1 Laboratoire Drouot, 75009 Paris, 2 Service d'Histologie-Embryologie-Cytogénétique et Biologie Cellulaire, Hôpital Bichat-Claude Bernard, 75018 Paris, 3 Laboratoire Roche, 38240 Meylan, 4 Service de Gynécologie-Obstétrique, Hôpital BichatClaude Bernard, 75018 Paris, 5 Departement de Biostatistique et d'Informatique Médicale, CHU Saint-Louis, 75010 Paris, France
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Abstract |
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Key words: hepatitis C virus/IVF/purified sperm/sperm
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Introduction |
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The aim of the present study was to report our first results regarding HCV RNA detection in the semen and sperm fractions of 35 HCV+ men. To increase the sensitivity of the technique used to detect HVC RNA and to decrease the influence of seminal amplification inhibitors, several changes were made in the technique originally used to detect HCV RNA in serum. We also report herein the preliminary results regarding the follow-up of the women after assisted reproduction treatment and children conceived.
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Materials and methods |
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HCV RNA tests
Cobas Amplicor HCV 2.0 Assay (Roche) was used for HCV RNA detection in semen after making four changes, mainly to the extraction of HCV RNA. First, an aliquot of 200 µl of semen was diluted in 400 µl phosphate-buffered saline (PBS) in order to dilute the amount of seminal inhibitory factors, since it has been reported that these factors are mainly present in seminal fluid (Semprini et al., 1998; Debono et al., 2000
). Second, the cell suspension was centrifuged at 28 000 g for 1 h at 8°C in a Heraeus ultracentrifuge (Kendro, Courtabeuf, France). Third, after discarding the supernatant, the pellet was lysed with 600 µl (rather than 400 µl) lysis buffer (Roche). After these three steps, the standardized Cobas Amplicor HCV 2.0 technique was performed. Briefly, the lysate was put into a warm dry oven for 10 min at 60°C and vortexed. Isopropanol (600 µl) was added for 2 min at room temperature. The solution was centrifuged for 15 min at 13 600 g at 20°C. The supernatant was discarded and 1 ml of 70° ethanol was added to the pellet. The resuspended pellet was again centrifuged at 13 600 g for 5 min. The supernatant was discarded and 200 µl of HCV diluant (Roche) was added to the pellet. Finally, a last modification of the technique was made: the pellet was disrupted, mixed for 10 s and centrifuged again at 28 000 g at 8°C for 5 min. HCV RNA was amplified and detected according to the manufacturer's instructions.
The following preliminary studies were conducted to validate the four changes. First, 100 µl of titrated serum containing 80 000 copies/ml of HCV RNA were added to 900 µl of pooled HCV- semen from HCV- men to obtain a sample titrated at 8000 copies/ml. This sample was diluted twice with the pooled HCV- semen at 1/5 and 1/10 to obtain viral loads respectively of 1600 and 800 copies/ml. For each load, 200 µl samples were centrifuged in triplicate at 1500 g for 15 min. Under these conditions, following the manufacturer's procedures for the serum HCV detection, the virus was never detected in any specimen including internal controls. We hypothesized that this result might be caused by either the presence of seminal inhibitory factors of amplification or the insufficient speed of centrifugation. Using the same stock preparations, we diluted the 200 µl sample with 400 µl of PBS and then increased the speed of the initial centrifugation from 1500 to 16 000 g for 10 min. In the absence of inhibition (all the internal controls were positive), the samples initially titrated at 8000 and 1600 copies/ml were positive but the 800 copies/ml samples were negative. Because the official and registered sensitivity for the Cobas Amplicor HCV 2.0 Assay applied to serum is 100 copies/ml, a third test was undertaken with an initial centrifugation in PBS at 24 000 g for 1 h. The three viral loads (8000, 1600 and 800 copies/ml) were then positive. To calculate the sensitivity of our adapted procedure, we prepared samples with three low viral loads, 100, 50 and 25 copies/ml, as follows. The sample titrated at 800 copies/ml was diluted at 1/8 with the pooled HCV- semen to obtain a viral load of 100 copies/ml which was diluted again at 1/2. The obtained sample titrated at 50 copies/ml was then diluted at 1/2 resulting in a viral load of 25 copies/ml. Each viral load from 8000 to 25 copies/ml was prepared in triplicate and each replica was tested three times for HCV RNA detection. The calculated sensitivity in semen was estimated to be between 50 and 100 copies/ml (Table I).
