treatment of intrauterine growth restriction with maternal growth hormone supplementation in sheep

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ASIC SCIENCE: OBSTETRICS

reatment of intrauterine growth restriction with maternalrowth hormone supplementation in sheep

endrina A. de Boo, PhD; Simona C. Eremia, MSc; Frank H. Bloomfield, FRACP, PhD;ark H. Oliver, PhD; Jane E. Harding, FRACP, DPhil

BJECTIVE: This study was undertaken to investigate whether maternalrowth hormone supplementation in pregnant sheep could reverse intra-terine growth restriction (IUGR) induced by placental embolization.

TUDY DESIGN: Animals were randomized into control, intrauterinerowth restriction � saline or intrauterine growth restriction � growthormone (twice daily injections of 0.1 mg/kg growth hormone) groups.ntrauterine growth restriction was induced by twice daily placental em-olization between 93 and 99 days’ gestation, and treatment was from00-128 days’ gestation (term � 147 days’ gestation).

ESULTS: Embolization reduced fetal growth rate and body weight

normal, pulsatile, GHoi: 10.1016/j.ajog.2008.04.035

ent significantly increased fetal growth rates and fat deposition,nd improved fetal body weight and length, but not liver weight.rowth hormone treatment produced hydranencephalic brain le-ions in some fetuses.

ONCLUSION: Maternal growth hormone treatment partially reversedntrauterine growth restriction caused by placental insufficiency. How-ver, the possible connection between growth hormone treatment andetal brain injury requires further investigation

ey words: brain, fetus, growth hormone, intrauterine growth
ut increased brain-to-liver weight ratio. Growth hormone treat- restriction, sheep
ite this article as: de Boo HA, Eremia SC, Bloomfield FH, et al. Treatment of intrauterine growth restriction with maternal growth hormone supplementation inheep. Am J Obstet Gynecol 2008;199:559.e1-559.e9.

ntrauterine growth restriction (IUGR)is a major cause of perinatal mortality

nd morbidity, and is associated withany health problems throughout life.1 In

urrent neonatal practice, perinatal man-gement of IUGR infants is primarilyimed at determining the right time for de-ivery of the infant, followed by neonatalietary intervention to achieve optimalrowth rates.2 It has been proposed thateversing some of the metabolic and endo-rine characteristics of IUGR in utero mayrevent some of its short- and long-termegative consequences.3 However, to date,

there are no available intrauterine treat-ments to improve the growth patterns ofthe growth-restricted fetus.

There are limited data showing thatIUGR in the sheep fetus can both be pre-vented and reversed by therapeutic manip-ulation of the fetus itself. Both intravenousand intragastric nutritional supplementa-tion of the sheep fetus prevented the onsetof experimentally induced IUGR,4,5 andboth fetal intravenous and intraamnioticsupplementation with insulin-like growthfactor-I (IGF-I) improved the fetal growthrate after IUGR was established.6 How-ever, these treatments require access to thefetal compartment, with the concomitantincreased risk of infection and preterm la-bor. Other studies have attempted to im-prove fetal growth by alterations in mater-nal nutrition.7 However, as most cases ofIUGR in the developed world are causedby placental factors, thus perturbing the fe-tal nutrient supply line,3 these attemptshave been largely unsuccessful.7

During human pregnancy, the pla-centa produces growth hormone (GH),which is continuously secreted into thematernal circulation and suppresses

maternal pituitary.8 The resulting in-crease in circulating GH causes relativeinsulin resistance in the mother, direct-ing maternal metabolism away from glu-cose use and toward lipolysis and proteinanabolism,9 thereby increasing the sub-strate availability for the fetus and pla-centa. Exogenous GH administrationduring gestation has been shown to fur-ther increase maternal insulin resistancein various species10-12 and to help redis-tribute available substrates between themother and fetus. In addition, GH sup-plementation in pregnant sheep in-creases placental capacity for simple dif-fusion,10 potentially further increasingsubstrate supply to the fetus.

Despite these effects of GH supple-mentation to increase substrate avail-ability for the fetus, previous attempts toimprove growth of the normal fetus bymaternal GH supplementation have hadvarying success. Although enhanced fetalgrowth has been reported in some stud-ies, the effects appear to depend at leastin part on maternal nutritional status aswell as the dose, duration, and timing ofthe GH supplementation.13-16 There

rom Liggins Institute, Faculty of Medicinend Health Services, University of Auckland,uckland, New Zealand.

eceived Sept. 4, 2007; revised Jan. 20, 2008;ccepted April 17, 2008.

eprints: Prof. J. E. Harding, The Ligginsnstitute, University of Auckland, Private Bag2019, Auckland, New Zealand. E-mail:

[email protected].

his study was funded by the Health Researchouncil of New Zealand.

002-9378/$34.002008 Mosby, Inc. All rights reserved.

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5

ng the potential use of maternal GHupplementation as a therapeutic ap-roach for the intrauterine treatment ofstablished IUGR. We therefore under-ook this study to test the hypothesis thatrolonged late gestation GH supplemen-ation in pregnant sheep will reverse therowth restriction induced by placentalmbolization.

