CircTHBS1 promotes trophoblast cell migration and invasion and inhibits trophoblast apoptosis by regulating miR-136-3p/IGF2R axis.

The precise molecular mechanism behind fetal growth restriction (FGR) is still unclear, although there is a strong connection between placental dysfunction, inadequate trophoblast invasion, and its etiology and pathogenesis. As a new type of non-coding RNA, circRNA has been shown to play a crucial role in the development of FGR. This investigation identified the downregulation of hsa_circ_0034533 (circTHBS1) in FGR placentas through high-sequencing analysis and confirmed this finding in 25 clinical placenta samples using qRT-PCR. Subsequent in vitro functional assays demonstrated that silencing circTHBS1 inhibited trophoblast proliferation, migration, invasion, and epithelial mesenchymal transition (EMT) progression and promoted apoptosis. Furthermore, when circTHBS1 was overexpressed, cell function experiments showed the opposite result. Analysis using fluorescence in situ hybridization revealed that circTHBS1 was primarily found in the cytoplasmic region. Through bioinformatics analysis, we anticipated the involvement of miR-136-3p and IGF2R in downstream processes, which was subsequently validated through qRT-PCR and dual-luciferase assays. Moreover, the inhibition of miR-136-3p or the overexpression of IGF2R partially reinstated proliferation, migration, and invasion abilities following the silencing of circTHBS1. In summary, the circTHBS1/miR-136-3p/IGF2R axis plays a crucial role in the progression and development of FGR, offering potential avenues for the exploration of biological indicators and treatment targets.

Decidual γδT cells of early human pregnancy produce angiogenic and immunomodulatory proteins while also possessing cytotoxic potential.

During pregnancy, the maternal immune system must allow and support the growth of the developing placenta while maintaining the integrity of the mother's body. The trophoblast's unique HLA signature is a key factor in this physiological process. This study focuses on decidual γδT cell populations and examines their expression of receptors that bind to non-classical HLA molecules, HLA-E and HLA-G. We demonstrate that decidual γδT cell subsets, including Vδ1, Vδ2, and double-negative (DN) Vδ1-/Vδ2- cells express HLA-specific regulatory receptors, such as NKG2C, NKG2A, ILT2, and KIR2DL4, each with varying dominance. Furthermore, decidual γδT cells produce cytokines (G-CSF, FGF2) and cytotoxic mediators (Granulysin, IFN-γ), suggesting functions in placental growth and pathogen defense. However, these processes seem to be controlled by factors other than trophoblast-derived non-classical HLA molecules. These findings indicate that decidual γδT cells have the potential to actively contribute to the maintenance of healthy human pregnancy.

High glucose-induced p66Shc mitochondrial translocation regulates autophagy initiation and autophagosome formation in syncytiotrophoblast and extravillous trophoblast.

BACKGROUND: p66Shc, as a redox enzyme, regulates reactive oxygen species (ROS) production in mitochondria and autophagy. However, the mechanisms by which p66Shc affects autophagosome formation are not fully understood. METHODS: p66Shc expression and its location in the trophoblast cells were detected in vivo and in vitro. Small hairpin RNAs or CRISPR/Cas9, RNA sequencing, and confocal laser scanning microscope were used to clarify p66Shc's role in regulating autophagic flux and STING activation. In addition, p66Shc affects mitochondrial-associated endoplasmic reticulum membranes (MAMs) formation were observed by transmission electron microscopy (TEM). Mitochondrial function was evaluated by detected cytoplastic mitochondrial DNA (mtDNA) and mitochondrial membrane potential (MMP). RESULTS: High glucose induces the expression and mitochondrial translocation of p66Shc, which promotes MAMs formation and stimulates PINK1-PRKN-mediated mitophagy. Moreover, mitochondrial localized p66Shc reduces MMP and triggers cytosolic mtDNA release, thus activates cGAS/STING signaling and ultimately leads to enhanced autophagy and cellular senescence. Specially, we found p66Shc is required for the interaction between STING and LC3II, as well as between STING and ATG5, thereby regulates cGAS/STING-mediated autophagy. We also identified hundreds of genes associated several biological processes including aging are co-regulated by p66Shc and ATG5, deletion either of which results in diminished cellular senescence. CONCLUSION: p66Shc is not only implicated in the initiation of autophagy by promoting MAMs formation, but also helps stabilizing active autophagic flux by activating cGAS/STING pathway in trophoblast.

