Progress in Biophysics and Molecular Biology

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1 JPBM_proof March 0 / Q Review Maternal diabetes, gestational diabetes and the role of epigenetics in their long term effects on offspring Q Ronald C.W. Ma a, b, c, *, Greg E. Tutino a, Karen A. Lillycrop d, Mark A. Hanson e, Wing Hung Tam f a Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong, China b Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China c Hong Kong Institute of Diabetes and Obesity, The Chinese University of Hong Kong, Hong Kong, China d Centre for Biological Sciences, Life Sciences Building, University of Southampton, Southampton, UK e Institute of Developmental Sciences, University of Southampton, Southampton, UK f Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong, China article Article history: Available online xxx Keywords: Gestational diabetes Maternal diabetes Childhood obesity Epigenetics Type diabetes Contents info Contents lists available at ScienceDirect Progress in Biophysics and Molecular Biology journal homepage: abstract There is a global epidemic of obesity and diabetes, and current efforts to curb the diabetes epidemic have had limited success. Epidemiological studies have highlighted increased risk of obesity, diabetes and cardiovascular complications in offspring exposed to maternal diabetes, and gestational diabetes increases the risk of diabetes in subsequent generations, thereby setting up a vicious cycle of diabetes begetting diabetes. This relationship between maternal hyperglycaemia and long-term health in the offspring is likely to become even more important with an increasing proportion of young woman being affected by diabetes, and the number of pregnancies complicated by hyperglycaemia continuing to rise. Animal models of gestational diabetes or maternal hyperglycaemia have highlighted long-term changes in the offspring with some instances of sex bias, including increased adiposity, insulin resistance, b-cell dysfunction, hypertension, as well as other structural and functional changes. Furthermore, several of these changes appear to be transmissible to later generations through the maternal line. Epigenetic changes play an important role in regulating gene expression, especially during early development. Recent studies have identified a number of epigenetic modifications in the offspring associated with maternal hyperglycaemia. In this review, we provide an overview of the epidemiological evidence linking maternal hyperglycaemia with adverse long-term outcome in the offspring, as well as of some of the studies that explore the underlying epigenetic mechanisms. A better understanding of the pathways involved may provide novel approaches for combating this global epidemic. 0 Published by Elsevier Ltd.. Introduction Maternal diabetes and gestational diabetes Maternal hyperglycaemia and effects on the offspring Maternal hyperglycaemia and obesity in the offspring: insights from epidemiological studies Maternal hyperglycaemia and glucose intolerance in the offspring Abbreviations: ACHOIS, Australian Carbohydrate Intolerance Study in Pregnancy Women; ALSPAC, Avon Longitudinal Study of Parents and Children; BMI, Body mass index; EPOCH, Exploring Perinatal Outcomes among Children; GDM, Gestational diabetes; GWAS, Genome-wide association studies; HAPO, Hyperglycaemia and Adverse Pregnancy Outcome; IADPSG, The International Association for Diabetes in Pregnancy Study Group; JADE, Joint Asia Diabetes Evaluation; NCD, Non-communicable diseases; OGTT, Oral glucose tolerance test; RCT, Randomized Clinical Trial; TD, Type Diabetes Mellitus. * Corresponding author. Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong. Tel.: þ ; fax: þ. address: rcwma@cuhk.edu.hk (R.C.W. Ma) / 0 Published by Elsevier Ltd. Progress in Biophysics and Molecular Biology xxx (0) e on offspring, Progress in Biophysics and Molecular Biology (0),

2 JPBM_proof March 0 / R.C.W. Ma et al. / Progress in Biophysics and Molecular Biology xxx (0) e Other effects of maternal hyperglycaemia on the offspring Insights from animal models of maternal hyperglycaemia/gdm Epigenetics and induction of metabolic risk Hyperglycaemia and epigenetic changes: evidence from experimental studies Human studies on epigenetics and maternal hyperglycaemia Transmission of induced traits between generations Other important contributing factors-maternal obesity, maternal diet, pregnancy weight gain and paternal effects Perspective on improving pregnancy outcome and prevention of NCD Acknowledgement References Introduction Diabetes has reached epidemic proportions globally, and is currently estimated to affect million people, with the number of individuals with type diabetes mellitus (TD) increasing in every country (International Diabetes Federation, 0). Diabetesrelated complications and morbidity are calculated to account for at least million USD in terms of healthcare expenditure worldwide. Whilst much of the current effort in combating diabetes is aimed at reducing the well-known risk factors for diabetes such as obesity, physical inactivity, poor diet, smoking, hypertension and hyperlipidaemia, these measures have only had limited success in reducing obesity and diabetes at a population level, at least in adults. Recent work has however shifted the focus to early development as a critical window of opportunity which can profoundly affect the risk of future non communicable diseases (NCDs) including diabetes (Godfrey et al., 0; Hanson et al., 0). In this regard, maternal hyperglycaemia has emerged as an important factor that may increase the risk of diabetes, obesity and cardiovascular disease in the offspring, thereby acting as a major contributor to the risk of NCD in a population (Yajnik, 00; Vohr et al., 00; Pinney et al., 0; Ma et al., 0, 0; Tutino et al., 0; El Hajj et al., 0) In the current global epidemic of diabetes, the age of onset has decreased significantly, with an increasing proportion of women of reproductive age being affected. For example, in the Joint Asia Diabetes Evaluation (JADE) Programme, which recruited,0 patients from over 00 clinics in countries across Asia, as much as % of patients with TD had age of onset below age 0 years (Yeung et al., 0). The importance of maternal diabetes was illustrated in the SEARCH study in the United States, where.% of diabetes in youth was thought to be attributed to maternal diabetes or obesity (Dabelea et al., 00). Likewise, it has been estimated in some indigenous populations such as the Canadian First Nation people that up to % of new cases of TD had their origin in gestational diabetes mellitus (GDM) in the previous generation (Osgood et al., 0).. Maternal diabetes and gestational diabetes There are a number of diabetogenic placental-derived hormones secreted during the third trimester of pregnancy resulting in a relative state of maternal insulin resistance that continues until delivery of the conceptus. The physiologic effect of this is to shunt maternal glucose to the rapidly developing foetus. If the consequent decline in insulin action cannot be compensated for by an increase in maternal insulin production, maternal hyperglycaemia and GDM may result (Di Cianni et al., 00). It has been estimated that the global prevalence of hyperglycaemia in pregnancy in women is.