Table 1.
Clinical characteristics of patients and newborns.
Figure 1.
Characterisation of exosome from maternal circulation.
Exosome were isolated from plasma of non-pregnant women and women uncomplicated pregnancies during first, second and third trimester by differential and buoyant density centrifugation (see Methods). (A–D) Representative vesicle size distribution isolated from maternal circulation across the pregnancy (first, second and third trimester) using a NanoSight NS500 instrument. Fractions 1 to 10, represent fractions collected after buoyant density centrifugation. (A) 200,000×g pellet (before sucrose density fractionation); (B) fractions 1–4; (C) fractions 5–8 enriched exosome population; (D) fractions 9–10. (E) percentage of vesicles before (white bar = starting solution: 100%) and after (blue: fractions 1–4; red: fractions 5–8 and orange: fractions 9–10) of the exosome purification using a continuos sucrose gradient. (F) Representative Western blot for exosome enriched markers: CD63, CD9 and CD81. Exosome density is represented as red square. (G) Representative electron micrograph of 200,000×g supernatant (1) and exosome fractions (pooled enriched exosome population from fractions 5 to 8). In G, Scale bar 100 nm.
Table 2.
vesicles size distribution during pregnancy.
Figure 2.
Exosome profiling across the pregnancy.
Enriched exosomal protein population and number of exosomes were quantified in in peripheral plasma of women in the first, second and third trimester of pregnancy and non-pregnant women using a colorimetric assay and ELISA kit, respectively. (A) Plasma exosome concentration presented as mg exosomal protein per ml plasma and (B) Number of exosomes across the pregnancy. Data are presented as aligned dot plot and values are mean ± SEM. In A and B, *p<0.01 versus all; In B, †p<0.05 versus ST trimester and ‡p<0.01 versus FT.
Figure 3.
Placenta-derived exosomes profile during pregnancy.
Enriched exosome vesicles were quantified in in peripheral plasma of women in the first, second and third trimester of pregnancy using an ELISA kit. (A) exosomal PLAP concentration across the normal pregnancy. (B) Same volume of enriched exosome pellet loaded and analyzed by Western Blot for PLAP and CD63 in exosomes (fractions 5 to 8 were pooled) from maternal plasma in the first, second and third trimester of pregnancy. Lowe panel: PLAP/CD63 ratio densitometries from data in top normalized to 1 (first trimester). Data are presented as aligned dot plot and values are mean ± SEM. In A and B, *p<0.01 versus ST and TT trimester; †p<0.05 versus ST trimester.
Figure 4.
Contribution of placental-derived exosomes into maternal circulation.
(A) Relationship between exosomal PLAP and NEV across the pregnancy. (B) Relationship between exosomal PLAP at TT of pregnancy and placental weight at delivery. (C and D) Relationship between exosomal PLAP and left and right Pulsatility Index (PI) across the pregnancy, respectively. Linear correlation (-). In A, n = 18 (2 missing data values). In B and C n = 49 (11 missing data values for PI across pregnancy).
Figure 5.
PLAP activity across the pregnancy.
The specific exosomal PLAP activity (defined as PLAP concentration per number of exosome vesicles) was quantified in plasma of women in the first, second and third trimester. (A) Changes in NEV and specific placental exosomes into maternal circulation during normal pregnancy. (B) Ratio of specific placental exosome and NEV. IN B, *p<0.05 versus FTand ST trimester.
Figure 6.
Maternal exosome effects on endothelial cells migration.
HUVEC were grown to confluence in PCM and a wound was made using 96 well WoundMaker (see Methods). HUVEC Migration was measured in absence or presence of 100 ug/ml of exosomes from first, second and third trimester for 24 h. (A) Graphical representation from a showing the calculation of initial wound width (black) and graphical representation of cell migration (gray) at the midpoint of the experiment. (B) Time course of wound closure for HUVEC expressed as relative wound density (%). (C) Area under curves from data in B. Data represent an n = 12 well each point with 6 different cells culture (i.e. biological replicates) of HUVEC isolated from first trimester pregnancies (see methods). Values are mean ± SEM. In C, *p<0.05 versus all condition; †P<0.005 versus second and third trimester exosomes.