Invasion Of Extravillous Trophoblast Cells Research Articles

Pigment epithelium‑derived factor inhibits proliferation, invasion and angiogenesis, and induces ferroptosis of extravillous trophoblasts by targeting Wnt‑β‑catenin/VEGF signaling in placenta accreta spectrum.

Placenta accreta spectrum (PAS) is one of the most dangerous complications in obstetrics, which can lead to severe postpartum bleeding and shock, and even necessitate uterine removal. The abnormal migration and invasion of extravillous trophoblast cells (EVTs) and enhanced neovascularization occurring in an uncontrolled manner in time and space are closely related to the abnormal expression of pro‑angiogenic and anti‑angiogenic factors. The pigment epithelium‑derived factor (PEDF) is a multifunctional regulatory factor that participates in several important biological processes and is recognized as the most efficient inhibitor of angiogenesis. The present study aimed to explore the effects of PEDF on EVT phenotypes and the underlying mechanisms in PAS. HTR‑8/SVneo cells were transfected to overexpress or knock down PEDF. Cell proliferation and invasion were assessed using Cell Counting Kit‑8, 5‑ethynyl‑2'‑deoxyuridine and Transwell assays. In vitro angiogenesis was analyzed using tube formation assays. The degree of ferroptosis was assessed by evaluating the levels of lipid reactive oxygen species, total iron, Fe2+, malondialdehyde and reduced glutathione using commercial kits. The expression levels of biomarkers of ferroptosis, angiogenesis, cell proliferation and Wnt signaling were examined by western blotting. PEDF overexpression decreased the proliferation, invasion and angiogenesis, and induced ferroptosis of EVTs. Activation of Wnt signaling with BML‑284 and overexpression of vascular endothelial growth factor (VEGF) reversed the PEDF overexpression‑induced suppression of cell proliferation, invasion and tube formation. PEDF overexpression‑induced ferroptosis was also decreased by Wnt agonist treatment and VEGF overexpression. It was predicted that PEDF suppressed the proliferation, invasion and angiogenesis, and increased ferroptosis in EVTs by decreasing Wnt‑β‑catenin/VEGF signaling. The findings of the present study suggested a novel regulatory mechanism of the phenotypes of EVTs and PAS.

Growth differentiation factor-11 upregulates matrix metalloproteinase 2 expression by inducing Snail in human extravillous trophoblast cells

The human extravillous trophoblast (EVT) cell invasion is an important process during placentation. Although the placenta is normal tissue, the EVT cells exhibit some features common to cancer cells, including high migratory and invasive properties. Snail and Slug are transcription factors that mediate the epithelial-mesenchymal transition (EMT), a crucial event for cancer cell migration and invasion. It has been shown that GDF-11-induced matrix metalloproteinase 2 (MMP2) expression is required for EVT cell invasion. Whether GDF-11 can regulate Snail and Slug expression in human EVT cells remains unknown. If it does, the involvement of Snail and Slug in GDF-11-induced MMP2 expression and EVT cell invasion must also be defined. In the present study, using the immortalized human EVT cell line, HTR-8/SVneo, and primary cultures of human EVT cells as experimental models, our results show that GDF-11 upregulates Snail and Slug expression. ALK4 and ALK5 mediate the stimulatory effects of GDF-11 on Snail and Slug expression. In addition, we demonstrate that SMAD2 and SMAD3 are required for the GDF-11-upregulated Snail expression, while only SMAD3 is involved in GDF-11-induced Slug expression. Moreover, our results reveal that Snail mediates GDF-11-induced MMP2 expression and cell invasion but not Slug. This study increases our understanding of the biological function of GDF-11 in human EVT cells and provides a novel mechanism for regulating MMP2 and EVT cell invasion.

3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages.

Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ("blastoids") that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate the defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick three-dimensional (3D) extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that the modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated invitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.

