Aspen/Snowmass Perinatal Biology Symposium: “Fetal Adaptations to Maternal and Placental Dysfunction”

August 24–27th 2013, Snowmass, Colorado Organizing Committee: Charles Ducsay, Ph.D. (co-chair), Loma Linda University Sean Limesand, Ph.D. (co-chair), University of Arizona Denise Hemmings, Ph.D., University of Alberta Anna Grazul-Bilska, Ph.D., North Dakota State University

The Aspen/Snowmass Perinatal Biology Conference is a multidisciplinary research meeting that brings together clinicians and scientists, established senior and junior beginning investigators, and graduate students to report and discuss their findings in an atmosphere conducive to frank yet amicable exchange. The overall goal is to develop effective trans-disciplinary scientific interactions that lead to productive translational perinatal science research endeavors in the areas of fetal programming of adult disease, preeclampsia, and maternal obesity and diabetes during pregnancy.

Sponsorship Statement

Publication of this supplement was sponsored by the Loma Linda University School of Medicine.

Poster Session 1—Fetus, Neonate, and Child

1-1. CHRONIC HYPERINSULINEMIA SUPPRESSES GLUCOSE-STIMULATED INSULIN SECRETION (GSIS), BUT NOT β-CELL MASS, IN THE OVINE FETUS

S Andrews¹, L Brown², S Thorn², M Davis³, S Limesand³, W Hay Jr.², and P Rozance²

Departments of ¹OB/Gyn and ²Pediatrics, University of Colorado, Aurora, CO, USA. ³School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, USA

Intrauterine growth restriction is associated with decreased fetal insulin secretion and β-cell mass. Insulin has autocrine actions that enhance insulin secretion and β-cell growth, but this has not been demonstrated in the normally growing fetus. We tested the hypothesis that chronic fetal hyperinsulinemia, with euglycemia, increases GSIS and β-cell mass in the ovine fetus. Singleton ovine fetuses at 125.6±0.5 days gestation (term=148) were intravenously infused with insulin (INS, HumulinR, n=8) to produce high physiological insulin concentrations and glucose to maintain euglycemia, or saline (SAL, n=9) for 6–10 days. Fetal arterial pH, oxygen content, glucose, insulin, cortisol, and norepinephrine concentrations were measured. On the final day, fetal GSIS was measured using a square-wave fetal hyperglycemic clamp. The pancreas was fixed for histological analysis. In four additional INS animals, GSIS was measured with and without concurrent pharmacologic adrenergic blockade. Glucose concentrations were similar (18.21±1.85 INS vs. 20.21±1.31 SAL, mg/dL). Oxygen content was lower (2.53±0.25 vs. 3.60±0.11 SAL, mmol/L, p<0.005) and norepinephrine higher (1159±115 INS vs. 553±155 SAL, pg/mL, p<0.01) in INS fetuses. Fetal arterial pH and cortisol were not different. GSIS was present in the SAL group (228% increase from baseline, p<0.05) but absent in the INS group (12% increase). Neither insulin-positive area (2.01±0.21 INS vs. 2.14±0.22 SAL, %) nor β-cell mass (53.16±5.19 INS vs. 62.34±10.99 SAL, mg) increased in the INS group. Pharmacologic adrenergic blockade restored GSIS in the INS fetuses (117% increase from baseline, p<0.05). Contrary to our hypothesis, chronic fetal insulin infusion decreased fetal oxygen and increased norepinephrine concentrations. β-cell mass was not increased. Pharmacologic adrenergic blockade demonstrated that increased norepinephrine concentrations were responsible for inhibiting GSIS. Increased adrenergic signaling in the β-cell mediates decreased GSIS in conditions characterized by fetal hypoxemia. (NIH R01 DK088139 and K08 HD060688)

1-2. CHRONIC FETAL HYPERINSULINEMIA FAILS TO ACTIVATE P70 AND S6 IN SHEEP SKELETAL MUSCLE

S Bourque¹, L Brown¹, S Andrews², S Thorn¹, W Hay Jr.¹, and P Rozance¹

Department of ¹Pediatrics and ²OB/GYN, University of Colorado, Aurora, CO, USA

Insulin is necessary for normal fetal growth. However, chronic experimental fetal hyperinsulinemia does not increase growth trajectory beyond normal. The mechanisms responsible for this paradox are unknown. The mTOR signaling pathway is central for regulating fetal growth, protein synthesis, and is activated by acute insulin stimulation; making this a pathway to study. Our objective was to determine if chronic fetal hyperinsulinemia would activate the mTOR pathway in fetal sheep skeletal muscle. Singleton sheep fetuses were infused with continuous intravenous insulin and glucose to maintain euglycemic hyperinsulinemia (INS n=7) or saline (SAL n=8) for 7–9 days. Fetal glucose, amino acids, insulin, norepinephrine, cortisol, IGF-1, pH, lactate, CO2, and O2 were measured. Biopsy of the biceps femoris hindlimb muscle was obtained under study conditions at 134 days (term 148 days). Muscle proteins were prepared and total and phosphorylated p70S6K, 4E-BP1, ribosomal protein S6 (rpS6), and actin were measured by western blotting. Insulin concentrations were 150% higher in the INS group compared to SAL (P<0.001). INS fetuses became hypoxemic and hypoaminoacidemic, but were not acidotic or hypercortisolemic. IGF-1 and norepinephrine concentrations increased 60% and 120%, respectively, in the INS group (P<0.05). There was no effect of insulin on total fetal and individual hindlimb muscle weights. There was 20% less p70S6K in the INS group (p<0.005), but no difference in the ratio of phosphorylated to total p70S6K or the ratio of phosphorylated to total rpS6. The ratio of phosphorylated to total 4E-BP1 was increased 115% in the INS group (p<0.005), but total 4E-BP1 did not change. Chronic fetal hyperinsulinemia increased the phosphorylation of 4E-BP1 but failed to increase phosphorylated p70S6K or rpS6 which may be responsible for the lack of enhanced muscle growth in the presence of chronic fetal hyperinsulinemia. We speculate that this is due to hypoxemia, hypercatecholinemia, and/or hypoaminoacidemia inhibiting mTOR signaling to P70S6K.

1-3. BODY MASS INDEX AND ETHNICITY AS DETERMINANTS OF BROWN ADIPOSE TISSUE FUNCTION AS MEASURED BY THERMAL IMAGING IN HEALTHY CHILDREN

H Budge, L Robinson, S Ojha, ME Symonds

Academic Division of Child Health, School of Medicine, University of Nottingham, Nottingham, NG7 2UH, United Kingdom

Brown adipose tissue (BAT) function is greatest in children but the main factors influencing it remain to be determined. Our study was, therefore, designed to determine whether body mass index (BMI) centile influenced skin temperature overlying BAT depots in the supraclavicular region (SCR) of healthy children. Infrared thermography was used to assess SCR skin temperature at baseline and following exposure to a mild cool stimulus (single hand immersion in water at 20.1°C) over a period of 5 minutes in young children aged 6 -11 years (n=55). There was a clear difference in SCR temperature between age matched healthy subjects who were either of low, or high, BMI. The primary indicator of baseline and stimulated SCR temperature was BMI centile for which there was an inverse relationship (R2 = 0.44; P = 0.009). Ethnicity was also a significant predictor of SCR temperature. We have demonstrated an inverse relationship between the temperature of the SCR, co-locating with the primary region of BAT, and BMI centile. Future studies aimed at determining the primary factors regulating BAT function in healthy children could be used to prevent excess white adipose tissue deposition in early life.

