Vitrification

The Cryotop® Method offers the best proven results on the market for vitrification of oocytes and embryos. The Cryotop®, the Vitrification and Thawing Media and its accessories allow our method to be applied efficiently to achieve the highest survival rates.

Vitrification has revolutionized ART practice and nowadays cryopreservation is an integral part of the current methods of assisted reproductive technology.

In the past two decades, slow freezing has been replaced worldwide by vitrification due to its association with improved survival rates and clinical outcomes comparable to fresh embryo transfers.



Since the introduction of vitrification, the number of frozen embryo cycles has increased continuously across the world, without the fear of complications for the patient such as ovarian hyperstimulation syndrome or multiple pregnancies.

Kitazato is recognized as one of the pioneering brands in driving and improving vitrification.
Its greatest contribution in this field has been the development of the renowned Cryotop® Method.

Kitazato’s Cryotop Method allows to work with oocytes and embryos at every stage of development, from PN to blastocyst.




Interview with Dr. Ana Cobo: How has vitrification changed your way of working in the clinic?

In this interview PhD. Ana Cobo, Director of the Cryobiology and cryopreservation Unit at the IVI Valencia Clinic, tells us how vitrification has changed her way of working in the clinic.

Its unique versatility makes Kitazato’s Cryotop® Method the only one that can be used for numerous highly efficient cryopreservation procedures.

EMBRYO VITRIFICATION

Surplus Embryos

When IVF was first introduced, fresh embryo transfer was the norm. However, to avoid risk of multiple pregnancies and to prevent wastage of surplus embryos, cryopreservation became an essential part of ART.

Segmentation of Cycle

Ovarian hyperstimulation (OHSS) is one of ART complications vitrification contributed to minimize. A planned ‘freeze all’ of the entire cohort of embryos in a fresh cycle and subsequent transfer in a frozen embryo cycle, contributes to decrease OHHS risk, allowing hormone levels on patients’ bodies return to normality before the embryo transfer.

‘Freeze All’

The objective of the ‘freeze all’ technique is to replace the embryos in a more favorable intrauterine environment, without possible adverse effects of hormone levels over endometrial receptivity. Studies show a substantial increase in the ongoing pregnancy rate when FET was performed compared to fresh ET.

PGT

Preimplantation genetic test involves testing an embryo for chromosomal or specific monogenic disorders before ET. This requires a biopsy of an embryo on Day 3 or Day 5. In the case of PGT programs it is essential to implement a successful vitrification technique to freeze all the biopsied embryos to allow sufficient time to obtain the genetic result.

Cryo-Pooling of Embryos:

In poor responder patients, embryos of 2–3 cycles are pooled to collect sufficient blastocysts to be transferred or tested by PGT.

OOCYTE VITRIFICATION

Fertility Preservation
for Medical Reasons

Chemotherapy and radiotherapy are generally gonadotoxic and can lead to premature ovarian failure in the future. Young women suffering from cancer can preserve their fertility by oocyte freezing if referred in time. Other than cancer, carriers of BRCA-1 and BRCA-2 requiring prophylactic oophorectomy, who suffer from autoimmune disorders and those prone to premature ovarian failure may be candidates for oocyte freezing.

Fertility Preservation
for Social Reasons

Oocyte freezing is a viable option for women who want to delay pregnancy for career or personal-related reasons, before their biological clock starts running out. As the rate of miscarriage also increases after 30 years of age, freezing eggs may give a woman a chance of having her own healthy genetic child whenever she is prepared for it.

Fertility Preservation
for Ethical Reasons

In some countries embryo vitrification is currently banned. In such circumstances, oocyte vitrification is a viable option rather than discarding surplus embryos.

No Sperm Retrieval

In cases of no sperm retrieval on the day of the egg collection, oocytes can be vitrified and then thawed at a later date when a sperm sample is obtained, or spermatozoa retrieved in a subsequent testicular biopsy.

Donor Oocyte Program/Egg Banking

Egg banking removes the need of synchronization between donor and recipient and hence reduces the anxiety of the patient. It also helps in giving the recipient wider options for selecting a donor without compromising pregnancy results.

