Valeria Savasi1 and Luca Mandia2
(1)
Unit of Obstetrics and Gynecology, Department of Biomedical and Clinical Sciences, Hospital “L. Sacco”, University of Milan, Milan, Italy
(2)
Department of Gynecology and Obstetrics, University of Milan, Milan, Italy
Valeria Savasi
Email: valeria.savasi@unimi.it
Keywords
SemenRiskFertilityReproductionVirusImmunity
Epidemiology of Hepatitis C Virus Infection
Hepatitis C virus (HCV) is a single-stranded RNA member of the Flaviviridae family. The virus has an extraordinary heterogeneity owing to its lack of ability to correct copying errors made during viral replication. Many of its nucleotide changes result in a non-functional genome or a replication-incompetent virus (lethal mutants), whereas others persist and account for the incredible viral diversity. Viral heterogeneity takes several forms depending upon the degree of diversity, such as quasispecies (families of different, but highly similar, strains that develop within an infected host over time, with a nucleotide sequence homology greater than 95 %) and over decades and centuries, the degree of HCV diversity has evolved into several distinct genotypes of the virus. Sequence homology among the genotypes is less than 80 %.
There are six genotypes and numerous subtypes of HCV. Globally, genotype 1 is the most common, accounting for 46 % of all infections; followed by genotypes 3 (22 %), and genotypes 2 and 4 (13 % each). Subtype 1b accounted for 22 % of all infections at the global level. There were significant variations across regions, with genotype 1 dominating in Australasia, Europe, Latin America and North America (53–71 % of all cases) and genotype 3 accounting for 40 % of all infections in Asia. Genotype 4 was most common (71 %) in North Africa and the Middle East, but when Egypt was excluded, it accounted for 34 %, whereas genotype 1 accounted for 46 % of infections across the same region. This heterogeneity is extremely important in the diagnosis of infection, pathogenesis of disease, and the response to treatment; it prevents the development of conventional vaccines, allows the virus to escape eradication by the host’s immune system, and affects the completeness of the response to antiviral therapies.
The virus has developed numerous strategies to impair immune responses and evade the host immune system, by delaying and reducing both the intrinsic and adaptive immune response arms. All these characteristics determine a great ability to establish a chronic infection, usually without producing striking symptoms, until the emergence of long-term complications, such as hepatic fibrosis, cirrhosis and hepatocellular carcinoma.
With the advent of new antivirals, HCV infection could in theory be curable in nearly all patients, and with focused strategies to screen and cure current infections in addition to preventing new HCV cases, the number of infections can be significantly lowered or eliminated within the next 15–20 years.
The just published “Global epidemiology and genotype distribution of the hepatitis C virus infection” [1] report includes data for anti-HCV prevalence from 87 countries—accounting for 88 % of the world’s adult population and 84 % of the estimated global anti-HCV population—and HCV viraemic rates for 54 countries—accounting for 77 % of the world’s adult population and 73 % of the estimated viraemic HCV population—the global prevalence of anti-HCV was estimated at 2.0 % (1.7–2.3 %) among adults and 1.6 % (1.3–2.1 %) for all ages, corresponding to 104 (87–124) million and 115 (92–149) million infections respectively. The viraemic prevalence was 1.4 % (1.2–1.7 %) among adults and 1.1 % (0.9–1.4 %) in all ages corresponding to 75 (62–89) million and 80 (64–103) respectively.
About one quarter of human immunodeficiency virus (HIV)-infected persons in the United States are also infected with HCV (CDC Data). Thus, coinfection with HIV and HCV is common (50–90 %) among HIV-infected injection drug users (IDUs) . The seroprevalence of anti-HCV is increased among promiscuous heterosexuals and men who have sex with men (MSM) . HCV infection is often prevalent among HIV-infected populations, with one-third of HIV-infected Americans, and seven million worldwide being coinfected. Chronic HCV infection is now the leading cause of death, after AIDS-related complications, among HIV-infected individuals in areas where highly active antiretroviral therapy (HAART) is available. HIV coinfection exacerbates HCV disease, increasing the likelihood of cirrhosis and HCV-related mortality. The prevalence of HCV coinfection varies, depending on the mode of HIV transmission [2].
