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1 Department of Obstetrics and Gynaecology, The University of Western Australia, Perth, WA 6009; 2 Womens and Infants Research Foundation, Perth, WA 6008; 4 Fetal and Neonatal Research Unit, Department of Physiology, Monash University, Victoria, Australia 3800; and 3 Medical Research Council Group in Fetal and Neonatal Health and Development, Department of Physiology, University of Toronto, Ontario, Canada M5S 1A8
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Our aim was to determine the postnatal effects of single and repeated glucocorticoid injections during late gestation. Repeated (104, 111, 118, 125 days) or single (104 days) injections of betamethasone or saline were given to the ewe or by ultrasound guided injection to the fetus (term 150 days). Lambs were born spontaneously and studied at 3 and 6 mo and 1 yr of age. Arterial pressure was measured at each age, and we performed intravenous glucose tolerance tests at 6 mo and 1 yr. Repeated maternal, but not single maternal or fetal, betamethasone injections prolonged gestation, reduced weight at birth and 3 mo, and was associated with low arterial pressure at 3 mo but not at 6 mo and 1 yr. Glucose metabolism was altered in all betamethasone treatment groups, regardless of the number or route of injections. Our data demonstrate that glucocorticoid-induced fetal growth restriction is associated with a transient reduction in postnatal arterial pressure, but glucocorticoid exposure with or without growth restriction alters glucose metabolism.
antenatal glucocorticoids; betamethasone; fetal intervention; intrauterine growth restriction
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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SINCE THE PIONEERING WORK of Liggins (15), meta analysis of randomized controlled trials has established that administration of a single course of glucocorticoids to women at risk of early preterm birth reduces the rates of intraventricular hemorrhage, necrotizing enterocolitis, and respiratory distress syndrome in their offspring (8). However, little is known about the duration of the beneficial effects of single glucocorticoid treatments (33), and it has become common for obstetricians to prescribe repeated doses for patients with recurring or persistent risk of preterm delivery (27). Experiments in sheep have demonstrated that repeated maternal glucocorticoid injections reduce fetal weight (10, 11, 21), although evidence for such an effect in humans is conflicting at this time (1, 2, 9, 19).
There is a well-established relationship between low birth weight and
diseases such as hypertension and diabetes during later life (13,
26). Recently, it has been suggested that this "programming" effect may be mediated by excess exposure of the developing fetus to
glucocorticoids (7, 29, 30). An issue that has not yet been addressed, however, is whether the programming effects of excess
prenatal glucocorticoid exposure are a consequence of the reduction in
fetal growth that generally accompanies glucocorticoid exposure
(30) or a direct effect on the fetus of the
glucocorticoids themselves. Administration of synthetic glucocorticoids
during the last week of pregnancy in rats decreases birth weight as
well as elevating adult blood pressure (14) and causing
glucose intolerance (24) in the adult offspring.
Increasing the exposure of rat fetuses to maternal glucocorticoids by
inhibiting 11
-hydroxysteroid dehydrogenase type 2 (11HSD2), an
enzyme that converts maternal glucocorticoids to inactive forms in the
placenta, thus preventing them from reaching the fetal circulation
(3), causes low birth weight accompanied by the postnatal
development of hyperglycemia and elevated blood pressure in the
offspring (16, 17). These effects of 11HSD2 inhibition
on birth weight and glucose tolerance are dependent on maternal
glucocorticoids because maternal adrenalectomy restores both normal
birth weight and normal glucose tolerance in the offspring of pregnant
rats treated with the 11HSD2 inhibitor carbenoxalone
(17). Although these studies clearly demonstrate a role
for glucocorticoids in the programming of arterial pressure and glucose
tolerance, at least in rats, they have been unable to discriminate
between effects due directly to elevated glucocorticoids or indirectly
as a result of intrauterine growth restriction (IUGR).
