Effect of using natural honey as a new cryoprotectant agent on production of reactive oxygen species in vitrified-warmed mouse blastocyst

Background and ObjectiveCryopreservation as an essential part of assisted reproductive technologies recently has received major success in clinical practice. Nowadays, vitrification is a common method for cryopreservation of oocyte and embryo. In this procedure, glasslike solidification is achieved by using a high concentration of permeable and non-permeable cryoprotectants and rapid cooling rate[1]. Exposure to the extreme conditions leads to various cryoinjuries and stresses including thermal, osmotic and oxidative stress due to overproduction and accumulation of reactive oxygen species (ROS) within the cell. The presence of carbohydrates as non-permeating cryoprotectants is an attempt in order to reduce these stresses. The main role of adding carbohydrates such as sucrose and trehalose in the vitrification media is to create an osmotic gradient across the cell membrane that enhances cell dehydration prior to freezing. Since the combination use of sugars leads to better results, in this project, natural honey which consist of a variety of monosaccharides, disaccharides and also rich in antioxidants and flavonoids, was applied as an alternative to sucrose in mouse blastocyst vitrification [2, 3].Materials and MethodsThe first part of the experiment was conducted to determine the optimum concentration of honey in vitrification and warming solutions. The second part was designed to compare the effect of honey-based selected solutions and sucrose-based one in terms of survival, hatching, implantation rate and relative intracellular peroxides level. ROS assay in each treatment group of blastocysts were evaluated using detectable fluorescent 2´,7´-dichlorodihydrofluorescein (DCF) by Cytation3 (Cell Imaging Multi-Mode Reader).FindingsAmong different concentrations of honey, optimum concentrations of 1 and 2 M were chosen for vitrification and thawing mediums, respectively. Results of the second part demonstrated that not only the survival rate can be equal with sucrose when using honey, but it also brings about as good results as the sucrose-based group regarding the hatching rate and more importantly the implantation rate. In addition, the increased expression level of reactive oxygen species in sucrose-based group is moderated by using the natural honey.ConclusionTherefore, we can propose natural honey as an appropriate substitute for sucrose to optimize the vitrification process and achieve higher post-thaw quality of embryos.

Keywords:

vitrification , non-permeating cryoprotectant , oxidative stress , antioxidant , reactive oxygen , natural honey , mouse blastocyst