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Maternal Growth Hormone Treatment Increases Placental Diffusion Capacity But Not Fetal or Placental Growth in Sheep¹

Research Centre for Developmental Medicine and Biology, Department of Paediatrics, University of Auckland, Auckland, New Zealand

Address all correspondence and requests for reprints to: Prof. J. E. Harding, Research Centre for Developmental Medicine and Biology, Department of Paediatrics, University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail: j.harding{at}auckland.ac.nz

	Abstract

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We tested the hypothesis that chronic maternal GH administration would increase fetal substrate supply, increase maternal and fetal insulin-like growth factor I (IGF-I) concentrations, and therefore enhance growth in the late gestation fetal sheep. Eleven ewes received bovine GH 0.1 mg/kg twice daily for 10 days, whereas 10 control ewes received saline. GH treatment increased placental capacity for simple diffusion (P < 0.01), with a trend toward an increase in placental capacity for facilitated diffusion (P = 0.07). GH treatment also lowered maternal and fetal blood urea concentrations, and there was a trend toward increased fetal protein oxidation (P = 0.07). Maternal but not fetal IGF-I and insulin concentrations increased. Fetal and placental growth were not altered by GH treatment. Maternal and fetal metabolic status was significantly affected by maternal food intake. We conclude that maternal GH treatment increases placental transport capacity, but that anabolic effects in the mother may limit fetal substrate supply and therefore prevent an increase in fetal growth.

	Introduction

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FETAL GROWTH in late gestation is limited largely by maternal substrate supply and mediated by a number of maternal and fetal hormones. Glucose is the major source of metabolic substrate for the late gestation fetus (1) and is supplied from the mother across the placenta. Birth size is proportional to cord blood insulin-like growth factor 1 (IGF-I) concentrations in a wide range of species (2), and gene deletion studies have shown clearly that fetal IGF-I is critical for fetal growth in late gestation (3, 4, 5).

Birth size is also related to maternal GH and perhaps IGF-I concentrations (6, 7, 8). GH and placental lactogen (PL) both induce relative insulin resistance in the mother, and thus are postulated to influence fetal growth by increasing maternal glucose levels and hence substrate supply available to the fetus (2). Nevertheless, previous studies of maternal GH administration have produced conflicting results regarding effects on fetal growth (9, 10, 11, 12, 13, 14, 15).

We have previously shown in pregnant sheep that short-term infusion of IGF-I to the mother results in metabolic changes consistent with an increase in substrate uptake from mother to fetus (16), whereas IGF-I infusion to the fetus results in fetal anabolic changes (17). We have therefore speculated that prolonged elevation of IGF-I concentrations on both sides of the placenta would be expected to lead to enhanced fetal growth (16).

We would therefore expect that conditions that both increased fetal glucose supply and increased maternal and fetal IGF-I concentrations would be most likely to enhance fetal growth. GH administration to the mother would be expected to increase maternal circulating blood glucose, fatty acid, and IGF-I concentrations. In the fetus, circulating IGF-I concentrations are regulated by fetal blood glucose and insulin concentrations (18, 19, 20). Thus, maternal GH administration, by increasing maternal and hence fetal blood glucose concentrations, would also be expected to increase both fetal and maternal IGF-I concentrations. We therefore undertook the current study to test the hypothesis that chronic maternal GH administration would chronically increase fetal substrate supply as well as maternal and fetal IGF-I concentrations and would therefore enhance the growth of the late gestation fetal sheep. Among the effects observed was an unanticipated action of GH to enhance placental diffusion capacity.

	Materials and Methods

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Animals
Twenty-one Coopworth-Border cross-bred ewes with singleton pregnancies of known gestational age were brought into the laboratory at approximately 105 days of gestation. They were housed in individual metabolic cages with free access to water and fed ad libitum once daily with weighed amounts of concentrates (NRM Multifeed sheep nuts, NRM Feeds, Ltd., Auckland, New Zealand) and chopped barley straw. The residual feed was weighed each day and the net feed intake recorded.

At 115 ± 4 days gestation (mean ± SEM) ewes underwent surgery as described previously (17). Briefly, hysterotomy was performed under general anaesthetic. Catheters were placed in the fetal femoral artery and vein via tarsal vessels and the common umbilical vein via a para-umbilical incision. Two growth measuring devices (growth catheters) were placed, one around each half of the fetal chest from sternum to spine (21). Catheters were also placed in the uterine vein via the utero-ovarian vein draining the pregnant horn, the maternal carotid artery and jugular vein and the maternal femoral artery and vein via the tarsal vessels.