%, affecting ~ million live births in 0 and this number is also increasing globally (Guariguata et al., 0). Hyperglycaemia during pregnancy can be due to pre-gestational diabetes where maternal hyperglycaemia antedates gravidity or, more commonly, GDM. GDM is defined as carbohydrate intolerance of any degree with onset or first recognition during pregnancy and its prevalence in a population is usually comparable with that of TD (ADA, 00; King, ). Recognized risk factors include previous history of GDM, advanced maternal age at conception, elevated body mass index (BMI ), family history of diabetes mellitus, and certain ethnicities (ACOG, 0) Importantly, diagnostic criteria have evolved with our understanding of the disorder and indeed currently reflect both maternal and fetal outcomes (Table ). Until recently, GDM was considerably less well characterized than its associated condition diabetes mellitus, where clinical features had been described as long as 00 years ago by the ancient Egyptians (Ahmed, 00). Prior to the discovery of insulin by Banting and Best in, there were relatively few women with diabetes who survived to reach reproductive age, consequently much of what we know about diabetes in pregnancy has accrued since the beginning of the 0th Century. Epidemiologic studies in the 's and 0's showed that women with diabetes gave birth to very large babies compared to normal mothers. Later in, Pedersen proposed that elevated maternal glucose crossed the placenta resulting in hyperinsulinaemia and excessive fetal growth (the Pedersen hypothesis) (Pedersen, ). In, Pedersen reported studies from births in Copenhagen between and in which data on the birth weight and length from infants born after the diagnosis of diabetes were collected. He observed that diabetic women gave birth to both large and small babies consistent with the population birth weight distribution curve, although the distribution for weight relative to length was shifted to the right. He also reported that babies of diabetic women were longer. Notably, a treatment effect was demonstrated where lower maternal plasma glucose was associated with lower mean offspring weight and length (Pedersen, ). In 0, Freinkel expanded upon the Pedersen hypothesis and proposed that excesses of mixed fuels including glucose, amino acids, lipids, ketones, and possibly altered concentrations of other nutrients, induce teratogenesis. Importantly, Freinkel suggested that teratogenic effects resulting from exposure to elevated levels of maternal mixed fuels, and the timing of such exposures, might have profound and long-term consequences for the foetus (Fig. ) (Freinkel, 0). Although we would not advocate the use of the term teratogenesis in foetuses that have a body composition which lies within the normal range (Hanson et al., 0), in this respect Freinkel was proposing somewhat more subtle effects to include fetal behavioural and anthropometric modifications. Indeed, with out of an eventual lifetime cycles of cell division occurring during the periconceptional period (Van Assche et al., 00), the intrauterine environment may have profound effects upon on offspring, Progress in Biophysics and Molecular Biology (0),

3 JPBM_proof March 0 / Table Historical summary of diagnostic cut-off and rationale for major diagnostic criteria for gestational diabetes mellitus. Criteria a GCT (mmol/l) OGTT glucose load (g) Fasting (mmol/l) offspring phenotype. Epidemiological evidence strongly suggests that, for example, pre-existing diabetes mellitus is associated with significant increased risk for fetal malformations versus the general population while data from GDM pregnancies has been conflicting (Allen et al., 00). Furthermore, the frequency of congenital malformations appears to be directly related to the severity of maternal hyperglycaemia (Bell et al., 0). Diabetes in pregnancy, both preexisting and GDM, may also be implicated in altered neurological development and has been associated with lower offspring cognition, educational attainment and disorders of attention deficit (Fraser et al., 0; Dionne et al., 00; Rizzo et al., ; Ornoy et al., ; Stenninger et al., ; Ornoy et al., 00). Both existing and emerging evidence suggest that effects may, in part, be sex specific with a resulting phenotypic gender dimorphism. Indeed, many common disorders such as diabetes and atherosclerosis display some degree of sexual bias. For example, males <0 years of age are more likely to be affected by metabolic syndrome with specific contributing metabolic components also differing by gender (Ervin, 00). Gender specific transgenerational inheritance can occur at a number of different levels, including maternal transmission during pregnancy and the immediate postnatal period; the sex of the transmitting parent via the germline; and the sex of the offspring displaying such effects (Vige et al., 00). Furthermore, gene expression occurs through a multitude of interconnected and interdependent pathways involving, DNA methylation; histone modifications; and RNA interference h (mmol/l) h (mmol/l) h (mmol/l) Diagnostic measures b Focus: maternal or fetal NDDG, 0 g. 0 g....0 Two of ¼ GDM Maternal Any ¼ Borderline Carpenter et al., ; ACOG, 00; 0 g. 0 g..0.. Two of ¼ GDM Maternal ADA, 000 Any ¼ Borderline WHO, ; Alberti et al., e g.0 e. e Either ¼ GDM Maternal <.0.e<.0 Either ¼ IGT IADPSG, 0; ADA, 0; WHO, 0; Ruchat et al., 0 e g..0. e Any ¼ GDM Maternal/Fetal a NDDG, National Diabetes Data Group; C & C, Carpenter & Coustan; WHO, World Health Organization; IADPSG, International Association of Diabetes and Pregnancy Study Groups. b Venous plasma glucose. Fig.. Possible developmental effects of exposure to maternal hyperglycaemia by trimester and potential progressive changes affecting offspring phenotype. Adapted from Freinkel N. Banting Lecture 0. Of pregnancy and progeny. Diabetes. 0 Dec;():e, American Diabetes Association, 0. Copyright and all rights reserved. Material from this publication has been used with the permission of American Diabetes Association. R.C.W. Ma et al. / Progress in Biophysics and Molecular Biology xxx (0) e discussed in more detail later in this review. Because these epigenetic processes occur at different periods during fetal development, any vicissitudes within the uterine environment may lead to gender based effects (Vige et al., 00). Epigenetic sex differences have been reported in various animal and human tissues (Yang et al., 00; Liu et al., 0). Five year old female offspring of GDM pregnancies had increased measures of adiposity and higher glucose and insulin concentrations compared to non GDM/paternal DM offspring. No such observations were present in male offspring or in children of diabetic fathers (Krishnaveni et al., 00). In a meta-analysis of diabetic pregnancy and offspring blood pressure in childhood, pre-existing diabetes and GSM were associated with elevated systolic blood pressure. Male offspring of GDM pregnancies had higher systolic and diastolic blood pressure compared to controls. There were no blood pressure differences in female offspring versus controls (Aceti et al., 0). Recently, genome-wide DNA methylation analysis of donor pancreatic islets revealed gender specific differences in DNA methylation, associated changes in levels of relevant micrornas, and altered gene expression and insulin secretion (Hall et al., 0). The term GDM was first used by O'Sullivan in. He recognised that the diabetogenic effects of pregnancy such as insulin resistance could allow a degree of gestational diabetes to develop long before it became clinically manifest. Between and, all prenatal care patients at two Boston, USA hospitals were screened and those meeting pre-specified criteria were administered an oral glucose tolerance test (OGTT). Subjects considered non-diabetic were subsequently given an OGTT during their pregnancy and if they met existing diabetes diagnostic criteria they were deemed to have GDM (unsuspected, asymptomatic diabetes in pregnancy) and were included in the analysis. From this early study, GDM prevalence was reported to be in pregnancies, compared to in 00e00 pregnancies for overt diabetes. It was observed that women with GDM had an incidence of conversion to diabetes mellitus of.% in the post-partum period and.% within the following. years (O'Sullivan, ). Further work by O'Sullivan and colleagues would lead to the first diagnostic criteria for GDM, largely based on maternal risk for future diabetes (O'Sullivan and Mahan, ). Over the subsequent five decades, the focus of GDM diagnosis has shifted from mainly maternal risk to fetal risk. The International Association for Diabetes in Pregnancy Study Group (IADPSG) provided the first GDM diagnostic criteria, which are based on the risk of adverse neonatal outcomes (from the Hyperglycaemia and Adverse Pregnancy Outcome (HAPO) study) (Metzger et al., 00), and its diagnostic cut-offs have recently been adopted by the World Health Organization (WHO) (Table ). Nevertheless, its ability to predict long-term metabolic risk in the offspring remains to be explored. Q on offspring, Progress in Biophysics and Molecular Biology (0),