IUGR is not simply associated with elevations in blood pressure during postnatal life. In humans (12, 25) and sheep (18, 28), the arterial pressure of low birth weight newborns is initially reduced during early postnatal life when compared with normally grown infants and lambs. Serial measurements made in humans (12) and sheep (28) have demonstrated that, following this initial period of relative hypotension, arterial pressure increases more in low birth weight subjects than in those with normal birth weights such that an inverse relationship between birth weight and blood pressure develops.
In a sheep model, maternal, but not fetal, injections of the synthetic glucocorticoid betamethasone restrict fetal growth (21) despite betamethasone levels being at least as high in the fetus after direct fetal administration (4). This differential effect of the route of administration of glucocorticoids on fetal growth provides an opportunity to discriminate between effects due to growth restriction induced by glucocorticoid exposure and the effects of glucocorticoids themselves in the absence of an associated growth effect. It was our aim to compare the effects of maternal and fetal injections of betamethasone on birth weight at term, and on postnatal growth, arterial pressure and glucose tolerance in sheep.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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All experimental procedures were approved by the Animal Experimentation Ethics Committee of the University of Western Australia.
Prenatal Treatments
Date-mated pregnant ewes bearing singleton fetuses (n = 157) were randomly allocated to receive either no treatment (untreated) or maternal or fetal injections of saline and/or betamethasone (Table 1).
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All ewes in the maternal and fetal injection protocols were injected intramuscularly with 150 mg of medroxyprogesterone acetate (MPA) (Depo Provera, Upjohn) at ~100 days of gestation. These ewes were then allocated into saline, single betamethasone, or repeated betamethasone treatment groups. Saline-treated animals were injected with normal saline at 104, 111, 118, and 125 days of gestation (maternal or fetal saline); single betamethasone animals were injected with betamethasone at 104 days of gestation and saline at 111, 118, and 125 days of gestation (maternal or fetal 1-beta). Repeated betamethasone animals were injected with betamethasone at 104, 111, 118, and 125 days of gestation (maternal or fetal 4-beta).
Maternal betamethasone (Celestone Chronodose, Schering Plough) injections were given intramuscularly in a dose of 0.5 mg/kg body wt; saline injections were of a comparable volume (5-6 ml). Fetal injections were given using an established technique (22). Briefly, the ewe was held in a sitting position, 70% ethanol was applied to the ewe's abdomen as a coupling medium, and the fetus was imaged using a 3.5-MHz sector transducer (Echo Camera SSD-500, Aloka). The fetal heart was imaged before aligning the transducer above the injection site. Betadine solution (Faulding) was applied to the injection site, and a 21-gauge 9-cm spinal needle (Terumo) was introduced through the maternal abdomen into the muscle of the fetal shoulder or rump. Betamethasone (0.5 mg/kg estimated fetal body wt; 1.4 kg at 104 days, 1.9 kg at 111 days, 2.2 kg at 118 days, 2.5 kg at 125 days) or an equal volume of saline (1 ml) was injected by an assistant; the needle tip was observed throughout the entire procedure. Betamethasone doses of 0.5 mg/kg (maternal or fetal weight) are the minimal doses required to consistently improve preterm lung function and have been shown previously to cause growth restriction if injected maternally but not directly to the fetus (21).
Delivery and Postnatal Procedures
Ewes were allowed to deliver their lambs spontaneously in a field environment and were not disturbed until the lamb was cleaned and standing. Within 12 h of birth, each lamb was weighed and crown-rump length was measured. Lambs were raised by their mothers and were monitored several times a day for the first few postnatal weeks. Two of the maternal 4-beta lambs were raised exclusively by hand or supplemented with powdered milk (Divetalact) when it became apparent that lactation was affected in some ewes from this group.At ~2 mo of age, lambs were immunized, their tails were cropped, and males were castrated. Six lambs from each of the seven treatment groups were chosen at random as subjects for measurement of arterial pressure at 3 and 6 mo and 1 yr of age. Supplementation of numbers in the maternal 4-beta group was required due to poor postnatal survival; these additional lambs were obtained from identically treated ewes originally enrolled in a parallel study. Weaning commenced after the 3-mo measurements had been made.
