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Endocrinology, doi:10.1210/en.2006-1701
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Endocrinology Vol. 148, No. 6 2963-2972
Copyright © 2007 by The Endocrine Society

Fetal and Amniotic Insulin-Like Growth Factor-I Supplements Improve Growth Rate in Intrauterine Growth Restriction Fetal Sheep

Simona C. Eremia, Hendrina A. de Boo, Frank H. Bloomfield, Mark H. Oliver and Jane E. Harding

Liggins Institute, University of Auckland, 1142 Auckland, New Zealand

Address all correspondence and requests for reprints to: Professor Jane E. Harding, Liggins Institute, University of Auckland, Private bag 92019, 1142 Auckland, New Zealand. E-mail: j.harding{at}auckland.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To date, there is no known prenatal treatment for intrauterine growth restriction (IUGR). IGF-I is an important regulator of fetal growth and circulating IGF-I concentrations are reduced in IUGR fetuses. We investigated whether any of three different methods of fetal IGF-I administration would reverse IUGR in sheep. Animals were randomized into five groups: control (n = 17), IUGR + saline (SAL, n = 17), IUGR + iv IGF-I (IGF-IV, n = 14), IUGR + intraamniotic IGF-I (IGF-AF, n = 14), or IUGR + intraamniotic IGF-I with nutrients (IGF-NUT, n = 16). Weekly IGF-I dose was 360 µg in each treatment group. IUGR was induced by placental embolization between 93 and 99 d and treatment was from 100–128 d gestation (term = 147 d). Embolization caused asymmetrical IUGR with reduced fetal growth rates and body and organ weights, but increased brain to liver weight ratio, at post mortem. Embolized fetuses were also hypoxemic and hypoglycemic and had reduced circulating IGF-I and insulin concentrations. IGF-AF and IGF-IV significantly increased fetal growth rates, but only IGF-AF significantly increased fetal liver weight, compared with saline-treated fetuses. Fetal weights and brain to liver weight ratios in all IGF-I-treated fetuses were intermediate between the control and SAL groups. Addition of nutrients reduced the effects of amniotic IGF-I treatment and increased fetal hemoglobin and lactate concentrations. Treatments did not change fetal plasma IGF-I and insulin concentrations. This is the first report of an intrauterine treatment significantly increasing fetal growth rate in established IUGR. Amniotic IGF-I administration may provide the basis for a clinically applicable prenatal treatment for the IUGR fetus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTRAUTERINE GROWTH RESTRICTION (IUGR) is a major cause of perinatal mortality and morbidity (1). Many IUGR infants continue to show impaired growth in childhood (2) and are at increased risk of developmental delay (3) and cognitive problems (4). Additionally, IUGR infants are more likely to develop adult onset diseases such as type 2 diabetes, hypertension, and heart disease (5).

The long-term effects of IUGR may be the result of fetal programing: the process whereby a stimulus or insult during a sensitive or critical period of development has irreversible long-term effects on development (6). It has therefore been proposed that a therapy designed to reverse the nutritional and endocrine characteristics of IUGR in utero may be successful in preventing both short- and long-term negative consequences (7). In sheep, the development of IUGR can be prevented by iv or intragastric nutrient supplementation (8, 9). However, to our knowledge, reversal of established IUGR has not yet been reported.

Late gestation fetal growth is predominantly regulated by endocrine and nutritional factors (10). The endocrine factor playing the most important role in the late gestation fetus is IGF-I, which stimulates fetal and placental substrate uptake and inhibits protein breakdown (11). Birth weight correlates with umbilical cord blood IGF-I levels and circulating fetal IGF-I levels are decreased in human IUGR pregnancies (12, 13, 14). Furthermore, deletion of the IGF-I gene causes severe growth restriction in humans (15) and mice (16). Regulation of IGF-I is strongly linked to nutrition. In sheep, maternal undernutrition causes growth restriction in the fetus associated with decreased fetal IGF-I levels (17), which are restored by fetal glucose supplementation (18). In addition, fetal pancreatectomy reduces circulating fetal IGF-I levels (19), and fetal insulin infusion increases them (18). Thus, growth in the late gestation ovine fetus is strongly reliant on the glucose-insulin-IGF-I axis.

