Mark Oliver

FETAL GROWTH AND PLACENTAL FUNCTION

Fetal growth is largely determined by the availability of nutrients to the fetus. The fetus is at the end of a supply line that ensures delivery of nutrients from the maternal/uterine circulation to the fetus via the placenta. However, this supply line can not be regarded as a linear relationship. Maternal undernutrition will not only reduce global nutrient availability but will also influence the maternal and fetal somatotrophic axis. Both endocrine systems react in a very similar way to limited substrate supply. The hormones of the fetal somatotrophic axis, and in particular insulin-like growth factor (IGF)-1, are important regulators of fetal growth. Placental function is pivotal to materno-fetal nutrient and metabolite transfer. Placental function in turn, is heavily influenced by the maternal and fetal growth hormone (GH)-IGF-1 system. The placenta itself is also an active endocrine organ and it produces a large number of hormones including GH and IGF-1 as well their corresponding receptors. Thus the placenta can no longer be considered merely a passive conduit for fetal nutrition. Rather, it is actively involved in the integration of nutritional and endocrine signals from the maternal and fetal somatotrophic axes.

Glucose but not a mixed amino acid infusion regulates plasma insulin-like growth factor-I concentrations in fetal sheep

ABSTRACT: The influence of fetal glucose and amino acid supply on the regulation of fetal plasma IGF-I levels was investigated in fetuses from starved ewes. Paired maternal and fetal blood samples were taken during an initial 2-d control period, after 48 h of maternal starvation, during a 24-h fetal infusion of glucose (n = 6) or an amino acid mixture (Synthamin 17, n = 5) with continued starvation, and after 48 h of maternal refeeding. After 48 h of starvation, maternal and fetal plasma IGF-I, insulin, and blood glucose fell significantly in both groups compared with control values (IGF-I for glucose group: maternal, −18.53 ± 6.60; fetal, −5.23 ± 1.81 nmol/L; amino acid group: maternal, −18.2 ± 6.97, fetal, −5.12 ± 1.61 nmol/L; both p < 0.05). Fetal glucose but not mixed amino acid infusion raised fetal plasma IGF-I, insulin, and blood glucose to near control values (glucose group fetal IGF-I, −1.77 ± 1.98; amino acid group, −5.93 ± 2.22 nmol/L; both p < 0.05). Maternal plasma IGF-I remained depressed during glucose infusion (−16.33 ± 8.32 nmol/L), but continued to fall in the amino acid group (−21.41 ± 8.20 nmol/L, p < 0.05). After 48 h of maternal refeeding, all values had returned to near control values for both groups (glucose group IGF-I: maternal, −5.2 ± 3.86; fetal, 0.01 ± 2.2 nmol/L; amino acid group: maternal, −11.66 ± 3.2; fetal, −0.70 ± 2.61 nmol/L). We conclude that in the ovine fetus glucose may have a more important role than amino acids in the regulation of fetal plasma IGF-I.

Epigenetic Changes in the Hypothalamic Proopiomelanocortin and Glucocorticoid Receptor Genes in the Ovine Fetus after Periconceptional Undernutrition

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.

Periconceptional Undernutrition of Ewes Impairs Glucose Tolerance in Their Adult Offspring

Maternal undernutrition throughout pregnancy can have long-term effects on the health of adult offspring. Undernutrition around the time of conception alters growth, metabolism, and endocrinology of the sheep fetus, but the impact on offspring after birth is largely unknown. We determined the effect of maternal periconceptional undernutrition in sheep on glucose tolerance in the offspring before and after puberty. Undernourished (UN) ewes were fed individually to maintain weight loss of 10–15% bodyweight from 61 d before until 30 d after mating. Offspring (24 UN, 30 control) underwent an i.v. glucose tolerance test at 4 and 10 mo of age. Glucose tolerance was similar in both groups at 4 mo. Insulin area under the curve increased by 33% between 4 and 10 mo (101 ± 8 versus 154 ± 12 ng · min · mL−1, p < 0.0001). At 10 mo, UN offspring had a 10% greater glucose area under the curve than controls (809 ± 22 versus 712 ± 20 mM · min, p < 0.01), a reduced first phase insulin response (p = 0.003) which was particularly apparent in females and in singletons, and a decreased insulin:glucose ratio (p = 0.01). We conclude that maternal undernutrition around the time of conception results in impaired glucose tolerance in postpubertal offspring.

