Neonatal outcomes after introduction of a national intrapartum fetal surveillance education program: a retrospective cohort study.

OBJECTIVE: To determine the impact of a multidisciplinary fetal surveillance education program (FSEP) on term neonatal outcomes. METHODS: A retrospective cohort study of term neonatal outcomes before (1998-2004) and after (2005-2010) introduction of a FSEP. Clinical data was collected for all term infants admitted to a neonatal intensive care unit (NICU) in Australia between 1998 and 2010. Infants with congenital abnormalities were excluded. Neonatal mortality and severe neonatal morbidity (admission to a NICU, respiratory support, hypoxic encephalopathy) were compared before and after the FSEP was introduced. The rates of operative delivery during this time were assessed. RESULTS: There were 3 512 596 live term births between 1998 and 2010. The intrapartum hypoxic death rate at term decreased from 2.02 to 1.07 per 10 000 total births. More neonates were admitted to NICU after 2005 (10.6 versus 14.6 per 10 000 live births), however fewer babies admitted to the neonatal unit had Apgar scores < 5 at five minutes (55.1-45.5%, RR 0.82, 95% CI 0.7-0.87); and rates of hypoxic ischemic encephalopathy fell from 36% to 30% (RR 0.83, 95% CI 0.76-0.90). There was no increase in rates of emergency in labour caesarean sections (11.7% pre versus 11.1% post, RR 0.95, 95% CI 0.95-0.96). CONCLUSIONS: Introduction of a national FSEP was associated with increased neonatal admissions but a reduction in intrapartum hypoxia, without increasing emergency caesarean section rates.

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.

New biomarkers for the prediction of spontaneous preterm labour in symptomatic pregnant women: a comparison with fetal fibronectin.

OBJECTIVE: To identify cervicovaginal fluid (CVF) biomarkers predictive of spontaneous preterm birth in women with symptoms of preterm labour. DESIGN: Retrospective cohort study. SETTING: Melbourne, Australia. POPULATION: Women with a singleton pregnancy admitted to the Emergency Department between 22 and 36 weeks of gestation presenting with symptoms of preterm labour. METHODS: Two-dimensional electrophoresis was used to analyse the CVF proteome. Validation of putative biomarkers was performed using enzyme-linked immunosorbent assay (ELISA) in an independent cohort. Optimal concentration thresholds of putative biomarkers were determined and the predictive efficacy for preterm birth was compared with that of fetal fibronectin. MAIN OUTCOME MEASURES: Prediction of spontaneous preterm labour within 7 days. RESULTS: Differentially expressed proteins were identified by proteomic analysis in women presenting with 'threatened' preterm labour without cervical change who subsequently delivered preterm (n = 12 women). ELISA validation using an independent cohort (n = 129 women) found albumin and vitamin D-binding protein (VDBP) to be significantly altered between women who subsequently experienced preterm birth and those who delivered at term. Prediction of preterm delivery within 7 days using a dual biomarker model (albumin/VDBP) provided 66.7% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 96.7% negative predictive value (NPV), compared with fetal fibronectin yielding 66.7, 87.9, 36.4 and 96.2%, respectively (n = 64). Using the maximum number of screened samples, the predictive utility of albumin/VDBP yielded a sensitivity of 77.8%, specificity and PPV of 100% and NPV of 98.0% (n = 109). CONCLUSIONS: The dual biomarker model of albumin/VDBP is more efficacious than fetal fibronectin in predicting spontaneous preterm delivery in symptomatic women within 7 days. A clinical diagnostic trial is required to test this model on a larger population to confirm these findings and to further refine the predictive values.

Haemodynamics in women with untreated pre-eclampsia.

