Effect of 3D and 2D cell culture systems on trophoblast extracellular vesicle physico-chemical characteristics and potency.

The growing understanding of the role of extracellular vesicles (EVs) in embryo-maternal communication has sparked considerable interest in their therapeutic potential within assisted reproductive technology, particularly in enhancing implantation success. However, the major obstacle remains the large-scale production of EVs, and there is still a gap in understanding how different culture systems affect the characteristics of the EVs. In the current study, trophoblast analogue human chorionic carcinoma cell line was cultivated in both conventional monolayer culture (2D) and as spheroids in suspension culture (3D) and how the cell growth environment affects the physical, biochemical and cellular signalling properties of EVs produced by them was studied. Interestingly, the 3D system was more active in secreting EVs compared to the 2D system, while no significant differences were observed in terms of morphology, size, and classical EV protein marker expression between EVs derived from the two culture systems. There were substantial differences in the proteomic cargo profile and cellular signalling potency of EVs derived from the two culture systems. Notably, 2D EVs were more potent in inducing a cellular response in endometrial epithelial cells (EECs) compared to 3D EVs. Therefore, it is essential to recognize that the biological activity of EVs depends not only on the cell of origin but also on the cellular microenvironment of the parent cell. In conclusion, caution is warranted when selecting an EV production platform, especially for assessing the functional and therapeutic potential of EVs through in vitro studies.

Secretory Proteomic Responses of Endometrial Epithelial Cells to Trophoblast-Derived Extracellular Vesicles.

Synchronized crosstalk between the embryo and endometrium during the periconception period is integral to pregnancy establishment. Increasing evidence suggests that the exchange of extracellular vesicles (EVs) of both embryonic and endometrial origin is a critical component of embryo-maternal communication during peri-implantation. Here, we investigated whether embryonic signals in the form of EVs can modulate the endometrial epithelial cell secretome. Receptive endometrial analog RL95-2 cells were supplemented with trophoblast analog JAr cell-derived EVs, and the secretory protein changes occurring in the RL95-2 cells were analyzed using mass spectrometry. EVs of non-trophoblastic origin (HEK 293 cells) were used as the control EV source to supplement endometrial cells. Trophoblast cell-derived EVs enriched endometrial epithelial cell secretions with proteins that support embryo development, attachment, or implantation, whereas control EVs were unable to induce the same effect. The present study suggests that embryonic signals in the form of EVs may prime receptive endometrial epithelial cells to enrich their secretory proteome with critical proteomic molecules with functional importance for periconception milieu formation.

The Extracellular Vesicles Proteome of Endometrial Cells Simulating the Receptive Menstrual Phase Differs from That of Endometrial Cells Simulating the Non-Receptive Menstrual Phase.

Successful embryo implantation into a receptive endometrium requires mutual endometrial-embryo communication. Recently, the function of extracellular vehicles (EVs) in cell-to-cell interaction in embryo-maternal interactions has been investigated. We explored isolated endometrial-derived EVs, using RL95-2 cells as a model of a receptive endometrium, influenced by the menstrual cycle hormones estrogen (E2; proliferative phase), progesterone (P4; secretory phase), and estrogen plus progesterone (E2P4; the receptive phase). EV sized particles were isolated by differential centrifugation and size exclusion chromatography. Nanoparticle tracking analysis was used to examine the different concentrations and sizes of particles and EV proteomic analysis was performed using shotgun label-free mass spectrometry. Our results showed that although endometrial derived EVs were secreted in numbers independent of hormonal stimulation, EV sizes were statistically modified by it. Proteomics analysis showed that hormone treatment changes affect the endometrial EV's proteome, with proteins enhanced within the EV E2P4 group shown to be involved in different processes, such as embryo implantation, endometrial receptivity, and embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs.

The role of extracellular vesicles in endometrial receptivity and their potential in reproductive therapeutics and diagnosis.

Extracellular vesicles (EVs) are small, nanometre sized, membrane-enclosed structures released by cells and are thought to be crucial in cellular communication. The cargo of these vesicles includes lipids, proteins, RNAs and DNA, and control various biological processes in their target tissues depending on the parental and receiver cell's origin and phenotype. Recently data has accumulated in the role of EVs in embryo implantation and pregnancy, with EVs identified in the uterine cavity of women, sheep, cows, horses, and mice, in which they aid blastocyst and endometrial preparation for implantation. Herein is a critical review to decipher the role of extracellular vesicles in endometrial receptivity and their potential in reproductive therapies and diagnosis. The current knowledge of the function of embryo and endometrial derived EVs and their cargoes, with regards to their effect on implantation and receptivity are summarized and evaluated. The findings of the below review highlight that the combined knowledge on EVs deriving from the endometrium and embryo have the potential to be translated to various clinical applications including treatment, a diagnostic biomarker for diseases and a drug delivery tool to ultimately improve pregnancy rates.

