Expression and function of macrophage-inducible C-type lectin (Mincle) in inflammation driven parturition in fetal membranes and myometrium.

The pivotal role of inflammatory processes in human parturition is well known, but not completely understood. We have performed a study to examine the role of macrophage-inducible C-type lectin (Mincle) in inflammation-associated parturition. Using human samples, we show that spontaneous labour is associated with up-regulated Mincle expression in the myometrium and fetal membranes. Mincle expression was also increased in fetal membranes and myometrium in the presence of pro-labour mediators, the proinflammatory cytokines interleukin (IL)-1B and tumour necrosis factor (TNF), and Toll-like receptor (TLR) ligands fsl-1, poly(I:C), lipopolysaccharide (LPS) and flagellin. These clinical studies are supported by mouse studies, where an inflammatory challenge in a mouse model of preterm birth increased Mincle expression in the uterus. Importantly, elimination of Mincle decreased the effectiveness of proinflammatory cytokines and TLR ligands to induce the expression of pro-labour mediators; namely, proinflammatory cytokines and chemokines, contraction-associated proteins and prostaglandins, and extracellular matrix remodelling enzymes, matrix metalloproteinases. The data presented in this study suggest that Mincle is required when inflammatory activation precipitates parturition.

A mobile health intervention promoting healthy gestational weight gain for women entering pregnancy at a high body mass index: the txt4two pilot randomised controlled trial.

OBJECTIVE: To determine the feasibility and effectiveness of an mHealth intervention promoting healthy diet, physical activity and gestational weight gain in pregnant women. DESIGN: Randomised controlled trial (RCT). SETTING: Australian tertiary obstetric hospital. POPULATION: One hundred pregnant women who were overweight or obese prior to pregnancy. METHODS: Women recruited at the first antenatal clinic visit were randomised to either an intervention or a control group. The intervention consisted of a tailored suite of strategies delivered (from first antenatal visit until 36 weeks' gestation) via multiple modalities available on mobile devices. MAIN OUTCOME MEASURES: The primary outcome was intervention feasibility and secondary outcomes were objectively measured changes in gestational weight gain (GWG) and self-reported dietary intake and physical activity. RESULTS: Ninety-one women completed the study. Delivery to protocol provides evidence of program feasibility. Most women engaged regularly with the program, with the majority (97.6%) reporting that the intervention was helpful. Secondary outcomes demonstrated a significantly lower GWG in the intervention group (7.8 kg ± 4.7 versus 9.7 kg ± 3.9; P =0.041) compared with the control group at intervention completion. Intervention group women reported significantly smaller reductions in total, light- and moderate-intensity physical activity from baseline to completion of the intervention (P = 0.001) compared with the control group, but no differences in consumption frequencies of key food groups. CONCLUSION: An intervention that aimed to deliver healthy diet, physical activity and GWG guidance utilising innovative technology can be feasibly implemented and produce positive physical activity and GWG outcomes. TWEETABLE ABSTRACT: txt4two mHealth study improved gestational weight gain and physical activity in pregnant women with high BMIs.

Postpartum IGF-I and IGFBP-2 levels are prospectively associated with the development of type 2 diabetes in women with previous gestational diabetes mellitus.

AIMS: Women with previous gestational diabetes mellitus (GDM) are at greater risk of developing type 2 diabetes. In the general population, the insulin-like growth factor (IGF) system has been implicated in the development of type 2 diabetes. The aim of this study was to determine if circulating IGF-I, IGF-II, IGFBP-1 and IGFBP-2 levels 12weeks following a GDM pregnancy are associated with an increased risk of developing type 2 diabetes. METHODS: IGF-I, IGF-II, IGFBP-1 and IGFBP-2 levels were measured in 98 normal glucose tolerant women, 12weeks following an index GDM pregnancy using enzyme immunoassay. Women were assessed for up to 10years for the development of overt type 2 diabetes. RESULTS: Among the 98 women with previous GDM, 21 (21%) developed diabetes during the median follow-up period of 8.5years. After adjusting for age and BMI, IGF-I and IGFBP-2 were significantly associated with the development of type 2 diabetes. In a clinical model of prediction of type 2 diabetes that included age, BMI, pregnancy fasting glucose and postnatal fasting glucose, the addition of IGF-I and IGFBP-2 resulted in an improvement in the net reclassification index of 17.8%. CONCLUSIONS: High postpartum IGF-I and low postpartum IGFBP-2 levels are a significant risk factor for the development of type 2 diabetes in women with a previous history of GDM. This is the first report that identifies IGF-I and IGFBP-2 as a potential biomarker for the prediction of type 2 diabetes in women with a history of GDM.

