Maternal food restriction is associated with the development of obesity in offspring. This study examined how maternal undernutrition in sheep affects the fetal hypothalamic glucocorticoid receptor (GR) and the appetite-regulating neuropeptides, proopiomelanocortin (POMC) and neuropeptide Y, which it regulates. In fetuses from ewes undernourished from -60 to +30 d around conception, there was increased histone H3K9 acetylation (1.63-fold) and marked hypomethylation (62% decrease) of the POMC gene promoter but no change in POMC expression. In the same group, acetylation of histone H3K9 associated with the hypothalamic GR gene was increased 1.60-fold and the GR promoter region was hypomethylated (53% decrease). In addition, there was a 4.7-fold increase in hypothalamic GR expression but no change in methylation of GR gene expression in the anterior pituitary or hippocampus. Interestingly, hypomethylation of both POMC and GR promoter markers in fetal hypothalami was also identified after maternal undernutrition from -60 to 0 d and -2 to +30 d. In comparison, the Oct4 gene, was hypermethylated in both control and underfed groups. Periconceptional undernutrition is therefore associated with marked epigenetic changes in hypothalamic genes. Increase in GR expression in the undernourished group may contribute to fetal programming of a predisposition to obesity, via altered GR regulation of POMC and neuropeptide Y. These epigenetic changes in GR and POMC in the hypothalamus may also predispose the offspring to altered regulation of food intake, energy expenditure, and glucose homeostasis later in life.

Although twins have lower birthweights than singletons, they may not experience the increased disease risk in adulthood reportedly associated with low birthweight. In contrast, another periconceptional event, maternal undernutrition, does not reduce birthweight but does affect fetal and postnatal physiology in sheep. We therefore studied maternal and fetal metabolism, growth and glucose-insulin axis function in late gestation in twin and singleton sheep pregnancies, either undernourished from 60 days before until 30 days after conception or fed ad libitum. We found that twin-bearing ewes had decreased maternal food intake in late gestation and lower maternal and fetal plasma glucose and insulin levels. Twin fetuses had fewer everted placentomes, grew slower in late gestation, and had a greater insulin response to a glucose challenge, but lesser response to arginine. In contrast, periconceptional undernutrition led to increased maternal food intake and a more rapid fall in maternal glucose levels in response to fasting. Periconceptional undernutrition increased the number of everted placentomes, and abolished the difference in insulin responses to glucose between twins and singletons. Thus, the physiology of twin pregnancy is quite different from that of singleton pregnancy, and is probably determined by a combination of factors acting in both early and late gestation. The inconsistency of the relationships between low birthweight and postnatal disease risk of twins may lie in their very different fetal development. These data suggest that twin pregnancy may be another paradigm of developmental programming, and indicate that twins and singletons must be examined separately in any study of fetal or postnatal physiology.

The relationships between reduced size at birth, increased activity of the hypothalamic-pituitary-adrenal (HPA) axis, and increased risk of disease in adulthood are well described in singletons but are much less clear in twins. This may be because the physiological processes underlying reduced size at birth are different in singletons and twins. Periconceptional undernutrition can cause altered activity of the fetal and postnatal HPA axis without altering size at birth. However, the independent effects of periconceptional undernutrition and twinning on activity of the maternal and fetal HPA axes are not well described. We therefore studied maternal and fetal HPA axis function during late gestation in twin and singleton sheep pregnancies, either undernourished around conception or fed ad libitum. We found that twinning led to suppressed baseline HPA axis function and decreased adrenal sensitivity to ACTH stimulation but increased fetal pituitary ACTH response both to direct stimulation by CRH (ACTH area under the curve response: 29.7 +/- 2.2 vs. 17.1 +/- 1.6 ng/min x ml, P < 0.01) and to decreased cortisol negative feedback. In contrast, periconceptional undernutrition resulted in a decreased pituitary response (ACTH area under the curve response: 19.4 +/- 1.6 vs. 26.1 +/- 2.2 ng/min x ml, P = 0.02) but no difference in adrenal response. Thus, the HPA axis function of twin sheep fetuses in late gestation is very different from that of control and undernourished singletons. If the HPA axis is an important mediator between fetal adaptations and adult disease, these data may help explain why the relationship between fetal growth and postnatal physiology and disease risk is inconsistent in twins.

Periconceptional undernutrition alters fetal growth and development. However, there are no data on separate effects of undernutrition before and after conception and few on underlying mechanisms. We determined the effects of mild periconceptional undernutrition on late gestation fetal growth, glucose-insulin axis, and maternal and fetal hypothalamic-pituitary-adrenal axes. Ewes were undernourished for 60 d before conception, 30 d after, or both, compared with well-nourished controls. Undernutrition before conception resulted in smaller, slower-growing fetuses with relatively larger placentae. Ewes that gained weight before, but lost weight after mating, or vice versa, had the smallest fetuses. Fetuses of ewes undernourished only before conception grew more slowly following instrumentation, and fetuses in both preconception undernutrition groups slowed their growth with a maternal fast. The fetal glucoseinsulin axes and maternal and fetal hypothalamic-pituitary-adrenal axis were not different among groups. Maternal undernutrition at different periods around conception has different effects on fetal growth trajectory that are not reflected in size in late gestation. Preconceptional undernutrition alone alters fetal growth responses to late gestation stressors, suggesting that maternal nutrition is important at both times, and that fetal effects are neither due solely to substrate limitation, nor to excess fetal glucocorticoid exposure at the time of undernutrition. (Pediatr Res 66: 605-613, 2009)

Maternal undernutrition before conception impairs adaptation of insulin-related metabolism during pregnancy in ways that affect fetal growth. This suggests a key mechanism whereby prepregnancy nutritional status influences pregnancy outcome.