Placental Trophoblast Research Articles

Ginsenoside Rg3 promotes the survival and migration of trophoblast cells in a rat model of gestational hypertension by regulating mir-100a/insulin growth factor-2 axis

Objective: Preeclampsia (PE) is a common complication during pregnancy. miR-100a is expressed in the placenta and regulates the survival and development of placental cells. Insulin growth factor-2 (IGF-2) may serve as its downstream target. This study investigated the protective mechanisms of ginsenoside Rg3 against PE in rat model. Materials and Methods: LPS-induced rat PE models were suitable for intravenous administration of the highly expressed miR-100a ginsenoside Rg3 lentiviral vector. Human trophoblasts were cultured in vitrofor JEG-3, and PE cell models were constructed. In vivoeffects on tumor growth and apoptosis were observed. Ginsenoside Rg3 was treated with different concentrations of shRNA, miR-100a analogs, inhibitors, or IGF-2. Autophagy and the expression of autophagy-related proteins were examined. Trophoblast activity and migration were determined using Cell Counting Kit-8 and Transwell assays. Both drugs strongly inhibited trophoblasts under normal conditions with some synergy between them. Double-luciferase return assay confirmed the binding affinity of miR-100a for IGF-2. Results: In response to Rg3, autophagy and the expression of autophagy-related proteins LC3-I/II, Beclin1, and SQSTM1 were reduced in PE rat placental trophoblasts. Rg3 inhibited autophagy in JEG-3 cells and promoted JEG-3 survival and migration in a concentration-dependent manner. miR-100a upregulated PE expression. These results suggested that autophagy was a vital signaling system. Rg3 intervention inhibited miR-100a expression and miR-100a downregulated IGF-2 expression in placental tissues and promoted autophagy, thereby inhibiting JEG-3 cell survival and migration. In rats, Rg3 inhibited PE development by regulating the activity of the miR-100a-IGF-2 signaling axis. Conclusion: Ginsenoside Rg3 positively regulates the miR-100a-IGF-2 axis and protects PE rats by inhibiting trophoblastic autophagy and promoting trophoblastic cell survival and migration.

Effect of Cyclophilin A on Biological Behavior of Placental Trophoblast Cells in Severe Preeclampsia by Regulating HIF-1 Alpha Signaling Pathway

Effect of Cyclophilin A on Biological Behavior of Placental Trophoblast Cells in Severe Preeclampsia by Regulating HIF-1 Alpha Signaling Pathway

Placental Trophoblast Cell Isolation from the Term Placenta.

Multiple cell lines have been utilized over time in studying placental biology. Still, most of them rely on choriocarcinoma cells or immortalized trophoblast cells that may not be entirely comparable with actual human placental trophoblast cells. Term placentas can be a source of primary villous trophoblasts. However, challenges remain in isolating them and maintaining them in extended culture. This manuscript describes our three-phase protocol utilizing enzymatic/mechanical digestion, modified Percoll gradient density separation, and immunopurification using magnetic beads. The resulting trophoblast culture remains viable for an extended period and highly pure after initial passaging.

Basic Research Advances in China on Embryo Implantation, Placentation, and Parturition

Abstract This review aimed to summarize the major progress in maternal-fetal medicine achieved by Chinese scientists in recent years. PubMed was systematically searched from January 2020 to November 2023. Publications that reported the progress in embryo implantation, placentation, and parturition made by Chinese scientists in the last 3 years were selected. The milestone events during gestation, embryo implantation, endometrial decidualization, placentation, and parturition are pivotal to a successful pregnancy. Embryo implantation requires intricate interactions between implantation-competent blastocysts and receptive endometrium. To adapt to pregnancy, endometrial stromal cells transform into specialized decidual cells, which occur spontaneously under the influence of ovarian hormones in humans but require the presence of embryos in mice. With embryonic development, the placenta forms to support fetal growth until parturition. The maternal-fetal interface is composed of diverse cell types, including endometrial decidual cells, placental trophoblast cells, endothelial cells, and various immune cells, a sophisticated interplay among which contributes to the maintenance of pregnancy. Near term, the uterus transitions from quiescence to contractility, in preparation for delivery. Disruptions to these events lead to pregnancy-related disorders such as repeated implantation failure, recurrent pregnancy loss, preeclampsia, fetal growth restriction, preterm birth, and infertility. In recent years, Chinese scientists have made prominent achievements in basic research on the aforementioned pregnancy events. Chinese scientists have made remarkable contributions to reproductive biology and maternal-fetal medicine research in recent years, highlighting future research directions in this field.