Oocyte Pooling
in Poor Responders

In patients with poor response to ovarian stimulation, oocytes from multiple stimulation cycles can be collected and vitrified. Once an adequate number of oocytes has been reached, ICSI can be performed to create an adequate number of embryos, giving better pregnancy rates.

Oocytes can be succesfully vitrified for fertility preservation in patients who want to postpone their maternity or for any medical indication. In addition, donor oocytes can also be vitrified and banked to provide better synchronization with recipients.

Furthermore, several studies demonstrate that embryos are able to keep their implantation potencial after vitrification allowing to cryopreserve surplus embryos, all embryo cohort for freeze-all cycles or PGT until genetic result is obtained.

In addition, cryotransfers of vitrified embryos generated from previously vitrified oocytes confirm the capacity of these embryos to sustain an additional vitrification process and to develop into healthy babies. In cases of PGT in which an additional biopsy is required and therefore a re-vitrification also show that double vitrification has no impact on delivery rates.

(1) Cobo A. et al. Outcome of cryotransfer of embryos developed from vitrified oocytes: double vitrification has no impact on delivery rates. http://dx.doi.org/10.1016/j.fertnstert.2013.01.106

Cells and the surrounding liquid solution are converted into a glass-like amorphous solid, free of any crystalline structures when plunged in liquid nitrogen (LN).

 

Vitrification requires high concentrations of cryoprotectant agents (CPA) and a very high cooling rate to produce the glassy state, nevertheless, a high warming rate is also crucial as ultimate survival of cryopreserved cells may be more dependent on the rate at which the specimen is warmed, rather than the rate at which it is cooled.

Before being plunged into LN, oocytes or embryos are exposed to a non-vitrifying CPA solution (ES – Equilibration Solution), containing only permeable CPAs that penetrate the cells, then to a vitrifying CPA solution (VS – Vitrification Solution), containing both permeable and non-permeable CPAs.

Upon exposure to the non-vitrifying CPAs, the cell immediately adjusts its osmolarity by losing water, and shrinks.

The CPAs then enter the cell more slowly, due to their low membrane permeability.

The time of exposure to the non-vitrifying solutions at a defined temperature is of critical importance, as it determines the intracellular concentration of CPA.

The vitrifying solution causes further cellular dehydration, which concentrates the intracellular CPA.

The overall objective is to create an intracellular environment more stable that will remain vitrified.

The movement of water and CPAs across cell membranes is crucial for cell survival during cryopreservation, and the degree of permeability depends upon the stage of development.

Oocytes have low surface/volume ratio and low permeability to CPA, so they are less efficient than embryos at losing water and taking up CPAs and thus are more susceptible to intracellular ice formation. Gradual addition of CPA during equilibration allows adequate permeation into the oocytes and ensures better survival outcome.

Permeability of cell membranes may also vary according to the temperature during vitrification and warming processes; therefore, it must be considered as a key factor for a proper exchange of CPAs and water.

Addition as well as removal of CPA causes successive phases of shrinkage and re-expansion due to movement of water and CPA across the cell membranes.

Kitazato protocol is designed to successfully induce less shrinkage-swelling stress to the cell during the procedures.

The movement of water and CPAs across cell membranes is crucial for cell survival during cryopreservation, and the degree of permeability depends upon the stage of development.

Oocytes have low surface/volume ratio and low permeability to CPA, so they are less efficient than embryos at losing water and taking up CPAs and thus are more susceptible to intracellular ice formation. Gradual addition of CPA during equilibration allows adequate permeation into the oocytes and ensures better survival outcome.

Permeability of cell membranes may also vary according to the temperature during vitrification and warming processes; therefore, it must be considered as a key factor for a proper exchange of CPAs and water.

Addition as well as removal of CPA causes successive phases of shrinkage and re-expansion due to movement of water and CPA across the cell membranes.

Kitazato protocol is designed to successfully induce less shrinkage-swelling stress to the cell during the procedures.