ATERIALS AND METHODSnimal preparationregnant Romney ewes carrying Dorsetrossbreed singleton fetuses were housedn individual cages with free access to wa-er and pelleted food. All experimentsere approved by the University ofuckland Animal Ethics Committee.After acclimatization to laboratory

onditions, the animals underwent sur-ery under halothane anesthesia on day0 of gestation (90 dGA). Streptopen250 mg procaine penicillin/250 mg di-ydrostreptomycin sulphate; Pittmanoore Ltd, Upper Hutt, New Zealand)as administered intramuscularly be-

ore surgery. Fetal carotid artery and jug-lar vein, maternal uterine arteries, ma-

ernal femoral artery and vein, andaternal carotid and jugular vein were

atheterized. Growth catheters were in-erted subcutaneously around the fetalhest, as described previously.10 Amni-tic catheters were inserted into the am-iotic sac, and 80 mg gentamicin (Phar-acia Pty Ltd, Bentley, Australia) was

dministered into the amniotic fluid.

mbolizationheep were allowed to recover for 3 daysfter surgery. Between 93 and 99 dGAterm � 145 dGA), growth restrictionas induced by twice daily embolizationf maternal uterine arteries with polysty-ene microspheres of 20-50 �m diameterSuperose 12 diluted 1:100; Pharmaciaiotech AB, Uppsala, Sweden). Emboli-ation was discontinued if fetal arterialaO2 fell below 19 mm Hg, fetal lactateoncentrations were more than 2 mM orf chest girth had not increased for 2 con-ecutive days.

tudy protocolefore surgery, sheep were randomly as-

igned to 1 of 3 experimental groups: t

59.e2 American Journal of Obstetrics & Gynecolo

Control (no embolization, no treat-ent, n � 10), saline (SAL) (IUGR �

hrice weekly injections of 2 mL salineolution into the amniotic fluid, n � 13),nd GH (IUGR � twice daily injectionsf 0.1 mg/kg bGH [American Cyanamido, Princeton, NJ] to the ewe intramus-

ularly, n � 9). Treatments commencedn 100 dGA and were continued for 28ays. The control and SAL animals haveeen described previously.6

Maternal and fetal blood and amnioticuid samples were collected into hepa-inized tubes on ice every 2-3 dayshroughout the study. Aliquots weresed to measure blood gases on a Chiron845 blood gas analyzer (Chiron Corp,

meryville, CA). Remaining samplesere centrifuged at 3000g at 4°C for 15inutes, and the plasma stored at �80°C

or further analysis.At 128-131 dGA, sheep were killedith an overdose of pentobarbitone. Theterus and its contents were weighed and

etal external body measurements wereaken. Fetal organs were dissected andeighed.

ssaysecause measuring all metabolites andormones at each time point for morehan 30 animals would have been im-ractical, only some samples were se-

ected for biochemical analysis. Metabo-ites were measured in samples taken athe beginning of the embolization andreatment periods (93 and 100 dGA), ev-ry 6 days during the treatment periodnd the last 3 samples before the experi-ent ended. Hormones were measured

n samples taken at the beginning of thembolization and treatment periods (93nd 100 dGA) and at 3 time points dur-ng the treatment period (106, 118, and27 dGA).Plasma glucose, lactate, urea, and free

atty acid (FFA) concentrations wereeasured on a metabolite analyser

Roche/Hitachi 9002 Analyser; Hitachiigh-Technologies Corporation, To-

yo, Japan).Insulin-like growth factor I (IGF-I)as measured by nonextraction double

ntibody radioimmunoassay, with theddition of IGF-II in excess for elimina-
ion of interference from binding pro- i
gy NOVEMBER 2008

eins.17 Rabbit anti-IGF-I serum wassed as the primary antibody and theecond antibody was 2% sheep antirab-it gamma globulin with 0.01% normalabbit serum and 8% PEG 6000 in 0.01

phosphate-buffered saline solutionPBS). The intraassay and interassay co-fficients of variation were 4.5% and8%, respectively. Insulin concentra-ions were measured by radioimmuno-ssay, with guinea pig antiovine insulins the first antibody and 8% PEG 6000,ith 0.5% sheep antiguinea pig and 0.1

normal guinea pig serum in 0.01 MBS as the second antibody.18 The in-

raassay and interassay coefficients ofariation for insulin were 7.3% and 16%,espectively.

ata analysisn case of fetal loss before completion ofhe study, data from the last 10 days be-ore death were excluded to avoid biaselated to the cause of death. Only ani-als that completed the study up to 127

GA were included in the analysis of theostmortem data.Growth catheter measurements were

ormalized for 100 dGA and analyzedeparately for embolization and treat-

ent periods by multiple linear regres-ion, with animal number nested withinreatment group and gestational age asndependent variables.19 Postmortemata were analyzed by factorial analysisf variance (ANOVA) with Fisher postoc correction.For analysis of blood gases and metab-

lites repeated measures ANOVA couldot be performed because of missingariables. Therefore, the entire study pe-iod was divided into 6 experimental pe-iods: 92-93 dGA (period 0, preemboli-ation), 98-100 dGA (period 1, lasteasurement of the embolization pe-

iod), 101-107 dGA (period 2), 108-115GA (period 3), 116-123 dGA (period), and 124-128 dGA (period 5). The re-ults were averaged for each individualnimal for each period.

Blood gas, hormone, and metaboliteata were analyzed separately for embo-

ization and treatment periods. The ef-ects of embolization were analyzed bysing repeated measures ANOVA, tak-

ng into account embolization (before vs

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www.AJOG.org Basic Science: Obstetrics Research

fter), group (control, saline solution, orH), and embolization � group interac-

ion. Treatment effects (periods 1-5)ere analyzed by factorial ANOVA withukey’s HSD corrections (P � .05) with

reatment group, time, and group �ime interactions as the statistics of inter-st. Time effects were also analyzed forach group individually by the samepproach.

All analyses were performed with Stat-iew or JMP (both SAS Institute, Cary,C). Data are presented as number,edian (range) or mean � SE, as

ppropriate.