1-4. KETAMINE SUPPRESSES INFLAMMATORY RESPONSE IN THE KIDNEY CORTEX OF LATE GESTATION OVINE FETUS EXPOSED TO ACUTE HYPOXIC STRESS

EI Chang and CE Wood

Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida, USA

Acute hypoxic hypoxia (HH) is a mild form of fetal stress that results in the shunting of combined ventricular output towards the fetal brain and heart, and away from the kidneys. A decrease in both renal blood flow and partial pressure of oxygen can cause ischemia to the kidneys, resulting in potential renal damage. Ketamine, an N-Methyl-D-aspartate receptor antagonist, is used in neonatal intensive care units (NICUs) as an anesthetic and analgesic. We have demonstrated that HH in late gestation causes brain inflammation, and this is ameliorated by treatment with ketamine. The purpose of this study was to evaluate the genomic response of fetal kidney cortex under HH with ketamine. In chronically catheterized fetal sheep (125±3 d), ketamine (3 mg/kg) was administered intravenously to the fetus 10 min prior to 30 min HH. Fetal kidney cortex was collected 24 hrs post onset of hypoxia; mRNA was extracted and analyzed using ovine Agilent 15.5k array and qPCR. Significant differences in gene expression between four groups (n=4/group): normoxic control, normoxia+ketamine, hypoxic control, hypoxia+ketamine, were determined (P<0.05). TLR2, CD14, NFKB, MYD88, CSF1, TNFa, IL6, IL1b, BCL3, CCL4, CCL5, CXCL10, and IRF1 mRNA expression were validated by qPCR. Enriched biological processes (BP) for the 405 up-regulated genes after hypoxia were immune and inflammatory response; 813 down-regulated genes were protein location and transportation. After ketamine, the BP for 274 up-regulated genes were DNA metabolic and macromolecule catabolic processes; 208 down-regulated genes were regulation of cell proliferation and apoptosis. qPCR validation showed increased mRNA expression after hypoxia, and blockade of these increases by ketamine. We conclude that HH activates inflammatory pathways and up regulates cytokines via toll-like receptors in the fetal kidney cortex, and that ketamine blocks this response. Ketamine could be a potential therapeutic therapy for pre-term infants in NICUs to reduce global inflammation as a consequence of hypoxic stress. (NIH grants: HD033053, HD057561, DK076541)

1-5. SURPRISING SIGNAL TRANSDUCTION CHANGES IN MATURING FETAL OVINE CARDIOMYOCYTES

NN Chattergoon^1,3, S Louey^1,2,3, PJ Stork^1,4, GD Giraud^1,2,3,5, KL Thornburg^1,2,3

¹Heart Research Center, ²Department of Physiology and Pharmacology, ³Knight Cardiovascular Institute, ⁴Vollum Institute, Oregon Health & Science University, Portland, OR, USA, ⁵Portland Veterans Affairs Medical Center, Portland, OR, USA

Maturation of fetal cardiomyocytes involves transition from a proliferating to a terminally differentiated non-proliferating state. Ovine fetal cardiomyocytes begin the maturation process after 100 days gestational age (dGA) such that at term (145dGA) ~60% of cardiomyocytes become binucleated. Rising near term thyroid hormone (T3) levels inhibit proliferation and drive myocyte maturation opposing the stimulation of proliferation by insulin-like growth factor-1 (IGF-1). However, the degree to which intracellular signaling patterns change with myocyte maturation when exposed to these hormones is unknown. We hypothesized that T3 and IGF-1 could activate extracellular regulatory kinase (ERK) and AKT signaling proteins similarly between 100 and 135dGA. Cardiomyocytes were enzymatically isolated from non-instrumented, control fetal sheep (n=11/ age, Ovis aries; mixed western breed) at 102 ± 3dGA and 135 ± 1dGA. Proliferation by BrdU uptake (10μM) was performed in the presence of T3 (0.37, 1.5nM), IGF-1 (1μg/ml) and pathway inhibitors (U0126 10μM, LY294002 10μM, Rapamycin 10nM). Western blot and immunohistochemistry were used to determine phosphorylation of ERK, AKT and p70S6K. IGF-1 stimulated proliferation at both ages; the addition of T3 suppressed mitotic activity to near basal levels (~50% reduction in 100dGA and ~75% reduction in 135dGA; p<0.001). IGF-1 and T3 independently stimulated phosphorylation of ERK, AKT, and p70S6K at both ages. In 135dGA cells, the combination of IGF-1 and T3 increased activation of ERK and AKT two-fold with the opposite being true in 100dGA cardiomyocytes. This was partially explained using immunohistochemical analysis: in most (~75%) 135dGA cardiomyocytes, both mono- and binucleated cells phosphorylated ERK and AKT when but only a small percentage were activated in the younger cells.

1-6. ELEVATED NOREPINEPHRINE PROTECTS FROM GREATER LOSS OF β-CELL MASS IN INTRAUTERINE GROWTH RESTRICTED OVINE FETUSES

M Davis, A Macko, M Anderson, and S Limesand

The University of Arizona, Tucson, AZ USA

Intrauterine growth restriction (IUGR) is associated with β-cell dysfunction and lower β-cell mass. IUGR fetuses with placental insufficiency are hypoglycemic and hypoxic. In response to these characteristics, norepinephrine is released from adrenal chromaffin cells, which is the sole fetal source for elevated plasma norepinephrine concentrations. Our objective was to determine the effect of chronic norepinephrine exposure on pancreatic endocrine cell area in near-term IUGR fetuses. The pancreas was collected following in vivo measurements from singleton fetuses at 134 days gestational age (term 150 days) for the following treatment groups: control and IUGR fetuses with either a sham or adrenal demedullation (AD) surgical procedure at 98 days. Immunofluorescent staining for pancreatic hormones was performed, and positive areas were measured and expressed as a percent of total pancreas area. As a result of hypoxemia, plasma norepinephrine concentrations were 4-fold greater (P<0.05) in sham IUGR fetuses compared to controls and AD IUGR fetuses, which were not different. Fetal weight was less (P<0.05) for sham and AD IUGR fetuses (1.58±0.3 kg and 2.4±0.4 kg) compared to sham and AD controls (3.2±0.1 kg and 3.2±0.2 kg). Fetal weight was greater (P<0.05) in AD IUGR than sham IUGR fetuses. Insulin positive area for sham control, AD control, sham IUGR, and AD IUGR fetuses were 2.15±0.20%, 1.78±0.26%, 1.53±0.15%, and 0.82±0.22%, respectively. In AD IUGR fetuses, insulin and glucagon positive areas were lower (P<0.05) than other treatment groups, although sham IUGR fetuses were also lower than controls. Lower β-cell areas in AD IUGR fetuses compared to sham IUGR fetuses indicates that norepinephrine to some extent protects β-cells from adverse conditions in placental insufficiency-induced IUGR. (Supported by R01-DK084842)

1-7. OVINE FETAL RENAL DEVELOPMENT IMPACTED BY MULTIPLE FETUSES AND UTERINE SPACE RESTRICTION

KM Meyer-Gesch, MY Sun, SE Blohowiak, R Magness, PJ Kling

Departments of Pediatrics/Obstetrics & Gynecology Neonatal/Perinatal Research Laboratories, University of Wisconsin, Madison, WI and Nova Southeastern University College of Osteopathic Medicine, USA

Intrauterine growth restriction (IUGR) from uteroplacental dysfunction causes impaired nephrogenesis and ultimately hypertension. It is unknown whether IUGR caused by impaired placental space seen in uterine anomalies and/or multifetal gestation also impairs nephrogenesis. It was hypothesized that limited uterine space causes fetoplacental metabolic adaptations that preserve a viable but compromised fetus with maladaptive renal development and function. Fetal renal development and metabolic status was studied in an ovine uterine space restriction (USR) model of limited space for placental formation and IUGR by combining unilateral horn surgical ligation with multifetal gestation. Reduced placental attachment sites and placental weight per fetus defined USR versus control nonrestricted (NSR) fetuses (Meyer, et al 2010). USR fetuses exhibited evidence for decreased plasma volume, with higher hematocrit and plasma albumin at GD120 (p<0.05). At GD130, USR developed IUGR, lower blood pO2 and oxygen content, and higher fetal osmolarity, BUN and creatinine (p<0.05 for all), but not higher plasma erythropoietin levels. In all fetuses, kidney weight relative to fetal weight was not constant, but inversely related to both fetal weight and fetal plasma creatinine levels (p<0.05 for both). Glomerular generations were directly related to cortical depth and inversely related to fetal plasma creatinine levels (p<0.05 for both). At GD130, kidney weights in USR fetuses were smaller, with decreased cortical depths and glomerular generations, with hyperplastic maculae densa (p<0.05 for all). Insufficient space for placental implantation negatively impacted fetal oxygenation, and kidney growth and development. USR-induced insufficiency in fetal renal excretory function was accompanied by an immature endocrine renal erythropoietin response to hypoxemia and a structurally immature nephrogenic zone with altered maculae densa. Using this model of limited placental development as is seen in multifetal gestation and/or uterine anomalies, we are poised to further investigate fetal renal adaptations responsible for programming of adult disease. (NIH HL49210, HD 38843, HL 087144)