4 JPBM_proof March 0 / R.C.W. Ma et al. / Progress in Biophysics and Molecular Biology xxx (0) e In this review, we aim to provide an overview of the epidemiological and experimental findings supporting a link between intra-uterine hyperglycaemia and adverse metabolic outcome in the offspring. We will take a bedside-to-bench and then back to bedside perspective, in order to illustrate how epidemiological observations have provided the foundations for this area of study, and how emerging experimental insights, especially on epigenetic regulation of gene expression, have informed the planning of recent translational and interventional studies.. Maternal hyperglycaemia and effects on the offspring.. Maternal hyperglycaemia and obesity in the offspring: insights from epidemiological studies The emphasis on maternal hyperglycaemia and related adverse outcomes in offspring began with intriguing observations reported in the Pima Indians of Arizona, located in the South-Western USA. Pettitt et al. noted in startling rates of obesity in the children of diabetic women in this population. At e years of age, % of offspring from diabetic pregnancies weighed in excess of 0% of their ideal body weight, compared to % from non-diabetic pregnancies. Children of diabetic pregnancies were found to be considerably more obese independent of their previous birth weight or maternal BMI (Pettitt et al.,, ). Obesity resulting from maternal diabetes was also prospectively reported by Silverman et al. in a cohort of year olds in Chicago, USA. By contrast, good to fair maternal metabolic control was achieved in this study. Nevertheless, at birth 0% of infants had weights >0th centile for gestational age. Interestingly, at months, weight and length were indistinguishable from the general population. By age, relative weight had increased noticeably and by years, half of the offspring of diabetic mothers had weights >0th centile. The authors noted that childhood obesity correlated with maternal pre-pregnancy weight and independently with amniotic fluid insulin at e weeks gestation (Silverman et al., ). Similarly, Hiller et al. reported among motherechild pairs in the Northwest USA that an increasing level of hyperglycaemia in pregnancy was associated with increased risk of obesity in offspring at age e years. Importantly, the risk may be modifiable by treating GDM, as offspring risk of obesity appeared decreased and was no longer significant after multivariate adjustment in the treated GDM group (Hillier et al., 00). More recently, Nehring et al. reported increased odds ratios for overweight and obesity at. years old after correction for maternal BMI and other confounders, and Baptiste-Roberts et al. prospectively at years after correction for maternal BMI (Nehring et al., 0; Baptiste-Roberts et al., 0). Furthermore, in the Avon Longitudinal Study of Parents and Children (ALSPAC), maternal GDM was associated with increased general and central obesity in offspring at age e, (Lawlor et al., 0) and increased adiposity measures at e years in Exploring Perinatal Outcomes among Children (EPOCH) Study, although observations in the latter were largely but not completely attenuated after adjustment for maternal pre-pregnancy BMI (Crume et al., 0a). In adult offspring, effects on obesity rates were described by Nielsen et al., who reported that army rejection rates due to adiposity in e0 year old Danish men were.% greater, and mean BMI. kg/m higher in those whose mothers had GDM (Nielsen et al., 0). Of note, the impact of maternal hyperglycaemia on offspring obesity may not be detected during the early phases of development. For example, in the EPOCH Study it was noted that offspring exposed to maternal hyperglycaemia appear to have accelerated growth trajectory, though this was most apparent between ages e (Crume et al., 0b). Taken together, these reports may be interpreted as substantial evidence for increased risk of offspring obesity as an outcome in pregnancies complicated by diabetes although effect size remains to be established... Maternal hyperglycaemia and glucose intolerance in the offspring In the same way, observations from epidemiological studies have strongly implicated the intrauterine environment in glucose intolerance in adolescent and adult offspring. Early evidence from the Pima Indians demonstrated that offspring aged e years and 0e years of women with abnormal glucose tolerance during pregnancy had a higher mean post-ogtt plasma glucose concentration and a significantly higher prevalence of diabetes. A mmol/l increase in maternal two hour glucose concentration after OGTT gave an odds ratio of. (% CI.0e.) for diabetes in the offspring at 0e years of age. The risk of developing diabetes was highest in offspring of women with diabetes at conception, followed by offspring of women who developed diabetes after pregnancy, then offspring of non-diabetic women (offspring diabetes prevalence: %,.%,.% respectively). Paternal diabetes status was not found to be a significant factor. Of particular importance was the observation that maternal -h glucose was a significant predictor of maternal glucose concentrations in female offspring when they in turn were pregnant, pointing towards a potentially intergenerational effect (Petitt et al., ; Pettitt et al.,, ) Indeed, the percentage of children aged e years who were exposed to hyperglycaemia in utero had increased greatly from % to almost % over years from the 0s to the 0s. Diabetes rates in the Pima Indian population doubled over the same time period from % to %. Much of the increase could be attributed to an increase in offspring BMI and in the frequency of intrauterine exposure to diabetes (Dabelea et al., ). Moreover, risk factors for the development of diabetes in the Pima Indians include the presence of diabetes in parents, certain genetic markers, obesity, poor diet, and the prenatal metabolic environment (Knowler et al., 0). Silverman et al. evaluated the long term effects of maternal hyperglycaemia on children from a population understood to have a lower risk of diabetes compared to the Pima Indians. Offspring of women with pre-gestational diabetes and GDM had plasma glucose and insulin measurements after fasting and two hours following. g/kg oral glucose when assessed on a yearly basis from. years of age. The prevalence of impaired glucose tolerance increased steadily with age from.% at < years,.% at e years, to.