Stella Liong

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

To identify cervicovaginal fluid (CVF) biomarkers predictive of spontaneous preterm birth in women with symptoms of preterm labour.

The Interplay of the Interleukin 1 System in Pregnancy and Labor

This work assessed the temporal coexpression of interleukin 1 (IL-1) and its inhibitor, IL-1 receptor antagonist (IL-1ra), in the cervicovaginal fluid (CVF) beyond 24 weeks gestation including women in spontaneous term labor. Two cohorts of women were recruited at 24 to 35 weeks’ gestation (n = 65) and in late pregnancy (>36 weeks’ gestation; n = 88). The CVF was serially collected either every 4 weeks between 24 and 35 weeks’ gestation (n = 123 samples) or weekly during late pregnancy (n = 240 samples). The IL-1 and IL-1ra were quantitated by enzyme-linked immunosorbent assay, and the effect of vaginal microflora and unprotected sexual intercourse were also investigated. The IL-1β and IL-1ra remain unaltered between 24 and 35 weeks’ gestation. At late pregnancy, IL-1α and β concentrations peak at 4 to 14 days prior to labor onset, while IL-1ra decreases with approaching spontaneous term labor (P < .05, 2-way analysis of variance). The IL-1 and IL-1ra were significantly correlated (P < .001, Pearson r). A combined biomarker model of IL-1α, IL-1β, and IL-1ra can predict term labor with 86% sensitivity and 92% specificity. This study indicates a shifting inflammatory balance in the gestational tissues prior to labor onset.

The Transcription Factor Interferon Regulatory Factor-1 (IRF1) Plays a Key Role in the Terminal Effector Pathways of Human Preterm Labor

ABSTRACT Preterm birth is the largest single cause of neonatal death and morbidity. By activating cytokine- and Toll-like receptor (TLR)-signaling pathways, infection and/or inflammation are strongly associated with preterm delivery. Interferon regulatory factor-1 (IRF1) is an important regulator of the inflammatory response. The aims of this study were to establish the effect of 1) labor on IRF1 expression in human fetal membranes and myometrium, 2) prolabor mediators on IRF1 expression and activity, and 3) IRF1 small interfering RNA on the expression of prolabor mediators. IRF1 expression was higher in fetal membranes and myometrium after spontaneous term labor and in preterm fetal membranes with infection. The proinflammatory cytokine IL1B, the bacterial product fsl-1, and viral analog polyinosinic:polycytidylic acid (poly [I:C]) significantly increased IRF1 mRNA expression and transcriptional activity in human primary myometrial cells. In addition, IL1B increased IRF1 activity in primary amnion cells. IRF1 silencing in myometrial cells decreased IL1B-, fsl-1-, and poly (I:C)-induced cytokine (IL6, TNF, IL1B) and chemokine (CXCL8, CCL2) mRNA expression and IL6, CXCL8, and CCL2 release. IL1B-, fsl-1-, and poly (I:C)-induced PTGS2 mRNA expression and IL1B-induced prostaglandin release was also decreased by IRF1 silencing. In conclusion, IRF1 upregulation in fetal membranes and myometrium after term labor indicates a proinflammatory role for IRF1 in human parturition. IRF1 is involved in TLR- and cytokine-mediated signaling in human myometrium. These data provide new insights into the mechanisms associated with inflammation- and infection-associated preterm birth. IRF1 inhibitors as therapeutics for the management of spontaneous preterm birth warrants further investigation.

Activation of AMPK improves inflammation and insulin resistance in adipose tissue and skeletal muscle from pregnant women

Gestational diabetes mellitus (GDM) is characterised by maternal peripheral insulin resistance and inflammation. Sterile inflammation and bacterial infection are key mediators of this enhanced inflammatory response. Adenosine monophosphate (AMP)-activated kinase (AMPK), which is decreased in insulin resistant states, possesses potent pro-inflammatory actions. There are, however, no studies on the role of AMPK in pregnancies complicated by GDM. Thus, the aims of this study were (i) to compare the expression of AMPK in adipose tissue and skeletal muscle from women with GDM and normal glucose-tolerant (NGT) pregnant women; and (ii) to investigate the effect of AMPK activation on inflammation and insulin resistance induced by the bacterial endotoxin lipopolysaccharide (LPS) and the pro-inflammatory cytokine IL-1β. When compared to NGT pregnant women, AMPKα activity was significantly lower in women with GDM as evidenced by a decrease in threonine phosphorylation of AMPKα. Activation of AMPK, using two pharmacologically distinct compounds, AICAR or phenformin, significantly suppressed LPS- or IL-1β-induced gene expression and secretion of pro-inflammatory cytokine IL-6, the chemokines IL-8 and MCP-1, and COX-2 and subsequent prostaglandin release from adipose tissue and skeletal muscle. In addition, activators of AMPK decreased skeletal muscle insulin resistance induced by LPS or IL-1β as evidenced by increased insulin-stimulated phosphorylation of IRS-1, GLUT-4 expression and glucose uptake. These findings suggest that AMPK may play an important role in inflammation and insulin resistance.