1-8. INFLUENCE OF PRENATAL ADENOVIRUS-VEGF GENE THERAPY ON mRNA EXPRESSION OF ANGIOGENIC RELATED GENES IN FETAL AND POSTNATAL OVINE INTESTINE

GQ Jia¹, JS Haring¹, RP Aitken², JS Milne², DJ Carr^2,3, AL David³, JM Wallace², and JS Caton¹

¹Center for Nutrition and Pregnancy, Animal Sciences Department, North Dakota State University, Fargo; ²Rowett Institute of Nutrition and Health, University of Aberdeen, Scotland, UK; ³Prenatal Cell and Gene Therapy Group, UCL Institute for Women’s Health

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. (Funded by the Wellcome Trust UK, Scottish Government, and AFRI Grant 2012-67016-19453 from USDA-NIFA).

1-9. VITAMIN D ATTENUATES LUNG INJURY AND IMPROVES SURVIVAL IN INFANT RATS AFTER ANTENATAL ENDOTOXIN EXPOSURE

EM Mandell, GJ Seedorf, J Gien, SH Abman

Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado, School of Medicine, Aurora, CO

Chorioamnionitis contributes to the pathogenesis of BPD. Vitamin D (Vit D) has immunomodulatory and anti-inflammatory properties, but whether Vit D can improve neonatal survival, preserve lung vascular and alveolar growth and prevent endothelial injury after exposure to antenatal inflammation is unknown. This study was designed to determine whether Vit D treatment could improve survival and preserve lung growth after antenatal exposure to endotoxin (ETX). Fetal rats were exposed to ETX (10μg), ETX + Vit D (1ng/ml), or saline via intra-amniotic (IA) injection and delivered two days later. Newborn pups exposed to IA ETX received daily injections of Vit D or saline for 2 weeks. To assess lung development, radial alveolar counts (RAC) were assessed at 14d. Fetal sheep pulmonary artery endothelial cells (PAEC) were assessed for growth and tube formation following treatment with Vit D (100-400nM) or ETX (1ng/ml) + Vit D. Growth of alveolar type II (AT II) cells treated with Vit D(100nm) or with ETX (10ng/ml) + Vit D was assessed. Prenatal Vit D treatment improved survival in rat pups exposed to IA ETX when compared to ETX exposure alone (100% vs. 60%; p<0.001) at DOL 14. In comparison to rats exposed to IA ETX, pups who also received either prenatal or postnatal Vit D showed improved RAC at 14 days by 54% (p <0.05). In control PAEC, Vit D increased cell growth and tube formation by 64 and 44% (p<0.001), respectively. ETX decreased PAEC growth and tube formation by 57% and 53% compared to controls (p<0.05), while co-treatment with Vit D preserved PAEC growth and tube formation (p<0.001). In addition, Vit D increased ATII cell growth by 26% (p<0.001) and attenuated ETX induced growth suppression by 73% (p<0.001). Vit D improved survival and enhanced lung structure after antenatal ETX exposure in vivo, and prevented ETX -induced PAEC and AT II cell dysfunction in vitro.

1-10. MATERNAL MELATONIN TREATMENT ALTERS EXPRESSION OF FETAL HYPOTHALAMIC 5α-REDUCTASE TYPE 1

LP Reynolds¹, JS Caton¹, PP Borowicz¹, KA Vonnahme¹, CO Lemley¹, and AJ Conley²

¹Center for Nutrition and Pregnancy, Animal Sciences Department, North Dakota State University, Fargo; ²Population Health and Reproduction Department, University of California-Davis

The enzyme 5α-reductase converts progesterone to 5α-dihydroprogesterone, a precursor to neurosteroids that suppress the fetal hypothalamic-pituitary-adrenal axis (HPAA). The effects of maternal dietary intake and melatonin treatment on fetal hypothalamic 5α-reductase expression were examined. Primiparous ewes carrying singletons were housed at constant temperature (14 °C) and 12:12-h light:dark (lights off at 19:00). On day 50 of gestation, ewes were assigned randomly in a 2 × 2 factorial to melatonin supplementation (0 or 5 mg) and maternal intake (100 or 60% of NRC requirements), resulting in four melatonin × dietary intake groups (n=6–8/group). The pelleted diet contained 15.8% crude protein and 2.66 Mcal/kg. Melatonin was delivered once daily at 14:00 by top-dressing the pellets (100 g) with melatonin in ethanol (5mg/kg). Maternal jugular serum was obtained at 23:00 on days 44 (baseline), 84, and 124 of gestation. At slaughter on d130, we obtained umbilical arterial serum, and fetal hypothalamic samples were frozen for qPCR. As previously reported (Lemley et al, Am J Physiol 302:R454-R467, 2012), maternal jugular melatonin concentrations were similar on d44 and on days 84 and 124 were unaffected by dietary intake but elevated (P<0.001) 2-fold in melatonin-treated compared with untreated ewes. Similarly, on day 130 umbilical arterial melatonin concentrations were unaffected by dietary intake but elevated (P<0.001) 50% in melatonin-treated compared with untreated ewes. For fetal hypothalamic mRNA expression of 5α-reductase type 1, there was a main effect (P<0.02) of melatonin (expression relative to 18S rRNA: 1.55±0.19 for melatonin-treated vs. 0.78±0.22 for untreated) but no effect of dietary intake and no melatonin-diet interaction. There was no effect of melatonin treatment or dietary intake on fetal hypothalamic mRNA expression of 5α-reductase type 2. These data suggest that diurnal changes in activity of the fetal HPAA may result from melatonin-induced changes in expression of 5α-reductase. mRNA. (Supported partly by a Lalor Foundation grant to COL)

1-11. FETAL HYPOGLYCEMIA INDUCES FETAL HEPATIC GLUCOSE OUTPUT

S Sekar, P Rozance, L Brown, W Hay, R Wilkening, S Thorn

University of Colorado School of Medicine, Aurora, CO, USA

This study was designed to measure fetal hepatic glucose production in response to hypoglycemia and determine the role of carbon substrates and hormones. Late gestation fetal sheep (n=5) had catheters placed in the abdominal aorta, umbilical vein, and left hepatic vein to simultaneously measure umbilical and hepatic substrate fluxes. Fetal glucose, lactate, pyruvate, amino acids (AA), hormones, and oxygen concentrations, and umbilical and hepatic blood flow rates were measured and umbilical and hepatic substrate and oxygen flux rates were calculated. Metabolic studies were done prior to (d0) and after (d1 and d5) induction of fetal hypoglycemia by a maternal insulin infusion. Fetal glucose concentrations and glucose uptake rates were 50% lower on d1 and d5 compared to d0. Net hepatic glucose output (d0: -14.6 µmol/min/100g liver) increased on d1 (1.5 µmol/min/100g liver) and d5 (16.1 µmol/min/100g liver). Hepatic lactate and pyruvate uptake rates were unchanged yet total hepatic AA uptake increased 50% on d1 compared to d0 and d5. Interestingly, on d0 the liver released glutamate and this was reduced by 33% on d1 and 56% on d5. Total hepatic carbon uptake and hepatic oxygen uptake increased on d1 compared to d0 and d5, suggesting an acute increase in hepatic metabolism. Total fetal carbon uptake, however, was reduced by 16% on d1 and 39% on d5 and became less than fetal oxygen uptake demonstrating decreased nutrient supply for growth and oxidative metabolism. Fetal insulin concentrations were 50% lower, and glucagon and cortisol concentrations were increased >50% on d5. The insulin:glucagon ratio correlated with hepatic glucose output (r2=0.568). Using hepatic catheterization, we demonstrate fetal hepatic glucose output in response to hypoglycemia. Increased hepatic provision of carbon from AA and glycogenolysis likely contribute to hepatic glucose output on d1. By d5, we speculate that hormonal changes favoring gluconeogenesis re-directed hepatic carbon output from glutamate to glucose. These findings are important for understanding the development of fetal hepatic glucose output in response to hypoglycemia. (NIH-T32-HD007186, NIH-K01-DK090199)

1-12. OVINE CLAVICULAR BROWN ADIPOSE TISSUE HAS A DIFFERENT ORIGIN FROM THE PERIRENAL DEPOT THAT IS INDICATIVE OF MUSCLE RELATED LINEAGE

ME Symonds, M Pope, M Birtwistle, V Perry and H Budge

Academic Division of Child Health, School of Medicine, University of Nottingham, Nottingham, NG7 2UH, United Kingdom

Multiple evidence from human studies indicates that the primary sites for brown adipose tissue in humans are the bilateral clavicular depots. In this study we aimed to identify an analogous region in the young sheep and whether it contained microRNA (MIR) specific to muscle or adipose tissue. This was compared with perirenal-abdominal adipose tissue which represents the major fat depot in young sheep. Four triplet-bearing mothers were entered into the study and a randomly selected triplet euthanased at 1, 7 or 28 days of age for adipose tissue sampling. Gene expression for target genes was determined by quantitative reverse transcriptase polymerase chain reaction. MIR 206 was highly abundant in skeletal muscle and was also detectable in BAT sampled from the clavicular but not perirenal depot at all sampling ages. This was despite an age related decline in UCP1 and related genes, whereas gene expression of RIP140 increased with age. Our study suggests a different cell lineage in clavicular adipose tissue compared with other BAT depots in the young sheep. Despite showing beige characteristics with increasing age, retention of some muscular origins may mean it is more response to environmental challenges aimed at promoting BAT function. The young sheep, therefore, provides a novel model for examining the developmental process of adipose transformation in early life.

1-13. EVIDENCE FOR INCREASED CORI CYCLE ACTIVITY IN IUGR FETUS

S Thorn, P Rozance, J Friedman, W Hay, L Brown

University of Colorado School of Medicine, Aurora, CO, USA

We previously showed that acute hyperinsulinemia fails to suppress the adaptive increase in hepatic glucose production and gluconeogenic gene expression in IUGR fetal sheep. IUGR fetuses also had normal to higher basal and insulin-stimulated glucose utilization rates, higher lactate, and lower oxygen concentrations, despite similar oxygen consumption rates. We hypothesize that skeletal muscle glucose utilization and subsequent lactate production is increased to fuel hepatic glucose production, defining an active Cori cycle in the IUGR fetus. Whole-body glucose metabolic studies were performed in late gestation control (CON, n=6) and IUGR fetuses (n=8) under basal and hyperinsulinemic-isoglycemic (3h) conditions. Liver and skeletal muscle samples were collected during hyperinsulinemia (n=11 CON, 12 IUGR) and from animals receiving a saline infusion (basal, n=6 CON, 7 IUGR) for gene expression analysis. Basal glucose oxidation rate was similar in CON and IUGR fetuses and increased by 50% during hyperinsulinemia. However, the fraction of glucose oxidized by the IUGR fetuses was 10% lower compared to CON fetuses during basal and hyperinsulinemia, suggesting decreased complete oxidation of glucose. IUGR fetuses also had lower lactate uptake rates from the placenta, indicating increased fetal production. IUGR muscle had higher basal expression of glycolytic genes, including phosphofructokinase (PFK1, 2-fold) and pyruvate kinase (PKM, 50%), yet higher pyruvate dehydrogenase (PDH) kinase 4 (PDK4, 10-fold, an inhibitor of PDH) and lower isocitrate dehydrogenase (IDH1, 50%), suggesting decreased glucose oxidation. IUGR liver had increased phosphoenolpyrvuvate carboxykinase, (PCK1, 30-fold), pyruvate carboxylase (PC, 2-fold), and PDK4 (5-fold) with decreased pyruvate kinase (PK, 50%) and IDH1 (50%) expression, supporting increased gluconeogenesis, increased lactate utilization, and decreased glucose oxidation. Hyperinsulinemia had little effect on these genes. These molecular and metabolic flux data support an active Cori cycle in the IUGR fetus. We speculate that increased lactate production from muscle resulting from increased glycolysis stimulates hepatic lactate utilization, a process which may play an important role for increased glucose production in the IUGR fetal liver. (NIH-DK090199)

1-14. INTRINSIC DEFICIENCIES IN MYOBLAST PROLIFERATION RESULT IN LESS DIFFERENTIATED MYOBLASTS AND SMALLER MYOFIBERS IN IUGR FETAL SHEEP

DT Yates, AR Macko, and SW Limesand

Department of Animal Sciences, The University of Arizona, Tucson, AZ, USA

Restricted skeletal muscle growth in late gestation and after birth is associated with intrauterine growth restriction (IUGR). The objective of this study was to determine whether deficient myoblast proliferation contributes to reduced skeletal muscle size in near-term IUGR fetal sheep. Compared to controls, IUGR fetuses were 67% lighter (P < 0.05) and had smaller (P < 0.05) mean semitendinosus fiber areas but similar fiber densities. Fewer (P < 0.05) nuclei stained positive for myogenin in IUGR semitendinosus and MyoD and myogenin mRNA concentrations were lower (P < 0.05) in semitendinosus muscle, which indicates less differentiated myoblasts. Furthermore, fewer (P < 0.05) Pax7-positive semitendinosus nuclei were also PCNA positive, which indicates reduced myoblast proliferation. Isolated myoblasts from both treatment groups incorporated less (P < 0.05) BrdU after being cultured in media containing IUGR fetal serum rather than control serum, which shows that IUGR serum components restrict myoblast proliferation. Moreover, IUGR myoblasts incorporated less (P < 0.05) BrdU and expressed less myoD and myogenin mRNA than control myoblasts after culture with either serum. This demonstrates that IUGR myoblasts possessed intrinsic functional deficiencies that impaired their capacity to proliferate and facilitate muscle growth. Additionally, myotubes formed in culture from IUGR myoblasts expressed less (P < 0.05) slow oxidative myosin heavy chain mRNA than myotubes from control myoblasts. Thus intrinsic deficiencies in IUGR myoblasts also alter the fiber phenotype. Together, these data begin to establish myoblast deficiencies as a cause of reduced muscle growth and impaired metabolic function in IUGR offspring. (Supported by NIH DK084842, SWL and NIFA-USDA 2012-67012-19855, DTY)

Poster Session 2—Maternal and Placental Regulation

2-1. DIET, BUT NOT ARGININE (ARG) TREATMENT, AFFECTS LEPTIN, INSULIN, AND PROGESTERONE (P4) SERUM CONCENTRATIONS IN NON-PREGNANT EWES

CS Bass¹, DA Redmer¹, SL Kaminski¹, DH Keisler², PP Borowicz¹, ST Dorsam¹, JS Haring¹ and AT Grazul-Bilska¹

¹Department of Animal Sciences, North Dakota State University, Fargo, ND, and ²University of Missouri, Columbia, MO, USA

The aim of this study was to determine the effects of diet with or without Arg supplementation on production of leptin, insulin and P4. Ewes were randomly assigned to three different nutrition groups; control (C; n=24; 2.14 Mcal/kg), overfed (O; n=27; 2xC), or underfed (U; n=31; 0.6xC), and were fed a specific diet for 60 days before Arg-treatment. Estrus was synchronized using CIDRs for 14 days. After CIDR withdrawal (one day before expected estrus; d0), ewes from each nutrition group were randomly assigned to one of two treatments: Arg (L-Arg HCl, 155 µmol/kgBW) or saline (~10 mL) administered 3 times/day from d0 through tissue collection. Blood samples were collected on d0 (estrus), 10 (mid-luteal phase), 21 (early-luteal phase) and 26 (mid-luteal phase) of Arg-treatment for evaluation of leptin, insulin and P4 serum concentration using radioimmunoassay or competitive chemiluminescent enzyme immunoassay. For leptin, there was a diet effect (P<0.01) for C, U, and O (5.40±0.81, 3.02±0.78 vs. 12.27±0.79 ng/ml, respectively) and a day effect (P<0.01) for d0, d10, d21 and d26 (8.23±0.57, 6.61±0.55, 6.91±0.73, vs. 5.83±0.71 ng/ml, respectively), and a diet x treatment x day interaction (P<0.02). For insulin, there was a diet effect (P<0.01) for C, U, and O (8.79±1.27, 4.18±1.13 vs. 15.49±1.21 µIU/ml, respectively). For P4, there was a diet effect (P<0.1) for C, U, and O (1.44±0.13, 1.22 ±0.11 vs. 1.57±0.12 ng/ml, respectively), a day effect (P<0.001) for d0, d10, d21 and d26 (0.22±0.04, 2.14±0.12, 1.13±0.14 vs. 2.16±0.14 ng/ml, respectively), and a diet x day interaction (P=0.05). These data reinforce the importance of dietary effects on endocrine function, and indicate that Arg supplementation does not alter serum leptin, insulin or P4 in ewes fed various nutritional planes. (Supported by USDA-AFRI grant 2011-67016-30174 to ATGB and DAR, and Hatch Project ND01748 to DAR).

2-2. TWO VARIANTS OF THE C-REACTIVE PROTEIN GENE ARE ASSOCIATED WITH RISK OF PRE-ECLAMPSIA IN AN AMERICAN INDIAN POPULATION

L Best¹, R Saxena², C Anderson³, G Falcon¹, C Martin¹, B Castillo⁴, K Keplin¹, N Pearson¹, F Lamb¹, S Bercier¹, B Keating⁴

¹Turtle Mountain Community College, Belcourt, ND, USA, ²Massachusetts General Hospital, Boston, MA, USA, ³University of North Dakota, Grand Forks, ND, USA, ⁴The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

The etiology of pre-eclampsia (PE) is unknown; but it is accepted that normal pregnancy represents a distinctive challenge to the maternal immune system. C-reactive protein (CRP) is a component of the innate immune system; and we previously reported an association between the CRP single nucleotide polymorphism (SNP), rs1205, and severe PE in this same cohort. Our aim was to explore the effects of additional CRP variants. This study recruited 136 cases of PE and 262 matched controls from an American Indian community. The Candidate Gene-Association Resource (CARe), 50K SNP, genotyping microarray was used and is focused on the pathophysiology of cardiovascular disease. The genotypes from 10 suitable CRP SNPs were analyzed by conditional logistic regression and other methods. A replication sample of 178 cases and 427 controls of European ethnicity was also genotyped. Nominally significant differences in the distribution of discordant matched pairs were seen for rs3093068 and rs876538. Odds ratios (ORs) in G allele dominant (rs3093068) and C allele recessive models (rs876538) were significant; but with adjustment for mother’s age, nulliparity and BMI the ORs attenuate to 1.78 (p=0.051, 95% CI 0.99 – 3.16) for rs876538. A risk score assessing either of the above two genotypes compared with neither gives an adjusted OR of 2.14 (p=0.018, 95% CI 1.16 – 4.66). The replication sample also demonstrated significant association of the rs876538 allele in an additive model (OR=1.55, p<0.01, 95% CI 2.16 – 1.10). The CRP variants rs876538 and rs3093068 are linked with cardiovascular disease phenotypes and are associated with pre-eclampsia in this American Indian population. (Supported by NIH grant P20 RR016741)

2-3. EFFECTS OF MATERNAL DIETARY TREATMENT ON UTERINE ARTERY BLOOD FLOW DURING LATE GESTATION IN BEEF COWS

LE Camacho¹, CO Lemley², KC Swanson¹, and KA Vonnahme¹

¹Department of Animal Sciences, North Dakota State University, Fargo ²Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA

The objective of this study was to examine the effects of maternal nutrient restriction followed by realimentation on uterine artery blood flow and resistance indices during late gestation. Gestating, multiparous beef cows were randomly assigned to dietary treatments: control (CCC; 100% NRC; n = 6) from day 30 until day 254 of gestation; nutrient restriction (RCC; 60% of control; n= 5) from day 30 to day 85, then realimented to control until day 254; or nutrient restriction (RRC; 60% of control; n = 6) from day 30 to 140, then realimented to control until day 254. Hemodynamic measurements of the uterine artery ipsilateral and contralateral to the conceptus were obtained via Doppler ultrasound including uterine artery blood flow (BF), pulsatility index (PI), and resistance index (RI) on day 210, 225, and 240 of gestation. There was no treatment by day interactions (P > 0.21) for any of the variables measured. Contralateral BF was not affected (P = 0.32) by dietary treatment; however, contralateral BF was increased (P < 0.01) during late gestation. Contralateral PI and RI were not affected (P > 0.65) by dietary treatment or day (P > 0.20) of gestation. Ipsilateral and total BF were increased (P < 0.05) in RCC vs. RRC and CCC cows. Ipsilateral and total BF were increased (P < 0.01) during late gestation. Ipsilateral PI was not affected (P > 0.09) by dietary treatment or day of gestation (P > 0.22); however, ipsilateral RI was decreased (P = 0.03) in RCC and RRC vs. CCC. Uterine artery BF was increased due to maternal nutrient restriction during early gestation followed by realimentation, suggesting that placental compensation can occur and augment fetal development (Funded by USDA grant 2009-65203-05812).

2-4. NITRATIVE STRESS INHIBITS HUMAN CHORIONIC GONADOTROPIN (HCG) SECRETION BY PLACENTAL CYTOTROPHOBLASTS

P Díaz, CP Sibley and SL Greenwood

Maternal and Fetal Health Research Centre, University of Manchester, Manchester, United Kingdom

Regulation of syncytiotrophoblast (STB) renewal by cellular turnover is essential for a successful pregnancy. hCG is an important autocrine/paracrine regulator of STB turnover. Pre-eclampsia is associated with abnormal STB renewal and increased nitrative stress. Here we test the hypothesis that chronic exposure to nitrative stress regulates cytotrophoblast hCG secretion and multinucleation. Cytotrophoblasts were isolated from normal term placentas (n=7) and cultured for 66h. This preparation recreates STB formation in vivo, as mononucleate cells (15h) fuse into multinucleate syncytia (66h) concomitant with elevated secretion of hCG. Cells were untreated (control) or treated at 3, 15 and 42h with ONOO- (0.1mM) or ONOO- generator 3-morpholinosydnonimine (SIN-1; 0.5mM). At 15, 42 and 66h, culture medium was collected to measure hCG (mIU/ml/mg protein) and cells fixed for immunofluorescence with anti-desmoplakin antibody to assess multinucleation (syncytial nuclei as % of total nuclei). Cytotrophoblast multinucleation increased 10-fold (p<0.0001) and hCG secretion 20-fold (p<0.0001), between 15 and 66h. Compared to controls, SIN-1 caused a significant reduction in hCG secretion of 23, 39 and 53% (all p<0.05) at 15, 42 and 66h respectively and this was not associated with any change in multinucleation. This inhibition was not caused by cytotoxicity as total nuclei and cell protein with SIN-1 did not differ from controls. Treatment with ONOO- did not affect hCG secretion/multinucleation. Generation of ONOO- by SIN-1 significantly inhibited hCG secretion from cytotrophoblasts in vitro. K+ channels are key regulators of endocrine secretion and we have previously demonstrated that 1) intermediate conductance Ca2+-activated K+ channels (IKCa) in cytotrophoblasts are activated by peroxynitrite (ONOO-) and 2) pharmacological activation of IKCa inhibits cytotrophoblast hCG secretion in vitro. We propose that nitrative stress inhibits hCG secretion through activation of IKCa. A reduction in hCG secretion could increase susceptibility to dysregulated STB renewal in pregnancy complications associated with nitrative stress. (Supported by CONICYT-Becas Chile 72090593 and Action Medical Research).

