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Untimely activation of the inflammatory response by sterile or infective insults in uterine tissues can result in preterm birth. Pro-inflammatory cytokines and pathogenic activation of toll-like receptors (TLRs) initiate a biochemical cascade of events leading to myometrial activation and contractility, cervical dilatation, and rupture of the chorioamniotic membranes. GIT2 is a signaling protein known to play a role in innate and adaptive immunity; however, its role in the inflammatory pathways of human labor is not known. In this article, we report that GIT2 expression is lower in human myometrium and fetal membranes with term labor, and in preterm amnion with histological chorioamnionitis. GIT2 knockdown by siRNA in primary myometrial and amnion cells exhibited reduced expression of pro-inflammatory cytokines and chemokines in response to inflammatory challenge by cytokines or TLR ligands. In addition, the pro-inflammatory cytokines IL1B and TNF could not induce the expression of extracellular matrix degrading enzymes in GIT2-deficient amnion cells. Myometrial activation in response to pro-inflammatory cytokines was also significantly suppressed in GIT2-deficient cells as evidenced by decreased prostaglandin release and expression of contraction-associated proteins. Further to this, collagen gel assays demonstrated that TNF had a reduced ability to induce myometrial contractility in situ in GIT2-deficient myometrial cells compared to control-transfected cells. In summary, the loss of GIT2 diminishes the effects inflammatory mediators have in promoting myometrial contraction and fetal membrane rupture in vitro, suggesting that GIT2 could be a possible target for preterm birth therapies., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Inflammation plays a central role in the terminal process of human labour and delivery, including myometrial contractions. RAF1 proto-oncogene serine/threonine-protein kinase (RAF1) can activate ERK (official gene symbol MAPK1) and/or nuclear factor-kappa B (NF-κB) to regulate genes involved in inflammation. There are, however, no studies on the role of RAF1 in the processes of human labour and delivery. Thus, the aims of this study were to determine the effect of i) human labour and pro-inflammatory cytokines interleukin 1 beta (IL1B) and tumour necrosis factor (TNF) alpha on RAF1 protein expression in myometrium and ii) siRNA knockdown of RAF1 on pro-inflammatory and pro-labour mediators in human myometrial primary cells. Term labour was associated with an increase in RAF1 protein expression. Furthermore, RAF1 protein expression was increased in myometrial cells treated with IL1B and TNF, two likely factors contributing to preterm birth. Knockdown of RAF1 by siRNA in primary myometrial cells significantly decreased IL1B- and TNF-induced IL1A, IL1B, IL6, (C-X-C motif) ligand 8 (CXCL8)and chemokine (C-C motif) ligand 2 (CCL2) mRNA abundance and IL6, IL8 and CCL2; prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels and prostaglandin PGF2 α release; and NF-κB activation. Furthermore, RAF1 knockdown was associated with decreased activation of ERK in the presence of IL1B but not TNF. Concordantly, the ERK inhibitor U0126 significantly decreased IL1B-induced IL6, CXCL8, CCL2 and PTGS2 mRNA abundance; IL6, CXCL8, CCL2 and PGF2 α release; and NF-κB activation. In conclusion, IL1B induces the expression and secretion of pro-labour mediators through the RAF1-MAPK1-NF-κB signalling pathway. TNF, on the other hand, regulates pro-labour mediators through the RAF1-NF-κB signalling pathway via an MAPK1-independent mechanism., (© 2016 Society for Reproduction and Fertility.)