Antibiotics [250 mg streptomycin and 250 mg penicillin (Streptopen, Pitman Moore, Upper Hutt, New Zealand)] im to the ewe and 80 mg gentamycin iv to the fetus) were administered at the time of surgery and daily for the next 3 days. Catheters were flushed on alternate days with saline containing 10 U/ml of heparin. Growth catheters were measured twice daily after the ewe had been standing quietly for several minutes, and the mean of the two measurements recorded. Every 5 days ewes were weighed and body condition scores recorded (22).

At the end of the experiment (134 ± 0.3 days) ewes were killed with an overdose of pentobarbitone. The uterus and its contents were dissected, weighed and measured. All procedures were approved by the institutional Animal Ethics Committee.

Experimental procedures
Beginning at 125 ± 0.3 days gestation, ewes were divided into GH treated and control groups. GH treated animals received 0.1 mg/kg recombinant bovine GH (courtesy of Dr. D. Chaleff, American Cyanamid, Princeton, NJ) sc twice daily for 10 days. Control animals received an equivalent volume of normal saline. Blood samples were taken from the fetus (2.5 ml) and the ewe (3 ml) in the morning before feeding on days 0, 3, 5, 7, and 10 for metabolic and hormone measurements.

On days 0, 5, and 10 feto-placental substrate uptakes were measured. A solution containing antipyrine 6.5 mg/ml, 3–O-[methyl-³H]glucose 40 µCi/ml and [¹⁴C]urea 7.3 µCi/ml in normal saline was infused through a 0.45 µm filter to the fetus via the fetal venous catheter at 3 ml/h for 3.5 h after a 3-ml bolus. After 150 min of infusion to establish steady state, five sets of blood samples were taken at 15-min intervals. Each set consisted of samples taken from the maternal artery (2.5 ml), uterine vein (2.5 ml), umbilical vein (1 ml), and fetal femoral artery (1.3 ml).

All blood samples for metabolic measurements were aliquoted into Eppendorf tubes on ice and immediately frozen at -70 C until assay. Blood for hormone measurements was centrifuged at 4 C and the plasma separated and frozen at -70 C until assay. Samples for blood gas measurement were kept in capped syringes on ice until the measurements were made within 30 min using a Radiometer ABL330 blood gas analyzer and Radiometer OSM2 hemoximeter (Radiometer, Copenhagen, Denmark).

Assays
Glucose (23) and urea (24) were measured by standard enzymatic colorimetric methods modified for assay using a 96-well plate reader (25). Lactate was measured in microplates using an enzymatic procedure based on the reduction of NAD. Amino nitrogen was assayed by colorimetric reaction with ß-napthoquinone sulfonate (26). Free fatty acids were measured by a modification of a commercial kit assay (27). Antipyrine was measured in duplicate by HPLC (28). [¹⁴C]urea and 3–O-[methyl-³H]glucose activities were determined in duplicate aliquots of blood deproteinized with sulfuric acid and sodium tungstate and counted in a dual channel liquid scintillation counter (Rak-Beta model 1219, LKB Wallac, Turku, Finland) with external standard quench correction for 10 min or to a dpm error of less than 3%.

IGF-I was measured by double antibody RIA after acid ethanol cryoprecipitation extraction validated for fetal sheep plasma (18, 29). Insulin (18) and ovine placental lactogen (30) were measured by RIA.

Data analysis
Blood oxygen content was calculated from measured hemoglobin, oxygen saturation, and pO2. Blood flows were calculated from measured antipyrine concentrations according to the Fick principle (31). Uptakes of oxygen, glucose, and lactate were calculated for the uterus and its contents (referred to as uterine uptake = maternal artery-uterine vein concentration difference x uterine blood flow), fetus (umbilical vein-femoral artery concentration difference x umbilical blood flow), and placenta (uterine uptake-fetal uptake). Clearances of [¹⁴C]urea and 3-O-[methyl-³H]glucose were calculated by steady state diffusion techniques. Fetal urea production rate was calculated as the product of [¹⁴C]urea clearance and the (fetal artery-maternal artery) urea concentration difference (32, 33).

All baseline measurements and postmortem measurements were compared using t tests. Not all data were available for every animal at each timepoint, largely because catheters failed to sample or there were technical problems with the assays. Furthermore, although all animals were fed ad libitum, there was a wide variation in food intake both between animals and from day to day in the same animal. Food intake in sheep is an important regulator both of fetal growth and of circulating metabolites such as glucose, lactate, and fatty acids. We therefore undertook all analyses using multiple linear regression, including daily metabolizable energy intake as well as treatment group and study day as independent variables. The effect of repeated samples was taken into account by including sheep number nested within treatment group in the regression model, and the effect of treatment was assessed by the treatment by study day interaction term.

Differences in growth rates were tested using multiple linear regression using a similar approach. To test for a significant change in growth rate, two time variables were used (before and during treatment) each with their own constant. The second time variable took values only during GH treatment. Animal number was nested within group as the independent variable and the treatment by study day interaction was the statistic of interest.

