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Low-dose antenatal betamethasone treatment achieves preterm lung maturation equivalent to that of the World Health Organization dexamethasone regimen but with reduced endocrine disruption in a sheep model of pregnancy

Open AccessPublished:July 02, 2022DOI:https://doi.org/10.1016/j.ajog.2022.06.058

      Background

      The intramuscular administration of antenatal steroids to women at risk of preterm delivery achieves high maternal and fetal plasma steroid concentrations, which are associated with adverse effects and may reduce treatment efficacy. We have demonstrated that antenatal steroid efficacy is independent of peak maternofetal steroid levels once exposure is maintained above a low threshold.

      Objective

      This study aimed to test, using a sheep model of pregnancy, whether the low-dose antenatal steroid regimen proposed as part of the Antenatal Corticosteroids for Improving Outcomes in Preterm Newborns trial would achieve preterm lung maturation equivalent to that of the existing World Health Organization dexamethasone treatment regimen, but with reduced risk of adverse outcomes.

      Study Design

      Following ethical review and approval, date-mated ewes with single fetuses received intramuscular injections of either (1) four 6-mg maternal intramuscular injections of dexamethasone phosphate every 12 hours (n=22), (2) 4 2-mg maternal intramuscular injections of betamethasone phosphate every 12 hours (n=21), or (3) 4 2-mL maternal intramuscular injections of saline every 12 hours (n=16). Of note, 48 hours after first injection, (124±1 day), lambs were delivered, ventilated for 30 minutes, and euthanized for sampling. Arterial blood gas, respiratory, hematological, and biochemical data were analyzed for between-group differences with analysis of variance according to distribution and variance, with P<.05 taken as significant.

      Results

      After 30 minutes of ventilation, lambs from both steroid-treated groups had significant and equivalent improvements in lung function relative to saline control (P<.05). There was no significant difference in arterial blood pH, pO2, pCO2, lung compliance, ventilator efficiency index, or lung volume at necropsy with a static pressure of 40 cmH2O. The messenger RNA expression of surfactant protein (Sp)a, Spb, Spc, Spd, aquaporin (Aqp)1, Aqp5, and sodium channel epithelial 1 subunit beta (Scnn1b) was equivalent between both steroid groups. Maternal and fetal plasma neutrophil, glucose, and fetal plasma C-peptide levels were significantly elevated in the dexamethasone group, relative to the betamethasone group. Fetal plasma insulin-like growth factor 1 was significantly reduced in the dexamethasone group compared with the betamethasone group (P<0.05). Fetal adrenocorticotropic hormone (r=0.53), maternal glucose value (r=−0.52), and fetal glucose values (r=−0.42) were correlated with maternal weight in the betamethasone group (P<.05), whereas fetal pCO2 and pO2 were not correlated. There was no significant difference between male and female lamb outcomes in any groups for any of the items evaluated.

      Conclusion

      This study reported that in preterm lambs, a low-dose treatment regimen of 8 mg betamethasone achieves lung maturation equivalent to that of a 24-mg dexamethasone-based regimen, but with smaller perturbations to the maternofetal hypothalamic-pituitary-adrenal axis. These data suggested that given steroid pharmacokinetic differences between sheep and humans, a betamethasone dose of 2 mg may remain above the minimum dose necessary for robust maturation of the preterm lung. Maternal weight–adjusted betamethasone doses might also be a key to reducing perturbations to the maternofetal hypothalamic-pituitary-adrenal axis.

      Key words

      Introduction

      Following the observations made in pregnant sheep,
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      Premature delivery of foetal lambs infused with glucocorticoids.
      and a subsequent clinical trial in New Zealand,
      • Liggins G.C.
      • Howie R.N.
      A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants.
      dexamethasone phosphate (Dex-P) and betamethasone phosphate (Beta-P) (as phosphate alone or in combination with the betamethasone acetate [Beta-Ac] form) have been used for several decades as antenatal corticosteroid (ACS) treatments for women considered at risk of impending preterm delivery. When targeted judiciously, ACS treatment decreases the incidence of respiratory distress syndrome (RDS) and other neonatal morbidities in preterm infants from 24 to 34 weeks of gestation.
      • Liggins G.C.
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      A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants.
      ,
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      • Dalziel S.R.
      Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth.
      Of note, 2 maternal intramuscular (IM) doses of 11.4 mg Beta-P and Beta-Ac or 12 mg Beta-P alone at a 24-hour interval are used preferentially in high-resource environments. In contrast, 4 maternal IM injections of 6 mg Dex-P at 12-hour intervals are the World Health Organization (WHO)-recommended regimen used predominantly in low-resource environments.
      Some meta-analyses of clinical trial data comparing Dex-P with Beta-P showed no difference in RDS or neonatal mortality.
      • Jobe A.H.
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      Choice and dose of corticosteroid for antenatal treatments.
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      • Quirk J.G.
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      Beta-P and Dex-P are rapidly dephosphorylated, resulting in high maternal and fetal concentrations. Here, we have demonstrated that in nonhuman primates and sheep, current ACS treatments expose the fetus to unnecessarily excessive amounts of steroids.
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      • Jobe A.H.
      Optimizing antenatal corticosteroid therapy.
      These elevated exposures seem not to benefit fetal lung maturation and, thus, may only contribute to adverse maternofetal side effects.
      • Jobe A.H.
      • Soll R.F.
      Choice and dose of corticosteroid for antenatal treatments.
      ,
      • Elimian A.
      • Garry D.
      • Figueroa R.
      • Spitzer A.
      • Wiencek V.
      • Quirk J.G.
      Antenatal betamethasone compared with dexamethasone (betacode trial): a randomized controlled trial.
      ,
      • Kemp M.W.
      • Saito M.
      • Usuda H.
      • et al.
      The efficacy of antenatal steroid therapy is dependent on the duration of low-concentration fetal exposure: evidence from a sheep model of pregnancy.
      • Schmidt A.F.
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      • Bridges J.P.
      • et al.
      Dosing and formulation of antenatal corticosteroids for fetal lung maturation and gene expression in rhesus macaques.
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      • et al.
      Immediate and delayed effects of antenatal corticosteroids on fetal heart rate: a randomized trial that compares betamethasone acetate and phosphate, betamethasone phosphate, and dexamethasone.
      A recent study with Dex-P in low-resource environments showed no benefit for small infants but showed increased mortality for large infants.
      • Althabe F.
      • Belizán J.M.
      • McClure E.M.
      • et al.
      A population-based, multifaceted strategy to implement antenatal corticosteroid treatment versus standard care for the reduction of neonatal mortality due to preterm birth in low-income and middle-income countries: the ACT cluster-randomised trial.
      ,
      • Althabe F.
      • Thorsten V.
      • Klein K.
      • et al.
      The Antenatal Corticosteroids Trial (ACT)’s explanations for neonatal mortality - a secondary analysis.
      A more recent study, again in low-resource jurisdictions, demonstrated the benefit of the use of Dex-P.
      • Oladapo O.T.
      • Vogel J.P.
      • et al.
      WHO ACTION Trials Collaborators
      Antenatal dexamethasone for early preterm birth in low-resource countries.
      A particular point of contrast between these 2 studies, and a potential explanation for the discordant findings, was the far higher quality and standardization of antenatal and neonatal care provided to most subjects in the latter study. Given this, more subtle adverse effects of ACS treatments, such as higher rate of maternal infection and hyperglycemia or fetal hypoglycemia and blunted adrenal function, may have a greater impact in low- and middle-income countries’ (LMICs) delivery settings with limited capacity to provide antenatal care and/or postpartum support to the mother and infant.
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      • Belizán J.M.
      • McClure E.M.
      • et al.
      A population-based, multifaceted strategy to implement antenatal corticosteroid treatment versus standard care for the reduction of neonatal mortality due to preterm birth in low-income and middle-income countries: the ACT cluster-randomised trial.
      ,
      • Althabe F.
      • Thorsten V.
      • Klein K.
      • et al.
      The Antenatal Corticosteroids Trial (ACT)’s explanations for neonatal mortality - a secondary analysis.
      ,
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      • Carter E.B.
      • Macones G.A.
      • Cahill A.G.
      • Tuuli M.G.
      13: Antenatal steroids and neonatal hypoglycemia.
      • Tegethoff M.
      • Pryce C.
      • Meinlschmidt G.
      Effects of intrauterine exposure to synthetic glucocorticoids on fetal, newborn, and infant hypothalamic-pituitary-adrenal axis function in humans: a systematic review.
      • Jain A.
      • Rutter N.
      • Cartlidge P.H.
      Influence of antenatal steroids and sex on maturation of the epidermal barrier in the preterm infant.
      • Willet K.E.
      • Jobe A.H.
      • Ikegami M.
      • et al.
      Postnatal lung function after prenatal steroid treatment in sheep: effect of gender.
      • Ramos-Navarro C.
      • Sánchez-Luna M.
      • Zeballos-Sarrato S.
      • Pescador-Chamorro I.
      Antenatal corticosteroids and the influence of sex on morbidity and mortality of preterm infants.
      Thus, optimizing the dosing for this very important therapy is one of the most pressing challenges that perinatal medicine is currently facing.

