Human First Trimester Placenta Research Articles

Acetyl-CoA metabolism maintains histone acetylation for syncytialization of human placental trophoblast stem cells

During pregnancy, placental-fetal nutrient allocation is crucial for fetal and maternal health. However, the regulatory mechanisms for nutrient metabolism and allocation in placental trophoblasts have remained unclear. Here, we used human first-trimester placenta samples and human trophoblast stem cells (hTSCs) to discover that glucose metabolism is highly active in hTSCs and cytotrophoblasts, but during syncytialization, it decreases to basal levels, remaining necessary for fueling acetyl-CoA and differentiation potential. Acetate supplementation could rescue syncytiotrophoblast fusion from glycolysis deficiency by replenishing acetyl-CoA and maintaining histone acetylation, thus rescuing the activation of syncytialization genes. Even brief glycolysis deficiency could permanently inhibit differentiation potential and promote inflammation, which could also be permanently rescued by brief acetate supplementation invivo. These results suggest that hTSCs retain only basal glycolytic acetyl-CoA metabolism during syncytialization to regulate cell fates via nutrient-responsive histone acetylation, with implications for our understanding of the balance between placental and fetal nutrition.

P-789 Trophoblast organoids as a 3D model for the study of oxygen concentration during in vitro preimplantation embryo culture

Abstract Study question To determine if trophoblast organoids could represent a relevant robust model to study the impact of oxygen concentration on human preimplantation embryo development. Summary answer Transcriptomic analysis of trophoblast organoids cultured in monophasic (5%) or biphasic (5-2%) O2 concentration identified 1,184 RNAs differentially expressed. What is known already Our team has previously demonstrated that a biphasic (5-2%) O2 concentration strategy during preimplantation embryo culture (from Day 0 to Day 6) significantly improved blastulation and live birth rates in in vitro fertilization (IVF). We also identified differentially expressed RNAs between monophasic and biphasic O2 strategies through whole transcriptome analysis on human embryos donated for research. However, the number of embryos donated to research is limited, they are all genetically different, and their use iethically controversial, representing a significative drawback to experimentation. Latest studies on trophoblast organoids indicate that they could represent a relevant model for studying preimplantation embryo development. Study design, size, duration A prospective monocentric study approved by the Institutional Review Board (I.R.B.) was conducted between February 2022 and September 2023: four early first-trimester human placentas were collected after surgical abortions and used for derivation of organoids in a 3D culture system. The protocol for organoid culture is described in the study of Sheridan and al, 2020. Organoids were cultured with either a monophasic or a biphasic oxygen concentration strategy. Participants/materials, setting, methods Trophoblast organoids were either exposed to 5% oxygen concentration during 5 days or to 5% O2 for 3 days and to 2% O2 for 2 days. Total RNA extraction of 21 organoids for each condition was performed. After whole transcriptome analysis using human Clariom D arrays (ThermoFisher Scientific®), microarray data were normalized and differentially expressed RNAs were identified using the Transcriptome Analysis Console software; the genetic network was generated with Ingenuity Pathway Analysis (Qiagen®). Main results and the role of chance Transcriptomic analysis of trophoblast organoids cultured in monophasic (5%) or biphasic (5-2%) O2 concentration identified 1,184 RNAs that were differentially expressed depending on the O2 strategy (fold change >2 or <-2, p < 0.05), with most of them (75.1%, n = 889/1,184) being downregulated in the biphasic (5-2%) O2 group. IPA analysis highlighted downregulation of transcripts involved in cell survival and differentiation as well as upregulation of transcripts involved in embryo angiogenesis and vascularization of extraembryonic tissue. The transcript HIF1a was upregulated in organoids cultured in biphasic strategy compared to monophasic strategy (fold change 2, p = 0.0129). Comparison between differentially expressed transcripts in monophasic and biphasic strategies highlighted 22 transcripts in common between organoids and embryos donated to research from our previous study. Limitations, reasons for caution Our study was limited by a low sample size. Wider implications of the findings This innovative model of trophoblast organoids could help us to overcome the rarity as well as the genetic/epigenetic heterogeneity associated with IVF human embryos donated to the research, thus leading to the realization of large-scale experimentations and promoting the identification of new non-invasive predictive biomarkers for IVF. Trial registration number not applicable

