Cell surface associated protein mucin 15 (MUC15) is elevated in preeclampsia.

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% O2) 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.

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

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% 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-[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-ß 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.

Circulating Chemerin Is Elevated in Women With Preeclampsia.

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.

Proinflammatory microenvironment promotes lymphoma progression in mice with high megakaryocyte and TPO levels.

Platelets have been shown to enhance the survival of lymphoma cell lines. However, it remains unclear whether they play a role in lymphoma. Here, we investigated the potential role of platelets and/or megakaryocytes in the progression of Eµ-myc lymphoma. Eµ-myc tumor cells were transplanted into recipient wild-type (WT) control, Mpl-/-, or TpoTg mice, which exhibited normal, low, and high platelet and megakaryocyte counts, respectively. TpoTg mice that underwent transplantation exhibited enhanced lymphoma progression with increased white blood cell (WBC) counts, spleen and lymph node weights, and enhanced liver infiltration when compared with WT mice. Conversely, tumor-bearing Mpl-/- mice had reduced WBC counts, lymph node weights, and less liver infiltration than WT mice. Using an Mpl-deficient thrombocytopenic immunocompromised mouse model, our results were confirmed using the human non-Hodgkin lymphoma GRANTA cell line. Although we found that platelets and platelet-released molecules supported Eµ-myc tumor cell survival in vitro, pharmacological inhibition of platelet function or anticoagulation in WT mice transplanted with Eµ-myc did not improve disease outcome. Furthermore, transient platelet depletion or sustained Bcl-xL-dependent thrombocytopenia did not alter lymphoma progression. Cytokine analysis of the bone marrow fluid microenvironment revealed increased levels of the proinflammatory molecule interleukin 1 in TpoTg mice, whereas these levels were lower in Mpl-/- mice. Moreover, RNA sequencing of blood-resident Eµ-myc lymphoma cells from TpoTg and WT mice after tumor transplantation revealed the upregulation of hallmark gene sets associated with an inflammatory response in TpoTg mice. We propose that the proinflammatory microenvironment in TpoTg mice promotes lymphoma progression.

Endothelial protein C receptor is increased in preterm preeclampsia and fetal growth restriction.

Placental dysfunction is the leading cause of both preeclampsia and fetal growth restriction. This study aimed to characterize endothelial protein C receptor (EPCR) in preterm preeclampsia, term preeclampsia, and fetal growth restriction (defined by delivery of a small for gestational age [SGA] infant [<10% birthweight centile]) and examine its regulation in primary syncytiotrophoblast. Placental EPCR mRNA and protein were significantly increased in patients with preterm preeclampsia (<34 weeks gestation) compared to gestation-matched controls (p < .0001). In the plasma, EPCR was also significantly elevated (p = .01) in established preterm preeclampsia while its substrate, protein C (PC) was significantly reduced (p = .0083). Placentas from preterm small for gestational age (SGA) cases, had elevated EPCR mRNA expression (p < .0001) relative to controls. At 36 weeks, no significant changes in plasma EPCR were detected in samples from patients destined to develop preeclampsia or deliver an SGA infant at term. In terms of syncytiotrophoblast, hypoxia significantly increased EPCR mRNA expression (p = .008), but Tumor Necrosis Factor Alpha (TNF-α) decreased EPCR mRNA. Interleukin-6 (IL-6) had no significant effect on EPCR mRNA expression. When isolated syncytiotrophoblast was treated with metformin under hypoxia (1% O2 ) or normoxia (8% O2 ), EPCR mRNA expression was significantly reduced (p = .008) relative to control. In conclusion, EPCR is markedly elevated in the placenta and the circulation of patients with established preterm preeclampsia and placental increases may be associated with hypoxia. Additionally, fetal growth-restricted pregnancies (as defined by the delivery of an SGA infant) also demonstrated elevated placental EPCR.

Placental galectin-3 is reduced in early-onset preeclampsia.

