Scientific Rationale for the Use of Antenatal Glucocorticoids to Promote Fetal Development

After completing this article, readers should be able to:Following publication of the report of the National Institutes of Health Consensus Development Conference on the Effect of Corticosteroids for Fetal Maturation in 1994, antenatal corticosteroid therapy has become routine practice in the United States and most other countries. This therapy had its origins in the pioneering observations of Liggins in 1969, who reported the unexpected viability of prematurely delivered lambs after treatment with corticosteroid in utero. Since then, much has been learned about the biochemical and physiologic effects as well as the mechanisms of corticosteroid action in the developing lung and other tissues. This review summarizes the evidence that constitutes the scientific rationale for clinical use of antenatal treatment and discusses pharmacokinetic issues related to the recommended doses of corticosteroid.The physiologic effects of glucocorticoids in the developing lung have been investigated extensively in various animal models (Table 1 ). In initial studies with rabbits and lambs, corticosteroid therapy was found to stimulate structural development of lung parenchyma involving both airspace lining cells (differentiation into surfactant-producing type II cells) and the mesenchyme (condensation with narrowing of saccular septae). When viewed by electron microscopy, the ultrastructure of airspace epithelial cells in glucocorticoid-treated cultured fetal lung resembles that of mature type II cells of adult lung, containing well-developed lamellar bodies and microvillae. Glucocorticoids also increase lung compliance and maximal volume independent of the effects of increased surface-active material, presumably reflecting the structural maturation. In addition, glucocorticoid treatment reduces protein leak from the pulmonary vasculature to airspace and appears to accelerate clearance of lung liquid before delivery. Enhanced structural maturity is likely the basis for the improved response of glucocorticoid-treated preterm animals and infants to surfactant therapy.The stimulatory effects of corticosteroids on the surfactant system have been examined in detail both in vivo and in cultured tissue (Table 2 ). It is particularly noteworthy that all known components of surfactant (lipids and surfactant-associated proteins) are induced by glucocorticoid treatment, resulting in increased content of surfactant that has normal composition and function. In cultured human fetal lung tissue, for example, dexamethasone treatment increases the content of mature, physiologically active surfactant proteins (SP) A, B, C, and D and stimulates production of fatty acid synthetase, cholinephosphate cytidylyltransferase, and lysophosphatidylcholine acyl CoA acyltransferase, key enzymes of phospholipid synthesis. These effects promote development of lamellar bodies that are secreted into the airspace lumen and increase the content of surface-active saturated phosphatidylcholine in lung tissue and alveolae. The spectrum of corticosteroid-induced proteins indicates that a variety of biochemical systems within the lung are regulated by glucocorticoids. In several animal species, glucocorticoids cause precocious increases in the activity of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase. Recently, glucocorticoids have been found to induce both Na+,K+-ATPase and the alpha subunit of the Na+ channel (ENaC) in rat lung, proteins that are involved in clearing lung fluid from airspaces. Each of these identified glucocorticoid effects represents precocious maturation of enzyme activity and, thus, presumably is protective in the transition to air breathing.High-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to examine the scope of hormonal effects on lung proteins. In a study with cultured fetal tissue, dexamethasone induced 15 proteins and repressed 4 proteins out of approximately 1,000 resolved proteins. One protein was induced only in tissue that was treated with both dexamethasone and cAMP. Among the regulated proteins resolved on 2-D PAGE, only SP-A and possibly precursor forms of SP-B were identified, based on their migration coordinates and isoform pattern. Some of the regulated proteins were enriched in either fibroblasts or type II cells isolated from the lung explants, consistent with cellular specificity of hormone action. These findings establish that glucocorticoids regulate a relatively small subset of proteins in lung cells, similar to their action on other target tissues. Presumably many of these proteins are important mediators of the physiologic effects of corticosteroids in the developing