Early moderate prenatal alcohol exposure and maternal diet impact offspring DNA methylation across species.

Alcohol consumption in pregnancy can affect genome regulation in the developing offspring but results have been contradictory. We employed a physiologically relevant murine model of short-term moderate prenatal alcohol exposure (PAE) resembling common patterns of alcohol consumption in pregnancy in humans. Early moderate PAE was sufficient to affect site-specific DNA methylation in newborn pups without altering behavioural outcomes in adult littermates. Whole-genome bisulfite sequencing of neonatal brain and liver revealed stochastic influence on DNA methylation that was mostly tissue-specific, with some perturbations likely originating as early as gastrulation. DNA methylation differences were enriched in non-coding genomic regions with regulatory potential indicative of broad effects of alcohol on genome regulation. Replication studies in human cohorts with fetal alcohol spectrum disorder suggested some effects were metastable at genes linked to disease-relevant traits including facial morphology, intelligence, educational attainment, autism, and schizophrenia. In our murine model, a maternal diet high in folate and choline protected against some of the damaging effects of early moderate PAE on DNA methylation. Our studies demonstrate that early moderate exposure is sufficient to affect fetal genome regulation even in the absence of overt phenotypic changes and highlight a role for preventative maternal dietary interventions.

Drinking excessive amounts of alcohol during pregnancy can cause foetal alcohol spectrum disorder and other conditions in children that affect their physical and mental development. Many countries advise women who are pregnant or trying to conceive to avoid drinking alcohol entirely. However, surveys of large groups of women in Western countries indicate that most women continue drinking low to moderate amounts of alcohol until they discover they are pregnant and then stop consuming alcohol for the rest of their pregnancy. It remains unclear how this common drinking pattern affects the foetus. The instructions needed to build and maintain a human body are stored within molecules of DNA. Some regions of DNA called genes contain the instructions to make proteins, which perform many tasks in the body. Other so-called 'non-coding' regions do not code for any proteins but instead have roles in regulating gene activity. One way cells control which genes are switched on or off is adding or removing tags known as methyl groups to certain locations on DNA. Previous studies indicate that alcohol may affect how children develop by changing the patterns of methyl tags on DNA. To investigate the effect of moderate drinking during the early stages of pregnancy, Bestry et al. exposed pregnant mice to alcohol and examined how this affected the patterns of methyl tags on DNA in their offspring. The experiments found moderate levels of alcohol were sufficient to alter the patterns of methyl tags in the brains and livers of the newborn mice. Most of the changes were observed in non-coding regions of DNA, suggesting alcohol may affect how large groups of genes are regulated. Fewer changes in the patterns of methyl tags were found in mice whose mothers had diets rich in two essential nutrients known as folate and choline. Further experiments found that some of the affected mouse genes were similar to genes linked to foetal alcohol spectrum disorder and other related conditions in humans. These findings highlight the potential risks of consuming even moderate levels of alcohol during pregnancy and suggest that a maternal diet rich in folate and choline may help mitigate some of the harmful effects on the developing foetus.

Low to moderate prenatal alcohol exposure and neurodevelopment in a prospective cohort of early school aged children.

Evidence is strong for adverse fetal effects of high level or chronic prenatal alcohol exposure (PAE), but many pregnant women continue to drink at lower levels. The 'Asking Questions about Alcohol in pregnancy' prospective cohort aimed to determine the neurodevelopmental consequences at 6-8 years of age of low to moderate PAE. 1570 women from seven public antenatal clinics in Melbourne, Australia, provided information on frequency and quantity of alcohol use, and obstetric, lifestyle and socio-environmental confounders at four gestation timepoints. PAE was classified into five trajectories plus controls. At 6-8 years, 802 of 1342 eligible families took part and completed a questionnaire (60%) and 696 children completed neuropsychological assessments (52%). Multiple linear regressions examined mean outcome differences between groups using complete case and multiple imputation models. No meaningful relationships were found between any of the PAE trajectories and general cognition, academic skills, motor functioning, behaviour, social skills, social communication, and executive function. Maternal education most strongly influenced general cognition and academic skills. Parenting behaviours and financial situation were associated with academic skills, behaviour, social skills and/or executive function. The lack of association between PAE and neurodevelopment at 6-8 years may partly be explained by cumulative positive effects of socio-environmental factors.