All values are given as the mean ± SE of the mean. All statistical analyses were carried out using commercial software packages, Statview (Abacus Concepts, Berkley, CA) and JMP (SAS Institute, Cary, NC).

	Results

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There was no difference between GH treated and control animals for any metabolic or endocrine measurements at the beginning of the treatment period (Tables 1–3).

Maternal GH treatment decreased maternal and fetal blood urea concentrations by approximately 45% (Table 2) but did not alter circulating concentrations of oxygen, glucose, lactate, amino nitrogen, or fatty acids in the maternal or fetal blood. Maternal and fetal blood urea concentrations and fetal blood oxygen content fell with increasing gestational age in both groups. Maternal and fetal fatty acid concentrations and fetal blood glucose concentrations were significantly affected by maternal feed intake independently of GH treatment (Table 2).

GH treatment did not alter uterine or umbilical blood flow, nor utero-placental uptake of oxygen, glucose, and lactate. Umbilical blood flow, fetal lactate uptake, and uterine and fetal oxygen uptakes all rose over the study period in both groups, with no differences between groups. Fetal protein oxidation, measured by fetal urea production, increased 3-fold in the GH treated group compared with 70% in controls (P = 0.07, Table 3). Fetal urea production and umbilical blood flow were also affected by maternal feed intake independently of GH treatment (Table 3).

Maternal GH treatment increased placental capacity for simple diffusion as measured by a 70% rise in [¹⁴C]urea clearance compared with a 20% rise in controls (P < 0.01, Table 3). Placental capacity for facilitated diffusion, measured by 3-O-[methyl-³H]glucose clearance, increased in both groups over the study period but the trend toward an increase with maternal GH treatment did not reach significance (P = 0.07, Table 3).

Fetuses of GH treated and control ewes had similar rates of growth over the study period, as indicated by similar rates of girth increment measured by growth catheters (Table 4). At post mortem there was no difference between groups in fetal weight (4261 ± 186 g in GH treated vs. 4278 ± 291 g in controls), crown-rump length (47.6 ± 0.8 vs. 46.0 ± 1.6 cm), girth (35.0 ± 0.6 vs. 34.7 ± 1.0 cm), placental weight (441 ± 36 vs. 411 ± 42 g), uterine weight (683 ± 45 vs. 668 ± 36 g), amniotic fluid volume (1495 ± 500 vs. 1116 ± 249 ml) or weights of any individual fetal organs (data not shown).

	Discussion

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In this study, maternal GH treatment increased placental capacity for simple diffusion but did not alter fetal or placental growth. To our knowledge, this is the first demonstration that any maternal hormone treatment is able to alter placental transport capacity. The effect was not due to a generalized increase in placental size, at least as measured by placental weight. Furthermore, because the effect on simple diffusion was greater than that on facilitated diffusion, it seems likely that GH treatment altered placental surface area or membrane thickness rather than activity of placental carrier proteins such as glucose transporters.

The mechanism for this effect of GH on placental function is not known. We have previously shown that short-term infusion of IGF-I into the maternal or fetal circulations alters placental metabolism, especially of lactate, suggesting that placental function can be influenced by maternal and fetal endocrine status (16, 17). In those studies, maternal IGF-I infusions sufficient to increase maternal plasma IGF-I levels 3-fold for 3 h approximately doubled placental lactate production and fetal lactate uptake. However, a similar metabolic effect was not seen in this study despite elevation in maternal IGF-I, albeit over a much longer time course.

We had hypothesized that maternal GH therapy, by increasing maternal and fetal substrate supply and circulating IGF-I concentrations, would increase fetal growth. Although the treatment did increase placental diffusion capacity, we found no evidence for enhanced fetal growth. This was despite our direct measurement of fetal growth by longitudinal assessment of fetal girth increment as well as indirect measurements by assessment of fetal and organ sizes at postmortem. There are a number of possible reasons for this.

Firstly, pregnancy is associated with relative GH resistance. Although maternal GH treatment did increase maternal IGF-I concentrations as expected, we did not achieve a significant rise in maternal blood glucose, and hence we did not see the postulated rise in fetal blood glucose and plasma IGF-I concentrations. GH treatment in the nonpregnant state increases blood glucose and fatty acid concentrations by inhibition of the effects of insulin leading to reduced peripheral glucose uptake and increased lipolysis (34). Pregnancy is associated with a state of relative insulin resistance at least partly due to high circulating concentrations of placental lactogen and GH of placental origin (35). This is suggested as the likely cause of resistance to the metabolic effects of the exogenous GH reported in pregnant rats (9). Thus, we may have found no increase in fetal growth because pregnancy associated GH resistance meant that GH treatment did not sufficiently increase the availability of substrates such as glucose and fatty acids to the fetus.

