The effects of alcohol on fetal development

Prenatal exposure to alcohol has profound effects on many aspects of fetal development. Although alterations of somatic growth and specific minor malformations of facial structure are most characteristic, the effects of alcohol on brain development are most significant in that they lead to substantial problems with neurobehavioral development. Since the initial recognition of the fetal alcohol syndrome (FAS), a number of important observations have been made from studies involving both humans and animals. Of particular importance, a number of maternal risk factors have been identified, which may well be of relevance relative to the development of strategies for prevention of the FAS as well as intervention for those who have been affected. These include maternal age >30 years, ethnic group, lower socioeconomic status, having had a previously affected child, maternal under-nutrition, and genetic background. The purpose of this review is to discuss these issues as well as to set forth a number of questions that have not adequately been addressed relative to alcohol's effect on fetal development. Of particular importance is the critical need to identify the full spectrum of structural defects associated with the prenatal effects of alcohol as well as to establish a neurobehavioral phenotype. Appreciation of both of these issues is necessary to understand the full impact of alcohol on fetal development.     

Risk for congenital anomalies associated with different sporadic and daily doses of alcohol consumption during pregnancy: a case-control study

BACKGROUND: The classic clinical criteria for the diagnosis of fetal alcohol syndrome (FAS) include a "characteristic" facial appearance, pre- and postnatal growth deficiency, microcephaly, mental retardation, and occasional major malformations. However, diagnostic constraints, especially in the newborn period, lead to an underestimate of their prevalence. We report an epidemiological study of the potential risk of congenital defects in the offspring of mothers who ingested different sporadic and daily amounts of alcohol during pregnancy. METHODS: The study was based on the data from the ECEMC hospital-based case-control study and surveillance system, with a methodology aimed not only at the surveillance of congenital anomalies, but also at investigating their characteristics, clustering, and causes. For the purposes of this study, we considered as exposed those infants whose mothers reported the ingestion of any amount of alcohol during gestation (4705 mothers of cases and 4329 mothers of controls), and classified them into five groups according to their levels of alcohol consumption. Two groups consisted of mothers who consumed increasing sporadic levels and the other three consisted of mothers who consumed increasing daily levels of alcohol. RESULTS: Our study showed that even low sporadic doses of alcohol consumption during pregnancy may increase the risk of congenital anomalies in the offspring and that this risk increases with increasing levels of alcohol exposure. CONCLUSIONS: The results of our study suggest that it is necessary to generalize the preventive norm and recommend complete abstinence from alcohol during gestation. Birth Defects Research (Part A), 2004.     

Fetal alcohol spectrum disorders among children in a Brazilian orphanage

Background: The objective was to investigate the frequency of fetal alcohol spectrum disorders (FASD) and ophthalmologic anomalies in orphanage children in Brazil. Methods: A prospective study was performed on 94 children living in an orphanage in Brazil. The children were examined by a multidisciplinary team consisting of specialists in pediatrics, neurology, psychology, neuropsychiatry, and ophthalmology. Results: The main reasons for living in the orphanage, in 61% of the children, were negligence, child abuse, and abandonment. Of all the children studied, 50% had mothers with known alcohol abuse and 47% had one or more diagnoses of neurodevelopmental/behavioral and/or cognitive deficits. General developmental delay was found in 18%, intellectual disability in 3%, cognitive impairment in 27%, attention‐deficit/hyperactivity disorder in 14%, and autism in 3%. Altogether 17% had FASD, comprising three children with fetal alcohol syndrome (FAS), six with partial FAS, and seven with alcohol‐related neurodevelopmental disorder. 16% had ophthalmological findings such as poor vision, strabismus, and dysmorphology of the optic nerves. Twenty‐eight children (30%) were adopted from the orphanage; of these, six had FASD (two FAS, three partial FAS, one alcohol‐related neurodevelopmental disorder), five had attention‐deficit/hyperactivity disorder, and eight had developmental delay. Conclusion: Nearly half of the children living in the orphanage had neurodevelopmental disorders and a considerable number showed signs of damage from prenatal alcohol exposure. A broader look at the problem of FASD in Brazil and other South American countries is desirable to document the burden of disease and provide data for targeting prevention efforts. Birth Defects Research (Part A) 103:178–185, 2015. © 2014 Wiley Periodicals, Inc.     

