The placenta is a fetal organ, which is lined by a single multinucleated cell the syncytiotrophoblast. The syncytiotrophoblast is bathed in maternal blood and sheds three sizes of extracellular vesicles (EV) into the maternal blood: macro-, micro-, and nano-EVs. These EVs carry so-called cell-free fetal DNA (cffDNA). We hypothesised that there is heterogeneity of cffDNA and its localisation in the three EV types.
Placental explant culture system and differential centrifugation were used to isolate placenta-derived macro-, micro-, nano-EVs (n=5). Qubit and Tapestation analyses allowed cffDNA quantification and fragment size determination. Combinations of triton X-100, proteinase-K, and DNase-1 were used to determine the localisation of cffDNA on each EV type.
Macro-EVs yielded 100 ng/gram placenta, and contained large DNA fragments sized (30000-13000 bp), micro-EVs contained 250ng/gram placenta while nano-EVs contained 500 ng/gram placenta, and both contained a mixture of large (12000-9000 bp) and smaller fragments (1700-400 bp). DNase-1 treatment reduced cffDNA associated with all EVs by 50%. The combination of DNase-1 with triton X-100 or proteinase-K did not further reduce cffDNA in macro-EV, but considerably reduced cffDNA in micro- and nano-EVs.
Differing cffDNA fragment sizes associated with the various placental EVs likely reflects separate biogenesis routes. All EVs were capable of carrying large cffDNA fragments, increasing the likelihood of carrying intact genes. The reduction of more than 50% cffDNA quantity by DNase-1 alone indicates exterior-bound cffDNA on all EVs. Further reduction by combining triton X-100 or proteinase-K indicates some cffDNA are protected inside the EVs or by a DNA-binding protein.