Transmission of Hepatitis C Virus
The transmission of HCV is a complex issue. HCV is transmitted mainly through percutaneous exposure to blood (transfusions, transplants), needle sticks, or the contamination of supplies shared among haemodialysis patients or intravenous drug abusers. Although most patients infected with HCV in the USA and Europe acquired the disease through intravenous drug use or blood transfusion, nowadays, very rare, non-identifiable risk factors for HCV infection can be hypothesized in cases with new infections. A potential mechanism of transmission in these instances is mucosal exposure to infectious blood or to body fluids containing blood, or inapparent percutaneous exposure through personal hygiene items (e.g. shared razors or toothbrushes). Then, in many situations, it is difficult to rule out the possibility that transmission resulted from common exposure to risk factors other than sexual exposure [3]. HCV RNA has also been detected in menstrual fluid and semen and both sexual and vertical transmissions have been suggested as alternative modes of transmission of HCV.
Epidemiological studies evaluated HCV sexual transmission. To our knowledge, three prospective cohort studies of monogamous heterosexual couples have been published [4–6]. The HCV Partners' study published in 2013 followed 500 monogamous heterosexual couples to provide quantifiable risk information for counselling long-term monogamous heterosexual couples in which one partner has a chronic HCV infection [5]. Criteria for study participation by each couple included having a heterosexual relationship for a minimum of 36 months, being monogamous for the duration of the relationship as reported by both partners and a minimum of three sexual contacts by the couple in the preceding 6 months. Couples were excluded if either partner had known HIV or hepatitis B virus (HBV) infection, had undergone previous organ transplantation, or was currently using antiviral or immunosuppressive therapy, or if both partners reported a history of IDU. Couples were interviewed separately for lifetime risk factors for HCV infection, within-couple sexual practices, and the sharing of personal grooming items. The median duration of the couples’ sexual relationships was 15 years (range, 2–52 years). Among the 500 partners of anti-HCV-positive index subjects, 20 (4 %) were confirmed to be anti-HCV-positive and 13 of the 20 partners were HCV RNA-positive. HCV genotyping/subtyping and HCV serotyping confirmed nine couples to be concordant, eight couples to be discordant and three couples to be of indeterminate status. Of the + genotype-concordant couples, both partners of six couples were viraemic, allowing phylogenetic analyses, showing that although the overall prevalence of HCV infection among the partners of anti-HCV-positive index subjects was 20 out of 500 (4 %), the prevalence of HCV infection among partners potentially attributable to sexual contact was 3 out of 500 (0.6 %; 95 % CI, 0.0–1.3 %) Based on 8377 person-years of follow-up, the maximum incidence rate of HCV transmission by sex was 0.07 % per year (95 % confidence interval, 0.01–0.13) or approximately 1 per 190,000 sexual contacts. No specific sexual practices were related to HCV positivity among couples.
Other authors suggest caution as the efficiency of HCV transmission by sexual intercourse is generally very low, but some studies showed an increase risk related to particular sexual practices, such as unprotected anal intercourse and vaginal intercourse during menstruation. Another large, prospective study included 895 monogamous, heterosexual partners of HCV-infected individuals who were followed for 10 years. The average weekly rate of sexual intercourse was 1.8. All couples denied practicing anal intercourse, having sex during menstruation, or using condoms. During follow-up, three patients developed HCV infection; however, molecular analysis showed that none had acquired it from their spouse. This risk seems to be related to the number of incidents of sexual intercourse [6].
The CDC Guidelines published in 2010 report that the sexual transmission of HCV has been considered to occur rarely, but CDC surveillance data indicate that 10 % of patients with acute HCV infection report having had contact with a known HCV-infected sexual partner as the only risk of infection (www.cdc.gov/std/treatment/2010/hepC.htm). Patients with acute or chronic HCV infection should be advised that transmission via sexual or household contacts is a possibility, although the risk is relatively low. It is likely that the use of condoms lowers the risk of sexual transmission further, similar to HBV and HIV. However, the United States Public Health Service and a consensus statement issued by the National Institutes of Health have not recommended barrier precautions between stable monogamous sexual partners.
Detection of Hepatitis C Virus in the Semen of Infected Men and Reproductive Assistance in HCV-Discordant Couples
During the past few years, assisted reproduction has been facing a new demand from patients requiring assisted reproductive technology (ART ) : couples at risk of partner-to-partner viral infection and mother-to-child transmission of viral infections—mainly HIV-1, HCV and HIV–HCV co-infected partners. The request for reproductive assistance in this context has been mainly for two medical reasons: either to overcome an infertility problem or to decrease the risk of horizontal transmission. This is directly correlated with three different scenarios: both partners being infected, female-only infection and male-only infection. The primary goal of assisting discordant couples is to protect the uninfected man or woman from viral horizontal transmission and consequently remove the risk of vertical transmission if the mother is unaffected. Also, in assisted reproduction, HCV transmission may pose a risk for the newborn, for gametes or embryos from non-contaminated parents and also for technicians.
