Unraveling the Multi-Omic Landscape of Extracellular Vesicles in Human Seminal Plasma
Abstract
:1. Introduction
2. Materials and Methods
2.1. Human Biological Sample Collection and Analysis
2.2. EV Isolation from Seminal Plasma
2.3. EV Characterization
2.3.1. Nanoparticle Tracking Analysis
2.3.2. Cholesterol Quantification Assay
2.3.3. Enzyme-Linked Immunosorbent Assay
2.3.4. Transmission Electron Microscopy Analysis
2.4. Proteomic Analysis—Gel-Free Analysis
2.4.1. EVs Preparation for Liquid Chromatography–Tandem Mass Spectrometry
2.4.2. LC-MS/MS Analysis
2.5. Proteomic Analysis—Gel-Based Analysis
2.5.1. EV Preparation for Gel-Based Proteomic Analysis
2.5.2. 2D Electrophoresis
2.5.3. MALDI-TOF-TOF MS—Protein Identification
2.5.4. Heatmap and PCA of Proteomic Gel-Based Results
2.5.5. Enrichment Analysis of Gel-Based and Gel-Free Proteomic Results by MetaCore
2.6. Transcriptome Profiling
2.6.1. Total RNA Isolation and Quality Assessment
2.6.2. Library Preparation and RNA-Seq
2.6.3. RNA-Seq Data Alignment and Identification of DEGs
2.7. Gene Expression Analysis by Droplets Digital PCR (ddPCR)
3. Results
3.1. Assessment of EV Recovery and Enrichment from Seminal Plasma
3.2. Characterization of a Normal Protein Cargo in Sperm EVs
3.3. Differential Proteomic Analysis of EVs from Subjects with Different Sperm Parameters
3.4. Differential Gene Expression Analysis of EVs from Subjects with Different Sperm Parameters
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
EVs | Extracellular Vesicles |
NORMO | Normozoospermia |
OAT | OligoAsthenoTeratozoospermia |
AZO | Azoospermia |
NTA | Nanoparticle Tracking Analysis |
PCR | Polymerase Chain Reaction |
GAPDH | Glyceraldehyde-3-Phosphate Dehydrogenase |
RNY4 | RNA, Ro60-Associated Y4 |
HSPA70 | Heat shock protein family A (Hsp70) member 4 |
CRISP1 | Cysteine Rich Secretory Protein 1 |
CRISP2 | Cysteine Rich Secretory Protein 2 |
CRISP3 | Cysteine Rich Secretory Protein 3 |
SPP1 | Secreted Phosphoprotein 1 |
MIF | Macrophage Migration Inhibitory Factor |
SPAM1 | Sperm Adhesion Molecule 1 |
ADAM2 | ADAM Metallopeptidase Domain 2 |
PGK2 | Phosphoglycerate Kinase 2 |
CLGN | Calmegin |
PAEP | Progestagen-Associated Endometrial Protein |
CLIC4 | Chloride intracellular channel 4 |
KLK3 | Kallikrein-related peptidase 3 |
IDH1 | Ocitrate dehydrogenase (NADP(+)) 1, cytosolic |
PSCA | Prostate stem cell antigen |
PARK7 | Parkinsonism-associated deglycase |
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Governini, L.; Haxhiu, A.; Shaba, E.; Vantaggiato, L.; Mori, A.; Bruttini, M.; Loria, F.; Zarovni, N.; Piomboni, P.; Landi, C.; et al. Unraveling the Multi-Omic Landscape of Extracellular Vesicles in Human Seminal Plasma. Biomolecules 2025, 15, 836. https://doi.org/10.3390/biom15060836
Governini L, Haxhiu A, Shaba E, Vantaggiato L, Mori A, Bruttini M, Loria F, Zarovni N, Piomboni P, Landi C, et al. Unraveling the Multi-Omic Landscape of Extracellular Vesicles in Human Seminal Plasma. Biomolecules. 2025; 15(6):836. https://doi.org/10.3390/biom15060836
Chicago/Turabian StyleGovernini, Laura, Alesandro Haxhiu, Enxhi Shaba, Lorenza Vantaggiato, Alessia Mori, Marco Bruttini, Francesca Loria, Natasa Zarovni, Paola Piomboni, Claudia Landi, and et al. 2025. "Unraveling the Multi-Omic Landscape of Extracellular Vesicles in Human Seminal Plasma" Biomolecules 15, no. 6: 836. https://doi.org/10.3390/biom15060836
APA StyleGovernini, L., Haxhiu, A., Shaba, E., Vantaggiato, L., Mori, A., Bruttini, M., Loria, F., Zarovni, N., Piomboni, P., Landi, C., & Luddi, A. (2025). Unraveling the Multi-Omic Landscape of Extracellular Vesicles in Human Seminal Plasma. Biomolecules, 15(6), 836. https://doi.org/10.3390/biom15060836