Objective
Glucocorticoid administration to women in preterm labor improves neonatal mortality and morbidity. Fetal exposure to glucocorticoid levels higher than those appropriate to the current gestational stage has multiple organ system effects. Some, eg, fetal hypertension, are maximal at lower than the clinical dose. We hypothesized that the clinical dose has supramaximal lung maturational effects.
Study Design
We evaluated the full, half, and quarter clinical betamethasone dose (12 mg/70 kg or 170 μg/kg intramuscularly twice 24 hours apart) on fetal sheep lung pressure volume curves (PVC) after 48 hours’ exposure at 0.75 gestation. We measured key messenger RNAs and protein products that affect lung function and total lung dipalmitoyl phosphatidyl choline.
Results
Full and half doses had similar PVC and total lung dipalmitoyl phosphatidyl choline effects. Messenger RNA for surfactant proteins A, B, and D and elastin increased in a dose-dependent fashion.
Conclusion
Half the clinical betamethasone dose produces maximal PVC improvement in fetal sheep at 0.75 gestation.
Preterm birth occurs in 10% of live births and is a major cause of neonatal mortality and morbidity that results primarily from pulmonary immaturity and intraventricular hemorrhage. The National Institutes of Health recommend routine glucocorticoid administration to women at risk of premature delivery between 24–34 weeks’ gestation to accelerate fetal lung maturation and decrease the incidence of complications of pulmonary immaturity. Antenatal glucocorticoid therapy reduces infant mortality approximately 30%, neonatal respiratory distress syndrome approximately 50%, and both intracranial hemorrhage and periventricular leukomalacia approximately 70%.
Glucocorticoids improve maximal neonatal lung volume by increasing lung compliance as a result of increased surfactant protein (SP) production and accelerated cytoarchitectural maturation. Lung compliance is primarily determined by the surface tension of the surfactant film and mechanical properties of the continuous fiber network forming the structural skeleton of the pulmonary interstitium. Maturation of the fetal lung involves the mesenchyme as well as the SP.
Since synthetic glucocorticoids rapidly cross the placenta, their administration to the pregnant mother inevitably exposes the fetus to glucocorticoid concentrations that are inappropriate for the current stage of fetal maturation. Indeed, this is the purpose of the treatment. Extensive studies have indicated that this exposure results in an altered trajectory of development of several fetal systems. Maternal administration of betamethasone (BM) to the pregnant baboon, in weight-adjusted doses equivalent to those given to women in premature labor, elevates fetal blood pressure and decreases key neuronal proteins in the fetal brain.
The present study determined in vivo changes following 48-hour exposure to BM at the full, half, and quarter dose administered to women in premature labor on fetal lung pressure volume curves (PVC) in sheep, the species in which the original observation was made by Liggins that glucocorticoids accelerate maturation of the fetal lung. We also evaluated changes in messenger RNA (mRNA) for SP A, B, C, and D and elastin and the glucocorticoid receptor and protein changes by Western analysis for SP A, C, and D. We hypothesized that the routine therapy of 2 12-mg doses of BM phosphate alone given intramuscularly to the mother 24 hours apart is supramaximal for fetal lung maturation.
Materials and Methods
Care and use of animals and tissue collection
We studied a total of 30 pregnant Rambouillet-Columbia ewes of known gestational age. BM phosphate (170 μg/kg, n = 8; 85 μg/kg, n = 8; 42.5 μg/kg, n = 7; or vehicle, n = 7 injected intramuscularly) was administered at 8:00 am on 2 consecutive days beginning at 110 days’ gestation. The 170-μg/kg starting point was chosen, as it represents the dose given to an “average” 70-kg woman (12 mg/70 kg). All procedures were approved by the University of Wyoming Institutional Animal Care and Use Committee.
Cesarean section
At 48 hours after the initial BM dose, the fetus was removed by cesarean section under general anesthesia. Pregnant sheep were tranquilized with ketamine hydrochloride (10 mg/kg). Following intubation, isoflurane (2%, 2 L/min) was administered to the ewe to maintain a surgical plane of anesthesia throughout surgery. The maternal abdomen and uterus were opened and the fetus was euthanized by exsanguination while still under isoflurane general anesthesia. In the cases of twin pregnancy the second fetus remained in utero until processing of the first was complete, at which time the second was euthanized in the same manner. The mother was euthanized by intravenous administration of euthanasia solution (390 mg pentobarbitone, 50 mg/mL phenytoin) at 10 mL/50 kg. In the 15 of the 30 ewes with twin fetuses, 1 fetus was chosen at random to be the first examined.
