Increased expression of alpha-enolase in cervico-vaginal fluid during labour

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Abstract

Objectives

The aim of this study was (i) to characterise differentially expressed proteins in cervico-vaginal fluid (CVF) at the time of preterm labour onset and (ii) to confirm these studies in human CVF samples taken from women before and during spontaneous labour.

Study design

Preterm labour was induced in sheep (n = 5) via fetal dexamethasone infusion (1 mg/24 h). CVF samples were taken prior to dexamethasone infusion (0 h), 28 h after the start of dexamethasone infusion, and immediately prior to delivery. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify differentially expressed proteins. For the human studies, paired CVF samples were taken 5–9 days before labour and during spontaneous labour onset (n = 7).

Results

There was a 4.2-fold increase in α-enolase protein expression in sheep CVF during labour. Likewise, α-enolase protein expression was significantly increased during spontaneous human labour at term.

Conclusions

Alpha-enolase is known to be bound to neutrophils and interact in the immune response, and thus may play a role in inflammation associated with human labour.

Introduction

One of the single most important factors contributing to neonatal and perinatal morbidity and mortality is preterm birth [1]. Further, there is a significant financial, emotional and societal burden associated with preterm birth. Methods currently available for predicting preterm labour, including cervical sonography [2], [3] and fetal fibronectin [4] are unreliable and with low sensitivity. Thus, there is an obvious need for effective and reliable diagnostic and prognostic indicators of imminent preterm delivery.

The protein composition of cervico-vaginal fluid (CVF) has the potential to provide unique insights into the biochemical pathways initiating human pregnancy and labour. Labour-associated changes in CVF proteins with imminent labour may reflect the biochemical changes occurring within the cervix and fetal membranes overlying the internal os. Studies have identified changes in cytokines, hormones, enzymes and inflammatory proteins in CVF during human pregnancy [5], [6] and in association with labour at term [7] and preterm [5], [8].

Further investigation of the CVF proteins implicated in the specific biochemical pathways of preterm labour using two-dimensional gel electrophoresis (2-DE) is warranted and may enhance our understanding of the complex biology involved in human labour. Thus, the aim of this study was to further characterise labour-associated differentially expressed CVF proteins using 2-DE protein display and matrix assisted laser desorption ionisation (MALDI) mass spectrometry and liquid chromatography–electrospray ionisation mass spectrometry (LC–ESI-MS). We will use a sheep model of labour as we have previously described [10]. Identification of differentially expressed proteins will then be confirmed using paired CVF samples taken from women during spontaneous labour onset (before rupture of human fetal membranes) and approximately 1 week before active labour.

Section snippets

Experimental animals

All animal experimental procedures were approved by the Physiology Animal Ethics Committee of Monash University. Five pregnant Border Leicester-Merino crossbred ewes underwent surgery at 126 days gestational age (GA) to insert maternal and fetal carotid artery and jugular vein catheters. An electromyographic (EMG) lead was sewn onto the uterus to measure contractile activity as labour progressed. A fetal intravenous dose of dexamethasone (1 mg/24 h) was administered at 135 days GA to induce

Two-dimensional gel electrophoresis

Protein expression was compared in CVF samples collected from ewes (n = 5) at each of the three time points: 0 h, 28 h and approximately 59 h after dexamethasone infusion. Differential expression of proteins was detected between all time points. Eight proteins were upregulated at delivery after preterm labour onset compared to the pre-induction samples (Fig. 1A, Table 1). In a previously published study, spot number 5004 was identified as bactenecin-1 [8], however, seven additional proteins could

Comment

Labour-associated changes in CVF proteins were characterised using 2-DE protein separation and mass spectrometry based peptide analysis. The data obtained establish that dexamethasone increased protein relative abundance of α-enolase, a neutrophil-derived glycolytic protein. Additionally, we used paired human CVF samples obtained from women before labour and during spontaneous labour onset to demonstrate increased expression of α-enolase during active labour.

Mammalian enolases are composed of

Funding

The work described in this manuscript was partly funded by a National Health and Medical Research Council (NHMRC) Project grant. Professor Greg Rice is a Research Fellow supported by the NHMRC. Dr. Martha Lappas is in recipient of a NHMRC RD Wright Fellowship (grant no. 454777).

Acknowledgements

We thank Alex Satragno from the Department of Physiology, Monash University for his surgical expertise. The authors also express their thanks to Gabrielle Fleming and Mardi Reeves (Mercy Hospital for Women) for obtaining study samples, and Dr. Harry Georgiou and Yujing Heng (Department of Obstetrics and Gynaecology, University of Melbourne) for providing four of the CVF samples.

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