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Background: As one of the most common and abundant internal modifications of eukaryotic mRNA, N6-methyladenosine (m6A) modifications are closely related to placental development. Ferroptosis is a newly discovered form of programmed cell death. During placental development, placental trophoblasts are susceptible to ferroptosis. However, the interactions of m6A and ferroptosis in trophoblast physiology and injury are unclear. Methods: Recurrent miscarriage (RM) was selected as the main gestational disease in this study. Published data (GSE76862) were used to analyze the gene expression profiles in patients with RM. The extent of m6A modification in total RNA of villous tissues between patients with RM and healthy controls (HC) was compared. ALKBH5 (encoding AlkB homolog 5, RNA demethylase) was selected as the candidate gene for further research. Quantitative real-time reverse transcription PCR, western blotting, and immunohistochemistry (IHC) confirmed the elevated expression of ALKBH5 in the cytotrophoblasts of patients with RM. Then, cell counting kit-8 assays, glutathione disulfide/glutathione quantification, 2',7'-dichlorfluorescein-diacetate staining, and malonaldehyde assays were used to explore the alterations of ferroptosis-related characteristics following RAS-selective lethal (RSL3) stimulation after overexpression of ALKBH5. Thereafter, we re-analyzed the published RNA sequencing data upon knockdown of ALKBH5, combined with published tissue RNA-seq data, and FTL (encoding ferritin light chain) was identified as the ferroptosis-related gene in cytotrophoblasts of patients with RM that is regulated by ALKBH5. Finally, western blotting and IHC confirmed the increased expression of FTL in the cytotrophoblasts from patients with RM. Results: Total m6A levels were decreased in patients with RM. The most significant differentially m6A-related gene was ALKBH5, which was increased in patients with RM. In vitro cell experiments showed that treatment with RSL3 resulted in increased cell death and upregulated ALKBH5 expression. Overexpression of ALKBH5 alleviated RSL3-induced HTR8 cell death and caused decreased levels of intracellular oxidation products. Published transcriptome sequencing revealed that FTL was the major ferroptosis-related gene regulated by ALKBH5 in the villous tissues of patients with RM. Consistent with the expression of ALKBH5, FTL was increased by RSL3-induction and increased in patients with RM. Conclusion: Elevated ALKBH5 alleviated RSL3-induced cytotrophoblast cell death by promoting the expression of FTL in patients with RM. Our results supported the view that ALKBH5 is an important regulator of the ferroptosis-related etiology of RM and suggested that ALKBH5 could be responsible for epigenetic aberrations in RM pathogenesis.
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Aborto Habitual , Desmetilasa de ARN, Homólogo 5 de AlkB , Ferroptosis , Trofoblastos , Humanos , Ferroptosis/genética , Femenino , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Aborto Habitual/genética , Aborto Habitual/metabolismo , Aborto Habitual/patología , Trofoblastos/metabolismo , Trofoblastos/patología , Embarazo , Adulto , Estudios de Casos y ControlesRESUMEN
Insufficient cytotrophoblast (CTB) migration and invasion into the maternal myometrium leads to pregnancy related complications like Intra-uterus Growth Restriction (IUGR), and pre-eclampsia (PE). We previously found that hydrogen sulfide (H2S) enhanced CTB migration without knowing the mechanism(s) and the pathophysiological significance. By studying human samples and cell line, we found that H2S levels were lower in PE patients' plasma; H2S synthetic enzyme cystathionine ß-synthetase (CBS) was reduced in PE extravillious invasive trophoblasts. GYY4137 (H2S donor, 1 µM) promoted CBS/H2S translocation onto mitochondria, preserved mitochondria functions, enhanced cell invasion and migration. CBS knockdown hindered the above functions which were rescued by GYY4137, indicating the vital roles of CBS/H2S signal. Disturbance of mitochondria dynamics inhibited cell invasion and migration. The 185 and 504 cysteines of Mitochondrial Rho GTPase 2 (Miro2C185/C504) were highly sulfhydrated by H2S. Knockdown Miro2 or double mutation of Miro2C185/C504 to serine fragmented mitochondria, and inhibited cell invasion and migration which can't be rescued by H2S. The present study showed that human cytotrophoblast receives low dose H2S regulation; CBS/H2S sustained mitochondria functions via Miro2C185/C504 sulfhydration to enhance cytotrophoblast mobility. These findings established a new regulatory pathway for cytotrophoblast functions, and provided new targets for IUGR and PE.
