Hula, N. | bioRxiv Science

bioRxiv2025

Gestational Chronic Intermittent Hypoxia Triggers Maternal Inflammation and Disrupts Placental Stress Responses

Gestational hypoxia is associated with placental cellular responses, including oxidative stress and inflammation. Circulating cell-free mitochondrial DNA (ccf-mtDNA) is a marker of cell stress, that can be transported within extracellular vehicles (EVs), eliciting proinflammatory responses. We hypothesized that systemic exposure to chronic intermittent hypoxia (CIH) during late pregnancy would increase maternal inflammation, alter circulating EV characteristics, and disrupt placental stress responses. Pregnant rats were exposed to CIH (n=8) or normoxia (n=9) during gestational days 15-20 (term 22-23 days). On GD20, ccf-mtDNA and EV-associated mtDNA (EV-mtDNA) were quantified with qRT-qPCR, while maternal circulating cytokines were quantified using a MILLIPLEX(R) cytokine array. Systemic oxidative stress was measured by plasma advanced oxidation protein products (AOPP). Placental stress responses were evaluated by examining the balance between proinflammatory and antioxidant gene expression and the activation of proteins involved in apoptotic and autophagic processes. CIH exposure increased placental weights (p=0.015) and reduced placental efficiency (p=0.0006) without affecting fetal biometrics (p>0.05). Absolute ccf-mtDNA and EV-mtDNA content were unchanged (p>0.05), but EV concentrations were reduced (p=0.011) in response to CIH, suggesting an increase in EV-mtDNA per EV. Maternal interleukin-18 (IL-18) concentrations increased in the CIH group (p=0.047). Placental mRNA expression of catalase (p=0.048) and sod2 (p=0.038) were upregulated, while autophagy-related proteins Beclin-1 (p=0.006) and p62 (p=0.023) were also increased in response to CIH, with no changes in LC3A/B expression (p>0.05). Gestational CIH disrupts maternal EV and inflammatory profiles, reduces placental efficiency, and modulates placental antioxidant and autophagic mechanisms, without impairing fetal growth in rats.

physiology↗

bioRxiv2024

Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA differs in pregnancies with placental dysfunction from healthy pregnancies and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA from non-placental cells; yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 M) or rotenone (0.2-50 M) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane bound, non-membrane bound, and vesicular-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (p<0.0001), induced cell necrosis (p=0.0004) but not apoptosis (p=0.6471) and was positively associated with release of membrane-bound and non-membrane bound mtDNA (p<0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicular-bound form; p=0.0019) and reduced autophagy marker expression (LC3A/B, p=0.0002; p62, p<0.001). Rotenone treatment did not influence mtDNA release or cell death (p>0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes non-apoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress. NEW & NOTEWORTHYThis is the first study to test whether trophoblast cells release mitochondrial DNA in response to oxidative stress and to identify mechanisms of release and biological forms of mtDNA from this cellular type. This research identifies potential cellular mechanisms that can be used in future investigations to establish the source and biomarker potential of circulating mitochondrial DNA in preclinical experimental models and humans.

cell biology↗