John S Milne

University of Aberdeen, Aberdeen, Scotland, United Kingdom

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Publications (62)228.25 Total impact

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    D J Carr · J S Milne · R P Aitken · C L Adam · J M Wallace
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    ABSTRACT: Intrauterine growth restriction (IUGR) and postnatal catch-up growth confer an increased risk of adult-onset disease. Overnourishment of adolescent ewes generates IUGR in ∼50% of lambs, which subsequently exhibit increased fractional growth rates. We investigated putative epigenetic changes underlying this early postnatal phenotype by quantifying gene-specific methylation at cytosine:guanine (CpG) dinucleotides. Hepatic DNA/RNA was extracted from IUGR [eight male (M)/nine female (F)] and normal birth weight (12 M/9 F) lambs. Polymerase chain reaction was performed using primers targeting CpG islands in 10 genes: insulin, growth hormone, insulin-like growth factor (IGF)1, IGF2, H19, insulin receptor, growth hormone receptor, IGF receptors 1 and 2, and the glucocorticoid receptor. Using pyrosequencing, methylation status was determined by quantifying cytosine:thymine ratios at 57 CpG sites. Messenger RNA (mRNA) expression of IGF system genes and plasma IGF1/insulin were determined. DNA methylation was independent of IUGR status but sexual dimorphism in IGF1 methylation was evident (MF (both P<0.001). IGF1 mRNA expression correlated negatively with IGF1 methylation (r=-0.507, P=0.002) and positively with plasma IGF1 (r=0.884, P<0.001). Carcass and empty body weights were greater in males (P=0.002-0.014) and this gender difference in early body conformation was mirrored by sexual dimorphism in hepatic IGF1 DNA methylation, mRNA expression and plasma IGF1 concentrations.
    08/2015; DOI:10.1017/S2040174415001415
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    ABSTRACT: Adverse nutritional effects on developing fetal hypothalamic appetitive pathways may contribute to programmed hyperphagia and obesity in intra-uterine growth-restricted, low birth weight offspring. Here, for the first time, hypothalamic gene expression for primary orexigenic and anorexigenic genes was examined in late gestation ovine fetuses (130 days; term = 145 days) whose mothers were undernourished (UN) or well-nourished (C) throughout pregnancy, or transferred from UN to C on day 90 (UN-C). Pregnancies resulted from singleton embryo transfer into adolescent growing ewes. Body weight, carcass fat content and perirenal adipose tissue (PAT) mass were all lower for UN (n = 9) than C (n = 7) and intermediate for UN-C fetuses (n = 6), with no effect of gender. PAT leptin gene expression (by RT-PCR) was lower in UN than C and UN-C groups, and lower in males than females. Gene expression (by in situ hybridisation with radiolabelled riboprobes) in the arcuate nucleus was greater in UN than C fetuses for neuropeptide Y (NPY), agouti-related peptide (AGRP) and leptin receptor (OBRb) but not different for pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript. Gene expression in UN-C fetuses was intermediate for NPY and AGRP and not different from C fetuses for OBRb. Gene expression for NPY, AGRP and OBRb correlated negatively with fetal carcass fat content and with PAT leptin gene expression across all groups. Males had greater mRNA expression for AGRP than females, with NPY and OBRb showing similar trends. Therefore maternal undernutrition throughout pregnancy increased orexigenic gene expression in the late gestation fetal hypothalamus, and expression levels were largely normalised by improved maternal nutrition in the last third of pregnancy. These findings may have implications for avoiding or correcting prenatal programming of postnatal hyperphagia and obesity. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Journal of Neuroendocrinology 07/2015; DOI:10.1111/jne.12302 · 3.51 Impact Factor
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    ABSTRACT: Low birthweight is a risk factor for neonatal mortality and adverse metabolic health, both associated with inadequate prenatal adipose tissue development. Here we investigated the impact of maternal undernutrition on expression of genes regulating fetal perirenal adipose tissue (PAT) development and function at gestation days 89 and 130 (term=145d). Singleton fetuses were taken from adolescent ewes fed control (C) intake to maintain adiposity throughout pregnancy or undernourished (UN) to maintain conception weight but deplete maternal reserves (n=7/group). Fetal weight was independent of maternal intake at day 89 but by day 130 fetuses from UN dams were 17% lighter with lower PAT mass containing fewer unilocular adipocytes. Relative PAT expression of IGF1, IGF2, IGF2R and peroxisome-proliferator-activated receptor-gamma (PPARG) mRNA was lower in UN than in C, predominantly at day 89. Independent of maternal nutrition, PAT gene expression of PPARG, glycerol-3-phosphate dehydrogenase, hormone sensitive lipase, leptin, uncoupling protein-1 and prolactin receptor increased and IGF1, IGF2, IGF1R, IGF2R decreased between 89 and 130 days. Fatty acid synthase and lipoprotein lipase (LPL) mRNAs were not influenced by nutrition or stage of pregnancy. Females had greater LPL and leptin mRNA than males, and LPL, leptin and PPARG mRNAs were decreased by UN at day 89 in females only. PAT gene expression correlations with PAT mass were stronger at day 89 than day 130. These data suggest that key genes regulating adipose tissue development and function are active from mid-gestation when they are sensitive to maternal undernutrition. This leads to reduced fetal adiposity by late pregnancy.