Physiological Studies at 3 mo of Age
Lambs chosen as subjects were taken from their mothers, weighed, and catheters (containing heparinized saline) were inserted ~10 cm into the femoral artery and vein during aseptic surgery while the lambs were under general anesthesia (20 mg/kg ketamine, 0.5 mg/kg xylazine; Troy Laboratories). Catheters were tunneled subcutaneously to an incision in the lamb's flank, attached to three-way stopcocks, and fastened in plastic bags underneath elasticized netting (Surgifix, 3M) that was fitted over the lamb's torso. All lambs were injected intramuscularly with penicillin (Benacillin, Troy Laboratories). After full recovery from anesthesia, lambs were returned to their mothers, and 2 days of recovery were allowed before measurements of arterial pressure were made. Catheters were removed after experiments were conducted. Where possible, the same lambs were studied at 6 mo and 1 yr of age; unavoidable losses from some groups necessitated supplementation of groups at 6 mo and 1 yr.Arterial pressure measurement.
Lambs were taken from their mothers in pairs and placed in ventral
recumbency in slings with leg holes. Body temperature was measured, and
an arterial blood sample (1 ml) was removed for measurement of arterial
pH, PCO2, PO2, and
concentrations of glucose, K+, Cl
,
Ca2+, and Na+ (Rapidlab 860, Chiron
Diagnostics). The arterial catheter was attached to a pressure
transducer (Ohmeda), and the signal was amplified (Octal Bridge Amp, AD
Instruments), sampled at 200 Hz (Powerlab 8e, AD Instruments), and
recorded onto a computer (Power Macintosh G3, Apple) using a
data-acquisition program (Chart v.3.5.7, AD Instruments). Arterial
pressure was recorded for at least 15 min while the lamb rested
quietly. Catheters were removed at the completion of arterial pressure measurements.
Physiological Studies at 6 mo of Age
At 6 mo of age, lambs were restudied after undergoing aseptic surgery (ketamine/xylazine anesthesia as at 3 mo) to implant vascular catheters. Lambs were allowed at least 24 h to recover before glucose tolerance tests were conducted. Arterial pressure measurements were made 2 days after glucose tolerance tests. Catheters were removed after the completion of experiments.Arterial pressure measurement. Arterial blood was sampled, and arterial pressure was recorded as described previously while lambs stood quietly in individual cages, in the company of other sheep, for at least 10 min.
Glucose tolerance tests. Blood samples (5 ml) were removed from the arterial catheter for measurement of whole blood glucose concentration and collection of plasma, 30 and 15 min and immediately before administration of a bolus injection of glucose (0.5 g/kg; Baxter) into the venous catheter. Further samples were removed at 5, 10, 20, 30, 60, 90, 120, 180, and 240 min after the glucose bolus.
Physiological Studies at 1 yr of Age
Sheep underwent aseptic surgery to implant vascular catheters (halothane anesthesia, 1-2% in O2, following induction with ketamine/xylazine) and were allowed at least 1 day to recover before measurements of glucose tolerance and arterial pressure were made (as at 6 mo).Data Analysis
Arterial pressure. Recordings of arterial pressure were scrutinized, and sections of the recording containing artifacts due to animal movement and vocalization were excluded from analysis. Arterial pressure recordings were analyzed on a beat-by-beat basis. Systolic, diastolic, and mean arterial pressures and instantaneous heart rate were determined from individual beats. These data were averaged for each animal to provide single values for statistical analysis.
Insulin assays. Plasma immunoreactive insulin concentrations were measured using a kit (Linco Research). The interassay coefficient of variation was 6%, and the mean assay sensitivity was 0.1 ng/ml. The insulin antibody cross-reacts 100% with rat, sheep, and porcine insulin; binding to rat C peptide, glucagon, somatostatin, pancreatic polypeptide, and insulin-like growth factor I is undetectable (Linco).
