Preterm birth (PTB) remains a significant global health challenge and a leading cause of neonatal mortality and morbidity. Despite advancements in neonatal care, the prediction of PTB remains elusive, in part due to complex etiologies and heterogeneous patient populations. This study aimed to validate and extend information on gene expression biomarkers previously described for predicting spontaneous PTB (sPTB) using maternal whole blood from the All Our Families pregnancy cohort study based in Calgary, Canada. The results of this study are two-fold: first, using additional replicates of maternal blood samples from the All Our Families cohort, we were unable to repeat the findings of a 2016 study which identified top maternal gene expression predictors for sPTB. Second, we conducted a secondary analysis of the original gene expression dataset from the 2016 study, including external validation using a pregnancy cohort based in Detroit, USA. While initial results of our machine learning model suggested promising performance (area under the receiver operating curve, AUC 0.90 in the training set), performance was significantly degraded on the test set (AUC 0.54), and further degraded in external validation (AUC 0.51), suggesting poor generalizability, likely due to overfitting exacerbated by a low feature-to-noise ratio. Prediction was not improved when using machine learning approaches over traditional statistical learning. These findings underscore the challenges in translating biomarker discovery into clinically useful predictive models for sPTB. This study highlights the critical need for rigorous methodological safeguards and external validation in biomarker research. It also emphasizes the impact of data noise and overfitting on model performance, particularly in high-dimensional omics datasets. Future research should prioritize robust validation strategies and explore mechanistic insights to improve our understanding and prediction of PTB.