This study aimed to compare the haemodynamics in healthy pregnant women with the haemodynamics in women with untreated pre-eclampsia, to determine the cardiovascular reason for hypertension in pre-eclampsia. 40 women with untreated pre-eclampsia, 40 matched healthy pregnant women and 20 non-pregnant women were studied using transthoracic echocardiography. Untreated pre-eclampsia demonstrated (mean (SD), healthy non-pregnant vs healthy pregnant vs untreated pre-eclampsia) increased cardiac output (3400 (752) vs 4109 (595) vs 4789 (1416) ml.min(-1), p=0.002), increased stroke volume (53 (10) vs 53 (8) vs 59 (13) ml, p=0.04), increased fractional shortening (35 (5) vs 35 (7) vs 41 (8) %, p=0.006), increased fractional area change (57 (7) vs 57 (9) vs 65 (9)%, p=0.002) and increased systemic vascular resistance (2116 (457) vs 1613 (315) vs 2016 (625) dyne.s.cm(-5), p=0.001). Mitral E/septal e' was higher (6.0 (1.1) vs 6.7 (1.3) vs 10.4 (2.4), p=0.002) and left atrial size increased (3.2 (0.3) vs 3.8 (0.4) vs 4.0 (0.4) cm, p=0.002). Hypertension in untreated pre-eclampsia is due to increased cardiac output and mild vasoconstriction, with increased inotropy and reduced diastolic function.

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.

Fulminant herpes simplex virus hepatic failure in pregnancy requiring liver transplantation.

Herpes simplex virus hepatitis is a rare but potentially fatal condition that usually affects the immunocompromised, including pregnant women. This case report details the course of fulminant hepatic failure in a woman at 31 weeks gestation resulting in emergent delivery of the fetus and liver transplant in the mother.

Expression and localisation of FoxO3 and FoxO4 in human placenta and fetal membranes.

Forkhead box O (FoxO) proteins regulate inflammation, extracellular matrix (ECM) remodelling and apoptosis. We have previously identified FoxO1 proteins in human gestational tissues, and demonstrated a link between FoxO1 and rupture of fetal membranes. There is, however, no data available on the expression and localisation of FoxO3 and FoxO4 in human intrauterine tissues. Thus the aim of this study was to characterise the localisation and expression of FoxO3 and FoxO4 in (i) human placenta and fetal membranes before term spontaneous labour onset, and (ii) supracervical site (SCS) and distal site (DS) fetal membranes from non-labouring women. Immunohistochemistry, Western blotting and quantitative RT-PCR (qRT-PCR) was used to localise and quantitate FoxO3 and FoxO4 protein and mRNA expressions. Cytoplasmic and nuclear FoxO3 was localised in the syncytiotrophoblast layer, chorionic trophoblasts, amnion epithelium and decidua. Cytoplasmic FoxO4 was localised in the syncytiotrophoblasts and chorionic trophoblasts. No or very little FoxO4 protein and mRNA was present in amnion epithelium. The intensity and extent of staining of FoxO3 and FoxO4 was greater in fetal membranes obtained from the SCS compared to DS. Presence of FoxO3 and FoxO4 are expected to contribute to apoptosis and/or cell cycle regulation associated with fetal membrane rupture.

Association between severe pandemic 2009 influenza A (H1N1) virus infection and immunoglobulin G(2) subclass deficiency.