Prediction of spontaneous preterm labour in at-risk pregnant women

The ability to recognise women who are at-risk of preterm labour (PTL) is often difficult. Over 50% of women who are identified with factors associated with an increased risk of preterm birth will ultimately deliver at term. The cervicovaginal fluid (CVF) comprises a range of proteins secreted by gestational tissues, making it an ideal candidate for the screening of differentially expressed proteins associated with PTL. CVF samples were collected from at-risk asymptomatic women. Two-dimensional gel electrophoresis techniques were used to examine the CVF proteome of women who spontaneously delivered preterm 11-22 days later compared with gestation-matched women who delivered at term. Five candidate biomarkers were selected for further validation in a larger independent cohort of asymptomatic women. Thioredoxin (TXN) and interleukin 1 receptor antagonist (IL1RN) concentrations in the CVF were found to be significantly reduced up to 90 days prior to spontaneous PTL compared with women who subsequently delivered at term. TXN was able to predict spontaneous PTL within 28 days after sampling with a high positive predictive value (PPV) and negative predictive value (NPV) of 75.0% and 96.4% respectively. IL1RN also showed comparable PPV and NPV of 72.7% and 95.7% respectively. The discovery of these differentially expressed proteins may assist in the development of a new predictive bedside test in identifying asymptomatic women who have an increased risk of spontaneous PTL.

Human cervicovaginal fluid biomarkers to predict term and preterm labor

Preterm birth (PTB; birth before 37 completed weeks of gestation) remains the major cause of neonatal morbidity and mortality. The current generation of biomarkers predictive of PTB have limited utility. In pregnancy, the human cervicovaginal fluid (CVF) proteome is a reflection of the local biochemical milieu and is influenced by the physical changes occurring in the vagina, cervix and adjacent overlying fetal membranes. Term and preterm labor (PTL) share common pathways of cervical ripening, myometrial activation and fetal membranes rupture leading to birth. We therefore hypothesize that CVF biomarkers predictive of labor may be similar in both the term and preterm labor setting. In this review, we summarize some of the existing published literature as well as our teams breadth of work utilizing the CVF for the discovery and validation of putative CVF biomarkers predictive of human labor. Our team established an efficient method for collecting serial CVF samples for optimal 2-dimensional gel electrophoresis resolution and analysis. We first embarked on CVF biomarker discovery for the prediction of spontaneous onset of term labor using 2D-electrophoresis and solution array multiple analyte profiling. 2D-electrophoretic analyses were subsequently performed on CVF samples associated with PTB. Several proteins have been successfully validated and demonstrate that these biomarkers are associated with term and PTL and may be predictive of both term and PTL. In addition, the measurement of these putative biomarkers was found to be robust to the influences of vaginal microflora and/or semen. The future development of a multiple biomarker bed-side test would help improve the prediction of PTB and the clinical management of patients.

Endoplasmic Reticulum Stress Is Increased after Spontaneous Labor in Human Fetal Membranes and Myometrium Where It Regulates the Expression of Prolabor Mediators

ABSTRACT Increasing evidence indicates that endoplasmic reticulum (ER) stress is involved in various diseases. In nongestational tissues, several markers of the unfolded protein response (UPR) have been shown to regulate the inflammatory response. Thus, the aim of this study was to determine the effect of human labor on markers of ER stress in fetal membranes and myometrium. In addition, the effect of ER stress inhibition on the expression and secretion of proinflammatory and prolabor mediators was also assessed. The markers of ER stress, GRP78, IRE1, and spliced XBP1 (XBP1s), were significantly increased in fetal membranes and myometrium after term and preterm labor compared to nonlaboring samples. Given that inflammation is considered to be one of the leading causes of spontaneous preterm birth, here we used bacterial endotoxin lipopolysaccharide (LPS) as a model for infection-induced preterm birth. In term nonlabored fetal membranes and myometrium, LPS induced UPR activation as evidenced by a significant increase in the expression of GRP78, IRE1, and XBP1s in fetal membranes and myometrium. The use of the chemical chaperones 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA) alleviated ER stress induced by LPS. 4-PBA and TUDCA also ameliorated the increase in LPS-induced prolabor mediators. Our data suggest that the UPR may regulate the inflammatory responses associated with labor or infection in fetal membranes and myometrium of pregnant term and preterm women. Thus, the use of ER stress inhibitors, in particular 4-PBA or TUDCA, may be a potential therapeutic strategy for the prevention of infection-mediated spontaneous preterm birth.

