Fetal sex shapes placental inflammatory responses to extracellular mitochondrial DNA

Aberrant changes in circulating cell-free mitochondrial DNA (ccf-mtDNA) across gestation are associated with adverse pregnancy outcomes. Given the inflammatory properties of ccf-mtDNA via pattern recognition receptors such as Toll-like receptor 9 (TLR9), we hypothesized that extracellular mtDNA induces placental inflammation via TLR9 signaling and that this response differs by fetal sex. Pregnant Sprague-Dawley rats were treated intravenously with purified mtDNA (300 g/kg), nuclear DNA (nDNA), saline, and/or the TLR9 antagonist ODN2088 across five studies. Placental responses were evaluated 4 h (Studies 1-3) and 24 h (Study 4) post-treatment; pregnancy and neonatal outcomes were assessed at delivery (Study 5). Exposure to mtDNA, but not nDNA, increased placental il1{beta}, tnf, and il10 mRNA (p < 0.05), establishing response specificity. mtDNA-induced placental inflammation was fetal sex-dependent: mtDNA increased il6 and il1{beta} mRNA in male placentas (p [&le;] 0.0004) but not female placentas, whereas ifn{gamma} was selectively induced in female placentas (p = 0.0004). TLR9 and MyD88 abundance increased in female but not male placentas, and TLR9 antagonism modified selected inflammatory responses with sex-specific patterns. The 4 h inflammatory transcriptional signature resolved by 24 h, whereas mtDNA exposure was associated with a sex-specific shift in antioxidant enzyme expression persisting to 24 h. Despite no effects on gestational length or neonatal biometrics, mtDNA exposure was associated with a higher estimated stillbirth count per litter (IRR = 4.23, 95% CI [0.89, 20.1], p = 0.069). These findings establish extracellular mtDNA as an acute, sex-differentiated placental inflammatory stimulus with partial TLR9 dependence and a potential impact on fetal viability. New & NoteworthyThis study demonstrates that acute exposure to extracellular mtDNA induces placental inflammatory responses in vivo. This response is specific to mtDNA, fetal-sex dependent, and partially mediated by TLR9, with male and female placentas engaging distinct inflammatory signals within hours of exposure. The biological effects extend beyond the initial inflammatory window, with mtDNA exposure producing lasting, sex-specific changes in antioxidant enzyme expression. mtDNA-exposed dams had higher expected stillbirth counts, suggesting extracellular mtDNA may affect fetal viability.