Results: We demonstrated that SPHK1, MMP1‐3, and TIMP1‐3 were highly expressed in HTR‐8/SVneo cells. We found that treatment of cells with SK1‐I, SKI‐II, and knockdown of SPHK1 or SPHK2 increased the expression of MMP1, MMP3, and TIMP3. The addition of extracellular S1P inhibits the upregulation of MMPs and TIMPs in treated cells. Conclusions: SPHKs negatively regulate the expression of MMP1, MMP3, and TIMP3. The level of intracellular S1P acts as a negative feedback switch for MMP1, MMP3, and TIMP3 expression in EVT cells. [ABSTRACT FROM AUTHOR]

An immune reaction is a protector of our body but a target to be overcome for all non-self-derived medicine. Extracellular Vesicles (EVs), noted as a primary alternative to cell therapy products that exhibit immune rejection due to mismatching-major histocompatibility complex (MHC), were discovered to have excellent curative effects through the delivery of various biologically active substances. Although EVs are sure to incur immune reaction by immunogenicity due to alloantigens from their parental cells, their immune rejection is rarely known. Hence, to develop cell lines and EVs as medicines with no immune rejection, we noted the immune tolerance where the foetus, as semi-allograft, is perfectly protected from the maternal immune system. We designed the ex-vivo culture systems to simulate in-vivo environmental factors inducing extravillous trophoblast (EVT)-specific Human Leukocyte Antigen-G (HLA-G) expression and secretion of HLA-G-bearing EVs at the mother-foetus interface. Using them, we confirmed that immune-tolerized stem cells (itSCs) continuously expressing and secreting HLA-G like EVTs during pregnancy can be induced. Also, EVs secreted from itSCs are verified as immune-tolerized EVs (itSC-EVs) containing HLA-G and not causing immune rejection through various analytical methods. These findings can provide a new perspective on the local and extensive immune tolerance environment where HLA-G is expressed and secreted by pregnancy-related hormones and different biological conditions. Furthermore, they show the new way to develop itSCs-EVs-based therapeutics that are free from time, space, and donor limitation causing immune rejection. CFSE: carboxyfluorescein succinimidyl ester; DC: dendritic cells; ELISA: enzyme-linked immunosorbent assay; EV: extracellular vesicles; EVT: extravillous trophoblast; FSH: follicle stimulating hormone; HA: hyaluronic acid; hCG: human chorionic gonadotropin; HLA-G: human leukocyte antigen G; iPSC: induced pluripotent stem cells; itSC-EVs: immune-tolerized extracellular vesicles from itSCs; itTBC-EVs: immune-tolerized extracellular vesicles from itTBCs; itSCs: immune tolerized stem cells; itTBCs: immune-tolerized trophoblast cells; LH: luteinizing hormone; MHC: major histocompatibility complex; MSC: mesenchymal stem cells; NK: natural killer cells; NTA: nanoparticle tracking analysis; PBMC: peripheral blood mononuclear cells; PHA: phytohemagglutinin; SP-IRIS: single particle interferometric reflectance imaging sensing; STB: syncytiotrophoblast [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV) is one of the most common intrauterine infection virus, which can cause intrauterine transmission through the placenta, resulting in abortion, stillbirth and congenital malformations. In this study, the co‐culture extravillous trophoblast (EVT) HTR8/SVneo cell model of CMV infection was established in vitro. The toxicity of CMV infected EVT was determined, and then, the cell invasion experiment was conducted to evaluate the effect on the invasion ability of EVT cell lines. Western blot and real‐time PCR were used to detect the related cytokines in the PI3K/AKT signalling pathway in cells. Flow cytometry was used to detect the immune function related factors of the supernatant of CMV culture on decidual NK cells. The TCID50 of CMV virus was 10−5.4. The results of immunofluorescence showed that a large number of fluorescent green of CMV pp65 antigen signals appeared in the cytoplasm of CMV infection group. CMV could infect and replicate EVT cells and inhibited cell proliferation. The expression of proteins PDK1, AKT‐S473 and AKT‐S308 was significantly increased in CMV infection group. The levels of IL‐17, IL‐4 and IFN‐γ were 8.7 ± 0.48%, 12.17 ± 0.61% and 6.66 ± 0.25%, respectively, in CMV infection group. The above results indicated that CMV infection inhibited EVT cells proliferation, weakened the invasion ability and inhibited the immune function of NK cells at the maternal‐fetal interface, resulting in the abnormal maternal‐fetal crosstalk. [ABSTRACT FROM AUTHOR]

