ИССЛЕДОВАНИЕ ВЛИЯНИЯ ВНЕКЛЕТОЧНЫХ ВЕЗИКУЛ НА МОРФОФУНКЦИОНАЛЬНОЕ СОСТОЯНИЕ ЭНДОМЕТРИЯ IN VITRO

Abstract

Despite the long history of assisted reproductive technologies (ART), the effectiveness of in vitro fertilization (IVF) programs remains at the level of 30-40%. Of great importance in the implantation process is the receptivity of the endometrium, that is, its ability to ensure the necessary stages of implantation.

In world practice, the search for ways to influence the endometrial tissue in order to improve the morphological and functional state, which could increase the effectiveness of assisted reproductive technology programs, continues. Extracellular vesicles can be used as a therapy with cell technology products. Their use is a promising approach to stimulating immunomodulation and tissue regeneration.

The purpose of the study is to assess the effect of extracellular vesicles on endometrial cell culture.

Materials and methods. The material was collected by endometrial pipelle biopsy. The resulting tissue fragments were placed in a phosphate-saline solution with the addition of penicillin-streptomycin, gentamicin and amphotericin for transportation. A combination of mechanical and chemical methods was used to isolate primary cells. Extracellular vesicles were isolated from follicular fluid by sequential ultracentrifugation. Vesicles were added to the culture medium at a concentration of 5 % and 10 %. The growth dynamics, morphological and morphometric parameters of endometrial cells were studied during cultivation in the presence of extracellular vesicles.

Biological material for isolating the primary endometrial cell culture and extracellular vesicles of follicular fluid was provided by the Ural Research Institute for Maternal and Child Health. The work was carried out at the Center for Biotechnology and Bioengineering of the Institute of Natural Sciences and Mathematics of the Ural Federal University in Yekaterinburg.

Results. In the course of the work, a technique for obtaining endometrial cell cultures was developed. The primary endometrial cell culture is heterogeneous and contains fibroblast-like stromal cells and glandular cells with epithelial-like morphology. After adding vesicles, the growth dynamics of the endometrial culture is not disturbed, the cells retain their natural morphology: stellate stromal cells with processes and small round epithelial-like cells are found. When adding vesicles, no critical changes in the structure of the cell cytoplasm were detected, it remains homogeneous. At the same time, the nuclear-cytoplasmic ratio in the endometrial cells increases dose-dependently, which indicates a change in the functional state of the cells in the culture. Under the influence of vesicles, the number of multinucleated endometrial cells increases. In the experimental groups, there were cells with up to 7 nuclei.

When staining with fluorescent dyes separately on the preparation, which was microscopically examined 2 hours after applying 200 μl of vesicles, cells surrounded by an areola of small spherical formations were found, which are extracellular vesicles.

Conclusion. In the course of the work, data were obtained on the effect of extracellular vesicles on cell growth in culture, on the size and number of their nuclei. It was noted that extracellular vesicles do not have a toxic effect on cell culture. Under the influence of vesicles, the number of cell nuclei increases. When vesicles are introduced into cell culture, the proliferative activity of cells increases, and morphofunctional changes in the cellular structure of endometrial tissue occur.