Characterization of Extracellular Vesicles Labelled with a Lipophilic Dye Using Fluorescence Nanoparticle Tracking Analysis.

Research on extracellular vesicles (EVs) has intensified over the past decade, including fluorescent membrane labeling of EVs. An optimal fluorescent method requires the size of EVs to be preserved after labeling. Lipophilic fluorescent dyes, such as CellMask™ Green (CMG), have been widely used for this purpose. Here, we investigated conditions affecting the optimum CMG labeling of EVs derived from human choriocarcinoma cells (JAr) and different biological fluids using fluorescence NTA (fl-NTA). The effect of CMG labeling on the size, concentration and zeta potential (ZP) on JAr EVs purified with different methods were measured along with biological fluid-derived EVs. With the increase of CMG dye concentration, a significant decrease in the mean size of fluorescent nanoparticles (fl-NPs) was observed. The ZP of fl-NPs originating from JAr cells with the lowest and highest dye concentrations showed a significant shift towards more and less negative ZP values, respectively. Differences in the concentration of fl-NPs were observed for JAr EVs purified using size-exclusion chromatography (SEC) alone and SEC in combination with tangential flow filtration. The proportion of CMG labeling of NPs varied across different biological sources. CMG labeling may be a reliable technique for the detection of EVs using fl-NTA.

Trophoblast derived extracellular vesicles specifically alter the transcriptome of endometrial cells and may constitute a critical component of embryo-maternal communication.

BACKGROUND: The period of time when the embryo and the endometrium undergo significant morphological alterations to facilitate a successful implantation-known as "window of implantation"-is a critical moment in human reproduction. Embryo and the endometrium communicate extensively during this period, and lipid bilayer bound nanoscale extracellular vesicles (EVs) are purported to be integral to this communication. METHODS: To investigate the nature of the EV-mediated embryo-maternal communication, we have supplemented trophoblast analogue spheroid (JAr) derived EVs to an endometrial analogue (RL 95-2) cell layer and characterized the transcriptomic alterations using RNA sequencing. EVs derived from non-trophoblast cells (HEK293) were used as a negative control. The cargo of the EVs were also investigated through mRNA and miRNA sequencing. RESULTS: Trophoblast spheroid derived EVs induced drastic transcriptomic alterations in the endometrial cells while the non-trophoblast cell derived EVs failed to induce such changes demonstrating functional specificity in terms of EV origin. Through gene set enrichment analysis (GSEA), we found that the response in endometrial cells was focused on extracellular matrix remodelling and G protein-coupled receptors' signalling, both of which are of known functional relevance to endometrial receptivity. Approximately 9% of genes downregulated in endometrial cells were high-confidence predicted targets of miRNAs detected exclusively in trophoblast analogue-derived EVs, suggesting that only a small proportion of reduced expression in endometrial cells can be attributed directly to gene silencing by miRNAs carried as cargo in the EVs. CONCLUSION: Our study reveals that trophoblast derived EVs have the ability to modify the endometrial gene expression, potentially with functional importance for embryo-maternal communication during implantation, although the exact underlying signalling mechanisms remain to be elucidated.

Measurement of the Size and Concentration and Zeta Potential of Extracellular Vesicles Using Nanoparticle Tracking Analyzer.

Extracellular vesicles (EVs) are membrane-bound nanoparticles that are secreted by most cell types with an emerging role in cellular communication and potential as biomarkers of disease. Nanoparticle tracking analysis (NTA) is a commonly used technique to measure the size and concentration of nanoparticles, such as EVs. Here, we present two protocols for the analysis of size profile concentration, and zeta potential (ZP) of well-characterized EVs derived from human choriocarcinoma JAr cells using NTA. These protocols describe how the size profile concentration, and ZP of JAr EVs are measured using optimized settings of NTA. With good experimental practices and consistent protocol, NTA measurements of EVs can provide reliable data that could potentially translate further uses of EVs for diagnostic and therapeutic applications.

Potential applicability of cytokines as biomarkers of disease activity in rheumatoid arthritis: Enzyme-linked immunosorbent spot assay-based evaluation of TNF-α, IL-1ß, IL-10 and IL-17A.