    Journal of Molecular Endocrinology 04/2015; 54(3). DOI:10.1530/JME-15-0048 · 3.62 Impact Factor
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    British Maternal and Fetal Medicine Society 17th Annual Meeting, London, UK; 04/2015
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    Society for Reproductive Investigation 62nd Annual Meeting, San Francisco, USA; 03/2015
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    J. M. Wallace · J. S. Milne · R. P. Aitken · C. L. Adam
    Proceedings of The Nutrition Society 01/2015; 74(OCE3). DOI:10.1017/S0029665115002220 · 4.94 Impact Factor
  • J. M. Wallace · J. S. Milne · R. P. Aitken · C. L. Adam
    Proceedings of The Nutrition Society 01/2015; 74(OCE3). DOI:10.1017/S0029665115002232 · 4.94 Impact Factor
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    ABSTRACT: Intrauterine growth restriction (IUGR) is a risk factor for obesity, particularly when offspring are born into an unrestricted nutritional environment. Herein we investigated the impact of IUGR and gender on circulating lipids and on adipogenic, lipogenic and adipokine gene expression in perirenal adipose tissue. Singleton lambs born to overnourished adolescent dams were normal birthweight (N) or IUGR (32% lower birthweight due to placental insufficiency). IUGR lambs exhibited increased fractional growth rates but remained smaller than N at necropsy (d77). At 48d, fasting plasma triglycerides, nonesterified fatty acids and glycerol were elevated predominantly in IUGR males. Body fat content was independent of prenatal growth but higher in females than males. In perirenal fat, relative to male lambs, females had larger adipocytes and lipoprotein lipase, fatty acid synthase and leptin mRNA expression levels were higher while IGF1, IGF2, IGF1R, IGF2R and hormone sensitive lipase mRNAs were lower, and all were independent of prenatal growth category; peroxisome-proliferator-activated receptor-γ and glycerol-3-phosphate dehydrogenase (G3PDH) mRNA expression were not affected by IUGR or gender. Adiposity indices were inversely related to G3PDH mRNA expression, and for the population as a whole the expression of IGF system genes in perirenal fat was negatively correlated with plasma leptin, fat mass and adipocyte size, and positively correlated with circulating IGF1. Higher plasma lipids in IUGR males may predict later adverse metabolic health and obesity, but in early postnatal life gender is the dominant influence on adipose tissue gene expression reflecting the already established sexual dimorphism in body composition.
    Journal of Molecular Endocrinology 06/2014; DOI:10.1530/JME-14-0123 · 3.62 Impact Factor
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    Perinatal Medicine 2014, Harrogate, UK; 06/2014
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    D. J. Carr · J. S. Milne · R. P. Aitken · J. M. Wallace
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    ABSTRACT: Early postnatal growth and body composition are variously influenced by fetal nutrient supply and gender. Thus relative to normal birth weight lambs (N), those categorised as fetal growth restricted (FGR, 30% lighter at birth) subsequently showed increased fractional growth but remained lighter at necropsy (77d postnatal). Moreover, gender was the dominant influence on size and body composition with females (F) lighter and fatter than males (M).(1) We investigated epigenetic changes underlying this early postnatal phenotype by quantification of methylation at cytosine: guanine (CpG) dinucleotides.
