Effects of moderate drinking during pregnancy on placental gene expression

Abstract

Many children adversely affected by maternal drinking during pregnancy cannot be identified early in life using current diagnostic criteria for fetal alcohol spectrum disorder (FASD). We conducted a preliminary investigation to determine whether ethanol-induced alterations in placental gene expression may have some utility as a diagnostic indicator of maternal drinking during pregnancy and as a prognostic indicator of risk for adverse neurobehavioral outcomes in affected offspring. Pregnant Long-Evans rats voluntarily consumed either a 0 or 5% ethanol solution 4 h each day throughout gestation. Ethanol consumption produced a mean maternal daily intermittent peak serum ethanol concentration of 84 mg/dL. Placentas were harvested on gestational day 20 for gene expression studies. Microarray analysis of more than 28,000 genes revealed that the expression of 304 known genes was altered twofold or greater in placenta from ethanol-consuming dams compared with controls. About 76% of these genes were repressed in ethanol-exposed placentas. Gene expression changes involved proteins associated with central nervous system development; organ morphogenesis; immunological responses; endocrine function; ion homeostasis; and skeletal, cardiovascular, and cartilage development. To date, quantitative real-time polymerase chain reaction analysis has confirmed significant alterations in gene expression for 22 genes, including genes encoding for three calcium binding proteins, two matrix metalloproteinases, the cannabinoid 1, galanin 2 and toll-like receptor 4, iodothyronine deiodinase 2, 11-β hydroxysteroid dehydrogenase 2, placental growth factor, transforming growth factor alpha, gremlin 1, and epithelial growth factor (EGF)-containing extracellular matrix protein. These results suggest that the expression of a sufficiently large number of placental mRNAs is altered after moderate drinking during pregnancy to warrant more detailed investigation of the placenta as a biomarker system for maternal drinking during pregnancy and as an early indicator of FASD. Furthermore, these results provide new insights into novel mechanisms on how ethanol may directly or indirectly mediate its teratogenic effects through alterations in placental function during pregnancy.

Fig. 1

A. Box whisker plots of placental gene expression in control and ethanol-exposed placentas representing the minimum (end of the bottom whisker), the first quartile (bottom border of the box), the median (line through the box), the third quartile (top border of the box), and the maximum (end of the top whisker) of the distribution. The separately drawn points are outliers. Points are regarded as outliers if the minimum of their distance to the first and the third quartile is greater than 1.5 times the interquartile range (IQR = third quartile–second quartile). B. Heat map plots illustrating unsupervised hierarchical clustering on similarity in expression profiles across conditions of 643 genes list, filtered by twofold expression changes and statistical significance (Student's t-test). CONT = saccharin control group and EtOH = 5% ethanol treatment group. The numbers below the group labels indicate the litter number from which the placenta tissue was harvested. Each heat map corresponds to the whisker plot directly above it.

Fig. 2

Selected enriched “biological processes” gene ontology (GO) level 5 categories. Data were analyzed using Gene Ontology Tree Machine GOTM software (Zhang et al., 2000). The distribution of differentially regulated genes (gray bars) in each GO category was compared with all genes on the Affymetrix Rat 230 2.0 array (black bars). Statistical significance was analyzed using the hypergeometric statistical test; *P < .05, **P < .005.

Fig. 3

Selected enriched “molecular function” gene ontology (GO) categories. Numbers in parenthesis after the molecular function denote the GO category level. Data were analyzed using Gene Ontology Tree Machine GOTM software (Zhang et al., 2000). The distribution of differentially regulated genes (gray bars) in each GO category was compared with all genes on the Affymetrix Rat 230 2.0 array (black bars). Statistical significance was analyzed using the hypergeometric statistical test; *P < .05, **P < .005, ***P < .0005.