The authors have declared that no competing interests exist.
Conceived and designed the experiments: SL HMG MKWD MP. Performed the experiments: SL. Analyzed the data: SL HMG MKWD MP. Contributed reagents/materials/analysis tools: HMG MKWD. Wrote the paper: SL HMG MKWD. Consented the subjects and collected samples: GF. Clinical guidance: MKWD MP.
Vitamin D binding protein (VDBP) has previously been identified in the amniotic fluid and cervicovaginal fluid (CVF) of pregnant women. The biological functions of VDBP include acting as a carrier protein for vitamin D metabolites, the clearance of actin that is released during tissue injury and the augmentation of the pro-inflammatory response. This longitudinal observational study was conducted on 221 healthy pregnant women who spontaneously laboured and delivered either at term or preterm. Serial CVF samples were collected and VDBP was measured by ELISA. Binary logistic regression analysis was performed to assess the utility of VDBP as a predictor of labour. VDBP in the CVF did not change between 20 and 35 weeks' gestation. VDBP measured in-labour was significantly increased 4.2 to 7.4-fold compared to 4–7, 8–14 and 15–28 days before labour (
Vitamin D binding protein (VDBP) is a 56–58 kDa plasma α-globulin that is primarily synthesised by hepatic parenchymal cells. Originally named “group-specific component” (Gc)
VDBP has other physiological functions not restricted to vitamin D transportation. One of the more extensively studied properties is its high-affinity to form complexes with globular (G)-actin monomers, therefore inhibiting actin polymerisation
Aside from serum, VDBP has been detected in other biological fluids such as cerebrospinal fluid, urine and breast milk
There are limited studies of VDBP in pregnancy. Bouillon and colleagues reported increased serum VDBP levels in pregnancy and in non-pregnant women using oestrogen therapy
It is well established that both term and preterm labour are associated with increased inflammation and extensive tissue remodelling of the cervix and fetal membranes. As such, it is hypothesised that the VDBP concentration in the CVF may serve as an indicator of up-regulated cell death and tissue remodelling involved in cervical ripening and subsequent labour onset. Therefore the aims of this observational study were: (i) to investigate the concentration of VDBP in the CVF during the second half of pregnancy; (ii) to investigate the temporal VDBP concentration in the CVF of women with spontaneous term and preterm labour outcomes; and (iii) to determine the utility of VDBP to predict spontaneous labour onset.
The collection of CVF from pregnant women was approved by the Mercy Health Human Research Ethics Committee (R06/56). All participants involved in this longitudinal observational study provided informed, written consent.
Healthy, pregnant women attending the Mercy Hospital for Women, Heidelberg, Australia were recruited by a research midwife or medical officer. Exclusion criteria for recruitment were: obstetric complications such as hypertension, diabetes mellitus, known fetal anomaly, triplet or higher-order pregnancy; cervical cerclage
Following the aims described above, the proposed objectives required two arms of subject recruitment as it was deemed inappropriate to subject women to repeated frequent CVF sampling from 20 weeks' gestation until term labour onset. Women were either recruited preterm, in order to establish the antenatal expression of VDBP in the CVF and to optimise the number of samples from women who subsequently spontaneously laboured preterm, or recruited in late pregnancy in order to establish VDBP expression in the CVF in association with ‘normal’ term labour. Gestational age was calculated from first trimester ultrasound assessment. Specific details of recruitment are listed below.
Women with either a singleton or twin pregnancy were recruited at 20–24 weeks' gestation, based on known risk-factor(s) for a preterm birth including: a history of spontaneous preterm labour; previous preterm pre-labour rupture of the fetal membranes; twin gestation; uterine anomaly; shortened cervix (<2.5 cm based ultrasonographic diagnosis); and a previous history of cervical cone biopsy. Recruited women had a CVF sample collected every four weeks until 35 completed weeks' gestation. Additional CVF samples were obtained from women presenting in spontaneous preterm labour, prior to rupture of the fetal membranes. The CVF samples were then retrospectively selected for analysis from women who experienced either spontaneous term labour or spontaneous preterm labour. All CVF samples were collected prior to any therapeutic modalities (e.g. maternal corticosteroids, tocolytics and antibiotics).
Women with a singleton pregnancy were recruited at approximately 36 weeks' gestation and had a CVF swab performed weekly and where possible, an in-labour swab was collected prior to rupture of the fetal membranes. CVF samples were then retrospectively selected for analysis from women who experienced spontaneous term labour. Labour was defined as regular painful contractions leading to subsequent effacement and dilatation of the cervix (3–7 cm).
At the time of sampling, all participants were asked whether they had had unprotected sexual intercourse in the preceding 48 h; experienced vaginal bleeding in the preceding 24 h; or had a transvaginal ultrasound or digital vaginal examination performed in the 6 h prior to CVF sample collection. Participants were also questioned on whether they were taking any medications (oral and/or vaginal).
