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Pregnant women are often prescribed or abuse opioid drugs. The placenta is likely the key to understanding how opioids cause adverse pregnancy outcomes. Maternal oxycodone (OXY) exposure of pregnant mice leads to disturbances in the layer of invasive parietal trophoblast giant cells (pTGC) that forms the interface between the placenta and uterus. These cells are analogous to extravillous trophoblasts of the human placenta. They are crucial to coordinating the metabolic needs of the conceptus with those of the mother and are primary participants in the placenta-brain axis. Their large nuclear size, however, has precluded both single-cell (sc) and single-nucleus (sn) RNA-seq analyses beyond embryonic day (E) 8.5. Here, we compared the transcriptomes of placentas from pregnant mice exposed to OXY with unexposed controls at E12.5, with particular emphasis on the pTGC. The nonfluidic Parse snRNA-seq approach permitted characterization of the nuclear transcriptomes of all the major placental cell lineages and their presumed progenitors at E12.5. OXY exposure had a negligible effect on components of the placental labyrinth, including the two syncytial cell layers, but caused transcriptomic changes consistent with metabolic stress throughout the spongiotrophoblast. Most notably, there was loss of the majority of pTGC, whose normal gene expression is consistent with elevated energy demand relating to biosynthesis of multiple secretory products, especially hormones, and endoduplication of DNA. This unusual sensitivity of pTGC presumably puts the pregnancy and future health of the offspring at particular risk to OXY exposure., Competing Interests: Competing interests statement:The authors declare no competing interest.

Benz[a]anthracene (BaA), a hazardous polycyclic aromatic hydrocarbon classified by the EPA, is a probable reproductive toxicant. Epidemiological studies suggest that BaA exposure may be a risk factor for recurrent miscarriage (RM). However, the underlying mechanisms are not well understood. This study identified DEC1 as a key gene through RNA-seq and single-cell RNA sequencing analysis. DEC1 expression was found to be downregulated in villous tissues from women with RM and in primary extravillous trophoblasts (EVTs) exposed to BaA. BaA suppressed DEC1 expression by promoting abnormal methylation patterns. Further analysis revealed that ARHGAP5 is a direct target of DEC1 in EVTs, where DEC1 inhibits trophoblast invasion by directly regulating ARHGAP5 transcription. Additionally, BaA destabilized matrix metalloproteinase 2 (MMP2) by activating the aryl hydrocarbon receptor (AhR) and promoting E3 ubiquitin ligase MID1-mediated degradation. In a mouse model, BaA induced miscarriage by modulating the DEC1/ARHGAP5 and MID1/MMP2 axes. Notably, BaA-induced miscarriage in mice was prevented by DEC1 overexpression or MID1 knockdown. These findings indicate that BaA exposure leads to miscarriage by suppressing the DEC1/ARHGAP5 pathway and enhancing the MID1/MMP2 pathway in human EVTs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sustained or chronic inflammation in the placenta can result in placental insufficiency, leading to adverse reproductive outcomes such as pregnancy loss. Branched-chain amino acid transaminase 1 (BCAT1) expresses in the placenta and is involved in the pathological inflammatory response, but its role in recurrent miscarriage (RM) has not been fully investigated. In the present study, we delved into the effects of BCAT1 on trophoblast inflammation induced by lipopolysaccharide (LPS) and a mouse model of pregnancy loss induced by LPS. In vitro, after the HTR-8/SVneo cells were treated with LPS and BCATc inhibitor 2 (a selective BCAT inhibitor), the cell apoptosis was verified by TUNEL assay, and the activity of caspase-3 and caspase-9 was detected. Real-time PCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) were used to determine the expression of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and inflammasomes (NLRP3 and ASC) in LPS-treated trophoblast cells. Western blot analysis was performed to verify the expression of phospho-IκBα (p-IκBα) in cells and NF-κB p65 in the nuclei. IF staining was used to detect the nuclear translocation of NF-κB p65. The DNA binding activity of NF-κB was detected by an electrophoretic mobility shift assay (EMSA). The results demonstrated that inhibition of BCAT1 reduced trophoblast apoptosis, suppressed the release of proinflammatory cytokines, and prevented NLRP3 inflammasome activation in response to LPS. Additionally, BCAT1 inhibition blocked the activation of the NF-κB pathway in trophoblasts. This study highlights the potential therapeutic role of targeting BCAT1 in preventing adverse reproductive outcomes associated with chronic placental inflammation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

ATP-binding cassette (ABC) transporters, the important transmembrane efflux transporters, play an irreplaceable role in the placenta barrier. The disposition and drug-drug interaction of clinical drugs are also closely related to the functions of ABC transporters. The trophoblast is a unique feature of the placenta, which is crucial for normal placentation and maintenance during pregnancy. ABC transporters are abundantly expressed in placental syncytiotrophoblast, especially P-gp, BCRP, and MRPs. However, due to the lack of appropriate modeling systems, the molecular mechanisms of regulation between ABC transporters and trophoblast remains unclear. In this report, trophoblast organoids were cultured from human placental villi and developed into three-dimension structures with cavities. Trophoblast organoids exhibited transporter expression and localization comparable to that in villous tissue, indicating their physiological relevance for modeling drug transport. Moreover, fluorescent substrates can accumulate in organoids and be selectively inhibited by inhibitors, indicating the efflux function of ABC transporters (P-gp, BCRP, MRP1, and MRP2) in organoids. Two commonly used hypertension drugs and three antipsychotics were chosen to further validate this drug transport model and demonstrate varying degrees of inhibitory effects on ABC transporters. Overall, a new drug transport model mediated by ABC transporter has been successfully established based on human trophoblast organoids, which can be used to study drug transport in the placenta., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Trophoblast cell surface antigen 2 expressed in several malignant cancers promotes tumor growth and metastasis via several signal transduction pathways. Trop2 is reputed as a prospective biomarker and therapeutic target. Trophoblast cell surface antigen 2-targeted agents, including antibodies, antibody conjugates and therapeutic combinations, could be utilized to fight cancers. To develop an effective drug targeting strategy, we resorted to a new trophoblast cell surface antigen 2-targeted anticancer treatment through aptamer conjugated with chemotherapeutic drug. This study identified trophoblast cell surface antigen 2-specific ssDNA aptamers using engineered trophoblast cell surface antigen 2 overexpression cells for cell-SELEX. The obtained ssDNA aptamer bound to trophoblast cell surface antigen 2 overexpressed cells with nanomolar affinity and was specific for several tumor cell types which express trophoblast cell surface antigen 2 abundantly. Significant cytotoxicity against HT29 cell by the conjugate of trophoblast cell surface antigen 2 aptamer and Emtansine was observed while resulting negligible therapeutic effect on human normal intestinal epithelial cell line HIEC in vitro, indicating that the conjugate shows potential as a promising therapeutic agent. Furthermore, the isolated aptamer demonstrated the ability for the targeted delivery, resulting excellent therapeutic effectiveness of aptamer-drug conjugate for xenograft tumor model of mice with human colorectal cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

