Molecular evidence of placental hypoxia in preeclampsia

Abstract

Background: Oxygen plays a central role in human placental pathologies including preeclampsia, a leading cause of fetal and maternal death and morbidity. Insufficient uteroplacental oxygenation in preeclampsia is believed to be responsible for the molecular events leading to the clinical manifestations of this disease.

Design: Using high-throughput functional genomics, we determined the global gene expression profiles of placentae from high altitude pregnancies, a natural in vivo model of chronic hypoxia, as well as that of first-trimester explants under 3 and 20% oxygen, an in vitro organ culture model. We next compared the genomic profile from these two models with that obtained from pregnancies complicated by preeclampsia. Microarray data were analyzed using the binary tree-structured vector quantization algorithm, which generates global gene expression maps.

Results: Our results highlight a striking global gene expression similarity between 3% O(2)-treated explants, high-altitude placentae, and importantly placentae from preeclamptic pregnancies. We demonstrate herein the utility of explant culture and high-altitude placenta as biologically relevant and powerful models for studying the oxygen-mediated events in preeclampsia.

Conclusion: Our results provide molecular evidence that aberrant global placental gene expression changes in preeclampsia may be due to reduced oxygenation and that these events can successfully be mimicked by in vivo and in vitro models of placental hypoxia.

FIG. 1.

Experimental strategy for microarray study. Flow chart represents sequential steps taken to increase reliability and reproducibility from expression profiling. Left, Pooled samples in each experimental condition (preeclamptic tissues depicted as example, other conditions include: first-trimester tissues, AMC, term C/S, MA, HA, 3- and 20%-treated explant). Right, Reference sample (originating from five normal term placentae) used in all experimental conditions as a normalization parameter. R, Reference sample; P1-P10, patient RNA samples (preeclamptic tissues).

FIG. 2.

BTSVQ analysis of microarray data. A, Unsupervised clustering of all experimental conditions. BTSVQ divided the sample experimental arrays into two biologically relevant groups (child 1: first-trimester tissues; child 2: third-trimester tissues). B (left), Gene assignment by a SOM. Depicted are hexagonal units of a component plane (array of nodes), listed with the labels of genes around clusters selected by the SOM algorithm using vector quantization. The color gradient represents gene expression values associated with individual units, projecting average of gene expressions in a given unit using this color scheme. Similarly expressed genes in the same area of component planes across various arrays have similar color. Differential gene expression results in color differences between the same areas of different arrays. B (right), SOM component planes of three different arrays hybridized with the age-matched control sample and the reference sample. Note a virtually identical gene expression pattern among the SOM visualizations of the three arrays. C, Quantitative RT-PCR for relative VEGF transcript expression in unpooled (individual samples: open bars) vs. pooled samples (black bars) from preterm AMC and PE tissues as well as from explants incubated under 20 and 3% oxygen. D, Quantitative RT-PCR for relative integrin-α6 transcript expression in control conditions (open bar; AMC, 20% ex-plant and MA) relative to the three hypoxia conditions (PE, 3% explant, HA). Analysis was performed in trip-licate. *, P < 0.05, Student’s t test.

FIG. 3.

Global gene expression similarities and differences in experimental groups. A, B, and C, SOMs of duplicate arrays run for each experimental condition. A, Global gene expression differences between first-trimester explants treated under 3 and 20% O₂ as well as strong expression similarities between in vivo first-trimester tissues (12 wk gestation) and explants treated under 20% O₂. B, Differential gene expression between MA and HA placental tissues. C, Global gene expression differences between PE and AMC samples as well as normal healthy C/S samples. D, Striking similarity in global gene profile between 3% O₂-treated explants, PE, and high-altitude placentae. E, Graphical pseudocolor representation of a correlation matrix, showing a relationship among representative arrays from the three hypoxia conditions (3% O₂-treated explants; PE; HA) and their respective controls (20% O₂-treated explants; AMC; MA). The color map corresponds to the scale of positive correlation coefficients. Noncorrelated data display a coefficient of zero (dark blue), and positive significant correlation ranges from green-yellow to red. The diagonal of the symmetric correlation matrix represents self-correlation and thus is equal to 1 (dark red).

FIG. 4.

Similarly expressed genes. Two conserved areas (A and B) between 3% O₂-treated explants, preeclamptic, and HA placentae are illustrated by black circles across all three conditions and their respective controls (20% O₂, AMC, and PE). The table lists all the similarly expressed (nonsignificant) genes within each area among the three conditions. The unigene ID and gene name are included for reference.

FIG. 5.

Differentially expressed genes. The table lists all the differentially expressed genes within areas A and B increasing in the three hypoxia conditions relative to their respective controls (significance, P < 0.05). The unigene ID, fold change, P values, and gene name are included for reference.