Abstract
We evaluated the effects of multiple fetal exposures to glucocorticoids on postnatal lung function and growth. Ewes were randomized to receive 1 to 4 doses of 0.5 mg/kg betamethasone or saline placebo at 7 d intervals from 104 d to 118 d and at 124 d gestation. All lambs were delivered preterm at 125 d gestation, and postnatal lung function was evaluated. There were sequential improvements in compliance, ventilation efficiency, and lung volumes for two, three, and four doses of betamethasone. The maximal effect was a 150% increase in compliance and a 4-fold increase in lung volume after fetal exposure to four doses of betamethasone. However, birth weights decreased (15% after one dose, 19% after two doses, and 27% after three and four doses). There were no changes in lung to body weight ratios, lung dry to wet weight ratios, lung protein to body weight ratios, or lung hyaluronan content. Prenatal glucocorticoid exposure also altered postnatal cortisol, thyroid, and catecholamine plasma levels. Repetitive 7-d interval exposures of fetal lambs to glucocorticoids progressively enhanced postnatal lung function and resulted in growth and endocrine abnormalities.
Prenatal glucocorticoid treatments of women at risk for preterm delivery decrease the risk of respiratory distress syndrome and other problems of prematurity such as intraventricular hemorrhage and patent ductus arteriosus (1). Although the recent National Institute of Health Consensus Conference recommended broad use of glucocorticoid therapy, a number of questions regarding clinical use could not be answered from the experimental literature or from the clinical data that were available (2). Information in the human concerning fetal responses at gestations that approach viability at 22–25 wk gestation is limited because most of the clinical trials of glucocorticoid for infants at risk of preterm labor were conducted before interventions to support extremely preterm infants were common. Growth retardation and changes in lung structure were noted at term following exposure of fetal monkeys to high dose glucocorticoids given at mid-gestation (3, 4). Possible effects of early fetal exposure to clinically relevant doses of glucocorticoids on subsequent growth and lung function have not been evaluated. Many fetuses at risk of preterm delivery do not deliver within 7 d of initial glucocorticoid treatment. The recently completed Australian collaborative trial of antenatal thyrotropin-releasing hormone (ACTOBAT) randomized 1,234 women at 24 to 32 wk gestation, and the average time from randomization to delivery was about 30 d (5). The duration of effect of an initial glucocorticoid treatment course was estimated to be 7 to 10 d (6). However, this estimate was for infants that were studied before 1972 and were more mature than are infants considered to be at high risk today. Currently, some obstetricians are using repetitive treatment courses of maternal glucocorticoid at 7- to 10-d intervals to try to maximize fetal responses (7), and woman receiving as many as 10 courses have delivered infants with a Cushing-like syndrome (8). Another concern is that fetal exposure of the primate to relatively short courses and low doses of glucocorticoid can alter brain development and cause growth failure (9). Growth failure is a uniform result of fetal glucocorticoid exposure in rodents (10). Although we found no effects on fetal growth 7 d after treatment of fetal sheep with 0.5 mg/kg betamethasone (11), multiple treatments have not been evaluated. We also demonstrated that a single fetal glucocorticoid exposure improved postnatal lung function of the preterm lambs that lasted for at least 7 d following treatment (11). However a second dose of glucocorticoid 24 h before preterm delivery did not further augment postnatal lung function (12). Therefore, we designed a multiple retreatment protocol beginning early in gestation to characterize postnatal lung function of the preterm lamb following up to four fetal exposures to betamethasone.
