1 Department of Obstetrics and Gynaecology, Hôpital Bichat, 75018 Paris, 2 Cecos, Hôpital Necker, 75007 Paris, 3 Centre de Fertilité, Institut Mutualiste Montsouris, 75014 Paris, 4 Department of Virology, Hôpital Bichat, 75018 Paris and 5 Department of Histo-embryology, Hôpital Bichat, 75018 Paris, France
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Abstract |
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Key words: assisted reproductive technology/cryopreservation/experimental study/HIV/straw
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Introduction |
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The aim of our experimental study was to use HIV-1 to test the impermeability of the three most common types of straws used for human gamete or embryo cryopreservation and currently stored in liquid nitrogen tanks in France.
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Materials and methods |
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Each sealed straw was cut into two equal halves and sealed according to the schedule shown in Table I. Each demi-straw was then filled with 100 µl of HIV-1-containing supernatant (reverse transcriptase activity: 15 000 c.p.m./50 µl). Unfortunately, because of the inability to displace air, every attempt to fill the PVC and PETG demi-straws with the open end sealed ultrasonically overflowed and contaminated the exterior of the straw; thus, only the demi-straws with the open end sealed with cotton were filled (Table I
). The IR straws, because of their larger calibre, did not present any problem. The cotton end of each PVC and PETG demi-straw and both ends of the IR demi-straws were submerged two-thirds deep into a Falcon tube containing 2 ml of RPMI medium (Bio-Whittaker, Boehringer, Belgium) and placed in an incubator at 37°C under a 5% CO2 atmosphere. A 1 ml sample of the surrounding RPMI was withdrawn on days 3, 7 and 11, each time being replaced with 1 ml of sterile RPMI medium, and frozen at 80°C until all samples were subjected to HIV-1 RNA detection simultaneously. As controls, one Falcon tube was filled with 100 µl of HIV-1-containing supernatant and 2 ml of RPMI medium, and another one was filled with 2 ml of RPMI medium. For testing, each sample was thawed and centrifuged for 1 h at 23 500 g. The pellet was resuspended in 200 µl of RPMI medium, and virus RNA was extracted, amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by nested PCR (Titan RT-PCR kit, Boehringer, Mannheim, Germany), using primers specific to HIV-1 protease gene and loaded on 1% Agarose gel. Sterile water was used as the negative PCR control.
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Results |
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Discussion |
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Our investigation is open to several criticisms. First, we tested only HIV-1 and we have no idea what could happen with other viruses. Although HIV-1 is the biggest compared to hepatitis B and C viruses, we do not know whether molecular weight alone is a sufficiently robust criterion to test the impermeability of the straws. Second, this study was conducted over a much briefer interval compared to that over which human gametes and embryos are usually frozen in liquid nitrogen. Third, it was conducted at 37°C and in liquid nitrogen. This point is particularly relevant for the IR straws because the material from which they are made was only intended for cryopreservation and might not be safe at 37°C.
Furthermore, doubt persists as to the impermeability of the ultrasonic seals of PVC and PETG straws. Since 1998, classical PVC straws can no longer be used in France for the packaging, storage and/or in-vivo transfer of biological products of human origin because they cannot be sterilized by radiation. Nonetheless, many remain in liquid nitrogen tanks as do the PETG straws currently being used. Lastly, intact PVC and PETG straws should have been filled with the virus-containing solution and then sealed ultrasonically, but this procedure risks contaminating the apparatus, which might prove difficult, if not impossible, to decontaminate.
It should be noted that the increased contamination of RPMI with prolonged duration of incubation of IR straws suggests that their composition is defective at 37°C. However, this possibility does not preclude possible leakage due to defective sealing procedures. Indeed, the potential transmission of infectious agents from packaged semen during storage for artificial insemination of non-domestic livestock was recently described (Russel et al., 1997). In their experimental study, leakage from the straws seemed to be dependent on the filling and sealing methods used. It has also recently been suggested that the storage of semen in cryovials placed in direct contact with liquid nitrogen presents a risk because a proportion of cryovials absorb liquid nitrogen through caps, even when a second skin was used to provide an adequate seal (Clarke et al., 1999
). All these remarks justify further examination of the medical security of cryopreservation tanks, and underline the need to establish the safety of straws and good-practice guidelines for packaging human gametes or embryos for cryopreservation.
In conclusion, under these experimental conditions (37°C), the unsealed cotton end of PVC and PETG demi-straws appeared effectively to prevent HIV-1 escape, as did the thermosoldered cotton end of IR demi-straws, but doubt persists regarding sealed and unsealed plastic plug ends and for unsealed cotton ends of IR demi-straws. Additional studies are needed, especially to test the ultrasonically sealed ends of the PVC and PETG straws, and ends of IR straws under cryopreservation conditions. Should these further experiments confirm the impermeability of all the straws, the risk of contaminating the contents of other straws stored in liquid nitrogen and by the latter would become negligible.