ESULTShirty-three animals were studied. Six of0 animals in the control group, 9 of 13nimals in the SAL group, and 5 of 10nimals in the GH group completed thetudy. Two ewes in the GH grouptopped eating and were withdrawnrom the study after 19 and 24 days ofreatment. Other losses were as follows: 1reterm delivery and 1 catheter failure inach of the 3 treatment groups, 2 fetaleaths in each of the control and SALroups, 1 fetal death in the GH group.

ffects of embolizationlacental embolization reduced fetal

FIGURE 1Fetal chest girth increments

-20 0

20 40 60 80

100 120 140 160 180

irth increment (mm)

93 100 107 114 121 128 Gestational age (d)

CG

alues are normalized for 93 dGA and expresseds mean � SE. � � control (n � 6); Œ �AL (n � 10); □ � GH (n � 5). The shadedorizontal bar indicates period of embolization,he white bar indicates the treatment period.

ean daily girth increments are reduced in bothAL and GH groups during the embolizationeriod (significance not indicated in figure). c P

.05 vs controls during the treatment period;P � .05 vs GH during the treatment period.

e Boo. Treatment IUGR with maternal growth hormoneupplementation in sheep. Am J Obstet Gynecol 2008.

rowth as shown by reductions in girth

ncrements in both embolized groupsuring the embolization period (93-99GA, controls 2.55 � 0.10 mm/d, SAL.79 � 0.15 mm/d, and GH 1.75 � 0.15m/d; SAL and GH both P � .05 com-

ared with control, Figure 1). Fetaleight and crown-rump length were re-uced at postmortem in the SAL groupTable 1). Fetal biparietal diameter, chestnd abdominal girths, and limb lengthst post mortem were not significantly re-uced by embolization. However, embo-

ization reduced weights of fetal liver,eart, thyroid, and perirenal fat and in-reased the brain-to-liver weight ratioTable 2).

Embolization reduced fetal PaO2 byore than 25% in both embolized

roups (Table 3). Fetal hemoglobin val-es increased over the embolization pe-iod in all groups. Embolization did notffect fetal plasma glucose concentra-ions, but maternal-to-fetal glucose ra-ios increased significantly in both em-olized groups. Fetal plasma ureaoncentrations and maternal-to-fetalrea ratios were not different amongroups. Fetal plasma lactate concentra-ions increased 2-fold after emboliza-ion. The increases in fetal plasma IGF-Ind insulin concentrations seen in theontrol animals over the embolizationeriod did not occur in the embolizedroups. Maternal plasma IGF-I and in-ulin concentrations increased to theame extent in all groups.

TABLE 1Gestational age and fetal body mea

Control (n � 6)

Gestational age (d) 127.2 � 1.6...................................................................................................................

Fetal weight (g) 3906 � 253...................................................................................................................

CRL (cm) 46.8 � 1.7...................................................................................................................

BPD (mm) 63 � 2...................................................................................................................

Chest girth (cm) 34.3 � 0.5...................................................................................................................

Abdominal girth (cm) 35.2 � 1.1...................................................................................................................

Forelimb length (cm) 27.7 � 0.7...................................................................................................................

Hindlimb length (cm) 33.7 � 1.1...................................................................................................................

Values are mean � SE.BPD, biparietal diameter; CRL, crown-rump lengtha P � .05 compared with control (factorial ANOVA with Fish

de Boo. Treatment IUGR with maternal growth hormone supp


ffects of growth hormonereatmentH treatment increased fetal chest girth

ncrements from 1.75 � 0.15 mm/d to.36 � 0.09 mm/d (P � .05); whereashest girths in the control or SAL groupsid not change over the treatment periodcontrol group from 2.55 � 0.10 mm/do 2.49 � 0.05 mm/d; SAL group from.79 � 0.15 mm/d to 2.19 � 0.08 mm/d;igure 1). Throughout the treatment pe-iod, girth increments in the GH groupere higher than those of the SAL group

2.36 � 0.09 mm/d and 2.18 � 0.08m/d, respectively; P � .05).At postmortem, fetal length andeight in the GH group were interme-iate between the control and SALroups (Table 1), but chest and ab-ominal girths and limb lengths wereot different among groups. Heart and

hyroid weights partially recovered anderirenal fat weights recovered com-letely after GH treatment but livereights were not affected (Table 2).rain weights in the GH group were

ignificantly lower than those of theAL and control groups, caused by hy-ranencephalic lesions in 3 of 5 GH fe-uses. On opening the skulls of theseetuses, the cerebral hemispheres im-

ediately collapsed. There was exten-ive bilateral loss of cerebral white

atter, which was replaced with clearuid; there was no evidence of intra-entricular hemorrhage. There was

rements at postmortemSAL (n � 9) GH (n � 5)

127.2 � 1.4 130.4 � 0.6..................................................................................................................

2994 � 279a 3251 � 144..................................................................................................................

42.7 � 1.1a 45.3 � 0.9..................................................................................................................

62 � 2 61 � 1..................................................................................................................

32.4 � 1.5 31.7 � 1.0..................................................................................................................

31.9 � 1.7 32.2 � 1.2..................................................................................................................

26.7 � 0.9 27.0 � 0.9..................................................................................................................

30.9 � 1.3 31.3 � 0.8..................................................................................................................

ost hoc correction).

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arked leucomalacia, with the soft tis-ues disintegrating on handling.

Fetal pH was significantly higher in theAL and GH groups than in the controlroup, with the GH group showing aarked decrease in fetal pH over the em-

olization period, which recovered toaseline values during the treatment pe-iod (Figure 2, A). The fetal PaO2 in theontrols remained higher than in bothmbolized groups throughout the treat-ent period (Figure 2, B). Fetal hemo-

lobin values were higher in the 2 embo-ized groups than in the control grouput did not change over the treatmenteriod (Figure 2, C). Maternal blood

TABLE 2Weights of fetal organs and uterop

Control

Fetal tissues (g)...................................................................................................................

n � 6...................................................................................................................

Liver 147.2 � 11.8...................................................................................................................

Heart 29.2 � 2.0...................................................................................................................

Lungs 113.7 � 18.7...................................................................................................................

Spleen 8.23 � 2.8...................................................................................................................

Pancreas 3.57 � 0.6...................................................................................................................

Brain 48.2 � 4.3...................................................................................................................

Pituitary 0.13 � 0.0...................................................................................................................

Thyroid 1.49 � 0.1...................................................................................................................

Thymus 15.9 � 2.6...................................................................................................................