      Why was this study conducted?

      Antenatal corticosteroid therapy is now widely used to improve preterm outcomes, with the principal desired benefit being precocious maturation of the fetal lung. Increasing evidence shows that current clinical regimens (ie, intramuscular injections of betamethasone acetate [Beta-Ac] and betamethasone phosphate [Beta-P] [12 mg, 2 doses at a 24-hour interval] or dexamethasone phosphate [Dex-P] [6 mg, 4 doses at 12-hour intervals]) achieve maternofetal steroid exposures well above those required to elicit lung maturation. We and others have previously suggested that these elevated exposures may increase the risk of harm. In support of the forthcoming Antenatal Corticosteroids for Improving Outcomes in Preterm Newborns trial (https://www.who.int/publications/m/item/the-who-action-iii-(antenatal-corticosteroids-for-improving-outcomes-in-preterm-newborns)-trial), we used a sheep model of pregnancy to study the lung maturation and pharmacodynamic effects of a low-dose treatment regimen employing Beta-P regimens (2 mg, 4 times at 12-hour intervals) and compared it with outcomes deriving from the current World Health Organization (WHO)-recommended regimen employing Dex-P (6 mg, 4 times at 12-hour intervals).

      Key findings

      The primary findings of this study were as follows: (1) the low-dose regimen (four 12-hourly doses of Beta-P at 2 mg: a total of 8 mg) achieved functional maturation of the ovine preterm lung equivalent to that of the current WHO-recommended dosing regimen (four 12-hourly doses of Dex-P at 6 mg: a total of 24 mg), and (2) the low-dose regimen resulted in less severe disruptions of the maternofetal hypothalamic-pituitary-adrenal axis, circulating immunocyte populations, plasma glucose, and insulin. Suppression of the key fetal growth factor, insulin-like growth factor 1, was seen only in the standard-dose Dex-P treatment group.

      What does this add to what is known?

      This study demonstrated that in a sheep model of pregnancy, low-dose antenatal steroid treatments (66% reduction in steroid administered) achieve equivalent lung maturation compared with standard-dose regimens currently in use. The low-dose regimen was predicted to achieve lower peak steroid exposures and an improved side effect profile (ie, fewer or more modest endocrine and immunocyte perturbations). Given the significant difference in maternofetal steroid gradient between sheep and humans, these data strongly suggested that a regimen based on doses of betamethasone significantly <2 mg will achieve similarly improved outcomes in humans.

      Study Aim

      This study aimed to use a sheep model of pregnancy to evaluate the use of a low-dose Beta-P regimen (four 2-mg doses given at 12-hour intervals; equivalent to 24 mg prednisone a day) against the current clinical treatment with Dex-P (four 6-mg doses given at 12-hour intervals; equivalent to 72 mg prednisone a day) in anticipation of the Antenatal Corticosteroids for Improving Outcomes in Preterm Newborns randomized control trial.
      World Health Organization
      Action; Published 2020. Accessed. The WHO ACTION-III (Antenatal CorticosTeroids for Improving Outcomes in preterm Newborns) Trial.
      As the interacting influence of ACS and sex on the singleton fetus is still controversial,
      • Jain A.
      • Rutter N.
      • Cartlidge P.H.
      Influence of antenatal steroids and sex on maturation of the epidermal barrier in the preterm infant.
      • Willet K.E.
      • Jobe A.H.
      • Ikegami M.
      • et al.
      Postnatal lung function after prenatal steroid treatment in sheep: effect of gender.
      • Ramos-Navarro C.
      • Sánchez-Luna M.
      • Zeballos-Sarrato S.
      • Pescador-Chamorro I.
      Antenatal corticosteroids and the influence of sex on morbidity and mortality of preterm infants.
      sex differences in baseline fetal lung function and hematological, biochemical and endocrinological responses to antenatal steroid treatment were also investigated.
      As responsiveness to steroids may vary with gestational age (GA) at the time of exposure and does alter with treatment to delivery interval, all fetuses were delivered at an equivalent treatment duration and GA-matched time point.
      • Kemp M.W.
      • Saito M.
      • Usuda H.
      • et al.
      The efficacy of antenatal steroid therapy is dependent on the duration of low-concentration fetal exposure: evidence from a sheep model of pregnancy.
      Exposure time and dose, delivery procedure, and postnatal management were all stringently controlled to minimize the effect of these variables on outcome measurements. Investigators responsible for the management of lambs undergoing ventilation were blinded to the treatment.

      Material and Methods

      Antenatal corticosteroid treatments

      The Animal Ethics Committee of Murdoch University reviewed and approved these studies before study initiation (R3330/21). Time-mated Merino ewes with singleton fetuses received an IM injection of 150 mg medroxyprogesterone acetate (Depo-Provera, Pfizer, New York, NY) at 117± 1 days of gestation to decrease the risk of steroid-induced premature labor. Antenatal progesterone has been previously reported not to impact fetal lung maturation in sheep.
      • Jobe A.H.
      • Newnham J.P.
      • Moss T.J.
      • Ikegami M.
      Differential effects of maternal betamethasone and cortisol on lung maturation and growth in fetal sheep.
      Animals were allocated to 1 of 3 groups, with the first treatment given at 122 ± 1 days of gestation (term=150 days of gestation): (1) saline control group, ewes received 4 maternal IM injections of sterile normal saline at 12-hour intervals; (2) dexamethasone group, ewes received 4 maternal IM injections of 6 mg Dex-PO4 (DBL dexamethasone sodium phosphate 4 mg/mL; Hospira NZ, New Zealand) at 12-hour intervals; or (3) betamethasone group, ewes received 4 maternal IM injections of 2 mg Beta-PO4 (Rinderon injection 4 mg/mL; Shionogi & Co, Ltd, Osaka, Japan) at 12-hour intervals. All animals were delivered 48 hours after the first injection at 124 ± 1 days of gestation. The experiment was structured so that at least 2 animals from each of the 3 groups were delivered on each study day to assist in controlling for confounding.

      Ventilatory assessment

      Before delivery, ewes received an intravenous bolus of midazolam (0.5 mg/kg) and ketamine (10 mg/kg) for the deep induction of anesthesia. A 3-mL injection of 2% (20 mg/mL) lidocaine was given at L6-L7 for spinal analgesia. The head of the fetus was delivered through abdominal and uterine incisions. A 4.5-mm endotracheal tube was secured by tracheostomy. The fetus was delivered, and the ewe euthanized under anesthesia with pentobarbital. The lamb was weighed, dried, and placed in a radiant warmer (Cozy Cot, Fisher & Paykel Healthcare, Auckland, New Zealand) bed under a plastic insulating wrap (NeoWrap, Fisher & Paykel Healthcare, Auckland, New Zealand). Mechanical ventilation (fabian HFO, Acutronic Medical Systems AG, Hirzel, Switzerland) was immediately started with the following settings: peak inspiratory pressure (PIP) of 35 cmH2O, positive end-expiratory pressure (PEEP) of 5 cmH2O, respiratory rate (RR) of 50 bpm, and inspiratory time of 0.5 seconds, using 100% heated and humidified oxygen. The standard use of 100% oxygen allows the comparison of oxygenation through the partial arterial pressure of oxygen among the groups. An umbilical artery was catheterized for blood sampling and administration of supplemental anesthesia with ketamine (5 mg/kg) if necessary. The tidal volume (VT) was continuously measured, and the PIP was adjusted to keep the VT between 7.0 and 8.0 mL/kg but with a maximal pressure limited of 35 cmH2O. At 10, 20, and 30 minutes of ventilation, we measured the temperature, blood pressure, and ventilator data (PIP, VT, and compliance) and performed blood gas measurements—pH, PCO2 (mm Hg), PO2 (mm Hg), O2 saturation (%), total hemoglobin (g/dL), and glucose (mg/dL) levels (Siemens RAPIDPoint 500, Munich, Germany). Dynamic compliance (Cdyn, mL/cmH2O/kg) was recorded as measured by the ventilator. The ventilation efficiency index (VEI) was calculated using the formula VEI=3800/(RR×[PIP−PEEP]×PCO2[mm Hg]).
      • Pillow J.J.
      • Musk G.C.
      • McLean C.M.
      • et al.
      Variable ventilation improves ventilation and lung compliance in preterm lambs.