Preeclampsia is a disease of pregnancy responsible for significant maternal and neonatal mortality. Galectin-3 is a ß-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.

Associations Between Soluble fms-Like Tyrosine Kinase-1 and Placental Growth Factor and Disease Severity Among Women With Preterm Eclampsia and Preeclampsia.

Background The angiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) are postulated to be pathogenic disease drivers of preeclampsia. If true, then circulating levels should become more deranged with increasing disease severity. Methods and Results We investigated the association between circulating sFlt-1 and PlGF levels and severe adverse maternal outcomes among 348 women with preeclampsia. Compared with 125 women with preeclampsia without severe features, 25 women with preeclampsia and any of hemolysis, elevated liver enzymes, low platelet count syndrome, disseminated intravascular coagulation, or severe renal involvement had sFlt-1 levels that were 2.63-fold higher (95% CI, 1.81-3.82), sFlt-1/PlGF levels that were 10.07-fold higher (95% CI, 5.36-18.91) and PlGF levels that were 74% lower (adjusted fold change, 0.26 [95% CI, 0.18-0.39]). Compared with 125 women with preeclampsia without severe features, 37 with eclampsia had sFlt-1 levels that were 2-fold higher (2.02 [95% CI, 1.32-3.09]), sFlt-1/PIGF levels that were 4.71-fold higher (95% CI, 2.30-9.66) and PIGF levels that were 63% lower (0.43-fold change [95% CI, 0.27-0.68]). Compared with those without severe features, preeclampsia with severe hypertension (n=146) was also associated with altered angiogenic levels (sFlt-1, 1.71-fold change [95% CI, 1.39-2.11]; sFlt/PlGF, 2.91 [95% CI, 2.04-4.15]; PlGF, 0.59 [95%CI 0.47-0.74]). We also found that sFlt-1 and PlGF levels were altered by the number of maternal complications experienced. Conclusions Further angiogenic imbalance among women with preeclampsia is likely a pathogenic disease driver responsible for the life-threatening maternal complications.

PSG7 and 9 (Pregnancy-Specific ß-1 Glycoproteins 7 and 9): Novel Biomarkers for Preeclampsia.

Background Preeclampsia is pregnancy specific, involving significant maternal endothelial dysfunction. Predictive biomarkers are lacking. We evaluated the biomarker potential, expression, and function of PSG7 (pregnancy-specific ß-1 glycoprotein 7) and PSG9 (pregnancy-specific ß-1 glycoprotein 9) in preeclampsia. Methods and Results At 36 weeks gestation preceding term preeclampsia diagnosis, PSG7 and PSG9 (in Australian cohorts of n=918 and n=979, respectively) were significantly increased before the onset of term preeclampsia (PSG7, P=0.013; PSG9, P=0.0011). In samples collected at 28 to 32 weeks from those with preexisting cardiovascular disease and at high risk of preeclampsia (Manchester Antenatal Vascular Service, UK cohort, n=235), both PSG7 and PSG9 were also significantly increased preceding preeclampsia onset (PSG7, P<0.0001; PSG9, P=0.0003) relative to controls. These changes were validated in the plasma and placentas of patients with established preeclampsia who delivered at <34 weeks gestation (PSG7, P=0.0008; PSG9, P<0.0001). To examine whether PSG7 and PSG9 are associated with increasing disease severity, we measured them in a cohort from South Africa stratified for this outcome, the PROVE (Preeclampsia Obstetric Adverse Events) cohort (n=72). PSG7 (P=0.0027) and PSG9 (P=0.0028) were elevated among patients who were preeclamptic with severe features (PROVE cohort), but not significantly changed in those without severe features or with eclampsia. In syncytialized first trimester cytotrophoblast stem cells, exposure to TNFα (tumor necrosis factor α) or IL-6 (interleukin 6) significantly increased the expression and secretion of PSG7 and PSG9. In contrast, when we treated primary endothelial cells with recombinant PSG7 and PSG9, we only observed modest changes in Flt-1 (FMS-like tyrosine kinase-1) expression and Plgf (placental growth factor) expression, and no other effects on proangiogenic/antiangiogenic or endothelial dysfunction markers were observed. Conclusions Circulating PSG7 and PSG9 are increased before preeclampsia onset and among those with established disease with their production and release potentially driven by placental inflammation.