Decidual stromal cells promote invasiveness of extravillous trophoblast cells: a potential role for osteopontin

Decidual stromal cells promote invasiveness of extravillous trophoblast cells: a potential role for osteopontin

WNK1 mediates amphiregulin-induced MMP9 expression and cell invasion in human extravillous trophoblast cells

The invasion of human extravillous trophoblast (EVT) cells is a critical event required for a successful pregnancy. Amphiregulin, a ligand of the epidermal growth factor receptor (EGFR), has been shown to stimulate cell invasion in an immortalized human EVT cell line, HTR-8/SVneo. The with-no-lysine kinase 1 (WNK1) is involved in regulating cell invasion. It is known that WNK1 is expressed in the human placenta, but its role in human EVT cells remains unknown. In the present study, we show that AREG treatment phosphorylated WNK1 at Thr60 in both HTR-8/SVneo and primary human EVT cells. The stimulatory effect of AREG on WNK1 phosphorylation was mediated by the activation of PI3K/AKT, but not the ERK1/2 signaling pathway. AREG upregulated matrix metalloproteinase 9 (MMP9) but not MMP2. In addition, cell invasiveness was increased in response to the treatment of AREG. Using the siRNA-mediated knockdown approach, our results showed that the knockdown of WNK1 attenuated the AREG-induced upregulation of MMP9 expression and cell invasion. Moreover, the expression of WNK1 was downregulated in the placentas with preeclampsia, a disease resulting from insufficiency of EVT cell invasion during pregnancy. This study discovers the physiological function of WNK1 in human EVT cells and provides important insights into the regulation of MMP9 and cell invasion in human EVT cells.

Snail mediates GDF-8-stimulated human extravillous trophoblast cell invasion by upregulating MMP2 expression

BackgroundExtravillous trophoblast (EVT) cell invasion is a tightly regulated process that requires for a normal pregnancy. The epithelial-mesenchymal transition (EMT) has been implicated in EVT cell invasion. Growth differentiation factor-8 (GDF-8), a member of the transforming growth factor-beta (TGF-β) superfamily, is expressed in the human placenta and promotes EVT cell invasion by upregulating the expression of matrix metalloproteinase 2 (MMP2). However, the underlying molecular mechanism of GDF-8-induced MMP2 expression remains undetermined. Therefore, the present study aims to examine the role of Snail and Slug, the EMT-related transcriptional regulators, in GDF-8-stimulated MMP2 expression and cell invasion in HTR-8/SVneo human EVT cell line and primary cultures of human EVT cells.MethodsHTR-8/SVneo and primary cultures of human EVT cells were used to examine the effect of GDF-8 on MMP2 expression and explore the underlying mechanism. For gene silencing and overexpression, the HTR-8/SVneo cell line was used to make the experiments more technically feasible. The cell invasiveness was measured by Matrigel-coated transwell invasion assay.ResultsGDF-8 stimulated MMP2 expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effect of GDF-8 on MMP2 expression was blocked by the inhibitor of TGF-β type-I receptors, SB431542. Treatment with GDF-8 upregulated Snail and Slug expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effects of GDF-8 on Snail and Slug expression were blocked by pretreatment of SB431542 and siRNA-mediated knockdown of SMAD4. Interestingly, using the siRNA knockdown approach, our results showed that Snail but not Slug was required for the GDF-8-induced MMP2 expression and cell invasion in HTR-8/SVneo cells. The reduction of MMP2 expression in the placentas with preeclampsia (PE) was also observed.ConclusionsThese findings discover the physiological function of GDF-8 in the human placenta and provide important insights into the regulation of MMP2 expression in human EVT cells.1EyNAiCz9UDZ9srU6xtfDxVideo

The Role of Interferon (IFN)-γ in Extravillous Trophoblast Cell (EVT) Invasion and Preeclampsia Progression.