Self-weighing and simple dietary advice for overweight and obese pregnant women to reduce obstetric complications without impact on quality of life: a randomised controlled trial.

OBJECTIVE: To determine the effect of serial weighing and dietary advice compared with standard antenatal care on obstetric outcomes. DESIGN: Randomised controlled clinical trial. SETTING: Australian tertiary obstetric hospital. POPULATION: Three hundred and eighty-two overweight or obese non-diabetic pregnant women at less than 20 weeks gestation with a singleton pregnancy. METHODS: Women were randomised to targeted, serial self-weighing and simple dietary advice, (intervention), or standard antenatal care (control). MAIN OUTCOMES MEASURES: The primary outcome was a reduction in a composite of obstetric complications: gestational hypertension, pre-eclampsia, diabetes, assisted or caesarean birth, shoulder dystocia, severe perineal trauma, postpartum haemorrhage and maternal high dependency care. Secondary outcomes were gestational weight gain at 36 weeks' gestation, quality of life (QOL) and maternal serum levels of 28-week leptin, adiponectin and C-reactive protein (CRP). RESULTS: There was no difference in the rate of the primary composite outcome of obstetric complications: 124/184 (67% control), 124/187 (66% intervention) [relative risk 0.98 (95% confidence interval (CI) 0.85-1.14)]. There was no difference in mean gestational weight gain [-0.9 kg (95% CI -2.0, 0.25)], QOL or leptin, adiponectin or CRP levels between intervention and control groups. CONCLUSIONS: This low-cost, pragmatic intervention failed to prevent obstetric complications or modify maternal biochemistry or gestational weight gain in overweight or obese pregnant women. Participation in the study did not impair participants' QOL. TWEETABLE ABSTRACT: Serial self-weighing and dietary advice failed to reduce obstetric complications in overweight pregnant women.

Decreased STAT3 in human idiopathic fetal growth restriction contributes to trophoblast dysfunction.

Abnormal trophoblast function is associated with fetal growth restriction (FGR). The JAK-STAT pathway is one of the principal signalling mechanisms by which cytokines and growth factors modulate cell proliferation, differentiation, cell migration and apoptosis. The expression of placental JAK-STAT genes in human idiopathic FGR is unknown. In this study, we propose the hypothesis that JAK-STAT pathway genes are differentially expressed in idiopathic FGR-affected pregnancies and contribute to abnormal feto-placental growth by modulating the expression of the amino acid transporter SNAT2, differentiation marker CGB/human chorionic gonadotrophin beta-subunit (ß-hCG) and apoptosis markers caspases 3 and 8, and TP53. Expression profiling of FGR-affected placentae revealed that mRNA levels of STAT3, STAT2 and STAT5B decreased by 69, 52 and 50%, respectively, compared with gestational-age-matched controls. Further validation by real-time PCR and immunoblotting confirmed significantly lower STAT3 mRNA and STAT3 protein (total and phosphorylated) levels in FGR placentae. STAT3 protein was localised to the syncytiotrophoblast (ST) in both FGR and control placentae. ST differentiation was modelled by in vitro differentiation of primary villous trophoblast cells from first-trimester and term placentae, and by treating choriocarcinoma-derived BeWo cells with forskolin in cell culture. Differentiation in these models was associated with increased STAT3 mRNA and protein levels. In BeWo cells treated with siRNA targeting STAT3, the mRNA and protein levels of CGB/ß-hCG, caspases 3 and 8, and TP53 were significantly increased, while that of SNAT2 was significantly decreased compared with the negative control siRNA. In conclusion, we report that decreased STAT3 expression in placentae may contribute to abnormal trophoblast function in idiopathic FGR-affected pregnancies.