Recruited women were subsequently excluded from analysis if they were diagnosed with bacterial vaginosis; presented with clinical signs of infection (e.g. chorioamnionitis, urinary tract infection); had a digital vaginal examination or transvaginal ultrasound within 6 h prior to CVF collection; had an induction of labour or an elective Caesarean section delivery.
All swabs were performed prior to the rupture of the fetal membranes in order to avoid amniotic fluid contamination of the CVF. CVF was collected from pregnant women during a routine speculum examination as previously described
Women recruited preterm had a high vaginal swab taken for microbiological assessment at the time of every CVF swab collection. Women recruited in late pregnancy had a single high vaginal swab taken on entry to the study. Microbiological assessment was performed according to standard laboratory methods.
The VDBP concentration in the CVF was measured using the VDBP DuoSet® ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturer's instructions. The linear detection range of the assay was 156.25 pg/mL to 1000 pg/mL. The sensitivity, inter-assay and intra-assay coefficients of variation for the assay were 23.86 pg/mL, 8.7% and 5.6%, respectively.
The VDBP concentrations presented in the Results section were normalised against the protein content of each CVF sample and are expressed as per milligram of protein. The total protein concentration was determined using the Bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL).
From the 221 women recruited to the study, 461 CVF samples were collected. Subanalyses were performed according to the flow diagram represented in
Eight subanalyses of VDBP expression in the CVF were performed and the number of samples utilised is indicated.
Binary logistic regression was performed to assess the utility of VDBP as a predictive biomarker of spontaneous labour. Samples from women who spontaneously laboured either at term or preterm were used in the analysis. Only VDBP values from women who provided two or more CVF samples were utilised. To account for multiple sampling from the same subject, the subject was entered as a categorical variable. Receiver operator characteristic (ROC) curves were generated and the prediction of labour within 3, 7 and 14 days was determined. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) are reported based on a classification cut-off of 0.5.
A description of the participant demographics is summarised in
Preterm labour | Term labour | |
(N = 48 women) | (N = 173 women) | |
Maternal age (years) | 32.28±5.1 | 31.69±4.6 |
Gravidity | N (%) | N (%) |
1 | 11 (23%) | 7 (4%) |
2–3 | 28 (58%) | 105 (61%) |
≥4 | 9 (19%) | 61 (35%) |
Parity | N (%) | N (%) |
Nulliparous | 19 (40%) | 27 (16%) |
1 | 23 (48%) | 103 (60%) |
2–3 | 4 (8%) | 36 (20%) |
≥4 | 2 (4%) | 7 (4%) |
Singleton (N (%)) | 23 (48%) | 166 (96%) |
Twins (N (%)) | 25 (52%) | 7 (4%) |
Delivery gestation (weeks) | 34.34±2.4 | 39.77±1.1 |
Baby weight (grams) | 2289±595 | 3505±497 |
Where the information was volunteered (53% of responses, N = 246 samples), the VDBP concentration was not significantly different in the samples obtained from women who had recent unprotected sexual intercourse compared with women who had not, irrespective of whether they delivered at term (N = 189 samples,
(A) Unprotected sexual intercourse within 48 h of CVF collection did not affect the VDBP concentration in women who delivered either term (
For analysis, the microbiology results were stratified into five groups: normal vaginal flora (NVF); NVF with Group B Streptococcus (GBS); NVF with
A total of 352 samples were analysed to assess the effect of advancing gestation on the concentration of VDBP in the CVF. The VDBP concentration was not significantly different between gestational groups at 20–23, 24–25, 26–27, 28–29, 30–31, 32–33 and 34–35 weeks' gestation in women with a singleton pregnancy that subsequently reached term gestation (
The * indicates significance in the VDBP concentration in gestational groups ≤30–31 weeks' compared with CVF samples collected ≥36 weeks' gestation. The † indicates significance in VDBP concentration between samples collected at 36 and 37 weeks' gestation compared with samples collected at 40 and 41 weeks' gestation. Statistical significance was defined as
The same 352 samples described above were analysed with respect to the effect of time-from-labour on the concentration of VDBP. There was no significant change in the concentration of VDBP between 29 to >90 days prior to spontaneous term labour onset (
The * indicates a significant difference in VDBP concentration in the CVF between the in-labour group and groups ≥4 days from labour. The † indicates a significant difference between the 0–3 day group versus groups ≥15 days from labour onset. The ‡ indicates a significant difference between the 4–7 day group versus groups ≥15 days from labour onset. The § indicates a significant difference between the 8–14 day group versus groups ≥29 days from labour onset. The ¤ indicates a significant difference between the 15–28 day group versus groups ≥29 days from labour onset. Statistical significance was defined as
In women who delivered at term, the VDBP concentration increased both in terms of advancing gestation (
To investigate VDBP expression in the CVF of women who delivered
VDBP was not significantly different between singleton and twin gestation in-labour (
Using the same 461 samples, a comparison of VDBP concentration between women who laboured at term and preterm was performed. Women with
(A) Women with
To assess the utility of VDBP to predict labour up to 14 days before onset, a total of 141 women representing 392 samples were analysed. To take into account the relatively wide variation in VDBP concentration between women, only women who provided two or more CVF samples were analysed by Binary Logistic Regression. Samples from both term and preterm deliveries were included, as these demonstrated no significant difference in this time frame (
Subjects who provided two or more samples were included in the analysis (N = 392), with the subject entered as a categorical factor in the model. Area under the curves were 0.974 at ≤3 days; 0.943 at ≤7 days; and 0.934 at ≤14 days prior to spontaneous labour onset. Optimal predictive utility was obtained at ≤3 days from spontaneous labour.
