BackgroundDespite extensive research, the identification of effective biomarkers for early prediction of preterm birth (PTB) continues to be a challenging endeavor. This study aims to identify amniotic fluid (AF) protein biomarkers useful for the early diagnosis of PTB.MethodsWe initially identified the protein expression profiles in the AF of women with PTB (n = 22) and full-term birth (FTB, n = 22), from the First People's Hospital of Yunnan Province who underwent amniocentesis from November 2019 to February 2020, using mass spectrometry employing the data-independent acquisition (DIA) technique, and then analyzed differentially expressed proteins (DEPs). Subsequently, the least absolute shrinkage and selection operator (LASSO) and random forest analysis were employed to further screen the key proteins for PTB biomarker identification. The receiver operating characteristic (ROC) analysis, calibration plots, and decision curve analyses (DCA) were utilized to assess the discrimination and calibration of the key biomarkers.ResultsA total of 25 DEPs were identified between the PTB and FTB groups, comprising 13 up-regulated and 12 down-regulated proteins. Three key protein biomarkers for early PTB diagnosis were identified: IL1RL1 (interleukin-1 receptor-like 1), APOE (apolipoprotein E), and NECTIN4 (nectin cell adhesion molecule 4). The results of the ROC analysis showed that the area under the curve (AUC) of the three proteins combined as a biomarker for early diagnosis of PTB was 0.913 (95% CI: 0.823-1.000), with a sensitivity of 0.864 and a specificity of 0.955, both superior to those of the individual biomarkers. Bootstrap internal validation revealed a concordance index (C-index) of 0.878, with a sensitivity of 0.812 and a specificity of 0.773, indicating the robust predictive performance of these biomarkers.ConclusionsWe identified three previously unexplored yet potentially useful protein biomarkers in AF for early PTB diagnosis: IL1RL1, APOE, and NECTIN4.