HnRNPL expression dynamics in the embryo and placenta

Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a conserved RNA binding protein (RBP) that plays an important role in the alternative splicing of gene transcripts, and thus in the generation of specific protein isoforms. Global deficiency in hnRNPL in mice results in preimplantation embryonic lethality at embryonic day (E) 3.5. To begin to understand the contribution of hnRNPL-regulated pathways in the normal development of the embryo and placenta, we determined hnRNPL expression profile and subcellular localization throughout development. Proteome and Western blot analyses were employed to determine hnRNPL abundance between E3.5 and E17.5. Histological analyses supported that the embryo and implantation site display distinct hnRNPL localization patterns. In the fully developed mouse placenta, nuclear hnRNPL was observed broadly in trophoblasts, whereas within the implantation site a discrete subset of cells showed hnRNPL outside the nucleus. In the first-trimester human placenta, hnRNPL was detected in the undifferentiated cytotrophoblasts, suggesting a role for this factor in trophoblast progenitors. Parallel in vitro studies utilizing Htr8 and Jeg3 cell lines confirmed expression of hnRNPL in cellular models of human trophoblasts. These studies [support] coordinated regulation of hnRNPL during the normal developmental program in the mammalian embryo and placenta.

Abstract PD10-03: PD10-03 Super-enhancer-oriented integrative bioinformatics identifies aberrant KLF4 signaling in endocrine-resistant breast cancer (BC)

Abstract Background: Recent studies suggest that enhancer reprogramming underlies heterogeneity and disease progression in estrogen receptor-positive (ER+) BC. Cell-type/state specific transcription is governed by high-order assemblies of master transcription factors (TFs) and epigenetically defined regulatory regions including super-enhancers (SEs). We previously showed that aberrant activation of the pioneer TF FOXA1 promotes enhancer and transcriptional reprogramming in endocrine-resistant BC, involving the ER and the AP-1 FRA1 and c-JUN TFs. As SEs maintain a robust cell-type/state specific core transcriptional regulatory circuitry (CRC) in developmental and tumorigenic processes, we sought to identify key additional TFs in SE/FOXA1-driven CRCs in endocrine resistance, which could serve as attractive therapeutic targets. Methods: TF binding motif at the shared SEs (mapped by H3K27ac ChIP-seq) between MCF7-parental (P) cells with ectopic FOXA1 overexpression (OE) and the endogenous FOXA1-amplifed tamoxifen-resistant (TamR) cells was analyzed by HOMER. ER-bound SEs distinguishing TamR vs. P cells were defined by integrating the SEs with our prior ER ChIP-seq data (PMID 28507152). KLF4 motif within these ER-bound SEs was scanned using FIMO and linked to nearby genes by intersection with the previously defined promoter-tethered regions (PTRs) (PMID 24141950). Differential gene expression in MCF7-TamR cells upon KLF4 knockdown (KD) by 3 unique siRNAs was analyzed using limma from edgeR. The biological and clinical significance of the KLF4-dependent genes was analyzed using Gene Ontology and survival modeling with METABRIC and the ER+ metastatic BC cohort (SABCS19-GS2-02). Cell migration was assessed by the wound-healing assay. Results: We identified KLF4 among the top enriched TF binding motifs at the shared SEs in FOXA1-overexpressing MCF7-P cells and the FOXA1-amplified TamR cells. Analysis of our prior RNA-seq data of MCF7-P and TamR cells upon OE or siRNA KD of FOXA1, FRA1, or c-JUN (PMIDs 27791031, 31826955, 32424275, SABCS21-PD1-05) revealed KLF4, the Yamanaka factor for induced pluripotent stem cells, as a common target activated by the FOXA1/FRA1/c-JUN axis. We next identified 44 genes commonly down-regulated upon KLF4 KD in the MCF7-TamR cells. This KLF4-dependent 44-gene set was enriched in biological processes of embryonic development and tumor progression, preferentially dependent on ER in MCF7-TamR vs. P cells, highly elevated in ER+ metastases vs. primary tumors, and associated with poor outcome in ER+ BC treated with endocrine therapy. KLF4 KD, using the 2 siRNAs that generated similar pathway perturbations in MCF7-TamR cells, reduced TamR cell migration. Notably, among the genes co-dependent on KLF4 and ER in TamR cells, PYGB was the only gene with a PTR residing in an ER-bound SE established in TamR but not P cells. Glycogen phosphorylase B, encoded by PYGB, is the rate-limiting enzyme in glycogen degradation and plays a role in the progression of various tumors. Expression of KLF4, FOXA1, and FRA1 are commonly activated during the differentiation of human embryonic stem cells into foregut endoderm and in the inner core of fibroblasts of first-trimester human placenta villi, suggesting a unique role of KLF4 in mediating lineage-specific CRC, possibly by engaging PYGB and the glycogen metabolic pathway in advanced ER+ disease. Conclusions: Using SE-oriented integrative bioinformatics, we identified KLF4 as a potential novel target in the FOXA1/AP-1 transcriptional axis. As KLF4 binding motif resides in the unique ER-bound SEs of TamR cells, KLF4 likely forms an auto-regulated loop amplifying CRC in transcriptional reprogramming, among which the PYGB/glycogen metabolic pathway merits further investigation in endocrine-resistant ER+ disease. Citation Format: Chia Chia Liu, Lanfang Qin, Shanunak Sathe, Sarmistha Nanda, Jamunarani Veeraraghavan, Ofir Cohen, Nikhil Wagle, Mothaffar Rimawi, C. Kent Osborne, Xiaoyong Fu, Rachel Schiff. PD10-03 Super-enhancer-oriented integrative bioinformatics identifies aberrant KLF4 signaling in endocrine-resistant breast cancer (BC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-03.