Most cases of IUGR in the developed world are caused by placental factors, resulting in reduced nutrient supply to the fetus (7, 20). It is therefore not surprising that attempts to improve growth of IUGR fetuses by alterations in maternal nutrition have been largely unsuccessful (21), and intrauterine treatments for IUGR that bypass the placenta may have better chance of success. One potential paraplacental route of treatment is via the amniotic fluid. The fetus swallows large quantities of amniotic fluid (22, 23) and makes use of the nutrients and growth factors ingested (24, 25, 26). Esophageal ligation in the sheep fetus, which inhibits ingestion of amniotic fluid, has been shown to cause IUGR, the onset of which can be prevented by intragastric infusion of IGF-I (27). Intragastric nutrient supplementation also prevents the development of IUGR in the ovine fetus during maternal nutrient restriction (9), and low-dose amniotic IGF-I supplementation reverses the effects of IUGR on fetal gut (28).

Studies in this and other laboratories have shown that the iv route can also be successfully used to manipulate fetal growth. Chronic iv nutrient supplementation prevents the onset of growth restriction in fetal sheep during placental embolization (8). Chronic high-dose IGF-I infusion to normally growing late-gestation fetal sheep results in selective organ growth without altering fetal weight (29), whereas IGF-I infusion at a lower dose does not alter fetal growth or metabolite uptake, although placental morphology and transport capacity are altered (30).

These experimental data suggest that IUGR may potentially be reversed by nutritional or hormonal supplementation via paraplacental routes. The current study was undertaken to test the hypothesis that IUGR could be reversed in utero by fetal iv or amniotic IGF-I supplementation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal preparation
Eighty-three pregnant Romney ewes carrying Dorset cross-breed singleton fetuses were housed in individual cages with free access to water and pelleted food. All experiments were approved by the University of Auckland Animal Ethics Committee.

After acclimatization to laboratory conditions animals underwent surgery under halothane anesthesia on d 90 of gestation (90 dGA, Fig. 1Go). Streptopen (250 mg procaine penicillin/250 mg dihydrostreptomycin sulfate; Pittman Moore Ltd., Upper Hutt, New Zealand) was administered im before surgery. Fetal carotid artery and jugular vein, maternal uterine arteries, maternal femoral artery and vein, and maternal carotid and jugular vein were catheterized. Growth catheters were inserted sc around the fetal chest, as described previously (31). Amniotic catheters were inserted into the amniotic sac and 80 mg gentamicin (Pharmacia Pty. Ltd., Bentley, Australia) was administered into the amniotic fluid.


Figure 1
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  		FIG. 1. Experimental design. Dark arrows indicate time of injections of IGF-I into the amniotic fluid in the amniotic treatment groups. PM, Postmortem.

 
Embolization
Sheep were allowed to recover for 3 d after surgery. Between 93 and 99 dGA (term = 145 dGA, Fig. 1Go), growth restriction was induced by twice daily embolization of maternal uterine arteries with polystyrene microspheres of 20–50 µm diameter (Superose 12 diluted 1:100; Pharmacia Biotech AB, Uppsala, Sweden). Embolization was discontinued if fetal arterial PaO2 fell less than 19 mm Hg, fetal lactate concentrations were greater than 2 mM, or chest girth had not increased for 2 consecutive days.

Study protocol
Before surgery, sheep were randomly assigned to one of five experimental groups: control (no embolization, no treatment), SAL (IUGR + thrice weekly injections of 2 ml saline into the amniotic fluid), IGF-IV [IUGR + continuous iv infusion to the fetus of 50 µg/d IGF-I (batch G117AZC983X; Gentech Inc., South San Francisco, CA)], IGF-AF (IUGR + thrice weekly injections of 120 µg IGF-I into the amniotic cavity), and IGF-NUT [IUGR + thrice weekly injections of 120 µg IGF-I combined with continuous infusion of glucose (1.5 g/kg·d and amino acids [10% Tropamine; Biomed, Auckland, New Zealand] and 0.4 g nitrogen/kg·d)] into the amniotic cavity). Glucose and amino acid infusion rates were adjusted weekly according to estimated fetal weights based on similarly instrumented fetuses in our laboratory over previous years. Administered doses were calculated to provide approximately 25% of daily fetal nutrient requirements. Weekly doses of IGF-I were the same for each IGF-I-treated group. Treatments commenced on 100 dGA and were continued for 28 d (Fig. 1Go).