Statistical Analysis
Data were summarized by calculating the mean and SE within each of the seven treatment groups. Comparisons of group means employed linear regression models (SAS and S-PLUS), allowing for differences in the distribution of males and females between the treatment groups. For glucose tolerance tests, comparisons between groups at two timepoints used the observed difference in successive measurements on each of the sheep as the outcome variable in models similar to those described above. Areas under glucose- and insulin-response curves were calculated using the trapezoid rule and compared between groups by ANOVA.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Prenatal Mortality, Gestation Length, Growth, and Survival
Table 2 shows the number of ewes enrolled initially, the total number of liveborn lambs, and pre- and postnatal loss rates for each of the seven treatment groups. Prenatal losses (due either to death in utero and/or abortion) occurred in the maternal 4-beta group and in all three fetal injection groups. The timing of fetal loss in the maternal 4-beta group is unknown, but loss in the fetal injection groups occurred at some time after the fourth injection (with only 1 exception). In addition to the fetal losses, the maternal 4-beta group also contained a large number of ewes (9 of 29) that failed to give birth. Postnatal losses were also high in the maternal 4-beta group (5 of 6 liveborn lambs died within the first 2-3 postnatal days), due to prior effects of repeated maternal betamethasone treatments in precipitating premature lactation and reducing milk supply after birth. To obtain sufficient numbers for postnatal studies, we supplemented numbers in this group with maternal 4-beta lambs from another, parallel, study. These extra animals were delivered by identically treated ewes that were from the same flock as those originally enrolled in our study and treated at the same time of year.
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Gestational lengths were not significantly different between treatment
groups (Fig. 1); however, the mean
gestational length of liveborn lambs in the maternal 4-beta group
(154 ± 1 days) was 3 days longer than that of untreated lambs
(151 ± 1 days; P = 0.06; these data do not
include gestation lengths from pregnancies in which labor did not
occur). Lambs from ewes in all treated groups had significantly lower
birth weights than untreated lambs (Fig. 1). Liveborn maternal 4-beta
lambs had significantly lower birth weights (3.05 ± 0.46 kg) than
maternal saline lambs (4.65 ± 0.35 kg, P = 0.001); weights of maternal 1-beta lambs (4.12 ± 0.27 kg) were
higher than maternal 4-beta lambs (P = 0.03) but were
not different from those of maternal saline lambs (P = 0.25). This effect of betamethasone on birth weight was not present
when injections were given directly to the fetus. Crown-rump length at
birth was not different between groups (Fig. 1).
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Postnatal Weight Gain
Body weights and ages of animals studied postnatally are shown in Table 3. At 3 mo of age, maternal 4-beta lambs weighed less than all other groups. By 6 mo of age, maternal 4-beta lambs had displayed catch-up growth, with weights not significantly different from other groups, although they tended to weigh less than untreated lambs (P = 0.07). At 1 yr of age, body weights were not significantly different between the seven groups.
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Arterial pH, PCO2, PO2, and Electrolyte Concentrations
Arterial blood gases and electrolyte concentrations at 3 and 6 mo and 1 yr of age are shown in Tables 4, 5, and 6. No consistent effects of prenatal treatments were observed on pHa, PaCO2, and PaO2 or electrolytes. Small, statistically significant differences were observed in some cases, but these were considered to be of no physiological significance.
