Preeclamptic Placentas Research Articles

Mitochondrial-targeted antioxidant attenuates preeclampsia-like phenotypes induced by syncytiotrophoblast-specific Gαq signaling.

Syncytiotrophoblast stress is theorized to drive development of preeclampsia, but its molecular causes and consequences remain largely undefined. Multiple hormones implicated in preeclampsia signal via the Gαq cascade, leading to the hypothesis that excess Gαq signaling within the syncytiotrophoblast may contribute. First, we present data supporting increased Gαq signaling and antioxidant responses within villous and syncytiotrophoblast samples of human preeclamptic placenta. Second, Gαq was activated in mouse placenta using Cre-lox and DREADD methodologies. Syncytiotrophoblast-restricted Gαq activation caused hypertension, kidney damage, proteinuria, elevated circulating proinflammatory factors, decreased placental vascularization, diminished spiral artery diameter, and augmented responses to mitochondrial-derived superoxide. Administration of the mitochondrial-targeted antioxidant Mitoquinone attenuated maternal proteinuria, lowered circulating inflammatory and anti-angiogenic mediators, and maintained placental vascularization. These data demonstrate a causal relationship between syncytiotrophoblast stress and the development of preeclampsia and identify elevated Gαq signaling and mitochondrial reactive oxygen species as a cause of this stress.

FRI375 Validation Of A Novel Mouse Placenta Explant Protocol For Screening Of Drugs Modulating Circadian Rhythms

Abstract Disclosure: J.S. Lee: None. H.M. Hoffmann: None. Introduction Pre-eclampsia is one of the leading causes of maternal, fetal, and neonatal morbidity and mortality, complicating about 4% of pregnancies. Recent work has identified PERIOD3 (PER3), a gene regulating 24-hour circadian rhythms, to be downregulated in the preeclamptic placenta. The role of circadian rhythms in the placenta largely remains unknown. Our goal is to establish an organotypic placental preparation to allow screening of drugs that regulate circadian rhythms. These studies are a first step towards understanding how circadian rhythm changes are caused in the placenta and will be the basis for future studies to understand how circadian rhythm disruption in the placenta cause pregnancy complications, such as preeclampsia. Methods We established a novel organotypic placenta preparation allowing to study circadian rhythms in a LumiCycle using gestation day (GD) 14 and GD18 placental explants from the validated circadian reporter mouse Per2::Luciferase. Using H&E we validated the explants to be >80% enriched in either decidua (DC) which is strictly of maternal origin, labyrinth (LB) which is strictly of fetal origin, and the junctional zone (JZ) which consists of binucleated cells fused of maternal and fetal cells. Results Through H&E staining, we validated by distinct morphology that the DC is enriched with neutrophils; the JZ contains spongiotrophoblasts and glycogen trophoblasts; whereas the LB has anucleated red blood cells. We next established a protocol to record Per2::Luciferase circadian rhythms for <6 days. We found that the Per2::Luciferase circadian rhythm shortens with increased gestational age, where the period in DC, LZ and LB in the GD14 placenta is ∼4h longer than in the GD18 placenta, showing an unexpected plasticity in circadian timekeeping in the placenta. Because PER3 is downregulated in the pre-eclamptic placenta, we asked if the explants would be useful for pharmacologically screenings. We applied PF670462 (1μM), which stabilizes PER1/2/3, or epidermal growth factor (EGF, 30ng/mL), a growth factor important in placental function and a PER2 enhancer. PF670462 lengthened Per2::Luciferase period in the DC and JZ, confirming the expected circadian rhythm stabilizing effect of PF670462. Surprisingly, EGF only modulated circadian rhythms in the GD14 placental, where EGF shortened Per2::Luciferase period in DC, and lengthen the period in the JZ and LB zones in the GD14 placenta. This data suggests EGF has gestational age and placenta zone specific effects. Conclusion We validated a novel ex vivo placental preparation to study circadian rhythms at GD14 and GD18. We identified a placenta zone and gestation age specific effect of EGF, an effect future studies will further expand on. This model will be useful to complete pharmacological screenings of drugs that can restore circadian rhythms in the preeclamptic placenta. Presentation: Friday, June 16, 2023

Comparison of placental expression of basic fibroblast growth factor and insulin-like growth factor-1 in placentae of normal, pregnancy-induced hypertension, and preeclamptic pregnancies in Iraqi mothers