Activation of AMPK in human fetal membranes alleviates infection-induced expression of pro-inflammatory and pro-labour mediators.

INTRODUCTION: In non-gestational tissues, the activation of adenosine monophosphate (AMP)-activated kinase (AMPK) is associated with potent anti-inflammatory actions. Infection and/or inflammation, by stimulating pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9, play a central role in the rupture of fetal membranes. However, no studies have examined the role of AMPK in human labour. METHODS: Fetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and after preterm pre-labour rupture of membranes (PPROM). AMPK activity was assessed by Western blotting of phosphorylated AMPK expression. To determine the effect of AMPK activators on pro-inflammatory cytokines, fetal membranes were pre-treated with AMPK activators then stimulated with bacterial products LPS and flagellin or viral dsDNA analogue poly(I:C). Primary amnion cells were used to determine the effect of AMPK activators on IL-1ß-stimulated MMP-9 expression. RESULTS: AMPK activity was decreased with term labour. There was no effect of preterm labour. AMPK activity was also decreased in preterm fetal membranes, in the absence of labour, with PROM compared to intact membranes. AMPK activators AICAR, phenformin and A769662 significantly decreased IL-6 and IL-8 stimulated by LPS, flagellin and poly(I:C). Primary amnion cells treated with AMPK activators significantly decreased IL-1ß-induced MMP-9 expression. DISCUSSION: The decrease in AMPK activity in fetal membranes after spontaneous term labour and PPROM indicates an anti-inflammatory role for AMPK in human labour and delivery. The use of AMPK activators as possible therapeutics for threatened preterm labour would be an exciting future avenue of research.

Insulin-like growth factor-binding protein 1 and 7 concentrations are lower in obese pregnant women, women with gestational diabetes and their fetuses.

OBJECTIVE: To determine the effect of pre-existing maternal obesity and gestational diabetes mellitus (GDM) on the circulating levels of insulin growth factor-binding protein (IGFBPs) in cord and maternal plasma. STUDY DESIGN: IGFBP-1-7 levels were measured on maternal and cord plasma from women with normal glucose tolerance (NGT) (30 non-obese and 36 obese) and GDM (44 non-obese and 26 obese) at the time of term elective cesarean section. RESULT: Maternal plasma IGFBP-1, IGFBP-6 and IGFBP-rP1 concentrations were significantly lower in NGT obese compared with NGT non-obese women and in non-obese GDM women compared with non-obese NGT women. In cord plasma, IGFBP-1-3 and IGFBP-rP1 concentrations were significantly lower in NGT obese compared with NGT non-obese women and in non-obese GDM women compared with non-obese NGT women. Significant positive correlations were observed between maternal and cord plasma IGFBP-1 and IGFBP-rP1 levels and maternal insulin resistance. In cord plasma, significant positive correlations were observed between IGFBP-1-3 and IGFBP-rP1 levels and fetal insulin resistance. Fetal birthweight was inversely correlated with maternal plasma IGFBP-1 levels and cord plasma IGFBP-1 and IGFBP-2 levels. When corrected for maternal body mass index, the only significant relationship that still existed was between cord plasma IGFBP-1 concentrations and fetal birthweight. CONCLUSION: At the time of term cesarean section, pre-existing maternal obesity and GDM are associated with lower IGFBP levels in maternal and cord plasma. Alterations in circulating IGF and IGFBPs may alter birthweight and/or neonatal adiposity. This may lead to alterations in optimal growth trajectory and lead to metabolic disorders later in life.

Myostatin in the placentae of pregnancies complicated with gestational diabetes mellitus.

