Effect of Ephrin-A1/EphA2 on Invasion of Trophoblastic Cells

The effect of axon guidance factors ephrin-A1/EphA2 on the invasion of trophoblastic cells and the possible mechanism were investigated in this study. The expression of EphA2 in vascular endothelial cells was detected by immunohistochemistry. The proliferation and invasion of TEV-1 cells （an extravillous trophoblastic cell line） in first trimester were determined by cell counting kit-8 （CCK-8） and Transwell invasion assay. Real-time PCR was used to detect the expression ofephrin-A1 in TEV-I cells treated with EphA2 at different concentrations （10, 50, 100, 500, 1000 and 5000 μg/L）. The results showed： （1） EphA2 was expressed in the vascular endothelial cells; （2） EphA2 could promote the proliferation of TEV-1 cells. The proliferative capacity reached a peak in TEV-1 cells treated with 100 μg/L EphA2 （P〈0.05）; （3） EphA2 could increase the invasion of TEV-1 cells. The invasive ability was the greatest in TEV-1 cells treated with 500 pg/L EphA2 （P〈0.05）; （4） in the presence of EphA2 （0-500 μg/L）, the expression of ephrin-A1 was increased concentration-dependently （P〈0.05）, but when the concentration of EphA2 was over 500 μg/L, the expression of ephrin-A 1 ceased to increase （P〉0.05）. It was concluded that EphA2 can promote the invasion and proliferation of the human extravillous trophoblastic cells probably by regulating the ephrin-A1 ligand.

Exosome-derived miR-146a-5p from decidual macrophages in preeclampsia inhibits the viability and invasive ability of trophoblast cells by targeting HIF1α

Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnant women were collected.Macrophages were obtained by the density gradient method and then flow cell sorting,then the exosomes were extracted.The structure of the exosomes was observed by transmission electron microscope.The expression of CD63,a marker protein of the exocrine body,was detected by western blot,and the exosomes were identified.CCK-8 was used to detect the effect of exosomes on trophoblast cell viability.Transwell migration experiment was used to detect the influence on migration ability.The expression of miR-146a-5p in exosomes was detected by qPCR.The effect of exosomes on the expression of HIF1αprotein in trophoblasts was detected by western blot and detection of the binding site between miR-146a-5p and HIF1αby double luciferase reporter gene was conducted.Results:The exosomes of macrophages present a"cake"structure with a middle depression about 30-130 nm in diameter,and CD63 is highly expressed,which conforms to the characteristics of exosomes.Compared with the normal group,the exosomes of decidual macrophages in the PE group inhibited the activity and migration of trophoblast cells(P<0.001).The expression of miR-146a-5p in the exosomes of decidual macrophages in the PE decreased significantly,and after exosomes of PE decidual macrophages treating trophoblast cells,the protein expression of HIF1αin trophoblast cells was significantly increased.There are targeted binding sites between miR-146a-5p and HIF1α.Conclusion:PE decidual macrophage exosomes can inhibit the viability and migration of trophoblast cells,which may be related to the decreased expression of miR-146a-5p in exosomes,thus promoting HIF1αprotein expression of trophoblast cells.

Expression and Immune Effect of Toll-Like Receptor 4 in Human Trophoblast Cells

This study investigated the expression and immune effect of TLR4 in human trophoblast cells. The expression level of TLR4 mRNA in normal and LPS-stimulated human term trophoblast cells （1 mg/L LPS, 12 h） was detected by RT-PCR. In LPS-stimulated human term trophoblast cells of TLR4-blocked group and non-TLR4-blocked group, and normal term trophoblast cells of blank control group, apoptosis rate was measured by flow cytometry （FCM）, and the level of TNF-α determined by using enzyme linked immunosorbent assay （ELISA） respectively. RT-PCR results showed that the expression level of TLR4 mRNA in LPS-stimulated human trophoblast cells was significantly higher than that in normal cells （P〈0.01）. FCM revealed that there was significant difference in apoptosis rate of LPS-stimulated human term trophoblast cells between TLR4-blocked group and non-TLR4-blocked group （P〈0.05）, or between TLR4 antibody-blocked group and blank control group. ELISA indicated that the level of TNF-α in LPS-stimulated human trophoblast cells also had statistical differences between TLR4 antibody-blocked group and non-TLR4 antibody-blocked group （P〈0.05）. Our results suggest that TLR4 plays an important role in the immunological mechanism of apoptosis and secretion of TNF-α of human term trophoblast cells stimulated by LPS.

