INTRODUCTION

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INTRAUTERINE GROWTH RETARDATION is an important cause of perinatal morbidity and mortality, with decreased oxygen delivery to the fetus over a prolonged period being widely thought to be a principal causal factor, a view that has received recent experimental support (6, 10, 24). Mechanisms by which the fetus adapts to both short- and longer-term hypoxemia have been well described by a number of investigators. These include marked changes in the secretion and plasma concentrations of the catecholamines epinephrine (Epi) and norepinephrine (NE) (10, 11, 15, 24, 28). Alterations in their production by the sympathetic nervous system and adrenal medulla are considered to play a very important role in the adaptation of the fetus to reduced oxygen availability (18), yet most knowledge of their role is based on extrapolation from short-term studies (i.e., <4 h) (17, 20, 26), although plasma Epi and NE concentrations remain elevated for periods greatly in excess of 24 h during chronic hypoxemia or intrauterine growth retardation (10, 11, 18, 24). Even continuous fetal infusion of Epi or NE for 24 h (5, 14) is too short to provide unequivocal evaluation of their effects on fetal growth. The ability of Epi and NE to influence fetal growth adversely during prolonged administration, either with or without concurrent hypoxemia, therefore remains unknown. Closer examination of the actions of Epi and NE during prolonged administration is evidently necessary if their proposed roles in counterregulation of chronic fetal hypoxemia are to be understood.

To examine the hypothesis that Epi and NE may adversely influence growth when their concentration in the fetus is increased, we have administered Epi and NE for periods of 7-12 days to chronically cannulated fetal sheep during late gestation. Changes in fetal blood gases and metabolite and hormone concentrations in the fetus were monitored throughout these prolonged infusions and compared with changes in saline-infused control twin fetuses subject to the same maternal environment. Effects of Epi and NE on fetal organ and tissue development were determined at the end of the infusion. The studies reported provide unequivocal evidence that both Epi and NE do bring about a severe and selective retardation in the growth of many fetal organs and tissues by mechanisms that do not depend on the presence of hypoxemia. Adverse effects on the growth and development of the fetal body are marked, with particularly severe effects on the growth of skeletal muscle being evident.

	METHODS

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Animal preparation. All surgical procedures and experimental protocols used in the studies reported below were carried out in accordance with a project license approved by the UK Home Office under the terms of the Animals (Scientific Procedures) Act 1986. Twenty Mule × Suffolk crossbred ewes, of accurately known gestational age mated with Polled Dorset rams and diagnosed as twin-pregnant by ultrasound scanning at 70-90 days gestation, were fasted for 24 h before surgery at 118-120 days gestation (full term is 145-147 days in these breeds). Anesthesia was induced by intravenous injection of 20 ml of 5% thiopentone sodium (Intraval sodium) and maintained after tracheal intubation with halothane (1.5% in oxygen). With the use of aseptic procedures and full sterile precautions, vinyl catheters (Dural Plastics, Auburn, Australia) were placed into the dorsal aorta (1.5 mm OD, single lumen) and inferior vena cava (2.5 mm OD, triple lumen) of each fetus via the femoral artery of one hindlimb and the recurrent tarsal vein of the other, and a 3-mm OD polyvinyl catheter was attached to the skin of the fetus between the hindlimbs to permit sampling of amniotic fluid. In two ewes, found at surgery to be carrying only a single fetus, an additional vinyl catheter (1.5 mm OD, single lumen) was also inserted into the common umbilical vein of the fetus. To provide an estimate of fetal size, the length of both hind feet (metatarsal) was measured against a ruler before returning the fetal hindlimbs to the uterus and closing the incision. Polyvinyl catheters (3 mm OD; Portex, Hythe, UK) were placed in the left carotid artery and jugular vein of the ewe. The surgical procedures used have been reported before (4). At surgery, 25 mg medroxyprogesterone acetate (Depo-provera; Upjohn, Crawley, UK) and prophylactic antibiotics consisting of 250 mg procaine penicillin and 250 mg dihydrostreptomycin were administered intramuscularly to the ewe. Each fetus was given 300 mg benzyl penicillin and 10 mg gentamycin intravenously after cannulation. Similar amounts of antibiotics were administered once daily for a further 3 days after surgery. Catheters were flushed daily with heparinized 0.9% sterile saline (250 U/ml) throughout the study. After surgery, ewes were housed in metabolism cages and offered water and hay ad libitum. Beginning 24-36 h after surgery, ewes were also offered 300 g of a barley-based concentrate mixture twice daily to provide sufficient metabolizable energy and protein for maintenance and pregnancy during the final month of gestation. Four days after surgery, daily blood sampling of the ewe and fetuses before the morning feed was begun and was continued until termination of the study to monitor blood gases and pH (1 ml) and to determine plasma metabolite and hormone concentrations (4 ml) during the experiments. Samples were collected into heparinized syringes and placed on ice immediately. Plasma was separated by centrifugation at 4°C and stored at 20°C until required for analysis. On the day before prolonged infusions were commenced, fetal arterial and amniotic catheters were connected to Devices pressure transducers (Lectromed, Letchworth, UK) in a box on the outside of the cage to permit collection of continuous records of fetal arterial blood pressure and heart rate. True fetal arterial pressures were obtained by subtracting amniotic pressure electronically, and these, together with heart rate, were output on a Devices multichannel recorder. Voltages were also output to a Squirrel data logger (Grant Instruments, Cambridge, UK) in most of the studies. Readings taken at 5-s intervals and averaged over a minute were stored for later transfer to a computer and calculation of heart rate and pressure values. Mean values over each hour, calculated after removal of out-of-range observations, are used in the results presented here.