INTRODUCTION: Gestational diabetes mellitus (GDM) is characterised by maternal glucose intolerance and insulin resistance during pregnancy. Myostatin, initially identified as a negative regulator of muscle development may also function in the regulation of placental development and glucose uptake. Myostatin expression in placentae of GDM complicated pregnancies is unknown. However, higher myostatin levels occur in placentae of pregnancies complicated with preeclampsia. We hypothesise that myostatin will be differentially expressed in GDM complicated pregnancies. METHODS: Myostatin concentrations (ELISA) were evaluated in plasma of presymptomatic women who later developed GDM and compared to plasma of normal glucose tolerant (NGT) women. Furthermore, myostatin protein expression (Western blot) was studied in placentae of pregnant women with GDM (treated with diet or insulin) compared to placentae of NGT women. RESULTS: No significant difference in myostatin concentration was seen in plasma of pre-symptomatic GDM women compared to NGT women. In placenta significant differences in myostatin protein expressions (higher precursor; p < 0.05and lower dimer: p < 0.005) were observed in GDM complicated compared to NGT pregnancies. Furthermore, placentae of GDM women treated with insulin compared to diet have higher dimer (p < 0.005) and lower precursor (p < 0.05). Compared to lean women, placentae of obese NGT women were lower in myostatin dimer expression (p < 0.05). DISCUSSION: Myostatin expression in placental tissue is altered under stress conditions (e.g. obesity and abnormal glucose metabolism) found in pregnancies complicated with GDM. We hypothesise that myostatin is active in these placentae and could affect glucose homoeostasis and/or cytokine production thereby altering the function of the placenta.

The transcription factor Nrf2 is decreased after spontaneous term labour in human fetal membranes where it exerts anti-inflammatory properties.

INTRODUCTION: Inflammation plays a central role in the terminal processes of human labour and delivery, including the rupture of fetal membranes. Recent studies show a role for the transcription factor Nrf2 (NF-E2-related factor 2) in regulating inflammation. The aims of this study were to determine the effect of human spontaneous term and preterm labour on Nrf2 expression in fetal membranes; and Nrf2 siRNA knockdown on pro-inflammatory cytokines. METHODS: Fetal membranes, from term and preterm, were obtained from non-labouring and labouring women. Primary amnion cells were used to determine the effect of Nrf2 siRNA knockdown on pro-inflammatory cytokines in the presence of inflammation or infection. RESULTS: Nrf2 mRNA expression and nuclear protein expression were significantly decreased after spontaneous term labour and delivery. There was, however, no effect of spontaneous preterm labour and delivery on Nrf2 mRNA expression and nuclear protein expression. On the other hand, Nrf2 gene expression was significantly lower in fetal membranes obtained from women at preterm with histologic chorioamnionitis compared to fetal membranes obtained from women at preterm without histologic chorioamnionitis. Nrf2 silencing by siRNA in primary amnion cells was associated with a significant increase in IL-6 and IL-8 mRNA expression and release induced by IL-1ß, TNF-α, flagellin and poly(I:C). DISCUSSION: Nrf2 has an anti-inflammatory effect in human fetal membranes. It is decreased with term labour and preterm chorioamnionitis; and Nrf2 silencing increases inflammation- and infection-induced pro-inflammatory cytokines. Further studies are required to determine if agents that can increase Nrf2 expression may be a potential therapeutic strategy in the treatment and management of infection-induced preterm labour.

The NR4A receptors Nurr1 and Nur77 are increased in human placenta from women with gestational diabetes.

INTRODUCTION: Members of the NR4A subfamily are involved in a wide range of diseases including obesity and diabetes. The aim of this study was to determine the effect of maternal obesity and gestational diabetes mellitus (GDM) on the expression of the NR4A receptors Nurr1, Nur77 and NOR1. METHODS: Human placenta was obtained at the time of term Caesarean section from (i) lean and obese and normal glucose tolerant (NGT) pregnant women; and (ii) women with GDM and BMI-matched NGT women (n = 16 patients). Primary trophoblast cells, isolated from human term placenta, were used to determine the effect of pro-inflammatory cytokines on NR4A protein expression. Primary trophoblast cells were also used to assess the effect of Nurr1, Nur77 and NOR1 siRNA knockdown on pro-inflammatory cytokines. RESULTS: There was no effect of pre-existing maternal obesity on Nurr1, Nur77 or NOR1 expression. Nurr1 and Nur77 expression were significantly higher in GDM placenta compared to NGT placenta, and in the presence of the pro-inflammatory cytokines TNF-α and IL-ß in primary trophoblast cells. Knockdown of Nurr1 and Nur77 in human primary trophoblast cells significantly decreased TNF-α induced expression and secretion of IL-6 and IL-8. DISCUSSION: Nurr1 and Nur77, which were increased in human placenta from women with GDM, are involved in TNF-α induced-expression of pro-inflammatory cytokines. Pro-inflammatory cytokines are known to play a role in placental nutrient transport. Thus, the regulation of pro-inflammatory cytokines by Nurr1 and Nur77 suggest that these proteins may play a role in placental function and transport mechanisms.

