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Background: Mucins are a family of proteins that protect the epithelium. A particular type of mucin, MUC15 is highly expressed in the placenta. This study aimed to characterise MUC15 in preeclampsia and investigate its role in placental stem cell biology., Methods: MUC15 mRNA and protein were measured in placentas from patients with early onset (<34 weeks' gestation) preeclampsia. Circulating serum MUC15 was measured via ELISA. MUC15 was localised in the placenta using in situ hybridisation. MUC15 mRNA expression was measured across differentiation of human trophoblast stem cells (hTSCs) to syncytiotrophoblast and extravillous trophoblasts. MUC15 was measured after syncytialised hTSCs were cultured in hypoxic (1% O 2 ) and proinflammatory (TNF α, IL-6) conditions. MUC15 secretion was assessed when syncytialised hTSCs were treated with brefeldin A (impairs protein trafficking) and batimastat (inhibits matrix metalloproteinases)., Results: MUC15 protein was significantly increased in the placenta (P = 0.0003, n = 32 vs n = 20 controls) and serum (P = 0.016, n = 32 vs n = 22 controls) of patients with preeclampsia, whilst MUC15 mRNA remained unchanged (n = 61 vs n = 18 controls). MUC15 mRNA (P = 0.005) and protein secretion (P = 0.006) increased following differentiation to syncytiotrophoblast cells. In situ hybridisation confirmed MUC15 localised to the syncytiotrophoblast cell within the placenta. Neither hypoxic or inflammatory conditions changed MUC15 mRNA expression or secretion. Brefeldin A treated hTSCs did not alter MUC15 secretion, whilst batimastat reduced MUC15 secretion (P = 0.044)., Conclusions: MUC15 is increased in early onset preeclampsia and is cleaved by matrix metalloproteinases. Increased MUC15 may reflect a protective mechanism associated with placental dysfunction. Further research will aid in confirming this., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare for this manuscript., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Preeclampsia 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., Methods: PEG10 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% O 2 ) 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-[Formula: see text] signalling and proliferation using luciferase and xCELLigence assays, respectively., Results: PEG10 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 [Formula: see text] (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., Conclusions: Placental 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-[Formula: see text] signalling, and thus may be involved in trophoblast dysfunction associated with this pathway., (© 2023. The Author(s).)

As the placenta develops across gestation, the mitochondria and other organelles like the endoplasmic reticulum (ER) must continue to adapt to stressors such as oxidative stress. As pregnancy approaches term, these stressors may contribute to placental aging, including mitochondrial changes leading to cellular senescence. When these processes are exacerbated, pregnancy pathologies arise. This study aimed to identify correlations between genes related to mitochondria, ER and cellular senescence in placentae complicated by pregnancy complications. Placental samples from pregnancies classified as preterm, term, post-term, preterm with foetal growth restriction (FGR), preterm with preeclampsia (PE) and preterm with PE and FGR were used to measure gene expression of TOMM20, MFN1, TFAM, MFN2, PARK2, PINK1, EIF2AK3, TP53 and ERN1. MetaboAnalyst 5.0 was used to generate heatmaps, principal component analysis plots, correlation graphs and receiver operating characteristic analysis. This study found that genes-related mitochondrial dynamics and aging undergo changes in placentae affected by pregnancy pathologies. The TOMM20/PARK2 ratio may be a promising marker to discriminate between healthy and unhealthy placental tissue. Future studies should explore circulating biomarkers of mitochondrial aging and dysfunction as indicators of placental health., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Activin A is aberrantly expressed by the preeclamptic placenta and circulating levels have been investigated as a potential biomarker for the disease. In a nested case-control study we measured Activin A levels in maternal plasma at 28- and 36-weeks' gestation preceding term preeclampsia diagnosis. At 28 weeks Activin A was not significantly altered (n = 73 destined to develop preeclampsia vs n = 191 controls). At 36 weeks' gestation Activin A was significantly increased in 40 women destined to develop preeclampsia relative to 201 controls (p < 0.0001). These findings provide further validation of Activin A as a potential biomarker for subsequent term preeclampsia., (Copyright © 2021 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) transcripts are elevated in the circulation of individuals whose pregnancies are complicated by preterm fetal growth restriction (FGR). In this paper, we show that the cases with preeclampsia (PE) have increased circulating NR4A2 transcripts compared to those with normotensive FGR. We aimed to establish whether the dysfunctional placenta mirrors the increase in NR4A2 transcripts and further, to uncover the function of placental NR4A2. NR4A2 expression was detected in preterm and term placental tissue; expressed higher at term. NR4A2 mRNA expression and protein were not altered in placentas from preterm FGR or PE pregnancies. Hypoxia (1% O 2 compared to 8% O 2 ) significantly reduced cytotrophoblast NR4A2 mRNA expression, but not placental explant NR4A2 expression. Silencing cytotrophoblast NR4A2 expression under hypoxia (via short interfering (si)RNAs) did not alter angiogenic Placental Growth Factor, nor anti-angiogenic sFlt-1 mRNA expression or protein secretion, but increased expression of cellular antioxidant, oxidative stress, inflammatory, and growth genes. NR4A2 expression was also not altered in a model of tumour necrosis factor-α-induced endothelial dysfunction, or with pravastatin treatment. Further studies are required to identify the origin of the circulating transcripts in pathological pregnancies, and investigate the function of placental NR4A2., (© 2021. The Author(s).)

Introduction: Preeclampsia is associated with reduced pro-angiogenic Placental Growth Factor (PlGF) and increased levels of anti-angiogenic soluble FMS like tyrosine kinase-1 (sFlt-1). We have previously shown that sFlt-1 secretion is positively regulated via the Epidermal Growth Factor Receptor (EGFR) and mitochondrial respiration pathways. We assessed whether these pathways also regulate endothelial and placental secretion of PlGF., Methods: Primary cytotrophoblast cells and primary human umbilical vein endothelial cells (HUVECs) were treated with EGFR inhibitor gefitinib, or small molecules that inhibit down-stream pathways of the receptor: U0126, PD98059 (ERK/MEK pathway inhibitors), ZM336372 (JAK/STAT inhibitor) or AG490 (JAK inhibitor). We inhibited mitochondrial respiration in primary cytotrophoblasts using mitochondrial complex inhibitors rotenone (complex I), antimycin (complex III) or oligomycin (complex IV). We then measured PlGF secretion in the condition media., Results: Three inhibitors of the EGFR pathway significantly increased PlGF secretion: gefitinib (p = 0.03), AG490 (p < 0.0001) and U0126 (p = 0.03) in primary cytotrophoblasts, while PD98059 reduced PlGF secretion (p = 0.002). In the same cells, neither gefitinib or UO126 altered PlGF mRNA expression, but AG490 significantly increased its expression (p = 0.02). Primary endothelial cell PlGF secretion was significantly reduced when treated with PD98059 and U0126 while ZM336372 had no effect. Rotenone significantly reduced cytotrophoblast PlGF secretion (p = 0.0005). Neither antimycin (p = 0.9) or oligomycin (p = 0.9) had an effect., Discussion: We have shown that PlGF secretion from primary cytotrophoblast and HUVECs is altered by inhibiting EGFR signaling and potentially mitochondrial respiration, coincident with reduced sFlt-1 secretion. This suggests that common pathways are regulating both pro and anti-angiogenic molecules that are changed in association with preeclampsia and provides insight into the pathogenesis of this serious disease., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: The human placenta has a defined lifespan and placental aging is a key feature as pregnancy progresses. Placental aging and mitochondrial dysfunction are known to play a key role in pregnancy pathophysiology. Premature aging of the placenta has also been linked with placental dysfunction resulting in poor fetal development and premature birth., Methods: The expression of key mitochondrial-related genes were analysed in a series of publicly available databases then expression changes were validated in placental samples collected from term, pre-term, post-term pregnancies and pregnancies complicated by fetal growth restriction (FGR). Gene and protein expression levels of MFN1, MFN2, TFAM, TOMM20, OPA3 and SIRT4 were measured in placental tissues via qPCR and western blotting., Results: Initial analysis found that key mitochondrial transcripts related to biogenesis, bioenergetics and mitophagy clustered by pregnancy trimester. A refined list of 13 mitochondrial-related genes were investigated in additional external datasets of pregnancy complications. In the new cohort, protein expression of MFN1 was decreased in FGR and MFN2 is decreased in post-term placenta. Analysis of placental tissues revealed that TOMM20 gene and protein expression was altered in FGR and post-term placenta., Discussion: MFN1 and MFN2 play a major role in mitochondrial dynamics, and alterations in these markers have been highlighted in early unexplained miscarriage. TOMM20 is an importer protein that plays a major role in mitophagy and changes have also been identified in age-related diseases. Significant changes in MFN1, MFN2 and TOMM20 indicate that mitochondrial regulators play a critical role in placental aging and placental pathophysiology., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Background We investigated the biomarker potential of growth differentiation factor 15 (GDF-15), a stress response protein highly expressed in placenta, to predict preeclampsia. Methods and Results In 2 prospective cohorts (cohort 1: 960 controls, 39 women who developed preeclampsia; cohort 2: 950 controls, 41 developed preeclampsia), plasma concentrations of GDF-15 at 36 weeks' gestation were significantly increased among those who developed preeclampsia ( P <0.001), area under the receiver operating characteristic curves (AUC) of 0.66 and 0.71, respectively. In cohort 2 a ratio of sFlt-1/PlGF (a clinical biomarker for preeclampsia) had a sensitivity of 61.0% at 83.2% specificity to predict those who will develop preeclampsia (AUC of 0.79). A ratio of GDF-15×sFlt-1/PlGF yielded a sensitivity of 68.3% at 83.2% specificity (AUC of 0.82). GDF-15 was consistently elevated across a number of international cohorts: levels were higher in placenta and blood from women delivering <34 weeks' gestation due to preterm preeclampsia in Melbourne, Australia; and in the blood at 26 to 32 weeks' gestation among 57 women attending the Manchester Antenatal Vascular Service (MAViS, UK) who developed preeclampsia ( P =0.0002), compared with 176 controls. In the Preeclampsia Obstetric adVerse Events biobank (PROVE, South Africa), plasma GDF-15 was significantly increased in women with preeclampsia with severe features ( P =0.02; n=14) compared to controls (n=14). Conclusions We conclude circulating GDF-15 is elevated among women more likely to develop preeclampsia or diagnosed with the condition. It may have value as a clinical biomarker, including the potential to improve the sensitivity of sFlt-1/PlGF ratio.

