Extracellular vesicles (EVs) are emerging as key factors in maintaining cellular homeostasis, critical mediators of intercellular communication, potential biomarkers, and therapeutic tools. While small EVs have been extensively characterized, the molecular signatures of large EVs (including those generated during regulated cell death pathways) remain poorly defined. Here, we investigated the characteristics of large EVs released during apoptosis and pyroptosis by human monocytic cell lines (THP-1 and U937). Apoptosis was induced by staurosporine and blocked using the pan-caspase inhibitor Q-VD-OPh, whereas pyroptosis was triggered by LPS/nigericin and inhibited with a selective NLRP3 inhibitor. We found that both forms of regulated cell death markedly enhanced the release of large EVs. Both apoptotic and pyroptotic large EVs showed increased Annexin V binding and decreased CD9 expression compared with those released by healthy cells. Large EVs derived from apoptotic and pyroptotic cells exhibited distinct proteomic profiles. Pyroptotic large EVs carried interacting protein networks of RNA-binding proteins and chromatin-associated proteins many of which are known damage-associated molecular patterns or alarmins. In contrast, we found that a subpopulation of apoptotic large EVs was characterized by the presence of dsDNA, and active caspase-3/7. Together, our data shed light on the specific protein cargo of large EVs released by cells during apoptosis and pyroptosis. This study identifies candidate markers of large EVs released by dying cells and may enhance our understanding of the role of EVs in regulated cell death.

Background: Due to the difficulty of identifying the window of receptivity, a molecular understanding of endometrial changes after fertilization is essential for improving the effectiveness of in vitro fertilization (IVF). This multi-level study examines the compo-sition of cytokines and extracellular vesicles (EVs) in uterine lavage samples obtainable via a noninvasive procedure in mice. The goal is to offer a new perspective on the tim-ing of embryo transfer. Methods: We performed flow cytometric analyses of inflamma-tory cytokines and EVs in uterine lavage samples collected from CD1 mice at the pre-receptive stage (2.5 days post coitum [dpc]) and the receptive stage (4.5 dpc). We also assessed endometrial progesterone receptor expression via immunohistochemistry and evaluated the expression of adhesion molecules related to implantation by RT-PCR. Re-sults: Significant differences were found in the concentrations of inflammatory cyto-kines (IL-6, IFN-γ, and MCP-1), the EV pattern in uterine lavage fluid, and the expres-sion of some genes related to implantation (CD44, CD81, and CD29). These results re-veal uterine lavage fluid markers associated with endometrial receptivity. Using these noninvasive markers can improve the effectiveness of IVF by enabling more precise timing of embryo transfer.