1 Departments of Obstetrics and Gynaecology and 2 Paediatrics, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, 90035003, Brasil
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Abstract |
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Key words: hepatopathy/infertility/in-vitro fertilization/non-A non-B hepatitis
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Introduction |
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Epidemiological and experimental studies indicate that the main route of HCV transmission is parenteral, through transfusion of blood and plasma derivatives (Genesca et al., 1991). Other routes of parenteral transmission include intravenous drug use, haemodialysis and organ transplantation (Ho, 1991
). In addition, healthcare workers have developed non-A, non-B hepatitis after accidental needle-stick exposure, as well as in the absence of apparent percutaneous exposure (Genesca et al., 1991
).
Non-parenteral HCV transmission is also frequent. At least 50% of patients with hepatitis C have had no parenteral exposure. Serological studies have also shown vertical HCV transmission (Stevens, 1994), and mother-to-child transmission of HCV in women co-infected with HCV, and human immunodeficiency virus (HIV) has been detected using PCR (Novati et al., 1992
). However, although viral RNA has been detected in saliva and semen, the role of sexual, perinatal, and other possible non-parenteral routes of hepatitis C transmission is still unclear (Tedder et al., 1991
; Kao et al., 1996
; McKee et al., 1996
; Alter et al., 1999
).
In assisted reproduction, HCV transmission may pose a risk for the baby, and also for technicians and gametes or embryos from non-contaminated parents (Levy et al., 2000). The exact risk for HCV transmission in this population is, however, unknown; therefore, specific guidelines to prevent HCV infection in reproductive medicine have not yet been established. It is important to determine HCV prevalence among infertile couples who seek assisted reproduction so that specific health policies can be devised and infertile patients with HCV infection can be counselled during ART cycles.
The aim of this study was to determine the prevalence of HCV infection in a population of infertile couples, and to search for risk factors associated with this condition.
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Materials and methods |
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Patients
All patients received counselling before and after being tested for hepatitis C. Those patients with positive results were submitted to gastroenterologic evaluation and management according to the National Institute of Health Consensus Statement on the Management of hepatitis C (National Institute of Health, 1997). Hepatitis B, HIV, and known risk factors associated with HCV transmission were also evaluated. Every patient was also tested for Chlamydia and Treponema infection, in addition to the investigation of possible causes for their infertility.
The couples answered a questionnaire concerning previous history of personal and familial hepatitis, sexual exposure, use of injectable drugs, and exposure to whole blood or plasma derivatives in several situations.
We included only infertile patients who agreed to participate (248 women and 161 men) and who, if HCV test positive, agreed to have their disease investigated and monitored. At the beginning of the study, all subjects involved gave their informed consent to participate and to have their blood samples tested. Only two women and one man refused to be included.
During the initial visit, all patients were submitted to a thorough examination. Previous medical history was evaluated and current use of any medication was recorded. Exclusion criteria were: age (>40 years) or having any acute health disorder. Blood contact was defined as any contact with blood (with or without protection) and surgery defined as any procedure (out-patient/in-patient) performed in a hospital.
Assays
The detection of antibodies to hepatitis C virus (anti-HCV) and to hepatitis B surface virus antigens (HBsAg) was included as part of routine infertility investigation. Sera from patients were screened for anti-HCV by ELISA (Ortho HCV 3.0; Ortho-Clinical Diagnostics, Nackargewünd, Germany), and for HBsAg by enzyme-linked fluorescent assay (ELFA) (Vidas HBsAg; bioMériex, Marcy-I'Etoile, France). HIV was also investigated by ELISA (Vironostike® HIV Uni-Form II plus O; Organon, Boxtel, Holland).
The specificity of the anti-HCV test (ELISA) in a low-risk population is 99.96% (4 units per 10 000 donations); the intra-assay coefficient of variation (CV) is 9.9% ; the inter-assay CV range 1.214.8%.
In patients who tested positive for anti-HCV (ELISA), RNA was extracted as described ( Chomcczynski and Sacchi, 1987). RTPCR was carried out to detect hepatitis C virus RNA using primers drawn from the 5' non-coding region of HCV (NS-5'R) for amplification (Simmonds et al., 1993
). The technique's detection limit was 200 copies/ml. To confirm the results, a second amplification was carried out using internal primers (nested PCR). HCV-genotyping was performed by restriction fragment length polymorphism (RFLP) analysis of the nested PCR products using three restriction enzymes in two separate digestion systems. Digested products were submitted to electrophoresis, stained, and separated on polyacrylamide gels. Band patterns for the different HCV genotypes were derived from those described (McOmish et al., 1994
) and from the 5'NCR (non-coding region) sequences obtained from gene databases.
