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

Abstract

Cryopreservation of human ovarian tissue is a technology for protection of reproductive potential in patients undergoing aggressive anticancer treatments. This technology includes the following stages: saturation by permeable cryoprotectants, freezing, thawing, removal of cryoprotectants, and tissues in vitro or in situ culture. The aim of our investigations was the evaluation of genetic risks and molecular alterations in human ovarian tissue during in vitro culture. 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-Seq. 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. DNA and RNA analyses after cultivation indicated a rise in gene fusion and alternative splicing events, potentially affecting gene expression and cellular functions. It was concluded that long-time in vitro culture of human ovarian tissue results in substantial changes in its morphology and genetic alteration.