Kidneys 22.9 � 2.9...................................................................................................................

Adrenals 0.51 � 0.1...................................................................................................................

Perirenal fat 14.7 � 1.3...................................................................................................................

Brain/liver 0.35 � 0.0...................................................................................................................

Uteroplacental tissues (g)...................................................................................................................

n � 5...................................................................................................................

Uterus 644 � 22...................................................................................................................

Membranes 265 � 43...................................................................................................................

Fetal fluids 1663 � 440...................................................................................................................

Placenta 513 � 104...................................................................................................................

Placenta/fetus 0.12 � 0.0...................................................................................................................

Values are mean � SE.a P � .01.b P � .001, compared with control.c P � .05.d Includes data from the 3 fetuses with morphologic brain abe P � .05, compared with SAL (factorial ANOVA with Fisher

de Boo. Treatment IUGR with maternal growth hormone

ases and pH did not change over the t


reatment period. Maternal hemoglobinalues in the GH group were higher thanhose of the SAL and control groups dur-ng the treatment period (Figure 2, D).

GH treatment markedly increased fe-al plasma glucose concentrations, al-hough these were very variable (Figure, A). Closer inspection of the data fromndividual animals showed that 3 of 5H-treated animals were hyperglycemicuring the treatment period (range, 1.6-.2 mM) whereas the other 2 were notrange, 0.56-0.97 mM). Fetal plasma glu-ose concentrations remained lower inhe SAL group than in the control grouphroughout the treatment period. Ma-

ental tissues at postmortemSAL GH

..................................................................................................................

n � 9 n � 5..................................................................................................................

84.8 � 10.0a 86.5 � 5.5b

..................................................................................................................

22.0 � 1.8a 26.9 � 1.2..................................................................................................................

85.9 � 3.6 94.9 � 8.6..................................................................................................................

5.37 � 0.76 7.39 � 0.87..................................................................................................................

2.71 � 0.33 3.92 � 0.22..................................................................................................................

40.9 � 2.0 29.9 � 9.0c,d..................................................................................................................

0.11 � 0.02 0.11 � 0.01..................................................................................................................

0.88 � 0.10a 1.27 � 0.17..................................................................................................................

10.0 � 1.8 14.6 � 2.2..................................................................................................................

19.4 � 2.1 20.5 � 0.7..................................................................................................................

0.52 � 0.08 0.53 � 0.07..................................................................................................................

10.9 � 0.9c 15.8 � 1.8e

..................................................................................................................

0.55 � 0.09c 0.36 � 0.10..................................................................................................................

..................................................................................................................

n � 7 n � 5..................................................................................................................

691 � 138 691 � 68..................................................................................................................

510 � 129 279 � 10..................................................................................................................

1305 � 193 1761 � 550..................................................................................................................

367 � 58 345 � 9..................................................................................................................

0.11 � 0.01 0.11 � 0.00..................................................................................................................

alities.

t hoc correction).


ernal plasma glucose concentrations in g

gy NOVEMBER 2008

he GH group were increased 1.6-foldompared with the control group (range:.9-10.8 mM, Figure 3, B) and correlatedith fetal plasma glucose concentrations

P � .0001, R2 � 0.62). Maternal-to-fe-al plasma glucose ratios were increasedn both embolized groups but were notifferent between embolized groupsFigure 3, C).

GH reduced both fetal and maternallasma urea concentrations in the firsteek of treatment (Figure 4, A and B,

espectively). This was followed by aradual increase in the subsequenteeks, though they remained lower than

n the control group. The maternal-to-etal urea concentration ratio did nothange (data not shown).

GH treatment suppressed maternaleed intake (Figure 5, A) but increased

aternal FFA concentrations (Figure 5,). Fetal plasma lactate concentrations

ncreased gradually throughout thereatment period in the control animals,ut they remained lower than those ofhe 2 embolized groups (Figure 5, C).H treatment did not change fetallasma lactate concentrations.Fetal plasma IGF-I concentrations re-ained lower in both embolized groups

han in the control group (Figure 6, A).etal plasma insulin concentrations inhe SAL group remained lower thanhose of the control group throughouthe treatment period (Figure 6, B). GHreatment increased maternal plasmaGF-I and insulin concentrations 4- and-fold, respectively (Figure 6, C and D,espectively).

OMMENTe have shown that maternal GH treat-ent in late gestation can improve fetal

rowth after IUGR caused by placentalnsufficiency. Twice daily maternal GHnjections after placental embolizationesulted in the restoration of fetal chestirth increments and body length, en-anced fetal fat deposition, and im-roved fetal weight. This is the first dem-nstration that a maternal hormonalreatment can reverse established IUGR,hus suggesting a potentially clinicallyeasible intrauterine treatment for the

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There may be several mechanisms un-erlying the effects of maternal GH sup-lementation on fetal growth in the cur-ent study. First, GH contributes to theegulation of nutrient supply to the fetusy altering maternal metabolism duringregnancy. Exogenous GH supplemen-ation increases peripheral insulin resis-ance and promotes lipolysis and proteinnabolism in sheep10,11,14 and pigs.12

his change in maternal metabolism ishought to increase the nutrient supplyo the fetus. Consistent with this, we ob-erved increased plasma glucose and in-ulin concentrations, increased plasmaFA concentrations, and decreasedlasma urea concentrations in GH-reated ewes. In sheep, circulating FFA

TABLE 3Embolization effects on fetal bloodmetabolite and hormone concentra

Control, n � 1

Before

Blood gases..........................................................................................................

FA PaO2 (mm Hg)a,b,c 5.5 � 2.1..........................................................................................................

FA hemoglobin (g/L)d 99 � 07...................................................................................................................

Metabolites..........................................................................................................

FA glucose (mM) 1.0 � 0.1..........................................................................................................

MA/FA glucose e,f,g 3.3 � 0.5..........................................................................................................

FA urea (mM) 4.1 � 1.0..........................................................................................................

