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.

Determining insulin sensitivity from glucose tolerance tests in sheep.

A mathematical model of the dynamics of insulin and glucose during a frequently sampled intravenous glucose tolerance test (IVGTT) in sheep was developed that characterizes the large second-phase insulin secretion response in sheep during IVGTT. The model was fit to measurements of the glucose and insulin dynamics during standard IVGTT ( = 42) and modified IVGTT ( = 40), where insulin was injected 60 min after the initiation of the IVGTT. The correlation between log insulin sensitivity determined by hyperglycemic clamps (HGC) and standard IVGTT was = 0.43 ( = 0.005). The correlation between log insulin sensitivity determined by HGC and modified IVGTT was = 0.51 ( = 0.002). The model, therefore, provides a method to determine insulin sensitivity through a cheaper and more easily performed IVGTT. We validated our estimation procedure using 2 independent experiments on the effect of 1) pregnancy and 2) being born preterm and exposed to dextrose or dextrose with insulin on HGC-derived insulin sensitivity. The IVGTT-derived insulin sensitivity was significantly greater in pregnant ewes than in prepregnant ewes (difference of 0.39 ± 0.12 log n ng mL; < 0.05), and this was consistent with the significantly greater hyperinsulinemic euglycemic clamp-derived insulin sensitivity in pregnant ewes than in prepregnant ewes (difference of 4.03 ± 0.66 µmol mL kg min ng; < 0.001). There was no significant effect of being born preterm on IVGTT/HGC-derived insulin sensitivity. Basal insulin, insulin sensitivity, insulin production, and insulin clearance were lower in prepregnant ewes ( < 0.05). That is, prepregnant ewes have a lower insulin equilibrium status and less responsive insulin turnover. There was also a significant effect of insulin therapy on the rate of insulin clearance in preterm lambs ( < 0.05). This effect was independently significant of its covariance with all other model parameters. Therefore, it can be interpreted as a direct effect on the rate of insulin clearance by the insulin treatment. All other parameter responses to the insulin treatment effect can be regarded as being due to the covariance between these parameters. These analyses demonstrate that treatment effects on insulin sensitivity can be detected using IVGTT experiments.

Maternal insulin sensitivity in midpregnancy does not determine birth weight after embryo transfer between large and small breed sheep.

Embryo transfer of large sheep breed embryos (Suffolk) into small breed ewes (Cheviot) constrains birth size, but the maternal factors influencing fetal growth restriction are unknown. We hypothesized that reciprocal embryo transfer crosses between breeds of divergent size would affect pregnancy-related development of maternal insulin resistance in midgestation, thereby influencing fetal growth. Following superovulation, embryos were surgically collected 6 d postmating and transferred to recipients on the same day. Between- and within-breed transfers were performed. Between 60 and 70 d of pregnancy overnight-fasted ewes underwent hyperinsulinemic-euglycemic clamps for assessment of insulin sensitivity. Maternal insulin sensitivity did not vary with transferred lamb breed. Overall, Cheviot ewes tended to have higher fasting glucose (P = 0.068), fasting insulin (P = 0.052), and steady-state glucose (P = 0.065) concentrations than Suffolk ewes at the stage of pregnancy studied. As expected, transferred between-breed Suffolk lambs were born lighter (P = 0.014), and transferred between-breed Cheviot lambs tended to be heavier at birth (P = 0.056) than respective lambs transferred within breed. Midgestation insulin sensitivity does not appear to be a major factor constraining growth of large breed sheep fetus transferred into smaller breed or a factor in releasing constraint in growth of a small breed fetus within a larger breed ewe. However, as embryo size is already different between transferred groups by 19 d, factors other than maternal gestational insulin resistance may determine fetal growth in this embryo transfer paradigm.

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.

Size at birth and adult fat mass in twin sheep are determined in early gestation.

