Understanding the Warming Procedure for Vitrified Embryos
By Elise Fiskum, BS

Embryo warming is a critical component in the successful application of cryopreservation techniques in ART. Understanding how temperature, osmolarity, and media components interact to restore cellular integrity is important when optimizing protocols, troubleshooting, and improving post-warming outcomes. Currently, vitrification is the most widely used form of gamete cryopreservation in ART. A thorough understanding of the post-vitrification warming procedure can improve this often final step in a patient’s family planning.

A typical warming kit includes three solutions: Thawing Solution (TS), Dilution Solution (DS), and Washing Solution (WS). TS contains extracellular cryoprotectants such as sucrose or trehalose, diluted to form a sticky 1.0 M solution for initial high-temperature warming^{3, 10, 12, 16, 17}.These sugars have a high molecular weight and are too large to cross the cell membrane, with trehalose being the larger and more effective of the two disaccharides⁷. DS contains the same extracellular cryoprotectants at 0.5 M concentration^{3, 10, 12, 16, 17}. WS primarily contains buffering agents like HEPES to maintain pH stability, mimicking culture media^{3, 10, 12, 16, 17}. In all three solutions, manufacturers may add additional proteins or antibiotics to support embryo re-expansion and minimize contaminates^{3, 10, 12, 16, 17}.

Warming should rapidly bring the embryo to culture temperature (37°C) to avoid ice crystal formation^{1, 5, 11, 13}. Warming at a rate exceeding 2170°C/min is recommended to minimize the time the embryo spends at water’s freezing point, bypassing ice crystal formation during the warming process^{5, 13}. While avoiding ice formation during vitrification is important, warming rate tends to have a greater impact on embryo survival than cooling rate¹². A rapid warming rate is necessary for post-vitrification embryo survival.

Though temperature must increase quickly, rehydration of embryos should be gradual during the warming process^{2, 4}. The addition of non-penetrating extracellular cryoprotectants to TS creates a high osmolarity solution, facilitating a moderate osmotic gradient during initial rehydration. Without these additions, water may surge into the embryo too quickly and cause cell membranes to burst with expansion, leading to cell death^{2, 4, 6}. A moderated difference in osmolarity allows diffusion to occur at an optimal rate to minimize damage during re-expansion^{2, 4}.

Subsequent steps of warming occur in DS and WS. These steps are performed at room temperature to minimize solution evaporation, which could affect the solution’s osmolarity and introduce undue stress.  Diffusion continues in DS, at a lower osmolarity. Media carry-over is utilized to maintain a gradual gradient, pooling TS around the embryo when moved to DS to slowly allow the embryo to adjust to the 0.5 M solution³. In WS, the same carry-over effect helps the embryo adapt to culture conditions without a rapid decrease in osmolarity³. The name “washing solution” is apt, as this step is also important in minimizing lingering cryoprotectants from freezing. Warming protocols often indicate multiple drops of WS first to allow slow acclimatization to culture conditions, and second to rinse away insulting compounds like DMSO or ethylene glycol. When moving the embryo to a culture drop, minimal freezing compounds that could cause progressive cell death in culture should remain in and around the embryo¹⁴. 

Embryologists and researchers continue to investigate warming mechanisms to determine the best protocol for embryo warming. Current research indicates that an ultra-fast thaw procedure may improve workflow without compromising embryo integrity, completing the warming process in just one minute using only TS^{8, 9, 15, 8}. Although gradual rehydration has been emphasized in past and current protocols, these new findings highlight the embryo's robust ability to self-regulate. The dramatic increase in osmotic pressure when moving an embryo from TS to culture media seems irrelevant to embryo survival, though sufficient washing through multiple culture drops to remove compounds associated with vitrification remains important. Warming rate and initial rehydration in high-osmolarity TS seem to be the primary factors affecting embryo survival.

References

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