Higher levels of several cytokines, representing Th1, Th2 and Th17 lineages, were associated with increased odds of PTD <35 weeks accompanied by severe HCA. These cytokines were not associated with other PTD <35 weeks, nor were they associated with later HCA-related PTD. This is the first report of an association between a biomarker from maternal circulation collected prior to onset of labor or PROM and placental findings of intrauterine infection.
Studies evaluating biochemical markers for spontaneous PTD in women prior to PTL or PROM require large prospective cohorts given the relative rarity of early spontaneous PTD. High levels of various proteins sampled from maternal circulation in mid-pregnancy have been associated with spontaneous PTD, but not with specific evidence of infection. Elevated C-reactive protein collected in early to mid-pregnancy in serum or plasma has been positively associated with PTD in several studies (Catov et al., 2007
; Lohsoonthorn et al., 2007
; Pitiphat et al., 2005
). High plasma G-CSF (Goldenberg et al., 2000
) and serum ferritin (Tamura et al., 1996
; Xiao et al., 2002
) were associated with increased risk of spontaneous PTD. A small study in a very high risk cohort consisting of women with prior PTD <30 weeks used a panel of cytokines similar to the one employed here, and found that twelve of eighteen (including IL-1β, IL-2, IL-5, IL-6, IL-8, IL-12, IL-18, TNF-α, soluble TNF receptor I, TGF-β, TREM-1 and GM-CSF) were associated with recurrent PTD (Vogel et al., 2007
). Recently, a study reported on five serum cytokines (IL-2, IL-6, TNF-α, IFN-γ and GM-CSF) collected prior to 22 weeks, but found little evidence that these markers were strong predictors of spontaneous PTD, although there were partial positive associations for IFN-γ and IL-6 (Curry et al., 2007
Limited evidence has linked markers in cervical or vaginal fluid among non-laboring women to infection. Low levels of IL-1β, IL-6 or IL-8 in vaginal fluid at 8–20 weeks’ gestation were associated with increased risk of clinical (i.e. symptomatic) chorioamnionitis (Simhan et al., 2003
). High cervicovaginal fetal fibronectin at 23–24 weeks was associated with PTD, and among a subset of women who delivered <32 weeks and had clinical placental pathology exams available, fetal fibronectin was associated with HCA (Goldenberg et al., 1996b
Many of the cytokines considered in this analysis were highly correlated, and these correlations were not limited to other cytokines within the same T-cell lineage. In particular, the 7 cytokines found to be associated with HCA and PTD <35 weeks were all highly correlated with one another and may mark the same pathway. Although cytokines of different T-cell lineages are theoretically up-regulated at different times in an infectious process, the precise time course of events in the immune response cannot be disentangled in this study, since each woman was only sampled at one time point. Although the tissues of origin of the circulating cytokines are unclear, the observed elevations across many cytokines among women who later delivered <35 weeks with HCA may represent a robust immune response to infection within gestational tissues.
It is possible that, by the time an occult infection has become sufficiently established to be detected in a maternal compartment, the process leading to parturition is well advanced. The lack of association between PTD and HCA near term (i.e. 35–36 weeks) suggests that, for these women, infection has not yet occurred or advanced to a point of detection in the maternal circulation at 15–27 weeks. These findings are consistent with published results on other prospective biomarkers from maternal circulation which find much stronger results for earlier PTD (Goldenberg et al., 1996a
; Lohsoonthorn et al., 2007
; Xiao et al., 2002
). Our finding of a modest association between several cytokines and spontaneous PTD occurring at 35–36 weeks without HCA could represent a separate pathway to PTD, perhaps related to inflammation in the maternal vascular system.
A major strength of this study is the ability to link upstream biomarker data collected prior to clinical manifestations of pregnancy complications to specific placental findings, which increases the specificity of the findings between prospectively collected biologic material and intrauterine infection. The placenta samples were assessed without knowledge of delivery timing or other clinical variables using a detailed descriptive, rather than diagnostic instrument, and the HCA definition was previously linked to PTD. Because the POUCH Study sampled from several communities and included women from urban, suburban and rural populations, external validity should be greater than studies from single clinical sites or high-risk practices. Oversampling of women with high MSAFP into the subcohort should not distort the analyses, since the sampling probability was taken into account using weighted analyses.
Although the cohort from which this sample was drawn was relatively large (N=3019), the number of women with PTD <35 completed weeks is relatively small, and further stratification on HCA status led to small numbers in some outcome categories. This resulted in sufficient power to detect effect sizes of 1.6 or greater in the <35 week with HCA group, but the power was not sufficient for effects of smaller magnitude. The sample size of PTD/HCA was not sufficient to test for effect modification by timing of sample collection within the 13-week enrollment window or to consider PTD <32 weeks. While the multiplex assay has some attendant error, misclassification should be non-differential because assay plates included PTD and term samples; thus, measurement error would be expected to bias results toward the null. We attempted to minimize error associated with interassay variability by standardizing cytokine values. Thirteen analytes were considered in this analysis, thus an increased level of type I error could produce spurious results by chance. In fact, 7 of 13 cytokines tested followed a similar pattern of elevation prior to PTD <35 weeks with severe HCA, and hence we find it unlikely that this pattern is attributable to chance alone.
Before predictive values can be developed, several studies need to replicate prospective association findings, linking a relevant group of cytokines to specific evidence of an infection PTD pathway. However, the results of this study give hope that women with HCA could be screened prior to labor onset using non-invasive means, and that interventions could be targeted to these high-risk women with the goal of preventing PTD.