Periconceptional Undernutrition Alters Growth Trajectory and Metabolic and Endocrine Responses to Fasting in Late-Gestation Fetal Sheep

Maternal undernutrition during pregnancy can lead to impaired metabolic regulation in postnatal offspring. The extent to which such abnormalities are determined in early gestation, and may already be present in fetal life, is unknown. We studied the effect of periconceptional undernutrition on fetal growth and metabolism in late gestation. Ewes were either well fed throughout pregnancy (N) or undernourished to reduce ewe weight by 15% from 61 d before until 30 d after mating (UN). At 121 d, one cohort of ewes and their singleton fetuses (N, n = 10; UN, n = 10) were fasted for 72 h, given 25 g of glucose i.v. over 8 h, and refed. Fetal and placental weights were not different at 131 d. UN ewes had higher glucose, amino nitrogen, and Hb concentrations than N ewes. UN fetuses had higher glucose and lactate, and lower amino nitrogen and O2 concentrations and pH. UN ewes had higher insulin and lower IGF-I concentrations, and UN ewes and fetuses had higher placental lactogen concentrations. A second cohort (N, n = 8; UN, n = 10) were studied until term. Chest girth increment was reduced in UN fetuses until delivery, but birth weights were not significantly reduced. These findings suggest that fetal growth, metabolic and endocrine status, and placental function in late gestation are influenced by maternal nutrition in the periconceptional period, independent of fetal size. Metabolic and endocrine adaptations in the mother may mediate some of these effects. Such adaptations may allow continued fetal growth and survival in the face of a potentially adverse nutritional environment.

Fetal exposure to excess glucocorticoid is unlikely to explain the effects of periconceptional undernutrition in sheep

Periconceptional undernutrition alters fetal growth, metabolism and endocrinology in late gestation. The underlying mechanisms remain uncertain, but fetal exposure to excess maternal glucocorticoids has been hypothesized. We investigated the effects of periconceptional undernutrition on maternal hypothalamic–pituitary–adrenal axis function and placental 11β‐hydroxysteroid dehydrogenase type 2 (11βHSD2) activity. Ewes received maintenance feed (N, n= 20) or decreased feed from −60 to +30 days from mating to achieve 15% weight loss after an initial 2‐day fast (UN, n= 21). Baseline plasma samples and arginine vasopressin (AVP)–corticotrophin‐releasing hormone (CRH) challenges were performed on days −61, −57, −29, −1, +29, 33, and 49 from mating (day 0). Maternal adrenal and placental tissue was collected at 50 days. Baseline plasma levels of adrenocorticotrophic hormone (ACTH) and cortisol decreased in the UN group (P < 0.0001). ACTH response to AVP–CRH was greater in UN ewes during undernutrition (P= 0.03) returning to normal levels after refeeding. Cortisol response to AVP–CRH was greater in UN ewes after the initial 2‐day fast, but thereafter decreased and was lower in UN ewes from mating until the end of the experiment (P= 0.007). ACTH receptor, StAR and p450c17 mRNA levels were down‐regulated in adrenal tissue from UN ewes. Placental 11βHSD2 activity was lower in UN than N ewes at 50 days (P= 0.014). Moderate periconceptional undernutrition results in decreased maternal plasma cortisol concentrations during undernutrition and after refeeding, and adrenal resistance to ACTH for at least 20 days after refeeding. Fetal exposure to excess maternal cortisol is unlikely during the period of undernutrition, but could occur later in gestation if maternal plasma cortisol levels return to normal while placental 11βHSD2 activity remains low.

