Neonatal fatty acid profiles are correlated with infant growth measures at 6 months.

Rapid weight gain in infancy and low levels of n-3 long chain polyunsaturated fatty acids (LCPUFA) at birth are associated with increased adiposity later in life. The association between placental LCPUFA delivery and weight gain in infancy is poorly understood. We sought to determine the relationships between maternal phenotype, placental fatty acid transporter expression and offspring growth patterns over the first 6 months. Placental tissue and cord blood were collected at term delivery from women with uncomplicated pregnancies. Offspring body composition measurements were recorded 1 day and 6 months after birth. Body mass index (BMI) z-scores were determined using World Health Organization 2006 reference data. Body phenotype patterns were compared among offspring who had an increase in BMI z-score and those who had a decrease. High skinfold thickness at birth and positive change in BMI z-scores during infancy were associated with low neonatal n-3 LCPUFA plasma levels (r=-0.46, P=0.046) and high saturated fatty acids levels (r=0.49, P=0.034). Growth of skinfolds over 6 months of age was associated with placental fatty acid transporter gene expression. Change in BMI z-score in the first 6 months of life correlated with arm muscle area growth, a measure of lean mass (r=0.62, P=0.003), but not with growth in skinfold thickness. Early infancy weight gain was associated with poor plasma LCPUFA status at birth, and fat deposition in infancy was related to changes in placental lipid handling. Thus, neonatal fatty acid profiles may influence the trajectory of infant growth and fat and lean mass deposition.

A review of fundamental principles for animal models of DOHaD research: an Australian perspective.

Epidemiology formed the basis of 'the Barker hypothesis', the concept of 'developmental programming' and today's discipline of the Developmental Origins of Health and Disease (DOHaD). Animal experimentation provided proof of the underlying concepts, and continues to generate knowledge of underlying mechanisms. Interventions in humans, based on DOHaD principles, will be informed by experiments in animals. As knowledge in this discipline has accumulated, from studies of humans and other animals, the complexity of interactions between genome, environment and epigenetics, has been revealed. The vast nature of programming stimuli and breadth of effects is becoming known. As a result of our accumulating knowledge we now appreciate the impact of many variables that contribute to programmed outcomes. To guide further animal research in this field, the Australia and New Zealand DOHaD society (ANZ DOHaD) Animals Models of DOHaD Research Working Group convened at the 2nd Annual ANZ DOHaD Congress in Melbourne, Australia in April 2015. This review summarizes the contributions of animal research to the understanding of DOHaD, and makes recommendations for the design and conduct of animal experiments to maximize relevance, reproducibility and translation of knowledge into improving health and well-being.

IFPA meeting 2015 workshop report II: mechanistic role of the placenta in fetal programming; biomarkers of placental function and complications of pregnancy.

Workshops are an integral component of the annual International Federation of Placenta Association (IFPA) meeting, allowing for networking and focused discussion related to specialized topics on the placenta. At the 2015 IFPA meeting (Brisbane, Australia) twelve themed workshops were held, three of which are summarized in this report. These workshops focused on various aspects of placental function, particularly in cases of placenta-mediated disease. Collectively, these inter-connected workshops highlighted the role of the placenta in fetal programming, the use of various biomarkers to monitor placental function across pregnancy, and the clinical impact of novel diagnostic and surveillance modalities in instances of late onset fetal growth restriction (FGR).

Overcoming birth weight: can physical activity mitigate birth weight-related differences in adiposity?

BACKGROUND: Individuals born at low or high birth weight (BW) have elevated adiposity. The extent to which physical activity can mitigate this risk is unknown. OBJECTIVES: The aim of this study was to determine if associations between BW and adiposity vary by self-reported moderate-to-vigorous physical activity (MVPA) among adolescents. METHODS: We used data on adolescents in the National Health and Nutrition Examination Survey (1999-2006; 12-15 years; n = 4064). Using gender-stratified linear regression, we modelled body mass index (BMI) and waist circumference (WC) z-scores as a function of low, normal and high BW, MVPA (weekly Metabolic Equivalent of Task hours) and MVPA*BW cross-product terms, adjusting for sociodemographics, diet and, in WC models, BMI. RESULTS: Among girls with low MVPA, those born with high BW had greater BMI than normal BW; this difference diminished with greater MVPA (coefficient [95% confidence interval]: low MVPA: 0.72 [0.29, 1.14]; high MVPA: -0.04 [-0.48, 0.39]; P for interaction = 0.05). Among boys, MVPA did not modify the associations between BW and BMI. WC was unrelated to BW, regardless of MVPA. CONCLUSIONS: Findings suggest that effects of high BW in total adiposity can be more easily modified with MVPA in adolescent girls than in boys.