Implication of viruses in the etiology of preeclampsia.

Preeclampsia is one of the most common disorders that poses threat to both mothers and neonates and a major contributor to perinatal morbidity and mortality worldwide. Viral infection during pregnancy is not typically considered to cause preeclampsia; however, syndromic nature of preeclampsia etiology and the immunomodulatory effects of viral infections suggest that microbes could trigger a subset of preeclampsia. Notably, SARS-CoV-2 infection is associated with an increased risk of preeclampsia. Herein, we review the potential role of viral infections in this great obstetrical syndrome. According to in vitro and in vivo experimental studies, viral infections can cause preeclampsia by introducing poor placentation, syncytiotrophoblast stress, and/or maternal systemic inflammation, which are all known to play a critical role in the development of preeclampsia. Moreover, clinical and experimental investigations have suggested a link between several viruses and the onset of preeclampsia via multiple pathways. However, the results of experimental and clinical research are not always consistent. Therefore, future studies should investigate the causal link between viral infections and preeclampsia to elucidate the mechanism behind this relationship and the etiology of preeclampsia itself.

Unlocking trophectoderm mysteries: In vivo and in vitro perspectives on human and mouse trophoblast fate induction.

In recent years, the pursuit of inducing the trophoblast stem cell (TSC) state has gained prominence as a compelling research objective, illuminating the establishment of the trophoblast lineage and unlocking insights into early embryogenesis. In this review, we examine how advancements in diverse technologies, including in vivo time course transcriptomics, cellular reprogramming to TSC state, chemical induction of totipotent stem-cell-like state, and stem-cell-based embryo-like structures, have enriched our insights into the intricate molecular mechanisms and signaling pathways that define the mouse and human trophectoderm/TSC states. We delve into disparities between mouse and human trophectoderm/TSC fate establishment, with a special emphasis on the intriguing role of pluripotency in this context. Additionally, we re-evaluate recent findings concerning the potential of totipotent-stem-like cells and embryo-like structures to fully manifest the trophectoderm/trophoblast lineage's capabilities. Lastly, we briefly discuss the potential applications of induced TSCs in pregnancy-related disease modeling.

Phenylbutyric acid inhibits hypoxia-induced trophoblast apoptosis and autophagy in preeclampsia via the PERK/ATF-4/CHOP pathway.

Preeclampsia (PE) is a common pregnancy complication with a high mortality rate. Abnormally activated endoplasmic reticulum stress (ERS) is believed to be responsible for the destruction of key placental cells-trophoblasts. Phenylbutyric acid (4-PBA), an ERS inhibitor, is involved in regulating the development of ERS-related diseases. At present, how 4-PBA affects trophoblasts and its mechanisms is still unclear. In this study, PE cell models were established by stimulating HTR-8/SVneo cells with hypoxia. To verify the underlying mechanisms of 4-PBA on PE, CCT020312, an activator of PERK, was also used. The results showed that 4-PBA restored hypoxia-induced trophoblast viability, inhibited HIF-1α protein expression, inflammation, and PERK/ATF-4/CHOP pathway. Hoechst 33342 staining and flow cytometry results confirmed that 4-PBA decreased hypoxia-induced apoptosis in trophoblasts. The results of the JC-1 analysis and apoptosis initiation enzyme activity assay also demonstrated that 4-PBA inhibited apoptosis related to the mitochondrial pathway. Furthermore, by detecting autophagy in trophoblasts, an increased number of autophagic vesicles, damaged mitochondria, enhanced dansylcadaverine fluorescence, enhanced levels of autophagy proteins Beclin-1, LC3II, and decreased p62 were seen in hypoxia-stimulated cells. These changes were reversed by 4-PBA. Furthermore, it was observed that CCT020312 reversed the effects of 4-PBA on the viability, apoptosis, and autophagosome number of hypoxia-induced trophoblasts. In summary, 4-PBA reduces autophagy and apoptosis via the PERK/ATF-4/CHOP pathway and mitochondrial pathway, thereby restoring the viability of hypoxic trophoblasts. These findings provide a solid evidence base for the use of 4-PBA in PE treatment and guide a new direction for improving the outcomes of patients with PE.

ENO1 promotes trophoblast invasion regulated by E2F8 in recurrent miscarriage.

