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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

Open AccessPublished:October 06, 2021DOI:https://doi.org/10.1016/j.ajog.2021.10.001

      Background

      Antenatal corticosteroid therapy is a standard of care for women at imminent risk of preterm labor. However, the optimal (maximum benefit and minimal risk of side effects) antenatal corticosteroid dosing strategy remains unclear. Although conveying overall benefit when given to the right patient at the right time, antenatal corticosteroid treatment efficacy is highly variable and is not risk-free. Building on earlier findings, we hypothesized that when administered in combination with slow-release betamethasone acetate, betamethasone phosphate and the high maternal-fetal betamethasone concentrations it generates are redundant for fetal lung maturation.

      Objective

      Using an established sheep model of prematurity and postnatal ventilation of the preterm lamb, we aimed to compare the pharmacodynamic effects of low-dosage treatment with betamethasone acetate only against a standard dosage of betamethasone phosphate and betamethasone acetate as recommended by the American College of Obstetricians and Gynecologists for women at risk of imminent preterm delivery between 24 0/7 and 35 6/7 weeks’ gestation.

      Study Design

      Ewes carrying a single fetus at 122±1 days’ gestation (term=150 days) were randomized to receive either (1) maternal intramuscular injections of sterile saline (the saline negative control group, n=12), (2) 2 maternal intramuscular injections of 0.25 mg/kg betamethasone phosphate+betamethasone acetate administered at 24-hour dosing intervals (the betamethasone phosphate+betamethasone acetate group, n=12); or (3) 2 maternal intramuscular injections of 0.125 mg/kg betamethasone acetate administered at 24-hour dosing intervals (the betamethasone acetate group, n=11). The fetuses were surgically delivered 48 hours after treatment initiation and ventilated for 30 minutes to determine functional lung maturation. The fetuses were euthanized after ventilation, and the lungs were collected for analysis using quantitative polymerase chain reaction and Western blot assays. Fetal plasma adrenocorticotropic hormone levels were measured in the cord blood samples taken at delivery.

      Results

      Preterm lambs were defined as either antenatal corticosteroid treatment responders or nonresponders using an arbitrary cutoff, being a PaCO2 level at 30 minutes of ventilation being more extreme than 2 standard deviations from the mean value of the normally distributed saline control group values. Compared with the animals in the saline control group, the animals in the antenatal corticosteroid treatment groups showed significantly improved lung physiological responses (blood gas and ventilation data) and had a biochemical signature (messenger RNA and surfactant protein assays) consistent with functional maturation. However, the betamethasone acetate group had a significantly higher treatment response rate than the betamethasone phosphate+betamethasone acetate group. These physiological results were strongly correlated to the amount of surfactant protein A. Birthweight was lower in the betamethasone phosphate+betamethasone acetate group and the fetal hypothalamic-pituitary-adrenal axis was suppressed to a greater extent in the betamethasone phosphate+betamethasone acetate group.

      Conclusion

      Low-dosage antenatal corticosteroid therapy solely employing betamethasone acetate was sufficient for fetal lung maturation. The elevated maternal-fetal betamethasone concentrations associated with the coadministration of betamethasone phosphate did not in addition improve lung maturation but were associated with greater fetal hypothalamic-pituitary-adrenal axis suppression, a lower antenatal corticosteroid treatment response rate, and lower birthweight—outcomes not desirable in a clinical setting. These data warranted a clinical investigation of sustained low-dosage antenatal corticosteroid treatments that avoid high maternal-fetal betamethasone exposures.

      Key words

      Introduction

      One of the most pressing challenges facing preterm neonates (born before 37 weeks’ gestation) is the transition to breathing room air. As such, perinatal care is significantly focused on improving preterm lung function.
      • Blencowe H.
      • Cousens S.
      • Chou D.
      • et al.
      Born too soon: the global epidemiology of 15 million preterm births.
      ,
      • Liu L.
      • Johnson H.L.
      • Cousens S.
      • et al.
      Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000.
      The introduction of antenatal corticosteroid (ACS) therapy, after Liggins and Howie reported the beneficial effects of ACS in their landmark 1972 paper, has resulted in an improved prognosis for a large cross-section of preterm-born neonates.
      • Liggins G.C.
      • Howie R.N.
      A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants.
      Today, ACS therapy is the standard of care for women at risk of imminent preterm delivery. The American College of Obstetricians and Gynecologists recommends the use of a single course of ACS for pregnant women between 24 to 33 weeks’ gestation who are at risk of preterm labor within 7 days.
      Committee on Obstetric Practice
      Committee Opinion No. 713: antenatal corticosteroid therapy for fetal maturation.
      Although ACS therapy has been used widely, and benefits demonstrated when given to the right women at the right time, concerns remain regarding an increased potential risk of adverse effects, including fetal growth restriction, neonatal hypoglycemia, and negative effects on the maternal and fetal hypothalamic-pituitary-adrenal (HPA) axis.
      • French N.P.
      • Hagan R.
      • Evans S.F.
      • Godfrey M.
      • Newnham J.P.
      Repeated antenatal corticosteroids: size at birth and subsequent development.
      • Murphy K.E.
      • Hannah M.E.
      • Willan A.R.
      • et al.
      Multiple courses of antenatal corticosteroids for preterm birth (MACS): a randomised controlled trial.
      • Murphy K.E.
      • Willan A.R.
      • Hannah M.E.
      • et al.
      Effect of antenatal corticosteroids on fetal growth and gestational age at birth.
      • Alexander N.
      • Rosenlöcher F.
      • Stalder T.
      • et al.
      Impact of antenatal synthetic glucocorticoid exposure on endocrine stress reactivity in term-born children.
      In addition, ACS efficacy per se has been shown to vary among individuals in similar environments. Several Cochrane reviews of outcomes from a single course of ACS therapy highlight a reduction in respiratory distress syndrome of approximately 40%.
      • Roberts D.
      • Brown J.
      • Medley N.
      • Dalziel S.R.
      Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth.
      A similar pattern has been observed in animal studies—both those initially published by Liggins and in more recent studies by our group, which demonstrate an ACS treatment efficacy in approximately 60% of preterm sheep administered an accurately timed dosage and ventilated under standard conditions for 30 minutes.
      • 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.

      Why was this study conducted?

      Antenatal corticosteroid (ACS) therapy is widely used to improve preterm outcomes, with the primary beneficial effect being precocious maturation of the fetal lung. Although in clinical use for >50 years, dosing is poorly optimized, and adverse effects remain of concern. We and others have previously suggested that the maternal-fetal steroid exposures achieved with current clinical dosing are excessive and may increase the risk of harm. Using a sheep model of pregnancy, we studied the lung maturation and pharmacodynamic effects of betamethasone acetate (Beta-Ac) when administered individually and in combination with betamethasone phosphate (Beta-P), which is widely used today.

      Key findings

      We demonstrated that 2 doses of 0.125 mg/kg Beta-Ac were associated with fetal lung maturation at least as effective as a standard clinical course of 2, 0.25 mg/kg doses of Beta-P+Beta-Ac. Surfactant protein A expression was strongly correlated with functional maturation of the fetal lung. The ACS therapy response rate was significantly higher in the low-dosage Beta-AC–only group than in the high-dosage Beta-P+Beta-Ac group. Maternal and cord plasma betamethasone levels were significantly higher in the combined treatment group, the elevation of which were associated with lower birthweight and a greater degree of hypothalamic-pituitary-adrenal (HPA) axis perturbation, consistent with the established dosage-dependent response to antenatal steroids.

      What does this add to what is known?