Genetic polymorphisms: impact on the risk of fetal alcohol spectrum disorders

Clinical reports on monozygotic and dizygotic twins provided the initial evidence for the involvement of genetic factors in risk vulnerability for fetal alcohol spectrum disorders (FASD) including fetal alcohol syndrome (FAS). Research with selectively bred and inbred rodents, genetic crosses of these lines and strains, and embryo culture studies have further clarified the role of both maternal and fetal genetics in the development of FASD. Research to identify specific polymorphisms contributing to FASD is still at an early stage. To date, polymorphisms of only one of the genes for the alcohol dehydrogenase enzyme family, the ADH1B, have been demonstrated to contribute to FASD vulnerability. In comparison with ADH1B*1, both maternal and fetal ADH1B*2 have been shown to reduce risk for FAS in a mixed ancestry South African population. ADH1B*3 appears to afford protection for FASD outcomes in African-American populations. Other candidate genes should be examined with respect to FASD risk, including those for the enzymes of serotonin metabolism, in particular the serotonin transporter. By its very nature, alcohol teratogenesis is the expression of the interaction of genes with environment. The study of genetic factors in FASD falls within the new field of ecogenetics. Understanding of the array of genetic factors in FASD will be enhanced by future genetic investigations, including case-control, family association, and linkage studies.     

Retinoic acid signaling reduction recapitulates the effects of alcohol on embryo size

Intrauterine growth restriction (IUGR) is commonly observed in human pregnancies and can result in severe clinical outcomes. IUGR is observed in Fetal Alcohol Syndrome (FAS) fetuses as a result of alcohol (ethanol) exposure during pregnancy. To further understand FAS, the severe form of Fetal Alcohol Spectrum Disorder, we performed an extensive quantitative analysis of the effects of ethanol on embryo size utilizing our Xenopus model. Ethanol‐treated embryos exhibited size reduction along the anterior–posterior axis. This effect was evident primarily from the hindbrain caudally, while rostral regions appeared refractive to ethanol‐induced size changes, also known as asymmetric IUGR. Interestingly, some embryo batches in addition to shortening from the hindbrain caudally also exhibited an alcohol‐dependent reduction of the anterior head domain, known as symmetric IUGR. To study the connection between ethanol exposure and reduced retinoic acid levels we treated embryos with the retinaldehyde dehydrogenase inhibitors, DEAB and citral. Inhibition of retinoic acid biosynthesis recapitulated the growth defects induced by ethanol affecting mainly axial elongation from the hindbrain caudally. To study the competition between ethanol clearance and retinoic acid biosynthesis we demonstrated that, co‐exposure to alcohol reduces the teratogenic effects of treatment with retinol (vitamin A), the retinoic acid precursor. These results further support the role of retinoic acid in the regulation of axial elongation.     

Prenatal alcohol exposure triggers ceramide-induced apoptosis in neural crest-derived tissues concurrent with defective cranial development

Fetal alcohol syndrome (FAS) is caused by maternal alcohol consumption during pregnancy. The reason why specific embryonic tissues are sensitive toward ethanol is not understood. We found that in neural crest-derived cell (NCC) cultures from the first branchial arch of E10 mouse embryos, incubation with ethanol increases the number of apoptotic cells by fivefold. Apoptotic cells stain intensely for ceramide, suggesting that ceramide-induced apoptosis mediates ethanol damage to NCCs. Apoptosis is reduced by incubation with CDP-choline (citicoline), a precursor for the conversion of ceramide to sphingomyelin. Consistent with NCC cultures, ethanol intubation of pregnant mice results in ceramide elevation and increased apoptosis of NCCs in vivo. Ethanol also increases the protein level of prostate apoptosis response 4 (PAR-4), a sensitizer to ceramide-induced apoptosis. Prenatal ethanol exposure is concurrent with malformation of parietal bones in 20% of embryos at day E18. Meninges, a tissue complex derived from NCCs, is disrupted and generates reduced levels of TGF-β1, a growth factor critical for bone and brain development. Ethanol-induced apoptosis of NCCs leading to defects in the meninges may explain the simultaneous presence of cranial bone malformation and cognitive retardation in FAS. In addition, our data suggest that treatment with CDP-choline may alleviate the tissue damage caused by alcohol.     

