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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140094	1/1	Homo sapiens	Blood plasma	(d)(U)C DG Filtration	Sarker S	2014	56%
Study summary Full title Placenta-derived exosomes continuously increase in maternal circulation over the first trimester of pregnancy All authors Sarker S, Scholz-Romero K, Perez A, Illanes SE, Mitchell MD, Rice GE, Salomon C Journal J Transl Med Abstract BACKGROUND: Human placenta releases specific nanovesicles (i.e. exosomes) into the maternal circulat (show more...)BACKGROUND: Human placenta releases specific nanovesicles (i.e. exosomes) into the maternal circulation during pregnancy, however, the presence of placenta-derived exosomes in maternal blood during early pregnancy remains to be established. The aim of this study was to characterise gestational age related changes in the concentration of placenta-derived exosomes during the first trimester of pregnancy (i.e. from 6 to 12 weeks) in plasma from women with normal pregnancies. METHODS: A time-series experimental design was used to establish pregnancy-associated changes in maternal plasma exosome concentrations during the first trimester. A series of plasma were collected from normal healthy women (10 patients) at 6, 7, 8, 9, 10, 11 and 12 weeks of gestation (n = 70). We measured the stability of these vesicles by quantifying and observing their protein and miRNA contents after the freeze/thawing processes. Exosomes were isolated by differential and buoyant density centrifugation using a sucrose continuous gradient and characterised by their size distribution and morphology using the nanoparticles tracking analysis (NTA; Nanosight™) and electron microscopy (EM), respectively. The total number of exosomes and placenta-derived exosomes were determined by quantifying the immunoreactive exosomal marker, CD63 and a placenta-specific marker (Placental Alkaline Phosphatase PLAP). RESULTS: These nanoparticles are extraordinarily stable. There is no significant decline in their yield with the freeze/thawing processes or change in their EM morphology. NTA identified the presence of 50-150 nm spherical vesicles in maternal plasma as early as 6 weeks of pregnancy. The number of exosomes in maternal circulation increased significantly (ANOVA, p = 0.002) with the progression of pregnancy (from 6 to 12 weeks). The concentration of placenta-derived exosomes in maternal plasma (i.e. PLAP+) increased progressively with gestational age, from 6 weeks 70.6 ± 5.7 pg/ml to 12 weeks 117.5 ± 13.4 pg/ml. Regression analysis showed that weeks is a factor that explains for >70% of the observed variation in plasma exosomal PLAP concentration while the total exosome number only explains 20%. CONCLUSIONS: During normal healthy pregnancy, the number of exosomes present in the maternal plasma increased significantly with gestational age across the first trimester of pregnancy. This study is a baseline that provides an ideal starting point for developing early detection method for women who subsequently develop pregnancy complications, clinically detected during the second trimester. Early detection of women at risk of pregnancy complications would provide an opportunity to develop and evaluate appropriate intervention strategies to limit acute adverse sequel. (hide) EV-METRIC 56% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 53.28 (pelleting) / 53.28 (washing) Protein markers EV: CD63 non-EV: Proteomics yes EV density (g/ml) 1.12-1.2 Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T8100 Pelleting: adjusted k-factor 53.28 Wash: volume per pellet (ml) 5 Wash: Rotor Type T8100 Wash: adjusted k-factor 53.28 Density gradient Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 200000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63 ELISA Detected EV-associated proteins CD63 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV140094
species	Homo sapiens
sample type	Blood plasma
condition	NAY
separation protocol	(d)(U)C DG Filtration
Exp. nr.	1
EV-METRIC %	56