Single-cell RNA sequencing reveals association of aberrant placental trophoblasts and FN1 reduction in late-onset fetal growth restriction

IntroductionFetal growth restriction (FGR) can lead to fetal mental development abnormalities, malformations, and even intrauterine death. Defects in the trophoblasts at the maternal-fetal interface may contribute to FGR. However, the impact of trophoblasts on FGR is still not well understood. Therefore, the objective of this study is to characterize the heterogeneity of placental cells at the single-cell level and investigate the role of trophoblast subtypes in the pathogenesis of FGR at the cellular and molecular levels. MethodsSingle-cell RNA sequencing was performed on the maternal side of placentas from two normal pregnant women and two pregnant women with FGR. Lentivirus transfection was used to establish a FN1 knockout model in trophoblast HTR-8-Svneo cells. The effect of FN1 knockout on cell migration and invasion of HTR-8-Svneo cells was assessed through wound healing and transwell assays. ResultsNine cell types were annotated in 39,161 cells derived from single-cell RNA sequencing. The FGR group exhibited a decrease in the percentage of trophoblasts, especially in subtype of extravillous trophoblasts (EVTs). The expression of FN1 was reduced in trophoblasts and EVTs. Furthermore, the protein expression levels of FN1 in the placentas of FGR patients were significantly lower than those of normal pregnant women. The cell migration and invasion ability of HTR-8-Svneo cells were inhibited after the knockdown of FN1. DiscussionThe dysregulation of the trophoblast subtype-EVTs is involved in placental dysplasia related to FGR. The association between aberrant placental trophoblasts and reduced FN1 expression may contribute to insufficient remodeling of spiral arteries and the formation of FGR.

The Role of DNMT1 and C/EBPα in the Regulation of CYP11A1 Expression During Syncytialization of Human Placental Trophoblasts.

Progesterone synthesized in the placenta is essential for pregnancy maintenance. CYP11A1 is a key enzyme in progesterone synthesis, and its expression increases greatly during trophoblast syncytialization. However, the underlying mechanism remains elusive. Here, we demonstrated that passive demethylation of CYP11A1 promoter accounted for the upregulation of CYP11A1 expression during syncytialization with the participation of the transcription factor C/EBPα. We found that the methylation rate of a CpG locus in the CYP11A1 promoter was significantly reduced along with decreased DNA methyltransferase 1 (DNMT1) expression and its enrichment at the CYP11A1 promoter during syncytialization. DNMT1 overexpression not only increased the methylation of this CpG locus in the CYP11A1 promoter, but also decreased CYP11A1 expression and progesterone production. In silico analysis disclosed multiple C/EBPα binding sites in both CYP11A1 and DNMT1 promoters. C/EBPα expression and its enrichments at both the DNMT1 and CYP11A1 promoters were significantly increased during syncytialization. Knocking-down C/EBPα expression increased DNMT1 while it decreased CYP11A1 expression during syncytialization. Conclusively, C/EBPα plays a dual role in the regulation of CYP11A1 during syncytialization. C/EBPα not only drives CYP11A1 expression directly, but also indirectly through downregulation of DNMT1, which leads to decreased methylation in the CpG locus of the CYP11A1 promoter, resulting in increased progesterone production during syncytialization.

Gene-network based analysis of human placental trophoblast subtypes identifies critical genes as potential targets of therapeutic drugs.