Developmental Programming: Priming Disease Susceptibility for Subsequent Generations.

Racial and/or ethnic minorities carry the highest burden of many adverse health outcomes intergenerationally We propose a paradigm in which developmental programming exacerbates the effects of racial patterning of adverse environmental conditions, thereby contributing to health disparity persistence. Evidence that developmental programming induces a heightened response to adverse exposures ("second hits") encountered later in life is considered. We evaluated the evidence for the second hit phenomenon reported in animal and human studies from three domains (air, stress, nutrition). Original research including a gestational exposure and a childhood or adulthood second hit exposure was reviewed. Evidence from animal studies suggest that prenatal exposure to air pollutants is associated with an exaggerated reaction to postnatal air pollution exposure, which results in worse health outcomes. It also indicates offspring exposed to prenatal maternal stress produce an exaggerated response to subsequent stressors, including anxiety and hyper-responsiveness of the hypothalamic-pituitary-adrenal axis. Similarly, prenatal and postnatal Western-style diets induce synergistic effects on weight gain, metabolic dysfunction, and atherosclerotic risk. Cross-domain second hits (e.g., gestational air pollution followed by childhood stressor) were also considered. Suboptimal gestational environments induce exaggerated offspring responses to subsequent environmental and social exposures. These developmental programming effects may result in enhanced sensitivity of ongoing, racially patterned, adverse exposures in race/ethnic minorities, thereby exacerbating health disparities from one generation to the next. Empirical assessment of the hypothesized role of priming processes in the propagation of health disparities is needed. Future social epidemiology research must explicitly consider synergistic relationships among social environmental conditions to which gestating females are exposed and offspring exposures when assessing causes for persistent health disparities.

Influence of high fat diet and resveratrol supplementation on placental fatty acid uptake in the Japanese macaque.

INTRODUCTION: Adequate maternal supply and placental delivery of long chain polyunsaturated fatty acids (LCPUFA) is essential for normal fetal development. In humans, maternal obesity alters placental FA uptake, though the impact of diet remains uncertain. The fatty fetal liver observed in offspring of Japanese macaques fed a high fat diet (HFD) was prevented with resveratrol supplementation during pregnancy. We sought to determine the effect of HFD and resveratrol, a supplement with insulin-sensitizing properties, on placental LCPUFA uptake in this model. METHODS: J. macaques were fed control chow (15% fat, n = 5), HFD (35% fat, n = 10) or HFD containing 0.37% resveratrol (n = 5) prior to- and throughout pregnancy. At ∼ 130 d gestation (term = 173 d), placentas were collected by caesarean section. Fatty acid uptake studies using (14)C-labeled oleic acid, arachidonic acid (AA) and docosahexanoic acid (DHA) were performed in placental explants. RESULTS: Resveratrol supplementation increased placental uptake of DHA (P < 0.05), while HFD alone had no measurable effect. Resveratrol increased AMP-activated protein kinase activity and mRNA expression of the fatty acid transporters FATP-4, CD36 and FABPpm (P < 0.05). Placental DHA content was decreased in HFD dams; resveratrol had no effect on tissue fatty acid profiles. DISCUSSION: Maternal HFD did not significantly affect placental LCPUFA uptake. Furthermore, resveratrol stimulated placental DHA uptake capacity, AMPK activation and transporter expression. Placental handling of DHA is particularly sensitive to the dramatic alterations in the maternal metabolic phenotype and placental AMPK activity associated with resveratrol supplementation.

The programming of cardiovascular disease.

In spite of improving life expectancy over the course of the previous century, the health of the U.S. population is now worsening. Recent increasing rates of type 2 diabetes, obesity and uncontrolled high blood pressure predict a growing incidence of cardiovascular disease and shortened average lifespan. The daily >$1billion current price tag for cardiovascular disease in the United States is expected to double within the next decade or two. Other countries are seeing similar trends. Current popular explanations for these trends are inadequate. Rather, increasingly poor diets in young people and in women during pregnancy are a likely cause of declining health in the U.S. population through a process known as programming. The fetal cardiovascular system is sensitive to poor maternal nutritional conditions during the periconceptional period, in the womb and in early postnatal life. Developmental plasticity accommodates changes in organ systems that lead to endothelial dysfunction, small coronary arteries, stiffer vascular tree, fewer nephrons, fewer cardiomyocytes, coagulopathies and atherogenic blood lipid profiles in fetuses born at the extremes of birthweight. Of equal importance are epigenetic modifications to genes driving important growth regulatory processes. Changes in microRNA, DNA methylation patterns and histone structure have all been implicated in the cardiovascular disease vulnerabilities that cross-generations. Recent experiments offer hope that detrimental epigenetic changes can be prevented or reversed. The large number of studies that provide the foundational concepts for the developmental origins of disease can be traced to the brilliant discoveries of David J.P. Barker.

