Clinical and Translational Physiology

Human Cervicovaginal Fluid Biomarkers to Predict Term and Preterm Labour Yujing Jan Heng, Stella Liong, Michael Permezel, Gregory Edward Rice, Megan Katherine Waterhouse Di_Quinzio and Harry Michael Georgiou

Journal Name:

Frontiers in Physiology

ISSN:

1664-042X

Article type:

Review Article

Received on:

16 Dec 2014

Accepted on:

27 Apr 2015

Provisional PDF published on:

28 Apr 2015

Frontiers website link:

www.frontiersin.org

Citation:

Heng YJ, Liong S, Permezel M, Rice GE, Di_quinzio MK and Georgiou HM(2015) Human Cervicovaginal Fluid Biomarkers to Predict Term and Preterm Labour. Front. Physiol. 6:151. doi:10.3389/fphys.2015.00151

Copyright statement:

© 2015 Heng, Liong, Permezel, Rice, Di_quinzio and Georgiou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

This Provisional PDF corresponds to the article as it appeared upon acceptance, after rigorous peer-review. Fully formatted PDF and full text (HTML) versions will be made available soon.

1

Human Cervicovaginal Fluid Biomarkers to Predict Term and Preterm Labour

Yujing J. Heng1; Stella Liong2,3; Michael Permezel2,3; Gregory E. Rice4; Megan K.W. Di Quinzio2,3; Harry M. Georgiou2,3*

1

Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center,

Harvard University, Boston, MA, USA; 2

Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, VIC,

Australia; 3

Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, VIC, Australia;

4

University of Queensland Centre for Clinical Research, Herston, QLD, Australia

*Corresponding author: Dr Harry Georgiou, PhD Department of Obstetrics and Gynaecology, University of Melbourne Mercy Hospital for Women 163 Studley Rd, Heidelberg 3084, Victoria Australia E-mail: [email protected] Tel: +61-3-8458 4368

Running Title: Biomarkers of term and preterm birth Key Words: Cervicovaginal fluid, Predictive biomarkers, Pregnancy, Labour, Preterm labour, Preterm birth, IL-1 receptor antagonist, Thioredoxin, Albumin, Vitamin D binding protein

2

Table of Contents 1 INTRODUCTION

5

2 PHYSIOLOGY OF HUMAN LABOUR

5

3 PRETERM BIRTH

6

4 PREDICTING HUMAN LABOUR

8

4.1 RISK SCORING 4.2 CERVICAL LENGTH 4.3 BIOCHEMICAL MARKERS 4.3.1 FETAL FIBRONECTIN 4.3.2 PHOSPHORYLATED INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1 4.3.3 OTHER BIOCHEMICAL MARKERS 4.4 SUMMARY OF COMMONLY USED SCREENING TESTS

8 8 9 9 10 11 12

5 CERVICOVAGINAL FLUID BIOMARKERS OF HUMAN LABOUR

12

5.1 CERVICOVAGINAL FLUID PROTEOME 5.2 PROTEOMIC STUDIES OF PRETERM BIRTH

13 14

6 BIOMARKERS PREDICTIVE OF HUMAN LABOUR

16

6.1 PATIENT GROUPS AND CERVICOVAGINAL FLUID COLLECTION 6.2 METHODS: BIOMARKER DISCOVERY USING 2D-PAGE AND 2D-DIGE 6.3 METHODS: BIOMARKER VALIDATION USING IMMUNOBASED TECHNIQUES

16 17 18

7 BIOMARKER DISCOVERY - TERM PARTURITION

19

8 BIOMARKER DISCOVERY - PRETERM PARTURITION

19

8.1 ASYMPTOMATIC PRETERM PARTURITION 8.2 ASYMPTOMATIC PRETERM PRELABOUR RUPTURE OF MEMBRANES 8.3 SYMPTOMATIC PRETERM PARTURITION

20 20 21

9 CANDIDATE BIOMARKER VALIDATION - TERM PARTURITION

21

10 CANDIDATE BIOMARKER VALIDATION - PRETERM PARTURITION

22

11 INFLUENCE OF VAGINAL MICROFLORA AND SEMEN

22

3

12 MULTIPLE BIOMARKER PREDICTION OF HUMAN PARTURITION AND ITS CHALLENGES

23

13 CONCLUDING REMARKS

26

14 ACKNOWLEDGEMENTS

27

15 REFERENCES

28

4

ABSTRACT

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 labour (PTL) share common pathways of cervical ripening, myometrial activation and fetal membranes rupture leading to birth. We therefore hypothesise that CVF biomarkers predictive of labour may be similar in both the term and preterm labour setting. In this review, we summarise some of the existing published literature as well as our team’s breadth of work utilising the CVF for the discovery and validation of putative CVF biomarkers predictive of human labour.