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Movimiento Celular , Cistationina betasintasa , Sulfuro de Hidrógeno , Mitocondrias , Dinámicas Mitocondriales , Compuestos Organotiofosforados , Preeclampsia , Trofoblastos , Proteínas de Unión al GTP rho , Humanos , Trofoblastos/metabolismo , Trofoblastos/patología , Sulfuro de Hidrógeno/metabolismo , Sulfuro de Hidrógeno/farmacología , Femenino , Embarazo , Cistationina betasintasa/metabolismo , Cistationina betasintasa/genética , Mitocondrias/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/genética , Preeclampsia/metabolismo , Preeclampsia/patología , Preeclampsia/genética , Compuestos Organotiofosforados/farmacología , Morfolinas/farmacología , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Transducción de Señal , AdultoRESUMEN
Preeclampsia (PE) is a common placental-origin complication of pregnancy and a major cause of morbidity and mortality among pregnant women and fetuses. However, its pathogenesis has not been elucidated. Effective strategies for prevention, screening, and treatment are still lacking. Studies have indicated that dysfunction of placental trophoblast cells, such as impaired syncytialization, proliferation, and epithelial-mesenchymal transition processes, plays a crucial role in the development of PE. Glucose transporter 1 (GLUT1) is a key protein regulating glucose transport in placental tissues. However, the effect of GLUT1 on the function of trophoblast cells in PE is not well understood. In this study, we found that GLUT1 expression is reduced in PE placental tissues. GLUT1 promotes the syncytialization process by increasing the glucose uptake ability of BeWo cells. Additionally, GLUT1 promotes the proliferation, migration, and invasion capabilities of HTR-8/SVneo cells by regulating MAPK and PI3K/AKT signaling pathways. Overall, these findings provide a new insight into understanding the biological functions of GLUT1, clarifying the pathogenesis of PE, and identifying diagnostic and therapeutic targets for PE.
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Movimiento Celular , Proliferación Celular , Transportador de Glucosa de Tipo 1 , Glucosa , Preeclampsia , Trofoblastos , Humanos , Trofoblastos/metabolismo , Trofoblastos/patología , Transportador de Glucosa de Tipo 1/metabolismo , Transportador de Glucosa de Tipo 1/genética , Embarazo , Femenino , Preeclampsia/metabolismo , Preeclampsia/patología , Glucosa/metabolismo , Línea Celular , Transducción de Señal , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adulto , Fosfatidilinositol 3-Quinasas/metabolismo , Placenta/metabolismo , Placenta/patologíaRESUMEN
Dysregulation of macrophage polarization can prevent the invasion of trophoblast cells and further limit spiral artery remodeling in preeclampsia (PE). However, its mechanism is obscure. HTR8-/Svneo cells were cultured under normoxic or hypoxic conditions and extracellular vesicles (EVs) in the culture supernatants were extracted. Next, the cells were incubated with those EVs to investigate their effects on trophoblasts. A co-culture system consisting of HTR8-/Svneo cells and macrophages was used to reveal how the trophoblast-derived EVs affected the macrophage subtype. Finally, a PE mouse model and miR-141-3p knockout mice were used to verify the function of miR-141-3p in PE. Hypoxia induced abnormal increases in the levels of miR-141-3p in HTR8-/Svneo cells and EVs. EVs from hypoxia-treated HTR8-/Svneo cells could downregulate PTEN, a potential target of miR-141-3p, and inhibit trophoblast mitophagy and invasion. However, HTR8-/Svneo cells transfected with an miR-141-3p inhibitor could attenuate the influence of EVs. In an HTR8-/Svneo cell plus macrophage co-culture system, hypoxia-pretreated cells promoted the transformation of macrophages into the M1-phenotye, and HTR8-/Svneo invasion was inhibited by the macrophages. MiR-141 from EVs could target and downregulate dual specificity phosphatase 1 (DUSP1) expression in macrophages, induce formation of the M1 macrophage phenotype in THP-1 cells, downregulate DUSP1 expression, and upregulate TAB2/TAK1 signaling. These results were also demonstrated in normal pregnant mice and PE pregnant mice. A hypoxic environment could upregulate miR-141 expression in the EVs of HTR8-/Svneo cells, and THP-1-derived macrophages could uptake EVs releasing miR-141 to downregulate DUSP1 expression and induce the formation of M1 macrophages, which can lead to the development of PE.
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Vesículas Extracelulares , Macrófagos , MicroARNs , Preeclampsia , Trofoblastos , MicroARNs/genética , MicroARNs/metabolismo , Femenino , Trofoblastos/metabolismo , Trofoblastos/patología , Preeclampsia/metabolismo , Preeclampsia/genética , Preeclampsia/patología , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Humanos , Animales , Embarazo , Ratones , Decidua/metabolismo , Decidua/patología , Ratones Noqueados , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Línea Celular , Fosfatasa 1 de Especificidad Dual/metabolismo , Fosfatasa 1 de Especificidad Dual/genéticaRESUMEN
BACKGROUND: Recurrent spontaneous abortion (RSA) is defined as two or more consecutive spontaneous abortions before 20 weeks with the same spouse [1]. However, approximately 50% of RSA cases of unknown cause are classified as unexplained recurrent spontaneous abortion (URSA). Potential factors include decreased trophoblast cell migration and invasion, leading to impaired placental implantation and maintenance of the normal maternal-fetal interface. However, the mechanism of this pathogenesis remains unknown. In this study, we investigated the potential role and mechanism of KLF4 in regulating URSA by influencing the invasion and migration ability of trophoblast cells. METHODS: We firstly identified 817 differentially expressed genes by performing a difference analysis of the dataset GSE121950 [2] related to recurrent abortion, and intersected the top 10 genes obtained respectively by the three algorithms: DMNC, MNC, and EPC using Venn Diagram.To detect the expression levels of core genes, villi samples were obtained from normal pregnant women and patients with URSA. RT-qPCR analysis revealed a significant difference in KLF4 mRNA expression and KLF4 was then analyzed. Trophoblast cell lines HTR8 and JEG3 were used to investigate the effect of KLF4 on trophoblastic function. Wound healing and transwell assays was performed to detect the invasion and migration of trophoblast cells. The expression of epithelial-mesenchymal transition(EMT) molecules were detected by RT-qPCR and western blot. Promoter detection and epigenetic modification were detected by chromatin immunoprecipitation (ChIP) assay. Molecular nuclear localization was detected by immunofluorescence and subcellular fractionation. Miscarried mice model was used to study the effects of KLF4 on URSA induced by reduced trophoblast invasion and migration. RESULTS: KLF4 is highly expressed in the villi of patients with URSA. KLF4 inhibits the expression level of H3R2ME2a in trophoblast cells by regulating the transcriptional level and nuclear translocation of PRMT6, thereby inhibiting the possible regulatory mechanism of trophoblastic invasion and providing a potential treatment strategy for URSA in vivo. CONCLUSIONS: The KLF4/PRMT6/H3R2ME2a axis regulates mechanisms associated with unexplained recurrent spontaneous abortion by regulating trophoblast function.