Unexpected maturation of PI3K and MAPK-ERK signaling in fetal ovine cardiomyocytes.

In the first two-thirds of gestation, ovine fetal cardiomyocytes undergo mitosis to increase cardiac mass and accommodate fetal growth. Thereafter, some myocytes continue to proliferate while others mature and terminally differentiate into binucleated cells. At term (145 days gestational age; dGA) about 60% of cardiomyocytes become binucleated and exit the cell cycle under hormonal control. Rising thyroid hormone (T3) levels near term (135 dGA) inhibit proliferation and stimulate maturation. However, the degree to which intracellular signaling patterns change with age in response to T3 is unknown. We hypothesized that in vitro activation of ERK, Akt, and p70(S6K) by two regulators of cardiomyocyte cell cycle activity, T3 and insulin like growth factor-1 (IGF-1), would be similar in cardiomyocytes at gestational ages 100 and 135 dGA. IGF-1 and T3 each independently stimulated phosphorylation of ERK, Akt, and p70(S6K) in cells at both ages. In the younger mononucleated myocytes, the phosphorylation of ERK and Akt was reduced in the presence of IGF-1 and T3. However, the same hormone combination led to a dramatic twofold increase in the phosphorylation of these signaling proteins in the 135 dGA cardiomyocytes-even in cells that were not proliferating. In the older cells, both mono- and binucleated cells were affected. In conclusion, fetal ovine cardiomyocytes undergo profound maturation-related changes in signaling in response to T3 and IGF-1, but not to either factor alone. Differences in age-related response are likely to be related to milestones in fetal cardiac development as the myocardium prepares for ex utero life.

Placental programming of chronic diseases, cancer and lifespan: a review.

Particular paths of fetal growth are now known to predict a range of disorders in adult life. This is thought to reflect fetal programming, the phenomenon whereby nutrition and other influences during development set the body's organs and systems for life. The thesis of this review is that normal variations in the processes of placental development lead to variations in the supply of nutrients to the fetus and programme a small number of key systems that are linked to later disease. A baby's growth and nutrition depend both on the function of the placenta, reflected in its gross morphology at birth, and on the mother's lifetime nutrition, reflected in her height and weight. In many studies, the effects of placental size and shape on later disease have been examined within different categories of mother's body size. The review shows that variations in gross placental morphology at birth predict a wide range of disorders in later life. Any particular placental phenotype seems to predict a limited number of diseases. Further research into the links between the processes of placentation and the morphology of the placenta at birth is now required. We need to know more about the relative importance of nutrient flow, nutrient balance and the timing of nutritional events in determining disorders in later life. We also need to understand why, compared to other placental mammals, the human placenta is so variable in its morphology and functional capacity.

Maternal cotyledons at birth predict blood pressure in childhood.

INTRODUCTION: A small placental surface at birth has been shown to be associated with the development of hypertension in later life. In this study we extend this observation by looking at the relationship between the number of placental cotyledons and blood pressure in childhood. Because the number of cotyledons is correlated with the surface area, we hypothesized that fewer cotyledons would be associated with higher blood pressure. METHODS: The Alspac study is a longitudinal study of 13,971 children born in Bristol. Their placentas were stored in formalin. We photographed the placentas of a sample of the children and related the number of maternal cotyledons to their blood pressure levels at age 9 years. RESULTS: Contrary to our hypothesis, a greater number of maternal cotyledons was associated with higher blood pressure. Among boys, a greater number of cotyledons was associated with higher systolic and diastolic pressure but not with higher pulse pressure. Diastolic pressure rose by 2.2 mmHg (95% CI 0.6 to 3.7, p = 0.007) for every 10 additional cotyledons. Among girls, a greater number of cotyledons was associated with higher systolic pressure and pulse pressure but not with higher diastolic pressure. Pulse pressure rose by 2.7 mmHg (1.1-4.3, p < 0.001) for every 10 additional cotyledons. These associations were little changed by adjustment for placental surface area. CONCLUSION: Our study has shown that a large number of maternal cotyledons is associated with raised blood pressure in childhood. The associations differ in the two sexes.