Protocols for these experiments were reviewed and approved by the Animal Use Committees at the Harbor-UCLA Medical Center and the Western Australian Department of Agriculture in accordance with U.S. Public Health Service and American Association for Accreditation of Laboratory Animal Care guidelines. Date-mated ewes with singletons confirmed by ultrasound at 60 d gestation were given 150 mg medroxyprogesterone (Depo-Provera® ; Upjohn Co., Kalamazoo, MI) by IM injection at 101 d gestation and 3 d before randomization to one of five treatment groups (Figure 1). The medroxyprogesterone was used to minimize the occurrence of preterm labor and abortion induced by glucocorticoid in sheep (13). At 104-d gestation, ewes were randomized to receive either an intramuscular saline injection or 0.5 mg/kg betamethasone intramuscularly (Celestone Chronodose® ; Schering, New South Wales, Australia) based on maternal weight at 101-d gestation. The animals then were allowed to freely feed in paddocks between subsequent saline or betamethasone injections at 7-d intervals (111 and 118-d gestation) At 124-d gestation, all animals again received saline or betamethasone injections followed by preterm delivery at 125-d gestation. All injections were based on the maternal weight at 101-d gestation, and the animals were not handled other than for the betamethasone or saline treatments.
Fig. 1. Protocol for gestational ages at maternal treatment with 0.5 mg/kg betamethasone or saline by intramuscular injection. All animals were delivered at 125-d gestation. Study groups will be referred to by the number of betamethasone doses given to the ewes.
[More] [Minimize]At 125-d gestation, the ewes were sedated (ketamine, 1 gm intramuscularly) and received spinal anesthesia (lidocaine, 2%, 4 ml). The fetal head was exposed by midline abdominal and uterine incisions and the fetus was sedated (ketamine, 10 mg/kg IM). The anterior fetal neck was anesthetized (2% lidocaine, subcutaneously) and, after tracheotomy, a 4.5 mm endotracheal tube was securely tied into place. The fetus was then delivered. An arterial blood sample was drawn from the placental cord for pH, blood gas, and hormone determinations.
After delivery the lamb was dried and ventilated with a time-cycled, pressure-limited infant ventilator set to deliver an Fi O2 of 1.0 at a respiratory rate of 40 breaths/min, an inspiratory time of 0.7 s and a positive end-expiratory pressure of 3 cm H2O (14). Peak inspiratory pressures were initially set at 35 cm H2O and subsequently adjusted over the 40 min postdelivery study period in an attempt to achieve arterial Pco 2, values of about 50 mm Hg. Peak inspiratory pressures were limited to 40 cm H2O to avoid pneumothorax. Other ventilator settings were not altered during the study period. The investigators initiating the changes in ventilator settings were blinded as to the treatment status of the animal being studied. An arterial catheter was advanced into the descending aorta through an umbilical artery and each lamb received pentobarbital (15 mg/kg) by slow arterial infusion. No spontaneous respirations were noted for the remainder of the study. The body temperature of each lamb was maintained at 39° C with a radiant warmer.
Tidal volumes were measured with a pneumotachometer and ventilatory pressures were calculated as peak pressure at the endotracheal tube minus end-expiratory pressure (3 cm H2O). Ventilation also was regulated by adjusting peak ventilatory pressure to achieve a tidal volume of about 10 ml/kg. Compliance was calculated by dividing the tidal volume by the ventilatory pressure, then normalized to body weight in kilograms (14, 15). Ventilatory efficiency index, an index that integrates ventilation with respiratory support (in the absence of spontaneous breathing) was calculated by the formula: Ventilatory efficiency index = 3,800 ÷ P · f · Pco 2 (16). In this equation, 3,800 is a carbon dioxide production constant (ml · mm Hg · kg−1 · min−1), P is ventilatory pressure, f is the ventilation rate (40 breaths/ min), and Pco 2 is the arterial partial pressure of carbon dioxide. After a final arterial blood sample 40 min after birth each lamb was given pentobarbital (30 mg/kg), the tracheal tube was clamped for 3 min to achieve atelectasis for the pressure–volume measurement and the body weight was recorded followed by exsanguination.
For measurement of the pressure–volume curve the chest of each lamb was opened, the lung was filled with air to 40 cm H2O pressure for 1 min and the volume recorded. Pressure was then progressively decreased to 20, 10, 5, and 0 cm H2O with volumes recorded after 30 s each pressure to characterize the deflation limb of the pressure–volume curve (14). The left lobe was homogenized and aliquots were used for measurements of protein (17) and DNA (18). A piece of the right lower lobe of each lung was weighed and then dried at 80 ° C for 5 d and reweighed for estimation of dry to wet lung weight ratios. A second weighed piece of the right lower lobe of each lung was used for assay of hyaluronan using the kit from Pharmacia Diagnostics (Uppsala, Sweden) according to Juul and coworkers (19).