Experimental procedures. To determine effects of prolonged intravenous administration of Epi or NE to the fetus on fetal development, infusions into the inferior vena cava of the fetus via one lumen of the triple-lumen catheter inserted into the recurrent tarsal vein were begun 7 ± 1.3 days (mean ± SD) after surgery and after collection of control blood samples for 3 or 4 days. Epi was infused into 12 fetuses (9 twins and 3 singles) for periods ranging from 7 to 12 days (9 ± 2.0 days, mean ± SD). The rate of infusion, initially 1 µg/min, was increased after either 48 or 72 h to 2 µg/min to counteract attenuation of responsiveness and was continued at this rate for the remainder of the study. Five experiments were terminated by premature onset of parturition or death of one fetus after 7 or more days of infusion. In the others, the infusion was switched off for 1-2 h before termination. NE was infused into five twin fetuses for 12 or 13 days before termination. The rate of infusion, initially 2 µg/min, was increased after 72 h to 4 µg/min for the remainder of the study. In one further experiment, NE infusion was terminated by premature onset of delivery after 7 days of infusion, and in another, observations on a saline-infused control twin fetus were continued for 11 days before termination after death of the NE-infused fetus.

Infusions were carried out using infusion lines covered in black tape attached to black 50-ml syringes and Braun Perfusor syringe pumps (Braun Medical). Infusion solutions were replaced daily using sterile solutions prepared freshly from commercial preparations (Epi: Adrenaline Injection, Antigen Pharmaceuticals, Roscrea, Ireland; NE: Levophed, Sanofi Winthrop, Guildford, UK) or from sterile stock solutions of L-epinephrine bitartrate or L-norepinephrine hydrochloride (Sigma Chemical, Poole, UK), diluted in 0.9% sterile saline containing 0.3% ascorbic acid and stored at 4°C. In each experiment on ewes with twin fetuses, only one fetus was infused with the drug, the other being infused with the diluent (0.3% ascorbic acid in 0.9% sterile saline). All infusions were given at the same rate (1.2 ml/h) via one lumen of the venous catheter. This approach was used to provide a directly comparable maternal environment for comparison of development and metabolism in control and drug-infused fetuses, because previous studies (4) had provided no evidence for effects of the -agonist ritodrine or Epi infused into one twin on metabolism or measures of cardiovascular function in the other saline-infused control twin. Choice of cannulated twin for infusion of catecholamine or of diluent was random.