MA/FA urea 0.8 � 0.2..........................................................................................................

FA lactate (mM) d,h,g 1.0 � 0.1...................................................................................................................

Hormones..........................................................................................................

FA IGF-I (ng/mL)d,e,i 34.2 � 7.3..........................................................................................................

MA IGF-I (ng/mL)d 71.2�18.5..........................................................................................................

FA Insulin (ng/mL)i,j 0.19 � 0.08..........................................................................................................

MA Insulin (ng/mL)b,j 0.21 � 0.08...................................................................................................................

Values are mean � SD.FA, fetal arterial; MA, maternal arterial.a P � .0001, for group effect.b P � .001 for embolization effect.c P � .001, for group by embolization interaction (repeatedd P � .0001, for embolization effect.e P � .05 for group effect.f P � .05 for embolization effect.g P � .05 for group by embolization interaction (repeated mh P � .01 for group effect.i P � .01 for group embolization interaction (repeated measj n � 5 for values in the GH group.

de Boo. Treatment IUGR with maternal growth hormone

re taken up by the liver and converted to f

etone bodies.20 In a normal sheep preg-ancy, hepatic ketogenesis is increasednd there is a significant uptake of ke-one bodies by the uterus. The increase in

aternal plasma FFA concentrationsherefore provides an alternate uteropla-ental and peripheral energy source forhe ewe, making more glucose availableo the fetus.

Maternal glucose reaches the fetal cir-ulation by crossing the placenta via fa-ilitated diffusion down a concentrationradient. Thus, we found the expectedncrease in fetal glucose concentrations,hich correlated with maternal concen-

rations. Although glucose is the mainutrient for the fetus, supraphysiologiclucose concentrations mainly stimulate

ses and fetal and maternalns in the 3 treatment groups

GH, n � 8

After Before After

.........................................................................................................................

27.2 � 4.7 23.9 � 3.5 17.6.........................................................................................................................

110 � 21 97 � 09 117 �.........................................................................................................................

n � 10 n � 7.........................................................................................................................

1.2 � 0.2 1.0 � 0.3 0.9.........................................................................................................................

3.0 � 0.5 3.5 � 0.8 4.4.........................................................................................................................

6.4 � 1.2 4.2 � 1.2 6.6.........................................................................................................................

0.9 � 0.0 0.7 � 0.1 0.8.........................................................................................................................

1.1 � 0.2 1.1 � 0.2 2.3.........................................................................................................................

n � 9 n � 7.........................................................................................................................

51.2 � 9.0 30.4 � 8.6 33.5.........................................................................................................................

100.1 � 18.9 72.3 � 20.8 99.3.........................................................................................................................

0.35 � 0.13 0.26 � 0.21 0.18.........................................................................................................................

0.35 � 0.09 0.24 � 0.16 0.38.........................................................................................................................

ures ANOVA).

res ANOVA).

ANOVA).


at deposition. Stevens et al21 showed r


hat chronic glucose infusion to theheep fetus resulted in an 18% increasen fetal body weight, but 47% and 360%ncreases in brown and white adipose tis-ue, respectively. Previous studies onate-gestation GH supplementation inregnant sheep also showed increasedelative fetal fat content and perirenal fatass.14 In our study, GH supplementa-

ion increased fetal perirenal fat deposi-ion, perhaps as a direct result of the in-reased glucose availability.

The improved fetal growth after GHreatment may also have been caused byhanges in fetal endocrine status. Inheep, maternal undernutrition causeseduced fetal growth associated with re-uced fetal IGF-I levels,22 which are

SAL, n � 13

Before After

..................................................................................................................

.4 24.6 � 3.3 17.8 � 3.7..................................................................................................................

92 � 08 117 � 18..................................................................................................................

n � 11..................................................................................................................

.2 1.0 � 0.3 0.9 � 0.2..................................................................................................................

.1 3.6 � 0.9 4.5 � 1.0..................................................................................................................

.5 6.1 � 7.0 5.4 � 1.8..................................................................................................................

.1 0.8 � 0.1 0.8 � 0.2..................................................................................................................

.0 1.1 � 0.4 2.1 � 0.8..................................................................................................................

n � 13..................................................................................................................

.9 32.1 � 7.2 35.8 � 12.1..................................................................................................................

3.6 76.6 � 17.1 104.4 � 14.1..................................................................................................................

0.08 0.20 � 0.10 0.15 � 0.10..................................................................................................................

0.11 0.22 � 0.14 0.39 � 0.17..................................................................................................................

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estored by fetal glucose supplementa-


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5

ion.23 Furthermore, fetal pancreatec-omy reduces fetal growth24 and circu-ating fetal IGF-I concentrations,25

hereas fetal insulin infusion increaseshem.23 Studies with IGF-I supplemen-ation to the fetus have shown varied ef-ects on fetal growth, depending on dose,uration, and route of administra-ion.26-28 In the current study, the in-reased fetal glucose concentrationsere not associated with increased fetal

nsulin or IGF-I concentrations. Indeed,oth insulin and IGF-I concentrationsemained significantly lower in the GHroup than in the control grouphroughout the treatment period, so thatmall changes in fetal hormone concen-rations that were not detected by our as-ays are unlikely to explain the observedhanges in fetal growth. However, in ourrevious study in sheep of the same ges-ational age, we also found improved fe-al growth after fetal intravenous or in-raamniotic IGF-I supplementationithout increasing circulating IGF-I

oncentrations.6 Nevertheless, it is pos-ible that there were local paracrine or

FIGURE 2Fetal pH and PaO2 and fetal and m

7.35 7.36 7.37 7.38 7.39 7.40 7.41 7.42 7.43

7.44etal rterial H

85 95

105 115 125 135 etal

rterial b

g/dL)

0 1 2 3 4 5

A

C

c

SG

SG

*

Experimental periods

, Fetal pH. B, Fetal PaO2. C, Fetal hemoglobiepeated measures ANOVA with Tukey post hoc

10-5); Œ � SAL (n � 13-6); □ � GH (neriod because of attrition. *P � .001; **P � .roup effects, and lower case letters represent po/cP � .05 vs control; S/sP � .05 vs SAL; G/gPe Boo. Treatment IUGR with maternal growth hormone sup

utocrine effects of GH on the fetal IGF p


ystem without measurable changes inirculating hormone levels.