25-hydroxycholesterol (25HC) is an oxysterol derived from cholesterol and plays a role in various cellular processes, such as lipid metabolism, inflammatory responses, and cell survival. Extravillous trophoblasts (EVTs) are a major cell type found in the placenta, which are highly energetic cells with proliferative and invasive properties. EVT dysfunction can lead to pregnancy complications, including preeclampsia and intrauterine growth restriction. This study investigated the effects and underlying mechanisms of action of 25HC on EVT proliferation. Swan 71 cells, an EVT cell line, were treated with different concentrations of 25HC. Next, cell proliferation was assessed. The mitochondrial reactive oxygen species (mtROS), mitochondrial membrane potentials (MMPs), lipid peroxidation (LPO), and glutathione (GSH) levels were measured. Apoptosis, ferroptosis, and autophagy were evaluated by western blotting and flow cytometry. The results revealed that 25HC significantly inhibited proliferation and decreased the metabolic activity of EVTs. Moreover, 25HC caused oxidative stress by altering mtROS, LPO, MMPs, and GSH levels. Additionally, 25HC induces apoptosis, ferroptosis, and autophagy through the modulation of relevant protein levels. Interestingly, pretreatment with Z-VAD-FMK, an apoptosis inhibitor, and ferrostatin-1, a ferroptosis inhibitor, partially restored the effects of 25HC on cell proliferation, oxidative stress, and cell death. In summary, our findings suggest that 25HC treatment inhibits EVT proliferation and triggers apoptosis, ferroptosis, and autophagy, which are attributable to oxidative stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Objective: Chemotherapy during pregnancy has a certain risk of causing a series of complications, such as miscarriage, premature birth, or fetal growth restriction, although the relationship between these complications and chemotherapy is currently unclear. This experiment focuses on the possible damage mechanism of the chemotherapeutic drug paclitaxel on placental trophoblast cells, and explores whether chemotherapy can affect pregnancy outcomes by directly damaging placental tissue., Methods: This study explored the mechanism of paclitaxel induced damage on placental trophoblast cell lines JEG-3 and BEWO through immunofluorescence staining, Western blot experiments, cell flow cytometry, Seahorese cell metabolism experiments, and mouse modeling verification., Results: The experiment found that paclitaxel could induce JEG-3 and BEWO cells to produce reactive oxygen species (ROS), and elevate the ratio of Bax/Bcl-2 expression. Besides, paclitaxel mediated the reduction of mitochondrial membrane potential in JEG-3 and BEWO cells, causing damage and leading to mitochondrial autophagy and the occurrence of unfolded protein response. Paclitaxel inhibited the glycolysis rate of JEG-3 and BEWO cells, and leaded to impaired mitochondrial function, including decreased basal respiratory values, decreased respiratory reserve capacity, and proton leakage. In pregnant mice with tumor modeling, paclitaxel could cause DNA damage in placental tissue cells, and might lead to apoptosis of chemotherapy mice placental tissue cells and impairment of normal physiological functions., Conclusion: Paclitaxel may directly or indirectly affect the normal physiological functions of placental trophoblast cells, including energy metabolism and protein synthesis dysfunction, which may be related to the adverse pregnancy outcomes caused by paclitaxel chemotherapy., (© 2024. The Author(s).)

Microcystin-leucine arginine (MC-LR) has been reported to exhibit placental toxicity, leading to potential adverse pregnancy outcomes. Placental abnormalities often coincide with congenital heart defects (CHD). However, the extent to which MC-LR-induced placental abnormalities contribute to CHD and the cellular mechanisms underlying this association remain unknown. In this study, we observed abnormal polarization of placental macrophages in pregnant mice exposed to MC-LR during pregnancy, and the embryos developed cardiac developmental defects that persisted into adulthood. Trophoblast-derived extracellular vesicles (T-EVs) increase in number during pregnancy and act as a critical signal in macrophage polarization. However, MC-LR significantly affected the miRNA expression profile of T-EVs. Upon internalization into macrophages, T-EV-derived miR-377-3p specifically targets the 3'UTR region of NR6A1 to inhibit gene expression. Silencing of transcription suppressor NR6A1 leads to abnormal activation of the downstream mTOR/S6K1/SREBP pathway, inducing metabolic reprogramming and ultimately leading to M1 polarization of macrophages. This study elucidated the placental mechanism underlying MC-LR-induced CHD for the first time, providing insights into the environmental risks associated with CHD., Competing Interests: Declaration of competing interest The authors declare they have no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 (PM 10 ) 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 PM 10 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 PM 10 soluble extract exposure exhibited protective effects against PM 10 soluble extract-induced damage, including inflammation inhibition and intracellular ROS suppression. Notably, CN-EtOAcF down-regulated PM 10 -induced miR-146a-5p expression, with SOX5 identified as a potential target. Overall, CN-EtOAcF demonstrated the potential to protect against PM 10 -induced harm in trophoblast cells, suggesting its possible application in future therapeutic approaches., (© 2024. The Author(s).)

Maternal exposure to nanoparticles during gestation poses potential risks to fetal development. The placenta, serving as a vital interface for maternal-fetal interaction, plays a pivotal role in shielding the fetus from direct nanoparticle exposure. However, the impact of nanoparticles on placental function is still poorly understood, primarily due to the absence of proper human placental models. In this study, we established a placenta-on-a-chip model capable of recapitulating nanoparticle exposure to assess potential nanotoxicity. The model was assembled by coculturing human trophoblast stem cells (hTSCs) and endothelial cells within a dynamic microsystem. hTSCs exhibited progressive differentiation into syncytiotrophoblasts under continuous fluid flow, forming a bilayered trophoblastic epithelium that mimicking both structural and functional aspects of human placental villi. Copper oxide nanoparticles (CuO NPs) were introduced into the trophoblastic side to simulate maternal blood exposure. Our findings revealed that CuO NPs hindered hTSCs differentiation, leading to diminished hormone secretion and impaired glucose transport. Subsequent analysis indicated that CuO NPs disrupted the autophagic flux in trophoblasts and induced apoptosis. Furthermore, the placenta-on-a-chip model exhibited inflammatory responses to CuO NP exposure, including maternal macrophage activation, inflammatory cytokine secretion, and endothelial barrier disruption. Dysfunction of the placental barrier and the ensuing inflammatory cascades may contribute to aberrant fetal development. Overall, our placenta-on-a-chip model offers a promising platform for assessing nanoparticle exposure-related risks and conducting toxicology studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Congenital Chagas disease (CCD) is a worldwide neglected problem with significant treatment limitations. This study aimed to evaluate the potential of Copaifera spp. oleoresins (ORs) against Trypanosoma cruzi infection in trophoblast cells (BeWo lineage) and human chorionic villous explants (HCVE). The cytotoxicity of ORs was investigated using LDH and MTT assays. T. cruzi (Y strain) proliferation, invasion and reversibility were assessed in OR-treated BeWo cells, and proliferation was evaluated in OR-treated HCVE. The ultrastructure of T. cruzi trypomastigotes and amastigotes treated with ORs were analyzed by scanning and transmission electronic microscopy. ROS production in infected and treated BeWo cells and cytokines in BeWo and HCVE were measured. The ORs irreversibly decreased T. cruzi invasion, proliferation and release in BeWo cells by up to 70 %, 82 % and 80 %, respectively, and reduced parasite load in HCVE by up to 80 %. Significant structural changes in treated parasites were observed. ORs showed antioxidant capacity in BeWo cells, reducing ROS production induced by T. cruzi infection. Also, T. cruzi infection modulated the cytokine profile in both BeWo cells and HCVE; however, treatment with ORs upregulated cytokines decreased by T. cruzi infection in BeWo cells, while downregulated cytokines increased by the T. cruzi infection in HCVE. In conclusion, non-cytotoxic concentrations of Copaifera ORs demonstrated promising potential for controlling T. cruzi infection in models of the human maternal-fetal interface., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Proper fetal development requires tight regulation of serotonin concentrations within the fetoplacental unit. This homeostasis is partly maintained by the placental transporter OCT3/SLC22A3, which takes up serotonin from the fetal circulation. Metformin, an antidiabetic drug commonly used to treat gestational diabetes mellitus, was shown to inhibit OCT3. We, therefore, hypothesized that its use during pregnancy could disrupt placental serotonin homeostasis. This hypothesis was tested using three experimental model systems: primary trophoblast cells isolated from the human term placenta, fresh villous human term placenta fragments, and rat term placenta perfusions. Inhibition of serotonin transport by metformin at three concentrations (1 μM, 10 μM, and 100 μM) was assessed in all three models. The OCT3 inhibitor decynium-22 (100 μM) and paroxetine (100 μM), a dual inhibitor of SERT and OCT3, were used as controls. In primary trophoblasts, paroxetine exhibited the strongest inhibition of serotonin uptake, followed by decynium-22. Metformin showed a concentration-dependent effect, reducing serotonin uptake by up to 57 % at the highest concentration. Its inhibitory effect was less pronounced in fresh villous fragments but remained statistically significant at all concentrations. In the perfused rat placenta, metformin demonstrated a concentration-dependent effect, reducing placental serotonin uptake by 44 % at the highest concentration tested. Our findings across all experimental models show inhibition of placental OCT3 by metformin, resulting in reduced serotonin uptake by the trophoblast. This sheds light on mechanisms that may underpin metformin-mediated effects on fetal development., Competing Interests: Declaration of Competing Interest none, (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Recurrent spontaneous abortion (RSA) has a complex pathogenesis with an increasing prevalence and is one of the most intractable clinical challenges in the field of reproductive medicine. Quercetin (QCT) is an effective active ingredient extracted from Semen Cuscutae and Herba Taxilli used in traditional Chinese medicine for tonifyng the kidneys and promoting fetal restoration. Although QCT helps improve adverse pregnancy outcomes, the specific mechanism remains unclear. The trophoblast cell line HTR-8/SVneo cultured in vitro was treated with different concentrations of QCT, and the cell counting kit-8 assay, wound healing assay, transwell assay, and western blotting were used to evaluate the effects and mechanisms of QCT on the proliferation, migration, and invasion of HTR-8/SVneo cells, respectively. To assess the expression levels of miR-149-3p and AKT serine/threonine kinase 1 (AKT1), quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis were performed. A dual-luciferase reporter assay was used to investigate the potential regulatory relationship between miR-149-3p and AKT1. Our results showed that QCT promoted the proliferation, migration, and invasion of trophoblast cells, promoted the expression of MMP2, MMP9, and vimentin, and downregulated the expression of E-cadherin. Mechanistically, QCT downregulated the expression of miR-149-3p and upregulated the expression of AKT1, and miR-149-3p directly targets AKT1, negatively regulating its expression. Overexpression of miR-149-3p and silencing of AKT1 counteracted the promotional effects of QCT on trophoblast proliferation, migration, and invasion. Taken together, QCT regulates the migration and invasion abilities of HTR-8/SVneo cells through the miR-149-3p/AKT1 axis, which may provide a promising therapeutic approach for RSA., (© 2024 The Author(s). The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Background: In response to the high-glucose environment in patients with gestational diabetes mellitus (GDM), trophoblast cells undergo a series of pathological changes. Gamma-aminobutyric acid type A receptor subunit pi (GABRP) is involved in the development of pregnancy-related diseases and regulation of blood glucose., Objectives: To explore the relationship between GABRP and hyperglycemia stimulation in GDM patients, and to provide preliminary experimental evidence for whether GABRP has the potential as a molecular target for the treatment of GDM., Material and Methods: Within 30 min after birth, placental samples were taken from 20 GDM patients and 20 pregnant women without GDM. Human chorionic trophoblast HTR-8/SVneo cells were utilized for in vitro experimental investigation. We explored changes in GABRP expression in placental samples and HTR-8/Svneo cells using western blot and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Cells in the high-glucose treatment group were exposed to medium containing 25 mM glucose. To explore the relationship between GABRP and high-glucose stimulation, GABRP was overexpressed in HTR-8/SVneo cells. We monitored the cell viability, invasion and migration abilities using Cell Counting Kit-8 (CCK-8), transwell and scratch assays, respectively., Results: We found that GABRP expression was significantly reduced in placental samples from GDM patients. Furthermore, high-glucose treatment decreased the expression level of GABRP in HTR-8/SVneo cells. High-glucose stimulation reduced the cell viability, invasion and migration abilities. GABRP overexpression reversed the biological dysfunction of the cells induced by high-glucose stimulation., Conclusions: Hyperglycemia in GDM patients downregulates the expression of GABRP, and overexpression of GABRP promotes the viability, migration and invasive ability of HTR8-/SVneo cells.