Fetal growth restriction is a leading cause of stillbirth that often remains undetected during pregnancy. Identifying novel biomarkers may improve detection of pregnancies at risk. This study aimed to assess syndecan-1 as a biomarker for small for gestational age (SGA) or fetal growth restricted (FGR) pregnancies and determine its molecular regulation. Circulating maternal syndecan-1 was measured in several cohorts; a large prospective cohort collected around 36 weeks' gestation (n = 1206), a case control study from the Manchester Antenatal Vascular service (285 women sampled at 24-34 weeks' gestation); two prospective cohorts collected on the day of delivery (36 + 3-41 + 3 weeks' gestation, n = 562 and n = 405 respectively) and a cohort who delivered for preterm FGR (< 34 weeks). Circulating syndecan-1 was consistently reduced in women destined to deliver growth restricted infants and those delivering for preterm disease. Syndecan-1 secretion was reduced by hypoxia, and its loss impaired proliferation. Matrix metalloproteinases and mitochondrial electron transport chain inhibitors significantly reduced syndecan-1 secretion, an effect that was rescued by coadministration of succinate, a mitochondrial electron transport chain activator. In conclusion, circulating syndecan-1 is reduced among cases of term and preterm growth restriction and has potential for inclusion in multi-marker algorithms to improve detection of poorly grown fetuses., (© 2021. The Author(s).)

Objectives: The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is upregulated in the maternal vasculature in preeclampsia, and contributes to oxidative stress and endothelial dysfunction. However, its function in the placenta is unclear. This paper investigated LOX-1 expression in models of placental dysfunction and preeclampsia, and whether candidate therapeutics for preeclampsia could alter its expression., Study Design: Placentas were collected from preterm pregnancies and cases of preterm preeclampsia and fetal growth restriction. Blood was collected from participants whose pregnancies were complicated by preterm fetal growth restriction and/or preeclampsia. Primary cytotrophoblast and placental explant tissue were cultured under hypoxic (1% O 2 ) or normoxic (8% O 2 ) conditions. Cytotrophoblast were exposed to 10% preeclamptic or control serum. Cytotrophoblast and preeclamptic explant tissue were treated with 100 µM esomeprazole, lansoprazole or rabeprazole., Main Outcome Measures: LOX-1 expression was assessed in all samples via qPCR., Results: LOX-1 expression was reduced in placentas from cases of preterm preeclampsia, but not fetal growth restriction, compared to controls. LOX-1 expression was reduced in cytotrophoblast under hypoxia, but not in explant tissue. Treatment with preeclamptic serum in vitro did not alter cytotrophoblast LOX-1 expression. Circulating LOX-1 mRNA was unaltered in patients with fetal growth restriction, preeclampsia, and fetal hypoxia, compared to controls. Treatment with esomeprazole or lansoprazole in vitro increased placental LOX-1 expression., Conclusions: LOX-1 expression is reduced in preeclamptic placentas and hypoxic cytotrophoblast. Esomeprazole and lansoprazole increase placental LOX-1 expression. These findings demonstrate a role for LOX-1 in the placenta, and improve our understanding of maternal adaptations in pregnancy complications., (Copyright © 2021 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Biomarkers for placental dysfunction are currently lacking. We recently identified SPINT1 as a novel biomarker; SPINT2 is a functionally related placental protease inhibitor. This study aimed to characterise SPINT2 expression in placental insufficiency. Circulating SPINT2 was assessed in three prospective cohorts, collected at the following: (1) term delivery ( n = 227), (2) 36 weeks ( n = 364), and (3) 24-34 weeks' ( n = 294) gestation. SPINT2 was also measured in the plasma and placentas of women with established placental disease at preterm (<34 weeks) delivery. Using first-trimester human trophoblast stem cells, SPINT2 expression was assessed in hypoxia/normoxia (1% vs. 8% O2), and following inflammatory cytokine treatment (TNFα, IL-6). Placental SPINT2 mRNA was measured in a rat model of late-gestational foetal growth restriction. At 36 weeks, circulating SPINT2 was elevated in patients who later developed preeclampsia ( p = 0.028; median = 2233 pg/mL vs. controls, median = 1644 pg/mL), or delivered a small-for-gestational-age infant ( p = 0.002; median = 2109 pg/mL vs. controls, median = 1614 pg/mL). SPINT2 was elevated in the placentas of patients who required delivery for preterm preeclampsia ( p = 0.025). Though inflammatory cytokines had no effect, hypoxia increased SPINT2 in cytotrophoblast stem cells, and its expression was elevated in the placental labyrinth of growth-restricted rats. These findings suggest elevated SPINT2 is associated with placental insufficiency.

Previously, we identified increased maternal circulating DAAM2 mRNA in pregnancies complicated by preterm fetal growth restriction (FGR). Here, we assessed whether circulating DAAM2 mRNA could detect FGR, and whether the DAAM2 gene, known to play roles in the Wnt signalling pathway is expressed in human placenta and associated with dysfunction and FGR. We performed linear regression analysis to calculate area under the ROC curve (AUC) for DAAM2 mRNA expression in the maternal circulation of pregnancies complicated by preterm FGR. DAAM2 mRNA expression was assessed across gestation by qPCR. DAAM2 protein and mRNA expression was assessed in preterm FGR placenta using western blot and qPCR. DAAM2 expression was assessed in term cytotrophoblasts and placental explant tissue cultured under hypoxic and normoxic conditions by qPCR. Small interfering RNAs were used to silence DAAM2 in term primary cytotrophoblasts. Expression of growth, apoptosis and oxidative stress genes were assessed by qPCR. Circulating DAAM2 mRNA was elevated in pregnancies complicated by preterm FGR [p < 0.0001, AUC = 0.83 (0.78-0.89)]. Placental DAAM2 mRNA was detectable across gestation, with highest expression at term. DAAM2 protein was increased in preterm FGR placentas but demonstrated no change in mRNA expression. DAAM2 mRNA expression was increased in cytotrophoblasts and placental explants under hypoxia. Silencing DAAM2 under hypoxia decreased expression of pro-survival gene, BCL2 and oxidative stress marker, NOX4, whilst increasing expression of antioxidant enzyme, HMOX-1. The increased DAAM2 associated with FGR and hypoxia implicates a potential role in placental dysfunction. Decreasing DAAM2 may have cytoprotective effects, but further research is required to elucidate its role in healthy and dysfunctional placentas.