Associations between low-moderate prenatal alcohol exposure and brain development in childhood.

BACKGROUND: The effects of low-moderate prenatal alcohol exposure (PAE) on brain development have been infrequently studied. AIM: To compare cortical and white matter structure between children aged 6 to 8 years with low-moderate PAE in trimester 1 only, low-moderate PAE throughout gestation, or no PAE. METHODS: Women reported quantity and frequency of alcohol consumption before and during pregnancy. Magnetic resonance imaging was undertaken for 143 children aged 6 to 8 years with PAE during trimester 1 only (n = 44), PAE throughout gestation (n = 58), and no PAE (n = 41). T1-weighted images were processed using FreeSurfer, obtaining brain volume, area, and thickness of 34 cortical regions per hemisphere. Fibre density (FD), fibre cross-section (FC) and fibre density and cross-section (FDC) metrics were computed for diffusion images. Brain measures were compared between PAE groups adjusted for age and sex, then additionally for intracranial volume. RESULTS: After adjustments, the right caudal anterior cingulate cortex volume (pFDR = 0.045) and area (pFDR = 0.008), and right cingulum tract cross-sectional area (pFWE < 0.05) were smaller in children exposed to alcohol throughout gestation compared with no PAE. CONCLUSION: This study reports a relationship between low-moderate PAE throughout gestation and cingulate cortex and cingulum tract alterations, suggesting a teratogenic vulnerability. Further investigation is warranted.

A meta-analysis of pre-pregnancy maternal body mass index and placental DNA methylation identifies 27 CpG sites with implications for mother-child health.

Higher maternal pre-pregnancy body mass index (ppBMI) is associated with increased neonatal morbidity, as well as with pregnancy complications and metabolic outcomes in offspring later in life. The placenta is a key organ in fetal development and has been proposed to act as a mediator between the mother and different health outcomes in children. The overall aim of the present work is to investigate the association of ppBMI with epigenome-wide placental DNA methylation (DNAm) in 10 studies from the PACE consortium, amounting to 2631 mother-child pairs. We identify 27 CpG sites at which we observe placental DNAm variations of up to 2.0% per 10 ppBMI-unit. The CpGs that are differentially methylated in placenta do not overlap with CpGs identified in previous studies in cord blood DNAm related to ppBMI. Many of the identified CpGs are located in open sea regions, are often close to obesity-related genes such as GPX1 and LGR4 and altogether, are enriched in cancer and oxidative stress pathways. Our findings suggest that placental DNAm could be one of the mechanisms by which maternal obesity is associated with metabolic health outcomes in newborns and children, although further studies will be needed in order to corroborate these findings.

A data driven approach to identify trajectories of prenatal alcohol consumption in an Australian population-based cohort of pregnant women.

Accurate information on dose, frequency and timing of maternal alcohol consumption is critically important when investigating fetal risks from prenatal alcohol exposure. Identification of distinct alcohol use behaviours can also assist in developing directed public health messages about possible adverse child outcomes, including Fetal Alcohol Spectrum Disorder. We aimed to determine group-based trajectories of time-specific, unit-level, alcohol consumption using data from 1458 pregnant women in the Asking Questions about Alcohol in Pregnancy (AQUA) longitudinal study in Melbourne, Australia. Six alcohol consumption trajectories were identified incorporating four timepoints across gestation. Labels were assigned based on consumption in trimester one and whether alcohol use was continued throughout pregnancy: abstained (33.8%); low discontinued (trimester one) (14.4%); moderate discontinued (11.7%); low sustained (13.0%); moderate sustained (23.5%); and high sustained (3.6%). Median weekly consumption in trimester one ranged from 3 g (low discontinued) to 184 g of absolute alcohol (high sustained). Alcohol use after pregnancy recognition decreased dramatically for all sustained drinking trajectories, indicating some awareness of risk to the unborn child. Further, specific maternal characteristics were associated with different trajectories, which may inform targeted health promotion aimed at reducing alcohol use in pregnancy.

Association of prenatal alcohol exposure with offspring DNA methylation in mammals: a systematic review of the evidence.

