The association between prenatal alcohol exposure and protein expression in human placenta

Abstract

Background: The need for earlier recognition of children at risk for neurobehavioral problems associated with prenatal ethanol exposure (PAE) has prompted investigations of biomarkers prognostic for altered fetal development. Here, we examined whether PAE alters the expression of angiogenesis-related proteins and cytokines in human placenta in subjects from an Ethanol, Neurodevelopment, Infant and Child Health prospective cohort.

Methods: PAE was ascertained by screening questionnaires, Time-line Follow-back interviews and a panel of ethanol biomarkers at two study visits. After delivery, placental tissue samples were collected for protein analysis.

Results: No significant differences in the prevalence of substance use, demographic or medical characteristics were observed between the No PAE and PAE groups. PAE was associated with significant reductions in placental expression of VEGFR2 and annexin-A4, while the levels of VEGFR1 and CCM-3 trended downward. A trend toward higher expression of the cytokines TNF-α and IL-13 was also observed in the PAE group. Receiver operating characteristic analyses of the data demonstrated a moderate-to-high degree of diagnostic accuracy for individual placental proteins. Combinations of proteins substantially increased their ability to differentiate between PAE and No PAE subjects.

Conclusions: These results establish the feasibility of harvesting placental tissue for protein analyses of PAE in a prospective manner. In addition, given the importance of vascular remodeling in both placenta and developing brain, the role of angiogenic and cytokine proteins in this process warrants further investigation for their utility for predicting alterations in brain development, as well as their mechanistic role in PAE-induced pathology.

Keywords: angiogenesis; annexin-A4; cytokines; fetal alcohol spectrum disorder; placenta; prenatal alcohol exposure; vascular endothelial growth factor receptor 2.

FIGURE 1

Impact of time of tissue collection after delivery on the stability of two placental proteins. The relatively high abundance Annexin-A4 protein and the lower abundance VEGFR2 protein. Data points represent the mean ± SEM of four samples, from two vaginal and two cesarean deliveries, from non-PAE samples separate from the main study. A one-way repeated-measures ANOVA revealed no statistically significant differences across collection time points for either protein.

FIGURE 2

Representative Western blots for three human placental proteins. 2A: Annexin-A4. 2B: CCM-3 2C: VEGFR2. The three proteins of interest are the green bands and β-Actin (45 kDa), the reference protein, is the red band on each blot. The molecular weights for each of the three proteins of interest are indicated above each blot.

FIGURE 3

Effect of prenatal alcohol exposure on human placental protein expression. The lower and upper boundary of each vertical box plot represent the 25^th and 75^th percentile, the horizontal bar indicates the median and the error bars represent the 10^th and 90^th percentile for the raw (unadjusted) placental protein data. Sample sizes are noted on the x-axis under each box plot.