During early pregnancy, extravillous trophoblasts (EVTs) play a crucial role in modifying the maternal uterine environment. Failures in EVT lineage formation and differentiation can lead to pregnancy complications such as preeclampsia, fetal growth restriction, and pregnancy loss. Despite recent advances, our knowledge on molecular and external factors that control and affect EVT development remains incomplete. Using trophoblast organoid in vitro models, we recently discovered that coordinated manipulation of the transforming growth factor beta (TGFβ) signaling is essential for EVT development. To further investigate gene networks involved in EVT function and development, we performed weighted gene co-expression network analysis (WGCNA) on our RNA-Seq data. We identified 10 modules with a median module membership of over 0.8 and sizes ranging from 1005 (M1) to 72 (M27) network genes associated with TGFβ activation status or in vitro culturing, the latter being indicative for yet undiscovered factors that shape the EVT phenotypes. Lastly, we hypothesized that certain therapeutic drugs might unintentionally interfere with placentation by affecting EVT-specific gene expression. We used the STRING database to map correlations and the Drug-Gene Interaction database to identify drug targets. Our comprehensive dataset of drug-gene interactions provides insights into potential risks associated with certain drugs in early gestation.

Placenta accreta on non-scarred uterus: About a case

Placenta accreta is an abnormally adherent placenta (abnormal adhesion of the placenta to the myometrium), due to the localized or diffuse absence of the decidua basalis. Its incidence increases with increasing cesarean section rates. Placenta accreta in a non-scarred uterus is very rare but remains possible due to other risk factors which are age, smoking, placenta preavia and finally invasive endouterine procedures. Among its consequences, we first note the hemorrhage of delivery which can be associated with significant maternal morbidity and mortality, uterine rupture and also the invasion of adjacent organs by the placental trophoblast. We report an interesting case of placenta accreta on a non-scarred uterus discovered during a planned cesarean section for placenta previa. This is a 41-year-old patient with no history of scarred uterus, 5th procedure, 3rd parity, with history of spontaneous miscarriage cured at 2 months, pregnant at 39 weeks, unmonitored pregnancy, admitted to the maternity emergency room for two days before for delivery method where the parturient benefited from a clinical examination and an obstetric ultrasound showing a completely covering anterior placenta, amniotic fluid of normal quantity and an estimate of the fetal weight at 2800g, then scheduled for a cesarean section. The diagnosis of placenta accreta is made during cesarean section. We opted for radical treatment. Placenta accreta is a pathology at risk of serious hemorrhagic complications during pregnancy and the postpartum period. This should encourage us to systematically search for ultrasound criteria for placenta accreta. The ultrasound report, in these patients, must explicitly mention this. Adequate care in the presence of a multidisciplinary team makes it possible to limit the mortality and morbidity associated with this pathology.

Glucose Regulates Glucose Transport and Metabolism via mTOR Signaling Pathway in Bovine Placental Trophoblast Cells.

It has been confirmed that improving the energy level of the diet contributed to the greater reproductive performance and birth weight of calves in periparturient dairy cows. To investigate the effect of glucose on nutrient transport during fetal development, the bovine placental trophoblast cells (BPTCs) were cultured in media with different glucose concentrations (1, 2, 4, 8, or 16 mg/mL). Subsequently, the BPTCs were cultured in media with 1, 8 mg/mL glucose and 8 mg/mL glucose plus 100 nmol/L rapamycin (the inhibitor of mTOR pathway). Compared with the 1 mg/mL glucose, the addition of 8 mg/mL glucose stimulated cell proliferation, upregulated the mRNA abundance of the glucose transporter GLUT1 and GLUT4, and increased the activity of glucose metabolism-related enzyme glucose-6-phosphate dehydrogenease (G6PD), lactate dehydrogenase (LDHA) and phosphoglycerate kinase 1 (PGK1), as well as adenosine-triphosphate (ATP) content (p < 0.05).Furthermore, compared with the treatment of 1 mg/mL glucose, adding 8 mg/mL of glucose-upregulated gene expression in the mTOR signaling pathway, including phosphatidylinositol3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) and 70 kDa ribosomal protein S6 kinase 2 (P70S6K) (p < 0.05).The supplementation of rapamycin downregulated the gene and protein expression of the mTOR signaling pathway, including mTOR, P70S6K, EIF4E-binding protein 1 (4EBP1), hypoxia-inducible factor 1-alpha (HIF-1α) and gene expression of glucose transporter upregulated by 8 mg/mL glucose (p < 0.05). Thus, these results indicated that the addition of 8 mg/mL glucose regulated the glucose transport and metabolism in BPTCs through the mTOR signaling pathway, thereby promoting the supply of nutrients to fetus.

