The relevance of extracellular vesicles (EVs) has grown exponentially, together with innovative basic research branches that feed medical and bioengineering applications. Such attraction has been fostered by the biological roles of EVs, as they carry biomolecules from any cell type to trigger systemic paracrine signaling or to dispose metabolism products. To fulfill their roles, EVs are transported through circulating biofluids, which can be exploited for the administration of therapeutic nanostructures or collected to intercept relevant EV-contained biomarkers. Despite their potential, EVs are ubiquitous and considerably heterogeneous. Therefore, it is fundamental to profile and identify subpopulations of interest. In this study, we optimized EV-labeling protocols on two different high-resolution single-particle platforms, the NanoFCM NanoAnalyzer (nFCM) and Particle Metrix ZetaView Fluorescence Nanoparticle Tracking Analyzer (F-NTA). In addition to the information obtained by particles’ scattered light, purified and non-purified EVs from different cell sources were fluorescently stained with combinations of specific dyes and antibodies to facilitate their identification and characterization. Despite the validity and compatibility of EV-labeling strategies, they should be optimized for each platform. Since EVs can be easily confounded with similar-sized nanoparticles, it is imperative to control instrument settings and the specificity of staining protocols in order to conduct a rigorous and informative analysis.

Current state‐of‐the‐art tools for analysing extracellular vesicles (EVs) offer either highly sensitive but unidimensional bulk measurements of EV components, or high‐resolution multiparametric single‐particle analyses which lack standardization and appropriate reference materials. This limits the accuracy of the assessment of marker abundance and overall marker distribution amongst individual EVs, and finally, the understanding of true EV heterogeneity. In this study, we aimed to define the standardized operating procedures and reference material for fluorescent characterization of EVs with two commonly used EV analytical platforms—nanoparticle tracking analysis (NTA) and nano‐flow cytometry (nFCM). We achieved quantitative fluorescence analyses on ZetaView NTA and NanoAnalyzer nFCM instruments, by utilizing yellow‐green FluoSpheres (FS) with assigned ERF (equivalent reference fluorophore) values. This standardization technique allowed for fluorescent EV signal to be expressed in ERF units (indicative of bound fluorescent antibodies per EV), thus enabling measurement of target protein marker abundance on individual EVs, and in the whole EV population. The NTA's and nFCM's limits of detection (LoD) were evaluated at 21 and 9 Alexa Fluor 488 (AF488) molecules, respectively. To complement the limited quantification of markers expressed in a few copies per single EV, in‐line bulk fluorescence measurements with a plate reader were performed. This provided absolute marker quantification and more insightful analyses of EV heterogeneity and marker stoichiometry. The standardization method outlined in this work unlocks the full analytical potential of NTA and nFCM, enabling cross‐platform data comparison. At the same time, it highlights some of the technical challenges and considerations and thus contributes to the ongoing efforts towards the development of EV analytical tools.

Stochiometric issues, encompassing both the quantity and heterogeneity of extracellular vesicles (EVs) derived from tumour or other tissues in blood, pose important challenges across various stages of biomarker discovery and detection, affecting the integrity of data, introducing losses and artifacts during blood processing, EV purification and analysis. These challenges shape the diagnostic utility of EVs especially within the framework of established and emerging methodologies. By addressing these challenges, we aim to delineate crucial parameters and requirements for tumour‐specific EV detection, or more precisely, for tumour identification via EV based assays. Our endeavour involves a comprehensive examination of the layers that mask or confound the traceability of EV markers such as nucleic acids and proteins, and focus on ‘low prevalence—low concentration’ scenario. Finally, we evaluate the advantages versus limitations of single‐particle analysers over more conventional bulk assays, suggesting that the combined use of both to capture and interpret the EV signals, in particular the EV surface displayed proteins, may ultimately provide quantitative information on their absolute abundance and distribution.

