Cell-free DNA release by mouse placental explants

Although suggested that “fetal” cell-free DNA (cfDNA) is derived from trophoblast cells, the exact origin is unclear. The studies in this report sought to demonstrate that placental tissue releases cfDNA in parallel with cell death, that the size range of cfDNA is similar to that found in maternal plasma, and that the cfDNA fragments are able to stimulate a proinflammatory cytokine response. Placentas were harvested from near term pregnant CD-1 mice and cultured in DMEM/Ham’s F12/FBS media in 8% or 21% O(2). After centrifugation to remove cells and cellular debris, the cfDNA was extracted from the media and quantified by DNA spectrophotometry. The cfDNA fragments were sized using a 1.5% TAE gel. Cell death was quantified by lactate dehydrogenase assay; and tissue homogenates were used to quantify caspase activity and BAX expression. Cultured RAW-264.7 macrophage cells were used to determine IL6 stimulation by cfDNA. The cfDNA levels released in 8% O(2) (placental normoxia) were not significantly different from explants cultured in 21% O(2) (placental hyperoxia). The cfDNA fragments ranged in size from < 100 –< 400 bp. The cfDNA release increased when cultured with LPS, whereas it decreased with trolox (vitamin E analog). Explant release of cfDNA increased in parallel with cell death. The cfDNA release and cell death of trophoblast appears to involve components of the apoptosis signaling pathway as suggested by LPS enhancement of placental caspase activity, suppression of cfDNA release by a pan-caspase inhibitor and the trend toward increased Bax protein expression. Studies with cultured macrophage cells confirmed the ability of cfDNA to stimulate an IL6 response. In summary, these studies have confirmed the ability of placental tissue to release significant amounts of cfDNA, a phenomenon that appears to be mediated, at least in part, by apoptosis; and that the cfDNA released by the placental explants is able to stimulate a significant proinflammatory response. Thus, these studies provide support for the hypothesis that cell-free fetal DNA released by placental tissue potentially plays a mechanistically important role during the events leading to the onset of parturition.