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Aborto Habitual , Movimiento Celular , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Trofoblastos , Adulto , Animales , Femenino , Humanos , Ratones , Embarazo , Aborto Habitual/metabolismo , Aborto Habitual/genética , Aborto Habitual/patología , Línea Celular , Movimiento Celular/genética , Vellosidades Coriónicas/metabolismo , Metilación de ADN/genética , Transición Epitelial-Mesenquimal/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Factor 4 Similar a Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Trofoblastos/metabolismo , Trofoblastos/patología , MasculinoRESUMEN
Placenta accreta spectrum (PAS) disorders are characterized by abnormal trophoblastic invasion into the myometrium, leading to significant maternal health risks. PAS includes placenta accreta (invasion < 50% of the myometrium), increta (invasion > 50%), and percreta (invasion through the entire myometrium). The condition is most associated with previous cesarean deliveries and increases in chance with the number of prior cesarians. The increasing global cesarean rates heighten the importance of early PAS diagnosis and management. This review explores genetic expression and key regulatory processes, such as apoptosis, cell proliferation, invasion, and inflammation, focusing on signaling pathways, genetic expression, biomarkers, and non-coding RNAs involved in trophoblastic invasion. It compiles the recent scientific literature (2014-2024) from the Scopus, PubMed, Google Scholar, and Web of Science databases. Identifying new biomarkers like AFP, sFlt-1, ß-hCG, PlGF, and PAPP-A aids in early detection and management. Understanding genetic expression and non-coding RNAs is crucial for unraveling PAS complexities. In addition, aberrant signaling pathways like Notch, PI3K/Akt, STAT3, and TGF-ß offer potential therapeutic targets to modulate trophoblastic invasion. This review underscores the need for interdisciplinary care, early diagnosis, and ongoing research into PAS biomarkers and molecular mechanisms to improve prognosis and quality of life for affected women.
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Biomarcadores , Placenta Accreta , Humanos , Placenta Accreta/metabolismo , Placenta Accreta/diagnóstico , Placenta Accreta/patología , Placenta Accreta/genética , Femenino , Embarazo , Transducción de Señal , Trofoblastos/metabolismo , Trofoblastos/patologíaRESUMEN
Scar tissue formation is a hallmark of wound repair in adults and can chronically affect tissue architecture and function. To understand the general phenomena, we sought to explore scar-driven imbalance in tissue homeostasis caused by a common, and standardized surgical procedure, the uterine scar due to cesarean surgery. Deep uterine scar is associated with a rapidly increasing condition in pregnant women, placenta accreta spectrum (PAS), characterized by aggressive trophoblast invasion into the uterus, frequently necessitating hysterectomy at parturition. We created a model of uterine scar, recapitulating PAS-like invasive phenotype, showing that scar matrix activates mechanosensitive ion channel, Piezo1, through glycolysis-fueled cellular contraction. Piezo1 activation increases intracellular calcium activity and Protein kinase C activation, leading to NF-κB nuclear translocation, and MafG stabilization. This inflammatory transformation of decidua leads to production of IL-8 and G-CSF, chemotactically recruiting invading trophoblasts towards scar, initiating PAS. Our study demonstrates aberrant mechanics of scar disturbs stroma-epithelia homeostasis in placentation, with implications in cancer dissemination.
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Cicatriz , Inflamación , Canales Iónicos , Placenta Accreta , Trofoblastos , Femenino , Embarazo , Humanos , Placenta Accreta/metabolismo , Placenta Accreta/patología , Cicatriz/metabolismo , Cicatriz/patología , Canales Iónicos/metabolismo , Canales Iónicos/genética , Animales , Inflamación/metabolismo , Inflamación/patología , Trofoblastos/metabolismo , Trofoblastos/patología , Decidua/patología , Decidua/metabolismo , Ratones , FN-kappa B/metabolismo , Cesárea/efectos adversos , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Interleucina-8/metabolismo , Útero/patología , Útero/metabolismoRESUMEN
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.
