Characterization of Ultra-Short plasma Cell-Free DNA in maternal blood and its preliminary potential as a screening marker for preeclampsia.

BACKGROUND: Cell-free DNA (cfDNA) in maternal blood is the foundation for non-invasive prenatal screening (NIPS). Recently, ultra-short cfDNA fragments, ranging from 30 to 70 base pairs (bp), have been identified and show promise in cancer diagnostics. However, the characteristics of ultra-short cfDNA in maternal blood during pregnancy remain unexplored. This study aims to characterize these fragments in pregnancy and evaluate their potential as biomarkers for preeclampsia. METHODS: Ultra-short cfDNA was isolated from the plasma of pregnant and non-pregnant women, and next-generation sequencing (NGS) was constructed. We performed deep sequencing to profile ultra-short cfDNA during pregnancy, including a cohort of women with preeclampsia, to identify distinct ultra-short peaks. These features were used to build a diagnostic model, which was validated in a separate testing cohort. RESULTS: Sequencing data revealed that ultra-short cfDNA in maternal blood primarily originates from accessible open chromatin regions in blood and placental cells. A significant proportion of these fragments exhibited potential G-quadruplex (G4) motifs on the antisense strand. Significant differences in ultra-short cfDNA features were observed between women with preeclampsia and healthy controls. Based on these features, the diagnostic model achieved an area under the curve (AUC) of 0.90 in the training cohort and 0.86 in the test cohort. CONCLUSIONS: This study comprehensively characterizes ultra-short cfDNA in maternal blood and suggests its preliminary potential as a diagnostic marker for early preeclampsia detection. TRIAL REGISTRATION: Retrospectively registered. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s10020-025-01307-1.