% at e years, all of which were significantly higher compared to control subjects (Silverman et al., ). Most notably, the risk of intergenerational transmission of TD appears to occur via the maternal line. In a study of more than 000 Chinese patients with TD, maternal history of diabetes was more prevalent than paternal history of diabetes (Lee et al., 000). Also, consistent with observations reported by Pettitt et al., Harder and Plagemann noted that there was aggregation of TD in mothers and grandmothers of women with GDM, but no aggregation of TD in fathers and grandfathers of women with GDM, suggesting that there is excess maternal transmission of TD over successive generations (Harder et al., 000). Exposure to diabetes in utero results in an earlier age of diagnosis in the offspring. An analysis from the SEARCH for Diabetes in Youth study, a multi-ethnic population with diabetes diagnosed under the age of 0, indicated that those children with type diabetic mothers whose diabetes was identified prior to pregnancy were diagnosed with diabetes earlier than those for whom maternal diabetes was not diagnosed until after the pregnancy. The timing of paternal diabetes onset was not associated with that of the offspring (Pettitt et al., 00). Furthermore, Stride et al on offspring, Progress in Biophysics and Molecular Biology (0),

5 JPBM_proof March 0 / R.C.W. Ma et al. / Progress in Biophysics and Molecular Biology xxx (0) e reported that patients with maturity-onset diabetes of the young (MODY) who are carriers of the HNF-a mutation develop diabetes at a younger age if their mother's diabetes was diagnosed before the pregnancy rather than after. The authors suggest that this is evidence that non-genetic effects are important determinants of age at diabetes diagnosis, even in those with a high genetic risk for diabetes (Stride et al., 00)... Other effects of maternal hyperglycaemia on the offspring In the Pima Indian studies, there was a strong association between obesity and diabetes in the parents and incidence in the offspring (Knowler et al., ). These associations highlight the challenges in separating the diabetogenic effects of maternal genetic risk from that of exposure to the intrauterine environment. An elegant sibling study examining these effects attempted to address this problem. The prevalence of diabetes and mean BMI in offspring born before and after the onset of maternal diabetes indicated that the former had a significantly higher odds ratio (., % CI.e.) for the development of diabetes and a greater BMI. Notably, there were no significant differences in risk of diabetes or in BMI between offspring born before and after onset of paternal diabetes (Dabelea et al., 00). Another well-designed study by Sobngwi et al. endeavoured to assess the effects of maternal hyperglycaemia independently of genetic influences. They measured insulin sensitivity and insulin secretion in response to oral and intravenous glucose in non-diabetic adult offspring of mothers with prepregnancy type diabetes and similar offspring of type diabetic fathers. One third of the offspring of diabetic mothers exhibited impaired glucose tolerance and defects in early insulin secretion following OGTT. No offspring of diabetic fathers had impaired glucose tolerance (Sobngwi et al., 00). These studies demonstrate that exposure to maternal hyperglycaemia in utero conveys an additional risk for the development of obesity and TD in the offspring, in addition to any genetic risk that may be present. Animal studies have demonstrated that offspring of diabetic mothers exhibit hyper secretion of insulin at birth followed by a rapid decline thereafter. b-cell function is also significantly impaired at all time points (Aerts et al., ; Aref et al., 0). Human data suggest generally similar findings. Cord blood samples from diet controlled GDM pregnancies were compared to non-gdm controls. Both fetal insulin and proinsulin were increased in offspring of GDM pregnancies compared to controls despite the fact that maternal and fetal glucose concentrations were not significantly different between GDM and controls (Wang et al., 0). In e year old subjects of GDM pregnancies, maternal glucose concentrations were associated with increased indices of b- cell response while in year old offspring of diabetic mothers whose onset occurred before the reference pregnancy, initial insulin secretory responses were lower compared to offspring where maternal diabetes occurred after the reference pregnancy (Bush et al., 0; Salbe et al., 00). In adult non diabetic offspring of type diabetic mothers, early insulin secretion was reduced compared to controls (offspring of type diabetic father), and in offspring of mothers with young-onset TD, early insulin response was similarly reduced compared to those with young-onset type diabetic fathers (Sobngwi et al., 00; Singh et al., 00). Animal and human observations suggest an exaggerated initial insulin response, followed by a precipitous decline thereafter. In addition to impaired b-cell function, glucose intolerance, diabetes and obesity, offspring exposed to hyperglycaemia in utero also have increased cardiovascular risk. In our local prospective follow-up study, offspring exposed to mild GDM have significantly higher blood pressure, and a worse lipid profile, compared to offspring of mothers with normal glucose tolerance during pregnancy (Tam et al., 00). This association between maternal hyperglycaemia and elevated blood pressure in the offspring has been consistently noted in several studies, and was confirmed in a recent meta-analysis of studies (Aceti et al., 0). Importantly, elevated umbilical cord insulin or C-peptide levels at birth was associated with increased risk of glucose intolerance in offspring at years, obesity and metabolic syndrome at years, as well as increased arterial stiffness in adolescents, highlighting the persistent effects of hyperglycaemia and hyperinsulinaemia in utero on subsequent obesity, glucose intolerance and cardiovascular risk in the offspring (Tam et al., 00, 0, 0) In another study from Asia, female offspring of mothers with GDM had higher adiposity, higher glucose and insulin concentrations by years of age. There was no similar association in offspring of diabetic fathers, again highlighting the potential importance of the intra-uterine environment (Krishnaveni et al., 00). There is an abundance of evidence implicating the intrauterine environment in developmental origins of disease, especially in relation to long term metabolic effects on the offspring including insulin resistance, obesity, and diabetes that can, in turn, lead to increased risk in subsequent generations. The seminal observations by Barker and Hales highlighted the relationship between low birth weight, as a proxy of in utero under-nutrition, and long-term metabolic risk (Barker and Osmond, ; Barker et al., ). There is now extensive experimental and epidemiological data linking early life factors and a diverse scope of outcomes ranging from metabolic, reproductive, immunological, neurodevelopmental and cancer risk, centred on the conceptual framework of developmental origins of health and disease (Gluckman et al., 0). Along with this theoretical interpretation, it is important to point out that although the initial focus of the field was on the relationship between in utero under-nutrition and adverse metabolic outcomes, epidemiological evidence has consistently demonstrated a U-shaped relationship between birth weight and subsequent metabolic disturbances in offspring, including increased risk for GDM in affected daughters (Dabelea et al., ; Rogvi et al., 0). With maternal hyperglycaemia and maternal obesity being the main factors which lead to elevated birth weight, these studies highlight that