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Statistical analysis
Serum viral loads in HCV+ semen and HCV- semen groups are expressed as means ± SD and were compared using the Wilcoxon rank sum test with the level of significance set at 5%.
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Results |
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Discussion |
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The presence of HVC RNA in semen has been investigated for 10 years with various results. Thus, the frequency of HCV RNA detection in semen of infected men ranged from 0% (Hsu et al., 1991; Fried et al., 1992
; Terada et al., 1992
; Caldwell et al., 1996
; Semprini et al., 1998
; Debono et al., 2000
), to 5% (2/39) (Levy et al., 2000
), 24% (4/17) (Liou et al., 1992
), 33% (2/6) (Leruez-Ville et al., 2000
), 57% (4/7) (Tang et al., 1996
) and even 100% (3/3) (Liu et al., 1994
). We detected HCV RNA in 14% of the 50 tested sperm. Our findings confirm that the presence of HCV in sperm from infected men is variable, and, even though we have never documented virus transmission to the woman or child, the possibility of a contamination by HCV+ sperm cannot be excluded. However, the HCV loads in the HCV RNA+ semen samples were below the quantification threshold. This result is in agreement with those of Leruez-Ville et al. who showed that semen HCV loads were low (Leruez-Ville et al., 2000
) and suggests that the risk of HCV sexual transmission is also probably very low, a point which is consistent with epidemiological data (Dienstag, 1997
). We also observed that the virus persists in semen, since two patients whose semen samples were again collected and tested after an interval of 4 or 9 months remained HCV+. This latter observation needs to be confirmed to determine whether the presence of HCV in semen is persistent or intermittent and if any such persistence increases with time the risk of contamination in assisted reproduction treatment or sexual transmission.
When the viral status of sperm fractions, obtained after gradient-density centrifugation followed (Pasquier et al., 2000) or not (McKee et al., 1996
; Levy et al., 2000
) by a swim-up step, were studied, no HCV RNA was found in the purified 90% fraction of sperm, which is the one used for assisted reproduction. That finding was confirmed in our series, since HCV RNA was found only in the 45% fraction of 1/7 HCV+ semen samples but never in the 90% fraction of purified sperm. The absence of virus in the latter could be explained by the association of low-speed centrifugation, routinely used to select sperm for assisted reproduction which does not concentrate HCV in the 90% fraction, and the very low HCV load in seminal fluid.
However, the risk of contamination does potentially exist both for the embryos resulting from an assisted reproduction performed in an HCV- couple but incubated in the same incubator as those resulting from an HCV infected couple, and for the biologists and technicians performing the different steps of assisted reproduction procedures. That is why, until February 2000 in France, inclusion in an assisted reproduction programme of infertile couples, in which one member was HCV+ with active viral replication, was prevented by the law. However, since February 2000, a multicentric study allowed assisted reproduction centres to include infertile couples with one or both HCV+ partners who wanted to attempt assisted reproduction treatment to procreate. Thus, it will be possible to evaluate whether assisted reproduction raises the risk of HCV contamination for newborns, compared with HCV+ women who become pregnant spontaneously. More recently, French law has again been modified, and since May 2001 all centres can include HCV+ couples with active viral replication in assisted reproduction programmes. These centres must separatein time or spacethe inclusion of infected and uninfected HCV couples and, in the case of HCV+ men, must freeze and test the semen or purified sperm before performing treatment, which can proceed only if HCV is not detected. Even though no HCV was detected in the purified fraction of sperm (McKee et al., 1996; Levy et al., 2000
; Pasquier et al., 2000
), the principle of precaution predominates in France since the HCV and HIV-1/2 blood contamination affair of the 1990s.
