Patients and drug administration

This was a planned ancillary study to the Maternal-Fetal Medicine Units Network randomized trial of women with twins who were receiving either intramuscular 17-OHPC or placebo. A total of 661 women with twins were recruited for the randomized trial. Subjects received masked weekly injections of either 250 mg of 17-OHPC in 1 mL of castor oil or 1 mL of castor oil alone from the time of enrollment (16 0/7 to 20 6/7 weeks) until 34 weeks 6 days or until delivery, whichever came first.

Blood sampling schedule

Consenting subjects were informed that 2 blood samples would be taken, 1 at 24-28 weeks (epoch 1) and 1 at 32-35 weeks (epoch 2) for determination of progesterone, 17-hydroxyprogesterone (17-OHP), 17-OHPC, CRP, and CRH concentrations. The timing of epoch 1 was intended to obtain a blood sample after a minimum of 4 injections would have been administered to allow steady state to be reached. The timing of the second epoch was intended to evaluate gestational age–related changes in pharmacodynamic parameters.

Sample analysis

For measurement of 17-OHPC, progesterone, 17-OHP, and CRP, blood was collected in 10-mL tubes with anticoagulant and centrifuged within 1 hour at 2500 rpm for 10 minutes. The supernatant plasma was aliquoted to 1-mL tubes and was frozen at −70°C until analysis. Quantification of 17-OHPC, progesterone, and 17-OHP concentration was performed using high-performance liquid chromatography with tandem mass spectrometry. The assay methodology has been reported elsewhere. The lower assay limit of detection for 17-OHPC was 1 ng/mL; interassay and intraassay variability at 10 ng/mL was 7.9% and 5.2%, respectively. For progesterone and 17-OHP, the lower limits of detection were 2 ng/mL and 1 ng/mL, respectively. The interassay and intraassay variabilities for progesterone (5 ng/mL) were 7.7% and 8.4%, respectively, and 12.3% and 9.5% for 17-OHP. At the time of the analysis, the analyst and the clinical centers involved in recruitment were blinded to the treatment assignment.

CRP in maternal plasma was measured with a commercially available enzyme-linked immunosorbent assay kit (Alpha Diagnostic International, San Antonio, TX), with a coefficient of variation of 3.0%. Separate linear standard curves were generated for each sample plate, and those values that were greater than the maximum concentration of the standard were repeated after progressive dilutions.

For CRH measurements, blood was collected in a 5-mL chilled tube with EDTA and aprotinin (500 KIU/mL blood), then centrifuged at 2500 rpm for 10 minutes at 4°C. Aliquots of the supernatant were transferred to a polypropylene tube then stored at −70°C until analyzed. CRH peptide was extracted from plasma using the method of Linton et al. The dried extracts were resuspended in 0.5 mL, 0.005 mol/L phosphate buffer, pH 7.3, containing 5.8 g/L sodium chloride, 9.5 g/L ethylenediamine tetracetate, 1.0 g/L sodium azide, and 1 mL/L Triton and transferred to a storage vial. Vials were rinsed with another 0.5-mL buffer to recuperate residual peptide. Pooled aliquots of the reconstituted extracts were stored at −80°C and assayed in batches. Each methanol extraction consisted of spiked plasma controls from a healthy male donor. Purified, CRH peptide (Peninsula Laboratories, San Carlos, CA) was adjusted to 5 ng/mL and 2.5 ng/mL in the donor plasma to serve as both assay controls and as an extraction reference standard. Because methanol extraction efficiency varies from batch to batch, all CRH test sample values were adjusted accordingly. For example, the 5-ng control following extraction was tested at 4.5 ng CRH (an extraction efficiency of 90%, assuming the detection method is complete). In this case, all test samples would be adjusted up by a factor of 1.11 in this batch to compensate for incomplete CRH extraction.

For the enzyme immunoassay (Peninsula Laboratories), anti-CRH antibodies were plated on a 96-well plate. In each well, a known concentration of biotinylated tracer was coincubated for 2 hours at 21-23°C with antihuman CRH and 50 μL of sample. Following a wash step, streptavidin-conjugated horseradish peroxidase was added to each well and the incubation continued for 1 hour. The wells were again washed and the substrate tetramethyl benzidine dihydrochloride was added. After 45 minutes, the reaction was stopped with 2 N hydrochloric acid and the absorbance was read at 450 nm. The data were plotted against a CRH standard S-shaped curve on a semi-log plot, which was provided as a spreadsheet (Excel; Microsoft, Redmond, WA) by the Bachem Group, a member of Peninsula Laboratories. In our laboratory, the intraassay and interassay variability of the CRH enzyme immunoassay is comparable to other commercial immunoassay kits: ≤10%.