Plasma samples were quickly recovered by centrifugation at 4° C from cord blood and blood samples taken at 40 min of age and were snap frozen. Cortisol, T3, and T4 levels were measured using chemiluminescent kits standardized for fetal samples (Nichols Institute, San Juan Capistrano, CA). Plasma epinephrine and norepinephrine levels were measured by radioenzymatic assay (20).
Group means were compared using ANOVA. When significant differences were noted, post hoc analysis was performed using the Student-Newman-Keuls or Kruskal-Wallis tests. Differences between cord and 40 min hormone values were evaluated by paired t-tests. Significance was defined as p < 0.05. All data are reported as mean ± SEM.
More animals were randomized to each treatment group than were studied because of potential losses due to glucocorticoid-induced abortion. An abortion was identified as an empty uterus, but the time of abortion was not known. Delivery order each study day was randomly assigned and animals from each group were delivered each day. Deliveries for a group were continued until 11 liveborn lambs had been studied. There were no losses for the saline injected controls or for the single dose group that received maternal betamethasone at 104-d gestation (Table 1). There was one abortion with two doses, four with three doses, and one with four doses betamethasone. Early fetal deaths were identified as small, macerated fetuses for the three and four dose groups. The causes of the fetal deaths were not known. The striking result was the decrease in birth weights of the live-born fetuses: 15% after one dose, 19% after two doses, and 27% after three and four maternal doses of betamethasone. The weight differences were not explained by the sex distributions of the newborns (Figure 2). Cord blood pH and gas values were similar for all groups of animals.
| Control | One Dose | Two Doses | Three Doses | Four Doses | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Outcome at delivery | ||||||||||
| Number of ewes evaluated | 11 | 11 | 12 | 18 | 15 | |||||
| Fetal deaths | 0 | 0 | 0 | 3 | 3 | |||||
| Abortions | 0 | 0 | 1 | 4 | 1 | |||||
| Liveborns studied | 11 | 11 | 11 | 11 | 11 | |||||
| Maternal weight at 101-d gestation, kg | 54 ± 2 | 53 ± 2 | 53 ± 2 | 55 ± 2 | 54 ± 1 | |||||
| Weight of newborn, kg* | 2.82 ± 0.11 | 2.40 ± 0.07 | 2.28 ± 0.08 | 2.09 ± 0.09 | 2.02 ± 0.13 | |||||
| Male/female | 6/5 | 5/6 | 3/8 | 6/5 | 1/10 | |||||
| Cord blood values | ||||||||||
| pH | 7.36 ± 0.01 | 7.33 ± 0.01 | 7.33 ± 0.01 | 7.33 ± 0.02 | 7.34 ± 0.01 | |||||
| Pco 2, mm Hg | 51 ± 1 | 53 ± 1 | 55 ± 2 | 54 ± 2 | 50 ± 1 | |||||
| Po 2, mm Hg | 22 ± 2 | 18 ± 2 | 17 ± 1 | 16 ± 1 | 21 ± 3 |
Fig. 2. Birth weights of male and female lambs. Mean body weights decreased after betamethasone exposure for both males and females, and there were no consistent diferences between the male and female lambs.
[More] [Minimize]All respiratory variables had become stable by 20 min of age (data not shown). At 40 min, the control lambs remained on the maximal ventilatory pressure (peak inspiratory minus end expiratory pressure) of 37 cm H2O, and each dose of betamethasone resulted in lower pressure requirements, although the differences were not significant until two doses had been given (Table 2). The lower ventilatory pressures for the groups that received two, three, and four doses of betamethasone resulted in higher tidal volumes that approximated the target value of 10 ml/kg. The Pco 2 values also progressively decreased with each additional dose of betamethasone, and pH values reflected the Pco 2 values. The lambs did not have significant metabolic acidosis. The Po 2 values did not change with betamethasone treatment.
