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Air pollution poses a significant global concern, notably impacting pregnancy outcomes through mechanisms such as DNA damage, oxidative stress, inflammation, and altered miRNA expression, all of which can adversely affect trophoblast functions. Cleistocalyx nervosum var. paniala, known for its abundance of anthocyanins with diverse biological activities including anti-mutagenic, antioxidant, and anti-inflammatory properties, is the focus of this study examining its effect on Particulate Matter 10 (PM10) soluble extract-induced trophoblast cell dysfunction via miRNA expression. The study involved the extraction of C. nervosum fruit using 70% ethanol, followed by fractionation with hexane, dichloromethane, and ethyl acetate. Subsequent testing for total phenolics, flavonoids, anthocyanins, and antioxidant activity revealed the ethyl acetate fraction (CN-EtOAcF) as possessing the highest phenolic and anthocyanin content along with potent antioxidant activity, prompting its selection for further investigation. In vitro studies on HTR-8/SVneo cells demonstrated that 5–10 µg/mL PM10 soluble extract exposure inhibited cell proliferation, migration, invasion, and induced apoptosis. However, pretreatment with 20–80 µg/mL CN-EtOAcF followed by 5 µg/mL PM10 soluble extract exposure exhibited protective effects against PM10 soluble extract-induced damage, including inflammation inhibition and intracellular ROS suppression. Notably, CN-EtOAcF down-regulated PM10-induced miR-146a-5p expression, with SOX5 identified as a potential target. Overall, CN-EtOAcF demonstrated the potential to protect against PM10-induced harm in trophoblast cells, suggesting its possible application in future therapeutic approaches. [ABSTRACT FROM AUTHOR]

Dysregulation of macrophage polarization can prevent the invasion of trophoblast cells and further limit spiral artery remodeling in preeclampsia (PE). However, its mechanism is obscure. HTR8-/Svneo cells were cultured under normoxic or hypoxic conditions and extracellular vesicles (EVs) in the culture supernatants were extracted. Next, the cells were incubated with those EVs to investigate their effects on trophoblasts. A co-culture system consisting of HTR8-/Svneo cells and macrophages was used to reveal how the trophoblast-derived EVs affected the macrophage subtype. Finally, a PE mouse model and miR-141-3p knockout mice were used to verify the function of miR-141-3p in PE. Hypoxia induced abnormal increases in the levels of miR-141-3p in HTR8-/Svneo cells and EVs. EVs from hypoxia-treated HTR8-/Svneo cells could downregulate PTEN, a potential target of miR-141-3p, and inhibit trophoblast mitophagy and invasion. However, HTR8-/Svneo cells transfected with an miR-141-3p inhibitor could attenuate the influence of EVs. In an HTR8-/Svneo cell plus macrophage co-culture system, hypoxia-pretreated cells promoted the transformation of macrophages into the M1-phenotye, and HTR8-/Svneo invasion was inhibited by the macrophages. MiR-141 from EVs could target and downregulate dual specificity phosphatase 1 (DUSP1) expression in macrophages, induce formation of the M1 macrophage phenotype in THP-1 cells, downregulate DUSP1 expression, and upregulate TAB2/TAK1 signaling. These results were also demonstrated in normal pregnant mice and PE pregnant mice. A hypoxic environment could upregulate miR-141 expression in the EVs of HTR8-/Svneo cells, and THP-1-derived macrophages could uptake EVs releasing miR-141 to downregulate DUSP1 expression and induce the formation of M1 macrophages, which can lead to the development of PE. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a common placental-origin complication of pregnancy and a major cause of morbidity and mortality among pregnant women and fetuses. However, its pathogenesis has not been elucidated. Effective strategies for prevention, screening, and treatment are still lacking. Studies have indicated that dysfunction of placental trophoblast cells, such as impaired syncytialization, proliferation, and epithelial-mesenchymal transition processes, plays a crucial role in the development of PE. Glucose transporter 1 (GLUT1) is a key protein regulating glucose transport in placental tissues. However, the effect of GLUT1 on the function of trophoblast cells in PE is not well understood. In this study, we found that GLUT1 expression is reduced in PE placental tissues. GLUT1 promotes the syncytialization process by increasing the glucose uptake ability of BeWo cells. Additionally, GLUT1 promotes the proliferation, migration, and invasion capabilities of HTR-8/SVneo cells by regulating MAPK and PI3K/AKT signaling pathways. Overall, these findings provide a new insight into understanding the biological functions of GLUT1, clarifying the pathogenesis of PE, and identifying diagnostic and therapeutic targets for PE. [ABSTRACT FROM AUTHOR]

Problem: Pregnancy complications such as spontaneous abortion, preeclampsia, and preterm birth persist, despite current interventions aimed at their prevention and treatment largely proving unsuccessful. Interleukin‐27 (IL‐27), composed of p28 and EBI3 subunits, binds to IL‐27R, which consists of gp130 and IL‐27Rα (also known as WSX‐1 or TCCR), and plays a pivotal role in tumor development and inflammation regulation. At the maternal‐fetal interface, IL‐27 expression has been detected in trophoblasts, endometrial stromal cells, and decidual cells. Abnormal levels of IL‐27/IL‐27R have been linked to adverse pregnancy outcomes, including spontaneous miscarriage, preeclampsia, and preterm birth. This review aims to explore the expression of IL‐27 at the maternal‐fetal interface and its signaling pathway, uncovering the complex role of IL‐27 in pregnancy complications. Method of Study: A comprehensive literature review was conducted using PubMed/Medline, Scopus, and Embase databases, analyzing studies on IL‐27 expression and its signaling pathways at the maternal‐fetal interface. The review focused on identifying the presence of IL‐27 in various cell types and linking abnormal IL‐27/IL‐27R expression to pregnancy complications such as spontaneous miscarriage, preeclampsia, and preterm birth. Discussion and Conclusion: IL‐27 plays a complex role at the maternal‐fetal interface, with abnormal expression linked to several pregnancy complications. These findings highlight the need for further research to elucidate IL‐27's mechanisms and develop targeted interventions. Future studies should aim to develop targeted interventions and improve therapeutic strategies for managing pregnancy complications. [ABSTRACT FROM AUTHOR]

In the first trimester of pregnancy the human placenta grows rapidly, making it sensitive to changes in the intrauterine environment. To test whether exposure to an environment in utero often associated with obesity modifies placental proteome and function, we performed un- targeted proteomics (LC-MS/MS) in placentas from 19 women (gestational age 35-48 days, i.e. 5+0-6+6 weeks). Maternal clinical traits (body mass index, leptin, glucose, C-peptide and insulin sensitivity) and gestational age were recorded. DNA replication and cell cycle pathways were enriched in the proteome of placentas of women with low maternal insulin sensitivity. Driving these pathways were the minichromosome maintenance (MCM) proteins MCM2, MCM3, MCM4, MCM5, MCM6 and MCM7 (MCM-complex). These proteins are part of the pre-replicative complex and participate in DNA damage repair. Indeed, MCM6 and γH2AX (DNA-damage marker) protein levels correlated in first trimester placental tissue (r = 0.514, P<0.01). MCM6and γH2AX co-localized to nuclei of villous cytotrophoblast cells, the proliferative cell type of the placenta, suggesting increased DNA damage in this cell type. To mimic key features of the intrauterine obesogenic environment, a first trimester trophoblast cell line, i.e., ACH-3P, was exposed to high insulin (10 nM) or low oxygen tension (2.5% O2). There was a significant correlation between MCM6 and γH2AX protein levels, but these were independent of insulin or oxygen exposure. These findings show that chronic exposure in utero to reduced maternal insulin sensitivity during early pregnancy induces changes in the early first trimester placental proteome. Pathways related to DNA replication, cell cycle and DNA damage repair appear especially sensitive to such an in utero environment. [ABSTRACT FROM AUTHOR]

Aims: Preterm birth (PTB), recognized as delivery before 37 weeks of gestation, is a multifactorial syndrome characterizing as the main cause of neonatal mortality. Reactive oxygen species (ROS) have been identified as proinflammatory factors to cause placental inflammation, thereby resulting in several pregnancy outcomes. To date, limited knowledge regarding the underlying mechanisms of ROS-induced PTB has been reported. In this study, we aimed to investigate the role of oxidative stress in PTB and the protective effects of mitochondria-targeted antioxidant MitoTEMPO (MT) on preterm labor and offspring mice. Results: In this study, we found that preterm placentas had abnormal mitochondrial function, oxidative stress, and inflammatory response. In the lipopolysaccharide (LPS)-induced PTB mouse model, MT inhibited PTB by ameliorating maternal oxidative stress and inflammation, especially in placentas, thus improving placental function to maintain pregnancy. Antenatal administration of MT prevented LPS-induced fetal brain damage in acute phase and improved long-term neurodevelopmental impairments. Furthermore, our in vitro investigations validated that MT retarded the ROS accumulation and inflammatory response in LPS-stimulated trophoblast cells by promoting Kelch-like ECH-associated protein 1 (Keap1) degradation and subsequently activating nuclear factor erythroid 2-related factor 2 (Nrf2). By inhibiting Nrf2 activation, we discovered that the anti-inflammation and protective characteristics of MT were Nrf2/ARE pathway dependent. Innovation and Conclusion: MT inhibited PTB and fetal brain injury by inhibiting maternal inflammation and improving placental function through Keap1/Nrf2/antioxidant response element signaling pathway. Our findings provide a novel therapeutic strategy for PTB. Antioxid. Redox Signal. 41, 597–615. [ABSTRACT FROM AUTHOR]

Intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin are cell adhesion molecules that play a significant role in inflammation and are implicated in the pathophysiology of preeclampsia development and HIV infection. More specifically, the immune expression of ICAM-1, VCAM-1, and E-selectin within cyto- and syncytiotrophoblast cells are dysregulated in preeclampsia, indicating their role in defective placentation. This study investigates the associations of ICAM-1, VCAM-1, and E-selectin gene variants (rs3093030, rs3783605, and rs1805193, respectively) with preeclampsia comorbid with HIV infection in women of African ancestry. It also examines the susceptibility to preeclampsia development and the effect of highly active antiretroviral therapy (HAART). A total of 405 women were enrolled in this study. Out of these women, 204 were preeclamptic and 201 were normotensive. Clinical characteristics were maternal age, weight, blood pressure (systolic and diastolic), and gestational age. Whole blood was collected, DNA was extracted, and genotyping of the ICAM-1 (rs3093030 C>T), VCAM-1(rs3783605 A>G), and E-selectin (rs1805193 A>C) gene polymorphisms was performed. Comparisons were made using the Chi-squared test. Our results demonstrated that preeclamptic women exhibited a higher frequency of analyzed variants, in contrast to those with the duality of preeclampsia and HIV infection. Additionally, the C allele of the ICAM-1 (rs3093030 C>T) and G allele of the VCAM-1 (rs3783605 A>G) genes were found to have a greater role in the co-morbidity and may be considered as a risk factor for preeclampsia development in women of African ancestry. In contrast, the SNP of rs1805193 of the E-selectin gene indicated that A>C was only significantly associated with HIV infection and not with preeclampsia. These findings highlight a strong association of the rs3093030 SNP of the ICAM-1 gene and of the VCAM-1 rs3783605 gene with the development of preeclampsia, indicating their role in the defective trophoblast invasion of preeclampsia. Sub-group analysis further reveals an association of the AA genotype with late-onset preeclampsia, a less severe form of disease indicating differing genetic predispositions between early and late-onset forms. [ABSTRACT FROM AUTHOR]

Introduction: Hyperglycemia is closely related to trophoblast dysfunction during pregnancy and results in suppressed invasion, migration, and pro‐inflammatory cell death of trophoblasts. Hyperglycemia is a dependent risk factor for gestational hypertension accompanied by decreased placental growth factor (PLGF), which is important for maternal and fetal development. However, there is currently a lack of evidence to support whether PLGF can alleviate trophoblast cell dysfunction caused by high blood sugar. Here, we aim to clarify the effect of hyperglycemia on trophoblast dysfunction and determine how PLGF affects this process. Methods: The changes in placental tissue histomorphology from gestational diabetes mellitus (GDM) patients were compared with those of normal placentas. HTR8/SVneo cells were cultured in different amounts of glucose to examine cellular pyroptosis, migration, and invasion as well as PLGF levels. Furthermore, the levels of pyroptosis‐related proteins (NLRP3, pro‐caspase1, caspase1, IL‐1β, and Gasdermin D [GSDMD]) as well as autophagy‐related proteins (LC3‐II, Beclin1, and p62) were examined by Western blotting. The GFP‐mRFP‐LC3‐II system and transmission electron microscopy were used to detect mitophagy levels, and small interfering RNAs targeting BCL2 Interacting Protein 3 (siBNIP3) and PTEN‐induced kinase 1 (siPINK1) were used to determine the role of mitophagy in pyroptotic death of HTR‐8/SVneo cells. Results: Our results show that hyperglycemia upregulates NLRP3, pro‐caspase1, caspase1, IL‐1β at the protein level in GDM patients. High glucose (HG, 25 mM) inhibits viability, invasion, and migration of trophoblast cells while suppressing superoxide dismutase levels and promoting malondialdehyde production, thus leading to a senescence associated beta‐gal‐positive cell burst. PLGF levels in nucleus and the cytosol are also inhibited by HG, whereas PLGF treatment inhibited pyroptosis‐related protein levels of NLRP3, pro‐caspase1, caspase1, IL‐1β, and GSDMD, Gasdermin D N‐terminal domain (GSDMD‐N). HG‐induced mitochondrial dysfunction and BNIP3 and PINK1/Parkin expression. Knocking down BINP3 and PINK1 abolished the protective role of PLGF by preventing mitophagy. Conclusion: PLGF inhibited hyperglycemia, while PLGF reversed hyperglycemic injury by promoting mitophagy via the BNIP3/PINK1/Parkin pathway. Altogether, these results suggest that PLGF may protect against trophoblast dysfunction in diabetes. [ABSTRACT FROM AUTHOR]

Congenital heart defects are leading causes of neonatal mortality and are often associated with placental abnormalities, but mechanisms linking placenta and heart development are poorly understood. Herein, we investigated a potential signaling network connecting the placenta and nascent heart in mice. We found that fetal hearts exposed to media conditioned by placental tissue or differentiated wild-type trophoblast stem (TS) cells, but not undifferentiated TS cells, showed increased heart rate and epicardial cell outgrowth. This effect was not observed when hearts were exposed to media from TS cells lacking OVO-Like 2, a transcription factor required for trophoblast differentiation and placental development. Trophoblasts released abundant extracellular vesicles into media, and these vesicles were sufficient to mediate cardio-promoting effects. Our findings provide a potential mechanism whereby the placenta communicates with the fetal heart to promote cardiac morphogenesis, and offers insight into the link between poor placentation and a higher incidence of heart defects. Poor development of the placenta is linked with heart problems. This study reveals that extracellular vesicles from placental trophoblasts promote heart development in mice, shedding light on how placental health affects cardiac formation. [ABSTRACT FROM AUTHOR]

Dysregulated progenitor cell populations may contribute to poor placental development and placental insufficiency pathogenesis. Side-population cells possess progenitor properties. Recent human trophoblast side-population isolation identified enrichment of 8 specific genes (CXCL8, ELL2, GATA6, HK2, HLA-DPB1, INTS6, SERPINE3 and UPP1) (Gamage et al. 2020, Stem Cell Rev Rep). We characterised these trophoblast side-population markers in human placenta and in placental insufficiency disorders: preeclampsia and fetal growth restriction (FGR). Trophoblast side-population markers localised to mononuclear trophoblasts lining the placental villous basement membrane in preterm control, preeclamptic and FGR placental sections (n = 3, panel of 3 markers/serial section). Analysis of single-cell transcriptomics of an organoid human trophoblast stem cell (hTSC) to extravillous trophoblast (EVT) differentiation model (Shannon et al. 2022, Development) identified that all side-population genes were enriched in mononuclear trophoblast and trophoblasts committed to differentiation under hTSC culture conditions. In vitro validation via 96 h time course hTSC differentiation to EVTs or syncytiotrophoblasts (n = 5) demonstrated ELL2 and HK2 increased with differentiation (p < 0.0024, p < 0.0039 respectively). CXCL8 and HLA-DPB1 were downregulated (p < 0.030, p < 0.011 respectively). GATA6 and INTS6 increased with EVT differentiation only, and UPP1 reduced with syncytialisation. SERPINE3 was undetectable. Trophoblast side-population marker mRNA was measured in human placentas (< 34-weeks' gestation; n = 78 preeclampsia, n = 30 FGR, and n = 18 gestation-matched controls). ELL2, HK2 and CXCL8 were elevated in preeclamptic (p = 0.0006, p < 0.0001, p = 0.0335 respectively) and FGR placentas (p = 0.0065, p < 0.0001, p = 0.0001 respectively) versus controls. Placental GATA6 was reduced in pregnancies with preeclampsia and FGR (p = 0.0014, p = 0.0146 respectively). Placental INTS6 was reduced with FGR only (p < 0.0001). This study identified the localisation of a unique trophoblast subset enriched for side-population markers. Aberrant expression of some side-population markers may indicate disruptions to unique trophoblast subtypes in placental insufficiency. [ABSTRACT FROM AUTHOR]

Fetal growth restriction (FGR) is a relatively common complication of pregnancy, and insufficient syncytialization in the placenta may play an important role in the pathogenesis of FGR. However, the mechanism of impaired formation of the syncytiotrophoblast layer in FGR patients requires further exploration. In the present study, we demonstrated that the level of syncytialization was decreased in FGR patient placentas, while the expression of connective tissue growth factor (CTGF) was significantly upregulated. CTGF was found to inhibit trophoblast fusion via regulating cell cycle progress of BeWo cells. Furthermore, we found that CTGF negatively regulates cell cycle arrest in a p21-dependent manner as overexpression of p21 could rescue the impaired syncytialization induced by CTGF-overexpression. Besides, we also identified that CTGF inhibits the expression of p21 through ITGB4/PI3K/AKT signaling pathway. Our study provided a new insight for elucidating the pathogenic mechanism of FGR and a novel idea for the clinical therapy of FGR. [ABSTRACT FROM AUTHOR]

Abnormal expression of long non-coding RNAs (lncRNAs) is associated with the dysfunctions of human trophoblast cells and the occurrence of miscarriage (abnormal early embryo loss). BBC3/PUMA (BCL2 binding component 3) plays significant roles in regulation of cell apoptosis. However, whether specific lncRNAs might regulate BBC3 in trophoblast cells and further induce apoptosis and miscarriage remains completely unclear. Through screening, we identified a novel lnc-HZ12, which was significantly highly expressed in villous tissues of recurrent miscarriage (RM) patients relative to their healthy control (HC) group. Lnc-HZ12 suppressed chaperone-mediated autophagy (CMA) degradation of BBC3, promoted trophoblast cell apoptosis, and was associated with miscarriage. In mechanism, lnc-HZ12 downregulated the expression levels of chaperone molecules HSPA8 and LAMP2A in trophoblast cells. Meanwhile, lnc-HZ12 (mainly lnc-HZ12-SO2 region in F2 fragment) and HSPA8 competitively bound with the 169RVLYNL174 patch on BBC3, which prevented BBC3 from interactions with HSPA8 and impaired the formation of BBC3-HSPA8-LAMP2A complex for CMA degradation of BBC3. Thus, lnc-HZ12 upregulated the BBC3-CASP9-CASP3 pathway and induced trophoblast cell apoptosis. In villous tissues, lnc-HZ12 was highly expressed, CMA degradation of BBC3 was suppressed, and the apoptosis levels were higher in RM vs HC villous tissues, all of which were associated with miscarriage. Interestingly, knockdown of murine Bbc3 could efficiently suppress placental apoptosis and alleviate miscarriage in a mouse miscarriage model. Taken together, our results indicated that lnc-HZ12 and BBC3 played important roles in trophoblast cell apoptosis and miscarriage and might act as attractive targets for miscarriage treatment. Abbreviation: 7-AAD: 7-aminoactinomycin D; BaP: benzopyrene; BBC3/PUMA: BCL2 binding component 3; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HC: healthy control; HSPA8: heat shock protein family A (Hsp70) member 8; IP: immunoprecipitation; LAMP2A: lysosomal associated membrane protein 2; LncRNA: long non-coding RNA; mRNA: messenger RNA; MT: mutant-type; NC: negative control; NSO: nonspecific oligonucleotide; PARP1: poly(ADP-ribose) polymerase 1; RIP: RNA immunoprecipitation; RM: recurrent miscarriage; TBP: TATA-box binding protein; WT: wild-type. [ABSTRACT FROM AUTHOR]

