Maternal alcohol consumption in pregnancy enhances arterial stiffness and alters vasodilator function that varies between vascular beds in fetal sheep.

While the impact of alcohol consumption by pregnant women on fetal neurodevelopment has received much attention, the effects on the cardiovascular system are not well understood. We hypothesised that repeated exposure to alcohol (ethanol) in utero would alter fetal arterial reactivity and wall stiffness, key mechanisms leading to cardiovascular disease in adulthood. Ethanol (0.75 g (kg body weight)(-1)) was infused intravenously into ewes over 1 h daily for 39 days in late pregnancy (days 95-133 of pregnancy, term ∼147 days). Maternal and fetal plasma ethanol concentrations at the end of the hour were ∼115 mg dl(-1), and then declined to apparent zero over 8 h. At necropsy (day 134), fetal body weight and fetal brain-body weight ratio were not affected by alcohol infusion. Small arteries (250-300 µm outside diameter) from coronary, renal, mesenteric, femoral (psoas) and cerebral beds were isolated. Endothelium-dependent vasodilatation sensitivity was reduced 10-fold in coronary resistance arteries, associated with a reduction in endothelial nitric oxide synthase mRNA (P = 0.008). Conversely, vasodilatation sensitivity was enhanced 10-fold in mesenteric and renal resistance arteries. Arterial stiffness was markedly increased (P = 0.0001) in all five vascular beds associated with an increase in elastic modulus and, in cerebral vessels, with an increase in collagen Iα mRNA. Thus, we show for the first time that fetal arteries undergo marked and regionally variable adaptations as a consequence of repeated alcohol exposure. These alcohol-induced vascular effects occurred in the apparent absence of fetal physical abnormalities or fetal growth restriction.

Association of maternal and nutrient supply line factors with DNA methylation at the imprinted IGF2/H19 locus in multiple tissues of newborn twins.

Epigenetic events are crucial for early development, but can be influenced by environmental factors, potentially programming the genome for later adverse health outcomes. The insulin-like growth factor 2 (IGF2)/H19 locus is crucial for prenatal growth and the epigenetic state at this locus is environmentally labile. Recent studies have implicated maternal factors, including folate intake and smoking, in the regulation of DNA methylation at this locus, although data are often conflicting in the direction and magnitude of effect. Most studies have focused on single tissues and on one or two differentially-methylated regions (DMRs) regulating IGF2/H19 expression. In this study, we investigated the relationship between multiple shared and non-shared gestational/maternal factors and DNA methylation at four IGF2/H19 DMRs in five newborn cell types from 67 pairs of monozygotic and 49 pairs of dizygotic twins. Data on maternal and non-shared supply line factors were collected during the second and third trimesters of pregnancy and DNA methylation was measured via mass spectrometry using Sequenom MassArray EpiTyper analysis. Our exploratory approach showed that the site of umbilical cord insertion into the placenta in monochorionic twins has the strongest positive association with methylation in all IGF2/H19 DMRs (p<0.05). Further, evidence for tissue- and locus-specific effects were observed, emphasizing that responsiveness to environmental exposures in utero cannot be generalized across genes and tissues, potentially accounting for the lack of consistency in previous findings. Such complexity in responsiveness to environmental exposures in utero has implications for all epigenetic studies investigating the developmental origins of health and disease.

The Peri/postnatal Epigenetic Twins Study (PETS).

The Peri/postnatal Epigenetic Twins Study (PETS) is a longitudinal cohort of 250 pairs of Australian twins and their mothers, who were recruited mid-way through pregnancy from January 2007 to September 2009. The study is centered on the developmental origins of health and disease paradigm (DOHaD) in which an adverse intrauterine environment predisposes the individual to complex disease in later life by reducing growth in utero and adversely altering developmental plasticity. Data concerning diet and lifestyle were collected from mothers during pregnancy, and samples of plasma and serum taken at 28 weeks' gestation. We attended 75% of all births, at which time we collected multiple biological samples including placenta, cord blood, and neonatal cheek cells, the latter from 91% of pairs. Chorionicity was recorded and zygosity was determined by DNA testing where necessary. Approximately 40% of the twins are monozygotic, two-thirds of which are dichorionic. Twins were seen again at 18 months of age and repeat blood and cheek swabs taken where possible. Studies of gene expression and the epigenetic marks of DNA methylation have so far revealed that twins exhibit a wide range of epigenetic discordance at birth, that one-third of the epigenome changes significantly between birth and 18 months; shared (maternal) environment, genetic factors, and non-shared intrauterine environment contribute to an increasing proportion of epigenetic variation at birth, respectively, and affect tissues differently, and that within-pair birth weight discordance correlates with epigenetic discordance in genes associated with lipid metabolism, supporting an epigenetic mechanism for DOHaD.