Review of published studies of kidney,liver, and gastrointestinal birth defects in fetal alcohol spectrum disorders

OBJECTIVE: Review of published studies of birth defects of the renal, liver, and gastrointestinal organ systems in subjects with fetal alcohol spectrum disorders (FASD). METHOD: We searched PubMed ( http://www.pubmed.gov ) using the following terms: fetal alcohol syndrome and: gastrointestinal tract, kidney, liver, and congenital abnormalities for all years and English only citations. RESULTS: We located 12 studies of FASD and defects of or functional impairments for the liver, 12 of renal abnormalities, and only two with gastrointestinal defects. We did not identify specific patterns of malformations or functional deficits for any of the three organ systems. The existing literature suggests a series of nonspecific outcomes in FASD. CONCLUSIONS: Fetal alcohol spectrum disorder includes a diagnostic category of alcohol‐related birth defects which is clinically difficult to apply. This study adds to the existing literature on birth defects in FASD which is still very limited. The categorical diagnosis of alcohol‐related birth defects requires additional research to determine if a specific pattern of organ specific abnormalities or functional deficits emerges in subjects with FASD. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Advances in the development of novel antioxidant therapies as an approach for fetal alcohol syndrome prevention

Ethanol is the most common human teratogen, and its consumption during pregnancy can produce a wide range of abnormalities in infants known as fetal alcohol spectrum disorder (FASD). The major characteristics of FASD can be divided into: (i) growth retardation, (ii) craniofacial abnormalities, and (iii) central nervous system (CNS) dysfunction. FASD is the most common cause of nongenetic mental retardation in Western countries. Although the underlying molecular mechanisms of ethanol neurotoxicity are not completely determined, the induction of oxidative stress is believed to be one central process linked to the development of the disease. Currently, there is no known effective strategy for prevention (other than alcohol avoidance) or treatment. In the present review we will provide the state of art in the evidence for the use of antioxidants as a potential therapeutic strategy for the treatment using whole‐embryo and culture cells models of FASD. We conclude that the imbalance of the intracellular redox state contributes to the pathogenesis observed in FASD models, and we suggest that antioxidant therapy can be considered a new efficient strategy to mitigate the effects of prenatal ethanol exposure. Birth Defects Research (Part A) 103:163–177, 2015. © 2014 Wiley Periodicals, Inc.     

Septo-optic dysplasia as a manifestation of valproic acid embryopathy.

BACKGROUND: The use of valproic acid during pregnancy has been associated with adverse fetal outcomes, including major and minor congenital malformations, intrauterine growth retardation (IUGR), hyperbilirubinemia, hepatotoxicity, transient hyperglycemia, and fetal and neonatal distress. In addition, intrauterine exposure to valproic acid has been associated with an increased risk of central nervous system abnormalities, primarily neural tube defects. Optic nerve hypoplasia has been reported in association with other prenatal anticonvulsant exposures, but the occurrence of septo-optic dysplasia as a manifestation of valproic acid embryopathy has not been reported previously. RESULTS: We report on a woman who received Depakote (valproic acid) throughout her pregnancy for the treatment of a seizure disorder. The patient presented with features typical of valproic acid embryopathy, including bitemporal narrowing, hypertelorism, short palpebral fissures, epicanthal folds, microphthalmia, a flat broad nasal bridge, small mouth, hypoplastic nails, mild clinodactyly, and camptodactyly. MRI showed hypoplasia of the optic chiasm and absence of the septum pellucidum. CONCLUSIONS: We report the first case of septo-optic dysplasia associated with maternal exposure to valproic acid throughout pregnancy. This case expands the clinical phenotype of valproate embryopathy.     

