1 Laboratoire de Bactériologie-Virologie and 2 Laboratoire de Biologie de la Reproduction, GIMAP, University Hospital of Saint-Etienne, 42055 Saint-Etienne, France
3 To whom correspondence should be addressed. e-mail: rachel.levy{at}chu-st-etienne.fr
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Abstract |
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Key words: assisted reproductive technology/cryopreservation/hepatitis C virus/straws/viral safety
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Introduction |
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When appropriate techniques are used, HCV RNA is detected in 1238% of the seminal plasma from HCV chronically infected subjects, depending whether they are co-infected or not by HIV (Leruez-Ville et al., 2000; Pasquier et al., 2000
; 2003
; Bourlet et al., 2002
; Pekler et al., 2003
).
Before its use in ART, the potentially HCV-infected semen has to be cryopreserved in straws stored in liquid nitrogen containers. The safety of the straws is of high importance, in order to avoid cross-contamination that could occur during filling and sealing steps, storage in liquid nitrogen and opening of the straws, as reported previously (Tedder et al., 1995; Fountain et al., 1997
).
High-security ionomeric resin (IR) straws (Cryo Bio System, LAigle, France) have recently been validated for the storage of semen containing HIV (Benifla et al., 2000; Letür-Könirsch et al., 2003
); however, no data are available regarding their safety for semen from HCV-infected subjects. The aim of this study was to evaluate the safety of IR straws when they are filled with seminal plasma containing HCV RNA, and of the procedures surrounding their use.
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Materials and methods |
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Design of the study
After thawing, seminal fractions were spiked with a blood plasma containing 5 x 106 IU/ml of HCV RNA taken from a chronically HCV-infected patient. High-security IR straws were filled with 100 µl of seminal plasma using a pump, as recommended by the manufacturer, and sealed at both ends using a thermo-solder (SYMSTM System; Cryo Bio System).
Before testing, the straws were submitted to different treatments: (i) disinfection of the extremity of the straw with no subsequent cryopreservation; (ii) no disinfection and no cryopreservation; (iii) disinfection before cryopreservation and before thawing; (iv) disinfection only before cryopreservation; (v) disinfection only before thawing; and (vi) cryopreservation without disinfection. The decontamination step was performed by soaking the straws in a 4% sodium hypochlorite solution for 10 min and bleaching with a sterile compress. Cryoconservation was performed for 8 days in liquid nitrogen after progressive freezing at 20°C and 80°C.
For each experiment, three HCV RNA concentrations were tested: 500 000, 50 000 and 5000 IU/ml. Control straws filled with semen spiked with blood plasma from HCV-negative subjects were tested in parallel.
After optimal treatment, the end of each straw was soaked in 1 ml of RNAse-free water for 10 min at room temperature. The soaking water was then frozen at 80°C in aliquots of 250 µl until use. Some of the straws containing semen spiked with HCV RNA-positive or -negative blood were opened using a heating wire system in order to analyse their contents.
To test the safety of the filling and sealing steps, an empty straw was sealed at the end of the study. A wipe-test was also performed on the sealing system and on the heating wire used to open the straws and on the forceps used for this. The straws and swabs were rinsed in RNase-free water and each water sample was frozen as described above.
Detection of HCV RNA by RTPCR assay
The qualitative detection of HCV RNA in rinse water was performed using the Cobas Amplicor HCV 2.0 assay (Roche Diagnostics). For semen samples, we used a modified sensitive protocol combining a high-centrifugation step and a silica-based RNA extraction followed by the Cobas assay amplification (Bourlet et al., 2002). The sensitivity of the test was of
50 IU HCV RNA per ml of seminal plasma.
Human protamine-2 gene amplification
To test for potential contamination of the sealing and opening systems by semen, the DNA and mRNA from the protamin-2 gene, a human seminal housekeeping gene (Miller et al., 1994), was investigated as described previously (Bourlet et al., 2002
).
Influence of the cryoprotectant on the RTPCR sensitivity
To evaluate the impact of the cryoprotectant on the sensitivity of the RTPCR, eight straws (two for each of the three concentrations of HCV RNA mentioned above and two negative controls) were filled with 100 µl of seminal plasma diluted 1:1 in glycerol containing cryoprotectant (MedicultTM; Lyon, France).
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Results |
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Material controls
The wipe-test performed on the sealing system tested negative for HCV RNA and for both DNA and mRNA of the protamine-2 gene, as did the rinse water from the end of the empty straw sealed at the end of the study (Figure 1, lanes 2 and 3).
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Discussion |
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Another important point stressed herein is the lack of inhibitory effect of the cryoprotectant on the RTPCR assay, allowing the detection of HCV RNA in all cryopreserved semen samples.
