Directed biomechanical compressive forces enhance fusion efficiency in model placental trophoblast cultures.

The syncytiotrophoblast is a multinucleated structure that arises from fusion of mononucleated cytotrophoblasts, to sheath the placental villi and regulate transport across the maternal-fetal interface. Here, we ask whether the dynamic mechanical forces that must arise during villous development might influence fusion, and explore this question using in vitro choriocarcinoma trophoblast models. We demonstrate that mechanical stress patterns arise around sites of localized fusion in cell monolayers, in patterns that match computational predictions of villous morphogenesis. We then externally apply these mechanical stress patterns to cell monolayers and demonstrate that equibiaxial compressive stresses (but not uniaxial or equibiaxial tensile stresses) enhance expression of the syndecan-1 and loss of E-cadherin as markers of fusion. These findings suggest that the mechanical stresses that contribute towards sculpting the placental villi may also impact fusion in the developing tissue. We then extend this concept towards 3D cultures and demonstrate that fusion can be enhanced by applying low isometric compressive stresses to spheroid models, even in the absence of an inducing agent. These results indicate that mechanical stimulation is a potent activator of cellular fusion, suggesting novel avenues to improve experimental reproductive modelling, placental tissue engineering, and understanding disorders of pregnancy development.

C-C motif chemokine ligand 2 regulates prostaglandin synthesis and embryo attachment of the bovine endometrium during implantation.

C-C motif chemokine ligand 2 (CCL2) has been reported to be expressed in the bovine endometrium during pregnancy. However, the details of its functions involved in the implantation mechanism are still not clear. The purpose of this study is to analyze the functional properties of CCL2 in the bovine endometrium and embryos. The expression of CCR2 was not different between the luteal phase and implantation phase of their endometrial tissues, but was significantly high in IFNa treated bovine endometrial stromal (BES) cells in vitro. The expressions of PGES1, PGES2, AKR1C4, and AKR1C4 were high at the implantation stage compared with the luteal stage. On the other hand, PGES2 and AKR1B1 in BEE and PGES3 and AKR1A1 in BES were significantly increased by CCL2 treatment, respectively. The expressions of PCNA and IFNt were found significantly high in the bovine trophoblastic cells (BT) treated with CCL2 compared to the control. CCL2 significantly increased the attachment rate of BT vesicles to BEE in in vitro co-culture system. The expression of OPN and ICAM-1 increased in BEE, and ICAM-1 increased in BT by CCL2 treatment, respectively. The present results indicate that CCL2 has the potential to regulate the synthesis of PGs in the endometrium and the embryo growth. In addition, CCL2 has the possibility to regulate the process of bovine embryo attachment to the endometrium by modulation of binding molecules expression.

Establishment and comparison of human term placenta-derived trophoblast cells†.

Research on the biology of fetal-maternal barriers has been limited by access to physiologically relevant cells, including trophoblast cells. In this study, we describe the development of a human term placenta-derived cytotrophoblast immortalized cell line (hPTCCTB) derived from the basal plate. Human-term placenta-derived cytotrophoblast immortalized cell line cells are comparable to their primary cells of origin in terms of morphology, marker expression, and functional responses. We demonstrate that these can transform into syncytiotrophoblast and extravillous trophoblasts. We also compared the hPTCCTB cells to immortalized chorionic trophoblasts (hFM-CTC), trophoblasts of the chorionic plate, and BeWo cells, choriocarcinoma cell lines of conventional use. Human-term placenta-derived cytotrophoblast immortalized cell line and hFM-CTCs displayed more similarity to each other than to BeWos, but these differ in syncytialization ability. Overall, this study (1) demonstrates that the immortalized hPTCCTB generated are cells of higher physiological relevance and (2) provides a look into the distinction between the spatially distinct placental and fetal barrier trophoblasts cells, hPTCCTB and hFM-CTC, respectively.

Hsa_circ_0001326 inhibited the proliferation, migration, and invasion of trophoblast cells via miR-145-5p/TGFB2 axis.