Pravastatin is a promising medication to treat preeclampsia. However, the appropriate dose of pravastatin for managing preeclampsia has not been established. In this in vitro study, we examined the effects of low concentrations of pravastatin (0.01 to 10 µM) under hypoxic conditions on two types of placental cells and found that pravastatin decreased sFlt-1 levels up to 34% in cytotrophoblast cells isolated from human term placentas. Furthermore, we showed that sFlt-1 levels in HTR-8/SVneo cells, a cell line derived from first trimester trophoblast cells, decreased after exposure to very low concentrations of pravastatin (0.01, 0.1 µM). We also examined the effects of pravastatin on uterine spiral artery remodeling-related events and showed in wound healing and tube formation assays that low concentrations of pravastatin upregulated cell migration and invasion in HTR-8/SVneo cells. These results demonstrated that a low dose of pravastatin has in vitro effects that suggest a potential for anti-preeclamptic effects in vivo., (© 2024. The Author(s).)

Introduction: 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., Methods: 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., Results: 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., Discussion: 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., Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest exists., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Abstract: Low molecular weight heparins (LMWH) are used to prevent or treat thromboembolic events during pregnancy. Although studies suggest an overall protective effect of LMWH in preeclampsia (PE), their use in PE remains controversial. LMWH may convey beneficial effects in PE independent of their anticoagulant activity, possibly by inhibiting inflammation. Here, we evaluated whether LMWH inhibit placental thromboinflammation and trophoblast NLRP3 inflammasome activation. Using an established procoagulant extracellular vesicle-induced and platelet-dependent PE-like mouse model, we show that LMWH reduces pregnancy loss and trophoblast inflammasome activation, restores altered trophoblast differentiation, and improves trophoblast proliferation in vivo and in vitro. Moreover, LMWH inhibits platelet-independent trophoblast NLRP3 (NLR family pyrin domain containing 3) inflammasome activation. Mechanistically, LMWH activates via heparin-binding epidermal growth factor (HBEGF) signaling the PI3-kinase-AKT pathway in trophoblasts, thus preventing inflammasome activation. In human PE placental explants, inflammasome activation and PI3-kinase-AKT signaling events were reduced with LMWH treatment compared with those without LMWH treatment. Thus, LMWH inhibits sterile inflammation via the HBEGF signaling pathway in trophoblasts and ameliorates PE-associated complications. These findings suggest that drugs targeting the inflammasome may be evaluated in PE and identify a signaling mechanism through which LMWH ameliorates PE, thus providing a rationale for the use of LMWH in PE., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Triphenyl phosphate (TPhP) is an organophosphate flame retardant that is widely used in many commercial products. The United States Environmental Protection Agency has listed TPhP as a priority compound that requires health risk assessment. We previously found that TPhP could accumulate in the placentae of mice and impair birth outcomes by activating peroxisome proliferator-activated receptor gamma (PPARγ) in the placental trophoblast. However, the underlying mechanism remains unknown. In this study, we used a mouse intrauterine exposure model and found that TPhP induced preeclampsia (PE)-like symptoms, including new on-set gestational hypertension and proteinuria. Immunofluorescence analysis showed that during placentation, PPARγ was mainly expressed in the labyrinth layer and decidua of the placenta. TPhP significantly decreased placental implantation depth and impeded uterine spiral artery remodeling by activating PPARγ. The results of the in vitro experiments confirmed that TPhP inhibited extravillous trophoblast (EVT) cell migration and invasion by activating PPARγ and inhibiting the PI3K-AKT signaling pathway. Overall, our data demonstrated that TPhP could activate PPARγ in EVT cells, inhibit cell migration and invasion, impede placental implantation and uterine spiral artery remodeling, then induce PE-like symptom and impair birth outcomes. Although the exposure doses used in this study was several orders of magnitude higher than human daily intake, our study highlights the placenta as a potential target organ of TPhP worthy of further research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed toward the relevance of hypoxia as modulator of trophoblast cell death. Previous reports have shown that leptin, a placental cytokine, promotes cell survival in both cell culture and placental explant models. The aim of this work is to establish the role of leptin in apoptosis under hypoxic condition in trophoblast cells. In this study, we evaluated the effect of cobalt chloride, a hypoxia mimicking agent that stabilizes the expression of hypoxia-inducible factor-1 alpha, on Swan-71 and human placental explants. Hypoxia chamber was also used to generate 2% oxygen. Apoptosis was determined by the presence of apoptotic nucleus, fragmentation of DNA and Caspase-3 and PARP-1 cleavage. The pro-apoptotic proteins BAX, BID, BAD, and BAK and the anti-apoptotic effectors BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1 were also analyzed. We found that hypoxia-inducible factor-1 alpha stabilization increased the appearance of apoptotic nucleus, fragmentation of DNA, and Caspase-3 and PARP-1 cleavage. Hypoxia mimicking conditions enhanced the expression of pro-apoptotic effectors BAX, BID, BAD, and BAK. Hypoxia-inducible factor-1 alpha stabilization also downregulated the level of BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1. All these apoptotic parameters changes were reversed with leptin treatment. Moreover, we showed that leptin action on apoptosis modulation involves PI3K and MAPK signaling pathways. Obtained data demonstrate that hypoxia-inducible factor-1 alpha stabilization induces apoptosis in human placenta and leptin counteracts this effect, reinforcing its role as a survival cytokine., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