BACKGROUND: . Severe pandemic 2009 influenza A virus (H1N1) infection is associated with risk factors that include pregnancy, obesity, and immunosuppression. After identification of immunoglobulin G(2) (IgG(2)) deficiency in 1 severe case, we assessed IgG subclass levels in a cohort of patients with H1N1 infection. METHODS: Patient features, including levels of serum IgG and IgG subclasses, were assessed in patients with acute severe H1N1 infection (defined as infection requiring respiratory support in an intensive care unit), patients with moderate H1N1 infection (defined as inpatients not hospitalized in an intensive care unit), and a random sample of healthy pregnant women. RESULTS: Among the 39 patients with H1N1 infection (19 with severe infection, 7 of whom were pregnant; 20 with moderate infection, 2 of whom were pregnant), hypoabuminemia (P < .001), anemia (P < .001), and low levels of total IgG (P= .01), IgG(1) (P= .022), and IgG(2) (15 of 19 vs 5 of 20; P= .001; mean value +/- standard deviation [SD], 1.8 +/- 1.7 g/L vs 3.4 +/- 1.4 g/L; P= .003) were all statistically significantly associated with severe H1N1 infection, but only hypoalbuminemia (P= .02) and low mean IgG(2) levels (P= .043) remained significant after multivariate analysis. Follow-up of 15 (79%) surviving IgG(2)-deficient patients at a mean (+/- SD) of 90 +/- 23 days (R, 38-126) after the initial acute specimen was obtained found that hypoalbuminemia had resolved in most cases, but 11 (73%) of 15 patients remained IgG(2) deficient. Among 17 healthy pregnant control subjects, mildly low IgG(1) and/or IgG(2) levels were noted in 10, but pregnant patients with H1N1 infection had significantly lower levels of IgG(2) (P= .001). CONCLUSIONS: Severe H1N1 infection is associated with IgG(2) deficiency, which appears to persist in a majority of patients. Pregnancy-related reductions in IgG(2) level may explain the increased severity of H1N1 infection in some but not all pregnant patients. The role of IgG(2) deficiency in the pathogenesis of H1N1 infection requires further investigation, because it may have therapeutic implications.

Peroxisome proliferator-activated receptors are altered in pathologies of the human placenta: gestational diabetes mellitus, intrauterine growth restriction and preeclampsia.

BACKGROUND: Common complications of pregnancy arise in part from dysfunctional placental development, and include gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR) and preeclampsia (PE). Peroxisome proliferator-activated receptors (PPARs), and their partner retinoid X receptor a (RXRalpha), mediate trophoblast differentiation and thus may offer insight into the pathophysiology of these diseases. METHODS: Human placentae were obtained from women at term with GDM and were compared to uncomplicated term placentae. Placentae from women who delivered preterm with IUGR, PE or co-existing PE and IUGR were compared to matched controls. Quantitative RT-PCR and Western blotting were used to examine mRNA and protein expression of PPARalpha, PPARdelta, PPARgamma and RXRalpha. DNA binding activity of PPAR isoforms were measured in nuclear protein extracts. RESULTS: GDM was associated with significantly lower placental PPARgamma mRNA and protein, PPARalpha protein and RXRalpha protein expression, while PPAR DNA binding activity remained unchanged. Placentae from women with PE did not demonstrate any changes in mRNA or protein expression or PPAR DNA binding activity, while IUGR/PE placenta showed significant increases in PPARalpha protein, PPARgamma mRNA and protein and RXRalpha mRNA and protein expression. Significantly elevated protein expression of PPARalpha and RXRalpha were associated with IUGR placentae. IUGR and IUGR/PE placentae had significantly higher PPARgamma DNA binding activity compared to controls. CONCLUSIONS: The data presented herein suggest that PPARs may be involved in the pathophysiology of GDM, PE and IUGR.

The management of epilepsy in pregnancy.

While most women with epilepsy can expect a normal pregnancy outcome, epilepsy remains a significant contributor to both maternal and perinatal morbidity. Pre-pregnancy planning must address reliable contraception and optimisation of antiepileptic drug (AED) regimens to minimise teratogenic risk while maintaining seizure control. The most recent data from the AED registries regarding malformations is presented in this review, as is the limited data on the newer AEDs and studies linking neurocognitive outcomes to AED exposure. During pregnancy, important considerations include; therapeutic drug monitoring, surveillance for obstetric complications and vigilance for seizures during the intrapartum and postpartum period.

Localisation and expression of FoxO1 proteins in human gestational tissues.