Maternal high-fat diet impacts endothelial function in nonhuman primate offspring.

OBJECTIVE: The link between maternal under-nutrition and cardiovascular disease (CVD) in the offspring later in life is well recognized, but the impact of maternal over-nutrition on the offspring's cardiovascular function and subsequent risk for CVD later in life remains unclear. Here, we investigated the impact of maternal exposure to a high-fat/calorie diet (HFD) during pregnancy and early postnatal period on endothelial function of the offspring in a nonhuman primate model. METHODS: Offspring, naturally born to either a control (CTR) diet (14% fat calories) or a HFD (36% fat calories) consumption dam, were breast-fed until weaning at about 8 months of age. After weaning, the offspring were either maintained on the same diet (CTR/CTR, HFD/HFD), or underwent a diet switch (CTR/HFD, HFD/CTR). Blood samples and arterial tissues were collected at necropsy when the animals were about 13 months of age. RESULTS: HFD/HFD juveniles displayed an increased plasma insulin level and glucose-stimulated insulin secretion in comparison with CTR/CTR. In abdominal aorta, but not the renal artery, acetylcholine-induced vasorelaxation was decreased remarkably for HFD/HFD juveniles compared with CTR/CTR. HFD/HFD animals also showed a thicker intima wall and an abnormal vascular-morphology, concurrent with elevated expression levels of several markers related to vascular inflammation and fibrinolytic function. Diet-switching animals (HFD/CTR and CTR/HFD) displayed modest damage on the abdominal vessel. CONCLUSION: Our data indicate that maternal HFD exposure impairs offspring's endothelial function. Both early programming events and postweaning diet contribute to the abnormalities that could be reversed partially by diet intervention.

Restriction of placental vasculature in a non-human primate: a unique model to study placental plasticity.

The limits of placental plasticity, i.e., the ability of the placenta to adapt and alter its growth trajectory in response to altered fetal requirements, are not known. We report fetal and placental hemodynamic adaptations in a novel non-human primate model in which the fetal inter-placental bridging vessels were surgically ligated. Doppler ultrasound studies showed that the rhesus placenta compensates for an approximate 40% reduction in functional capacity by increased growth and maintenance of umbilical volume blood flow. This unique experimental animal model has applications for mechanistic studies of placental plasticity and the impact on fetal development.

Mother's lifetime nutrition and the size, shape and efficiency of the placenta.

BACKGROUND: Studies have shown that the shape and size of the placenta at birth predict blood pressure in later life. The influences that determine placental morphology are largely unknown. We have examined the role of mother's body size. METHODS: We studied 522 neonates who were born in a maternity hospital in Mysore, South India. The weight of the placenta and the length and breadth of its surface, were measured after delivery. RESULTS: Higher maternal fat mass predicted a larger placental surface (p = 0.02), while larger maternal head circumference predicted a more oval placental surface (p = 0.03). Higher maternal fat mass and larger maternal head circumference were associated with greater placental efficiency, indicated by lower ratios of the length (p = 0.0003 and p = 0.0001 respectively) and breadth (p = 0.0002 and p < 0.0001) of the surface to birthweight. In a sub-sample of 51 mothers whose own birthweight was available, higher maternal birthweight was related to lower ratios of the length and breadth of the surface to birthweight (p = 0.01 and 0.002). Maternal height was unrelated to placental size or shape. CONCLUSIONS: Higher maternal fat mass, reflecting the mother's current nutritional state, and larger maternal head circumference, reflecting the mother's fetal/infant growth, are associated with changes in the shape and size of the placental surface and greater placental efficiency. We suggest that these associations reflect effects of the mother's nutrition at different stages of her lifecourse on the development of the placenta and on materno-placento-fetal transfer of nutrients.

The lifespan of men and the shape of their placental surface at birth.