Maternal and fetal blood and amniotic fluid samples were collected into heparinized tubes on ice every 2–3 d throughout the study. Aliquots were used to measure blood gases on a Chiron M845 blood gas analyzer (Chiron Corp., Emeryville, CA). Remaining samples were centrifuged at 3000 rpm at 4 C for 15 min and the plasma stored at –80 C for further analysis.

At 128 dGA, sheep were killed with an overdose of pentobarbitone. The uterus and its contents were weighed and fetal external body measurements were taken. Fetal organs were dissected and weighed. Placentomes were dissected and individually weighed and classified according to Vatnick et al. (32).

Assays
Because measuring all metabolites and hormones at each time point for more than 30 animals would have been impractical, only some samples were selected for biochemical analysis. Metabolites were measured in samples taken at the beginning of the embolization and treatment periods (93 and 100 dGA) every 6 d during the treatment period and the last three samples before the experiment ended. Hormones were measured in samples taken at the beginning of the embolization and treatment periods (93 and 100 dGA) and at three time points during the treatment period (106, 118, and 127 dGA).

Glucose, lactate, and urea concentrations were measured on a metabolite analyzer (Roche/Hitachi 9002 analyzer; Hitachi High-Technologies Corp., Tokyo, Japan).

IGF-I was measured by nonextraction double-antibody RIA, with addition of IGF-II in excess for elimination of interference from binding proteins (33). Rabbit anti-IGF-I serum was used as the primary antibody and the second antibody was 2% sheep antirabbit {gamma}-globulin with 0.01% normal rabbit serum and 8% PEG 6000 in 0.01 M PBS. The intra- and interassay coefficients of variation were 4.5 and 18%, respectively. Insulin concentrations were measured by RIA, with guinea pig anti ovine-insulin as the first antibody and 8% PEG 6000, with 0.5% sheep anti-guinea pig and 0.1 M normal guinea pig serum in 0.01 M PBS as the second antibody (34). The intra- and interassay coefficients of variation for insulin were 7.3 and 16%, respectively.

Data analysis
Data from animals that did not complete the first half of the study (up to 114 dGA) were excluded from analysis. In case of fetal loss after this time, data from the last 10 d before death were excluded to avoid bias related to the cause of death.

Growth catheter measurements were normalized for 100 dGA and analyzed separately for embolization (93–100 dGA) and treatment (100–128 dGA) periods by multiple linear regression, with animal number nested within treatment group and gestational age as independent variables (35). The effects of embolization on fetal weights and measurements were analyzed by Student’s t test, comparing control and saline groups. Treatment effects were then analyzed for all five treatment groups by factorial ANOVA with Fisher’s post hoc correction.

For analysis of blood gases and metabolites repeated-measures ANOVA could not be performed due to missing variables. Therefore, the entire study period was divided into six experimental periods: 92–93 dGA (period 0, preembolization); 98–100 dGA (period 1, last measurement of the embolization period); 101–107 dGA (period 2); 108–115 dGA (period 3); 116–123 dGA (period 4); and 124–128 dGA (period 5; Fig. 1Go). The results were averaged for each individual animal for each period.

Blood gas, hormone, and metabolite data were analyzed separately for embolization and treatment periods. Factorial ANOVA with Fisher’s post hoc corrections were used to determine differences among treatment groups before and at the end of the embolization period. There were no differences among any of the treatment groups before embolization or among the embolized groups after embolization. Therefore, the results of all embolized groups were combined for the analysis of embolization effects using two-way ANOVA with Fisher’s post hoc correction for repeated measures, comparing control and embolized groups, before and after embolization. Treatment effects (periods 1–5) were analyzed by factorial ANOVA with Tukey’s honestly significant difference corrections (P ≤ 0.05) with treatment group, time, and group x time interactions as the statistics of interest. Time effects were also analyzed for each group individually by the same approach.

