Introduction:
The human placenta releases EVs, including microvesicles and exosomes, into the maternal blood for feto-maternal communication. The concentration of placenta-secreted exosomes in the maternal plasma increases as gestation progresses. Placental CRH mRNA levels rise with gestation and have been reported to increase in complicated pregnancies. Our study aimed to (i) isolate, purify and characterise placenta-secreted EVs in placental explants and maternal plasma, (ii) determine whether placental EVs contain CRH mRNA and (iii) determine whether CRH mRNA containing placentally-derived EVs were detectible in maternal plasma.
Methods:
Placental EVs (microvesicles and exosomes) were isolated from human term placentas via villus washing (N=6) and from explant cultures (N=6) supernatants by differential centrifugation and then purified on a continuous sucrose gradient (0.25-2.5M). EVs were characterised by western blotting using placenta- and exosome-specific markers and the morphology was studied by Transmission Electron Microscopy (TEM). EVs from maternal plasma (gestational weeks 38-41, N=9) were isolated by ultracentrifugation and Fluorescent Activated Cell Sorting (FACS)using fluorescent-labelled PLAP antibody. CRH mRNA was measured in EVs isolated from the placental washes, explants and maternal plasma using real-time PCR.
Results:
Placental (PLAP) and exosomal markers (CD63, TSG, LAMP-2, Calreticulin) were observed in purified exosomes at a density of 1.16g/ml. TEM images showed microvesicles and exosomes with sizes ranging from 200-500 nm and 100-150 nm, respectively. The PCR data showed the presence of CRH mRNA in placenta-derived EVs from both placental washes and explants. In blood plasma, size-specific placental EVs (100-500 nm) were sorted by FACS using PLAP-Ab. CRH mRNA was detected in EVs obtained from maternal blood plasma.
Conclusion:
EVs released by the human placenta can be isolated and purified by ultracentrifugation and FACS from ex-vivo explant culture media and maternal blood. These placenta-secreted EVs contain CRH mRNA. However, the physiological role of CRH mRNA in maternal plasma remains unknown.