Neonatal DNA methylation profile in human twins is specified by a complex interplay between intrauterine environmental and genetic factors, subject to tissue-specific influence.

Comparison between groups of monozygotic (MZ) and dizygotic (DZ) twins enables an estimation of the relative contribution of genetic and shared and nonshared environmental factors to phenotypic variability. Using DNA methylation profiling of ∼20,000 CpG sites as a phenotype, we have examined discordance levels in three neonatal tissues from 22 MZ and 12 DZ twin pairs. MZ twins exhibit a wide range of within-pair differences at birth, but show discordance levels generally lower than DZ pairs. Within-pair methylation discordance was lowest in CpG islands in all twins and increased as a function of distance from islands. Variance component decomposition analysis of DNA methylation in MZ and DZ pairs revealed a low mean heritability across all tissues, although a wide range of heritabilities was detected for specific genomic CpG sites. The largest component of variation was attributed to the combined effects of nonshared intrauterine environment and stochastic factors. Regression analysis of methylation on birth weight revealed a general association between methylation of genes involved in metabolism and biosynthesis, providing further support for epigenetic change in the previously described link between low birth weight and increasing risk for cardiovascular, metabolic, and other complex diseases. Finally, comparison of our data with that of several older twins revealed little evidence for genome-wide epigenetic drift with increasing age. This is the first study to analyze DNA methylation on a genome scale in twins at birth, further highlighting the importance of the intrauterine environment on shaping the neonatal epigenome.

Maternal vitamin D predominates over genetic factors in determining neonatal circulating vitamin D concentrations.

BACKGROUND: There are multiple potential regulators of neonatal vitamin D status of environmental, genetic, and epigenetic origins. The relation between these factors and circulating neonatal vitamin D has yet to be fully characterized. OBJECTIVE: The aim of this study was to examine the relative contribution of genetic factors, maternal circulating 25-hydroxyvitamin D [25(OH)D] concentrations, and the placental methylation level of the gene that encodes the primary catabolic enzyme of active vitamin D [25(OH)D-24-hydroxylase encoded by CYP24A1] to neonatal 25(OH)D concentrations. DESIGN: We used the classical twin study design to determine the genetic contribution to neonatal 25(OH)D. A total of 86 twin pairs (32 monozygotic and 54 dizygotic twin pairs) were included in this study. Serum 25(OH)D was measured by using a 25(OH)D kit. CYP24A1 promoter DNA methylation was measured by means of matrix-assisted laser desorption time-of-flight mass spectrometry. RESULTS: Maternal and neonatal 25(OH)D showed a strong association (R² = 0.19). Monozygotic and dizygotic within-pair serum 25(OH)D correlations were similar (R² = 0.71 and 0.67, respectively), which suggested no genetic effect. Placental CYP24A1 methylation did not show an association with maternal or neonatal 25(OH)D concentrations. CONCLUSIONS: Our results suggest that maternal circulating 25(OH)D is the most significant regulator of neonatal circulating 25(OH)D concentrations, with underlying genetic factors playing a limited role. The placental methylation of the CYP24A1 promoter appears subject to a genetic influence, although no evidence of a relation between the methylation level of this gene and circulating maternal or neonatal 25(OH)D was apparent.

Twinship influence on morbidity and mortality across the lifespan.