lung, although the identity and function of most of these regulated proteins currently is not known. Future studies with DNA microarray chip technology will provide a more definitive understanding of the genes regulated by glucocorticoid in lung cells.The clinical observations of multisystem beneficial effects of antenatal corticosteroid therapy for the newborn are consistent with data from animal studies investigating glucocorticoid treatment, hypophysectomy, or adrenalectomy. Glucocorticoids stimulate cytodifferentiation and cause precocious changes in developmentally regulated proteins in at least 16 different tissues, accelerating the rate of differentiation without altering the sequence of developmental events that normally occur. By contrast, adrenalectomy or hypophysectomy delays the differentiation process.Some of the earliest observations regarding the maturational effects of glucocorticoids are related to the gut. In the intestines of fetal and suckling animals, glucocorticoids enhance maturation of glands and villi, induce digestive enzymes (eg, sucrase), and improve digestive functions, while decreasing absorption of intact proteins. In the fetal liver, glucocorticoid treatment accelerates glycogen synthesis and deposition, hastens the appearance of bile canaliculi and disappearance of hematopoetic cells, and induces a variety of metabolic enzymes. Glucocorticoids induce a number of enzymes in the fetal pancreas, increase the content of zymogen granules, and enhance the insulin response to glucose. The skin of the fetal rat and embryonic chick undergoes both cytodifferentiation and keratinization in response to corticosteroid therapy.Recent evidence indicates that glucocorticoids regulate levels of Na+,K+-ATPase in the developing kidney and increase glomerular filtration rate and sodium reabsorption. In the fetal adrenal, corticosteroids enhance adrenocorticotropic hormone (ACTH) responsiveness of the cortex and increase the content of epinephrine following induction of phenylethanolamine-N-methyl transferase, resulting in maturation of catecholamine responses to birth in the preterm animal. The effects on the adrenal medulla are likely very important in adapting to extrauterine life because of the known effects of catecholamines on cardiovascular perfusion, glucose mobilization, and nonshivering thermogenesis. Myocardial adenylyl cyclase activity is increased in corticosteroid-treated animals and helps to account for their improved respiratory, cardiovascular, and metabolic responses. In addition, corticosteroid may act as the “molecular switch” in the developmental expression of Na+,K+-ATPase alpha-isoforms that contribute to maturation of cardiovascular function. Of particular relevance to clinical use, glucocorticoid treatment of fetal lambs decreases responsiveness of the ductus arteriosus to the relaxing action of prostaglandin E2, facilitating closure after birth. These examples illustrate the generality of glucocorticoid effects in preparing organisms for the major developmental transitions of birth and weaning, and they provide a scientific basis for the use of antenatal steroid treatment to accelerate development of various organ systems of the human fetus before preterm delivery.The modulating effects of glucocorticoids on tissue differentiation likely involve interactions with other fetal hormones. Studies in the fetal lamb suggest that prolactin is needed for a maximal response of the fetal lung to glucocorticoid treatment and that there are additive or synergistic interactions between glucocorticoids and both thyroid hormones and catecholamines. In culture systems, treatment with glucocorticoid plus thyroid hormone has an additive effect on phospholipid synthesis, and the combination of corticosteroid plus cAMP additively stimulates synthesis of phospholipid, SP-A, and SP-B.These observations prompted trials of combination prenatal therapy with glucocorticoids plus thyrotropin-releasing hormone, but this therapy has not proven efficacious in reducing the incidence of respiratory distress syndrome (RDS) or chronic lung disease. Stimulatory or inhibitory effects on components of surfactant also occur in response to treatment with insulin, epidermal growth factor, interferon-gamma, transforming growth factor-beta, bombesin, and stimulators of protein kinase C and testosterone, although neither their interactions with glucocorticoid nor their in vivo effects are well defined. Thus, it is possible that developmental deficiencies (or excesses) of other hormones or growth factors contribute to the variable responses to corticosteroids seen among preterm infants.Data from animal studies and from clinical observations provide strong support for the