      We and others have demonstrated that functional lung maturation can be achieved at exposures significantly lower than those derived from current clinical dosing protocols. This report demonstrates that Beta-P is redundant when used in combination with Beta-Ac to precociously mature the preterm lung. Our data suggested that the elevated fetal betamethasone levels deriving from the use of Beta-P are associated with a greater risk of lower birthweight and a greater degree of HPA axis perturbation. Critically, these data also suggested that compared with using Beta-Ac alone, combining Beta-P with Beta-Ac may increase the risk of ACS nonresponsiveness.
      Although there is room for improvement in ACS treatment efficacy, reliability, and optimal patient selection, sizable geographic variation in the usage of ACS remains. It is clear that an optimal ACS treatment strategy is yet to be determined and that studies informing a regimen to improve efficacy and durability while minimizing the risk of adverse effects are warranted.
      This study was undertaken with this objective in mind. We focused on the differential pharmacodynamics of betamethasone phosphate (Beta-P) and betamethasone acetate (Beta-Ac), which are commonly used in combination as an ACS therapy. These 2 agents have distinct pharmacokinetic profiles after intramuscular (IM) injection.
      • 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.
      Beta-P can be absorbed quickly after IM administration, which leads to high peak concentration and a short half-life. Beta-Ac slowly dissolves before diffusing into the vascular space. This signature enables Beta-Ac to have a far longer half-life, with a much lower maximum concentration and delayed peak concentration time.
      • Samtani M.N.
      • Lohle M.
      • Grant A.
      • Nathanielsz P.W.
      • Jusko W.J.
      Betamethasone pharmacokinetics after two prodrug formulations in sheep: implications for antenatal corticosteroid use.
      ,
      • Schmidt A.F.
      • Kemp M.W.
      • Rittenschober-Böhm J.
      • et al.
      Low-dose betamethasone-acetate for fetal lung maturation in preterm sheep.
      We have previously demonstrated that once a low fetal plasma betamethasone threshold has been achieved (approximately 1–4 ng/mL), further elevations in maternal and fetal plasma betamethasone concentrations do not in addition benefit fetal lung maturation.
      • Kemp M.W.
      • Saito M.
      • Usuda H.
      • et al.
      Maternofetal pharmacokinetics and fetal lung responses in chronically catheterized sheep receiving constant, low-dose infusions of betamethasone phosphate.
      Furthermore, we have shown that constant exposure of ≥26 hours is required for lung maturation in preterm lambs delivered 48 hours after ACS treatment initiation.
      • Kemp M.W.
      • Saito M.
      • Schmidt A.F.
      • et al.
      The duration of fetal antenatal steroid exposure determines the durability of preterm ovine lung maturation.
      Given the evidence showing a biphasic glucocorticoid signaling response in key lung maturation determinants (eg, surfactant protein A),
      • Iannuzzi D.M.
      • Ertsey R.
      • Ballard P.L.
      Biphasic glucocorticoid regulation of pulmonary SP-A: characterization of inhibitory process.
      we hypothesized that the high fetal betamethasone levels achieved by the Beta-P component of combined Beta-P and Beta-Ac therapy would be redundant for driving preterm lung maturation.
      To test this hypothesis, we used a preterm sheep model to explore the pharmacodynamic differences between a single course of combined Beta-P and Beta-Ac used clinically in Australia and the United States and a single course of Beta-Ac alone.

      Materials and Methods

      Animal work

      All protocols were reviewed and approved by the animal ethics committee of The University of Western Australia (RA/3/100/1702). All animals used were obtained from a single supplier. Experiments were performed in the same place and within 2 weeks during the normal breeding season. Notably, 36 date-mated ewes carrying a singleton fetus were randomized to 1 of 3 groups (n=12/each group): (1) a saline control group receiving maternal IM saline injections only, (2) a single-course Beta-P+Beta-Ac group receiving 2 maternal IM injections of 0.25 mg/kg Beta-P+Beta-Ac (Celestone Chronodose, Merck Sharp & Dohme, Australia) administered at 24-hour dosing intervals on 121 and 122 days’ gestation, or (3) a single-course Beta-Ac group receiving 2 maternal IM injections of 0.125 mg/kg Beta-Ac (Hovione, Portugal) on 122 and 123 days’ gestation. Each ewe received an IM injection of 150 mg medroxyprogesterone acetate (Depo-Ralovera; Pfizer, West Ryde, New South Wales, Australia) at least 5 days before steroid or control treatments to reduce the risk of steroid-induced preterm labor (term=150 days). This treatment has been previously shown not to influence ovine fetal lung maturation.
      • 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.
      Injectable 3 mg/mL solutions of active pharmaceutical ingredient Beta-Ac (Hovione, Portugal) were prepared immediately before the study commencing by Oxford Compounding (Perth, Western Australia) and tested for sterility, potency, and the absence of endotoxin contamination. An overview of the experimental design is provided in Figure 1. In keeping with good ethical practice, the animals in the saline control group were shared with a separate study and, as such, received a total of 4 maternal IM saline injections. The administration of maternal saline injections does not change fetal lung maturation status.
      Figure thumbnail gr1
      Figure 1Timing of interventions in each group
      A, The animals in the saline control group received 4 maternal IM injections of 2 mL of saline at 115-, 116-, 117-, and 118-days’ gestation. The fetuses were delivered at 123 days’ gestation. B, The animals in the Beta-P+Beta-Ac group received 2 maternal IM injections of 0.25 mg/kg of Beta-P+Beta-Ac at 121 and 122 days’ gestation. The fetuses were delivered at 123 days’ gestation, 48 hours after commencement of the intervention. C, The animals in the Beta-Ac group received 2 maternal IM injections of 0.125 mg/kg of Beta-Ac at 122- and 123-days’ gestation. The fetuses were delivered at 124 days’ gestation, 48 hours after commencement of the intervention.
      Beta-Ac, betamethasone acetate; Beta-P, betamethasone phosphate; GD, gestational day; IM, intramuscular.
      Takahashi et al. Betamethasone phosphate combined with betamethasone acetate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights. Am J Obstet Gynecol 2022.

      Delivery and ventilation

      All animals were delivered at 123 or 124 days’ gestation, with all steroid-treated animals delivered precisely 48 hours after receiving their first steroid treatment. At delivery, pregnant ewes received an intravenous injection of midazolam (0.5 mg/kg) and ketamine (10 mg/kg) followed by spinal injection of 3 mL lidocaine (20 mg/mL). Lambs had a tracheostomy to insert and secure a 4.5F endotracheal tube. Next, lambs were delivered, weighed, dried, and placed on a temperature-controlled radiant warmer (CosyCot, Fisher & Paykel Healthcare, New Zealand). Mechanical ventilation was performed using Acutronic Fabian infant ventilators (Acutronic Medical Systems, Hirzel, Switzerland), with ventilation commencing immediately after delivery and maintained for 30 minutes, initially with the following parameters: peak inspiratory pressure (PIP) of 35 cm H2O, positive end-expiratory pressure (PEEP) of 5 cm H2O, respiratory rate of 50 breaths per minute, inspiratory time of 0.5 seconds, and 100% heated and humidified oxygen. Tidal volume (Vt) was maintained between 7.0 and 8.0 mL/kg by adjustment of the PIP only but with maximal PIP limited to 35 cm H2O. An umbilical artery catheter was placed to allow measurement of arterial blood pH, pO2, pCO2, heart rate (HR), and blood pressure. Ventilation data, including PIP, Vt, and compliance, were recorded, and the ventilation efficacy index (VEI), an integrated assessment of ventilation and gas exchange, was calculated as follows: VEI=3800/(respiratory rate [PIP–PEEP]PaCO2 [mm Hg]).
      • Notter R.H.
      • Egan E.A.
      • Kwong M.S.
      • Holm B.A.
      • Shapiro D.L.
      Lung surfactant replacement in premature lambs with extracted lipids from bovine lung lavage: effects of dose, dispersion technique, and gestational age.
      Noting a 1-day difference in gestational age at delivery among the groups, fetal growth curves (y=0.0011x2−0.1557x+5.6144, where x equals gestational age and y equals delivery weight) generated from Western Australian merino cross sheep provided by our livestock supplier and adjusted at our facilities were used to standardize gestational age across groups.

      Necropsy and measurement of static lung compliance

      Lambs were euthanized and weighed after 30 minutes of ventilation. The chest was opened to allow measurement of the lung pressure-volume (PV) relationship with air inflation of the lung to a pressure of 40 cm H2O followed by deflation. The volume was standardized by lung weight. The right lower lobe was dissected free and frozen for molecular analysis.