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Acknowledgments |
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Notes |
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References |
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Bagasra, O., Farzadegan, H., Seshamma, T. et al. (1994) Detection of HIV-1 proviral DNA in sperm from HIV-1 infected men. AIDS, 12, 16691674.
Bahadur, G. and Tedder, R.S. (1997a) Safety during sperm banking (letter). Hum. Reprod., 12, 198.[ISI][Medline]
Bahadur, G. and Tedder, R.S. (1997b) Quarantine and cryopreservation (letter). Hum. Reprod., 12, 25792580.
Balet, R., Lower, A.M., Wilson, C. et al. (1998) Attitudes towards routine human immunodeficiency virus (HIV) screening and fertility treatment in HIV positive patients a UK survey. Hum. Reprod., 13, 10851087.[Abstract]
Clarke, G.N. (1999) Sperm cryopreservation: is there a significant risk of cross-contamination? Hum. Reprod., 14, 29412943.
Dulioust, E., Tachet, A., De Almeida, M. et al. (1998) Detection of HIV-1 in seminal plasma and seminal cells of HIV-1 seropositive men. J. Reprod. Immunol., 41, 2740.[ISI][Medline]
Eron, J.J., Vernazza, P.L., Johnston, D.M. et al. (1998) Resistance of HIV-1 to antiretroviral agents in blood and seminal plasma: implications for transmission. AIDS, 15, 181189.
Janssens, P.M.W. (1997) Safety during sperm banking (letter). Hum. Reprod., 12, 2579.
Luizzi, G., Chirianni, A., Clementi, M. et al. (1998) Differences in the genotypic resistance pattern of HIV between semen and plasma samples from HIV-infected subjects and evidence for viral compartmentalization. Drug Therapy in HIV Infection, Poster Abstracts, AIDS, European Meeting, Glasgow, P232, S75.
Marina, S., Marina, F., Alcolea, R. et al. (1998) Human immunodeficiency virus type 1-serodiscordant couples can bear healthy children after undergoing intrauterine insemination. Fertil. Steril., 70, 3539.[ISI][Medline]
Muciaccia, B., Filippini, A., Ziparo, E. et al. (1998) Testicular germ cells of HIV-1 seropositive asymptomatic men are infected by the virus. J. Reprod. Immunol., 41, 8193.[ISI][Medline]
Nuovo, G.J., Becker, J., Simsir, A. et al. (1994) HIV nucleic acids localize to the spermatogonia and their progeny. A study by polymerase chain reaction in situ hybridization. Am. J. Pathol., 144, 11421148.[Abstract]
Olaitan, A., Reid, W., Mocroft, A. et al. (1996) Infertility among human immunodeficiency virus-positive women: incidence and treatment dilemmas. Hum. Reprod., 11, 27932796.[Abstract]
Russel, P.H., Lyaruu, V.H., Millar, J.D. et al. (1997) The potential transmission of infectious agents by semen packaging during storage for artificial insemination. Anim. Reprod. Sci., 47, 337342.[ISI][Medline]
Semprini, A.E., Fiore, S. and Pardi, G. (1997) Reproductive counselling for HIV-discordant couples. Lancet, 349, 14011402.[ISI][Medline]
Stewart, G.J., Tyler, J.P.P., Cunningham, A.L. et al. (1985) Transmission of human T-cell lymphotropic virus type III (HTLV-III) by artificial insemination by donor. Lancet, 14, 581585.
Tedder, R.S., Zuckerman, M.A., Goldstone, A.H. et al. (1995) Hepatitis B transmission from contaminated cryopreservation tank. Lancet, 346, 137140.[ISI][Medline]
Vernazza, P.L., Troiani, L., Flepp, M. et al. (1998) Potent antiretroviral treatment (ART) results in marked suppression of seminal HIV-RNA and -DNA shedding. Drug Therapy in HIV Infection, Poster Abstracts, AIDS, European Meeting, Glasgow, OP63: S15.
Zagury, D., Bernard, J., Leibowitch, J. et al. (1984) HTLV-III in cells cultured from semen of two patients with AIDS. Science, 226, 449451.[ISI][Medline]
Zhang, H., Dornadula, G., Beumont, M. et al. (1998) Human immunodeficiency virus type 1 in the semen of men receiving highly active antiretroviral therapy. N. Engl. J. Med., 339, 18031809.
Submitted on January 4, 2000; accepted on July 5, 2000.