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

The inflammatory process plays a pivotal role during the pathogenesis of human labour, both at term and preterm. Visfatin levels increase during normal human pregnancy and in infection associated preterm labour. The effects of visfatin in the processes of human labour and delivery, however, are not known. The aim of this study was to determine the effect of visfatin on the expression and release of pro-labour mediators in human placenta. Samples were obtained from normal pregnancies at the time of Caesarean section. Human placenta was incubated in the absence (basal control) or presence of a 50 ng/ml visfatin for 24 h (n=6). Inflammatory gene expression was analysed by quantitative RT-PCR (qRT-PCR), the medium was collected and cytokine, prostaglandin and 8-isoprostane (marker of oxidative stress) release was quantified by ELISA, and secretory protease activity by zymography. Visfatin significantly increased IL-6 and IL-8 gene expression and secretion, COX-2 expression and resultant prostaglandin (PG) E(2) and PGF(2α) release, and 8-isoprostane release. There was, however, no effect of visfatin on pro MMP-9 enzyme activity. These actions of visfatin were elicited via the nuclear factor-κB (NF-κB) pathway as visfatin induced the degradation of IκB-α (inhibitor of NF-κB) whilst increasing NF-κB p65 DNA binding activity. Further to this, visfatin-induced pro-labour responses were abrogated by treatment with the NF-κB inhibitor BAY 11-7082. Collectively, these data indicate that visfatin activates pro-inflammatory cytokine release and phospholipid metabolism in human placenta via activation of the NF-κB pathway. Thus, visfatin represents a novel cytokine linked to the events of human labour initiation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: The aim of this study was to determine (i) the effect of maternal obesity and gestational diabetes mellitus (GDM) on (i) the circulating levels of omentin-1 in cord and maternal plasma, and (ii) gene expression and release of omentin-1 from human placenta and adipose tissue. The effect of pregnancy on circulating omentin-1 levels was also determined., Design: Omentin-1 levels were measured in maternal and cord plasma from obese and non-obese normal glucose tolerant women (NGT; n = 44) and women with GDM (n = 39) at the time of term elective Caesarean section. Placenta and adipose tissue expression and release of omentin-1 was measured from 22 NGT and 22 GDM women collected at the time of term elective Caesarean section. Omentin-1 levels were also measured in maternal plasma from 13 NGT women at 11 and 28 weeks gestation and 7 weeks postpartum., Results: Maternal obesity was associated with significantly lower omentin-1 levels in maternal plasma; however, there was no effect of maternal obesity on cord omentin levels. Omentin-1 gene expression was lower in placenta and adipose tissue obtained from women with pre-existing obesity. In addition to this, adipose tissue release of omentin-1 was significantly lower from obese pregnant women. Omentin-1 levels were significantly lower in non-obese GDM compared to non-obese NGT women. However, there was no difference in omentin-1 levels between obese NGT and obese GDM women. There was no effect of GDM on cord omentin levels, and placental and adipose tissue omentin-1 expression. Maternal omentin-1 levels were negatively correlated with fetal birthweight and fetal ponderal index., Conclusions: The data presented in this study demonstrate that pre-existing maternal obesity is associated with lower omentin-1 expression in placenta, adipose tissue and maternal plasma. Alteration in omentin-1 in pregnancy may influence the development of metabolic disorders in offspring later in life.

In response to oxidative stress, gestational diabetes mellitus (GDM) placenta releases less 8-isoprostane and tumour necrosis factor (TNF) alpha. The effect of oxidative stress on other cytokines and antioxidant gene expressions are unknown. The aim of this study is to further explore the antioxidant status and effect of oxidative stress in GDM tissue. Human placenta, omental and subcutaneous adipose tissue from women with and without GDM were exposed to hypoxanthine (HX)/xanthine oxidase (XO). Cytokine release was analysed by ELISA and cytokine and antioxidant gene expression by RT-PCR. Catalase (CAT) and glutathione reductase (GSR) mRNA expression was higher in GDM (n=18) compared with normal (n=23) placenta. There was no difference in glutathione peroxidase and superoxide dismutase mRNA expression. Antioxidant gene expression was unaltered between normal (n=18) and GDM (n=10) adipose tissue. HX/XO treatment significantly stimulated cytokine release (13/16 cytokines) and cytokine mRNA expression, and decreased antioxidant gene expression (CAT and GSR) in human placenta from normal pregnant women. In GDM placenta, HX/XO only significantly increased the release of 3/16 cytokines, while there was no effect on antioxidant gene expression. In normal and GDM adipose tissues, HX/XO increased proinflammatory cytokine and 8-isoprostane release, while there was no change in antioxidant gene expression. GDM placenta is characterised by increased antioxidant gene expression, and is less responsive to exogenous oxidative stress than tissues obtained from normal pregnant women. This may represent a protective or adaptive mechanism to prevent damage from further oxidative insult in utero as indicated by increased tissue antioxidant expression.

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.