      Measurement of RNA transcript expression changes in the fetal lung

      Messenger RNA (mRNA) was extracted from fetal lung tissue (right lower lobe) using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The concentration of extracted mRNA was determined by a broad range nucleic acid quantitation kit and a Qubit 2.0 Fluorometer (both Life Technologies, Carlsbad, CA). All mRNA extracts were diluted in nuclease-free water (Life Technologies, Carlsbad, CA) to achieve a final mRNA concentration of 25 ng/μL.
      Quantitative polymerase chain reaction (PCR) cycling was performed with ovine-specific TaqMan probe and primer sets (Applied Biosystems, Foster City, CA) with a OneStep Real-Time PCR System according to the manufacturer’s instructions. The mRNA transcripts for genes aquaporin 1 (AQP-1), aquaporin 5 (AQP-5), epithelial sodium channel subunit B (ENaC-B), elastin (ELN), nuclear receptor subfamily 3 group c member 1 (NR3C1; also known as the glucocorticoid receptor, GR), surfactant protein A (SP-A), surfactant protein B (SP-B), surfactant protein C (SP-C), and surfactant protein D (SP-D) were measured. AQP-1, AQP-5, and ENaC-B expressions were associated with improved fluid clearance in the lung. A key function of surfactant proteins (SP-A, SP-B, SP-C, and SP-D) is alveolar stabilization and the reduction of surface tension, whereas elastin plays a key role in alveolar structure and mechanical properties under shear stress.
      • Zelenina M.
      • Zelenin S.
      • Aperia A.
      Water channels (aquaporins) and their role for postnatal adaptation.
      ,
      • Wittekindt O.H.
      • Dietl P.
      Aquaporins in the lung.
      In addition, 18s ribosomal protein was used as an internal reference to normalize the amplification data for each gene. Delta quantification cycle values were used to determine the relative expressions of the transcripts.

      Western Blot

      Notably, 20 mg of fetal lung tissue was added into 400 μL of RIPA Lysis and Extraction Buffer or T-PER Tissue Protein Extraction Reagent (both Thermo Fisher Scientific, Waltham, MA) containing complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland) at the ratio of 1 tablet per 10 mL of lysis buffer. Samples were prepared in Precellys 2 mL Tissue Homogenizing Mixed Bead Kit (Bertin Instruments, Montigny-le-Bretonneux, France) and were homogenized at 6500 rpm for 30 seconds using a Precellys 24 Tissue Homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). Samples were incubated for 90 minutes at 4°C to reduce foaming before they were centrifuged at 10,000×RCF for 5 minutes. The supernatant was collected, and protein concentrations were measured by Pierce Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA).
      Protein in RIPA buffer was used for GR measurements, and protein in T-PER buffer was used for surfactant protein A, B, and C (SP-A, SP-B, and SP-C) measurements. An XCell SureLock Mini-Cell Electrophoresis System (Life Sciences) was used for electrophoresis and transfers. Samples were reduced, and 15 or 20 μg of protein for GR assay or SP-A and SP-C, respectively, were applied to each well in NuPAGE Bis-Tris Mini Gel (Invitrogen, Waltham, MA). In addition, 20 μg of nonreduced samples were used for SP-B assay. NuPAGE Bis-Tris Mini Gels of 10% concentration were used. Electrophoresis was run for 50 minutes at 200 V constant with NuPAGE MOPS SDS Running Buffer (Invitrogen, Waltham, MA). Protein was transferred to low-fluorescence polyvinylidene fluoride transfer membranes at 30 V constant for 1 hour as per the manufacturer’s protocol. Membranes were incubated with No-Stain Protein Labelling Reagent (Invitrogen, Waltham, MA) to normalize total protein. Membranes were incubated with Blocker FL Fluorescent Blocking Buffer (Thermo Fisher Scientific, Waltham, MA) for 30 minutes followed by overnight primary antibody incubation at 4°C. Primary antibodies were diluted into SuperSignal Western Blot Enhancer (Thermo Fisher Scientific, Waltham, MA) as follows: antiglucocorticoid receptor antibody (ab225886, abcam, Cambridge, United Kingdom) at 1/2000, antisurfactant protein A/PSAP antibody (ab115791, abcam) at 1/1,000, antimature surfactant protein B antibody (WRAB-88912, Seven Hills Bioreagents, OH, kindly provided by Professor Jeffrey Whitsett, Cincinnati Children’s Hospital, Cincinnati, OH) at 1/5000, and antiprosurfactant protein C antibody (ab40879) at 1/10,000. Washed membranes were incubated with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 800 (Invitrogen) at 1/10,000 in wash buffer (phosphate-buffered saline with 0.05% Tween 20; both Sigma-Aldrich, St. Louis, MO) for 60 minutes.
      Membranes were analyzed using an iBright FL1000 Imaging System (Invitrogen, Waltham, MA), and target band concentrations were measured and normalized by total protein concentration. To normalize the difference between membranes, standard quality control samples were transferred to each membrane and probed. A concentration-dependent densitometry response was confirmed with serially diluted lung protein (Supplemental Figure). Thymus extracts were used as negative control samples to confirm surfactant protein band specificity.

      Correlation and regression analysis of surfactant protein and physical data

      Regression analysis was performed to explore a relationship between surfactant protein expression and ventilation data. Multiple linear regression was used to predict V40 based on SP-A (protein), SP-B (protein), and SP-C (protein). A simple linear regression model was used to predict PaCO2 based on V40.

      Hematological analysis from maternal and fetal blood

      Maternal and fetal umbilical artery cord blood collected at delivery was assayed for plasma cortisol, adrenocorticotropic hormone (ACTH), and betamethasone concentrations. Cortisol and ACTH levels were measured by an independent clinical pathology laboratory (Vetpath, Perth, Australia). The detection limit was 5.5 nmol/L for cortisol levels and 5 pg/mL for ACTH levels. For statistical analyses, we assumed a 5 pg/mL ACTH level for samples that were found to be below the limit of detection. Betamethasone concentrations were measured with mass spectrometry 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.
      • 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.

      Definition of antenatal corticosteroid response

      We defined animals as either ACS treatment responders or nonresponders as described previously.
      • Roberts D.
      • Brown J.
      • Medley N.
      • Dalziel S.R.
      Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth.
      ,
      • 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.
      Briefly, we used normally distributed saline control group data and set an arbitrary cutoff, based on cord arterial PaCO2 levels after 30 minutes of ventilation. Those animals considered to have responded to treatment (responder subgroup) were defined as having a PaCO2 level more extreme than 2 standard deviations (SDs) below the mean value of the saline control group’s PaCO2 value. Animals were defined as being ACS treatment nonresponders (nonresponder subgroup) when having a 30-minute ventilation PaCO2 within 2 SDs of the control group mean value.

      Statistical analysis

      Statistical analysis was performed using IBM SPSS Statistics for Windows (version 25.0; IBM Corp, Armonk, NY). One-way analysis of variance followed by Tukey or Games-Howell posthoc tests was used for multiple group comparisons as appropriate. Hematological data in ACS-treated groups were tested for significance with t tests or Mann-Whitney U tests. A chi-square test was used to compare the rate of ACS responsiveness between the Beta-P+Beta-Ac group and Beta-Ac group.