Recurrent miscarriage (RM) is related to the dysfunction of extravillous trophoblast cells (EVTs), but the comprehensive mechanisms remain largely unexplored. We analyzed single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing and microarray datasets obtained from Gene Expression Omnibus (GEO) database to explore the hub genes in the mechanisms of RM. We identified 1724 differentially expressed genes (DEGs) in EVTs from the RM, and they were all expressed along the trajectory of EVTs. These DEGs were associated with hypoxia and glucose metabolism. Single-cell Regulatory Network Inference and Clustering (SCENIC) analysis revealed that E2F transcription factor (E2F) 8 (E2F8) was a key transcription factor for these DEGs. And the expression of ENO1 can be positively regulated by E2F8 via RNA sequencing analysis. Subsequently, we performed immunofluorescence assay (IF), plasmid transfection, western blotting, chromatin immunoprecipitation (ChIP), real-time quantitative polymerase chain reaction (qRT-PCR), and transwell assays for validation experiments. We found that the expression of alpha-Enolase 1 (ENO1) was lower in the placentas of RM. Importantly, E2F8 can transcriptionally regulate the expression of ENO1 to promote the invasion of trophoblast cells by inhibiting secreted frizzled-related protein 1/4 (SFRP1/4) to activate Wnt signaling pathway. Our results suggest that ENO1 can promote trophoblast invasion via an E2F8-dependent manner, highlighting a potential novel target for the physiological mechanisms of RM.

Nox2 inhibition reduces trophoblast ferroptosis in preeclampsia via the STAT3/GPX4 pathway.

AIMS: Ferroptosis, a novel mode of cell death characterized by lipid peroxidation and oxidative stress, plays an important role in the pathogenesis of preeclampsia (PE). The aim of this study is to determine the role of Nox2 in the ferroptosis of trophoblast cells, along with the underlying mechanisms. METHODS: The mRNA and protein levels of Nox2, STAT3, and GPX4 in placental tissues and trophoblast cells were respectively detected by qRT-PCR and western blot analysis. CCK8, transwell invasion and tube formation assays were used to evaluate the function of trophoblast cells. Ferroptosis was evaluated using flow cytometry and the lipid peroxidation assay. Glycolysis and mitochondrial respiration were investigated by detecting the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using Seahorse extracellular flux technology. The t-test or one-way ANOVA was used for statistical analysis. KEY FINDINGS: Nox2 was up-regulated while STAT3 and GPX4 were down-regulated in PE placental tissues. Nox2 knockdown inhibited ferroptosis in trophoblast cells, which was shown by enhanced proliferation and invasion, decreased ROS and lipid peroxide levels, and reduced glycolysis and mitochondrial dysfunction. Nox2 negatively correlated with MVD in PE placentas, and Nox2 knockdown restored ferroptosis-inhibited tube formation. Nox2 could interact with STAT3. Inhibiting Nox2 restored ferroptosis-induced alterations in the mRNA and protein levels of STAT3 and GPX4. SIGNIFICANCE: Nox2 may trigger ferroptosis through the STAT3/GPX4 pathway, subsequently leading to regulation of mitochondrial respiration, transition of glycolysis, and inhibition of placental angiogenesis. Therefore, targeted inhibition of Nox2 is expected to become a new therapeutic target for PE.

Cannabidiol Disrupts Mitochondrial Respiration and Metabolism and Dysregulates Trophoblast Cell Differentiation.

Trophoblast differentiation is a crucial process in the formation of the placenta where cytotrophoblasts (CTs) differentiate and fuse to form the syncytiotrophoblast (ST). The bioactive components of cannabis, such as Δ9-THC, are known to disrupt trophoblast differentiation and fusion, as well as mitochondrial dynamics and respiration. However, less is known about the impact of cannabidiol (CBD) on trophoblast differentiation. Due to the central role of mitochondria in stem cell differentiation, we evaluated the impact of CBD on trophoblast mitochondrial function and differentiation. Using BeWo b30 cells, we observed decreased levels of mRNA for markers of syncytialization (GCM1, ERVW1, hCG) following 20 µM CBD treatment during differentiation. In CTs, CBD elevated transcript levels for the mitochondrial and cellular stress markers HSP60 and HSP70, respectively. Furthermore, CBD treatment also increased the lipid peroxidation and oxidative damage marker 4-hydroxynonenal. Mitochondrial membrane potential, basal respiration and ATP production were diminished with the 20 µM CBD treatment in both sub-lineages. mRNA levels for endocannabinoid system (ECS) components (FAAH, NAPEPLD, TRPV1, CB1, CB2, PPARγ) were altered differentially by CBD in CTs and STs. Overall, we demonstrate that CBD impairs trophoblast differentiation and fusion, as well as mitochondrial bioenergetics and redox homeostasis.