BACKGROUND: Prenatal alcohol exposure (PAE) is associated with a range of adverse offspring neurodevelopmental outcomes. Several studies suggest that PAE modifies DNA methylation in offspring cells and tissues, providing evidence for a potential mechanistic link to Fetal Alcohol Spectrum Disorder (FASD). We systematically reviewed existing evidence on the extent to which maternal alcohol use during pregnancy is associated with offspring DNA methylation. METHODS: A systematic literature search was conducted across five online databases according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed, Web of Science, EMBASE, Google Scholar and CINAHL Databases were searched for articles relating to PAE in placental mammals. Data were extracted from each study and the Risk of Bias in Non-Randomized Studies of Interventions (ROBINS-I) was used to assess the potential for bias in human studies. RESULTS: Forty-three articles were identified for inclusion. Twenty-six animal studies and 16 human studies measured offspring DNA methylation in various tissues using candidate gene analysis, methylome-wide association studies (MWAS), or total nuclear DNA methylation content. PAE dose and timing varied between studies. Risk of bias was deemed high in nearly all human studies. There was insufficient evidence in human and animal studies to support global disruption of DNA methylation from PAE. Inconclusive evidence was found for hypomethylation at IGF2/H19 regions within somatic tissues. MWAS assessing PAE effects on offspring DNA methylation showed inconsistent evidence. There was some consistency in the relatively small number of MWAS conducted in populations with FASD. Meta-analyses could not be conducted due to significant heterogeneity between studies. CONCLUSION: Considering heterogeneity in study design and potential for bias, evidence for an association between PAE and offspring DNA methylation was inconclusive. Some reproducible associations were observed in populations with FASD although the limited number of these studies warrants further research. Trail Registration: This review is registered with PROSPERO (registration number: CRD42020167686).

Cohort profile: early school years follow-up of the Asking Questions about Alcohol in Pregnancy Longitudinal Study in Melbourne, Australia (AQUA at 6).

PURPOSE: The Asking Questions about Alcohol in Pregnancy (AQUA) study, established in 2011, is a prebirth cohort of 1570 mother and child pairs designed to assess the effects of low to moderate prenatal alcohol exposure and sporadic binge drinking on long-term child development. Women attending general antenatal clinics in public hospitals in Melbourne, Australia, were recruited in their first trimester, followed up three times during pregnancy and at 12 and 24 months postpartum. The current follow-up of the 6-8-year-old children aims to strengthen our understanding of the relationship between these levels of prenatal alcohol exposure and neuropsychological functioning, facial dysmorphology, brain structure and function. PARTICIPANTS: Between June 2018 and April 2021, 802 of the 1342 eligible AQUA study families completed a parent-report questionnaire (60%). Restrictions associated with COVID-19 pandemic disrupted recruitment, but early school-age neuropsychological assessments were undertaken with 696 children (52%), and 482 (36%) craniofacial images were collected. A preplanned, exposure-representative subset of 146 children completed a brain MRI. An existing biobank was extended through collection of 427 (32%) child buccal swabs. FINDINGS TO DATE: Over half (59%) of mothers consumed some alcohol during pregnancy, with one in five reporting at least one binge-drinking episode prior to pregnancy recognition. Children's craniofacial shape was examined at 12 months of age, and low to moderate prenatal alcohol exposure was associated with subtle midface changes. At 2 years of age, formal developmental assessments showed no evidence that cognitive, language or motor outcome was associated with any of exposure level. FUTURE PLANS: We will investigate the relationship between prenatal alcohol exposure and specific aspects of neurodevelopment at 6-8 years, including craniofacial shape, brain structure and function. The contribution of genetics and epigenetics to individual variation in outcomes will be examined in conjunction with national and international collaborations.

Sex- and tissue-specific effects of binge-level prenatal alcohol consumption on DNA methylation at birth.

Background: Binge-level prenatal alcohol exposure (PAE) causes developmental abnormalities, which may be mediated in part by epigenetic mechanisms. Despite this, few studies have characterised the association of binge PAE with DNA methylation in offspring. Methods: We investigated the association between binge PAE and genome-wide DNA methylation profiles in a sex-specific manner in neonatal buccal and placental samples. Results: We identified no differentially methylated CpGs or differentially methylated regions (DMRs) at false discovery rate <0.05. However, using a sum-of-ranks approach, we identified a DMR in each tissue of female offspring. The DMR identified in buccal samples is located near regions with previously-reported associations to fetal alcohol spectrum disorder (FASD) and binge PAE. Conclusion: Our findings warrant further replication and highlight a potential epigenetic link between binge PAE and FASD.