Embryo implantation is a tightly regulated process, critical for a successful pregnancy. After attachment of the blastocyst to the surface epithelium of the endometrium trophoblast migrate from the trophectoderm and invade into the stromal component of endometrium. Alterations on either process will lead to implantation failure or miscarriage. Volatile organic compounds (VOCs) such as benzene induce pregnancy complications, including preterm birth and miscarriages. The mechanism of this effect is unknown. The objective of this study was to elucidate the impact of benzene metabolite, Hydroquinone, on trophoblast function. We tested the hypothesis that Hydroquinone activates the Aryl hydrocarbon receptor (AhR) pathway modulating trophoblast migration and invasion. First-trimester trophoblast cells (Sw.71) were treated with hydroquinone (6 and 25 μM). Trophoblast migration and invasion was evaluated using a 3D invasion/migration model. Gene expression was quantified by q-PCR and Western blot analysis. Hydroquinone impairs trophoblast migration and invasion. This loss is associated with the activation of the AhR pathway which reduced the expression of Twist1and IFITM1. IFITM1 overexpression can rescue impaired trophoblast migration. Our study highlights that hydroquinone treatment induces the activation of the AhR pathway in trophoblast cells, which impairs trophoblast invasion and migration. We postulate that activation of the AhR pathway in trophoblast suppress Twist1 and a subsequent IFITM1. Thus, the AhR-Twist1-IFITM1 axis represent a critical pathway involved in the regulation of trophoblast migration and it is sensitive to benzene exposure. These findings provide crucial insights into the molecular mechanisms underlying pregnancy complications induced by air pollution. [Display omitted] • Benzene exposure affects pregnancy outcome. • Hydroquinone treatment inhibits trophoblast migration and invasion. • The AhR pathway is functional in trophoblast cells. • Hydroquinone activates the Ahr pathway in trophoblast. • Twist1 and IFITM1 are major regulators of trophoblast migration. [ABSTRACT FROM AUTHOR]

Women with polycystic ovary syndrome (PCOS) exhibit sustained elevation in circulating androgens during pregnancy, an independent risk factor linked to pregnancy complications and adverse outcomes in offspring. Yet, further studies are required to understand the effects of elevated androgens on cell type‐specific placental dysfunction and fetal development. Therefore, a PCOS‐like mouse model induced by continuous androgen exposure is examined. The PCOS‐mice exhibited impaired placental and embryonic development, resulting in mid‐gestation lethality. Co‐treatment with the androgen receptor blocker, flutamide, prevents these phenotypes including germ cell specification. Comprehensive profiling of the placenta by whole‐genome bisulfite and RNA sequencing shows a reduced proportion of trophoblast precursors, possibly due to the downregulation of Cdx2 expression. Reduced expression of Gcm1, Synb, and Prl3b1 is associated with reduced syncytiotrophoblasts and sinusoidal trophoblast giant cells, impairs placental labyrinth formation. Importantly, human trophoblast organoids exposed to androgens exhibit analogous changes, showing impaired trophoblast differentiation as a key feature in PCOS‐related pregnancy complications. These findings provide new insights into the potential cellular targets for future treatments. [ABSTRACT FROM AUTHOR]

Background: The syncytiotrophoblast (SCT) layer in the placenta serves as a crucial physical barrier separating maternal-fetal circulation, facilitating essential signal and substance exchange between the mother and fetus. Any abnormalities in its formation or function can result in various maternal syndromes, such as preeclampsia. The transition of proliferative villous cytotrophoblasts (VCT) from the mitotic cell cycle to the G0 phase is a prerequisite for VCT differentiation and their fusion into SCT. The imprinting gene P57Kip2, specifically expressed in intermediate VCT capable of fusion, plays a pivotal role in driving this key event. Moreover, aberrant expression of P57Kip2 has been linked to pathological placental conditions and adverse fetal outcomes. Methods: Validation of STK40 interaction with P57Kip2 using rigid molecular simulation docking and co-immunoprecipitation. STK40 expression was modulated by lentivirus in BeWo cells, and the effect of STK40 on trophoblast fusion was assessed by real-time quantitative PCR, western blot, immunofluorescence, and cell viability and proliferation assays. Co-immunoprecipitation, transcriptome sequencing, and western blot were used to determine the potential mechanisms by which STK40 regulates P57Kip2. Results: In this study, STK40 has been identified as a novel interacting protein with P57Kip2, and its expression is down-regulated during the fusion process of trophoblast cells. Overexpressing STK40 inhibited cell fusion in BeWo cells while stimulating mitotic cell cycle activity. Further experiments indicated that this effect is attributed to its specific binding to the CDK-binding and the Cyclin-binding domains of P57Kip2, mediating the E3 ubiquitin ligase COP1-mediated ubiquitination and degradation of P57Kip2. Moreover, abnormally high expression of STK40 might significantly contribute to the occurrence of preeclampsia. Conclusions: This study offers new insights into the role of STK40 in regulating the protein-level homeostasis of P57Kip2 during placental development. [ABSTRACT FROM AUTHOR]

Visfatin is an adipokine involved in energy metabolism, insulin resistance, inflammation, and female reproduction. Due to limited data about its action in the human placenta, the aims of our studies included the analysis of visfatin expression and immunolocalization in trophoblast cell lines JEG-3 and BeWo as well as in human placentas from normal and pathological pregnancies. Moreover, we also checked the hormonal regulation of visfatin levels and the molecular mechanism of observed changes in JEG-3 cells. Cell culture and placental fragments collection along with statistical analysis were performed using standard laboratory procedures also described in our previous papers. We demonstrated an increased gene and protein expression of visfatin in JEG-3, BeWo cells, while variable expression in maternal and fetal parts of normal/ pathological pregnancy placentas. In addition, the immunolocalization of visfatin was observed in the cytoplasm of both cell lines, the capillary epithelium of the maternal part and syncytiotrophoblasts of the placental fetal part; in all tested pathologies, the signal was also detected in decidual cells. Furthermore, we demonstrated that hormones: progesterone, estradiol, human chorionic gonadotropin, and insulin increased the visfatin levels in JEG-3 cells with the involvement of specific signaling pathways. Taken together, differences in the expression and localization of visfatin between normal and pathological placentas suggested that visfatin may be a potential marker for the diagnosis of pregnancy disorders. In addition, we found that placental levels of visfatin can be regulated by hormones known to modulate the function of placental cells. [ABSTRACT FROM AUTHOR]

Background: The syncytiotrophoblast (SCT) layer in the placenta serves as a crucial physical barrier separating maternal-fetal circulation, facilitating essential signal and substance exchange between the mother and fetus. Any abnormalities in its formation or function can result in various maternal syndromes, such as preeclampsia. The transition of proliferative villous cytotrophoblasts (VCT) from the mitotic cell cycle to the G0 phase is a prerequisite for VCT differentiation and their fusion into SCT. The imprinting gene P57Kip2, specifically expressed in intermediate VCT capable of fusion, plays a pivotal role in driving this key event. Moreover, aberrant expression of P57Kip2 has been linked to pathological placental conditions and adverse fetal outcomes. Methods: Validation of STK40 interaction with P57Kip2 using rigid molecular simulation docking and co-immunoprecipitation. STK40 expression was modulated by lentivirus in BeWo cells, and the effect of STK40 on trophoblast fusion was assessed by real-time quantitative PCR, western blot, immunofluorescence, and cell viability and proliferation assays. Co-immunoprecipitation, transcriptome sequencing, and western blot were used to determine the potential mechanisms by which STK40 regulates P57Kip2. Results: In this study, STK40 has been identified as a novel interacting protein with P57Kip2, and its expression is down-regulated during the fusion process of trophoblast cells. Overexpressing STK40 inhibited cell fusion in BeWo cells while stimulating mitotic cell cycle activity. Further experiments indicated that this effect is attributed to its specific binding to the CDK-binding and the Cyclin-binding domains of P57Kip2, mediating the E3 ubiquitin ligase COP1-mediated ubiquitination and degradation of P57Kip2. Moreover, abnormally high expression of STK40 might significantly contribute to the occurrence of preeclampsia. Conclusions: This study offers new insights into the role of STK40 in regulating the protein-level homeostasis of P57Kip2 during placental development. [ABSTRACT FROM AUTHOR]

Galectins are a class of lectins that are extensively expressed in all organisms. Galectins are involved in a range of functions, including early development, tissue regeneration, cancer and inflammation. It has been shown that galectin-8 is expressed in the villous and extravillous trophoblast (EVT) cells of the human placenta; however, its physiological role in pregnancy establishment has not been elucidated. Taking these factors into account, we investigated the functional role of galectin-8 in HTR-8/SVneo cells—a human EVT cell line—and human primary cytotrophoblast cells isolated from a first-trimester placenta. We analyzed the effects of recombinant human galectin-8 (rh galectin-8) on the adhesion, migration and invasion of HTR-8/SVneo cells. We used qPCR, cell-based ELISA (cELISA) and gelatin zymography to study the effects of galectin-8 on mediators of these processes, such as integrin subunits alpha-1 and beta-1 and matrix metalloproteinases (MMPs)-2 and -9, on the mRNA and protein levels. Further, we studied the effects of galectin-8 on primary cytotrophoblast cells' invasion. Galectin-8 stimulated the adhesion, migration and invasion of HTR-8/SVneo cells, as well as the invasion of primary cytotrophoblasts. In addition, the MMP-2 and -9 levels were increased, while the expression of integrins alpha-1 and beta-1 was not affected. Galectin-8 has the ability to positively affect EVTs' invasion, so it can be considered a significant factor in the trophoblast cell invasion process. [ABSTRACT FROM AUTHOR]