Cellular inhibitors of apoptosis (cIAP) 1 and 2 are increased in placenta from obese pregnant women.

INTRODUCTION: Independent of their role in apoptosis, cellular inhibitors of apoptosis (cIAP) 1 and 2, have emerged as regulators of inflammation. Obesity in pregnancy is characterised by maternal and placental inflammation. Thus, the aim of this study was to determine the effect of maternal obesity and pro-inflammatory mediators on cIAP expression in human placenta. METHODS: The expression of cIAP was assessed in human placenta from lean (n = 15) and obese (n = 14) patients by qRT-PCR and Western blotting. Primary trophoblast cells were used to determine the effect of pro-inflammatory cytokines on cIAP expression, and the effect of cIAP siRNA on pro-inflammatory cytokines. RESULTS: cIAP1 and cIAP2, gene and protein expression were significantly higher in placenta from women with pre-existing maternal obesity compared to placenta form lean women. Additionally, bacterial endotoxin LPS and the pro-inflammatory cytokines tumour necrosis factor (TNF)-α and interleukin (IL)-1ß significantly increased the expression of both cIAP1 and cIAP2 in primary trophoblast cells isolated from human term placenta. Knockdown of cIAP1 or cIAP2 in human primary trophoblast cells significantly decreased TNF-α induced expression and secretion of pro-inflammatory cytokines IL-6 and IL-8 and of matrix metalloproteinase (MMP)-9. DISCUSSION: cIAP1 and cIAP2 expression is increased in placenta from women with pre-existing maternal obesity and in response to treatment with pro-inflammatory cytokines. Functional studies in placental trophoblast cells revealed that cIAPs are involved in TNF-α induced-expression of pro-inflammatory cytokines. Given the central role of pro-inflammatory cytokines in placental nutrient transport, this data suggest that cIAP1 and cIAP2 may play a role in fetal growth and development.

The effect of pre-existing maternal obesity and diabetes on placental mitochondrial content and electron transport chain activity.

INTRODUCTION: Mitochondria dysfunction has been extensively implicated in the progression of these metabolic disorders, their role in placental tissue of diabetic and/or obese pregnant women is yet to be investigated. The aim of this study was to determine the effect of pre-existing type 1 and type 2 diabetes mellitus (DM), and pre-existing maternal obesity on placental mitochondrial function as assessed by mitochondrial content, electron transport chain (ETC) complex activities and oxidative stress. METHODS: Human placenta was obtained at the time of term Caesarean section from (i) non-obese (n = 19) and obese (n = 23) normal glucose tolerant (NGT) pregnant women; (ii) women with type 1 DM (n = 14) and BMI-matched NGT women (n = 14); and (iii) women with type 2 DM (n = 11) and BMI-matched NGT women (n = 11). The following endpoints were assessed: placental mitochondrial content by citrate synthase activity and mitochondrial DNA (mtDNA content); mitochondrial respiratory chain activity (complexes I, II, II & III, III and IV), and mitochondrial ROS (as assessed by mitochondrial hydrogen peroxide (H2O2) levels). RESULTS: When compared to placenta from NGT non-obese women, there was significantly lower mitochondrial DNA (mtDNA) content and electron transport chain complex I activity, and significantly higher mitochondrial H2O2 levels in placenta from NGT obese women (P < 0.05). Placental tissue from type 1 DM women showed significant reductions in ETC complex I, II & III, and III activity and increased H2O2 levels when compared to BMI-matched NGT women (P < 0.05). Type 2 DM women only exhibited significantly reduced ETC complex II & III activity when compared to BMI-matched NGT women (P < 0.05). DISCUSSION AND CONCLUSIONS: Women with pre-existing obesity or diabetes have decreased placental mitochondrial respiratory chain enzyme activities which may have detrimental consequences on placental function and therefore fetal growth and development.