Extra villous trophoblast-derived PDL1 can ameliorate macrophage inflammation and promote immune adaptation associated with preeclampsia

IntroductionSevere preeclampsia (sPE) is a systemic syndrome that may originate from chronic inflammation. Maintaining maternal-fetal hemostasis by the co-inhibitory molecule programmed death ligand 1 (PDL1) can be favorable for ameliorating inflammation from immune cells. Apart from programmed death 1 (PD1) expression, decidual macrophages (dMs) produce inflammatory cytokines, in response to cells which express PDL1. However, strong evidence is lacking regarding whether the PDL1/PD1 interaction between trophoblasts and decidual macrophages affects inflammation during sPE development. MethodsTo determine whether the trophoblast-macrophage crosstalk via the PDL1/PD1 axis modulates the inflammatory response in sPE-like conditions, at first, maternal-fetal tissues from sPE and normal patients were collected, and the PDL1/PD1 distribution was analyzed by Western blot, immunohistochemistry/ immunofluorescence and flow cytometry. Next, a coculture system was established and flow cytometry was used to identify how PDL1 was involved in macrophage-related inflammation under hypoxic stress. Transcriptional analysis was performed to clarify the inflammation-associated pathway induced by the PDL1/PD1 interaction. Finally, the Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) mouse model was used to examine the effect of PDL1 on macrophage-related inflammation by measuring PE-like symptoms. ResultsIn maternal-fetal tissue from sPE patients, placental extravillous trophoblasts (EVTs) and dMs had a surprisingly increase of PDL1 and PD1 expression, respectively, accompanied by a higher percentage of CD68 +CD86 + dMs. In vitro experiments showed that trophoblast-derived PDL1 under hypoxia interacted with PD1 on CD14 +CD80 +macrophages, leading to suppression of inflammation through the TNFα-p38/NFκB pathway. Accordingly, the PE-like mouse model showed a reversal of PE-like symptoms and a reduced F4/80 + CD86 + macrophage percentage in the uterus in response to recombinant PDL1 protein administration, indicating the protective effect of PDL1. DiscussionOur results initially explained an immunological adaptation of trophoblasts under placental hypoxia, although this protection was insufficient. Our findings suggest the possible capacity of modulating PDL1 expression as a potential therapeutic strategy to target the inflammatory response in sPE.

Bisphenol A induces placental ferroptosis and fetal growth restriction via the YAP/TAZ-ferritinophagy axis