| Control | One Dose | Two Doses | Three Doses | Four Doses | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Ventilatory | ||||||||||
| Inspiratory pressure − PEEP, cm H2O* | 37 ± 0 | 35 ± 1 | 31 ± 2 | 29 ± 2 | 24 ± 2 | |||||
| Tidal volume, ml/kg† | 6.5 ± 0.3 | 7.7 ± 0.5 | 8.9 ± 0.5 | 9.8 ± 0.3 | 9.6 ± 0.3 | |||||
| Blood gas and pH | ||||||||||
| pH† | 7.02 ± 0.03 | 7.06 ± 0.04 | 7.17 ± 0.03 | 7.21 ± 0.02 | 7.28 ± 0.03 | |||||
| Pco 2, mm Hg ‡ | 98 ± 8 | 89 ± 7 | 73 ± 6 | 64 ± 4 | 54 ± 3 | |||||
| Po 2, mm Hg | 109 ± 29 | 82 ± 19 | 95 ± 35 | 139 ± 35 | 101 ± 26 |
Total thoracic compliance increased by 28% after one dose, by 77% after two doses, by 100% after three doses, and by 150% after four doses (Figure 3). However, the response to one dose was not different from the control by ANOVA. The ventilatory efficiency index, an integrated measurement of gas exchange, increased following two and three doses of betamethasone and increased more than three-fold from the control value after four doses of betamethasone. Therefore, postnatal lung function improved progressively following the second dose of betamethasone given at 111-d gestation.
Fig. 3. Lung function following prenatal betamethasone. The number of doses of betamethasone given to the ewes before preterm delivery is on the horizontal axis. Both compliance and ventilatory efficiency index increased following repeated fetal exposure to betamethasone. *p < 0.05 versus 2, 3 and 4 dose groups; **p < 0.01 versus 2, 3 and 4 dose groups; tp < 0.05 versus 4 doses; ttp < 0.01 versus 4 doses.
[More] [Minimize]The gas volumes of the lungs were measured by pressure volume curves (Figure 4). Maximal lung volumes measured at 40 cm H2O were increased 2.8-fold by two doses, 3.5-fold by three doses, and 4-fold by four doses of betamethasane. The 53% increase after a single dose of betamethasone at 104-d gestation was not different from the controls by ANOVA. The deflation limbs of the pressure–volume curves also demonstrated more volume retention following the multiple dose betamethasone exposures.
Fig. 4. Pressure-volume curves. The deflation pressure-volume curves demonstrated increased lung volumes for lambs exposed to two or more doses of betamethasone. *p < 0.05 versus 2, 3, 4 dose groups; tp < 0.05 versus 4 dose group.
[More] [Minimize]The betamethasone treatments resulted in decreased body weights (Table 1) and smaller lungs, but the decreases in lung weights were proportionate to the decreases in body weights (Table 3). The dry to wet ratios for these lungs also did not change after betamethasone treatment. Measurements of total lung protein relative to body weight were not influenced by the betamethasone treatments (Table 3). However there was a decrease in lung DNA after four doses relative to one and two doses of betamethasone. Hyaluronan content of the lungs did not change with prenatal betamethasone treatments.
| Control | One Dose | Two Doses | Three Doses | Four Doses | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Lung weight/body weight, g/kg | 38.6 ± 1.0 | 42.8 ± 2.1 | 39.2 ± 1.0 | 41.5 ± 1.4 | 37.6 ± 1.6 | |||||
| Lung dry weight/wet weight, g/g | 0.094 ± 0.0002 | 0.089 ± 0.003 | 0.096 ± 0.001 | 0.090 ± 0.003 | 0.089 ± 0.003 | |||||
| Protein/body weight, g/kg | 1.53 ± 0.06 | 1.58 ± 0.08 | 1.41 ± 0.06 | 1.56 ± 0.06 | 1.41 ± 0.06 | |||||
| DNA/body weight, mg/kg | 179 ± 12 | 223 ± 16* | 240 ± 28* | 176 ± 18 | 131 ± 14 | |||||
| Hyaluronan, μg/g lung | 63.3 ± 5.9 | 62.3 ± 8.8 | 74.8 ± 6.6 | 56.7 ± 6.7 | 69.2 ± 6.4 |
The primary outcome variables of compliance, ventilatory efficiency index and lung volume at 40 cm H2O pressure increased as body weight decreased because of the parallel effects of increasing doses betamethasone on weight and the lung performance variables. However, within each dose group, there were significant correlations by linear regression for compliance (r = 0.66) and lung volume (r = 0.75) with body weight only for lambs that had received two doses of betamethasone. The lack of correlations for lambs receiving three or four doses of betamethasone indicated that severity of growth retardation did not predict lung function. There also were no significant differences within each treatment group for compliance, ventilatory efficiency index or lung volume between male and female lambs, although the female lambs tended to have higher values for the measurements.