Experiments were terminated by rapid intravenous administration of 20 ml of a barbiturate anesthetic (Euthatal; Rhône Mérieux, Harlow, UK) to the ewe. The uterus was removed with catheters still attached, so that control and drug-infused fetuses could be identified. Fetuses were removed, towelled dry, and weighed before dissection to recover and weigh the principal organs. In one ewe from the NE infusion group, a third uncannulated fetus was found at autopsy. This was treated as other fetuses and used to provide additional control information on organ and carcass tissue development. When premature delivery occurred, lambs were killed by rapid intravenous administration of a barbiturate anesthetic (Euthatal) as soon after delivery as possible. Before dissection, the length of both fetal hindlimbs was measured to provide an index of fetal limb growth since the time of surgery. Fetal carcasses were frozen and stored at 20°C for later dissection.

To obtain a more specific evaluation of carcass tissue development, carcasses were thawed to permit removal of a selection of muscles or muscle groups [biceps femoris, semitendinosus, semimembranosus, adductor femoris, gastrocnemius, and lateral extensors (extensor medialis, extensor longus, peroneus longus, peroneus tertius and tibialis anterior)] and bones (pelvis, femur, tibia and metatarsal) from one hindlimb, the longissimus (L.) dorsi from the lumbar and thoracic region of the spine, and dorsal muscles [L. costarum, L. capitis et atlantis, L. dorsi (cervical part), complexus, spinalis, and multifidus dorsi] from the cervical region of the spine. To avoid possible adverse effects of femoral arterial cannulation on limb development, only the hindlimb that had contained the venous catheter was used. After removal of the skin and before removal of any muscles, the crown-rump length of each fetus was measured. Muscles were dissected free of adherent fat and other tissues after isolation and before weighing. Bones were separated from attached muscle, ligaments, and tendons and then weighed before measurement of length and, where applicable, minimum diameters of the bone shaft with vernier calipers. It is acknowledged that measurements on tissues that had been frozen and thawed would inevitably lead to some underestimation of their fresh weight because of loss of liquid during thawing. To assess the magnitude of this loss, muscles from carcasses of 13 other fetal sheep, which had been dissected and weighed before being frozen for storage, were thawed and weighed again after blotting to remove surface liquid. Regression analysis showed thawed weight was 94.8 ± 0.93% of the fresh muscle weight (range 4-40 g), and the values were highly correlated (r² = 0.995, n = 50).

Analyses. Blood arterial partial O₂ (Pa_O2) and CO₂ (Pa_CO2) pressure and pH values were determined at 39°C using a Corning 168 blood gas analyzer (Corning Medical and Scientific, Halstead, Essex, UK); blood hemoglobin, arterial O₂ saturation (Sa_O2), and O₂ content were determined with an OSM2 Hemoximeter (Radiometer, Crawley, UK); and hematocrits were determined using a microhematocrit centrifuge (Hawksley, Lancing, UK). Plasma glucose, lactate, nonesterified fatty acids (NEFA), and -amino nitrogen (-AN) concentrations were measured using the same spectrophotometric methods as used in earlier studies (4), and tissue concentrations of protein, DNA, and RNA were measured by procedures used by Fletcher and Bassett (8). Also, as in earlier studies (4), plasma insulin was determined by radioimmunoassay using antiporcine insulin antiserum, a monocomponent ovine insulin standard (Lilly), ¹²⁵I-labeled porcine insulin, and a dextran-coated charcoal separation. Plasma growth hormone (GH) was determined using National Institutes of Health standards [ovine GH (NIDDK-oGH-I-4)], antiserum (NIDDK-anti-oGH-2), ¹²⁵I-oGH (NIDDK-oGH-I-4), labeled locally, and a dextran-coated charcoal separation.

Calculations. Adopting an approach similar to that of Boyle et al. (6) and Santucci et al. (27), we used fetal body weight at autopsy and measurements of metatarsal length on the hind- limb without an arterial catheter from 46 control fetuses in the present experiments, as well as other recent related investigations, to calculate the relationship between metatarsal length and fetal body weight in our flock. The significant relationship (Eq. 1) obtained (r² = 0.656, n = 46) provided estimates for the weight of fetuses at surgery more like those calculated from the Santucci equation (27) than those calculated using the Boyle equation (6)

(1)

This equation was therefore used to calculate estimates for the weight at surgery of all fetuses used in the present investigations. The values obtained were in the same range as body weights of twin Mule × Suffolk crossbred fetal lambs measured at 118-120 days gestation in earlier studies at Oxford (13). With the added assumption, which appears justified by the observations of Harding et al. (12), that the increase in live weight from surgery until autopsy was linear in saline-infused control fetuses and would have been similar in control and Epi or NE-infused fetuses before the start of infusion, we calculated an estimate of the daily rate of body weight increase during the period of catecholamine or diluent infusion for each individual fetus using Eq. 2

(2)

where W_F is the fetal body weight at autopsy (kg), W_Ie is the estimated initial body weight, RG_c is the mean daily rate of gain for control fetuses, D_S-I is the time from surgery to the start of infusion (days), and D_I is the duration of the infusion (days).