Since the first mouse induced pluripotent stem cells (iPSCs) was derived, the in vitro culture of domestic iPSCs functionally and molecularly comparable with mouse iPSCs has been a challenge. Here, we established dairy goat iPSCs (giPSCs) from goat ear fibroblast cells with mouse iPSCs morphology, the expression of pluripotent markers and differentiation ability in vitro delivered by piggyBac transposon with nine Dox-inducible exogenous reprogramming factors. These reprogramming factors were bOMSK (bovine OCT4 , CMYC , SOX2 , and KLF4), pNhL (porcine NANOG and human LIN28), hRL (human RARG and LRH1), and SV40 Large T. Notably, AF-giPSCs (induced in activin A and bFGF condition) were capable of differentiation in embryoid bodies in vitro and could contribute to interspecies chimerism in mouse E6.5 embryos in vitro , demonstrating that AF-giPSCs have the developmental capability to generate some embryonic cell lineages. Moreover, Wnt/β-catenin signaling has an important role in driving goat induced trophoblast-like stem cells (giTLSCs) from Dox-independent giPSCs. This study will support further establishment of the stable giPSC lines without any integration of exogenous genes. • The goat induced pluripotent stem cells (giPSCs) expressed pluripotent markers and have differentiation ability in vitro. • The AF-giPSCs contributed to mouse E6.5 embryos in vitro. • The goat induced trophoblast like stem cells (giTLSCs) were firstly derived from Dox-independent giPSCs. • We found Sox2 co-expressed with Cdx2, and as well as with Eomes in the nucleus indicated the potency of giTLSC. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) are presented in the uterine lumen of many mammals, and in vitro experiments have determined that several miRNAs are important for the regulation of endometrial and trophoblast functions. Our aim was to identify and contrast the miRNAs present in extracellular vesicles (EVs) in the uterine lumen fluid (ULF) at the onset of attachment in cattle pregnancies (gestation d 18) initiated by artificial insemination (AI) or by the transfer of an in vitro-produced blastocyst (IVP-ET). A third group had no conceptus after the transfer of an IVP embryo. Results: The abundance of 263 annotated miRNAs was quantified in the EVs collected from ULF. There was an increase in the transcript abundance of 20 miRNAs in the ULF EVs from the AI pregnant group, while 4 miRNAs had a lower abundance relative to the group not containing a conceptus. Additionally, 4 miRNAs were more abundant in ULF EVs in the AI pregnant group relative to IVP-ET group (bta-mir-17, bta-mir-7-3, MIR7-1, MIR18A). Specific miRNAs in the ULF EVs were co-expressed with messenger RNAs expressed in extra-embryonic tissues and endometrium, including genes that are known to be their targets. Conclusions: The results provide biological insights into the participation of miRNAs in the regulation of trophoblast proliferation and differentiation, as well as in endometrium receptivity. The knowledge that in vitro cultured embryos can contribute to the altered abundance of specific miRNAs in the uterine lumen can lead to the development of corrective approaches to reduce conceptus losses during the first month of pregnancy in cattle. [ABSTRACT FROM AUTHOR]

Decidual macrophages residing at the maternal-fetal interface have been recognized as pivotal factors for maintaining normal pregnancy; however, they are also key target cells of Toxoplasma gondii (T. gondii) in the pathology of T. gondii-induced adverse pregnancy. Trem2, as a functional receptor on macrophage surface, recognizes and binds various kinds of pathogens. The role and underlying mechanism of Trem2 in T. gondii infection remain elusive. In the present study, we found that T. gondii infection downregulated Trem2 expression and that Trem2-/- mice exhibited more severe adverse pregnancy outcomes than wildtype mice. We also demonstrated that T. gondii infection resulted in increased decidual macrophages, which were significantly reduced in the Trem2-/- pregnant mouse model as compared to wildtype control animals. We further described the inhibited proliferation, migration, and invasion functions of trophoblast cell by T. gondii antigens through macrophages as an "intermediate bridge", while this inhibition can be rescued by Trem2 agonist HSP60. Concurrently, Trem2 deficiency in bone marrow-derived macrophages (BMDMs) heightened the inhibitory effect of TgAg on the migration and invasion of trophoblast cells, accompanied by higher pro-inflammatory factors (IL-1β, IL-6 and TNF-α) but a lower chemokine (CXCL1) in T. gondii antigens-treated BMDMs. Furthermore, compelling evidence from animal models and in vitro cell experiments suggests that T. gondii inhibits the Trem2-Syk-PI3K signaling pathway, leading to impaired function of decidual macrophages. Therefore, our findings highlight Trem2 signaling as an essential pathway by which decidual macrophages respond to T. gondii infection, suggesting Trem2 as a crucial sensor of decidual macrophages and potential therapeutic target in the pathology of T. gondii-induced adverse pregnancy. Author summary: T. gondii is a critically important intracellular protozoan that is a major cause of adverse pregnancy outcomes in humans and livestock. The pathology of adverse pregnancy induced by T. gondii involves the dysfunction of decidual macrophages. Trem2, a functional immune receptor on the surface of decidual macrophages, governs its survival and phagocytosis. Here, we sought to elucidate whether Trem2 signaling is involved in the pathology of T. gondii-induced adverse pregnancy. We found that T. gondii infection led to the downregulation of Trem2 expression in macrophages, both in vivo and in vitro. Trem2-/- mice exhibit more severe adverse pregnancy outcomes after T. gondii infection. We further described the inhibited proliferation, migration, and invasion functions of trophoblast cell by T. gondii antigens through macrophages as an "intermediate bridge", while this inhibition can be rescued by Trem2 agonist HSP60. In parallel, Trem2 deficiency in BMDMs heightened the inhibitory effect of TgAg on the migration and invasion of trophoblast cells. Our work uncovers an important factor involved in the pathological mechanism of T. gondii-induced adverse pregnancy, and research on Trem2 may provide new preventive and therapeutic targets for toxoplasmosis. [ABSTRACT FROM AUTHOR]

Glucose metabolism produces lactate and hydrogen ions in an anaerobic environment. Fetuses with intrauterine growth restriction are considered to become progressively lactacidemic as well as hypoxic. Roles of lactate in the placenta in the presence of fetal growth restriction (FGR) remain to be clarified. Immunohistochemical localization of lactate-related substances, such as a receptor for lactate (hydroxy-carboxylic acid 1 receptor (HCA1 receptor/GPR81)), monocarboxylate transporters (MCTs) for lactate, lactate dehydrogenases (LDHs), and proteins expressed in syncytiotrophoblasts or cytotrophoblasts was examined in placentas of appropriate weight for gestational age (AGA) fetus and those showing FGR. Immunoreactivity for the HCA1 receptor was present in the cytoplasm of some trophoblasts, predominantly localized to their basal (fetus-facing) side, and was frequently colocalized with that for E-cadherin or serine peptidase inhibitor, Kunitz type 1 (SPINT1), a marker protein of cytotrophoblasts. Immunoreactivity for MCT1 and MCT4 was present on the basal and the microvillous (maternal-facing) membranes of trophoblasts in both groups, respectively. Clear immunoreactivity for LDHA and LDHB was also observed in the cytoplasm of trophoblasts, mainly localized to their basal side. However, there were no significant differences in immunohistochemically stained areas of lactate-related substances between AGA and late-onset FGR groups. On the other hand, there were correlations between coefficients of the presence of chorioamnionitis and the values of LDHB and E-cadherin. Immunohistochemical localization of the HCA1 receptor was predominantly observed in the cytoplasm located on the basal side of trophoblasts, suggesting a role of lactate in human placental development, including syncytialization. • Immunoreactivity for the HCA1 receptor is in the cytoplasm of cytotrophoblasts. • Immunoreactivity for MCT1 is in the basal membrane of trophoblasts. • Immunoreactivity for MCT4 is in the microvillous membrane of trophoblasts. • Localization of lactate receptor/transporters are comparable between AGA and FGR. [ABSTRACT FROM AUTHOR]

The maternal–fetal interface is composed of the placenta, which is affiliated with the fetus, and the maternal decidua. During pregnancy, the placenta is mainly responsible for nutrient transport and immune tolerance maintenance, which plays a key role in fetal growth and development and pregnancy maintenance. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that exists in various cell types at the maternal–fetal interface and is involved in multiple cellular processes. Recent studies have highlighted the role of AhR in regulating various physiological processes, including glucose and lipid metabolism, as well as tryptophan metabolism and immune responses, within non-pregnant tissues. This review shifts focus towards understanding how AhR modulation impacts metabolism and immune regulation at the maternal-fetal interface. This may implicate the development of pregnancy-related complications and the potential target of the AhR pathway for therapeutic strategies against poor pregnancy outcomes. • AhR during pregnancy is spatio-temporal specific at the maternal-fetal interface. • AhR plays a role in immunity and metabolism at the maternal-fetal interface. • AhR may be a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Objective • Gestational diabetes mellitus (GDM) is a prevalent metabolic disorder caused by abnormal glucose metabolism during pregnancy. Trophoblast dysfunction induced by hyperglycemia during pregnancy is the main factor leading to the development of GDM. In this study, we evaluated the expression of miR-942-5p in the placenta of patients with GDM and its regulation of trophoblast cell biological function. Methods • HTR-8/SVneo trophoblast cells were incubated with glucose to establish in vitro models, and miR-942-5p mimics transfected cells were added. The expression levels of miR-942-5p, CCAAT-enhancer-binding protein alpha (CEBPA) and N-cadherin in tissues and cells were detected by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), protein blotting and immunohistochemistry (IHC). MTT, flow cytometry and Transwell assays were used to determine changes in cell proliferation, apoptosis and invasion. Dual-luciferase reporter assay and Pearson analysis were used to confirm the association between miR-942-5p and CEBPA. Results • miR-942-5p and N-cadherin were decreased in placental tissue and in human placental trophoblast cells (HTR-8/SVneo) exposed to high glucose (HG) conditions, while CEBPA was increased in placental tissue and HTR-8/ SVneo exposed to HG conditions. Elevated levels of miR942-5p suppressed apoptosis induced by HG and facilitated the proliferative and invasive capacities of HTR-8/SVneo. Mechanistically, we confirmed that miR-942-5p overexpression directly targeted CEBPA and suppressed CEBPA expression, while upregulating N-cadherin expression, which is involved in the EMT process of trophoblast cells and alleviated the dysfunction of trophoblast cells induced by HG in GDM. Conclusion • Overexpression of miR-942-5p promotes proliferation, invasion and EMT of trophoblast cells by targeting and negatively regulating CEBPA. These findings offer novel understanding regarding the treatment of GDM. [ABSTRACT FROM AUTHOR]