Establishment of human induced trophoblast stem cells via reprogramming of fibroblasts.

During early mammalian embryonic development, trophoblast cells play an essential role in establishing cell-cell interactions at the maternal-fetal interface to ensure a successful pregnancy. In a recent study, we showed that human fibroblasts can be reprogrammed into induced trophoblast stem (iTS) cells by transcription factor-mediated nuclear reprogramming using the Yamanaka factors OCT4, KLF4, SOX2 and c-MYC (OKSM) and a selection of TS cell culture conditions. The derivation of TS cells from human blastocysts or first-trimester placenta can be limited by difficulties in obtaining adequate material as well as ethical implications. By contrast, the described approach allows the generation of iTS cells from the adult cells of individuals with diverse genetic backgrounds, which are readily accessible to many laboratories around the world. Here we describe a step-by-step protocol for the generation and establishment of human iTS cells directly from dermal fibroblasts using a non-integrative reprogramming method. The protocol consists of four main sections: (1) recovery of cryopreserved human dermal fibroblasts, (2) somatic cell reprogramming, (3) passaging of reprogramming intermediates and (4) derivation of iTS cell cultures followed by routine maintenance of iTS cells. These iTS cell lines can be established in 2-3 weeks and cultured long term over 50 passages. We also discuss several characterization methods that can be performed to validate the iTS cells derived using this approach. Our protocol allows researchers to generate patient-specific iTS cells to interrogate the trophoblast and placenta biology as well as their interactions with embryonic cells in health and diseases.

Human placental lipid content and lipid metabolic enzyme abundance in obesity and across gestation.

Changes in placental lipid metabolism influence the delivery of lipids critical for fetal development and fetal requirements for lipids change across gestation. We hypothesized that placental lipid content and metabolic enzyme protein levels increase across gestation and are elevated in obesity. Placentas (4-40 weeks' gestation) were collected from control (body mass index, BMI = 18.5-24.9, n=37) and obese (BMI > 30, n=19) pregnant women. Trophoblast villous tissue was homogenized and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) for phospholipid and triacylglycerol (TAG) analysis and western blot for protein quantification. The placental content of TAG species and nine of 35 identified phosphatidylcholines (PC) were significantly higher (P<0.05) in first trimester (28-79%, 10-47%, respectively). Furthermore, two TAG and three PC differed by maternal BMI and were significantly increased (P<0.05) in the obese group in first trimester (72-87%, 88-119%, respectively). Placental protein abundance of glycerol-2-phosphate (GPAT3) and 1-acyl-sn-glycerol-3-phosphate acyltransferase 2 (AGPAT2), involved in de novo synthesis of PC and TAG, were higher (P<0.05) in the first trimester (66 and 74%, respectively). The protein abundance of the PC-remodeling enzyme PLA2G4c was also higher (63%) in first trimester (P<0.05). In conclusion, the placental content of many phospholipid and TAG species and the protein level of associated synthesis enzymes are higher in first-trimester human placenta. The high PC content may be related to the rapid membrane expansion in early pregnancy and the low placental oxygen tension may promote the accumulation of tissue TAGs in first trimester. Maternal obesity had only limited impact on placental lipid content and metabolic enzyme protein abundance.

