The human placenta is lined by a single multinucleated cell, syncytiotrophoblast. The syncytiotrophoblast releases three sizes of extracellular vesicles (EV) that carry cell-free fetal DNA (fDNA) into the maternal circulation; 1) macro-, 2) micro-, and 3) nano-EVs. We hypothesised that EVs released from first trimester, term, and pre-eclamptic placenta have varying cargos of fetal DNA including intact fetal genes.
Placental explant cultures followed by differential centrifugation were used to isolate macro-, micro-, and nano-EVs from first-trimester, term, and pre-eclamptic placentae (n=3-5). Following DNA extraction from the EVs, long-range PCR for Csh1 (7150 bp) and Vegfa (18008 bp) were conducted to confirm the presence of intact genes, and Tapestation analysis was used to determine prominent DNA fragment sizes.
Long-range PCR indicated all three EV types from first-trimester, term, and pre-eclamptic placentae contained intact Csh1 and Vegfa genes and their 5’ regulatory regions. While accurate quantification is not possible using this method, the amplicons from first trimester macro-EVs were markedly stronger than those from term macro-EVs. In contrast, the amplicons from macro-EVs from preeclamptic placentae were comparable to those from first trimester.
Tapestation analysis confirmed that all EVs sizes contained abundant long DNA fragments, but the long fragments in term macro-EVs were on average shorter (9 - 13 kb) than those from either first-trimester (13 – 30 kb) or pre-eclamptic macro-EVs (9 - 25 kb). There were no obvious differences in DNA fragment size in micro- or nano-EVs from first trimester, term or preeclamptic placentae.
All placental EVs we examined, regardless of EV size or placenta of origin contained full-length genes encoding the model genes Csh1 and Vegfa. We have previously hypothesised that placental EVs have the capacity to transfect maternal cells with fetal genes and this demonstration that all sizes of placental EVs carry intact fetal genes supports that hypothesis.