The Impact of SLC2A8 RNA Interference on Glucose Uptake and the Transcriptome of Human Trophoblast Cells.

While glucose is the primary fuel for fetal growth, the placenta utilizes the majority of glucose taken up from the maternal circulation. Of the facilitative glucose transporters in the placenta, SLC2A8 (GLUT8) is thought to primarily function as an intracellular glucose transporter; however, its function in trophoblast cells has not been determined. To gain insight into the function of SLC2A8 in the placenta, lentiviral-mediated RNA interference (RNAi) was performed in the human first-trimester trophoblast cell line ACH-3P. Non-targeting sequence controls (NTS RNAi; n = 4) and SLC2A8 RNAi (n = 4) infected ACH-3P cells were compared. A 79% reduction in SLC2A8 mRNA concentration was associated with an 11% reduction (p ≤ 0.05) in ACH-3P glucose uptake. NTS RNAi and SLC2A8 RNAi ACH-3P mRNA were subjected to RNAseq, identifying 1525 transcripts that were differentially expressed (|log2FC| > 1 and adjusted p-value < 0.05), with 273 transcripts derived from protein-coding genes, and the change in 10 of these mRNAs was validated by real-time qPCR. Additionally, there were 147 differentially expressed long non-coding RNAs. Functional analyses revealed differentially expressed genes involved in various metabolic pathways associated with cellular respiration, oxidative phosphorylation, and ATP synthesis. Collectively, these data indicate that SLC2A8 deficiency may impact placental uptake of glucose, but that its likely primary function in trophoblast cells is to support cellular respiration. Since the placenta oxidizes the majority of the glucose it takes up to support its own metabolic needs, impairment of SLC2A8 function could set the stage for functional placental insufficiency.

Pigment epithelium­derived factor inhibits proliferation, invasion and angiogenesis, and induces ferroptosis of extravillous trophoblasts by targeting Wnt­ß­catenin/VEGF signaling in placenta accreta spectrum.

Placenta accreta spectrum (PAS) is one of the most dangerous complications in obstetrics, which can lead to severe postpartum bleeding and shock, and even necessitate uterine removal. The abnormal migration and invasion of extravillous trophoblast cells (EVTs) and enhanced neovascularization occurring in an uncontrolled manner in time and space are closely related to the abnormal expression of pro­angiogenic and anti­angiogenic factors. The pigment epithelium­derived factor (PEDF) is a multifunctional regulatory factor that participates in several important biological processes and is recognized as the most efficient inhibitor of angiogenesis. The present study aimed to explore the effects of PEDF on EVT phenotypes and the underlying mechanisms in PAS. HTR­8/SVneo cells were transfected to overexpress or knock down PEDF. Cell proliferation and invasion were assessed using Cell Counting Kit­8, 5­ethynyl­2'­deoxyuridine and Transwell assays. In vitro angiogenesis was analyzed using tube formation assays. The degree of ferroptosis was assessed by evaluating the levels of lipid reactive oxygen species, total iron, Fe2+, malondialdehyde and reduced glutathione using commercial kits. The expression levels of biomarkers of ferroptosis, angiogenesis, cell proliferation and Wnt signaling were examined by western blotting. PEDF overexpression decreased the proliferation, invasion and angiogenesis, and induced ferroptosis of EVTs. Activation of Wnt signaling with BML­284 and overexpression of vascular endothelial growth factor (VEGF) reversed the PEDF overexpression­induced suppression of cell proliferation, invasion and tube formation. PEDF overexpression­induced ferroptosis was also decreased by Wnt agonist treatment and VEGF overexpression. It was predicted that PEDF suppressed the proliferation, invasion and angiogenesis, and increased ferroptosis in EVTs by decreasing Wnt­ß­catenin/VEGF signaling. The findings of the present study suggested a novel regulatory mechanism of the phenotypes of EVTs and PAS.

MicroRNA­155­5p inhibits trophoblast cell proliferation and invasion by disrupting centrosomal function.

