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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV231005	8/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C SEC (non-commercial)	Barranco I	2024	63%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 63% (75th 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 Seminal plasma Sample origin Entire ejaculate Focus vesicles large extracellular vesicles 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 (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type 24 x 1.5/2.0mL Pelleting: speed (g) 20000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Not detected contaminants Albumin Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 241 EM EM-type Transmission-EM Image type Wide-field

	EV231005	2/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C SEC (non-commercial)	Barranco I	2024	50%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 50% (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 Seminal plasma Sample origin First 10 mL of sperm rich fraction Focus vesicles large extracellular vesicles 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 (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type 24 x 1.5/2.0mL Pelleting: speed (g) 20000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 235 EM EM-type Transmission-EM Image type Wide-field

	EV231005	4/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C SEC (non-commercial)	Barranco I	2024	50%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 50% (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 Seminal plasma Sample origin Remaining sperm rich fraction Focus vesicles large extracellular vesicles 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 (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type 24 x 1.5/2.0mL Pelleting: speed (g) 20000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 244 EM EM-type Transmission-EM Image type Wide-field

	EV231005	6/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C SEC (non-commercial)	Barranco I	2024	50%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 50% (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 Seminal plasma Sample origin Post sperm rich fraction Focus vesicles large extracellular vesicles 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 (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type 24 x 1.5/2.0mL Pelleting: speed (g) 20000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 267 EM EM-type Transmission-EM Image type Wide-field

	EV231005	7/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C Filtration UF SEC (non-commercial)	Barranco I	2024	50%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 50% (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 Seminal plasma Sample origin Entire ejaculate Focus vesicles small extracellular vesicles 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 (Differential) (ultra)centrifugation Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Not detected contaminants Albumin Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 142 EM EM-type Transmission-EM Image type Wide-field

	EV231005	1/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C Filtration UF SEC (non-commercial)	Barranco I	2024	38%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 38% (15th 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 Seminal plasma Sample origin First 10 mL of sperm rich fraction Focus vesicles small extracellular vesicles 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 (Differential) (ultra)centrifugation Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 124 EM EM-type Transmission-EM Image type Wide-field

	EV231005	3/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C Filtration UF SEC (non-commercial)	Barranco I	2024	38%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 38% (15th 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 Seminal plasma Sample origin Remaining sperm rich fraction Focus vesicles small extracellular vesicles 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 (Differential) (ultra)centrifugation Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 129 EM EM-type Transmission-EM Image type Wide-field

	EV231005	5/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C Filtration UF SEC (non-commercial)	Barranco I	2024	38%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 38% (15th 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 Seminal plasma Sample origin Post sperm rich fraction Focus vesicles small extracellular vesicles 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 (Differential) (ultra)centrifugation Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 149 EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV231005
species	Sus scrofa domesticus
sample type	Seminal plasma
condition	Entire ejaculate	First 10 mL of sperm rich fraction	Remaining sperm rich fraction	Post sperm rich fraction	Entire ejaculate	First 10 mL of sperm rich fraction	Remaining sperm rich fraction	Post sperm rich fraction
separation protocol	dUC/ Size-exclusion chromatography (non-commercial)	dUC/ Size-exclusion chromatography (non-commercial)	dUC/ Size-exclusion chromatography (non-commercial)	dUC/ Size-exclusion chromatography (non-commercial)	dUC/ Filtration/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Filtration/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Filtration/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Filtration/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)
vesicle related term	large EVs	large EVs	large EVs	large EVs	small EVs	small EVs	small EVs	small EVs
Exp. nr.	8	2	4	6	7	1	3	5
EV-METRIC %	63	50	50	50	50	38	38	38

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