All analyses were done using Statview or JMP (both SAS Institute, Cary, NC). Data are presented as number, median (range) or mean ± SE, as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Animal losses
A total of 78 animals entered the experiment and underwent surgery. Of these animals, 44 were lost before completion of the study (Table 1Go). In total 34 animals completed the study: six in the controls; nine in the SAL group; five in the IGF-IV group; seven in the IGF-AF group, and seven in the IGF-NUT group. There were no differences in survival rates among treatment groups (Table 1Go).


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  		TABLE 1. Distribution of experimental outcomes among treatment groups

 
Growth
Embolization effects.
Fetal forelimb length, biparietal diameter, and chest circumference were not different among groups at surgery (data not shown).

Embolization reduced fetal growth rate, measured by chest girth increment, by 22–30%, compared with controls (Table 2Go and Fig. 2Go), and reduced fetal weight in the SAL group at postmortem by a similar amount (Table 3Go). Crown-rump length was reduced by 10%, but biparietal diameter, total chest, and abdominal girth and limb length were not significantly altered. Weights of the fetal liver, heart, pituitary, thyroid, and perirenal fat were reduced in absolute terms (Table 4Go) but not relative to body weight (data not shown). Brain to liver weight ratios were increased. Embolization did not significantly affect the weights of the uterus, fetal membranes, fetal fluids, or placenta. There was also no difference in placental to fetal weight ratio.


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  		TABLE 2. Effects of embolization and treatment on daily fetal girth increment

 

Figure 2
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  		FIG. 2. Fetal chest girth increments. Values are normalized for 93 dGA and expressed as mean ± SE. bullet, Control (n = 6); {circ}, SAL (n = 10); {blacksquare}, IGF-AF (n = 5); {square}, IGF-NUT (n = 6); {blacktriangleup}, IGF-IV (n = 5). The gray horizontal bar indicates period of embolization. Mean daily girth increments are reduced in embolized groups during the embolization period (Table 2Go, not indicated in figure). Upper-case letters indicate group effects during the treatment period (SP ≤ 0.05 vs. SAL; NP ≤ 0.05 vs. IGF-NUT).

 

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  		TABLE 3. Gestational age and fetal body measurements at postmortem

 

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  		TABLE 4. Weights of fetal organs and uteroplacental tissues at postmortem

 
Treatment effects.
During the treatment phase, fetal chest girth increment remained 12% lower in the SAL group than in controls. Chest girth increment increased in the IGF-IV and IGF-AF groups to be significantly higher than that of the SAL group, but remained low in the IGF-NUT group (Table 2Go and Fig. 2Go).

Fetal weights in all IGF-I treatment groups were intermediate between control and SAL groups and not significantly different from either (Table 3Go). Crown-rump length remained lower only in the IGF-IV group. There were no differences in biparietal diameter, chest girth, abdominal girth, and fore- or hindlimb lengths among treatment groups.

Despite this increase in fetal growth rate and partial restoration of fetal weight in all IGF-I treatment groups, effects on organ weights were different among treatment groups (Table 4Go). Liver weight was significantly greater in the IGF-AF fetuses than in saline-treated fetuses, but this effect was abolished by the addition of nutrients (IGF-NUT group); liver weights were intermediate in the IGF-IV group. Both amniotic treatments (IGF-AF and IGF-NUT), but not IGF-IV treatment, significantly increased perirenal fat weight, compared with saline, and partially restored the weight of the fetal lungs to values intermediate between control and SAL groups. None of the treatments affected fetal thyroid weight or the weight of the placenta, uterus, membranes, or fetal fluids. When organ weights were expressed per kilogram body weight, only the reduced lung weight in the IGF-IV group remained significant (data not shown).

Blood gases
Embolization effects.
In embolized fetuses, but not controls, there was a decrease in arterial PaO2 and an increase in hemoglobin concentrations over the embolization period (Table 5Go). In embolized and control fetuses, there was a small decrease in fetal pH (data not shown), a small increase in maternal PaO2, and a decrease in maternal hemoglobin and pH over period of embolization but with no change in fetal or maternal PaCO2 (Table 5Go).