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Hiperglucemia , Pravastatina , Preeclampsia , Trofoblastos , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Pravastatina/farmacología , Pravastatina/uso terapéutico , Humanos , Preeclampsia/patología , Preeclampsia/metabolismo , Preeclampsia/tratamiento farmacológico , Femenino , Embarazo , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo , Trofoblastos/patología , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Movimiento Celular/efectos de los fármacos , Fenotipo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor de Crecimiento Placentario/metabolismo , Glucosa/farmacología , Endoglina/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 1 de Activador Plasminogénico/genéticaRESUMEN
Studies on DNA methylation alterations associated with pre-eclampsia (PE) have improved our understanding of the mechanisms underlying this disorder. However, differentially methylated cytosines (DMCs) have not been adjusted for cell-type heterogeneity, hampering the identification of alterations that drive disease risk. Using a reference-based, cell-type deconvolution approach, we estimated the nuclear proportions of 335 placental samples based on DNA methylation data. We found that the nuclei of total trophoblast lineages accounted for more than 80% of the placental samples, with a significant increase in PE placentas. The nuclear proportions of stromal and Hofbauer cells decreased in PE placentas. Our nuclear proportion estimation reflected previous histological knowledge on the changes in cell type proportions in PE placentas. We corrected 2125 DMCs associated with early-onset PE for cell-type heterogeneity by adjusting for the nuclear proportions and observed a notable reduction in the association signals, with 145 probes not reaching epigenome-wide significance. After correction, the top 200 significant DMCs were strongly enriched in active enhancers in trophoblast lineages, whereas 145 non-significant probes were enriched in regions with a quiescent state of chromatin. Our results suggest that future epigenetic studies of PE should focus on functional regulatory sequences.
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Núcleo Celular , Metilación de ADN , Epigénesis Genética , Placenta , Preeclampsia , Preeclampsia/genética , Preeclampsia/metabolismo , Preeclampsia/patología , Humanos , Femenino , Embarazo , Placenta/metabolismo , Placenta/patología , Núcleo Celular/metabolismo , Núcleo Celular/genética , Trofoblastos/metabolismo , Trofoblastos/patología , AdultoRESUMEN
INTRODUCTION: Preeclampsia is a pregnancy-specific disorder characterized by de novo development of hypertension and proteinuria over 20 weeks gestation that has been associated with the dysfunction of trophoblasts. Current evidence suggests that syncytin-1 plays an important role in the non-fusogenic biological activity of trophoblasts, except for specific fusogenic function. However, the underlying mechanism remains unclear. METHODS: The expression and location of syncytin-1 in normal and the late-onset preeclampsia placentas were detected by quantitative real-time PCR, western blotting and immunofluorescence. Morphological and apoptosis analysis were processed in placentas. The ex vivo extravillous explant culture model was used to explore the effect of syncytin-1 on EVT outgrowths. Real-time quantitative PCR and immunoblotting were used to calculate syncytin-1 levels in the trophoblast cells before and after syncytin-1 knockdown or overexpression. CCK-8 assay was used to detect the cell viability. TUNEL staining and immunoblotting were processed in trophoblast cells. Transwell assays and wound healing assays were utilize to assess the invasion and migration of trophoblastic cells. Conditional knockout of syncytin-a mouse model was conducted to present the change of placentas in vivo. The ex vivo extravillous explant culture model was used to explore the effect of syncytin-1 on EVT outgrowths. Western blotting was used to identify the key proteins of PI3K/Akt pathways and invasion-related proteins in trophoblast cells. RESULTS AND DISCUSSION: Here, reduced syncytin-1 was identified in the late-onset preeclampsia placentas. Reduced syncytin-1 may attenuates the EMT process by promoting apoptosis, inhibiting proliferation and invasion by suppressed PI3K/Akt pathway in trophoblast cells. Our findings provide novel insights into the non-fusogenic biological function of reduced syncytin-1 that may be involves in the pathogenesis of preeclampsia.
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Apoptosis , Productos del Gen env , Preeclampsia , Proteínas Gestacionales , Trofoblastos , Femenino , Preeclampsia/metabolismo , Preeclampsia/patología , Preeclampsia/genética , Embarazo , Trofoblastos/metabolismo , Trofoblastos/patología , Apoptosis/fisiología , Proteínas Gestacionales/metabolismo , Proteínas Gestacionales/genética , Humanos , Animales , Productos del Gen env/metabolismo , Productos del Gen env/genética , Ratones , Placenta/metabolismo , Placenta/patología , Adulto , Ratones Noqueados , Movimiento Celular/fisiología , Transducción de Señal/fisiologíaRESUMEN
A comparative study was carried out to analyze the number of syncytial knots and the intensity of expression of vascular endothelial growth factor (VEGF) in the villi of the monochorionic diamniotic placenta in pregnancies complicated by the syndrome of selective fetal growth restriction (sFGR). We performed a morphological analysis of 32 monochorionic diamniotic placentas after term delivery which were divided into two groups. The main group included the placentas of 15 puerperas whose pregnancies were complicated by sFGR. The control group included twin placentas of 17 puerperas without signs of sFGR. The number of syncytial knots was determined by histological studies, and the levels of VEGF expression in syncytiotrophoblast and capillary endotheliocytes of the placental villi were determined by immunohistochemical studies. The study showed an increase in the number of syncytial knots in the villi of the placental part of the fetus with sFGR which indicated the development of preplacental hypoxia. A significant increase in the level of VEGF expression in the syncytiotrophoblast and vascular endothelium of the villi should be considered as a manifestation of a compensatory adaptational response to hypoxia, though it is insufficient to prevent the development of sFGR.