BACKGROUND: Studies in twins may be questioned with respect to their representativeness of the general population, not least considering the potential importance of the fetal environment for future health and disease. To better understand the influence twinning may have on health, we investigated long-term health outcomes of twins, their singleton siblings and singletons from the population. METHODS: Morbidity and mortality in twins was contrasted to that of their singleton siblings. These singletons from families with twins were then compared with singletons of the population to further reveal potential twin family influences on health. Familial relations were identified through the Swedish Multi-Generation Register. Among individuals born between 1932 and 1958, the number of twins and their singleton siblings identified were 49,156 and 35,277, respectively. Outcomes were incident overall cancer, cardiovascular disease (CVD) and death, identified in national registers. Standardized survival functions were estimated using Cox proportional hazards regression and the corresponding cumulative risks plotted against age. RESULTS: Cumulative risks of cancer, CVD and death in twins did not differ from singletons of families with twins, who in turn were found to be similar to singletons of families without twins. As could be expected from these findings, no differences in risks were found when twins were compared with singletons of the population. CONCLUSIONS: Despite their adverse intrauterine experience, twins do not seem to fare worse than singletons with respect to adult morbidity and mortality. The findings indicate that the unique experience of twinning does not lead to adverse long-term health outcomes.

Propensity scores: from naive enthusiasm to intuitive understanding.

Estimation of the effect of a binary exposure on an outcome in the presence of confounding is often carried out via outcome regression modelling. An alternative approach is to use propensity score methodology. The propensity score is the conditional probability of receiving the exposure given the observed covariates and can be used, under the assumption of no unmeasured confounders, to estimate the causal effect of the exposure. In this article, we provide a non-technical and intuitive discussion of propensity score methodology, motivating the use of the propensity score approach by analogy with randomised studies, and describe the four main ways in which this methodology can be implemented. We carefully describe the population parameters being estimated - an issue that is frequently overlooked in the medical literature. We illustrate these four methods using data from a study investigating the association between maternal choice to provide breast milk and the infant's subsequent neurodevelopment. We outline useful extensions of propensity score methodology and discuss directions for future research. Propensity score methods remain controversial and there is no consensus as to when, if ever, they should be used in place of traditional outcome regression models. We therefore end with a discussion of the relative advantages and disadvantages of each.

Vitamin D insufficiency is associated with impaired vascular endothelial and smooth muscle function and hypertension in young rats.

Increasing evidence links vitamin D deficiency and cardiovascular dysfunction in human adults. There is a worldwide increase in the prevalence of vitamin D deficiency in women of reproductive age, particularly dark-skinned and/or veiled women and their infants. We used a rat model to determine the functional impact of vitamin D deficiency during intra uterine and early life on resistance artery reactivity and blood pressure in the offspring as young adults. Rat dams were maintained on vitamin D deficient or replete chow before and during pregnancy and lactation. The offspring were maintained on the same chow until studied at 7-8 weeks of age. Conscious blood pressure was measured. Endothelial and smooth muscle function were tested in mesenteric arteries on a pressure myograph. Vitamin D deficient male and female offspring had a 10-fold lower serum 25-hydroxyvitamin D (P < 0.0001) and markedly elevated blood pressures (11-20 mmHg, P < 0.001) and heart rates (21-40 beats min(-1), P < 0.02) than control fed offspring. Serum calcium was unchanged. Mesenteric artery myogenic tone was doubled in vitamin D deficiency. Endothelium-derived nitric oxide-evoked dilation was halved in arteries from vitamin D deficient males and dioestrous females. Dilation attributed to endothelium-derived hyperpolarizing factor was all but abolished in vitamin D deficient oestrous females. Nitroprusside-evoked dilation was unaltered in arteries from males, but was markedly reduced in vessels of vitamin D deplete females. In conclusion, early life vitamin D deficiency is associated with endothelial vasodilator dysfunction, and this is likely to contribute to the accompanying elevation in blood pressure and an increased cardiovascular disease risk.

Expression discordance of monozygotic twins at birth: effect of intrauterine environment and a possible mechanism for fetal programming.

