Preliminary evidence on the safety of ICSI with testicular spermatozoa in HCV-infected male: a case report

M. Manno1,3, E. Marchesan1, M. Crovatto2, P. Martelli2, F. Tomei1 and V. Adamo1

1 Service of Physiopathology of Human Reproduction, Obstetrics and Gynaecology Unit, Maternal–Paediatric 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
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
An infertile couple with a hepatitis C (HCV) RNA-positive male came to our attention. We were not able to perform an assisted reproduction technology (ART) procedure with ejaculated spermatozoa free of HCV RNA using gradient centrifugation and swim up, due to retrograde ejaculation and severe male factor. ICSI with testicular spermatozoa was the most rational therapeutic approach. The couple was informed about the lack of adequate data on the safety of this therapeutic approach. The risks of this procedure were accepted by the couple. Testicular sperm aspiration combined with ICSI (TESA-ICSI) was performed. After a negative result for an HCV RNA PCR on the embryos’ culture medium, four embryos were transferred to the uterine cavity on the third day. The procedure resulted in an ongoing clinical pregnancy, and HCV antibody determinations performed in the mother at the 12th and 24th week of pregnancy were negative. The pregnancy ended at the 39th week due to endo-uterine death. No malformation or hepatic pathologies were found in the conceptus. A second TESA-ICSI cycle is ongoing. This preliminary evidence suggests that, in HCV sero-positive males, ICSI with testicular spermatozoa may be a safe procedure. However, we need more observations to clarify if ART is really able to reduce horizontal and vertical transmission of HCV in sero-discordant couples (only the male infected) in comparison with natural conception.

Key words: HCV/hepatitis/ICSI/male/TESA


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
Apart from ovarian stimulation and surgical complications, the major risks associated with treatment in any assisted reproductive treatment (ART) laboratory are genetic and infective ones. Although there is a general agreement on the optimal behaviour to prevent genetic risks, it is not clear, as yet, what should be the best strategy to reduce the risk of transmitting a viral disease to the partner, other patients, offspring or laboratory staff. At present, different protocols are observed in different IVF laboratories in human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV) infections, due to the lack of worldwide accepted indications. There are several cases of ART procedures performed in couples with HCV RNA-positive males using ejaculated spermatozoa [intrauterine inseminations (IUIs) and classic IVF] but, to the best of our knowledge, no data on the safety of ICSI with testicular spermatozoa are available.


    Case report
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
We observed a man with retrograde ejaculation due to a retro-peritoneal lymph node dissection for rectal cancer, elevated gonadotrophin (FSH 11 and LH 15 mIU/ml) with detectable HCV antibody (sample rate/cut-off ratio: 82.23 and 77.28) and positive HCV RNA after one cycle of PCR (estimated viral load >850 000 IU/ml). The infection probably resulted from a viral infection picked up during a blood transfusion after cardiac surgery for Fallot’s tetralogy. A pharmacological trial with imipramine failed to produce anterograde ejaculation. Due to substantially increased gonadotrophin levels, the option of IUI with spermatozoa, retrieved after urine alkalinization in this patient who had evident tubular dysfunction, was excluded. Moreover, due to previous cardiac surgery, we chose to avoid the risk of bacteraemia trying to retrieve motile spermatozoa in culture medium with bladder catheterization. We also preferred to avoid further damage to spermatozoa of an extreme male factor patient which could result from extracting them from diluted and alkalinized urine with the risk of an undesirable reduction of the fertilization rate in the following ICSI. A classic surgical testicular biopsy with cryopreservation, before the ICSI cycle, was excluded due to the patient’s refusal of such an aggressive approach, an estimated higher risk of sample contamination with infected blood and possible additional cryodamage to testicular spermatozoa and a further reduction in their fertilizing capacity (Chan et al., 2000Go; Wood et al., 2002Go). Therefore, we preferred to make a diagnostic testicular fine needle aspiration that confirmed an important hypo-spermatogenesis in agreement with the patient’s increased FSH levels (sertolian index 185 dx and 193 sx testis). Then a TESA-ICSI with HCV RNA PCR determination on the zygotes’/embryos’ culture medium before transfer was proposed to the couple. We were not able to find in the literature any data on the safety of ART treatment performed with testicular spermatozoa. In such cases, blood contamination increases the viral load of the sample and the risk of HCV transmission in comparison with procedures done with ejaculated spermatozoa purified by gradient selection and swim up. Therefore, the couple, refusing ART with donor sperm, were informed about the risk of natural conception and the lack of adequate data on the safety of this kind of ART, and signed a specific informed consent.

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 {beta}-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
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
Epidemiological evidence indicates that the most frequent vehicle for HCV transmission is blood. However, subsequent observations have shown that the route of HCV transmission may also be sexual (Thomas et al., 1995Go; Scotto et al., 1996Go). It has been estimated that ~5% of all HCV cases are sexually transmitted. Female partners of anti-HCV-positive males are 3.7 times more likely to have anti-HCV antibodies than female partners of anti-HCV-negative males (Thomas et al., 1995Go). As yet, no active or passive immunization is available to protect the sexual partner, babies or laboratory staff. A recent epidemiological study found a prevalence of anti-HCV antibodies in 3.2% of all females and 3.7% of all males attending a Brazilian infertility clinic (Passos et al., 2002Go). These data raise the following questions. Is ART a good medical practice for HCV RNA-positive infertile couples? What kind of assisted reproduction should we choose for these couples to avoid infection of their partner, baby and risks for other patients and the IVF laboratory staff? Some authors support the point of view that in couples where there is an HCV RNA-positive male, a natural conception could be considered acceptable both medically and ethically (Steyaert et al., 2000Go). However, in comparison with natural conception, ART produces more responsibilities: of the physician towards the conceptus, of the non-infected partner, of the other couples and of the employer towards the IVF laboratory staff. A general agreement on this issue is lacking, and different IVF laboratories adopt different behaviours. Some laboratories prudentially denied any treatment to such couples; others have admitted the couples to their IVF programmes but, unfortunately, have transmitted HCV RNA from serum-positive to serum-negative women (Lesourd et al., 2000Go).

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. (1998Go) 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., 2000Go; Levy et al., 2002Go). 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., 2000Go, 2002; Semprini et al., 2001Go). However, recently, some authors (Meseguer et al., 2002Go) 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. (2001Go) 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., 2002Go) 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-{alpha} plus ribavirin) or utilization of spermatozoa from a serum-negative donor.


    Acknowledgements
 
The authors gratefully acknowledge the excellent technical assistance of Daniele Cicutto


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 Introduction
 Case report
 Discussion
 References
 
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Submitted on October 17, 2002; resubmitted on March 24, 2003; accepted on April 30, 2003.