Betamethasone

The expected rate of the primary outcome in the placebo group was estimated from a pilot study of infants born in the late preterm period, adjusted downward to account for the proportion of women at risk for late preterm delivery who deliver at term (20-40%), as preterm delivery cannot reliably be predicted. We estimated that a sample size of 2,800 women would be required to detect a one third decrease in the primary outcome rate from 9.5% in the placebo arm to 6.3% in the steroid arm, with type 1 error of 5%, 2-sided, and power of at least 85%. Details are in the Supplementary Appendix.
Analyses were performed according to the intention-to-treat principle. We compared continuous variables using the Wilcoxon test and categorical variables using the chi-square test, and Fisher's exact test. An independent data and safety monitoring committee monitored the trial. A group sequential method was used to control the Type I error rate using the Lan-DeMets characterization of the O'Brien-Fleming boundary.^{14 (link)} Two interim analyses were performed; in the final analysis of the primary outcome, a two-tailed P value of less than 0.048 was considered to indicate statistical significance. Since the adjustment is minimal we report the 95% confidence interval for the relative risk. For all secondary outcomes, a nominal P value of less than 0.05 was considered to indicate statistical significance, without adjustment for multiple comparisons; relative risks and 95% confidence intervals are reported. To determine whether there was a differential effect of betamethasone for the primary and severe respiratory composite outcomes within the pre-specified subgroups, the Breslow–Day interaction test was conducted; where a nominal P value less than 0.05 was considered to indicate statistical significance.

Eligible and consenting women were randomly allocated in a 1:1 ratio to a course of two intramuscular injections containing either 12 mg of betamethasone or matching placebo, 24 hours apart. The randomization sequence was prepared by the independent data coordinating center according to the simple urn method,¹² with stratification by clinical site and gestational age category (34 to 35 weeks versus 36 weeks). Each participant's supply of study medication was packaged according to this sequence. Neither the participants nor the investigators were aware of treatment assignments. During the trial, we changed the company manufacturing the placebo and packaging the study medication. This resulted in suspension of recruitment until a new company was identified.
After administration of the study medication, the women were managed clinically according to local practice including discharge home if delivery did not occur and the patient was considered stable. For those enrolled because of an indication for preterm delivery, labor inductions were expected to start by 36 weeks 5 days, and cesarean deliveries were to be scheduled by 36 weeks 6 days and not before 24 hours after randomization. Trained and certified research staff abstracted information from maternal and neonatal charts including demographic information, medical, obstetric, and social history, and outcome data. All infants discharged on oxygen were followed up at 28 days of age to determine whether there was a continuing need for oxygen supplementation.

On P10 or on P15, the offspring prenatally primed with betamethasone or exposed to saline were injected with a single i.p. NMDA injection. For some experiments, NMDA was administered repeatedly on P12, P13, and P15. The NMDA dose was 5.0 mg/kg in P10 rats, 7.5 mg/kg in P12 rats, 12 mg/kg in P13 rats, and 15 mg/kg for P15 rats, based on pilot experiments and our previous publications (Velíšek, et al. 2010 (link), Velíšek, et al. 2007 (link)). Immediately after the NMDA injection, the rats were observed for 90 minutes because our pilot experiments demonstrated that the spasms would completely disappear within this time frame. Latency to onset of spasms was recorded. The number of complete spasms was counted to indicate severity of spasms. In some experiments in P10 and P15 rats, we recorded a time for each spasm for illustration of the temporal clustering of spasms.