Secondly, maternal GH treatment may have actually reduced the availability to the fetus of substrates that are potentially limiting to fetal growth. GH has anabolic effects on protein metabolism, increasing amino acid uptake and protein synthesis while reducing urea synthesis (36). This effect is reflected in our finding of a marked reduction in maternal blood urea concentrations. The fall in fetal urea concentrations is most likely to be secondary to the fall in maternal concentrations because urea produced by the fetus is excreted to the mother across the placenta by simple diffusion down a concentration gradient. Despite this, GH treatment was associated with a trend toward an increase in fetal urea production, a direct measure of fetal protein oxidation. Amino acids are normally taken up across the placenta by the late gestation fetal sheep in considerable excess of requirements for tissue accretion (37) and may normally account for some 20–30% of fetal oxidative substrate requirements (32). However, some amino acids are taken up at rates very close to those required for tissue accretion, and the margin of safety of these amino acids for fetal growth is very small (37). Maternal GH treatment, by increasing maternal tissue amino acid uptake, may have reduced the availability of some amino acids to the fetus. This could in turn limit overall amino acid incorporation into protein in the fetus and result in their disposal by oxidation to lead to the observed increase in fetal urea production.

This explanation is consistent with previous observations that GH treatment of pregnant rats may inhibit fetal growth if the dams are poorly nourished (9). In contrast, treatment of pregnant rats with GH antibody inhibited maternal muscle protein synthesis and increased fetal weight (38), suggesting that inhibition of maternal GH effects on maternal protein accretion improved fetal substrate supply and hence fetal growth. Furthermore, we have shown that maternal IGF-I infusion reduces the circulating concentration of essential amino acids in the fetal circulation, as well as lowering both essential and nonessential amino acid concentrations in the maternal circulation (39). This would be consistent with the reduction in the supply of amino acids to the fetus that we postulate under the circumstances of the present study.

Thirdly, any effect of GH therapy on fetal growth and metabolism may have been overshadowed in our studies by the powerful effects of maternal nutrition on all the parameters of interest. Although all ewes in this study were considered to be in good condition and were fed ad libitum, their feed intake varied widely both between animals and from day to day in the same animal. In ruminants, maternal feed intake directly influences the circulating concentrations of glucose and fatty acids, and hence of insulin and IGF-I. This influence is reflected in the independent effect of feed intake on several of these parameters in our study in multivariate analysis. This wide variation in food intake may have been large enough to obscure any relatively small effects of GH treatment.

A number of studies of the effects of maternal GH therapy on fetal growth have previously been reported, with variable results. Zamenhof and colleagues (10) reported that administration of large doses (3 mg/day) of bovine GH to pregnant rats from days 7 to 20 of gestation increased the brain but not body weight of the offspring. Clendinnen and Eayrs (11) used a more highly purified preparation of bovine GH from days 7 to 19 and found increased birthweight as well as altered brain structure and function in the offspring. Similar findings were obtained using doses of 0.1–3 mg/day of purified porcine GH from days 7 to 20 (12). More recently, Gargosky and colleagues (13) used recombinant human GH administered by osmotic minipump at 2.4 mg/kg·day from day 11 but failed to show any effect of this treatment on maternal IGF-I concentrations or fetal or placental size on day 21. Treatment of pregnant pigs with purified porcine GH from days 28 to 40 of gestation also increased fetal length but not weight (14). Treatment of pregnant sheep with GRF from days 130 to 140 of gestation increased lamb weight and cord blood IGF-II but not IGF-I concentrations (15). These differing findings may in part be a result of differing maternal nutritional planes and ages (38), resulting in differing effects on nutrient partitioning between mother and fetus, as well as different GH preparations and dose regimens. We are not aware of any previous studies of maternal GH administration in large, chronically catheterized animals that allow in vivo evaluation of the metabolic and endocrine changes in mother and fetus during treatment to clarify these effects.

We conclude that maternal GH therapy in late gestation pregnant sheep increases placental diffusion capacity but does not increase fetal growth. This suggests that placental diffusion capacity is not limiting on fetal growth under these circumstances. Although a rise in maternal IGF-I concentrations may be expected to increase placental substrate uptake, the anabolic effects of GH in the mother may counteract any potential benefit to the fetus by reducing fetal supply of limiting substrates such as amino acids. It seems likely, therefore, that enhancement of fetal growth will require enhanced substrate supply as well as an appropriate endocrine milieu and placental transport capacity.

	Acknowledgments

 
We would like to thank Dr. B. Breier for the hormone assays. Fiona Ffolliott-Powell, Christine Gibson, Sam Rossenrode, and Eric Thorstensen provided excellent technical assistance. Dr. D. Chaleff of American Cyanamid provided the GH for these studies.

	Footnotes

 
¹ This work was supported by the Auckland Medical Research Foundation and the Health Research Council of New Zealand.

Received June 24, 1997.

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