It is interesting to note the lack of in-rease in fetal insulin concentrations inesponse to the increased glucose con-entrations. As some fetuses had a morehan 3-fold increase in plasma glucoseoncentrations, increases in plasma in-ulin concentrations of similar magni-ude could be expected, and these woulde readily detectable using our currentssays. It has been shown previously thathronic, sustained hyperglycemia in theetal sheep suppresses basal and glucose-nduced insulin secretion.29 Thus, theack of increase in insulin concentrationsn the current study may be the result ofersistent hyperglycemia caused by theH supplementation. It is therefore of

ome interest that there was a trend to-ard increased pancreas weight in theH group compared with the SAL group

P � .07), although this was unchangedelative to fetal body weight.

The observed increase in fetal growthay also have been caused by changes in

lacental transport capacity. We have

rnal hemoglobin concentrations

14

16

18

20

22

24

26

28

30l ial 2

Hg)

B sg

sg s s

s

**

85

90

95

100

105

110

115

120ernalrial

L)

0 1 2 3 4 5

D c

SC


SG

, Maternal hemoglobin. Data are analyzed byt and values are mean � SE. � � control (n-5). Animal numbers decreased over the study1 for time effects; upper case letters representoc significance of the group � time interaction.05 vs GH.

entation in sheep. Am J Obstet Gynecol 2008.

reviously shown that maternal GH sup- s

gy NOVEMBER 2008

lementation in sheep increases placen-al capacity for simple diffusion,10 po-entially increasing substrate supply tohe fetus. In the current study, we did not

easure placental transport directly, butid not find any changes in maternal-to-

etal concentration ratios for glucose andrea, or in fetal plasma lactate concen-

rations, to suggest altered placentalunction. However, we did find that GHupplementation caused a 4-fold in-rease in maternal IGF-I levels, and ma-ernal IGF-I supplementation has been

FIGURE 3Fetal and maternal plasmaglucose concentrations andmaternal/fetal glucoseconcentration ratio

0.50

1.00

1.50

2.00

2.50 etal lasma lucose (mM)

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5

/F plasma lucose ratio

0 1 2 3 4 5

2.5 3.5 4.5 5.5 6.5 7.5 aternal

lasma lucose (mM)

A

B

C

cs

cs

S

CS

cs

cs s

CS*

**

CG


, Fetal plasma glucose concentration. B, Ma-ernal plasma glucose concentration. C, Mater-al to fetal glucose concentration ratio. Data arenalyzed by repeated measures ANOVA withukey post hoc test and values are mean � SE.

� control (n � 10-5); Œ � SAL (n �3-6); □ � GH (n � 9-5). Animal numbersecreased over the study period because ofttrition. *P � .05; **P � .0001 for time effects;pper case letters represent group effects, andower case letters represent post hoc signifi-ance of the group � time interaction C/cP �

05 vs control; S/sP � .05 vs SAL; G/gP � .05s GH.e Boo. Treatment IUGR with maternal growth hormoneupplementation in sheep. Am J Obstet Gynecol 2008.

ate

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plem

hown in sheep and guinea pigs to alter

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lacental metabolism and enhance glu-ose delivery to the fetus.30,31 In humanregnancy, correlations have also been

ound between maternal IGF-I concen-rations and fetal size.32

Although GH supplementation im-roved fetal carcass growth and fat dep-sition, it had no clear effect on therowth of the visceral organs. Placentalmbolization reduced fetal liver weight,hich was not restored by GH treat-ent. Weights of the fetal heart were also

educed by embolization and were onlyartially restored by GH supplementa-ion. Previous studies have shown in-reased fetal heart and liver weights in adibitum-fed pregnant adolescent sheepn response to maternal GH supplemen-ation.11 The effect on heart weight waseduced in animals fed a moderated diet,

FIGURE 4Fetal and maternal plasmaurea concentrations

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

aternal lasma rea (mM)

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

etal lasma rea (mM)

0 1 2 3 4 5

cs

g

g g

SG

SG

G

*

**

A

B


, Fetal plasma urea concentration. B, Maternallasma urea concentration. Data are analyzed byepeated measures ANOVA with Tukey post hocest and values are mean � SE. � � control (n

10-5); Œ � SAL (n � 13-6); □ � GH (n9-5). Animal numbers decreased over the

tudy period because of attrition. *P � .001; **P.0001 for time effects; upper case letters

epresent group effects, and lower case lettersepresent post hoc significance of the group �ime interaction C/cP � .05 vs control; S/sP �

05 vs SAL; G/gP � .05 vs GH.e Boo. Treatment IUGR with maternal growth hormoneupplementation in sheep. Am J Obstet Gynecol 2008.

nd there was no effect on fetal liver s

eight in these animals. It was con-luded that the increased growth of theetal visceral organs in the ad libitum-ed, GH-supplemented animals was theesult of the increased fetal glucose con-entrations in these animals and the as-ociated increase in fetal insulin concen-rations. Although our animals were alsoed ad libitum and had increased fetallucose concentrations, this was not as-ociated with an increase in fetal insulinoncentrations, which could explain theack of effect on fetal liver growth.

