Postnatal effects of intrauterine treatment of the growth-restricted ovine fetus with intra-amniotic insulin-like growth factor-1.

KEY POINTS: Fetal growth restriction increases the risk of fetal and neonatal mortality and morbidity, and contributes to increased risk of chronic disease later in life. Intra-amniotic insulin-like growth factor-1 (IGF1) treatment of the growth-restricted ovine fetus improves fetal growth, but postnatal effects are unknown. Here we report that intra-amniotic IGF1 treatment of the growth-restricted ovine fetus alters size at birth and mechanisms of early postnatal growth in a sex-specific manner. We also show that maternal plasma C-type natriuretic peptide (CNP) products are related to fetal oxygenation and size at birth, and hence may be useful for non-invasive monitoring of fetal growth restriction. Intrauterine IGF1 treatment in late gestation is a potentially clinically relevant intervention that may ameliorate the postnatal complications of fetal growth restriction. ABSTRACT: Placental insufficiency-mediated fetal growth restriction (FGR) is associated with altered postnatal growth and metabolism, which are, in turn, associated with increased risk of adult disease. Intra-amniotic insulin-like growth factor-1 (IGF1) treatment of ovine FGR increases growth rate in late gestation, but the effects on postnatal growth and metabolism are unknown. We investigated the effects of intra-amniotic IGF1 administration to ovine fetuses with uteroplacental embolisation-induced FGR on phenotypical and physiological characteristics in the 2  weeks after birth. We measured early postnatal growth velocity, amino-terminal propeptide of C-type natriuretic peptide (NTproCNP), body composition, tissue-specific mRNA expression, and milk intake in singleton lambs treated weekly with 360 µg intra-amniotic IGF1 (FGRI; n = 13 females, 19 males) or saline (FGRS; n = 18 females, 12 males) during gestation, and in controls (CON; n = 15 females, 22 males). There was a strong positive correlation between maternal NTproCNP and fetal oxygenation, and size at birth in FGR lambs. FGR lambs were ∼20% lighter at birth and demonstrated accelerated postnatal growth velocity. IGF1 treatment did not alter perinatal mortality, partially abrogated the reduction in newborn size in females, but not males, and reduced accelerated growth in both sexes. IGF1-mediated upregulation of somatotrophic genes in males during the early postnatal period could suggest that treatment effects are associated with delayed axis maturation, whilst treatment outcomes in females may rely on the reprogramming of nutrient-dependent mechanisms of growth. These data suggest that the growth-restricted fetus is responsive to intra-amniotic intervention with IGF1, and that sex-specific somatotrophic effects persist in the early postnatal period.

Intrauterine Intervention for the Treatment of Fetal Growth Restriction.

Fetal growth restriction (FGR) is associated with an increased incidence of fetal and neonatal death, and of neonatal morbidity. Babies born following FGR also are at risk of a range of postnatal complications, which may contribute to an increased incidence of disease later in life. There currently are no effective clinical interventions which improve perinatal survival, intrauterine growth and later outcomes of the FGR baby. Postnatal interventions aimed at promoting or accelerating growth in FGR babies to improve outcome, particularly neurodevelopmental outcomes, may further increase the risk of metabolic dysregulation and, therefore, the risk of developing chronic disease in adulthood. An intrauterine intervention to improve nutrition and growth in the FGR fetus may have the potential to decrease mortality and improve long-term outcomes by delaying preterm delivery and mitigating the need for and risks of accelerated postnatal growth.

Antenatal glucocorticoids: where are we after forty years?

Since their introduction more than forty years ago, antenatal glucocorticoids have become a cornerstone in the management of preterm birth and have been responsible for substantial reductions in neonatal mortality and morbidity. Clinical trials conducted over the past decade have shown that these benefits may be increased further through administration of repeat doses of antenatal glucocorticoids in women at ongoing risk of preterm and in those undergoing elective cesarean at term. At the same time, a growing body of experimental animal evidence and observational data in humans has linked fetal overexposure to maternal glucocorticoids with increased risk of cardiovascular, metabolic and other disorders in later life. Despite these concerns, and somewhat surprisingly, there has been little evidence to date from randomized trials of longer-term harm from clinical doses of synthetic glucocorticoids. However, with wider clinical application of antenatal glucocorticoid therapy there has been greater need to consider the potential for later adverse effects. This paper reviews current evidence for the short- and long-term health effects of antenatal glucocorticoids and discusses the apparent discrepancy between data from randomized clinical trials and other studies.

Mothers of babies enrolled in a randomized trial immediately after birth report a positive experience.