Biomarkers play a pivotal role in the management of rheumatoid arthritis (RA) by facilitating early diagnosis and 'treat to the target.' However, no gold standard biomarker has been identified for monitoring the disease activity in RA. Cytokines, a diverse group of small protein molecules secreted by peripheral blood mononuclear cells (PBMCs), play a pivotal role in pathogenesis and disease progression in RA. Research is currently underway to find out the applicability of cytokines as biomarkers in RA. This study aimed to quantify the PBMCs that secrete four types of cytokines; TNF-α, IL-1ß, IL-10 and IL-17A in two cohorts of active RA patients (early RA patients and established RA patients), compared to healthy controls (HC), using the enzyme-linked immunosorbent spot (ELISPOT) assay, and to assess their association with measures of disease activity of RA. Patients were recruited from outpatient rheumatology clinics, and the disease activity was assessed using single and composite measures of disease activity. The cytokine expression was evaluated using freshly separated PBMCs from whole blood of RA patients using the ELISPOT assay. The number of PBMCs (counted as spot-forming cells (SFCs) per 105 PBMCs) that secreted the cytokine of interest were statistically significantly higher in early RA patients, compared to HC, for IL-17A (P<0.05). Such an increased number of SFCs was not observed in the established RA group, compared to controls, for any of the cytokines tested. The correlation analysis showed that IL-17A is having a moderate correlation (Spearman`s ρ, p <0.05) with five clinical measures of disease activity, including disease activity score 28 (DAS28). According to the multivariable linear regression models, IL17A was a good predictor of both the disease activity score 28 (DAS28) and clinical disease activity index (CDAI). In conclusion, IL-17A has potential applicability as a biomarker of disease activity of RA.

Oviduct as a sensor of embryo quality: deciphering the extracellular vesicle (EV)-mediated embryo-maternal dialogue.

Embryo-derived extracellular vesicles (EVs) may play a role in mediating the embryo-maternal dialogue at the oviduct, potentially carrying signals reflecting embryo quality. We investigated the effects of bovine embryo-derived EVs on the gene expression of bovine oviductal epithelial cells (BOECs), and whether these effects are dependent on embryo quality. Presumptive zygotes were cultured individually in vitro in culture medium droplets until day 8 while their development was assessed at day 2, 5 and 8. Conditioned medium samples were collected at day 5 and pooled based on embryo development (good quality embryo media and degenerating embryo media). EVs were isolated from conditioned media by size exclusion chromatography and supplemented to primary BOEC monolayer cultures to evaluate the effects of embryo-derived EVs on gene expression profile of BOEC. Gene expression was quantified by RNA-seq and RT-qPCR. A total of 7 upregulated and 18 downregulated genes were detected in the BOECs supplemented with good quality embryo-derived EV compared to the control. The upregulated genes included interferon-τ-induced genes, such as OAS1Y, MX1 and ISG15, which have previously been reported as upregulated in the oviductal epithelial cells in the presence of embryos. Of the upregulated genes, OAS1Y and MX1 were validated with RT-qPCR. In contrast, only one differentially expressed gene was detected in BOECs in response to degenerating embryo-derived EVs, suggesting that oviductal responses are dependent on embryo quality. Our results support the hypothesis that embryo-derived EVs are involved in embryo-maternal communication at the oviduct and the oviductal response is dependant on the embryo quality. KEY MESSAGES: • Extracellular vesicles (EVs) released by individually cultured pre-implantation bovine embryos can alter the gene expression of primary oviductal epithelial cells. • The oviductal response, in terms of gene expression, to the bovine embryo-derived EVs varied depending on the embryo quality. • In vivo, the oviduct may have the ability to sense the quality of the pre-implantation embryos. • The observed effect of embryo-derived EVs on oviductal epithelial cells could serve as a non-invasive method of evaluating the embryo quality.

Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability.

Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions. Zeta potential (ZP) is a popular method to measure the surface potential of EVs, while used as an indicator of surface charge, and colloidal stability influenced by surface chemistry, bioconjugation, and the theoretical model applied. Here, we investigated the effects of such factors on ZP of well-characterized EVs derived from the human choriocarcinoma JAr cells. The EVs were suspended in phosphate-buffered saline (PBS) of various phosphate ionic concentrations (0.01, 0.1, and 1 mM), with or without detergent (Tween-20), or in the presence (10 mM) of different salts (NaCl, KCl, CaCl2, and AlCl3) and at different pH values (4, 7, and 10) while the ZP was measured. The ZP changed inversely with the buffer concentration, while Tween-20 caused a significant (p < 0.05) lowering of the ZP. Moreover, the ZP was significantly (p < 0.05) less negative in the presence of ions with higher valency (Al3+/Ca2+) than in the presence of monovalent ones (Na+/K+). Besides, the ZP of EVs became less negative at acidic pH, and vice versa. The integrated data underpins the crucial role of physicochemical attributes that influence the colloidal stability of EVs.

Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers.

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 µl droplets of culture media, and 50 µl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential.