1 Service of Physiopathology of Human Reproduction, Obstetrics and Gynaecology Unit, MaternalPaediatric Department and 2 Microbiology Unit, Laboratory Medicine Department, Pordenone Hospital, Italy
3 To whom correspondence should be addressed. e-mail: serpma{at}aopn.fvg.it
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: HCV/hepatitis/ICSI/male/TESA
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Case report |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The female was subjected to ovarian stimulation (long protocol with 2925 IU of rFSH) and the male was subjected to fine needle testicular aspiration. Surgical and laboratory staff used gloves and masks to prevent mucocutaneous and percutaneous infection. Only samples without macroscopic blood contamination were used for the preparation of cytological suspensions for TESA-ICSI. The cytological material was processed in a vertical flow cabinet for handling of biohazardous materials, washed in human tubule fluid (HTF) medium (1 ml) and centrifuged (8 min at 300 g) twice, and after the second treatment the pellet was suspended in 20 µl of the same medium. We postulated that not only in density gradients but also in simple culture medium, sample centrifugation may produce an effective separation of spermatozoa from viral particles due to their different mass, leaving the virus in the supernatant and spermatozoa in the pellet. The specimen was then distributed in many small (5 µl) droplets of culture medium under oil, and spermatozoa retrieved from these droplets by an injecting pipette were rinsed repeatedly in 7% polyvinylpyrrolidone (PVP) solution with human albumin and then injected into the womans oocytes; from 12 oocytes injected, five embryos were obtained and were cultured at 37°C with 5% CO2 in air in medium droplets under oil. The residual cytological sample was insufficient for a reliable HCV RNA PCR determination with our method and its dilution was excluded to avoid the risk of a misleading false-negative result. A negative PCR HCV RNA determination on the zygotes/embryos culture medium immediately before transfer was obtained (sensitivity of the method: 50 IU/ml WHO standard Cobas Amplicor HCV test, version 2.0, Roche). The zygotes/embryos culture medium was not changed from microinjection to the transfer in order to avoid a dilution effect on the possible viral load that would be responsible for any HCV RNA PCR false-negative determination. On the third day, after the negative HCV RNA result after one cycle of PCR on culture medium, four embryos were transferred, and 14 days after retrieval -hCG was 20 mIU/ml. Six weeks after retrieval, transvaginal ultrasound (TVUS) showed an ongoing pregnancy (uterine sack with fetal heartbeat). HCV antibody investigation (MEIA) in the mother on the 12th and 24th week of pregnancy gave negative results. The pregnancy ended at the 39th week with intrauterine death. Pathological examination of the conceptus failed to find macroscopic cardiac malformations or hepatic damage. A second TESA-ICSI cycle is ongoing.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clinical observations suggest that HCV transmission between women during an IVF cycle is a real possibility (two cases in the same laboratory already described) even with good medical and biological practice. Thus the ART eligibility of HCV-infected woman must be carefully weighed against the risk of HCV infection transmission to other women. The widespread treatment of such patients by all ART laboratories in cycles temporally or spatially dedicated to couples with the same viral infection is a possible solution which needs a future careful evaluation. Probably, a case by case approach to the couple, based on which partner is HCV RNA positive, is preferable.
In HCV RNA-positive males, the sperm sample may also be positive. If so, what is the risk for partner and baby? Is the vertical and horizontal virus transmission risk with assisted procreation lower than with natural conception? A general agreement is lacking on this issue: Semprini et al. (1998) were not able to detect HCV RNA in semen samples. However, HCV RNA was detected by other authors, with a different method, in
30% of sperm samples of patients with serological HCV RNA positivity (Leruez-Ville et al., 2000
; Levy et al., 2002
). This discrepancy may be due to the different methods used and/or to the presence of PCR inhibitors in the ejaculate. However, HCV RNA-positive sperm samples, in most studies, showed undetectable HCV RNA levels, after one round of PCR, after seminal plasma and white blood cells were removed by gradient selection and swim-up (Levy et al., 2000
, 2002; Semprini et al., 2001
). However, recently, some authors (Meseguer et al., 2002
) have found HCV RNA with nested PCR in previously PCR-negative samples after a single cycle. So a residual viral load in the treated sample cannot be excluded. Previously, Semprini et al. (2001
) carried out 2300 ART procedures (IVF and IUI), using sperm samples selected by gradient centrifugation followed by swim-up, in HIV-positive men, 62% of whom were co-infected with HCV, and not a single woman sero-converted either for HIV or HCV. Moreover, other authors (Levy et al., 2002
) have recently reported a pregnancy in an HCV RNA sero-discordant couple (male positive) without female partner sero-conversion after a classic IVF with cryopreserved HCV RNA-negative spermatozoa obtained with gradient selection and subsequent swim up.