Lay abstract Women who binge-drink alcohol in pregnancy are more likely to have children with health and behavioural problems. It is possible that this happens through changes to the 'epigenetic' switches that control our genes. Yet few have tried to prove this. To test this idea, we designed a study called 'Asking QUestions about Alcohol in pregnancy'. We carefully measured levels of drinking across all trimesters in volunteers who were pregnant. Then we measured one type of 'gene switch' in cells from the placenta and from the cheeks of their babies. We did not find strong evidence that maternal binge drinking changed 'epigenetic' gene switches in babies. However, when we looked at female offspring only, we did find some evidence. The genes that we found had been seen by others in similar studies. Our findings have no immediate medical application but provide evidence for conducting larger studies.

Placental DNA methylation signatures of maternal smoking during pregnancy and potential impacts on fetal growth.

Maternal smoking during pregnancy (MSDP) contributes to poor birth outcomes, in part through disrupted placental functions, which may be reflected in the placental epigenome. Here we present a meta-analysis of the associations between MSDP and placental DNA methylation (DNAm) and between DNAm and birth outcomes within the Pregnancy And Childhood Epigenetics (PACE) consortium (N = 1700, 344 with MSDP). We identify 443 CpGs that are associated with MSDP, of which 142 associated with birth outcomes, 40 associated with gene expression, and 13 CpGs are associated with all three. Only two CpGs have consistent associations from a prior meta-analysis of cord blood DNAm, demonstrating substantial tissue-specific responses to MSDP. The placental MSDP-associated CpGs are enriched for environmental response genes, growth-factor signaling, and inflammation, which play important roles in placental function. We demonstrate links between placental DNAm, MSDP and poor birth outcomes, which may better inform the mechanisms through which MSDP impacts placental function and fetal growth.

Cognitive and Behavioural Attention in Children with Low-Moderate and Heavy Doses of Prenatal Alcohol Exposure: a Systematic Review and Meta-analysis.

Attention problems are thought to be a hallmark feature of prenatal alcohol exposure (PAE). Despite decades of research however, these findings have never been pooled to assess the association between PAE and the different domains of attention functioning. This systematic review and meta-analysis aimed to examine the relationships between low-moderate, binge and heavy PAE with domains of attention functioning (encode, focus, shift, sustain and behavioural) in children. Thirteen studies compared children with PAE to abstinent controls. A significant adverse effect of any PAE on shifting attention (Cohen's d = -0.61), and a trend towards an adverse effect of heavy PAE on encoding attention (Cohen's d = -0.79) were identified. Compared to controls, there were trends showing that low-moderate PAE (odds ratio = 1.21) was associated with greater odds of behavioural attention problems. Remaining analyses were limited by insufficient studies or were non-significant. In summary, a vulnerability of higher-level attention skills to PAE was found. Urgent investigation into the effects of low to moderate PAE is needed given the prevalence of this drinking pattern, trends towards behavioural attention problems, the lack of comprehensive and high-quality research and the known impacts of attention difficulties on academic and social development in children.

Influences on drinking choices among Indigenous and non-Indigenous pregnant women in Australia: A qualitative study.

Despite women's awareness that drinking alcohol in pregnancy can lead to lifelong disabilities in a child, it appears that an awareness alone does not discourage some pregnant women from drinking. To explore influences on pregnant women's choices around alcohol use, we conducted interviews and group discussions with 14 Indigenous Australian and 14 non-Indigenous pregnant women attending antenatal care in a range of socioeconomic settings. Inductive content analysis identified five main influences on pregnant women's alcohol use: the level and detail of women's understanding of harm; women's information sources on alcohol use in pregnancy; how this information influenced their choices; how women conceptualised their pregnancy; and whether the social and cultural environment supported abstinence. Results provide insight into how Indigenous Australian and non-Indigenous pregnant women understand and conceptualise the harms from drinking alcohol when making drinking choices, including how their social and cultural environments impact their ability to abstain. Strategies for behaviour change need to: correct misinformation about supposed 'safe' timing, quantity and types of alcohol; develop a more accurate perception of Fetal Alcohol Spectrum Disorder; reframe messages about harm to messages about optimising the child's health and cognitive outcomes; and develop a holistic approach encompassing women's social and cultural context.