The involvement of the immune system in pregnancy is a controversial subject. The functions of T helper (Th) 1 and Th2 cells have been proposed, that Th1 cytokines promoting allograft rejection may impair pregnancy, whereas Th2-type cytokines suppressing Th1 responses improve allograft tolerance and hence embryonic survival. Maternal-fetal tolerance begins in the uterus; therefore, optimal adaptation to the fetus is the result of a complex interference. The invasion of extravillous trophoblast cells (EVTs) into the decidua and the inner third of the myometrium is essential for a healthy pregnancy. The mechanisms that influence trophoblast invasion are unknown; however, cytokines from uterine natural killer (uNK) cells, NKT cells, macrophages, and T cells appear to be involved. All these cells are major sources of interferon gamma (IFN-γ). Recent studies have shown that IFN-γ can inhibit EVT invasion via a mechanism dependent on an increase in EVT apoptosis and a decrease in matrix metalloproteinases (MMPs). Regarding controversies in this context, this study aimed to comprehensively review the role of IFN-γ and IFN-γ-producing cells in EVT invasion, successful pregnancy, and preeclampsia.

Conservation at the uterine–placental interface

The hemochorial placentation site is characterized by a dynamic interplay between trophoblast cells and maternal cells. These cells cooperate to establish an interface required for nutrient delivery to promote fetal growth. In the human, trophoblast cells penetrate deep into the uterus. This is not a consistent feature of hemochorial placentation and has hindered the establishment of suitable animal models. The rat represents an intriguing model for investigating hemochorial placentation with deep trophoblast cell invasion. In this study, we used single-cell RNA sequencing to characterize the transcriptome of the invasive trophoblast cell lineage, as well as other cell populations within the rat uterine-placental interface during early (gestation day [gd] 15.5) and late (gd 19.5) stages of intrauterine trophoblast cell invasion. We identified a robust set of transcripts that define invasive trophoblast cells, as well as transcripts that distinguished endothelial, smooth muscle, natural killer, and macrophage cells. Invasive trophoblast, immune, and endothelial cell populations exhibited distinct spatial relationships within the uterine-placental interface. Furthermore, the maturation stage of invasive trophoblast cell development could be determined by assessing gestation stage-dependent changes in transcript expression. Finally, and most importantly, expression of a prominent subset of rat invasive trophoblast cell transcripts is conserved in the invasive extravillous trophoblast cell lineage of the human placenta. These findings provide foundational data to identify and interrogate key conserved regulatory mechanisms essential for the development and function of an important compartment within the hemochorial placentation site that is essential for a healthy pregnancy.

The m6A methyltransferase METTL3 promotes trophoblast cell invasion by regulating MYLK expression

IntroductionThe progression of placental diseases such as preeclampsia is closely related to trophoblast dysfunction. Recent studies indicated the dysregulation of N6-methyladenosine (m6A) RNA modification in trophoblast disorders, while the function of METTL3, a methyltransferase of m6A, in trophoblasts remains to be studied. MethodsThe expression of METTL3 was determined by real-time PCR and immunoblotting. METTL3 expression in trophoblast cell lines HTR-8/SVneo and JEG-3 was knocked down using shRNA. The invasion of trophoblast cells in Matrigel was determined using xCELLigence. The m6A-containing transcripts was determined by m6A-sequencing in HTR-8/SVneo cells. The myosin light chain kinase (MYLK) gene was transfected into HTR-8/SVneo cells. ResultsThe expression of METTL3 was downregulated in preeclamptic placentae compared to normal placentae. Knockdown of METTL3 repressed the invasion of extravillous trophoblast cells. Mechanistically, METTL3 promoted the stability of MYLK mRNA through m6A modification. Overexpression of MYLK rescued retarded cell invasion by METTL3 depletion. DiscussionCollectively, our results highlight an essential role of METTL3-MYLK axis in trophoblast invasion.