Proinflammatory prostaglandins and cytokines are involved in the initiation of human labor and delivery. Although cyclopentenone prostaglandins regulate the formation of these prolabor mediators via nuclear factor-kappaB (NF-kappaB) and/or peroxisome proliferator-activated receptor-gamma, recent evidence suggests that they do not exist in vivo. Cyclopentenone isoprostanes (IsoPs), which are highly reactive structural isomers of bioactive cyclopentenone prostaglandins, do exist physiologically and have been shown to inhibit the inflammatory response in macrophages. Therefore the aim of this study was to determine the effect of the synthetic cyclopentenone IosP 15-A(2)-IsoP on the expression of prolabor mediators in human gestational tissues. Human placenta and gestational membranes (n=5) were incubated in the absence or presence of 12.5, 25, and 50 microM 15-A(2)-IsoP with 10 microg/ml lipopolysaccharide (LPS). Treatment of placenta and fetal membranes with 15-A(2)-IsoP caused a dose-dependent decrease in LPS-stimulated release of the cytokines IL-1beta, IL-6, IL-8, and TNF-alpha and the prostaglandins PGE(2) and PGF(2)alpha. NF-kappaB p65 DNA binding activity was significantly inhibited by treatment with 50 microM 15-A(2)-IsoP. Collectively, these data suggest that 15-A(2)-IsoP exhibits antiinflammatory properties via antagonism of NF-kappaB activity. Cyclopentenone IsoPs may serve as negative feedback regulators of the inflammatory response in 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.

Beyond their effects on central metabolic functions, leptin, resistin, and adiponectin have profound effects on a number of other physiologic processes, including immune function and inflammation. Although leptin, resistin, and adiponectin are produced in human placenta and adipose tissue, their immunoregulatory actions in these tissues are not known. Therefore, the aim of this study was to determine the effect of leptin, resistin, and adiponectin on the release of proinflammatory mediators in human placenta and sc adipose tissue. Samples were obtained from normal pregnancies at the time of cesarean section. Tissue explants (n = 5) were incubated in the absence (basal control) or presence of a leptin (1, 10, and 100 ng/ml), resistin (1, 10, and 100 ng/ml), and adiponectin (0.1 and 0.5 microg/ml). After 6 h incubation, the medium was collected, and the release of IL-1beta, IL-6, TNFalpha, prostaglandin (PG)F2alpha and PGE2 was quantified by ELISA. There was no effect of resistin on proinflammatory cytokine or prostaglandin release; however, leptin at 100 ng/ml and adiponectin at 0.1 and/or 0.5 microg/ml significantly increased the release of IL-1beta, IL-6, TNFalpha, and PGE2 from human placenta and adipose tissue. Although both leptin and adiponectin significantly increased PGF2alpha release from human placenta, there was no effect of these hormones on PGF2alpha release from adipose tissue. Furthermore, this leptin- and adiponectin-induced proinflammatory response could be abrogated by treatment with the antiinflammatory ERK1/2 MAPK inhibitor U0126, the peroxisomal proliferator-activated receptor-gamma ligand troglitazone, and the nuclear factor-kappaB inhibitor BAY 11-7082. Collectively, these data indicate that leptin and adiponectin activate proinflammatory cytokine release and phospholipid metabolism in human placenta and adipose tissue, and antiinflammatory agents can abrogate leptin- and adiponectin-induced inflammation.

The aim of this study was to 1) profile the basal release of TNF-alpha, IL-6, IL-8, and 8-isoprostane (a marker of oxidative stress); and 2) investigate the effect of stimulation on the release of cytokines from sc adipose tissue and skeletal muscle from normal pregnant women and women with gestational diabetes mellitus (GDM). Placenta, sc adipose tissue, and skeletal muscle were incubated in the absence (control) or presence of lipopolysaccharide (LPS; 10 microg/ml), TNF-alpha (10 ng/ml), IL-6 (10 ng/ml), or IL-8 (10 ng/ml). After an 18-h incubation, the medium was collected, and the release of TNF-alpha, IL-6, IL-8, and 8-isoprostane was quantified by ELISA. In all three tissues, 8-isoprostane release was greater in women with GDM, and stimulation with LPS increased 8-isoprostane release from adipose and skeletal muscle, but not placenta, obtained from women with GDM. However, in tissues obtained from normal pregnant women, LPS stimulation increased 8-isoprostane release in placenta and had no effect in adipose tissue and skeletal muscle. Their was no difference in the release of TNF-alpha, IL-6, and IL-8 from placenta, adipose tissue, and skeletal muscle obtained from normal pregnant women and women with GDM. Stimulation of placenta, adipose tissue, and skeletal muscle with LPS and TNF-alpha resulted in greater release of IL-6 and IL-8, whereas only LPS increased TNF-alpha release from all three tissues. The data presented in this study demonstrate that there is a differential release of 8-isoprostane from fetal (placenta) and maternal (adipose tissue and skeletal muscle) tissues obtained from normal pregnant women and women with GDM. These data are consistent with the hypothesis that oxidative stress may be involved in the progression and/or pathogenesis of GDM.