What do pregnant women eat, and are they meeting the recommended dietary requirements for pregnancy?

OBJECTIVE: To compare the dietary intake of pregnant women to the 2013 Australian Dietary Guidelines and explore factors associated with inadequate intake. DESIGN: Dietary intake data were collected between July 2011 and July 2012 (n = 1570) using a 74-item food frequency questionnaire. SETTING: Metropolitan public health hospitals in Melbourne, Australia. PARTICIPANTS: Pregnant women, at least 16 years of age, with a singleton pregnancy, and literate in English. MEASUREMENTS AND FINDINGS: The highest proportion of women met the recommended daily servings for fruit (65.7%), followed by dairy products (55.2%), meat/meat alternatives (31.1%), vegetables (10.3%), and then grain foods (1.8%). A majority of women (83.8%) regularly consumed up to 2.5 serves of discretionary foods per day. Only one woman met the minimum recommended daily servings for all five food groups. Women who were obese were more likely to consume an inadequate diet (Adj. OR 2.13, 95% CI 1.53, 2.95); and having a university degree was associated with a lower odds of consuming an inadequate diet (Adj. OR 0.63, 95% CI 0.50, 0.78). KEY CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: Pregnancy care providers need to be aware of women's low compliance with the national dietary guidelines, particularly regarding the poor intake of vegetables and grain foods; targeted as well as population-based approaches may be required.

Engaging pregnant women in observational research: a qualitative exploratory study.

BACKGROUND: Recruitment of pregnant women to population health research can be challenging, especially if the research topic is sensitive. While many pregnant women may be inherently interested in research about pregnancy, there is the possibility that the nature and timing of the project may give rise to anxiety in some women, especially if the topic is sensitive or it brings about new awareness of potential pregnancy complications. Research staff undertaking recruitment need to be skilled at strategies to manage the environment, and have well developed communication and interpersonal skills to explain and promote the study and facilitate each woman's informed decision-making regarding participation. However, the skills needed by recruitment staff to successfully engage pregnant women with a research topic are not well understood. This study aimed to address this evidence gap by providing insight into the dynamics between a pregnant woman and recruitment staff at the time of the offer to participate in an observational study about alcohol use in pregnancy. METHODS: Naturalistic inquiry guided a qualitative exploratory descriptive approach. Experienced recruitment staff from the Asking Questions about Alcohol in Pregnancy (AQUA) study (Muggli et al., BMC Pregnancy Childbirth 14:302, 2014) participated in individual semi-structured interviews and were asked about their experiences and approaches to engaging pregnant women. Interviews were transcribed verbatim and analysed using inductive content analysis. RESULTS: Pregnant women brought with them an inherent interest or disinterest in alcohol research, or in research in general, which formed the basis for engagement. Women responded favourably to the invitation to participate being delivered without pressure, and as part of a two-way conversation. Engagement with a sensitive topic such as alcohol use in pregnancy was facilitated by a non-judgmental and non-targeted approach. Influences such as privacy, distractions, partner's opinion, time factors and level of clinical support either facilitated or hindered a woman's engagement with the research. CONCLUSIONS: These results provide an in-depth explanation of barriers and enablers to recruitment of pregnant women in antenatal clinics to studies that may inform strategies and the training of recruitment staff.

Time- and sex-dependent associations between prenatal alcohol exposure and placental global DNA methylation.

AIM: Epigenetic changes, in particular in the placenta, may mediate the effects of prenatal alcohol exposure (PAE) on children's health. We examined the relationship between PAE patterns, based on dose and timing, and placental global DNA methylation. METHODS: Using linear regression analysis, we examined the association between different PAE categories and placental global DNA methylation (n = 187), using the proxy measure of Alu-interspersed repeats. RESULTS: Following adjustment for important covariates, we found no evidence of an association between PAE and placental global DNA methylation overall. However, when stratifying by newborn sex, PAE throughout pregnancy was associated with higher placental global DNA methylation (1.5%; p = 0.01) of male newborns. CONCLUSION: PAE may have sex-specific effects on placental global DNA methylation if alcohol is consumed throughout pregnancy.

Alcohol consumption in a general antenatal population and child neurodevelopment at 2 years.