OBJECTIVE: Randomized trials are essential for improving outcomes, but researchers can be hesitant about undertaking clinical trials in newborn babies because of perceived vulnerability of the baby and risk of increasing parental anxiety. There is a paucity of evidence about the parental experience. We investigated mothers' experiences of having their newborn baby participate in a randomized double-blind placebo-controlled trial soon after birth. STUDY DESIGN: Eligible mothers had consented to their baby's participation in the Sugar Babies Study. Mothers of potentially eligible babies were invited to join the study antenatally, but others were approached postnatally. Babies were enrolled in the study soon after birth and remained in the study for 48 h. After 2 weeks the birth mothers were interviewed by phone about their experience. RESULT: Four hundred and eighty-one mothers were enrolled, of whom 310 (64%) gave consent antenatally. All mothers were contacted and 477 (99%) were interviewed. The majority of mothers (458, 96%) reported they would consent to participating again, if they had another eligible baby, and 460 mothers (96%) reported they would recommend participation to family and friends. Nineteen mothers (4%) reported they did not like the heel lance blood tests, which were part of routine clinical care and not part of the trial protocol. CONCLUSION: Most mothers reported the experience of having their newborn baby participate in a clinical trial as positive. Most negative responses were related to aspects of routine care rather than the trial protocol.

Fetal growth factors and fetal nutrition.

Optimal fetal growth is important for a healthy pregnancy outcome and also for lifelong health. Fetal growth is largely regulated by fetal nutrition, and mediated via the maternal and fetal glucose/insulin/insulin-like growth factor axes. Fetal nutrition may reflect maternal nutrition, but abnormalities of placental function can also affect fetal growth, as the placenta plays a key intermediary role in nutritional signalling between mother and fetus. Fetal nutrition also impacts on the development of key fetal endocrine systems such as the glucose-insulin and insulin-like growth factor axes. This is likely to contribute to the link between both fetal growth restriction and fetal overgrowth, and increased risks of obesity and impaired glucose tolerance in later life. This review focuses on the associations between maternal and fetal nutrition, fetal growth and later disease risk, with particular emphasis on the role of insulin-like growth factors and the importance of the periconceptional period.

Optimum feeding and growth in preterm neonates.

Approximately 10% of all babies worldwide are born preterm, and preterm birth is the leading cause of perinatal mortality in developed countries. Although preterm birth is associated with adverse short- and long-term health outcomes, it is not yet clear whether this relationship is causal. Rather, there is evidence that reduced foetal growth, preterm birth and the long-term health effects of both of these may all arise from a suboptimal intrauterine environment. Further, most infants born preterm also experience suboptimal postnatal growth, with potential adverse effects on long-term health and development. A number of interventions are used widely in the neonatal period to optimise postnatal growth and development. These commonly include supplementation with macronutrients and/or micronutrients, all of which have potential short-term risks and benefits for the preterm infant, whereas the long-term health consequences are largely unknown. Importantly, more rapid postnatal growth trajectory (and the interventions required to achieve this) may result in improved neurological outcomes at the expense of increased cardiovascular risk in later life.

Periconceptional undernutrition in sheep leads to decreased locomotor activity in a natural environment.

Maternal undernutrition during pregnancy increases offspring obesity and metabolic disease risk. We hypothesized that periconceptional undernutrition in sheep from 60 days before conception through to day 30 of gestation (UN) would decrease voluntary locomotor activity in adult offspring. Distance travelled was measured at 18 months of age for ∼48 h in the paddock. Data were analysed using multiple regression analysis, with explanatory variables including sex, nutrition group, birth weight, average time between GPS measurements and percentage of time during the measurement period spent in daylight. Mean (±s.e.) distance walked (m/h) was greater for control (CON) than UN animals, and greater for females than males [110.2 (6.5), CON females; 110.7 (6.3), CON males; 105.1 (5.3), UN females and 95.5 (5.8), UN males; P = 0.02 for nutrition group effect and for sex effect]. Periconceptional undernutrition may lead to a significant decrease in voluntary physical activity in adult offspring.

Periconceptional undernutrition suppresses cortisol response to arginine vasopressin and corticotropin-releasing hormone challenge in adult sheep offspring.

Poor maternal nutrition during pregnancy can result in increased disease risk in adult offspring. Many of these effects are proposed to be mediated via altered hypothalamo-pituitary-adrenal axis (HPAA) function, and are sex and age specific. Maternal undernutrition around the time of conception alters HPAA function in foetal and early postnatal life, but there are limited conflicting data about later effects. The aim of this study was to investigate the effect of moderate periconceptional undernutrition on HPAA function of offspring of both sexes longitudinally, from juvenile to adult life. Ewes were undernourished from 61 days before until 30 days after conception or fed ad libitum. HPAA function in offspring was assessed by arginine vasopressin plus corticotropin-releasing hormone challenge at 4, 10 and 18 months. Plasma cortisol response was lower in males than in females, and was not different between singles and twins. Periconceptional undernutrition suppressed offspring plasma cortisol but not adrenocorticotropic hormone responses. In males, this suppression was apparent by 4 months, and was more profound by 10 months, with no further change by 18 months. In females, suppression was first observed at 10 months and became more profound by 18 months. Maternal undernutrition limited to the periconceptional period has a prolonged, sex-dependent effect on adrenal function in the offspring.

