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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200029	1/2	Bos taurus	Bovine embryo culture medium	qEV	Pavani KC	2022	75%
Study summary Full title Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts. All authors Pavani KC, Meese T, Pascottini OB, Guan X, Lin X, Peelman L, Hamacher J, Van Nieuwerburgh F, Deforce D, Boel A, Heindryckx B, Tilleman K, Van Soom A, Gadella BM, Hendrix A, Smits K Journal Proc Natl Acad Sci U S A Abstract SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less l (show more...)SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation. (hide) EV-METRIC 75% (50th 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 Bovine embryo culture medium Sample origin Control condition Focus vesicles extracellular vesicle 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 Commercial method Protein markers EV: TSG101/ CD63/ CD9 non-EV: AGO2/ APOA1 Proteomics no Show all info Study aim Biomarker Sample Species Bos taurus Sample Type Bovine embryo culture medium Separation Method Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101 Detected contaminants APOA1 Not detected contaminants AGO2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 94.5±1.7 E08 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV200029	2/2	Bos taurus	Bovine embryo culture medium	qEV	Pavani KC	2022	75%
Study summary Full title Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts. All authors Pavani KC, Meese T, Pascottini OB, Guan X, Lin X, Peelman L, Hamacher J, Van Nieuwerburgh F, Deforce D, Boel A, Heindryckx B, Tilleman K, Van Soom A, Gadella BM, Hendrix A, Smits K Journal Proc Natl Acad Sci U S A Abstract SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less l (show more...)SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation. (hide) EV-METRIC 75% (50th 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 Bovine embryo culture medium Sample origin Control condition Focus vesicles extracellular vesicle 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 Commercial method Protein markers EV: TSG101/ CD63/ CD9 non-EV: AGO2/ APOA1 Proteomics no Show all info Study aim Biomarker Sample Species Bos taurus Sample Type Bovine embryo culture medium Separation Method Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101 Detected contaminants APOA1 Not detected contaminants AGO2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 76.5±8.2 E08 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV200029
species	Bos taurus
sample type	Bovine embryo culture medium
condition	Control condition
separation protocol	qEV	qEV
Exp. nr.	1	2
EV-METRIC %	75	75