To the Editor:
We read with interest the recent article by Criado et al. (1) regarding the assessment of contamination of a single-straw ultravitrification closed carrier (Ultravit). They observed no contamination in any microdrops of medium contained in the closed devices, whereas the bacteria they used for forced contamination of liquid nitrogen (LN2) were present in 45% of open carriers’ strip (Cryotop).
The authors describe their novel single-straw carrier, as a very thin straw specifically designed to load oocytes/embryos with minimum volume of cryoprotectant solution. They state that this vitrification system determines just 5 seconds’ contact of this microcapillary tube with the LN2 and that the cell position inside the microcapillary tube avoids direct contact of cells and of the cryoprotective medium with the LN2. After 5 seconds the microcapillary tube is passed to the protective sheath and enclosed with an ultrasound sealer.
In our opinion, this system could not avoid the transmission of microorganisms in the culture medium during the warming procedure due to the previous direct contact during vitrification between the external surface of the microcapillary and the contaminated LN2. This obvious problem has hampered scientist to make an analogue device since the middle of the 90’s, and required a separate warming and dilution procedure for similar tools including Cryotip (Irvine Scientific, Santa Ana, California, USA) and Cryopette (Origio, Jyllinge, Denmark).
Furthermore, we guess that the protective sheath is pre-cooled in LN2, in that case its internal surface can be contaminated by nitrogen vapour (2). Accordingly, even after sealing microorganisms can pass from the internal surface of the protective sheath to the external surface of the microcapillary tube. For these reasons the authors should check in accurate way the contamination of the external surface of the microcapillary tube as well as the surface of the Cryotop strip by using highly sensitive methods and various microorganisms. Even a completely negative result may be insufficient to exclude the danger that theoretically exists, and to pass through the rigorous legislation procedure of authorities.
Lodovico Parmegiani M.Sc.Reproductive Medicine Unit
GynePro Medical Centers
Gábor Vajta M.D., Ph.D., D.Sc.
BGI Ark Biotechnology
1. Criado E, Moalli F, Polentarutti N, Albani E, Morreale G, Menduni F, Levi-Setti PE. Experimental contamination assessment of a novel closed ultravitrification device. Fertil Steril. 2011 ;1777-1779.
2. Parmegiani L, Cognigni GE, Filicori M. Vitrification with UV-sterilized super-cooled air. Fertil Steril 2011; in press.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.04.036
The Authors Respond:
I would like to thank Mr Parmegiani and Dr. Vajta for their stimulating comments.
I would like to emphasize that all the closed devices, like Cryotip (Irvine Scientific, Santa Ana, California, USA) or Cryopette (Orygio, Jyllinge, Denmark), that are accepted by the FDA and other authorities, are immersed in the warming solutions without contamination risk. In our study (1), we did not find contamination in the microdrops into which we emptied the contents of the microcapillary, also submerging the end of this (0.2mm diameter, 0.01mm of wall thickness and 1 mm in contact with the warming drop). There is a great difference between thawing with an open device (in which the entire strip is submerged in the warming solution with a surface of 42-50 mm2) and thawing with Ultravit with 95.5% less of surface in contact with the warming drop (1.7-1.9 mm2). With Ultravit protocol, only the end of the microcapillary touches the base of the dish used to thaw, but at no time does the external part touch the warming drop.
Our work has demonstrated that the microorganisms that may be in the cooling solution, on the outside of the microcapillary, cannot come into contact with ultravitrified cells inside due to the loading procedure, the contact time with the cooling solution and the diameter and surface of Ultravit make it a secure device. I would like to remind Mr Parmegiani that his group demonstrated UV sterilization using the same bacteria that we have used to demonstrate the sterility of Ultravit. 45% of contamination with open devices and 0% of contamination with Ultravit is enough to exclude the theoretical danger of contamination.
Before entering discussions regarding the sterility in nitrogen used for vitrification, we should debate the use of communal containers, which is where cross-contamination can be found, as there is a possibility that the “contaminated cells” could come into contact with each other, and where a number of viruses and bacteria are found, which would never be found in the commercial liquid nitrogen (2).
Enrique Criado, B.Sc.
Department of Gynecology and Reproductive Medicine,
Istituto Clinico Humanitas
Rozzano, Milan, Italy
1. Criado E, Moalli F, Polentarutti N, Albani E, Morreale G, Menduni F, Levi-Setti PE.
Experimental contamination assessment of a novel closed ultravitrification device.
Fertil Steril. 2011;1777-9.
2. Bielanski A and Vajta G. Risk of contamination of germplasm during cryopreservation and cryobaking in IVF units. Human Reproduction. 2003; 2457-67.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.04.037