To the Editor:
We read with interest the recent article by Larman and Gardner (1) regarding their alternative technique for vitrifying oocytes/embryos with the carrier Rapid-i. The authors proposed inserting the Rapid-i into the super-cooled air of a straw for instantaneous vitrification and then sealing the open end of the straw (postsealing method) in order to avoid direct contact with liquid nitrogen (LN2). We would like to point out that the super-cooled air inside the straws will be basically composed of nitrogen vapors, due to the nitrogen’s rapid evaporation and molecular weight. For this reason any microorganism accidentally present in the LN2 can also pass to the nitrogen vapor phase (2), and this may lead to the hypothetical contamination of the oocyte/embryos and the inner carrier of Rapid-I.
The authors stated that contamination issues associated with LN2 can be avoided using super-cooled air, and suggest its use for human gamete and embryo vitrification. But we would recommend that they sterilize the LN2 before evaporation using ultraviolet (UV) radiation to ensure clinical application in humans. Our group has recently demonstrated that decontamination of a small volume of LN2 via UV irradiation is feasible and straightforward (3-5).
We agree with the authors that, to date, there have been no reports of human infection attributed to the use of contaminated cryostored germplasm; furthermore, the outer straw of Rapid-i assures a safe hermetical cryostorage. However, any techniques preventing hypothetical risk of contaminations during cooling procedure must be welcome, especially in the light of probable new regulations worldwide.
Lodovico Parmegiani, M.Sc.
Graciela Estela Cognigni, M.D.
Marco Filicori, M.D.
Reproductive Medicine Unit
GynePro Medical Centers
1. Larman MG, Gardner DK. Vitrification of mouse embryos with super-cooled air. Fertil Steril 2011;95:1462-1466.
2. Grout BW, Morris GJ. Contaminated liquid nitrogen vapour as a risk factor in pathogen transfer. Theriogenology 2009;71:1079-1082.
3. Parmegiani L, Cognigni GE, Filicori M. Ultra-violet sterilization of liquid nitrogen prior to vitrification. Hum Reprod 2009;24:2969.
4. Parmegiani L, Accorsi A, Cognigni GE, Bernardi S, Troilo E, Filicori M. Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos. Fertil Steril 2010;94:1525-1528.
5. Parmegiani L, Cognigni GE, Filicori M. Efficacy of ultraviolet sterilization of liquid nitrogen. Reprod Biomed Online 2011;22:320.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.03.057
The Authors Respond:
We would like to thank Parmegiani et al. for their comments. However, given that 78% of the air inside the straw is already comprised of atmospheric nitrogen, the authors would challenge the statement (on the principles of diffusion) that “the super-cooled air inside the straws will be basically composed of nitrogen vapors (from the liquid nitrogen).”
Furthermore, although UV-sterilized liquid nitrogen could certainly be used in combination with the Rapid-i, the authors would like to point out that the IVF laboratory is not a truly sterile environment. Consequently, if we are to entertain discussions of hypothetical contaminations, then systems that employ sterilized liquid nitrogen will have to ensure sustained sterility during the entirety of the vitrification procedure.
Mark G. Larman, Ph.D.
David K. Gardner, Ph.D.
Department of Zoology
University of Melbourne
Melbourne, Victoria, Australia
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.03.075