premise that increasing the availability of endogenous corticosteroids during fetal development modulates the rate of differentiation of numerous tissues. In a variety of animal models (rabbit, rat, sheep, rhesus monkey) the concentration of circulating unbound corticosteroid (the biologically active fraction) increases during late gestation when lung and other tissues acquire differentiated functions. The mechanism for increased corticosteroid availability varies between species and includes increased fetal production of corticosteroids, decreased levels of corticosteroid-binding globulin, increased conversion of inactive 11-oxysteroid to active 11-hydroxysteroid (eg, cortisone to cortisol) by target tissues, and increased concentration of glucocorticoid receptors, which enhances responsiveness. In experimental studies of adrenal function ablation by decapitation or hypophysectomy, lung maturation is delayed, as indicated by indices such as morphology, maximal lung volume, and content of saturated phosphatidylcholine. In fetal sheep, these effects were reversed by ACTH treatment.In the human, concentrations of amniotic fluid corticoid conjugates, which are primarily derived from fetal cortisol, increase approximately five-fold during the third trimester in parallel with the lecithin/sphingomyelin ratio (Fig. 1 ). By contrast, levels of cortisol, which is primarily of maternal origin, increase steadily throughout gestation (2.5-fold). A developmental increase also is observed in cord blood cortisol obtained both at birth and by fetal sampling, which avoids stress-related effects of labor and delivery. Clinically, untreated infants who have RDS are expected to begin their recovery 48 to 72 hours after birth, a timing consistent with accelerated lung maturation in response to the elevation in cortisol that occurs with the strain of labor, delivery, and respiratory distress. Indeed, postnatal administration of hydrocortisone to infants who have RDS does not improve their clinical course or outcome, suggesting that the postnatal elevation in endogenous levels of cortisol is sufficient to induce lung maturity. Thus, prenatal corticosteroid therapy is believed to mimic the exposure to endogenous corticosteroids that occurs in utero as well as the physiologic stress levels that occur postnaally.The mechanism of glucocorticoid effects on fetal lung development has been examined in isolated cells and explant culture of fetal lung tissue. The latter system has been particularly useful because lung tissue is cultured in the absence of serum, cell-cell relationships are maintained, and both biochemical and morphological responses to glucocorticoids resemble those observed in vivo. With the availability of molecular probes and reagents, many studies have focused on regulation of the surfactant components and related enzymes. In each case, properties of the induction process indicate increased gene expression mediated by glucocorticoid receptor. For SP-B and SP-C, which are critical for normal surfactant function, glucocorticoids enhance both synthesis of protein (via increased messenger RNA [mRNA] production) and processing of precursor proteins to mature SP-B and SP-C. Supporting evidence for glucocorticoid receptor involvement includes the time course of response (increased rate of gene transcription within 1 h, maximal increase in mRNA and protein content by 24 and 48 h, respectively); reversibility of induction consistent with disassociation of the steroid from the receptor after hormone is removed; responsiveness to both synthetic and natural glucocorticoids and not to estrogens, androgens, or progestins; and dose-response characteristics. Maximal induction of lung proteins or their mRNAs occurs at nanomolar concentrations of dexamethasone and at approximately 10-fold higher concentrations of cortisol than predicted by the relative affinity of the two steroids for glucocorticoid receptor. Responses plateau at higher glucocorticoid concentrations because receptor sites are nearly fully occupied. Extrapolated to the in vivo situation, these observations indicate that physiologic stress levels of endogenous cortisol (eg, 30 mcg/dL, equivalent to approximately 300 nM unbound steroid) or peak concentrations of administered synthetic corticosteroid (eg, 20 ng/mL dexamethasone, equivalent to approximately 30 nM unbound steroid) should be sufficient to increase the rate of synthesis of target proteins in fetal tissues to near maximal levels.The complete reversibility of glucocorticoid effects in cultured lung tissue is of interest for clinical management. Levels of induced SP-B and SP-C mRNAs decline to control levels within 2 to 3 days after removal of cortisol. The same pattern