BACKGROUND: Tall men generally lead longer lives than short men. Within the Helsinki Birth Cohort, however, there is a group of boys among whom being tall when they entered school was associated with reduced lifespan. These boys had birthweights and maternal heights above the median for the cohort; but they tended to be lighter at birth than their mother's body mass index (weight/height(2)) in pregnancy predicted. We suggested that, while they had grown rapidly in utero, their growth had faltered at some point; and their tallness at age seven was the result of a resumption during infancy of their rapid growth trajectory. We here examine the size and shape of their placentas at birth to gain further insight into their path of fetal growth. METHODS: We examined all cause mortality in the 1217 men who had birthweights and maternal heights above the median for the cohort. Their birth measurements included placental weight and the length and breadth of the placental surface. RESULTS: Shorter length of the placental surface was associated with increased mortality (p = 0.002). There was no similar trend with the breadth. Mortality rose as the difference between the length and breadth decreased, that is as the surface became rounder. The hazard ratio was 1.10 (1.03-1.18, p = 0.007) for every cm decrease in the difference. Among men with a round placental surface (length-breadth difference 2 cm or less) increased mortality was associated with lower birthweight (p = 0.03 or 0.005 allowing for mother's body mass index) and shorter gestation, but not with lower head circumference or length. CONCLUSION: Reduced lifespan among men is associated with a particular path of early growth. After rapid growth in early gestation, associated with tall maternal stature, soft tissue growth falters in mid-gestation. Rapid growth resumes in late gestation and continues through infancy.

Insignificant response of the fetal placental circulation to arterial hypotension in sheep.

Infusion of the angiotensin-converting enzyme inhibitor enalaprilat into fetal sheep caused a profound arterial hypotension within days. Five fetal lambs were infused with enalaprilat for 8 days starting at day 128 of gestation. Total accumulated dose was 0.30 ± 0.11 mg/kg. Arterial pressure decreased from 43.6 to 25.6 mmHg; venous pressure did not change. Biventricular output was not statistically significantly changed; placental blood flow decreased almost in proportion to the decrease in pressure but the increase in somatic flow was not statistically significant. There were no significant changes in pressure 30 min after the initial 50-µg loading dose of enalaprilat. However, the arterial pressure responses to test doses of ANG I were largely abolished. After 1 day, however, there was a significant decrease in somatic vascular resistance, which became stronger with time, but almost no decrease in the placental resistance. We conclude that the fetal somatic circulation exhibits a slow but strong decrease in resistance but that the response to hypotension is weak or absent in the fetal placenta, possibly because it is already fully relaxed.

Atrial natriuretic peptide inhibits angiotensin II-stimulated proliferation in fetal cardiomyocytes.

The role of atrial natriuretic peptide (ANP) in regulating fetal cardiac growth is poorly understood. Angiotensin II (Ang II) stimulates proliferation in fetal sheep cardiomyocytes when growth is dependent on the activity of the mitogen-activated protein kinase (MAPK) and phosphoinositol-3-kinase (PI3K) pathways. We hypothesized that ANP would suppress near-term fetal cardiomyocyte proliferation in vitro and inhibit both the MAPK and PI3K pathways. Forty-eight hour 5-bromodeoxyuridine (BrdU) uptake (used as an index of proliferation) was measured in cardiomyocytes isolated from fetal sheep (135 day gestational age) in response to 100 nm Ang II with or without ANP (0.003-100 nm) or 1 microm 8-bromo-cGMP. The effects of these compounds on the MAPK and PI3K pathways were assessed by measuring extracellular signal-regulated kinase (ERK) and AKT phosphorylation following 10 min of treatment with Ang II, ANP or 8-bromo-cGMP. In right ventricular myocytes (RV), the lowest dose of ANP (0.003 nm) inhibited Ang II-stimulated BrdU uptake by 68%. Similarly, 8-bromo-cGMP suppressed Ang II-stimulated proliferation by 62%. The same effects were observed in left ventricular (LV) cardiomyocytes but the RV was more sensitive to the inhibitory effects of ANP than the LV (P < 0.0001). Intracellular cGMP was increased by 4-fold in the presence of 100 nm ANP. Ang II-stimulated ERK and Akt phosphorylation was inhibited by 100 nm ANP. The activity of ANP may in part be cGMP dependent, as 8-bromo-cGMP had similar effects on the cardiomyocytes.

Reduced systolic pressure load decreases cell-cycle activity in the fetal sheep heart.

The fetal heart is highly sensitive to changes in mechanical load. We have previously demonstrated that increased cardiac load can stimulate cell cycle activity and maturation of immature cardiomyocytes, but the effects of reduced load are not known. Sixteen fetal sheep were given either continuous intravenous infusion of lactated Ringer solution (LR) or enalaprilat, an angiotensin-converting enzyme inhibitor beginning at 127 days gestational age. After 8 days, fetal arterial pressure in the enalaprilat-infused fetuses (23.8 +/- 2.8 mmHg) was lower than that of control fetuses (47.5 +/- 4.7 mmHg) (P < 0.0001). Although the body weights of the two groups of fetuses were similar, the heart weight-to-body weight ratios of the enalaprilat-infused fetuses were less than those of the LR-infused fetuses (5.6 +/- 0.5 g/kg vs. 7.0 +/- 0.6 g/kg, P < 0.0001). Dimensions of ventricular myocytes were not different between control and enalaprilat-infused fetuses. However, there was a significant decrease in cell cycle activity in both the right ventricle (P < 0.005) and the left ventricle (P < 0.002) of the enalaprilat-infused fetuses. Thus, we conclude a sustained reduction in systolic pressure load decreases hyperplastic growth in the fetal heart.