In non-gestational tissues, emerging data indicate that the FoxO1 family of Forkhead transcription factors play diverse roles in many cellular processes coordinating programs of gene expression that regulate apoptosis, oxidative stress resistance, and immune cell homeostasis. Successful outcome of human parturition rely on many of these processes, however there is no data available on FoxO1 proteins in human intrauterine tissues, nor their role in pregnancy complications such as pre-eclampsia. Thus the aim of this study was (i) to characterises the localisation and expression of FoxO1, acetylated (ac)-FoxO1 and phosphorylated (p)-FoxO1 in human placenta and fetal membranes obtained from term Caesarean sections (n=5); and (ii) to compare the expression of FoxO1 proteins in term placental samples from normal and pre-eclamptic pregnancies (n=5 per group). In placenta, weak FoxO1 staining was localised to the syncytiotrophoblast layer, whereas ac-FoxO1 and p-FoxO1 staining was mainly localised in the syncytiotrophoblasts and cytotrophoblasts. In fetal membranes, FoxO1, ac-FoxO1 and p-FoxO1 were localised to the trophoblast layer of the chorion, amnion epithelium and decidual cells. Quantitative RT-PCR (qRT-PCR) analysis showed a 6-fold and 12-fold higher mRNA expression in the choriodecidua compared to placenta and amnion, respectively. In both amnion and choriodecidua, FoxO1 protein expression was higher in the cytoplasmic fractions than in the nuclear fractions. On the otherhand, ac-FoxO1 and p-FoxO1 protein expression was higher in the nuclear fractions for all three tissues. There was no difference in the mRNA or protein expression of FoxO1 proteins in placental samples from normal and pre-eclamptic term pregnancies. The exact role of FoxO1 proteins in human pregnancy are unknown, however the finding that they are expressed in human gestational tissues warrants further research into their function in these tissues.

Peroxisome proliferator-activated receptors and retinoid X receptor-alpha in term human gestational tissues: tissue specific and labour-associated changes.

Peroxisome proliferator-activated receptors (PPARs) and their transcriptional partner retinoid X receptor (RXR) are involved in transcriptionally regulating the events that contribute to the control of parturition in humans. Definitive data, however, are lacking with respect to PPAR and RXR expression and activation during term labour in human gestational tissues. The aim of this study, therefore, was to identify tissue and labour-associated changes of PPAR isoforms (alpha, delta and gamma) and RXRalpha in placenta, amnion and choriodecidua. Gestational tissues from term non-labouring women were used for immunohistochemistry localisation and confirmation studies of PPAR isoforms (alpha, delta and gamma) and RXRalpha. Human gestational tissues were then collected from term women not-in-labour (NIL) (elective Caesarean section), in-labour (IL) (emergency Caesarean section) and post-labour (PL) (normal vaginal delivery). Quantitative RT-PCR (qRT-PCR) and Western blotting were employed to study mRNA and protein expression profiles respectively. Significantly higher mRNA expression was observed in placental tissues taken from women in labour (PPARdelta, PPARgamma and RXRalpha). Elevated PPARdelta and RXRalpha mRNA expression in fetal membranes was also associated with being in labour. In contrast, PPARgamma mRNA in the amnion was decreased with term PL compared to NIL. In placenta, PPARalpha, PPARdelta and PPARgamma protein expression was significantly increased in the IL group compared to the NIL or PL group. There was no significant difference in PPAR or RXRalpha protein expression in both amnion and choriodecidua between the three labour groups. PPAR (alpha and gamma) transcription factor DNA binding activity was found to decline IL compared to NIL and PL in the placenta. PPARdelta DNA binding activity also decreased in the choriodecidua IL compared to PL. In amnion, PPARalpha DNA binding activity was found to be higher IL compared to NIL. In conclusion, term human labour is associated with changes in expression and activity of PPAR isoforms and its transcription partner, RXRalpha. This data is consistent with the hypothesis that PPAR:RXR are involved in regulating of the processes of human term parturition.

Pre-labour fetal membranes overlying the cervix display alterations in inflammation and NF-kappaB signalling pathways.