PROBLEM: Preeclampsia (PE) is an obstetric disease involving multiple systems, which account for maternal and fetal complications and increased mortality. Circular RNAs (circRNAs) were recently deemed to associate with the pathogenesis of PE. This study aims to clarify the correlation between circRNA hsa_circ_0001326 and PE and explore its biological function in PE. METHOD OF STUDY: The expression of hsa_circ_0001326 in PE placentas was detected by real-time quantitative PCR (qRT-PCR). After overexpressing or inhibiting hsa_circ_0001326 in trophoblast cells, the cell growth, migration, and invasion were evaluated by Cell Counting Kit-8 (CCK-8) and transwell assays. Western blot assay was applied to detect the epithelial-mesenchymal transition (EMT) proteins, E-cadherin and Vimentin. Furthermore, a dual-luciferase reporter assay was applied to verify the binding sites of hsa_circ_0001326, miR-145-5p, and transforming growth factor beta 2 (TGFB2). RESULTS: Hsa_circ_0001326 was found to be higher expressed in PE placentas than in normal placentas. Furthermore, hsa_circ_0001326 played a negative regulating role in trophoblast cell viability, migration, and invasion. Overexpression of hsa_circ_0001326 inhibited the viability, migration, and invasion of trophoblast cells, while inhibition of hsa_circ_0001326 showed opposite effects. Mechanistically, hsa_circ_0001326 sponged miR-145-5p to elevate TGFB2 expression in trophoblast cells. CONCLUSION: This study provided evidence that the up-regulated hsa_circ_0001326 in PE restrained trophoblast cells proliferation, migration, and invasion by sponging miR-145-5p to elevate TGFB2 expression. Our results might provide a novel insight into the role of hsa_circ_0001326 in the pathogenesis of PE.

MiR-30a-3p Targeting FLT1 Modulates Trophoblast Cell Proliferation in the Pathogenesis of Preeclampsia.

Preeclampsia (PE) may pose significant adverse effects on pregnant women. Dysregulation of angiogenesis, trophoblast invasion, and proliferation are known to be associated with PE development and progression. Fms related tyrosine kinase 1 (FLT1), an anti-angiogenic factor, is consistently upregulated in PE patients. Recent papers highlight that aberrant miR-30a-3p expression contributes to PE development. More effects are needed to assess the biological function of placental miR-30a-3p in PE. The soluble FLT1 (sFLT1) and FLT1 levels were tested by ELISA assay and Western blotting assay. mRNA levels were measured by RT-qPCR assay. Colony formation and MTT assays were applied to assess the effect of miR-30a-3p on trophoblast cell proliferation. The serum sFLT1 and placental FLT1 levels were substantially high in patients with PE. Using miRNA microarray assay, we identified miR-30a-3p upregulation in PE patients' placenta tissues. We further confirmed that miR-30a-3p binds to the 3'-UTR of FLT1 gene and positively regulate its expression. Forcing miR-30a-3p expression inhibited trophoblast cell proliferation and vice versa. In conclusion, persistent high levels of FLT1 and miR-30a-3p may pose adverse effects on angiogenesis and trophoblast proliferation in placenta of PE patients. Therefore, targeting FLT1 and miR-30a-3p may serve as ideal strategies for managing patients with PE.

Downregulation of CDC42 inhibits the proliferation and stemness of human trophoblast stem cell via EZRIN/YAP inactivation.

Placental dysplasia increases the risk of recurrent spontaneous abortion (RSA). However, the underlying mechanism regulating placental development remains unclear. In this study, we showed that the expression of CDC42 was decreased in the villous tissue of RSA samples compared to healthy controls. Further examination demonstrated that CDC42 deficiency led to the differentiation of human trophoblast stem cells (hTSCs) and inhibited their proliferation. Genetic manipulation of YAP and EZRIN in hTSCs revealed that CDC42 regulates the stemness and proliferation of hTSCs; this is dependent on EZRIN, which translocates YAP into the nucleus. Moreover, the expression pattern of EZRIN, YAP, and Ki67 was also abnormal in the villous tissue of RSA samples, consistent with in vitro experiments. In summary, these findings suggest that the CDC42/EZRIN/YAP pathway plays an important role in placental development.

Bone marrow stromal cell antigen 2(BST2) suppresses the migration and invasion of trophoblasts in preeclampsia by downregulating matrix metallopeptidase 2(MMP2).