Prediction | 3 d before onset | 7 d before onset | 14 d before onset |
Samples (N) | ≤3 days; N = 41 | ≤7 days; N = 71 | ≤14 days; N = 111 |
>3 days; N = 351 | >7 days; N = 321 | >14 days; N = 281 | |
Area under curve | 0.974 | 0.943 | 0.934 |
95% confidence interval | 0.958–0.989 | 0.921–0.966 | 0.912–0.956 |
<0.001 | <0.001 | <0.001 | |
Classification cut-off value | 0.5 | 0.5 | 0.5 |
Sensitivity (%) | 58.5 | 57.7 | 71.2 |
Specificity (%) | 98.6 | 96.6 | 87.9 |
Positive Predictive Value (%) | 82.8 | 78.8 | 69.9 |
Negative Predictive Value (%) | 95.3 | 91.2 | 88.5 |
To our knowledge, this is a novel study characterising the temporal changes of VDBP in the CVF in association with pregnancy and spontaneous labour. The presence of VDBP in the CVF during pregnancy is thought to be the result of plasma transudate. In the final four weeks of a term singleton pregnancy, we found that the VDBP concentration in the CVF was significantly elevated at 40–41 weeks' compared to 36–37 weeks' gestation (
The significant increase of VDBP in the CVF collected in-labour and at 0–3 days pre-labour may be attributed to the increased permeability of blood vessels as a result of the inflammatory process of labour leading to cervical remodelling and subsequent fetal membrane rupture. Indeed, synthesis of VDBP can be augmented by the presence of the pro-inflammatory cytokine IL-6,
The CVF expression of VDBP was similar in women who spontaneously laboured at term and preterm. In particular, the most dynamic change occurs in the final 4 weeks of pregnancy in both term and preterm cases. A significantly increased concentration of VDBP was found in samples collected from women >28 days from labour who spontaneously delivered preterm, compared to women who delivered at term (
There have been no previous temporal studies of VDBP expression in the CVF during pregnancy. A study conducted in Belgium by Bouillon
The aetiology of spontaneous preterm birth is multifactorial. To strengthen this study, we have attempted to account for various potential confounders including risk factor status, vaginal flora, recent sexual activity, twin gestation and time from spontaneous labour. For women who delivered at term (both singleton and twin pregnancies), unprotected intercourse had no effect on the VDBP concentration in the CVF. For women who delivered preterm, again no difference was observed, however the sample size was underpowered to confirm this finding with confidence.
Multifetal gestation is a risk factor for preterm birth and this study included a proportion of women with a twin gestation. We recognise this is a limitation of the study however we included twin pregnancies in order to increase the preterm sample size. The increased VDBP concentration with respect to spontaneous labour onset (i.e. time from labour) was achieved using data derived from singleton pregnancies. We subsequently compared singleton and twin groups and found no significant difference in the VDBP concentration (both term and preterm outcomes) although twin sample sizes were small, particularly those less than 28 days from labour (
On the basis of the promising preterm data the utility of VDBP to predict spontaneous labour was investigated. The prediction of spontaneous labour within both 3 and 7 days compares favourably with fetal fibronectin (fFN). fFN remains the gold standard test for the prediction of labour
The role of VDBP and the contribution of its various isoforms in pregnancy is not fully understood. Activated B and T lymphocytes secrete enzymes that are able to process the O-linked side chain of VDBP
In conclusion, we have characterised VDBP in the CVF from mid- to late-pregnancy and report a significant increase in the final four weeks of pregnancy. Increased concentration of VDBP in the CVF was associated with spontaneous labour, both at term and preterm. The increase in VDBP may be indicative of increased inflammation and tissue damage that are integral to cervical remodelling and fetal membrane weakening with approaching labour onset. VDBP was able to predict spontaneous labour onset with high positive and negative predictive values up to 14 days from sampling. Further investigation to include other biomarkers (e.g. cervical length, fFN, IL-1ra
The authors would like to acknowledge Dr Yujing Heng (former PhD student), research midwives and medical officers involved in the recruitment and sample collection for this study. Microbiological assessment was performed by Austin Pathology, Austin Health, Heidelberg, Victoria, Australia or the Department of Microbiology at St Vincents Hospital, Fitzroy, Victoria, Australia.