Exposure to bisphenol A (BPA) during gestation leads to fetal growth restriction (FGR), whereby the underlying mechanisms remain unknown. Here, we found that FGR patients showed higher levels of BPA in the urine, serum, and placenta; meanwhile, trophoblast ferroptosis was observed in FGR placentas, as indicated by accumulated intracellular iron, impaired antioxidant molecules, and increased lipid peroxidation products. To investigate the role of ferroptosis in placental and fetal growth, BPA stimulation was performed both in vivo and in vitro. BPA exposure during gestation was associated with FGR in mice; also, it induces ferroptosis in mouse placentas and human placental trophoblast. Pretreatment with ferroptosis inhibitor ferritin-1 (Fer-1) alleviated BPA-induced oxidative damage and cell death. Notably, BPA reduced the trophoblastic expression of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), which regulated tissue growth and organ size. YAP or TAZ siRNA enhanced BPA-induced ferroptosis, suggesting that trophoblast ferroptosis is dependent on YAP/TAZ downregulation after BPA stimulation. Consistently, the protein levels of YAP/TAZ were also reduced in FGR placentas. Further results revealed that silencing YAP/TAZ promoted BPA-induced ferroptosis through autophagy. Pretreatment with autophagy inhibitor chloroquine (CQ) attenuated BPA-induced trophoblast ferroptosis. Ferritinophagy, an autophagic degradation of ferritin (FTH1), was observed in FGR placentas. Similarly, BPA reduced the protein level of FTH1 in placental trophoblast. Pretreatment with iron chelator desferrioxamine (DFO) and NCOA4 (an autophagy cargo receptor) siRNA weakened the ferroptosis of trophoblast after exposure to BPA, indicating that autophagy mediates ferroptosis in BPA-stimulated trophoblast by degrading ferritin. In summary, ferroptosis was featured in BPA-associated FGR and trophoblast injury; the regulation of ferroptosis involved the YAP/TAZ-autophagy-ferritin axis.

Suppression of circular RNA serum and glucocorticoid-induced kinase 1 elevates antioxidant molecules and angiogenesis in trophoblast cells to attenuate preeclampsia via microRNA-508-3p to target and restrain PUM homolog 1.

Preeclampsia (PE) is a pregnancy-specific syndrome characterized by hypertension and proteinuria. Recently, multiple circular RNAs (circRNAs) were considered latent clinical diagnostic markers or therapeutic targets. This study was to explore the impact of circRNA serum and glucocorticoid-induced kinase 1 (SGK1) on PE via influencing the microRNA (miR)-508-3p/PUM homolog 1 (PUM1) axis. Placental tissues of 34 pregnant women with PE and 34 normal pregnant women were collected to measure circRNA SGK1 (circSGK1), miR-508-3p, and PUM1. Human placental trophoblasts HTR-8/SVneo were transfected with plasmids, thereafter to observe proliferation, migration, invasion, and apoptosis, analyze antioxidant molecules Troxerutin (TXN), Glutamate-cysteine ligase catalytic subunit (GCLC), NAD (P) H-quinone oxidoreductase 1 (NQO1), and determine angiogenesis. After the construction of the PE rat model, antioxidant molecules TXN, GCLC, and NQO1, vascular-associated factor vascular endothelial growth factor A (VEGF-A), and histopathological conditions were tested. Examination of the binding of circSGK1 and miR-508-3p with PUM1 was performed. Our data showed that circSGK1 expression was elevated in the placenta of patients with PE. Silenced circSGK1 or elevated miR-508-3p promoted the growth and antioxidant molecules and angiogenesis in trophoblast cells; CircSGK1 combined with miR-508-3p, and miR-508-3p targeted PUM1. In summary, suppression of circSGK1 augments antioxidant molecules and angiogenesis in trophoblast cells to attenuate PE via miR-508-3p to target PUM1.

B-Flow/Spatiotemporal Image Correlation M-Mode and Contrast-Enhanced/Microbubble Ultrasonography Quantification of Spiral Artery Distensibility and Placental Intervillous Perfusion in the First Trimester in a Primate Model of Impaired Spiral Artery Remodeling.