Introduction: Tissue Factor Pathway Inhibitor (TFPI) is a part of the extrinsic coagulation pathway, and highly expressed in the placenta. We aimed to assess its potential as a preeclampsia biomarker., Methods: Maternal plasma was prospectively collected at 36 weeks' gestation. Circulating TFPI was measured in a nested case-control group (39 women who developed preeclampsia, 98 controls), before being measured in a larger independent cohort along with Placental Growth Factor (PlGF; 41 who developed preeclampsia, 954 controls). Circulating TFPI was then measured in women with underlying vascular disease, and also assessed in the plasma and placentas from women with preterm preeclampsia (delivered at <34 weeks)., Results: Circulating TFPI was significantly increased in women destined to develop preeclampsia in the case-control study, a finding that validated in Cohort 2, with median TFPI in the preeclampsia group being 42.3 ng/ml (IQR 30-51 ng/ml) compared to 30 ng/ml (IQR 23.1-38.6 ng/ml) in controls (p < 0.0001). The area under the receiver operator characteristic curve (AUC) was 0.70. PlGF was significantly reduced in the preeclampsia group, and a ratio of TFPI/PlGF had an improved AUC of 0.78. In women with underlying vascular disease who were later diagnosed with early onset preeclampsia, circulating TFPI was significantly increased with a 0.29 (95% CI 0.13-0.44) increase in logTFPI (adjusted for gestation and hypertensive status). Circulating and placental TFPI were significantly increased in women with preterm preeclampsia., Discussion: Circulating TFPI is increased in women preceding diagnosis of preeclampsia (at 36 weeks) and in women with preterm disease. TFPI may beneficially contribute to a multi-marker blood test to predict preeclampsia., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: The antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG) are elevated in preeclampsia and have been implicated in its pathogenesis. We have previously demonstrated metformin and sulfasalazine independently reduce antiangiogenic factor secretion. Here we examined whether combining metformin and sulfasalazine may be more effective than either alone in reducing placental expression and secretion of antiangiogenic and angiogenic factors and the expression of markers of endothelial dysfunction., Methods: We performed functional experiments using primary human placenta to explore the effect of metformin and sulfasalazine, at lower doses than previously explored, individually and in combination, on sFlt-1 and sENG secretion and placental growth factor (PlGF) and vascular endothelial growth factor (VEGFα) expression. Using primary endothelial cells we induced dysfunction using cytokine tumor necrosis factor-α (TNF-α) and assessed the effect of low dose combination treatment on the expression of vascular cell adhesion molecule-1 (VCAM-1) and Endothelin-1 (a potent vasoconstrictor)., Results: We demonstrated combination metformin and sulfasalazine was additive in reducing sFlt-1 secretion from cytotrophoblasts and placental explants. Combination treatment was also additive in reducing sENG secretion from placental explants. Furthermore, combination treatment increased cytotrophoblast VEGFα mRNA expression. Whilst combination treatment increased PlGF mRNA expression this was similar to treatment with sulfasalazine alone. Combination therapy reduced TNFα induced endothelin-1 mRNA expression however did not change VCAM expression., Discussion: Low dose combination metformin and sulfasalazine reduced cytotrophoblast sFlt-1 and sENG secretion, increased VEGFα expression and reduced TNFα induced endothelin-1 expression in primary endothelial cells. Combination therapy has potential to treat preeclampsia., Competing Interests: Declaration of competing interest The authors report no conflict of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Introduction: Oxidative damage and biochemical ageing are implicated in placental dysfunction and potentially fetal death. Cellular senescence may play a role in the pathophysiology of fetal growth restriction (FGR) and preeclampsia (PE). Aurora kinases (AURKA, B and C) are important regulators of cellular division in mitosis and meiosis with implications in cellular senescence. We aimed to investigate whether aurora kinase expression is altered with placental dysfunction or placental ageing., Methods: Placenta and blood was obtained across gestation from pregnancies complicated by PE, FGR or both PE and FGR, as well as gestation-matched control samples. Expression of AURKA, B and C mRNA was examined using real time qPCR in both the placenta and maternal circulation., Results: Placental aurora kinase expression decreased as gestation progressed: AURKA and AURKB were significantly reduced at 37-40 weeks, whereas AURKC was significantly reduced at 34-37 weeks, when compared to <34 weeks. In the maternal circulation, the mRNA level of AURKB was significantly reduced at >40 weeks compared to <34 weeks gestation. A significant reduction in AURKC was seen in FGR pregnancies <34 weeks compared to gestation-matched controls., Conclusion: Placental AURK expression is reduced with increased gestation. Circulating AURKB mRNA reduces at >40 weeks gestation, when compared to <34 weeks. AURKC is significantly reduced in placentas from pregnancies complicated by severe early onset (<34 weeks) FGR compared with gestation-matched controls. The functional role of aurora kinase in the placenta and in gestational age warrants further investigation., (Copyright © 2020. Published by Elsevier Ltd.)

Placental insufficiency can cause fetal growth restriction and stillbirth. There are no reliable screening tests for placental insufficiency, especially near-term gestation when the risk of stillbirth rises. Here we show a strong association between low circulating plasma serine peptidase inhibitor Kunitz type-1 (SPINT1) concentrations at 36 weeks' gestation and low birthweight, an indicator of placental insufficiency. We generate a 4-tier risk model based on SPINT1 concentrations, where the highest risk tier has approximately a 2-5 fold risk of birthing neonates with birthweights under the 3 rd , 5 th , 10 th and 20 th centiles, whereas the lowest risk tier has a 0-0.3 fold risk. Low SPINT1 is associated with antenatal ultrasound and neonatal anthropomorphic indicators of placental insufficiency. We validate the association between low circulating SPINT1 and placental insufficiency in two other cohorts. Low circulating SPINT1 is a marker of placental insufficiency and may identify pregnancies with an elevated risk of stillbirth.

Introduction: Recently, Delta-like homolog 1 (DLK1) was identified as a potential marker of small-for-gestational-age (SGA; <10th centile) fetuses; mouse studies suggest reduced levels may represent a fetal stress signal. We sought to measure DLK1 in a large independent cohort of maternal blood samples, correlate levels with measures of placental insufficiency and assess whether DLK1 might be placental derived., Methods: The Fetal Longitudinal Assessment of Growth (FLAG) study was a prospective blood collection from 2000 women. We assessed a case-control cohort at 28 and 36 weeks from the first 1000 FLAG women, before validating changes in the entire second 1000. A subgroup of FLAG participants underwent ultrasound examinations, and 137 neonates, body composition assessment (PEAPOD). DLK1 secretion was assessed from human placentas ex vivo., Results: Circulating DLK1 was significantly reduced at 28 and 36 weeks' gestation in women destined to deliver a SGA fetus and associated with birthweight centile (n = 999, p < 0.0001), and placental weight (n = 96, p = 0.0064). Ex vivo, DLK1 was abundantly released from human placenta and significantly reduced under hypoxia (n = 7, p < 0.05). We found no relationship between circulating DLK1 and estimated fetal weight, cerebroplacental ratio, uterine artery or umbilical artery pulsatility index. Nor was there a relationship between DLK1 and neonatal fat or lean mass (n = 137)., Conclusion: We confirmed circulating DLK1 is reduced at both 28 and 36 weeks' gestation preceding delivery of a SGA infant, shown that it is not significantly associated with clinical measures of placental insufficiency, and provide new data demonstrating it may be placenta-derived in humans., Competing Interests: Declaration of competing interest TMM, SPW, ST and TKL are listed as inventors for molecules to identify placental insufficiency unrelated to DLK1., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: Death associated protein kinase-1 (DAPK-1) is highly expressed in the placenta relative to all other human tissues. We examine whether it is differentially expressed with preeclampsia., Methods: We examined samples from a large prospective collection of plasma from 2002 women. We split the samples into two cohorts: Cohort 1 (n = 1000) and Cohort 2 (n = 1002). We first measured circulating DAPK-1 at 36 weeks' gestation in a nested case-control group (from Cohort 1) of 39 women who developed preeclampsia and 98 controls. We then validated our findings by measuring circulating levels in all samples from both cohorts. We also measured DAPK-1 in the circulation and placentas of women who were diagnosed with preterm preeclampsia or delivered a growth restricted infant at <34 weeks' gestation., Results: In the case-control study, circulating DAPK-1 was significantly increased in women destined to develop preeclampsia (p < 0.01). We validated this by measuring circulating levels in Cohorts 1 and 2. Again, circulating DAPK-1 was significantly higher (p < 0.001) among women destined to develop preeclampsia (Cohort 1, Area under the receiver operator characteristic curve (AUC) = 0.66; Cohort 2 AUC = 0.67). Circulating DAPK-1 was also significantly elevated in women with established preterm preeclampsia. Placental DAPK-1 mRNA and protein expression were elevated in women with established preeclampsia., Discussion: DAPK-1 is a novel placenta-enriched molecule that is elevated in the circulation of women preceding the diagnosis of preeclampsia and is likely to be secreted from the placenta., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: Preeclampsia is a hypertensive disorder of pregnancy with no available medical treatment. We recently reported sulfasalazine, an anti-inflammatory medication, to be a candidate therapeutic for preeclampsia. We showed sulfasalazine decreases placental secretion of soluble Fms-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic factor strongly implicated in the pathogenesis of preeclampsia. However, the cellular mechanism(s) by which sulfasalazine reduces placental sFlt-1 are yet to be determined. Recently we also reported that both the mitochondria and the epidermal growth factor receptor (EGFR) signalling pathways regulate secretion of placental sFlt-1. In this study we sought to assess directly whether sulfasalazine's capacity to reduce sFlt-1 secretion may be mediated via EGFR or the mitochondria., Methods and Results: Using primary cytotrophoblast cells, we confirmed sulfasalazine reduced sFlt-1 secretion. Interestingly, when we measured the mRNA expression of EGFR, we found a reduction in EGFR expression which closely mirrored the changes in sFlt-1 secretion. At the protein level, sulfasalazine significantly reduced phosphorylated and active EGFR (phosphorylated/total) expression. Additionally, sulfasalazine significantly reduced the protein expression of ERK1/2 and STAT3 which are key adaptor molecules downstream of EGFR. Next, we assessed mitochondrial respiration following sulfasalazine treatment and found no effect on basal respiration, ATP production, proton leak or maximal respiration., Conclusion: Sulfasalazine reduces EGFR and down-stream signalling molecule expression coincident with reduced sFlt-1 secretion. EGFR signalling is a potential mechanism by which sulfasalazine decreases placental secretion of sFlt-1. Further interrogation of the EGFR may identify new candidate treatments for preeclampsia., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Preeclampsia is a hypertensive disorder of pregnancy characterized by maternal endothelial dysfunction and end-organ damage. The antiangiogenic factor, sFlt-1 (soluble FMS-like tyrosine kinase-1) has been strongly implicated in the pathogenesis of preeclampsia. sFlt-1 is secreted into the maternal circulation where it antagonizes VEGF (vascular endothelial growth factor) and ultimately disrupts vascular homeostasis. However, the upstream mechanisms regulating release of sFlt-1 are poorly characterized. We investigated the roles of key prosurvival pathways, EGFR (epidermal growth factor receptor) signaling, and the mitochondria, in regulating sFlt-1 production. We initially found that the mRNA and protein of EGFR and downstream adaptor molecules were significantly increased in preeclamptic placental tissue relative to normotensive controls. Inhibiting the EGFR signaling cascade using siRNA (small interfering ribonucleic acid) or small molecule inhibitors significantly reduced sFlt-1 release from primary cytotrophoblast (placental cells). Additionally, inhibiting the mitochondrial electron transport chain or activating downstream energy-sensing molecules (AMPK [AMP-activated kinase], SIRT1 [sirtuin 1 ], and PGC1α [PPAR-γ co-activator 1]) also significantly reduced sFlt-1 secretion from cytotrophoblast cells, without affecting EGFR signaling. In vivo, treating pregnant mice with gefitinib (an EGFR inhibitor) or resveratrol (perturbs mitochondrial function) significantly reduced circulating murine sFlt-1 compared with vehicle control. Furthermore, treating primary cytotrophoblasts with therapeutics which have been previously found to reduce sFlt-1 secretion, either reduced EGFR signaling (esomeprazole and statins) or perturbed mitochondrial function (esomeprazole, resveratrol, and metformin). Additionally, targeting both pathways simultaneously produced additive reductions in sFlt-1 secretion. Thus, we have identified 2 key survival pathways that seem to be overactive in preeclampsia and involved in the regulation of placental sFlt-1 secretion.

