Abstract
BackgroundDuring human pregnancy, placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation. Trophoblasts also give rise to cell-free DNA (cfDNA) in maternal blood, and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of DNA in the EVs (evDNA) of maternal blood, and compared evDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases.MethodsMaternal blood from 20 euploid pregnancies, 9 T21 pregnancies, 3 T18 pregnancies, 1 T13 pregnancy, and 2 pregnancies with FGFR3 mutations were obtained. EVs were separated from maternal plasma, and confirmed by transmission electronic microscopy (TEM), western blotting, and flow cytometry (FACS). evDNA was extracted and its fetal origin was confirmed by quantitative PCR (qPCR). Pair-end (PE) whole genome sequencing was performed to characterize evDNA, and the results were compared with that of cfDNA. The fetal risk of aneuploidy and monogenic diseases was analyzed using the evDNA sequencing data.ResultsEVs separated from maternal plasma were confirmed with morphology by TEM, and protein markers of CD9, CD63, CD81 as well as the placental specific protein placental alkaline phosphatase (PLAP) were confirmed by western blotting or flow cytometry. EvDNA could be successfully extracted for qPCR and sequencing from the plasma EVs. Sequencing data showed that evDNA span on all 23 pairs of chromosomes and mitochondria, sharing a similar distribution pattern and higher GC content comparing with cfDNA. EvDNA showed shorter fragments yet lower fetal fraction than cfDNA. EvDNA could be used to correctly determine fetal gender, trisomies, and de novo FGFR3 mutations.ConclusionsWe proved that fetal DNA could be detected in EVs separated from maternal plasma. EvDNA shared some similar features to plasma cfDNA, and could potentially be used to detect genetic diseases in fetus.
Highlights
During human pregnancy, placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation
Baig et al showed that in the blood of pregnant women with preeclampsia, there was an elevated concentration of syncytiotrophoblast microvesicles, and the proteomic and lipidomic profiles of the microvesicles were considerably changed comparing with normal pregnant women [11, 12]
Detection of fetal trisomies and monogenic diseases by Extracellular vesicle DNA (evDNA) sequencing Since evDNA demonstrated some similar characteristics and fetal origin to cell-free DNA (cfDNA), we explored the feasibility of detecting fetal diseases using evDNA
Summary
Placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation. Trophoblasts give rise to cell-free DNA (cfDNA) in maternal blood, and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of DNA in the EVs (evDNA) of maternal blood, and compared evDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases. Placental trophoblasts can release EVs into maternal circulation, and the concentration of EVs positively correlates with gestation weeks [5, 6]. Luo et al reported that human villous trophoblasts secreted placenta-specific microRNA into maternal plasma via exosomes, and may confer antiviral ability though communicating with target cells [10]. Baig et al showed that in the blood of pregnant women with preeclampsia, there was an elevated concentration of syncytiotrophoblast microvesicles, and the proteomic and lipidomic profiles of the microvesicles were considerably changed comparing with normal pregnant women [11, 12]
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