Preeclampsia is a grievous pregnancy-related complication with an incidence of approximately 5∼7% in pregnant women. Placental abnormalities and decreased placental perfusion associated with impaired trophoblast invasion are early pathological findings of preeclampsia. BST2 is a multifunctional transmembrane protein that plays critical roles in physiological and pathological processes, but its impacts and mechanisms of action in preeclampsia are inadequately understood. The aim of this manuscript was to investigate the functional impacts of BST2 and MMP2 on the biological behavior of trophoblast cells in preeclampsia. The expression of these proteins and their genes was analyzed by qRT-PCR, western blotting and immunohistochemistry. The results showed that the expression of BST2 and MMP2 was significantly downregulated in preeclampsia. The migration and invasion capacities of HTR-8/SVneo and JAR cells with overexpression or knockdown of BST2 were detected by wound healing assay and Transwell assays. It was found that BST2 overexpression could up-regulate MMP2 expression, and enhance the migration and invasion capacity of HTR-8/SVneo and JAR cells. BST2 knockdown could reverse these effects. MMP2 knockdown could downregulate the invasion capacity of HTR-8/SVneo cells, and MMP2 overexpression reversed these effects. Pearson correlation analysis demonstrated that the expression of MMP2 and BST2 were positively correlated. These results indicate that the downregulation of BST2 lowers MMP2 expression and restraint trophoblast functions, which probably explain its role in the pathogenesis of preeclampsia.

Cleavage-stage human embryo arrest, is it embryo genetic composition or others?

Embryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.

Transcriptome regulation of extracellular vesicles derived from porcine uterine flushing fluids during peri-implantation on endometrial epithelial cells and embryonic trophoblast cells.

The extracellular vesicles (EVs) in uterine fluids play a vital role in embryo implantation by mediating intrauterine communication between conceptus and maternal endometrium in pigs. However, the regulatory mechanism of EVs in uterine fluids is largely unclear. In order to understand the effect of EVs in uterine flushing fluids (UFs) during embryo implantation on endometrial epithelial cells (EECs) and embryonic trophoblast cells (PTr2 cells). The UFs-EVs on day 13 of pregnancy (D13) were added to the culture medium of EECs and PTr2 cells. It was found that PKH-67 labeled UFs-EVs could be taken up in EECs and PTr2 cells. Transcriptome sequencing analysis showed that a total of 1793 and 6279 genes were differentially expressed in the EECs and PTr2 cells after the treatment of UFs-EVs on D13, respectively. Among these genes, real-time quantitative PCR (RT-qPCR) results indicated that ID2, ITGA5, CXCL10 and CXCL11 genes were differentially expressed in both EECs and PTr2 cells after treatment. Bioinformatics analysis showed that the differentially expressed (DE) genes in EECs and PTr2 cells after treatment are involved in immune regulation, cell migration, cell adhesion and the secretion and uptake of EVs. Our research offers novel insight into the regulation mechanism of UFs-EVs on D13 in EECs and PTr2 cells.

Prothioconazole induces cell cycle arrest by up-regulation of EIF4EBP1 in extravillous trophoblast cells.

Prothioconazole (PTC) is a new broad-spectrum triazole antibacterial agent that is being widely used in agriculture. PTC has been linked to a number of reproductive outcomes including embryo implantation disorder; however, the exact mechanism underlying this relationship has yet to be determined. Proper trophoblast proliferation and migration is a prerequisite for successful embryo implantation. To elucidate the underlying molecular perturbations, we detect the effect of PTC on extravillous trophoblast cells proliferation and migration, and investigate its potential mechanisms. Exposure to different concentrations of PTC (0-500 µM) significantly inhibited the cell viability and migration ability (5 µM PTC exposure), and also caused the cell cycle arrest at the lowest dose (1 µM PTC exposure). Transcriptome analysis revealed that PTC exposure disturbed multiple biological processes including cell cycle and apoptosis, consistent with cell phenotype. Specifically, eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1, 4E-BP1) was identified as up-regulated in PTC exposure group and knockdown of EIF4EBP1, and attenuated the G1 phase arrest induced by PTC exposure. In summary, our data demonstrated that 4E-BP1 participated in PTC-induced cell cycle arrest in extravillous trophoblast cells by regulating cyclin D1. These findings shed light on the potential adverse effect of PTC exposure on the embryo implantation.