During early human pregnancy, placental trophoblasts remodel spiral arteries into distensible low-resistance vessels to promote placental perfusion. We have established a model of impaired spiral artery remodeling (SAR) by elevating estradiol levels in the first trimester of baboon pregnancy. In the present study, B-flow/spatiotemporal image correlation (STIC) M-mode ultrasonography, a non-Doppler technology for sharp rendering of vessel dimensions, was used to determine whether spiral artery distensibility was altered in SAR-suppressed baboons. Contrast-enhanced ultrasound/microbubble imaging was also performed to determine whether it detected changes in placenta intervillous space perfusion in SAR-suppressed baboons. The two imaging procedures were performed in the first trimester in baboons not treated or treated with estradiol to suppress SAR. Spiral artery distensibility, that is, luminal diameter at systole minus diameter at diastole, and volume flow as quantified by B-flow/STIC M-mode were 26% (p=0.03) and 55% (p=0.059) lower, respectively, in SAR-suppressed baboons. However, placental intervillous space flow rate and video intensity plateau levels reflecting blood perfusion, quantified by contrast-enhanced ultrasound/microbubble imaging, were unaltered in SAR-suppressed baboons. The results indicate that B-flow/STIC M-mode ultrasonography provides a non-invasive method to detect reduced distensibility and, thus, function of spiral arteries across the cardiac cycle in the first trimester in a primate model of impaired SAR. This study represents a first step in determining whether B-flow/STIC M-mode detects a similar defect in SAR early in adverse human pregnancy. This would provide an avenue to develop therapeutic modalities to prevent the devastating consequences of impaired SAR.

Trophoblast-Targeted Liposomes for Placenta-Specific Drug Delivery.

A major challenge in developing potential treatments for pregnancy complications is minimizing adverse effects to the fetus and mother. Placenta-targeted drug delivery could reduce the risks of drug treatments in pregnancy by targeting tissue where most pregnancy complications originate and decreasing dosages. We previously developed a tool for the targeted delivery of drug-carrying nanoparticles to the placenta using a synthetic placental chondroitin sulfate A-binding peptide (plCSA-BP) derived from the malarial protein VAR2CSA, which binds a distinct type of chondroitin sulfate A (CSA) exclusively expressed by placental trophoblasts. Liposomes are a type of nanoparticle already approved for use in humans by the Food and Drug Administration (FDA) and used successfully for the treatment of a wide range of diseases. Here, we present a detailed method to create plCSA-BP-decorated liposomes that can be used to deliver drugs specifically to placental trophoblasts. Liposomes are first generated by the standard film method and then conjugated to plCSA-BPs using the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysulfosuccinimide (EDC/NHS) bioconjugate technique. This protocol may facilitate bench-to-bedside translation of drug discovery for the treatment of pregnancy disorders by reducing risks of side effects, and enabling rapid and scalable production.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transducing Force into Procoagulation: PIEZO1-TMEM16F Functional Coupling in Blood Lining Cells

Thrombosis is a major global disease burden that accounts for 1 in 4 deaths worldwide. Hypertension and preeclampsia greatly increase thrombotic risks by imposing high blood pressure and abnormal hemodynamic shear stress on blood-lining cells, namely endothelial cells (ECs) and placental syncytiotrophoblasts (STBs) in pregnant women. Nonetheless, it remains elusive how abnormal mechanical forces under hypertensive conditions promote coagulation. PIEZO1 mechanosensitive ion channel is a major force sensor in a wide variety of cells, including red blood cells (RBCs) and endothelial cells (ECs), where it transduces mechanical forces into chemical signals through its ion permeability to calcium. On the other hand, TMEM16F is a calcium-activated lipid scramblase (CaPLSase) that translocates phosphatidylserine (PS), an important procoagulant factor, to the cell surfaces of blood cells and endothelial cells. We recently discovered that TMEM16F is the sole CaPLSase in placental trophoblasts and RBCs. Our preliminary results show that PIEZO1 and TMEM16F are functionally coupled in RBCs, and calcium entry through PIEZO1 activates TMEM16F, leading to PS exposure. Based on all these, we hypothesize that in the blood lining cells, PIEZO1-TMEM16F coupling is responsible for transducing mechanical forces into PS exposure, a critical procoagulant signal for blood coagulation. By employing immunostaining, patch clamp electrophysiology, and a fluorescence microscopy-based scrambling assay, we show that PIEZO1 is functionally expressed in placental trophoblasts, including STBs. We further demonstrate that PIEZO1 activation, either stimulated by its agonist Yoda1 or by shear stress, induces PS exposure in BeWo human trophoblast cell line and human umbilical vein endothelial cell line (HUVEC) but not the TMEM16F deficient cell lines. In addition, using chromogenic tenase and prothrombinase, and fluorescence fibrin assays, we show that PIEZO1 activation by its agonist, Yoda1, triggered coagulation activities in a TMEM16F-dependent fashion in trophoblasts. Our study thus discovers the functional expression of the PIEZO1 mechanosensitive channel in placental trophoblasts and elucidates a novel force-induced procoagulant mechanism in blood-lining cells. In this mechanism, PIEZO1-TMEM16F coupling transduces mechanical forces into procoagulant PS exposure on the blood lining cells. Enhanced PIEZO1-TMEM16F coupling may contribute to hypercoagulable states in hypertension and preeclampsia, and breaking the coupling is therefore a potential therapeutic strategy to prevent thrombotic risks associated with these diseases.