Inverse relationship between gestational weight gain and glucose uptake in human placenta from female foetuses.

BACKGROUND: Maternal obesity and gestational weight gain (GWG) have a significant impact on the in utero environment, and thus on foetal development and the health of the offspring later in life. OBJECTIVE: The aim of this study was to determine the effect of maternal pre-existing obesity and maternal GWG on glucose uptake from placentas from male and female offspring. METHODS: Total glucose uptake was measured in placental explants using radio-labelled glucose. RESULTS: In the female placentas (n = 36), GWG and glucose uptake were significantly negatively correlated (r = -0.7, P < 0.0001; n = 36), and customized birthweight centile correlated with placental glucose uptake (r = 0.36, P = 0.03) but not GWG. In the male placentas (n = 45), GWG and glucose uptake were not related, and customized birthweight centile correlated with GWG (r = 0.34, P = 0.02; n = 45), but not placental glucose uptake. CONCLUSIONS: The female placenta can adapt glucose uptake in the face of excessive GWG. The male placenta showed no evidence of changing glucose uptake in response to maternal GWG.

The expression of the let-7 miRNAs and Lin28 signalling pathway in human term gestational tissues.

INTRODUCTION: Labour and delivery are processes associated with inflammation within intrauterine and cervical tissues. The mechanisms that induce labour-associated changes and, in particular, the role of microRNAs (miRNAs) remain to be elucidated. MiRNAs are small non-coding RNAs that repress gene expression via mRNA degradation and translational repression. Let-7 miRNAs are negatively regulated by RNA-binding protein, Lin28, and both function downstream of NF-κB signalling. In non-gestational tissues, let-7 and Lin28 reportedy function as negative and positive regulators of IL-6 expression. We hypothesised that labour-associated inflammation involves the downregulation of let-7 miRNAs and upregulation of Lin28 expression. AIM: To determine the expression of Lin28 protein and let-7 miRNA in human gestational tissue obtained before and after labour. METHOD: Gestational tissues were collected from women at term by Caesarean section with and without labour and following normal vaginal delivery (n = 6 per group). Protein and RNA was extracted and Lin28 and let-7 miRNA expression was measured by Western blotting and real-time PCR. RESULTS: The data obtained established that let-7 miRNA and Lin28 display tissue-specific expression: Lin28 was strongly expressed in the placenta and choriodecidua, but not measurable in amnion; and let-7b and -7c expression were significantly lower in choriodecidua compare to placenta and amnion, whereas the amnion expressed less let-7d and -7f than other tissues. CONCLUSION: While the expression of Lin28 protein and let-7 miRNA did not vary significantly with labour onset and delivery, changes in their bioactivity and impact on nuclear signalling pathways in human gestational tissues remain to be established.

Circulating RNA coding genes regulating apoptosis in maternal blood in severe early onset fetal growth restriction and pre-eclampsia.

OBJECTIVE: To determine whether the intrinsic apoptosis pathway is differentially expressed in placenta and maternal blood in severe preterm fetal growth restriction (FGR) and pre-eclampsia (PE), and to examine whether circulating RNA in maternal blood may be potential biomarkers. STUDY DESIGN: Maternal blood samples and placental biopsies were collected from women with preterm: FGR (n=20), PE without FGR (n=8) and controls (n=20). Real-time PCR examined the expression of genes in the intrinsic apoptosis pathway in FGR and PE, stratified according to the severity of placental insufficiency. RESULT: Severe preterm FGR, with or without PE, was associated with increased expression of BCL2, BCL-XL, BIM, BAD and Survivin in both the placenta and maternal blood (1.6 to 3.3-fold, P<0.05). In preterm PE, but not FGR, there was increased placental expression of BCL-XL and BCL2 (1.6 to 2.5-fold, P<0.05), but only BCL2 was significantly increased in the maternal blood (1.8-fold, P<0.05). Increased expression of genes of the intrinsic apoptosis pathway reflected the severity of FGR as determined by deteriorations in umbilical artery Doppler velocimetry. CONCLUSION: In severe early onset FGR there was increased expression of genes regulating intrinsic apoptosis in both the placenta and maternal blood. Circulating RNA regulating placenta apoptosis may be used to develop noninvasive novel biomarkers for FGR.