Recurrent miscarriage is used to refer to more than three pregnancy failures before 20 weeks of gestation. Defective trophoblast cell growth and invasion are frequently observed in recurrent miscarriage. Several microRNAs (miRs), including miR­155­5p, are aberrantly upregulated in recurrent miscarriage; however, the underlying molecular mechanisms remain unclear. The centrosome orchestrates microtubule networks and coordinates cell cycle progression. In addition, it is a base for primary cilia, which are antenna­like organelles that coordinate signaling during development and growth. Thus, deficiencies in centrosomal functions can lead to several disease, such as breast cancer and microcephaly. In the present study, the signaling cascades were analyzed by western blotting, and the centrosome and primary cilia were observed and analyzed by immunofluorescence staining. The results showed that overexpression of miR­155­5p induced centrosome amplification and blocked primary cilia formation in trophoblast cells. Notably, centrosome amplification inhibited trophoblast cell growth by upregulating apoptotic cleaved­caspase 3 and cleaved­poly (ADP­ribose) polymerase in miR­155­5p­overexpressing trophoblast cells. In addition, overexpression of miR­155­5p inhibited primary cilia formation, thereby inhibiting epithelial­mesenchymal transition and trophoblast cell invasion. All phenotypes could be rescued when cells were co­transfected with the miR­155­5p inhibitor, thus supporting the role of miR­155­5p in centrosomal functions. It was also found that miR­155­5p activated autophagy, whereas disruption of autophagy via the depletion of autophagy­related 16­like 1 alleviated miR­155­5p­induced apoptosis and restored trophoblast cell invasion. In conclusion, the present study indicated a novel role of miR­55­5p in mediating centrosomal function in recurrent miscarriage.

Protective role of complement factor H against the development of preeclampsia.

Pregnancy is an immunologically regulated, complex process. A tightly controlled complement system plays a crucial role in the successful establishment of pregnancy and parturition. Complement inhibitors at the feto-maternal interface are likely to prevent inappropriate complement activation to protect the fetus. In the present study, we aimed to understand the role of Factor H (FH), a negative regulator of complement activation, in normal pregnancy and in a model of pathological pregnancy, i.e. preeclampsia (PE). The distribution and expression of FH was investigated in placental tissues, various placental cells, and in the sera of healthy (CTRL) or PE pregnant women via immunohistochemistry, RT-qPCR, ELISA, and Western blot. Our results showed a differential expression of FH among the placental cell types, decidual stromal cells (DSCs), decidual endothelial cells (DECs), and extravillous trophoblasts (EVTs). Interestingly, FH was found to be considerably less expressed in the placental tissues of PE patients compared to normal placental tissue both at mRNA and protein levels. Similar results were obtained by measuring circulating FH levels in the sera of third trimester CTRL and PE mothers. Syncytiotrophoblast microvesicles, isolated from the placental tissues of PE and CTRL women, downregulated FH expression by DECs. The present study appears to suggest that FH is ubiquitously present in the normal placenta and plays a homeostatic role during pregnancy.

HLA-G isoforms, HLA-C allotype and their expressions differ between early abortus and placenta in relation to spontaneous abortions.

INTRODUCTION: Spontaneous abortion (SAB) affects approximately 10% of clinically recognized pregnancies. Fetal trophobalst invasion and remodeling of maternal spiral arteries is reported to be dependent on crosstalk between HLA-C/HLA-G expressed on extra villous trophoblast (EVTs)and Killer cell Immunoglobin like receptors (KIRs) of decidual NK (dNK). Immune dysfunction in decidua contributes to early miscarriage. METHODOLOGY: The study used mother neonate paired cord blood and term placenta samples (n = 46), elective abortus (n = 17,gestational age = 10-12 weeks of pregnancy) and SAB abortus (n = 24, gestational age = 12-15 weeks of pregnancy) for HLA-G, KIR2D and HLA-C. In addition, term placenta was collected from women with history of spontaneous pregnancy loss (n = 24) and women with history of live birth (n = 32). SSP-PCR was used for genotyping, RT-PCR for gene expression, copy number variation (CNVs) and HLA-C allotyping and ELISA for protein expression studies. RESULTS: Membrane bound HLA-G4 isoform proportion was higher 39.28%, p = 0.02) in term placenta. SAB abortus had higher proportion of HLA-G3 (50%),while elective abortus exhibited higher proportion of soluble isoforms (HLA-G5, = 5.9, HLA-G6 = 5.9%, HLA-G7 = 11.8%). Higher inhibitory KIR2DL1 content and copy numbers with lower HLA-C2 in SAB contrasted with higher copy numbers of KIR2DS1(p = 0.001), KIR2DS1+/2DL1+- HLA-C2 combined genotype in healthy placenta. Elevated KIR2D protein levels (p = 0.001), and concurrently, HLA-C levels were upregulated in healthy placenta. CONCLUSION: Our data supports lower cognate receptor ligand KIR2DS1+/2DL1+ HLA-C2 together with predominance of HLA-G3 isoform in SAB as confounding factors in spontaneous pregnancy loss. HLA-G isoforms and expression differed between first trimester abortus and term placenta suggesting temporal modulation and marks novelty of the study.