Hypoxia Promotes Extravillous Trophoblast Cell Invasion through the Hypoxia-Inducible Factor Urokinase-Type Plasminogen Activator Receptor Pathway

Objectives: Hypoxia is a common feature of extravillous trophoblast (EVT) cells that promote invasion during the early stages of human placentation. This study aimed to examine whether hypoxia-induced an invasive phenotype in EVT cells in vitro and explore the underlying molecular mechanisms. Design: The invasiveness of primary EVT cells isolated from the first trimester placental tissues during weeks 5–8 of gestation was examined under hypoxic (5% O₂) and normoxic (20% O₂) conditions. Methods: Invasiveness was determined by transwell and wound-healing invasion assays using the IncuCyte ZOOM™ Live-Cell Imaging System. Protein expression of the urokinase plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor of hypoxia or normoxia-treated cells was measured using Western blot analysis. Knockdown of hypoxia-inducible factor-1 alpha (HIF-1α) was assessed using small interfering RNA (siRNA). Results: Hypoxia enhanced EVT cell invasion but did not affect apoptosis. The stimulatory effect of hypoxia on EVT cell invasiveness was associated with induction of the uPA-uPAR pathway. The synthetic inhibitor of uPAR significantly inhibited hypoxia-induced EVT cell invasion. Silencing of HIF-1α by siRNA abolished the stimulatory effect of hypoxia and inhibited the upregulation of uPAR expression, suggesting that the HIF-1α-uPAR signal is the key mediator for hypoxia-induced EVT cell invasion. Further experiments need to be performed to elucidate the HIF-1α-uPAR signal pathways. Conclusions: The low oxygen-regulated early events of EVT invasion may be mediated by the HIF-1α-uPAR pathway.

Nickel nanoparticles affect the migration and invasion of HTR-8/SVneo cells by downregulating MMP2 through the PI3K/AKT pathway

Proper migration and invasion of extravillous trophoblast cells into the endometrium in early gestation is essential for successful embryo implantation. The development of nanotechnology has led to the emergence of nickel nanoparticles (Ni NPs), for which attendant health concerns are widespread. Ni NPs are known to affect reproduction and be embryotoxic, but whether they affect the migration and invasion functions of trophoblast cells is unclear. We investigated the effects of Ni NPs on the migration and invasion of HTR-8/SVneo in extravillous trophoblast cells and explored the possible role of the PI3K/AKT/MMP2 signaling pathway in this regard. Results showed that the migration and invasion of cells was significantly inhibited by the exposure of Ni NPs. The protein and mRNA levels of PI3K/AKT/MMP2 signaling pathway were significantly reduced with the increase in Ni NPs concentration. The presence of the PI3K activator 740Y-P partially attenuated the inhibition of cell migration and invasion by Ni NPs, confirming the involvement of this pathway. Thus, Ni NPs inhibit migration and invasion of human trophoblast HTR-8/SVneo cells by downregulating the PI3K/AKT/MMP2 signaling pathway. This study is important for the development of safety evaluation criteria for Ni NPs.

Upregulation of circRNA hsa_circ_0008726 in Pre-eclampsia Inhibits Trophoblast Migration, Invasion, and EMT by Regulating miR-345-3p/RYBP Axis

Accumulating evidence shows that impaired spiral artery remodeling, placental dysfunction, and insufficient trophoblast infiltration contribute to the etiology and pathogenesis of pre-eclampsia (PE). circRNAs are a class of endogenous non-coding RNAs implicated in the pathogenesis of many diseases, including PE. This study aims to investigate the role of circRNA hsa_circ_0008726 in regulating the migration and invasion of extravillous trophoblast cells. RNase R assay was performed to confirm that circ_0008726 was a circular transcript. The expression of circ_0008726, RYBP, and miR-345-3p was examined by qRT-PCR. The functional interaction between miR-345-3p and circ_0008726 or RYBP was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP). Cell migration and invasion ability was analyzed by Transwell assays. Western blot was used for the quantification of RYBP protein level. Circ_0008726 expression was significantly increased in PE placenta tissues as compared with normal placenta tissues. Circ_0008726 was resistant to RNase R digestion and was predominately located in the cytoplasm of HTR-8/SVneo cells. Silencing circ_0008726 promoted cell migration and EMT (epithelial-mesenchymal transition), while circ_0008726 overexpression suppressed these processes. Mechanistically, circ_0008726 sponged miR-345-3p to negatively regulate its expression, and miR-345-3p negatively modulated the expression of RYBP. In PE samples, the expression level of circ_0008726 was negatively correlated with miR-345-3p level, but was positively correlated with RYBP expression. Transfection of miR-345-3p mimic or RYBP knockdown counteracted the effects of circ_0008726 overexpression on cell migration and EMT. Our data demonstrate the upregulation of circ_0008726 in PE placenta, which inhibits the migration, invasion, and EMT of HTR-8/SVneo cells by targeting miR-345-3p/RYBP axis. These data suggest that circ_0008726 could be a potential biomarker and therapeutic target for PE.