With the further assumption that the proportion of body weight represented by muscle and bone did not change significantly in control fetuses during the period of late gestation covered by the studies, this equation has also been used to provide an estimate for growth during Epi or NE infusion of the selected group of muscles and bones measured in the investigation.

Statistical analysis. Concentrations of lactate, NEFA, insulin, and GH were converted to logarithms before calculation of means or any other statistical procedures because absolute values are not normally distributed and changes in concentration are usually multiplicative. Values reported in the text are geometric means, and logarithmic scales are used for graphical presentation of means and SEs throughout. Because of misdiagnosis, the loss of one twin before completion of the study, or great disparity in initial size of twin pairs, it was not possible to evaluate effects of Epi or NE treatment on the fetus within twin pairs. General linear model (GLM) ANOVA procedures and covariance have therefore been used for statistical evaluation of fetal responses to Epi or NE infusion. Effects on fetal development were assessed using a GLM ANOVA for unequal subclass numbers, with groups as the independent factor and the estimate of fetal weight at surgery as a covariate, to adjust for differences in initial fetal size in all analyses on measures of fetal development. To evaluate effects of Epi or NE infusion on blood gases, mean arterial pressure, heart rate, and the plasma concentration of hormones and metabolites by a similar ANOVA procedure, a mean value, calculated for each individual for the period from 2 days after the start of infusion until termination, was used. A mean control value, calculated from values before the start of infusion in each fetus, was used as a covariate in each analysis. Pairwise comparisons between estimated marginal means for each treatment group were used for post hoc assessment of differences between groups. To evaluate differences among Epi, NE, and control fetuses in responses of blood gases and metabolite and hormone concentrations during the first week of infusion, results were analyzed using a repeated-measures GLM procedure for unequal subclass numbers, with groups and time as factors. The Bonferroni modified t-test was used for post hoc comparisons among groups at each time point. Probabilities less than 0.05 were regarded as significant. Correlation coefficients and linear regression equations were calculated using least-squares methods. SPSS for Windows, Advanced Statistics 7.5, was used for all calculations.

	RESULTS

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Effects on fetal growth and development. There were no significant differences among control fetuses and those to be infused with Epi or NE in their mean initial body weight estimated from the metatarsal length measured at surgery (Table 1). However, initial fetal weight had a highly significant influence (P < 0.001) on fetal body weight and all other measurements of fetal tissue and organ sizes at autopsy and so was included as a covariate in all statistical analyses evaluating the effects of Epi or NE on fetal growth. Figure 1 illustrates how the effects of Epi or NE infusion on fetal weight and the length of the metatarsal measurement at autopsy in individual fetuses were influenced by their relationship to estimated size at surgery. Even so, the mean body weight of fetuses infused with either Epi or NE for 8-12 days (3.2 ± 0.16 kg, n = 12, and 3.6 ± 0.33 kg, n = 6) was significantly less at autopsy (P < 0.01) than that of control fetuses (4.3 ± 0.14 kg, n = 15), and the effect of the two catecholamines did not differ. Calculation of weight gain during the actual period of infusion (Table 1) shows both Epi and NE prevented any significant increase, whereas controls continued to grow at a mean rate approaching 100 g/day. The metatarsal measurement was also significantly less at autopsy (P < 0.05) in fetuses infused with either Epi (12.7 ± 0.15 cm) or NE (13.1 ± 0.28 cm) than that of control fetuses (13.6 ± 0.06 cm), and calculations showed the increase during Epi or NE infusion was reduced to 40% of that in controls (Table 1). Crown-rump length measured at autopsy was only significantly decreased by Epi infusion, but the weight-to-length ratio was significantly decreased in fetuses infused with either Epi or NE (Table 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Body weights (top) and metatarsal length measurements (bottom) of epinephrine (Epi; )- or norepinephrine (NE; )-infused fetuses and of saline-infused control twins (Epi,  or NE, ) relative to their estimated body weight at surgery. Lines shown represent calculated regressions for the relationships y = 0.99x + 1.71 and y = 1.23x + 10.3 for body weight and metatarsal measurement at autopsy in saline-infused control fetuses (broken line) and y = 0.87x + 1.02 and y = 1.64x + 8.43 in Epi- or NE-infused fetuses (solid line).

