Megan K. W. Di Quinzio

Proteomic analysis and characterisation of human cervico-vaginal fluid proteins

Aim:  Cervico‐vaginal fluid (CVF) may provide insight into the biochemical pathways of human reproduction and parturition. The aim of this study was to establish a 2‐D electrophoretic map of human CVF in healthy, pregnant women at term.

Proteomic Analysis of Human Cervico-Vaginal Fluid Displays Differential Protein Expression in Association with Labor Onset at Term

Human labor is characterized by dramatic physiological and structural alterations of the cervix and overlying fetal membranes, leading to myometrial activation and delivery. To investigate the potential mechanism of these changes, we performed 2D PAGE proteomic analysis on serial cervico-vaginal fluid samples obtained from women during late pregnancy and spontaneous labor. We identified 9 protein spots that were significantly altered ( p < 0.05) in association with spontaneous term labor. Eight protein spots were definitively characterized by electrospray ion-trap mass spectrometry yielding 7 different proteins: cystatin-A, interleukin-1 receptor antagonist, glutathione S-transferase P, peroxiredoxin-2, thioredoxin, copper-zinc superoxide dismutase, and epidermal fatty-acid binding protein. These proteins are involved in protease inhibition, anti-inflammatory cytokine activity, and oxidative stress defense. These findings may provide an insight into the biochemical processes and timing associated with extracellular matrix remodelling of the cervix, supracervical fetal membranes, and myometrial activation in association with spontaneous term labor. Application of these findings may lead to development of predictive biomarkers of labor onset.

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.

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.

Temporal proteomic analysis of human cervicovaginal fluid with impending term labor.

This study utilized 2D-PAGE and MALDI-ToF to investigate labor-associated temporal protein changes in the human cervicovaginal fluid (>25 kDa). Monocyte/neutrophil elastase inhibitor, squamous cell carcinoma antigen 1, annexin A3, collagen and albumin demonstrated differential expression prior to and/or during term labor. These findings provide further insight into the extracellular matrix remodeling of the cervix and overlying fetal membranes and may be useful to develop predictive tools for labor.

Predicting Preterm Labour: Current Status and Future Prospects

Preterm labour and birth are a major cause of perinatal morbidity and mortality. Despite modern advances in obstetric and neonatal management, the rate of preterm birth in the developed world is increasing. Yet even though numerous risk factors associated with preterm birth have been identified, the ability to accurately predict when labour will occur remains elusive, whether it is at a term or preterm gestation. In the latter case, this is likely due to the multifactorial aetiology of preterm labour wherein women may display different clinical presentations that lead to preterm birth. The discovery of novel biomarkers that could reliably identify women who will subsequently deliver preterm may allow for timely medical intervention and targeted therapeutic treatments aimed at improving maternal and fetal outcomes. Various body fluids including amniotic fluid, urine, saliva, blood (serum/plasma), and cervicovaginal fluid all provide a rich protein source of putative biochemical markers that may be causative or reflective of the various pathophysiological disorders of pregnancy, including preterm labour. This short review will highlight recent advances in the field of biomarker discovery and the utility of single and multiple biomarkers for the prediction of preterm birth in the absence of intra-amniotic infection.

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.

Temporal Investigation of Matrix Metalloproteinases and Their Inhibitors in Human Cervicovaginal Fluid in Late Pregnancy and Labor

Temporal expression of matrix metalloproteinase (MMP)-1, -2, -3, -7, -8, -9, -12, and -13, and tissue inhibitors of metalloproteinases (TIMPs)-1 and -2 in human cervicovaginal fluid (CVF) in term pregnancy and labor was investigated. Term parous women provided CVF samples that were grouped into labor, 1 to 3, 6 to 8, and 12 to 16 days before labor onset. Both MMPs and TIMPs (n = 60) were quantified using multiplex solution array and enzyme-linked immunosorbent assays, respectively. Further analysis of TIMP-1 (n = 180) was undertaken. All MMPs and TIMPs except MMP-12 and -13 were detected in the CVF. Matrix metalloproteinase 7, TIMP-1, and TIMP-2 were significantly increased in labor. Tissue inhibitors of metalloproteinase 1 was significantly increased up to 7 days before spontaneous labor onset. The data suggest a role of MMP-7 in the remodeling and rupture of fetal membranes and may reflect the homeostatic regulation of extracellular matrix remodeling of MMP-7 by TIMP-1 and TIMP-2.

Temporal expression of antioxidants in human cervicovaginal fluid associated with spontaneous labor.

Proteomic analysis of human cervicovaginal fluid (CVF) by 2D electrophoresis revealed significant differential expression of several major antioxidant enzymes during late pregnancy and term labor. Temporal quantitative changes of total antioxidant capacity (TAC), Cu,Zn superoxide dismutase (Cu,Zn SOD) and thioredoxin-1 (Trx-1) with impending term labor were investigated, and the potential of these biomarkers as individual and multiple predictors of labor was determined. The TAC of CVF (n = 193) was 8-fold significantly lower in labor, and approximately 2-fold significantly lower at 0-7, 8-14, 15-21, and 22-28 days, compared with >or=29 days prior to labor onset (p < 0.001). The expression of Cu,Zn SOD (n = 170) was 1.5- to 1.9-fold significantly decreased in labor (p < 0.001). Trx-1 (n = 163) was 2.8- to 5.1-fold significantly lower in labor (p = 0.002). The combination of TAC and Cu,Zn SOD produced the best predictive efficacy with 74% sensitivity and 95% specificity to predict term labor within 3 days of onset. These findings suggest that labor is associated with increased oxidative stress well before its onset and is reflected in the human CVF. The biomarkers identified in this study could serve as predictors of labor and offer potential strategies for novel therapeutics.