Molecular mechanisms in IL-1β-mediated decorin production by decidual cells.

Decorin, a small leucine-rich proteoglycan produced by decidual cells restrains trophoblast differentiation, migration and invasiveness of extra-villous trophoblast cells. Decidual overproduction of decorin is associated with preeclampsia, and elevated decorin levels in maternal plasma are a predictive biomarker of preeclampsia. Furthermore, decorin plays an autocrine role in maturation of human endometrial stromal cells into decidual cells. Thus, a balanced decorin production by the decidua is critical for healthy pregnancy. However, the molecular mechanisms regulating decorin production by the decidua are unclear. Interleukin-1 beta is an inflammation-associated multi-functional cytokine, and is reported to induce decidualization in primates. Hence, the present study was designed: (i) to test if exogenous Interleukin-1 beta stimulated decorin production by human endometrial stromal cells; and if so, (ii) to identify the cellular source of Interleukin-1 beta in first trimester decidual tissue; (iii) to identify the downstream molecular partners in Interleukin-1 beta mediated decorin production by human endometrial stromal cells. Results revealed that (i) amongst multiple pro-inflammatory cytokines tested, Interleukin-1 beta alone stimulated decorin production by these cells; (ii) both macrophages and decidual cells in first trimester decidua produced Interleukin-1 beta; (iii) Interleukin-1 beta mediated decorin production was dependent on Interleukin-1 receptor activation, followed by activation and nuclear translocation of nuclear factor kappa B and its binding to the decorin promoter. These results reveal that Interleukin-1 beta plays a novel role in inducing decorin production by human endometrial stromal cells by activating nuclear factor kappa B.

Matrix Metalloproteinases in Human Decidualized Endometrial Stromal Cells.

Cyclic changes, such as growth, decidualization, shedding, and regeneration, in the human endometrium are regulated by the reciprocal action of female hormones, such as estradiol (E2), and progesterone (P4). Matrix metalloproteases (MMPs) and tissue inhibitors of MMPs (TIMPs) control the invasion of extravillous trophoblast cells after implantation. Several MMPs and TIMPs function in the decidua and endometrial stromal cells (ESCs). Here, we aimed to systematically investigate the changes in MMPs and TIMPs associated with ESC decidualization. We evaluated the expression of 23 MMPs, four TIMPs, and four anti-sense non-coding RNAs from MMP loci. Primary ESC cultures treated with E2 + medroxyprogesterone acetate (MPA), a potent P4 receptor agonist, showed significant down-regulation of MMP3, MMP10, MMP11, MMP12, MMP20, and MMP27 in decidualized ESCs, as assessed by quantitative reverse transcription PCR. Further, MMP15 and MMP19 were significantly upregulated in decidualized ESCs. siRNA-mediated silencing of Heart and Neural Crest Derivatives Expressed 2 (HAND2), a master transcriptional regulator in ESC decidualization, significantly increased MMP15 expression in untreated human ESCs. These results collectively indicate the importance of MMP15 and MMP19 in ESC decidualization and highlight the role of HAND2 in repressing MMP15 transcription, thereby regulating decidualization.