In vitro Study on Human Trophoblast Cells Infected with HCMV

Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient.Cells and purity were identified by using immunocytochemistry assay with anti-CK7,Vim and β-hCG antibodies.HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection(p.i.).The results showed tha...

The Effects of Anordrin on Luteal Cells, DecidualCellsand TrophoblastCells in Vitro

Division of Reproductive Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China *DEPT. Of Pharmacology, Shanghai Tiedao University, Medical College Shanghai 200070, China By using the morphology and the viability of cells index, the direct effects of anordrin on serum free primary cultures of rat luteal cells, human decidual cells and trophoblast cells were observed.Meanwhile, the effect of anordrin on the secretive function of rat luteal cells was also observed. The results indicated that (1) anordrin has damaging effects on rat luteal cells, human decidual cells and trophoblast cells. The LD 50 s were 14.34±0.9 μg/ml, 17.33±4.1 μg/ml and 34.87±4.9 μg/ml respectively. (2) With nonlethal dose (5 μg/ml), the activity of progesterone secretion of rat lutein cells which was stimulated by hCG and pregnenolone was not influenced by anordrin while the stimulating activity of forskolin was inhibited remarkably.The results suggest that luteolytic action is the main mechanism of the termination of early pregnancy by anordrin and the direct damaging effects of anordrin on decidua and cytotrophoblasts also play a role in the termination of early pregnancy.

Relationship between Insuline-like Growth Factor-I and Progesterone Secretion of Cultured Human Trophoblast Cells in Vitro

Objective To investigate the effect of insuline-like growth factor-Ⅰ （IGF-Ⅰ） on progesterone genesis and regulation. Methods Cytotrophoblast cells were collected by trypsin-collagenase digestion and percoll gradient centrifugation for primary culture. After stimulated with different concentrations（100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 μg/ml） of IGF-Ⅰ at the same time and with different duration（12 h,24 h,48 h, 72 h） of IGF-Ⅰ with the same concentration, progesterone levels in the media were measured by radioimmunoassay. Simultaneously, semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） was applied to determine the expression of low density lipoprotein receptor （LDLR） mRNA. Results Progesterone levels correlated positively with IGF-Ⅰ along with the IGF-Ⅰ concentration increasing, progesterone level began to increase at 12 h, and reached the climax at 48 h when cultured with 100 μg/L IGF-Ⅰ. The expression of LDLR mRNA was detectable in every group and accordant with variation of progesterone level. Conclusion Progesterone secretion has time- and dose-dependent effect on IGF-Ⅰ, and IGF-1 can up-regulate the expression of LDLR mRNA. IGF-Ⅰ may play an important role in promoting secretion of progesterone in trophoblast cells.

The expression of TUSC3 in Preeclampsia and the function in trophoblast cell

Objective:In this study,we aimed to explore the expression of TUSC3 in Preeclampsia and to research the potential function of TUSC3 in placental trophoblast cells.Methods:We collected 10 cases of normal placental tissues and preeclampsia placental tissues,respectively.These parturient received treatment at the First Affiliated Hospital of Hainan Medical University between June 1,2020,and December 31,2022.The expression of TUSC3 in placenta was detected by immunohistochemistry.The effect of TUSC3 on the migration and invasion of HTR8/SVneo cells was analyzed by migration assay and Transwell assay.Results:The expression of TUSC3 was slightly increased in placental villis in preeclampsia.Immunohistochemistry and qRT-PCR were used to detect the expression of TUSC3 protein and mRNA in placental tissues.TUSC3 was markedly upregulated in PE placental tissues(P<0.01).The results of migration assay and Transwell assay showed that the migration rate and the number of invasive cells were significantly decreased in HTR8 overexpressing TUSC3(P<0.01).Conclusions:TUSC3 was markedly increased in PE placental tissues and inhibited trophoblast cells migration and invasion.

Enzyme-linked immunosorbent assays for quantification of MMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosorbent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were successfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation between serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias%ranged from -12.2% to -5.2%,precision ranged from -12.4% to -1.4%,and the relative standard deviation(RSD)was less than 6.6% and 8.7%,respectively.The total error was less than 20.4%.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.

Effect of Leptin on Cytotrophoblast Proliferation and Invasion

The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated. Immunohistochemistry was used to determine the placental expression of leptin in first-trimester pregnancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, invasiveness and migration was assessed by MTT, Transwell invasion assay and migration assay respectively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that： （1） Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear mem- brane of cytotrophoblast; （2） Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly （P〈0.01）; （3） After culture with different concentrations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls （P〈0.05）. It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotro- phoblast invasion and migration activity.