Our team established an efficient method for collecting serial CVF samples for optimal 2dimensional gel electrophoresis resolution and analysis. We first embarked on CVF biomarker discovery for the prediction of spontaneous onset of term labour using 2Delectrophoresis 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.

5

1 INTRODUCTION A human pregnancy has a gestational period between 37 and 42 weeks. Births before 37 completed weeks of gestation are classified as preterm. Preterm birth (PTB) remains a major obstetric complication that leads to an increased risk of adverse neonatal morbidity and mortality (1). Currently, the ability to predict PTB is disappointing. Biomarkers that can predict PTB with improved sensitivity and specificity are required. In pregnancy, the human cervicovaginal fluid (CVF) proteome reflects the biochemical milieu and physiological changes of the vagina, cervix and adjacent overlying fetal membranes. Although the initiating triggers may differ, the physiology of human parturition at term and preterm gestations share the common pathways of cervical ripening, myometrial activation and membrane rupture. In this review, we describe our team’s breadth of work utilising the CVF for the discovery and validation of biomarkers predictive of human labour.

2 PHYSIOLOGY OF HUMAN LABOUR Human parturition is characterised by regular and painful uterine contractions that increase in duration, intensity and frequency. With each successive contraction, the fetus progressively descends and exerts pressure on the cervix and overlying fetal membranes. This promotes remodelling of the cervix leading to effacement and dilatation (2, 3), weakening and rupture of fetal membranes (4, 5) and the subsequent delivery of the neonate. In general, the spontaneous onset of uterine contractions and cervical remodelling precede fetal membrane rupture (6). However, the spontaneous rupture of the fetal membranes can also occur in the absence of uterine contractions and/or cervical dilatation at term and preterm gestations, i.e. prelabour rupture of membranes (PROM). PROM may arise from a different physiological pathway than that of spontaneous term and preterm labour (PTL; (7)).

Parturition is a complex systemic physiological event involving multiple reproductive or gestational tissues (fetal membranes, uterus, cervix and vagina) and various biochemical and physical pathways including: hormonal regulation (e.g. activation of the fetal hypothalamic-pituitary-adrenal axis (8), progesterone (9, 10), oestrogen (11) and

6

prostaglandin (12, 13)); inflammation (14-22); mechanical stretch (23-30); oxidative stress (31-37); apoptosis (38-43); lipid metabolism (44-46); and extracellular matrix (ECM) remodelling (47-53).

3 PRETERM BIRTH Globally, approximately 15 million babies are born prematurely each year. PTB rates are highest (15% or higher) in developing nations such as Pakistan, Indonesia and sub-Saharan Africa (54). In 2010, there were 22,952 PTB out of 297,357 live births (8.3%) in Australia (55). PTB rates in the United States have reached 12% and have remained consistently high between 1990 and 2010 (54, 56, 57). The rising incidence of PTB, especially late PTB between 34 and 36 weeks of gestation, is thought to be the result of obstetric complications and the increase in multifetal pregnancies associated with modern fertility treatments (58).

PTB contributes to the 3.1 million neonatal deaths worldwide annually (35%) (54). The survival rate of premature babies correlates with advancing gestational age. In England the survival rate for preterm neonates born at 23 weeks is 19% with the rate increasing to 77% if birth occurs at 26 weeks (59). Although advances in perinatal care have improved survival rates, cognitive impairment and other life-long disabilities associated with preterm birth remain significant (60-64). It is not surprising that PTB carries an immense financial burden particularly in developed countries where advanced medical resources are widely available. Premature babies require a longer hospital stay, have medical complications necessitating multiple hospital admissions and the use of medical equipment and operative procedures (65-67). The annual economic cost of PTB is estimated at USD$26 billion in the United States (68) and ₤3 billion in the United Kingdom (69).