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.

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.

Effects of twinning and periconceptional undernutrition on late-gestation hypothalamic-pituitary-adrenal axis function in ovine pregnancy.

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.

Cardiovascular adaptations to pregnancy in sheep and effects of periconceptional undernutrition.

The objective of this study was to describe the effects of pregnancy on blood volume and uterine blood flow in sheep, and to test the hypothesis that the effects of periconceptional undernutrition on the late-gestation fetus are mediated by alterations in these parameters. Singleton-bearing ewes that had been undernourished preconception, postconception, both, or neither, underwent estimation of blood volume in mid and late gestation, and measurement of uterine blood flow in late gestation. Seven non-pregnant ewes were also studied. Pregnancy resulted in a 31% greater red cell volume in mid-gestation (21.0+/-1.3 vs 16.1+/-0.8ml/kg, p<0.05), but no significant change in plasma or blood volume. However maternal blood volume was correlated with uterine blood flow (r(2)=0.22, p=0.05) and fetal size (r(2)=0.20, p=0.02). Uterine blood flow was 13% greater in the undernourished groups than controls (1847+/-100 vs 1641+/-79ml/min, p<0.01). The large increase in maternal blood volume integral to a successful human pregnancy was not present in sheep. The increased uterine blood flow after periconceptional undernutrition suggests that nutritional signals before and in early pregnancy influence fetal nutrient supply in late gestation.

A method for assessment of blood volume parameters in pregnant sheep using fluorescein-labelled dextran.

The assessment of blood volume parameters in clinical and research settings has been limited by methods that involve radioactivity, complex assays or are unreliable. We aimed to design a method for measuring blood volume parameters that was non-radioactive, simple, cheap and reliable. We have used a commercially available fluorescein-labelled 250kDa dextran, a large inert molecule, and have measured dilution of this through the intravascular space of pregnant ewes. From this estimation of plasma volume and measured hematocrit, we have calculated blood volume and red cell volume. The blood volume results are 6% lower than those obtained using radiolabelled red cells, but there is no significant difference in red cell volume between methods. The coefficient of variation for repeated measurements of plasma volume measurements is 3.8%. This is a simple, reliable, cheap and non-radioactive method for estimating blood volume parameters in pregnant sheep, and may prove useful in other settings.

Repeat doses of prenatal corticosteroids for women at risk of preterm birth for preventing neonatal respiratory disease.

BACKGROUND: It is not clear whether there is benefit in repeating the dose of prenatal corticosteroids for women who remain at risk of preterm birth after an initial course. OBJECTIVES: To assess the effectiveness and safety of a repeat dose(s) of prenatal corticosteroids. SEARCH STRATEGY: We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (February 2007), the Cochrane Central Register of Controlled Trials (The Cochrane Library 2006, Issue 4), MEDLINE (1965 to November 2006), EMBASE (1988 to November 2006) and Current Contents (1997 to November 2006). SELECTION CRITERIA: Randomised controlled trials of women who have already received a single course of corticosteroids seven or more days previously and are still considered to be at risk of preterm birth; outcomes compared for women randomised to receive a repeat dose(s) of prenatal corticosteroids, with women given no further prenatal corticosteroids. DATA COLLECTION AND ANALYSIS: We assessed trial quality and extracted the data independently. MAIN RESULTS: Five trials, involving over 2000 women between 23 and 33 weeks' gestation, are included. Treatment with repeat dose(s) of corticosteroid was associated with a reduction in occurrence (relative risk (RR) 0.82, 95% confidence interval (CI) 0.72 to 0.93, four trials, 2155 infants) and severity of any neonatal lung disease (RR 0.60, 95% CI 0.48 to 0.75, three trials, 2139 infants) and serious infant morbidity (RR 0.79, 95% CI 0.67 to 0.93, four trials, 2157 infants).Mean birthweight was not significantly different between treatment groups (weighted mean difference (WMD) -62.07 g, 95% CI -129.10 to 4.96, four trials, 2273 infants), although in one trial, treatment with repeat dose(s) of corticosteroid was associated with a reduction in birthweight Z score (WMD) -0.13, 95% CI -26 to 0.00, 1 trial, 1144 infants), and in two trials, with an increased risk of being small for gestational age at birth (RR 1.63, 95% CI 1.12 to 2.37, two trials, 602 infants). No statistically significant differences were seen for any of the other primary outcomes that included other measures of respiratory morbidity, fetal and neonatal mortality, periventricular haemorrhage, periventricular leukomalacia and maternal infectious morbidity. Treatment with repeat dose(s) of corticosteroid was associated with a significantly increased risk of caesarean section (RR 1.11, 95% CI 1.01 to 1.22, four trials, 1523 women). AUTHORS' CONCLUSIONS: Repeat dose(s) of prenatal corticosteroids reduce the occurrence and severity of neonatal lung disease and the risk of serious health problems in the first few weeks of life. These short-term benefits for babies support the use of repeat dose(s) of prenatal corticosteroids for women at risk of preterm birth. However, these benefits are associated with a reduction in some measures of weight, and head circumference at birth, and there is still insufficient evidence on the longer-term benefits and risks.