STUDY QUESTION: Does the molecular composition of uterine fluid extracellular vesicles (UF-EVs) reflect endometrial tissue changes across the menstrual cycle? SUMMARY ANSWER: Concordance between endometrial tissue and UF-EVs exists on miRNA and mRNA levels along the menstrual cycle phases and UF-EV surface proteomic signatures suggest EVs originate from several major endometrial cell populations. WHAT IS KNOWN ALREADY: The clinical value of endometrial receptivity testing is restricted by invasiveness and the use of only one omics level of input. There is promising evidence that UF-EVs can reflect changes in mid-secretory endometrium, highlighting the potential to establish endometrial receptivity testing right before embryo transfer. However, the dynamic changes of UF-EVs molecular cargo have not been directly compared to endometrial tissue on multiple omics levels. STUDY DESIGN, SIZE, DURATION: This cross-sectional study included fertile women from four menstrual cycle phases: proliferative and early-, mid-, and late-secretory phases. In total, 26 paired samples of UF and endometrial tissue were collected. mRNA and miRNA were sequenced, and differential analysis was performed on consecutive phases. UF-EVs were profiled for various protein surface markers associated with different cell types. EVs from epithelial endometrial organoid-conditioned culture media were used as a reference of pure epithelial endometrial EVs. PARTICIPANTS/MATERIALS, SETTING, METHODS: Paired UF and endometrial tissue samples were collected from 26 fertile, reproductive-age women. EV isolation from UF was validated using electron microscopy and western blotting, and particle numbers were measured by nanoparticle tracking analysis. The transcriptome and miRNome of UF-EVs and endometrial tissue were sequenced, and differential expression analysis was conducted on consecutive phases of the menstrual cycle. Bead-based EV flow cytometry targeting 37 surface protein markers was used to characterize EVs from UF and endometrial organoids. MAIN RESULTS AND THE ROLE OF CHANCE: Surface proteome analysis revealed that UF-EVs from the mid-secretory phase had significantly increased expression of natural killer cell marker CD56 (P < 0.005), pan-leukocyte marker CD45 (P < 0.005), pan-T-cell marker CD3 (P < 0.005), and coagulation-related protein CD142 (P < 0.005) compared to those from the proliferative phase, whereas markers associated with endometrial epithelial cells (CD29, CD133, and CD326) did not significantly change across the menstrual cycle. Transcriptomic analysis highlighted differential expression of histone and metallothionein genes that correlated between paired UF-EVs and endometrial tissues in each tested menstrual cycle phase. Principal component analysis of miRNomes of paired UF-EVs and endometrial tissue samples resulted in similar clustering patterns, where mid- and late-secretory samples clustered closely, and proliferative and early-secretory phase samples clustered separately. Half of the differentially expressed miRNAs in each phase in UF-EVs were also differentially expressed in the endometrium. Importantly, nine mid-secretory phase UF-EV DE miRNAs were identified, five of which were common between UF-EVs and endometrial biopsies, including hsa-miR-30d-5p and hsa-miR-200b-3p, both of which were previously implicated in implantation. Notably, three of the nine miRNAs, hsa-miR-200b-3p, hsa-miR-141-3p, and hsa-miR-200a-3p, were predicted to regulate mRNAs in the endometrial tissue and the pre-implantation embryo trophectoderm. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: The clinical dating of the menstrual cycle phase is based on the first day of menstruation and the time of the LH peak, which does not exclude the possibility that the expected endometrial phase was not reached. The wider limitation of our study is the lack of standardized procedures for collecting UF samples in gynaecological practice, which could challenge the replication of our findings. WIDER IMPLICATIONS OF THE FINDINGS: Evidence that UF-EVs reflect endometrial phases of menstrual cycle supports the use of UF-EVs in endometrial receptivity testing. Additionally, further studies of UF-EVs in endometrial pathologies could be beneficial for diagnostics, considering that more invasive tissue biopsies only reflect the biopsy site and not the full endometrium. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the European Regional Development Fund Enterprise Estonia’s Applied Research Program under the grant agreement number 2014-2020.4.02.21-0398 (EVREM), the Estonian Research Council (grant nos. PRG1076 and PSG1082), the Horizon Europe NESTOR grant (grant no. 101120075) of the European Commission, the Swedish Research Council (grant no. 2024-02530), the Novo Nordisk Fonden (grant no. NNF24OC0092384), and the National Recovery and Resilience Plan of the Republic of Bulgaria, project number BG-RRP-2.004-0001-C01. A.S.L. received funding from the Becas Fundación Ramón Areces para Estudios Postdoctorales. All the authors declare no conflict of interest.

Current state-of-the-art tools for analyzing extracellular vesicles (EVs) offer either highly sensitive but unidimensional bulk measurements of EV components, or high-resolution multiparametric single particle analyses which lack standardization and appropriate reference materials. This limits the accuracy of assessment of marker abundance and overall marker distribution among individual EVs, and finally, the understanding of true EV heterogeneity. In this study, we aimed to define the standardized operating procedures and reference material for fluorescent characterization of EVs with two commonly used EV analytical platforms - nanoparticle tracking analysis (NTA) and nano-flow cytometry (nFCM). For the first time, we achieved quantitative fluorescence analyses on ZetaView NTA and NanoAnalyzer nFCM instruments, by utilizing yellow-green FluoSpheres (FS) with assigned ERF (equivalent reference fluorophore) values. This standardization technique allowed for fluorescent EV signal to be expressed in ERF units (indicative of bound fluorescent antibodies per EV), thus enabling measurement of target protein marker abundance on individual EVs, and in the whole EV population. The NTA’s and nFCM’s limits of quantification (LoQ) were evaluated at 115 and 75 Alexa Fluor 488 (AF488) molecules, respectively. To complement these shortcomings, in-line bulk fluorescence measurements in a plate reader were performed. This provided absolute marker quantification, and more insightful analyses of EV heterogeneity and marker stoichiometry. The standardization method outlined in this work unlocks the full analytical potential of NTA and nFCM, enabling cross-platform data comparison. At the same time, it highlights some of the technical challenges and considerations, and thus contributes to the ongoing efforts towards development of EV analytical tools.