There are no effective therapies to prevent preeclampsia (PE). Pravastatin shows promise by attenuating processes associated with PE such as decreased cytotrophoblast (CTB) migration, aberrant angiogenesis, and increased oxidative stress. This study assesses the effects of pravastatin on hyperglycemia-induced CTB dysfunction., Methods: Human CTB cells were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 h. Some cells were pretreated with pravastatin (1 µg/mL), while others were cotreated with pravastatin and glucose. The expression of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) mRNA, vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble endoglin (sEng) were measured. CTB migration was assayed using a CytoSelect migration assay kit. Statistical comparisons were performed using an analysis of variance with Duncan's post hoc test., Results: The hyperglycemia-induced downregulation of uPA was attenuated in CTB cells pretreated with pravastatin at glucose levels > 200 mg/dL and cotreated at glucose levels > 300 mg/dL ( p < 0.05). Hyperglycemia-induced decreases in VEGF and PlGF and increases in sEng and sFlt-1 were attenuated in both the pretreatment and cotreatment samples regardless of glucose dose ( p < 0.05). Pravastatin attenuated hyperglycemia-induced dysfunction of CTB migration., Conclusions: Pravastatin mitigates stress signaling responses in hyperglycemic conditions, weakening processes leading to abnormal CTB migration and invasion associated with PE in pregnancy.

Exposure to environmental BaP or its metabolite BPDE causes trophoblast cell dysfunctions to induce miscarriage (abnormal early embryo loss), which might be generally regulated by lncRNAs. IL1B, a critical inflammatory cytokine, is closely associated with adverse pregnancy outcomes. However, whether IL1B might cause dysfunctions of BaP/BPDE-exposed trophoblast cells to induce miscarriage, as well as its specific epigenetic regulatory mechanisms, is completely unexplored. In this study, we find that BPDE-DNA adducts, trophoblast cell dysfunctions, and miscarriage are closely associated. Moreover, we also identify a novel lnc-HZ06 and IL1B, both of which are highly expressed in BPDE-exposed trophoblast cells, in villous tissues of recurrent miscarriage patients, and in placental tissues of BaP-exposed mice with miscarriage. Both lnc-HZ06 and IL1B suppress trophoblast cell migration/invasion and increase apoptosis. In mechanism, lnc-HZ06 promotes STAT4-mediated IL1B mRNA transcription, enhances IL1B mRNA stability by promoting the formation of METTL3/HuR/IL1B mRNA ternary complex, and finally up-regulates IL1B expression levels. BPDE exposure promotes TBP-mediated lnc-HZ06 transcription, and thus up-regulates IL1B levels. Knockdown of either murine lnc-hz06 (which down-regulates Il1b levels) or murine Il1b could alleviate miscarriage in BaP-exposed mice. Collectively, this study not only discovers novel biological mechanisms and pathogenesis of unexplained miscarriage but also provides novel potential targets for treatment against BaP/BPDE-induced miscarriage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: This study aimed to explore the association between ferroptosis, a newly identified type of cell death, and the role of retinoic acid in developing pregnancy complications. Therefore, the effects of all-trans retinoic acid (ATRA) on ferroptosis susceptibility in BeWo cells were assessed to understand abnormal placental development., Methods: BeWo cells were used as surrogates for cytotrophoblasts. The effect of ATRA on ferroptosis sensitivity was assessed on BeWo cells pretreated with ATRA or dimethyl sulfoxide (DMSO; control), following which the LDH-releasing assay was performed. The effects of ATRA pretreatment on the antioxidant defense system (including glutathione [GSH], mitochondrial membrane potential, and heme oxygenase-1 [HMOX1]) in BeWo cells were assessed using assay kits, RT-qPCR, and HMOX1 immunostaining. To evaluate the effect of ATRA on BeWo cells, HMOX1 was silenced in BeWo cells using shRNA., Results: ATRA pretreatment increased ferroptosis resistance in BeWo cells. Although with pretreatment, qPCR indicated upregulation of HMOX1, no significant change was observed in the GSH levels or mitochondrial membrane potential. This was corroborated by intensified immunostaining for heme oxygenase-1 protein (HO-1). Notably, the protective effect of ATRA against ferroptosis was negated when HO-1 was inhibited. Although HMOX1-silenced BeWo cells exhibited heightened ferroptosis sensitivity compared with controls, ATRA pretreatment counteracted ferroptosis in these cells., Discussion: ATRA pretreatment promotes BeWo cell viability by suppressing ferroptosis and upregulating HMOX1 and this can be used as a potential therapeutic strategy for addressing placental complications associated with ferroptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024. Published by Elsevier Ltd.)

Exposures to complex environmental chemical mixtures during pregnancy reach and target the feto-placental unit. This study investigates the influence of environmental chemical mixtures on placental bioenergetics. Recognizing the essential role of the epidermal growth factor receptor (EGFR) in placental development and its role in stimulating glycolysis and mitochondrial respiration in trophoblast cells, we explored the effects of chemicals known to disrupt EGFR signaling on cellular energy production. Human primary cytotrophoblasts (hCTBs) and a first-trimester extravillous trophoblast cell line (HTR-8/SVneo) were exposed to a mixture of EGFR-interfering chemicals, including atrazine, bisphenol S, niclosamide, PCB-126, PCB-153, and trans-nonachlor. An RNA sequencing approach revealed that the mixture altered the transcriptional signature of genes involved in cellular energetics. Next, the impact of the mixture on cellular bioenergetics was evaluated using a combination of mitochondrial and glycolytic stress tests, ATP production, glucose consumption, lactate synthesis, and super-resolution imaging. The chemical mixture did not alter basal oxygen consumption but diminished the maximum respiratory capacity in a dose-dependent manner, indicating a disruption of mitochondrial function. The respiratory capacity and ATP production were increased by EGF, while the Chem-Mix reduced both EGF- and non-EGF-mediated oxygen consumption rate in hCTBs. A similar pattern was observed in the glycolytic medium acidification, with EGF increasing the acidification, and the Chem-Mix blocking EGF-induced glycolytic acidification. Furthermore, direct stochastic optical reconstruction microscopy (dSTORM) imaging demonstrated that the Chem-Mix led to a reduction of the mitochondrial network architecture, with findings supported by a decrease in the abundance of OPA1, a mitochondrial membrane GTPase involved in mitochondrial fusion. In conclusion, we demonstrated that a mixture of EGFR-disrupting chemicals alters mitochondrial remodeling, resulting in disturbed cellular bioenergetics, reducing the capacity of human cytotrophoblast cells to generate energy. Future studies should investigate the mechanism by which mitochondrial dynamics are disrupted and the pathological significance of these findings., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Almudena Veiga-Lopez reports financial support was provided by the National Institute of Environmental Health Sciences, National Institutes of Health, United States. Almudena Veiga-Lopez reports financial support was provided by Michael Reese Foundation. Ying Hu reports financial support was provided by National Institute of General Medical Sciences, National Institutes of Health, United States. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Benzo(a)pyrene (BaP) or benzo (a) pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE) exposure causes trophoblast cell dysfunctions and induces miscarriage, which is generally epigenetically regulated. Homologous recombination (HR) repair of DNA double strand break (DSB) plays a crucial role in maintenance of genetic stability and cell normal functions. However, whether BaP/BPDE might suppress HR repair in human trophoblast cells to induce miscarriage, as well as its epigenetic regulatory mechanism, is largely unclear. In this study, we find that BaP/BPDE suppresses HR repair of DSB in trophoblast cells and eventually induces miscarriage by up-regulating lnc-HZ08. In mechanism, lnc-HZ08 (1) down-regulates the expression levels of FOXA1 (forkhead box A1) and thus suppresses FOXA1-mediated mRNA transcription of BRCA1 (Breast cancer susceptibility gene 1) and CtIP (CtBP-interacting protein), (2) impairs BRCA1 and CtIP protein interactions by competitive binding with CtIP through lnc-HZ08-1 fragment, and also (3) suppresses BRCA1-mediated CtIP ubiquitination without affecting CtIP stability, three of which eventually suppress HR repair in human trophoblast cells. Supplement with murine Ctip could efficiently restore (i.e. increase) HR repair and alleviate miscarriage in BaP-exposed mouse model. Collectively, this study not only reveals the association and causality among BaP/BPDE exposure, the defective HR repair, and miscarriage, but also discovers novel mechanism in lnc-HZ08-regulated BRCA1/CtIP-mediated HR repair, bridging epigenetic regulation and genetic instability and also providing an efficient approach for treatment against BaP/BPDE-induced unexplained miscarriage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2024. The Author(s).)

