Most protocols to cryopreserve ovarian tissue utilize the permeable cryoprotective agents (CPAs) in 1.5 M concentration. However the issues related to the ability to use higher concentrations of CPAs have remained open. The research aim was to assess the efficiency of media containing osmotically active sugars (sucrose, mannitol) at stepwise adding/removing of 3 M dimethylsulphoxide (DMSO) and propanediol (PROH) on ovarian tissue integrity. After the CPAs adding/removing the ovarian tissue injury was histologically examined, as well as the oocyte volume in tissue structure was assessed. It has been found, that after adding/removing of PROH and DMSO solutions the maximum amount of normal follicles made 67-93% when using Dulbecco's Modified Eagle Medium (DMEM) with 200 mM sucrose. Assessment of tissue damage after adding/removing of CPAs has demonstrated that the percentage of normal follicles was 83-87% using DMSO in presence of both sucrose and mannitol as the dilution media components. While removing PROH the level of follicles preservation was 2.5× higher when using mannitol compared with sucrose. Our results indicate that the ovarian tissue injury was minimal during adding 3 M CPAs using DMEM, containing sucrose and following application of mannitol at removing both DMSO and PROH.

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