Cryopreservation Techniques in Assisted Reproductive Technologies

Cryopreservation has become integral to assisted reproductive technologies, enabling long-term storage of gametes and embryos for fertility preservation, donor banking and cycle optimisation. Two principal methods coexist: slow freezing, which employs controlled cooling rates and low concentrations of cryoprotectants to minimise osmotic shock, and vitrification, an ultrarapid cooling approach that solidifies cellular water into a glass-like state without ice formation. Vitrification has largely supplanted slow freezing for oocytes and embryos, owing to higher post-thaw survival, improved developmental competence and reduced cryoprotectant exposure times. Recent refinements include closed carriers to prevent contamination, laser-assisted warming to enhance survival, automated handling systems to reduce operator variability, and dehydration-based exposure protocols that shorten warming and cooling steps. These advances have global significance, offering safer, more efficient workflows and expanded clinical applications in oncofertility, elective preservation and resource-limited settings.

Research from Nature Portfolio

Recent studies have demonstrated that a two-minute dehydration-based protocol can achieve the critical intracellular solute concentration necessary for successful vitrification of human oocytes and zygotes. By rapidly equilibrating cells in stepwise hypertonic solutions, this approach minimises exposure to cytotoxic cryoprotectants and suboptimal thermal conditions without compromising post-warming survival or the ability to resume cell division. Complementary mechanistic work has elucidated the kinetics of water efflux and cryoprotectant permeation, confirming that intracellular dehydration reaches its nadir within one minute, thereby supporting streamlined clinical workflows.

Cryopreservation Techniques in Assisted Reproductive Technologies publication trend

The graph below shows the total number of articles in cryopreservation techniques in assisted reproductive technologies across all publications each year (not limited to Nature Index journals).

Technical terms

Vitrification: An ultrarapid cryopreservation method that transforms water into a glass-like solid state, preventing ice crystal formation.

Cryoprotectant: A chemical agent (for example, dimethyl sulfoxide or ethylene glycol) that protects cells from cryoinjury by reducing ice nucleation and osmotic stress.

Slow freezing: A controlled-rate cooling process that gradually lowers temperature in the presence of low-concentration cryoprotectants to avoid intracellular ice formation.

Oocyte: A female gamete or egg cell used in assisted reproductive procedures, whose viability is critical for successful fertilisation and embryo development.

Osmotic stress: Cellular strain caused by rapid changes in extracellular solute concentration, leading to water movement that can damage membranes and organelles.

References

1. Human oocytes and zygotes are ready for ultra-fast vitrification after 2 minutes of exposure to standard CPA solutions. Scientific Reports (2019).
2. Ultra-Fast Vitrification: Minimizing the Toxicity of Cryoprotective Agents and Osmotic Stress in Mouse Oocyte Cryopreservation. International Journal of Molecular Sciences (2024).
3. Automation in vitrification and thawing of mouse oocytes and embryos. Frontiers in Cell and Developmental Biology (2023).
4. Efficiency and safety of vitrification of surplus oocytes following superovulation: a comparison of different clinical indications of oocyte cryopreservation in IVF/ICSI cycles. Frontiers in Endocrinology (2023).
5. Refined Techniques for Enabling Long-Term Cryo-Repository Using Vitrification and Laser Warming. Bioengineering (2023).