Several studies have suggested that iron status may have a unique, modifying influence upon the clinical outcomes in FASD and this work provides the first direct demonstration for this hypothesis. We have demonstrated that maternal nutritional status, and specifically maternal iron insufficiency, is an important and powerful modifier of neurobehavior and growth in this rat model of FASD. The alcohol-exposed offspring born to mothers with low iron reserves had significant reductions in somatic growth, associative learning, myelination, and neuronal survival. In contrast, when maternal iron status was adequate, alcohol’s neurodevelopmental damage was mitigated and comparatively modest deficits in learning, body growth, and neuroanatomy were found. For several measures the impact of alcohol and ID was synergistic and not additive. Importantly, the deficits in the ID-alcohol animals represent part of the diagnostic criteria of Fetal Alcohol Syndrome, which represents the severe end of the FASD diagnostic spectrum 
. These characteristics include reduced somatic growth trajectories, enhanced neuronal and myelin losses, and deficits in associative learning. It is indisputable that alcohol is a potent neurodevelopmental teratogen and these data show that comorbid conditions can significantly influence the final outcome. Although there is a long-standing assumption that maternal nutritional status has a significant contribution to FASD 
, this hypothesis has not been systematically analyzed. Alcohol abuse is associated with higher risk for several nutritional deficiencies including iron, zinc, copper, thiamin, vitamin A, and perhaps choline; alcohol may also alter nutrient metabolism and requirements. More work is needed to identify additional nutritional factors that could affect FASD outcome. However, because ID is the most common nutritional deficiency in women of child-bearing age 
, an ID-alcohol interaction likely represents a significant portion of those receiving a FASD or FAS diagnosis. We propose that the treatment of maternal iron inadequacy and normalization of iron status will substantially attenuate the damage caused by prenatal alcohol exposure in a significant percentage of children at risk for FASD.
These findings have significant public health implications given the high rates of both alcohol abuse and ID in childbearing-age women of both industrialized and less-developed nations 
. For male and post-menopausal female alcoholics, iron overload is common due to alcohol’s dysregulation of hepcidin and subsequent enhancement of liver iron stores 
. However, the situation differs for women of child-bearing age due to their regular iron losses from menses and pregnancy; moreover, alcohol’s ability to enhance dietary iron absorption is irrelevant if the diet is iron-inadequate. Iron deficiency is more common in alcohol-abusing pregnancies than currently appreciated 
. In an alcohol-abusing U.S. cohort 
, the pregnant women with the highest drinking rates (>8 drinks/day) had the poorest iron status and 75% (9/12) of those women had iron-deficient anemia. In a South African FASD cohort 72% of children born to binge-drinking women had both iron-deficient anemia (42% in non-drinkers) and the most pronounced growth retardation, a hallmark of FAS 
. In addition to overt anemia, our data show that ID without anemia also poses a significant risk for adverse outcome. As others have shown and our own work confirms, when iron is limiting the mother cannot supply sufficient iron to meet the growing offspring’s needs. Thus the child has a greater risk for ID, anemia and low iron reserves as compared to its mother 
. In monitoring iron status, hematological indicators change slowly relative to iron stores and can significantly overestimate them, failing to identify those pregnancies with poor iron status and increased ID risk. For this reason, we endorse the adoption of more sensitive indicators of iron status such as the ratio of zinc protoporphyrin to heme, as these identify at-risk pregnancies more accurately than do hemoglobin, serum ferritin, or transferrin saturation, which are affected by factors other than iron stores 
. Increased attention to iron status during pregnancy is a novel and likely successful strategy to address the significant public health problem of FASD and FAS.
Our data may also explain recent controversial findings that children born to light-drinking mothers have lower FASD risk compared with abstainers or high drinkers 
. This light-drinking group also had higher income and education compared with the other cohorts. The prevalence of gestational ID decreases as income and education rise, partly because of increased iron supplement use 
. Thus light-drinking is likely associated with reduced risk for gestational ID. Similarly, our findings also inform the significant, positive associations between FAS and parity. A term pregnancy requires 1040 mg iron 
. When iron intake is limiting, it is difficult to rebuild iron reserves during the interpregnancy interval and thus multiparous women have increased ID risk 
. Shorter interpregnancy intervals are associated with low income and high parity 
. We suggest that maternal iron reserves and iron supplement use, and the environmental factors that modify these, partly explain the impact of parity, age, and socioeconomic status upon FASD risk.