To the best of our knowledge, our preliminary results showed for the first time that standard IVF or ICSI techniques, including oocyte washing and changing embryo culture media, do not increase the risk of contamination for couples. Indeed, none of the tested embryo culture media inseminated with the 90% fractions of the seven HCV+ semen samples was HCV RNA+. Furthermore, none of the women nor the only newborn were infected after these assisted reproduction attempts. Therefore, these preliminary data strongly suggest that the HCV contamination risk is very low for infertile couples with HCV-infected men included in assisted reproduction programmes. Should this conclusion prove true for a larger population, freezing sperm from HCV infected men could become unnecessary. It is known that freezing sperm results in decreased motility, vitality and number of sperm cells (Donnelly et al., 2001) with, as an eventual consequence, poor oocyte fertilization and pregnancy rates during assisted reproduction treatment. Consequently, it is not obvious that freezing sperm is necessary in HCV+ men because we observed, like all the other previous studies (McKee et al., 1996
; Levy et al., 2000
; Pasquier et al., 2000
), that routine technical means can eliminate the virus. However, the possible presence of HCV in semen implies a potential risk of nosocomial contamination. Thus, to prevent this risk, safety rules must be strictly respected (Steyaert et al., 2000
) in all assisted reproduction laboratories that include infertile couples with HCV+ men.
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Acknowledgements |
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Notes |
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References |
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Caldwell, S.H., Sue, M., Bowden, J.H., Dickson, R.C., Driscoll, C.J., Yeaton, P., Stevenson, W.C., Ishitani, M.B., McCullough, C.S., Pruett, T.L. et al. (1996) Hepatitis C virus in body fluids after liver transplantation. Liver Transplant. Surg., 2, 124129.[Medline]
Debono, E., Halfon, P., Bourliere, M., Gerolami-Santandrea, V., Gastaldi, M., Castellani, P., Cartouzou, G., Botta-Fridlund, D., Cau, P. and Gauthier, A. (2000) Absence of hepatitis C genome in semen of infected men by polymerase chain reaction, branched DNA and in situ hybridization. Liver, 20, 257261.[ISI][Medline]
Dienstag, J.L. (1997) Sexual and perinatal transmission of hepatitis C. Hepatology, 26 (Suppl. 1), 6670.[ISI]
Donnelly, E.T., Steele, E.K., McClure, N. and Lewis, S.E. (2001) Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum. Reprod., 16, 11911199.
Fried, M.W., Shindo, M., Fong, T., Fox, P.C., Hoofnagle, J.H. and Di Bisceglie, A.M. (1992) Absence of hepatitis C viral RNA from saliva and semen of patients wth chronic hepatitis C. Gastroenterology, 102, 13061308.[ISI][Medline]
Hsu, H.H., Wright, T.L., Luba, D., Martin, M., Feinstone, S.M., Garcia, G. and Greenberg, H.B. (1991) Failure to detect hepatitis C virus genome in human secretions with the polymerase chain reaction. Hepatology, 14, 763767.[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.
Liou, T., Chang, T.T., Young, K.C., Lin, X.Z., Lin, C.Y. and Wu, H.L. (1992) Detection of HCV RNA in saliva, urine, seminal fluid and ascites. J. Med. Virol., 37, 197202.[ISI][Medline]
Liu, F.H., Tian, G.S. and Fu, X.X. (1994) Detection of plus and minus strand hepatitis C virus RNA in peripheral blood mononuclear cells and spermatid. Zhonghua Yixue Zazhi, 74, 284286.[Medline]
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]
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]
Steyaert, S.R., Leroux-Roels, G.G. and Dhont, M. (2000) Infections in IVF: review and guidelines. Hum. Reprod. Update, 6, 432441.
Tang, Z., Yang, D., Hao, L., Tang. Z., Huang, Y. and Wang, S. (1996) Detection and significance of HCV RNA in saliva, seminal fluid and vaginal discharge in patients with hepatitis C. J. Tongji. Med. Univ., 16, 1113.[Medline]
Terada, S., Kawanishi, K. and Katayama, K. (1992) Minimal hepatitis C infectivity in semen. Ann. Intern. Med., 117, 171172.
Submitted on March 1, 2002; resubmitted on June 13, 2002; accepted on August 25, 2002.