Hippo signaling cofactor, WWTR1, at the crossroads of human trophoblast progenitor self-renewal and differentiation

Healthy progression of human pregnancy relies on cytotrophoblast (CTB) progenitor self-renewal and its differentiation toward multinucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, the underlying molecular mechanisms that fine-tune CTB self-renewal or direct its differentiation toward STBs or EVTs during human placentation are poorly defined. Here, we show that Hippo signaling cofactor WW domain containing transcription regulator 1 (WWTR1) is a master regulator of trophoblast fate choice during human placentation. Using human trophoblast stem cells (human TSCs), primary CTBs, and human placental explants, we demonstrate that WWTR1 promotes self-renewal in human CTBs and is essential for their differentiation to EVTs. In contrast, WWTR1 prevents induction of the STB fate in undifferentiated CTBs. Our single-cell RNA sequencing analyses in first-trimester human placenta, along with mechanistic analyses in human TSCs revealed that WWTR1 fine-tunes trophoblast fate by directly regulating WNT signaling components. Importantly, our analyses of placentae from pathological pregnancies show that extreme preterm births (gestational time ≤28 wk) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish the critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal versus differentiation. It plays positive instructive roles in promoting CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from attaining the STB fate.

Maternal Fatty Acid Metabolism in Pregnancy and Its Consequences in the Feto-Placental Development.

During pregnancy, maternal plasma fatty acids are critically required for cell growth and development, cell signaling, and the development of critical structural and functional aspects of the feto-placental unit. In addition, the fatty acids modulate the early stages of placental development by regulating angiogenesis in the first-trimester human placenta. Preferential transport of maternal plasma long-chain polyunsaturated fatty acids during the third trimester is critical for optimal fetal brain development. Maternal status such as obesity, diabetes, and dietary intakes may affect the functional changes in lipid metabolic processes in maternal-fetal lipid transport and metabolism. Fatty acids traverse the placental membranes via several plasma membrane fatty acid transport/binding proteins (FAT, FATP, p-FABPpm, and FFARs) and cytoplasmic fatty acid-binding proteins (FABPs). This review discusses the maternal metabolism of fatty acids and their effects on early placentation, placental fatty acid transport and metabolism, and their roles in feto-placental growth and development. The review also highlights how maternal fat metabolism modulates lipid processing, including transportation, esterification, and oxidation of fatty acids.

Dysregulated BMP2 in the Placenta May Contribute to Early-Onset Preeclampsia by Regulating Human Trophoblast Expression of Extracellular Matrix and Adhesion Molecules

Many pregnancy disorders, including early-onset preeclampsia (EOPE), are associated with defects in placental trophoblast cell invasion and differentiation during early placental development. Bone morphogenetic protein 2 (BMP2) belongs to the TGF-β superfamily and controls various physiological and developmental processes. However, the expression of BMP2 in the placenta and underlying molecular mechanisms of how BMP2 regulates trophoblast function remain unclear. In this study, we analyzed several publicly available microarray and RNA-seq datasets and revealed differences in expression of TGF-β superfamily members between gestational age-matched non-preeclamptic control and EOPE placentas. Importantly, BMP2 levels were significantly reduced in EOPE placentas compared with controls, and RNAscope in situ hybridization further demonstrated BMP2 expression was disrupted in EOPE placental villi. To explore the molecular mechanisms of BMP2-regulated early trophoblast differentiation, we examined BMP2 expression in first-trimester human placenta and found it to be localized to all subtypes of trophoblasts and the decidua. RNA-seq analysis on control and BMP2-treated primary human trophoblast cells identified 431 differentially expressed genes, including several canonical TGF-β/BMP signaling targets (BAMBI, ID1, INHBA, IGFBP3). Gene ontology annotations revealed that differentially expressed genes were involved in cell adhesion and extracellular matrix organization. Furthermore, we identified adhesion molecule with IgG-like domain 2 (AMIGO2) as a novel target for BMP2 that contributed to BMP2-induced trophoblast invasion and endothelial-like tube formation. Overall, our findings provide insight into the molecular processes controlled by BMP2 during early placental development that may contribute to the pathogenesis of EOPE.