Role of CircCHD2 in the pathogenesis of gestational diabetes mellitus by regulating autophagy via miR-33b-3p/ULK1 axis

Gestational diabetes mellitus (GDM) is a common pregnancy complication with a high incidence in women; however, its pathophysiology remains unknown. Our previous study suggested that the circCHD2/miR-33b-3p/ULK1 axis may be involved in GDM pathogenesis. However, the mechanism through which circCHD2 regulates GDM development requires further investigation. We found that high-glucose (HG, 25 mmol/L) significantly induced the expression of circCHD2, increased autophagy and apoptosis, and decreased cell viability in human placental trophoblast HTR-8/SVneo cells. In contrast, the downregulation of circCHD2 significantly attenuated the effects of HG on HTR-8/SVneo cells. MiR-33b-3p downregulated in the placenta of GDM patients was reduced by HG and detected as a target of circCHD2 using bioinformatics analysis, a dual-luciferase reporter assay, and qRT-PCR assay. Further studies showed that the inhibition of miR-33b-3p significantly blocked the effects of circCHD2 downregulation on cell viability, apoptosis, and autophagy in HG-treated HTR-8/SVneo cells. ULK1 is a target of miR-33b-3p, based on bioinformatics analysis, a dual-luciferase reporter assay, qRT-PCR assay, and Western blot analysis. Compared to miR-33b-3p, ULK1 is upregulated in the placenta of GDM patients. ULK1 overexpression notably blocked the effects of miR-33b-3p mimics on cell viability, apoptosis, and autophagy in HG-treated HTR-8/SVneo cells. These findings suggested that circCHD2 acts as an autophagy promoter via the miR-33b-3p/ULK1 axis to induce apoptosis in HTR-8/SVneo cells, suggesting that circCHD2 is a potential diagnostic and therapeutic target for GDM.

Gestational exposure to 1-NP induces ferroptosis in placental trophoblasts via CYP1B1/ERK signaling pathway leading to fetal growth restriction

Fetal growth restriction (FGR) is a prevalent complication in obstetrics, yet its exact aetiology remains unknown. Numerous studies suggest that the degradation of the living environment is a significant risk factor for FGR. 1-Nitropyrene (1-NP) is a widespread environmental pollutant as a representative substance of nitro-polycyclic aromatic hydrocarbons. In this study, we revealed that 1-NP induced FGR in fetal mice by constructing 1-NP exposed pregnant mice models. Intriguingly, we found that placental trophoblasts of 1-NP exposed mice exhibited significant ferroptosis, which was similarly detected in placental trophoblasts from human FGR patients. In this regard, we established a 1-NP exposed cell model in vitro using two human trophoblast cell lines, HTR8/SVneo and JEG-3. We found that 1-NP not only impaired the proliferation, migration, invasion and angiogenesis of trophoblasts, but also induced severe cellular ferroptosis. Meanwhile, the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively rescued 1-NP-induced trophoblast biological function impairment. Mechanistically, we revealed that 1-NP regulated ferroptosis by activating the ERK signaling pathway. Moreover, we innovatively revealed that CYP1B1 was essential for the activation of ERK signaling pathway induced by 1-NP. Overall, our study innovatively identified ferroptosis as a significant contributor to 1-NP induced trophoblastic functional impairment leading to FGR and clarified the specific mechanism by which 1-NP induced ferroptosis via the CYP1B1/ERK signaling pathway. Our study provided novel insights into the aetiology of FGR and revealed new mechanisms of reproductive toxicity of environmental pollutants.

