Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET.

Currently, there is a need to improve the cryopreservation process for male gametes, especially for patients with low cryotolerance during sperm cryopreservation. Methods: The content and size of donor extracellular vesicles (EVs) in seminal plasma (SP) were assessed using nanoparticle tracking analysis (NTA), CD marker analysis, and transmission electron microscopy (TEM). Patient ejaculates were exposed to cryopreservation with or without prior co-culture with SP EVs and were not exposed to cryopreservation. The interaction of SP EVs with spermatozoa was assessed by TEM. Apoptotic, necrotic and late apoptotic cells, and mitochondrial functional activity were detected by flow cytometry. Results: NTA showed the highest concentration of SP EVs with a size of 80 nm, corresponding to small EVs. The binding of SP EVs to spermatozoa occurred along the entire plasma membrane, with an increased concentration of SP EVs at the neck and upper third of the sperm head. A significant increase in sperm motility was observed in the EVs co-culture group after cryopreservation/thawing. Flow cytometry showed a significant difference in the JC-1 Red/JC-1 Green ratio, indicating a higher mitochondrial membrane potential in the EVs exposure group. Conclusions: SP EVs have a protective function during human sperm cryopreservation.

Background. Biological fluids contain diverse extracellular vesicle (EV) populations differing in size, origin, composition, and function. This study pioneers asymmetric depth filtration for isolating seminal plasma (SP) EVs, bypassing ultracentrifugation (current gold standard). Objective. Validate the novel asymmetric depth filtration method for SP EV isolation as an alternative to ultracentrifugation. Design and methods. Twelve samples of seminal plasma were collected from young men and patients of advanced reproductive age with mild teratozoospermia. EVs were isolated from the SP using the proposed method. Western blot analysis for vesicle membrane markers CD9, CD63, and CD81 confirmed the presence of SP EVs in the studied samples. Transmission electron microscopy further validated the presence of SP EVs. Morphological characteristics of SP EVs from men of different age groups were described. Quantitative characteristics of the SP EV samples were assessed using Nanoparticle Tracking Analysis (NTA). Results. The obtained particle size and concentration distributions revealed age-related changes in SP EVs: in the advanced reproductive age group, a shift in the dis tribution peak toward larger particles was observed compared to the peak distribution in the young men group. Conclusion. The results provide a basis for further investigation of SP EVs and exploration of their potential clinical applications in assisted reproductive technologies, such as in vitro fertilization programs.

Extracellular vesicles are membrane structures of cellular origin secreted by various types of cells and glands into both external and internal environments. These vesicles play an important role in intercellular communication and many health- and disease-related processes. Although extracellular vesicles have been isolated from nearly all specialized tissues of the male reproductive tract, their composition and function are best characterized in the epididymis and seminal fluid. Extracellular vesicles enhance fertility by promoting cell sperm development and function as well as influencing cellular physiology in the female reproductive tract. This review synthesizes current evidence on the role of extracellular vesicles in stabilizing sperm function and highlights their significance as key regulators of male fertility. Keywords: seminal plasma, extracellular vesicles, exosomes, microvesicles, epididymosomes, intercellular communication, assisted reproductive technologies, sperm