Age and Sex-Related Changes in Human First-Trimester Placenta Transcriptome and Insights into Adaptative Responses to Increased Oxygen.

Physiological oxygen tension rises dramatically in the placenta between 8 and 14 weeks of gestation. Abnormalities in this period can lead to gestational diseases, whose underlying mechanisms remain unclear. We explored the changes at mRNA level by comparing the transcriptomes of human placentas at 8–10 gestational weeks and 12–14 gestational weeks. A total of 20 samples were collected and divided equally into four groups based on sex and age. Cytotrophoblasts were isolated and sequenced using RNAseq. Key genes were identified using two different methods: DESeq2 and weighted gene co-expression network analysis (WGCNA). We also constructed a local database of known targets of hypoxia-inducible factor (HIF) subunits, alpha and beta, to investigate expression patterns likely linked with changes in oxygen. Patterns of gene enrichment in and among the four groups were analyzed based on annotations of gene ontology (GO) and KEGG pathways. We characterized the similarities and differences between the enrichment patterns revealed by the two methods and the two conditions (age and sex), as well as those associated with HIF targets. Our results provide a broad perspective of the processes that are active in cytotrophoblasts during the rise in physiological oxygen, which should benefit efforts to discover possible drug-targeted genes or pathways in the human placenta.

Induction of Human Trophoblast Stem Cells from Somatic Cells and Pluripotent Stem Cells

Human trophoblast stem cells (hTSCs) derived from blastocysts and first-trimester cytotrophoblasts offer an unprecedented opportunity to study the placenta. However, access to human embryos and first-trimester placentas is limited, thus preventing the establishment of hTSCs from diverse genetic backgrounds associated with placental disorders. Here, we show that hTSCs can be generated from numerous genetic backgrounds using post-natal cells via two alternative methods: (1) somatic cell reprogramming of adult fibroblasts with OCT4, SOX2, KLF4, MYC (OSKM) and (2) cell fate conversion of naive and extended pluripotent stem cells. The resulting induced/converted hTSCs recapitulated hallmarks of hTSCs including long-term self-renewal, expression of specific transcription factors, transcriptomic signature, and the potential to differentiate into syncytiotrophoblast and extravillous trophoblast cells. We also clarified the developmental stage of hTSCs and show that these cells resemble day 8 cytotrophoblasts. Altogether, hTSC lines of diverse genetic origins open the possibility to model both placental development and diseases in a dish.

GDF8 Promotes the Cell Invasiveness in Human Trophoblasts by Upregulating the Expression of Follistatin-Like 3 Through the ALK5-SMAD2/3 Signaling Pathway.

Growth differentiation factor 8 (GDF8) and its antagonist follistatin-like 3 (FSTL3) are expressed in the placenta during early pregnancy. These two factors may have a role to play in the regulation of normal placentation. However, whether GDF8 can regulate the expression of FSTL3 in human trophoblasts remains to be elucidated. In this study, we aimed to investigate the effects of GDF8 on the expression of FSTL3 and the underlying molecular mechanisms using human trophoblasts as a study model. Our results showed that GDF8 significantly upregulates the expression and production of FSTL3, which further promotes cell invasiveness in immortalized extravillous cytotrophoblast cells and primary extravillous cytotrophoblast cells obtained from human first-trimester placentae. Additionally, using an siRNA-mediated knockdown approach, we found that this regulatory effect is most likely mediated by the ALK5-Sma- and Mad-related protein (SMAD)2/3-induced signaling pathway. These findings deepen our understanding of the functional roles of GDF8 and FSTL3 in the regulation of cell invasiveness of trophoblasts.

Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta.