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Vellosidades Coriónicas , Retardo del Crecimiento Fetal , Placenta , Trofoblastos , Factor A de Crecimiento Endotelial Vascular , Humanos , Femenino , Embarazo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Retardo del Crecimiento Fetal/metabolismo , Retardo del Crecimiento Fetal/patología , Vellosidades Coriónicas/metabolismo , Vellosidades Coriónicas/patología , Trofoblastos/metabolismo , Trofoblastos/patología , Placenta/metabolismo , Placenta/patología , AdultoRESUMEN
STUDY QUESTION: Do obstetric and perinatal complications vary according to different blastocyst developmental parameters after frozen-thawed single-blastocyst transfer (SBT) cycles? SUMMARY ANSWER: Pregnancies following the transfer of a blastocyst with a grade C trophectoderm (TE) were associated with an increased risk of placenta previa compared to those with a blastocyst of grade A TE. WHAT IS KNOWN ALREADY: Existing studies investigating the effect of blastocyst morphology grades on birth outcomes have mostly focused on fetal growth and have produced conflicting results, while the risk of obstetric complications has rarely been reported. Additionally, growing evidence has suggested that the appearance of TE cells could serve as the most important parameter for predicting implantation and live birth. Given that the TE ultimately develops into the placenta, it is plausible that this independent predictor may also impact placentation. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study at a tertiary-care academic medical center included 6018 singleton deliveries after frozen-thawed SBT cycles between January 2017 and December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Singleton pregnancies were grouped into two groups according to blastocyst developmental stage (Days 5 and 6), four groups according to embryo expansion (Stages 3, 4, 5, and 6), three groups according to inner cell mass (ICM) quality (A, B, and C), and three groups according to TE quality (A, B, and C). The main outcomes included pregnancy-induced hypertension, preeclampsia, gestational diabetes mellitus, preterm premature rupture of membrane, placenta previa, placental abruption, placenta accreta, postpartum hemorrhage, preterm birth, low birth weight, small for gestational age, and birth defects. Multivariate logistic regressions were performed to evaluate the effect of blastocyst developmental stage, embryo expansion stage, ICM grade, and TE grade on measured outcomes adjusting for potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: No association was found between blastocyst developmental stage and obstetric or perinatal outcomes both before and after adjusting for potential confounders, and similar results were found with regard to embryo expansion stage and ICM grade. Meanwhile, the incidence of placenta previa derived from a blastocyst with TE of grade C was higher compared with those derived from a blastocyst with TE of grade A (1.7%, 2.4%, and 4.0% for A, B, and C, respectively, P = 0.001 for all comparisons). After adjusting for potential covariates, TE grade C blastocysts had 2.81 times the likelihood of resulting in placenta previa compared to TE grade A blastocysts (adjusted odds ratio 2.81, 95% CI 1.11-7.09). No statistically significant differences were detected between any other measured outcomes and TE grades both before or after adjustment. LIMITATIONS, REASONS FOR CAUTION: The study is limited by its retrospective, single-center design. Additionally, although the sample size was relatively large for the study group, the sample size for certain subgroups was relatively small and lacked adequate power, particularly the ICM grade C group. Therefore, these results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: The study extends our knowledge of the potential downstream effect of TE grade on placental abnormalities. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Key Research and Development Program of China (2023YFC2705500, 2023YFC2705501, 2023YFC2705505, 2019YFA0802604); National Natural Science Foundation of China (82130046, 82320108009, 82371660, 32300710); Shanghai leading talent program, Innovative research team of high-level local universities in Shanghai (SHSMU-ZLCX20210201, SHSMU-ZLCX20210200, SHSMU-ZLCX20180401), Shanghai Jiaotong University School of Medicine Affiliated Renji Hospital Clinical Research Innovation Cultivation Fund Program (RJPY-DZX-003), Science and Technology Commission of Shanghai Municipality (23Y11901400), Shanghai's Top Priority Research Center Construction Project (2023ZZ02002), and Three-Year Action Plan for Strengthening the Construction of the Public Health System in Shanghai (GWVI-11.1-36). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.
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Criopreservación , Placenta Previa , Humanos , Femenino , Embarazo , Placenta Previa/epidemiología , Adulto , Estudios Retrospectivos , Transferencia de un Solo Embrión , Blastocisto , Trofoblastos/patologíaRESUMEN
A key aspect of preeclampsia pathophysiology is the reduced invasiveness of trophoblasts and the impairment of spiral artery remodelling. Understanding the causes of altered trophoblast function is critical to understand the development of preeclampsia. B7-H4, a checkpoint molecule, controls a wide range of processes, including T-cell activation, cytokine release, and tumour progression. Our previous findings indicated that B7-H4 levels are elevated in both maternal blood and placental villous tissue during the early stages of preeclampsia. Here, we investigated the function of B7-H4 in trophoblast physiology. Recombinant B7-H4 protein was used to treat human SGHPL-5 extravillous trophoblast cells. Biological functions were investigated using MTT, wound healing, and transwell assays. Signalling pathways were analysed by immunoblotting and immunofluorescence. The functionality of B7-H4 was further confirmed by immunoblotting and immunohistochemical analysis in placental tissues from control and preeclamptic patients following therapeutic plasma exchange (TPE) or standard of care treatment. This study showed that B7-H4 inhibited the proliferation, migration, and invasion capacities of SGHPL-5 extravillous cells while promoting apoptosis by downregulating the PI3K/Akt/STAT3 signalling pathway. These results were consistently confirmed in placental tissues from preterm controls compared to early-onset preeclamptic placental tissues from patients treated with standard of care or TPE treatment. B7-H4 may play a role in the development of preeclampsia by inhibiting essential functions of extravillous trophoblast cells during placental development. One possible mechanism by which TPE improves pregnancy outcomes in preeclampsia is through the elimination of B7-H4 amongst other factors.