2-5. CORTISOL AND DHEA SYNTHESIS IN ADULT OFFSPRING FOLLOWING MATERNAL DEXAMETHASONE TREATMENT IN THE SPINY MOUSE

H Dickinson¹, T Quinn¹, D Walker^1,2

¹The Ritchie Centre, Monash Institute of Medical Research, ²Department of Obstetrics and Gynecology, Monash University, Australia

Antenatal stress can increase maternal plasma glucocorticoids and disturb development of the fetal hypothalamic-pituitary-adrenal axis. However, long-term effects on adrenal steroidogenesis have not been elucidated. We recently reported fetal adrenal production of dehydroepiandrosterone (DHEA) in the precocial spiny mouse (Acomys cahirinus)¹. We therefore determined the effect of brief maternal exposure to high glucocorticoids (dexamethasone, DEX) at mid and late pregnancy on adrenal structure and cortisol/DHEA secretions in adult offspring. Pregnant spiny mice were treated for 60 h with either 125µg/kg DEX or saline s.c by osmotic minipump at day 20 (0.5 term) or 30 (0.75 term) of gestation. Protein expression of tyrosine hydroxylase (TOH), steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3βHSD), 17-hydroxylase and 17-20lyase (p450c17), and cytochrome b5 (cytb5) were determined by immunohistochemistry in adrenal glands at 20-25 weeks of age. DHEA and cortisol were measured by radioimmunoassay. Maternal DEX at 20 days gestation did not affect adrenal structure, but expression of StAR, p450c17, and cytb5 were significantly reduced in the adult adrenal zona reticularis (ZR), with significantly greater change in male vs female offspring (p<0.05). Plasma DHEA was significantly decreased in male offspring from DEX-treated (3.8 ± 0.6 ng/ml) vs saline-treated (8.6 ± 0.8 ng/ml; p=0.001) dams; plasma cortisol was unaffected in either sex, so that the DHEA:cortisol ratio was significantly lower in males only (p<0.05). DEX treatment at 30 days of gestation had no effect on adrenal steroid enzyme expression or plasma cortisol or DHEA in either sex at 20-25 weeks of age. Brief exposure to excess glucocorticoid has a long-term impact on the ZR and adrenal steroidogenesis, affecting secretion of DHEA in male offspring, an effect produced at 0.5 but not 0.75 gestation. DHEA is important for brain development, and its suppression in adult life might contribute to the neurobehavioral pathologies that can arise after illness and stress during pregnancy. ¹Quinn et al, Endocrinology 154:1190-201.

2-6. MATERNAL DIETARY CREATINE SUPPLEMENTATION PREVENTS LONG-TERM DEFICITS CAUSED BY BIRTH ASPHYXIA

H Dickinson¹, D LaRosa¹, S Ellery¹, E Pereleshina¹, Z Ireland², R Snow³, P Temple-Smith⁴ and D Walker^1,4

¹The Ritchie Centre, Monash Institute for Medical Research, ²University of Queensland Centre for Clinical Research, Brisbane, ³Centre for Physical Activity and Nutrition Research, Deakin University, ⁴Department of Obstetrics and Gynaecology, Monash University

Using a model of birth asphyxia (BA) in the spiny mouse, we have shown that a creatine-enriched diet from mid-pregnancy improves postnatal survival and growth¹, and prevents brain², diaphragm³ and kidney⁴ injuries at 24h of age. This study assessed the potential for maternal creatine supplementation to provide long-term protection to these organs, and also investigated the effects of BA on the ovary. Pregnant dams were fed a control or 5% creatine-supplemented diet from gestation day 20 (term 39 days). On day 38, pups were delivered by c-section, or after BA induced by placing the excised uterus containing all fetuses in a saline bath for 7.5-8mins, after which time the fetuses were expelled and resuscitated. Surviving neonates were cross-fostered to a nursing dam until 33 days of age when isolated diaphragm muscle and intact heart function was assessed in vitro, and ovarian follicle and nephron number were determined by unbiased stereology. BA resulted in increased fatigue and reduced maximum tetanic force of diaphragm muscle, and impaired cardiac contractility with end diastolic pressures remaining at ~20mmHg, compared to age-matched controls. BA reduced ovarian follicular reserve by 32%, an effect minimized (20%) in females from creatine-fed mothers. Preliminary nephron number calculations show a significant deficit after BA, the effects of creatine remain to be determined. Asphyxia induced compromise of diaphragm and cardiac function, nephron number and ovarian follicular reserve persists until at least 33 days of age, effects prevented/minimized by maternal creatine loading during pregnancy. ¹Ireland Z, et al. Am J Obstet Gynecol 2008;198:431 e1-6; ²Ireland Z, et al. Neuroscience 2011;194:372-9; ³Cannata DJ, et al. Pediatr Res 2010;68:393-8; ⁴Ellery S, et al Pediatr Res 2013;73(2):201-8.

2-7. RUMEN-PROTECTED ARGININE SUPPLEMENTATION ALTERS CAROTID ARTERY HEMODYNAMICS IN EWES

K Egeland¹, JL Peine¹, EE Gaspar¹, ME Kapphahn¹, NM Chapel¹, PP Borowicz¹, AM Meyer², LP Reynolds¹, and JS Caton¹

¹Department of Animal Sciences and Center for Nutrition and Pregnancy North Dakota State University, Fargo ²Department of Animal Science, University of Wyoming, Laramie

Our hypothesis was that rumen-protected arginine supplementation would alter ovine carotid artery hemodynamics in a manner consistent with increased tissue perfusion. To test this hypothesis, nulliparous Rambouillet ewes (n = 60; 51 ± 1.4 kg body weight) were allocated to one of 4 treatments including rumen-protected arginine (RP-ARG) supplementation at 0, 90, 180, or 360 mg/kg BW fed once daily in a 50 g fine ground corn carrier. Following supplement consumption, ewes were offered a complete pelleted diet. Ewes were penned individually in a temperature-controlled facility and adapted to the pelleted diet. Before initiation of RP-ARG treatments, baseline carotid hemodynamic measurements were acquired via color Doppler ultrasound. These measurements were replicated beginning 6 d post-treatment initiation and continued every 3 d for a total of 4 post-baseline measurements. Data were expressed as percentage change from baseline measures and normalized to the control RP-ARG treatment 0. No period × treatment interactions were observed. Contrasts were used to address 3 specific questions; 1) was there an effect of RP-ARG, 2) was the effect of RP-ARG linear, and 3) was the effect of RP-ARG quadratic. Pulsatility index (PI) and resistance index (RI) percentage changes from baseline were decreased (P ≤ 0.02) by RP-ARG and the response was quadratic (P ≤ 0.03) with increasing dose of RP-ARG (0.00, -6.99, -8.30, -5.10 ± 2.53% for PI, and 0.00, -3.28, -3.60, -0.92 ± 0.93% for RI, respectively). Heart rate, end diastolic velocity, and mean velocity were increased (P ≤ 0.02), while peak systolic velocity tended (P = 0.08) to increase in ewes fed RP-ARG. Additionally, these responses were linear (P ≤ 0.06) with increasing RP-ARG. Flow volume and cardiac output were not altered by treatment (P > 0.60). These results confirm our hypothesis that RP-ARG alters ovine carotid artery hemodynamics.