The human placenta is essential for successful reproduction. There is great variation in the anatomy and development of the placenta in different species, meaning that animal models provide limited information about human placental development and function. Until recently, it has been impossible to isolate trophoblast cells from the human placenta that proliferate in vitro. This has limited our ability to understand pregnancy disorders. Generating an in vitro model that recapitulates the unique features of the human placenta has been challenging. The first in vitro model system of human trophoblast that could be cultured long term and differentiated to syncytiotrophoblast (SCT) and extravillous trophoblast (EVT) was a two-dimensional (2D) culture system of human trophoblast stem cells. Here, we describe a protocol to isolate trophoblast from first-trimester human placentas that can be grown long term in a three-dimensional (3D) organoid culture system. Trophoblast organoids can be established within 2-3 weeks, passaged every 7-10 d, and cultured for over a year. The structural organization of these human trophoblast organoids closely resembles the villous placenta with a layer of cytotrophoblast (VCT) that differentiates into superimposed SCT. Altering the composition of the medium leads to differentiation of the trophoblast organoids into HLA-G+ EVT cells which rapidly migrate and invade through the Matrigel droplet in which they are cultured. Our previous research confirmed that there is similarity between the trophoblast organoids and in vivo placentas in their transcriptomes and ability to produce placental hormones. This organoid culture system provides an experimental model to investigate human placental development and function as well as interactions of trophoblast cells with the local and systemic maternal environment.

TEAD4 ensures postimplantation development by promoting trophoblast self-renewal: An implication in early human pregnancy loss

Early pregnancy loss affects ∼15% of all implantation-confirmed human conceptions. However, evolutionarily conserved molecular mechanisms that regulate self-renewal of trophoblast progenitors and their association with early pregnancy loss are poorly understood. Here, we provide evidence that transcription factor TEAD4 ensures survival of postimplantation mouse and human embryos by controlling self-renewal and stemness of trophoblast progenitors within the placenta primordium. In an early postimplantation mouse embryo, TEAD4 is selectively expressed in trophoblast stem cell-like progenitor cells (TSPCs), and loss of Tead4 in postimplantation mouse TSPCs impairs their self-renewal, leading to embryonic lethality before embryonic day 9.0, a developmental stage equivalent to the first trimester of human gestation. Both TEAD4 and its cofactor, yes-associated protein 1 (YAP1), are specifically expressed in cytotrophoblast (CTB) progenitors of a first-trimester human placenta. We also show that a subset of unexplained recurrent pregnancy losses (idiopathic RPLs) is associated with impaired TEAD4 expression in CTB progenitors. Furthermore, by establishing idiopathic RPL patient-specific human trophoblast stem cells (RPL-TSCs), we show that loss of TEAD4 is associated with defective self-renewal in RPL-TSCs and rescue of TEAD4 expression restores their self-renewal ability. Unbiased genomics studies revealed that TEAD4 directly regulates expression of key cell cycle genes in both mouse and human TSCs and establishes a conserved transcriptional program. Our findings show that TEAD4, an effector of the Hippo signaling pathway, is essential for the establishment of pregnancy in a postimplantation mammalian embryo and indicate that impairment of the Hippo signaling pathway could be a molecular cause for early human pregnancy loss.

LncRNA MEG3 inhibits trophoblast invasion and trophoblast‐mediated VSMC loss in uterine spiral artery remodeling

Extravillous trophoblasts (EVTs) migrate into uterine decidua and induce vascular smooth muscle cell (VSMC) loss through mechanisms thought to involve migration and apoptosis, achieving complete spiral artery remodeling. Long noncoding RNA maternally expressed gene 3 (MEG3) can regulate diverse cellular processes, such as proliferation and migration, and has been discovered highly expressed in human placenta tissues. However, little is known about the role of MEG3 in modulating EVT functions and EVT-induced VSMC loss. In this study, we first examined the location of MEG3 in human first-trimester placenta by in situ hybridization. Then, exogenous upregulation of MEG3 in HTR-8/SVneo cells was performed to investigate the effects of MEG3 on EVT motility and EVT capacity to displace VSMCs. Meanwhile, the molecules mediating EVT-induced VSMC loss, such as tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) were detected at transcriptional and translational levels. Finally, VSMCs were cocultured with MEG3-upregulated HTR-8/SVneo to explore the role of MEG3 on EVT-mediated VSMC migration and apoptosis. Results showed that MEG3 was expressed in trophoblasts in placental villi and decidua, and MEG3 enhancement inhibited HTR-8/SVneo migration and invasion. Meanwhile, the displacement of VSMCs by HTR-8/SVneo and the expression of TNF-α, FasL and TRAIL in HTR-8/SVneo were reduced following MEG3 overexpression in HTR-8/SVneo. Furthermore, HTR-8/SVneo with MEG3 upregulation impaired VSMC migration and apoptosis. The PI3K/Akt pathway, which is possibly downstream, was inactivated in MEG3-upregulated HTR-8/SVneo. These findings suggest that MEG3 might be a negative regulator of spiral artery remodeling via suppressing EVT invasion and EVT-mediated VSMC loss.