      Results

      One animal from the Beta-Ac group was delivered before the protocol commenced (before steroid treatment) and was removed from further analyses. All animals from the saline control group and Beta-P+Beta-Ac groups were delivered at 123 days’ gestation, and all animals of the Beta-Ac group were delivered at 124 days’ gestation. There was no significant intergroup difference in sex, cord blood pH, cord blood PaCO2, or lung weight per body weight. Although there was no significant difference in birthweight between animals in the saline control group and animals in the Beta-Ac group, the animals in the Beta-P+Beta-Ac group had significantly lower birthweights than the animals in the saline control group (Table 1; Figure 2). The statistically significant difference in birthweight we identified was maintained when the 1-day difference in gestational age was controlled by adjustment with the fetal growth curve. In addition, there was no difference in birthweight between genders in each treated group (data not shown).
      Table 1Summary of delivery data
      VariableControlBeta-P+Beta-AcBeta-Ac
      n121211
      Gestational age (d)123123124
      Birthweight (kg)2.8±0.32.4±0.3
      Significantly lower than the control group (P<.01).
      2.7±0.3
      Sex (male/female)5/78/46/5
      Cord pH7.33±0.157.36±0.157.37±0.15
      Cord blood PaCO2 (mm Hg)53.1±4.848.4±4.246.8±4.5
      Lung weight (g/kg)35.6±3.834.3±3.733.3±3.2
      Beta-Ac, betamethasone acetate; Beta-P, betamethasone phosphate.
      Takahashi et al. Betamethasone phosphate combined with betamethasone acetate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights. Am J Obstet Gynecol 2022.
      a Significantly lower than the control group (P<.01).
      Figure thumbnail gr2
      Figure 2Birthweight, blood gas measurement, and physiological parameters
      Birthweight, arterial blood gas measurements, and physiological parameters at 30 minutes of preterm lamb ventilation. A, Birthweight (saline control vs Beta-P+Beta-Ac: mean difference, −0.43 [95% CI, −0.75 to −0.11]; P=.007; saline control vs Beta-Ac: mean difference, −0.13 [95% CI, −0.46 to 0.20]; P=.619; Beta-P+Beta-Ac vs Beta-Ac: mean difference, 0.30 [95% CI, −0.03 to 0.63]; P=.078). B, pH (saline control vs Beta-P+Beta-Ac: mean difference, 0.26 [95% CI, 0.14–0.38]; P<.001; saline control vs Beta-Ac: mean difference, 0.33 [95% CI, 0.22–0.44]; P<.001; Beta-P+Beta-Ac vs Beta-Ac: mean difference, 0.07 [95% CI, −0.07 to 0.21]; P=.420). C, PaCO2 (saline control vs Beta-P+Beta-Ac: mean difference, −35.8 [95% CI, −57.3 to −14.3]; P=.001; saline control vs Beta-Ac: mean difference, −54.0 [95% CI, −76.0 to −32.0]; P<.001; Beta-P+Beta-Ac vs Beta-Ac: mean difference, −18.2 [95% CI, −40.2 to 3.8]; P=.119). D, pO2 (saline control vs Beta-P+Beta-Ac: mean difference, 36.3 [95% CI, −1.0 to 73.5]; P=.057; saline control vs Beta-Ac: mean difference, 117.9 [95% CI, 25.4–210.4]; P=.014; Beta-P+Beta-Ac vs Beta-Ac: mean difference, 81.6 [95% CI, −14.2 to 177.4]; P=.100). E, HR (saline control vs Beta-P+Beta-Ac: mean difference, −52.2 [95% CI, −78.8 to −25.5]; P<.001; saline control vs Beta-Ac: mean difference, −31.5 [95% CI, −58.8 to −4.3]; P=.021; Beta-P+Beta-Ac vs Beta-Ac: mean difference, 20.6 [95% CI, −6.6 to 47.9]; P=.167). F, mBP (no difference). The asterisk indicates a significant difference among the groups. Error bars represent ±1 standard deviation.
      Beta-Ac, betamethasone acetate; Beta-P, betamethasone phosphate; CI, confidence interval; HR, heart rate; mBP, mean blood pressure.
      Takahashi et al. Betamethasone phosphate combined with betamethasone acetate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights. Am J Obstet Gynecol 2022.

      Ventilation data (30 minutes)

      Arterial blood gas measurements and key physiological parameters after 30 minutes of ventilation are shown in Figure 2. Both ACS treatment groups had significantly improved pH and PaCO2 compared with the saline control group. Only the Beta-Ac group had a PaO2 value significantly higher than that of the saline control group. HR was significantly lower in both the Beta-P+Beta-Ac and Beta-Ac groups than the saline control group, but there was no significant difference in mean blood pressure among the groups.
      Based on a saline control group PaCO2 mean and a standard division after 30 minutes of ventilation (117.8±20.8 mm Hg, respectively), we set an arbitrary cutoff for determining ACS responder and nonresponder animals at 76.1 mm Hg. Accordingly, 33.3% of animals (4/12) in the Beta-P+Beta-Ac group were classified as responders, compared with 81.8 % of animals (9/11) in the Beta-Ac group (Figure 3). The responder rate was significantly higher in the Beta-Ac group than in the Beta-P+Beta-Ac group (P=.036). There was no animal from the saline control group assigned to the responder subgroup.
      Figure thumbnail gr3
      Figure 3Response rate to ACS treatment
      Preterm lambs were divided into a responder group or nonresponder group based on an arbitrary cutoff. The cutoff was set at 76.1 mm Hg in PaCO2 at 30 minutes ventilation, which was 2 standard deviations below the PaCO2 average in the control group at 30 minutes ventilation. A, All animal PaCO2 at 30 minutes ventilation data were plotted. The dashed line shows the arbitrary cutoff at 76.1 mm Hg. The animals under the dashed line were assigned to the responder group. Animals above the line were assigned to the nonresponder group. B, The graph shows the response rate in both ACS-treated groups. The asterisk represents the Beta-Ac group showed a significantly higher response rate at 81.8% than the Beta-P+Beta-Ac group at 33.3%. (χ2[1]=3.69; P=.036).
      ACS, antenatal corticosteroid; Beta-Ac, betamethasone acetate; Beta-P, betamethasone phosphate.
      Takahashi et al. Betamethasone phosphate combined with betamethasone acetate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights. Am J Obstet Gynecol 2022.
      Ventilation data collected 30 minutes into the procedure are presented in Figure 4. There was no difference in PIP between the ACS-treated groups. Both the Beta-P+Beta-Ac and Beta-Ac groups showed significantly higher dynamic compliance, Vt, and VEI than the saline control group. There was no difference in the parameters between the Beta-P+Beta-Ac and Beta-Ac groups. Figure 5 shows static lung compliance during necropsy. As shown in the ventilation data, lung gas volume at 40 mm H2O was significantly higher in the Beta-P+Beta-Ac and Beta-Ac groups than in the saline control group. PV curve showed both treatment groups had larger volume loops than the saline control group.
      Figure thumbnail gr4
      Figure 4Ventilation data
      Ventilation data at 30 minutes. A, PIP (saline control vs Beta-P+Beta-Ac: mean difference, −1.66 [95% CI, −3.94 to 0.60]; P=.163; saline control vs Beta-Ac: mean difference, −0.81 [95% CI, −1.73 to 0.11]; P=.087; Beta-P+Beta-Ac vs Beta-Ac: mean difference, 0.86 [95% CI, −1.50 to 3.21]; P=.620). B, Dynamic compliance (saline control vs Beta-P+Beta-Ac: mean difference, 0.22 [95% CI, 0.05–0.39]; P=.012; saline control vs Beta-Ac: mean difference, 0.30 [95% CI, 0.20 0.41]; P<.001; Beta-P+Beta-Ac vs Beta-Ac: mean difference, 0.08 [95% CI, −0.10 to 0.27]; P=.515). C, Vt (saline control vs Beta-P+Beta-Ac: mean difference, 2.78 [95% CI, 1.68–3.87]; P<.001; saline control vs Beta-Ac: mean difference, 3.40 [95% CI, 2.46–4.35]; P<.001; Beta-P+Beta-Ac vs Beta-Ac: mean difference, 0.63 [95% CI, −0.69 to 1.94]; P=.463). D, VEI (saline control vs Beta-P+Beta-Ac: mean difference, 0.016 [95% CI, 0.0003–0.0312]; P=.045; saline control vs Beta-Ac: mean difference, 0.022 [95% CI, 0.008–0.036]; P=.004; Beta-P+Beta-Ac vs Beta-Ac: mean difference, 0.006 [95% CI, −0.013 to 0.026]; P=.680). The asterisk indicates a significant difference among the groups. Error bars represent ±1 standard deviation.
      Beta-Ac, betamethasone acetate; Beta-P, betamethasone phosphate; CI, confidence interval; PIP, peak inspiratory pressure; VEI, ventilation efficiency index; Vt, tidal volume.
      Takahashi et al. Betamethasone phosphate combined with betamethasone acetate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights. Am J Obstet Gynecol 2022.
      Figure thumbnail gr5
      Figure 5Lung maturation analysis
      A, Static lung gas volumes measured at a maximal pressure of 40 cm H2O. (saline control vs Beta-P+Beta-Ac: mean difference, 726 [95% CI, 358–1092]; P=.001; saline control vs Beta-Ac: mean difference, 1103 [95% CI, 805–1401]; P<.001; Beta-P+Beta-Ac vs Beta-Ac: mean difference, 377 [95% CI, −56 to 812]; P=.095) B, Pressure-volume relationship for air inflation and deflation of the lung at necropsy. The higher line from 0 cm H2O to 40 cm H2O of pressure in each loop is the inflation arm, and the lower line from 40 cm H2O to 0 cm H2O of pressure is the deflation arm. The asterisk indicates a significant difference among the groups. Error bars represent ±1 standard deviation.
      Beta-Ac, betamethasone acetate; Beta-P, betamethasone phosphate; CI, confidence interval.
      Takahashi et al. Betamethasone phosphate combined with betamethasone acetate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights. Am J Obstet Gynecol 2022.