Abstract Background: Many pathological insults are associated with elevation of blood pressure levels during pregnancy resulting in a difficult pregnancy and a poor outcome on both mother and baby. Objective: In this study, we examine the histological and immunohistochemical markers of the placentae in cases of hypertension in pregnancy and preeclampsia and compared them to a placenta of normal pregnancy among a random sample of Iraqi pregnant women. Materials and Methods: Ninety women divided into three groups, 30 in each, selected with straightforward pregnancies (Group A), mothers with pregnancy-induced hypertension (Group B), and preeclamptic mothers (Group C) were chosen from the indoor patients of the gynecology and obstetrics department of Al-Khansaa teaching hospital in Mosul for placental tissues examination. Histological examination was done by using hematoxylin and eosin stain (H & E), and immunohistochemistry was achieved by using immunohistochemical markers named: insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (b-FGF) markers, which are expressed in placental tissues. Results: Different changes were observed in the placentae affected when compared with normal one, such as syncytial knots formation, thickening of trophoblastic basement membrane, cytotrophoblastic cellular proliferation, fibrinoid necrosis, endothelial proliferation, calcified and hyalinised villous spots, villous edema, and atherosis of the uteroplacental arteries. Significant immunohistochemical changes were obtained when compared with normal placentae where elevation of both b-FGF and IGF-1 in preeclamptic placentae was observed when compared to hypertensive and control cases. Conclusion: Significant changes appeared in the placentae of hypertensive and preeclamptic mothers, both in histological and immunohistochemical examinations.

Apelin/APJ system protects placental trophoblasts from hypoxia-induced oxidative stress through activating PI3K/Akt signaling pathway in preeclampsia

BackgroundPreeclampsia is a placentally induced syndrome with diverse clinical presentation that currently has no cure. Oxidative stress is a potent inducer of placental dysfunction. The apelin receptor (APJ) system is a pleiotropic pathway with a potential for therapeutic targeting in preeclampsia. This study examines the alteration of circulating apelin levels and placental APJ expression in preeclampsia and investigates whether apelin/APJ system can protect placental trophoblast from hypoxia-induced oxidative stress injury through PI3K/AKT signaling pathway. ResultsOur results confirmed that maternal apelin concentration was increased in women with preeclampsia, but APJ expression was reduced in the preeclamptic placentas. Apelin-13 treatment not only specifically attenuated CoCl2-induced superoxide production, but also prevented CoCl2-induced reduction of SOD activity and SOD1 expression. In addition, apelin-13 suppressed CoCl2-induced apoptosis by increasing the expression of bcl-2/bax ratio and by decreasing the expression of active caspase-3 in placental trophoblasts. Furthermore, we found that apelin-13 binding APJ activated the PI3K and AKT kinases and inhibition of PI3K kinase significantly blocked the anti-oxidative effects of apelin-13 in placental trophoblasts. ConclusionsDecrease of placental APJ expression is associated with oxidative stress-induced placental dysfunction in preeclampsia, and increased circulating apelin could be a moderately successful marker to differentiate subjects with preeclampsia from healthy pregnant women. Inhibition of superoxide production and caspase-3 cleavage, together with upregulation of SOD activity/expression and bcl-2/bax ratio, could be the potential molecular mechanisms by which apelin-13/APJ protects placental trophoblasts from oxidative stress injury.

Orchestrated feedback regulation between melatonin and sex hormones involving GPER1-PKA-CREB signaling in the placenta.

Maintaining placental endocrine homeostasis is crucial for a successful pregnancy. Pre-eclampsia (PE), a gestational complication, is a leading cause of maternal and perinatal morbidity and mortality. Aberrant elevation of testosterone (T0 ) synthesis, reduced estradiol (E2 ), and melatonin productions have been identified in preeclamptic placentas. However, the precise contribution of disrupted homeostasis among these hormones to the occurrence of PE remains unknown. In this study, we established a strong correlation between suppressed melatonin production and decreased E2 as well as elevated T0 synthesis in PE placentas. Administration of the T0 analog testosterone propionate (TP; 2 mg/kg/day) to pregnant mice from E7.5 onwards resulted in PE-like symptoms, along with elevated T0 production and reduced E2 and melatonin production. Notably, supplementation with melatonin (10 mg/kg/day) in TP-treated mice had detrimental effects on fetal and placental development and compromised hormone synthesis. Importantly, E2 , but not T0 , actively enhanced melatonin synthetase AANAT expression and melatonin production in primary human trophoblast (PHT) cells through GPER1-PKA-CREB signaling pathway. On the other hand, melatonin suppressed the level of estrogen synthetase aromatase while promoting the expressions of androgen synthetic enzymes including 17β-HSD3 and 3β-HSD1 in PHT cells. These findings reveal an orchestrated feedback mechanism that maintains homeostasis of placental sex hormones and melatonin. It is implied that abnormal elevation of T0 synthesis likely serves as the primary cause of placental endocrine disturbances associated with PE. The suppression of melatonin may represent an adaptive strategy to correct the imbalance in sex hormone levels within preeclamptic placentas. The findings of this study offer novel evidence that identifies potential targets for the development of innovative therapeutic strategies for PE.