Preeclampsia is associated with intermittent placental hypoxia, inflammation and the release of antiangiogenic factors, namely sFLT-1 and sEng. These factors cause maternal endothelial dysfunction and the manifestation of clinical disease. Disulfiram is a dehydrogenase inhibitor used to treat alcoholism and has been suggested as a proteasome inhibitor. Inhibiting the proteasome has been previously shown to reduce FLT-1 gene expression. Thus, we aim to investigate whether disulfiram alters the secretion of sFLT-1 and sEng and reduces endothelial dysfunction. METHODS AND RESULTS: We assessed the effects of disulfiram on primary cytotrophoblast and human umbilical vein endothelial cells (HUVECs). Disulfiram significantly reduced mRNA expression of membrane bound FLT-1 and sFLT-1 variants in primary cytotrophoblasts, which translated into a significant reduction in the protein secretion of sFLT-1. Additionally, sFLT-1 was reduced in primary HUVECs treated with disulfiram, whilst sEng was only reduced in primary cytotrophoblasts. Next, we investigated the effect of disulfiram on endothelial dysfunction using primary HUVECs treated with 5% preeclamptic serum ± disulfiram. Serum from preeclamptic women induced endothelial dysfunction evidenced by increased mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs. The addition of disulfiram reduced VCAM-1 mRNA expression, however did not affect the adhesion of PBMCs to endothelial cells. Lastly, we assessed the effects of disulfiram on the 20S subunit of the proteasome and found disulfiram did not inhibit this subunit in either primary cytotrophoblast or HUVECs. CONCLUSIONS: Disulfiram quenches sFLT-1 and sEng via mechanisms independent of the 20S subunit of the proteasome. Understanding of the mechanisms by which disulfiram inhibits antiangiogenic secretion may reveal insights into the pathogenesis and potential therapeutic targets for preeclampsia., (Copyright © 2018 International Society for the Study of Hypertension in Pregnancy. All rights reserved.)

The genetic deletion of apelin receptor early endogenous ligand (Elabela; official name APELA) produces a preeclampsia-like phenotype in mice. However, evidence linking ELABELA with human disease is lacking. Therefore, we measured placental mRNA and circulating ELABELA in human samples. ELABELA mRNA (measured by RNA sequencing) was unchanged in 82 preeclamptic placentas compared with 82 matched controls (mean difference, 0.53%; 95% CI, -25.9 to 27.0; P = 0.78). We measured circulating ELABELA in 32 women with preterm preeclampsia (delivered at <34 weeks' gestation) and 32 matched controls sampled at the same gestational age. There was no difference in circulating ELABELA concentrations in the preeclamptic cohort compared with controls (median, 28.5 pg/mL; 95% CI, 5.3 to 63.2 versus median, 20.5 pg/mL; 95% CI, 9.2 to 58.0, respectively); the median difference was 8.0 pg/mL (95% CI, -17.7 to 12.1; P = 0.43). In contrast, soluble FLT1 (a protein with an established association with preeclampsia) mRNA was increased in placental tissue (mean difference, 34.9%; 95% CI, 16.6 to 53.1; P = 0.001), and circulating concentrations were 16.8-fold higher among the preeclamptic cohort (P < 0.0001). In conclusion, we were able to recapitulate the association between circulating soluble FLT1 and preeclampsia, but there was no association with ELABELA. The speculated clinical relevance of observations in the murine model linking ELABELA to preeclampsia likely are incorrect., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Preeclampsia is one of the most serious complications of pregnancy. Currently there are no medical treatments. Given placental oxidative stress may be an early trigger in the pathogenesis of preeclampsia, therapies that enhance antioxidant pathways have been proposed as treatments. Melatonin is a direct free-radical scavenger and indirect antioxidant. We performed in vitro assays to assess whether melatonin 1) enhances the antioxidant response element genes (heme-oxygenase 1, (HO-1), glutamate-cysteine ligase (GCLC), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), thioredoxin (TXN)) or 2) alters secretion of the anti-angiogenic factors soluble fms-like tyrosine kinase-1 (sFLT) or soluble endoglin (sENG) from human primary trophoblasts, placental explants and human umbilical vein endothelial cells (HUVECs) and 3) can rescue TNF-α induced endothelial dysfunction. In primary trophoblast melatonin treatment increased expression of the antioxidant enzyme TXN. Expression of TXN, GCLC and NQO1 was upregulated in placental tissue with melatonin treatment. HUVECs treated with melatonin showed an increase in both TXN and GCLC. Melatonin did not increase HO-1 expression in any of the tissues examined. Melatonin reduced sFLT secretion from primary trophoblasts, but had no effect on sFLT or sENG secretion from placental explants or HUVECs. Melatonin did not rescue TNF-α induced VCAM-1 and ET-1 expression in endothelial cells. Our findings suggest that melatonin induces antioxidant pathways in placenta and endothelial cells. Furthermore, it may have effects in reducing sFLT secretion from trophoblast, but does not reduce endothelial dysfunction. Given it is likely to be safe in pregnancy, it may have potential as a therapeutic agent to treat or prevent preeclampsia.

Ectopic pregnancies complicate 1-2 pregnancies and are a leading cause of maternal death. An effective oral drug therapy that replaces surgery might make its treatment safer, cheaper, simpler and therefore more widely accessible. The only current medical treatment offered to women is intramuscular methotrexate, but this only reliably resolves smaller ectopic pregnancies. As such, many ectopic pregnancies require surgical excision. We show that vinorelbine, an orally available chemotherapeutic agent, potently induced placental cell death but did not harm fertility in mice. Vinorelbine was 100-1000 times more potent than methotrexate in inducing placental cell death in vitro, and more potent than combination methotrexate and gefitinib (another proposed treatment for ectopic pregnancy being evaluated in phase III trials). Mechanistically, it caused microtubule condensation, blocked mitosis and activated the apoptosis cascade in placental cells. Vinorelbine was more efficacious than methotrexate±gefitinib in reducing the volume of placental cell tumors xenografted subcutaneously in SCID mice. Mice exposed to vinorelbine and allowed to breed, following a four week washout period, displayed normal fertility, however long-term fertility was not assessed. Human Fallopian tubes treated with vinorelbine did not exhibit up-regulation of apoptosis molecules. Our findings show that placental cells appear sensitive to vinorelbine and it has potential as a tablet-only approach to treat ectopic pregnancy., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Preeclampsia is a major pregnancy complication associated with poor placental perfusion and placental hypoxia. Systemic and placental inflammation and elevated placental secretion of the antiangiogenic factors sFlt-1 (soluble fms-like tyrosine kinase 1) and sEng (soluble endoglin) are hallmarks of preeclampsia, causing endothelial dysfunction and multiorgan injury. A molecule that links placental hypoxia, inflammation, and antiangiogenic factor release has not been described. ATF3 (activating transcription factor 3) is highly expressed in placenta. We assessed whether placental ATF3 is dysregulated in preterm preeclampsia, is altered by hypoxia, and regulates proinflammatory cytokine and antiangiogenic factor production. ATF3 mRNA and protein expression was significantly reduced in preterm preeclamptic placentas compared with gestation-matched controls. Hypoxia reduced ATF3 expression in primary cytotrophoblast and placental explants. Silencing ATF3 in primary cytotrophoblast increased proinflammatory cytokine (IL-6 [interleukin 6], TNF-α [tumor necrosis factor α]) and NF-κB (nuclear factor κB) expression. In silico analysis identified an ATF3-binding site in the promoter of Flt-1 (the transcript from which sFlt-1 is produced). Silencing ATF3 increased sFlt-1 and sEng secretion from primary cytotrophoblast possibly by increasing Rab11a and Arf1, cargo proteins that facilitate exosomal release of sFlt-1. ATF3 knockout mice did not have a preeclampsia phenotype, suggesting that these pathways may be specific to humans (preeclampsia is a uniquely human condition). To conclude, we have shown that ATF3 is decreased in preeclamptic placentas and that this decrease is likely to occur after prolonged hypoxia. We show that ATF3 is a regulator of placental proinflammatory cytokines and antiangiogenic factors sFlt-1 and sEng. Therefore, reduced ATF3 may be centrally involved in the pathology of preeclampsia., (© 2017 American Heart Association, Inc.)

