**7. Contamination of LN2: Open versus closed systems**

There are many potential advantages of vitrification in that it is an easy, cheap, fast and an apparently successful cryopreservation method; however, there is one issue that is still up for debate. It has been shown that fungi, bacteria and viruses are able to survive in liquid nitrogen (LN2) (Tedder *et al*, 1995; Fountain *et al,* 1997; Bielanski *et al,* 2000; 2003; Kyuwa *et al*, 2003; Letur-Konirsch *et al*, 2003). Given the direct exposure of the human cells as they are directly plunged into LN2 during the vitrification process, this therefore raises the question as to whether the LN2 has to be sterilized, as it may be a possible source of contamination for those cells. To this point there has been no fungal, viral or bacterial contamination that has been described from about 400 publications related to vitrification since the first report in 1985. Bielanski and colleagues (2000) demonstrated a viral transmission rate of 21 % to human embryos stored in open freezing containers under experimental conditions of extremely elevated viral presence; while in contrast all embryos stored in sealed freezing containers were free from contamination. Based on this observation they proposed that the sealing of freezing containers appears to prevent exposure to potential contaminants. Commercial systems to purify LN2 by filtration have been developed, however this technology to date has received little practical application in IVF laboratories that have active cryopreservation programs. While it is not totally clear that contamination is a real risk in everyday use of LN2, nevertheless it may be prudent to consider routine sterilization of LN2 when open carrier systems are used for vitrification, followed by a sealing of that system for cryo-storage. Further there are currently at least three 'closed' sealed vitrification systems that are commercially available, with FDA clearance, that represent successful alternatives to open systems for embryo vitrification (Liebermann, 2009, 2011)

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