Bone Morphogenetic Protein 2 Increases Human Trophoblast Invasion by Up-Regulating Integrin Beta3

The adequate invasion of extravillous trophoblast cells (EVTs) is indispensable for the implantation of embryos and subsequent remodeling of uterine spiral arteries in early human gestation. Bone morphogenetic protein 2 (BMP2), which is abundantly expressed at the maternal-fetal interface, has been shown to promote the human EVT invasion process (1). Integrin switching (i.e., a switch from α6β4 to αvβ3) plays essential roles in cell-extracellular matrix adhesion and has been reported to influence EVT migration and invasion (2). Moreover, integrin β3 has been found to promote the adhesion, invasion, and migration abilities of embryonic trophoblasts (3). However, whether integrin β3 participates in BMP2 signaling and mediates BMP2-increased-human trophoblast invasion remains unknown. The purpose of our study was to explore the effects of BMP2 on integrin αvβ3 expression and the possible mediation role of integrin β3 in BMP2-regulated human trophoblast invasion. We used immortalized human trophoblast cell line (HTR8/SVneo) and primary human extravillous trophoblast cells (EVTs) isolated from first-trimester villi as study models. RT-qPCR and Western blot assay were respectively utilized to detect the messenger RNA and protein levels of intergrin αv and β3. The function of the target protein was studied by siRNA knockdown, and the trophoblast invasion ability was checked by Matrigel-coated transwell invasion assays. Our results demonstrated that the mRNA and protein levels of integrin β3, rather than integrin αv, were up-regulated after BMP2 treatment in HTR8/SVneo and primary EVT cells. Importantly, siRNA-mediated down-regulation of integrin β3 significantly inhibited basal and BMP2-induced HTR8/SVneo cell invasionas measured by transwell invasion assay. In conclusion, we findings support that BMP2 promotes human trophoblast cell invasion by up-regulating integrin β3 expression, benefiting the in-depth understanding of the pro-invasive effect of BMP2 on human trophoblasts during early pregnancy. Reference: (1) Hong-Jin Zhao et al., Cell Death Dis 2018;9:174. (2) Damsky, C.H. et al, Development 1994; 120, 3657-3666. (3) Dong-Mei He et al., Reproduction 2019;157:423-430.

LncRNA H19-Derived miR-675-5p Accelerates the Invasion of Extravillous Trophoblast Cells by Inhibiting GATA2 and Subsequently Activating Matrix Metalloproteinases.

The invasion of extravillous trophoblast (EVT) cells into the maternal decidua, which plays a crucial role in the establishment of a successful pregnancy, is highly orchestrated by a complex array of regulatory mechanisms. Non-coding RNAs (ncRNAs) that fine-tune gene expression at epigenetic, transcriptional, and post-transcriptional levels are involved in the regulatory mechanisms of EVT cell invasion. However, little is known about the characteristic features of EVT-associated ncRNAs. To elucidate the gene expression profiles of both coding and non-coding transcripts (i.e., mRNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs)) expressed in EVT cells, we performed RNA sequencing analysis of EVT cells isolated from first-trimester placentae. RNA sequencing analysis demonstrated that the lncRNA H19 and its derived miRNA miR-675-5p were enriched in EVT cells. Although miR-675-5p acts as a placental/trophoblast growth suppressor, there is little information on the involvement of miR-675-5p in trophoblast cell invasion. Next, we evaluated a possible role of miR-675-5p in EVT cell invasion using the EVT cell lines HTR-8/SVneo and HChEpC1b; overexpression of miR-675-5p significantly promoted the invasion of both EVT cell lines. The transcription factor gene GATA2 was shown to be a target of miR-675-5p; moreover, small interfering RNA-mediated GATA2 knockdown significantly promoted cell invasion. Furthermore, we identified MMP13 and MMP14 as downstream effectors of miR-675-5p/GATA2-dependent EVT cell invasion. These findings suggest that miR-675-5p-mediated GATA2 inhibition accelerates EVT cell invasion by upregulating matrix metalloproteinases.