      Lung assessment

      After 30 minutes of ventilation, lambs were euthanized with pentobarbital and disconnected from the ventilator, and the endotracheal tube was clamped for 2 minutes to achieve atelectasis by oxygen absorption. The lambs were weighed, and the chest was opened for visual evaluation of gross lung injury—pulmonary hemorrhage, pulmonary interstitial emphysema, gas pockets within the lung, or subpleural dissection—performed by the same investigator. A deflation pressure-volume curve was measured after air inflation of the lungs to a pressure of 40 cmH2O. Volume at a pressure of 40 cmH2O was calculated using fetal weight after ventilation (kg) as V40 (mL/kg).

      Definition of antenatal steroid responder and nonresponder subgroups

      Steroid-treated animals were analyzed as a group and subdivided into responder or nonresponder subgroups to confirm the overall treatment benefit as described previously.
      • Takahashi T.
      • Saito M.
      • Schmidt A.F.
      • et al.
      Variability in the efficacy of a standardized antenatal steroid treatment was independent of maternal or fetal plasma drug levels: evidence from a sheep model of pregnancy.
      ,
      • Takahashi T.
      • Fee E.L.
      • Takahashi Y.
      • et al.
      Betamethasone phosphate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights when administered to pregnant sheep in combination with betamethasone acetate.
      A value of 2 standard deviations (2 SDs) below the average of normally distributed PaCO2 value at 30 minutes of ventilation from the saline control group of animals was used as an arbitrary, a priori cutoff for subgroup distribution.
      • Takahashi T.
      • Saito M.
      • Schmidt A.F.
      • et al.
      Variability in the efficacy of a standardized antenatal steroid treatment was independent of maternal or fetal plasma drug levels: evidence from a sheep model of pregnancy.
      ,
      • Takahashi T.
      • Fee E.L.
      • Takahashi Y.
      • et al.
      Betamethasone phosphate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights when administered to pregnant sheep in combination with betamethasone acetate.
      The arterial pCO2 value (±2 SD) for the saline control group of animals to determine the responders-to-nonresponders ratio was 152.3±34.2. Accordingly, the responder subgroups were defined as animals with a PaCO2 level more extreme than 2 SDs below the saline control group mean. Conversely, the nonresponder subgroups were defined as animals with a PaCO2 level less extreme than 2 SDs below the saline control group mean.

      Hematological and biochemical data acquisition

      Plasma isolated from maternal and fetal blood samples collected at delivery was used for hematological analyses, including white blood cell (WBC) counts (/μL), differential leukocyte counts (%), and biochemical parameters; cortisol (nmol/L), adrenocorticotropic hormone (ACTH; pg/mL), insulin-like growth factor 1 (IGF-1; μg/dL), glucose (mg/dL), and C-peptide (ng/mL). Those analyses were performed by an independent clinical pathology laboratory (VetPath, Perth, Australia). Cortisol values under the limit of detection (<5.52 nmol/L) were allocated a value of 2.76 nmol/L for statistical analyses. ACTH values under the limit of detection (<5.0 pg/mL) were allocated a value of 2.5 pg/mL for statistical analyses.

      Quantitation of messenger RNA

      RNA was extracted from frozen lung tissue (right lower lobe) using RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The concentration of extracted RNA was determined using a broad-range acid quantitation kit and a Qubit 2.0 Fluorometer (both supplied by Life Technologies, Carlsbad, CA). All RNA extracts were diluted in nuclease-free water (Life Technologies, Carlsbad, CA) to yield a final RNA concentration of 25 ng/μL.
      Quantitative polymerase chain reaction (PCR) cycling was performed using ovine-specific TaqMan probe and primer sets (Applied Biosystems, Foster City, CA) on a StepOne Real-Time PCR System following the manufacturer’s instructions. Messenger RNA (mRNA) transcripts surfactant protein (Sp)a, Spb, Spc, Spd, aquaporin (Aqp)1, Aqp5, and sodium channel epithelial 1 subunit beta (Scnn1b) were measured. In addition, 18s ribosomal protein RNA was used as an internal reference to normalize the amplification data for each gene. Delta quantification cycle values were used to determine the relative expression of transcripts for statistical analyses. Final data were expressed as a fold increase over the control value.

      Statistical analysis

      Statistical analyses were performed using IBM SPSS Statistics for Windows (version 25.0; IBM Corp, Armonk, NY). A chi-square test was used to test the differences in nominal values among groups. All numeric data were tested for normality with Shapiro-Wilk tests. Extreme outliers were tested for exclusion with Smirnov-Grubbs tests. In the comparison of 2 groups (males and females, respectively, in the dexamethasone, betamethasone, and saline control groups), between-group differences in parametric data were tested for significance with t tests, whereas Mann-Whitney U tests were used for nonparametric data. In the comparison of 3 groups (dexamethasone, betamethasone, and saline control group), between-group differences in parametric data were tested for significance with a 1-way analysis of variance, whereas Kruskal-Wallis tests were used for nonparametric data. Multiple posthoc comparisons were performed with Tukey tests. The Spearman correlation coefficient was calculated to assess the relationship with maternal weight in the dexamethasone and betamethasone groups. All P values of <.05 were accepted as significant and indicated with an asterisk.

      Results

      In the following description, significant differences among experimental groups were expressed as group comparison with P values, mean differences, and 95% confidence intervals (CIs). Statistical differences between males and females in each experimental group were expressed as group with P value.

      Delivery data and responder rates

      The number of animals, sex distribution, responder rates, GA at delivery, maternal weights, and fetal birthweights are given in Table 1. There was no significant difference in sex distribution, days of gestation, maternal weights and fetal birthweights, or lung injury (ie, emphysema) among the 3 groups. There was no significant difference in treatment response ratio between the dexamethasone and the betamethasone groups. The average weight (±2 SD) of animals in the steroid-treated groups was 68.8±12.8 kg.
      Table 1Comparison of delivery data and responder ratio among groups
      VariableDexamethasone (6 mg, 4 times)Betamethasone (2 mg, 4 times)SalineTestP value
      Total number222116
      Sex (male/female)10/1213/811/5Chi-square.316
      Responder/nonresponder (responder ratio in total number)16/6 (72.7)16/5 (76.2)Chi-square.795
      Gestational age124.1±0.6123.9±0.6123.6±0.4Kruskal-Wallis.219
      Maternal weight67.8±7.069.9±5.569.4±7.21-way ANOVA.909
      Fetal birthweight3.0±0.32.9±0.43.0±0.31-way ANOVA.564
      Data are presented as number of responders/number of nonresponders (percentage) or group mean±SD, unless otherwise indicated. A P value of <.05 was considered significant. The responder subgroup was defined as a PCO2 level more extreme than 2 SDs below the control group mean; the nonresponder subgroup was defined as a PCO2 level within 2 SDs of the control group mean.
      ANOVA, analysis of variance; SD, standard deviation.
      Usuda. A low-dose antenatal betamethasone regimen matures the fetal lung and minimizes hypothalamic-pituitary-adrenal axis disruption. Am J Obstet Gynecol 2022.
      Delivery values as a function of fetal sex were assessed for each group (Table 2). There was no significant difference in days of gestation, maternal weights, and fetal birthweights between male and female lambs in any of the 3 groups. There was no significant difference in response ratio between male and female lambs between the steroid treatment groups.
      Table 2Comparison of delivery data and responder ratio between male and female in the respective groups
      VariableDexamethasone (6 mg, 4 times)P valueBetamethasone (2 mg, 4 times)P valueSalineP valueTest
      MaleFemaleMaleFemaleMaleFemale
      Number1012138115
      Responder/nonresponder (responder ration in total number)7/3 (70)9/3 (75).79310/3 (76.9)6/2 (75).92Chi-square
      Gestational age124.1±0.7124.1±0.9.963123.8±0.8124.0±0.9.562123.7±0.7123.4±0.5.454Mann-Whitney U test
      Maternal weight67.6±6.567.9±5.8.92169.2±6.771.0±4.6.49867.3±5.874±6.7.092t test
      Fetal birthweight3.0±0.33.0±0.3.7062.9±0.43.0±0.3.3513.0±0.33.0±0.3.975t test
      Data are presented as number of responders/number of nonresponders (percentage) or group mean±SD, unless otherwise indicated. A P value of <.05 was considered significant. The responder subgroup was defined as a PCO2 level of <2 SDs of the control group mean, and the nonresponder subgroup was defined as a PCO2 level within 2 SDs of the control group mean.
      SD, standard deviation.
      Usuda. A low-dose antenatal betamethasone regimen matures the fetal lung and minimizes hypothalamic-pituitary-adrenal axis disruption. Am J Obstet Gynecol 2022.