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Arterial Pressure and Heart Rate
Mean arterial pressures and heart rates of lambs aged 3 and 6 mo and 1 yr of age are shown in Table 3.At 3 mo of age, mean arterial pressure of maternal 4-beta lambs (75.3 ± 2.4 mmHg) was lower than that of untreated lambs (83.8 ± 3.3 mmHg; P = 0.02) and tended to be lower than in maternal 1-beta lambs (82.8 ± 2.4 mmHg; P = 0.06). However, mean arterial pressure was not significantly different between maternal saline (79.9 ± 2.2 mmHg) and maternal 4-beta lambs at this age (P = 0.22). Mean arterial pressure was similar between untreated and fetal saline, 1-beta, or 4-beta groups. Mean arterial pressure tended to be lower in maternal 4-beta lambs than in fetal 4-beta lambs (P = 0.07). Heart rates at 3 mo of age were not significantly different between any of the treatment groups but tended to be lower than untreated lambs (99.8 ± 5.6 beats/min) in maternal saline (87.6 ± 4.5 beats/min; P = 0.09) and fetal 1-beta (85.4 ± 5.3 beats/min; P = 0.06) groups.
At 6 mo of age, mean arterial pressures were not different between untreated lambs and any of the maternal treatment groups. Fetal saline and fetal 1-beta groups had mean arterial pressures (86.2 ± 3.9 and 86.7 ± 3.8 mmHg, respectively) that were significantly higher than that of the untreated group at 6 mo (77.0 ± 1.6 mmHg; P = 0.04 for both groups), but values in fetal 4-beta lambs were not significantly different from untreated lambs. Mean arterial pressure at 6 mo was not different between maternal and fetal injection groups. Heart rate at 6 mo was not affected by fetal or maternal saline or betamethasone treatments.
At 1 yr of age, there were no differences in arterial pressure and heart rate between treatment groups.
Glucose Tolerance
Blood glucose and plasma insulin concentrations, from samples collected during glucose tolerance tests conducted in maternal saline, maternal 1-beta, maternal 4-beta, fetal saline, fetal 1-beta, and fetal 4-beta lambs, at 6 mo and 1 yr of age, are shown in Figs. 2 and 3. Data from the untreated group are not illustrated; glucose and insulin concentrations were not significantly different between untreated and maternal saline or fetal saline groups at any time points.
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At 6 mo of age, blood glucose concentrations before and after
administration of 0.5 mg/kg glucose were not different between groups.
Plasma insulin concentrations before glucose administration were not
different between groups. Insulin concentrations were elevated
(P < 0.05) in maternal 1-beta lambs at 10, 20 (P = 0.06), and 60 min and in maternal 4-beta lambs at
10 (P = 0.06), 60, 120 (P = 0.06), and
180 min after glucose administration (compared with maternal saline
lambs). Insulin concentrations in fetal 1-beta lambs were elevated 5 and 20 min after glucose administration (compared with fetal saline
lambs). Fetal 4-beta lambs had higher insulin concentrations 10 (P = 0.09) and 20 min after the glucose bolus (compared
with fetal saline lambs). Areas under glucose-response curves were not
different between groups, but areas under insulin curves tended to be
greater in maternal 1-beta (P = 0.06) and maternal
4-beta (P = 0.1) groups than in controls (Fig.
4). Insulin-to-glucose ratios were
significantly greater in maternal 1-beta and maternal 4-beta groups
than in maternal saline lambs.
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At 1 yr of age, blood glucose concentrations were higher at rest
(before glucose administration) in maternal 4-beta lambs (4.01 ± 0.13 mmol/dl) than in maternal saline lambs (3.59 ± 0.12 mmol/dl); blood glucose concentrations were not different between other
groups. Maternal 1-beta lambs had greater (P < 0.05)
blood glucose levels 5, 10, 20 (P = 0.06), 30, and 60 min after glucose injection, and maternal 4-beta lambs had elevated
glucose levels 180 min after the glucose bolus (Fig. 3). Before glucose
administration, plasma insulin concentrations were higher in maternal
4-beta lambs than in untreated (P = 0.06) and maternal
1-beta lambs (P = 0.05). Insulin concentrations were
higher (P < 0.05) in maternal 4-beta lambs than
maternal saline lambs 5, 10 (P = 0.06), and 30 min after the glucose bolus but were not different between maternal 1-beta
and maternal saline lambs (Fig. 3). Fetal 1-beta and 4-beta lambs had
initial increases in blood glucose concentrations that were similar to
fetal saline lambs, but these two groups had lower (P < 0.05) blood glucose concentrations 20 (fetal 4-beta,
P = 0.09), 30, 60, and 90 (fetal 4-beta,
P = 0.06) min after the glucose injection (Fig. 3).