Circulating SPINT1 Is Reduced in a Preeclamptic Cohort with Co-Existing Fetal Growth Restriction.

Fetal growth restriction (FGR), when undetected antenatally, is the biggest risk factor for preventable stillbirth. Maternal circulating SPINT1 is reduced in pregnancies, which ultimately deliver small for gestational age (SGA) infants at term (birthweight < 10th centile), compared to appropriate for gestational age (AGA) infants (birthweight ≥ 10th centile). SPINT1 is also reduced in FGR diagnosed before 34 weeks' gestation. We hypothesised that circulating SPINT1 would be decreased in co-existing preterm preeclampsia and FGR. Plasma SPINT1 was measured in samples obtained from two double-blind, randomised therapeutic trials. In the Preeclampsia Intervention with Esomeprazole trial, circulating SPINT1 was decreased in women with preeclampsia who delivered SGA infants (n = 75, median = 18,857 pg/mL, IQR 10,782-29,890 pg/mL, p < 0.0001), relative to those delivering AGA (n = 22, median = 40,168 pg/mL, IQR 22,342-75,172 pg/mL). This was confirmed in the Preeclampsia Intervention 2 with metformin trial where levels of SPINT1 in maternal circulation were reduced in SGA pregnancies (n = 95, median = 57,764 pg/mL, IQR 42,212-91,356 pg/mL, p < 0.0001) compared to AGA controls (n = 40, median = 107,062 pg/mL, IQR 70,183-176,532 pg/mL). Placental Growth Factor (PlGF) and sFlt-1 were also measured. PlGF was significantly reduced in the SGA pregnancies, while ratios of sFlt-1/SPINT1 and sFlt1/PlGF were significantly increased. This is the first study to demonstrate significantly reduced SPINT1 in co-existing FGR and preeclamptic pregnancies.

Circulating Activin A is elevated at 36 weeks' gestation preceding a diagnosis of preeclampsia.

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.

A disintegrin and metalloproteinase 12 (ADAM12) is reduced at 36 weeks' gestation in pregnancies destined to deliver small for gestational age infants.

First trimester circulating ADAM12 is reduced in fetal growth restriction (FGR) and preeclampsia. We measured plasma ADAM12 at 36 weeks' gestation preceding diagnosis of term preeclampsia or delivery of a small for gestational age (SGA; birthweight <10th centile) infant in two independent cohorts (Cohort 1 90 SGA, 41 preeclampsia, 862 controls; Cohort 2121 SGA 23 preeclampsia; 190 controls). ADAM12 was reduced with SGA in both cohorts (p = 0.0015 and 0.011 respectively), and further reduced with birthweight <5th centile (p = 0.0013 and 0.0058 respectively). This validates ADAM12 as an SGA biomarker near term. Circulating ADAM12 preceding preeclampsia was not consistently altered.

Placental growth factor is negatively regulated by epidermal growth factor receptor (EGFR) signaling.

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.

Circulating Growth Differentiation Factor 15 Is Increased Preceding Preeclampsia Diagnosis: Implications as a Disease Biomarker.

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.

Circulating syndecan-1 is reduced in pregnancies with poor fetal growth and its secretion regulated by matrix metalloproteinases and the mitochondria.

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.

Elevated Circulating and Placental SPINT2 Is Associated with Placental Dysfunction.

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.

Maternal circulating SPINT1 is reduced in small-for-gestational age pregnancies at 26 weeks: Growing up in Singapore towards health outcomes (GUSTO) cohort study.

