Human oocytes and zygotes are ready for ultra-fast vitrification after 2 minutes of exposure to standard CPA solutions

Vitrification of human oocytes and embryos in different stages of development is a key element of daily clinical practice of in vitro fertilization treatments. Despite the cooling and warming of the cells is ultra-fast, the procedure as a whole is time consuming. Most of the duration is employed in a long (8-15 minutes), gradual or direct exposure to a non-vitrifying cryoprotectant solution, which is followed by a short exposure to a more concentrated vitrifying solution. A reduction in the duration of the protocols is desirable to improve the workflow in the IVF setting and reduce the time of exposure to suboptimal temperature and osmolarity, as well as potentially toxic cryoprotectants. In this work it is shown that this reduction is feasible. In silico (MatLab program using two-parameter permeability model) and in vitro observations of the oocytes' osmotic behaviour indicate that the dehydration upon exposure to standard cryoprotectant solutions occurs very fast: the point of minimum volume of the shrink-swell curve is reached within 60 seconds. At that point, intracellular water ejection is complete, which coupled with the permeation of low molecular weight cryoprotectants results in similar intracellular and extracellular solute concentrations. This shows that prolonging the exposure to the cryoprotectant solutions does not improve the cytosolic glass forming tendency and could be avoided. To test this finding, human oocytes and zygotes that were donated for research were subjected to a shortened, dehydration-based protocol, consisting of two consecutive exposures of one-minute to two standard cryoprotectant solutions, containing ethylene glycol, dimethyl sulfoxide and sucrose. At the end of this two-minute dehydration protocol, the critical intracellular solute concentration necessary for successful vitrification was attained, confirmed by the post-warming survival and ability to resume cytokinesis of the cells. Further studies of the developmental competency of oocytes and embryos would be necessary to determine the suitability of this specific dehydration protocol for clinical practice, but based on our results, short times of exposure to increasingly hypertonic solutions could be a more time-efficient strategy to prepare human oocytes and embryos for vitrification.