    Perinatal Medicine 2014, Harrogate, UK; 06/2014
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    ABSTRACT: Introduction: Fetal growth restriction (FGR) occurs in ~8% of pregnancies and is a major cause of perinatal mortality and morbidity. There is no effective treatment. FGR is characterised by reduced uterine blood flow (UBF). In normal sheep pregnancies, local uterine artery (UtA) adenovirus (Ad) mediated over-expression of vascular endothelial growth factor (VEGF) increases UBF. Herein we evaluated Ad.VEGF therapy in the overnourished adolescent ewe, an experimental paradigm in which reduced UBF from mid-gestation correlates with reduced lamb birthweight near term. Materials and Methods: Singleton pregnancies were established using embryo transfer in adolescent ewes subsequently offered a high-intake (n=45) or control-intake (n=12) of a complete diet to generate FGR or normal fetoplacental growth, respectively. High-intake ewes were randomised mid-gestation to receive bilateral UtA injections of 5x1011 particles Ad.VEGF-A165 (n=18), control vector Ad.LacZ (n=14) or control saline (n=13). Fetal growth/wellbeing were evaluated using serial ultrasound. UBF was monitored using indwelling flowprobes until necropsy at 0.9 gestation. Vasorelaxation, neovascularisation within the perivascular adventitia and placental mRNA expression of angiogenic factors/receptors were examined using organ bath analysis, anti-vWF immunohistochemistry and qRT-PCR, respectively. Results: Ad.VEGF significantly increased ultrasonographic fetal growth velocity at 3-4 weeks post-injection (p=0.016-0.047). At 0.9 gestation fewer fetuses were markedly growth-restricted (birthweight >2SD below contemporaneous control-intake mean) following Ad.VEGF therapy. There was also evidence of mitigated fetal brain sparing (lower biparietal diameter to abdominal circumference and brain to liver weight ratios). No effects were observed on UBF or neovascularisation, however Ad.VEGF-transduced vessels demonstrated strikingly enhanced vasorelaxation. Placental efficiency (fetal to placental weight ratio) and FLT1/KDR mRNA expression was increased in the maternal but not fetal placental compartments, suggesting downstream effects on placental function. Conclusion: Ad.VEGF gene therapy improves fetal growth in a sheep model of FGR, although the precise mechanism of action remains unclear.
    Human Gene Therapy 03/2014; 25(4):375-384. DOI:10.1089/hum.2013.214 · 3.62 Impact Factor
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    ABSTRACT: Maternal uterine artery adenovirus VEGF (Ad.VEGF-A165) gene therapy has been shown to increase pre- and early postnatal growth velocity in an overnourished adolescent ovine model of fetal growth restriction. Herein our objective was to investigate the effect of prenatal Ad.VEGF on fetal and postnatal intestinal mRNA expression of angiogenic factors and their receptors (n=18 genes). In Exp. 1, first parity ewe lamb recipients were offered a control (CON; n=12) or high (H; n=45, 2 x CON) quantity of the same diet after singleton embryo transfer. At 89±1.5 d of gestation overnourished H-ewes were randomly allocated to receive 1 of 3 injections into both uterine arteries: a) Ad.VEGF-A165 (5 × 1011 particles; n=17); b) control Ad vector containing the β galactosidase reporter gene, Ad.LacZ (n=14); c) saline (n=14). CON received saline. In Exp. 2, overnourished ewes received no injection (n=6), Ad.VEGF-A165 (n=16) or saline (n=15) as described above. Ewes lambed normally at 141±0.4 d gestation and offspring were raised until weaning. Fetal (d 130) and postnatal (d 84) intestinal tissues were harvested and mRNA expression relative to 18s determined. In Exp. 1, no differences (P≥0.16) were observed in mRNA expression. In Exp. 2, maternal Ad.VEGF-A165 increased (P≤0.04) mRNA expression of FLT1, KDR, NRP1, FGF2R, TIE2, NOS3, and sGC, and tended (P≤0.10) to increase ANG1, ANG2, NRP2, FGF2, and HIF1A. Thus the previously reported benefits of this prenatal therapy on birth weight and early postnatal growth were mirrored by increased expression of a cadre of angiogenic genes. These may in turn influence intestinal vascularity and thereby absorptive capacity and nutrient uptake.