To generate new insights into the process of fetal membrane rupture, we have developed a technique whereby fetal membranes overlying the cervix (i.e. supracervical site, SCS) are tagged in women undergoing term elective Caesarean section. The specific aim is to determine the effect of supracervical apposition on the release of inflammatory mediators and NF-kappaB signalling in pre-labour fetal membranes. Fetal membranes were collected from both the SCS and from a distal site (DS). The level of NF-kappaB proteins and its transcriptional co-activator protein CBP and p300 was determined by Western blotting and/or immunohistochemistry (IHC), and cytokine and prostaglandin release was quantified by enzyme immunoassay. Tissues obtained before labour at term possess an area of the fetal membranes (i.e. supracervical site) that exhibit decreased release of IL-1beta, IL-6, IL-8, TNF-alpha and PGE(2). IHC revealed that NF-kappaB signalling proteins, CBP and p300 were significantly increased in SC fetal membranes compared to distal membranes. In summary, data from this study confirm that supracervical fetal membranes display altered structural and biochemical characteristics.

Mitogen-activated protein kinase proteins regulate LPS-stimulated release of pro-inflammatory cytokines and prostaglandins from human gestational tissues.

The role of pro-inflammatory cytokines and prostaglandins in human labour is well established. Many of the mRNAs stabilised by the MAPK pathway encode inflammatory mediators, suggesting that this kinase pathway plays a major role in the regulation of inflammation. The aim of this study was to determine if the MAPK pathway regulates the inflammatory response in human gestational tissues. Placenta and fetal membranes (n=5) obtained from pregnant women undergoing Caesarean section before the onset of labour were exposed to LPS, and co-incubated in the absence or presence of 12.5, 25 and 50 microM U0126 (ERK 1/2 inhibitor), SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). After 18 h incubation, tissues were collected and ERK 1/2, p38 MAPK, and JNK total and phosphorylated protein expression was assessed by ELISA and/or Western blotting. The incubation medium was collected and TNF-alpha, IL-1beta, IL-6, IL-8, PGE(2) and PGF(2alpha) release was quantified by ELISA. Treatment of placenta and fetal membranes with LPS activated all three MAPK proteins. Co-incubation with U0126, SP600125 and SB202190 significantly suppressed LPS-stimulated activation of ERK 1/2, JNK, and p38 MAPK, respectively. All cytokine and prostaglandin release was significantly suppressed by all concentrations of U0126. LPS-stimulated IL-6, TNF-alpha, PGE(2) and PGF(2alpha) release was significantly suppressed by treatment with all concentrations of SB202190, whereas ILS-stimulated IL-1beta release was only significantly inhibited in the presence of 50 microM SB202190 and there was no effect of SB202190 on LPS-stimulated IL-8 release. SP600125 significantly repressed LPS-stimulated release of IL-1beta and TNF-alpha at all concentrations, whereas LPS-stimulated IL-6, PGE(2) and PGF(2alpha) release were inhibited at 25 and 50 microM. In conclusion, the MAPK inhibitors used in this study demonstrated differential activity against a range of sequelae commonly associated with inflammation, supporting the therapeutic potential of MAPK inhibitors in pregnancy complications associated with an aberrant inflammatory response.

15-Deoxy-Delta(12,14)-prostaglandin J(2) and troglitazone regulation of the release of phospholipid metabolites, inflammatory cytokines and proteases from human gestational tissues.