PTB can be categorised as either spontaneous or iatrogenic. About 75% of PTB are spontaneous, resulting from spontaneous PTL with intact membranes (45%) or spontaneous preterm prelabour rupture of fetal membranes (preterm PROM; 30%). The remaining 25% of PTB are iatrogenic due to maternal (e.g. diabetes), placental (e.g. pre-eclampsia) or fetal disorders (e.g. intra-uterine growth restriction) that necessitate delivery of a compromised fetus and/or mother (70, 71).

7

The aetiology of PTB is multifactorial. Multiple pregnancy, previous PTB, ethnicity (especially of African descent (72, 73)) and second- or third-trimester vaginal bleeding are most consistently associated with PTB. Other factors associated with the initiation of PTL include: poor maternal sociobiological variables (stress, malnutrition, smoking, low socioeconomic status, heavy physical work and substance abuse); fetal physiological stress (malformation, intra-uterine growth restriction); infection; preterm PROM; cervical dysfunction; placental abruption; uterine over distension (polyhydramnios or multifetal gestation); and genetic predisposition (familial factors and genetic mutation) (28, 71, 74-79).

The treatment of PTL involves the use of tocolytic agents (e.g. β-adrenergic receptor agonists such as ritodrine or calcium channel blockers such as nifedipine) to inhibit uterine contractions (80, 81). Tocolytics may stop contractions temporarily but rarely prevent PTB (82). Thus, the current application of tocolytics is largely directed at prolonging the pregnancy for up to 48 hours to augment fetal lung maturity after maternal corticosteroid administration and arrange transport of the woman to a tertiary hospital with appropriate neonatal facilities (83).

The prevention of PTB in women with known risk factors may be divided into two approaches: (i) to reduce clinical risk factors by antenatal education (e.g. optimal nutrition (84, 85)) and reducing physical and emotional stress and substance abuse; and (ii) optimise appropriate use of pharmacological agents such as the use of progesterone. Progesterone has been clinically trialled to prevent PTB with encouraging results (reviewed by (86)). The prevention of PTB is more challenging for women in their first pregnancy who have no known risk factors. For all women, screening programs to detect early cervical change by ultrasonography are of limited benefit. Alternatively, diagnostic screening tools (biochemical markers) to reliably predict the onset of (preterm) labour (82) are required. The prognostic value of the current biochemical markers to predict PTB is disappointing. The relatively poor sensitivity and positive predictive value of these tests often lead to costly and unnecessary interventions.

8

4 PREDICTING HUMAN LABOUR Given the challenges of preventing PTB, the ability to reliably predict who will deliver preterm may aid in the clinical management of asymptomatic or symptomatic women (i.e. in threatened PTL with uterine contractions without cervical dilatation). The accurate prediction of PTL is difficult due to the multifactorial aetiology of PTB as various biochemical triggers may result in different clinical presentations of early onset labour. Therefore, a single predictor of PTB may yield varying utility in symptomatic women compared to asymptomatic women without or with risk factors for PTL (e.g. previous history of PTB or preterm PROM, multifetal gestation, cervical incompetence etc.).

4.1 RISK SCORING Risk scoring is a quantitative approach to identify women as low-, medium- or high-risk for PTB within the obstetric population (87). A combined Australia and New Zealand cohort study of nulliparous women reported maternal demographics (ethnicity, low body mass index, smoking, drug use and anxiety) and obstetric characteristics (>3 months to conceive, shortened cervical length, gestational diabetes, mild gestational hypertension and preeclampsia) as risk factors for spontaneous PTB and/or preterm PROM (88). Other factors associated with a higher risk of PTB include multifetal gestation (89), bacterial vaginosis (90) and previous cervical cone biopsy (91). However the use of risk scoring alone is often unreliable as more than 50% of women who experience PTB have no recognisable risk factor (76, 88, 92).

4.2 CERVICAL LENGTH Transvaginal ultrasonography was introduced in the 1980s as a screening tool to assess the cervical morphology during pregnancy (measurement of cervical length and evaluate the dilated internal cervical os (93, 94)). It is a more reliable and reproducible method than digital cervical examination to predict PTB (87). A study by Iams et al. (95) reported increasing risk of PTB (