Forkhead-box C1 attenuates high glucose-induced trophoblast cell injury during gestational diabetes mellitus via activating adenosine monophosphate-activated protein kinase through regulating fibroblast growth factor 19.

Gestational diabetes mellitus (GDM) is a complication developed during pregnancy and recover after childbirth. The purpose of this study was to investigate the protective role of FOXC1 during GDM and the underlying mechanism. FOXC1 was downregulated in GDM placental tissues and HG-treated HTR-8/SVneo cells. Overexpression of FOXC1 prevented HG-induced inhibition of cell proliferation, migration and invasion. FOXC1 suppressed HG-induced cell apoptosis in HTR-8/SVneo cells. The apoptosis-related proteins: cleaved caspase-3, cleaved caspase-9 and BAX, were also downregulated by FOXC1 overexpression. FOXC1 increased glucose uptake and improved insulin sensitivity. The expression of FOXC1 was positively correlated with FGF19 expression. FOXC1 regulated the expression of FGF19 and phosphorylation of AMPK. Inhibition of FGF19 attenuated the biological functions of FOXC1 through inactivation of AMPK. In conclusion, this study demonstrates that FOXC1 attenuates HG-induced trophoblast cell injury through upregulating FGF19 to activate the AMPK signaling pathway during GDM, suggesting that FOXC1 is a potential therapeutic target for drug discovery in the future.

Interference of KLF9 relieved the development of gestational diabetes mellitus by upregulating DDAH2.

Gestational diabetes mellitus (GDM) is a situation where glucose intolerance is found in pregnant women without a previous diagnosis of diabetes. The role of Kruppel-like factor 9 (KLF9) has not been investigated in GDM, which constituted the aim of our study. HTR8/SVneo cells were induced by high glucose (HG) and pregnant mice were treated with streptozocin (STZ) to establish GDM model in vitro and in vivo, respectively. The expression level of KLF9 was detected by real-time PCR, immunohistochemical staining, and Western blot. Cell viability, apoptosis, inflammation, and oxidative stress were investigated by cell counting kit-8 (CCK-8), TUNEL, enzyme-linked immunosorbent assay (ELISA) and oxidative stress detection kits, respectively. The interaction of KLF9 with dimethylarginine dimethylaminohydrolase 2 (DDAH2) was predicted by bioinformatic tools and confirmed by luciferase reporter assay and chromatin immunoprecipitation (ChIP). The expression of KLF9 was increased in the placental tissues of GDM patients and HG-induced HTR8/SVneo cells. Silencing of KLF9 increased cell viability, reduced cell apoptosis, and suppressed inflammation and oxidative stress in HG-induced HTR8/SVneo cells. KLF9 could bind to DDAH2 promoter and negatively regulate DDAH2 expression. Inhibition of DDAH2 partly weakened the effects of KLF9 silencing on cell apoptosis, inflammation, and oxidative stress. The suppressive effects of KLF9 silencing on blood glucose and insulin concentration in vivo were also abolished by DDAH2 knockdown. In conclusion, we provided evidence that interference of KLF9 could hinder the development of GDM by alleviating cell apoptosis, inflammation, and oxidative stress through upregulating DDAH2, which might instruct the targeting therapies against GDM.Abbreviations: KLF9: Kruppel-like factor 9; DDAH2: dimethylarginine dimethylaminohydrolase 2 ; GDM: gestational diabetes mellitus; ELISA: enzyme-linked immunosorbent assay; CCK-8: cell counting kit-8; ChIP: chromatin immunoprecipitation; sh: short hairpin; HG: high glucose; PBS: phosphate-buffered saline; DAPI: 4, 6-diamidino-2-phenylindole; IL-6: Interleukin-6; TNF-α: tumor necrosis factor-α; ROS: reactive oxygen species; MDA: malondialdehyde; SOD: superoxide dismutase; wt: wild-type; mut: mutant.

