Sperm cryopreservation: is there a significant risk of cross-contamination?

Gary N.Clarke were contaminated during the cryostorage process, probably from the contaminated liquid nitrogen. Thus, there is no doubt Andrology Department, The Royal Women’s Hospital, Carlton, that a range of microbes, including hepatitis B, can survive Victoria 3053, Australia direct exposure to liquid nitrogen, and under certain conditions result in cross-infection. Given the strength of the evidence of This opinion was previously published on Webtrack 85, September 10, 1999 liquid nitrogen contamination by microbes and cross-infection in certain situations, we felt that the possibility of contamination Interest in the subject of sperm cryopreservation has a long or cross-contamination during semen cryopreservation should history, with thousands of published articles in the scientific/ be taken seriously. medical literature. Clinical pregnancy rates using donor Traditionally, semen storage protocols have not involved insemination can now approach natural pregnancy rates (Clarke sterile techniques. In particular, filling of straws by the ‘dip et al., 1997b). However, an issue which has received very and wipe’ procedure (Russell et al., 1997), or cracking of little attention in the literature is the subject of microbiological overfilled straws during freezing would have significant potencross-contamination in sperm banks. This subject obviously tial to contaminate the liquid nitrogen. Straws which were concerns many sperm bank managers, who have instituted inadequately sealed could absorb the contaminated nitrogen, several approaches aimed at reducing the perceived risks. leading to a potential cross-infection episode during subsequent One approach is to screen all patients or donors for human clinical use of the thawed semen. There is an additional immunodeficiency virus (HIV) status, hepatitis B and C and risk of cross-contamination during semen processing prior syphilis prior to cryopreservation of their semen. Another to freezing in those laboratories which use containers of approach involves the use of quarantine tanks to hold samples polyvinylalcohol (PVA) sealing powder for multiple patients until screening results are obtained, before placing the cleared or donors. The PVA powder could accumulate microbes from samples into the main storage system. The samples of patients a number of individuals, which are then introduced to the with positive screening results are then transferred to specially inside of straws of another patient or donor. The storage of designated tanks. Both of these approaches have obvious semen in cryovials placed in direct contact with liquid nitrogen drawbacks because of the incubation period of HIV and the also presents a serious risk because a significant proportion of fact that the screening procedures do not cover all potential cryovials absorb liquid nitrogen through caps which do not pathogens. This issue therefore needs to be addressed from maintain their seal under these conditions. Although the another perspective in order to estimate the likelihood of its manufacturer strongly recommends the use of a second ‘skin’ occurrence and to determine what measures, if any, might be called Cryoflex (Product No. 343958; Nunc Nalge Internaconsidered appropriate to prevent or minimize it. tional, Roskilde, Denmark) to provide an adequate seal, it is The occurrence of cross-contamination during liquid nitrocommon practice to store naked vials in liquid nitrogen. Recent gen storage of biological material and subsequent crosstests conducted in my laboratory showed that 45% of Cryovials infection of patients has previously been demonstrated (Tedder (Product No. 340711; Nunc Nalge International) without an et al., 1995). They reported on a cluster of hepatitis B infection O-ring and 85% of Iwaki Cryovials (Iwaki, Japan) with an which was definitively traced to cross-contamination during O-ring absorbed liquid nitrogen during 3 h immersion in liquid bone marrow storage using nucleotide sequence analysis nitrogen. In contrast, there was no evidence of liquid nitrogen (Hawkins et al. 1996). Other viruses have previously been condensation inside the vials when stored in the vapour phase found to survive direct exposure to liquid nitrogen, including for 16–24 h. Thus, in some instances, cryovials stored in the vesicular stomatitis virus (Schafer et al., 1976), herpes simplex liquid phase could absorb up to 1 ml of potentially contaminated virus, adenovirus (Jones and Darville, 1989), and papillomaliquid nitrogen. Depending on the length of storage and the virus (Goodman, 1960; Charles and Sir, 1971). There is also exact thawing protocol, any microbes in the absorbed nitrogen evidence of contamination of liquid nitrogen by other micromay have settled onto the semen interface and be left there organisms, including a wide range of bacterial and fungal when the nitrogen boiled off. Clinical use of this semen, species (Fountain et al., 1997). In the latter laboratory, one particularly when multiple vials may be inseminated during a liquid nitrogen tank was found to be heavily contaminated by treatment cycle, could pose an increased risk of cross-infection. an Aspergillus species which is a potential pathogen. These Some diseases such as hepatitis B require very small amounts of virus to transmit infection. authors reported that 1–2% of thawed bone-marrow samples