Our observation provides preliminary evidence that ICSI with testicular spermatozoa retrieved by fine needle aspiration may be safe in HCV sero-discordant couples due to low levels of blood contamination of the sample after any macroscopically bloody samples were discarded, and separation of spermatozoa and virus particle by repeated centrifugation and repeated washing in the PVP solution before spermatozoa injection in oocytes. Therefore, in the treated sample, the residual viral load, if present, is extremely reduced in comparison with untreated blood or sperm. If confirmed by other observations, the reduced risk for HCV-infected males of vertical and horizontal virus transmission by assisted reproduction in comparison with natural conception should be underlined with sero-discordant couples requesting homologous assisted reproduction, not only for treatment performed with ejaculated (IUI, IVF or ICSI), but also with testicular spermatozoa (TESA-ICSI). Probably nested PCR before treated sperm sample cryopreservation may, if well standardized, be the best method to select couples at almost null risk for horizontal and vertical HCV transmission. HCV RNA PCR on embryo culture medium before transfer may increase the safety of ART in these couples. At present, for couples refusing the low residual risk of homologous ART, we can only suggest deferment of the IVF cycle until a negative HCV RNA determination after pharmacological treatment (interferon- plus ribavirin) or utilization of spermatozoa from a serum-negative donor.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.[CrossRef][ISI][Medline]
Lesourd, F., Izopet, J., Mervan, C., Payen, J.L., Sandres, K., Monrozies, X. and Parinaud, J. (2000) Transmissions of hepatitis C virus during the ancillary procedures for assisted conception. Hum. Reprod., 15, 10831085.
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, 810816.
Levy, R., Bourlet, T., Maertens, A., Salle, B., Lornage, J., Laurent, J.L., Pozzetto, B. and Guerin, J.F. (2002) Pregnancy after safe IVF with hepatitis C virus RNA-positive sperm. Hum. Reprod., 17, 26502653.
Meseguer, M., Garrido, N., Gimeno, C., Remohì, J., Simon, C. and Pellicer, A. (2002) Comparison of polymerase chain reaction-dependent methods for determining the presence of human immunodeficiency virus and hepatitis C virus in washed sperm. Fertil. Steril., 78, 11991202[CrossRef][ISI][Medline]
Passos, E.P., Silveira, T.R., Salazar, C.C., Facin, A.C., Souza, C.A., Guerin, Y.L, Gratao, A.A. and Cunhafilho, J.S. (2002) Hepatitis C virus infection and assisted reproduction. Hum. Reprod., 17, 20852088.
Scotto, G., Savastano, A.M., Fazio, V., Conte, P.E., Ferrara, S., Mangano, A. and Tantimonaco, G. (1996) Sexual transmission of hepatitis C virus infection. Eur. J. Epidemiol., 13, 241244.[CrossRef]
Semprini, A.E., Persico, T., Thiers, V. et al. (1998) Absence of HCV in semen. J. Infect. Dis., 177, 848854.[ISI][Medline]
Semprini, A.E., Vucetich, A. and Persico, T. (2001) Hepatitis C detection [letter]. Lancet, 357, 557.
Steyaert, S.R., Leroux-Roels, G.G. and Dhont, M. (2000) Infections in IVF: review and guidelines. Hum. Reprod. Update, 6, 432441.
Thomas, D.L., Zenilman, J.M., Alter, H.J., Shih, J.W., Galai, N., Carella, A.V. and Quinn, T.C. (1995) Sexual transmission of hepatitis C virus among patients attending sexually transmitted diseases clinics in Baltimorean analysis of 309 sex partnerships. J. Infect. Dis., 171, 768775.[ISI][Medline]
Wood, S., Kevin, T., Schnauffer, K., Troup, S., Kingsland, C. and Lewis-Jones, I. (2002) Reproductive potential of fresh and cryopreserved epididymal and testicular spermatozoa in consecutive intracytoplasmic sperm injection cycles in the same patients Fertil. Steril., 77, 11621166.[CrossRef][ISI][Medline]
Submitted on October 17, 2002; resubmitted on March 24, 2003; accepted on April 30, 2003.