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Movimiento Celular , Proliferación Celular , Preeclampsia , Transducción de Señal , Trofoblastos , Inhibidor 1 de la Activación de Células T con Dominio V-Set , Humanos , Preeclampsia/patología , Preeclampsia/metabolismo , Femenino , Trofoblastos/metabolismo , Trofoblastos/patología , Embarazo , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismo , Apoptosis , Línea Celular , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores Inmunológicos/metabolismo , Factor de Transcripción STAT3/metabolismo , Placenta/metabolismo , Adulto , Fosfatidilinositol 3-Quinasas/metabolismo , Trofoblastos ExtravellososRESUMEN
Chronic histiocytic intervillositis (CHI) is a recurrent placental lesion where maternal macrophages infiltrate the intervillous space. Its cause is unknown, though due to similarities to rejected allografts one hypothesis is that CHI represents maternal-fetal rejection. Here, virtual crossmatching was applied to healthy pregnancies and those with a history of CHI. Anti-HLA antibodies, measured by Luminex, were present in slightly more controls than CHI (8/17 (47.1%) vs 5/14 (35.7%)), but there was no significant difference in levels of sensitisation or fetal specific antibodies. Quantification of immunohistochemical staining for HLA-Class II was increased in syncytiotrophoblast of placentas with CHI (Grade 0.44 [IQR 0.1-0.7]) compared to healthy controls (0.06 [IQR 0-0.2]) and subsequent pregnancies (0.13 [IQR 0-0.3]) (P = 0.0004). HLA-Class II expression was positively related both to the severity of CHI (r = 0.67) and C4d deposition (r = 0.48). There was no difference in overall C4d and HLA-Class I immunostaining. Though increased anti-HLA antibodies were not evident in CHI, increased expression of HLA-Class II at the maternal-fetal interface suggests that they may be relevant in its pathogenesis. Further investigation of antibodies immediately after diagnosis is warranted in a larger cohort of CHI cases to better understand the role of HLA in its pathophysiology.
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Antígenos de Histocompatibilidad Clase II , Humanos , Femenino , Embarazo , Adulto , Antígenos de Histocompatibilidad Clase II/metabolismo , Placenta/patología , Placenta/metabolismo , Placenta/inmunología , Regulación hacia Arriba , Enfermedades Placentarias/patología , Enfermedades Placentarias/inmunología , Enfermedades Placentarias/metabolismo , Vellosidades Coriónicas/metabolismo , Vellosidades Coriónicas/patología , Vellosidades Coriónicas/inmunología , Trofoblastos/metabolismo , Trofoblastos/patología , Trofoblastos/inmunología , Enfermedad CrónicaRESUMEN
Preeclampsia (PE) is a life-threatening pregnancy-specific complication with controversial mechanisms and no effective treatment except delivery is available. Currently, increasing researchers suggested that PE shares pathophysiologic features with protein misfolding/aggregation disorders, such as Alzheimer disease (AD). Evidences have proposed defective autophagy as a potential source of protein aggregation in PE. Endoplasmic reticulum-selective autophagy (ER-phagy) plays a critical role in clearing misfolded proteins and maintaining ER homeostasis. However, its roles in the molecular pathology of PE remain unclear. We found that lncRNA DUXAP8 was upregulated in preeclamptic placentae and significantly correlated with clinical indicators. DUXAP8 specifically binds to PCBP2 and inhibits its ubiquitination-mediated degradation, and decreased levels of PCBP2 reversed the activation effect of DUXAP8 overexpression on AKT/mTOR signaling pathway. Function experiments showed that DUXAP8 overexpression inhibited trophoblastic proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Moreover, pathological accumulation of swollen and lytic ER (endoplasmic reticulum) was observed in DUXAP8-overexpressed HTR8/SVneo cells and PE placental villus trophoblast cells, which suggesting that ER clearance ability is impaired. Further studies found that DUXAP8 overexpression impaired ER-phagy and caused protein aggregation medicated by reduced FAM134B and LC3II expression (key proteins involved in ER-phagy) via activating AKT/mTOR signaling pathway. The increased level of FAM134B significantly reversed the inhibitory effect of DUXAP8 overexpression on the proliferation, migration, and invasion of trophoblasts. In vivo, DUXAP8 overexpression through tail vein injection of adenovirus induced PE-like phenotypes in pregnant rats accompanied with activated AKT/mTOR signaling, decreased expression of FAM134B and LC3-II proteins and increased protein aggregation in placental tissues. Our study reveals the important role of lncRNA DUXAP8 in regulating trophoblast biological behaviors through FAM134B-mediated ER-phagy, providing a new theoretical basis for understanding the pathogenesis of PE.