Upregulation of autotaxin by oxidative stress via Nrf2 activation: A novel insight into the compensation mechanism in preeclamptic placenta

The response of autotaxin (ATX)–lysophosphatidic acid (LPA) signaling system to placental oxidative stress (OS) and its significance to preeclampsia were investigated. For this purpose, oxidative stress index (OSI) and ATX levels were measured in the serum of pregnant women with preeclampsia. The expression levels of ATX and LPA receptors were assessed in trophoblast cells under high OS and glucose deprivation/re-oxygenation (OGD/R) conditions, with particular emphasis on the antioxidative nuclear factor erythroid 2-related factor 2 (NRF2) pathway. The influence of ATX–LPA signaling on cell migration was also evaluated using the wound healing assay. ATX concentrations and OSI in the serum were found to be elevated in preeclamptic women. The serum ATX levels were also positively correlated with OSI. Trophoblast cells responded to OS by increasing ATX mRNA expression concomitantly with intranuclear translocation of Nrf2, whereas inhibition of Nrf2 activation reverted this effect. The ATX–LPA signaling pathway facilitated trophoblast cell motility after Nrf2 activation. In conclusion, OS accumulation in preeclamptic placenta may activate the ATX–LPA system in trophoblasts via the Nrf2 pathway to sustain trophoblast functionality.

Rosiglitazone-Mediated Activation of PPARγ Restores HO1 Expression in the Human Preeclamptic Placenta.

Preeclampsia is a hypertensive disorder of pregnancy characterized by chronic placental ischemia and suppression of proangiogenic proteins, causing oxidative stress, hypertension, and maternal systemic organ damage. The transcription factor, PPARγ (peroxisome proliferator-activated receptor-γ) promotes healthy trophoblast differentiation but is dysregulated in the preeclampsia placenta. Our study identifies the beneficial impact of Rosiglitazone-mediated PPARγ-activation in the stressed preeclampsia placenta. We used first trimester placentas, preeclamptic and preterm control placentas, and human trophoblast cell lines to study PPARγ activation. Induction of PPARγ activates cell growth and antioxidative stress pathways, including the gene, heme oxygenase 1 (Hmox1). Protein expression of both PPARγ and HO1 (heme oxygenase 1) are reduced in preeclamptic placentas, but Rosiglitazone restores HO1 signaling in a PPARγ-dependent manner. Restoring disrupted pathways by PPARγ in preeclampsia offers a potential therapeutic pathway to reverse placental damage, extending pregnancy duration, and reduce maternal sequelae. Future research should aim to understand the full scope of impaired PPARγ signaling in the human placenta and focus on compounds for safe use during pregnancy to prevent severe perinatal morbidity and mortality.

The endocannabinoid system and its vascular effect in healthy and preeclamptic placentas

The endocannabinoid system and its vascular effect in healthy and preeclamptic placentas

Placental transfer and metabolism of betamethasone in healthy and preeclamptic human placentas

Placental transfer and metabolism of betamethasone in healthy and preeclamptic human placentas

Lipidomic profiling of preeclamptic placenta according to severe features

Lipidomic profiling of preeclamptic placenta according to severe features

Investigation of Beclin 1 and TNF-α expressions in preeclampsia placentas: Immunohistochemical study.

Preeclampsia is a pregnancy complication Aim of this study was to investigate expression of Beclin1 and tumor necrosis factor (TNF)-α in normotensive and preeclamptic placentas of pregnant women patients. Twenty normotensive and 20 preeclamptic patients placentas were dissected for paraffin- wax processing. Placental samples were embedded in parafin blocks. Sections were stained with Hematoxylin-Eosin staining and TNF-α and Beclin1 immunostaining. In control group, root and floating villi were normal in histological perspectives, syncytial node number was low, vessels were normal with connective tissue. No hemorrhage was observed in the intervillous area. In preeclampsia group, decidual cell degeneration and fibrinoid accumulation increased. Vascular dilatation and congestion with mononuclear cell infiltration were observed. Beclin1 reaction was generally negative in control group. In preeclampsia group, Beclin1 reaction was increased in decidual cells, syncytial nodes and bridges and in chorionic villi and in some Hoffbauer cells. In control group, TNF-α expression was mainly negative but only in some decidual cells. In preeclampsia, TNF-α reaction was observed in degenerated decidua cells, in leukocytes and in villi. In preeclampsia placentas, degenerated decidua cells and inflammation increased. It was thought that Beclin1 and TNF-α signals could be used as a marker in affecting the fetal structure of blood flow in preeclamptic placentas.