      Quantitative polymerase chain reaction analysis of transcript expression changes in the fetal lung

      AQP-1 and AQP-5 mRNA transcripts were significantly elevated in the Beta-P+Beta-Ac group than in the saline control group. There was no significant difference in these values between the Beta-Ac and saline control groups (Table 2). Both steroid treatment groups had significant increases in transcripts for ENaC-B, SP-A, SP-B, SP-C, and ELN compared with the saline control group. The Beta-P+Beta-Ac group showed a higher fold change in ENaC-B than the Beta-Ac group. GR transcript levels were significantly lower in the Beta-Ac group than in both the saline control and Beta-P+Beta-Ac groups. There was no significant difference in GR transcript levels between the control group and Beta-P+Beta-Ac group. SP-D was not different among groups.
      Table 2Lung messenger RNA and protein quantification in fold change and control animals
      TissueTarget mRNAFold change (vs control)
      ControlBeta-P+Beta-AcBeta-Ac
      LungAQP-11.00 (0.81–1.24)1.40 (1.11–1.78)
      Significantly different than the control group (P<.05)
      1.13 (0.89–1.44)
      AQP-51.00 (0.72–1.39)1.76 (1.27–2.45)
      Significantly different than the control group (P<.05)
      1.35 (0.96–1.88)
      ENaC-B1.00 (0.73–1.37)2.34 (1.77–3.10)
      Significantly different than the control group (P<.05)
      1.50 (1.13–2.00)
      Significantly different than the control group (P<.05)
      ,
      Significant difference between the Beta-P+Beta-Ac and Beta-Ac groups (P<.05).
      ELN1.00 (0.84–1.20)2.45 (1.87–3.21)
      Significantly different than the control group (P<.05)
      2.08 (1.46–2.95)
      Significantly different than the control group (P<.05)
      GR1.00 (0.84–1.19)0.90 (0.73–1.11)0.70 (0.57–0.87)
      Significantly different than the control group (P<.05)
      ,
      Significant difference between the Beta-P+Beta-Ac and Beta-Ac groups (P<.05).
      SP-A1.00 (0.57–1.75)2.63 (1.58–4.37)
      Significantly different than the control group (P<.05)
      2.12 (1.26–3.57)
      Significantly different than the control group (P<.05)
      SP-B1.00 (0.71–1.40)1.82 (1.32–2.50)
      Significantly different than the control group (P<.05)
      1.47 (1.06–2.04)
      Significantly different than the control group (P<.05)
      SP-C1.00 (0.69–1.45)2.25 (1.52–3.34)
      Significantly different than the control group (P<.05)
      1.92 (1.28–2.87)
      Significantly different than the control group (P<.05)
      SP-D1.00 (0.65–1.45)1.27 (0.72–2.23)0.78 (0.44–1.39)
      TissueTarget proteinControlBeta-P+Beta-AcBeta-Ac
      LungGR1.00 (0.79–1.21)0.54 (0.31–0.76)
      Significantly different than the control group (P<.05)
      0.73 (0.48–0.97)
      SP-A1.00 (0.78–1.22)1.85 (1.37–2.32)
      Significantly different than the control group (P<.05)
      2.43 (1.93–2.94)
      Significantly different than the control group (P<.05)
      SP-B1.00 (0.52–1.47)2.33 (1.52–3.12)
      Significantly different than the control group (P<.05)
      1.73 (1.03–2.43)
      SP-C1.00 (0.70–1.30)1.88 (1.36–2.39)
      Significantly different than the control group (P<.05)
      1.99 (1.55–2.44)
      Significantly different than the control group (P<.05)
      The data are presented as average (95% confidence interval).
      AQP-1, aquaporin 1; AQP-5, aquaporin 5; Beta-Ac, betamethasone acetate; Beta-P, betamethasone phosphate; ELN, elastin; ENaC-B, epithelial sodium channel subunit B; GR, glucocorticoid receptor; NR3C1, nuclear receptor subfamily 3 group c member 1; SP-A, surfactant protein A; SP-B, surfactant protein B; SP-C, surfactant protein C; SP-D, surfactant protein D.
      Takahashi et al. Betamethasone phosphate combined with betamethasone acetate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights. Am J Obstet Gynecol 2022.
      a Significantly different than the control group (P<.05)
      b Significant difference between the Beta-P+Beta-Ac and Beta-Ac groups (P<.05).

      Western blot analysis of lung tissue

      GR, SP-A, mature SP-B, and pro–SP-C bands were confirmed at 90, 35 to 37, 18, and 19 kDa, respectively (Supplemental Figure). Band volumes were determined to be proportional to total protein concentration. SP-A is highly heterogeneous on immunoblotting analyses because of the presence of multiple glycosylation and acetylation sites and multimeric forms resistant to reduction.
      • Rubio S.
      • Lacaze-Masmonteil T.
      • Chailley-Heu B.
      • Kahn A.
      • Bourbon J.R.
      • Ducroc R.
      Pulmonary surfactant protein A (SP-A) is expressed by epithelial cells of small and large intestine.
      In this analysis, SP-A was detected in separate bands at 35 and 37 kDa. We recognized both bands as SP-A–specific because of their absence in protein extracts from the ovine thymus, which does not express SP-A at discernible levels.
      • Madsen J.
      • Tornoe I.
      • Nielsen O.
      • Koch C.
      • Steinhilber W.
      • Holmskov U.
      Expression and localization of lung surfactant protein A in human tissues.
      As the 37 kDa SP-A band volume was unaffected by ACS, we have only reported band volumes for the 35 kDa SP-A form in this study. SP-B showed 2 bands at 18 and 25 kDa. As the band at 18 kDa is a homodimer of mature SP-B and the band at 25 kDa is likely pro–SP-B, only mature SP-B was analyzed.
      • Wert S.E.
      • Whitsett J.A.
      • Nogee L.M.
      Genetic disorders of surfactant dysfunction.
      GR protein concentration was significantly lower in the Beta-P+Beta-Ac group than in the saline control group. There was no difference between the saline control group and Beta-Ac group (Table 2). Both the Beta-P+Beta-Ac and Beta-Ac groups showed significantly high SP-A and pro–SP-C concentrations compared with the control group. There was no difference between the Beta-P+Beta-Ac and Beta-Ac groups for GR, SP-A, or pro–SP-C concentrations. Only the Beta-P+Beta-Ac group showed significantly higher SP-B concentrations than the saline control group.

      Regression analysis of surfactant protein and physical data

      Figure 6, A and B, shows the predicted correlations between SP-A (protein) concentration and V40 and between V40 and 30-minute cord arterial blood PaCO2 values. Although SP-A (protein) and SP-B (protein) were significant predictors of V40 (SP-A: β=0.66, P<.001; SP-B: β=0.23; P<.05), SP-C (protein) was not (β=0.04; P=.72) (R2=0.734). V40 was a significant predictor of PaCO2 (β=−0.82; P<.001; R2=0.683) (Figure 6, C). A single regression analysis was calculated to predict V40 based on SP-A (protein). It showed that SP-A (protein) was a significant predictor of V40 (β=0.84; P<.001; R2=0.719) (Figure 6, A).
      Figure thumbnail gr6
      Figure 6Relationship between surfactant protein and lung physical maturation
      A, The graphs show correlations between protein amounts of SP-A and V40 as static lung compliance. Each group was plotted with different color. B, The graph shows a correlation between V40 and PaCO2 at 30 minutes ventilation. Each group was plotted with different color. C, Structural equation modeling from surfactant proteins to lung functional maturation. The observed variables were presented by a square box, and latent or unmeasured variables were presented by a circle. A number along with each arrow is the standardized partial regression coefficient. The asterisks indicate significant predictors.
      Takahashi et al. Betamethasone phosphate combined with betamethasone acetate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights. Am J Obstet Gynecol 2022.