First trimester placental development comprises some of the most critical yet understudied events that impact fetal development. Improper placentation leads to a host of health issues that not only impact the fetal period but also influence offspring throughout their lives. Thus, a paradigm to study early placental development is necessary, and this has spurred on the pursuit of new in vitro model systems that recapitulate specific aspects of placentation. One of the most complex and translationally valid models to arise are organoids, three-dimensional structures comprising multiple differentiated cell types that originate from a common progenitor population. Trophoblasts are the progenitor cells of the placenta, serving as the proliferative base for placental development. Recent advances have enabled the derivation of organoids from primary tissue, yet access to first trimester human samples is ethically constrained; derivation from established trophoblast stem cell lines is an alternative source. Organoids have already proven useful in generating insights into molecular events that underlie trophoblast differentiation, with the identification of new cell subtypes that are primed to differentiate down different paths. In this review, (1) we recap early pregnancy development events, (2) provide an overview of the cellular complexity of the placenta, (3) discuss the generation of organoids from tissue versus cellular sources, (4) highlight the value of translational animal models, and (5) focus on the complexities of the molecular regulation of trophoblast organoid development, differentiation, and function. [ABSTRACT FROM AUTHOR]

Oxidative stress (OS) and apoptosis are critical factors in placental development and function. Their interplay influences trophoblast proliferation, differentiation, and invasion, as well as vascular development. An imbalance between these processes can lead to pregnancy-related disorders such as preeclampsia, intrauterine growth restriction, and even spontaneous abortion. Our study seeks to elucidate the associations between preventive antioxidant/protective OS response factors—glutathione (GSH), MutT Homolog 1 (MTH1), and apoptotic regulation modulators—tumor protein p53 and B-cell lymphoma (Bcl-2) transcripts, in the context of spontaneous abortion (30 samples) versus elective termination of pregnancy (20 samples), using immunohistochemistry (IHC) to determine their proteomic expression in chorionic villi within abortive fetal placenta tissue samples. Herein, comparative statistical analyses revealed that both OS response factors, GSH and MTH1, were significantly under-expressed in spontaneous abortion cases as compared to elective. Conversely, for apoptotic regulators, p53 expression was significantly higher in spontaneous abortion cases, whereas Bcl-2 expression was significantly lower in spontaneous abortion cases. These findings suggest that a strong pro-apoptotic signal is prevalent within spontaneous abortion samples, alongside reduced anti-apoptotic protection, depleted antioxidant defenses and compromised oxidative DNA damage prevention/repair, as compared to elective abortion controls. Herein, our hypothesis that OS and apoptosis are closely linked processes contributing to placental dysfunction and spontaneous abortion was thus seemingly corroborated. Our results further highlight the importance of maintaining redox homeostasis and apoptotic regulation for a successful pregnancy. Understanding the mechanisms underlying this interplay is essential for developing potential therapies to manage OS, promote placentation, and avoid unwanted apoptosis, ultimately improving pregnancy outcomes. Antioxidant supplementation, modulation of p53 activity, and the enhancement of DNA repair mechanisms may represent potential approaches to mitigate OS and apoptosis in the placenta. Further research is needed to explore these strategies and their efficacy in preventing spontaneous abortion. [ABSTRACT FROM AUTHOR]

Purpose: Physiological decidual senescence promotes embryo implantation, whereas pathological decidual senescence causes many pregnancy pathologies. The aim of this study was to evaluate the effect of rapamycin on decidual cell subpopulations and endometrial function in physiological and induced senescence and to investigate the decidual cell subpopulations present in physiological conditions during early pregnancy and implantation in mice. Methods: Control, physiological decidualization (0.5 mM cAMP and 1 μM MPA added), and induced senescence (0.1 mM HU added) models with and without 200 nM rapamycin treatment were established using a human endometrial stromal cell line, and decidual cell subpopulations were analyzed by immunofluorescence and flow cytometry. The human extravillous trophoblast cell line AC-1M88 was also cultured in decidualization models, and spheroid expansion analysis was performed. In in vivo studies, decidual cell subpopulations were analyzed by immunofluorescence during early mouse pregnancy. Results: The results revealed that rapamycin decreased DIO2 and β-GAL expressions in physiological and induced senescence without FOXO1. Notably, in induced senescence, increased fragmentation was observed in AC-1M88 cells, and rapamycin treatment successfully attenuated the fragmentation of spheroids. We showed that the FOXO1-DIO2 signaling axis can trigger decidual senescence during early gestation and days of implantation in mice. Conclusions: Our study underlines the importance of rapamycin in modulating decidual cell subpopulations and endometrial tissue function during decidual senescence. The information obtained may provide insight into the pathologies of pregnancy seen due to decidual senescence and guide better treatment strategies for reproductive problems. [ABSTRACT FROM AUTHOR]

The significance of hypoxia at the maternal–fetal interface is proven to be self-explanatory in the context of pregnancy. During the first trimester, low oxygen conditions play a crucial role in processes such as angiogenesis, trophoblast invasion and differentiation, and immune regulation. Recently, there has been increasing research on decidual macrophages, which contribute to the maintenance of immune tolerance, placental and fetal vascular development, and spiral artery remodeling, to investigate the effects of hypoxia on their biological behaviors. On these grounds, this review describes the dynamic changes in oxygen levels at the maternal–fetal interface throughout gestation, summarizing current knowledge on how the hypoxic environment sustains a successful pregnancy by regulating retention, differentiation and efferocytosis of decidual macrophages. Additionally, we explore the relationship between spontaneous miscarriages and an abnormal hypoxia–macrophage axis, shedding light on the underlying mechanisms. However, further studies are essential to elucidate these pathways in greater detail and to develop targeted interventions that could improve pregnancy outcomes. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is an arbovirus with maternal, sexual, and TORCH-related transmission capabilities. After 2015, Brazil had the highest number of ZIVK-infected pregnant women who lost their babies or delivered them with Congenital ZIKV Syndrome (CZS). ZIKV triggers an immune defense in the placenta. This immune response counts with the participation of interleukins and transcription factors. Additionally, it has the potential involvement of human endogenous retroviruses (HERVS). Interleukins are immune response regulators that aid immune tolerance and support syncytial structure development in the placenta, where syncytin receptors facilitate vital cell-to-cell fusion events. HERVs are remnants of ancient viral infections that integrate into the genome and produce syncytin proteins crucial for placental development. Since ZIKV can infect trophoblast cells, we analyzed the relationship between ZIKV infection, HERV, interleukin, and transcription factor modulations in the placenta. To investigate the impact of ZIKV on trophoblast cells, we examined two cell types (BeWo and HTR8) infected with ZIKV-MR766 (African) and ZIKV-IEC-Paraíba (Asian–Brazilian) using Taqman and RT2 Profiler PCR Array assays. Our results indicate that early ZIKV infection (24–72 h) does not induce differential interleukins, transcription factors, and HERV expression. However, we show that the expression of a few of these host defense genes appears to be linked independently of ZIKV infection. Future studies involving additional trophoblastic cell lineages and extended infection timelines will illuminate the dynamic interplay between ZIKV, HERVs, interleukins, and transcription factors in the placenta. [ABSTRACT FROM AUTHOR]

This review describes in detail the morphological, cytoskeletal and gene expression events leading to the gene regulatory network bifurcation point of trophoblast and inner cell mass cells in a variety of mammalian preimplantation embryos. The interrelated processes of compaction and polarity establishment are discussed in terms of how they affect YAP/WWTR activity and the location and fate of cells. Comparisons between mouse, human, cattle, pig and rabbit embryos suggest a conserved role for YAP/WWTR signalling in trophoblast induction in eutherian animals though the mechanisms for, and timing of, YAP/WWTR activation differs among species. Downstream targets show further differences, with the trophoblast marker GATA3 being a direct target in all examined mammals, while CDX2-positive and SOX2-negative regulation varies. [Display omitted] • Onset of compaction relative to polarisation varies among mammalian embryos. • De novo polarisation in embryos transits from apical domain to apical ring stage. • Restriction of nuclear YAP correlates to apical ring stage and trophoblast specification. • Hippo/Lats signals regulate YAP activity in mammalian embryos but not exclusively. • Variations in YAP/TEAD regulation of trophoblast lineage factors CDX2 and GATA3. [ABSTRACT FROM AUTHOR]