both in utero undernutrition, as well as over-nutrition, contribute to intergenerational metabolic risk via the maternal lineage, and that both of these components can operate in populations and even within families depending on the nutritional state of the mother at the time. Although the precise mechanisms of these inter-generational effects remain to be elucidated, epigenetic processes are suspected to be key mediators in the process. Better understanding of the underlying mechanisms, as well as identification of markers of this risk, may enable earlier detection, and potentially bring about interventions to reduce its transgenerational passage... Insights from animal models of maternal hyperglycaemia/gdm Animal models of hyperglycaemia during pregnancy have been explored for the last few decades in studies on the long-term effects of maternal hyperglycaemia. These studies provided evidence in support of the observations made in clinical studies, and have provided the opportunity to explore the underlying mechanisms. These models involved chemical destruction of b-cells using drugs such as streptozotocin (STZ), or continuous infusion of glucose (Aerts and Van Assche, 00; Fujisawa et al., 00). These studies reported that hyperglycaemia in the pregnant mother was associated with increased glucose delivery to the foetus via facilitated transport (Aerts et al., 0), a process which does not appear to be saturable, unlike many other placental transport mechanisms. Towards the end of gestation, when the fetal pancreas is able to on offspring, Progress in Biophysics and Molecular Biology (0),

6 JPBM_proof March 0 / R.C.W. Ma et al. / Progress in Biophysics and Molecular Biology xxx (0) e produce insulin, exposure to hyperglycaemia leads to b-cell hyperplasia, enlargement of the endocrine pancreas, as well as increased insulin biosynthetic activity (Aerts et al., 0). This increased fetal insulin production and insulin action, along with availability of nutrients in the form of excess glucose, will therefore lead to fetal macrosomia. In addition to increased adiposity, fetal hyperinsulinaemia is also associated with a greater uptake of amino acids and increased protein synthesis (Aerts et al., 00). Whilst these changes in fetal insulin production have been observed for mild maternal hyperglycaemia, it is worthwhile to note that marked hyperglycaemia in experimental settings is associated with over-stimulation of pancreatic b-cells, disruption of endocrine pancreas development and microsomia (Aerts et al., 00). In these models, adult offspring of diabetic animals were noted to have normal development of the endocrine pancreas (Aerts et al., ; Ma et al., 0). However, they develop glucose intolerance and impaired insulin response to glucose challenge, and display insulin resistance, mainly in the liver and muscle, highlighting the presence of both insulin resistance and b-cell dysfunction (Aerts et al., ; Holemans et al., a,b). The key role of the intrauterine environment was demonstrated by a series of embryo transfer experiments, which showed that the diabetes risk in a low genetic risk strain can be substantially increased by the hyperglycaemic environment of a dam with a high genetic risk of diabetes (Gill-Randall et al., 00). Offspring of severely hyperglycaemic mothers have also been seen to develop elevated triglyceride and cholesterol levels, along with increased blood pressure at weeks (Ma et al., 0), in line with clinical observations. In a rat model of STZ-induced diabetes, offspring exposed to maternal hyperglycaemia have salt-sensitive hypertension and impaired renal function in adulthood, thought to be due to effects on fetal renal tubular development (Nehiri et al., 00). Animal models of GDM have demonstrated that metabolic abnormalities in offspring exposed to maternal hyperglycaemia appear to persist into the third generation through the maternal lineage (Aerts et al.,, 00), again recapitulating the intergenerational effects through maternal transmission seen in epidemiological studies (O'Sullivan and Mahan, ). Recently, a mouse model of in utero under-nutrition has been shown not only to lead to progressive obesity and impaired glucose tolerance in the F male offspring (Jimenez-Chillaron et al., 00), but also transmission of the phenotype to subsequent generations (Jimenez- Chillaron et al., 00). For example, it was found that reduced birth weight progressed to F offspring through the paternal line, whereas obesity was transmitted to F offspring through the maternal line, with impaired glucose tolerance transmitted to subsequent generations through both parental lineages (Jimenez- Chillaron et al., 00). This has provided novel insights into the mechanisms of transgenerational metabolic effects, and provides experimental evidence in support of the inter-linked effects of both nutrient-mediated teratogenesis (under-nutrition) and fuelmediated teratogenesis (over-nutrition) that are likely to be operational in developing countries (Yajnik, 00). Several hypotheses have been put forward to explain the underlying mechanism linking exposure to maternal hyperglycaemia and adverse outcome in the offspring. Pederson hypothesized that increasing maternal glucose concentration will lead to exposure of the foetus to hyperglycaemia, via facilitated transport, and thereby stimulate the fetal pancreas to produce insulin. The growthpromoting effects of fetal insulin may then promote fetal growth, macrosomia and increase the risk of later obesity (Pedersen,). In addition, Dorner and Plagemann have suggested that hyperinsulinaemia during fetal and early development may program brain development during this critical window, and may lead to permanent alteration of hypothalamic organization of appetite and metabolism pathways (D orner et al., ). Embryos exposed to diabetes in pregnancy were found to have significantly altered gene expression profile, in particular, the expression of key transcription factors, suggesting that critical developmental pathways can be altered following exposure to intrauterine hyperglycaemia (Pavlinkova et al., 00). Whilst a combination of different mechanisms may contribute to the increased risk of obesity and metabolic disorders in offspring exposed to maternal hyperglycaemia, increasing evidence has accumulated that epigenetic changes may be involved in developmental induction of metabolic risk (Gluckman et al., 00; Pinney et al., 0), and may potentially explain the inter-generational effects of intrauterine hyperglycaemia.. Epigenetics and induction of metabolic risk Epigenetic changes refer to heritable changes in gene expression which do not involve changes in DNA sequences. Several epigenetic mechanisms have been found to regulate gene expression. Whilst the most studied mechanism relates to DNA methylation, other changes, including histone modifications and non-coding RNAs, also play an important role, and can be transmitted from one generation to the next. DNA methylation involves the addition of methyl groups to DNA, mainly at CpG sites, which converts cytosine to -methylcytosine, and usually lead to transcriptional silencing of genes (Smallwood et al., 0). Traditionally, studies have focused on the analysis of methylation at CpG islands in gene promoter regions, which are usually demethylated. Many of the fundamental studies related to these changes involved in cancer. However, it is increasingly appreciated that DNA methylation occurs in different genomic contexts, including methylation of gene bodies and regulatory elements, and may play an important role in modulating the risk of diseases other than cancer (Jones, 0; Lou et al., 0). The