Blood glucose concentrations in fetal 1-beta and fetal 4-beta lambs
were higher 120 min after glucose injection than in fetal saline lambs.
Insulin levels following glucose injection were not different between
fetal 1-beta, 4-beta, or fetal saline lambs (Fig. 3). The area under
the glucose-response curve was greater in maternal 1-beta lambs than in
maternal saline lambs (P = 0.01). Area under the
insulin-response curve tended to be higher in maternal 4-beta lambs
than in maternal saline lambs (P = 0.15), and
insulin-to-glucose ratios also tended to be higher in this group
(P = 0.08; Fig. 5).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We have demonstrated that antenatal glucocorticoids have lasting postnatal effects on glucose metabolism in the offspring of ovine pregnancies, independent of glucocorticoid effects on prenatal growth. Our data suggest that programming of arterial pressure and glucose tolerance are not the result of a common mechanism and have important implications for obstetric practice.
Repeated courses of glucocorticoids are commonly given to women at risk of early preterm birth (27). As reported previously (21) and confirmed by our present study, clinically relevant repeated maternal injections of betamethasone cause IUGR in sheep. This effect of repeated glucocorticoid treatments has been demonstrated in retrospective studies of human subjects (2, 9) but has yet to be confirmed by randomized controlled trials in human infants (32).
All of our prenatal glucocorticoid treatments resulted in postnatal insulin insensitivity, as indicated by increased insulin responses to glucose challenge (6). This is the first time that the effects on postnatal glucose metabolism of prenatal glucocorticoid exposure and impaired fetal growth have been separated, and our findings indicate that programming of glucose metabolism may result from increased prenatal exposure to glucocorticoids, rather than simply from an effect of reduced fetal growth. The initial observational studies we have described are unable to identify possible mechanisms by which antenatal glucocorticoids alter postnatal glucose metabolism, but our betamethasone-treated sheep may be affected in a similar way to rats treated in late gestation with glucocorticoids. Adult rats exposed to late-gestational dexamethasone (which results in prenatal growth restriction and glucose intolerance during adult life) increase hepatic expression of glucocorticoid receptor and phosphoenopyruvate carboxykinase (PEPCK) (24). PEPCK is the rate-limiting enzyme for gluconeogenesis, and an increase in hepatic PEPCK may increase glucose production by the liver, contributing to glucose intolerance (24). However, the glucose and insulin profiles displayed by our betamethasone-treated sheep in response to glucose challenge were somewhat different to those that have been observed in rats exposed to late-gestation dexamethasone (24). At 6 mo of age, lambs that were exposed to prenatal glucocorticoids had increased insulin responses to glucose challenge but normal glucose responses (when compared with saline-treated controls); the rats studied (24) had elevated insulin and glucose responses. It is possible that this difference is related to the different dose or duration of exposure to glucocorticoids or the fact that our sheep were less mature than the adult rats that have been studied. We speculate that, with increasing age, the lambs that were exposed to prenatal glucocorticoids will display elevations in glucose concentrations at rest and following glucose challenge. It appears, from our data, that this pattern is emerging at 12 mo: maternal 4-beta lambs displayed elevated glucose concentrations at rest and maternal 1-beta lambs had higher blood glucose concentrations following glucose challenge. The reason for the lower blood glucose response to glucose challenge in fetal 1- and 4-beta lambs at 1 yr is unknown.