It has been well acknowledged that maternal exposure to fine particulate matters (PM 2.5 ) might lead to poor pregnancy outcomes including the intrauterine growth restriction (IUGR) by interfering with the placental development. Our previous studies have demonstrated that maternal PM 2.5 exposure induces IUGR, accompanied with increased maternal circulating TNFα level and impaired extravillous trophoblast cells (EVTs) invasion in mice. In this study, HTR8/SVneo cells, the immortalized human EVTs line, were used to assess effects and the underlying molecular mechanisms of nicotinamide on the impaired EVTs invasion. Our results showed that, the placental FLT1 protein level was significantly increased whereas maternal serum nicotinamide concentration was remarkably decreased in PM 2.5 -exposured pregnant mice at GD17.5 (vaginal plug day=GD0.5), compared to that in normal GD17.5 pregnant mice. FLT1 expression in HTR8/SVneo cells was significantly up-regulated by TNFα treatment, and the down-regulated FLT1 expression effectively abated the inhibitory effects of TNFα on HTR8/SVneo cells migration and invasion. Meanwhile, TNFα promoted reactive oxygen species (ROS) production and NF-κB signaling pathway activation in HTR8/SVneo cells in a dose-dependent manner. Nicotinamide treatment significantly reversed the effects of TNFα on cell migration and invasion, as well as the FLT1 expression, ROS production and NF-κB pathway activation. In summary, increased TNFα induced by PM 2.5 exposure inhibits EVTs invasion by activating the ROS/NF-κB/FLT1 signaling pathway, and this adverse effect could be attenuated by nicotinamide treatment, suggesting a potential application in the clinical intervention of PM 2.5 -induced IUGR., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The placenta experiences a low-oxygen stage during early pregnancy. Aspirin is an effective preventative treatment for preeclampsia if applied early in pregnancy. Elevation of fibronectin (FN) level has been reported to be associated with preeclampsia; however, the role of FN in the physiological hypoxic phase and whether aspirin exerts its effect on FN at this hypoxic stage remain unknown. We determined pregnancy outcomes by injecting saline or recombinant FN protein into C57BL/6 pregnant mice and one group of FN-injected mice was fed aspirin. The effects of FN, the underlying pathways on trophoblast biology, and cilia formation under hypoxia were investigated in FN-pretreated or FN-knockdown HTR-8/SVneo cells in a hypoxic chamber (0.1 % O 2 ). Preeclampsia-like phenotypes, including blood pressure elevation and proteinuria, developed in FN-injected pregnant mice. The fetal weight of FN-injected mice was significantly lower than that of non-FN-injected mice (p < 0.005). Trophoblast FN expression was upregulated under hypoxia, which could be suppressed by aspirin treatment. FN inhibited trophoblast invasion and migration under hypoxia, and this inhibitory effect occurred through downregulating ZEB1/2, MMP 9 and the Akt and MAPK signaling pathways. Ciliogenesis of trophoblasts was stimulated under hypoxia but was inhibited by FN treatment. Aspirin was shown to reverse the FN-mediated inhibitory effect on trophoblast invasion/migration and ciliogenesis. In conclusion, FN overexpression induces preeclampsia-like symptoms and impairs fetal growth in mice. Aspirin may exert its suppressive effect on FN upregulation and FN-mediated cell function in the hypoxic stage of pregnancy and therefore provides a preventative effect on preeclampsia development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Evidence suggests that increasing salt intake in pregnancy lowers blood pressure, protecting against preeclampsia. We hypothesized that sodium (Na + ) evokes beneficial placental signals that are disrupted in preeclampsia., Methods: Blood and urine were collected from nonpregnant women of reproductive age (n=26) and pregnant women with (n=50) and without (n=55) preeclampsia, along with placental biopsies. Human trophoblast cell lines and primary human trophoblasts were cultured with varying Na + concentrations., Results: Women with preeclampsia had reduced placental and urinary Na + concentrations, yet increased urinary angiotensinogen and reduced active renin, aldosterone concentrations, and osmotic response signal TonEBP (tonicity-responsive enhancer binding protein) expression. In trophoblast cell cultures, TonEBP was consistently increased upon augmented Na + exposure. Mechanistically, inhibiting Na + /K + -ATPase or adding mannitol evoked the TonEBP response, whereas inhibition of cytoskeletal signaling abolished it., Conclusions: Enhanced Na + availability induced osmotic gradient-dependent cytoskeletal signals in trophoblasts, resulting in proangiogenic responses. As placental salt availability is compromised in preeclampsia, adverse systemic responses are thus conceivable., Competing Interests: None.

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., (© 2024. The Author(s).)

Resmethrin, a type I pyrethroid insecticide, is frequently used globally in residential and farmland areas to control pests. Owing to the repeated administration of resmethrin, and particularly because of its lipophilic nature, residues have been detected in various environments, crops, and livestock. Previous studies have shown the adverse effects of resmethrin, including neurotoxicity and hepatotoxicity. However, the toxic effects of resmethrin on the female reproductive system have rarely been investigated. In the present study, we used two cell types, porcine trophectoderm (pTr) and porcine uterine luminal epithelial (pLE) cells, to examine the toxic effects of resmethrin on implantation and its mechanisms. Our study showed that resmethrin exposure induced apoptosis and inhibited cell cycle progression, thereby reducing the viability of both cell types. In addition, calcium homeostasis was disrupted following resmethrin treatment, and disrupted calcium homeostasis impaired the mitochondrial membrane potential and mitochondrial respiration. In addition to mitochondrial dysfunction, GRP75 and ER stress-related proteins were upregulated. Furthermore, the AKT and MAPK cascades were altered, and reactive oxygen species production and inflammation occurred after resmethrin treatment. Ultimately, through various mechanisms, resmethrin decreased the migratory abilities, and it could diminish the crosstalk between the two cell lines and lower the probability of successful implantation. Overall, we demonstrated that resmethrin interfered with the implantation process by triggering various toxic mechanisms. This study presents, for the first time, evidence regarding the mechanisms through which resmethrin exerts toxic effects on the female reproductive system, thereby raising awareness regarding the potential implications of its widespread use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: With the decline of global fertility, drug therapeutic of ectopic pregnancy is of great significance. Lithospermum erythrorhizon is using for embryo killing as herbal medicine. Shikonin is the critical nucleus of Lithospermum erythrorhizon ; however, the mechanism is still unclear. The study aimed to explore the mechanism of shikonin against ectopic pregnancy., Material and Methods: In this study, we examined the viability and LDH release of HTR-8/SVneo cells by assays, observed pore formation in cell membranes by microscopy imaging and PI staining, and IL-1β release by WB and ELISA assay kit. Then, we used network pharmacology to analyse the potential interaction between shikonin, ectopic pregnancy and pyroptosis and used molecular docking techniques to verify interactions between shikonin and core common targets. Finally, western blotting and immunofluorescence assay were used to explore the mechanism of shikonin-inducing pyroptosis of HTR-8/SVneo cells., Results: Shikonin could cause a significant inhibition of HTR-8/SVneo cell viability in a concentration- and time-dependent manner. In HTR-8/SVneo cells, shikonin-induced cell swelling, bubble formation, an increase in the release of lactate dehydrogenase (LDH) and up-regulation of several pyroptosis-associated factors. And network pharmacology showed that The main targets of shikonin-ectopic pregnancy-pyroptosis were IL-1β and caspase-1, and molecular docking results showed that shikonin can closely bind to IL-1β, caspase-1 and GSDMD. Additionally, the necroptosis inhibitor GSK'872 could not suppress the expression of mature-IL-1β and prevent the pyroptosis phenotype from developing. However, the nucleotide oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inhibitor MCC-950 could downregulate the expression of pyroptosis-associated factors and prevent the pyroptosis phenotype from developing. Shikonin led to an elevation in the expression of cathepsin B (CTSB), and the CTSB inhibitor CA-074 abolished pyroptosis induced by shikonin; however, the NLRP3 inhibitor MCC-950 could not inhibit the expression of CTSB., Conclusions: Our results suggest that shikonin activates CTSB to induce NLRP3-dependent pyroptosis in HTR-8/SVneo cells. This study has important clinical implications for the treatment of ectopic pregnancy.