    Aspen/Snowmass Perinatal Biology Symposium, Colorado, USA; 08/2013
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    ABSTRACT: Intra-uterine growth restriction (IUGR) is involved in developmental metabolic programming and here we test the hypothesis that IUGR affects the developing hypothalamic energy balance regulatory pathways in a sex-specific manner. This experiment investigated early postnatal hypothalamic gene expression for six primary leptin- and insulin-sensitive neuropeptides and receptors in male and female IUGR (n=8 and 9, respectively) and normal (N) birth weight lambs (n=8 per gender) gestated and suckled by overnourished mothers. IUGR lambs were smaller at birth, had increased fractional growth rates (FGR), lower final body weight (11 weeks) and similar body fat content compared with N lambs, while males had higher final body weight and insulinemia but lower body fat and leptinemia than females. In situ hybridization revealed greater gene expression in the hypothalamic arcuate nucleus at 11 weeks for anorexigenic genes in females and orexigenic genes in males, with no effect of IUGR. Leptinemia correlated with gene expression for neuropeptide Y (NPY, negatively) in both sexes and pro-opiomelanocortin (POMC, positively) in females but with leptin receptor (negatively) only in males. Current FGR for girth correlated negatively with gene expression for NPY in males and POMC in females. Neither IUGR nor gender affected suckling activity (proxy for appetite) assessed at 3 weeks, but final NPY gene expression correlated with suckling weight gain in males. This study has revealed no effect of IUGR on early postnatal hypothalamic energy balance gene expression but a major effect of gender associated with major sex differences in adiposity and leptinemia.
    International journal of developmental neuroscience: the official journal of the International Society for Developmental Neuroscience 08/2013; 31(7). DOI:10.1016/j.ijdevneu.2013.07.005 · 2.92 Impact Factor
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    ABSTRACT: Intrauterine growth restriction is a major cause of perinatal mortality and morbidity. Injection of an adenovirus (Ad) vector containing the vascular endothelial growth factor-A165 (VEGF-A165) gene into the uterine arteries increased growth velocity and birth weight (trend) in growth restricted fetal sheep (1,2). Ad.VEGF-A165 treatment increased mRNA levels of the VEGF receptor FLT1/KDR in maternal but not fetal placental tissues and placental efficiency near term (trend) (1,2). We measured late gestation placental expression of genes regulating growth (insulin-like growth factor receptor 1, IGF-1R), lipid handling (lipoprotein lipase, LPL) and amino acid transport (TAT1) in normal and growth-restricted pregnancies with and without Ad.VEGF-A165 treatment. Single embryos (from superovulated single sire inseminated donor ewes) were transferred into adolescent recipient ewe uteri under general inhalational anaesthesia (isoflurane in O2/NO). Recipients were fed control (C n=12) or high (H n=45) intake of complete diet from 4-131 days' gestational age (dGA term=145dGA) to induce normal and restricted fetal growth, respectively (3). At 89dGA under general anaesthesia (induction with propofol IV then as above) both uterine arteries were injected with 5x1011 particles of Ad.VEGF-A165 (H-Ad.VEGF, n=18) or control vector Ad.LacZ/control saline (H-Ad.LacZ/saline, n=27; C-saline, n=12). At 131dGA ewes were killed (pentobarbitone overdose IV). RNA was extracted from separated fetal and maternal placental compartments (frozen at -80C) and real-time quantitative PCR was performed to measure mRNA levels of LPL, TAT1 and IGFR-1 (normalised to GAPDH and βactin geometric mean). Data are mean±SEM and were analysed by two-way ANOVA (placental compartment and group [C-saline; H-Ad.VEGF; H-Ad.LacZ/saline]) then Least Squares Difference correction. Regardless of placental side TAT1 and LPL mRNA levels were lower in H-Ad.VEGF compared to H-Ad.LacZ/saline (TAT1, 0.54±0.08 vs. 0.75±0.06, P=0.02; LPL, 0.77±0.09 vs. 0.97±0.07, P=0.06) and compared to C-saline (TAT1, 0.54±0.08 vs. 0.76±0.09, P=0.07; LPL, 0.77±0.09 vs. 1.03±0.10, P=0.06). Regardless of group LPL, IGFR-1 and TAT1 mRNA levels were greater in the fetal compared to maternal placental side (LPL: 1.22±0.07 vs. 0.66±0.07; IGFR-1: 1.19±0.07 vs. 0.99±0.07; TAT1: 1.24±0.07 vs. 0.13±0.06; P <0.05). These data suggest that increased fetal growth velocity following Ad.VEGF-A165 is independent of altered IGFR-1 gene expression. Decreased TAT1 and LPL gene expression following Ad.VEGF-A165 is unexpected but may reflect complex amino acid and lipid transport regulation in these animals which requires further molecular analysis. Higher expression of genes related to nutrient transfer in the fetal-facing placental portion may indicate membrane specific placental nutrient transport mechanisms.