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  		TABLE 5. Effects of embolization on fetal and maternal blood gases and fetal, maternal, and amniotic fluid metabolite and hormone concentrations

 
Treatment effects.
In the control group, fetal PaO2 remained higher than in any of the treatment groups throughout the treatment period (Fig. 3AGo). In all embolized groups, fetal PaO2 recovered transiently but then gradually decreased over the treatment period. The IGF-IV group had higher fetal PaO2 and lower fetal PaCO2 than the other treatment groups (Fig. 3Go, A and B). Fetal hemoglobin increased gradually over the treatment period and was significantly higher in the IGF-NUT group than in the other groups (Fig. 3CGo). Fetal pH did not change over time or among groups (data not shown).


Figure 3
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  		FIG. 3. Fetal and maternal blood gases and hemoglobin concentrations. A, Fetal PaO2. B, Fetal PaCO2. C, Fetal hemoglobin. D, Maternal PaO2. E, Maternal PaCO2. F, Maternal hemoglobin. Data are analyzed by repeated measures ANOVA with Tukey post hoc test and values are mean ± SE. bullet, Control (n = 10–5); {circ}, SAL (n = 13–6); {blacksquare}, IGF-AF (n = 11–5); {square}, IGF-NUT (n = 13–6), {blacktriangleup}, IGF-IV (n = 11–5). Animal numbers decreased over the study period due to attrition (see Table 1Go). *, P ≤ 0.05, **, P ≤ 0.01, ****, P ≤ 0.0001 for time effects; upper-case letters represent group effects, and lower-case letters represent post hoc significance of the group x time interaction. C/cP ≤ 0.05 vs. control; S/sP ≤0.05 vs. SAL; A/aP ≤ 0.05 vs. IGF-AF; N/nP ≤ 0.05 vs. IGF-NUT; I/iP ≤ 0.05 vs. IGF-IV; ALL/allP ≤ 0.05 vs. all other groups.

 
In the IGF-IV group, maternal PaO2 was higher and maternal hemoglobin concentrations lower than in other groups (Fig. 3Go). Maternal PaCO2 was lower in the IGF-IV group than in the controls. There were no other changes in maternal blood gases over time or among groups.

Metabolites
Embolization effects.
Fetal plasma glucose concentrations increased in the control animals but decreased in the embolized animals over the embolization period (Table 5Go). Fetal plasma lactate and urea concentrations increased in all groups over this period. Maternal plasma glucose and urea concentrations increased and maternal lactate concentrations were unchanged in all groups over the embolization period. In the amniotic fluid, urea and lactate concentrations increased and glucose concentrations decreased in all groups over this period.

Treatment effects.
During the treatment period, fetal plasma glucose concentrations were higher in the controls than in the other groups (Fig. 4AGo). Fetal plasma lactate concentrations increased in control, IGF-AF, and IGF-NUT groups over the treatment period and were higher in the SAL and IGF-NUT groups than in the controls (Fig. 4BGo). Fetal plasma urea concentrations did not change over time but were lower in the SAL group than control and IGF-IV groups and lower in IGF-NUT than in controls (Fig. 4CGo).


Figure 4
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  		FIG. 4. Fetal and maternal arterial and amniotic fluid metabolite concentrations. A, Fetal glucose. B, Fetal lactate. C, Fetal urea. D, Maternal glucose. E, Maternal lactate. F, Maternal urea. G, Amniotic fluid glucose. H, Amniotic fluid lactate. I, Amniotic fluid urea. Data are analyzed by repeated-measures ANOVA with Tukey post hoc test and values are mean ± SE. bullet, Control (n = 10–6); {circ}, SAL (n = 12–7); {blacksquare}, IGF-AF (n = 12–6); {square}, IGF-NUT (n = 11–7); {blacktriangleup}, IGF-IV (n = 11–5). Animal numbers decreased throughout the study period due to attrition (see Table 1Go). *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001, ****, P ≤ 0.0001 for time effects; upper-case letters represent group effects, and lower-case letters represent post hoc significance of the group x time interaction. C/cP ≤ 0.05 vs. control; S/sP ≤ 0.05 vs. SAL; A/aP ≤ 0.05 vs. IGF-AF; N/nP ≤ 0.05 vs. IGF-NUT; I/iP ≤ 0.05 vs. IGF-IV; ALL/allP ≤ 0.05 vs. all other groups.