Statistical analysis

For all analyses, we excluded women who did not receive all of their scheduled injections up to the time of the first blood draw (epoch 1). For analyses involving data from the second blood draw, we also excluded women who did not receive all of their injections between the first and second blood draw. For analyses related to gestational age at delivery, we excluded all women who did not receive all their scheduled injections up to the time of delivery or until 35 weeks gestation. Rate of change for each analyte was calculated as the difference between the first and second epoch value divided by time elapsed in weeks between blood draws. The χ ² Wilcoxon tests were used to make univariable 2-group comparisons for categorical and continuous variables, respectively. Spearman rank correlation coefficients were calculated to assess the pairwise correlation between continuous variables. Proportional hazards regression was used to analyze the relationship between the analytes and gestational age at delivery, adjusting for gestational age at the time of blood draw, as well as for race/ethnicity and body mass index (BMI), which may impact plasma 17-OHPC concentrations. Indicated deliveries were censored at the time of delivery. For the multivariable analysis, monochorionic-diamniotic pregnancies were excluded because placentation, progesterone levels, and endogenous steroid hormones could affect gestational age at delivery compared with dichorionic-diamniotic pregnancies. For the proportional hazards models assessing gestational age at delivery vs CRP and vs progesterone, 17-OHPC was also included as a covariate because 17-OHPC concentration increases with gestational age as a consequence of greater number of castor oil depots seen with weekly injections. Generalized R ² values were calculated to estimate the proportion of explained variation. Scatter plots were used to demonstrate relationships visually. For all statistical tests, nominal 2-sided P values are reported with statistical significance defined as a P value < .05. No corrections were made for multiple comparisons. SAS Software (SAS Institute, Cary, NC) was used for these analyses.

Characteristics of the study population

Among the 661 subjects enrolled in the main trial, 217 agreed to the study, received all scheduled injections, and had blood drawn at epoch 1; 118 in the placebo group and 99 in the 17-OHPC group. Table 1 compares demographic parameters of these subjects at enrollment according to treatment group. Gestational age at delivery is also provided. There were no significant differences between women treated with 17-OHPC or placebo. A total of 92 of these women in the placebo group and 72 in the 17-OHPC group had received all of their injections and had blood drawn by epoch 2. A median of 7 injections had been administered by epoch 1 and 14 by epoch 2 in each of the 2 groups. The gestational age at which blood samples were obtained was similar in the 2 treatment groups at the first sampling but there was a significant ( P = .04) difference of 4 days in gestational age at the second blood sampling.

Comparison of plasma CRH, CRP, progesterone, and 17-OHP concentrations by treatment group

Table 2 compares plasma CRH, CRP, progesterone, and 17-OHP concentrations at each of the 2 epochs and the rate of change of these hormones between the 2 sampling times according to treatment group. The plasma concentrations of 17-OHPC at each epoch are also provided.

CRH concentrations and the rate of change of CRH concentration were not significantly different. CRP was significantly higher in epoch 1 in the 17-OHPC group when compared with placebo. At epoch 2 however, plasma CRP concentrations were similar in the 2 treatment groups. Progesterone and 17-OHP concentrations were not significantly different at epochs 1 and 2 between the 17-OHPC and placebo groups and neither were the rates of change. Concentrations of 17-OHPC increased slightly from epoch 1 to epoch 2. The drug was not detected in women receiving placebo.

Relationship of 17-OHPC with selected biomarkers of preterm birth

We evaluated the relationship between 17-OHPC concentration and CRH, CRP, progesterone, and 17-OHP. Table 3 summarizes these relationships. A highly significant positive monotonic relationship is seen between 17-OHPC concentration and progesterone concentration, as well as 17-OHPC concentration and 17-OHP at both sample times. These relationships at epoch 2 are depicted in Figure 1 for progesterone and its metabolite 17-OHP. The rate of change in progesterone concentration likewise is positively related to the concentration of 17-OHPC.

Relationship of 17-OHPC concentration with progesterone and 17-OHP concentrations

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