Effects of ethanol on the primitive streak stage mouse embryo

Recent studies of mouse models have suggested that malformations associated with the fetal alcohol syndrome (FAS) are caused by the effects of ethanol on early embryos during gastrulation and neurulation. A study of Xenopus laevis embryos showed that exposure of gastrula stage amphibian embryos to ethanol inhibits migration of the mesodermal cells, causes formation of small neural plates, and subsequently causes hypoplastic craniofacial malformations in tadpoles. We now report effects of ethanol on the primitive streak stage mouse embryos. An ethanol solution (25%) was injected intraperitonealy twice into mice of 6.5-7.0 days of pregnancy at a dose of 0.015 ml/gm of body weight. Histological and morphometric examinations of 7.5-day embryos, 20 hr after the second injection, showed that the epiblast layer was disorganized and shrunk with formation of many blebs. In addition, formation of the mesodermal cell layer was retarded in the ethanol-treated embryos, suggesting that exposure of gastrula stage embryos to ethanol causes similar abnormalities in mouse and Xenopus embryos. These results suggest that the inhibition of the morphogenetic movements during gastrulation may be the primary effect of ethanol in causing major craniofacial malformations of FAS.     

Genesis of alcohol-induced craniofacial dysmorphism

The initial diagnosis of fetal alcohol syndrome (FAS) in the United States was made because of the facial features common to the first cohort of patients. This article reviews the development of an FAS mouse model whose craniofacial features are remarkably similar to those of affected humans. The model is based on short-term maternal treatment with a high dosage of ethanol at stages of pregnancy that are equivalent to Weeks 3 and 4 of human gestation. At these early stages of development, alcohol's insult to the developing face is concurrent with that to the brain, eyes, and inner ear. That facial and central nervous system defects consistent with FAS can be induced by more "realistic" alcohol dosages as illustrated with data from an oral alcohol intake mouse model in which maternal blood alcohol levels do not exceed 200 mg/dl. The ethanol-induced pathogenesis involves apoptosis that occurs within 12 hrs of alcohol exposure in selected cell populations of Day 7, 8, and 9 mouse embryos. Experimental evidence from other species also shows that apoptosis underlies ethanol-induced malformations. With knowledge of sensitive and resistant cell populations at specific developmental stages, studies designed to identify the basis for these differing cellular responses and, therefore, to determine the primary mechanisms of ethanol's teratogenesis are possible. For example, microarray comparisons of sensitive and resistant embryonic cell populations have been made, as have in situ studies of gene expression patterns in the populations of interest. Studies that illustrate agents that are effective in diminishing or exacerbating ethanol's teratogenesis have also been helpful in determining mechanisms. Among these agents are antioxidants, sonic hedgehog protein, retinoids, and the peptides SAL and NAP.     

Evaluating the effects of maternal alcohol consumption on murine fetal brain vasculature using optical coherence tomography

Prenatal alcohol exposure (PAE) can result in a range of anomalies including brain and behavioral dysfunctions, collectively termed fetal alcohol spectrum disorder. PAE during the 1st and 2nd trimester is common, and research in animal models has documented significant neural developmental deficits associated with PAE during this period. However, little is known about the immediate effects of PAE on fetal brain vasculature. In this study, we used in utero speckle variance optical coherence tomography, a high spatial‐ and temporal‐resolution imaging modality, to evaluate dynamic changes in microvasculature of the 2nd trimester equivalent murine fetal brain, minutes after binge‐like maternal alcohol exposure. Acute binge‐like PAE resulted in a rapid (<1 hour) and significant decrease (P < .001) in vessel diameter as compared to the sham group. The data show that a single binge‐like maternal alcohol exposure resulted in swift vasoconstriction in fetal brain vessels during the critical period of neurogenesis.      