Background: Asiaticoside (AS) has been reported to improve the changes induced by high glucose stimulation, and it may have potential therapeutic effects on gestational diabetes mellitus (GDM). This study aims to explore the effect of AS on the cell model of GDM and the action mechanism of the PI3K/AKT pathway., Methods: The GDM model was established in HTR-8/Svneo cells with a high glucose (HG) medium. After the cytotoxicity assay of AS, cells were divided into the control group, HG group and HG + AS group to conduct control experiment in cells. The cell proliferation and migration were detected by CCK-8 assay and scratch test, respectively. The mRNA levels of PI3K, AKT2, mTORC1, and GLUT4 in PI3K/AKT signalling pathway were measured by RT-PCR, and the protein expressions of these signalling molecules were monitored by western blot., Results: AS showed a promotion effect on the cell proliferation rate of HTR-8/Svneo cells, and 80 μmol/L AS with a treatment time of 48 h had no cytotoxicity. The cell proliferation rate, migration rate, mRNA levels and protein expressions of PI3K, AKT2, mTORC1, and GLUT4 in the HG group were significantly lower than those in the control group, which were significantly increased in the HG + AS group ( p  < 0.05)., Conclusions: AS can facilitate the cell proliferation and migration in the cell model of GDM, and might play a role in GDM treatment via PI3K/AKT pathway.

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., (© 2024. The Author(s).)

Embryonic stem cells (ESCs) can differentiate into all cell types of the embryonic germ layers. ESCs can also generate totipotent 2C-like cells and trophectodermal cells. However, these latter transitions occur at low frequency due to epigenetic barriers, the nature of which is not fully understood. Here, we show that treating mouse ESCs with sodium butyrate (NaB) increases the population of 2C-like cells and enables direct reprogramming of ESCs into trophoblast stem cells (TSCs) without a transition through a 2C-like state. Mechanistically, NaB inhibits histone deacetylase activities in the LSD1-HDAC1/2 corepressor complex. This increases acetylation levels in the regulatory regions of both 2C- and TSC-specific genes, promoting their expression. In addition, NaB-treated cells acquire the capacity to generate blastocyst-like structures that can develop beyond the implantation stage in vitro and form deciduae in vivo. These results identify how epigenetics restrict the totipotent and trophectoderm fate in mouse ESCs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the role of angiotensin-converting enzyme 1 (ACE1) in H2O2-induced trophoblast cell injury and the potential molecular mechanisms. Oxidative stress was modeled by exposing HTR-8/SVneo cells to 200 μM H2O2. Western blot and real-time quantitative PCR methods were used to detect protein and mRNA expression level of ACE1 in chorionic villus tissue and trophoblast HTR-8/SVneo cell. Inhibition of ACE1 expression was achieved by transfection with small interfering RNA. Then flow cytometry, Cell Counting Kit-8, and Transwell assay was used to assess apoptosis, viability, and migration ability of the cells. Reactive oxygen species (ROS) were detected by fluorescent probes, and malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) activities were determined by corresponding detection kits. Angiotensin-converting enzyme 1 expression was upregulated in chorionic villus tissue of patients with missed abortion (MA) compared with individuals with normal early pregnancy abortion. H2O2 induced elevated ACE1 expression in HTR-8/SVneo cells, promoted apoptosis, and inhibited cell viability and migration. Knockdown of ACE1 expression inhibited H2O2-induced effects to enhance cell viability and migration and suppress apoptosis. Additionally, H2O2 stimulation caused increased levels of ROS and MDA and decreased SOD and GSH activity in the cells, whereas knockdown of ACE1 expression led to opposite changes of these oxidative stress indicators. Moreover, knockdown of ACE1 attenuated the inhibitory effect of H2O2 on the Nrf2/HO-1 pathway. Angiotensin-converting enzyme 1 was associated with MA, and it promoted H2O2-induced injury of trophoblast cells through inhibiting the Nrf2 pathway. Therefore, ACE1 may serve as a potential therapeutic target for MA., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A limited number of accessible and representative models of human trophoblast cells currently exist for the study of placentation. Current stem cell models involve either a transition through a naïve stem cell state or precise dynamic control of multiple growth factors and small-molecule cues. Here, we demonstrated that a simple five-day treatment of human induced pluripotent stem cells with two small molecules, retinoic acid (RA) and Wnt agonist CHIR 99021 (CHIR), resulted in rapid, synergistic upregulation of CDX2. Transcriptomic analysis of RA + CHIR-treated cells showed high similarity to primary trophectoderm cells. Multipotency was verified via further differentiation towards cells with syncytiotrophoblast or extravillous trophoblast features. RA + CHIR-treated cells were also assessed for the established criteria defining a trophoblast cell model, and they possess all the features necessary to be considered valid. Collectively, our data demonstrate a facile, scalable method for generating functional trophoblast-like cells in vitro to better understand the placenta., (© 2024. The Author(s).)

The placenta is crucial for fetal development, yet the impact of environmental stressors such as arsenic exposure remains poorly understood. We apply single-cell RNA sequencing to analyze the response of the mouse placenta to arsenic, revealing cell-type-specific gene expression, function, and pathological changes. Notably, the Prap1 gene, which encodes proline-rich acidic protein 1 (PRAP1), is significantly upregulated in 26 placental cell types including various trophoblast cells. Our study shows a female-biased increase in PRAP1 in response to arsenic and localizes it in the placenta. In vitro and ex vivo experiments confirm PRAP1 upregulation following arsenic treatment and demonstrate that recombinant PRAP1 protein reduces arsenic-induced cytotoxicity and downregulates cell cycle pathways in human trophoblast cells. Moreover, PRAP1 knockdown differentially affects cell cycle processes, proliferation, and cell death depending on the presence of arsenic. Our findings provide insights into the placental response to environmental stress, offering potential preventative and therapeutic approaches for environment-related adverse outcomes in mothers and children., (© 2024. The Author(s).)

Scope: This study investigates the impact of extracts derived from Antarctic fish species, Trematomus newnesi and Trematomus bernacchii, on the migration of human placental trophoblast JEG-3 cells, which is a crucial aspect of successful pregnancy., Methods and Results: The extracts, obtained from the muscles of these fish, significantly enhance the migration and invasion of JEG-3 cells in in vitro wound healing, Transwell, and collagen invasion assays. These effects are accompanied by an increase in matrix metalloproteinase (MMP) 9 activity, as demonstrated by zymography. Furthermore, the extracts activated Akt and protein phosphatase 1, resulting in the dephosphorylation of β-catenin at Ser33/37/Thr41, as confirmed by western blot analysis. Consequently, MMP9 is upregulated, while metallopeptidase inhibitor 1/3 is downregulated, as verified by western blot and qRT-PCR analyses. Finally, utilizing ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, followed by matching with the Global Natural Product Social Molecular Networking library, the study annotates the compound responsible for the observed migratory activity as taurocholic acid. Importantly, the study confirms that taurocholic acid enhances cell migration in JEG-3 cells., Conclusion: The results of this study emphasize the potential of Antarctic fish extracts in promoting extravillous trophoblast migration and invasion, which are critical for successful pregnancy., (© 2024 The Author(s). Molecular Nutrition & Food Research published by Wiley‐VCH GmbH.)