Sex-Specific Gene Expression Differences Are Evident in Human Embryonic Stem Cells and During In Vitro Differentiation of Human Placental Progenitor Cells

The placenta is a short-lived tissue required for embryonic growth and survival, and it is fetal derived. Fetal sex influences gestation, and many sexual dimorphic diseases have origins in utero. There is sex-biased gene expression in third-trimester human placentas, yet the origin of sex-specific expression is unknown. Here, we used an in vitro differentiation model to convert human embryonic stem cells (hESCs) into trophoblastic progenitor cells of the first-trimester placenta, which will eventually become mature extravillous trophoblasts and syncytiotrophoblasts. We observed significant sex differences in transcriptomic profiles of hESCs and trophoblastic progenitors, and also with the differentiation process itself. Male cells had higher dosage of X/Y gene pairs relative to female samples, supporting functions for Y-linked genes beyond spermatogenesis in the hESCs and in the early placenta. Female-specific differentiation altered the expression of several thousand genes compared with male cells, and female cells specifically upregulated numerous autosomal genes with known roles in trophoblast function. Sex-biased upregulation of cellular pathways during trophoblast differentiation was also evident. This study is the first to identify sex differences in trophoblastic progenitor cells of the first-trimester human placenta, and reveal early origins for sexual dimorphism.

XCL1-XCR1 pathway promotes trophoblast invasion at maternal-fetal interface by inducing MMP-2/MMP-9 activity.

Certain chemokines with their receptors can promote or inhibit trophoblast cell migration and invasion in human first-trimester placenta. Whether the lymphotactin (Lptn; XCL1)-XC chemokine receptor 1 (XCR1) chemokine pathway affects trophoblast cell migration and invasion in human first-trimester placenta remains unclear. The expression pattern of chemokine XCL1 and its receptor XCR1 was detected in human first-trimester by qRT-PCR, and the effect of recombinant human XCL1 (rhXCL1) on trophoblast cell function was tested by wound healing and Transwell assays. Matrix metalloproteinase (MMP) activity in trophoblast cells treated with rhXCL1 was assessed via qRT-PCR and gelatin zymography. Abundant XCR1 mRNA was expressed in the first-trimester decidua and villi. XCL1 and XCR1 mRNA were expressed at a higher level in the first-trimester than in the term placenta. RhXCL1 promoted trophoblast cell migration and invasion by increasing MMP-9 and MMP-2 activity and that of the MMP-2/tissue inhibitor of metalloproteinases 2 (TIMP-2) complex via the phosphatidylinositol 3-kinase (PI3K)/AKT kinase (AKT), mitogen-activated protein kinase (MEK), and JUN N-terminal kinase (JNK) signaling pathways. XCL1-XCR1 chemokine pathway promotes trophoblast invasion by increasing matrix metalloproteinase activity in human first-trimester placenta.

Zika Virus Replicates in Proliferating Cells in Explants From First-Trimester Human Placentas, Potential Sites for Dissemination of Infection.

Maternal Zika virus (ZIKV) infection with prolonged viremia leads to fetal infection and congenital Zika syndrome. Previously, we reported that ZIKV infects primary cells from human placentas and fetal membranes. Here, we studied viral replication in numerous explants of anchoring villi and basal decidua from first-trimester human placentas and midgestation amniotic epithelial cells (AmEpCs). Explants and AmEpCs were infected with American and African ZIKV strains at low multiplicities, and ZIKV proteins were visualized by immunofluorescence. Titers of infectious progeny, cell proliferation, and invasiveness were quantified. In anchoring villus, ZIKV replicated reproducibly in proliferating cytotrophoblasts in proximal cell columns, dividing Hofbauer cells in villus cores, and invasive cytotrophoblasts, but frequencies differed. Cytotrophoblasts in explants infected by Nicaraguan strains were invasive, whereas those infected by prototype MR766 largely remained in cell columns, and titers varied by donor and strain. In basal decidua, ZIKV replicated in glandular epithelium, decidual cells, and immune cells. ZIKV-infected AmEpCs frequently occurred in pairs and expressed Ki67 and phosphohistone H3, indicating replication in dividing cells. ZIKV infection in early pregnancy could target proliferating cell column cytotrophoblasts and Hofbauer cells, amplifying infection in basal decidua and chorionic villi and enabling transplacental transmission.