      Hematological analyses of maternal and fetal blood

      Maternal and fetal plasma levels of cortisol and ACTH were measured with maternal blood and umbilical cord blood collected at delivery. Cortisol levels were below the limit of detection (5.5 nmol/L) in all but 3 maternal blood samples from the Beta-Ac group (16.7, 23.2, 80 nmol/L) and 1 fetal blood sample from the Beta-Ac group (11 nmol/L); accordingly, group differences in cortisol levels were not analyzed. Plasma ACTH levels are shown in Figure 7, A. ACTH levels in 2 maternal samples and 1 fetal sample from the Beta-P+Beta-Ac group and 1 maternal sample from the Beta-Ac group were below the limit of detection at 5 pg/mL. We arbitrarily replaced them with a value of 5 pg/mL to allow the analysis as outlined above. No difference was seen in maternal ACTH values between the Beta-P+Beta-Ac and Beta-Ac groups. Fetal plasma ACTH levels were significantly lower in the Beta-P+Beta-Ac group than in the Beta-Ac group. Figure 7, B, shows the betamethasone concentrations in maternal and fetal plasma at delivery. Maternal betamethasone concentrations were 6 times lower and fetal concentrations 3 times lower in the Beta-Ac group than concentrations in the Beta-P+Beta-Ac group.
      Figure thumbnail gr7
      Figure 7Hematological results from maternal and fetal blood
      The graphs show the ACTH and betamethasone concentrations in maternal and fetal plasma at delivery. A, Maternal or fetal plasma ACTH levels. In (A), 3 samples (2 in the Beta-P+Beta-Ac group and 1 in the Beta-Ac group) from maternal plasma and 1 sample from fetal plasma in the Beta-P+Beta-Ac group showed very low ACTH levels to be detected. Maternal ACTH (no difference; P=.104; Mann-Whitney U test), fetal ACTH (significant difference; P=.002; Mann-Whitney U test). B, Maternal or fetal betamethasone concentration at delivery. Maternal betamethasone concentration (Beta-P+Beta-Ac vs Beta-Ac: mean difference, −5.73 [95% CI, −7.63 to 3.83]; P<.001; t test) and fetal betamethasone concentration (significant difference; P<.001; Mann-Whitney U test). The parenthesis indicates the number of samples that could not detect ACTH. The asterisk indicates a significant difference among the groups. Error bars represent ±1 standard deviation.
      ACTH, adrenocorticotropic hormone; Beta-Ac, betamethasone acetate; Beta-P, betamethasone phosphate; CI, confidence interval.
      Takahashi et al. Betamethasone phosphate combined with betamethasone acetate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights. Am J Obstet Gynecol 2022.

      Comment

      Principal findings

      The primary findings of this study were as follows: (1) a single course (2 doses at 0.125 mg/kg) of Beta-Ac achieved more consistent functional maturation of the ovine preterm lung than the combined Beta-Ac+Beta-P in 2, 0.25 mg/kg doses and (2) a single course of combined Beta-P and Beta-Ac resulted in higher maternal and fetal plasma betamethasone concentrations in association with a greater degree of fetal HPA axis suppression and statistically significant reductions in birthweight than Beta-Ac alone. On the basis of these observations, it may be concluded that for deliveries occurring 48 hours after treatment initiation, the coadministration of Beta-P with Beta-Ac not only fails to in addition benefit fetal ovine lung maturation but may also suppress GR-driven maturational signaling in the lung, compared with that elicited by the sole administration of Beta-Ac at a lower total dosage. Based on the pharmacokinetics and mode of action of the agents used, the root cause of these differences in treatment outcomes is likely the elevated maternal-fetal betamethasone concentrations derived from the use of Beta-P. Additional studies with a specific focus on molecular mechanisms of GR signaling are necessary to validate this theory.
      Overall, both treated groups had improved lung maturation compared with the saline control group. Favorable arterial blood gas data (pH, PaO2, and PaCO2), ventilation data (dynamic compliance, Vt, and VEI) and static compliance (V40 and PV curves) data all demonstrate that both ACS regimens could mature the preterm lung structurally, leading to more efficient gas exchange. In addition, HR was significantly reduced in both treated groups, suggesting that both ACS therapies could stabilize the cardiovascular system, potentially by improving cardiac performance and reducing vascular permeability. Furthermore, evidence for preterm lung maturation independent of Beta-P use was provided by our mRNA transcript analyses. Here, both ACS-treatment groups showed significantly increased mRNA transcript for ENaC-B, SP-A, SP-B, SP-C, and ELN compared with the saline control group animals. However, there was no difference in these mRNA transcripts apart from ENaC-B and GR between the ACS-treatment groups. Although there was no significant difference, these values seem to be correlated with the total amount of ACS administered, as shown in the case of ENaC-B. Other than for SP-B, these differences in mRNA expression did not equate to increased protein expression.
      Although the changes in PaCO2 values between the ACS treatment groups were not significantly different, when animals were classified into ACS responders and nonresponders based on an arbitrary cutoff, derived from the saline control group values, there was a clear difference in the interanimal variability of the 2 steroid regimens. It is important to note that PaCO2 levels were used instead of PaO2 levels (which were significantly different between the ACS treatment groups) because of the potential for PaO2 values to be confounded by alterations in the patency of the ductus arteriosus.
      • 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.
      ,
      • Schmidt A.F.
      • Kemp M.W.
      • Kannan P.S.
      • et al.
      Antenatal dexamethasone vs. betamethasone dosing for lung maturation in fetal sheep.
      Although not directly comparable with a clinical outcome, such as respiratory distress syndrome, it is interesting to note that a significant degree of ACS nonresponsiveness is regularly reported in human randomized control trials of ACS therapy, even when other variables (ie, successful administration of treatment course, delivery within 7 days of treatment) are controlled. It would be of significant interest, and great potential importance, to determine whether a constant, low-concentration maternal-fetal ACS exposure (via the use of either Beta-Ac only or another appropriate regiment) similarly reduced the variability of treatment efficacy, yielding more favorable numbers needed to treat values for outcomes, including perinatal death and respiratory distress syndrome.
      In seeking to explore the basis for the difference in ACS outcomes identified in this study, it is important to explore the pharmacokinetics and pharmacodynamics of Beta-Ac and Beta-P. It is well known from both animal and human studies that adverse ACS effects have a clear dosage-dependent risk profile, including risks of fetal growth restriction, impairment of HPA axis function, and neurodevelopmental effects.
      • Waljee A.K.
      • Rogers M.A.
      • Lin P.
      • et al.
      Short term use of oral corticosteroids and related harms among adults in the United States: population based cohort study.
      • Savoy C.
      • Ferro M.A.
      • Schmidt L.A.
      • Saigal S.
      • Van Lieshout R.J.
      Prenatal betamethasone exposure and psychopathology risk in extremely low birth weight survivors in the third and fourth decades of life.
      • Busada J.T.
      • Cidlowski J.A.
      Mechanisms of glucocorticoid action during development.
      • Braun T.
      • Sloboda D.M.
      • Tutschek B.
      • et al.
      Fetal and neonatal outcomes after term and preterm delivery following betamethasone administration.
      Given that lower maternal-fetal steroid exposures are desirable, it is remarkable that the Beta-Ac group, which received a much lower dosage of glucocorticoids, had lung maturation that was at least as good as that seen in the higher-dosage Beta-P+Beta-Ac group. Although data were limited to effects within 48 hours of ACS treatment, it is apparent that the lower-dosage ACS caused less disruption to fetal growth and the HPA axis.
      Dissecting the differences in the Beta-P+Beta-Ac and Beta-Ac treatment protocols that contribute to the difference in treatment effects and observed adverse outcomes in this study is of particular importance. It is important to note that the Beta-P+Beta-Ac group received a much larger total dosage of betamethasone and that this treatment conveyed a substantially higher maternal-fetal plasma betamethasone concentration. Because of its high solubility, the phosphate ester of betamethasone generates a higher peak concentration (around 5 times that of Beta-Ac alone in the sheep) that is rapidly cleared.
      • Roberts D.
      • Brown J.
      • Medley N.
      • Dalziel S.R.
      Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth.
      Our previous results have shown that a sustained, low-magnitude betamethasone exposure is far more effective in maturing the preterm ovine lung than a brief, high exposure pulse of betamethasone.
      • 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.
      ,
      • Kemp M.W.
      • Saito M.
      • Schmidt A.F.
      • et al.
      The duration of fetal antenatal steroid exposure determines the durability of preterm ovine lung maturation.
      ,
      • 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.

