Highly efficient vitrification for cryopreservation of human oocytes and embryos

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Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method Kuwayama, M Theriogenology 2007

Dr. Daiter’s summary of this research article
Target:  Review literature regarding the latest achievements in vitrification for cryopreservation in embryology, especially the Cryotop method.

Purpose: Outline the role of vitrification, including methods using the Cryotop technology,  in human assisted reproductive technologies.

Introductory Comments:  (1)The biological clock of live objects can be stopped for an unlimited (or practically unlimited) period of time by cooling them to temperatures below –150C.  (2) The smaller the sample size, and the simpler the structural complexity of the sample, the better the survival rates with current cryopreservation methods.  (3) In embryology the sample sizes are generally relatively small (eg, spermatozoa, oocytes, embryos) however seemingly negligible variations in size have been seen to cause significant differences in survival rates. (4) Mammalian embryos and especially oocytes that have been cryopreserved appear to benefit from vitrification more than from the traditional slow-rate freezing methods. (5) The Cryotop vitrification method has resulted in the highest number of babies born after vitrification of human embryos and oocytes worldwide.

Vitrification:  (1) Vitrification can be defined as a vitreous, transparent, ice-free solidification of water-based solutions at subzero temperatures.  (2) Vitrification for cryopreservation of mammalian embryos and oocytes was generally considered “experimental” (2007) (3) Vitrification may result from the use of extremely high cryoprotectant concentrations and/or extremely high cooling rates (lower cryoprotectant concentrations can be used if cooling rates are increased, and visa versa). (4)  Extremely high cryoprotectant concentrations may have toxic or osmotic effects on the frozen sample.  (5) Direct contact between the sample and liquid nitrogen to increase cooling rates may result in contamination of the sample by substances within the liquid nitrogen.  (6) New technology in vitrification may minimize or eliminate the exposure to risk from extremely high cryoprotectant concentrations (eg., limit time of exposure to the highest concentrations at temperatures when risk is greatest)  and/or contamination due to direct exposure to liquid nitrogen (eg., sealed cryostraws, filtered or UV sterilized liquid nitrogen).

Minimum volume vitrification methods:  (1) Minimizing the volume of vitrification solution used to cryopreserve the embryos or oocytes increases the cooling and warming rates as well as decreases the chance of ice crystal formation.  (2) Chilling injury is significant for samples between +15 to +25C and –5C so increasing the cooling rate through these temperatures should improve survival (some research suggests a cooling rate of 20,000C/min best allows a reduction in cryoprotectant concentration and time in the most sensitive temperature ranges).  (3) The thickness of the cold liquid nitrogen vapor layer over the surface of the solution and the speed at which the sample passes through this layer may have significant impact on the final outcome, so a liquid nitrogen slush for cooling with a commercially available device (Vitmaster) has been suggested to decrease both chilling injury and cryoprotectant concentrations. (4) Minimum volume vitrification may also reduce or eliminate zona pellucida and embryo fracture damage that often occurs when samples are cryopreserved in standard insemination straws and warmed rapidly.  (5) Minimum volumes are usually considered <<1 mcL for direct dropping of samples into liquid nitrogen, with droplets of 0.1-0.5 mcL prepared on glass coverslip strips and immersed into liquid nitrogen or liquid nitrogen slush having a 50% reduction in cryoprotectant concentration and good outcomes. (6) Storage in cryovials or straws suggest liquid nitrogen vapor and often strongly discourage submerging the vials in liquid nitrogen since the vials may explode due to extreme pressure changes during evaporation of liquid nitrogen that may enter the vials during cooling and warming.

Cryotop Method:  (1) Minimum volume vitrification using a narrow, thin film strip (0.4mm wide x 20mm long x 0.1mm thick) attached to hard plastic holder; a 3cm long plastic tube cap can be attached to cover the film part for storage; and commercially available solutions for both vitrification and warming.  (2) There is a two step equilibration in a vitrification solution (ethylene glycol, DMSO and sucrose).  (3) Following a two step equilibration in a vitrification solution (ethylene glycol, DMSO, sucrose) the sample is loaded using a narrow glass capillary onto the top of the film strip (<0.1 mcL volume), most of the liquid is removed from the embryos and/or oocytes, the sample is immersed in liquid nitrogen, the plastic cap is pulled over the film part, and the sample is stored under liquid nitrogen.  (4) To warm, the protective plastic cap is removed from the Cryotop under liquid nitrogen (while submerged), the polypropylene strip is immersed directly into a 37C medium containing sucrose (to reduce the osmotic shock caused by the permeable cryoprotectants within the cells), the samples are sequentially incubated in diluent solutions, and then they are placed in media for tissue culture or embryo transfer. (5) Ethylene glycol is a highly permeable cryoprotectant with moderate toxicity that is commonly used in vitrification solutions and DMSO reportedly increases the permeability of ethylene glycol.  The minimal volume approach increases cooling and warming rates (up to 40,000 C/min) which may improve survival rates for the samples

Results with Cryotop vitrification in humans

(1) Vitrification of 5881 human pronuclear stage embryos resulted in 100% survival, 93% cleavage (growth), and 52% blastocyst formation (significantly better than with slow cooling).  (2) Vitrification of 6328 human blastocysts resulted in 90% survival, 5659 transferred blastocysts, a 53% clinical pregnancy rate, and 45% live birth rate (highly significantly better than previous reported results).  (3) Vitrification of 111 Metaphase II human oocytes resulted in 95% survival, 91% fertilization after ICSI, 50% developed to blastocyst in vitro, 29 embryo transfers (mean 2.2 embryos per transfer) had a 41% pregnancy rate

Dr. Daiter’s review of this research article

Masashige Kuwayama, Scientific Director of the Kato Ladies’ Clinic (7-20-3 Nishishinjuku, Shinjuku, Tokyo, Japan), one of the world’s largest IVF centers, is a recognized world class pioneer in the field of human embryology and theriogenology.   Dr. Kuwayama developed vitrification procedures and tools (a technique of cryopreservation that rapidly freezes samples) to improve success with human oocyte (egg) and human embryo (fertilized egg) cryopreservation that are now widely used throughout the world.

This important article presents the Cryotop Method of vitrification that Dr. Kuwayama developed in Japan, which appears to provide improved reproductive success for both human oocytes and human embryos (including pronuclear stage embryos and blastocysts) as compared to the more conventional “slow freezing” methods commonly used throughout the USA.  The simplified reason for this improvement in reproductive survival and function seems to be that vitrification, especially when using the Cryotop tool, allows for extraordinarily fast cooling rates in excess of 20,000C/min so that “chilling injury” as the samples pass between +15-25C and -5C is reduced.  It is important to note that the Cryotop tool, as an open system, should also allow for much faster warming during thawing (slow thawing is where exposure to a great deal of chilling injury is thought to occur).   ​


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