Fetal growth restriction arising from placental insufficiency is a leading cause of stillbirth. We recently identified low maternal circulating SPINT1 concentrations as a novel biomarker of poor fetal growth. Here we measured SPINT1 in a prospective cohort in Singapore. Circulating SPINT1 concentrations were significantly lower among 141 pregnant women destined to deliver small-for-gestational age infants (birthweight <10th centile), compared to 772 controls (p < 0.01) at as early as 26 weeks' gestation. There were no correlations between infant body composition and circulating SPINT1 concentrations at 26 weeks. This provides validation that low maternal SPINT1 concentration is associated with poor fetal growth.

Circulating Tissue Factor Pathway Inhibitor (TFPI) is increased preceding preeclampsia diagnosis and in established preeclampsia.

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.

MicroRNAs 363 and 149 are differentially expressed in the maternal circulation preceding a diagnosis of preeclampsia.

Preeclampsia is a pregnancy complication associated with angiogenic dysbalance, maternal endothelial dysfunction and end-organ injury. A predictive test to identify those who will develop preeclampsia could substantially decrease morbidity and mortality. MicroRNAs (miRs) are small RNA molecules involved in post-transcriptional gene regulation. We screened for circulating miRs differentially expressed at 36 weeks' gestation in pregnancies before the development of preeclampsia. We used a case-control group (198 controls, 34 pre-preeclampsia diagnosis) selected from a prospective cohort (n = 2015) and performed a PCR-based microarray to measure the expression of 41 miRs. We found six circulating miRs (miRs 363, 149, 18a, 1283, 16, 424) at 36 weeks' had significantly reduced expression (p < 0.0001-0.04). miR363 was significantly downregulated at 28 weeks' gestation, 10-12 weeks before the onset of clinical disease. In the circulation of another cohort of 34 participants with established preterm preeclampsia (vs 23 controls), we found miRs363, 18a, 149 and 16 were significantly down regulated (p < 0.0001-0.04). Combined expression of miRs149 and 363 in the circulation at 36 weeks' gestation provides a test with 45% sensitivity (at a specificity of 90%) which suggests measuring both miRs may have promise as part of a multi-marker test to predict preeclampsia.

Esomeprazole and sulfasalazine in combination additively reduce sFlt-1 secretion and diminish endothelial dysfunction: potential for a combination treatment for preeclampsia.

Development and repurposing of therapies that show promise in the prevention or treatment of preeclampsia would be a major advance for the obstetrics field. We recently identified esomeprazole and sulfasalazine as potential candidates for the treatment of preeclampsia. Both reduce placental and endothelial secretion of sFlt-1 and sENG and mitigate endothelial dysfunction in vitro. Here we assessed whether esomeprazole and sulfasalazine in combination would additively attenuate the elevated release of anti-angiogenic factors and markers of endothelial dysfunction, key characteristics of preeclampsia. Primary placental tissue and cells, and primary endothelial cells were treated with esomeprazole and sulfasalazine alone and in combination. We assessed secretion of sFlt-1 and sENG and performed in vitro assays of endothelial dysfunction. Combining esomeprazole and sulfasalazine in lower concentrations caused an additive reduction in sFlt-1 secretion in primary cytotrophoblasts, placental explants and endothelial cells. No additive reduction was observed in sENG secretion when esomeprazole and sulfasalazine were combined. Together, esomeprazole and sulfasalazine additively reduced TNF-α-induced VCAM and ET-1 mRNA expression, and monocyte adhesion to endothelial cells. In conclusion, combining esomeprazole and sulfasalazine additively reduced secretion of sFlt-1 and markers of endothelial dysfunction. Combined administration of esomeprazole and sulfasalazine may provide a more effective treatment or prevention for preeclampsia compared to either as single agents.

Circulating SPINT1 is a biomarker of pregnancies with poor placental function and fetal growth restriction.

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 3rd, 5th, 10th and 20th 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.