      Clinical implications

      This work has 2 important implications for clinical ACS use. The first relates to efforts to improve the safety and efficacy of combined Beta-Ac and Beta-P therapy based on the protocol used by Liggins and Howie in their landmark clinical trial that is now widely employed in the United States, Australia, and parts of Europe.
      • Liggins G.C.
      • Howie R.N.
      A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants.
      The benefits of combined therapy, when administered to the right women at the right time, are supported by multiple randomized control trials.
      • Roberts D.
      • Brown J.
      • Medley N.
      • Dalziel S.R.
      Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth.
      However, these same data make it clear that treatment responsiveness is extremely variable, and several additional studies have reported an increased risk of harm in association with Beta-Ac and Beta-P use.
      • Murphy K.E.
      • Hannah M.E.
      • Willan A.R.
      • et al.
      Multiple courses of antenatal corticosteroids for preterm birth (MACS): a randomised controlled trial.
      • Murphy K.E.
      • Willan A.R.
      • Hannah M.E.
      • et al.
      Effect of antenatal corticosteroids on fetal growth and gestational age at birth.
      • Alexander N.
      • Rosenlöcher F.
      • Stalder T.
      • et al.
      Impact of antenatal synthetic glucocorticoid exposure on endocrine stress reactivity in term-born children.
      • Roberts D.
      • Brown J.
      • Medley N.
      • Dalziel S.R.
      Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth.
      • 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.
      In addition, the treatment variability and ACS dosage-dependent reduction in birthweight seen clinically in association with combined Beta-Ac and Beta-P use were identified in this study. Given that the exclusive use of Beta-Ac reduced HPA axis disruption, lessened the effects on fetal growth, and improved overall treatment success rate, it is reasonable to suggest that clinical studies to explore the use of a Beta-Ac-only therapy (or therapy with another glucocorticoid so delivered to replicate the constant, low-amplitude exposure given by Beta-Ac dosing) are now justified. In contrast, variable responsiveness to ACS treatment per se remains unclear. Once we have a better understanding of the critical mechanisms driving steroid-induced lung maturation, there may be a chance to identify those likely not to respond early in pregnancy. Furthermore, we may be capable of tailoring therapies to take these (likely) genetic differences into account.
      Secondly, this work has implications for ACS dosing regimens based on the sole use of Beta-P (the United Kingdom and Japan) or dexamethasone phosphate (ie, the widely employed World Health Organization–recommended protocol).
      World Health Organization
      WHO recommendations on interventions to improve preterm birth outcomes.
      Whether administering 2, 12 mg doses of Beta-P every 24 hours, or 4 6 mg doses of dexamethasone phosphate every 12 hours, these 2 protocols each generate a pulsatile pattern of exogenous glucocorticoid exposure, characterized by comparatively high concentration peaks (notably in the 12 mg Beta-P protocol) immediately after administration, rapidly followed by concentration troughs immediately before the subsequent administration. Our earlier work has demonstrated the importance of a constant steroid exposure and that the concentration threshold for an efficacious threshold is comparably low, approximately 1 to 4 ng betamethasone per milliliter of fetal plasma, and certainly much lower than the concentration peaks generated by contemporary dexamethasone and Beta-P concentrations.
      • Roberts D.
      • Brown J.
      • Medley N.
      • Dalziel S.R.
      Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth.
      ,
      • 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.
      Based on these findings, and the new data presented herein, we suggest that a betamethasone or dexamethasone regimen based on frequent lower-dosage treatments (perhaps as low as 0.05 mg/kg) may constitute an efficacious ACS treatment regimen absent of the high concentration peaks that we have shown to be both redundant for fetal lung maturation and potentially causative of harm.

      Research implications

      Unpacking the molecular mechanisms driving the differential treatment effects identified in this study will be of particular importance to the future optimization of this important therapy. A particular focus will likely be on why a higher maternal-fetal steroid exposure correlates with increased variability in treatment efficacy. The regulation of surfactant protein A provides some insight into 1 explanation for this phenomenon. Although not essential for lung function, SP-A is implicated in tubular myelin formation, in the formation of surfactant films, and in phospholipid cycling.
      • Cañadas O.
      • Olmeda B.
      • Alonso A.
      • Pérez-Gil J.
      Lipid-protein and protein-protein interactions in the pulmonary surfactant system and their role in lung homeostasis.
      • Klein J.M.
      • McCarthy T.A.
      • Dagle J.M.
      • Snyder J.M.
      Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro.
      • Khubchandani K.R.
      • Snyder J.M.
      Surfactant protein A (SP-A): the alveolus and beyond.
      Here, SP-A protein rather than SP-B or SP-C showed a strong correlation with V40, which represented static lung compliance (Figure 6, A). Although both SP-A and SP-B are essential for normal lung function, SP-A protein expression is likely quite informative in assessing ACS treatment response and lung maturation status. In addition, V40 was correlated to PaCO2 at 30 minutes of ventilation (Figure 6, B).
      Ballard et al
      • Iannuzzi D.M.
      • Ertsey R.
      • Ballard P.L.
      Biphasic glucocorticoid regulation of pulmonary SP-A: characterization of inhibitory process.
      have previously demonstrated that SP-A is exquisitely responsive to GR stimulation. Unlike other surfactant proteins, such as SP-B, which seem to exhibit a linear response to GR activation, SP-A seemed to have a pronounced biphasic response, wherein maximal transcript expression occurs at a low exogenous steroid concentration, and was reduced (apparently via both negative feedback and increased mRNA turnover) at higher steroid concentrations. Bridges et al
      • Bridges J.P.
      • Sudha P.
      • Lipps D.
      • et al.
      Glucocorticoid regulates mesenchymal cell differentiation required for perinatal lung morphogenesis and function.
      have recently demonstrated the role of GR activation in the modulation of Wingless-related integration site, Janus kinase-signal transducers and activators of transcription, and vascular endothelial growth factor signaling in the fetal lung, leading to matrix fibroblast differentiation and mature alveolar type I and II cell transformation. It is tempting to speculate that, similar to the situation observed with SP-A responses, one or more key regulatory elements in these pathways has a “goldilocks” response to GR signaling, where too little (in terms of both magnitude and/or duration) or too much exposure results in a suboptimal maturation response.

      Strengths and limitations

      Several limitations should be taken into account when assessing the translatability of the data presented herein. Although the sheep is an excellent translational model to study ACS therapy, it should be remembered that the data are from an animal study rather than a human clinical study and sex-linked differences were not accounted. Furthermore, this study used a small number of animals. The study was adequately powered to explore a potential difference between ACS treatments and saline control but was not designed to assess any (likely more subtle) differences between ACS treatment groups. A much larger study (group sizes of approximately 30) may allow for the identification of treatment differences between ACS groups and assist in the identification of any statistically significant difference in delivery weight between the Beta-Ac and saline control groups.
      There were 2 limitations of the study design; as appropriate for good ethical practice and the reduction of animals used in research studies, the animals in the saline control group were shared with a separate protocol and received 4 maternal saline injections on different days compared with animals treated with ACS. Based on a comparison with earlier data (not shown), 2 additional injections of saline do not alter fetal lung development. Secondly, there was a 1-day difference in gestational age between the animals in the Beta-Ac group and animals in the saline control and Beta-P+Beta-Ac groups because of limitations in our sheep mating capacity. To ensure the observed difference in birthweight was not confounded by this difference, we corrected for 1 day of growth using fetal weight medical records previously developed by our group. In performing a correction, we found that the observed difference in weights retained statistical significance. Given this, and the strong body of evidence linking a dose-dependent relationship between fetal glucocorticoid exposure and growth restriction, we are confident that the observed difference is a function of the treatment received rather than a small difference in gestational age.

      Conclusion

      We hypothesized that the high fetal betamethasone levels achieved by the Beta-P component of combined Beta-P and Beta-Ac therapy would be redundant in driving preterm lung maturation. The study results supported our hypothesis and strongly suggested that lower-dosage treatment with Beta-Ac, avoiding high maternal-fetal steroid exposures, is both safer and more effective than combined Beta-P and Beta-Ac therapy. These findings add further impetus to the undertaking of clinical trials to optimize the agent of choice and dosing regimen for ACS therapies.

      Acknowledgments

      The authors wish to acknowledge Sara, and Andrew Ritchie, (Icon Agriculture, Darkan, Western Australia) for their expertise in supplying date-mated sheep, Siemens Australia for the kind donation of RAPIDPoint 500 consumables, Medtronic Australia for the generous donation of suture materials, Fisher & Paykel New Zealand for the kind donation of infant warmers and circuit humidifiers, and Hovione for the generous donation of betamethasone acetate. The authors thank Professor Dorota Doherty, PhD, and Dr Liz Nathan, PhD (Women and Infants Research Foundation Biostatistics Unit) for their assistance with the birthweight correction analysis. Lastly, the authors would like to acknowledge the significant generosity of the late Mr Alan Hale (owner, A&M Medical), whose donations of ventilation equipment and technical support made much of the foundation work for this study possible.