      Arterial blood gas and respiratory physiological data under ventilation

      Arterial blood gas and respiratory physiological data at 30 minutes of ventilation was assessed (Figure 1, A–F). Arterial blood gas values for both the dexamethasone and the betamethasone groups of animals (Figure 1) were significantly different from those of the saline control group of animals. There was no significant difference in these values between the dexamethasone and the betamethasone groups of animals: pH (dexamethasone group vs saline control group: 0.29 [95% CI, 0.18–0.40; P<.001]; betamethasone group vs saline control group: 0.23 [95% CI, 0.12–0.34; P<.001]; dexamethasone group vs betamethasone group: P=.366), PO2 (dexamethasone group vs saline control group: 22.4 [95% CI, 9.4–35.1; P<.001], betamethasone group vs saline control group: 32.2 [95% CI, 19.7–45.0; P<.001]; dexamethasone group vs betamethasone group: P=.122), VEI (dexamethasone group vs saline control group: 0.018 [95% CI, 0.007–0.030; P=.001]; betamethasone group vs saline control group: 0.017 [95% CI, 0.006–0.029; P=.002]; dexamethasone group vs betamethasone group: P=.977), Cdyn (dexamethasone group vs saline control group: 0.11 [95% CI, 0.06–0.16; P<.001]; betamethasone group vs saline control group: 0.10 [95% CI, 0.05–0.16; P<.001]; dexamethasone group vs betamethasone group: P=.923), and V40 (dexamethasone group vs saline control group: 9.0 [95% CI, 1.7–14.6; P=.009]; betamethasone group vs saline control group: 9.3 [95% CI, 1.1–15.0; P=.018]; dexamethasone group vs betamethasone group: P=.969).
      Figure thumbnail gr1
      Figure 1Arterial blood gas and respiratory physiological data under ventilation
      A, Arterial pH. B, Arterial PO2. C, Arterial PCO2. D, Ventilation efficacy index. E, Dynamic compliance. F, Lung volume at 40 cmH2O. All values are presented as bar charts with the group mean and with whiskers representing standard deviation. Differences of values among the groups were tested for significance using 1-way analysis of variance or Kruskal-Wallis tests according to statistic distribution followed by Tukey tests as posthoc with P value of <.05 accepted as significant. † indicates P<.01. Asterisk indicates P<.05. Differences of values between male and female in the respective groups were tested for significance using t tests or Mann-Whitney U tests according to statistic distribution with P value of <.05 accepted as significant. The ventilation efficiency index is calculated as 3800/(RR×[PIP−PEEP]×PCO2 [mm Hg]).
      Beta, betamethasone; Dex, dexamethasone.
      Usuda. A low-dose antenatal betamethasone regimen matures the fetal lung and minimizes hypothalamic-pituitary-adrenal axis disruption. Am J Obstet Gynecol 2022.
      Although PCO2 values of both the dexamethasone and the betamethasone groups of animals were significantly lower than that of the saline control group of animals, there was no significant difference between those in the dexamethasone group of animals and those in the betamethasone group of animals (dexamethasone group vs saline control group: −65.5 [95% CI, −89.1 to −41.9; P<.001]; betamethasone group vs saline control group: −61.2 [95% CI, −85.0 to −37.4; P<.001]; dexamethasone group vs betamethasone group: P=.885).
      There was no significant difference in any of the following items between male and female lambs in the 2 steroid-treated groups: pH (dexamethasone group, P=.411; betamethasone group, P=.431; saline control group, P=.391), PO2 (dexamethasone group, P=.732; betamethasone group, P=.545; saline control group, P=.650), PCO2 (dexamethasone group, P=.574; betamethasone group, P=.651; saline control group, P=.753), VEI (dexamethasone group, P=.510; betamethasone group, P=.469; saline control group, P=.804), Cdyn (dexamethasone group, P=0.203; betamethasone group, P=.385; saline control group, P=.336), and V40 (dexamethasone group, P=.367; betamethasone group, P=.541; saline control group, P=.693).

      Messenger RNA quantitation of lung tissue

      Fold changes in the expression of lung maturation–associated transcripts in both the dexamethasone and the betamethasone groups of animals (Figure 2) were significantly higher than those of the saline control group of animals. There was no significant difference in any of the items between the dexamethasone and the betamethasone groups of animals: Sp-a (dexamethasone group vs saline control group: 3.37 [95% CI, 2.53–4.21; P<.001]; betamethasone group vs saline control group: 2.66 [95% CI, 1.81–3.51; P<.001]; dexamethasone group vs betamethasone group: P=.082), Sp-b (dexamethasone group vs saline control group: 1.87 [95% CI, 1.34–2.40; P<.001]; betamethasone group vs saline control group: 1.66 [95% CI, 1.11–2.19; P<.001]; dexamethasone group vs betamethasone group: P=.562), Sp-c (dexamethasone group vs saline control group: 2.08 [95% CI, 1.35–2.81; P<.001]; betamethasone group vs saline control group: 2.19 [95% CI, 1.46–2.93; P<.001]; dexamethasone group vs betamethasone group: P=.911), Sp-d (dexamethasone group vs saline control group: 0.46 [95% CI, 0.09–0.83; P=.012]; betamethasone vs saline control group: 0.52 [95% CI, 0.14–0.90; P=.004]; dexamethasone group vs betamethasone group: P=.909), Aqp-1 (dexamethasone group vs saline control group: 1.09 [95% CI, 0.64–1.55; P<.001]; betamethasone group vs saline control group: 0.66 [95% CI, 0.20–1.12; P=.003]; dexamethasone group vs betamethasone group: P=.054), Aqp-5 (dexamethasone group vs saline control group: 1.06 [95% CI, 0.69–1.48; P<.001]; betamethasone vs saline control group: 0.94 [95% CI, 0.54–1.34; P<.001]; dexamethasone group vs betamethasone group: P=.603), and Scnn1b (dexamethasone group vs saline control group: 1.70 [95% CI, 0.81–2.60; P<.001]; betamethasone vs saline control group: 1.86 [95% CI, 0.96–2.76; P<.001]; dexamethasone group vs betamethasone group: P=.889).
      Figure thumbnail gr2
      Figure 2Messenger RNA quantitation of lung tissue
      A, SP-A. B, SP-B. C, SP-C. D, SP-D. E, AQP-1. F, AQP-5. G, SCNN 1B. All values are presented as bar charts with the group mean and with whiskers representing standard deviation. Differences of values among the groups were tested for significance using 1-way analysis of variance or Kruskal-Wallis tests according to statistic distribution followed by Tukey tests as posthoc with P value of <.05 accepted as significant. Dagger indicates P<.01. Asterisk indicates P<.05. Differences of values between male and female in the respective groups were tested for significance using t tests or Mann-Whitney U tests according to statistic distribution with P value of <.05 accepted as significant.
      Beta, betamethasone; Dex, dexamethasone; SP, surfactant protein; AQP, aquaporin; SCNN 1B, sodium channel epithelial 1 subunit beta.
      Usuda. A low-dose antenatal betamethasone regimen matures the fetal lung and minimizes hypothalamic-pituitary-adrenal axis disruption. Am J Obstet Gynecol 2022.
      There was no significant difference in any of the assessed items between male and female lambs in any of the 3 groups: Sp-a (dexamethasone group: P=.950; betamethasone group: P=.562; saline control group: P=.126), Sp-B (dexamethasone group: P=.680; betamethasone group: P=.787; saline control group: P=.245), Sp-c (dexamethasone group: P=.169; betamethasone group: P=.426; saline control group: P=.983), Sp-d (dexamethasone group: P=.323; betamethasone group: P=.335; saline control group: P=.978), Aqp-1 (dexamethasone group: P=.065; betamethasone group: P=.562; saline control group: P=.462), Aqp-5 (dexamethasone group: P=.658; betamethasone group: P=.469; saline control group: P=.262), and Scnn1b (dexamethasone group: P=.333; betamethasone group: P=.311; saline control group: P=.975).