The debate on HCV-discordant couples requiring assisted reproduction is still open today. As most of the HCV patients enrolled in ART programmes are coinfected with HIV, in this scenario, coinfection is a crucial point when dealing with the sexual transmission of HCV, as the latter seems to be more frequent in patients coinfected by HIV, as shown by Filippini et al. [7] and Briat et al. [8].
The presence of the virus in the sperm is not a controversial issue. Several studies have analysed semen samples of HCV-positive men to define the possible presence of HCV RNA in semen fractions with controversial results. The majority of the papers published in the literature reported a prevalence of seminal HCV RNA varying from 0 to 30 % using different PCR techniques . These contradictory findings can be explained by differences in the collection and/or storage of samples and in the sensitivity of the assays designed to detect HCV RNA. An additional critical point is that HCV viral loads in seminal plasma show dramatic and rapid variations through time. Anyway, today we can assert that viral particles may be found in seminal plasma and in the other cell fractions, but not in spermatozoa [9].
The study by Briat et al. [8] included 120 HCV-positive men, 82 of them coinfected with HIV-1, and demonstrated that HCV RNA was more frequently found in the semen of men coinfected with HIV-1 (37.8% in men coinfected with HIV-1vs 18.4% in those with only HCV infection).
The presence of the HIV virus in semen could therefore be a bias for the presence of HCV. However, other studies [10] reported that HIV-positive status does not influence the presence of HCV RNA in the semen, and inversely, the presence of HIV RNA in the seminal plasma is not influenced by the HCV status. Only one paper by Bourlet et al. in 2009 [11] reported a 4-year follow-up French multicentre study that enrolled 86 HCV-serodiscordant couples. All the men enrolled were chronically infected by HCV and 10 of them by HIV. From the 58 couples effectively enrolled in the ART programmes of various reproductive centres, 24 pregnancies and 28 newborns were achieved. All of them tested negative for HCV RNA in blood.
Our group conducted a prospective study to assess prospectively the viral risk for HCV-discordant couples in an ART programme with the aims:
1.
2.
3.
All couples completed the immuno-virological and fertility triage, and were treated according to our protocols. Couples were advised to use a condom during intercourse throughout the period of the ART programme. After sperm collection we performed “sperm-washing ” to separate motile spermatozoa from non-sperm cells, immotile spermatozoa and seminal plasma. After swim-up , a supernatant volume of 500 μl was recovered and used for intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI) ovulation induction using gonadotrophins. We simultaneously performed an HCV assay for the qualitative and quantitative detection of HCV RNA in male blood plasma, but we decided to not perform PCR to detect HCV RNA in the final swim-up after sperm-washing, because previous papers have demonstrated that the HCV virus is not able to infect spermatozoa after swim-up. The status of the female partner was confirmed by HCV antibody testing measurements during the 2 weeks before each ART attempt. These tests were repeated 6 months after treatment and again at delivery for pregnant women. The children born were tested once after birth for HCV RNA and after 18 months to check HCV antibodies.
The risk factors for HCV infection were intravenous drug usage in 4 (11 %), blood transfusion in 1 (3 %) and undetermined in 30 (86 %). The range of viral load for HCV was between 1742 and 360911 million of copies/ml in blood plasma. None of the males had ever received any treatment with antiviral drugs. Infertility was originally in the female or male in 29 % or 71 % of the cases respectively. The main cause of female infertility was fallopian tube occlusion (10 patients) and the main cause of male infertility was abnormal semen parameters. None of the seminal samples analysed in our study had normal values in all seminal parameters (number, motility, morphology), but the role of HCV in male infertility is still to be defined. Bourlet et al. [11, 13] and Garrido et al. [14] supported the findings that HCV does not influence male infertility. In disagreement with Bourlet, who mainly performed ICSI in his study because of the use of frozen–thawed semen, we performed either IUI or ICSI on the unique basis of the infertility problems of the couples.
