Assessment of molecular and morphological dynamics during long-time in vitro cultivation of cryopreserved human ovarian tissue: risk of genetic alterations

Background Cryopreservation of human ovarian tissue is a technology for patients undergoing aggressive anticancer treatments. This technology includes the following stages: saturation by permeable cryoprotectants, freezing, thawing, removal of cryoprotectants, as well as tissues in vitro or in situ culture. Objective Evaluation of quality of tissue after cryopreservation and in vitro culture with the aim of detection of genetic and molecular changes in cells. Methods Ovarian tissue was frozen in 6% ethylene glycol and 6% dimethyl sulfoxide with speed of cooling 0.3°C/min and thawed at 100°C. After removal of cryoprotectants tissue fragments were in vitro cultured with the soluble extract of basement membrane protein (Matrigel) 3-D culture system for 7 days. Morphological and functional assessments were conducted using microscopic observation and RNA-Sequencing. Comparative analysis of tissue morphology before and after culture was performed with bioinformatics for gene expression and variant analysis, including functional annotation and study of protein-protein interaction. Results DNA and RNA analyses after cultivation indicated a rise in gene fusion and alternative splicing events, potentially affecting gene expression and cellular functions. Conclusion Long-time in vitro culture of human ovarian tissue results in substantial changes in its morphology and genetic alteration.