      Hematological and biochemical data

      Adrenocorticotropic hormone and cortisol concentrations of maternal plasma at delivery

      ACTH concentrations in the dexamethasone group of ewes were significantly lower than those of ewes in both the betamethasone and the saline control groups (Figure 3, A and B). ACTH concentrations in the betamethasone group of ewes were significantly lower than that of the saline control group of ewes (dexamethasone group vs saline control group; −497 [95% CI, −679 to −314]; P<.001; betamethasone group vs saline control group: −307 [95% CI, −491 to −123; P=.001]; dexamethasone group vs betamethasone group: −190 [95% CI, −359 to −21; P=.024]).
      Figure thumbnail gr3
      Figure 3Maternal and fetal plasma concentrations of ACTH, cortisol, and IGF-1
      A, Maternal ACTH. B, Maternal cortisol. C, Fetal ACTH. D, Fetal cortisol. E, Fetal IGF-1. All values are presented as bar charts with the group mean and with whiskers representing standard deviation. Differences of values among the groups were tested for significance using 1-way analysis of variance or Kruskal-Wallis tests according to statistic distribution followed by Tukey tests as posthoc with P value of <.05 accepted as significant. Dagger indicates P<.01. Asterisk indicates P<.05. Differences of values between male and female in the respective groups were tested for significance using t tests or Mann-Whitney U tests according to statistic distribution with P value of <.05 accepted as significant.
      ACTH, adrenocorticotropic hormone; Beta, betamethasone; Dex, dexamethasone; IGF-1, insulin-like growth factor 1.
      Usuda. A low-dose antenatal betamethasone regimen matures the fetal lung and minimizes hypothalamic-pituitary-adrenal axis disruption. Am J Obstet Gynecol 2022.
      Maternal cortisol concentrations in both the dexamethasone and the betamethasone group of ewes were significantly lower than that of the saline control group of ewes. There was no significant difference between those of the dexamethasone group of ewes and those of the betamethasone group of ewes (dexamethasone group vs saline control group: −103, [95% CI, −123 to −84; P<.001]; betamethasone group vs saline control group: −96, [95% CI, −116 to −76; P<.001]; dexamethasone group vs betamethasone group: P=.614).

      Adrenocorticotropic hormone, cortisol, and insulin-like growth factor 1 concentrations in fetal plasma at delivery

      Both ACTH and cortisol concentrations in the dexamethasone and betamethasone group of fetuses (Figure 3, C–E) were significantly lower than that of the saline group of animals. There was no significant difference between dexamethasone and betamethasone groups of fetuses for either ACTH (dexamethasone group vs saline control group: −363 [95% CI, −513 to −211; P<.001]; betamethasone group vs saline control group: −352 [95% CI, −505 to −200; P<.001]; dexamethasone group vs betamethasone group: P=.983) or cortisol (dexamethasone group vs saline control group: −7.5 [95% CI, −9.9 to −5.2; P<.001]; betamethasone group vs saline control group: −7.0 [95% CI, −9.3 to −4.6; P<.001]; dexamethasone group vs betamethasone group: P=.807).
      Fetal plasma IGF-1 concentrations in the dexamethasone group of fetuses were significantly lower than that of both the betamethasone and the saline control group of fetuses. There was no significant difference in fetal plasma IFG-1 levels between the betamethasone and the saline control groups of fetuses (dexamethasone group vs saline control group: −25.5 [95% CI, 49.2 to −1.8; P=.032]; betamethasone group vs saline control group: P=.202; dexamethasone group vs betamethasone group: −42.7 [95% CI, −64.6 to −20.7; P<.001]).
      There was no significant difference in any of the items assessed between male and female lambs across all 3 groups: ACTH concentration (dexamethasone group: P=.471; betamethasone group: P=.266; saline control group: P=.562), cortisol concentration (dexamethasone group: P=.998; betamethasone group: P=.911; saline control group: P=.446), and IGF-1 concentration (dexamethasone group: P=.171; betamethasone group: P=.880: saline control group: P=.412).

      Maternal hematological data at delivery

      There was no significant intergroup difference in total WBC counts (dexamethasone group vs saline control group: P=.876; betamethasone group vs saline control group: P=.998; dexamethasone group vs betamethasone group: P=.887) (Figure 4, A and B).
      Figure thumbnail gr4
      Figure 4Maternal and fetal hematological data at delivery
      A, Maternal WBC counts. B, Maternal neutrophil percentage. C, Fetal WBC counts. D, Fetal neutrophil percentage. All values are presented as bar charts with the group mean and with whiskers representing standard deviation. The yellow bars indicate the Dex group. The green bars indicate the Beta group. The blue bars indicate the saline control group. Differences of values among the groups were tested for significance using 1-way analysis of variance or Kruskal-Wallis tests according to statistic distribution followed by Tukey tests as posthoc with P value of <.05 accepted as significant. Dagger indicates P<.01. Asterisk indicates P<.05. Differences of values between male and female in respective group were tested for significance using t tests or Mann-Whitney U tests according to statistic distribution with P value of <.05 accepted as significant.
      Beta, betamethasone; Dex, dexamethasone; WBC, white blood cell.
      Usuda. A low-dose antenatal betamethasone regimen matures the fetal lung and minimizes hypothalamic-pituitary-adrenal axis disruption. Am J Obstet Gynecol 2022.
      Neutrophils in the dexamethasone group of ewes were significantly higher than that of both the betamethasone and the saline control group of ewes. There was no significant difference in neutrophil counts between the betamethasone and saline control groups of ewes (dexamethasone group vs saline control group: 22.0 [95% CI, 10.3–33.7; P<.001]; betamethasone group vs saline control group: P=.118; dexamethasone group vs betamethasone group: 12.1 [95% CI, 1.2–22.9; P=.026]).

      Fetal hematological data at delivery

      There was no significant difference among any group of fetuses in total WBC counts (dexamethasone group vs saline control group: P=.415; betamethasone group vs saline control group: P=.824; dexamethasone group vs betamethasone control group: P=.747) (Figure 4, C and D).
      Neutrophils in the dexamethasone group of fetuses were significantly higher than those in both the betamethasone and saline control group of fetuses. Neutrophils in the betamethasone group of fetuses were significantly higher than those in the saline control group of fetuses (dexamethasone group vs saline control group: 38.3 [95% CI, 28.0–48.7; P<.001]; betamethasone group vs saline control group: 22.4 [95% CI, 11.9–32.9; P<.001]; dexamethasone group vs betamethasone group: 15.9 [95% CI, 6.3–25.6; P=.001]). There was no significant difference between male and female fetuses in all groups for total WBC counts (dexamethasone group: P=.190; betamethasone group: P=.701; saline control group: P=.261) and neutrophil (dexamethasone group: P=.529; betamethasone group: P=.659; saline control group: P=.428).

      Glucose levels in maternal plasma

      Maternal glucose levels in the dexamethasone group of animals were significantly higher than those in both the betamethasone and the saline control group of animals (Figure 5, A). There was no significant difference between glucose levels in the betamethasone and saline control groups of ewes (dexamethasone group vs saline control group: 28.6 [95% CI, 14.9–42.3; P<.001]; betamethasone group vs saline control group: P=.058; dexamethasone group vs betamethasone group: 15.1 [95% CI, 2.4–27.8; P=.016]).
      Figure thumbnail gr5
      Figure 5Maternal plasma glucose level and fetal plasma concentrations of glucose and C-peptide
      A, Maternal glucose level. B, Fetal glucose level. C, Fetal C-peptide concentration. All values are presented as bar charts with the group mean and with whiskers representing standard deviation. Differences of values among the groups were tested for significance using 1-way analysis of variance or Kruskal-Wallis tests according to statistic distribution followed by Tukey tests as posthoc with P value of <.05 accepted as significant. Dagger indicates P<.01. Asterisk indicates P<.05. Differences of values between male and female in the respective groups were tested for significance using t tests or Mann-Whitney U tests according to statistic distribution with P value of <.05 accepted as significant.
      Beta, betamethasone; Dex, dexamethasone.
      Usuda. A low-dose antenatal betamethasone regimen matures the fetal lung and minimizes hypothalamic-pituitary-adrenal axis disruption. Am J Obstet Gynecol 2022.

      Glucose and C-peptide concentrations in fetal plasma at delivery

      Fetal glucose levels in the dexamethasone group of fetuses were significantly higher than those in both the betamethasone and the saline control group of fetuses (Figure 5, B and C). Plasma glucose concentrations in the betamethasone group of fetuses were also significantly higher than those in the saline control group of fetuses (dexamethasone group vs saline control group: 20.4 [95% CI, 14.0–26.7; P<.001]; betamethasone group vs saline control group: 11.5 [95% CI, 5.1–17.9; P<.001]; dexamethasone group vs betamethasone group: 8.9 [95% CI, 3.0–14.8; P=.002]).
      Fetal C-peptide concentrations in the dexamethasone group of fetuses were significantly higher than those in both the betamethasone and saline control group of fetuses. There was no significant difference in C-peptide levels between the betamethasone and the saline control groups of fetuses (dexamethasone group vs saline control group: 0.021 [95% CI, 0.006–0.037; P=.004]; betamethasone group vs saline control group: P=.606; dexamethasone group vs betamethasone group: 0.028 [95% CI, 0.013–0.042; P<.001]). There was no significant difference between male and female fetal glucose concentrations (dexamethasone group: P=.274; betamethasone group: P=.923; saline control group: P=.531) or C-peptide concentrations (dexamethasone group: P=.889; betamethasone group: P=.306; saline control group: P=.782) among groups.