Within-pair comparison of monozygotic (MZ) twins provides an ideal model for studying factors that regulate epigenetic profile, by controlling for genetic variation. Previous reports have demonstrated epigenetic variability within MZ pairs, but the contribution of early life exposures to this variation remains unclear. As epigenetic marks govern gene expression, we have used gene expression discordance as a proxy measure of epigenetic discordance in MZ twins at birth in two cell types. We found strong evidence of expression discordance at birth in both cell types and some evidence for higher discordance in twin pairs with separate placentas. Genes previously defined as being involved in response to the external environment showed the most variable expression within pairs, independent of cell type, supporting the idea that even slight differences in intrauterine environment can influence expression profile. Focusing on birthweight, previously identified as a predisposing factor for cardiovascular, metabolic and other complex diseases, and using a statistical model that estimated association based on within-pair variation of expression and birthweight, we found some association between birthweight and expression of genes involved in metabolism and cardiovascular function. This study is the first to examine expression discordance in newborn twins. It provides evidence of a link between birthweight and activity of specific cellular pathways and, as evidence points to gene expression profiles being maintained through cell division by epigenetic factors, provides a plausible biological mechanism for the previously described link between low birthweight and increased risk of later complex disease.

Birthweight and mortality in adulthood: a systematic review and meta-analysis.

BACKGROUND: Small birth size may be associated with increased risk of cardiovascular diseases (CVD), whereas large birth size may predict increased risk of obesity and some cancers. The net effect of birth size on long-term mortality has only been assessed in individual studies, with conflicting results. METHODS: The Meta-analyses of Observational Studies in Epidemiology (MOOSE) guidelines for conducting and reporting meta-analysis of observational studies were followed. We retrieved 22 studies that assessed the association between birthweight and adult mortality from all causes, CVD or cancer. The studies were systematically reviewed and those reporting hazard ratios (HRs) and 95% confidence intervals (95% CIs) per kilogram (kg) increase in birthweight were included in generic inverse variance meta-analyses. RESULTS: For all-cause mortality, 36,834 deaths were included and the results showed a 6% lower risk (adjusted HR = 0.94, 95% CI: 0.92-0.97) per kg higher birthweight for men and women combined. For cardiovascular mortality, the corresponding inverse association was stronger (HR = 0.88, 95% CI: 0.85-0.91). For cancer mortality, HR per kg higher birthweight was 1.13 (95% CI: 1.07-1.19) for men and 1.04 (95% CI: 0.98-1.10) for women (P(interaction) = 0.03). Residual confounding could not be eliminated, but is unlikely to account for the main findings. CONCLUSION: These results show an inverse but moderate association of birthweight with adult mortality from all-causes and a stronger inverse association with cardiovascular mortality. For men, higher birthweight was strongly associated with increased risk of cancer deaths. The findings suggest that birthweight can be a useful indicator of processes that influence long-term health.

DNA methylation analysis of multiple tissues from newborn twins reveals both genetic and intrauterine components to variation in the human neonatal epigenome.

Mounting evidence from both animal and human studies suggests that the epigenome is in constant drift over the life course in response to stochastic and environmental factors. In humans, this has been highlighted by a small number of studies that have demonstrated discordant DNA methylation patterns in adolescent or adult monozygotic (MZ) twin pairs. However, to date, it remains unclear when such differences emerge, and how prevalent they are across different tissues. To address this, we examined the methylation of four differentially methylated regions associated with the IGF2/H19 locus in multiple birth tissues derived from 91 twin pairs: 56 MZ and 35 dizygotic (DZ). Tissues included cord blood-derived mononuclear cells and granulocytes, human umbilical vein endothelial cells, buccal epithelial cells and placental tissue. Considerable variation in DNA methylation was observed between tissues and between unrelated individuals. Most interestingly, methylation discordance was also present within twin pairs, with DZ pairs showing greater discordance than MZ pairs. These data highlight the variable contribution of both intrauterine environmental exposures and underlying genetic factors to the establishment of the neonatal epigenome of different tissues and confirm the intrauterine period as a sensitive time for the establishment of epigenetic variability in humans. This has implications for the effects of maternal environment on the development of the newborn epigenome and supports an epigenetic mechanism for the previously described phenomenon of 'fetal programming' of disease risk.

The utility of neonatal dried blood spots for the assessment of neonatal vitamin D status.