Environmental changes can trigger endoplasmic reticulum (ER) stress and misfolded protein accumulation, potentially leading to pre-eclampsia (PE). Amyloid-β (Aβ) is a crucial misfolded protein that can overactivate autophagy. Our study assessed the expression of Aβ 1-42 and autophagic activity in PE placental tissues and trophoblasts under ER stress. Placental tissues were surgically collected from normal pregnant women (NP) and pregnant women with late-onset PE (LOPE) delivering through cesarean section. The expression levels of Aβ 1-42 were detected in both PE and NP placental tissues, as well as in tunicamycin (TM)-induced HTR-8/SVneo cells. Autophagy-related proteins, such as Beclin-1, the ratio of LC3-II to LC3-I, ATG5, and SQSTM1/p62 in the placental tissues and HTR-8/SVneo cells were measured by Western blot. The number and morphology of autophagosomes were observed using transmission electron microscopy (TEM). Potential targets associated with the unfolded protein response (UPR) in the placental tissues of NP and PE cases were screened using PCR Arrays. The misfolded protein was significantly upregulated in the PE group. In both PE placental tissues and TM-induced HTR-8/SVneo cells, not only was Aβ 1-42 upregulated, but also Beclin-1, ATG5, and LC3BII/I were significantly increased, accompanied by an increase in autophagosome count, while SQSTM1/P62 was downregulated. A total of 17 differentially expressed genes (DEGs) associated with the UPR were identified, among which elevated calnexin (CANX) was validated in the placenta from both PE and TM-induced HTR-8/SVneo cells. Autophagy is significantly upregulated in PE cases due to ER stress-induced Aβ 1-42 accumulation, likely mediated by autophagy-related proteins involved in the UPR., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Copper pollution has attracted global environmental concern. Widespread Cu pollution results in excessive Cu accumulation in human. Epidemiological studies and animal experiments revealed that Cu exposure might have reproductive toxicity. Cuproptosis is a recently reported Cu-dependent and programmed cell death pattern. However, the mechanism by which copper exposure might cause cell cuproptosis is largely unknown. We chose trophoblast cells as cell model and found that copper exposure causes trophoblast cell cuproptosis. In mechanism, copper exposure up-regulates lnc-HZ11 expression levels, which increases intracellular Cu 2+ levels and causes trophoblast cell cuproptosis. Knockdown of lnc-HZ11 efficiently reduces intracellular Cu 2+ levels and alleviate trophoblast cell cuproptosis, which could be further alleviated by co-treatment with DC or TEPA. These results discover novel toxicological effects of copper exposure and also provide potential target for protection trophoblast cells from cuproptosis in the presence of excessive copper exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Altersolanol A, a fungus-derived tetrahydroanthraquinone, has shown cytotoxic effects on multiple cancer cells. However, its reproductive toxicity in humans has not been well-addressed. The present study was aimed at investigating the cytotoxicity of altersolanol A on human placental trophoblasts including choriocarcinoma cell line JEG-3 and normal trophoblast cell line HTR-8/SVneo in vitro. The results showed that altersolanol A inhibited proliferation and colony formation of human trophoblasts, and the choriocarcinoma cells were more sensitive to the compound than the normal trophoblasts. Altersolanol A induced cell cycle arrest at G2/M phase in JEG-3 cells and S phase in HTR-8/SVneo cells, downregulated the expression of cell cycle-related checkpoint proteins, and upregulated the p21 level. Altersolanol A also promoted apoptosis in human trophoblasts via elevating the Bax/Bcl-2 ratio and decreasing both caspase-3 and caspase-9 levels. Meanwhile, altersolanol A suppressed the mitochondrial membrane potential and induced ROS production and cytochrome c release, which activated the mitochondria-mediated intrinsic apoptosis. Moreover, migration and invasion were inhibited upon altersolanol A exposure with downregulation of matrix metalloproteinase (MMP)-2 in JEG-3 cells and MMP-9 in HTR-8/SVneo cells. Mechanically, altersolanol A supplement decreased the phosphorylation of JNK, ERK, and p38, manifesting the inactivation of MAPK signaling pathway in the human trophoblasts. In conclusion, altersolanol A exhibited potential reproductive cytotoxicity against human trophoblasts via promoting mitochondrial-mediated apoptosis and inhibiting the MAPK signaling pathway., (© 2024. The Author(s) under exclusive licence to Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature.)

Pyrethroid insecticides, such as beta-cyfluthrin, are used extensively globally, including in households and agriculture, and have been detected in the milk and urine of humans and cattle. Beta-cyfluthrin exhibits toxic effects, including neurotoxicity and male reproductive toxicity; however, few studies have investigated female reproductive toxicity despite its wide environmental distribution. The present study investigates effects of beta-cyfluthrin on implantation in porcine cells (pTr from the trophectoderm and pLE from the endometrial luminal epithelium). To identify the various physiological changes induced by beta-cyfluthrin, such as apoptosis and lipid peroxidation, flow cytometry analysis and immunofluorescence were performed with various reagents. In addition, the expression of genes and proteins associated with intracellular changes was confirmed using qRT-PCR and western blotting. Beta-cyfluthrin induced cell-cycle arrest and altered intracellular calcium flux. It also disrupted the mitochondrial function and promoted reactive oxygen species (ROS) production, leading to lipid peroxidation. Moreover, ROS induced by beta-cyfluthrin altered mitogen-activated protein kinase (MAPK) pathways and decreased cell migration capability. The expression levels of genes that are significant during early pregnancy were altered by beta-cyfluthrin in both cell lines. The changes resulted in apoptosis and diminished cell proliferation of pTr and pLE. Collectively, the results imply that beta-cyfluthrin disrupts the implantation process by affecting the physiology of the trophectoderm and endometrial luminal epithelial cells. The present study is the first to reveal the cellular mechanisms of beta-cyfluthrin on the female reproductive system and highlights the need for further in-depth research into its hazards., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Vitamin C (VC) serves as a pivotal nutrient for anti-oxidation process, metabolic responses, and stem cell differentiation. However, its precise contribution to placenta development and gestation remains obscure. Here, we demonstrated that physiological levels of VC act to stabilize Hand1, a key bHLH transcription factor vital for the development trajectory of trophoblast giant cell (TGC) lineages, thereby promoting the differentiation of trophoblast stem cells into TGC. Specifically, VC administration inactivated c-Jun N-terminal kinase (JNK) signaling, which directly phosphorylates Hand1 at Ser48, triggering the proteasomal degradation of Hand1. Conversely, a loss-of-function mutation at Ser48 on Hand1 not only significantly diminished both intrinsic and VC-induced stabilization of Hand1 but also underscored the indispensability of this residue. Noteworthy, the insufficiency of VC led to severe defects in the differentiation of diverse TGC subtypes and the formation of labyrinth's vascular network in rodent placentas, resulting in failure of maintenance of pregnancy. Importantly, VC deficiency, lentiviral knockdown of JNK or overexpression of Hand1 mutants in trophectoderm substantially affected the differentiation of primary and secondary TGC in E8.5 mouse placentas. Thus, these findings uncover the significance of JNK inactivation and consequential stabilization of Hand1 as a hitherto uncharacterized mechanism controlling VC-mediated placentation and perhaps maintenance of pregnancy., (© 2024. The Author(s).)

Background: Combination antiretroviral therapy (cART) has been reported to reduce perinatal transmission of human immunodeficiency virus (HIV) and improve maternal survival outcomes. Recent studies have associated in-utero exposure to cART drugs with adverse outcomes such as pre-eclampsia, preterm delivery, low birth weight and small-for-gestational-age births. However, the exact molecular mechanisms underlying cART-induced adverse pregnancy outcomes remain poorly defined., Objectives: To investigate the effects of cART drugs on trophoblast proliferation in the HTR-8/SVneo cell line., Study Design: HTR-8/SVneo cells were exposed to tenofovir (0.983-9.83 µM), emtricitabine (0.809-8.09 µM) and efavirenz (0.19-1.09 µM), the individual drugs of the first-line single tablet cART regimen termed 'Atripla ' , and zidovudine (1.12-1.12 µM), lamivudine (0.65-6.5 µM), lopinavir (0.32-3.2 µM) and ritonavir (0.69-6.9 µM), the individual drugs of the second-line single tablet cART regimen termed 'Aluvia ' . The cells were treated for 24, 48, 72 and 96 h, and trophoblast proliferation was assessed using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltretrazolium bromide assay., Results: Two-way analysis of variance showed a significant dose-dependent decrease (p < 0.05) in trophoblast proliferation in response to individual and combined drug components of first- and second-line antiretroviral therapy., Conclusions: First- and second-line cART drugs inhibit trophoblast proliferation, and may contribute to placenta-mediated adverse pregnancy outcomes in patients with HIV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

