1 Laboratoire de Biologie de la Reproduction and 2 Laboratoire de Virologie, Hôpital Nord, Saint-Etienne, 42055 and 3 Laboratoire de Biologie de la Reproduction, 8, Avenue Rockefeller, 69373 Lyon Cedex 08, France
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Abstract |
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Key words: ART/HCV RNA/semen/sperm
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Introduction |
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Case report |
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The patient tested positive for the presence of HCV RNA in blood and negative for HIV antibodies and hepatitis B virus (HBs) antigen. He was non-symptomatic with active chronic hepatitis according to biochemical and histological data. The risk factor for HCV contamination was from i.v. drug addiction >10 years ago. Semen parameters were normal according to the criteria of the World Health Organization (World Health Organization, 1992)
Samples
A total of 12 consecutive samples was tested. Semen characteristics are reported in Table I. The patient had not been taking any antiviral medication during the period in which the first nine semen samples were obtained, due to the possible severe adverse effects of ribavirin on the germinal cell lines (Narayana et al., 2002
). Once IVF succeeded, three additional semen samples were obtained after a bitherapy combining alpha interferon and ribavirin had been started.
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HCV RNA detection and quantification in blood serum and seminal plasma
The qualitative and quantitative detection of HCV RNA in blood serum and seminal plasma were performed using the Cobas Amplicor® HCV assay 2.0 and the Cobas Monitor® HCV assay 2.0 (Roche Diagnostics, France) respectively. For seminal plasma, a modified protocol was performed for the RNA extraction step including a 1:2 sample dilution in PBS, a centrifugation at 24 000x g for 1 h at 4°C and the use of the QIAmp Viral RNA kit® (Qiagen, France) as previously described (Bourlet et al., 2002).
HCV RNA detection in sperm fractions
An aliquot of 500 000 cells was tested for the presence of HCV RNA. The RNA extraction step was carried out using the RNA Easy Mini Kit® (Qiagen). Reverse transcription and qualitative PCR were performed using the Cobas Amplicor HCV assay 2.0
HCV genotyping
The genotypes of HCV strains were analysed in blood and semen samples by sequencing the 5' non coding region of HCV using the TruGene® HCV assay (Visible Genetics, France).
Follow-up of HCV RNA detection in blood and semen
HCV RNA in blood could be quantified for five samples, before antiviral therapy, with a mean viral load of 1.62x106copies/ml (6.20 log copies/ml) (range: 1.42.0x106 copies/ml) (6.156.30 log copies/ml) (Table I). The threshold of our qualitative assay in seminal plasma was estimated at
40 copies/ml (1.6 log copies/ml) by using serial dilutions of quantified blood plasma from an HCV-infected patient diluted in an HCV negative seminal plasma (Bourlet et al., 2002
). No PCR inhibitor was detected in any semen sample. All the nine sequential seminal plasma samples collected before antiviral bitherapy were found positive for the presence of HCV RNA, with viral loads ranging from <401450 copies/ml (<1.603.18 log copies/ml) (Table I
). The average difference between blood and seminal viral loads was roughly 3 log 10. The HCV strains isolated from both seminal and blood samples belonged to genotype 3. During the course of the follow-up, two of the motile sperm fractions obtained after gradient selection showed the presence of HCV RNA (Table I
). However, when the sperm recovered after the three-layer gradient were submitted to an additional 30 min swim-up step, no HCV RNA was detected in the final motile sperm suspension. Following the detection of HCV RNA in both the seminal plasma and the motile sperm fraction of these two semen samples, the programmed IVF attempt with fresh semen was immediately stopped. As no HCV RNA was detected in the sperm fraction obtained from a further semen sample, we decided to freeze the motile sperm selected by swim-up migration in CBS® highly secure straws (CryoBiosystem, France) which were stored in a separated cryotank.
IVF and pregnancy follow-up
IVF was carried out using a single straw after washing the thawed HCV RNA negative sperm by centrifugation at 400 g for 10 min. The post-thawing recovery rate was 50%. After the ovarian stimulation treatment, 27 oocytes were collected by means of an ultrasound-guided vaginal follicular puncture. Twenty-four embryos resulted from the IVF. Two days post-fertilization, two grade I embryos were transferred to the woman and 14 grade I embryos were frozen in secure straws which were stored in a separate container. Two weeks after the embryo transfer, the ß-hCG concentration indicated pregnancy, which was confirmed 2 weeks later by an ultrasound scan. A further ultrasound examination revealed the presence of twins, with normal fetal heart activity. The woman tested negative for HCV antibodies at the time of the transfer, and again 1 month later. She also tested negative for HCV RNA detection at 8 and 20 weeks following the embryo transfer. The woman and her children will be tested again for HCV antibodies and RNA at birth and 3 months later, according to the informed consent approved by the parents.
Patients antiviral treatment
Antiviral treatment combining pegylated interferon and ribavirin was given to the male infected partner after the pregnancy was confirmed. Two weeks after treatment, the viral load decreased to 14500 copies/ml in blood and to between 40100 copies/ml in seminal plasma (see Table I). Ten weeks after the treatment was started, no HCV RNA was detectable in the seminal plasma.
