Trophoblast cells exposed to hypoxia and oxidative stress release mitochondrial DNA and undergo apoptosis




Blessing, Alexandra
Cushen, Spencer
Chaudhari, Sarika
Patil, Shruti
Phillips, Nicole R.
Goulopoulou, Styliani


Journal Title

Journal ISSN

Volume Title



  1. Purpose Preeclampsia is a syndrome of high blood pressure with end organ damage diagnosed after the 20th week of pregnancy. This syndrome is associated with placental ischemia, oxidative stress, and exaggerated rates of placental cell death. Mitochondrial DNA (mtDNA) that is released in the extracellular space due to mitochondrial damage or cell death has potent pro-inflammatory and pro-immunogenic properties. Circulating mtDNA is increased in plasma from women with preeclampsia compared to healthy pregnant women; however, the cellular origin of mtDNA is unknown. This leads to our hypothesis that trophoblast cells exposed to hypoxic and oxidative stress release mitochondrial DNA and undergo apoptosis.
  2. Methods Human trophoblast cells (BeWo choriocarcinoma cell line) were grown to 80-90% confluency before treatment with: 1) hypoxia (1% O2) vs. normoxia (21% O2) for 6, 15, or 24 h, or 2) an oxidative stress inducer (H2O2, 200 μM for 4 h or 24 h), a mitochondrial complex I inhibitor (Rotenone, 5 μM for 4 h), or untreated control. Absolute real-time PCR quantification of mtDNA was measured on total nucleic acid extracts (Omega Mag-Bind® Blood & Tissue DNA HDQ Kit) from cell culture supernatants using TaqMan® probes and chemistry. Apoptosis was quantified via double staining for Annexin V and propidium iodide using flow cytometry.
  3. Results Concentrations of mtDNA were higher in supernatants from BeWo cells exposed to hypoxia than those exposed to normoxia for 15 h (normoxia: 14.2 pg/μL ± 1.2, n = 3 vs. hypoxia: 20.6 pg/μL ± 0.4, n = 3; p = 0.02). BeWo cells treated with H2O2 showed no increase in mtDNA release after 4 or 24 h (p ≥ 0.34). Cells exposed to hypoxia did not exhibit increased apoptosis after 6, 15, or 24 h (p ≥ 0.25). Incubation with H2O2 for 18 h resulted in increased apoptosis (Untreated control: 15.6%, n = 1 vs. H2O2: 32.5%, n = 1). Treatment with rotenone resulted in increased BeWo cell apoptosis (Untreated control: 18.1% ± 2.6, n = 2 vs. Rotenone: 35.5% ± 2.5, n = 2).
  4. Conclusions Extracellular mtDNA was increased in a trophoblast cell culture exposed to hypoxia, but not when exposed to H2O2. Apoptosis was not increased when cells were exposed to hypoxia but was increased after exposure to inducers of oxidative stress. Future studies will investigate the mechanism by which hypoxia results in release of mtDNA, with focus on determining whether mtDNA release is independent of apoptosis.