Phospholipid-derived mediators, inflammatory cytokines and extracellular matrix remodelling enzymes are all involved in the initiation of human labour and delivery. We have previously demonstrated that natural and synthetic PPAR-gamma ligands regulate LPS-stimulated pro-inflammatory cytokine release from human gestational tissues, however, the effect of these ligands on the basal and/or LPS-induced expression of prostaglandins and proteases is not known. Therefore, the aim of this study was to determine the effects of 15d-PGJ(2) and troglitazone on the expression of basal and LPS-stimulated inflammatory mediators in human gestational tissues. Human placenta, amnion and choriodecidua (n=5) were incubated in the presence or absence of 15 microM 15d-PGJ(2) and 30 microM troglitazone under basal and LPS-stimulated (10 microg/ml) conditions. Treatment of placenta, amnion and choriodecidua with both 15d-PGJ(2) and troglitazone decreased basal and LPS-stimulated IL-1beta, IL-6, IL-10 and TNF-alpha release. Basal type II PLA(2) release from placental tissues was also significantly decreased by 15d-PGJ(2) and troglitazone. There was no effects of 15d-PGJ(2) and troglitazone on cPLA(2) protein expression. Both 15d-PGJ(2) and troglitazone significantly decreased basal and LPS-stimulated PGE(2) and PGF(2alpha) release from placenta and amnion. However, in choriodecidua, although 15d-PGJ(2) decreased basal and/or LPS-stimulated PGE(2) and PGF(2alpha) release, there was an increase in PGE(2) and PGF(2alpha) release in the presence of troglitazone. 15d-PGJ(2) and troglitazone inhibited MMP-9 release from human amnion. NF-kappaB DNA binding activity and NF-kappaB p65 protein expression was inhibited by treatment with 15d-PGJ(2) in human amnion. There was no effect of 15d-PGJ(2) or troglitazone on PPAR-gamma protein, and GW9662 failed to alleviate 15d-PGJ(2) and troglitazone inhibition of IL-6 and TNF-alpha release in placenta, amnion and choriodecidua. The data demonstrated in this study suggest that the 15d-PGJ(2) and troglitazone exhibit anti-inflammatory properties in human gestational tissues via PPAR-gamma independent actions.

Lipopolysaccharide and TNF-alpha activate the nuclear factor kappa B pathway in the human placental JEG-3 cells.

Up-regulation of pro-inflammatory cytokines, cyclooxygenase (COX-2) and prostaglandins is a critical factor driving human term labour and inflammation-associated preterm labour. Nuclear factor kappa B (NF-kappaB) is activated in response to a number of inflammatory mediators, including cytokines and lipopolysaccharide (LPS). The aim of this study was (i) to investigate if TNF-alpha and LPS activate the NF-kappaB pathway; and (ii) to use short interfering RNA (siRNA) against inhibitor kappaB kinase (IKK)-beta to confirm the role of the NF-kappaB pathway in the regulation of pro-inflammatory mediators in human placental JEG-3 cells. JEG-3 cells (3 independent experiments) were (i) incubated in the presence or absence of 10 microg/ml LPS or 20 ng/ml TNF-alpha, or (ii) transfected with 100 nM IKK-beta siRNA. Incubation of JEG-3 cells with LPS and TNF-alpha increased the expression of cytoplasmic IKK-beta and phosphorylated IkappaB-alpha, and nuclear NF-kappaB proteins p50 and p65. This was associated with a concurrent increase in COX-2 protein, and IL-6 and PGF2alpha release from JEG-3 cells. Treatment of cells with BAY 11-7082 at 50 microM significantly inhibited basal, LPS- and TNF-alpha-induced NF-kappaB and COX-2 expression, and IL-6 and PGF2alpha release. Transfection of JEG-3 cells with IKK-beta siRNA significantly decreased IL-6 and PGF2alpha release. The data presented in this study demonstrate that pro-inflammatory mediators regulate the NF-kappaB transcription pathway in human JEG-3 cells, and the IKK-beta/NF-kappaB pathway is a regulator of inflammatory mediators in placental JEG-3 cells.

Factors affecting umbilical venous perfusion during experimental cord knotting.