Increased oxidative stress in human fetal membranes overlying the cervix from term non-labouring and post labour deliveries.

Enzymatic breakdown of the collagen-rich extracellular matrix (ECM) that connects the amnion and chorion layers of the fetal membranes is one of the key events leading to rupture of membranes. Oxidant stress caused by increased formation of reactive oxygen species and/or reduced antioxidant capacity may predispose to membrane rupture, a major cause of preterm birth. The aim of this study was to determine the effect of human labour and supracervical (SC) apposition on antioxidant enzymes and 8-isoprostane (a marker of lipid peroxidation). To determine the effect of human labour on oxidative stress status, fetal membranes from the SC site (SCS) were collected from women at term Caesarean section (no labour), and from the site of membrane rupture (SOR) after spontaneous labour onset and delivery (post labour). To determine the effect of SC apposition on oxidative stress status, amnion was collected from the SCS and a distal site (DS) in women at term Caesarean section in the absence of labour. The release of 8-isoprostane was significantly higher in amnion from the SCS compared to DS, and in fetal membranes from the SOR compared to the SCS. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were lower in amnion from the SC compared to DS. SOD gene expression and enzyme activity were lower in fetal membranes after labour. There was no difference in expression or activity in catalase, GPx and glutathione reductase (GSR) between no labour and post labour fetal membranes. In primary amnion cells, SOD supplementation significantly augmented IL-1ß induced MMP-9 expression and activity. In summary, non-labouring SC fetal membranes are characterised by reduced antioxidant enzyme activity when compared to distal membranes, and, as such, may be more susceptible to oxidative damage and thus membrane rupture.

Human labour is associated with decreased cytoplasmic FoxO4.

Forkhead box O (FoxO) proteins function primarily as transcription factors in the nucleus where they bind to their cognate DNA targeting sequences. FoxO regulated genes include those involved in cellular stress responses, inflammation and apoptosis; all of which are involved in the processes of human labour and delivery. We have previously identified Forkhead box O4 (FoxO4) proteins in human gestational tissues; there is, however, no data is available on the role of FoxO4 in the processes of human labour and delivery. Thus the aim of this study was to determine the effect of (i) human labour, preterm chorioamnionitis and pro-inflammatory stimuli on the expression of FoxO4 in human placenta and fetal membranes; and (ii) FoxO4 knockdown by siRNA on the expression of pro-labour mediators. Quantitative RT-PCR (qRT-PCR), immunohistochemistry and/or Western blotting was used to analyse the expression of FoxO4 (n = 6 per group). Human labour and preterm chorioamnionitis significantly decreased cytoplasmic FoxO4 expression in placenta and/or choriodecidua. Knockdown of FoxO4 mRNA and protein in JEG-3 cells using siRNA was associated with decreased COX-2 mRNA expression concomitant with lower PGF(2α) secretion. However, in BeWo cells, siRNA inhibition of FoxO4 was not associated with inflammation, oxidative stress or apoptosis. In summary, human term labour and chorioamnionitis is characterised by lower FoxO4 mRNA and/or protein expression in placenta and/or choriodecidua. Although the exact role of FoxO4 in human pregnancy remains to be fully elucidated, our data demonstrate that it can regulate COX-2 expression and subsequent prostaglandin expression.

MAPK and AP-1 proteins are increased in term pre-labour fetal membranes overlying the cervix: regulation of enzymes involved in the degradation of fetal membranes.