    37th Congress of the International Union of Physiological Sciences, Birmingham, UK; 07/2013
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    ABSTRACT: Uterus didelphys is a rare congenital abnormality of the reproductive tract. Although it occurs in various species, there are no published reports describing pregnancy outcome in association with this abnormality. Herein we describe a case of successful unilateral singleton pregnancy in a ewe incidentally found to have uterus didelphys during the course of a biomedical research study. The pregnancy was established using assisted reproductive techniques and interrupted in late gestation, at which point the abnormality was identified. Serial ultrasound assessment of foetal biometry revealed a normal foetal growth trajectory. Despite a 45% reduction in placentome number, total placentome weight was near normal secondary to compensatory placentome growth and development. To our knowledge, this is the first detailed report of normal foetal growth in an animal with uterus didelphys and illustrates the ability of the ovine placenta to adapt to a reduced number of placentomes and maintain foetal nutrient supply.
    Reproduction in Domestic Animals 06/2013; 48(5):e78-80. DOI:10.1111/rda.12191 · 1.18 Impact Factor
  • Reproduction Fertility and Development 05/2013; DOI:10.1071/RD13090 · 2.58 Impact Factor
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    ABSTRACT: Introduction: We previously showed that adenovirus (Ad) mediated over-expression of vascular endothelial growth factor (VEGF) in the uteroplacental circulation increases fetal growth velocity in the overnourished adolescent sheep paradigm of fetal growth restriction (1,2). Lambs born following Ad.VEGF therapy also demonstrated accelerated lean tissue accretion and enhanced insulin secretion following glucose challenge (3). Herein we examined for putative epigenetic changes underlying these postnatal effects by quantification of methylation at cytosine:guanine (CpG) dinucleotides. Methods: DNA was extracted from liver samples obtained at 82±0.2d postnatal age in 29 lambs born after prenatal treatment with Ad.VEGF (n=16: 8×Male/8×Female) or Saline (n=13: 7×Male/6×Female). After incubation with sodium bisulphite, PCR was performed using custom-designed primers targeting 9 CpG islands across 5 genes: insulin, growth hormone, insulin-like growth factor (IGF)1, IGF2 and H19. Using pyrosequencing, methylation status was determined by quantifying cytosine:thymine ratios at 35 individual CpG sites. Plasma IGF1/insulin levels were measured by RIA. Results: There were no significant differences in DNA methylation between Ad.VEGF and Saline groups, except at 1 of 4 CpG dinucleotides preceding IGF2 exon-6 (3.8±0.59 vs. 1.9±0.60%, p=0.029). Irrespective of treatment, insulin gene methylation was greater in males than females (88.7±0.23 vs. 87.0±0.50%, p=0.007) and negatively correlated with fasting insulin levels (r = –0.431, n=28, p=0.022). By contrast, IGF1 methylation tended to be lower in males than females (84.3±0.16 vs. 84.8±0.22%, p=0.053) and was unrelated to plasma IGF1 levels. Conclusion: Increased postnatal growth rates in Ad.VEGF-treated lambs most likely reflect their relative size advantage at birth rather than altered epigenetic status of key somatotropic genes. References 1. Carr DJ, Aitken RP, Milne JS, et al. Maternal Ad.VEGF gene therapy increases fetal growth velocity in growth restricted sheep fetuses. BJOG 2011;118:1008-1009. 2. Carr DJ, Aitken RP, Milne JS, et al. Prenatal gene therapy increases fetal growth velocity and alters uterine artery vascular reactivity in the absence of a measurable effect on uterine blood flow in a sheep model of fetal growth restriction. Archives of Disease in Childhood: Fetal and Neonatal Edition 2012;97(Suppl_1):A1. 3. Carr DJ, Aitken RP, Milne JS, et al. Alterations in postnatal growth and metabolism following prenatal treatment of intrauterine growth restriction with Ad.VEGF gene therapy in the sheep. Archives of Disease in Childhood: Fetal and Neonatal Edition 2011;96(Suppl1):Fa7.