Effect of interaction between long non-coding RNA malat1 and microrna-146a on trophoblast function in preeclampsia

Objective:To explore the interaction of long noncoding RNA (LncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) with microRNA (miRNA)-146a on the effect and mechanism of preeclampsia (PE) trophoblast function.Methods: Choriocarcinoma cell line JEG-3 were cultured in vitro, and JEG-3 cells were transfected into four groups, namely Control, sh-MALAT1, miR-146a-5p inhibitor and sh-MALATI+miR-146a-5p inhibitor group. The sh-MALAT1 group was transfected with sh-MALAT1, the miR-146a-5p inhibitor group was transfected with miR-146a-5p inhibitor, the sh-MALAT1+inhibitor group was co-transfected with sh-MALAT1 and miR-146a-5p inhibitor, and Isometric empty vector was added in to the Control group. The mRNA level was detected by qPCR;the target relationship between MALAT1 and miR-146a-5p was predicted by bioinformation;the proliferation ability of JEG-3 cells was detected by CCK8 experiment after the four groups of plasmids were transfected;Western blot was used to detect the expression of protein in JEG-3 cells after different treatments.Results: sh-MALATl significantly decreased sh-MALATl and increased the mRNA level of miR-146a-5p in the choriocarcinoma cell line JEG-3. sh-MALATl inhibited the proliferation of JEG-3 cells, miR-146a-5p inhibitor promoted the proliferation of JEG-3 cells and weakened the effect of sh-MALATl production. At the same time, sh-MALATl attenuates the expression of TRAF6, VEGR, and MMP2 proteins in trophoblast cells, while miR-146a-5p inhibitor can enhance its expression and reduce the inhibitory effect of sh-MALATl.Conclusion: MALATl and miR-146a can interact to affect the biological behavior of trophoblast cells in preeclampsia.

Salvianolic acid B promotes the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating matrix metalloproteinase-9 via the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B pathway

OBJECTIVE:To elucidate the regulatory effects of salvianolic acid B(Sal B)on trophoblast cells in preeclampsia(PE).METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)assays were used to detect the viability of human extravillous trophoblast HTR-8/Svneo cells induced by H_(2)O_(2)following treatment with different concentrations of Sal B.The levels of oxidative stressrelated molecules,including superoxide dismutase,glutathione-Px and malondialdehyde were detected using corresponding kits.Cell apoptosis was detected using a Terminal deoxynucleotidyl transferase(Td T)d UTP NickEnd Labeling(TUNEL)assay,and the expression of apoptosis-related proteins was detected using western blot analysis.In the present study,wound healing and Transwell assays were performed to measure the levels of cell invasion and migration.Western blot analysis was also used to detect the expression levels of epithelialmesenchymal transition-related proteins.The mechanisms underlying Sal B were further investigated using reverse transcription-quantitative real-time polymerase chain reaction(RT-q PCR)and western blot analysis,to determine the expression levels of matrix metallopeptidase 9(MMP-9)and phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(Akt).RESULTS:Sal B increased the activity of HTR-8/Svneo cells,inhibited oxidative damage and promoted the invasion and migration of trophoblast cells induced by H_(2)O_(2).Furthermore,the expression levels of MMP-9 and members of the PI3K/Akt signaling pathway were significantly decreased.The pathway agonist,LY294002,and MMP-9 inhibitor,GM6001,reversed the effects of Sal B on H_(2)O_(2)-induced cells.CONCLUSIONS:Sal B promoted the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating MMP-9 via the PI3K/Akt signaling pathway.

Decidual stromal cells recruit Th 17 cells into decidua to promote proliferation and invasion of human trophoblast cells by secreting IL-17

T helper 17 （Th17） cells have both regulatory and protective roles in physiological conditions. The Th17 subset and the cytokine interleukin-17A （IL-17A） have been implicated in the pathogenesis of certain autoimmune diseases, several types of cancer and allograft rejection. However, the role of Th17 cells at the maternal/fetal interface remains unknown. Here, we demonstrate that Th17 cells are present in decidua and are increased in the peripheral blood of 10 clinically normal pregnancies based on intracellular cytokine analysis. Our results suggest a potential role of Th17 cells in sustaining pregnancy in humans. Furthermore, we demonstrate that decidual stromal cells （DSCs） but not trophoblast cells recruit peripheral Th17 cells into the decidua by secreting CCL2. The recruited Th17 cells promote proliferation and invasion and inhibit the apoptosis of human trophoblast cells by secreting IL-17 during the first trimester of pregnancy. These findings indicate a novel role for Th17 cells in controlling the maternal-fetal relationship and placenta development.