In addition, the present study validated the safety of the cryopreservation step of IR straws, as illustrated by the absence of HCV RNA in the rinse water and in the control seminal plasma from straws kept in liquid nitrogen. This is of importance, since cryovials were shown to be permeable to liquid nitrogen during storage (Clark, 1999). In fact, liquid nitrogen is an excellent survival medium for most infectious agents, as illustrated by straws contaminated by hepatitis B virus (Tedder et al., 1995
) and tanks by Aspergillus sp. (Fountain et al., 1997
). This contamination of the straw content can originate from outside the straw (during the filling or sealing step), or during storage, due to a permeability defect. Another potential source of contamination is the step of opening the straw, after thawing, by splashing of the straw content on and around the material. In contrast with the previous steps, the opening system using a heating wire was at high risk of contamination in this study, as demonstrated by the detection of both HCV RNA and protamine-2 gene products in the rinse water of the wipe-tests carried out on the system itself and on the forceps used to handle the straw. These results, not yet reported by others, indicate that this opening system, and others similar to it, should be eliminated and replaced by disposable scissors.
In conclusion, this study validates the safety of the filling procedure of the IR straws and of their cryopreservation in liquid nitrogen tanks when used with samples containing high loads of HCV RNA. The availability of DNase- and RNase-free straws is an additional benefit, since their content has to be tested by molecular technology. These straws are particularly recommended for use by laboratories performing ART with semen from subjects with chronic viral diseases. In addition, in order to satisfy the rules of standard precautions, these recommendations should be extended to all re-usable biological material stored in liquid nitrogen.
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References |
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Benifla JL, Letur-Konirsch H, Collin G, Devaux A, Kuttenn F, Madelenat P, Brun-Vezinet F and Feldmann G (2000) Safety of cryopreservation straws for human gametes or embryos: a preliminary study with human immunodeficiency virus-1. Hum Reprod 15,21862189.
Bourlet T, Levy R, Maertens A, Tardy JC, Grattard F, Cordonier H, Laurent JL, Guerin JF and Pozzetto B (2002) Detection and characterization of hepatitis C virus RNA in seminal plasma and spermatozoon fractions of semen from patients attempting medically assisted conception. J Clin Microbiol 40,32523255.
Bourlet T, Levy R, Laporte S, Blachier S, Bocket L, Cassuto G, Chollet L, Leruez-Ville M, Maertens A, Mousnier F et al. (2003) Multicenter quality control for the detection of hepatitis C virus RNA in seminal plasma specimens. J Clin Microbiol 41,789793.
Clark GN (1999) Sperm cryopreservation: is there a significant risk of cross-contamination? Hum Reprod 14,29412943.
Fountain D, Ralston M, Higgins N, Gorlin JB, Uhl L, Wheeler C, Antin JH, Churchill WH and Benjamin RJ (1997) Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 37,585591.[CrossRef][Medline]
Leruez-Ville M, Kunstmann JM, De Almeida M, Rouzioux C and Chaix ML (2000) Detection of hepatitis C virus in the semen of infected men. Lancet 356,4243.[CrossRef][Medline]
Letür-Könirsch H, Collin G, Sifer C, Devaux A, Kuttenn F, Madelenat P, Brun-Vezinet F, Feldmann G and Benifla JL (2003) Safety of cryopreservation straws for human gametes or embryos: a study with human immunodeficiency virus-1 under cryopreservation conditions. Hum Reprod 18,140144.
Miller D, Tang PZ, Skinner and Lilford R (1994) Differential RNA fingerprinting as a tool in the analysis of spermatozoal gene expression. Hum Reprod 9,864869.[Abstract]
Pasquier C, Daudin M, Righi L, Berges L, Thauvin L, Berrebi A, Massip P, Puel J, Bujan L and Izopet J (2000) Sperm washing and virus nucleic acid detection to reduce HIV and hepatitis C virus transmission in serodiscordant couples wishing to have children. AIDS 14,20932099.[CrossRef][Medline]
Pasquier C, Bujan L, Daudin M, Righi L, Berges L, Thauvin L, Berrebi A, Massip P, Puel J and Izopet J (2003) Intermittent detection of hepatitis C virus (HCV) in semen from men with human immunodeficiency virus type 1 (HIV-1) and HCV. J Med Virol 69,344349.[CrossRef][Medline]
Pekler V, Robbins W, Nyamathi A, Yashina T, Leak B and Robins T (2003) Use of Versant TM TMA and bDNA 3.0 assays to detect and quantify hepatitis C virus in semen. J Clin Lab Anal 17,264270.[CrossRef][Medline]
Tedder RS, Zuckerman MA, Goldstone AH, Hawkins AE, Fielding A, Briggs EM, Irwin D, Blair S, Gorman AM, Patterson KG et al. (1995) Hepatitis B transmission from contaminated cryopreservation tank. Lancet 346,137140.[Medline]
Submitted on February 5, 2004; accepted on March 24, 2004.
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