 
Maternal plasma glucose concentrations were similar in all groups and decreased significantly in the SAL group over the treatment period (Fig. 4DGo), whereas maternal plasma lactate concentrations increased in control, SAL, and IGF-IV groups (Fig. 4EGo). Maternal plasma urea concentrations were lower in the SAL group than control and IGF-IV groups (Fig. 4FGo).

Hormones
Embolization effects.
Fetal plasma insulin concentrations increased over the embolization period in controls but decreased in embolized animals (Table 5Go). Fetal plasma IGF-I concentrations increased to a significantly greater extent in the controls than in the embolized animals (50 and 8%, respectively). Maternal plasma insulin and IGF-I concentrations and IGF-I concentrations in amniotic fluid increased in all animals over the embolization period.

Treatment effects.
During the treatment period, fetal plasma IGF-I concentrations increased steadily in controls, but increased and then declined again in the embolized groups (Fig. 5AGo). Fetal plasma IGF-I and insulin (Fig. 5BGo) concentrations remained higher in the controls than in all other groups throughout the treatment period. None of the treatments altered fetal plasma IGF-I or insulin levels. Maternal plasma IGF-I and insulin concentrations were higher in the IGF-IV group than in the IGF-NUT group (Fig. 5Go, C and D). Amniotic fluid IGF-I concentrations decreased over the treatment period in the CON and SAL groups and increased 11- and 5-fold in the IGF-AF and IGF-NUT groups, respectively (Fig. 5EGo).


Figure 5
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  		FIG. 5. Fetal and maternal arterial and amniotic fluid IGF-I and insulin concentrations. A, Fetal IGF-I. B, Fetal insulin. C, Maternal IGF-I. D, Maternal insulin. E, Amniotic fluid IGF-I. Data are analyzed by repeated-measures ANOVA with Tukey post hoc test and values are mean ± SE. bullet, Control (n = 9–4); {circ}, SAL (n = 13–7); {blacksquare}, IGF-AF (n = 12–6); {square}, IGF-NUT (n = 11–6); {blacktriangleup}, IGF-IV (n = 10–5). Animal numbers decreased over the study period due to attrition (see Table 1Go). *, P ≤ 0.05, **, P ≤ 0.01, ****, P ≤ 0.0001 for time effects; upper-case letters represent group effects, and lower-case letters represent post hoc significance of the group x time interaction. C/cP ≤ 0.05 vs. control; S/sP ≤ 0.05 vs. SAL; A/aP ≤ 0.05 vs. IGF-AF; N/nP ≤ 0.05 vs. IGF-NUT; I/iP ≤ 0.05 vs. IGF-IV; ALL/allP ≤ 0.05 vs. all other groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IUGR is a major complication of pregnancy with many short- and long-term consequences for the baby. As yet there are no clinically applicable means of treating IUGR in utero. It has been shown previously that the development of IUGR can be prevented by intravenous or intragastric nutrient supplementation of the fetus (8, 9). However, to our knowledge, this is the first report of an intrauterine treatment significantly increasing fetal growth rate in established experimental IUGR.

The current study was designed to investigate the effects of fetal iv or amniotic IGF-I supplementation (alone or in combination with nutrients) on the growth, metabolism, and endocrine milieu of ovine fetuses with growth restriction by placental embolization. Embolization was successful in the current study, producing the expected changes in fetal growth patterns and hormone and metabolite levels consistent with those reported previously (28, 36). We found that all methods of fetal IGF-I supplementation investigated caused some degree of reversal of the IUGR caused by placental embolization. Thus, chest girth increments decreased in all groups after embolization but normalized to control values after IGF-AF and IGF-IV treatment. All treatments improved fetal weights at postmortem to values intermediate between control and SAL groups and not significantly different from either. A single measure of fetal weight at postmortem is a much less sensitive measure of fetal growth than repeated, direct, and precise measures of growth rate. The lack of a significant increase in fetal weight at postmortem after IGF-I treatment may therefore reflect the lack of power to detect a difference in this measurement.