occurs for SP-A and inducible enzymes of lipid synthesis, although the kinetics vary with relative stabilities of the mRNAs. This response is consistent with the properties of glucocorticoid action in adult tissues where circadian increases in endogenous corticosteroid produce a cyclic stimulation of inducible enzymes.The kinetics of corticosteroid effects in vivo have been examined in the lamb model. In these studies, pregnant ewes were treated with betamethasone at various times before delivery of preterm lambs, and surfactant content and lung function were assessed. Some of these findings for animals delivered 2 to 14 days after treatment are summarized in Table 3 . There are four findings of particular interest. First, treatment 48 hours before delivery improves lung function, as measured by dynamic compliance and the maximal lung volume at 40 cm H2O, and these changes occur without any significant increase in surfactant components in lung lavage fluid. In other studies with this animal model, lung function improved as early as 15 hours after treatment. It can be speculated that the improved pulmonary function reflects morphologic maturation of the lung that occurred after corticosteroid treatment, apparently independent of changes in the amount of pulmonary surfactant. Second, 1 to 2 weeks after treatment, surfactant components are increased many-fold in lung lavage fluid, and there is a more modest but significant increase in tissue levels. At this time, pulmonary function is maximally improved, probably reflecting the contribution of increased surfactant in airspaces. Third, benefit from corticosteroid treatment remains 2 weeks after the last dose, although not at the same levels as at 1 week. If a similar time course of responses occurs in the human fetus, these observations suggest that the benefit of antenatal corticosteroid occurs rapidly (ie, by 24 h) and may extend beyond a 1-week interval. Finally, strong correlations (r >0.6) were found between concentrations of SP-A, SP-B, and saturated phosphatidylcholine in lavage samples of individual animals, consistent with a coordinated induction of surfactant components by glucocorticoid treatment. Despite the impressive induction, the levels of surfactant proteins and lipid in lavage fluid achieved after 2 weeks’ exposure to betamethasone are only about 20% and 44% of the level at term, respectively. Thus, even with optimal prenatal therapy, many preterm infants may have inadequate surfactant activity for fully normal lung function.The time course for induction of SP mRNAs in fetal sheep are summarized in Figure 2 . Maximal increases of two- to three-fold occur about 24 hours after treatment, and levels of all three mRNAs return close to control levels by 7 days. This pattern parallels responses in cultured tissue and establishes that the inductive effects of glucocorticoid are fully reversible in vivo. The longer persistence of elevated levels of surfactant proteins and lipid (Table 3 ) reflects longer half-lives of the induced proteins compared with their mRNAs.One adverse effect associated with corticosteroid treatment of the sheep was a dose-dependent decrease in birthweight at both preterm and term delivery. This finding is consistent with the known effects of glucocorticoids to inhibit DNA synthesis in many tissues and to cause premature senesence of the in The of treatment in the human fetus have not been fully but there is initial evidence for adrenal and from early lung on clinical use of exposure to elevated glucocorticoid levels in utero also may and increase the of to of circulating corticosteroids have been in and their infants after exposure to prenatal corticosteroid The of Liggins used a of betamethasone plus betamethasone with two doses administered 24 hours This exposure to betamethasone by of the plus a more exposure from of the With this therapy, maximal betamethasone by a was found 1 after treatment in the and 1 to 2 hours in the fetus 20 ng/mL in cord was from the maternal with a of approximately hours and was not 48 hours after the last The was longer in the fetal cord levels were than in maternal samples and betamethasone was not in infants who were delivered more than 40 hours after the last of different corticosteroids for prenatal therapy is by their relative glucocorticoid from to fetus, and rate of clearance from the synthetic corticosteroids have a affinity for glucocorticoid than cortisol and betamethasone are and consistent with their both in and in vivo. corticosteroids also have longer half-lives in which increases their relative to cortisol. corticosteroids not to proteins the from to fetus and are for cellular synthetic dexamethasone and betamethasone not are not by