Perturbations to extravillous trophoblast (EVT) cell migration and invasion are associated with the development of placenta-mediated diseases. Phytochemicals found in the lowbush blueberry plant (Vaccinium angustifolium) have been shown to influence cell migration and invasion in models of tumorigenesis and noncancerous, healthy cells, however never in EVT cells. We hypothesized that the phenolic compounds present in V. angustifolium leaf extract promote trophoblast migration and invasion. Using the HTR-8/SVneo human EVT cell line and Boyden chamber assays, the influence of V. angustifolium leaf extract (0 to 2 × 104  ng/ml) on trophoblast cell migration (n = 4) and invasion (n = 4) was determined. Cellular proliferation and viability were assessed using immunoreactivity to Ki67 (n = 3) and trypan blue exclusion assays (n = 3), respectively. At 20 ng/ml, V. angustifolium leaf extract increased HTR-8/SVneo cell migration and invasion (p < .01) and did not affect cell proliferation or viability. Chlorogenic acid was identified as a major phenolic compound of the leaf extract and the most active compound. Evidence from Western blot analysis (n = 3) suggests that the effects of the leaf extract and chlorogenic acid on trophoblast migration and invasion are mediated through an adenosine monophosphate-activated protein (AMP) kinase-dependent mechanism. Further investigations examining the potential therapeutic applications of this natural health product extract and its major chemical compounds in the context of placenta-mediated diseases are warranted. [ABSTRACT FROM AUTHOR]

Autophagy is a well-conserved mechanism in cells from yeast to mammals, and autophagy maintains homeostasis against stress. The role of autophagy was originally shown to be a mechanism of energy production under starvation. In fact, multiple lines of evidence reveal that autophagy has numerous functions, such as protection from stress, energy regulation, immune regulation, differentiation, proliferation, and cell death. In the field of reproduction, the role of autophagy in implantation, embryogenesis, placentation, and delivery has become clearer. In addition, recent study has elucidated that the placenta has the ability to protect extraplacental cells from virus infection by activating autophagy. During resent research into autophagy, several issues have occurred in the interpretation of the autophagy status. In this review, we discuss the relation between autophagy and reproductive events, and show the importance of autophagy for placentation and pre-eclampsia. [ABSTRACT FROM AUTHOR]

The process of uterine spiral artery remodeling in the first trimester of human pregnancy is an essential part of establishing adequate blood perfusion of the placenta that will allow optimal nutrient/waste exchange to meet fetal demands during later development. Key regulators of spiral artery remodeling are the uterine natural killer cells and the invasive extravillous trophoblasts. The functions of these cells as well as regulation of their activation states and temporal regulation of their localization within the uterine tissue are beginning to be known. In this review, we discuss the roles of these two cell lineages in arterial remodeling events, their interaction/influence on one another and the outcomes of altered temporal, and spatial regulation of these cells in pregnancy complications. [ABSTRACT FROM AUTHOR]

Regulatory T cells ( Tregs) have been identified as immunomodulatory cells, which induce tolerance. Evidence from numerous recent studies implicates these cells as critical for maternal tolerance to the fetal-derived placenta, proper functioning of which is required for normal gestation and growth and development of the fetus in utero. This review focuses on the recent studies of Tregs at the maternal-fetal interface in pregnancy complications, as well as their identification in specific inflammatory lesions in the placenta. [ABSTRACT FROM AUTHOR]