Liver growth was promoted in the IGF-AF group, as reflected by the larger liver weight at postmortem. This effect was not seen when the IGF-I supplementation was combined with nutrients or given iv. It is possible that these effects of IGF-I supplementation on liver growth may be at least in part dose related. We have previously reported reductions in fetal liver, spleen, and thymus weights after amniotic IGF-I supplementation (28), using less than 40% of the dose used in the current study, whereas postnatal enteral IGF-I supplementation is reported to increase liver weight in other species (37, 38). Similarly, iv fetal IGF-I supplementation was reported to increase liver weight when given at a dose some 50 times higher than the dose used in this experiment (29) but not when given at a lower dose (30).

Perirenal fat weights were reduced by embolization, and this reduction was also reversed by amniotic IGF-I, both with and without nutrients, but not by iv IGF-I supplementation. IGF-I is important for the mitogenesis (39) and differentiation (40) of fetal brown adipocytes in rats. It is not clear why the same dose of IGF-I administered iv did not have the same effect on organ growth as amniotic IGF-I, but this finding may indicate that portal venous administration is more effective in promoting visceral organ growth for reasons yet to be determined.

The effects of IGF-I treatment on fetal carcass or organ growth were not accompanied by corresponding increases in fetal circulating IGF-I concentrations in any of the treatment groups. Embolization decreased fetal circulating IGF-I levels in all embolized groups, consistent with previous findings (28, 36). Although there was an initial increase in fetal plasma IGF-I concentrations in the first week of treatment in all embolized groups, this increase was more likely the result of the cessation of embolization than of IGF-I treatment because it was also seen in the SAL group. After the initial increase, fetal plasma IGF-I concentrations decreased again despite IGF-I supplementation. The lack of effect on circulating IGF-I concentrations after amniotic supplementation is consistent with the study of Bloomfield et al. (28), in which amniotic IGF-I supplementation resulted, paradoxically, in suppression of fetal circulating IGF-I concentrations. In that study, IGF-I mRNA levels in liver, muscle, and placenta were also significantly decreased in IGF-I-treated fetuses, suggesting that exogenous IGF-I may have suppressed endogenous IGF-I production (41). We previously demonstrated that radiolabeled IGF-I administered into amniotic fluid is taken up intact across the fetal gut into the portal circulation and hence would be expected to reach the fetal liver directly (24). Others have also reported suppression of hepatic IGF-I mRNA expression after much higher doses of iv IGF-I given for 10 d (42). Suppression of hepatic mRNA and protein expression may therefore represent the molecular basis for the lack of effects on circulating IGF-I concentrations in our IGF-I treatment groups.

Another possible mechanism for the growth-promoting effects of amniotic IGF-I treatment in the absence of altered circulating IGF-I levels may be via the release of yet unknown mediators from the fetal gut in response to the IGF-I supplementation. One possibility is ghrelin, a GH secretagogue first identified from rat stomach (43). We confirmed that ghrelin is also expressed in the fetal sheep stomach (our unpublished data). Ghrelin appears to be involved in the regulation of fetal growth and could be a possible candidate for the mediation of fetal growth in the absence of other endocrine stimuli (44, 45). Other potential gut peptides include the incretins, such as glucagon-like peptide-1 and -2 (46).

Interestingly, maternal circulating IGF-I concentrations increased in the IGF-IV group. Maternal IGF-I affects placental development and fetal growth (47). The effects of iv IGF-I supplementation on fetal carcass growth may therefore have been mediated by maternal IGF-I concentrations despite the absence of change in fetal circulating IGF-I. However it seems likely that ewes in the IGF-IV group differed from the other groups by chance because they also had higher PaO2 and insulin concentrations and lower PaCO2 than other groups, and none of these differences are readily explained by fetal IGF-I supplementation.