    British Maternal and Fetal Medicine Society 16th Annual Meeting, Dublin, Ireland; 04/2013
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    ABSTRACT: Introduction: We previously showed that adenovirus (Ad) mediated overexpression of vascular endothelial growth factor (VEGF) in the uteroplacental circulation increases fetal growth velocity in the over nourished adolescent sheep model of fetal growth restriction. Lambs born following Ad.VEGF therapy also demonstrated accelerated lean tissue accretion and enhanced insulin secretion after glucose challenge. Herein we examined for putative epigenetic changes underlying these postnatal effects by quantification of methylation at cytosine:guanine (CpG) dinucleotides. Methods: DNA was extracted from liver samples obtained at 82±0.2d postnatal age in 29 lambs born after prenatal treatment with Ad.VEGF (n=16: 8 male, 8 female) or Saline (n=13: 7 male, 6 female). After incubation with sodium bisulphite, PCR was performed using custom-designed primers targeting 14 CpG islands in 5 genes: insulin, growth hormone, insulin-like growth factor (IGF)1, IGF2 and H19. Using pyrosequencing, % methylation was determined by quantifying cystosine:thymine ratios at 29 individual CpG sites. Plasma IGF1/insulin levels were measured by RIA. Results: The table shows mean DNA methylation for each gene of interest. There were no significant differences between Ad.VEGF and Saline groups, except at 1 of 4 CpG dinucleotides preceding IGF2 exon 6 (3.8±0.59 vs. 1.9±0.60%, p=0.029). Irrespective of treatment, insulin gene methylation was greater in males than females (88.7±0.23 vs. 87.0±0.50%, p=0.007) and negatively correlated with fasting insulin levels (r=-0.431, n=28, p=0.022). By contrast, IGF1 methylation tended to be lower in males than females (84.3±0.16 vs. 84.8±0.22%, p=0.053) and was unrelated to plasma IGF1 levels. Saline (n=13) versus Ad.VEGF (n=16): GH: 64.8±1.90% vs. 61.9±1.28% (p=0.209) IGF1: 84.6±0.28% vs. 84.5±0.14% (p=0.564) IGF2: 20.9±0.51% vs. 20.4±0.45% (p=0.475) H19: 40.2±0.66% vs. 39.8±0.41% (p=0.536) Insulin: 88.3±0.43% vs. 87.6±0.42% (p=0.312) Conclusion: Increased postnatal growth rates in Ad.VEGF-treated lambs most likely reflect their relative size advantage at birth rather than altered epigenetic status of key somatotropic genes.