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Autofagia , Retículo Endoplásmico , Preeclampsia , Proteínas Proto-Oncogénicas c-akt , ARN Largo no Codificante , Transducción de Señal , Serina-Treonina Quinasas TOR , Trofoblastos , Adulto , Animales , Femenino , Humanos , Embarazo , Ratas , Autofagia/genética , Línea Celular , Movimiento Celular/genética , Proliferación Celular/genética , Retículo Endoplásmico/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Preeclampsia/genética , Preeclampsia/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Serina-Treonina Quinasas TOR/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patología , MasculinoRESUMEN
INTRODUCTION: Gestational cholestasis, also known as intrahepatic cholestasis of pregnancy (ICP) or obstetric cholestasis, is a liver disease that can manifest in late pregnancy. Trophoblast cell surface antigen (TROP2) is a type I transmembrane glycoprotein identified in placental trophoblast cells that plays a critical role in trophoblast invasion of the decidua upon implantation into the placenta. Our study aims to investigate the role of TROP2 in pregnancy cholestasis. MATERIALS AND METHODS: Study groups: Group 1 (control group) (n = 10): consists of healthy normal pregnant women without any disease, Group 2 (cholestasis group) (n = 10): consists of pregnant women diagnosed with cholestasis. After routine histological follow-up, hematoxylin and eosin staining and TROP2 immunostaining were performed and scored. RESULTS: In the cholestasis group, in contrast to the control group, thrombus structures were observed in the intervillous space. In the cholestasis group compared to the control group, villus mesenchymal connective tissue cells, capillary endothelium and trophoblasts around the villus showed significantly stronger anti-TROP2 staining (p < 0.05). DISCUSSION: Cholestasis, a condition that may manifest during pregnancy, may be associated not only with observable pathological changes in placental tissues at the light microscopic level, but also with an increase in TROP2 expression. Given the critical role of TROP2 in trophoblast invasion during placental implantation, we hypothesize that TROP2 may serve as a key marker of the cholestatic processes occurring during pregnancy.
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Antígenos de Neoplasias , Moléculas de Adhesión Celular , Colestasis Intrahepática , Placenta , Complicaciones del Embarazo , Humanos , Femenino , Embarazo , Colestasis Intrahepática/metabolismo , Colestasis Intrahepática/patología , Antígenos de Neoplasias/metabolismo , Complicaciones del Embarazo/metabolismo , Complicaciones del Embarazo/patología , Placenta/metabolismo , Placenta/patología , Adulto , Moléculas de Adhesión Celular/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patología , Estudios de Casos y ControlesRESUMEN
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 ß-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.
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Senescencia Celular , Decidua , Implantación del Embrión , Endometrio , Sirolimus , Femenino , Decidua/efectos de los fármacos , Decidua/metabolismo , Sirolimus/farmacología , Implantación del Embrión/efectos de los fármacos , Ratones , Embarazo , Animales , Humanos , Senescencia Celular/efectos de los fármacos , Endometrio/efectos de los fármacos , Endometrio/patología , Endometrio/metabolismo , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo , Trofoblastos/patología , Transducción de Señal/efectos de los fármacos , Línea Celular , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genéticaRESUMEN
The aberrant invasive capability of trophoblast cells is widely acknowledged as a primary mechanism underlying RSA. Recently, IGF2BP3 has been implicated in various cancers due to its influence on cellular invasion and migration. However, whether IGF2BP3 involve in the occurrence of RSA and the specific functions it assumes in the development of RSA remain elusive. In our study, we firstly collected villous tissues from RSA and those with normal pregnancies individuals to performed Protein sequencing and then detected the expression of IGF2BP3 through Western blot, qRT-PCR and immunohistochemistry. Secondly, we analyzed the single-cell data (GSE214607) to assess the expression of IGF2BP3 in invasive EVT trophoblasts. Thirdly, we utilized lentivirus technology to establish HTR-8/SVneo cell lines with stable IGF2BP3 knockdown and RNA-seq analysis was employed to investigate the GO functional pathway enrichment of IGF2BP3. Meanwhile, the effect of IGF2BP3 knockdown on trophoblast cells apoptosis, migration, and ferroptosis was evaluated through functional experiments. Additionally, LPS-induced abortion animal model was constructed to evaluate IGF2BP3 expression in placental tissues. A significant downregulation of IGF2BP3 was observed in the villous tissues of RSA patient, a finding corroborated by subsequent single cell sequencing results. Furthermore, it suggested that IGF2BP3 may be involved in the migration and apoptotic processes of trophoblast cells. Mechanistic research indicated that IGF2BP3 knockdown could compromise GPX4 mRNA stability, leading to the promotion of ferroptosis. Finally, our investigation observed the down-regulation of IGF2BP3 expression in placental villous tissues of an LPS-induced abortion animal model. Our findings revealed that IGF2BP3 was downregulated in the villous tissues of RSA patients. Mechanically, down-regulation of IGF2BP3 may induce RSA by promoting GPX4-mediated ferroptosis and inhibiting trophoblast invasion and migration. Our study may provide new targets and research directions for the pathogenesis of RSA.
