Dynamic Temporal Changes in Urinary Proteome of Pregnant Wild-Type Mice Carrying Heterozygous 3xTg-AD Fetuses

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with insidious onset. At present, effective early diagnostic biomarkers are scarce, and few studies have explored ultra-early molecular alterations during embryonic development. Urinary proteomics boasts unique strengths including complete non-invasiveness, repeatable sequential sampling and high detection sensitivity, which provides a potential technical strategy for prenatal monitoring of congenital disorders. In this study, we constructed an experimental group by mating wild-type female mice with 3xTg transgenic male mice to obtain pregnant dams carrying heterozygous AD-susceptible fetuses, while wild-type male-female mating was set as the blank control. Urine samples were consecutively collected at 10 time points from gestational day 1 (D1) to D19. Label-free quantitative proteomics was adopted to screen differentially expressed proteins, and Gene Ontology (GO) enrichment analysis was carried out to interpret temporal biological processes. The results showed that stable intergroup differential proteins could be detected as early as the implantation stage (D1), and differential protein profiles existed throughout the whole gestation period. The quantity and expression trend of differential proteins exhibited obvious temporal dynamics, and permutation tests verified that the intergroup differences were not random noise. Paternally inherited AD-causing mutations could trigger systematic molecular responses in maternal mice at the early embryonic stage. This study for the first time characterized the dynamic urinary proteomic landscape of maternal mice that reflects fetal AD susceptibility. It demonstrates that maternal urine can mirror molecular signatures related to fetal AD development, offering fundamental animal experimental data for subsequent screening of prenatal non-invasive monitoring biomarkers and research on the embryonic origin of AD.