Cannabidivarin (CBDV) and cannabigerol (CBG) are minor phytocannabinoids from Cannabis sativa, whose health benefits have been reported. However, studies about the impact of these cannabinoids on fundamental cellular processes in placentation are scarce. Placental development involves physiological endoplasmic reticulum (ER) stress, however when exacerbated it can lead to altered angiogenesis and pregnancy disorders, such as intrauterine growth restriction and preeclampsia. In this work, the effects of CBDV and CBG (1–10 µM) on placental extravillous trophoblasts were studied, using the in vitro model HTR-8/SVneo cells. Both cannabinoids induced anti-proliferative effects and reactive oxygen/nitrogen species generation, which was dependent on transient receptor potential vanilloid 1 (TRPV1) activation. Moreover, CBDV and CBG significantly upregulated, in a TRPV-1 dependent manner, the gene expression of HSPA5/Glucose-regulated protein 78 (GRP78/BiP), a critical chaperone involved in ER stress and unfolded protein response (UPR) activation. Nevertheless, the UPR pathways were differentially activated. Both cannabinoids were able to recruit the IRE branch, while only CBDV enhanced the expression of downstream effectors of the PERK pathway, namely p-eIF2α, ATF4 and CHOP. It also augmented the activity of the apoptotic initiator caspases-8 and -9, though the effector caspases-3/-7 were not activated. TRB3 expression was increased by CBDV, which may hinder apoptosis termination. Moreover, both compounds upregulated the mRNA levels of the angiogenic factors VEGFA, PGF and sFLT1, and disrupted the endothelial-like behavior of HTR-8/SVneo cells, by reducing tube formation. Thus, CBDV and CBG treatment interferes with EVTs functions and may have a negative impact in placentation and in pregnancy outcome. [ABSTRACT FROM AUTHOR]

Abnormal functions of trophoblast cells are associated with the pathogenesis of preeclampsia (PE). Nuclear receptor subfamily 2 group F member 1 (NR2F1) acts as a transcriptionally regulator in many diseases, but its role in PE remains unknown. Hypoxia/reoxygenation (H/R)-stimulated HTR-8/SVneo cells were used to mimic PE injury in vitro. NR2F1 overexpression alleviated trophoblast apoptosis, as evidenced by the decreased number of TUNEL-positive cells and the downregulation of caspase 3 and caspase 9 expression in cells. NR2F1 overexpression increased the invasion and migration ability of HTR-8/SVneo cells, accompanied by increased protein levels of matrix metalloproteinase (MMP)-2 and MMP-9. mRNA-seq was applied to explore the underlying mechanism of NR2F1, identifying growth differentiation factor 15 (GDF15) as the possible downstream effector. Dual-luciferase reporter, ChIP-qPCR, and DNA pull-down assays confirmed that NR2F1 bound to the promoter of GDF15 and transcriptionally inhibited its expression. GDF15 overexpression increased apoptosis and decreased the ability of invasion and migration in HTR-8/SVneo cells expressing NR2F1. MAPK pathway was involved in the regulation of PE. Administration of p38 inhibitor, ERK inhibitor, and JNK inhibitor reversed the effect of simultaneous overexpression NR2F1 and GDF15 on trophoblast apoptosis, invasion, and migration. Our findings demonstrated that NR2F1 overexpression inhibited trophoblast apoptosis and promoted trophoblast invasion and migration. NR2F1 might negatively regulate GDF15 expression by binding to its promoter region, which further inhibited MAPK signaling pathway in PE. Our study highlights that NR2F1 might sever as a potential target in PE. [ABSTRACT FROM AUTHOR]

Insufficient trophoblast migration and impaired uterine spiral artery remodeling are implicated in the pathogenesis of preeclampsia, contributing to inadequate placentation. However, the molecular mechanism underlying this process remains unclear. Aerobic glycolysis, which produces substantial lactate, is crucial for establishing a favorable microenvironment for early uterine preparation and supporting embryo implantation and trophoblast migration. In the present study, we have demonstrated that SORBS2, an RNA-binding protein, regulated aerobic glycolysis and significantly improved trophoblast migration in vitro. Our results showed that SORBS2 expression was significantly reduced in human PE placentas and trophoblasts during hypoxia. Overexpression of SORBS2 enhanced cell proliferation and migration, whereas knockdown of SORBS2 decreased these functions in HTR-8/SVneo cells. Mechanistic studies have demonstrated that SORBS2 directly interacts with the 3' untranslated regions (UTRs) of key glycolysis-related genes, specifically HK2. This interaction results in enhanced stability of HK2 and activation of glycolysis. Moreover, silencing HK2 abrogated the enhancement of proliferation and migration of HTR-8/SVneo cells induced by SORBS2. In conclusion, our findings suggest that the downregulation of SORBS2 may contribute to the pathogenesis of preeclampsia by regulating mRNA stability and inhibiting trophoblast migration during placentation. [ABSTRACT FROM AUTHOR]

Objective: Bisphenol A and phthalate are known endocrine disruptors and capable of inducing epigenetic changes in the human population. However, their impact on the placenta is less well studied. Our objective was to measure the effect of exposure to bisphenol A and benzyl butyl phthalate in first-trimester HTR8-SVneo and third-trimester 3A-sub E trophoblast cells by profiling the DNA methylation pattern of the imprinting control region of the IGF2 (insulin-like growth factor) and H19 genes. Methods: Human placental HTR8-SVneo and 3A-sub E cell lines were treated with two sub-lethal concentrations of bisphenol A and benzyl butyl phthalate. Demethylating agent, 5-azacytidine, was used as a positive control. Cells were harvested on post-treatment days 1 and 4. The methylation profile of six CpG dinucleotide sites, part of the CTCF 6 binding site of the IGF2/H19 imprinting control region, was determined by pyrosequencing. Results: In the first-trimester HTR8-SVneo cell line, we observed a significant increased methylation of the CpG sites 3, 4 when treated with a high concentration of bisphenol A or benzyl butyl phthalate while increased methylation at site 6 for both high and low dose treatment on day 4. Demethylation of the CpG sites 1, 4, and 6 was observed when treated with 5-azacytidine on day 4. In the third-trimester 3A-sub E cell line, no significant changes in the methylation profile were observed under any treatment conditions. Conclusions: The results of this study demonstrate the capability of epigenetic changes in human placenta cells induced by bisphenol A and benzyl butyl phthalate. The observed methylation changes only in the first-trimester HTR8-SVneo cells phthalate may reflect a window of epigenetic susceptibility related to these environmental toxicants. [ABSTRACT FROM AUTHOR]

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Abstract Air pollution poses a significant global concern, notably impacting pregnancy outcomes through mechanisms such as DNA damage, oxidative stress, inflammation, and altered miRNA expression, all of which can adversely affect trophoblast functions. Cleistocalyx nervosum var. paniala, known for its abundance of anthocyanins with diverse biological activities including anti-mutagenic, antioxidant, and anti-inflammatory properties, is the focus of this study examining its effect on Particulate Matter 10 (PM10) soluble extract-induced trophoblast cell dysfunction via miRNA expression. The study involved the extraction of C. nervosum fruit using 70% ethanol, followed by fractionation with hexane, dichloromethane, and ethyl acetate. Subsequent testing for total phenolics, flavonoids, anthocyanins, and antioxidant activity revealed the ethyl acetate fraction (CN-EtOAcF) as possessing the highest phenolic and anthocyanin content along with potent antioxidant activity, prompting its selection for further investigation. In vitro studies on HTR-8/SVneo cells demonstrated that 5–10 µg/mL PM10 soluble extract exposure inhibited cell proliferation, migration, invasion, and induced apoptosis. However, pretreatment with 20–80 µg/mL CN-EtOAcF followed by 5 µg/mL PM10 soluble extract exposure exhibited protective effects against PM10 soluble extract-induced damage, including inflammation inhibition and intracellular ROS suppression. Notably, CN-EtOAcF down-regulated PM10-induced miR-146a-5p expression, with SOX5 identified as a potential target. Overall, CN-EtOAcF demonstrated the potential to protect against PM10-induced harm in trophoblast cells, suggesting its possible application in future therapeutic approaches.

Abstract Dysregulation of macrophage polarization can prevent the invasion of trophoblast cells and further limit spiral artery remodeling in preeclampsia (PE). However, its mechanism is obscure. HTR8-/Svneo cells were cultured under normoxic or hypoxic conditions and extracellular vesicles (EVs) in the culture supernatants were extracted. Next, the cells were incubated with those EVs to investigate their effects on trophoblasts. A co-culture system consisting of HTR8-/Svneo cells and macrophages was used to reveal how the trophoblast-derived EVs affected the macrophage subtype. Finally, a PE mouse model and miR-141-3p knockout mice were used to verify the function of miR-141-3p in PE. Hypoxia induced abnormal increases in the levels of miR-141-3p in HTR8-/Svneo cells and EVs. EVs from hypoxia-treated HTR8-/Svneo cells could downregulate PTEN, a potential target of miR-141-3p, and inhibit trophoblast mitophagy and invasion. However, HTR8-/Svneo cells transfected with an miR-141-3p inhibitor could attenuate the influence of EVs. In an HTR8-/Svneo cell plus macrophage co-culture system, hypoxia-pretreated cells promoted the transformation of macrophages into the M1-phenotye, and HTR8-/Svneo invasion was inhibited by the macrophages. MiR-141 from EVs could target and downregulate dual specificity phosphatase 1 (DUSP1) expression in macrophages, induce formation of the M1 macrophage phenotype in THP-1 cells, downregulate DUSP1 expression, and upregulate TAB2/TAK1 signaling. These results were also demonstrated in normal pregnant mice and PE pregnant mice. A hypoxic environment could upregulate miR-141 expression in the EVs of HTR8-/Svneo cells, and THP-1-derived macrophages could uptake EVs releasing miR-141 to downregulate DUSP1 expression and induce the formation of M1 macrophages, which can lead to the development of PE.