Microcystin-LR prenatal exposure drives preeclampsia-like changes in mice by inhibiting the expression of TGF-β and VEGFA

Microcystin-leucine-arginine (MC-LR) is widespread in the water and food, which has suspected to be associated with adverse pregnancy outcomes. In the present study, we aim to assess the interaction between MC-LR exposure and preeclampsia development and elucidate the molecular events involved. After exposure to MC-LR during pregnancy, the mice developed hypertension and proteinuria, the typical symptoms of preeclampsia. This was associated with decreased invasiveness of placental trophoblast and vascular dysplasia caused by MC-LR through down-regulating VEGFA and TGF-β expression via AKT/m-TOR/HIF-1α pathway. In addition, this conclusion has been confirmed in a case-control study. Significantly, the addition of Deferoxamine (DFM), a phosphorylated serine-threonine protein kinases (p-AKT) specific agonist, can antagonize the inhibitory effect of MC-LR on the expression of related proteins, which further ameliorate the migration and invasion ability of HTR-8/Svneo cells. To sum up, our study revealed the pathologic mechanism by which MC-LR lead to preeclampsia and emphasized the importance of pregnancy management.

PSV-9 Cold Exposure Impacts Dna Methylation Patterns of Genes in Cattle Sperm Involved in Early Embryonic and Bone Development

Abstract DNA methylation and other epigenetic modifications are indispensable for maintaining sperm quality, fertilization capacity, and embryonic and postnatal development. In mice, environmental factors, such as stress, nutrition, or exposure to the cold condition, have been demonstrated as factors that alter methylation marks of sperm that are passed to the subsequent generation through transgenerational inheritance and genomic imprinting. Breeding bulls from northern regions of the USA are exposed to extreme cold for 2 to 3 months during the winter; however, no single study has analyzed the consequences of this cold exposure on the methylation pattern of bull sperm, which constitutes a significant knowledge gap. Therefore, this study aimed to explore the effects of cold exposure on the overall and gene-specific methylation status in sperm. We collected semen from 5 bulls during winter seasons after cold exposure and at normal temperatures during late spring. Whole Genome Bisulfite Sequencing (WGBS) was conducted to obtain the DNA methylation profile of these semen samples and to identify unique genes that have differentially methylated regions due to cold exposure (Figure 1). Cold exposure did not change the overall methylation level between the two groups but induced 438 Differentially methylated regions (DMRs) that overlapped with promoters, introns, exons, intergenic regions, shores, and shelves of CpG island (CGI) in 186 unique genes. We also identified nine unique differentially methylated genes (DMGs) (Pax6, Macf1, Mest, Ubqln1, Smg9, Trappc9, Ctnnb1, Lsm4, Peg10) involved in embryonic development and nine unique DMGs (Prmt6, Nipal1, C21h15orf40, Slc37a3, Fam210a, Raly, Rgs3, Lmbr1, Gan) involved in osteogenesis (Table 1). DMRs were located in the promoter regions and introns of these genes which preferentially involve gene silencing and alternative splicing. Among the DMGs involved in embryonic development, Mest and Peg10 are two paternally imprinted genes where only the paternal allele expresses. Peg10, required for optimal placental growth and trophoblast proliferation, overlapped with a hypermethylated DMR in the promoter region. Mest, another paternally imprinted gene, the downregulation of which results in embryonic growth retardation, had hypermethylation in introns. Moreover, Methylation-specific PCR (MS-PCR) verified the methylation changes in the identified regions. It was the first study to investigate the effect of cold exposure on DNA methylation of cattle sperm and suggest its association with altered sperm DNA methylation profiles. Differential methylation appears to alter gene expression and affect early embryonic development, osteogenic activity, and overall offspring growth performance through the imprinting effects.