Increased cell‐free fetal DNA release after apoptosis and sterile inflammation in human trophoblast cells

Abstract

Problem Cell‐free fetal DNA (cffDNA) shed from the placenta can be detected in maternal blood and increases incrementally during gestation. Concentrations are further elevated with pregnancy complications. Specific activators of cffDNA release in such complications have not been identified. Here, we use trophoblast cells from early and term placenta to examine cffDNA release following apoptosis, infection, and sterile inflammatory stress. Method of Study HTR8/SVneo cells were used to model first‐trimester trophoblasts, and term cytotrophoblasts (CTBs) were isolated from placentae collected after uncomplicated deliveries. Trophoblasts were treated with varying concentrations of doxorubicin (DOX), lipopolysaccharide (LPS), or high‐mobility group box protein 1 (HMGB1) for 18 h. Cells or supernatants were quantified for caspase‐3/7 cleavage, pro‐inflammatory cytokine secretion, and cffDNA release. Results Both HTR8/SVneo and CTBs underwent caspase‐3/7 cleavage following DOX treatment, with HTR8/SVneo cells more sensitive to apoptosis than term CTBs. Apoptotic cells released more cffDNA in a dose‐dependent manner. Treatment with LPS resulted in an increase in pro‐inflammatory IL‐6 release, particularly in term CTBs compared to early trophoblasts; however, LPS did not affect cffDNA release. Lastly, while neither cell released more TNF‐α following stimulation with HMGB1, both HTR8/SVneo and CTBs released significantly more cffDNA in the presence of HMGB1. Conclusions These data show that apoptosis and sterile inflammation induced by DOX and HMGB1, respectively, cause an increase in cffDNA concentrations in both first‐trimester and term trophoblasts. Understanding physiologic release of cffDNA during healthy and complicated pregnancy can identify new targets for the diagnosis and treatment of gestational complications.

Fetal DNA Causes Sex-Specific Inflammation From Human Fetal Membranes

“…It is known that cffDNA can stimulate inflammation in several tissues and cell types ( Goldfarb et al, 2018 ; Kazemi et al, 2021 ; Yeganeh Kazemi et al, 2021 ) but its effects have not yet been studied on the human FM. In addition to this, it is also known that the sex of the fetus can influence both the magnitude and the resultant cytokine profile of the inflammatory response, depending on the specific circumstances ( Burns et al, 2015 ; Mitchell et al, 2017 ).…”

“…Increased circulating levels of cffDNA have also been associated with adverse pregnancy outcomes and pathologies such as; preeclampsia (Martin et al, 2014;Kwak et al, 2020), gestational diabetes (Hopkins et al, 2020), and preterm birth (Gomez-Lopez et al, 2020). cffDNA from the trophoblast is also known to increase by sterile inflammation, via HMGB1 (Yaganeh Kazemi et al, 2021), but its ability to generate inflammation itself is contentious. Some studies have clearly shown that it is able to interact with toll-like receptor 9 (TLR9) (Goldfarb et al, 2018) and that immune cells respond to it by producing inflammatory cytokines (Yeganeh Kazemi et al, 2021).…”

“…cffDNA from the trophoblast is also known to increase by sterile inflammation, via HMGB1 (Yaganeh Kazemi et al, 2021), but its ability to generate inflammation itself is contentious. Some studies have clearly shown that it is able to interact with toll-like receptor 9 (TLR9) (Goldfarb et al, 2018) and that immune cells respond to it by producing inflammatory cytokines (Yeganeh Kazemi et al, 2021). However, others have not been able to confirm this (van Boeckel et al, 2020).…”

“…cffDNA released from the apoptotic and necrotic cells of placental origin into the maternal circulation has been studied more extensively ( Kazemi et al, 2021 ). These DNA fragments are <313 bp ( Sin et al, 2020 ) in length and present within the maternal circulation as early as 35 days gestation.…”

Increased fetal fraction is associated with the incidence of birth weight discordance and selective fetal growth restriction：a retrospective cohort study

“…The liberation of cffDNA can be affected by doxorubicin and high-mobility group box protein-1, which are inflammatory agents. Conversely, lipopolysaccharide does not affect the release of early or term cffDNA [16] , suggesting that cffDNA is involved in sterile inflammation as opposed to infectious processes. Sterile inflammation may lead to placental dysfunction and consequently cause pregnancy complications.…”