Quantification of intra-amniotic inflammation in late preterm prelabour rupture of membranes

Abstract

Preterm prelabour rupture of membranes (PPROM) complicated by intra-amniotic inflammation (IAI) represents a substantial proportion of preterm birth cases. Currently, IAI is frequently defined as amniotic fluid IL-6 concentration above 2,600 pg/mL. However, the amniotic fluid IL-6 concentration was never correlated with the global response of other proinflammatory proteins to the ongoing IAI. In this cross-sectional study, protein quantification was performed using mass spectrometry (MS) analysis followed by target quantification of selected proinflammatory proteins. Levels of amniotic fluid proteins determined by MS were put into the correlation with IL-6 concentration determined by electrochemiluminescence immunoassay method (ECLIA). In total, 925 proteins were efficiently quantified and differential expression analysis revealed 378 proteins upregulated towards IL-6 concentration above 10,000 pg/mL. Four proteins (LCN2, MMP8, MPO, and S100A12) were selected to verify the achieved results and IL-6 concentration of 10,000 pg/mL was determined as the cut-off value for global IAI response.

Fig. 1

The change of amniotic fluid proteome along with the IL-6 concentration > 10,000 pg/mL. (A) The hierarchical clustering was designed based on reported log₂FC values in differential expression analysis. (B) The mean of relative levels determined by LC-MS/MS of significantly changed amniotic fluid proteins (n = 378) in collected late PPROM samples stratified according to IL-6 concentration. The level of those proteins is significantly upregulated in the group with IL-6 concentrations above 10,000 pg/mL compared to late PPROM groups with IL-6 concentration under 5,000 pg/mL. The differences among late PPROM groups were compared using one-way ANOVA on the significance level 0.05 (p value) and it is marked by asterisk (* p < 0.05, ** p < 0.01).

Fig. 2

1D enrichment of significantly changed amniotic fluid proteins. Significantly upregulated proteins were associated with cellular metabolism, chromosome organization, nucleus and cellular migration, whereas collagens and extracellular matrix were downregulated. 1D enrichment was performed with B-H correction on the FDR level 0.05.

Fig. 3

The selection of proinflammatory proteins to verification of proteomic results. The volcano graphs were designed to illustrate the difference of the late PPROM group with IL-6 concentrations above 10,000 pg/mL from the other late PPROM groups based on differential expressional analysis using modified LIMMA t-test and reported log₂FC values along with FDR p-values adjusted by B-H correction. The selected proteins are highlighted by gene names and blue colour.

Fig. 4

Quantification of selected amniotic fluid proteins using ELISA. The concentration of selected proteins MMP8, MPO, LCN2, and S100A12 was determined and the differences among late PPROM groups stratified according to IL-6 were compared using nonparametric Kruskal-Wallis test and Dunn’s post hoc test. Significance is marked by asterisk (* p < 0.05, ** p < 0.01, *** p < 0.001) and medians of each group are highlighted as red lines.

Fig. 5

Sensitivity and specificity of IL-6 concentration to recognize the upregulation of other proinflammatory proteins (MMP8, MPO, LCN2, and S100A12). ROC curves were designed to find appropriate cutoff value of amniotic fluid IL-6 concentration to recognize IAI defined as global change of proinflammatory proteins in amniotic fluid. ROC curves were compared using DeLong’s method and the significance was expressed as p value.