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Trimethylamine N\u2010oxide (TMAO) is a gut microbial metabolite closely associated with pyroptosis and PE. This study aims to investigate the role of TMAO in trophoblast cell pyroptosis and explore possible mechanisms.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Western blot and qRT\u2010polymerase chain reaction (PCR) were used to detect the expression levels of ten\u2010eleven translocation 2 (TET2), cytochrome b (CYTB), pyroptosis\u2010related molecules, and mitochondrial proteins. The level of mitochondrial reactive oxygen species (mtROS) was detected by fluorescent probe DCFH\u2010DA. Immunofluorescence was used to measure the level of 5\u2010hydroxymethylcytosine (5hmC). TET2 overexpression\/silencing and CYTB overexpression\/silencing lentiviruses were transfected into HTR8\/SVneo cells, respectively. MitoTEMPO was used to reduce mtROS. TMAO levels in placental tissues were quantified by liquid chromatography\u2010tandem mass spectrometry (LC\u2013MS\/MS), and representative extracted ion chromatograms were analyzed for retention times and peak areas. ELISA was used to further validate TMAO concentrations in placental tissues.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>TMAO is capable of enhancing the expression of proteins related to pyroptosis (including NLRP3, GSDMD, GSDMD\u2010N, Caspase\u20101) as well as inflammatory factors (such as IL\u20101\u03b2, IL\u201018) in HTR8\u2010S\/Vneo cells. Meanwhile, the positive rate of propidium iodide (PI), mtROS levels, and intracellular Ca2+ levels all increased. Electron microscopy results showed an increase in mitochondrial membrane pore numbers, abnormal mitochondrial morphology, and downregulation of the expression levels of mitochondrial proteins nuclear respiratory factor 1 (NRF1), NRF2, peroxisome proliferator\u2010activated receptor gamma coactivator 1\u2010alpha (PGC\u20101\u03b1), and NADH dehydrogenase subunit 2 (ND2). LC\u2013MS\/MS and ELISA analyses revealed significantly elevated TMAO levels in PE placental tissues compared to normal tissues, further supporting the role of TMAO accumulation in placental dysfunction associated with PE. Overexpression of CYTB inhibited TMAO\u2010induced pyroptosis and mitochondrial dysfunction (MDF) in HTR8\u2010S\/Vneo cells, while silencing of CYTB promoted pyroptosis and MDF in HTR8\u2010S\/Vneo cells, but this condition could be partially reversed by MitoTEMPO. TMAO inhibited the expression of TET2 and CYTB and downregulated the level of 5hmc. The results of TET2 overexpression\/knockout indicated that the expression of CYTB was regulated by TET2, and overexpression of TET2 alleviated TMAO\u2010induced pyroptosis and MDF as well as the decrease in 5hmc levels in HTR8\u2010S\/Vneo cells, but this condition could be partially reversed by silencing CYTB.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>In summary, these findings suggest that TMAO induces pyroptosis in HTR8\/SVneo cells through the TET2\u2010CYTB\u2010mtROS pathway, contributing to mitochondrial dysfunction and inflammation. The significant elevation of TMAO levels in PE placental tissues further supports its role in the pathophysiology of PE. Targeting the TET2\u2010CYTB\u2010mtROS pathway may provide a novel therapeutic strategy for the treatment of PE.<\/jats:p><\/jats:sec>","DOI":"10.1111\/jog.16237","type":"journal-article","created":{"date-parts":[[2025,2,17]],"date-time":"2025-02-17T08:50:46Z","timestamp":1739782246000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Trimethylamine N\u2010oxide induces pyroptosis in HTR8\u2010S\/Vneo cells through the ten\u2010eleven translocation 2\u2010cytochrome b\u2010reactive oxygen species pathway"],"prefix":"10.1111","volume":"51","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5753-0358","authenticated-orcid":false,"given":"Lingli","family":"Liang","sequence":"first","affiliation":[{"name":"Institute of Cardiovascular Disease, Key Laboratory for Arteriosclerology of Hunan Province, Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Hengyang Medical College 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