Placental DAPK1 and autophagy marker LC3B-II are dysregulated by TNF-\u03b1 in a gestational age-dependent manner

Autophagy, a cell-survival process responsible for degradation of protein aggregates and damaged organelles, is increasingly recognized as another mechanism essential for human placentation. A substantial body of experiments suggests inflammation and oxidative stress as the underlying stimuli for altered placental autophagy, giving rise to placenta dysfunction and pregnancy pathologies. Here, the hypothesis is tested whether or not pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α are able to influence the expression profile of autophagy genes in human first-trimester villous placenta. Autophagy-focused qPCR arrays identified substantial downregulation of death-associated protein kinase 1 (DAPK1) in first-trimester placental explants in response to IL-6 and TNF-α, respectively. Immunohistochemistry of placental explants detected considerable DAPK1 staining in placental macrophages, villous cytotrophoblasts and less intense in the syncytiotrophoblast. Both immunohistochemistry and Western blot showed decreased DAPK1 protein in TNF-α-treated placental explants compared to control. On cellular level, DAPK1 expression decreased in SGHPL-4 trophoblasts in response to TNF-α. Observed changes in the expression profile of autophagy-related genes were reflected by significantly decreased lipidation of autophagy marker microtubule-associated protein light chain 3 beta (LC3B-II) in first trimester placental explants in response to TNF-α. Analysis of TNF-α-treated term placental explants showed decreased DAPK1 protein, whereas in contrast to first-trimester LC3B expression and lipidation increased. Immunohistochemistry of placental tissues from early-onset preeclampsia (PE) showed less DAPK1 staining, when compared to controls. Accordingly, DAPK1 mRNA and protein were decreased in primary trophoblasts isolated from early-onset PE, while LC3B-I and -II were increased. Results from this study suggest that DAPK1, a regulator of apoptosis, autophagy and programmed necrosis, decreases in human placenta in response to elevated maternal TNF-α, irrespective of gestational age. In contrast, TNF-α differentially regulates levels of autophagy marker LC3B in human placenta over gestation.

Gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor are expressed at tubal ectopic pregnancy implantation sites

To investigate whether gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) are expressed at tubal ectopic pregnancy sites, and to study the potential role of GnRH signaling in regulating immortalized human trophoblast cell viability. Immunohistochemical and experimental studies. Academic research laboratory. Fallopian tube implantation sites (n = 25) were collected from women with ectopic pregnancy. First-trimester human placenta biopsies (n = 5) were obtained from elective terminations of pregnancy. None. GnRH and GnRHR expression was examined by means of immunohistochemistry and histoscoring. Trophoblastic BeWo choriocarcinoma and immortalized extravillous trophoblast (HTR-8/SVneo) cell viability was examined by means of cell counting after incubation with GnRH and/or GnRH antagonist (Antide). GnRH and GnRHR immunoreactivity was detected in cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast in all women with tubal pregnancy. GnRH immunoreactivity was higher and GnRHR immunoreactivity lower in syncytiotrophoblast compared with cytotrophoblast. GnRH and GnRHR immunoreactivity was detected in adjacent fallopian tube epithelium. Whereas neither GnRH nor Antide altered HTR-8/SVneo cell viability, treatment with GnRH significantly increased the overall cell viability of BeWo cells at 48 and 72hours, and these effects were abolished by pretreatment with Antide. GnRH and GnRHR are expressed in trophoblast cell populations and fallopian tube epithelium at tubal ectopic pregnancy sites. GnRH increases BeWo cell viability, an effect mediated by the GnRHR. Further work is required to investigate the potential role of GnRH signaling in ectopic pregnancy.