2-8. EFFECT OF TRICLOSAN ON GLOBAL GENE EXPRESSION IN THE LATE GESTATION OVINE PLACENTA

A Grapes, MB Rabaglino and CE Wood

Department of Animal Sciences, Department of Physiology and Functional Genomics, University of Florida Colleges of Agriculture and Life Sciences and Medicine

Triclosan is an antibacterial agent found ubiquitously in man-made health care products, plastics, and textiles. It has been detected in maternal serum (1-2 ng/mL), breast milk (60-300ng/g lipid), amniotic fluid (8.3-8.7 ng/mL), fetal plasma (0.2-9.2 ng/mL) and placental tissue (1-104.9ng/mL). Like the similar compounds bisphenol-A (BPA) and polychlorinated biphenyl (PCB), triclosan has been shown to act as a xenoestrogen. For example, triclosan increases uterine weight and advances the onset of puberty in rats. It has also been shown to disturb placental steroidogenesis in vitro by increasing estradiol (E2) and progesterone concentrations. Triclosan is also a potent inhibitor of E2 and estrone sulfonation in vitro. This study was designed to test the hypothesis that triclosan would disrupt estrogen-sensitive pathways important to late gestation because of its ability to inhibit estrogen sulfonation and thus, HPA maturation and activation. We chronically catheterized sheep at 120 days of gestation and after the recovery period, each fetus was subjected to a continuous two-day infusion of either 200µg/kg triclosan (n=6), 500µg/kg estradiol (n=4), or saline (n=5). After the infusion period, fetal tissues were collected, including the perirenal fat, hypothalamus and placental cotyledons. An Agilent ovine array containing 15,744 probes and annotated with homologous human protein names was used for global transcriptomic analysis of the placenta. Compared to the control, triclosan and E2 differentially expressed 957 and 895 genes, respectively (p < 0.05). Of these genes, the two treatment groups only differentially expressed 7.4% (128) of the same genes, signifying a potent non-estrogenic effect of triclosan. KEGG pathway analysis reveals an up-regulation of adipocytokine signaling and pro-inflammatory cytokines, as well as altered lipid, sterol, and glucose biosynthesis and metabolism. Triclosan expression patterns are similar to those seen in a gestational diabetes mellitus placenta - including changes related to villus branching, NO signaling, energy balance, inflammation, insulin sensitivity, and nutrient metabolism.

2-9. ANALYSIS OF EXPRESSED BREAST MILK PROTEIN CONTENT PREDICTS ACCUMULATED PROTEIN DEFICIT AFTER PRETERM BIRTH

S Khaira, A Maraglino, K Harvey-Wilkes, M Volpe

Department of Newborn Medicine, Floating Hospital for Children at Tufts Medical Center, Boston, Massachusetts, United States

Expressed Breast Milk (EBM) fortification for preterm infants assumes equal EBM protein content using published mean protein values. Studies show that EBM protein content is highly variable between mothers and is often below published levels. Since EBM protein actually decreases with advancing weeks of lactation the current approach may result in significant protein deficit over time. The objective of this study was to determine if measured EBM protein content defines a significant degree of accumulated protein deficit over time after preterm birth. The Julie Z7 milk analyzer (Scope Electric Ltd, Germany) was used to determine EBM protein content at day of life (DOL) 10 and every 2 weeks thereafter from mothers of 10 preterm infants at 24-29 6/7 weeks gestational age at birth. The anticipated protein deficit based on milk analysis was calculated up to 12 weeks postnatal age and compared to presumed protein intake using current published mean EBM protein levels (1.4 g/dl). EBM protein content was 1.3 g/dl ± 0.04 (mean ± SD) at DOL 10 which is 7% below published estimates for EBM protein content. Assuming protein content from EBM analysis is not utilized but current published mean EBM protein levels are used for protein fortification, these infants will accumulate an average protein deficit of 25.2 grams by 12 weeks postnatal age. Without use of specific EBM protein analysis, preterm infants will accumulate significant protein deficit over time, potentially compromising growth and neurodevelopment. We speculate that individualized EBM fortification may better optimize nutrition and facilitate ideal growth and neurodevelopmental outcomes in preterm infants. A prospective, randomized, blinded and gestational age stratified trial is underway to test this hypothesis.

2-10. SEXUAL DIMORPHISM IN THE EFFECT OF MATERNAL ADIPOSITY ON PLACENTAL miR-210 EXPRESSION

S Muralimanoharan, C Guo, L Myatt, and A Maloyan

Center for Pregnancy and Newborn Research, Dept of Ob/Gyn, University of Texas Health Science Center San Antonio, Texas, USA

Obesity in pregnancy, which leads to developmental programming, is associated with oxidative stress and mitochondrial dysfunction in the placenta. MicroRNAs (miRs) regulate various physiological and pathological events in placenta. We demonstrated increased placental miR-210 with preeclampsia and a causative role for miR-210 in mitochondrial dysfunction in trophoblast. We tested the hypothesis that miR-210 expression is dysregulated in the placenta with increasing maternal adiposity. Placentas of both male and female fetuses were collected from healthy lean (LN: BMI 19-24.9), overweight (OW: BMI 25-29.9) and obese (OB: BMI 30-45) women (n=6 each sex/group) after C-section at term with no labor. Expression of miR-210 and its mitochondrial target genes were analyzed by RT-PCR and Western Blot. No differences in placental miR-210 expression were seen with increasing adiposity with a male fetus. However, in placentas of female fetuses miR-210 expression was upregulated 3-fold in OW and 2-fold in OB compared to LN women (p<0.05). Correspondingly miR-210 target genes, Iron Sulfur Cluster assembly proteins, NADH dehydrogenase (ubiquinone) 1α subcomplex 4 and succinate dehydrogenase complex, subunit B, were significantly reduced in female placentas with increasing maternal adiposity (p<0.05). No statistically significant changes in these genes were found in male placentas. No changes were detected in placental HIF-1α, previously shown to regulate miR-210. Rather, we found a significant increase in NFκB p65 protein expression in both male and female placentas of OW and OB women vs. LN (p<0.05). ChIP analysis confirmed miR-210 expression can be regulated by binding of NFκB p65 to miR-210 promoter. We found a 2-fold increase in p65 binding to the miR-210 promoter in female but not male placentas of OW and OB vs. LN women (p<0.05). We show a sexual dimorphism in placental miR-210 expression with increasing maternal adiposity, which may at least be partially regulated by the NFκB pathway. (Supported by NIH HD075297)

2-11. INFLUENCE OF MATERNAL ADIPOSITY ON HUMAN PLACENTAL METHYLOME

L Myatt¹, A Parker¹, L Liu², J Ruan² and K Mitsuya¹

¹Center for Pregnancy and Newborn Research, Department of Ob/Gyn, University of Texas Health Science Center at San Antonio, Texas, USA, ²Department of Computer Science, University of Texas at San Antonio, Texas, USA

Over 60% of women of child bearing age in US are overweight or obese. Maternal obesity has both immediate effects on pregnancy outcome but also programs the offspring for obesity, metabolic syndrome and cardiovascular disease in adult life. The programming effect of the adverse intrauterine environment is transduced through the placenta. The interaction of environmental factors such as maternal diet with the genome may cause epigenetic changes that alter placental function. We hypothesized that increased maternal adiposity would affect the placental epigenome at the level of DNA methylation. Villous tissue was collected following cesarean section at term prior to labor from normal weight (pre-pregnant or 1st trimester BMI=23.4±2.3) and obese (BMI=34.0±2.9) women (n=10 each group). Five random samples of tissue were collected from each placenta and flash frozen. Random tissue samples were combined, genomic DNA isolated from each of the 10 placentas per group, pooled into two separate groups (normal and obese) and subjected to MeDIP-chip analysis using NimbleGen 2.1M human DNA methylation array. Initially we isolated differential methylation in the regions -500 to +500bp from transcriptional start sites. MeDIP-chip analysis identified 23,082 and 24,399 methylated regions in normal and obese placentas, respectively. We identified 21 genes where DNA methylation was significantly altered (19 increased and 2 decreased methylation) in response to maternal adiposity. The differentially methylated genes identified include those involved in placental development, cell migration, vascular remodeling, increasing BMI, lipid transport, energy metabolism, immune and/or inflammatory responses. qRT-PCR analysis demonstrated down-regulation of the differentially methylated genes that are located at the growth hormone and pregnancy-specific glycoprotein gene clusters on chromosomes 17q and19q, respectively.

