Assisted Conception Unit, Birmingham Women's Hospital, Edgbaston, Birmingham B15 2TG, UK
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Abstract |
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Key words: cryopreservation/nitrogen vapour/safety/sperm banks
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Introduction |
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Although primarily aimed at bone marrow stem cells, the document suggested that similar principles should apply to other cryopreserved cells and tissues, and indeed the HFEA working group recommended that the new code of practice address this specifically for gametes and embryos. Within the Blood Transfusion Service there has been a drive towards liquid nitrogen vapour storage as the `safer' alternative, however, members of the assisted reproductive technology (ART) community are understandably keen to avoid a `knee-jerk' reaction and follow suit. There are obviously a number of issues related to vapour storage, which need careful consideration:
Safety
At present there is no direct evidence of cross-contamination in a cryobank within a fertility clinic setting. There is however evidence of the presence of the hepatitis C virus in different ejaculates of the same semen donor (Mckee et al., 1996) and these are known to survive in the liquid phase. The safe cryopreservation of infected samples from infertile hepatitis C virus male patients has previously been highlighted (Massey et al., 1996
).
Cost
The smallest automated nitrogen vapour vessels cost more than £5000 (UD$7660). In addition, many units with smaller storage facilities would find it difficult to make use of the relatively large capacity offered by the vapour storage systems currently available, an inefficient and expensive use of freezer space. Adaptation of current liquid nitrogen vessels would significantly affect storage capacity.
Viability of spermatozoa and embryos
With the existence of temperature gradients within vapour storage vessels, there is a general concern that viability of spermatozoa and embryos after nitrogen vapour storage would not be comparable with that of storage in the liquid phase.
The aim of this article is to discuss these issues one by one. We use the small amount of experience we have had with vapour storage in our clinic, discussing the advantages and potential pitfalls. We discuss the reasons for changing our procedures and specifically address the issues of safety, quality control, cost and cell viability.
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The sperm bank |
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In our own cryostore, at least four vessels were in need of replacement. Coupled to the high cost of fitting alarms to a further six, it was decided to replace the entire system with two vapour cryostores (Taylor Wharton 10K), supplied by a single 240 l vessel using hoses connected on a T-piece. The capacity of each, depending on the inventory design is 50008000 1.2 ml vials or 32 000 0.75 ml straws. Our inventory was designed using combinations of towers, containing vial boxes and canisters, which can take either straws in goblets or vials on canes. Although this does not necessarily make the best use of freezer space, it was necessary to accommodate 16 000 existing patient straws as well as cryovials. Moving the entire `bank' also provides a further opportunity for audit, although this can significantly lengthen the process of transferring samples from liquid to the vapour freezers. If this is planned, then thought should be given to liquid nitrogen consumption, as the exercise increased nitrogen consumption by almost 90%. This figure has reduced since completion to around 50%, roughly 150 l per week.
Apart from the obvious increase in capacity, several integral features of an automated system soon become apparent, all of which lend themselves to quality control and help to reduce the chances of loss or damage to valuable biological material, due to human error. They include autofilling (a fill cycle commences when the liquid nitrogen level is unable to maintain the temperature under the lid at 140°C or below, or when the lid is replaced for fast temperature recovery) and alarms (generation of local and remote alarms for high temperatures, lid open, fault finding, over/underfilling). Data logging of all events, including filling activity, temperature, nitrogen levels and alarms. All logged events can be printed using a chart recorder or `down-loaded' to a computer.
Although extremely useful, total reliance on automation would be foolish. Auto-fill systems, for example are a potential hazard. If moisture is allowed into the nitrogen supply hoses, ice can accumulate and migrate to the solenoid-valves which control the flow of nitrogen. In a worst case scenario, the valve can be `frozen open' allowing a constant flow of nitrogen, quickly emptying the supply vessel and turning the vapour phase freezer into an `overflowing liquid' phase freezer. Therefore, a secondary `back-up' solenoid should be fitted to any auto-fill system. Additional operating procedures need to be in place to ensure that the freezers are performing as they should. Procedures for regular checking of the controller settings, independent temperature monitoring and regular servicing are essential.
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Safety of vapour storage |
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There is an obvious difference in risk between viral transmission from blood products (which are then transfused) and transmission by insemination/ART. From a European cohort, the risk of HIV infection from a single act of unprotected intercourse has been calculated at 2 in 1000 (de Vincenzi, 1994) and it remains unclear as to whether or not sexual transmission is a major route of the spread of hepatitis C (Semprini et al., 1998
; Neumayr et al., 1999
; Wejstal, 1999
). If we also bear in mind that sperm preparation for use in ART significantly lowers the viral load of the inseminate (Kim et al., 1999
; A.Semprini, personal communication), and that there is a relatively low uptake/usage of stored patient samples (710%), it could take many years and thousands of inseminations before an incident of cross-contamination in a sperm bank becomes apparent.
The fact that a cross-contamination incident has not yet occurred may well have provided us with a false sense of security. Clearly, we must assume that such an incident is possible and must take as many practical steps as we can to prevent transmission to a patient. Is vapour storage therefore inherently safer than storage in liquid? It would seem so, as a vector for viral transmission cannot be identified. The honest answer however is that we cannot say for sure. Fountain et al. (1997) demonstrated growth of a number of skin and other environmental micro-organisms from both liquid and vapour vessels. Although more species were grown from liquid, potentially pathogenic Aspergillus spp. were commonly found in the vapour vessels. However, when swabs from blood bags were cultured, transmission in vapour could not be demonstrated, even after a further 2 weeks of culture. The suggestion that liquid is a more effective transport medium for infectious material is not incredible. Any individual who has had the misfortune of having to empty and clean a liquid nitrogen vessel will be well aware of the veritable `biological soup' or detritus at the bottom. Storage in the gaseous phase may well eliminate much of this obvious risk and understandably is now standard practice in the Blood Transfusion Service.
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Other safety measures |
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Is sperm and embryo viability maintained in vapour storage? |
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Vapour storage has been in use in our clinic for >2 years and we have attempted to monitor performance in that time using a number of simple parameters. Firstly, to examine very short term storage, post-thaw results from donor ejaculates (n = 40) split between liquid and vapour storage were examined. Perhaps, understandably, `short-term' viability of spermatozoa was unaffected with no differences observed between groups with respect to percentage reduction on motility (36% liquid compared with 39% vapour). This is not perhaps surprising since samples can be adequately stored for up to 3 weeks in a dry shipping vessel. Examination of the clinic's donor insemination results as yet has not revealed any adverse affects of storage in vapour (Table I). We have also examined the viability of mouse embryos in vapour storage. 1-cell mouse embryos (n = 30) were kept in storage for 1 year; 25 survived thawing and they were all cultured to the blastocyst stage. Out of the 25 that survived, 23 formed normal blastocysts (92%), comparable with any results obtained from liquid nitrogen storage (Shaw et al., 1991
).
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Discussion |
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Notes |
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This debate was previously displayed on Webtrack, August 25, 2000
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References |
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