Background: It is not known whether fasting affects levels of circulating placenta-specific transcripts., Objective: To assess whether a glucose load affects circulating placenta-specific transcripts., Method: RNA was extracted from paired blood samples (fasting and 1-h post 75 g oral glucose) from 22 women. Placenta-specific genes were measured by RT-qPCR., Results: There was no change in ADM, CSH1, PAPPA2, PSG1 or TAC3 expression between fasting and post-glucose states. However, HTRA1 decreased after glucose load., Conclusion: Maternal fasting state does not influence expression of the majority of placenta-specific genes but may need to be accounted for when validating biomarkers of placental disease.

Objective: sFLT-1 e15a is a recently described sFlt-1 variant that is placental and primate specific. As such, it may have potential as a biomarker. Using a newly developed ELISA, we measured maternal plasma sFLT-1 e15a levels in women with fetal growth restriction and pre-eclampsia., Method: We performed a nested case-control study where we measured total sFLT-1 and sFLT-1 e15a plasma protein concentrations. Samples, selected from a prospective cohort study, consisted of 87 healthy controls, 11 cases that developed term preeclampsia and 20 cases where there was fetal growth restriction. We also measured sFLT-1 and sFLT-1 e15a plasma concentrations in a separate cohort: 15 cases of preterm preeclampsia and 24 healthy controls., Results: The prospective case-control cohort demonstrated significantly increased sFLT-1 e15a among cases with term fetal growth restriction (p < 0.05). We also observed that total sFLT-1 (this ELISA indiscriminately detects all variants) was significantly increased in term preeclampsia (p < 0.0001), but not fetal growth restriction. The separate cohort of early-onset preeclamptics showed significantly increased sFLT-1 e15a levels (p < 0.0001)., Conclusion: Plasma sFLT-1 e15a is significantly increased in early-onset preeclampsia and term fetal growth restriction. Further assessment of the benefit for sFLT-1 e15a testing in prediction or diagnosis of these disease states is warranted.

Introduction: Preeclampsia (PE) and fetal growth restriction (FGR) are among the leading causes of perinatal morbidity and mortality. Placental insufficiency is central to these conditions. The mechanisms underlying placental insufficiency are poorly understood. Apoptosis has long been considered the only form of regulated cell death, recent research has identified an alternate process of programmed cell death known as necroptosis [1]. Necroptosis is distinct from apoptosis, relying on the deacetylase sirtuin-2 [2], receptor interacting kinases RIPK1 and 3, and the pseudokinase MLKL [3]. We aimed to determine whether these key necroptosis effector molecules were present in human placenta and whether they are differentially expressed in severe preterm (PT) PE and FGR., Methods: PT placentas from severe early onset (<34 weeks) PE (n = 30), FGR (n = 12) and control (18) pregnancies were collected. SIRT2 and RIPK1 localization and quantitation was determined by immunohistochemistry and western blot. Immunocytochemistry was used to detect SIRT2 and RIPK1 in trophoblastic debris from first trimester, term control and PE pregnancies. Expression of SIRT2, RIPK1, RIPK3 and MLKL was examined by qPCR., Results: SIRT2 and RIPK1 were localized to the syncytiotrophoblast, villous leukocytes and vasculature in all PT placentas. A significant reduction in SIRT2 protein expression in both PE and FGR placentas was identified. RIPK1 mRNA expression was significantly increased in PE placentas. Immunofluorescence identified both SIRT2 and RIPK1 in the cytotrophoblast cytoplasm., Discussion: We have identified the presence of activators of necroptosis in human placenta. Interestingly, there is differential expression in major pregnancy complications. We conclude necroptosis may contribute to placental pathophysiology that underlies serious pregnancy complications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: Preeclampsia is a serious complication affecting 5-8% of pregnancies. Central to its pathogenesis is placental hypoxia and inflammation which leads to secretion of soluble fms-like tyrosine kinase 1 (sFlt-1). sFlt-1 causes widespread endothelial dysfunction. The molecular mechanisms regulating sFlt-1 production remain poorly understood. Recently, a binding site for the nuclear factor activated T cells (NFAT) transcription factor has been found on fms-like tyrosine kinase 1 (FLT-1) promoter., Methods: We assessed whether inhibiting NFAT impacts FLT-1, sFlt-1 and cytokine expression, as well as sFlt-1 secretion in primary cytotrophoblasts, placental explants and human umbilical vein endothelial cells (HUVECs). We investigated whether NFAT is regulated by hypoxia in primary cytotrophoblasts. We characterised the expression of NFAT1-4 in preterm preeclamptic compared to gestationally matched placentas., Results: Inhibiting NFAT reduced FLT-1 and sFlt-1 splice variant e15a transcription, concordant with reduced total sFlt-1 and sFlt-1 e15a secretion from primary human cytotrophoblasts. This effect appeared tissue specific as inhibiting NFAT did not change sFlt-1 secretion from endothelial cells. Inhibiting NFAT also reduced transcription of inflammatory cytokines IL-1β and IL-10 in primary cytotrophoblasts. NFAT1 and NFAT3 mRNA expression were significantly increased under hypoxia (1% O 2 ). Inhibiting NFAT under hypoxia significantly reduced FLT-1 and sFlt-1 e15a transcription, but did not reduce sFlt-1 secretion. NFAT mRNA and protein localisation was not different in preeclamptic compared to gestationally matched placenta., Discussion: NFAT positively regulates placental FLT-1 and sFlt-1 e15a, secretion of sFlt-1 and inflammatory cytokine expression. It may be involved in the pathophysiology of preeclampsia., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Preeclampsia is a serious complication of pregnancy affecting 5% of pregnancies. Our team identified 137 genes highly expressed in placenta relative to other human tissues. Here, we have explored a role for steroid sulfatase (STS) in preeclampsia by characterising STS expression and the functional effects of STS on primary placental trophoblasts., Methods: Characterisation of STS was performed on preterm preeclamptic and gestation-matched normotensive preterm controls who delivered at <34 weeks gestation. We characterised placental and maternal whole blood STS mRNA and placental protein expression via qRT-PCR, immunohistochemistry and Western Blot. To assess whether STS is involved in sFlt1 secretion and syncytialisation, we administered siRNA to silence STS in primary trophoblasts before measuring sFlt1 and hCG secretion and E-Cadherin expression., Results: A custom array containing 45 placental specific genes identified 10 genes significantly altered in the placentas of preeclamptic patients relative to normotensive gestation-matched controls. Of these genes, qRT-PCR and western blot on a larger cohort confirmed that the expression of STS was significantly elevated in preeclamptic placentas (n = 44) relative to gestation matched controls (n = 26). Given placental RNA leaks in to the maternal circulation, we also assessed STS mRNA expression in the whole blood of patients with preeclampsia and found it was significantly increased relative to normotensive controls. siRNA knockdown of STS in primary trophoblast resulted in a modest but significant reduction in sFlt1 secretion, but had no affect on hCG secretion or E-Cadherin protein expression., Discussion: STS is increased in preeclamptic placentas and maternal whole blood. Our data suggests that STS may affect sFlt1 secretion by regulating sFlt1-i13 transcription, and not via alterations in syncytialisation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Preeclampsia is associated with the placental release of soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sENG). These anti-angiogenic factors cause hypertension and multi-organ injury. Pravastatin decreases placental secretion of sFlt-1 in vitro and is currently being examined in clinical trials as a potential treatment for preeclampsia. However, it is possible that different classes of statins may be more potent at decreasing sFlt-1 secretion. We compared the relative potency of three different generations of statins on sFlt-1 and sENG secretion from human endothelial cells, trophoblast cells, and placenta explants., Methods: We performed functional experiments using primary human umbilical vein endothelial cells, trophoblast cells and preterm preeclamptic placental explants to assess the affect of simvastatin, rosuvastatin and pravastatin on sFlt-1 and sENG secretion and compared the relative potency of each statin at reducing these factors (Inhibitory Concentration 50). Furthermore we assessed the effect of each statin on the antioxidant and cytoprotective enzyme, heme-oxygenase 1., Results: All statins reduced sFlt-1 secretion from endothelial cells, trophoblasts and preterm preeclamptic placental explants. Simvastatin was the most potent inhibitor of sFlt-1 secretion from endothelial cells (IC 50 3.2 μM), trophoblast cells (IC 50 61.4 μM) and placental explants. Simvastatin was 28 times and 3 times more potent at reducing sFlt-1 secretion from endothelial cells and 85 times and 33 times more potent at reducing sFlt-1 secretion from trophoblast cells than pravastatin or rosuvastatin respectively. All statins increased sENG secretion from endothelial cells however did not change secretion from placental explants. While all statins up-regulated heme-oxygenase 1 in endothelial cells, only simvastatin up-regulated its expression in placenta from patients with preterm preeclampsia., Conclusion: Simvastatin may be a more potent inhibitor of sFlt-1 secretion from endothelial cells, trophoblast cells and placenta from women with preterm preeclampsia than either pravastatin or rosuvastatin.