2-12. GENERATION OF CYTOTROPHOBLAST-LIKE CELLS FROM HUMAN EMBRYONIC STEM CELLS IN DEFINED MEDIA

MM Parast, Y Li, MM Zita, F Soncin, A Wakeland, LC Laurent

Departments of Pathology and Reproductive Medicine, UC San Diego, La Jolla, CA, USA

Human trophoblast lineage-specific differentiation is difficult to study, due to a lack of a multipotent “trophoblast stem cell” (TSC) model. First trimester human cytotrophoblast (CTB) are bipotential, with the ability to differentiate into both hCG-secreting syncytiotrophoblast (STB) and HLAG-positive extravillous trophoblast (EVT); however, access to large numbers of such cells is limited. Human embryonic stem cells (hESCs) can be differentiated into trophoblast using BMP4 in presence of feeder-conditioned media (FCM); however, these conditions are inherently inconsistent and often result in a heterogeneous population of cells. We set out to find defined media for differentiation of hESCs into bipotential CTBs. Feeder-free hESCs (WA09/H9) were cultured in StemPro+bFGF. To differentiate the cells into CTB, cells were switched to minimal media for two days, then treated with BMP4 (10 ng/ml) for an additional four days. RNA was isolated and subjected to microarray analysis, alongside RNA from first trimester CTB, placental stroma, amnion epithelial cells, JEG3, and BeWo cells. Treated hESCs were also evaluated by immunostaining for markers of pluripotency and CTB. After four days in minimal media plus BMP4, cells downregulated the pluripotency marker OCT4, and induced CTB markers KRT7, EGFR, and p63. Microarray analysis revealed these cells to cluster, along with JEG3 and BeWo cells, away from amnion and placental stroma, and close to primary CTBs. These cells could also be replated, and when cultured in FCM plus BMP4, they proceeded to differentiate into both hCG-secreting STB, and HLAG-positive EVT. In summary, we have successfully established a reproducible in vitro model, using defined media, to derive CTB-like cells from hESCs. These cells offer a much improved alternative to the aneuploid human trophoblast cell lines, and, with the ability to differentiate into both STB and EVT, can be used as a model for investigating trophoblast lineage-specific differentiation. (Supported by CIRM grant RN2-00931).

2-13. NUTRIENT RESTRICTION DURING EARLY AND MID-GESTATION FOLLOWED BY REALIMENTATION ALTERS COTYLEDONARY ARTERIAL VASOREACTIVITY IN RESPONSE TO BRADYKININ IN BEEF COWS DURING LATE GESTATION

A Reyaz¹, MS Sane², F Yao³, LE Camacho¹, CO Lemley⁴, KC Swanson¹, ST O’Rourke², and KA Vonnahme¹

¹Departments of Animal Sciences and ²Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND, USA ³Department of BSOM Pharmacology, East Carolina University, Greenville, NC, USA ⁴Department of Animal and Dairy Sciences, Mississippi

This study was designed to examine the effects of maternal nutrient restriction during early and mid-gestation followed by realimentation on cotyledonary artery vasoreactivity in response to bradykinin (BK) in beef cows. Cows received 100% (CCC; n=5) of recommended nutrients from d 30 to 254 (gestation length = 280 d), 60% (RCC; n=4) of CCC from d 30 to 85 with realimentation on d 85 until 254, or 60% (RRC; n=5) of CCC from d 30 to 140 with realimentation until d 254 of gestation. At d 254 of gestation, cows were euthanized and arteries terminating into the fetal portion of the placentome were used for in vitro vasoreactivity assay using wire myography. Endothelium intact rings were contracted with U46619 (10-6 M) and a concentration response curve to bradykinin (BK) was obtained for rings incubated for 30 min with inhibitors to: 1) BK2 receptors, HOE-140 (10-6); 2) large conductance Ca2+-activated K+ channels (BKCa), iberiotoxin (IbTX;10-7 M); and 3) endogenous nitric oxide (NO) synthase; N(nitro)-L-arginine (NLA; 10-5). By d 254, maternal diet had no effect (P>0.10) on COT arterial sensitivity to BK induced relaxations. When incubated with HOE-140, BK-induced relaxation was blocked in all cows. Relaxation in CCC and RRC cows was not blocked (P≥0.81) by IbTX. However there was a treatment x dose interaction (P=0.01) observed in RCC cows, where IbTX blocked BK-induced vasodilation at greater BK doses. NLA blocked (P<0.01) BK-induced relaxation in all treatments. Data suggests that BK-induced vasorelaxation is being mediated by NO and there may be a role for BKCa channels in RCC cows. (Supported by Grant no. 2009-65203-05812 from the USDA National Institute of Food and Agriculture)

2-14. IN VIVO STUDIES OF GLUCOSE TRANSPORT OVER THE HUMAN PLACENTA

MCP Roland¹, AM Holme¹, B Lorentzen¹, TM Michelsen¹, T Henriksen^1,2

¹Department of Obstetrics, Oslo University Hospital, Norway, ²University of Oslo, Norway

With the aim to perform studies of functional properties of placenta in the human we have established a method to obtain arterial and venous blood samples from both sides of placenta in vivo. We present our initial data by describing glucose concentrations and gradients on both sides of placenta. The study include women (n=39) with uncomplicated pregnancies undergoing planned caesarean section. Fasting blood samples were obtained from the radial artery (substitute for uterine artery) and the uterine vein just before uterine incision, and the umbilical artery and vein immediately after cord clamping. Mean glucose concentration in the radial artery was 4.71 mmol/L (SD 0.48), in the uterine vein 4.41 mmol/L (SD 0.45), in the umbilical vein 3.87 mmol/L (SD 0.41), and in the umbilical artery 3.48 mmol/L (SD 0.45). The maternal arteriovenous glucose gradient was 0.29 mmol/L (SD 0.23), p<0.001. The glucose gradient on the fetal side was 0.38 mmol/L (SD 0.31), p<0.001. The gradient between the radial artery and the umbilical vein (maternal-fetal gradient) was 0.84 mmol/L (SD 0.25), p<0.001.Based on the measured concentrations and calculated gradients, the placenta extracted 6% of maternal arterial glucose levels. At the same time, the fetus consumed 10% of available glucose in the umbilical vein. There was a significant correlation between maternal arterial glucose concentration and the glucose concentration in the umbilical vein (r=0.86, p<0.001), whereas the correlation between maternal arterial glucose concentration and the glucose gradient on the fetal side was non-significant (r=0.30, p=0.07). Assuming a utero-placental blood flow of 1.0 L/min and a feto-placental flow of 0.4 l/min approximately 50 % of the glucose extracted from the maternal circulation was retained in the placenta. We have established a human in vivo model which provides novel data on placental glucose transport. (Supported by Norwegian Resource Centre for Women’s Health, Oslo University Hospital, Norwegian Health Association and Norwegian Extrafoundation for Health and Rehabilitation)

2-15. DIFFERENTIAL DNA METHYLATION IN GENES ASSOCIATED WITH IMMUNE FUNCTION IN PREGNANT WOMEN WITH GROUP B STREPTOCOCCUS COLONIZATION

ML Wright¹, CM Anderson¹, and JE Ohm²

¹College of Nursing and Professional Disciplines, ²School of Medicine and Health Science, University of North Dakota, Grand Forks, North Dakota, USA

In order to determine the feasibility of evaluating DNA methylation patterns as a contributing factor for group B Streptococcus (GBS) colonization, we analyzed genome wide DNA methylation patterns in peripheral white blood cells of pregnant women with and without GBS colonization for association with immune response. Secondary analysis of DNA methylation data was completed to identify potential differences in pregnant women with and without GBS colonization in early pregnancy. A delta-beta criteria of >0.2 or <-.02 was used to determine a significant gain or loss in methylation, representing a 20% change respectively. Significance in mean methylation changes was determined at p<0.05. To identify potential for clinically relevant findings, the function of differentially methylated genes was categorized using DAVID Bioinformatics Resources v6.7. DNA methylation in >1,000 CpG sites differed by 20% or more in GBS positive versus GBS negative women. 350 CpG sites remained significantly different when a more stringent 30% change in methylation was applied. Genes with methylation changes were associated with immune response, histocompatibility, immunoglobulin, cytokine production, and the intestinal immune network for IgA production. The preliminary data suggest DNA methylation changes in genes associated with immune function may contribute to GBS colonization susceptibility. (Supported by International Society of Nurses in Genetics Research Grant & Midwest Nursing Research Society Dissertation Grant (MLW); Robert Wood Johnson Nurse Faulty Scholar Award (CMA, 64202))