MNSFβ Promotes the Proliferation and Migration of Human Extravillous Trophoblast Cells and the Villus Expression Level of MNSFβ Is Decreased in Recurrent Miscarriage Patients

Aims: The invasion of extravillous trophoblast (EVT) cells into maternal decidua is essential for the establishment and maintenance of pregnancy. Derangement of EVT cell invasion might cause pregnancy complications including recurrent miscarriage (RM). We previously reported that deficiency of monoclonal nonspecific suppressor factor beta (MNSFβ) led to the early pregnancy failure in mice and the decidual MNSFβ expression level in RM patients was significantly decreased, but the underlying molecular mechanism of the role that MNSFβ played at the maternal-fetal interface remains unclear. Thus, in the present study, we determined effects of downregulated MNSFβ expression on human EVT cell activities. Methods: The MNSFβ expression in first-trimester human decidual and placental villus tissues was detected, respectively, by immunofluorescence or immunohistochemical analyses. The MNSFβ expression level in the immortalized first-trimester human EVT cell line HTR8/SVneo was downregulated by transfecting the small interfering RNA against MNSFβ and upregulated by transfecting the recombinant pDsRed-MNSFβ plasmids. The proliferation, migration, invasion, and apoptosis activities of HTR8/SVneo cells were, respectively, determined by cytometry assay, scratch test, transwell assay, and FITC/PI staining. The expression levels of P53, RhoA, Bcl-2, Bax, and MMP-9 in HTR8/SVneo cells, as well as the expression levels of MNSFβ and RhoA in placental villi of RM patients and physically normal pregnant women (NP), were examined by Western blot analysis. Results: MNSFβ protein signals were observed in first-trimester human villus and extravillous trophoblast cells. The downregulated MNSFβ expression significantly attenuated the proliferation, migration, and invasion abilities of HTR8/SVneo cells, accompanied with the obviously decreased expression levels of P53, RhoA, Bcl-2, Bax, and MMP-9, whereas the upregulated MNSFβ expression in HTR8/SVneo cells represented the inverse effects. Furthermore, expression levels of MNSFβ and RhoA in first-trimester human placental villus tissues of RM patients were significantly decreased compared to that of NP women. Conclusion: These data suggested that MNSFβ promotes proliferation and migration of human EVT cells, probably via the P53 signaling pathway, and the deficiency of MNSFβ in placental villi might lead to early pregnancy loss by reducing proliferation and invasion activities of EVTs.

Increased expression of IFN-γ in preeclampsia impairs human trophoblast invasion via a SOCS1/JAK/STAT1 feedback loop.

The weakening of extravillous trophoblast (EVT) invasion results in shallow placenta implantation. In HTR8/SVneo cells, IFN-γ can activate STAT1 and reduce cell invasion, and suppressor of cytokine signaling (SOCS) is an important negative regulatory protein in the Janus kinase (JAK)/STAT activator pathway and has a negative feedback function on JAK/STAT1. The aim of the present study was to elucidate how SOCS1 feedback regulates JAK/STAT1 and affects EVT cell invasion, which in turn affects the development of preeclampsia (PE). MTT and Annexin V/phosphatidylserine (PS) assays were performed to evaluate the viability and apoptosis of HTR8/SVneo cells treated with IFN-γ, respectively. Wound healing and invasion assays were also conducted to measure the migratory and invasive abilities of IFN-γ-treated HTR8/SVneo cells. The mRNA and protein expression levels of genes were detected using reverse transcription-quantitative PCR and western blot analysis. Small interfering RNA knockdown of SOCS1 was used to verify the role of feedback regulation in the IFN-γ-activated JAK/STAT1 signaling pathway. IFN-γ can inhibit HTR8/SVneo migration and invasion, and promote apoptosis by increasing the expression of phosphorylated (p)-JAK, p-STAT1 and caspase3, and reducing the expression of platelet-derived growth factor receptor A and Ezrin. Furthermore, SOCS1 may negatively regulate JAK/STAT1 and affect HTR-8/SVneo invasiveness. Evaluation of clinical samples demonstrated that the expression levels of SOCS1 and IFN-γ were higher in patients with PE compared with the healthy group. Collectively, the present results indicated that IFN-γ reduced the invasion of HTR-8/SVneo cells by activating JAK/STAT1, concurrently leading to an increase in SOCS1, which negatively regulates JAK/STAT1 and eliminates the pro-inflammatory effects of IFN-γ, thus forming a feedback loop.