High level 27-HC impairs trophoblast cell invasion and migration via LXR in pre-eclampsia.

INTRODUCTION: To explore the potential impact of 27-hydroxycholesterol (27-HC) on trophoblast cell function in pre-eclampsia. RESULTS: The levels of 27-HC and the expression of CYP27A1 are upregulated in clinical samples of PE. Furthermore, high concentrations of 27-HC can inhibit the invasion and migration ability of trophoblast cells in vitro, and this inhibitory effect is weakened after LXR silencing. In HTR8/SVneo cells treated with 27-HC, the expression of ABCA1/ABCG1 are increased. Finally, we established a mouse model of PE using l-NAME (N-Nitro-l-Arginine Methyl Ester). We found an increase in the levels of 27-HC in the peripheral blood serum of the PE mouse model, and an upregulation of CYP27A1 and LXR expressions in the placenta of the PE mouse model. CONCLUSION: 27-HC inhibits the invasion and migration ability of trophoblast cells by activating the LXR signaling pathway, which is involved in the pathogenesis of Pre-eclampsia(PE).

Isolation of serum-derived placental/amnio-chorionic extracellular vesicles across pregnancy by immunoaffinity using PLAP and HLA-G.

In brief: Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G. Abstract: Extracellular vesicles (EVs) are membrane-bound nanovesicles secreted from the cells into extracellular space and body fluids. They are considered 'fingerprints of parent cells', which can reflect their physiological and functional states. During pregnancy, EVs are produced by the syncytiotrophoblasts and extravillous trophoblasts and are released into the maternal bloodstream. In the present study, placental alkaline phosphatase (PLAP)-specific extracellular vesicles were isolated from maternal serum-derived EVs (SDE) across pregnancy. Transmission electron microscopy and dynamic light scattering analysis showed that the isolated EVs exhibited a spherical morphology with ~30-150 nm size range. Nanoparticle tracking analysis indicated that the concentration of PLAP+ serum-derived EVs (PLAP+-SDE) increased across the gestation. PLAP+-SDE contained DNA with LINE1 promoter methylation pattern. C19 miRNA cluster miRNAs (miR 515-5p, 519e and 520f) were present in PLAP+-SDE along with other miRNAs (miR-133-3p, miR210-3p and miR-223-3p). PLAP+-SDE confirmed the presence of EV markers (CD63 and CD9), along with placental proteins (PLAP and cullin 7). A modified novel strategy to extract an enriched population of circulating placental/amniochorionic EVs was devised employing an additional marker of extravillous trophoblasts, human leukocyte antigen G (HLA-G), along with PLAP. The isolated pooled placental/amniochorionic (PLAP+&HLA-G+) serum-derived EVs (PP-SDE) showed ~two-fold increased protein levels of HLA-G in the third-trimester pregnant women compared to the non-pregnant controls. Future studies will be focused on validation of this novel strategy to isolate an enriched population of placental/amniochorionic EVs to facilitate a better understanding of placental physiology and pathophysiology.

Endogenous retrovirus HERVH-derived lncRNA UCA1 controls human trophoblast development.