      Correlations with maternal weight in the dexamethasone group of animals

      Fetal arterial PCO2, PO2, maternal ACTH, fetal ACTH, maternal neutrophils, fetal neutrophils, maternal glucose values, and fetal glucose values were calculated to assess the relationship with maternal weight in the dexamethasone group (data not shown). There was no significant correlation with any of PCO2 (r=−0.07; P=.75), PO2 (r=−0.32; P=.150), maternal ACTH (r=0.13; P=.558), fetal ACTH (r=.15; P=.519), maternal neutrophil (r=−0.07; P=.766), fetal neutrophil (r=−0.08; P=.716), maternal glucose value (r=−0.25; P=.256), and fetal glucose value (r=−0.04; P=.868).

      Correlations with maternal weight in the betamethasone group of animals

      Fetal arterial PCO2, PO2, maternal ACTH, fetal ACTH, maternal neutrophils, fetal neutrophils, maternal glucose values, and fetal glucose values were calculated to assess the relationship with maternal weight in the betamethasone group (Figure 6, A–G). There were significant correlations with fetal ACTH (r=0.53; P=.013), maternal glucose value (r=−0.52; P=.015), and fetal glucose value (r=−0.42; P=.048), whereas there was no significant correlation with PCO2 (r=0.40; P=.071), PO2 (r=−0.10; P=.656), maternal ACTH (r=0.39; P=.081), maternal neutrophil (r=−0.27; P=.238), and fetal neutrophil (r=−0.38; P=.093).
      Figure thumbnail gr6
      Figure 6Correlations with maternal weight in the betamethasone group of animals
      A, Fetal ACTH and maternal weight. B, Maternal glucose and maternal weight. C, Fetal glucose and maternal weight.
      ACTH, adrenocorticotropic hormone.
      Usuda. A low-dose antenatal betamethasone regimen matures the fetal lung and minimizes hypothalamic-pituitary-adrenal axis disruption. Am J Obstet Gynecol 2022.

      Comment

      Principal findings

      The primary findings of this study were as follows: (1) an antenatal steroid regimen based on four 2-mg doses of Beta-P given at 12-hour intervals (a total of 8 mg) achieved functional maturation of the ovine preterm lung equivalent to that of four 6-mg doses of Dex-P, total 24 mg (Table 1; Figures 1 and 2); (2) relative to standard-dose dexamethasone treatment, lower-dose betamethasone treatment reduced the scope and magnitude of potentially important disruption to HPA axis signaling and circulating immunocyte populations (Figure 3, Figure 4, Figure 5, Figure 6); and (3) neither of the 2 dosing regimens employed elicited differential effects because of the sex of the fetus. It is important to note that the regimens administered to the conservative (Dex-P) and experimental (Beta-P) groups in the present study had quite distinct pharmacokinetic profiles from that of the current standard of care (combined Beta-P and Beta-Ac) used in Australia, the United States, and much of Europe. As such, additional comparative studies against this regimen are warranted in the future.

      Clinical implications—functional lung maturation

      Fetal pulmonary oxygenation (pO2), diffusion capacity (pCO2 and VEI), compliance, and lung capacity (Cdyn and V40) were equivalent between the steroid-treatment groups. There was no difference in the treatment response rate (Figure 1). The expressions of mRNA markers of lung maturation were also equivalent between the 2 steroid-treatment groups (Figure 2).
      • Rosemarie C.T.
      • Machiko I.
      • Alan H.J.
      • Li Yuan Y.
      • Fred P.
      • Philip L.B.
      Developmental and glucocorticoid regulation of surfactant protein mRNAs in preterm lambs.
      ,
      • Schmidt A.F.
      • Jobe A.H.
      • Kannan P.S.
      • et al.
      Oral antenatal corticosteroids evaluated in fetal sheep.
      Overall, these findings demonstrated that the 2 treatments tested were equivalent in terms of enhancing preterm lung function in the sheep; for deliveries occurring 48 hours after treatment initiation, we concluded that a substantially lower dose of Beta-P may be used in place of a standard-dose of Dex-P without any discernable difference in treatment efficacy. For future studies, it would be of interest to explore the impact of a longer treatment-to-delivery interval on lung function outcomes.

      Clinical implications—suppression of hypothalamic-pituitary-adrenal axis

      Both the maternal and the fetal HPA axes were suppressed by ACS treatments (Figure 3). This is perhaps not unexpected given that the biological half-life of Dex-P and Beta-P (which relates to its effect on the HPA axis) is 36 to 54 hours and recovery time from suppression of HPA is approximately 60 to 72 hours in the human
      • Jobe A.H.
      • Milad M.A.
      • Peppard T.
      • Jusko W.J.
      Pharmacokinetics and pharmacodynamics of intramuscular and oral betamethasone and dexamethasone in reproductive age women in India.
      • Jobe A.H.
      • Kemp M.
      • Schmidt A.
      • Takahashi T.
      • Newnham J.
      • Milad M.
      Antenatal corticosteroids: a reappraisal of the drug formulation and dose.
      • Schmidt A.F.
      • Kemp M.W.
      • Rittenschober-Böhm J.
      • et al.
      Low-dose betamethasone-acetate for fetal lung maturation in preterm sheep.
      and that even the dose of glucocorticoid used in the betamethasone group was still sizable and within a range of 7.5 to 30.0 mg prednisone equivalent a day if viewed as a conventional anti-inflammatory dose.
      • Becker D.E.
      Basic and clinical pharmacology of glucocorticosteroids.
      ,
      • Buttgereit F.
      • Da Silva J.A.
      • Boers M.
      • et al.
      Standardised nomenclature for glucocorticoid dosages and glucocorticoid treatment regimens: current questions and tentative answers in rheumatology.
      Nevertheless, the use of a low-dose betamethasone regimen resulted in less perturbation of maternal ACTH concentrations than the use of the standard-dose Dex-P treatment modeled on the current WHO-recommended regimen. Similar improvements were seen in the fetal HPA axis and IGF-1 concentrations in the betamethasone group of lambs. It is reasonable to conclude that an ANS therapy that minimizes as much as possible the impact on the HPA axis would be desirable.