Effects of cytomegalovirus infection on extravillous trophoblast cells invasion and immune function of NK cells at the maternal-fetal interface.

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.

Dysregulated Kisspeptin/Receptor Expression at the Maternal‐Fetal Interface in the Preeclamptic‐like BPH/5 Mouse

Preeclampsia (PE) is a hypertensive pregnancy syndrome estimated to cause the death of approximately 63,000 women worldwide annually. Insufficient invasion of extravillous trophoblast cells (EVT) into the maternal decidua and inadequate placentation are key adverse events in the pathogenesis of PE. Kisspeptins (KP), a family of small peptides derived from the kiss1 gene product, have been implicated as physiologic inhibitors of EVT invasion, a mechanism associated with upregulation of Tissue Inhibitor of Metalloproteinases (TIMP) and downregulation of Matrix Metalloproteinases (MMP). In PE, placental dysregulation of kiss1 and the KP receptor (kiss1r) have been proposed. Based on in vitro studies and evaluation of term PE placentas, it has been suggested that kiss1 and kiss1r mRNA are overexpressed at the maternal‐fetal interface during early gestation and excessively impair EVT invasion. However, the innate challenges involving human sample collection and early diagnosis of PE prevent a holistic understanding of KP role. Therefore, the objective of this study was to utilize the preeclamptic‐like BPH/5 mouse to investigate uteroplacental KP dysregulation throughout gestation. We have previously shown that pregnant BPH/5 females spontaneously develop poor placentation with decreased trophoblast invasion and inadequately remodeled decidual vessels. Hence, we hypothesized that uteroplacental expression of kiss1/kiss1r would be temporally dysregulated in the BPH/5 compared to the control C57 mice. Furthermore, we hypothesized that higher kiss1/kiss1r levels would be associated with upregulation of TIMP2, an important endogenous inhibitor of MMPs and cellular migration. To test our hypothesis, implantation sites and placentas of BPH/5 and C57 mice were collected at the embryonic days (e) 4.5, 7.5, 12.5, 14.5 and 18.5. The kiss1, kiss1r and TIMP2 mRNA levels were measured using RT‐PCR and comparisons were performed using unpaired t‐test. A similar expression pattern of kiss1 and kiss1r was seen throughout gestation in BPH/5 and control groups, with levels increasing from early (e4.5) to mid‐gestation (e14.5) and decreasing thereafter (e14.5 and e18.5). During the peak of decidualization (e7.5), BPH/5 mice presented 2‐fold higher kiss1 and kiss1r compared to C57 mice (n = 6; p ≤ 0. 05). In accordance with the proposed KP role, a 10‐fold increase in TIMP2 was also seen in BPH/5 mice at e7.5 (n = 6, p ≤ 0.05). In mid‐gestation (e14.5) kiss1r levels were 2‐fold lower in BPH/5 (n = 5) than in C57 (n = 6; p < 0.05), whereas kiss1 levels were not different among groups (n = 6; p > 0.05). In late gestation (e18.5), kiss1 and kiss1r were 3‐ and 2.5‐fold lower respectively in placentas of BPH/5 mice (n = 6; p ≤ 0.05). To the author’s knowledge, this is the first report of translational studies using an animal model of PE to investigate the role of KP in PE‐associated poor placental development. A kiss1/kiss1r dysregulation is reported, with higher expression levels in the preeclamptic‐like mouse in the peak of decidualization, and concurrent upregulation of TIMP2. Further studies are needed to investigate KP‐related upstream and downstream signaling pathways in the context of PE.