The reduction in fetal growth due to Epi or NE infusion also involved differential effects on the weight of individual organs and tissues, as shown in Table 2. Brain weight was not altered by infusion of either and actually increased relative to body weight, the increase reaching significance in Epi-infused fetuses. The weight of most other organs decreased significantly, but the reduction was proportional to the decrease in fetal body weight with the exception of the thymus and spleen, which weighed <50% of control values and were significantly smaller relative to fetal weight. Adrenal and thyroid weights were not altered.

There were marked differences between muscle and bone in the magnitude of Epi or NE effects on their development (Tables 3 and 4), although there were no differences among the individual muscle groups. Muscle growth, as assessed by an ~27% decrease in the total weight of the selected muscles isolated, from 145 ± 6.4 g in the 15 controls to 97 ± 4.8 g or 106 ± 6.9 g in the 12 Epi- and 6 NE-infused fetuses (P < 0.001), was more severely influenced than growth of the fetus as a whole or that of the bones measured, where the decrease was ~13%. The differing magnitude of the effects of Epi or NE on the relationship of the weight of these muscles and hindlimb bones to estimated body weight at surgery is illustrated in Fig. 2. Calculation of muscle weight gain shows that Epi or NE infusion resulted in a complete cessation of muscle growth during the actual period of infusion and may have caused muscles to lose weight (Table 3). Despite these differences in growth, there were no significant effects on mean concentrations of protein (89 ± 6.9 vs. 93 ± 7.6 mg/g wet wt), DNA (2.0 ± 0.13 vs. 2.2 ± 0.23 mg/g wet wt), or RNA (4.4 ± 0.19 vs. 3.9 ± 0.27 mg/g wet wt) in muscles from control or Epi- and NE-infused fetuses.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Total weights of the selected muscles and bones dissected from the carcasses of individual Epi-infused () and NE-infused () fetal sheep and of saline-infused control twins (Epi,  and NE, ) relative to their estimated weight at surgery. Lines shown represent calculated regressions for the relationships y = 42.5x + 38.9 and y = 19.2x + 26.4 for muscle and bone in saline-infused control fetuses (broken line) and y = 27.1x + 28.7 and y = 21.8x + 3.66 in Epi- or NE-infused (solid line) fetuses.

Mean weights of each of the four hindlimb bones measured were all significantly less in fetuses infused with Epi or NE than in control fetuses (Table 4). Calculation of bone growth during the period of infusion suggests Epi and NE reduced the rate of weight gain in the hindlimb bones greatly (Table 4). On the other hand, bone length continued to increase at ~55% of the control rate during infusion, and this differential effect is evident in a significant decrease in the weight-to-length ratio for each of the four bones from fetuses infused with Epi or NE (Table 4). The diameter of the shaft of the femur, tibia, and metatarsal also decreased (P < 0.01) in fetuses infused with Epi or NE (results not shown). Despite this, length-to-diameter ratios for both the tibia and the metatarsal increased by ~7.3% (P < 0.05) in fetuses infused with Epi or NE.