C1q/tumor necrosis factor related protein 6 (CTRP6) regulates the phenotypes of high glucose-induced gestational trophoblast cells via peroxisome proliferator-activated receptor gamma (PPARγ) signaling.

Multiple studies have confirmed that adipokines are compactly relevant to insulin resistance and participate in the pathogenesis of gestational diabetes mellitus (GDM). This paper aimed to study the effects of C1q/tumor necrosis factor related protein (CTRP)6 on the phenotypes of trophoblast cells, covering cell proliferation, invasion and migration, and initially explore the mechanism. High glucose was used to induce trophoblast cells to establish an in vitro model. The expression levels of CTRP6 were firstly determined, and then the effects of CTRP6 knockdown on cell viability, apoptosis, migration and invasion were assessed using CCK8, TUNEL, wound healing, Transwell assays. Moreover, the role of peroxisome proliferator-activated receptor gamma (PPARγ), probable target of CTRP6, was evaluated through co-transfection with PPARγ overexpression vector. The results of the present study revealed that CTRP6 and PPARγ were both upregulated in high glucose-induced cells. And CTRP6 knockdown could significantly elevate the abilities of cell viability, migration and invasion, and avoid cell apoptosis. In addition, PPARγ overexpression was found to restrain the protective effects of CTRP6 knockdown on the above aspects, indicating CTRP6 played a role in trophoblast cells via inhibiting PPARγ expression. In conclusion, CTRP6 regulated the viability, migration and invasion of high glucose-induced gestational trophoblast cells through PPARγ signaling.

T-Lymphocyte proliferative activity in early pregnancy and outside pregnancy state.

T-lymphocytes are present in the endometrium before pregnancy and their number varies depending on menstrual cycle stage. Despite T-lymphocyte population heterogeneity, there is no clear vision of general mechanisms of decidua T-lymphocyte pool formation. One of the assumed variants is T-lymphocyte proliferation in situ. The study objective is to evaluate variations of peripheral blood T-lymphocyte proliferative activity in the presence of trophoblast cells. The peripheral blood was sampled from healthy nonpregnant women in the proliferative (n = 29) and secretory (n = 32) menstrual cycle phases and also from women on 6-7 weeks stage of physiological pregnancy (n = 30). Jeg-3 (ATCC) line cells were applied as trophoblast cells within in vitro model system. T-lymphocyte proliferation was determined by estimating the Ki-67 expression and T-lymphocyte relative number. It was established that trophoblast cells perform inhibiting effect on Ki-67 by T-lymphocytes in all groups of examined women both in course of PBMC cultivation and in case of preliminarily isolated T-lymphocytes. During cultivation in the presence of IL-2 and trophoblasts, PBMC T-lymphocytes in pregnant women are more resistant to trophoblast cells inhibition than in nonpregnant women. In case of isolated T-lymphocytes, decreased T-lymphocyte proliferation during pregnancy was observed as compared to the proliferative cycle phase hence pointing to necessity of T-lymphocyte contact with microenvironment cells for self-support.

Murine Trophoblast Stem Cells and Their Differentiated Cells Attenuate Zika Virus In Vitro by Reducing Glycosylation of the Viral Envelope Protein.

Zika virus (ZIKV) infection during pregnancy can cause devastating fetal neuropathological abnormalities, including microcephaly. Most studies of ZIKV infection in pregnancy have focused on post-implantation stage embryos. Currently, we have limited knowledge about how a pre-implantation stage embryo deals with a viral infection. This study investigates ZIKV infection on mouse trophoblast stem cells (TSCs) and their in vitro differentiated TSCs (DTSCs), which resemble the cellular components of the trophectoderm layer of the blastocyst that later develops into the placenta. We demonstrate that TSCs and DTSCs are permissive to ZIKV infection; however, ZIKV propagated in TSCs and DTSCs exhibit substantially lower infectivity, as shown in vitro and in a mouse model compared to ZIKV that was generated in Vero cells or mouse embryonic fibroblasts (MEFs). We further show that the low infectivity of ZIKV propagated in TSCs and DTSCs is associated with a reduced level of glycosylation on the viral envelope (E) proteins, which are essential for ZIKV to establish initial attachment by binding to cell surface glycosaminoglycans (GAGs). The decreased level of glycosylation on ZIKV E is, at least, partially due to the low-level expression of a glycosylation-related gene, Hexa, in TSCs and DTSCs. Furthermore, this finding is not limited to ZIKV since similar observations have been made as to the chikungunya virus (CHIKV) and West Nile virus (WNV) propagated in TSCs and DTSCs. In conclusion, our results reveal a novel phenomenon suggesting that murine TSCs and their differentiated cells may have adapted a cellular glycosylation system that can limit viral infectivity by altering the glycosylation of viral envelope proteins, therefore serving as a unique, innate anti-viral mechanism in the pre-implantation stage embryo.