Bioinformatic analysis reveals lysosome-related biomarkers and molecular subtypes in preeclampsia: novel insights into the pathogenesis of preeclampsia.

Background: The process of lysosomal biogenesis and exocytosis in preeclamptic placentae plays a role in causing maternal endothelial dysfunction. However, the specific lysosome-associated markers relevant to preeclampsia (PE) are not well-defined. Our objective is to discover new biomarkers and molecular subtypes associated with lysosomes that could improve the diagnosis and treatment of PE. Methods: We obtained four microarray datasets related to PE from the Gene Expression Omnibus (GEO) database. The limma package was utilized to identify genes that were differentially expressed between individuals with the disease and healthy controls. The logistic regression analysis was used to identify core diagnostic biomarkers, which were subsequently validated by independent datasets and clinical samples. Additionally, a consensus clustering method was utilized to distinguish between different subtypes of PE. Following this, functional enrichment analysis, GSEA, GSVA, and immune cell infiltration were conducted to compare the two subtypes and identify any differences in their functional characteristics and immune cell composition. Results: We identified 16PE-specific lysosome-related genes. Through regression analysis, two genes, GNPTG and CTSC, were identified and subsequently validated in the external validation cohort GSE60438 and through qRT-PCR experiment. A nomogram model for the diagnosis of PE was developed and evaluated using these two genes. The model had a remarkably high predictive power (AUC values of the training set, validation set, and clinical samples were 0.897, 0.788, and 0.979, respectively). Additionally, two different molecular subtypes (C1 and C2) were identified, and we found notable variations in the levels of immune cells present in the two subtypes. Conclusion: Our results not only offered a classification system but also identified novel diagnostic biomarkers for PE patients. Our findings offered an additional understanding of how to categorize PE patients and also highlighted potential avenues for creating treatments for individuals with PE.

Paternal Expressed Gene 10 (PEG10) is decreased in early-onset preeclampsia

BackgroundPreeclampsia is a severe complication of pregnancy which is attributed to placental dysfunction. The retrotransposon, Paternal Expressed Gene 10 (PEG10) harbours critical placental functions pertaining to placental trophoblast cells. Limited evidence exists on whether PEG10 is involved in preeclampsia pathogenesis. This study characterised the expression and regulation of PEG10 in placentas from patients with early-onset preeclampsia compared to gestation-matched controls.MethodsPEG10 expression was measured in plasma and placentas collected from patients with early-onset preeclampsia (< 34 weeks’) and gestation-matched controls using ELISA (protein) and RT-qPCR (mRNA). First-trimester human trophoblast stem cells (hTSCs) were used for in vitro studies. PEG10 expression was measured during hTSC differentiation and hTSC exposure to hypoxia (1% O2) and inflammatory cytokines (IL-6 and TNFα) using RT-qPCR. Functional studies used PEG10 siRNA to measure the effect of reduced PEG10 on canonical TGF-beta signalling and proliferation using luciferase and xCELLigence assays, respectively.ResultsPEG10 mRNA expression was significantly reduced in placentas from patients with early-onset preeclampsia (< 34 weeks’ gestation) relative to controls (p = 0.04, n = 78 vs n = 18 controls). PEG10 protein expression was also reduced in preeclamptic placentas (p = 0.03, n = 5 vs n = 5 controls, blinded assessment of immunohistochemical staining), but neither PEG10 mRNA nor protein could be detected in maternal circulation. PEG10 was most highly expressed in hTSCs, and its expression was reduced as hTSCs differentiated into syncytiotrophoblasts (p < 0.0001) and extravillous trophoblasts (p < 0.001). Trophoblast differentiation was not altered when hTSCs were treated with PEG10 siRNA (n = 5 vs n = 5 controls).PEG10 was significantly reduced in hTSCs exposed to hypoxia (p < 0.01). PEG10 was also reduced in hTSCs treated with the inflammatory cytokine TNF alpha (p < 0.01), but not IL-6. PEG10 knocked down (siRNA) in hTSCs showed reduced activation of the canonical TGF-β signalling effector, the SMAD binding element (p < 0.05) relative to controls. PEG10 knockdown in hTSCs however was not associated with any significant alterations in proliferation.ConclusionsPlacental PEG10 is reduced in patients with early-onset preeclampsia. In vitro studies suggest that hypoxia and inflammation may contribute to PEG10 downregulation. Reduced PEG10 alters canonical TGF-beta signalling, and thus may be involved in trophoblast dysfunction associated with this pathway.