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Discussion |
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For two successive semen samples, HCV RNA was detected in both the seminal plasma and the motile sperm obtained after PureSperm centrifugation, suggesting that a risk of contamination by HCV cannot be excluded through the use of ART. The presence in these fractions of a very low number of leukocytes cannot be definitely excluded. As it is extremely unlikely that sperm support HCV replication, another valuable explanation could rely on a passive adsorption to the cell membrane of HCV RNA or of virions present in the seminal plasma, as suggested by the association between the positivity of the sperm fractions for HCV RNA and high viral loads in seminal plasma (see Table I). This adsorption could impair the motility of the sperm, as illustrated by the absence of HCV RNA in the corresponding swim-up fraction.
Before using sperm from a patient exhibiting HCV RNA in seminal plasma for ART, the following recommendations could be given: (i) raw semen and corresponding motile sperm selected after gradient and swim-up migration must be cryopreserved in highly secure straws in liquid nitrogen until ART; (ii) the corresponding seminal plasma and motile sperm must be stored at 80°C for HCV RNA testing; (iii) only specimens corresponding to sperm tested negative for HCV RNA must be considered for ART. Furthermore, we recommend the use of highly secure straws for semen freezing, as previously suggested for potentially infectious samples (Benifla et al., 2000) and the storage of these straws in a separate cryotank dedicated to samples with infectious hazard. These guidelines would seem to prevent the mother and the embryos from a potential HCV infection, as illustrated by the preliminary negative results of HCV RNA and antibody detection from the pregnant woman.
Another interesting finding of this case report is the rapid decrease in the HCV viral load in seminal plasma after introduction of an antiviral treatment combining alpha interferon and ribavirin. Unfortunately, it is not possible to recommend the use of such a treatment before starting ART, in order to reduce the risks of HCV transmission, since ribavirin has been shown to induce serious damage to spermatogenesis (Narayana et al., 2002).
In view of the case reported herein, we consider IVF to be a safe method for achieving pregnancies in HCV serodiscordant infertile couples on condition that: (i) an attempt to discover the presence of HCV RNA is performed in semen and motile sperm; and (ii) strict secure measures of security are observed for the storage of infected material.
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Acknowledgements |
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Notes |
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References |
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Bourlet, T., Cazorla, C., Berthelot, P., Grattard, F., Cognasse, F., Fresard, A., Defontaine, C., Lucht, F.R., Genin, C. and Pozzetto, B. (2001) Compartmentalization of HIV-1 according to antiretroviral therapy: viral loads are correlated in blood and semen but poorly in blood and saliva. AIDS, 15, 284285.[ISI][Medline]
Bourlet, T., Levy, R., Maertens, A., Tardy, J.C., Grattard, F., Cordonier, H., Laurent, J.L., Guerin, J.F. and Pozzetto, B. (2002) Detection and characterization of hepatitis C virus RNA in seminal plasma and spermatozoa fractions of semen from patients attempting medically-assisted conception. J. Clin. Microbiol., in press.
Dore, G.J. and Kaldor, J.M. (2000) Detection of HCV RNA in semen. Lancet, 356, 1520.[Medline]
Fiore, R.J., Potenza, D., Monno, L., Appice, A., Di Stefano, M., Giannelli, A., La Grasta, L., Romanelli, C., Di Bari, C. and Pastore, G. (1995) Detection of HCV RNA in serum and seminal fluid from HIV-1 co-infected intravenous drug addicts. J. Med. Virol., 46, 364367.[ISI][Medline]
Fried, M.W., Shindo, M., Fong, T.L., Fox, P.C., Hoofnagle, J.H. and Di Bisceglie, A.M. (1992) Absence of hepatitis C viral RNA from saliva and semen of patients with chronic hepatitis C. Gastroenterology, 102, 13061308.[ISI][Medline]
Leruez-Ville, M., Kunstmann, J.M., De Almeida, M., Rouzioux, C. and Chaix, M.L. (2000) Detection of hepatitis C virus in the semen of infected men. Lancet, 356, 4243.[ISI][Medline]
Levy, R., Tardy, J.C., Bourlet, T., Cordonier, H., Mion, F., Lornage, J. and Guerin, J.F. (2000) Transmission risk of hepatitis C virus in assisted reproductive techniques. Hum. Reprod., 15, 10831085.
McKee, T.A., Avery, S., Majid, A. and Brinsden, P.R. (1996) Risks for transmission of hepatitis C virus during artificial insemination. Fertil. Steril., 66, 161163.[ISI][Medline]
Narayana, K., DSouza, U.J. and Seetharama Rao, K.P. (2002) Ribavarin-induced spem shape abnormalities in Wistar rat. Mutat. Res., 15, 193196.
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.[ISI][Medline]
Semprini, A.E., Persico, T., Thiers, V., Oneta, M., Tuveri, R., Serafini, P., Boschini, A., Giuntelli, S., Pardi, G. and Brechot, C. (1998) Absence of hepatitis C virus and detection of hepatitis G virus/GB virus C RNA sequences in the semen of infected men. J. Infect. Dis., 177, 848854.[ISI][Medline]
Tachet, A., Dulioust, E., Salmon, D., De Almeida, M., Rivalland, S., Finkielsztejn, L., Heard, I., Jouannet, P., Sicard, D. and Rouzioux, C. (1999) Detection and quantification of HIV-1 in semen: identification of a subpopulation of men at high potential risk of viral sexual transmission. AIDS, 13, 823831.[ISI][Medline]
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. Cambridge University Press, Cambridge, UK.
Submitted on December 10, 2002; resubmitted on March 8, 2002; accepted on June 6, 2002.