epigenome is particularly susceptible to internal and external influence during specific stages, for example during early development (Reik et al., 00; Hochberg et al., 0), when environmental cues can be transduced and lead to altered development and physiology. DNA methylation patterns are copied by DNA methyltransferase during DNA replication, thereby providing a mechanism whereby environmentally induced changes can be transmitted to subsequent cell generations through mitosis without altering the genetic code. Epigenetic processes represent a key characteristic of cell differentiation and play an important role in maintenance of the differentiated state (Smallwood et al., 0). During germ cell specification and development, there is global erasure of DNA methylation, followed by extensive epigenetic reprogramming of methylation and histone structure although it is not now believed that this erasure is complete, allowing marks to be transmitted between generations. Following fertilisation, there is a new wave of demethylation, which occurs more rapidly in the paternal compared to the maternal genome (Smallwood et al., 0). Several imprinted genes undergo allele-specific methylation during gametogenesis and embryogenesis (Smith et al., 0). For example, IGF- is a maternally imprinted gene which is key to human development and growth. It has been shown that methylation of IGF- is relatively stable in adult life, suggesting that early life factors may play a major role in determining variation in IGF- methylation (Heijmans et al., 00). That the intra-uterine environment can impact on long-term metabolic risk via epigenetic changes have been demonstrated in several animal models relating to maternal nutritional manipulation. One of the first demonstrations came from models with restricted protein intake of pregnant rats, which was associated with reduced methylation, and corresponding increased expression of peroxisome proliferator-activated receptor-a (PPARa) and the on offspring, Progress in Biophysics and Molecular Biology (0),

7 JPBM_proof March 0 / R.C.W. Ma et al. / Progress in Biophysics and Molecular Biology xxx (0) e glucocorticoid receptor in the liver, which can be prevented with folic acid supplementation (Lillycrop et al., 00). In islets of growth-restricted foetuses due to maternal under-nutrition, a series of epigenetic changes at different stages of development have been noted, with decrease in H and H acetylation at the promoter region of PDX-, a key transcription factor for islet development, reduced PDX- transcription, and later on, decreased HK trimethylation and increased HK dimethylation. This series of epigenetic changes appeared to lead to remodelling towards an inactive chromatin state in the islet and transcriptionally silencing of PDX-, which was associated with b-cell dysfunction and glucose intolerance (Park et al., 00; Pinney et al., 0). The insulin promoter has been found to be methylated in mouse embryonic stem cells, becoming demethylated only as the cells differentiate into insulinproducing cells. Together with demethylation of the human and mouse insulin promoter in pancreatic b-cells, these studies suggest a potential role of epigenetic regulation in b-cell maturation and islet insulin expression (Kuroda et al., 00). Feeding a high fat diet during pregnancy and lactation has also been shown to induce epigenetic and phenotypic changes in the offspring. Vucetic et al. showed increased expression of the m- opioid receptor (MOR) and preproenkephalin (PENK) in the nucleus accumbens, prefrontal cortex, and hypothalamus of mice from dams that consumed the high fat diet during pregnancy and this was accompanied by the hypomethylation of the promoter regions of these genes (Vucetic et al., 0). Recent studies have highlighted the potential importance of the one-carbon cycle, also referred to as -C metabolism, in the developmental origins of diabetes and obesity (Finer et al., 0). This one-carbon cycle includes the methylation cycle that produces S-adenosylmethionine from methionine and adenosine triphosphate (ATP). These pathways deliver methyl groups via the linked folateemethionine cycles for use in critical processes such as DNA synthesis and phospholipid and protein biosynthesis (Steegers- Theunissen et al., 0). For example, early animal studies have revealed that alterations of the cellular provision of methyl groups in early life may disrupt DNA methylation, and induce long-term phenotypic changes (Burdge et al., 00; Hoile et al., 0). One important study in sheep by Sinclair et al. demonstrated that deficiencies in specific B vitamins including vitamin B and folate, and methionine within normal physiological ranges, led to phenotypic changes in adults such as increased body weight and adiposity as well as changes in immune responses to antigenic challenge. Male animals were reported to also be insulin-resistant and had elevated blood pressure (Sinclair et al., 00). These experimental studies are in line with clinical observations that altered maternal vitamin B and folate levels during pregnancy are associated with long-term metabolic outcome in the offspring (Yajnik et al., 00). For instance, vitamin B, folate and homocysteine concentrations were measured at weeks gestation in a cohort of Indian women. Maternal homocysteine concentrations were inversely associated with all neonatal anthropometric measurements and positively associated with offspring HOMA-IR at. and. years of age (Krishnaveni et al., 0). An inverse relationship between homocysteine concentrations and birth weight has also been reported by Yajnik C et al. after analysis of two large fetal growth studies, thereby adding strength to the argument for causality of the association between maternal -C metabolism and fetal growth (Yajnik et al., 0)... Hyperglycaemia and epigenetic changes: evidence from experimental studies In a mouse model of maternal hyperglycaemia induced by STZ, it was noted that offspring of diabetic mothers had increased methylation of the H-IGF- imprinted region, and had reduced expression if IGF- and lower birth weight (Shao et al., 00). In a GDM mouse model, it was noted that the expression of IGF and H was down regulated in pancreatic islets of both F and F offspring, and was associated with altered methylation, impaired islet ultrastructure and function (Ding et al., 0). Other epigenetic changes have also been observed in pregnancies complicated by hyperglycaemia. For example, a recent genome-wide survey of histone acetylation in mouse embryos exposed to maternal diabetes noted an imbalance in embryonic histone acetylation patterns (Salbaum et al., 0). In addition to changes following exposure to intra-uterine hyperglycaemia, epigenetic changes have also been noted in other experimental settings of hyperglycaemia. For example, increased DNA methylation has been described for the promoter region of the peroxisome proliferator-activated receptor-g (PPARg) coactivator-a gene (PPARGCA) in diabetic islets (Ling et al., 00). Similar hypermethylation in the promoter region of the PPARGCA gene has been noted in the skeletal muscle from diabetic patients, and correlated with mitochondrial content (Barres et al., 00). Epigenetic changes have also been suggested to be responsible for the legacy effect of reduced risk of vascular complications after a period of sustained tight glucose control, or metabolic memory of transient hyperglycaemia and increased risk of diabetic vascular injury (Pirola et al., 0). Histone methylation variations have been noted in monocytes cultured in high glucose, as well as blood monocytes of diabetic patients (Miao et al., 00). In a series of