Evidence suggests that low concentrations of 25-hydroxyvitamin D(3) (25OHD3) during gestation may be associated with a range of adverse health outcomes in later life. Retrospective estimation of perinatal vitamin D status using questionnaires is extremely unreliable and stored serum samples are rarely available. We aimed to validate the use of dried blood spots (DBS) to estimate perinatal vitamin D status and to determine whether inter-group differences in cord serum 25OHD3 are reflected in DBS. We examined 25OHD3 in 4-year-old frozen cord sera and matched DBS from neonates born at a hospital in Melbourne, Australia (n = 100). We examined the correlation between these values and also investigated whether the expected seasonal (winter/spring vs. summer/autumn) difference in serum 25OHD3 was reflected in DBS values. 25OHD3 was assayed in triplicate using tandem mass spectroscopy in both a 3 microL sample of cord serum and in matched 3 mm punches from archived DBS. 25OHD3 concentrations in neonatal cord serum and DBS were highly correlated (r = 0.85, P < 0.0001). As expected, serum 25OHD3 concentrations were higher in neonates born in summer/autumn (December to March) vs. winter/spring (April to November) (median 46.6 vs. 23.7 nmol/L, P < 0.0001). A comparable difference was seen in DBS values (17.8 vs. 10.5 nmol/L, P = 0.0001). Archived DBS samples provided a valid measure of perinatal vitamin D status and identified inter-seasonal differences in perinatal 25OHD3 concentrations. They could be used for case-control studies investigating the association between perinatal vitamin D status and later health outcomes.

DNA methylation-mediated down-regulation of DNA methyltransferase-1 (DNMT1) is coincident with, but not essential for, global hypomethylation in human placenta.

The genome of extraembryonic tissue, such as the placenta, is hypomethylated relative to that in somatic tissues. However, the origin and role of this hypomethylation remains unclear. The DNA methyltransferases DNMT1, -3A, and -3B are the primary mediators of the establishment and maintenance of DNA methylation in mammals. In this study, we investigated promoter methylation-mediated epigenetic down-regulation of DNMT genes as a potential regulator of global methylation levels in placental tissue. Although DNMT3A and -3B promoters lack methylation in all somatic and extraembryonic tissues tested, we found specific hypermethylation of the maintenance DNA methyltransferase (DNMT1) gene and found hypomethylation of the DNMT3L gene in full term and first trimester placental tissues. Bisulfite DNA sequencing revealed monoallelic methylation of DNMT1, with no evidence of imprinting (parent of origin effect). In vitro reporter experiments confirmed that DNMT1 promoter methylation attenuates transcriptional activity in trophoblast cells. However, global hypomethylation in the absence of DNMT1 down-regulation is apparent in non-primate placentas and in vitro derived human cytotrophoblast stem cells, suggesting that DNMT1 down-regulation is not an absolute requirement for genomic hypomethylation in all instances. These data represent the first demonstration of methylation-mediated regulation of the DNMT1 gene in any system and demonstrate that the unique epigenome of the human placenta includes down-regulation of DNMT1 with concomitant hypomethylation of the DNMT3L gene. This strongly implicates epigenetic regulation of the DNMT gene family in the establishment of the unique epigenetic profile of extraembryonic tissue in humans.

Maternal alcohol intake and offspring pulse wave velocity.

BACKGROUND: Intrauterine exposure to alcohol may affect cardiovascular development, increasing risk of cardiovascular malformations. Intrauterine exposure to light maternal alcohol intake has been reported to affect human umbilical arterial contractility, and adult sheep exposed in utero have had altered cerebrovascular reactivity. In human adults, alcohol intake affects arterial stiffness. OBJECTIVES: We investigated whether intrauterine exposure to alcohol was associated with childhood pulse wave velocity (PWV), a measure of arterial stiffness. METHODS: On postnatal day 4, mothers of 147 twin pairs born in Tasmania from 1991 to 1993 reported alcohol intake during each trimester of pregnancy. At 9 years, child PWV was assessed over carotid-femoral and femoral-dorsalis pedis arterial segments by applanation tonometry. RESULTS: Carotid-femoral PWV was 0.2 m/s (95% CI 0.06, 0.4) higher (indicating stiffer vessels) in children whose mothers drank alcohol in the 2nd trimester rather than abstained, after adjusting for potential confounding factors. A similar effect was not seen for femoral-dorsalis pedis PWV. Findings were independent of child blood pressure which correlated strongly with PWV. Alcohol intake varied little between trimesters, so it was not possible to assess the effect of timing of exposure. CONCLUSIONS: Carotid-femoral PWV in adults is predictive of cardiovascular morbidity and mortality. The degree of continuity between childhood and adulthood PWV is unknown, but as we found an association between prenatal alcohol exposure and carotid-femoral PWV at 9 years, a permanent change in vessel wall structure or function is possible. These findings need to be confirmed in other and larger cohorts, and mechanistic animal studies are needed.