Human trophoblast cells induced MDSCs from peripheral blood CD14＋ myelomonocytic cells via elevated levels of CCL2

Successful human pregnancy requires the maternal immune system to recognize and tolerate the semi-allogeneic fetus. Myeloid-derived suppressor cells （MDSCs）, which are capable of inhibiting T-cell responses, are highly increased in the early stages of pregnancy. Although recent reports indicate a role for MDSCs in fetal-maternal tolerance, little is known about the expansion of MDSCs during pregnancy. In the present study, we demonstrated that the trophoblast cell line HTR8/SVneo could instruct peripheral CD14＋ myelomonocytic cells toward a novel subpopulation of MDSCs, denoted as CD 14 ＋ H LA-DR-/=~w cells, with suppressive activity and increased expression of I DO 1, ARG- 1, a nd COX2. After interaction with HTR8/SVneo cells, CD14＋ myelomonocytic cells secrete high levels of CCL2, promoting the expression of signal transducer and activator of transcription 3. We utilized a neutralizing monoclonal antibody to reveal the prominent role of CCL2 in the induction of CD14＋HLA-DR-/low MDSCs. In combination, the results of the present study support a novel role for the cross-talk between the trophoblast cell line HTR8/SVneo and maternal CD14＋ myelomonocytic cells in initiating MDSCs induction, prompting a tolerogenic immune response to ensure a successful pregnancy.

The transcriptional activator Klf5 recruits p300-mediated H3K27ac for maintaining trophoblast stem cell pluripotency

The effective proliferation and differentiation of trophoblast stem cells(TSCs)is indispensable for the development of the placenta,which is the key to maintaining normal fetal growth during pregnancy.Kruppel-like factor 5(Klf5)is implicated in the activation of pluripotency gene expression in embryonic stem cells(ESCs),yet its function in TSCs is poorly understood.Here,we showed that Klf5 knockdown resulted in the downregulation of core TSC-specific genes,consequently causing rapid differentiation of TSCs.Consistently,Klf5-depleted embryos lost the ability to establish TSCs in vitro.At the molecular level,Klf5 preferentially occupied the proximal promoter regions and maintained an open chromatin architecture of key TSC-specific genes.Deprivation of Klf5 impaired the enrichment of p300,a major histone acetyl transferase of H3 lysine 27 acetylation(H3K27ac),and further reduced the occupancy of H3K27ac at promoter regions,leading to decreased transcriptional activity of TSC pluripotency genes.Thus,our findings highlight a novel mechanism of Klf5 in regulating the self-renewal and differentiation of TSCs and provide a reference for understanding placental development and improving pregnancy rates.

Immune Regulation at Maternal-fetal Interface in Early Pregnancy

Decidual immune cells(DICs),including T-cells,regulatory T-cells,macrophages/dendritic cells,natural killer cells,and neutrophils,are resident at the maternal-fetal interface,and play vital roles in regulating trophoblast migration,decidual angiogenesis,immune tolerance,placentation,and decidualization during the early pregnancy.Extensive researches have revealed that these maternal DICs cooperated with each other,or with maternal decidual stromal cells,or with fetal-derived trophoblasts,and further formed a special maternal-fetal cross talk at the maternal-fetal interface,which was essential for the construction and maintenance of physiological pregnancy.Once aberrant cross talk and immune regulation arise,many pregnancy complications will inevitably occur,such as spontaneous abortion,intrauterine growth restriction(IUGR),preeclampsia(PE),and preterm birth.Here,we reviewed how critical immune cells are either enriched or excluded from the decidua,how their function is regulated within the decidua,and how they variously contribute to pregnancy success or failure.