landmark experiments, it was shown that endothelial cells exposed to short-term hyperglycaemia had persistently increased expression of the NF-kB active subunit p, and was associated with increased promoter HKme and occupancy by the histone monomethyltransferase SET/. In addition, transient hyperglycaemia was also associated with sustained reduction of HK methylation on the NF-kB p promoter, as well as recruitment of lysine-specific demethylase (LSD) (El-Osta et al., 00; Brasacchio et al., 00). LSD has also been found to regulate HK methylation in vascular smooth muscle cells in hyperglycaemic conditions, and may mediate the vascular inflammation (Reddy et al., 00). Other epigenetic mechanisms including micrornas and long noncoding RNAs have also been implicated in the pathogenesis of diabetic complications (Kato et al., 0)... Human studies on epigenetics and maternal hyperglycaemia One of the earliest studies that linked adverse intra-uterine environment, epigenetic changes and long-term adverse outcome came from studies of individuals who were exposed to the Dutch Hunger Winter in e (Heijmans et al., 00). In this study, adults who were exposed to famine in the periconception period as much as decades earlier were found to have significantly reduced methylation in the IGF- DMR when compared to their unexposed, same-sex siblings (Heijmans et al., 00). A subsequent study using the same cohort examined differential methylation of loci implicated in growth and development, and found significant differences in methylation of IL, LEP, ABCA, GNASAS and MEG (Tobi et al., 00). These studies provided the scientific evidence for epigenetic changes being a potential mechanistic link between in utero environment and long-term metabolic changes in humans. In two birth cohorts (also replicated twice in one cohort) from Southampton, UK, it was found that methylation at one CpG site in the retinoid X receptor-a (RXRA) gene in DNA extracted from umbilical cord tissue was associated with childhood adiposity, as well as maternal diet in early gestation (Godfrey et al., 0). Interestingly, these neonatal epigenetic changes were estimated to account for more than % of the variance in child's adiposity at or years on offspring, Progress in Biophysics and Molecular Biology (0),

8 JPBM_proof March 0 / R.C.W. Ma et al. / Progress in Biophysics and Molecular Biology xxx (0) e of age, highlighting the potential importance of early epigenetic changes in shaping phenotypic traits. In comparison, genetic variants identified through recent genome-wide association studies (GWAS) are thought to explain only less than % of the variance of obesity (McCarthy, 0). A recent study has identified increased methylation at HIFA to be associated with adult obesity, though it is suggested that changes in this locus are a consequence of obesity, rather than causal (Dick et al., 0). Recent studies have explored the role of epigenetic changes in offspring exposed to maternal hyperglycaemia. In an earlier candidate-gene study of placental tissues and maternal and cord blood samples from women with and without GDM, it was found that maternal glucose level was correlated with placental leptin gene DNA methylation, and decreased leptin gene expression, thereby providing a potential link between maternal hyperglycaemia, fetal programming and long-term risk of obesity and diabetes (Bouchard et al., 0). Similarly, DNA methylation of the adiponectin gene in placental DNA was associated with maternal glucose levels in a graded manner across a wide range. Subsequent application of genome-wide approach using a microarray that evaluated methylation at >0,000 CpG sites identified more than 00 genes in cord tissue and cord blood that are differentially methylated between offspring of GDM pregnancies and nonexposed offspring (Bouchard et al., 0). Among these differentially methylated genes, a significant number were also associated with birth weight. Notably, many of the genes which exhibited differential methylation were classified by Ingenuity Pathway Analysis (IPA) as belonging to metabolic disease pathways, including genes implicated in diabetes mellitus, consistent with epidemiological observations linking GDM and long-term risk of diabetes and other cardiometabolic abnormalities in the offspring (Ruchat et al., 0). The ATP-binding cassette transporter- (ABCA) gene is an important regulator of cholesterol efflux and atherosclerosis. Cord blood methylation of ABCA was found to be negatively correlated with maternal glucose levels, providing a potential link between maternal hyperglycaemia and cardiovascular risk in the offspring (Houde et al., 0). In another study, cord blood and placental samples with or without exposure to GDM were examined for changes in methylation levels of candidate genes for metabolic programming using quantitative pyrosequencing (El Hajj et al., 0). This revealed significantly different methylation in MEST and the glucocorticoid receptor NRC between GDM and controls in two tissues, and altered methylation in at least one tissue in other metabolic genes including OCT, PPARA and NESPAS. Furthermore, ALU repeats were found to be significantly hypomethylated in both cord blood and placental tissues, suggesting that maternal hyperglycaemia interferes with mechanisms controlling methylation patterns both at specific loci and at interspersed repeats (El Hajj et al., 0). Similar studies have been carried out in offspring of women with diabetes. A recent study compared the methylation pattern in peripheral blood leukocytes from non-diabetic adolescent Pima Indians who were either offspring of diabetic mothers or offspring of non-diabetic mothers. Differential methylated regions were determined using a Me-DIP-chip assay. Subsequent KEGG pathway analysis identified MODY, TD and Notch signalling as the top enriched pathways with differentially methylated genes. These pathways include genes which are important in pancreatic development, b-cell response to glucose as well as insulin secretion. This highlights the potential impact of intrauterine hyperglycaemia on methylation of genes implicated in b-cell function, thereby predisposing the offspring to increased risk of diabetes later in life (del Rosario et al., 0). Although these proof-of-concept studies provide exciting insights into possible epigenetic mechanisms that may underpin the developmental origins of obesity and metabolic disorders later in life, one has to bear in mind their limitations. The early studies in general investigated only a small sample, lacked independent replication, and the methylation changes detected through the hypothesis-free genome-wide approach often do not reach biological levels of significance. Additional considerations include the use of tissues that are not embryonic in origin (e.g. placental tissue), tissues that contain a mixture of different cell types (e.g. umbilical cord or cord blood) as well as tissue of mixed maternal or fetal origin (placenta again). Therefore, epigenetic changes in the tissues studied thus far may not represent the full spectrum, or the most relevant epigenetic changes associated with maternal hyperglycaemia and its metabolic consequences, given the difficulty of investigating relevant metabolic tissues such as the pancreatic islet, muscle, liver, adipose tissue and brain. It is expected that some of the changes present in accessible tissue such as cord blood may also be present in other tissues, though the relationship between epigenetic markers in different tissues remains to be clarified because epigenetic marks are likely to be tissue- and context-specific. Recent studies suggest there are some consistent