Birth Weight Rather Than Maternal Nutrition Influences Glucose Tolerance, Blood Pressure, and IGF-I Levels in Sheep

Both small size at birth in humans and maternal undernutrition in experimental animals are associated with impaired glucose tolerance and increased blood pressure in the adult offspring. However it is not clear whether maternal undernutrition in late gestation can influence postnatal phenotype, and whether this might occur independent of size at birth. We severely undernourished pregnant ewes for 10 d (UN10) or 20 d (UN20) from 105 d gestation (term = 146 d) and studied the offspring in comparison to those born to ad libitum fed ewes (AL20). Glucose tolerance tests and blood pressure recordings were performed at 5 mo of age and repeated at 30 mo together with insulin tolerance tests and GH challenges. UN20 lambs were lighter at birth (UN20, 4.9 ± 0.1 kg, n = 23; UN10, 5.3 ± 0.1 kg, n = 26; AL20, 5.6 ± 0.2 kg, n = 26) but not from weaning onward. Plasma glucose area under the curve at 5 mo and plasma insulin at 30 mo increased with current weight and decreased with increasing birth weight. Blood pressure also increased with current weight and decreased with increasing birth weight at 5 but not 30 mo. Plasma IGF-I concentrations increased with current weight at all ages and decreased with increasing birth weight at 30 mo. Plasma insulin response to GH challenge increased with current weight. Nutrition group was not related to any of the outcomes measured when birth weight and current weight were taken into account. These data suggest that size at birth is more closely related to processes that determine postnatal phenotype than is maternal nutrition in late gestation.

Effects of twin pregnancy and periconceptional undernutrition on maternal metabolism, fetal growth and glucose–insulin axis function in ovine pregnancy

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.

Intrauterine feeding of the growth retarded fetus: can we help?

Intrauterine feeding of the growth retarded fetus appears an attractive therapeutic possibility. However the factors which determine the reversibility of intrauterine growth retardation are poorly understood. While fetal substrate supply is the final common pathway by which many factors restrict fetal growth, improving fetal substrate supply does not always lead to improved fetal growth. Similarly, fetal substrate supply is an important regulator of fetal endocrine status, such as circulating IGF-1 levels, but again, improving fetal substrate supply does not always alter fetal endocrine status or fetal growth. The relationship between substrate supply, endocrine status and growth is regulated in a complex way by placental function. Understanding the role of the placenta in this regulation is essential if in the future we are to help the growth retarded fetus.

Effects of sex, litter size and periconceptional ewe nutrition on offspring behavioural and physiological response to isolation

Maternal periconceptional undernutrition alters fetal hypothalamic-pituitary-adrenal (HPA) axis development. However, the effects of this early nutritional insult on postnatal HPA axis function and stress-related behaviours are unknown. We investigated in sheep the effects of different periods of undernutrition, and of sex and litter size, on offspring behavioural and cortisol responses to isolation stress. We studied four nutritional groups: controls well nourished throughout pregnancy (n=39), or ewes undernourished (UN, 10-15% body weight reduction) before mating (-60 to 0d, n=26), after mating (-2 to +30d, n=20) or both (-60 to +30d, n=36). At 4 and 18months of age, offspring were isolated for 5min, their behaviour video recorded, and plasma cortisol concentrations measured. Offspring of all undernourished groups demonstrated 50% fewer escape attempts than controls at 4 months of age, and offspring of UN-60+30 ewes had 20% lower plasma cortisol area under the curve in response to isolation at 18months. Females had higher cortisol concentrations and vocalised more than males at 4 and 18months, and were more active at 18months. After isolation, UN-2+30 males had higher cortisol concentrations than UN-2+30 females whereas in all other groups males had lower concentrations than females. Singleton males made more escape attempts than females, whereas in twins females made more escape attempts than males. These findings suggest that maternal periconceptional undernutrition in sheep can suppress behavioural reactions and cortisol secretion in response to isolation stress in the offspring into adulthood, and that these effects differ between males and females.