Maternal Alcohol Consumption Increases Sphingosine Levels in the Brains of Progeny Mice

The effect of ‘binge’ alcohol upon sphingolipid metabolism in the fetal alcohol syndrome (FAS) was examined in pregnant mice (C57BL/6J) by administering a single dose of alcohol during the third trimester (gestational day 15–16). The control mice were administered a sucrose solution of equal caloric value. Brains from progeny at postnatal days 5, 15, 21 and 30 were dissected into three regions, and sphingolipid concentrations of the brain regions were determined including assay of monoglycosylceramide, ceramide, sphingosine and sphingomyelin. We found that a single dose of ethanol induces an elevation of sphingosine (2–3.5-fold) in the brain of progeny. The level of brain ceramide at a dose of 1.5 g/kg was significantly higher than control. Alcohol consumption during pregnancy induces neuronal loss in progeny brains. Our result suggests that the elevation of sphingosine in progeny brain induced by maternal alcohol consumption may be responsible for observed neuronal loss in FAS. Special issue in honor of Naren Banik.     

Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels

Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental malformations caused by alcohol consumption during pregnancy. Fetal Alcohol Syndrome (FAS), the strongest manifestation of FASD, results in short stature, microcephally and facial dysmorphogenesis including microphthalmia. Using Xenopus embryos as a model developmental system, we show that ethanol exposure recapitulates many aspects of FAS, including a shortened rostro-caudal axis, microcephally and microphthalmia. Temporal analysis revealed that Xenopus embryos are most sensitive to ethanol exposure between late blastula and early/mid gastrula stages. This window of sensitivity overlaps with the formation and early function of the embryonic organizer, Spemann's organizer. Molecular analysis revealed that ethanol exposure of embryos induces changes in the domains and levels of organizer-specific gene expression, identifying Spemann's organizer as an early target of ethanol. Ethanol also induces a defect in convergent extension movements that delays gastrulation movements and may affect the overall length. We show that mechanistically, ethanol is antagonistic to retinol (Vitamin A) and retinal conversion to retinoic acid, and that the organizer is active in retinoic acid signaling during early gastrulation. The model suggests that FASD is induced in part by an ethanol-dependent reduction in retinoic acid levels that are necessary for the normal function of Spemann's organizer.     

Major limb malformations following intrauterine exposure to ethanol: two additional cases and literature review

Two children are reported in whom major limb malformations were identified and whose mothers had consumed large quantities of alcohol in the first trimester of pregnancy. In one there was complete amelia of the upper limbs, while the other had preaxial polydactyly of both hands. These cases, taken together with previously reported instances of major limb anomalies following intrauterine ethanol exposure, as well as animal investigations that have demonstrated virtually identical limb malformations following ethanol administration, suggest that maternal ethanol abuse may be casually related to these limb malformations. We suggest that interruption of blood supply to the developing limb may be caused by ethanol exposure and may result in all of the various limb malformations described.     

Prenatal alcohol exposure and fetal programming: effects on neuroendocrine and immune function

Alcohol abuse is known to result in clinical abnormalities of endocrine function and neuroendocrine regulation. However, most studies have been conducted on males. Only recently have studies begun to investigate the influence of alcohol on endocrine function in females and, more specifically, endocrine function during pregnancy. Alcohol-induced endocrine imbalances may contribute to the etiology of fetal alcohol syndrome. Alcohol crosses the placenta and can directly affect developing fetal cells and tissues. Alcohol-induced changes in maternal endocrine function can disrupt maternal-fetal hormonal interactions and affect the female's ability to maintain a successful pregnancy, thus indirectly affecting the fetus. In this review, we focus on the adverse effects of prenatal alcohol exposure on neuroendocrine and immune function, with particular emphasis on the hypothalamic-pituitary-adrenal (HPA) axis and the concept of fetal programming. The HPA axis is highly susceptible to programming during fetal development. Early environmental experiences, including exposure to alcohol, can reprogram the HPA axis such that HPA tone is increased throughout life. We present data that demonstrate that maternal alcohol consumption increases HPA activity in both the maternal female and the offspring. Increased exposure to endogenous glucocorticoids throughout the lifespan can alter behavioral and physiologic responsiveness and increase vulnerability to illnesses or disorders later in life. Alterations in immune function may be one of the long-term consequences of fetal HPA programming. We discuss studies that demonstrate the adverse effects of alcohol on immune competence and the increased vulnerability of ethanol-exposed offspring to the immunosuppressive effects of stress. Fetal programming of HPA activity may underlie some of the long-term behavioral, cognitive, and immune deficits that are observed following prenatal alcohol exposure.     