Abstract Background The syncytiotrophoblast (SCT) layer in the placenta serves as a crucial physical barrier separating maternal-fetal circulation, facilitating essential signal and substance exchange between the mother and fetus. Any abnormalities in its formation or function can result in various maternal syndromes, such as preeclampsia. The transition of proliferative villous cytotrophoblasts (VCT) from the mitotic cell cycle to the G0 phase is a prerequisite for VCT differentiation and their fusion into SCT. The imprinting gene P57Kip2, specifically expressed in intermediate VCT capable of fusion, plays a pivotal role in driving this key event. Moreover, aberrant expression of P57Kip2 has been linked to pathological placental conditions and adverse fetal outcomes. Methods Validation of STK40 interaction with P57Kip2 using rigid molecular simulation docking and co-immunoprecipitation. STK40 expression was modulated by lentivirus in BeWo cells, and the effect of STK40 on trophoblast fusion was assessed by real-time quantitative PCR, western blot, immunofluorescence, and cell viability and proliferation assays. Co-immunoprecipitation, transcriptome sequencing, and western blot were used to determine the potential mechanisms by which STK40 regulates P57Kip2. Results In this study, STK40 has been identified as a novel interacting protein with P57Kip2, and its expression is down-regulated during the fusion process of trophoblast cells. Overexpressing STK40 inhibited cell fusion in BeWo cells while stimulating mitotic cell cycle activity. Further experiments indicated that this effect is attributed to its specific binding to the CDK-binding and the Cyclin-binding domains of P57Kip2, mediating the E3 ubiquitin ligase COP1-mediated ubiquitination and degradation of P57Kip2. Moreover, abnormally high expression of STK40 might significantly contribute to the occurrence of preeclampsia. Conclusions This study offers new insights into the role of STK40 in regulating the protein-level homeostasis of P57Kip2 during placental development.

When and why did variations in placental structure and function evolve? Such questions cannot be addressed without a reliable version of mammalian phylogeny. Twenty-five years ago, the mammalian tree was reshaped by molecular phylogenetics. Soon it was shown, in contrast to prevailing theories, that the common ancestor of placental mammals had invasive placentation. Subsequently, evolution of many other features of extraembryonic membranes was addressed. This endeavour stimulated research to fill gaps in our knowledge of placental morphology. Last year the mammalian tree was again revised based on a large set of genomic data. With that in mind, this review provides an update on placentation in the nineteen orders of placental mammals, incorporating much recent data. The principal features such as shape, interdigitation, the interhaemal barrier and the yolk sac are summarized in synoptic tables. The evolution of placental traits and its timing is then explored by reference to the revised mammalian tree. Examples are the early appearance of epitheliochorial placentation in the common ancestor of artiodactyls, perissodactyls, pangolins and carnivores (with reversion to invasive forms in the latter) and later refinements such as the binucleate trophoblast cells and placentomes of ruminants. In primates, the intervillous space gradually evolved from the more basic labyrinth whereas trophoblast invasion of the decidua was a late development in humans and great apes. Only seldom can we glimpse the "why" of placental evolution. The best examples concern placental hormones, including some striking examples of convergent evolution such as the chorionic gonadotropins of primates and equids. In concluding, I review current ideas about what drives placental evolution and identify significant gaps in our knowledge of placentation, including several relevant to the evolution of placentation in primates. [Display omitted] • Placental evolution in mammals is interpreted from the newly revised phylogenomic tree. • Traits with a deep origin include the four-chambered allantoic sac of afrotherians • Traits with a late origin include the intervillous space of haplorrhine primates. • Some morphological traits can be linked to hormone secretion in ruminants, horses, rodents and primates. [ABSTRACT FROM AUTHOR]

Introduction: In human placental development, the trophectoderm (TE) appears in blastocysts on day 5 post-fertilization and develops after implantation into three types of trophoblast lineages: cytotrophoblast (CT), syncytiotrophoblast (ST), and extravillous trophoblast (EVT). CDX2/Cdx2 is expressed in the TE, and Cdx2 expression is upregulated by knockdown of Foxo1 in mouse ESCs. However, the significance of FOXO1 in trophoblast lineage differentiation during the early developmental period remains unclear. In this study, we examined the effect of FOXO1 inhibition on the differentiation of naive human induced pluripotent stem cells (iPSCs) into TE and trophoblast lineages. Methods: We induced TE differentiation from naive iPSCs in the presence or absence of a FOXO1 inhibitor, and the resulting cells were subjected to trophoblast differentiation procedures without the FOXO1 inhibitor. The cells obtained in these processes were assessed for morphology, gene expression, and hCG secretion using phase-contrast microscopy, reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (RT-qPCR), RNA-seq, immunochromatography, and a chemiluminescent enzyme immunoassay. Results: In the induction of trophoblast differentiation from naive iPSCs, treatment with a FOXO1 inhibitor resulted in the enhanced expression of TE markers, CDX2 and HAND1, but conversely decreased the expression of ST markers, such as ERVW1 (Syncytin-1) and GCM1, and an EVT marker, HLA-G. The proportion of cells positive for an early TE marker TACSTD2 and negative for a late TE marker ENPEP was higher in FOXO1 inhibitor-treated cells than in non-treated cells. The expressions of ERVW1 (Syncytin-1), ERVFRD-1 (Syncytin-2), and other endogenous retrovirus (ERV)-associated genes that have been reported to be expressed in trophoblasts were suppressed in the cells obtained by differentiating the TE cells treated with FOXO1 inhibitor. Conclusions: Treatment with a FOXO1 inhibitor during TE induction from naive iPSCs promotes early TE differentiation but hinders the progression of differentiation into ST and EVT. The suppression of ERV-associated genes may be involved in this process.

Abstract Background Placenta accreta spectrum disorders (PAS) are a severe complication characterized by abnormal trophoblast invasion into the myometrium. The underlying mechanisms of PAS involve a complex interplay of various cell types and molecular pathways. Despite its significance, both the characteristics and intricate mechanisms of this condition remain poorly understood. Methods Spatial transcriptomics (ST) and single-cell RNA sequencing (scRNA-seq), were performed on the tissue samples from four PAS patients, including invasive tissues (ST, n = 3; scRNA-seq, n = 4), non-invasive normal placenta samples (ST, n = 1; scRNA-seq, n = 2). Three healthy term pregnant women provided normal myometrium samples (ST, n = 1; scRNA-seq, n = 2). ST analysis characterized the spatial expression landscape, and scRNA-seq was used to identify specific cellular components in PAS. Immunofluorescence staining was conducted to validate the findings. Results ST slices distinctly showed the myometrium in PAS was invaded by three subpopulations of trophoblast cells, extravillous trophoblast cells, cytotrophoblasts, and syncytiotrophoblasts, especially extravillous trophoblast cells. The pathways enriched by genes in trophoblasts, smooth muscle cells (SMC), and immune cells of PAS were mainly associated with immune and inflammation. We identified elevated expression of the angiogenesis-stimulating gene PTK2, alongside the cell proliferation-enhancing gene EGFR, within the trophoblasts of PAS group. Trophoblasts mainly contributed the enhancement of HLA-G and EBI3 signaling, which is crucial in establishing immune escape. Meanwhile, SMC regions in PAS exhibited upregulation of immunomodulatory markers such as CD274, HAVCR2, and IDO1, with CD274 expression experimentally verified to be increased in the invasive SMC areas of the PAS group. Conclusions This study provided information of cellular composition and spatial organization in PAS at single-cell and spatial level. The dysregulated expression of genes in PAS revealed a complex interplay between enhanced immune escape in trophoblasts and immune tolerance in SMCs during invasion in PAS. These findings will enhance our understanding of PAS pathogenesis for developing potential therapeutic strategies.

Key points Heavy metals disrupt mitochondrial function and activate the NOD‐like receptor pyrin‐containing 3 (NLRP3) inflammasome. We investigated the effect of lead (Pb)/cadmium (Cd) on mitochondrial function and NLRP3 inflammasome activation in human trophoblast under normoxic, hypoxic and pro‐inflammatory conditions. JEG‐3, BeWo and HTR‐8/SVneo cells were exposed to Pb or Cd for 24 h in the absence or presence of hypoxia or pro‐inflammatory lipopolysaccharide (LPS) or poly(I:C). Then, we evaluated cell viability, apoptosis, mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨ), NLRP3 inflammasome proteins and interleukin (IL)‐1β secretion. Although our data showed that Pb, Cd, hypoxia, poly(I:C) and LPS decreased mtDNAcn in the three cell lines, the effects of these treatments on other biomarkers were different in the different cell lines. We found that hypoxia decreased ΔΨ and promoted apoptosis in JEG‐3 cells, increased ΔΨ and prevented apoptosis in BeWo cells, and did not change ΔΨ and apoptosis in HTR‐8/SVneo cells. Moreover, Pb under hypoxic conditions reduced ΔΨ and promoted apoptosis of BeWo cells. Exposure of BeWo and HTR‐8/SVneo cells to hypoxia, Pb or Cd alone upregulated the expression of NLRP3 and pro‐caspase 1 but did not activate the NLRP3 inflammasome since cleaved‐caspase 1 and IL‐1β were not increased. To conclude, Pb and Cd affected trophoblast mitochondrial function and NLRP3 proteins in trophoblast cell lines, but in a cell line‐specific way. The objective of this work was an understanding of the effect of lead (Pb) and cadmium (Cd) on mitochondrial function and NLRP3 inflammasome activation in human trophoblast cell lines under normoxic, hypoxic and pro‐inflammatory conditions. Apoptosis of JEG‐3 cells was increased by hypoxia, while in BeWo cells, apoptosis was decreased by hypoxia, and in HTR‐8/SVneo, apoptosis was not affected by hypoxic treatment. Exposure to either Pb or Cd decreased mtDNAcn in three human placental trophoblast cell lines. However, Pb under hypoxia induced a decrease of ΔΨ and promoted apoptosis of BeWo cells, but Cd did not induce a reduction in ΔΨ in the three trophoblast cell lines under any conditions. Exposure to hypoxia, Pb or Cd increased NLRP3 and pro‐caspase 1 in BeWo and HTR‐8/SVneo cells. Our findings highlight that Pb and Cd affected trophoblast mitochondrial function and NLRP3 proteins in trophoblast cell lines but in a cell line‐specific way. [ABSTRACT FROM AUTHOR]