Effects on fetal blood gases, pH, blood pressure, and heart rate. Infusion of Epi or NE increased mean Pa_O2, Sa_O2, and oxygen content steadily during the first 48 h and decreased mean Pa_CO2 values over the same period (Fig. 3). Repeated-measures ANOVA using the mean preinfusion concentration as a covariate showed these changes were already significant 24 h (P < 0.05) after the start of infusion. Subsequently, Pa_O2 concentrations stabilized and mean values during the rest of the period of infusion remained significantly greater than values in control fetuses (Table 5), as were mean blood oxygen content and Sa_O2, whereas mean Pa_CO2 was decreased. There were, however, no consistent differences between Epi and NE in the magnitude of any of these changes. The increase in mean Pa_O2 above the preinfusion value was inversely correlated with the rate of weight gain in the muscles isolated (r = 0.76; P < 0.001) and with the rate of increase in the metatarsal measurement (r = 0.56; P < 0.01) during the period of infusion in the 31 fetuses where both measurements were made. Neither Epi nor NE significantly altered fetal arterial blood pH, either 24 h after the start of infusion or later (results not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Mean ± SE values for PaO2, PaCO2, O2 content, and O2 saturation in femoral arterial blood from fetal sheep infused with Epi (; n = 12) or NE (; n = 6) and in control twin fetuses infused with saline diluent only (; n = 15). Arrows and horizontal bars show period of infusion. Rates of Epi and NE infusion are given in METHODS.

Mean arterial pressure and heart rates increased to a similar degree during infusion of Epi and NE at these rates. Mean arterial pressure increased by ~25% to a maximum 11.7 ± 1.68 mmHg higher in fetuses infused with Epi (n = 11) and 10.6 ± 2.02 mmHg in fetuses infused with NE (n = 6) than the value in control twins (42 ± 1.1 mmHg; n = 13) 12-24 h after the start of infusion (P < 0.001). After this, arterial pressure declined in fetuses infused with Epi or NE. Despite a brief increase after doubling of the infusion rate, mean arterial pressure after the second day of Epi or NE infusion did not differ significantly from that of control twins (Table 5). Heart rates in fetuses infused with Epi or NE increased 14 ± 4.4 beats/min (n = 11) or 11 ± 5.5 beats/min (n = 6) above the mean of 170 ± 3.0 beats/min (n = 13) in saline-infused control twins during the first 6 h of infusion, and although there was some decline, heart rate remained significantly higher in fetuses infused with Epi or NE than in control twins throughout infusion (Table 5).

Effects on fetal blood metabolite and hormone concentrations. During the first 3 days of infusion, Epi increased fetal plasma concentrations of NEFA, lactate, and glucose significantly more than NE did (P < 0.001), despite the twofold greater rate of NE infusion. These results are presented in greater detail elsewhere (3). The acute effects of Epi were largely attenuated within 48 h, and there was no response to doubling of the infusion rate. Throughout the remainder of the experiment, effects of the two catecholamines were statistically indistinguishable at the infusion rates used. During this time, Epi and NE increased fetal plasma glucose concentration by ~77 and 53% in comparison with that of control twins (Table 6). In contrast, plasma -AN concentration tended to decrease, but only the effect of Epi was significant. Lactate and NEFA concentrations in fetuses infused with Epi or NE did not differ from those in controls over this period (Table 6).

Both Epi and NE decreased plasma insulin concentration dramatically within the first 24 h of infusion (P < 0.001), and there was little evidence for any recovery subsequently (Fig. 4). Geometric mean insulin concentrations from day 2 until the end of infusion were only ~30% of those in control fetuses during the same period (Table 6). By contrast with insulin, plasma GH concentrations of fetuses infused with Epi or NE increased (Fig. 4), so geometric mean concentrations after the first 48 h of infusion were almost 50% higher in Epi- and NE-infused fetuses than in controls (Table 6).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Geometric mean ± SE plasma insulin (top) and GH (bottom) concentrations in arterial blood from fetal sheep infused with Epi (; n = 11) or NE (; n = 6) and in control twins infused with saline (; n = 13). Arrows and horizontal bars show period of infusion. Rates of Epi and NE infusion are given in METHODS.

	DISCUSSION

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Increased fetal plasma Epi and NE concentrations have been observed in experimental models of intrauterine growth retardation (18) as well as during fetal hypoxemia (10, 11, 15, 24, 28), and it has generally been considered that the increases in catecholamine concentrations play an important role in the process of adaptation by the fetus to inadequate oxygen and metabolic substrate availability. The results of the present experiments are the first to provide unequivocal evidence that the cardiovascular, endocrine, and metabolic changes brought about by Epi or NE, which permit such adaptation, do also result in tissue-selective retardation of fetal growth in the absence of any externally imposed limitation to oxygen or nutrient supply. This suggests that elevation of plasma catecholamine concentrations is the primary determinant of adverse changes in fetal tissue growth observed during prolonged uterine blood flow reduction and hypoxemia, a conclusion opposite to that drawn from studies of Epi or NE infusion for 24 h only (5).