Altered placental ion channel gene expression in preeclamptic high-altitude pregnancies.

High-altitude (>2,500 m) residence increases the risk of pregnancy vascular disorders such as fetal growth restriction and preeclampsia, each characterized by impaired placental function. Genetic attributes of highland ancestry confer relative protection against vascular disorders of pregnancy at high altitudes. Although ion channels have been implicated in placental function regulation, neither their expression in high-altitude placentas nor their relationship to high-altitude preeclampsia has been determined. Here, we measured the expression of 26 ion-channel genes in placentas from preeclampsia cases and normotensive controls in La Paz, Bolivia (3,850 m). In addition, we correlated gene transcription to maternal and infant ancestry proportions. Gene expression was assessed by PCR, genetic ancestry evaluated by ADMIXTURE, and ion channel proteins localized by immunofluorescence. In preeclamptic placentas, 11 genes were downregulated (ABCC9, ATP2A2, CACNA1C, KCNE1, KCNJ8, KCNK3, KCNMA1, KCNQ1, KCNQ4, PKD2, and TRPV6) and two were upregulated (KCNQ3 and SCNN1G). KCNE1 expression was positively correlated with high-altitude Amerindian ancestry and negatively correlated with non-high altitude. SCNN1G was negatively correlated with African ancestry, despite minimal African admixture. Most ion channels were localized in syncytiotrophoblasts (Cav1.2, TRPP2, TRPV6, and Kv7.1), whereas expression of Kv7.4 was primarily in microvillous membranes, Kir6.1 in chorionic plate and fetal vessels, and MinK in stromal cells. Our findings suggest a role for differential placental ion channel expression in the development of preeclampsia. Functional studies are needed to determine processes affected by these ion channels in the placenta and whether therapies directed at modulating their activity could influence the onset or severity of preeclampsia.

H3K27me3-modulated Hofbauer cell BMP2 signalling enhancement compensates for shallow trophoblast invasion in preeclampsia.

Preeclampsia (PE) is a common hypertensive pregnancy disorder associated with shallow trophoblast invasion. Although bone morphogenetic protein 2 (BMP2) has been shown to promote trophoblast invasion invitro, its cellular origin and molecular regulation in placenta, as well as its potential role in PE, has yet to be established. Additionally, whether BMP2 and/or its downstream molecules could serve as potential diagnostic or therapeutic targets for PE has not been explored. Placentas and sera from PE and healthy pregnant women were subjected to multi-omics analyses, immunoblots, qPCR, and ELISA assays. Immortalized trophoblast cells, primary cultures of human trophoblasts, and first-trimester villous explants were used for invitro experiments. Adenovirus expressing sFlt-1 (Ad Flt1)-induced PE rat model was used for invivo studies. We find globally decreased H3K27me3 modifications and increased BMP2 signalling in preeclamptic placentas, which is negatively correlated with clinical manifestations. BMP2 is derived from Hofbauer cells and epigenetically regulated by H3K27me3 modification. BMP2 promotes trophoblast invasion and vascular mimicry by upregulating BMP6 via BMPR1A-SMAD2/3-SMAD4 signalling. BMP2 supplementation alleviates high blood pressure and fetal growth restriction phenotypes in Ad Flt1-induced rat PE model. Our findings demonstrate that epigenetically regulated Hofbauer cell-derived BMP2 signalling enhancement in late gestation could serve as a compensatory response for shallow trophoblast invasion in PE, suggesting opportunities for diagnostic marker and therapeutic target applications in PE clinical management. National Key Research and Development Program of China (2022YFC2702400), National Natural Science Foundation of China (82101784, 82171648, 31988101), and Natural Science Foundation of Shandong Province (ZR2020QH051, ZR2020MH039).

Healthy and diseased placental barrier on-a-chip models suitable for standardized studies.