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Aborto Habitual , Ferroptosis , Proteínas de Unión al ARN , Trofoblastos , Humanos , Femenino , Ferroptosis/inmunología , Embarazo , Aborto Habitual/metabolismo , Aborto Habitual/patología , Aborto Habitual/inmunología , Trofoblastos/metabolismo , Trofoblastos/patología , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ratones , Movimiento Celular/genética , Línea Celular , Placenta/metabolismo , Placenta/patología , Placenta/inmunología , Adulto , Apoptosis/inmunologíaRESUMEN
BACKGROUND: Krüppel-like factor 15 (KLF15) has been reported to be involved in ischemia injury of multiple types of diseases. Nevertheless, the roles and underlying mechanisms of KLF15 in preeclampsia (PE) are still unclear. METHODS: In this study, the expression of KLF15 in placenta tissues and hypoxia/reoxygenation (H/R)-induced HTR8/SVneo cells was evaluated by GSE66273 database, qRT-PCR and western blot assay. CCK-8 assay was employed to detect cell proliferation. Wound healing assay and transwell assay were used to detect cell migration and invasion. Cell oxidative stress was measured by DCFH-DA staining and kits. Cell apoptosis was evaluated by TUNEL assay and western blot assay. The JASPAR database was used to analyze the binding site of KLF15 and insulin-like growth factor-1 receptor (IGF1R) promoter region. The luciferase reporter assay was used to detect IGF1R promoter activity and ChIP assay was used to verify the combination of KLF15 and IGF1R promoter. Moreover, western blot was employed to measure the expressions of PI3K/Akt-related proteins. RESULTS: The data showed that the expression of KLF15 was significantly downregulated in GSE66273 database, tissues and HTR8/SVneo cells. KLF15 overexpression increased H/R-induced HTR8/SVneo cell proliferation, invasion and migration, and inhibited oxidative stress and cell apoptosis. In addition, IGF1R was highly expressed in H/R-induced HTR8/SVneo cells after KLF15 overexpression, and the binding of KLF15 and IGF1R promoter was verified. Silencing of IGF1R reversed the effects of KLF15 overexpression on H/R-induced HTR8/SVneo cell proliferation, migration, invasion, oxidative stress and cell apoptosis. Moreover, KLF15 overexpression and IGF1R silencing regulated the expressions of PI3K/Akt-related proteins in H/R-induced HTR8/SVneo cells. CONCLUSION: In conclusion, KLF15 overexpression promoted the proliferation and metastasis, and suppressed oxidative stress and cell apoptosis of H/R-induced HTR8/SVneo cells through mediating the PI3K/Akt pathway, which may provide a promising target for the treatment of preeclampsia.
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Apoptosis , Movimiento Celular , Factores de Transcripción de Tipo Kruppel , Estrés Oxidativo , Receptor IGF Tipo 1 , Trofoblastos , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Apoptosis/genética , Movimiento Celular/genética , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 1/genética , Trofoblastos/metabolismo , Trofoblastos/patología , Estrés Oxidativo/genética , Femenino , Embarazo , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Línea Celular , Fosfatidilinositol 3-Quinasas/metabolismo , Preeclampsia/metabolismo , Preeclampsia/patología , Preeclampsia/genética , Proliferación Celular/genéticaRESUMEN
Dysregulated progenitor cell populations may contribute to poor placental development and placental insufficiency pathogenesis. Side-population cells possess progenitor properties. Recent human trophoblast side-population isolation identified enrichment of 8 specific genes (CXCL8, ELL2, GATA6, HK2, HLA-DPB1, INTS6, SERPINE3 and UPP1) (Gamage et al. 2020, Stem Cell Rev Rep). We characterised these trophoblast side-population markers in human placenta and in placental insufficiency disorders: preeclampsia and fetal growth restriction (FGR). Trophoblast side-population markers localised to mononuclear trophoblasts lining the placental villous basement membrane in preterm control, preeclamptic and FGR placental sections (n = 3, panel of 3 markers/serial section). Analysis of single-cell transcriptomics of an organoid human trophoblast stem cell (hTSC) to extravillous trophoblast (EVT) differentiation model (Shannon et al. 2022, Development) identified that all side-population genes were enriched in mononuclear trophoblast and trophoblasts committed to differentiation under hTSC culture conditions. In vitro validation via 96 h time course hTSC differentiation to EVTs or syncytiotrophoblasts (n = 5) demonstrated ELL2 and HK2 increased with differentiation (p < 0.0024, p < 0.0039 respectively). CXCL8 and HLA-DPB1 were downregulated (p < 0.030, p < 0.011 respectively). GATA6 and INTS6 increased with EVT differentiation only, and UPP1 reduced with syncytialisation. SERPINE3 was undetectable. Trophoblast side-population marker mRNA was measured in human placentas (< 34-weeks' gestation; n = 78 preeclampsia, n = 30 FGR, and n = 18 gestation-matched controls). ELL2, HK2 and CXCL8 were elevated in preeclamptic (p = 0.0006, p < 0.0001, p = 0.0335 respectively) and FGR placentas (p = 0.0065, p < 0.0001, p = 0.0001 respectively) versus controls. Placental GATA6 was reduced in pregnancies with preeclampsia and FGR (p = 0.0014, p = 0.0146 respectively). Placental INTS6 was reduced with FGR only (p < 0.0001). This study identified the localisation of a unique trophoblast subset enriched for side-population markers. Aberrant expression of some side-population markers may indicate disruptions to unique trophoblast subtypes in placental insufficiency.