Protein metabolism in preterm infants with particular reference to intrauterine growth restriction.

There is growing evidence that neonatal and long-term morbidity in preterm infants, particularly those born before 32 weeks' gestation, can be modified by attained growth rate in the neonatal period. Guidelines for optimal growth and the nutritional intakes, particular of protein, required to achieve this are not well defined. Due to delays in postnatal feeding and a lack of energy stores developed in the last trimester of pregnancy, preterm infants often suffer early postnatal catabolism until feeding is established. There are indications that infants born with intrauterine growth restriction have perturbations in protein metabolism. Therefore, they may have different protein requirements than appropriate for gestational age infants. This review summarises what is known about protein requirements and metabolism in the fetus and preterm infant, with particular emphasis on the distinct requirements of the growth-restricted infant.

The effects of maternal nutrition around the time of conception on the health of the offspring.

The incidence of prematurity, diabetes and cardiovascular disease have been increasing in both the developed and developing world. Increasing numbers of human studies suggest that these serious health outcomes may have developmental origins originating from nutritional deficits in the periconceptional period, with maternal nutrition around the time of conception now shown to have important effects on the length of gestation, trajectory of fetal growth and on postnatal growth and health. Biomedical research using the pregnant sheep has been widely employed to gain a deeper understanding of the underlying mechanisms involved. There is growing awareness that this field of research has major implications for the livestock production industry. From our own studies on sheep we have evidence that maternal undernutrition during the periconceptional period results in altered fetal hypothalamic-pituitary-adrenal axis (HPAA) development, an increased rate of premature birth, altered fetal pancreatic function, insulin signalling and amino acid metabolism, and also alterations in maternal adaptation to pregnancy. We are currently studying the postnatal consequences of these changes. Other research groups have shown that restricted nutrition of sheep in the early part of pregnancy alters postnatal muscle development, fat deposition, cardiovascular regulation and HPAA function. One aim of this review is to illustrate how biomedical research using animals such as the sheep has been used to gain a better understanding of the consequences of reduced maternal nutrition during the periconceptional period. We suggest that there are equally important consequences of this research for the livestock production industries.

Urea production and arginine metabolism are reduced in the growth restricted ovine foetus.

Urea production may be impaired in intrauterine growth restriction (IUGR), increasing the risk of toxic hyperammonaemia after birth. Arginine supplementation stimulates urea production, but its effects in IUGR are unknown. We aimed to determine the effects of IUGR and arginine supplementation on urea production and arginine metabolism in the ovine foetus. Pregnant ewes and their foetuses were catheterised at 110 days of gestation and randomly assigned to control or IUGR groups. IUGR was induced by placental embolisation. At days 120 and 126 of gestation, foetal urea production was determined from [14C]-urea kinetics and arginine metabolism was determined from the appearance of radioactive metabolites from [3H]-arginine, both at baseline and in response to arginine or an isonitrogenous mixed amino acid supplementation. Urea production decreased with gestational age in the embolised animals (13.9 ±  3.1 to 11.2 ±  3.0 µmol/kg per min, P ≤ 0.05) but not in the controls (13.3 ±  3.5 to 14.8 ±  6.0 µmol/kg per min). Arginine supplementation increased urea production in both groups, but only at 126 days of gestation (control: 15.0 ±  8.5 to 17.0 ±  9.4 µmol/kg per min; embolised: 11.7 ±  3.1 to 14.3 ±  3.1 µmol/kg per min, P ≤ 0.05). Embolisation reduced foetal arginine concentrations by 20% ( P ≤ 0.05) while foetal arginine consumption was reduced by 27% ( P ≤ 0.05). The proportions of plasma citrulline and hydroxyproline derived from arginine were reduced in the embolised animals. These data suggest that foetal urea production and arginine metabolism are perturbed in late gestation after placental embolisation.