BACKGROUND: Prenatal alcohol exposure (PAE) is a community health problem with up to 50% of pregnant women drinking alcohol. The relationship between low or sporadic binge PAE and adverse child outcomes is not clear. This study examines the association between PAE in the general antenatal population and child neurodevelopment at 2 years, accounting for relevant contributing factors. METHODS: This prospective population-based cohort recruited 1570 pregnant women, providing sociodemographic, psychological and lifestyle information and alcohol use for five time periods. PAE categories were 'low', 'moderate/high', 'binge', in trimester 1 or throughout pregnancy. Measures of cognitive, language and motor development (Bayley Scales of Infant and Toddler Development) were available for 554 children, while measures of sensory processing (Infant/Toddler Sensory Profile) and social-emotional development (Brief Infant Toddler Social Emotional Assessment) were available for 948. RESULTS: A positive association in univariate analysis with low-level PAE throughout pregnancy and cognition (ß=4.1, 95% CI -0.02 to 8.22, p=0.05) was attenuated by adjusting for environmental/social deprivation risk factors (ß=3.06 (-1.19 to 7.30), p=0.16). Early binge drinking, plus continued PAE at lower levels, was associated with the child being more likely to score low in sensation avoidance (adjusted OR 1.88 (1.03 to 3.41), p=0.04). CONCLUSION: Early binge exposure, followed by lower-level PAE, demonstrated an increase in sensation-avoiding behaviour. There were, however, no significant associations between PAE and neurodevelopment following adjustment for important confounders and modifiers. Follow-up is paramount to investigate subtle or later onset problems.

Association Between Prenatal Alcohol Exposure and Craniofacial Shape of Children at 12 Months of Age.

Importance: Children who receive a diagnosis of fetal alcohol spectrum disorder may have a characteristic facial appearance in addition to neurodevelopmental impairment. It is not well understood whether there is a gradient of facial characteristics of children who did not receive a diagnosis of fetal alcohol spectrum disorder but who were exposed to a range of common drinking patterns during pregnancy. Objective: To examine the association between dose, frequency, and timing of prenatal alcohol exposure and craniofacial phenotype in 12-month-old children. Design, Setting, and Participants: A prospective cohort study was performed from January 1, 2011, to December 30, 2014, among mothers recruited in the first trimester of pregnancy from low-risk, public maternity clinics in metropolitan Melbourne, Australia. A total of 415 white children were included in this analysis of 3-dimensional craniofacial images taken at 12 months of age. Analysis was performed with objective, holistic craniofacial phenotyping using dense surface models of the face and head. Partial least square regression models included covariates known to affect craniofacial shape. Exposures: Low, moderate to high, or binge-level alcohol exposure in the first trimester or throughout pregnancy. Main Outcomes and Measures: Anatomical differences in global and regional craniofacial shape between children of women who abstained from alcohol during pregnancy and children with varying levels of prenatal alcohol exposure. Results: Of the 415 children in the study (195 girls and 220 boys; mean [SD] age, 363.0 [8.3] days), a consistent association between craniofacial shape and prenatal alcohol exposure was observed at almost any level regardless of whether exposure occurred only in the first trimester or throughout pregnancy. Regions of difference were concentrated around the midface, nose, lips, and eyes. Directional visualization showed that these differences corresponded to general recession of the midface and superior displacement of the nose, especially the tip of the nose, indicating shortening of the nose and upturning of the nose tip. Differences were most pronounced between groups with no exposure and groups with low exposure in the first trimester (forehead), moderate to high exposure in the first trimester (eyes, midface, chin, and parietal region), and binge-level exposure in the first trimester (chin). Conclusions and Relevance: Prenatal alcohol exposure, even at low levels, can influence craniofacial development. Although the clinical significance of these findings is yet to be determined, they support the conclusion that for women who are or may become pregnant, avoiding alcohol is the safest option.

Maternal micronutrient consumption periconceptionally and during pregnancy: a prospective cohort study.