Pathologies associated with uteroplacental hypoxia, such as preeclampsia are among the leading causes of maternal and perinatal morbidity in the world. Its fundamental mechanisms are yet poorly understood due to a lack of good experimental models. Here we report an in vitro model of the placental barrier, based on co-culture of trophoblasts and endothelial cells against a collagen extracellular matrix in a microfluidic platform. The model yields a functional syncytium with barrier properties, polarization, secretion of relevant extracellular membrane components, thinning of the materno-fetal space, hormone secretion, and transporter function. The model is exposed to low oxygen conditions and perfusion flow is modulated to induce a pathological environment. This results in reduced barrier function, hormone secretion, and microvilli as well as an increased nuclei count, characteristics of preeclamptic placentas. The model is implemented in a titer plate-based microfluidic platform fully amenable to high-throughput screening. We thus believe this model could aid mechanistic understanding of preeclampsia and other placental pathologies associated with hypoxia/ischemia, as well as support future development of effective therapies through target and compound screening campaigns. STATEMENT OF SIGNIFICANCE: : The human placenta is a unique organ sustaining fetus growth but is also the source of severe pathologies, such as Preeclampsia. Though leading cause of perinatal mortality in the world, preeclampsia remains untreatable due to a lack of relevant in vitro placenta models. To better understand the pathology, we have developed 3D placental barrier models in a microfluidic device. The platform allows parallel culture of 40 perfused physiological miniaturized placental barriers, comprising a differentiated syncytium and endothelium that have been validated for transporter functions. Exposure to a hypoxic and ischemic environment enabled the mimicking of preeclamptic characteristics in high-throughput, which we believe could lead to a better understanding of the pathology as well as support future effective therapies development.

The Impact of Hydroxychloroquine on Primary Feto-Placental Endothelial Cells from Healthy and Early-Onset Preeclamptic Placentas.

Hydroxychloroquine (HCQ), an anti-malarial drug, is suggested as a promising candidate for the treatment of pregnancy-related disorders associated with endothelial activation, among which there is preeclampsia (PE). Arterial feto-placental endothelial cells (fpECAs) were isolated from control (CTR) and early-onset preeclamptic (EO-PE) placentas. The aim of this study was to test potential protective effects of HCQ in an in vitro model of endothelial activation as well as in cells isolated from EO-PE placentas. To mimic PE conditions, CTR fpECAs were exposed to a pro-inflammatory environment consisting of tumor necrosis factor α (TNF-α), interleukin (IL)-6 and IL-1β (furtherly referred as MIX) with or without varying concentrations of HCQ (1 µg/mL and 10 µg/mL). Their effect on wound healing and endothelial barrier integrity was analyzed. Variations in the expression of IL-8 and leukocyte adhesion molecules (LAM) on both mRNA and protein levels were determined between CTR and PE fpECAs in the presence or absence of HCQ. MIX decreased wound healing and stability of the endothelial barrier, but HCQ did not affect it. Significant differences between CTR and EO-PE fpECAs were observed in IL-8 mRNA, protein secretion, and vascular cell adhesion protein 1 (VCAM-1) mRNA expression levels. After challenging CTR fpECAs with MIX, upregulation of both mRNA and protein levels was observed in all molecules. Combined treatment of HCQ and MIX slightly lowered VCAM-1 total protein amount. In CTR fpECAs, treatment with low concentrations of HCQ alone (1 µg/mL) reduced basal levels of IL-8 and VCAM-1 mRNA and secretion of IL-8, while in EO-PE fpECAs, a higher (10µg/mL) HCQ concentration slightly reduced the gene expression of IL-8. Conclusion: These results provide additional support for the safety of HCQ, as it did not adversely affect endothelial functionality in control fpECAs at the tested concentration. Furthermore, the observed limited effects on IL-8 secretion in EO-PE fpECAs warrant further investigation, highlighting the need for clinical trials to assess the potential therapeutic effects of HCQ in preeclampsia. Conducting clinical trials would offer a more comprehensive understanding of HCQ's efficacy and safety, allowing us to explore its potential benefits and limitations in a real-world clinical setting.