How do alcohol and ID interact to produce the neurodevelopmental deficits seen here? This likely involves several mechanisms as both alcohol and ID have multiple effects upon the developing brain. These include reduced mitochondrial energy generation, reduced neuronal survival and synaptogenesis, and altered neurotransmitter activity 
. We show here that an important mechanism is a significant interaction between ID-alcohol to reduce myelination. There is growing evidence that white matter deficits including myelination problems are a significant hallmark of FASD. Diffusion tensor imaging of individuals with FASD reveals white matter deficits in multiple brain regions including the cerebellum, corpus callosum, brainstem, temporal lobe, and thalamus 
. These changes correlate with specific cognitive impairments affecting executive function, math processing, visual-perception, visuomotor integration, and of special relevance to our findings, associative learning. The reduced MBP immunostaining seen in our alcohol-exposed animals supports both these clinical observations and animal FAS studies reporting reduced MBP, delayed myelination and altered axonogenesis 
. The present work highlights the vulnerability of cerebellar myelination to alcohol’s damage during the third trimester equivalent. Iron also plays a prominent role in myelination; oligodendrocytes are iron-enriched and require iron for maturation into the myelin sheath 
. The significantly reduced myelination in our ID animals confirms that work and endorses the importance of perinatal iron adequacy for normal cerebellar development. Importantly, the addition of gestational ID during the alcohol exposure substantially exacerbates the myelination deficits as compared with either treatment individually. Thus maternal ID heightens the vulnerability of myelination to alcohol-mediated damage. While additional study is needed to ascertain if these MBP losses represent disrupted myelin formation, axon formation, or both, the present findings identify iron status as a significant contributor to the white matter deficits associated with FASD. Our data suggest that the most pronounced white matter losses in FASD may represent alcohol-exposed pregnancies in which iron deficiency was also present.
These myelination deficits likely contributed to the impaired associative learning of the ID-alcohol rats. Cerebellar white matter tracts, including those within Lobule VIa 
, are essential participants in the delay ECC learning studied here and link Purkinje cells with granule cells in the cerebellar cortex, completing the circuit with the ocular musculature 
. The substantial myelin losses seen within the Lobule VIa GCL of the ID-alcohol animals implies that the major afferent signals with cerebellar Purkinje cells involved in basic motor learning were compromised by the ID-alcohol combination. Such reductions could contribute to the poor learning of these animals in the delay ECC task. One caveat is that the overall timing of these animals’ CRs was not altered. However, the delay ECC learning task used here is an optimal ECC learning task and is acquired with relative ease compared with more difficult ECC tasks. Thus the lack of timing deficits may be the result of a myelin reduction threshold not being reached. Our conclusions are also supported by the recent clinical demonstration of similar, close associations between prenatal alcohol exposure, cerebellar white matter deficits, and poor learning performance using a trace ECC task similar to that studied here 
. As that clinical population also experiences significant gestational ID as compared with non-alcohol-exposed controls within that community 
, the authors speculated that poor iron status may have heightened the offspring’s vulnerability to the adverse effects of prenatal alcohol exposure, perhaps through effects on myelination. Our data directly support this hypothesis and inform its mechanistic basis by showing a significant interaction between ID and alcohol to reduce cerebellar MBP content including regions that contribute to associative learning.
Children with FASD from this same community also show a strong association between poor iron status and reduced body growth 
, an association that was observed in this rat model. Gender has an additional modifying effect upon body weight in those with FASD, and pubertal males retain their smaller stature while females achieve normal weights through accumulation of fat mass 
. A similar gender effect was seen here, and ID-alcohol interacted to reduce body growth in male but not female offspring. Males are more sensitive to ID than females due to their more rapid growth and increased muscle mass, both of which increase iron needs. The greater growth reductions in the ID-alcohol males vs. females may reflect this difference and endorses the conclusion by Carter et al. 
that limiting iron stores may magnify the growth reductions of FASD, especially in males. Taken together, findings herein directly demonstrate that poor iron status heightens the offspring’s vulnerability to the growth, cognitive, and brain microstructural deficits caused by prenatal alcohol-exposure.
The critical period for the protective effects of iron repletion in the alcohol-exposed offspring was likely the alcohol exposure period itself, because the ID-alcohol offspring were iron-sufficient when the behavioral testing was performed at adolescence. Normalization of iron status post-weaning did not reverse their learning deficits. Under normal nutriture, the neonate receives most of its iron stores during the late third trimester and these stores cannot meet postnatal needs when maternal iron stores are limiting 
. Thus it is especially important to resolve iron deficiency prior to this critical period. Fortunately, there are proven, low-cost methods to enhance maternal and offspring iron status, including aggressive screening for women having low iron status but lacking overt anemia, the use of slow-release or low-dose iron supplements, delayed cord clamping at delivery, and increased breast-feeding 
. The significantly improved outcomes of the IS-alcohol over the ID-alcohol animals suggest that maternal iron supplements are accessible to the offspring despite the alcohol-exposure. This issue is important because clinical trials are underway to directly test the ability of nutritional supplements, including iron, to improve FASD outcomes 
. However, an important caveat to such interventions is that alcohol abuse alters iron homeostasis in the adult 
. Perinatal alcohol exposure may similarly alter iron homeostasis and brain iron availability in the offspring 
, and studies are underway in our laboratory to examine this further. Although our understanding of how alcohol alters iron homeostasis is incomplete, our data support the importance of normalizing maternal iron status in alcohol-exposed pregnancies. In conclusion, our data demonstrate that aggressive screening and treatment of gestational ID in alcohol-using women, in the presence or absence of overt anemia, offers a safe and clinically achievable approach to substantially ameliorate the devastating consequences of FASD.