Plasma concentrations of Epi and NE established by the infusions were not determined, but the infusion rates used (0.25-0.35 µg Epi · kg¹ · min¹ and 0.5-0.7 µg NE · kg¹ · min¹ based on estimated initial fetal body weight) were chosen for consistency with earlier short-term studies of Epi and NE actions in the late-gestation fetal lamb (17, 26), as well as to establish plasma concentrations in the range observed during hypoxemia. Hooper et al. (14) showed plasma Epi and NE concentrations do increase to values comparable to those observed in fetal hypoxemia when Epi or NE are infused at rates directly comparable with those used in the present study, suggesting that the responses reported here are of physiological relevance. The possibility that altered clearance of Epi and NE during prolonged administration might contribute to attenuation of responses cannot be ruled out, but transfer of infused Epi or NE to the other twin fetus or to the mother seems unlikely. Our studies provide no evidence for measurable effects of the infusions on any cardiovascular, metabolic, or endocrine parameter monitored in control twins or the mother, and the measured differences in growth between saline-infused control twins and the Epi- or NE-infused twins are distinct.

Recalculation of Boyle's results (6) by the approach used in the present study suggests the rate of fetal body weight growth was actually reduced by ~50% during the 7 days of experimental hypoxemia, even though fetal weight at autopsy decreased by <10%. In a longer study (24), repeated placental embolization for 21 days reduced fetal weight at autopsy by 28%, a reduction directly comparable to that observed in pancreatectomized fetal sheep (9) and probably indicative of a substantially greater reduction in the rate of growth during the experimental period itself. At the rates of Epi or NE infusion used, effects on fetal weight gain in the present study are, therefore, of comparable severity to those resulting from hypoxemia in late pregnancy or fetal pancreatectomy, but may be less severe than those resulting from the very prolonged restriction of fetal nutrient and oxygen supply due to very small placental size (12). The changes in body proportions resulting from disproportionate effects on the growth of individual tissues in each of these different models of fetal growth retardation are, in general, remarkably similar. Brain and skeletal development are preserved at the expense of soft tissue growth, with striking retardation in development of some individual organs such as the spleen and thymus in each (9, 10, 12). The reduction in size of the liver relative to body weight, observed after 24 h of Epi or NE infusion (5), was not evident when infusion was maintained for longer. The present study shows that, quantitatively, the most important reduction in growth during Epi or NE infusion is that in skeletal muscle, where the magnitude of retardation is comparable to that in the thymus and spleen. Growth of skeletal muscle appears to cease, with little evidence of variation in this among the muscles. In bone, although length growth continues, albeit at a reduced rate, the increase in weight is affected almost as severely as that of muscle. In contrast, prolonged infusion of Epi or NE did not cause significant mobilization of lipid from the perirenal brown adipose tissue depots or from depots elsewhere within the carcass. A more detailed examination of the perirenal adipose tissue, reported separately (3), showed that thermogenesis had not been activated by Epi or NE infusion, even though infusion of a ₂-adrenergic agonist, ritodrine, for a comparable period did result in activation of thermogenic enzymes and severe depletion of fetal lipid stores.

The mechanisms by which the changes in fetal growth are brought about during Epi or NE infusion have not been established by our studies. Effects of prolonged increases in plasma Epi and NE concentrations on the distribution of cardiac output in the absence of hypoxemia have not been reported, but redistribution of cardiac output away from carcass tissues during hypoxemia is well known (16, 28, 29) and is considered to result from increased -adrenergic receptor stimulation consequent on elevated levels of Epi and NE in fetal blood. The increases in mean arterial pressure and heart rate observed during early stages of Epi or NE infusion are consistent with earlier observations (5, 17, 20, 26) and with increased vascular resistance in peripheral tissues and redistribution of cardiac output, but attenuation of these effects after 24 h of infusion makes it questionable how long such redistribution may be maintained. In this context, some reports indicate that blood flow to carcass tissues remains unchanged during prolonged hypoxemia (11, 29), so there must be uncertainty whether oxygen or glucose delivery to carcass tissues has actually been reduced when blood oxygen and glucose concentrations increase, as they do during prolonged Epi or NE infusion. Marked differences between Epi and NE in the magnitude of their initial effects on plasma concentrations of glucose, lactate, and NEFA and the complete attenuation of these effects within 3 days, despite continued infusion, must raise questions about the importance of ₂-adrenergic receptor mechanisms in bringing about the prolonged increases in fetal plasma glucose, oxygen, and GH concentrations or the selective changes in tissue growth observed. On the other hand, prolonged infusion of the selective ₂-adrenergic receptor agonist ritodrine also results in growth retardation in fetal sheep (3), despite marked attenuation of its actions on metabolite and hormone concentrations and evidence that there is attenuation of -adrenergic receptor-mediated responses to the natural catecholamines too (4).