Introduction: Preeclampsia is a serious pregnancy complication for which there are no medical treatments. Soluble endoglin is an anti-angiogenic factor implicated in the pathogenesis of the disease, however little is known about its molecular regulation. The PI3K/Akt pathway is down regulated in preeclamptic placentas and decreased PI3K/Akt signaling has been linked to increased soluble endoglin release from endothelial cells. MMP14 is a key protease that functions to release soluble endoglin from the placental surface., Objective: This study aimed to determine whether reduced placental PI3K/Akt causes elevated release of soluble endoglin via MMP14., Study Design: Akt mRNA and protein expression were assessed in early onset preeclamptic and preterm control placentas (delivered <34weeks gestation). PI3K/Akt inhibition was achieved by administering PI3K inhibitor wortmannin, a specific Akt inhibitor or Akt siRNA to primary human umbilical vein endothelial cells, primary trophoblast and placental explants. The effect of PI3K/Akt inhibition on soluble endoglin release, MMP14, endoglin and TIMP-3 mRNA expression was determined., Results: We identified significantly reduced pAkt and total Akt in preeclamptic placentas relative to preterm control. Inhibition of PI3K/Akt resulted in significantly elevated soluble endoglin release from HUVECs, had no effect on MMP14 mRNA expression but resulted in significantly reduced TIMP3. In contrast inhibiting PI3K/Akt in placental explants or primary trophoblast did not change soluble endoglin release., Conclusion: This study confirms that the PI3K/Akt cell protection pathway is down regulated in preeclampsia, but demonstrates that this dysregulation is unlikely to be responsible for the excessive placental soluble endoglin release characteristic of preeclampsia., (Copyright © 2016 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

The anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT-1), plays a central role in preeclamptic pathophysiology. A splice variant of FLT-1 (VEGF receptor 1), sFLT-1 is released in excessive amounts from the preeclamptic placenta into the maternal circulation, where it causes endothelial dysfunction manifesting as end-organ disease. However, the mechanisms regulating its production within the placenta remain poorly understood. Recently it was shown in endothelial cells that Jumonji domain containing protein 6 (JMJD6) hydroxylates U2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65, a component of the splicesome). The hydroxylation by JMJD6 is oxygen dependent. Under hypoxia, JMJD6 is less able to hydroxylate U2AF65, and this unhydroxylated form of U2AF65 biases splicing of FLT-1 to sFLT-1. We assessed whether oxygen-sensing JMJD6 is differentially expressed in preeclamptic placenta and regulates sFLT-1 splicing in placenta via U2AF65. JMJD6 protein expression was significantly reduced in preterm preeclamptic placenta (P < 0.0001; n = 21) relative to preterm controls (n = 10). Exposing both placental and endothelial cells to hypoxia significantly reduced JMJD6 mRNA and increased sFLT-1 mRNA and protein expression. Silencing JMJD6 in primary endothelial and trophoblast cells significantly increased sFLT-1 secretion. Next, we examined whether these molecules may be directly interacting. We demonstrated that placental U2AF65 colocalized with JMJD6. In turn, we found JMJD6 directly interacts with U2AF65, which in turn produces sFLT-1 mRNA transcripts. Taken together, our findings provide evidence that JMJD6 may play a role in regulating the production of sFLT-1 in the preeclamptic placenta. Decreased placental JMJD6 expression may be an important component to the pathophysiology of preeclampsia., (© 2016 by the Society for the Study of Reproduction, Inc.)

In preeclampsia, the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFLT-1) is released from placenta into the maternal circulation, causing endothelial dysfunction and organ injury. A recently described splice variant, sFLT-1 e15a, is primate specific and the most abundant placentally derived sFLT-1. Therefore, it may be the major sFLT-1 isoform contributing to the pathophysiology of preeclampsia. sFLT-1 e15a protein remains poorly characterized: its bioactivity has not been comprehensively examined, and serum levels in normal and preeclamptic pregnancy have not been reported. We generated and validated an sFLT-1 e15a-specific ELISA to further characterize serum levels during pregnancy, and in the presence of preeclampsia. Furthermore, we performed assays to examine the bioactivity and antiangiogenic properties of sFLT-1 e15a protein. sFLT-1 e15a was expressed in the syncytiotrophoblast, and serum levels rose across pregnancy. Strikingly, serum levels were increased 10-fold in preterm preeclampsia compared with normotensive controls. We confirmed sFLT-1 e15a is bioactive and is able to inhibit vascular endothelial growth factor signaling of vascular endothelial growth factor receptor 2 and block downstream Akt phosphorylation. Furthermore, sFLT-1 e15a has antiangiogenic properties. sFLT-1 e15a decreased endothelial cell migration, invasion, and inhibited endothelial cell tube formation. Administering sFLT-1 e15a blocked vascular endothelial growth factor induced sprouts from mouse aortic rings ex vivo. We have demonstrated that sFLT-1 e15a is increased in preeclampsia, antagonizes vascular endothelial growth factor signaling, and has antiangiogenic activity. Future development of diagnostics and therapeutics for preeclampsia should consider targeting placentally derived sFLT-1 e15a., (© 2015 American Heart Association, Inc.)

Elevated placental release of the antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG), is central to the pathophysiology of preeclampsia. It is widely accepted that heme oxygenase-1 (HO-1) is decreased in preeclamptic placenta and negatively regulates sFlt-1 and sENG production. We set out to verify these contentions. There was no difference in HO-1 mRNA or protein levels in preterm preeclamptic placentas (n=17) compared with gestationally matched controls (n=27). In silico analysis of microarray studies did not identify decreased placental HO-1 expression in preeclamptic placenta. Silencing HO-1 in primary trophoblasts did not affect sFlt-1 protein secretion after 24 or 48 hours. Silencing nuclear factor (erythroid-derived 2)-like 2 (transcription factor that upregulates HO-1) in trophoblasts also did not affect sFlt-1 secretion. Administering tin protoporphyrin IX dichloride (HO-1 inhibitor) or cobalt protoporphyrin (HO-1 inducer) into placental explants did not affect sFlt-1 or sENG secretion. Silencing HO-1 in 2 types of primary endothelial cells (human umbilical vein endothelial and uterine microvascular endothelial cells) significantly increased sFlt-1 secretion but not sENG secretion. However, HO-1 silencing selectively increased mRNA expression of sFlt-1 i13 (generically expressed sFlt-1 variant) but not of sFlt-1 e15a (sFlt-1 variant mainly expressed in placenta). Furthermore, adding tin protoporphyrin IX dichloride decreased sFlt-1, whereas adding HO-1 inducers (cobalt protoporphyrin, dimethyl fumarate, and rosiglitazone) either had no effect or increased sFlt-1 or sENG secretion (these trends are opposite to what is expected). We conclude that HO-1 expression is not decreased in preeclamptic placenta and HO-1 does not negatively regulate placental sFlt-1 and sENG secretion in placental or endothelial cells., (© 2015 American Heart Association, Inc.)

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are major obstetric health problems. Higher levels of T-helper (Th) 1 (proinflammatory) cytokines have been observed in pregnancies complicated with PE and IUGR; this is in contrast to the predominant Th2 (anti-inflammatory) cytokine environment found in uncomplicated pregnancies. Myostatin is best known as a negative regulator of muscle development and reportedly has a role in fat deposition, glucose metabolism, and cytokine modulation (outside the placenta). Myostatin concentrations in plasma and protein expression in placental tissue are significantly higher in women with PE. Expression of myostatin in IUGR and PE-IUGR and the effect of this protein on the cytokine production from the placenta is unknown. In the current study, significant differences were identified in the expression of myostatin in pregnancies complicated with IUGR, PE, and PE with IUGR. Furthermore, cytokine production by first-trimester placental tissues was altered following myostatin treatment., (© The Author(s) 2015.)