The placenta links feto‐maternal circulation for exchanges of nutrients, gases, and metabolic wastes between the fetus and mother, being essential for pregnancy process and maintenance. The allantois and mesodermal components of amnion, chorion, and yolk sac are derived from extraembryonic mesoderm (Ex‐Mes), however, the mechanisms contributing to distinct components of the placenta and regulation the interactions between allantois and epithelium during chorioallantoic fusion and labyrinth formation remains unclear. Isl1 is expressed in progenitors of the Ex‐Mes and allantois the Isl1 mut mouse line is analyzed to investigate contribution of Isl1+ Ex‐Mes / allantoic progenitors to cells of the allantois and placenta. This study shows that Isl1 identifies the Ex‐Mes progenitors for endothelial and vascular smooth muscle cells, and most of the mesenchymal cells of the placenta and umbilical cord. Deletion of Isl1 causes defects in allantois growth, chorioallantoic fusion, and placenta vessel morphogenesis. RNA‐seq and CUT&Tag analyses revealed that Isl1 promotes allantoic endothelial, inhibits mesenchymal cell differentiation, and allantoic signals regulated by Isl1 mediating the inductive interactions between the allantois and chorion critical for chorionic epithelium differentiation, villous formation, and labyrinth angiogenesis. This study above reveals that Isl1 plays roles in regulating multiple genetic and epigenetic pathways of vascular morphogenesis, provides the insight into the mechanisms for placental formation, highlighting the necessity of Isl1 for placenta formation/pregnant maintenance. [ABSTRACT FROM AUTHOR]

Chorion trophoblasts (CTCs) and immune cell-enriched decidua (DECs) comprise the maternal-fetal membrane interface called the chorio-decidual interface (CDi) which constantly gets exposed to maternal stressors without leading to labor activation. This study explored how CTCs act as a barrier at CDi. The roles of human leukocyte antigen (HLA)-G and progesterone receptor membrane component 2 (PGRMC2) in mediating immune homeostasis were also investigated. The CDi was recreated in a two-chamber microfluidic device (CDi-on-chip) with an outer chamber of primary DECs and immune cell line-derived innate immune cells and an inner chamber of wild-type or PGRMC2 or HLA-G knockout immortalized CTCs. To mimic maternal insults, DECs were treated with lipopolysaccharide, poly(I:C), or oxidative stress inducer cigarette smoke extract. Expression levels of inflammation and immunity genes via targeted RNA sequencing, production of soluble mediators, and immune cell migration into CTCs were determined. In CDi-on-chip, decidua and immune cells became inflammatory in response to insults while CTCs were refractory, highlighting their barrier function. HLA-G and PGRMC2 are found to be vital to immune homeostasis at the CDi, with PGRMC2 serving as an upstream regulator of inflammation, HLA-G expression, and mesenchymal-epithelial transition, and HLA-G serving as a frontline immunomodulatory molecule, thus preventing fetal membrane compromise. An organ-on-chip model of the maternal-fetal membrane interface reveals that HLA-G and PGRMC2 produced by chorion trophoblasts modulate inflammation, thus serving as immunological barrier to prevent membrane compromise. [ABSTRACT FROM AUTHOR]

Prenatal exposure to toxins can adversely affect long-term health outcomes of the offspring. Though chemotherapeutics are now standard of care for treating cancer patients during pregnancy, certain compounds are known to cross the placenta and harm placental tissue. The consequences for the fetus are largely unexplored. Here we examined the responses of newborn cord blood mononuclear cells in tissue culture to two chemotherapeutic drugs, cyclophosphamide and epirubicin, when either directly exposed to these drugs, or indirectly after crossing a placenta trophoblast bilayer barrier. Cord blood mononuclear cells exposed to the conditioned media obtained from cyclophosphamide-exposed trophoblast barriers showed a significant 2.4-fold increase of nuclear ROS levels compared to direct exposure to cyclophosphamide. Indirect exposure to epirubicine-exposed trophoblast barriers not only enhanced nuclear ROS levels but also significantly increased the fraction of cord blood cells with double strand breaks, relative to directly exposed cells. Neither apoptosis nor proliferation markers were affected in cord mononuclear blood cells upon direct or indirect exposure to cyclophosphamide or epirubicin. Our data suggests that trophoblast cells exposed to cyclophosphamide or epirubicine may induce an indirect 'bystander' effect and can aggravate genotoxicity in the fetal compartment. [ABSTRACT FROM AUTHOR]

F. nucleatum, involved in carcinogenesis of colon carcinomas, has been described as part of the commensal flora of the female upper reproductive tract. Although its contribution to destructive inflammatory processes is well described, its role as commensal uterine bacteria has not been thoroughly investigated. Since carcinogenesis shares similar mechanisms with early pregnancy development (including proliferation, invasion, blood supply and the induction of tolerance), these mechanisms induced by F. nucleatum could play a role in early pregnancy. Additionally, implantation and placentation require a well-balanced immune activation, which might be suitably managed by the presence of a limited amount of bacteria or bacterial residues. We assessed the effect of inactivated F. nucleatum on macrophage-trophoblast interactions. Monocytic cells (THP-1) were polarized into M1, M2a or M2c macrophages by IFN-g, IL-4 or TGF-b, respectively, and subsequently treated with inactivated fusobacteria (bacteria:macrophage ratio of 0.1 and 1). Direct effects on macrophages were assessed by viability assay, flow cytometry (antigen presentation molecules and cytokines), qPCR (cytokine expression), in-cell Western (HIF and P-NF-kB) and ELISA (VEGF secretion). The function of first trimester extravillous trophoblast cells (HTR-8/SVneo) in response to macrophage-conditioned medium was microscopically assessed by migration (scratch assay), invasion (sprouting assay) and tube formation. Underlying molecular changes were investigated by ELISA (VEGF secretion) and qPCR (matrix-degrading factors and regulators). Inflammation-primed macrophages (M1) as well as high bacterial amounts increased pro-inflammatory NF-kB expression and inflammatory responses. Subsequently, trophoblast functions were impaired. In contrast, low bacterial stimulation caused an increased HIF activation and subsequent VEGF-A secretion in M2c macrophages. Accordingly, there was an increase of trophoblast tube formation. Our results suggest that a low-mass endometrial/decidual microbiome can be tolerated and while it supports implantation and further pregnancy processes. [ABSTRACT FROM AUTHOR]

The trophoblast lineage differentiation represents a rate‐limiting step in successful embryo implantation. Adhesion, invasion and migration processes within the trophoblast are governed by several transcription factors. Among them, CDX2 is a critical regulator shaping the destiny of the trophoblast. While its altered expression is a linchpin initiating embryo implantation in mice, the precise influence of CDX2 on the functionality and lineage differentiation of early human trophoblast remains unclear. In this study, we employed well‐established human trophoblast stem cell (hTSC) lines with CDX2 overexpression coupled with a 3D in vitro culture system for early human embryos. We revealed that the downregulation of CDX2 is a prerequisite for syncytialization during human embryo implantation based on immunofluorescence, transcriptome analysis, CUT‐tag sequencing and the construction of 3D human trophoblast organoids. While CDX2 overexpression inhibited syncytialization, it propelled hTSC proliferation and invasive migration. CDX2 exerted its influence by interacting with CGA, PTGS2, GCM1, LEF1 and CDH2, thereby hindering premature differentiation of the syncytiotrophoblast. CDX2 overexpression enhanced the epithelial–mesenchymal transition of human trophoblast organoids. In summary, our study provides insights into the molecular characteristics of trophoblast differentiation and development in humans, laying a theoretical foundation for advancing research in embryo implantation. [ABSTRACT FROM AUTHOR]

Preeclampsia is a complex pregnancy-related hypertensive disorder which poses significant risks for both maternal and fetal health. Preeclampsia affects 5–8% of pregnancies in the United States, causing a significant public health and economic burden. Despite extensive research, the etiology and pathogenesis of preeclampsia remain elusive, but have been correlated with maternal conditions such as obesity. In recent decades, the incidence of preeclampsia increased along with the prevalence of obesity among women of reproductive age. Maternal obesity has been shown to negatively affect pregnancy in almost all aspects. However, the precise mechanisms by which obesity influences preeclampsia are unclear. Ankyrin repeat and SOCS Box Containing protein 4 (ASB4) is an E3 ubiquitin ligase that can promote the degradation of a wide range of target proteins. ASB4-null mice display a full spectrum of preeclampsia-like phenotypes during pregnancy including hypertension, proteinuria, and decreased litter size. Furthermore, maternal obesity induced by a high-fat diet aggravates preeclampsia-like phenotypes in pregnant mice lacking ASB4. Variants in the ASB4 gene have been associated with obesity in humans, and a functional connection between the ASB4 gene and obesity has been established in mice. This review discusses the connections between preeclampsia, obesity, and ASB4. [ABSTRACT FROM AUTHOR]

A key aspect of preeclampsia pathophysiology is the reduced invasiveness of trophoblasts and the impairment of spiral artery remodelling. Understanding the causes of altered trophoblast function is critical to understand the development of preeclampsia. B7-H4, a checkpoint molecule, controls a wide range of processes, including T-cell activation, cytokine release, and tumour progression. Our previous findings indicated that B7-H4 levels are elevated in both maternal blood and placental villous tissue during the early stages of preeclampsia. Here, we investigated the function of B7-H4 in trophoblast physiology. Recombinant B7-H4 protein was used to treat human SGHPL-5 extravillous trophoblast cells. Biological functions were investigated using MTT, wound healing, and transwell assays. Signalling pathways were analysed by immunoblotting and immunofluorescence. The functionality of B7-H4 was further confirmed by immunoblotting and immunohistochemical analysis in placental tissues from control and preeclamptic patients following therapeutic plasma exchange (TPE) or standard of care treatment. This study showed that B7-H4 inhibited the proliferation, migration, and invasion capacities of SGHPL-5 extravillous cells while promoting apoptosis by downregulating the PI3K/Akt/STAT3 signalling pathway. These results were consistently confirmed in placental tissues from preterm controls compared to early-onset preeclamptic placental tissues from patients treated with standard of care or TPE treatment. B7-H4 may play a role in the development of preeclampsia by inhibiting essential functions of extravillous trophoblast cells during placental development. One possible mechanism by which TPE improves pregnancy outcomes in preeclampsia is through the elimination of B7-H4 amongst other factors. [ABSTRACT FROM AUTHOR]