Our finding that postnatal arterial pressure was affected only in lambs with reduced birth weight, and not those that received glucocorticoid treatments without accompanying IUGR (i.e., maternal 1-, fetal 1-, and 4-beta groups), suggests that some factor(s) associated with restricted prenatal growth, rather than a direct effect of the glucocorticoids, cause changes in postnatal arterial pressure. This is an important distinction, because glucocorticoid-mediated effects have been suggested as possible mechanisms whereby low birth weight may program postnatal blood pressure (28, 29). Our data suggest that physical alterations accompanying low birth weight per se may be the cause of postnatal hypertension. Celsi et al. (5) have provided evidence that physical alterations accompanying prenatal growth restriction may alter arterial pressure during postnatal life. They showed that oligonephronia (a physical effect of IUGR) was associated with impaired postnatal glomerular function and hypertension (5). However, this fails to explain why arterial pressure should be decreased during early postnatal life in low birth weight individuals.
Low arterial pressure has been observed during early postnatal life in IUGR lambs born after carunclectomy (28) or placental embolization (18), as we have demonstrated in lambs that were growth restricted as a result of repeated maternal betamethasone treatment. Such data complement those from low birth weight human subjects, which show a positive relationship soon after birth between birth weight and arterial pressure (i.e., low birth weight is associated with lower arterial pressure) (12, 25). Robinson et al. (28) found, using their carunclectomy model, that this relative hypotension was transient, with arterial pressure increasing above control levels by 2 mo of age; in contrast, the lambs studied after placental embolization (18) remained relatively hypotensive at 2 mo of age. Our low birth weight lambs had low arterial pressures at 3 mo of age, which had not increased above control levels by 1 yr. The reasons for these different effects on postnatal arterial pressure observed using the carunclectomy, placental embolization, and glucocorticoid models of fetal growth restriction are unclear at this time. Although the degree of growth restriction was similar for each model, other physical, endocrine, and metabolic effects, which may affect postnatal arterial pressure, are likely to exist.
An interesting and unexpected finding of our study was that gestation length was increased in maternal 4-beta lambs and parturition failed to occur in 9 of 29 of these sheep. This may be due to effects of the prenatal betamethasone treatments on the fetal hypothalamic-pituitary-adrenal axis. It has been shown that repeated maternal betamethasone injections cause circulating fetal ACTH and cortisol levels to be elevated at 145 days of gestation (31), indicating that absence of the normal preparturient rise in cortisol is not likely to be responsible for the failure of parturition. Rather, it seems the endocrine mechanisms responsible for the initiation of labor (23) in maternal 4-beta sheep have a reduced responsiveness to circulating cortisol, the underlying causes of which remain to be investigated.
The necessity, in our study, to administer MPA before glucocorticoid treatments provides a potential complication to the interpretation of our data, but we are confident our findings are not greatly influenced by the MPA treatment. Although progesterone binds to glucocorticoid receptors, its affinity is much weaker than that of betamethasone and would therefore be expected to have little effect through this mechanism. Many fetal endocrine effects of glucocorticoid administration are unaffected by MPA treatment (20). Gestation length in rabbits is prolonged by progesterone treatment (33), but we observed no difference in gestational age at delivery between untreated (no MPA treatment) and maternal saline (treated with MPA) treatment groups. Other variables were not significantly different between untreated and maternal saline groups. A practical decision to minimize animal numbers prevented the inclusion of other treatment groups (e.g., MPA only, maternal saline injections without MPA) in the current study. Our own previous experience (with 30 ewes) has shown that repeated maternal betamethasone injections without MPA result in 100% pregnancy loss. Practicality also required the castration of the male lambs in our study. We are unaware of any data that would suggest effects due to castration might interact with our treatments to alter long-term outcomes.