The production of reactive oxygen species (ROS), prostaglandins (PGs), proinflammatory cytokines, and proteases has been implicated in the pathogenesis of term and preterm labor. The nuclear factor-kappaB (NF-kappaB) transcription pathway is activated by ROS and is a key regulator of PGs, proinflammatory cytokine release, and protease activity. N-Acetyl-cysteine (NAC) is an antioxidant that through its ability to scavenger ROS suppresses NF-kappaB DNA-binding activity and resultant gene expression. The aim of this study was to elucidate the effect of NAC on NF-kappaB DNA-binding activity, phospholipid metabolism, cytokine release, and protease activity from human fetal membranes. Human amnion and choriodecidua (n = 9 separate placentas) were treated with 0 (control), 5, 10, or 15 mM NAC in the presence of 10 micro g/ml lipopolysaccharide. After 6-h incubation, the tissues were collected, NF-kappaB DNA binding activity was assessed by gel shift binding assays, and matrix metalloproteinase-9 and urokinase-type plasminogen activator activity were determined by zymography. The incubation medium was collected and assayed for type II phospholipase A(2) tissue content, IL-6, IL-8, TNFalpha, and 8-isoprostane release by ELISA. The release of PGF(2alpha) was measured by RIA. Treatment of fetal membranes with NAC significantly suppressed lipopolysaccharide-stimulated type II phospholipase A(2) release and content; PGF(2alpha), IL-6, IL-8, TNFalpha, and 8-isoprostane release; and matrix metalloproteinase-9 and urokinase-type plasminogen activator enzyme activity and suppressed NF-kappaB DNA-binding activity (by ANOVA, P < 0.05). The data presented in this study demonstrate that NAC inhibits an NF-kappaB-activated pathway and subsequent phospholipid metabolism, proinflammatory cytokine release, and protease activity in human fetal membranes.

Proinflammatory cytokines are implicated in the initiation and progression of human labor and delivery, particularly in relation to infection-induced preterm labor. In nongestational tissues, the nuclear factor kappa B (NF-kappaB) transcription pathway is a key regulator of proinflammatory cytokine release. In these tissues, sulfasalazine (SASP), through its ability to inhibit NF-kappaB activation, inhibits release of interleukin (IL)-2, IL-12, and tumor necrosis factor (TNF)-alpha. Therefore, the aim of this study was to investigate whether or not NF-kappaB activation regulates the formation of proinflammatory cytokines in human gestational tissues. Human placenta, amnion, and choriodecidua (n = 9 separate placentas) were incubated with 10 microg/ml of lipopolysaccharide (LPS) in the absence (control) or presence of SASP (0.1, 1, 5, or 10 mM). After 6 h of incubation, the tissues were collected, and NF-kappaB DNA binding activity in nuclear extracts was assessed by electromobility shift binding assay. The incubation medium was collected and the release of IL-6, IL-8, and TNF-alpha was quantified by ELISA. Treatment of placenta, amnion, and choriodecidua with SASP at concentrations 5 mM or greater significantly inhibited the release of IL-6, IL-8, and TNF-alpha, and NF-kappaB activation (ANOVA, P < 0.05). The data presented in this study demonstrate that the NF-kappaB transcription pathway is a key regulator of LPS-stimulated IL-6, IL-8, and TNF-alpha release from human gestational tissues. The control of NF-kappaB activation may therefore provide an alternative therapeutic strategy for reducing the release of proinflammatory mediators in infection associated preterm labor.