PVC
The fetal chest was opened by sectioning the ribs on both sides of the sternum. The thoracic cavity was then flushed with cold saline at 4°C to slow mRNA and protein degradation. The fetal trachea was cannulated with polyvinyl tubing that was advanced until the tip was just above the carina. The fetal lungs were inflated with air to produce 5-cm increments in pressure until 40 cm H 2 O were reached. The air volume required to produce the increase in pressure on the inflation curve was recorded in 5-cm pressure differences. The process was reversed for deflation. Lung samples were then taken from the middle right lobe for molecular biology and protein analysis. The left lobe was fixed in 4% phosphate-buffered paraformaldehyde solution.
Quantitative RT-polymerase chain reaction
Lung total RNA was isolated from each sample using Tri Reagent (Sigma, St. Louis, MO). RNA concentration was determined spectrophotometrically at 260 and 280 nm. Primers for amplification of the 6 genes studied were designed using Beacon Designer software (Bio-Rad, Hercules, CA) from GenBank human complimentary DNA (cDNA) sequences. Primer uniqueness was established using BLAST (National Center for Biotechnology Information, NCBI). Table 1 shows each gene name, gene identification, GenBank accession number, forward and reverse primer sequences (5–3′) with the primer first base number, polymerase chain reaction (PCR) product length, primer melting temperature, and primer annealing temperature.
| Gene | Gene identification | Accession no. | Primer sequence | Length | Tm | Ta |
|---|---|---|---|---|---|---|
| Elastin | ELN | M26188 | 27-CTCGGAGTTGGAGGACTG | 203 | 91 | 61 |
| 229-AAGGGCTTGGGAGGTTTG | ||||||
| SP A | SFTPA1 | AF076633 | 563-CCATTACCAGCATCGTGAAG | 200 | 87 | 61 |
| 762-CAGGCAGTTCTTGTCATTCC | ||||||
| SP B | SFTPB | AF107544 | 293-AGGAGTGCGATGTTCTTC | 200 | 89 | 61 |
| 492-CATCTTGTCCAGCAGAGG | ||||||
| SP C | SFTPC | AF076634 | 124-ATCGTGGTTGTGGTTGTG | 260 | 89.5 | 61 |
| 383-ATAATGTAGCAGCAGGTTCC | ||||||
| SP D | SFTPD | AJ133002 | 13-CGGAGTGTCGGGAAGAAG | 184 | 89 | 54 |
| 196-TGGTATCGGTCATGCTCAG | ||||||
| GR | NR3C1 | S44554 | 268-TTGGCAGGAGAGGATGATTC | 396 | 85 | 61 |
| 663-AACAGGAATTGGTGGAATGAC |
First-strand synthesis was performed using 250 ng of total RNA, 100 ng of random hexamers, and 50 U of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). A control reaction lacking reverse transcriptase was included. After cDNA synthesis, RNA was removed by incubation with 2 U of RNase H at 37°C for 20 minutes. Quantitative PCR was performed as described previously using an iCycler iQ real-time PCR detection system (Bio-Rad). All samples were run in triplicate. The 18S rRNA was used as an internal control (Ambion, Austin, TX).
PCR products were size fractionated in 1.3% agarose gels to confirm amplification of a single product of the expected size. PCR product sequences were validated by excision from the gel, purification using QIAquick gel extraction kit (QIAGEN, Valencia, CA), and sequencing by the New York University DNA sequencing facility. Sequences were subjected to a BLAST search (NCBI) to confirm identity with the corresponding GenBank sheep sequences ( Table 1 , accession numbers).
Western blot analysis of SP A, C, and D
Immunoblots for detection of SP A, C, and D were performed as detailed previously using 50 μg of total lung protein. Images were optically scanned (Hewlett-Packard 5200C with HP PrecisionScan software v2.02; Hewlett-Packard, Houston, TX) and digitized. Antigen signal was quantified by pixel density (Un Scan It gel v5.1; Silk Scientific, Orem, UT). Quantification was performed only after linearity was established between amount of protein and film exposure time. Pixel density data from all images are presented as the ratio of SP to β-actin.
Measurement of dipalmitoyl phosphatidyl choline
Dipalmitoyl phosphatidyl choline (DPPC) analysis was analyzed as in previous studies.
Statistical analysis
Measurements performed in twins were averaged to represent the pregnancy prior to analysis. Differences in treatment groups were compared using analysis of variance and Bonferroni multiple comparison tests comparing BM dose to saline control. Preliminary comparisons showed no differences according to fetal sex, so data were pooled. The distribution of singletons and twins in each treatment group were: control: n = 7 (3,4); 42.5 μg/kg: n = 7 (2,5); 85 μg/kg: n = 8 (2,6); and 170 μg/kg: n = 8 (3,5). Pearson correlations were used to assess the relationship between BM dose and quantitative reverse transcription-PCR (QRT-PCR) data from SP. Data are presented as mean ± SEM. Significance was set at P < .05.
Results
Effects of fetal exposure to BM on fetal body and organ weights
Table 2 provides the fetal bodyweight and organ weights for the 4 groups of animals. There were no significant differences among the groups.