Endogenous retroviruses (ERVs) are frequently reactivated in mammalian placenta. It has been proposed that ERVs contribute to shaping the gene regulatory network of mammalian trophoblasts, dominantly acting as species- and placental-specific enhancers. However, whether and how ERVs control human trophoblast development through alternative pathways remains poorly understood. Besides the well-recognized function of human endogenous retrovirus-H (HERVH) in maintaining pluripotency of early human epiblast, here we present a unique role of HERVH on trophoblast lineage development. We found that the LTR7C/HERVH subfamily exhibits an accessible chromatin state in the human trophoblast lineage. Particularly, the LTR7C/HERVH-derived Urothelial Cancer Associated 1 (UCA1), a primate-specific long non-coding RNA (lncRNA), is transcribed in human trophoblasts and promotes the proliferation of human trophoblast stem cells (hTSCs), whereas its ectopic expression compromises human trophoblast syncytialization coinciding with increased interferon signaling pathway. Importantly, UCA1 upregulation is detectable in placental samples from early-onset preeclampsia (EO-PE) patients and the transcriptome of EO-PE placenta exhibits considerable similarities to that of the syncytiotrophoblasts differentiated from UCA1-overexpressing hTSCs, supporting up-regulated UCA1 as a potential biomarker of this disease. Altogether, our data shed light on the versatile regulatory role of HERVH in early human development and provide a unique mechanism whereby ERVs exert a function in human placentation and placental syndromes.

Culture and Maintenance of Immune Cells to Model Innate Immune Status at the Feto-maternal Interface.

The inflammatory process leading to human labor is mostly facilitated by immune cells, which can be studied by isolating and characterizing primary immune cells from the feto-maternal interface. However, difficulty and inconsistency in sampling approaches of immune cells and short lifespan in vitro prevent their usage in mechanistic studies to understand the maternal-fetal immunobiology. To address these limitations, existing cell line models can be differentiated into immune-like cells for use in reproductive biology experiments. In this chapter, we discussed cell culture methods of maintaining and differentiating HL-60, THP-1, and NK-92 cells to obtain neutrophil-like, macrophage-like, and decidual natural killer-like cells, respectively, which can then be used together with intrauterine cells to elucidate and investigate immune mechanisms that contribute to parturition.

SFRP2 suppresses trophoblast cell migration by inhibiting the Wnt/ß­catenin pathway.

The present study investigates the role of Secreted Frizzled­Related Protein 2 (SFRP2) in trophoblast cells, a key factor in preeclampsia (PE) progression. Elevated levels of Secreted Frizzled­Related Protein 1/3/4/5 (SFRP1/3/4/5) are associated with PE, but the role of SFRP2 is unclear. We analyzed SFRP2 expression in PE placental tissue using the GSE10588 dataset and overexpressed SFRP2 in JEG­3 cells via lentiviral transfection. The viability, migration, apoptosis, and proliferation of SFRP2­overexpressing JEG­3 cells were assessed using Cell Counting Kit­8, Transwell assays, flow cytometry, and EdU staining. Additionally, we evaluated the impact of SFRP2 overexpression on key proteins in the Wnt/ß­catenin pathway and apoptosis markers (Bax, cleaved­caspase 3, BCL­2, MMP9, E­cadherin, Wnt3a, Axin2, CyclinD1, c­Myc, p­ß­catenin, ß­catenin, phosphorylated Glycogen Synthase Kinase 3 beta (p­GSK3ß), and GSK3ß) through western blotting. Results showed high SFRP2 mRNA and protein expression in PE placenta and JEG­3 cells post­transfection. SFRP2 overexpression significantly reduced JEG­3 cell viability, proliferation, and migration, while increasing apoptosis. It also altered expression levels of Wnt pathway proteins, suggesting SFRP2's potential as a therapeutic target for PE by inhibiting trophoblast cell migration through the Wnt/ß­catenin signaling cascade.

Unraveling the molecular mechanisms driving enhanced invasion capability of extravillous trophoblast cells: a comprehensive review.

Precise extravillous trophoblast (EVT) invasion is crucial for successful placentation and pregnancy. This review focuses on elucidating the mechanisms that promote heightened EVT invasion. We comprehensively summarize the pivotal roles of hormones, angiogenesis, hypoxia, stress, the extracellular matrix microenvironment, epithelial-to-mesenchymal transition (EMT), immunity, inflammation, programmed cell death, epigenetic modifications, and microbiota in facilitating EVT invasion. The molecular mechanisms underlying enhanced EVT invasion may provide valuable insights into potential pathogenic mechanisms associated with diseases characterized by excessive invasion, such as the placenta accreta spectrum (PAS), thereby offering novel perspectives for managing pregnancy complications related to deficient EVT invasion.