Quantification of intra-amniotic inflammation in late preterm prelabor rupture of membranes associated with the response of amniotic fluid proteome

Introduction: Preterm prelabor rupture of membranes is associated with the risk of intra-amniotic inflammation (IAI) even beyond 34 weeks of gestation (late PPROM). Currently, interleukin-6 (IL-6) concentration of 2500 pg/mL in amniotic fluid is frequently discussed as a marker of the presence of IAI in PPROM. However, there is a lack of information about the level of IL-6 associated with the global change of amniotic fluid proteome as a response to IAI. The correlation of IL-6 with the global change of amniotic fluid proteome might better define the cut-off value for IAI and help reveal women with the highest risk of excessive inflammatory response in late PPROM.

Methods: In total, 60 amniotic fluid samples were collected and the concentration of IL-6 was determined in each of them using Cobas ECL analyzer. Protein quantification was based on Tandem Mass Tag (TMT) isobaric peptide labeling followed by 2D nanoLC-MS/MS. To verify proteomic data, target quantification (ELISA) of selected proteins (S100A12, MMP8, MPO, Lipocalin-2) was performed.

Results: Using 2D TMT nanoLC-MS/MS, 925 proteins were efficiently quantified in all samples and multiple comparison analysis (ANOVA, B-H FDR 5%) revealed 254 proteins mostly upregulated towards IL-6 concentration above 10 000 pg/mL, which is far above the concentration of IL-6 2500 – 3000 pg/mL being previously presumed as the cut-off value for IAI. As examples, proteins associated with oxidative burst and redox imbalance (MPO, SOD2, TXN, PRDX1, PRDX5, GAPDH), neutrophil antimicrobial activity (CAMP, DEFA3, ENO1, histones, HSP, HMGB2, MMP9, MMP8, LCN2), and neutrophil metabolic activity (GPI, LDHA, LDHB, MDH1) were found as upregulated in the group of samples with IL-6 concentration higher than 10 000 pg/mL. ROC curves designed based on ELISA protein quantification confirmed that the amniotic fluid proteome shift was associated with IL-6 concentration higher than 5000 pg/mL.

Novel aspect: Correlation of global amniotic fluid proteome and IL-6 concentration was used to quantify IAI in late PPROM.