Abstract: 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 PGF2α 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. [Copyright &y& Elsevier]

Abstract: 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-κB signalling in pre-labour fetal membranes. Fetal membranes were collected from both the SCS and from a distal site (DS). The level of NF-κB 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-1β, IL-6, IL-8, TNF-α and PGE2. IHC revealed that NF-κB 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. [Copyright &y& Elsevier]

Abstract: Within the discipline of reproductive biology, our understanding of one of the most fundamental biological processes is lacking—the cellular and molecular mechanisms that govern birth. This lack of understanding limits our ability to reduce the incidence of labour complications. The incidence of labour complications including: preterm labour; cervical incompetence; and post-date pregnancies has not diminished in decades. The key to improving the management of human labour and delivery is an understanding of how the multiple processes that are requisite for a successful labour and delivery are coordinated to achieve a timely birth. Processes of human labour include the formation of: contraction associated proteins; inflammatory mediators (e.g. cytokines); uterotonic phospholipid metabolites (e.g. prostaglandins); and the induction of extracellular matrix (ECM) remodelling. Increasingly, it is becoming evident that labour onset and birth are the result of cross-talk between multiple components of an integrated network. This hypothesis is supported by recent data implicating various upstream regulatory pathways in the control of key labour-associated processes, including the activity of enzymes involved in the formation of prostaglandins and extracellular matrix remodelling, and mediators of inflammation. Clearly, the biochemical pathways involved in the formation of these mediators represent potential sites for intervention that may translate to therapeutic interventions to delay or prevent preterm labour and delivery. Available data strongly implicate the nuclear factor-κB (NF-κB) family as candidate upstream regulators of multiple labour-associated processes. Not only do these data warrant further detailed analysis of the involvement of these pathways in the process of human labour but also promise new insights into the key mechanisms that trigger birth and the identification of new therapeutic interventions that will improve the management of labour. [Copyright &y& Elsevier]

Abstract: 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-γ 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-PGJ2 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 15μM 15d-PGJ2 and 30μM troglitazone under basal and LPS-stimulated (10μg/ml) conditions. Treatment of placenta, amnion and choriodecidua with both 15d-PGJ2 and troglitazone decreased basal and LPS-stimulated IL-1β, IL-6, IL-10 and TNF-α release. Basal type II PLA2 release from placental tissues was also significantly decreased by 15d-PGJ2 and troglitazone. There was no effects of 15d-PGJ2 and troglitazone on cPLA2 protein expression. Both 15d-PGJ2 and troglitazone significantly decreased basal and LPS-stimulated PGE2 and PGF2α release from placenta and amnion. However, in choriodecidua, although 15d-PGJ2 decreased basal and/or LPS-stimulated PGE2 and PGF2α release, there was an increase in PGE2 and PGF2α release in the presence of troglitazone. 15d-PGJ2 and troglitazone inhibited MMP-9 release from human amnion. NF-κB DNA binding activity and NF-κB p65 protein expression was inhibited by treatment with 15d-PGJ2 in human amnion. There was no effect of 15d-PGJ2 or troglitazone on PPAR-γ protein, and GW9662 failed to alleviate 15d-PGJ2 and troglitazone inhibition of IL-6 and TNF-α release in placenta, amnion and choriodecidua. The data demonstrated in this study suggest that the 15d-PGJ2 and troglitazone exhibit anti-inflammatory properties in human gestational tissues via PPAR-γ independent actions. [Copyright &y& Elsevier]

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-κB) 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-α and LPS activate the NF-κB pathway; and (ii) to use short interfering RNA (siRNA) against inhibitor κB kinase (IKK)-β to confirm the role of the NF-κB 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 10μg/ml LPS or 20ng/ml TNF-α, or (ii) transfected with 100nM IKK-β siRNA. Incubation of JEG-3 cells with LPS and TNF-α increased the expression of cytoplasmic IKK-β and phosphorylated IκB-α, and nuclear NF-κB proteins p50 and p65. This was associated with a concurrent increase in COX-2 protein, and IL-6 and PGF2α release from JEG-3 cells. Treatment of cells with BAY 11-7082 at 50μM significantly inhibited basal, LPS- and TNF-α-induced NF-κB and COX-2 expression, and IL-6 and PGF2α release. Transfection of JEG-3 cells with IKK-β siRNA significantly decreased IL-6 and PGF2α release. The data presented in this study demonstrate that pro-inflammatory mediators regulate the NF-κB transcription pathway in human JEG-3 cells, and the IKK-β/NF-κB pathway is a regulator of inflammatory mediators in placental JEG-3 cells. [Copyright &y& Elsevier]