There is considerable evidence that Epi and NE adversely influence glucose utilization in skeletal muscle by attenuating its responsiveness to administered insulin (19, 23). The prolonged inhibition of insulin secretion by Epi or NE observed in the fetal sheep undoubtedly would also reduce glucose and oxygen utilization by insulin-sensitive fetal tissues, such as skeletal muscle (23), so the increase in fetal blood oxygenation and decline in blood Pa_CO2 observed, as well as the maintenance of fetal plasma glucose at concentrations comparable to those of pancreatectomized fetal sheep (9), could reflect decreased utilization consequent on the reduced level of insulin. However, the inhibition of insulin release could be a feature of more general relevance, because administration of insulin to fetal sheep at rates within the physiological range increases heart rate and the proportion of cardiac output distributed to the carcass tissues (22), as well as increasing glucose and oxygen utilization (23). These effects are accentuated by establishment of hyperinsulinemia (28) and involve significant reductions in blood pressure and vascular resistance in the carcass and other tissues, changes that are not overcome by superimposed hypoxemia (28). Hemodynamic effects of insulin also make a quantitatively important contribution to its action on glucose utilization in adult humans, through selective stimulation of skeletal muscle blood flow by a nitric oxide-dependent pathway (2). Inhibition of insulin secretion by Epi or NE during fetal hypoxemia could be crucial to the successful redistribution of cardiac output necessary for preservation of oxygen and substrate supply to the brain and myocardium.

Because insulin is an important inhibitory regulator of insulin-like growth factor (IGF)-binding protein (IGFBP)-1, as well as a stimulator of IGF-I (7), inhibition of its secretion by Epi and NE must also contribute to the increase in plasma IGFBP-1 concentration and its hepatic synthesis observed during Epi or NE administration (14), hypoxemia (21), and undernutrition (25) in fetal sheep. An increase in IGFBP-1 concentration, through its inhibitory effect on IGF-I action, together with the decreased plasma insulin concentrations observed and reductions in IGF-I and IGF-II, like those observed in hypoxemic (21) and growth-retarded (25) fetal sheep, may make important contributions to the selective reduction in DNA synthesis seen in fetal tissues after 24 h of hypoxemia (13) and to the growth retardation in muscle and other tissues observed during Epi or NE infusion as well as that occurring after prolonged hypoxemia (24). The increases in fetal plasma GH concentration observed during Epi and NE infusions are also consistent with a reduction in plasma IGF-I and its inhibitory feedback on GH release.

With the recognition that growth retardation during fetal development may influence physiological function and disease in adult life (1), much emphasis has been placed on the disproportionate effects of undernutrition or hormonal manipulation on organ and tissue growth in the fetus. The extent to which any of these reflect significant changes from the normal allometry of tissue and organ growth in the fetus and its response to altered nutrient availability characteristic of mammalian development remains far from clear. The similarity of differential fetal tissue growth responses to administration of either Epi or NE or pancreatectomy (9) to those associated with prolonged undernutrition (12) has already been noted and suggests that catecholamines and insulin acting directly or indirectly play major roles in regulating allometric relations between organs and tissues during normal and abnormal growth. On the other hand, the marked myocardial hypertrophy observed during prolonged experimental hypoxia (24) differs from the failure of Epi or NE administration to influence the size of the heart in normoxemic fetuses and does indicate that reduced oxygen availability may have additional direct effects on development of the heart that cannot be attributed directly to changes in blood pressure or distribution of the circulation resulting from increased catecholamine concentrations.

Perspectives