Endoplasmic reticulum stress is increased in adipose tissue of women with gestational diabetes.

Maternal obesity and gestational diabetes mellitus (GDM) are two increasingly common and important obstetric complications that are associated with severe long-term health risks to mothers and babies. IL-1β, which is increased in obese and GDM pregnancies, plays an important role in the pathophysiology of these two pregnancy complications. In non-pregnant tissues, endoplasmic (ER) stress is increased in diabetes and can induce IL-1β via inflammasome activation. The aim of this study was to determine whether ER stress is increased in omental adipose tissue of women with GDM, and if ER stress can also upregulate inflammasome-dependent secretion of IL-1β. ER stress markers IRE1α, GRP78 and XBP-1s were significantly increased in adipose tissue of obese compared to lean pregnant women. ER stress was also increased in adipose tissue of women with GDM compared to BMI-matched normal glucose tolerant (NGT) women. Thapsigargin, an ER stress activator, induced upregulated secretion of mature IL-1α and IL-1β in human omental adipose tissue explants primed with bacterial endotoxin LPS, the viral dsRNA analogue poly(I:C) or the pro-inflammatory cytokine TNF-α. Inhibition of capase-1 with Ac-YVAD-CHO resulted in decreased IL-1α and IL-1β secretion, whereas inhibition of pannexin-1 with carbenoxolone suppressed IL-1β secretion only. Treatment with anti-diabetic drugs metformin and glibenclamide also reduced IL-1α and IL-1β secretion in infection and cytokine-primed adipose tissue. In conclusion, this study has demonstrated ER stress to activate the inflammasome in pregnant adipose tissue. Therefore, increased ER stress may contribute towards the pathophysiology of obesity in pregnancy and GDM.

Proteomic analysis of human cervicovaginal fluid collected before preterm premature rupture of the fetal membranes.

A significant obstetric complication facing contemporary materno-fetal medicine is preterm premature rupture of the fetal membranes (preterm PROM), which occurs in 30% of all preterm births. The objective of this study was to identify differentially expressed proteins in the cervicovaginal fluid of asymptomatic women before the clinical manifestation of preterm PROM. The preterm PROM group comprised of women with samples collected 6-23 days before PROM, who subsequently delivered preterm (n=5). Women who spontaneously delivered at term served as gestation-matched controls (n=10). Two-dimensional difference in-gel electrophoresis was used to distinguish differential expression between the pooled groups and fold changes were subsequently confirmed by two-dimensional PAGE of individual samples. Spots of interest were identified by mass spectrometry. Proteins that were significantly reduced with impending preterm PROM included the following: thioredoxin (2.7-fold), interleukin 1 receptor antagonist (1.7-fold), fatty acid-binding protein 5 (2.1-fold), cystatin A (dimer; 1.9-fold), monocyte/neutrophil elastase inhibitor (1.6-fold), squamous cell carcinoma antigen-1 (2.1-fold) and γ-glutamyl cyclotransferase (3.0-fold). By contrast, annexin A3 (3.7-fold) and vitamin D binding protein (3.9-fold) were significantly increased with impending preterm PROM. Western blot analysis was also performed on an independent cohort of preterm PROM and control samples to validate these candidate biomarkers. These proteins have known biological functions in oxidative balance, anti-inflammatory activity, metabolism or protease inhibition that may facilitate membrane rupture.

The Stress-responsive Heme Oxygenase (HO)-1 Isoenzyme is Increased in Labouring Myometrium where it Regulates Contraction-associated Proteins

Sterile inflammation through activation of cytokine receptor signalling pathways and viral or bacterial infection via activation of Toll‐like receptors (TLRs) induces a cascade of events that leads to myometrial contractions and spontaneous preterm delivery. In non‐pregnant tissues, heme oxygenase‐1 (HO‐1) is thought to play a central role in regulating the inflammatory response. Thus, the aims of this study were to determine the effect of human term labour on HO‐1 expression in human myometrium and to investigate the role of HO‐1 in myometrial primary cells in response to cytokine‐ and TLR ligand‐induced inflammation.