G protein-coupled estrogen receptor stimulates human trophoblast cell invasion via YAP-mediated ANGPTL4 expression.

Insufficient invasion of trophoblast cells into the uterine decidua is associated with preeclampsia (PE). G protein-coupled estrogen receptor (GPER) is a membrane estrogen receptor involved in non-genomic estrogen signaling. GPER is expressed in human trophoblast cells and downregulated GPER levels are noted in PE. However, to date, the role of GPER in trophoblast cells remains largely unknown. Here, we applied RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to G1, an agonist of GPER, and identified angiopoietin-like 4 (ANGPTL4) as a target gene of GPER. Treatment of trophoblast cells with G1 or 17ß-estradiol (E2) activated Yes-associated protein (YAP), the major downstream effector of the Hippo pathway, via GPER but in a mammalian STE20-like protein kinase 1 (MST1)-independent manner. Using pharmacological inhibitors as well as loss- and gain-of-function approaches, our results revealed that YAP activation was required for GPER-stimulated ANGPTL4 expression. Transwell invasion assays demonstrated that activation of GPER-induced ANGPTL4 promoted cell invasion. In addition, the expression levels of GPER, YAP, and ANGPTL4 were downregulated in the placenta of patients with PE. Our findings reveal a mechanism by which GPER exerts its stimulatory effect on human trophoblast cell invasion by upregulating YAP-mediated ANGPTL4 expression.

Chorionic villus-derived mesenchymal stem cells induce E3 ligase TRIM72 expression and regulate cell behaviors through ubiquitination of p53 in trophoblasts.

Preeclampsia is a significant contributor for maternal or fetal morbidity and mortality, which is characterized by reduced invasion capacity of trophoblasts and is regulated by extracellular matrix (ECM). It is still under investigation whether chorionic villus-derived mesenchymal stem cells (CVMSC) could affect the functionality of trophoblasts. In this study, CVMSC-derived exosomes were isolated; their effect on trophoblasts was investigated based on the CCK8 assay, migration assay, and apoptosis detection. And the underlying mechanism of this effect was investigated using mRNA sequencing, western blot, co-immunoprecipitation, luciferase report assay, and ubiquitination assay. The results show that CVMSC-derived exosomes promote migration and proliferation of trophoblasts, and also reduce cell apoptosis. mRNA sequencing confirmed that after treatment of CVMSC-derived exosomes, Tripartite Motif Containing 72 (TRIM72) expression was upregulated and Tumor Protein P53 (P53) expression was downregulated, both significantly in trophoblasts. Subsequent study confirms that TRM72 can directly interact with P53 and promote P53 ubiquitination and proteasomal degradation, reducing apoptosis rate and elevating proliferation and migration in trophoblasts. Our study confirms that CVMSC-derived exosomes promote trophoblast migration and proliferation by upregulating TRIM72 expression, and subsequently advance P53 ubiquitination and proteasomal degradation.

Crosstalk Between Trophoblast and Macrophage at the Maternal-Fetal Interface: Current Status and Future Perspectives.

The immune tolerance microenvironment is crucial for the establishment and maintenance of pregnancy at the maternal-fetal interface. The maternal-fetal interface is a complex system containing various cells, including lymphocytes, decidual stromal cells, and trophoblasts. Macrophages are the second-largest leukocytes at the maternal-fetal interface, which has been demonstrated to play essential roles in remodeling spiral arteries, maintaining maternal-fetal immune tolerance, and regulating trophoblast's biological behaviors. Many researchers, including us, have conducted a series of studies on the crosstalk between macrophages and trophoblasts at the maternal-fetal interface: on the one hand, macrophages can affect the invasion and migration of trophoblasts; on the other hand, trophoblasts can regulate macrophage polarization and influence the state of the maternal-fetal immune microenvironment. In this review, we systemically introduce the functions of macrophages and trophoblasts and the cell-cell interaction between them for the establishment and maintenance of pregnancy. Advances in this area will further accelerate the basic research and clinical translation of reproductive medicine.