We found that maternal GH supple-entation markedly reduced ad libitum

ood intake. In 2 of the GH-treated ani-als, this was so severe that the ewes had

o be withdrawn from the study. This ef-ect of GH supplementation on appetiten sheep has been observed before in adibitum-fed pregnant sheep.11,14 Foodntake is strongly related to changes inirculating glucose concentrations inoth humans33 and rats.34 In humans itas shown that hyperglycemia was re-

ated to reduced appetite.35,36 The re-uced appetite in our GH-treated ewesay therefore have been related to the

bserved hyperglycemia. However, notll our animals were hyperglycemic, andood intake was also reduced in the nor-

oglycemic animals. Thus, the suppres-ion of maternal appetite is unlikely toave been a simple result of the changes

n circulating glucose concentrations.Nevertheless, the effect of GH supple-entation on maternal food intake may

e important in determining its effect onetal growth. In undernourished rats,H supplementation did not affect fetaleight37 and in some studies even re-uced fetal weight,38 even though thehange in maternal metabolism was stillbserved.37 In adolescent pregnantheep, nutrients are directed toward ma-ernal fat deposition rather than fetalrowth, causing fetal growth restrictionhen the ewes are overnourished,39 an

ffect not seen in adult ewes.40 GH treat-ent in late gestation normalized fetal

rowth in these overnourished adoles-ent ewes.14 GH treatment in early ges-ation also decreased maternal adipositynd increased maternal protein deposi-ion regardless of maternal nutritional
tatus, but only improved uteroplacental t

rowth when the ewes were overnour-shed.11 Thus, the decrease in maternalood intake caused by GH supplementa-ion may limit the extent to which fetalrowth can be enhanced by thisreatment.

GH supplementation also increasedaternal hemoglobin concentrations.H has long been known to stimulate

rythropoiesis in multiple species,41 bytimulating erythropoietin produc-

FIGURE 5Maternal feed intake, plasmaFFA, and fetal plasmalactate concentrations

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4aternal

eed intake kg/d)

0 1 2 3 4

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 etal

lasma actate mM)

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 aternal

lasma FA mM)

g

g A

B

C

GS

G

sc

CS*

c

SG**


0 1 2 3 4 5

0 1 2 3 4 5

, Maternal feed intake. B, Maternal plasma FFAoncentrations. C: Fetal plasma lactate concen-rations. Data are analyzed by repeated measuresNOVA with Tukey post hoc test and values areean � SE. � � control (n � 10-5); Œ �AL (n � 13-6); □ � GH (n � 9-5). Animalumbers decreased over the study period be-ause of attrition. *P � .05; **P � .0001 forime effects; upper case letters represent groupffects, and lower case letters represent post-oc significance of the group x time interaction/cP � .05 vs control; S/sP � .05 vs SAL; G/gP

.05 vs GH.e Boo. Treatment IUGR with maternal growth hormoneupplementation in sheep. Am J Obstet Gynecol 2008.

ion.42 However, it seems unlikely that


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he potentially increased oxygen carry-ng capacity of the maternal blood as aesult of the increased hemoglobin con-entrations contributed to the enhancedetal growth. Ewes in the control and SALroups were not anemic or hypoxemic,nd improved placental oxygen supplyy administration of oxygen to theother has not been shown to improve

rowth of the IUGR fetus in rat43 or hu-an44 pregnancy.GH treatment did not cause an in-

rease in fetal loss caused by infection,reterm labor, or other, unidentifiedeasons in our experiment. However, se-ere brain lesions were found in 3 of 5 ofhe GH-treated fetuses at post mortem.t is unclear what caused these lesionsnd why they were only seen in the GH-reated fetuses. We induced IUGR andssociated fetal hypoxemia by placentalmbolization, but fetuses in the GH-reated group were not more hypoxemichan those in the SAL group. Bilateral ca-otid artery ligation can cause hydranen-ephalic brain lesions in the ovine fe-us.45 In our study we only ligated 1 fetal

FIGURE 6Fetal and maternal plasma IGF-I an

20 30

40

50

60

70

80etal ng/ml)

50 100 150 200 250 300 350 400 450aternal

ng/ml)

93 100 106 118 127

s

cs cs cs

Time (dGA)

1-FGIA

C

, Fetal plasma IGF-I. B, Fetal plasma insulin. C,ata are analyzed by repeated measures ANOVAE. � � control (n � 10-5); Œ � SAL (necreased over the study period because of attriase letters represent group effects, and lowerroup � time interaction C/cP � .05 vs controe Boo. Treatment IUGR with maternal growth hormone sup

arotid artery, but we found similar t


rain lesions to those described afterilateral ligation. Many studies haveuggested that hyperglycemia may ex-cerbate hypoxic/ischemic brain in-ury,46-49 and it is possible that the hy-erglycemia found in the GH-treated

etuses contributed to the develop-ent of the brain lesions, in combina-

ion with the hypoxemia and ischemiaaused by placental embolization andarotid artery ligation. However, in 1f the 3 affected animals neither eweor fetus were hyperglycemic, so that

he exact nature and cause of the brainnjury remains uncertain.

In summary, our study showed thataternal GH treatment improves fetal

rowth after IUGR caused by placentalnsufficiency, and may therefore providehe basis for a clinically feasible prenatalreatment for the IUGR fetus. However,evere hydrancephalic brain lesions oc-urred in some treated fetuses. The pos-ible relationship between maternal GHreatment and fetal brain injury requiresurther investigation if this potential new

insulin concentrations

*

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0cs

cs

cs

CS

S

Time (dGA)

ilusnI

93 100 106 118 127

D

B n

ternal plasma IGF-I. D, Maternal plasma insulin.th Tukey post hoc test and values are mean �3-6); □ � GH (n � 9-5). Animal numbers. *P � .05; **P � .001 for time effects; upper

e letters represent post-hoc significance of the/sP � .05 vs SAL; G/gP � .05 vs GH.entation in sheep. Am J Obstet Gynecol 2008.

reatment is to be further developed. f L

gy NOVEMBER 2008

CKNOWLEDGMENTSe acknowledge the technical expertise of Toniitchell, Pierre van Zijl, Eric Thorstensen, andhris Keven. The growth hormone was pro-ided by Dr Baumbach (American Cyanamid,rinceton, NJ). The Lion Foundation of Newealand contributed toward laboratoryxpenses.