Fetal membranes overlying the cervix (i.e. supracervical site, SCS) are characterised by increased extracellular matrix (ECM) degradation. In non-gestational tissues, the mitogen activated protein kinase (MAPK) and activator protein (AP)-1 family are involved in the regulation of the ECM degrading enzyme metalloproteinase (MMP)-9. The aims of this study were (i) to compare the expression of AP-1 proteins in fetal membranes from the SCS and a distal site (DS), and (ii) determine if the MAPK/AP-1 pathway is involved in the regulation of MMP-9. Fetal membranes overlying the cervix were identified in situ in women undergoing term elective Caesarean section. Immunohistochemistry (n = 6) was used to localise the expression of the MAPK proteins ERK (total and phosphorylated), JNK (total and phosphorylated) and p38 MAPK (total and phosphorylated), and the AP-1 proteins JunB, cJun (total and phosphorylated), JunD, cFos and FosD. There was no difference in JNK, p38 MAPK, FosB, cJun and JunD protein expression between SC and distal fetal membranes. However, when compared to DS, the intensity and/or extent of staining of ERK, p-ERK, p-JNK, p-p38 MAPK, cFos, JunB and p-cJun were greater in amnion and chorion obtained from the SCS. In order to elucidate a role for these proteins in ECM degradation, pharmacological inhibitors of MAPK protein activation were utilised in primary amnion cells. The ERK inhibitor U0126, JNK inhibitor SP600125 and p38 MAPK inhibitor SB202190 all significantly decreased IL-ß-induced MMP-9 gene expression and pro MMP-9 in human primary amnion cells. In summary, at term, non laboured SC fetal membranes are characterised by increased expression of MAPK and AP-1 proteins. MMP-9 expression and production was significantly suppressed by inhibitors of three key enzymes in the signalling cascades leading to AP-1 formation, ERK 1/2, JNK and p38 MAPK. Thus, the MAPK/AP-1 pathway may play a role in the degradation of the ECM at the SCS making it more susceptible to membrane rupture.

Lower circulating levels of complement split proteins C3a and C4a in maternal plasma of women with gestational diabetes mellitus.

AIM: The aim of this study was to determine the effect of gestational diabetes mellitus on maternal circulating levels of C3a, C4a and C5a. METHODS: Anaphylatoxin C3a, C4a and C5a levels were measured in maternal and cord plasma from 42 women with normal glucose tolerance and 40 women with gestational diabetes at the time of term elective Caesarean section. Maternal plasma C3a, C4a and C5a concentrations were determined by enzyme-linked immunoassay. RESULTS: Maternal C3a and C4a concentrations were significantly lower in women with gestational diabetes compared with women with normal glucose tolerance at the time of term delivery (P < 0.05, Student's t-test). There was, however, no difference in maternal circulating C5a levels between the two groups. Additionally, there was no difference in C3a, C4a and C5a levels in cord plasma obtained from women with normal glucose tolerance and those with gesational diabetes. However, a significant positive correlation was observed between maternal and cord complement split levels. CONCLUSIONS: Gestational diabetes is characterized by lower levels of C3a and C4a in the maternal circulation at the time of term Caesarean delivery.

Opposing effects of monomeric and pentameric C-reactive protein on endothelial progenitor cells.

C-reactive protein (CRP) has been linked to the pathogenesis of atherosclerosis. The dissociation of native, pentameric (p)CRP to monomeric (m)CRP on the cell membrane of activated platelets has recently been demonstrated. The dissociation of pCRP to mCRP may explain local pro-inflammatory reactions at the site of developing atherosclerotic plaques. As a biomarker, pCRP predicts cardiovascular adverse events and so do reduced levels and function of circulating endothelial progenitor cells (EPCs). We hypothesised that mCRP and pCRP exert a differential effect on EPC function and differentiation. EPCs were treated with mCRP or pCRP for 72 h, respectively. Phenotypical characterisation was done by flow cytometry and immunofluorescence microscopy, while the effect of mCRP and pCRP on gene expression was examined by whole-genome gene expression analysis. The functional capacity of EPCs was determined by colony forming unit (CFU) assay and endothelial tube formation assay. Double staining for acetylated LDL and ulex lectin significantly decreased in cells treated with pCRP. The length of tubuli in a matrigel assay with HUVECs decreased significantly in response to pCRP, but not to mCRP. The number of CFUs increased after pCRP treatment. RNA expression profiling demonstrated that mCRP and pCRP cause highly contradictory gene regulation. Interferon-responsive genes (IFI44L, IFI44, IFI27, IFI 6, MX1, OAS2) were among the highly up-regulated genes after mCRP, but not after pCRP treatment. In conclusion, EPC phenotype, genotype and function were differentially affected by mCRP and pCRP, strongly arguing for differential roles of these two CRP conformations. The up-regulation of interferon-inducible genes in response to mCRP may constitute a mechanism for the local regulation of EPC function.