A number of complications were experienced in our study due to the pre- and postnatal mortality we observed in some of our treatment groups. Fetal loss rates in maternal 4-beta ewes are consistent with our experience using this model and are caused by glucocorticoid-induced abortion (15, 23), despite our administration of MPA. The reason for the prenatal loss of fetuses injected directly with saline or betamethasone is unknown. These losses were seemingly not an acute effect of the fetal injections per se, because they occurred some time after the final fetal injection in all but one case. Ultrasound-guided percutaneous needle introduction and blood sampling from fetal sheep result in only a mild transient stress response (as indicated by increases in catecholamines) in the fetus (22), and the effects of fetal intramuscular injections such as those given in the present study are likely to have a similarly small acute effect. Comparison of data from our postnatal studies of the offspring that survived following direct fetal saline injections and untreated lambs suggests that there is little, if any, long-term effect of fetal intramuscular injection per se. Further experimentation is required to determine the causes(s) of fetal loss experienced following fetal injection in the present study. Investigation of the effects of direct fetal injection is particularly necessary because of the potential for such an approach to be used clinically (21). As a result of fetal loss in the maternal 4-beta group and in all fetal treatment groups, the lambs that we were able to study postnatally may have been differently affected than those fetuses that did not survive to term or were not born (in the case of the maternal 4-beta group). The lambs that survived and were studied may therefore be those least affected by the prenatal glucocorticoid treatments.
Postnatal survival was not adversely affected by prenatal treatments, other than in the maternal 4-beta group, where neonatal mortality was due to the precipitation of early lactation causing a reduced supply of milk at birth. Initially, these lambs appeared as healthy as those in other treatment groups and were distinguishable from other lambs only by their size and weight. The maternal 4-beta lambs that died postnatally became gradually weak and less active the day after birth as a result of not being able to feed. Once this problem was identified, surrogate or supplemental feeding ensured survival in lambs born after repeated maternal betamethasone treatments. In all other groups, postnatal mortality (in between ages at which measurements were made) was due to suspected predation or euthanasia following accidental injury. Replacing these animals, to obtain sufficient numbers of subjects for meaningful statistical comparisons between treatment groups, removed the opportunity to use repeated-measures ANOVA for the analysis of our data, resulting in the use of more conservative statistical tests.
In conclusion, the results of our study indicate that postnatal glucose tolerance and arterial pressure are influenced by prenatal exposure to glucocorticoids with the effects on arterial pressure but not glucose metabolism being dependent on the fetal growth restriction. Our findings suggest that physical consequences of IUGR are more likely causes of altered postnatal arterial pressure than programming of the hypothalamic-pituitary-adrenal axis. An important implication of our study is the potential for antenatal glucocorticoid treatment in humans to result in postnatal alterations in insulin sensitivity and the subsequent development of glucose intolerance.
Perspectives
A mechanistic role for glucocorticoids has been proposed in the programming of fetal development, whereby impaired growth before birth is associated with the incidence of adult-onset disease. We have demonstrated that antenatal glucocorticoids have lasting postnatal effects on glucose metabolism in the offspring of ovine pregnancies, regardless of glucocorticoid effects on prenatal growth. Randomized controlled trials of single vs. repeated courses of antenatal glucocorticoids are currently underway in Australia and the United States and are planned for Canada and Britain. If these trials fail to show a deleterious effect of repeated glucocorticoids on the birth weights of infants, our data demonstrate that glucose metabolism may still be affected. Assessment of glucose tolerance in infants born after repeated prenatal glucocorticoid treatments is necessary to address this issue.| |
ACKNOWLEDGEMENTS |
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We thank E. Newnham for assistance with performing fetal injections and A. Jonker for assistance with postnatal experiments. We also thank A. M. Moss for assistance with both fetal injections and postnatal experimentation.
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FOOTNOTES |
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This research was funded by the National Health and Medical Research Council (Australia), project grant 980578 and by the Lotteries Commission of Western Australia.
Address for reprint requests and other correspondence: T. J. M. Moss, Lotteries Commission Perinatal Research Laboratories, Dept. of Obstetrics and Gynaecology, The Univ. of Western Australia, 35 Stirling Highway, Crawley 6009 WA, Australia (E-mail: tmoss{at}cyllene.uwa.edu.au).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 August 2000; accepted in final form 23 May 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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