Toll-Like Receptor 4/p65 Signalling Pathway Mediates Recurrent Spontaneous Abortion by Regulating Forkhead Box Protein P3

Background: Although multiple factors contribute to recurrent spontaneous abortion (RSA), recent studies have highlighted a role for regulatory T cells (Tregs) in maintaining immune tolerance during pregnancy. Understanding the intricate relationship between RSA and Tregs offers promising avenues for diagnosis and therapeutic strategies in reproductive medicine. Methods: We successfully established a mouse model of RSA, and all animals were euthanized on gestational day 13. Uterine horns were examined upon dissection to determine the number of viable fetuses and resorption sites, after which the post-implantation loss rate (abortion rate) was calculated. Placental morphology was evaluated via hematoxylin and eosin staining, whereas gene expression was analysed by immunohistochemistry. Treg abundance was determined by flow cytometry. Gene expression and cytokine levels in EL4 cells and patient samples were evaluated by quantitative real-time polymerase chain reaction (qPCR) and Western blotting. Results: The abortion rate of RSA mice was 31.64%. In addition, Treg numbers were reduced in both RSA mice and patients, while the expression of Toll-like receptor 4 (TLR4) and p65 was upregulated, further suppressing forkhead box protein P3 (FOXP3) expression. Blocking p65 expression with small interfering RNA (siRNA) targeting p65 prevented the lipopolysaccharide (LPS)-induced downregulation of FOXP3 in EL4 cells. The TLR4 inhibitor IAXO102 was ineffective at increasing FOXP3 expression in EL4 cells following p65 overexpression p65. Conclusions: The results of this study suggest that activation of the TLR4/p65 signalling in RSA inhibits FOXP3 expression and contributes to abortion. Importantly, the findings indicate that Treg suppression in RSA is p65-dependent.