Remodeling of maternal spiral arteries by invasion of extravillous trophoblast (EVT) is crucial for an adequate blood supply to the fetus. EVT cells that migrate through the decidual tissue destroy the arterial muscular lining from the outside (interstitial invasion), and those that migrate along the arterial lumen displace the endothelium from the inside (endovascular invasion). Numerous factors including cytokines/growth factors, chemokines, cell adhesion molecules, extracellular matrix-degrading enzymes, and environmental oxygen have been proposed to stimulate or inhibit the differentiation/invasion of EVT. Nevertheless, it is still difficult to depict overall pictures of the mechanism controlling perivascular and endovascular invasion. Potential factors that direct interstitial trophoblast towards maternal spiral artery are relatively high oxygen tension in the spiral artery, maternal platelets, vascular smooth muscle cells, and Eph/ephrin system. On the other hand, very little is understood about endovascular invasion except for the involvement of endothelial apoptosis in this process. Only small numbers of molecules such as polysialylated neural cell adhesion molecules and CCR1 have been suggested as specific markers for the endovascular trophoblast. Therefore, an initial step to approach the mechanisms for endovascular invasion could be more detailed molecular characterization of the endovascular trophoblast. [ABSTRACT FROM AUTHOR]

Toth B, Haufe T, Scholz C, Kuhn C, Friese K, Karamouti M, Makrigiannakis A, Jeschke U. Placental Interleukin-15 expression in recurrent miscarriage. Am J Reprod Immunol 2010; 64: 402-410 Cytokines play a fundamental role at the feto-maternal interface. A dysregulation of the cytokine profile could be linked with fetal rejection. This study investigated differences in the expression of the Th1 cytokine interleukin-15 (IL-15) of normal and disturbed pregnancy. Analysis of IL-15 expression by immunohistochemistry and real-time RT-PCR in placental tissue from normal pregnancies, spontaneous miscarriage and recurrent miscarriage (RM) was performed. Expression of IL-15 was upregulated on mRNA and protein level in the placenta of miscarriage patients, especially in patients with RM. Immunohistochemistry and immunofluorescence double staining revealed extravillous trophoblast cells (EVT) as IL-15-expressing cells. Staining results were confirmed by real-time PCR (TaqMan). Increased expression of IL-15 in the decidua of patients with RM may be linked to disturbed implantation and vascularization with consecutive placental and fetal rejection. [ABSTRACT FROM AUTHOR]

At the time of blastocyst implantation the uterine spiral arteries have already undergone morphological changes in the absence of any extravillous trophoblast invasion. Only 2 weeks after implantation, extravillous trophoblast cells develop and come into first contact with decidual tissues. Invading through the decidual interstitium, extravillous trophoblasts potentially reach and transform spiral arteries into uteroplacental arteries. Spiral arterial erosion starts at about mid-first trimester, whereas flow of maternal blood into the intervillous space is continuously established only at the beginning of the second trimester. One key regulator of the number of extravillous trophoblasts is oxygen. The steep gradient in oxygen concentration within the first trimester placenta is diminished with the onset of maternal blood flow. This gradient is used by the trophoblast to generate a large number of invasive cells to adapt the arterial vasculature in the placental bed to the growing needs of the fetus. Changes in oxygen concentrations or other factors leading to alterations in the rates of proliferation and/or apoptosis of extravillous trophoblast clearly impact on the remodelling of the vessels. The respective consequences of a failure in trophoblast invasion are growth restrictions of the baby and perhaps other pregnancy complications. [ABSTRACT FROM AUTHOR]

Human extravillous trophoblasts (EVT) invade maternal deciduas and reconstructed maternal spiral arteries during early placentation. However, the precise regulatory mechanisms to induce EVT invasion toward arteries and/or to protect EVT from further invasion have not been well understood. Recently, it was found that EVT that had already ceased their invasion, specifically expressed cluster of differentiation (CD9) and dipeptidyl peptidase IV (DPPIV) on their cell surface. In addition, EVT migrating to maternal spiral arteries expressed CC chemokine receptor type-1 (CCR-1), which is a chemokine receptor for regulated on activation normal T cell expressed and secreted (RANTES) and so on. CD9 is associated with integrin molecules on the cell surface and is considered to modulate integrin function. In contrast, DPPIV is a cell surface peptidase that can metabolize RANTES at extracellular sites before its accessing to the chemokine receptors. In vitro functional assay showed that CD9, DPPIV and RANTES are involved in the regulation for EVT invasion. From these findings, it can be proposed that CD9 and DPPIV, including chemokines, are new regulatory factors for human extravillous trophoblasts. (Reprod Med Biol 2005; 4: 189–195) [ABSTRACT FROM AUTHOR]