Vitrification procedure

 

The Repro Plate is a six conic-shaped well dish, exclusively designed to follow The Cryotop® Method and vitrification with comfort. The Repro Plate has high transparency and great visibility, and also offers two slots to support the Cryotop®, allowing those who wish to carry out loading specimens statically.

Each well must be filled with 300µl of the solutions allowing to use the proper volume of media to minimize water evaporation and osmolality change, and thus preventing cell disruption. Furthermore, the conic-shaped well of the Repro Plate allows gradual and stepwise addition of the solutions during oocyte vitrification, which is the preferred and recommended method for this stage of development(2). It has a flat base, which allows the use of traceability labels.

 

The oocytes and embryos are exposed to the solutions with increasing concentrations of cryoprotectant agents (CPAs). The CPAs will replace the intracellular water and prevent ice crystal formation during the transition to the glassy state.

The specimens are loaded on the Cryotop® and transferred to the LN at a very high cooling speed. The Cryotop® is a minimum volume device and so-called open system as it goes into direct contact with the LN during vitrification and storage.

Samples vitrified can be safely stored in LN or nitrogen vapor phase for a very long period of time.

(2) The Alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reprod BioMed Online. 2012 Aug, 25 (2): 146-167.

Kitazato Vitrification Media

Kitazato Vitrification Media have evolved by replacing HSA (Human Serum Albumin) with HPC (Hydroxypropiyl Cellulose) eliminating the possible risk of pathogens transmition from blood products. Furthermore, it has been shown that HPC supplementation in combination with trehalose has certain advantages (4). HPC gives higher viscosity to the solutions, which increases the probability of a successful vitrification and, therefore, to obtain better survival and further embryo development. In addition, HPC allows specimens to detach easily from the Cryotop during warming, reducing mechanical stress and increasing survival rates even in hatched blastocysts.

Kitazato Vitrification Media contain DMSO and EG as permeable cryoprotectants and trehalose as non-permeable cryoprotectant. The combination of DMSO and EG in our Vitrification media avoids excess cellular toxicity and reduce osmotic pressure that can damage oocytes and embryos during dehydration.

(4)Coello A. et al. A combination of hydroxypropyl cellulose and trehalose as supplementation for vitrification of human oocytes: a retrospective cohort study. J Assist Reprod Genet. 2016 Mar, 33(3): 413-421.

For the warming procedure, the Repro Plate is set up with Thawing Media.

The Cryotop is immersed in the first warming solution pre-heated at 37ºC achieving a very high warming speed. The oocytes and embryos are exposed to different solutions with a gradual decrease in CPA concentration preventing osmotic shock and enabling good and safe cell rehydration.

The non-permeant cryoprotectant in Kitazato Thawing Media is trehalose, which has a greater capacity to protect cell membranes compared to sucrose. As cells have a limited tolerance to contraction and swelling, the rehydration protocol must ensure a minimum of contractions and re-expansions of the cytoplasm in order not to alter cell functioning.

For the warming procedure, the Repro Plate is set up with Thawing Media.

The Cryotop is immersed in the first warming solution pre-heated at 37ºC achieving a very high warming speed. The oocytes and embryos are exposed to different solutions with a gradual decrease in CPA concentration preventing osmotic shock and enabling good and safe cell rehydration.

The non-permeant cryoprotectant in Kitazato Thawing Media is trehalose, which has a greater capacity to protect cell membranes compared to sucrose. As cells have a limited tolerance to contraction and swelling, the rehydration protocol must ensure a minimum of contractions and re-expansions of the cytoplasm in order not to alter cell functioning.

The IVF Lifecycle

Kitazato offers a well curated selection of quality products that maximize success at every step of the IVF Lifecycle. Learn more about the products involved in each IVF procedure.

View all
Oocyte Retrieval
More info
Andrology
More info
Culture
More info
Vitrification
More info
Embryo Transfer
More info
Oocyte Retrieval
More info
Andrology
More info
Culture
More info
Vitrification
More info
Embryo Transfer
More info