Validation of microarray data.

<p>(<b>A</b>) Gene data derived from the microarray analysis was firstly examined using unsupervised principal components analysis (PCA). Inspection of the PCA score plot revealed clear first component separation of activated (yellow) and low-activated (red) amnion samples. (<b>B</b>) Similarly, hierarchical clustering demonstrated grouping within both the activated and non-activated amnion groups. Collectively these results indicate that gene expression profiles of activated samples are similar to each other, yet distinct to non-activated samples. OTR (<b>C</b>) and IL-8 (<b>D</b>) mRNA levels were then assessed in 6 amnion samples post microarray analysis. Consistent with the microarray data, three samples displayed low levels of OTR and IL-8 mRNA whilst the other three samples displayed characteristically high levels indicative of amnion activation.</p

Ingenuity Pathway Analysis (IPA) of gene pathways driving amnion activation- network map 1.

<p>Network one comprising of upregulated genes (>2.5-fold) involved predominately in cell death, cancer and morphology. A total of 25 focus genes were used in the construction of the network. ADM (adrenomedullin/progesterone biosynthetic process), CTGF (connective tissue growth factor/angiogenesis), CYR61 (cysteine-rich angiogenic inducer/patterning of blood vessels), EMP2 (epithelial membrane protein 2/cell death), ETS1 (v-ets erythroblastosis virus E26 oncogene homolog 1 (avian)/DNA dependent transcription), ETS2 (DNA dependent transcription), HMGCS1 (transporter 2, ATP-binding cassette, sub-family B (MDR/TAP)/protein complex assembly), IL1RAP (interleukin 1 receptor accessory protein/transmembrane receptor activity involved in the inflammatory response), ITGA6 (integrin, alpha 6/cell adhesion and calcium ion binding), KLF4 (Kruppel-like factor 4 (gut)/DNA dependent transcription and nucleic acid binding), KLF6 (Kruppel-like factor 6/DNA dependent transcription and nucleic acid binding), NET1 (neuroepithelial cell transforming gene 1/regulation of Rho protein signal transduction), NME1 (non-metastatic cells 1, protein (NM23A)/nucleic acid binding), ODC1 (ornithine decarboxylase 1/polyamine biosynthetic process), PIM1 (pim-1 oncogene/protein serine/threonine kinase activity), PSAT1 (phosphoserine aminotransferase 1/phosphoserine transaminase activity), RND3 (Rho family GTPase 3/cell adhesion), SGK (serum/glucocorticoid regulated kinase/serine/threonine kinase activity), SLC7A1 (solute carrier family 7, (cationic amino acid transporter, y+ system) member 11/amino acid transport activity), SPRR1B (small proline-rich protein 1B (cornifin)/epidermis development), TAF5L (TAF5-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa/DNA dependent transcription), THBS1 (Thrombospondin 1/cell motility, adhesion in response to inflammation), TUBB3 (tubulin, beta 3/microtubule-based movement), TUBB2A (tubulin, beta 2A/microtubule-based movement), UGT1A6 (UDP glucuronosyltransferase 1 family/xenobiotic metabolic process).</p

Inflammatory genes significantly upregulated in activated amnion.

<p>A list for inflammatory genes upregulated in activated amnion samples compared to non-activated samples was generated. In total, 8 significant inflammatory genes were identified (<i>P</i><0.01).</p

Correlations between key components of the canonical, non-canonical and atypical NFκB signalling pathways.

<p>Protein levels of activated NFκB were examined in each sample by immunoblotting as previously described. Levels of both nuclear p65 and nuclear phospho-p65 were shown to correlate highly with nuclear levels with Rel-B (A and B, R² = 0.8145 and R² = 0.6288 respectively). No correlation was detected between nuclear levels of p65 and p50 (C, R² = 0.06856), nuclear Rel-B and p52 (D, R² = 0.00008) or nuclear p65 and IκBα (E, R² = 0.0077). Collectively these results suggest that neither the canonical, non-canonical nor the atypical signalling pathways are responsible for the observed differences in NFκB activation.</p

Interactions between p65 and Rel-B exist in pre-labour, human amnion.

<p>(<b>A</b>) Whole cell lysate from unstimulated and IL-1β stimulated pre-labour amnion epithelial cells was incubated p65 conjugated beads. The lysate was recaptured under denaturing conditions and Western immunoblotting performed with either anti-p65, anti-phospho-p65 or anti-Rel-B antibodies. Non-stimulated amnion was shown to contain both p65-pp65 and to a greater extent, p65-Rel-B dimers. When stimulated with IL-1β, p65-pp65 dimer levels increased maximally at 30 min and then decreased gradually over 24 h. Dimers of p65-Rel-B were maintained at high levels throughout the time series. (<b>B</b>) Non-radioactive DNA binding assay kit (TRANSAM perbioscience) measuring the binding of Rel-B to the NFκB consensus binding sequence in response to IL-1β showed an increase from the unstimulated state at 30 min before dropping slightly 1 h. Binding peaked at 4 h before again subsiding at 24 h.</p

Characterisation of amnion activation for microarray analysis.

<p>Prior to microarray analysis, levels of COX-2 mRNA (<b>A</b>) and COX-2 protein were assessed in 6 amnion samples by western blotting (<b>B</b>) and subsequent densitometric assessment of immuno-reactive bands (<b>C</b>). Three samples characteristically displayed high levels of both COX-2 gene and protein consistent with highly activated amnion. Three samples displaying low levels of COX-2 were also selected as non-activated samples. The six characterised samples were subsequently used for microarray analysis to examine genes involved in amnion activation.</p

Pyl A has no effect on NF-κB p65 activity in amniocytes and myocytes.

<p>Protein was extracted from IL-1β stimulated and Pyl treated cells and levels of nuclear p65 and phosphorylated p65 (p-p65) were examined using immunoblotting. A dose response of 0.1–32 µM of Pyl A was used. Representative immunoblots are shown for amniocytes, (<b>A</b>) and myocytes (<b>B</b>). Immunoblots were re-probed for β-actin as an internal loading control. Densitometric analysis of the immunoblots was conducted revealing no effect of Pyl A on p65 or p-p65 in amniocytes (<b>C</b>, <b>E</b>) or myocytes (<b>D</b>, <b>F</b>). NS = non-stimulated (non-IL-1β treated cells). Effect of treatment was examined for statistical significance using ANOVA of repeated measures with Bonferroni’s multiple comparison test; *<i>P</i><0.05.</p

The primer sequences used for amplification of CRTH2.

<p>The primer sequences used for amplification of CRTH2.</p

An overview of the mean fluorescence intensities and percentages in the gated cells during detection of endogenous CRTH2 in lymphocytes, amniocytes and myocytes.

<p>An overview of the mean fluorescence intensities and percentages in the gated cells during detection of endogenous CRTH2 in lymphocytes, amniocytes and myocytes.</p

Additional file 2 of Meta-analysis reveals the vaginal microbiome is a better predictor of earlier than later preterm birth

Additional file 2: List S1. Results of Lactobacillus species BLAST against sequences from cultured Lactobacillus strains