Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination

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Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Kuwayama, M et al Reprod BioMed Online 2005

Dr. Daiter’s summary of this research article

Target:  human embryos from minimal-mild stimulation IVF cycles, comparing methods for cryopreservation
Goal: Determine the reproductive outcomes (survival and development) of human embryos that were frozen with the more traditional “slow cooling” systems versus vitrification using either the earlier open system (Cryotop) or the newly introduced (2005) closed system (Cryotip).

Vitrification can be described as an extreme elevation in viscosity that can allow solidification of solutions without ice crystal formation at low temperature, either by applying an extreme cooling rate or by using high concentrations of cryoprotectant solutions.  Methods of vitrification in embryology use a combination of these two techniques.  By reducing ice crystal formation during the cryopreservation process the exposure of injury due to these ice crystals is reduced and survival and the subsequent development of embryos may be improved.  However, exposure of the embryos to high concentrations of cryoprotectants may be cause osmotic damage to the embryos or may be toxic.  Also, and maybe most importantly, the vitrification process directly exposes the embryos in their cryoprotectant solution to liquid nitrogen so there is a potential risk of disease transmission through contaminated liquid nitrogen during cooling and storage.

One of the goals of this study is to determine if the newer (2005) closed system (Cryotip), which effectively eliminates the exposure to risk of disease transmission through contaminated liquid nitrogen, is comparable in terms of reproductive potential to the earlier open system (Cryotop), which directly exposes the embryos and their cryoprotectants to liquid nitrogen.

Protocol:

(1) embryos for vitrification were transferred into Equilibration Solution (7.5% (v/v) ethylene glycol and 7.5% DMSO dissolved in TCM199 supplemented with 20% SSS at 27C for 5-15 minutes,

(2) after initial shrinkage, embryos regained their original volume and were transferred into 3 x 20 microL drops of Vitrification Solution (15% (v/v) ethylene glycol and 15% (v/v) DMSO and 0.5 mol/L sucrose dissolved in TCM199 supplemented with 20% SSS,

(3) after incubation for 20 seconds in each drop respectively, embryos were loaded into Cryotips (Irvine Scientific) or on Cryotops and plunged into liquid nitrogen.

CRYOTIP

The cryotip is a plastic straw with a thin part (250 microm inner diameter, 20 microm wall thickness, 3 cm length) connected to a thick part (2000 microm inner diameter, 150 microm wall thickness, 4.5 cm length) and equipped with a movable protective metal sleeve.

Embryos are loaded in approximately 1 microL solution into the narrow part without any air bubbles by aspiration of medium, embryo and medium, and medium by a connected syringe.  Then the straw is heat sealed at both ends, the protective sleeve is moved over the narrow part and the device is plunged into liquid nitrogen (time to load, seal, adjust sleeve, and plunge should not exceed 90 seconds).

For warming the cryotip is removed from the liquid nitrogen, plunged into a 37C water bath for 3 seconds, wiped with 70% ethanol and a towel, and the sealed ends were cut with a sterile scissor.  A syringe adjusted to the thick end of the straw is used to expel the contents onto a sterile Petri dish.  Solutions for further manipulations were at 27C.  A 1 mcL aliquot of Thawing Solution (1.0 mol/L sucrose in TCM199 plus 20% SSS was placed adjacent to the expelled drop and merged subsequently with the other drop containing the embryos.  After one minute, the embryos were retrieved and transferred to a second drop of Thawing Solution for 1 minute, then transferred to 2 x 20 mcL drops of Dilution Solution (0.5 mol/L sucrose in TCM199 plus 20% SSS for 2 minutes each).  After 3 subsequent washings through three successive 20 mcL drops of Washing Solution (TCM199 supplemented with 20% SSS) for 3 minutes each, and then embryos were transferred into 100 mcL droplets of Blastocyst medium (Irvine Scientific) and cultured.

CRYOTOP

The cryotop consists of a 0.4 mm wide x 20 mm long x 0.1 mm thick polypropylene strip attached to a plastic handle and equipped with a cover straw.

With Cryotops, after equilibration described above, individual embryos were picked up in an extremely small volume (<0.1 mcL) of Vitrification Solution, placed on the strip, and submerged into filtered liquid nitrogen.  Subsequently, under liquid nitrogen the plastic cover was placed over the strip to provide protection during storage.

For warming, the protective cover was removed in liquid nitrogen and the end of the polypropylene strip was immersed directly into 4.5 mL of Thawing Solution at 37C for 1 minute.  Embryos were then transferred into 4.5 mL of Dilution Solution for 3 minutes, and then washed twice in Washing Solution for 5 minutes each.  Then the embryos were cultured per cell culture protocol.

The survival and development of human pronuclear stage, 4-cell stage, and blastocyst stage embryos was significantly improved when cryopreservation using vitrification methods was compared to the traditional “slow cooling” methods.  However, there was no significant difference seen in the pregnancy or live birth rates between these two different methods of cryopreservation.

The survival and development, pregnancy rates, and live birth rates of human embryos that were cryopreserved using Cryotip versus Cryotop methods did not show any significant differences when these two methods were compared to one another.

Conclusion:  Vitrification is a relatively simple, inexpensive and safe alternative to traditional slow cooling that results in higher survival and in-vitro developmental rates for human embryos at the pronuclear, 4-cell and blastocyst stages.  The Cryotip method eliminates the exposure to risk from contamination within the liquid nitrogen while maintaining the identified reproductive benefits of the Cyrotop method.

 

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 compares the Cryotop (open) Method of vitrification to the Cryotip (closed) Method of vitrification that were developed in Japan by Dr. Kuwayama.  The open method uses the Cryotop straw that exposes the sample being frozen directly to liquid nitrogen and consequently theoretically exposes the sample to the risk of contamination (infectious or other) if there are contaminants (eg., viruses such as HIV) contained in the liquid nitrogen during the rapid cooling or during storage in liquid nitrogen with other samples.  Of note, there are no known reported cases of actual infectious or other contamination with respect to the Cryotop as of January, 2016.  The closed method uses the Cryotip straw that does not expose the sample being frozen directly to liquid nitrogen, thereby minimizing or eliminating the risk of contamination by the liquid nitrogen, yet has somewhat slower rates of cooling than the open method.  The reproductive parameters that were examined in this article when comparing the benefits of the open method to the closed method suggest that they are comparable to one another, so both techniques appear to provide the same advantages in terms of reproductive survival and function.

 

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