OBJECTIVE: To examine overall micronutrient intake periconceptionally and throughout pregnancy in a population-based cohort of Australian women. DESIGN: In a prospective cohort study, micronutrient dosages were extracted from self-reported maternal supplement use, recorded pre-conception, and for each trimester of pregnancy. A food frequency scale (DQESv2) captured usual maternal diet for gestational weeks 14-26. The influence of sociodemographic and lifestyle factors associated with supplement use was examined using logistic regression, and changes in micronutrient intakes prior to and throughout pregnancy were assessed using repeated-measures ANOVA analyses. SETTING: Metropolitan hospital sites in Melbourne, Australia. SUBJECTS: Women with a viable singleton pregnancy were recruited at less than 19 weeks' gestation (n 2146). RESULTS: Compared with non-users, women using supplements during pregnancy were more likely to have planned their pregnancy, be >25 years old, primiparous, Caucasian, non-smokers, have a tertiary education and be consuming a folate-rich diet. Intakes of folate, Fe and Zn were significantly lower in the periconceptional period, compared with other periods (P<0·001). Intakes below Recommended Daily Intake levels were common both periconceptionally and throughout pregnancy, with 19-46 % of women not meeting the Recommended Daily Intake for folate, 68-82 % for Fe and 17-36 % for Zn. Conversely, 15-19 % of women consumed beyond the recommended Upper Limit for folate and 11-24 % for Fe. CONCLUSIONS: The study highlights the need for improved public health education on nutritional needs during pregnancy, especially among women with lower educational achievements and income.

"Did you ever drink more?" A detailed description of pregnant women's drinking patterns.

BACKGROUND: This paper presents drinking patterns in a prospective study of a population-based cohort of 1570 pregnant women using a combination of dose and timing to give best estimates of prenatal alcohol exposure (PAE). Novel assessments include women's special occasion drinking and alcohol use prior to pregnancy recognition. METHODS: Information on up to nine types of alcoholic drink, with separate frequencies and volumes, including drinking on special occasions outside a 'usual' pattern, was collected for the periconceptional period and at four pregnancy time points. Weekly total and maximum alcohol consumption on any one occasion was calculated and categorised. Drinking patterns are described in the context of predictive maternal characteristics. RESULTS: 41.3 % of women did not drink during pregnancy, 27 % drank in first trimester only; most of whom stopped once they realised they were pregnant (87 %). When compared to women who abstained from alcohol when pregnant, those who drank in the first trimester only were more likely to have an unplanned pregnancy and not feel the effects of alcohol quickly. Almost a third of women continued to drink alcohol at some level throughout pregnancy (27 %), around half of whom never drank more than at low or moderate levels. When compared with abstainers and to women who only drank in trimester one, those who drank throughout pregnancy tended to be in their early to mid-thirties, smoke, have a higher income and educational attainment. Overall, almost one in five women (18.5 %) binge drank prior to pregnancy recognition, a third of whom were identified with a question about 'special occasion' drinking. Women whose age at first intoxication was less than 18 years (the legal drinking age in Australia), were significantly more likely to drink in pregnancy and at binge levels prior to pregnancy recognition. CONCLUSIONS: We have identified characteristics of pregnant women who either abstain, drink until pregnancy awareness or drink throughout pregnancy. These may assist in targeting strategies to enhance adherence to an abstinence policy and ultimately allow for appropriate follow-up and interpretation of adverse child outcomes. Our methodology also produced important information to reduce misclassification of occasional binge drinking episodes and ensure clearly defined comparison groups.

Osteomyelitis of the hyoid bones in two calves.

Two calves were referred because of ptyalism and difficulty opening the mouth (Calf 1) and for elective umbilical hernia surgery under inhalation anaesthesia (Calf 2). Additional clinical signs were increased breath sounds and swelling in the region of the mandibular angle in Calf 1. Ultrasonography and endoscopy revealed oral inflammation and abscessation in the area of the base of the tongue in both calves. Infection of the hyoid apparatus was suspected based on ultrasonographic findings and confirmed by means of computed tomography. In Calf 1, there was no response to treatment with systemic antibiotics, nonsteroidal anti-inflammatory drugs and local lavage, and Calf 2 was not treated. Both calves were euthanized because of a poor prognosis and the diagnoses confirmed during postmortem examination. In Calf 1, the abscess was associated with complete destruction of the left epihyoid bone and partial destruction of the left stylohyoid and ceratohyoid bones. In Calf 2, the abscess was located at the distal end of the right stylohyoid bone near the epihyoid bone. Stomatitis or laryngeal and pharyngeal abscessation caused by sharp feed particles are common in cattle and infection of the hyoid apparatus should be included in the differential diagnosis.