Statistics
Data were stored and analysed using the EPI-INFO 6.02 software (Atlanta, USA). Statistical analysis was carried out by estimation of relative risk (RR) and by the Fisher's exact test.
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Results |
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The clinical and demographic characteristics of studied patients are presented in Table I. Treponema infection was negative in all investigated cases. The prevalence of Chlamydia infection was 15% in HCV-negative patients and 17% in HCV-positive patients (P = not significant, Fisher's exact test). Considering other infertility factors: 103 patients (41.5%) presented with tubal occlusion; 43 (17 .3%) with endometriosis; and 17 (7%) with ovulatory dysfunctions. In 23 (9.2%) the infertility was idiopathic, and in 62 cases (25%), male infertility was present.
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One HIV-positive man also presented with anti-HCV positivity. Two patients (one man and one woman) were HBsAg-positive; both were anti-HCV-negative.
From the 14 anti-HCV positive patients, two were lost to follow-up. Serum was collected from the remaining 12 patients (7 women and 5 men) for assessment of viraemia levels (HCV RNA). Among those patients, only one male patient presented high viraemia levels (20x106 RNA copies/ml). The patients' genotype and levels of viraemia are described in Table II.
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Discussion |
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We showed that the prevalence of HCV infection was lower in infertile patients than in the general population. In the presence of infection, semen viraemia was extremely low. Our data is in accordance with other reports (Levy et al., 2000) in which a low viral load of HCV was detected in semen of infertile men using the same methodology employed by us (RNA detection).
HCV is found in the semen of HCV-positive patients in very low or of undetectable levels by the available methodology; however, these data must be interpreted with care, since semen is known for its ability to inhibit PCR (Debono et al., 1996; Semprini et al., 1998
; Levy et al., 2000
). Moreover, the importance of HCV virus in semen can be related to virus concentration, since viral infections might contribute to male infertility by causing an inflammatory/immunologic reaction or by a direct toxic effect (Keck et al., 1998
).
The risk for sexual HCV transmission is estimated at 5% (Dienstag, 1997) and in women, the risk for infection by HCV-positive partners is higher in relationships lasting more than 20 years. In these cases, transmission is more feasible if exposure is repeated and long-lasting (Caporaso et al., 1996
; Kao et al., 1996
). We also found a positive association between HCV-positivity in males and risk for HCV-positivity in their female partners. Some investigators have demonstrated that sexual transmission is more probable in cases with co-infection by HIV, suggesting an association with the elevated HCV charge produced by immune suppression (Eyster et al., 1991
; Soto et al., 1994
).
It is known that HCV transmission occurs essentially by the parenteral route, with a huge part of HCV-serum positive patients reporting a history of blood transfusion and use of drugs in the past (Diago et al., 1996). In our population, we found a relative risk of 23.24 for HCV infection in male patients who were users of injectable drugs. It is also known that in 4050% of hepatitis C cases the parenteral risk factor is not identified, suggesting the importance of other possible routes (Everhart et al., 1990
).
Vertical HCV transmission is less frequent, except in cases of mothers with high viraemia levels (HCV RNA ± 106 copies/ml), including those co-infected with HIV (Lin et al., 1994; Ohto et al., 1994
). In our study, none of the female patients had HCV RNA levels >3x106 copies/ml. Although current evidence indicates that the risk for sexual and perinatal transmission is low, the prognosis of neonates born with HCV infection remains unknown. In addition, there are few data concerning prevalence of HCV within infertile populations, repercussion of HCV infection in reproductive health and risk for transmission by assisted reproductive procedures.
Another unsolved question concerns the variety of viral genotypes and their relation with the vertical or laboratory risk for transmission. Some types of HCV virus could be more aggressive and should be managed differently.
In conclusion, our results show that most HCV-positive patients in this population of infertile couples had undetectable or low levels of HCV RNA. We should consider the inclusion of serum typing for HCV in the investigation of the infertile couple, to allow the definition of management and risks associated with viraemic levels when one or both partners are infected with HCV.
These data may be useful for counselling and management of couples who seek assisted reproduction, and also for further studies analysing the risk for HCV transmission in infertile couples.
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Acknowledgements |
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Notes |
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References |
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Submitted on September 4, 2001; resubmitted on January 2, 2002; accepted on April 9, 2002.