Leukocyte mtDNA methylation mediates the association between prenatal ozone exposure and gestational diabetes mellitus: Modification by residential greenness

Prenatal ozone (O 3 ) exposure has been associated with an increased risk of gestational diabetes mellitus (GDM), but the role of mitochondrial epigenetic alterations remains unclear. This nested case–control study investigated the association between prenatal O 3 exposure and GDM, and evaluated the mediating role of mitochondrial DNA (mtDNA) methylation and the modifying effect of residential greenness. A total of 500 pregnant women in Tianjin were included, comprising 250 GDM cases and 250 age-matched controls. Prenatal O₃ exposure was estimated using a spatiotemporal model, and residential greenness levels were characterized by the Normalized Difference Vegetation Index (NDVI). Leukocyte mtDNA methylation was quantified using bisulfite pyrosequencing. Conditional logistic regression was used to assess the associations. Joint effects of O 3 exposure and NDVI were evaluated by dichotomizing both variables at the median. Mediation and effect modification analyses were also conducted. Each 10 μg/m³ increase in O₃ exposure during the 1st and 2nd trimesters was associated with 34% ( OR = 1.34, 95% CI: 1.17–1.55) and 34% ( OR = 1.34, 95% CI: 1.17–1.53) higher odds of GDM, respectively, whereas higher NDVI was associated with reduced risk. Women exposed to high O 3 and low NDVI had a higher risk of GDM. Mediation analyses suggested that MT-COX1-Pos.1 and MT-COX3-Pos.1 methylation accounted for 7.93% and 5.87%, respectively, of the association between 2nd trimester O 3 exposure and GDM. NDVI modified the association between MT-COX1-Pos.1 methylation and GDM. Leukocyte mtDNA methylation may be a potential mediator of the association between prenatal O 3 exposure and GDM, while greenness may attenuate this association.