Review: The placenta is a programming agent for cardiovascular disease.

Cardiovascular disease remains the number one killer in western nations in spite of declines in death rates following improvements in clinical care. It has been 20 years since David Barker and colleagues showed that slow rates of prenatal growth predict mortality from ischemic heart disease. Thus, fetal undergrowth and its associated cardiovascular diseases must be due, in part, to placental inadequacies. This conclusion is supported by a number of studies linking placental characteristics with various adult diseases. A "U" shaped relationship between placental-to-fetal weight ratio and heart disease provides powerful evidence that placental growth-regulating processes initiate vulnerabilities for later heart disease in offspring. Recent evidence from Finland indicates that placental morphological characteristics predict risks for coronary artery disease, heart failure, hypertension and several cancers. The level of risk imparted by placental shape is sex dependent. Further, maternal diet and body composition strongly influence placental growth, levels of inflammation, nutrient transport capacity and oxidative stress, with subsequent effects on offspring health. Several animal models have demonstrated the placental roots of vulnerability for heart disease. These include findings that abnormal endothelial development in the placenta is associated with undergrown myocardial walls in the embryo, and that placental insufficiency leads to depressed maturation and proliferation of working cardiomyocytes in the fetal heart. Together these models suggest that the ultimate fitness of the heart is determined by hemodynamic, growth factor, and oxygen/nutrient cues before birth, all of which are influenced, if not regulated by the placenta.

Beyond birthweight: the maternal and placental origins of chronic disease.

New findings on the maternal and placental programming of chronic disease lead to four conclusions: (1) Growth of the placental surface is polarized from the time of implantation, so that growth along the major axis, the length, is qualitatively different from growth along the minor axis, the breadth. (2) The human fetus may attempt to compensate for undernutrition by expansion of the placental surface along its minor axis. This only occurs if the mother was well nourished before conception, and may have long-term costs that include hypertension. (3) The effects of placental size on long-term health are conditioned by the mother's nutritional state, as indicated by her socio-economic status, height and body mass index. (4) The maternal-placental programming of chronic disease differs in boys and girls. Boys invest less than girls in placental growth but more readily expand the placental surface if they become undernourished in mid-late gestation. Boys are more responsive to their mothers' current diets while girls respond more to their mothers' lifetime nutrition and metabolism.

Restriction of placental function alters heart development in the sheep fetus.

Placental insufficiency, resulting in restriction of fetal substrate supply, is a major cause of intrauterine growth restriction (IUGR) and increased neonatal morbidity. Fetal adaptations to placental restriction maintain the growth of key organs, including the heart, but the impact of these adaptations on individual cardiomyocytes is unknown. Placental and hence fetal growth restriction was induced in fetal sheep by removing the majority of caruncles in the ewe before mating (placental restriction, PR). Vascular surgery was performed on 13 control and 11 PR fetuses at 110-125 days of gestation (term: 150 +/- 3 days). PR fetuses with a mean gestational Po(2) < 17 mmHg were defined as hypoxic. At postmortem (<135 or >135 days), fetal hearts were collected, and cardiomyocytes were isolated and fixed. Proliferating cardiomyocytes were counted by immunohistochemistry of Ki67 protein. Cardiomyocytes were stained with methylene blue to visualize the nuclei, and the proportion of mononucleated cells and length and width of cardiomyocytes were measured. PR resulted in chronic fetal hypoxia, IUGR, and elevated plasma cortisol concentrations. Although there was no difference in relative heart weights between control and PR fetuses, there was an increase in the proportion of mononucleated cardiomyocytes in PR fetuses. Whereas mononucleated and binucleated cardiomyocytes were smaller, the relative size of cardiomyocytes when expressed relative to heart weight was larger in PR compared with control fetuses. The increase in the relative proportion of mononucleated cardiomyocytes and the relative sparing of the growth of individual cardiomyocytes in the growth-restricted fetus are adaptations that may have long-term consequences for heart development in postnatal life.