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GF Res 1999;9:11-7.4. Wallace JM, Matsuzaki M, Milne J, Aitken R.

d

SG*

*

CS*

Mawi

� 1tioncasl; S

plem

ate but not early gestational maternal growth

ho21Bmsg1Gts1dsf1f11tdn2Hb12pbl2mai12Pals2pJ2dpf12dl

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aE3gdo3ei14ooN4mp4ociJ4PpuF4tpR4Kdp4ofo4hr14Pis4Sp


ormone treatment increases fetal adiposity invernourished adolescent sheep. Biol Reprod006;75:231-9.5. Gatford KL, Owens JA, Campbell RG,oyce JM, Grant PA, De Blasio MJ, et al. Treat-ent of underfed pigs with GH throughout the

econd quarter of pregnancy increases fetalrowth. J Endocrinol 2000;166:227-34.6. Costine BA, Inskeep EK, Wilson ME.rowth hormone at breeding modifies concep-

us development and postnatal growth inheep. J Anim Sci 2005;83:810-5.7. Breier BH, Gallaher BW, Gluckman PD. Ra-ioimmunoassay for insulin-like growth factor-I:olutions to some potential problems and pit-alls. J Endocrinol 1991;128:347-57.8. Blum WF, Breier BH. Radioimmunoassays

or IGFs and IGFBPs. Growth Regul 1994;4:1-9.9. Harding JE. Prior growth rate determineshe fetal growth response to acute maternal un-ernutrition in fetal sheep of late gestation. Pre-at Neonatal Med 1997;2:300-9.0. Heitmann RN, Dawes DJ, Sensenig SC.epatic ketogenesis and peripheral ketoneody utilization in the ruminant. J Nutr987;117:1174-80.1. Stevens D, Alexander G, Bell AW. Effect ofrolonged glucose infusion into fetal sheep onody growth, fat deposition and gestation

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nd IGF-II are regulated differently by glucose ornsulin in the sheep fetus. Reprod Fertil Dev996;8:167-72.3. Oliver MH, Harding JE, Breier BH, EvansC, Gluckman PD. Glucose but not a mixedmino acid infusion regulates plasma insulin-

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ike growth factor I alters placental function but n

oes not affect fetal growth. Reprod Fertil Dev002;14:393-400.7. Bloomfield FH, Bauer MK, van Zijl PL,luckman PD, Harding JE. Amniotic IGF-I sup-lements improve gut growth but reduce circu-

ating IGF-I in growth-restricted fetal sheep.m J Physiol 2002;282:E259-69.8. Lok F, Owens JA, Mundy L, Robinson JS,wens PC. Insulin-like growth factor I promotesrowth selectively in fetal sheep in late gesta-ion. Am J Physiol 1996;270:R1148-55.9. Carver TD, Anderson SM, Aldoretta PA, Es-

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ers feto-placental carbohydrate and proteinetabolism in pregnant sheep. Endocrinology994;135:895-900.1. Sferruzzi-Perri AN, Owens JA, Standen P,aylor RL, Robinson JS, Roberts CT. Earlyreatment of the pregnant guinea pig with IGFsromotes placental transport and nutrient par-itioning near term. Am J Physiol 2007;292:668-76.2. Mirlesse V, Frankenne F, Alsat E, Poncelet, Hennen G, Evain-Brion D. Placental growth

ormone levels in normal pregnancy and inregnancies with intrauterine growth retarda-ion. Pediatr Res 1993;34:439-42.3. Campfield LA, Smith FJ, Rosenbaum M,irsch J. Human eating: evidence for a physio-

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ree-feeding rats. Obes Res 1996;4:497-500.5. Chapman IM, Goble EA, Wittert GA, MorleyE, Horowitz M. Effect of intravenous glucosend euglycemic insulin infusions on short-termppetite and food intake. Am J Physiol998;274:R596-603.6. Gielkens HA, Verkijk M, Lam WF, LamersB, Masclee AA. Effects of hyperglycemia andyperinsulinemia on satiety in humans. Metab-lism 1998;47:321-4.7. Woodall SM, Breier BH, Johnston BM, Bas-ett NS, Barnard R, Gluckman PD. Administra-ion of growth hormone or IGF-I to pregnant ratsn a reduced diet throughout pregnancy does
ot prevent fetal intrauterine growth retardation S

nd elevated blood pressure in adult offspring. Jndocrinol 1999;163:69-77.8. Chiang MH, Nicoll CS. Administration ofrowth hormone to pregnant rats on a reducediet inhibits growth of their fetuses. Endocrinol-gy 1991;129:2491-5.9. Wallace JM, Aitken RP, Cheyne MA. Nutri-nt partitioning and fetal growth in rapidly grow-

ng adolescent ewes. J Reprod Fertil 1996;07:183-90.0. Wallace JM, Milne JS, Aitken RP. The effectf overnourishing singleton-bearing adult ewesn nutrient partitioning to the gravid uterus. Br Jutr 2005;94:533-9.1. Golde DW, Bersch N, Li CH. Growth hor-one: species-specific stimulation of erythro-oiesis in vitro. Science 1977;196:1112-3.2. Sohmiya M, Ishikawa K, Kato Y. Stimulationf erythropoietin secretion by continuous sub-utaneous infusion of recombinant human GH

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ects of exogenous glucose on brain ischemia invine fetuses. Pediatr Res 2004;56:621-9.7. Dietrich WD, Alonso O, Busto R. Moderateyperglycemia worsens acute blood-brain bar-ier injury after forebrain ischemia in rats. Stroke993;24:111-6.8. Pulsinelli WA, Waldman S, Rawlinson D,lum F. Moderate hyperglycemia augments

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treatment of intrauterine growth restriction with maternal growth hormone supplementation in sheep

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