This study aimed to investigate the effect of cyclosporine A (CsA) on secretion of Th1 and Th2 cytokines by decidual stromal cells (DSCs) mediated by galectin (Gal)-9.HTR8/SVneo cells and primary trophoblasts were used for in vitro studies. Gal-9 expression was measured using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, CsA was used to regulate Gal-9 expression in trophoblasts. DSCs were treated with trophoblast supernatant and changes in Th1 and Th2 cytokine levels were analyzed. Changes in DSC levels of the T-cell immunoglobulin mucin receptor 3 (TIM-3) levels in DSCs after treatment with Gal-9 were assessed. Western blotting and ERK and AKT inhibitors were used to assess the involvement of the corresponding signaling pathways. Gal-9 was expressed by both primary trophoblasts and HTR8/SVneo cells. CsA treatment increased Gal-9 secretion by trophoblasts, which in turn increased IL-6 (Th2 cytokine) and decreased TNF-α and IFN-γ (Th1 cytokines) secretion in DSCs. Upon downregulation of trophoblast Gal-9 secretion, DSCs secreted lower levels of Th2 cytokines and higher levels of Th1 cytokines, and the effect was reversed by addition of CsA. TIM-3 expression changed in parallel with Gal-9 secretion. CsA treatment upregulated expression of Gal-9 in trophoblasts, promoted secretion of Th2 cytokines, and inhibited secretion of Th1 cytokines via ERK signaling., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Introduction: Cultured mouse trophoblast stem cells (mTSC) maintain proliferation/normal stemness (NS) under FGF4, which when removed, causes normal differentiation (ND). Hypoxic, or hyperosmotic stress forces trophoblast giant cells (TGC) differentiate. Hypoxic, hyperosmotic, and genotoxic benzo(a)pyrene (BaP), which is found in tobacco smoke, force down-regulation of inhibitor of differentiation (Id)2, enabling TGC differentiation. Hypoxic and hyperosmotic stress induce TGC by SAPK-dependent HAND1 increase. Here we test whether BaP forces mTSC-to-TGC while inducing SAPK and HAND1., Methods: Hand1 and SAPK activity were assayed by immunoblot, mTSC-to-TGC growth and differentiation were assayed at Tfinal after 72hr exposure of BaP, NS, ND, Retinoic acid (RA), or sorbitol. Nuclear-stained cells were micrographed automatically by a live imager, and assayed by ImageJ/FIJI, Biotek Gen 5, AIVIA proprietary artificial intelligence (AI) software or open source, CellPose artificial intelligence/AI software., Results: BaP (0.05-1μM) activated SAPK and HAND1 without diminishing growth. TSC-to-TGC differentiation was assayed with increasingly accuracy for 2-4 N cycling nuclei and >4 N differentiating TGC nuclei, using ImageJ/FIJI, Gen 5, AIVIA, or CellPose AI software. The AIVIA and Cellpose AI software matches human accuracy. The lowest BaP effects on SAPK activation/HAND1 increase are >10-fold more sensitive than similar effects for mESC. RA induces 44-47% 1st lineage TGC differentiation, but the same RA dose induces only 1% 1st lineage mESC differentiation., Discussion: First, these pilot data suggest that mTSC can be used in high throughput screens (HTS) to predict toxicant exposures that force TGC differentiation. Second, mTSC differentiated more cells than mESC for similar stress exposures, Third, open source AI can replace human micrograph quantitation and enable a miscarriage-predicting HTS., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Adverse pregnancy outcomes have been associated with the presence of glyphosate (G) in umbilical cord, serum, and urine samples from pregnant women. Our aim was to study the effect of G on blastocyst implantation using an in vitro mouse model, and the migration and acquisition of endothelial phenotype of the human trophoblastic HTR8/SVneo (H8) cells. In mouse blastocysts, no differences in attachment time and implantation outgrowth area were observed after G exposure. H8 cell migration was stimulated by 0.625 μM G without cytotoxicity. After 6 h, the mRNA expression of vascular endothelial growth factor (VEGF) and C-C motif chemokine ligand 2 (CCL2) was upregulated in H8 cells exposed to 1.25 μM G when compared vehicle-treated cells (p ≤ 0.05). No differences were observed in interleukin 11, VEGF receptor 1, and coagulation factor II thrombin receptor in H8 cells exposed to different concentrations of G for 6 h compared to the vehicle. Interestingly, exposure to G did not alter angiogenesis as measured by a tube formation assay. Taken all together, these results suggest that G exposure may contribute as a risk factor during pregnancy, due to its ability to alter trophoblast migration and gene expression., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Environmental benzo(a)pyrene (BaP) and itsmetabolite benzo(a)pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE), classic endocrine disrupting chemical and persistent organic pollutant, could cause miscarriage. However, the detailed mechanisms are still largely unclear and should be further explored. In this study, we discovered that exposure of trophoblast cells with BPDE could suppressed cell invasion/migration by inhibiting MEST/VIM (Vimentin) pathway. Moreover, BPDE exposure also increased lnc-HZ01 expression level, which further inhibited MEST/VIM pathway and then suppressed invasion/migration. Knockdown of lnc-HZ01 or overexpression of MEST could efficiently rescue invasion/migration of BPDE-exposed Swan 71 cells. Furthermore, lnc-HZ01 was highly expressed and MEST/VIM were lowly expressed in recurrent miscarriage (RM) villous tissues compared with healthy control (HC) group. Finally, we also found that BaP exposure inhibited murine Mest/Vim pathway in placental tissues and induced miscarriage in BaP-exposed mice. Therefore, the regulatory mechanisms were similar in BPDE-exposed human trophoblast cells, RM villous tissues, and placental tissues of BaP-exposed mice with miscarriage, building a bridge to connect BaP/BPDE exposure, invasion/migration, and miscarriage. This study provided novel insights in the toxicological effects and molecular mechanisms of BaP/BPDE-induced miscarriage, which is helpful for better elucidating the toxicological risks of BaP/BPDE on female reproduction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In our previous study, we uncovered that ghrelin promotes angiogenesis in human umbilical vein endothelial cells (HUVECs) in vitro by activating the Jagged1/Notch2/VEGF pathway in preeclampsia (PE). However, the regulatory effects of ghrelin on placental dysfunction in PE are unclear. Therefore, we applied Normal pregnant Sprague-Dawley (SD) rats, treated with lipopolysaccharide (LPS), to establish a PE-like rat model. The hematoxylin-eosin (HE) staining method and immunohistochemistry (IHC) technology were used to detect morphological features of the placenta. IHC and Western blot were applied to examine Bax and Bcl-2 expression levels. The concentrations of serum soluble fms-like tyrosine kinase-1 (sFlt1) and placental growth factor (PIGF) were assessed by enzyme-linked immunosorbent assay (ELISA) kit. In addition, the apoptosis rates of JEG-3 and HTR-8/SVneo trophoblast cells were determined by Annexin V-FITC/PI apoptosis detection kit. Cell migratory capacities were assessed by scratch-wound assay, and RNA-sequencing assay was used to determine the mechanism of ghrelin in regulating trophoblast apoptosis. It has been found that ghrelin significantly reduced blood pressure, urinary protein, and urine creatinine in rats with PE, at the meanwhile, ameliorated placental and fetal injuries. Second, ghrelin clearly inhibited placental Bax expression and circulating sFlt-1 as well as elevated placental Bcl-2 expression and circulating PIGF, restored apoptosis and invasion deficiency of trophoblast cells caused by LPS in vitro. Finally, transcriptomics indicated that nuclear factor kappa B (NF-κB) was the potential downstream pathway of ghrelin. Our findings illustrated that ghrelin supplementation significantly improved LPS-induced PE-like symptoms and adverse pregnancy outcomes in rats by alleviating placental apoptosis and promoting trophoblast migration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Oxidative stress has been implicated in numerous pregnancy-related disorders. Biologically active plant secondary metabolites, which are present in everyday diet, could prove effective therapeutic agents in preventing these disorders. This study evaluated effects of taxifolin (dihydroquercetin) on ROS production, markers of oxidative damage to lipids and proteins, activity of antioxidant enzymes and production of pro-inflammatory cytokines in H 2 O 2 -induced oxidative stress in trophoblast HTR-8/SVneo cells. Taxifolin in 10 µM and 100 µM concentrations attenuated oxidative damage to lipids and proteins, as evidenced by a decrease in MDA content, extracellular LDH activity, carbonyl groups and nitrite contents. A reduction in the activity of antioxidant enzymes SOD, CAT and GPx in cells pre-treated with taxifolin, prior to H 2 O 2 exposure, was also observed, along with a reduction in intracellular ROS production. Both evaluated concentrations of taxifolin showed anti-inflammatory activity in trophoblast cells, by reducing production of pro-inflammatory cytokines IL-1β and IL-6. In this model of H 2 O 2 -induced oxidative stress, taxifolin showed marked antioxidative and anti-inflammatory activities in trophoblast cells, adding further evidence of its protective effects and showing potential as a therapeutic agent in preventing adverse pregnancy outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)