Hypoxia-induced DEC1 mediates trophoblast cell proliferation and migration via HIF1α signaling pathway.

In early pregnancy, hypoxia is a typical extrinsic factor that regulates EVT functions including proliferation, migration and invasion which are essential for a successful pregnancy. Human differentiated embryonic chondrocyte-expressed gene 1 (DEC1), a hypoxia-regulated gene, has been reported to be overexpressed in several types of cancers. Given that the placenta and the cancer share several similarities with respect to their capacity to proliferate and invade adjacent tissues, we focused on the role of DEC1 on trophoblast function in a physiologically hypoxic environment, which may be associated with unexplained recurrent spontaneous abortion (URSA).In our study, we measured the expression of HIF-1α and DEC1 in first-trimester villi through real-time-PCR (RT-PCR) and immunohistochemical analysis. in vitro, DEC1 expression was downregulated in trophoblast cells via DEC1-specific shRNA plasmid transfection. The expression of DEC1 and HIF-1α was detected via western blotting and RT-PCR analysis. The proliferation and migration of HTR-8/SVneo cells were assayed using CCK-8 and Transwell migration assays, respectively.Our results indicated that the expression of DEC1 was significantly reduced in villi of URSA compared to that in normal pregnant women. in vitro, hypoxia induced the expression of HIF-1ɑ and DEC1 and upregulated proliferation and migration of the HTR-8/SVneo cells. Knockdown of DEC1 inhibited proliferation and migration of HTR-8/SVneo cells exposure to hypoxia. Furthermore, inhibition of HIF1α expression resulted in a significant decrease in DEC1. These findings illustrate that hypoxia-induced DEC1 expression promotes trophoblast cell proliferation and migration through the HIF1α signaling pathway, which plays an important role during placentation.

MicroRNA-3935 promotes human trophoblast cell epithelial-mesenchymal transition through tumor necrosis factor receptor-associated factor 6/regulator of G protein signaling 2 axis.

BACKGROUND: Insufficient migration and invasion during trophoblast epithelial-mesenchymal transition (EMT) results in the occurrence and development of preeclampsia (PE), and our previous study has screened 52 miRNAs, whose expression levels are altered in the placental samples from PE patients, compared with the normal group. Among those, miR-3935 is one of the miRNAs being most significantly down-regulated, indicating its involvement in PE. However, the exact effect and molecular mechanisms remain unknown. METHODS: In the present study, we investigate the roles and underlying mechanisms of miR-3935 in trophoblast EMT by use of the human extra-villous trophoblast cell line HTR-8/SVneo as well as human placental tissues and maternal blood samples obtained from 15 women with normal pregnancies and 15 women with PE. Experimental methods include transfection, quantitative reverse transcription-PCR (qRT-PCR), western blot, immunofluorescence staining, dual-luciferase assays, in vitro invasion and migration assays, RNA-Seq analysis, bisulfite sequencing and immunohistochemistry staining. RESULTS: MiR-3935 expression is significantly decreased in both placentas and peripheral blood specimens of PE, and functionally, miR-3935 promotes EMT of trophoblast cells. Mechanistically, TRAF6 is identified to be a direct target of miR-3935 and TRAF6 exerts its negative effect on EMT of trophoblast cells by inhibition of RGS2, which down-regulates the methylation status of promoter of CDH1 gene that encodes E-Cadherin protein through induction of ALKBH1, resulting in increase of E-Cadherin and subsequently insufficient trophoblast EMT. CONCLUSIONS: Together these results uncover a hitherto uncharacterized role of miR-3935/TRAF6/RGS2 axis in the function of human trophoblasts, which may pinpoint the molecular pathogenesis of PE and may be a prognostic biomarker and therapeutic target for such obstetrical diseases as PE.