Unexpectedly, the growth-promoting effects of amniotic IGF-I supplementation on fetal growth rate and liver weight were apparently reduced by the addition of nutrients. Fetal PaO2 was not reduced in the IGF-NUT group, suggesting that the nutrient load we administrated in this study did not exceed the metabolic capacities of the fetus. The growth-inhibitory effect of the nutrients may therefore have been mediated by other mechanisms, i.e. via alterations in IGF-I binding capacity. There is some evidence for nutritional regulation of IGFBPs in fetal life (48), although direct effects of nutrients on IGFBP activity have not been demonstrated. In sheep, IGFBP-3 seems to be the most abundant IGF binding protein in amniotic fluid (24), and fetal IGFBP-3 production is regulated by nutrients, insulin, and IGF-I (49). An increase in amniotic IGFBP concentrations may have reduced the bioavailability of the amniotic IGF-I, thus decreasing its uptake by the fetus and consequently fetal growth.

Alternatively, it is possible that the additional nutrient administration down-regulated the umbilical nutrient uptake. Charlton and Reis (50) showed that intragastric glucose administration to the ovine fetus suppressed umbilical glucose uptake. However, the dose of glucose administered in that study was 15–35 times higher than in our study and increased the fetal arterial glucose content. This would have reduced the maternal/fetal concentration gradient and hence diminished the umbilical uptake. In our study, fetal arterial glucose concentrations were not changed by nutrient administration. Furthermore, even a reduction in umbilical glucose uptake would not explain the difference in growth pattern between the two amniotic treatment groups because they received the same amount of IGF-I, and fetal glucose uptake would be expected to be reduced across the placenta only to the extent that it was increased across the gut.

Fetal hemoglobin values gradually increased over the treatment period in the IGF-NUT group. Polycythemia can be caused by prolonged hypoxia (51), and fetal glucose administration has been reported to increase fetal oxygen demand (26). Although the IGF-NUT fetuses were not hypoxemic during the treatment period, as shown by fetal PaO2 and oxygen content, they did have elevated plasma and amniotic lactate concentrations, and it is possible that these findings reflect a small but longstanding increase in oxidative metabolism in response to the additional nutrient supply over the treatment period.

There was a relatively high rate of fetal loss in this experiment. Most losses were due to preterm labor, fetal infective death, or other fetal deaths, and there was no evidence that any one of the IGF-I treatments was associated with higher loss rates. Because the study involved chronically instrumented sheep fetuses studied over a 5-wk period, it seems likely that the losses are largely the inevitable consequences of the prolonged instrumentation. Many of the fetuses that died from causes other than infection had abnormalities at postmortem such as hydrops, pleural and peritoneal effusions, or hepatosplenomegaly, which are all consequences of the embolization that we observed before. Other causes of fetal death were umbilical cord occlusion by the indwelling amniotic catheters or direct effects of the embolization itself. Because there were no differences in loss rates, or cause of loss, among groups, there is no indication that the relatively high loss rates affected the outcome of the study. Furthermore, because few authors report loss rates from before surgery in experiments involving chronically catheterized fetal sheep, it is entirely possible, and perhaps even very likely, that the rates reported here are in fact similar to those in many other comparable studies.

This is the first report of an intrauterine treatment resulting in a significant increase in fetal growth rate in fetuses with established experimental IUGR. Although all IGF-I treatments partially ameliorated the effects of embolization on fetal weight, the most promising results were found after amniotic supplementation of IGF-I, without added nutrients. This treatment significantly improved fetal growth rate, liver and perirenal fat weights, and carcass growth without altering fetal blood gases, metabolite or IGF-I, and insulin concentrations. The exact mechanisms underlying the different effects of different treatments remain to be further elucidated and may include effects on the fetal gut, liver, or placental function. Amniotic IGF-I administration may provide the basis for a clinically applicable prenatal treatment for the IUGR fetus.


   Acknowledgments
 
We acknowledge the technical expertise of Toni Mitchell, Pierre van Zijl, Eric Thorstensen, and Chris Keven. The IGF-I was a gift from Kabi Pharmacia (Stockholm, Sweden). The Lion Foundation of New Zealand contributed toward laboratory expenses.


   Footnotes
 
First Published Online March 8, 2007

Abbreviations: dGA, Day of gestation; IUGR, intrauterine growth restriction.

This work was supported by the Health Research Council of New Zealand.

The authors have nothing to disclose.

Received December 19, 2006.

Accepted for publication February 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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