Circular RNA expression profiles and circRNA-miRNA-mRNA crosstalk in pre-eclamptic placenta

Objective: To identify the expression profile of circular RNA (circRNA) in placenta of pre-eclampsia (PE) pregnant women by high-throughput sequencing, and to construct the circRNA-microRNA (miRNA)-messenger RNA (mRNA) interaction network, so as to reveal the related pathways and regulatory mechanisms of PE. Methods: The clinical data and placentas of 42 women with PE (PE group) and 30 normal pregnant women (control group) who delivered in West China Second University Hospital from November 2019 to June 2021 were collected. (1) High-throughput sequencing was used to establish the differentially expressed circRNA profiles in placental tissues of 5 pairs of PE group and the control group. (2) Real-time quantitative PCR (qRT-PCR) was used to verify the expression levels of 6 differentially expressed circRNAs in placental tissues of PE group and control group. (3) Bioinformatics analysis was used to predict the target miRNA and analyze the co-expressed mRNA to construct a competitive endogenous RNA (ceRNA) network. The differentially expressed circRNAs were analyzed by Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. (4) Logistic regression analysis, Pearson correlation and Kendall's tau-b correlation analysis were used to test the correlation between the three differentially expressed circRNAs and the risk of PE and clinical characteristics. (5) circRNA_05393 was selected for subsequent functional study. Small interfering RNA (siRNA) and overexpression plasmid were used to knock down or increase the expression level of circRNA_05393 in trophoblast cell line HTR-8/SVneo cells, respectively. Transwell assay was used to detect the migration and invasion ability of the trophoblasts in vitro. Cell counting kit-8 assay was used to detect the proliferation ability of the trophoblasts. Results: (1) Seventy-two differentially expressed circRNAs were identified by high-throughput sequencing, of which 35 were up-regulated and 37 were down-regulated. (2) qRT-PCR showed that compared with the control group, circRNA_00673 (1.306±0.168 vs 2.059±0.242; t=2.356, P=0.021) and circRNA_07796 (1.275±0.232 vs 1.954±0.230; t=2.018, P=0.047) were significantly increased, while circRNA_05393 (1.846±0.377 vs 0.790±0.094; t=3.138, P=0.002) was significantly decreased. (3) The circRNA-miRNA-mRNA interaction network contained 3 circRNAs, 8 miRNAs and 53 mRNAs. GO functional annotation analysis showed that the biological process was mainly enriched in iron ion homeostasis, membrane depolarization during action potential and neuronal action potential. In terms of cellular components, they were mainly enriched in cytoskeleton and membrane components. In terms of molecular function, they were mainly enriched in the activity of voltage-gated sodium channel and basic amino acid transmembrane transporter. KEGG pathway enrichment analysis showed that mRNAs in the interaction network were mainly enriched in complement and coagulation cascade, glycine, serine and threonine metabolism, p53 signaling pathway and peroxisome proliferators-activated receptors (PPAR) signaling pathway. (4) Logistic regression analysis showed that down-regulation of circRNA_05393 expression was a risk factor for PE (OR=0.044, 95%CI: 0.003-0.596; P=0.019). Correlation analysis showed that circRNA_05393 was significantly correlated with systolic blood pressure and diastolic blood pressure in PE pregnant women (both P<0.05). (5) Knock down or overexpression of circRNA_05393 significantly reduced or increased the migration and invasion abilities of HTR-8/SVneo cells (all P<0.05), but had no significant effect on the ability of tube formation and proliferation (all P>0.05). Conclusions: The construction of circRNA expression profile in placenta and the exploration of circRNA-miRNA-mRNA interaction network provide the possibility to reveal the regulatory mechanism of specific circRNA involved in PE. Inhibition of circRNA_05393 may induce the progression of PE by reducing the migration and invasion of trophoblasts.

P-319 Transcriptomic analysis reveals deregulated molecular mechanisms related to miscarriage, preeclampsia, and pregnancy complications during gestational endometrial phase in adenomyosis