There is recent interest for uniform placental collection protocols so laboratories can share samples. However, concerns have been raised a wash step at collection causes significant loss of sFlt-1 and soluble endoglin, among the most studied proteins in placentology. We measured Flt-1 and endoglin mRNA and protein in 10 preeclamptic placentas that were washed, or left unwashed. Reassuringly, there was no significant change in the Flt-1, sFlt-1-e15a or sFlt-1-i13 mRNA or Flt-1 or sFlt-1 protein expression or localization. There was also no change in endoglin mRNA expression or protein localization. Washing preeclamptic placental samples does not alter anti-angiogenic factor expression., (Copyright © 2015 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Preeclampsia is a serious complication of pregnancy with no medical treatment. It is caused by intermittent placental hypoxia and release of sFlt-1 and soluble endoglin, leading to wide spread maternal endothelial dysfunction and multisystem organ injury. YC-1 is a guanylyl cyclase activator and HIF1α inhibitor developed for use in hypertension and atherosclerosis. We examined whether YC-1 reduces sFlt-1 and sENG secretion and reverses endothelial dysfunction in primary human tissues. YC-1 significantly reduced sFlt-1 and sENG secretion from human umbilical vein endothelial cells, purified primary trophoblast cells and placental explants taken from patients with preterm preeclampsia. This was concordant with reduced HIF1α expression. YC-1 also reversed TNFα induced endothelial dysfunction, including reduced vascular cell adhesion molecule 1 expression and monocyte adhesion to primary endothelial cells. We conclude YC-1 decreases placental production of sFlt-1 and sENG and decreases endothelial dysfunction. It is a novel therapeutic candidate for preeclampsia., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: Preeclampsia is a major pregnancy complication where excess placental release of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin causes maternal endothelial and multisystem organ injury. Clinical trials have commenced examining whether pravastatin can be used to treat preeclampsia. However, the preclinical evidence supporting pravastatin as a treatment is limited to animal models, with almost no studies in human tissues. Therefore, we examined whether pravastatin reduced sFlt-1 and soluble endoglin secretion and decreased endothelial dysfunction in primary human tissues. Pravastatin reduced sFlt-1 secretion from primary endothelial cells, purified cytotrophoblast cells, and placental explants obtained from women with preterm preeclampsia. It increased soluble endoglin secretion from endothelial cells but did not change secretion from placental explants. The regulation of sFlt-1 by pravastatin seemed to be mediated via the 3-hydroxy-3-methylglutaryl-coenzyme A reductase cholesterol synthesis pathway. Pravastatin also reduced markers of endothelial dysfunction, including vascular cell adhesion molecule-1 expression and leukocyte adhesion on endothelial cells and increased endothelial cell migration and invasion. We also treated 4 patients with preterm preeclampsia presenting at <30 weeks of gestation with daily pravastatin. Pravastatin seemed to stabilize blood pressure, proteinuria, and serum uric acid levels. Furthermore, serum sFlt-1 levels decreased. We collected the placentas at delivery and found that pravastatin reduced sFlt-1 secretion. These results indicate that pravastatin reduced sFlt-1 and soluble endoglin production and decreased endothelial dysfunction in primary human tissues. We also present pilot data, suggesting that pravastatin can stabilize clinical and biochemical features of preterm preeclampsia. Our data obtained in human tissues support the concept that pravastatin is a candidate therapeutic for preeclampsia., Clinical Trial Registration: URL: http://www.anzctr.org.au. Unique identifier: ACTRN12613000268741., (© 2015 American Heart Association, Inc.)

Context: Preeclampsia (PE), a pregnancy-specific disorder closely associated with endothelial dysfunction and capillary leakage, is responsible for substantial maternal/fetal morbidity and mortality. PE can be classified as early-onset (<34 wk) and late-onset (>34 wk); the two subsets differ in presentation and pathogenesis., Objectives: The objectives of the study were to examine serine protease high-temperature requirement factor A4 (HtrA4) expression in the placenta and other human tissues and in early-onset vs late-onset PE, to determine serum HtrA4 levels in normal pregnancy and in PE subtypes, and to investigate the effect of high levels of HtrA4 on endothelial integrity and function., Methods: Microarray data analysis and RT-PCR determined HtrA4 expression in the human placenta, various tissues, and cell lines. The serum HtrA4 protein levels were analyzed by an ELISA. The potential impact of excessive circulating HtrA4 on the maternal vasculature was determined by in vitro endothelial tube and permeability assays., Results: Human HtrA4 expression was restricted to the placenta and significantly up-regulated in early-onset but not late-onset PE. The serum HtrA4 levels in normal pregnancy increased significantly between the first and second trimesters and then remained constant. Women with early-onset but not late-onset PE showed significantly higher HtrA4 levels in serum compared with gestational age-matched controls. In cell models, high levels of HtrA4 disturbed endothelial cell tube formation and permeability in a dose-dependent manner, and this was linked to alterations in junctional proteins and microtubule organization., Conclusions: HtrA4 represents a novel placenta-specific serine protease that is altered specifically in early-onset PE with potential causal roles in endothelial dysfunction and disease development.

Fetal growth restriction (FGR) is a major obstetric complication stemming from poor placental development. We have previously demonstrated that paternal obesity in mice is associated with impaired embryo development and significantly reduced fetal and placental weights. We hypothesised that the FGR observed in our rodent model of paternal diet-induced obesity is associated with alterations in metabolic, cell signalling and stress pathways. Male C57BL/6 mice were fed either a normal or high-fat diet for 10 weeks before sperm collection for IVF and subsequent embryo transfer. On embryonic day 14, placentas were collected and RNA extracted from both male and female placentas to assess mRNA expression of 24 target genes using custom RT-qPCR arrays. Peroxisome proliferator-activated receptor alpha (Ppara) and caspase-12 (Casp12) expression were significantly altered in male placentas from obese fathers compared with normal (P<0.05), but not female placentas. PPARA and CASP12 proteins were localised within the placenta to trophoblast giant cells by immunohistochemistry, and relative protein abundance was determined by western blot analysis. DNA was also extracted from the same placentas to determine methylation status. Global DNA methylation was significantly increased in female placentas from obese fathers compared with normal (P<0.05), but not male placentas. In this study, we demonstrate for the first time that paternal obesity is associated with changes in gene expression and methylation status of extraembryonic tissue in a sex-specific manner. These findings reinforce the negative consequences of paternal obesity before conception, and emphasise the need for more lifestyle advice for prospective fathers., (© 2015 Society for Reproduction and Fertility.)

Severe early onset pre-eclampsia is a serious pregnancy complication, believed to arise as a result of persistent placental hypoxia due to impaired placentation. Pregnancy-associated plasma protein A2 (PAPPA2) is very highly expressed in the placenta relative to all other tissues. There is some evidence that PAPPA2 mRNA and protein are increased in association with pre-eclampsia. The aim of the present study was to characterise the mRNA and protein expression, as well as localisation, of PAPPA2 in an independent cohort of severe early onset pre-eclamptic placentas. We also examined whether exposing placental explants to hypoxia (1% oxygen) changed the expression of PAPPA2. Expression of PAPPA2 mRNA and protein was upregulated in severe early onset pre-eclamptic placentas compared with preterm controls and localised to the syncytiotrophoblast. Interestingly, protein localisation was markedly reduced in term placenta. Syncytialisation of BeWo cells did not change PAPPA2 expression. However, hypoxia upregulated PAPPA2 mRNA and protein expression in primary placental explants. Together, our data suggest that PAPPA2 may be upregulated in severe pre-eclampsia and, functionally, this may be mediated via increased placental hypoxia known to occur with this pregnancy disorder.

Objective: Placenta has the highest expression of epidermal growth factor (EGF) receptor of all tissues, a cell signaling pathway promoting survival and growth. Therefore, EGF receptor inhibition could potentially treat ectopic pregnancy. We undertook preclinical studies to examine whether gefitinib (orally available EGF receptor inhibitor) with or without methotrexate inhibits placental cell growth., Methods: Gefitinib and methotrexate were added to placental cells and their ability inhibit cell growth, block EGF receptor signaling, and induce apoptosis (programmed cell death) was examined. They were also administered to two animal mouse models to examine their effects on placental tissue in vivo., Results: Epidermal growth factor receptor was highly expressed in placental tissue from ectopic pregnancies. Combining gefitinib with methotrexate potently inhibited growth of placental cells, including placental cell lines (JEG3, BeWo cells) and cells isolated from first-trimester placenta. These drugs were additive in blocking EGF receptor signaling and inducing apoptosis. Gefitinib and methotrexate administered together were more potent in decreasing the volume of human placental cells xenografted subcutaneously onto mice compared with either alone. By day 19 after xenografting, mean (± standard error of the mean), xenograft volumes were: 821 (± 68) mm after gefitinib treatment, 901 (± 204) mm after methotrexate treatment, and 345 (±137) mm after both drugs were given (P<.01 for both comparisons of single therapy compared with combination therapy). Combining these agents doubled rates of fetal resorption in pregnant mice compared with each drug alone., Conclusion: Combining gefitinib with methotrexate potently inhibits placental cell growth in vitro and in mouse models. The combination may have potential in treating ectopic pregnancies.

