Characterization of the transcriptome of chorioamniotic membranes at the site of rupture in spontaneous labor at term

Abstract

Objective: The purpose of this study was to compare the transcriptome between the site of membrane rupture and the chorioamniotic membranes away from the site of rupture.

Study design: The transcriptome of amnion and chorion (n=20 each) from and distal to the site of rupture from women with spontaneous labor and vaginal delivery at term after spontaneous rupture of membranes was profiled with Illumina HumanHT-12 microarrays. Selected genes were validated with the use of quantitative reverse transcription-polymerase chain reaction.

Results: Six hundred seventy-seven genes were differentially expressed in the chorion between the rupture and nonrupture sites (false discovery rate<0.1; fold change>1.5). Quantitative reverse transcription-polymerase chain reaction confirmed the differential expression in 10 of 14 genes. Enriched biological processes included anatomic structure development, cell adhesion and signal transduction. Extracellular matrix-receptor interaction was the most impacted signaling pathway.

Conclusion: The transcriptome of fetal membranes after spontaneous rupture of membranes in term labor is characterized by region- and tissue-specific differential expression of genes that are involved in signature pathways, which include extracellular matrix-receptor interactions.

FIGURE 1. Sampling sites of the fetal membranes

The image of the placenta demonstrates the representative sampling sites of the gross site of rupture (yellow box) and the site away from rupture (white box).

FIGURE 2. Transcriptomic analysis of chorion specimens

The volcano plot shows probability values of all probes in the microarray plotted against the fold changes. In this Figure, the log (base 10) of the false discovery rate–adjusted probability values are plotted against the log (base 2) ratio of fold changes between ruptured site chorion and nonruptured site chorion. On the Y-axis, values higher than the grey-line threshold represent significant probes with an adjusted probability value of < .1. On the X-axis, values outside the red lines represent fold-changes of ≥1.5 between ruptured site chorion and nonruptured site chorion. The 815 differentially expressed probes are labeled blue in the upper outer quadrants.

FIGURE 3. Heat maps of the clustering of genes

A, A heat map displays the hierarchical clustering of the 200 probes whose expression had the largest variance across all amnion and chorion specimens. The red and green colors in the heat map correspond with high and low gene expression, respectively. The labels at the top indicate individual patient samples and their membrane region designation. Two main clusters are seen that predominantly represent amnion from the rupture site and nonrupture site on the left and chorion from the rupture site and nonrupture site in the middle. A smaller cluster of mixed samples are on the right. B, A heat map displays the hierarchical clustering of the 200 probes whose expression had the largest variance across chorion specimens. Two separate cluster regions represent the rupture site on the right and non-rupture site on the left.

FIGURE 4. Significantly impacted pathways in chorion specimens

Two-dimensional plot illustrates the relationship between the 2 types of evidence considered by signaling pathway impact analysis. The X-axis shows the overrepresentation evidence (−log[P value]); the Y-axis shows the perturbation evidence (−log[P value]). Each pathway is represented by a point. Pathways above the oblique red line (red dots) are significant at 5% after Bonferroni correction; the pathways (blue dots and red dots) above the oblique blue line are significant at 5% after false discovery rate correction. The vertical and horizontal thresholds represent the same corrections for the 2 types of evidence considered individually.

FIGURE 5. Quantitative reverse transcriptase–polymerase chain reaction results

Results of quantitative reverse transcriptase–polymerase chain reaction assay of selected genes in a new set of specimens. The shaded boxes represent genes that are statistically significant (P< .05); the white boxes represent nonsignificant genes. The data are presented as the difference between the −ΔCt of rupture site chorion and the −ΔCt of non-rupture site chorion: positive values correlate with increased expression and negative values correlate with decreased expression of the gene in the rupture site chorion compared with the nonrupture site chorion. The boxes encompass 50% of the data from the 1st quartile to the 3rd quartile. The middle line represents the median value (50%) quartile. The whiskers extend to the most extreme data point but do not exceed values of >1.5 times the inter-quartile range from the box. The circles represent outliers.