Statistical analysis
We compared groups according to study stages and sub-categories of
preterm birth using Welch’s One-way Analysis of Variance (ANOVA) or
Kruskal-Wallis rank-sum test according to variable distribution, and
Mann-Whitney U for term and preterm delivery groups. Categorical
variables and frequencies were compared between groups using chi-squared
tests. A p-value <0.05 was established as the threshold for
statistical significance. For details on variable transformation and
imputation for benefit in models’ assumptions, see Supporting
Information.
To characterize a cytokine profile in CVF that describes clinical
manifestations in labor stages, a multiple principal-component analysis
(PCA) was used using cytokine concentrations centered with mean 0 and
standard deviation of 1. To choose the components to retain we used the
scree plot criterion.
We selected cytokines which better described stages and principal
component scores were extracted to develop explanatory models (linear
mixed-effects models) to predict pro-inflammatory signaling associated
with the onset of labor using stages as independent variable and
gestational age (sample collection
week) as the moderating variable.
Model diagnostics were conducted using R2 and
minimization of the Bayesian Information Criterion (BIC);
multicollinearity was assessed using tolerance and Variance Inflation
Factor (VIF). Predictors were subjected to homoscedasticity and
linearity tests; model diagnostics were performed by assessing normality
of the residuals. Model parameters were expressed using β coefficients
and 95% CI.
To test IL-6 diagnostic performance for identification of spontaneous
labor, we calculated an optimal cut-point using maximization of Youden’s
J index, and estimated the respective sensitivity, specificity,
predictive values, and likelihood ratios using theOptimalCutpoints R package. In addition, we evaluated their
time-varying diagnostic performance using time-dependent ROC curves
applying the Kaplan-Meier estimator at different time-points using thetimeROC R package.
Intervals between sampling and delivery were calculated and the
estimated cut-point was evaluated by Kaplan–Meier analysis and log-rank
test using the survminer R package. Cox regression analysis was
used to assess whether IL-6 was associated with risk of delivery at any
gestational age. Schoenfeld residuals were used to test the proportional
hazards assumption. The predictors were tested on homoscedasticity and
linearity assumptions. Finally, a post-estimation simulation of the Cox
models was performed to evaluate adjusted hazard ratio estimates across
IL-6 values using the simPH R package. All statistical analyses
were performed using R statistical software (Version 4.0.2).