Problem: Dysregulation of extravillous trophoblast (EVT) invasion leads to pregnancy complications, such as pre‐eclampsia, fetal growth restriction, and placenta accreta. The aim of this study was to explore the role of SIRT1 in EVT invasion and its underlying mechanism. Method of study: SIRT1‐specific siRNA was transfected into Swan 71 cells, an immortalized first trimester trophoblast cell line. The Boyden chamber invasion assay, the scratch wound healing assay, and cell proliferation assay were performed. The expression levels of epithelial‐to‐mesenchymal transition (EMT) markers, matrix metalloproteinase‐2 (MMP‐2), MMP‐9, p‐Akt, Akt, p‐p38MAPK, p38MAPK, p‐ERK, ERK, p‐JNK, JNK, Fas, and Fas ligand (FasL) were examined by western blot. Tube formation assay was conducted by using Matrigel. Results: SIRT1 knockdown by siRNA significantly enhanced invasion and migration as well as the expression of MMP‐2, MMP‐9, and EMT markers in Swan 71 cells, but reduced proliferation. The effects of SIRT1 knockdown on invasion, migration, proliferation, and endothelial‐like tube formation in Swan 71 cells were reversely regulated by blockade of Akt and p38MAPK signaling. In addition, SIRT1 knockdown markedly promoted colocalization of Swan 71 cells to human umbilical vein endothelial cell (HUVEC) networks and induced reduction in Fas and enhancement of FasL. Conditioned media of SIRT1 knockdown‐Swan 71 cells caused reduction in cell proliferation and augmentation of cytotoxicity along with increased Fas expression in HUVECs. Conclusion: Our results suggest that SIRT1 may be associated with placental development by controlling EVT invasion and spiral artery remodeling via modulation of EMT, MMP‐2, MMP‐9, Akt/p38MAPK signaling, and Fas/FasL. [ABSTRACT FROM AUTHOR]

Problem: During pregnancy, the decidual spiral arterioles (SpAs) that supply maternal blood to the placenta undergo a series of changes to optimise the transfer of nutrients and oxygen to the developing foetus. Recent studies have shown that initiation of SpA transformation coincides with decidual leucocyte infiltration. Leucocytes are known to be a source of matrix metalloproteinases (MMPs); however, the complete profile of MMPs expressed by decidual NK cells (dNK) and macrophages has not been characterised. We hypothesised that leucocyte‐derived MMPs contribute to SpA remodelling. Methods: Decidual NK cells and macrophages were isolated from first trimester decidua and their MMP repertoire profiled by qRT‐PCR (n = 10; 5‐11 weeks). Dual immunofluorescence was used to localise MMP expression in situ (n = 3; 5‐12 weeks). Gelatin zymography was carried out to assess whether leucocyte‐derived MMPs can degrade ECM. In situ zymography and immunofluorescence identified MMP activity in tissue‐resident dNK and macrophages. Results: Decidual NK cells cells and macrophages expressed MMP2, ‐7, ‐9, ‐11, ‐16, ‐19 and tissue inhibitors of metalloproteinase‐1, ‐2, and ‐3. Both cell types degraded gelatin using MMP2 and MMP9 and broke down collagen in an in vitro model of the SpA. Extravillous trophoblasts (EVTs) expressed a similar repertoire of MMPs. Conclusion: We suggest that matrix remodelling in SpA is initiated by infiltrating leucocytes, while EVTs become involved at later stages. [ABSTRACT FROM AUTHOR]