Early pregnancy peripheral blood gene expression and risk of preterm delivery: a nested case control study

Abstract

Background: Preterm delivery (PTD) is a significant public health problem associated with greater risk of mortality and morbidity in infants and mothers. Pathophysiologic processes that may lead to PTD start early in pregnancy. We investigated early pregnancy peripheral blood global gene expression and PTD risk.

Methods: As part of a prospective study, ribonucleic acid was extracted from blood samples (collected at 16 weeks gestational age) from 14 women who had PTD (cases) and 16 women who delivered at term (controls). Gene expressions were measured using the GeneChip(R) Human Genome U133 Plus 2.0 Array. Student's T-test and fold change analysis were used to identify differentially expressed genes. We used hierarchical clustering and principle components analysis to characterize signature gene expression patterns among cases and controls. Pathway and promoter sequence analyses were used to investigate functions and functional relationships as well as regulatory regions of differentially expressed genes.

Results: A total of 209 genes, including potential candidate genes (e.g. PTGDS, prostaglandin D2 synthase 21 kDa), were differentially expressed. A set of these genes achieved accurate pre-diagnostic separation of cases and controls. These genes participate in functions related to immune system and inflammation, organ development, metabolism (lipid, carbohydrate and amino acid) and cell signaling. Binding sites of putative transcription factors such as EGR1 (early growth response 1), TFAP2A (transcription factor AP2A), Sp1 (specificity protein 1) and Sp3 (specificity protein 3) were over represented in promoter regions of differentially expressed genes. Real-time PCR confirmed microarray expression measurements of selected genes.

Conclusions: PTD is associated with maternal early pregnancy peripheral blood gene expression changes. Maternal early pregnancy peripheral blood gene expression patterns may be useful for better understanding of PTD pathophysiology and PTD risk prediction.

Figure 1

Volcano plot of gene expression differences between cases and controls. Volcano plot distributions of fold change (log2 [fold change]) (X-axis) and Student's t-test p-values (-log10 [p-value]) (Y-axis)

Figure 2

Principal component analyses. Principal component analysis results of all samples (14 cases and 16 controls) using 209 differentially expressed genes. (Red/right: cases, Blue/left: controls).

Figure 3

Heat map illustration of samples and selected differentially expressed genes. Selected genes (N = 209) differentially expressed (upregulated: shades of red and downregulated: shades of green) in whole blood associated with preterm delivery (rows) and participants (columns, cases = pink and controls = green) grouped according to level and nature of gene expression (genes) and similarity of expression profiles (participants) and subjected to hierarchical tree clustering.

Figure 4

Promoter analysis results of differentially expressed genes. Inferred network of differentially expressed genes (Red = up regulated and Green = down regulated) in preterm delivery and transcription factors (White). Transcription factors were identified by their binding to over expressed promoter sequences in the differentially expressed genes.

Figure 5

Protein domain interactions of differentially expressed genes. Predicted domain interactions of differentially expressed genes: For instance, EGR1 can potentially interact (not only with its targets gene motifs) with the domain of many of the genes forming the inferred network genes. The thickness of the edge shows extent of interaction.

Figure 6

Fold changes of selected genes in microarray and RT-QPCR experiments.