changes in methylation that are observed in blood and other tissues such as brain, signifying that peripheral blood may be useful for identifying functionally relevant epigenetic pathways in disease-relevant tissues (Davies et al., 0). Another important issue is the need for prospective studies to eliminate the effect of reverse causality. This has been more of a problem in epigenetic studies in other disciplines, but less so in the field of developmental origins of health and disease, where there are large numbers of well-characterized longitudinal birth cohorts with longterm follow-up and a variety of biological specimens collected. We recently conducted a genome-wide analysis of GDM methylation changes by comparing offspring of mothers with GDM or controls from our longitudinal follow-up study (Tam et al., 00, 0). We found several consistent differentially methylated regions between GDM-offspring and non-exposed offspring at and years, suggesting that, at least for some of these markers, once the epigenetic changes are set they may persist through adolescence and beyond (Luan et al., 0)... Transmission of induced traits between generations There is some evidence that epigenetic and phenotypic traits induced by early life environment can be passed from one generation to the next. Although it is important to distinguish between intergenerational effects (in utero exposure of the developing embryo and its germline to the particular nutritional, hormonal, metabolic or other environment) from truly transgenerational effects in offspring and later progeny who have not been exposed to the initial environmental trigger. Several mechanisms may confound what appears to be transgenerational inheritance, including cryptic genetic variation, heritable changes in behaviour, effects on the microbiome and transmission of metabolites (Heard et al., 0). Epidemiological studies into transgenerational effects often could not rule out the impact of postnatal nutritional environment, or do not adequately adjust for confounding environmental or behavioural factors (Heard et al., 0). In human studies, diabetes mortality increased if the paternal grandfather was exposed to a surfeit of food during his slow growth period (Kaati et al., 00). Exposure of female offspring to the Dutch Famine in utero has been shown to induce higher neonatal ponderal index in their offspring than their mothers (Painter et al., 00). Animal studies have shown that feeding a protein restricted diet to pregnant rats in the F 0 generation resulted in elevated blood pressure and endothelial dysfunction in both the F and F generations, despite the fact that the dams in the F generation were given a standard control diet during pregnancy in the F generation on offspring, Progress in Biophysics and Molecular Biology (0),

9 JPBM_proof March 0 / (Torrens et al., 00). Hypomethylation of the hepatic GR and PPARa promoters was also observed in both the F and F male offspring (Burdge et al., 00) suggesting that transmission of a phenotype induced in the F generation to the F generation may be mediated through an epigenetic mechanism. However since the germ cells which gave rise to the F offspring were exposed to the original nutritional challenge within the F0 dam, such effects may simply reflect a direct effect of the challenge on the F germ cells. True transgenerational transmission of an epigenetic or phenotypic traits at least down the female line therefore requires assessment of the F offspring.. Other important contributing factors-maternal obesity, maternal diet, pregnancy weight gain and paternal effects Although this article has focused on the role of maternal diabetes and GDM on long-term metabolic outcome in the offspring, it is important to note that maternal obesity, gestational weight gain, or maternal consumption of a high fat diet have also been linked with adverse pregnancy outcomes and increased risk of childhood obesity (Rooney et al., 0; Poston and 0). There also appears to be variable response to these maternal factors according to ethnicity (Bowers et al., 0). Detailed discussion of these effects are beyond the scope of this current review, though there are notable similarities with the effects of maternal hyperglycaemia (Fig. ). Studies have linked these examples of in utero overnutrition with epigenetic changes in the offspring in both experimental models as well as in human studies (Nicholas et al., 0; Liu et al., 0). Of note is a recent study which compared infants of mothers with GDM, as well as those with intrauterine growth R.C.W. Ma et al. / Progress in Biophysics and Molecular Biology xxx (0) e restriction, with infants with normal postnatal growth, which noted that similar epigenetic modifications may underpin different prenatal exposures, and thereby contribute to increased risk of diabetes and obesity in the offspring (Quilter et al., 0). We have so far focused the discussion on maternal influences and their long-term effects on metabolic risk, though there is increasing evidence of an important biological role of fathers in metabolic programming of offspring as well. For example, paternal high fat diet in rats was found to be associated with b-cell dysfunction and glucose intolerance in female offspring, with altered gene expression and epigenetic modification within the islet of offspring (Ng et al., 0). Paternal obesity was associated with hypomethylation at the IGF DMR in DNA from cord blood leukocytes (Soubry et al., 0a). Paternal obesity has also been linked with hypomethylation of MEST in cord blood, suggesting that metabolic risk can be transmitted to the next generation through epigenetic signature in sperm (Soubry et al., 0b). A number of studies are currently underway to identify epigenetic modifications in sperm to unravel the mechanisms underlying paternal transmission of metabolic risk. These studies highlight the importance of optimizing nutrition and health for both future mothers and fathers in order to optimize pregnancy outcomes and reduce the burden of obesity and diabetes in future generations (Table ).. Perspective on improving pregnancy outcome and prevention of NCD While debate still exists surrounding the long term consequences on offspring resulting from exposure to hyperglycaemia in utero (Kim et al., 0; Donovan et al., 0), an increasing number Fig.. Conceptual framework of the role of epigenetics in the inter-generational passage of metabolic risk following exposure to maternal diabetes/gestational diabetes. Maternal hyperglycaemia, partly exacerbated by unbalanced diet, maternal obesity and gestational weight gain, contributes to in utero over-nutrition and metabolic changes including fetal hyperinsulinaemia. There can also be effects on placental function. Exposure to the adverse in utero environment may result in epigenetic changes in key metabolic and other genes during development, resulting in altered structure and function of a range of organs and control systems in the offspring. This may contribute towards the increased obesity, glucose intolerance, hypertension, dyslipidaemia and altered cardiovascular risk among offspring exposed to maternal hyperglycaemia. This increased risk may be further passed to subsequent generations through several mechanisms, including altered phenotype of the offspring, as well as persistent epigenetic changes which can be transmitted. This may result in progressive increase in the NCD burden in subsequent generations. MEST e Mesoderm-specific transcript homolog; PPARA e Peroxisome proliferator-activated receptor alpha; ABCA e ATP-binding cassette transporter. Q on offspring, Progress in Biophysics and Molecular Biology (0),