VACTERL association and maternal diabetes: A possible causal relationship?

BACKGROUND: Some factors(s)/features(s) of maternal insulin‐dependent diabetes mellitus are considered common human teratogens. Although the variable association of cardiac, renal, and skeletal anomalies are commonly observed in infants from diabetic mothers, the relationship between VACTERL (i.e., the association of vertebral and cardiac defects, tracheo‐esophageal fistula, renal/radial malformations, and other limb anomalies) and maternal diabetes has not been sufficiently emphasized in the literature. CASE: We report on a 3‐year‐old boy presenting with a constellation of blastogenetic malformations strongly suggestive of VACTERL association. His mother was affected by insulin‐dependent diabetes since she was 7 years old and pregnancy history disclosed very high glucose and HbA1c levels, especially during the first 2 gestational months. CONCLUSIONS: In an attempt to properly counsel the parents, we reviewed the literature and identified four additional patients with VACTERL and first trimester exposure to maternal diabetes mellitus. Although this evidence does not strongly support a causal relationship between these two conditions, additional arguments may substantiate this hypothesis. The pathogenesis of diabetic embryopathy in relation to the VACTERL phenotype is also discussed. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Biomarkers for detection of prenatal alcohol exposure: a critical review of fatty acid ethyl esters in meconium

BACKGROUND: The objective of this study was a review of published studies utilizing measurement of fatty acid ethyl esters (FAEE) in meconium as biomarkers for prenatal alcohol exposure. METHODS: We completed a literature search of PubMed using the terms meconium, fatty acid ethyl esters, biomarkers, and prenatal alcohol exposure. We included only peer reviewed studies utilizing analysis of meconium for the presence of FAEE in humans through the year 2007. RESULTS: We found 10 articles reporting on original research examining the relationship of FAEE from meconium and prenatal alcohol exposure (PAE). The 10 articles used six different PAE assessment strategies and four different analytical techniques for determining FAEE endpoints. The articles included 2,221 subjects (range 4 to 725) with 455 (20.5%) subjects identified as exposed using the methods stated in the articles. FAEE levels above the studies' respective cutoffs were reported for 502 (22.6%) subjects. CONCLUSIONS: The accurate identification of alcohol‐exposed pregnancies represents a significant challenge in the development of FAEE detection cutoffs to maximize the sensitivity and specificity of the test. We present several options for the improvement of exposure assessment in future studies of FAEE as biomarkers for PAE. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Ethanol Alters Proliferation and Differentiation of Normal and Chromosomally Abnormal Human Embryonic Stem Cell‐Derived Neurospheres

Ethanol is a powerful substance and, when consumed during pregnancy, has significant psychoactive and developmental effects on the developing fetus. These abnormalities include growth retardation, neurological deficits, and behavioral and cognitive deficiencies, commonly referred to as fetal alcohol spectrum disorder. The effect of ethanol has been reported to affect cellular development on the embryonic level, however, not much is known about mutations contributing to the influence of ethanol. The purpose of our study was to determine if mutation contribute to changes in differentiation patterning, cell‐cycle regulatory gene expression, and DNA methylation in human embryonic stem cells after ethanol exposure. We exposed human embryonic stem cells (with and without know DNA mutations) to a low concentration (20 mM) of ethanol and measured neurosphere proliferation and differentiation, glial protein levels, expression of various cell‐cycle genes, and DNA methylation. Ethanol altered cell‐cycle gene expression between the two cell lines; however, gene methylation was not affected in ether lines.. Birth Defects Res (Part B) 98:283–295, 2013. © 2013 Wiley Periodicals, Inc.