The aim was to determine experimentally the factors that increase the risk of venous occlusion by applying a standardised tightening force to isolated perfused umbilical cords tied in a true knot in vitro. Umbilical cords were collected from patients undergoing Caesarean section. Cords were clamped, isolated and studied within 15 min. The umbilical vein was cannulated, the cord tied in a true knot and traction was applied using standard weights. The umbilical vein was perfused with modified Krebs solution at a constant pressure of 40 mmHg and the attached weight increased until perfusion ceased. The cord mass index (weight/length), hydration index/100-[(dry weight/wet weight)x100], and coiling index (coils/length) were determined. Cord morphometric analysis was performed on 193 cords. Intra uterine growth restriction was associated with decreased cord mass index (p=0.002) and increased coiling index (p=0.002). Venous perfusion experiments were performed on 75 cords. Using multivariate regression analysis, cord morphometric factors that increased the risk of cord occlusion were decreased cord mass index (p=0.008), decreased cord hydration index (p=0.004), and low venous flow capacity (p=0.001). During experimental cord knotting with applied traction, the susceptibility to venous occlusion was increased with low cord mass index, low cord hydration index and low venous flow capacity. These cord characteristics were associated with low fetal body weight and intrauterine growth restriction. An increased susceptibility to cord occlusion may contribute to the higher perinatal morbidity and mortality in growth restricted pregnancies.

Effect of nuclear factor-kappa B inhibitors and peroxisome proliferator-activated receptor-gamma ligands on PTHrP release from human fetal membranes.

Parathyroid hormone-related protein (PTHrP) has been implicated in many processes during normal and pathological pregnancies. In the human fetal membranes, PTHrP exhibits cytokine-like actions. We have recently shown that inhibitors of the nuclear factor-kappa B (NF-kappaB) and activators of the peroxisome proliferator-activated receptor (PPAR)-gamma signalling pathways down-regulate cytokine release from human gestational tissues. Therefore, the aim of this study was to determine whether NF-kappaB and PPAR-gamma also regulate PTHrP release from human fetal membranes. Human amnion and choriodecidua explants were incubated in the absence (control) or presence of two known NF-kappaB inhibitors (1, 5 and 10 mM sulphasalazine (SASP) or 5, 10 and 15 mM N-acetyl-cysteine (NAC)), and two PPAR-gamma ligands (15 and 30 microM 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) or 15 and 30 microM troglitazone), under basal conditions. After 18 h incubation, the tissues were collected and NF-kappaB p65 DNA binding activity in nuclear extracts was assessed by ELISA, and the incubation medium was collected and the release of PTHrP was quantified by RIA. Treatment of amnion and choriodecidual tissues with SASP concentrations greater than 5 mM, 15 mM NAC, 30 microM 15d-PGJ(2) and 30 microM troglitazone significantly reduced the release of PTHrP (p < 0.05). This study demonstrates that PTHrP release from human fetal membranes is regulated by inhibitors of NF-kappaB, and ligands of PPAR-gamma.

Altered placental oxidative stress status in gestational diabetes mellitus.

Oxidative stress has been clearly linked to type 2 diabetes mellitus, however, limited data are available on the involvement of oxidative stress in gestational diabetes mellitus (GDM), a disease of similar pathophysiology. The aim of this study was to investigate the status of placental oxidative stress in healthy pregnant women and women with GDM. The hypothesis to be tested was that tissue markers of oxidative stress are significantly increased in GDM compared to normal placental tissues. Markers of oxidative stress measured were the release of 8-isoprostane (8-epi-prostaglandin F(2alpha)) from human term placental explants (n=11), the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase (n=10), and protein carbonyl content (n=12). Placental release of 8-isoprostane was 2-fold greater from women with GDM (P<0.001) compared to healthy pregnant women. Superoxide dismutase activity and protein carbonyl content were elevated in placentae obtained from women with GDM (P<0.04 and P<0.004 respectively), whilst there was no significant difference in the activity of glutathione peroxidase. These data demonstrate the presence of oxidative stress in the placenta from women with GDM, in addition to the induction of a key antioxidant, collectively indicating a state of existing oxidative stress in this condition.