To evaluate effects of glucocorticoid treatment on hormone systems which might influence fetal status and lung function, plasma cortisol, thyroid hormones, and catecholamine levels were measured. Plasma cortisol levels in cord blood were similar for all the groups (Figure 5). There was an increase in cortisol at 40 min of age for control lambs that approached significance (p = 0.07) but no postnatal increase for any of the betamethasone exposed groups of lambs. Therefore betamethasone exposure at 104-d gestation seemed to blunt the postnatal increase in cortisol following delivery 21 d later.
Fig. 5. Cortisol and thyroid hormone values. Measurements are for plasma from cord blood and for samples after 40 min of ventilation. *p < 0.001 versus all other cord and 40 min values; tp < 0.001 versus cord values.
[More] [Minimize]Plasma T4 levels were similar for cord blood and at 40 min for all groups of lambs except after four doses of betamethasone (Figure 5). This final dose of betamethasone 24 h before delivery depressed cord and 40 min T4 levels by about 50%. Prenatal betamethasone also did not affect cord T3 levels or the postnatal surge in T3 except after the fourth dose. There were two-fold increases in cord and 40 min T3 levels 24 h after the 4th betamethasone dose. These effects of the fourth dose of glucocorticoid on thyroid hormones probably are acute responses to the final treatment 24 h before delivery.
Cord plasma epinephrine was remarkably low for lambs 24 h after the 4th dose of betamethasone and remained low at 40 min (Figure 6). The normal postnatal increase in epinephrine occurred in control lambs. An increase also occurred after one, two, and three doses of the betamethasone, but the increase was less than for control lambs. Norepinephrine values increased after delivery for lambs treated with saline, one and two doses of betamethasone. The cord norepinephrine levels were increased after three and four doses of betamethasone about 2-fold, but the postnatal increases from these elevated levels were not significant.
Fig. 6. Epinephrine and norepinephrine values cord plasma and at 40 min of age. The cord and 40 min epinephrine levels were decreased after four doses of epinephrine relative to all other groups. *p < 0.05 cord values relative to cord values for 4 dose group; tp < 0.05 for 40 min values relative to cord values; †p < 0.05 relative to 40 min value for control group; ‡p < 0.05 relative to one dose group.
[More] [Minimize]This study demonstrates that repetitive single exposures to maternal betamethasone given at 7-d intervals resulted in progressive improvements in postnatal lung function in prematurely delivered lambs. However, this enhanced lung function was accompanied by a decrease in the body weights of the newborns. The dosing protocol was developed to test if multiple doses of maternal betamethasone would benefit postnatal lung function of the premature because of the common obstetric practice of administering prenatal corticosteroid at 7- to 10-d intervals to women at continued risk for preterm delivery (7). We previously found comparable augmentation of postnatal lung function in preterm lambs treated with 0.5 mg/kg betamethasone given by direct fetal injection for treatment to delivery intervals varying from 15 h to 7 d (11, 21). However, a second dose of betamethasone given to the fetus 6 d following an initial dose and 24 h before delivery did not further improve postnatal lung function of lambs delivered at 128 d gestation (12). We also have reported that single maternal or fetal treatments resulted in similar improvements in postnatal lung function (22). The progressive enhancement of postnatal lung function after two, three, or four doses of betamethasone reported here may result from route of treatment (maternal rather than fetal), the gestational age at initiation of treatment (104 rather than 121-d gestation) or the interval between doses required to achieve the cumulative effect.
