Background: Prenatal alcohol exposure (PAE) can result in developmental defects that include growth restriction, craniofacial anomalies, and cognitive behavioral deficits, though the presence and severity of these adverse outcomes can vary dramatically among exposed individuals. Preclinical animal models have demonstrated that the dose and timing of PAE account for much, but not all, of this phenotypic variation, suggesting that additional factors mitigate the effects of PAE. Here, we used a mouse model to investigate whether maternal age modulates the effects of PAE on the severity and variation in offspring growth and craniofacial outcomes.Methods: Nulliparous C57BL/6N dams received either an intraperitoneal injection of ethanol (EtOH) or vehicle solution on gestational day 7.5. Dams were divided into four groups: (1) EtOH-treated young dams (6 to 10 weeks); (2) control young dams; (3) EtOH-treated old dams (6 to 7 months); and (4) old control dams. Neonate offspring growth restriction was measured through body mass and organ-to-body mass ratios, while skeletal craniofacial features were imaged using micro-CT and analyzed for size, shape, and variation.Results: PAE and advanced maternal age each increased the risk of low birthweight and growth restriction in offspring, but these factors in combination changed the nature of the growth restriction. Similarly, both PAE and advanced maternal age individually caused changes to craniofacial morphology such as smaller skull size, dysmorphic skull shape, and greater skull shape variation and asymmetry. Interestingly, while the combination of PAE and advanced maternal age did not affect mean skull shape or size, it significantly increased the variation and asymmetry of those measures. Conclusion:Our results indicate that maternal age modulates the effects of PAE, but that the effects of this combination on offspring outcomes are more complex than simply scaling the effects of either factor.

Prenatal alcohol exposure (PAE) can result in fetal alcohol spectrum disorder (FASD), defined as a continuum of developmental defects including growth restriction, cognitive‐behavioral deficits, and craniofacial anomalies. Despite the known consequences of PAE, there is great variation in craniofacial and behavioral outcomes of affected children even when considering timing and quantity of exposure; phenotypic variation which could arise from maternal differences. In the present study, we asked: Does maternal age influence the nature and extent of growth restriction and craniofacial defects of PAE in offspring? To address this question, we used a C57BL/6 mouse model, as it enables precise control of dosing, gestational timing and maternal age. Dams were divided into four groups: 1. ethanol‐treated young dams (6–10 weeks); 2. control young dams; 3. ethanol‐treated old dams (6–7 months); and 4. control old dams. All dams were injected with either ethanol (treatment groups) or vehicle solution (control groups) on gestational day 7.5. Offspring growth restriction was analyzed by measures of body mass, liver mass, and body‐to‐liver mass ratio at birth. Micro‐CT scans of newborn soft tissue and skeletal craniofacial features underwent 3D analyses of shape and size to compare craniofacial phenotypes among treatment groups. Initial results of young ethanol‐treated and control groups demonstrated that ethanol‐treated offspring were born prematurely and showed significant growth restriction. Both soft tissue and skeletal craniofacial features were altered in the ethanol‐treated group, consistent with the FASD phenotype. Surprisingly, craniofacial shape of ethanol‐treated offspring was less variable than controls. Data collection for the old dam groups is ongoing, and we predict that offspring of old dams will have more severe growth and phenotypic consequences from PAE, exhibiting greater phenotypic variation compared to offspring of young dams. As many women are delaying childbirth, there is an increasing need to carryout preclinical studies with appropriate experimental controls to better understand the relative risk of PAE and maternal age, and ultimately contribute to public health policy for pregnant women. Support or Funding Information Funded by Children’s Health Research Institute, Internal Research Grant Fund to KEW “The Effects of Moderate Prenatal Alcohol Exposure on Craniofacial Morphology and Development” R5211A04