      Clinical implications—immunosuppression

      Glucocorticoids are known to increase the circulating WBC count acutely after administration.
      • Nakagawa M.
      • Terashima T.
      • D’Yachkova Y.
      • Bondy G.P.
      • Hogg J.C.
      • van Eeden S.F.
      Glucocorticoid-induced granulocytosis: contribution of marrow release and demargination of intravascular granulocytes.
      • Burton J.L.
      • Kehrli Jr., M.E.
      • Kapil S.
      • Horst R.L.
      Regulation of L-selectin and CD18 on bovine neutrophils by glucocorticoids: effects of cortisol and dexamethasone.
      • Waisman D.
      • Van Eeden S.F.
      • Hogg J.C.
      • Solimano A.
      • Massing B.
      • Bondy G.P.
      L-selectin expression on polymorphonuclear leukocytes and monocytes in premature infants: reduced expression after dexamethasone treatment for bronchopulmonary dysplasia.
      • Weber P.S.
      • Toelboell T.
      • Chang L.C.
      • et al.
      Mechanisms of glucocorticoid-induced down-regulation of neutrophil L-selectin in cattle: evidence for effects at the gene-expression level and primarily on blood neutrophils.
      • Cox G.
      Glucocorticoid treatment inhibits apoptosis in human neutrophils. Separation of survival and activation outcomes.
      • Haslett C.
      • Lee A.
      • Savill J.S.
      • Meagher L.
      • Whyte M.K.
      Apoptosis (programmed cell death) and functional changes in aging neutrophils. Modulation by inflammatory mediators.
      The increase in circulating WBC count predominantly reflects an increase in circulating neutrophils. The biological effects that contribute to this increase in circulating neutrophils include demargination of neutrophils from the endovascular lining (approximately 60%) and delayed migration of neutrophils into tissue and a slower rate of apoptosis (approximately 30%).
      • Nakagawa M.
      • Terashima T.
      • D’Yachkova Y.
      • Bondy G.P.
      • Hogg J.C.
      • van Eeden S.F.
      Glucocorticoid-induced granulocytosis: contribution of marrow release and demargination of intravascular granulocytes.
      • Burton J.L.
      • Kehrli Jr., M.E.
      • Kapil S.
      • Horst R.L.
      Regulation of L-selectin and CD18 on bovine neutrophils by glucocorticoids: effects of cortisol and dexamethasone.
      • Waisman D.
      • Van Eeden S.F.
      • Hogg J.C.
      • Solimano A.
      • Massing B.
      • Bondy G.P.
      L-selectin expression on polymorphonuclear leukocytes and monocytes in premature infants: reduced expression after dexamethasone treatment for bronchopulmonary dysplasia.
      • Weber P.S.
      • Toelboell T.
      • Chang L.C.
      • et al.
      Mechanisms of glucocorticoid-induced down-regulation of neutrophil L-selectin in cattle: evidence for effects at the gene-expression level and primarily on blood neutrophils.
      • Cox G.
      Glucocorticoid treatment inhibits apoptosis in human neutrophils. Separation of survival and activation outcomes.
      • Haslett C.
      • Lee A.
      • Savill J.S.
      • Meagher L.
      • Whyte M.K.
      Apoptosis (programmed cell death) and functional changes in aging neutrophils. Modulation by inflammatory mediators.
      These effects contribute to the action of glucocorticoids in inducing immunosuppressive effects at inflamed sites.
      • Nakagawa M.
      • Terashima T.
      • D’Yachkova Y.
      • Bondy G.P.
      • Hogg J.C.
      • van Eeden S.F.
      Glucocorticoid-induced granulocytosis: contribution of marrow release and demargination of intravascular granulocytes.
      • Burton J.L.
      • Kehrli Jr., M.E.
      • Kapil S.
      • Horst R.L.
      Regulation of L-selectin and CD18 on bovine neutrophils by glucocorticoids: effects of cortisol and dexamethasone.
      • Waisman D.
      • Van Eeden S.F.
      • Hogg J.C.
      • Solimano A.
      • Massing B.
      • Bondy G.P.
      L-selectin expression on polymorphonuclear leukocytes and monocytes in premature infants: reduced expression after dexamethasone treatment for bronchopulmonary dysplasia.
      • Weber P.S.
      • Toelboell T.
      • Chang L.C.
      • et al.
      Mechanisms of glucocorticoid-induced down-regulation of neutrophil L-selectin in cattle: evidence for effects at the gene-expression level and primarily on blood neutrophils.
      • Cox G.
      Glucocorticoid treatment inhibits apoptosis in human neutrophils. Separation of survival and activation outcomes.
      • Haslett C.
      • Lee A.
      • Savill J.S.
      • Meagher L.
      • Whyte M.K.
      Apoptosis (programmed cell death) and functional changes in aging neutrophils. Modulation by inflammatory mediators.
      Here, a low-dose betamethasone regimen blunted the elevation of neutrophils compared with the dexamethasone regimen (Figure 4, B and D). The data suggested that the use of a lower-dose ACS regimen might ameliorate the immunosuppressive effects associated with current regimens, resulting in a reduced risk of infection after ACS treatments—a risk of concern in LMICs.
      • Althabe F.
      • Belizán J.M.
      • McClure E.M.
      • et al.
      A population-based, multifaceted strategy to implement antenatal corticosteroid treatment versus standard care for the reduction of neonatal mortality due to preterm birth in low-income and middle-income countries: the ACT cluster-randomised trial.
      ,
      • Althabe F.
      • Thorsten V.
      • Klein K.
      • et al.
      The Antenatal Corticosteroids Trial (ACT)’s explanations for neonatal mortality - a secondary analysis.

      Clinical implications—fetal hypoglycemia

      The association of ACS treatments with neonatal hypoglycemia, especially for late preterm infants (34–36 weeks of gestation in humans) is an issue of considerable contemporary interest.
      • Althabe F.
      • Belizán J.M.
      • McClure E.M.
      • et al.
      A population-based, multifaceted strategy to implement antenatal corticosteroid treatment versus standard care for the reduction of neonatal mortality due to preterm birth in low-income and middle-income countries: the ACT cluster-randomised trial.
      ,
      • Althabe F.
      • Thorsten V.
      • Klein K.
      • et al.
      The Antenatal Corticosteroids Trial (ACT)’s explanations for neonatal mortality - a secondary analysis.
      ,
      • Deshmukh M.
      • Patole S.
      Antenatal corticosteroids for impending late preterm (34-36+6 weeks) deliveries-a systematic review and meta-analysis of RCTs.
      Maternal hyperglycemia leads to fetal hyperglycemia, resulting in fetal pancreatic beta-cell hyperplasia and/or fetal hyperinsulinemia (elevation of plasma C-peptide), resulting in subsequent neonatal hypoglycemia after delivery.
      • Pettit K.E.
      • Tran S.H.
      • Lee E.
      • Caughey A.B.
      The association of antenatal corticosteroids with neonatal hypoglycemia and hyperbilirubinemia.
      • Sifianou P.
      • Thanou V.
      • Karga H.
      Metabolic and hormonal effects of antenatal betamethasone after 35 weeks of gestation.
      • Tuohy J.F.
      • Bloomfield F.H.
      • Crowther C.A.
      • Harding J.E.
      Maternal and neonatal glycaemic control after antenatal corticosteroid administration in women with diabetes in pregnancy: a retrospective cohort study.
      Here, animals in the low-dose betamethasone group had reduced elevations of maternal glucose, fetal glucose, and C-peptide compared with those treated with standard doses of dexamethasone (Figure 5, A–C). These data suggested that assuming a similar degree of sensitivity between sheep and humans, a lower-dose ACS regimen might reduce the risk of steroid-induced fetal hypoglycemia after birth.

      Clinical implications—fetal growth restriction

      Preclinical and clinical pieces of evidence demonstrate that ACS treatments may exacerbate growth restriction and that an inverse relationship has been demonstrated between the number of corticosteroid courses and fetal growth.
      • Romejko-Wolniewicz E.
      • Teliga-Czajkowska J.
      • Czajkowski K.
      Antenatal steroids: can we optimize the dose?.
      Moreover, IGF-1 plays central roles in normal fetal growth, stimulating fetal cell proliferation, differentiation, and protein and glycogen syntheses, and it has been noted that reduced serum IGF-1 is correlated with reduced fetal growth and brain development.
      • Owens J.A.
      Endocrine and substrate control of fetal growth: placental and maternal influences and insulin-like growth factors.
      • Agrogiannis G.D.
      • Sifakis S.
      • Patsouris E.S.
      • Konstantinidou A.E.
      Insulin-like growth factors in embryonic and fetal growth and skeletal development (review).
      • Netchine I.
      • Azzi S.
      • Le Bouc Y.
      • Savage M.O.
      IGF1 molecular anomalies demonstrate its critical role in fetal, postnatal growth and brain development.
      • Joseph D’Ercole A.
      • Ye P.
      Expanding the mind: insulin-like growth factor I and brain development.
      In contrast to the postnatal situation, the initial driver of fetal IGF-1 regulator is not only growth hormone
      • Gluckman P.D.
      • Gunn A.J.
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      • et al.
      Congenital idiopathic growth hormone deficiency associated with prenatal and early postnatal growth failure. The International Board of the Kabi Pharmacia International Growth Study.
      • Klempt M.
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      • Breier B.H.
      • Baumbach W.R.
      • Gluckman P.D.
      Tissue distribution and ontogeny of growth hormone receptor messenger ribonucleic acid and ligand binding to hepatic tissue in the midgestation sheep fetus.
      • Breier B.H.
      • Ambler G.R.
      • Sauerwein H.
      • Surus A.
      • Gluckman P.D.
      The induction of hepatic somatotrophic receptors after birth in sheep is dependent on parturition-associated mechanisms.
      but also fetal insulin, which, in turn, is predominantly under regulation by fetal glucose availability.
      • Oliver M.H.
      • Harding J.E.
      • Breier B.H.
      • Gluckman P.D.
      Fetal insulin-like growth factor (IGF)-I and IGF-II are regulated differently by glucose or insulin in the sheep fetus.
      ,
      • Oliver M.H.
      • Harding J.E.
      • Breier B.H.
      • Evans P.C.
      • Gluckman P.D.
      Glucose but not a mixed amino acid infusion regulates plasma insulin-like growth factor-I concentrations in fetal sheep.
      It is also reported that the IGF-1 levels of fetuses exposed to synthetic corticosteroids are likely to be reduced.
      • Sifianou P.
      • Thanou V.
      • Karga H.
      Metabolic and hormonal effects of antenatal betamethasone after 35 weeks of gestation.
      ,
      • Verhaeghe J.
      • Vanstapel F.
      • Van Bree R.
      • Van Herck E.
      • Coopmans W.
      Transient catabolic state with reduced IGF-I after antenatal glucocorticoids.
      Although the underlying mechanisms of growth restriction because of ACS treatments are still unclear, the IGF-1 system and glucose metabolism are possibly implicated. Here, although there was no difference in birthweights among the groups, fetal plasma IGF-1 levels were reduced in the standard-dose dexamethasone group, but not in the lower-dose betamethasone group (Figure 3, E). Any regimen that conveys a reduced risk of even transient reductions in fetal weight or growth would seem to be a significant improvement over the existing treatments.

