Evidence of perturbations of the cytokine network in preterm labor

Abstract

Objective: Intraamniotic inflammation/infection is the only mechanism of disease with persuasive evidence of causality for spontaneous preterm labor/delivery. Previous studies about the behavior of cytokines in preterm labor have been largely based on the analysis of the behavior of each protein independently. Emerging evidence indicates that the study of biologic networks can provide insight into the pathobiology of disease and improve biomarker discovery. The goal of this study was to characterize the inflammatory-related protein network in the amniotic fluid of patients with preterm labor.

Study design: A retrospective cohort study was conducted that included women with singleton pregnancies who had spontaneous preterm labor and intact membranes (n = 135). These patients were classified according to the results of amniotic fluid culture, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry, and amniotic fluid concentration of interleukin (IL)-6 into the following groups: (1) those without intraamniotic inflammation (n = 85), (2) those with microbial-associated intraamniotic inflammation (n = 15), and (3) those with intraamniotic inflammation without detectable bacteria (n = 35). Amniotic fluid concentrations of 33 inflammatory-related proteins were determined with the use of a multiplex bead array assay.

Results: Patients with preterm labor and intact membranes who had microbial-associated intraamniotic inflammation had a higher amniotic fluid inflammatory-related protein concentration correlation than those without intraamniotic inflammation (113 perturbed correlations). IL-1β, IL-6, macrophage inflammatory protein (MIP)-1α, and IL-1α were the most connected nodes (highest degree) in this differential correlation network (degrees of 20, 16, 12, and 12, respectively). Patients with sterile intraamniotic inflammation had correlation patterns of inflammatory-related proteins, both increased and decreased, when compared to those without intraamniotic inflammation (50 perturbed correlations). IL-1α, MIP-1α, and IL-1β were the most connected nodes in this differential correlation network (degrees of 12, 10, and 7, respectively). There were more coordinated inflammatory-related protein concentrations in the amniotic fluid of women with microbial-associated intraamniotic inflammation than in those with sterile intraamniotic inflammation (60 perturbed correlations), with IL-4 and IL-33 having the largest number of perturbed correlations (degrees of 15 and 13, respectively).

Conclusions: We report for the first time an analysis of the inflammatory-related protein network in spontaneous preterm labor. Patients with preterm labor and microbial-associated intraamniotic inflammation had more coordinated amniotic fluid inflammatory-related proteins than either those with sterile intraamniotic inflammation or those without intraamniotic inflammation. The correlations were also stronger in patients with sterile intraamniotic inflammation than in those without intraamniotic inflammation. The findings herein could be of value in the development of biomarkers of preterm labor.

Keywords: chemokine; chorioamnionitis; correlation network; interactome; intraamniotic infection; network analysis; sterile inflammation.

Figure 1

Differential correlation analysis. The figure shows log₂ concentration (pg/ml) of IL-1β (left panel) and MIP1α (middle panel) as a function of gestational age at amniocentesis in patients with microbial-associated intra-amniotic inflammation (red) and those without intra-amniotic inflammation (black). A linear model was fit to the log₂ concentration of each analyte as a function of gestational age in each group and residuals were used to compute partial correlations between analytes (right panel). The partial correlation of residuals was positive and significant in the microbial-associated intra-amniotic inflammation group but negative and significant in patients without intra-amniotic inflammation, resulting in a significant differential correlation between groups.

Figure 2

Network of perturbed inflammatory related protein concentration correlations between groups of preterm labor with intact membranes. Each node (sphere) represents one of the 33 analytes, with a link (line) between two nodes representing a significantly perturbed correlation. The node color represents the direction of concentration change (red=increased; blue=decreased; white=no change in the first group compared to the second/reference group of the comparison). The color of links gives the direction of correlation change (red = increased correlation; blue = decreased correlation) while the type of line denotes the nature of the link (solid line= within module link; dashed line= cross-module link). Thick radial lines separate the modules as well as the set of unconnected nodes, as labeled in the Figure. The numbers inside/outside the dotted black circle represent the node degree/average absolute difference in correlations. A: Network of perturbed inflammatory-related protein concentration correlations between sterile intra-amniotic inflammation and no intra-amniotic inflammation. B: Network of perturbed inflammatory-related protein concentration correlations between microbial-associated intra-amniotic inflammation and no intra-amniotic inflammation. C: Network of perturbed inflammatory-related protein concentration correlations between microbial-associated intra-amniotic inflammation and sterile intra-amniotic inflammation.