      Supplementary Data

      Appendix

      Figure thumbnail fx1
      Supplemental FigureWestern blot analysis for target protein and total protein
      Serially diluted lung proteins every 5 μg from 5 μg to 25 μg were analyzed with GR, SP-A, SP-B, and pro–SP-C antibodies. The thymus was used as a negative control for SP-A, SP-B, and pro–SP-C antibodies. The blue bands show the total protein, and the target bands are shown as green bands indicated with arrows or a square bracket. Gray scale bands show target bands. Band volume of both total protein bands (blue) and target bands (green) were correlated to the amount of applied protein.
      Takahashi et al. Betamethasone phosphate combined with betamethasone acetate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights. Am J Obstet Gynecol 2022.

      References

        • Blencowe H.
        • Cousens S.
        • Chou D.
        • et al.
        Born too soon: the global epidemiology of 15 million preterm births.
        Reprod Health. 2013; 10: S2
        • Liu L.
        • Johnson H.L.
        • Cousens S.
        • et al.
        Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000.
        Lancet. 2012; 379: 2151-2161
        • Liggins G.C.
        • Howie R.N.
        A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants.
        Pediatrics. 1972; 50: 515-525
        • Committee on Obstetric Practice
        Committee Opinion No. 713: antenatal corticosteroid therapy for fetal maturation.
        Obstet Gynecol. 2017; 130: e102-e109
        • French N.P.
        • Hagan R.
        • Evans S.F.
        • Godfrey M.
        • Newnham J.P.
        Repeated antenatal corticosteroids: size at birth and subsequent development.
        Am J Obstet Gynecol. 1999; 180: 114-121
        • Murphy K.E.
        • Hannah M.E.
        • Willan A.R.
        • et al.
        Multiple courses of antenatal corticosteroids for preterm birth (MACS): a randomised controlled trial.
        Lancet. 2008; 372: 2143-2151
        • Murphy K.E.
        • Willan A.R.
        • Hannah M.E.
        • et al.
        Effect of antenatal corticosteroids on fetal growth and gestational age at birth.
        Obstet Gynecol. 2012; 119: 917-923
        • Alexander N.
        • Rosenlöcher F.
        • Stalder T.
        • et al.
        Impact of antenatal synthetic glucocorticoid exposure on endocrine stress reactivity in term-born children.
        J Clin Endocrinol Metab. 2012; 97: 3538-3544
        • Roberts D.
        • Brown J.
        • Medley N.
        • Dalziel S.R.
        Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth.
        Cochrane Database Syst Rev. 2017; 3: CD004454
        • 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.
        Am J Obstet Gynecol. 2020; 223: 921.e1-921.e10
        • 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.
        Clin Transl Sci. 2020; 13: 391-399
        • Samtani M.N.
        • Lohle M.
        • Grant A.
        • Nathanielsz P.W.
        • Jusko W.J.
        Betamethasone pharmacokinetics after two prodrug formulations in sheep: implications for antenatal corticosteroid use.
        Drug Metab Dispos. 2005; 33: 1124-1130
        • Schmidt A.F.
        • Kemp M.W.
        • Rittenschober-Böhm J.
        • et al.
        Low-dose betamethasone-acetate for fetal lung maturation in preterm sheep.
        Am J Obstet Gynecol. 2018; 218: 132.e1-132.e9
        • Kemp M.W.
        • Saito M.
        • Usuda H.
        • et al.
        Maternofetal pharmacokinetics and fetal lung responses in chronically catheterized sheep receiving constant, low-dose infusions of betamethasone phosphate.
        Am J Obstet Gynecol. 2016; 215: 775.e1-775.e12
        • Kemp M.W.
        • Saito M.
        • Schmidt A.F.
        • et al.
        The duration of fetal antenatal steroid exposure determines the durability of preterm ovine lung maturation.
        Am J Obstet Gynecol. 2020; 222: 183.e1-183.e9
        • Iannuzzi D.M.
        • Ertsey R.
        • Ballard P.L.
        Biphasic glucocorticoid regulation of pulmonary SP-A: characterization of inhibitory process.
        Am J Physiol. 1993; 264: L236-L244
        • 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.
        Am J Obstet Gynecol. 2003; 188: 22-28
        • Notter R.H.
        • Egan E.A.
        • Kwong M.S.
        • Holm B.A.
        • Shapiro D.L.
        Lung surfactant replacement in premature lambs with extracted lipids from bovine lung lavage: effects of dose, dispersion technique, and gestational age.
        Pediatr Res. 1985; 19: 569-577
        • Zelenina M.
        • Zelenin S.
        • Aperia A.
        Water channels (aquaporins) and their role for postnatal adaptation.
        Pediatr Res. 2005; 57: 47R-53R
        • Wittekindt O.H.
        • Dietl P.
        Aquaporins in the lung.
        Pflugers Arch. 2019; 471: 519-532
        • Rubio S.
        • Lacaze-Masmonteil T.
        • Chailley-Heu B.
        • Kahn A.
        • Bourbon J.R.
        • Ducroc R.
        Pulmonary surfactant protein A (SP-A) is expressed by epithelial cells of small and large intestine.
        J Biol Chem. 1995; 270: 12162-12169
        • Madsen J.
        • Tornoe I.
        • Nielsen O.
        • Koch C.
        • Steinhilber W.
        • Holmskov U.
        Expression and localization of lung surfactant protein A in human tissues.
        Am J Respir Cell Mol Biol. 2003; 29: 591-597
        • Wert S.E.
        • Whitsett J.A.
        • Nogee L.M.
        Genetic disorders of surfactant dysfunction.
        Pediatr Dev Pathol. 2009; 12: 253-274
        • Schmidt A.F.
        • Kemp M.W.
        • Kannan P.S.
        • et al.
        Antenatal dexamethasone vs. betamethasone dosing for lung maturation in fetal sheep.
        Pediatr Res. 2017; 81: 496-503
        • Waljee A.K.
        • Rogers M.A.
        • Lin P.
        • et al.
        Short term use of oral corticosteroids and related harms among adults in the United States: population based cohort study.
        BMJ. 2017; 357: j1415
        • Savoy C.
        • Ferro M.A.
        • Schmidt L.A.
        • Saigal S.
        • Van Lieshout R.J.
        Prenatal betamethasone exposure and psychopathology risk in extremely low birth weight survivors in the third and fourth decades of life.
        Psychoneuroendocrinology. 2016; 74: 278-285
        • Busada J.T.
        • Cidlowski J.A.
        Mechanisms of glucocorticoid action during development.
        Curr Top Dev Biol. 2017; 125: 147-170
        • Braun T.
        • Sloboda D.M.
        • Tutschek B.
        • et al.
        Fetal and neonatal outcomes after term and preterm delivery following betamethasone administration.
        Int J Gynaecol Obstet. 2015; 130: 64-69
        • 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.
        Am J Obstet Gynecol. 2018; 219: 301.e1-301.e16
        • World Health Organization
        WHO recommendations on interventions to improve preterm birth outcomes.
        (Available at:) (Accessed March 1, 2021)
        • Cañadas O.
        • Olmeda B.
        • Alonso A.
        • Pérez-Gil J.
        Lipid-protein and protein-protein interactions in the pulmonary surfactant system and their role in lung homeostasis.
        Int J Mol Sci. 2020; 21: 3708
        • Klein J.M.
        • McCarthy T.A.
        • Dagle J.M.
        • Snyder J.M.
        Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro.
        Am J Physiol Lung Cell Mol Physiol. 2002; 282: L386-L393
        • Khubchandani K.R.
        • Snyder J.M.
        Surfactant protein A (SP-A): the alveolus and beyond.
        FASEB J. 2001; 15: 59-69
        • Bridges J.P.
        • Sudha P.
        • Lipps D.
        • et al.
        Glucocorticoid regulates mesenchymal cell differentiation required for perinatal lung morphogenesis and function.
        Am J Physiol Lung Cell Mol Physiol. 2020; 319: L239-L255