      Clinical implications—sex effects

      Although the influence of ACS and sex on fetus is still controversial,
      • Jain A.
      • Rutter N.
      • Cartlidge P.H.
      Influence of antenatal steroids and sex on maturation of the epidermal barrier in the preterm infant.
      • Willet K.E.
      • Jobe A.H.
      • Ikegami M.
      • et al.
      Postnatal lung function after prenatal steroid treatment in sheep: effect of gender.
      • Ramos-Navarro C.
      • Sánchez-Luna M.
      • Zeballos-Sarrato S.
      • Pescador-Chamorro I.
      Antenatal corticosteroids and the influence of sex on morbidity and mortality of preterm infants.
      there was no sex difference identified in baseline fetal lung function and maternal and fetal hematological, biochemical, and endocrinological responses in either treatment regimen. This is an important observation, as it suggests that reducing the dose of steroids used in antenatal regimens does not increase the risk of reduced efficacy as a function of fetal sex.

      Research implications

      The current study raised several important questions for further research efforts in this area. Firstly, it is important to note that the ratio of the maternal-fetal steroid gradient in humans is approximately 3:1, whereas it is approximately 10:1 in sheep.
      • Kemp M.W.
      • Saito M.
      • Usuda H.
      • et al.
      The efficacy of antenatal steroid therapy is dependent on the duration of low-concentration fetal exposure: evidence from a sheep model of pregnancy.
      ,
      • Ballard P.L.
      • Ballard R.A.
      Scientific basis and therapeutic regimens for use of antenatal glucocorticoids.
      Assuming a broadly equivalent degree of responsiveness to dexamethasone or betamethasone stimulation between the ovine and the human lungs, these findings suggested that a treatment regimen using further reduced doses of Beta-P would likely be effective when used in humans. Additional studies in nonhuman primates to confirm these findings would make a valuable addition to our understanding of antenatal steroid dosing. There is a wealth of data to demonstrate that, broadly speaking, adverse effects concerning steroid therapy are dose dependent.
      • Buttgereit F.
      • Da Silva J.A.
      • Boers M.
      • et al.
      Standardised nomenclature for glucocorticoid dosages and glucocorticoid treatment regimens: current questions and tentative answers in rheumatology.
      ,
      • Andrews M.H.
      • Wood S.A.
      • Windle R.J.
      • Lightman S.L.
      • Ingram C.D.
      Acute glucocorticoid administration rapidly suppresses basal and stress-induced hypothalamo-pituitary-adrenal axis activity.
      As such, in the context of treating the developing fetus, it is important to design a treatment regimen employing the lowest dose of agent possible to reliably induce lasting maturation of the preterm lung.
      Several “at-risk” pregnancies remain undelivered more than 7 days after ACS treatment and are viewed at elevated risk of adverse outcomes relative to age-matched, untreated peers. In addition, recent data have highlighted the potential for increased harm in off-target use of antenatal steroids.
      • Althabe F.
      • Belizán J.M.
      • McClure E.M.
      • et al.
      A population-based, multifaceted strategy to implement antenatal corticosteroid treatment versus standard care for the reduction of neonatal mortality due to preterm birth in low-income and middle-income countries: the ACT cluster-randomised trial.
      ,
      • Althabe F.
      • Thorsten V.
      • Klein K.
      • et al.
      The Antenatal Corticosteroids Trial (ACT)’s explanations for neonatal mortality - a secondary analysis.
      It would be of particular interest to determine the durability of the 2 steroid treatments tested in this protocol at extended treatment to delivery intervals (ie, 7 days and 14 days after the first dose)—again to identify the optimal intersection of the lowest efficacious dose and the longest possible period of benefit.
      Determining if it is possible to personalize antenatal steroid dosing based on maternal weight may also offer the potential to improve treatment efficacy. In the current study, the distribution of ACS-treated maternal weights was 56.0 to 81.6 kg. There was no correlation between HPA axis markers and maternal weight in the dexamethasone group. It is tempting to speculate that this derives from the higher dexamethasone dose remaining above a threshold or plateau value for reduced suppression over the material weight range (ie, even at the heaviest maternal weight, the drug dose was still large enough to suppress HPA function). This concept is supported by the fact that there was an inverse correlation between maternal weight and HPA axis disruption in the much lower-dose betamethasone group—with lighter maternal weights (and thus higher milligrams of steroid and kilograms of maternal weight doses) correlating with greater glucose disruption (Figure 6).
      Low-dose ACS regimens may alleviate suppressive effects on the HPA axis. Data from human studies show that current ACS treatments reduce fetal and infant HPA activities.
      • Tegethoff M.
      • Pryce C.
      • Meinlschmidt G.
      Effects of intrauterine exposure to synthetic glucocorticoids on fetal, newborn, and infant hypothalamic-pituitary-adrenal axis function in humans: a systematic review.
      ,
      • Niwa F.
      • Kawai M.
      • Kanazawa H.
      • et al.
      Limited response to CRH stimulation tests at 2 weeks of age in preterm infants born at less than 30 weeks of gestational age.
      Although reduced basal HPA function seems to recover within the first 2 weeks after delivery, there is preliminary evidence that blunted HPA axis reactivity to pain-related stress persists throughout the first 4 months of life.
      • Tegethoff M.
      • Pryce C.
      • Meinlschmidt G.
      Effects of intrauterine exposure to synthetic glucocorticoids on fetal, newborn, and infant hypothalamic-pituitary-adrenal axis function in humans: a systematic review.
      ,
      • Niwa F.
      • Kawai M.
      • Kanazawa H.
      • et al.
      Limited response to CRH stimulation tests at 2 weeks of age in preterm infants born at less than 30 weeks of gestational age.
      Thus, evaluating fetal recovery from HPA suppression and the potential differences deriving from low-dose vs standard-dose (ie, contemporary) ACS treatments will be an important line of future investigation.

      Limitations

      Some limitations should be considered when assessing the translatability of these data. Although the sheep is an excellent translational model to study ACS therapy and its effect on lung maturation and fetal development, it should be noted that there are several differences between sheep and humans, including length of gestation, maternal-fetal steroid pharmacokinetics, and distribution of maternal weight between human and sheep.,
      • Ballard P.L.
      • Ballard R.A.
      Scientific basis and therapeutic regimens for use of antenatal glucocorticoids.
      Furthermore, the functional lung assessment reported in this study was limited to 30 minutes after delivery. Although this is an adequate period to assess functional lung maturation, it might not necessarily be sufficient to assess interactions between ACS use and complications, such as chronic lung injury in preterm babies, or adverse effects, such as fetal hypoglycemia and blunted adrenal function. Moreover, the treatment to delivery intervals and GAs were tightly controlled in this study—a significant difference from the clinical setting of ACS use.

      Conclusion

      We reported that in a sheep model of pregnancy, a low-dose treatment regimen of four 2-mg betamethasone given in equal parts in a 36-hour period achieves lung maturation equivalent to that of a four 6-mg dexamethasone-based regimen, but with smaller perturbations to the maternofetal HPA axis. Further optimization of lower-dose treatments using betamethasone might be effective in driving fetal lung maturation with a reduced risk of maternal and fetal adverse effects. At a standardized 48-hour treatment to delivery interval (and setting aside drug cost and availability), a low-dose Beta-P treatment regimen should be favored vs the current standard-dose Dex-P regimen.
      ACS: antenatal corticosteroid
      ACTH: adrenocorticotropic hormone
      Beta-P: betamethasone phosphate
      Cdyn: dynamic compliance
      Dex-P: dexamethasone phosphate
      Glucocorticoids: synthetic steroid hormones that exert pleiotropic signaling activity via activation of the glucocorticoid receptor and via non-genomic signaling. Widely used in obstetrics to precociously mature the preterm lung in anticipation of preterm delivery.
      IGF-1: insulin-like growth factor 1
      IM: intramuscular
      mRNA: messenger RNA
      PCR: polymerase chain reaction
      PEEP: positive end-expiratory pressure
      PIP: peak inspiratory pressure
      RDS: respiratory distress syndrome
      VEI: ventilation efficiency index
      VT: tidal volume
      WBC: white blood cell
      WHO: World Health Organization

      Supplementary Data

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