Three macrophage subsets are identified in the uterus during early human pregnancy

Macrophages are crucial for a successful pregnancy, and malfunctions of decidual macrophages correlate with adverse pregnancyoutcomes, such as spontaneous abortion and preeclampsia. Previously, decidual macrophages were often thought to be a singlepopulation. In the present study, we identified three decidual macrophage subsets, CCR2−CD11cLO (CD11clow, ~80%), CCR2−CD11cHI (CD11chigh, ~5%), and CCR2+CD11cHI (CD11chigh, 10–15%), during the first trimester of human pregnancy by flowcytometry analysis. CCR2−CD11cLO macrophages are widely distributed in the decidua, while CCR2−CD11cHI and CCR2+CD11cHImacrophages are primarily detected close to extravillous trophoblast cells according to immunofluorescence staining. According toRNA sequencing bioinformatics analysis and in vitro functional studies, these three subsets of macrophages have differentphagocytic capacities. CCR2+CD11cHI macrophages have pro-inflammatory characteristics, while the CCR2−CD11cHI population issuggested to be anti-oxidative and anti-inflammatory due to its high expression of critical heme metabolism-related genes,suggesting that these two subsets of macrophages maintain an inflammatory balance at the leading edge of trophoblast invasionto facilitate the clearance of pathogen infection as well as maintain the homeostasis of the maternal-fetal interface. The presentstudy physiologically identifies three decidual macrophage subsets. Further clarification of the functions of these subsets willimprove our understanding of maternal-fetal crosstalk in the maintenance of a healthy pregnancy.

Placental Development and Pregnancy-Associated Diseases

Serving as the interface between the fetal and maternal environments during gestation,the placenta plays critical roles in the protection of the developing fetus and the maintenance of maternal health.The placenta is primarily derived from the embryonic trophectoderm which differentiates into various subtypes of trophoblast cells through villous and extravillous pathways.The interactions among trophoblasts and multiple decidual cells and immune cells at the maternal-fetal interface fundamentally form the functional units of the placenta,which are responsible for blood perfusion and maternal-fetal material exchange,immune tolerance,and the regulation of pregnancy adaptation.Defects in placental development and functional maintenance are in tight association with adverse pregnancy outcomes such as preeclampsia.In this article,we review recent advances on human trophoblast cell differentiation and the construction of placental functional units and discuss the placental and maternal factors that may contribute to the occurrence of preeclampsia.

Galectin-9 Promotes Human Trophoblast Cell Invasion through Matrix Metalloproteinase-2 and p38 Signaling Pathway

Objective:Adequate extravillous trophoblast(EVT)invasion plays a crucial role in the establishment of successful pregnancy.Insufficient trophoblast migration and invasion can result in defective placentation,which is associated with a number of clinical pathological conditions of pregnancy including spontaneous abortion and preeclampsia.Galectin-9(Gal-9)has a wide variety of regulatory functions in innate and adaptive immunity during infection,tumor growth,and organ transplantation.Methods:We utilized immortalized human first-trimester EVT cells(HTR8/SVneo)for our functional study.We examined the effects of Gal-9 on viability,proliferation,and invasion of HTR8/SVneo cells,as well as on matrix metalloproteinase-2(MMP-2)production in HTR8/SVneo cells.Furthermore,we observed the effects of different MAPK-signaling pathway inhibitors on the stimulatory functions of Gal-9 on HTR8/SVneo cells’invasion.Results:We verified the secretion of Gal-9 by trophoblasts and detected a correlation between low levels of Gal-9 and spontaneous abortion.Gal-9 promoted the invasion of HTR8/SVneo cells through its interaction with Tim-3,not CD44,and subsequently increased MMP-2 production.Blockade of p38 signaling pathway inhibited Gal-9 activities in HTR8/SVneo cells.Conclusion:Gal-9 promotes human trophoblast cell invasion through MMP-2 and p38 signaling pathway in a Tim-3-dependent manner.

Frontier Progress in the Establishment of Trophoblast Stem Cell and the Identification of New Cell Subtypes at the Maternal-Fetal Interface

Proper development of the human placenta is of vital importance for a successful pregnancy,and a series of pregnancy complications are considered originating from dysfunctional placentas.Like other organ system development,placentation requires large numbers of co-regulators,while the underlying molecular mechanisms orchestrating the placental formation and function are poorly understood.Although we have made many signs of progress in understanding the placental architectures and developments using mouse models,the species-specific differences impede our progress due to the lack of appropriate model systems.In the past few years,major progress has been made by the establishment of novel in-vitro self-renewing stem cell models,as well as identifying the full picture of the cellular organization of the maternal and fetal interface.Providing the tools for the investigation of placentation and reproductive-related regulation mechanism.In this review,we focus on the detailed progress of the human trophoblast stem cells culturing system,and the cellular and molecular terrain at the maternal-fetal interface,respectively,thus providing new insights into placental development.