Midgestation treatment of primates with either high dose corticosteroids for 3 d or lower dose glucocorticoids for 13 d resulted in lungs that were smaller and that contained fewer alveoli at term (3, 4). Single prenatal glucocorticoid treatments in sheep result in decreased alveolar wall thickness and increased lung volumes (23). We measured hyaluronan because it is a major extracellular matrix component that increases with severity of respiratory distress syndrome in preterm monkeys (19). We anticipated that the changes in lung structure and the decreased severity of respiratory failure would correlate with a decreased hyaluronan content in lung tissue following glucocorticoid exposure. The lack of change in hyaluronan indicates that this matrix component did not participate in the changes in postnatal lung function.
Midgestation exposure of the primate fetus to glucocorticoid also caused growth retardation following preterm or term delivery. Fetal treatment of sheep with glucocorticosteroid has not been reported to cause growth retardation, perhaps because the preterm labor that accompanies fetal exposure of sheep to glucocorticoids was thought to make such experiments impractical (13). We found no growth effects following single fetal treatments with 0.5 mg/kg betamethasone given 7 d before preterm delivery or 20 d before term delivery (11, 12, 24). Therefore, the 15% decrease in fetal size resulting from the single maternal dose of betamethasone given at 104-d gestation may indicate a sensitivity of the more immature fetus to a very short exposure to glucocorticoid. There was no selective effect of the growth retardation on the lungs. Fetal exposure to dexamethasone can decrease growth globally and decrease DNA synthesis in multiple organs (25). Lung weight decreased proportionately to body weight, the same result found after a 13 d consecutive treatment of pregnant monkeys with glucocorticoids (3). No effects on fetal growth or subsequent lung or somatic growth of infants exposed to prenatal corticosteroids have been reported (26-28), although long-term follow up is available only for individuals who were not extremely preterm and who were not exposed to multiple treatment courses of glucocorticoid. Many of the very preterm infants being salvaged today are small based on the available fetal growth curves. Potential growth effects of prenatal glucocorticoids in early gestation humans have not been evaluated carefully.
This experiment was designed as a five group study and therefore was analyzed using five group comparison procedures. The modest improvements in postnatal lung function at 125-d gestation resulting from the single maternal treatment with betamethasone at 104-d gestation were not different from saline controls. However, comparison of this single dose group with the controls by t-test demonstrated significant increases in compliance and maximal lung volume, indicating a modest improvement in postnatal lung function after a treatment to delivery interval of 20 d. The hormone measurements also indicated residual effects of this single dose of betamethasone at 104 d gestation. We previously found that prenatal glucocorticoid exposure suppressed postnatal cortisol responses for only about 4 d in more mature lambs (11). In contrast there was a persistent suppression of the postnatal increases in cortisol and epinephrine in all groups in the present study. Therefore, a single exposure of early gestation fetal sheep to glucocorticoid resulted in persistent effects on growth, lung function, and hormone regulation.
Repeated doses of maternal betamethasone resulted in more striking changes in cord and postnatal hormone values. Most remarkably the fourth dose of betamethasone 24 h before preterm delivery severely suppressed cord and postnatal epinephrine, and decreased T4 but increased T3. These changes demonstrate multiple alternations in the hormone regulation of postnatal adaptation (29). Such changes could persist and be antecedents for changes in the incidence of high blood pressure and cardiovascular disease in later life that have been associated with low birth weights (30, 31).
Our results using the preterm lamb model indicate that multiple 7 d interval exposures of the fetal sheep to glucocorticoid remarkably improved postnatal lung function. However, the growth and endocrine effects suggest potential concerns with repetitive exposure of the very preterm fetus to glucocorticoids. Randomized clinical trials will be required to demonstrate the efficacy and safety of repetitive glucocorticoid treatments of the very preterm human fetus at risk for preterm delivery.
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