    Society for Gynecologic Investigation 60th Annual Scientific Meeting, Orlando, USA; 03/2013
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    ABSTRACT: Background: Fetal growth restriction (FGR) is an important obstetric condition that is commonly associated with reduced uterine blood flow. In normal sheep pregnancies, adenovirus (Ad) mediated overexpression of vascular endothelial growth factor (VEGF) in the uterine arteries increases uterine blood flow. We hypothesised that enhancing uterine blood flow would improve fetal substrate delivery in a sheep model of FGR that is characterised by reduced uterine blood flow from mid-gestation. Methods: Singleton pregnancies were established with embryo transfer in 90 adolescent ewes subsequently overnourished to generate FGR (n=78) or control fed (n=12). In study 1, at a mean gestational age of 89 days (SE 0·2), 45 overnourished ewes were randomised to receive Ad-VEGF (n=18), Ad-LacZ (control vector, n=14), or saline (n=13) injected into each uterine artery. Control-fed ewes (n=12) received saline. Uterine blood flow was monitored with indwelling flowprobes until necropsy at 131 days’ gestation (0·6). Placental mRNA expression of VEGF and its receptors, Flt1/KDR, was assessed in the maternal and fetal placental compartments by quantitative reverse transcription PCR. In study 2, at 88 days’ gestation (0·7), 33 overnourished ewes received Ad-VEGF (n=17) or saline (n=16) and were allowed to deliver spontaneously at 141 days’ gestation (0·4). In both studies fetal growth and wellbeing were evaluated (blind) using serial ultrasound. Findings: In both studies ultrasound measurements of abdominal circumference (AC) were greater in Ad-VEGF-treated FGR fetuses than in control-treated FGR fetuses at 21 days (0·3) and 28 days (0·5) post injection (p<0·001—0·047). In study 1, fewer fetuses were markedly growth-restricted (weight >2 SD below control mean) in Ad-VEGF compared with Ad-LacZ plus saline groups (5/18 vs 18/27, p=0·038). Indices of head sparing (ultrasound biparietal diameter:AC ratios, relative brain weight, and brain:liver weight ratios) were lower (p=0·001—0·046) and maternal placental Flt1/KDR mRNA expression higher (p=0·028/0·034) in Ad-VEGF than in Ad-LacZ plus saline groups, but no measurable differences in uterine blood flow were detected. In study 2, Ad-VEGF-treated lambs tended to be heavier (p=0·081) with increased placental efficiency (p=0·074). There were no maternal or fetal complications or significant differences in the level of neonatal care required to ensure lamb survival. Interpretation: Ad-VEGF safely increases fetal growth in this ovine model of FGR. Funding: Wellbeing of Women.
    Academy of Medical Sciences Spring Meeting for Clinician Scientists in Training, London, UK; 02/2013
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    ABSTRACT: Introduction: We previously showed that adenovirus (Ad) mediated overexpression of vascular endothelial growth factor (VEGF) in the uteroplacental circulation increases fetal growth velocity in the overnourished adolescent sheep paradigm of fetal growth restriction. Lambs born following Ad.VEGF therapy also demonstrated accelerated lean tissue accretion and enhanced insulin secretion following glucose challenge. Herein we examined for putative epigenetic changes underlying these postnatal effects by quantification of methylation at cytosine:guanine (CpG) dinucleotides. Methods: DNA was extracted from liver samples obtained at 82�0.2d postnatal age in 29 lambs born after prenatal treatment with Ad.VEGF (n = 16: 8xMale/8xFemale) or Saline (n = 13: 7xMale/6xFemale). After incubation with sodium bisulphite, PCR was performed using custom designed primers targeting 9 CpG islands across five genes: insulin, growth hormone, insulin-like growth factor (IGF)1, IGF2 and H19. Using pyrosequencing, methylation status was determined by quantifying cystosine:thymine ratios at 35 individual CpG sites. Plasma IGF1/insulin levels were measured by RIA. Results: There were no significant differences in DNA methylation between Ad.VEGF and Saline groups, except at 1 of 4 CpG dinucleotides preceding IGF2 exon-6 (3.8�0.59 vs. 1.9�0.60%, P = 0.029). Irrespective of treatment, insulin gene methylation was greater in males than females (88.7�0.23 vs. 87.0�0.50%, P = 0.007) and negatively correlated with fasting insulin levels (r = �0.431, n = 28, P = 0.022). By contrast, IGF1 methylation tended to be lower in males than females (84.3�0.16 vs. 84.8�0.22%, P = 0.053) and was unrelated to plasma IGF1 levels. Conclusion: Increased postnatal growth rates in Ad.VEGF-treated lambs most likely reflect their relative size advantage at birth rather than altered epigenetic status of key somatotropic genes.
    Royal College of Obstetricians and Gynaecologists Annual Academic Meeting, London, UK; 12/2012

Publication Stats

641 Citations
228.25 Total Impact Points

Institutions

  • 2009–2015
    • University of Aberdeen
      • Rowett Institute of Nutrition and Health
      Aberdeen, Scotland, United Kingdom
  • 2005–2008
    • North Dakota State University
      • Department of Animal Sciences
      Fargo, ND, United States
  • 2007
    • University of Nottingham
      • School of Veterinary Medicine and Science
      Nottingham, ENG, United Kingdom
    • Hirosaki University
      Khirosaki, Aomori, Japan
    • University of Colorado
      • Perinatal Research Center
      Denver, Colorado, United States