Maternal vitamin D deficiency leads to cardiac hypertrophy in rat offspring.

The aim of this study was to determine the effect of vitamin D deficiency from conception until 4 weeks of age on the development of the heart in rat offspring. Sprague-Dawley (SD) rats were fed either a vitamin D deplete or vitamin D-replete diet for 6 weeks prior to pregnancy, during pregnancy and throughout lactation. Cardiomyocyte number was determined in fixed hearts of offspring at postnatal day 3 and 4 weeks of age using an optical disector/fractionator stereological technique. In other litters, cardiomyocytes were isolated from freshly excised hearts to determine the proportion of mononucleated and binucleated cardiomyocytes. Maternal vitamin D deficiency had no effect on cardiomyocyte number, cardiomyocyte area, or the proportion of mononucleated/binucleated cardiomyocytes in 3-day-old male and female offspring. Importantly, however, vitamin D deficiency led to an increase in left ventricle (LV) volume that was accompanied by an increase in cardiomyocyte number and size, and in the proportion of mononucleated cardiomyocytes at 4 weeks of age. Our findings suggest that exposure to vitamin D deficiency in utero and early life leads to delayed maturation and subsequent enhanced growth (proliferation and hypertrophy) of cardiomyocytes in the LV. This may lead to altered cardiac function later in life.

A convergent model for placental dysfunction encompassing combined sub-optimal one-carbon donor and vitamin D bioavailability.

We hypothesise that the risk of placental dysfunction/insufficiency rises cumulatively in response to several interdependent risk factors that convergently regulate 1,25-dihydroxyvitamin D (the biologically active form of vitamin D, [1,25-(OH)(2)D]) levels at the feto-maternal interface. These factors include; (i) disturbances in genetic or epigenetic regulation of one-carbon metabolism and/or vitamin D metabolism and (ii) insufficiency in maternal vitamin D or in dietary intake of micronutrients that are involved in one-carbon donation. We predict that the sub-optimal functioning of folate and vitamin D metabolic pathways, in concert, represents a potential novel risk pathway for adverse pregnancy outcomes. We base this prediction on five observations: In order to test this model, future epidemiological studies aimed at identifying risk factors for disorders linked to sub-optimal placental development and functioning, should: (a) measure circulating precursor molecules (including folate, vitamin B12, homocysteine, and vitamin D) in maternal and cord blood; (b) collect samples for examination of genotypic variation in both one-carbon and vitamin D regulatory genes and, (c) collect samples for examination of epigenetic status of genes regulating vitamin D homeostasis and action in the placenta.

Early diet and general cognitive outcome at adolescence in children born at or below 30 weeks gestation.

OBJECTIVE: To test the hypothesis that effects of early diet on cognition observed at age 8 years persist in adolescents born preterm at < or = 30 weeks gestational age. STUDY DESIGN: A subgroup from a preterm infant cohort recruited for a randomized trial studying the effects of early dietary intervention was assessed at age 16 years. IQ scores were compared between those assigned a high-nutrient diet (n = 49) or standard-nutrient diet (n = 46) in infancy at both 8 and 16 years. RESULTS: At age 8 years, the high-nutrient group had higher mean Verbal IQ (VIQ; P = .03), Performance IQ (P = .01), and Full-Scale IQ (P = .02) scores compared with the standard-nutrient group; the VIQ difference persisted at adolescence (P = .02). This effect was accounted for principally by a significant difference in the mean Verbal Comprehension Index score (P < .008). CONCLUSIONS: A brief period of dietary intervention after preterm birth, principally between 26 and 34 weeks of gestation, affected IQ at age 16 years. A standard-nutrient diet was associated with lower VIQ, accounted for mainly by differences in verbal comprehension, which persisted after control of social factors.