Size at birth is related to adult health outcomes. Twins are born smaller than singletons; this has been assumed to be secondary to limited nutrient supply in late gestation.We hypothesised that growth trajectory in twins, and the adult consequences of being conceived a twin, are determined in early gestation. Twin pregnancies in sheep were randomised to reduction of one twin on day 42 of a 148 day pregnancy by intra-thoracic KCl (Reductions, n =46) or a sham procedure (Twins, n =22). Singleton-bearing ewes also underwent a sham procedure (n =27). Ewes lambed spontaneously. Linear measures of size at birth were similar in Twins and Reductions, and significantly less than in Singletons. Birthweight was lower in Twins and Reductions than in Singletons, and less in Twins than in Reductions (means (SEM): Singletons, liveborn n =23: 6.59 (0.17) kg; Twins, liveborn n =36: 5.23 (0.16) kg; Reductions, liveborn n =27: 5.76 (0.15) kg; all comparisons P <0.05). Reductions grew most rapidly between birth and weaning (Singletons, 20.0 (0.4) g kg⁻¹ day⁻¹; Twins, 20.0 (0.3) g kg⁻¹ day⁻¹; Reductions, 21.0 (0.3) g kg⁻¹ day⁻¹, P <0.05) and were of similar weight as Singletons by weaning; Twins remained smaller by weaning but grew most rapidly thereafter (Singletons, 1.6 (0.1) g kg⁻¹ day⁻¹; Twins, 2.1 (0.1) g kg⁻¹ day⁻¹; Reductions, 1.6 (0.1) g kg⁻¹ day⁻¹, P <0.01), so that all groups had similar weight at 2 years. However, Twins and Reductions had greater percentage fat mass than Singletons at 2 years (Singletons, 11.1 (1.1)%; Twins, 14.8 (1.2)%; Reductions, 15.5 (1.1)%, P <0.05). Thus, in twins, fetal growth trajectory, linear size at birth and adult fat mass are largely determined in early gestation. If this is also true in humans, there are important implications for interventions aimed at optimising fetal growth and pregnancy outcome.

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.

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.

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 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) 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 11betaHSD2 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 11betaHSD2 activity remains low.

Randomized, controlled trial comparing once daily and three times daily gentamicin in children with urinary tract infections.

OBJECTIVE: To undertake population pharmacokinetic modeling and to determine the safety and efficacy of once daily (OD) gentamicin dosing in children with severe urinary tract infections (UTI). METHODS: An open, randomized, controlled trial comparing OD with three times daily (TD) gentamicin dosing in hospitalized children ages 1 month to 12 years with UTI. Daily doses (milligrams per kg per day) of gentamicin in both groups were 7.5 (<5 years old), 6.0 (5 to 10 years old) and 4.5 (>10 years old). RESULTS: There were 179 children enrolled (90 OD, 89 TD). Baseline clinical characteristics and pathogens were similar, except that circulatory compromise and renal cortical scintigraphic defects were more common in the OD group. Median gentamicin treatment durations were 3.0 (OD) and 2.7 (TD) days. Mean peak gentamicin concentrations were 17.3 (OD) vs. 6.4 (TD) mg/l; 99% of peak concentrations were >7 mg/l in the OD group whereas 16% of peak concentrations were <5 mg/l in the TD group. Mean trough concentrations were 0.35 (OD) vs. 0.55 (TD) mg/l. In the OD group 4% of trough concentrations were > or = 2 mg/l, whereas in the TD group only 0.7% were > or = 2 mg/l. Age or prior elevated peak concentrations did not predict high trough concentrations. Population pharmacokinetic modeling of the data fitted a one-compartment model with first order elimination. There were no clinical or bacteriologic failures. The two disease-related complications were confined to the OD group. No nephro- or ototoxicity was identified. CONCLUSIONS: With age-appropriate dosing and measurement of serum trough concentrations before the second dose, OD gentamicin is safe and effective for the treatment of UTI requiring parenteral treatment in children aged 1 month to 12 years.

Fatal granulomatous amoebic encephalitis caused by Balamuthia mandrillaris.

Granulomatous amoebic encephalitis caused by Balamuthia mandrillaris is rare (63 human) cases reported worldwide) and fatal. We report a case in a five-year-old boy who had previously been well. For 18 months, he had had a slowly progressive, granulomatous mid facial lesion, but despite extensive investigation definitive diagnosis was made only with the acute onset of neurological signs in the last two weeks of life, when a brain biopsy specimen revealed amoebic trophozoites and cysts. Infection with B. mandrillaris should be considered in the differential diagnosis of chronic skin lesions with non-specific granulomatous histopathology and negative microbiological test results.