Abstract Study question Is there any molecular deregulation in the gestational endometrium of women with adenomyosis responsible for adenomyosis-related pregnancy disorders? Summary answer Women with adenomyosis show deregulated molecular mechanisms in their gestational phase endometrium involved in miscarriage, preeclampsia, and pregnancy complications, causing adenomyosis-related infertility. What is known already Women with adenomyosis are characterized by having defective decidualization, altered endometrial receptivity, impaired embryo-maternal communication, implantation failure and higher risk of preeclampsia and miscarriage. However, molecular mechanisms underlying these infertility-related conditions remain unknown, mainly due to the inaccessibility of obtaining and maintaining these endometrial tissues. We have previously established adenomyosis endometrial organoids and differentiated them into gestational phase, demonstrating the maintenance of disease-specific traits. For this reason, here we use the already established adenomyosis organoid platform to describe the molecular mechanisms deregulated during gestational phase involved in adenomyosis-associated pregnancy disorders, which could be possible infertility therapeutical targets. Study design, size, duration Endometrial organoids from eutopic endometrium of adenomyosis and non-disease (control) women (n = 15/group) were established and differentiated into gestational phase (ADENO-GESTorg and CONTROL-GESTorg) by supplementation with ovarian and pregnancy hormones. An RNA-sequencing was performed. DESeq2 was used to identify differentially expressed genes (DEGs), FDR &amp;lt; 0.05. GO and KEGG analysis were performed to study DEGs implication at biologically functional level. QIAGEN Ingenuity Pathway Analysis (IPA) was used to further identify upregulated and downregulated pathways. Participants/materials, setting, methods Endometrial biopsies were obtained by hysteroscopy from adenomyosis and control patients (n = 15/group) at IVI Valencia Clinics. Patients between 18≤45 years old and BMI≤28 kg/m2 diagnosed with adenomyosis (adenomyosis group) or without adenomyosis nor other uterine pathologies (control group) were included in the study. Main results and the role of chance We identified 1999 DEGs (153 upregulated and 1846 downregulated) in ADENO- compared to CONTROL-GESTorg. Between upregulated genes, CXCL14 restricts trophoblast invasion and outgrowth, CYP24A1 is increased in spontaneous miscarriage and preeclamptic placentas, and PTAFR induces preterm delivery in mice (log2 Fold Change [log2FC] = 1.6, 2.4, 0.8). Among downregulated genes, deregulated PGR expression has been related to severe preeclampsia and recurrent pregnancy loss, whereas defects on ZWINT, ESCO2 and MCM6 expression are associated with high incidence of aneuploidy, leading to miscarriage, infertility, and newborn disorders (log2FC = −2.21, -2.57, -2.53, -2.48). Functional analysis showed deregulated functions as blastocyst growth, in utero embryonic development, developmental maturation and response to oxidative stress, standing out for their possible involvement in pregnancy disorders. IPA predicted a significant inhibition of D-myo-inositol metabolism (z score [z] = −0.7) affecting oocyte and embryo quality, VEGF signaling (z = −1.5) disturbing trophoblast adaptation to hypoxia, ERK/MAPK signaling (z = −4.0) causing embryonic lethality due to placental alterations; and significant activation of PPARα/RXRα (z = 0.5) that gives rise to recurrent miscarriage and preeclampsia, p53 signaling (z = 1.1) related with preeclampsia-complicated pregnancies, and RHOGDI and PTEN signaling (z = 3.6, 3.4) both promoting preeclampsia. Limitations, reasons for caution Although organoid platforms faithfully recapitulate tissue of origin characteristics as well as disease-specific traits, they are still in vitro models that need to be translated into in vivo studies to further corroborate the results obtained. Wider implications of the findings Molecular mechanisms involved in impaired embryo development, pregnancy loss, preeclampsia and placental defects are deregulated in gestational phase endometrium of women with adenomyosis. These deregulated mechanisms could be therapeutic target to develop pharmacological treatments to ameliorate adenomyosis-related infertility. Trial registration number not applicable

Investigation of CD56, ADAM17 and FGF21 Expressions in the Placentas of Preeclampsia Cases.

Objective: In the present study, we investigated the expression of CD56, ADAM17 and FGF21 antibodies (Ab), which we think have an effect on the pathophysiology of preeclampsia (PE), in pregnant patients with healthy placentas and placentas with PE. The expression of these antibodies has been investigated in a limited amount of former research, but their role in PE has not yet been clarified. With this study, we aimed to contribute to the elucidation of the pathophysiology of PE and the detection of new target molecules for treatment. Materials and Methods: Parturients with singleton pregnancy at 32 weeks or above without any maternal or fetal pathology who were admitted to the Department of Obstetrics and Gynecology, Zonguldak Bülent Ecevit University Practice and Research Hospital between 11 January 2020 and 7 January 2022 were included in the present study. Pregnant women with coexisting disease or a pathology related to the placenta (ablation placenta, vasa previa, hemangioma, etc.) were excluded. CD56, ADAM17 and FGF21 antibodies were histopathologically and immunohistochemically detected in 60 placentas with PE (study group) and 43 healthy placentas (control group). Results: CD56, ADAM17 and FGF21 proteins were all more intensely expressed in preeclamptic placentas and a statistically significant difference was found between the two groups for all three antibodies (p < 0.001). Deciduitis, perivillous fibrin deposition, intervillous fibrin, intervillous hemorrhage, infarct, calcification, laminar necrosis and syncytial node were found to be significantly more common in the study group (p < 0.001). Conclusions: We observed that CD56, ADAM17 and FGF21 expressions increased in preeclamptic placentas. These Ab may be responsible for the pathogenesis of PE, which can be illuminated with further studies.