corticosteroid-binding cortisol is Thus, about of dexamethasone and betamethasone not by is for By contrast, circulating cortisol physiologic from to the data for and of corticosteroids for and corticosteroid globulin, unbound glucocorticoid activity can be by levels of endogenous cortisol and administered Figure 3 the time course pattern of unbound glucocorticoid activity in the human fetus after exposure to betamethasone 24 h) or betamethasone Maximal levels occur after each of betamethasone unbound cortisol and about a five-fold increase the untreated of The of elevated glucocorticoid activity with two doses of betamethasone is about levels are for the than the dose, and the of increased glucocorticoid concentrations is The time of glucocorticoid in target tissues is longer than for levels because of the relatively disassociation of steroid from receptor and the relatively half-lives of induced proteins. A course of treatment a of maternal and fetal adrenal function, although treated infants to newborn stress with a cortisol (Table 4 ). Maximal levels of circulating glucocorticoid activity after antenatal betamethasone treatment are in the physiologic stress equivalent to that in untreated infants who have or RDS (Table 4 ). In contrast, the of dexamethasone used much levels of circulating for many days or It is that of glucocorticoid in target cells increases from than in the untreated to a maximal of or following antenatal betamethasone treatment, which should provide a induction of genes in fetal target tissues. In a study to their and Liggins found increased benefit from the of betamethasone 24 Thus, the corticosteroid and by Liggins in initial clinical study to be optimal for Consensus recommended treatment of either two doses of betamethasone plus 24 hours or four doses of dexamethasone hours that the of dexamethasone as the are similar to those found for betamethasone the two recommended should in levels of circulating glucocorticoid activity in the treated fetus similar to those in Figure 3 . These similar of the two in accelerating fetal therapy with has been in two studies and was in reducing the incidence of The was at a which produce a very peak level of glucocorticoid activity after each 20 times that of and clearance of approximately 1 Thus, treated not the elevation of glucocorticoid for induction of proteins and be to levels of doses of hydrocortisone to 1,000 to h) were used in early clinical The maximal unbound glucocorticoid activity in infants to these is to higher than occurs with betamethasone There is evidence for increased of these higher doses for such doses are not needed for and doses increase the of adverse effects by higher or more doses of dexamethasone or betamethasone are not indicated and are by the Consensus of steroid either or by into the fetus has not been for or and is not recommended at this treatment both and fetus to higher initial concentrations of hormone than and the of adverse responses. corticosteroid from to fetus treatment of the fetus not be for therapy and the with an exposure of the to corticosteroid be after fetal than maternal treatment, the to the following corticosteroid are scientific basis for the use of prenatal corticosteroid therapy in preparing the human fetus for preterm delivery is and Studies in numerous animal species have precocious maturation of lung and other tissues after administration of glucocorticoids to the fetus, and ablation a of endogenous corticosteroid organ maturation. The timing of lung differentiation well with the developmental increase in circulating fetal the that endogenous corticosteroids, with other are important physiologic of tissue maturation and that prenatal steroid therapy the of endogenous glucocorticoids. In fetal lung, the response to glucocorticoid induction of a number of all the components of surfactant and key enzymes. These effects are mediated by glucocorticoid receptors, occur with physiologic concentrations of hormone, and are primarily at the level of gene responses occur rapidly and are These observations that a relatively exposure of the human fetus to glucocorticoid accelerates the normal developmental process in lung and other tissues and reduces the of to developmental treatment with either betamethasone or dexamethasone at the recommended doses similar physiologic stress levels of glucocorticoid activity in fetal sufficient to provide a of and induction of target proteins. glucocorticoid levels are during the course of treatment, and return to normal within 2 days after the last The of the Consensus of corticosteroid therapy for at of preterm delivery, with is well by evidence to glucocorticoid and for the to this for in the of the and the and of in by