Context: Fetal growth restriction (FGR) is a leading cause of perinatal mortality, yet no reliable screening test exists. Placental specific mRNA in the maternal circulation may reflect changes in the placental transcriptome in FGR and could be a novel biomarker for FGR., Objective: The aim of the study was to identify placental specific RNA detectable in the maternal circulation and examine whether they are differentially expressed in severe preterm FGR., Design: In silico screening was used to identify placental specific RNAs. Their expression in cases of severe FGR vs controls was examined in both maternal blood and placenta by microarray, RT-PCR, and in situ hybridization., Results: Via in silico analysis, we identified 137 genes very highly expressed in the placenta relative to other tissues. Using microarray, we found that they were detectable in the maternal blood and were globally dysregulated with preterm FGR; 75 genes (55%) had a ≥1.5-fold differential expression compared to controls. Eight genes (ERVWE-1, PSG1, PLAC4, TAC3, PLAC3, CRH, CSH1, and KISS1) were validated by RT-PCR to be significantly increased in both maternal blood and placenta in a larger cohort of severe FGR compared to controls. In situ hybridization confirmed PAPPA2 and ERVWE-1 localized to the syncytiotrophoblast., Conclusion: There is global differential expression of placental specific mRNA in the maternal blood in pregnancies complicated by severe preterm FGR. Placental specific mRNA in maternal blood may represent a new class of biomarkers for preterm FGR.

Background: Preeclampsia is a severe complication of pregnancy. Chemerin is an adipokine secreted from adipose tissue and highly expressed in placenta. This study evaluated the biomarker potential of circulating chemerin to predict preeclampsia. Methods: Maternal plasma and placenta were collected from women with early-onset preeclampsia (<34 weeks), with preeclampsia and eclampsia, or before preeclampsia diagnosis (36 weeks). Human trophoblast stem cells were differentiated into syncytiotrophoblast or extravillous trophoblasts across 96 hours. Cells were cultured in 1% O2 (hypoxia) or 5% O2 (normoxia). Chemerin was measured by enzyme-linked immunosorbent assay (ELISA) and RARRES2 (gene coding chemerin) by reverse transcription-quantitative polymerase chain reaction. Results: Circulating chemerin was increased in 46 women with early-onset preeclampsia (<34 weeks) compared to 17 controls (P <.0006). Chemerin was increased in placenta from 43 women with early-onset preeclampsia compared to 24 controls (P <.0001). RARRES2 was reduced in placenta from 43 women with early-onset preeclampsia vs 24 controls (P <.0001). Chemerin was increased in plasma from 26 women with established preeclampsia (P =.006), vs 15 controls. Circulating chemerin was increased in 23 women who later developed preeclampsia vs 182 who did not (P = 3.23 × 10−6). RARRES2 was reduced in syncytiotrophoblast (P =.005) or extravillous trophoblasts (P <.0001). Hypoxia increased RARRES2 expression in syncytiotrophoblast (P =.01) but not cytotrophoblast cells. Conclusions: Circulating chemerin was elevated in women with early-onset preeclampsia, established preeclampsia, and preceding preeclampsia diagnosis of preeclampsia. RARRES2 was dysregulated in placenta complicated by preeclampsia and may be regulated through hypoxia. Chemerin may have potential as a biomarker for preeclampsia but would need to be combined with other biomarkers. [ABSTRACT FROM AUTHOR]

Background: Dishevelled Associated Activator Of Morphogenesis 2 (DAAM2) levels are elevated in the maternal circulation and placenta in pregnancies complicated by fetal growth restriction. However, placental DAAM2 levels in cases of preeclampsia have not previously been explored. Here, we examined placental DAAM2 in pregnancies complicated by preterm preeclampsia, and whether candidate preeclampsia therapeutics altered its expression.Methods: DAAM2 mRNA and protein levels were assessed in placental tissue from cases of preterm preeclampsia and gestation-matched controls (delivering ≤ 34 weeks; qPCR and western blot respectively). Short interfering RNAs were used to silence DAAM2 in isolated primary cytotrophoblast under normoxic (8 % O2) and hypoxic (1 % O2) conditions, and expression of anti-angiogenic sFLT-1, angiogenic PGF, antioxidant, fetal growth, and inflammatory genes assessed. DAAM2 expression was measured in placental explant tissue from pregnancies complicated by preeclampsia, treated with three proton pump inhibitors (100 µM esomeprazole, lansoprazole, and rabeprazole).Results: DAAM2 expression was significantly reduced in preeclamptic placental tissue compared to controls, but protein production was unchanged. Silencing DAAM2 in hypoxic cytotrophoblast increased sFLT-i13 isoform expression, but did not alter sFLT-e15a or PGF expression, or sFLT-1 secretion. DAAM2 knockdown did not alter expression of antioxidant (NQO-1, TXN, GCLC), fetal growth (SPINT1), or inflammasome (NLRP3) genes. Esomeprazole and lansoprazole, but not rabeprazole, increased DAAM2 expression in placental explant tissue from cases of preeclampsia.Conclusion: Placental DAAM2 protein is not significantly altered in placental tissue in cases of preeclampsia, and its suppression does not alter sFLT-1 secretion. Hence, placental DAAM2 is unlikely to drive the pathogenesis associated with preeclampsia. [ABSTRACT FROM AUTHOR]

Preeclampsia is a disease of pregnancy responsible for significant maternal and neonatal mortality. Galectin-3 is a β-Galactoside binding protein. This study aimed to characterise galectin-3 in women with preeclampsia and human trophoblast stem cells (hTSCs). Galectin-3 was measured in placental lysates and plasma collected from patients with early-onset preeclampsia (delivered <34 weeks' gestation) and gestation matched controls. Placental galectin-3 protein was significantly reduced in 43 women with early-onset preeclampsia compared to 21 controls. mRNA expression of LGALS3 (galectin-3 encoding gene) was reduced in 29 women with early-onset preeclampsia, compared to 18 controls (p = 0.009). There was no significant difference in plasma galectin-3 protein in 46 women with early-onset preeclampsia compared to 20 controls. In a separate cohort of samples collected at 36 weeks' gestation, circulating galectin-3 was not altered in 23 women who later developed preeclampsia, versus 182 who did not. In syncytialised hTSCs, hypoxia increased mRNA expression of LGALS3 (p = 0.01). Treatment with inflammatory cytokines (TNF-α and IL-6) had no effect on LGALS3 mRNA expression. However, TNF-α treatment caused an increase in mRNA expression of LGALS3BP (galectin-3 binding protein encoding gene) in hTSCs (p = 0.03). This study showed a reduction of galectin-3 in placenta from pregnancies complicated by early-onset preeclampsia. LGALS3 mRNA expression was dysregulated by hypoxia exposure in placental stem cells. [ABSTRACT FROM AUTHOR]

Previously, we demonstrated that the proton pump inhibitor, esomeprazole magnesium hydrate (MH), could have potential as a repurposed treatment against preeclampsia, a serious obstetric condition. In this study we investigate the difference in the preclinical effectiveness between 100 µM of esomeprazole MH and its hydration isomer, esomeprazole magnesium trihydrate (MTH). Here, we found that both treatments reduced secretion of sFLT-1 (anti-angiogenic factor) from primary cytotrophoblast, but only esomeprazole MH reduced sFLT-1 secretion from primary human umbilical vein endothelial cells (assessed via ELISA). Both drugs could mitigate expression of the endothelial dysfunction markers, vascular cell adhesion molecule-1 and endothelin-1 (via qPCR). Neither esomeprazole MH nor MTH quenched cytotrophoblast reactive oxygen species production in response to sodium azide (ROS assay). Finally, using wire myography, we demonstrated that both compounds were able to induce vasodilation of human omental arteries at 100 µM. Esomeprazole is safe to use in pregnancy and a candidate treatment for preeclampsia. Using primary human tissues and cells, we validated that esomeprazole is effective in enhancing vascular relaxation, and can reduce key factors associated with preeclampsia, including sFLT-1 and endothelial dysfunction. However, esomeprazole MH was more efficacious than esomeprazole MTH in our in vitro studies. [ABSTRACT FROM AUTHOR]

Previously, we identified elevated transcripts for the gene Oleoyl-ACP Hydrolase (OLAH) in the maternal circulation of pregnancies complicated by preterm fetal growth restriction. As placental dysfunction is central to the pathogenesis of both fetal growth restriction and preeclampsia, we aimed to investigate OLAH levels and function in the human placenta. We assessed OLAH mRNA expression (qPCR) throughout pregnancy, finding placental expression increased as gestation progressed. OLAH mRNA and protein levels (Western blot) were elevated in placental tissue from cases of preterm preeclampsia, while OLAH protein levels in placenta from growth-restricted pregnancies were comparatively reduced in the preeclamptic cohort. OLAH expression was also elevated in placental explant tissue, but not isolated primary cytotrophoblast cultured under hypoxic conditions (as models of placental dysfunction). Further, we discovered that silencing cytotrophoblast OLAH reduced the expression of pro- and anti-apoptosis genes, BAX and BCL2, placental growth gene, IGF2, and oxidative stress gene, NOX4. Collectively, these findings suggest OLAH could play a role in placental dysfunction and may be a therapeutic target for mitigating diseases associated with this vital organ. Further research is required to establish the role of OLAH in the placenta, and whether these changes may be a maternal adaptation or consequence of disease. [ABSTRACT FROM AUTHOR]