Fourteen couples underwent superovulation and IUI . The mean age (±SD) was 38 ± 4 for female partners. Basal FSH in women was 6.9 ± 2.9 IU/l. Couples had a mean of three treatments. Seventy per cent of pregnancies were obtained in the first three attempts. Twenty-one couples were treated using second-level ART procedures (mean number of cycles per couple 1.8). The mean age was (±SD) 37 ± 3 for female partners. The pregnancy rate in the IUI group (15 %) is higher than the rate reported for infertile couples by the Italian National register, which collects the ART data from all Italian infertility centres (11 %). This difference is reasonably due to the small number of procedures (47 IUIs) performed. The pregnancy rate for ICSI was 18 % and the mean age of the women was 38 years. The Italian National register and Bourlet et al. [11] reported similar pregnancy rates in uninfected infertile couples. The fertilisation rate was high (88 %), supporting the hypothesis that the spermatozoa of HCV-infected patients might be competent. The pregnancy outcome of all the babies was good, with no preterm labour.
It is currently unclear why HCV should be less transmissible than HIV by sexual contact, similar in frequency for vertical transmission, and probably more infectious through parenteral exposure. It has been argued that the low infectivity of HCV by sexual contact is related to its low titre in genital secretions, but titres of free HIV are also low. It is possible that the presence of HIV-infected lymphocytes might be more relevant for the transmission of HIV. However, interesting data by Azzari et al. [15] show that maternal peripheral blood mononuclear cell culture (PBMNC) infection by HCV and viral replicative activity in PBMNCs are important factors in the transmission of HCV from mother to child. It has been suggested that HCV might infect not only hepatocytes, but also mononuclear lymphocytes, including B cells that express the CD81 molecule, a putative HCV receptor. Thus, HCV is able to enter into lymphocytes and replicate itself, as demonstrated by the presence of a negative strain of HCV RNA, a marker of its replication. The role of PBMNC infection by HCV is controversial, but it has been demonstrated that lymphocytes may act as an HCV reservoir and play a key role in the relapse of HCV disease after liver transplantation [8] or after the discontinuation of interferon therapy. Only one author has analysed the quasispecies of the virus in blood and semen and did not find any differences [8]. The author observed that some men with a low blood viral load had detectable HCV RNA in their semen and conversely some men with a high blood viral load did not . Such discrepancies could be explained by local replication in leukocytes of the male genital tract, as reported in blood lymphocytes and monocytes [16–18]. Anyway, phylogenetic comparison of HCV quasispecies in blood and in semen showed no evidence of HCV replication in genital leukocytes; however, a phylogenetic structure was observed between compartments and Briat et al. [8] suggested that HCV particles in semen might originate from passive passage from the blood, with preferential transfer of some variants.
There is also an additional risk to consider. The risk of HCV transmission during reproductive procedures is not documented and there is only a prospective multicentre paper reporting assisted reproduction in 56 HCV-serodiscordant couples. The aim of this study was to evaluate if the sperm-washing method was able to reduce levels of HCV in semen and the risk of HCV transmission to the newborn [11]. It is open to discussion whether or not infertile HCV-discordant couples should be treated as infected couples, and whether it is necessary to apply specific laboratory precautions. Since the publication of a case report that described the transmission of HCV from an infected patient undergoing IVF to two non-infected patients undergoing IVF within the same clinic during the same time period (ASRM), there is great concern among clinicians. Although data on laboratory/nosocomial transmission of HCV during assisted reproduction are both limited and controversial, showing that transmission of viral hepatitis in assisted reproduction is possible, but with a risk of unknown magnitude, it is necessary for infertile HCV-discordant couples to be included in protocols of controlled assisted reproduction procedures to avoid any risk of HCV transmission to the partner and to the staff preparing the sample, and laboratory contamination of the gametes of other non-infected couples through cryopreservation and manipulation [22].
Indeed, in assisted reproduction HCV transmission raises some questions. One of these is that specific guidelines establishing the behaviour of physicians in reproductive medicine have not yet been established. Should we treat HCV-discordant couples who require reproductive assistance? Most researchers believe that a sequential preparation with density–gradient centrifugation–washing–swim-up is recommended for HCV-positive men, similar to HIV semen preparation [11, 19, 20]. Other authors do not consider HCV to be a sexually transmitted disease and for this reason believe that it is unnecessary to perform sperm-washing (http://www.sginf.ch). The second main question is: should fertile HCV-discordant couples be treated? We believe that it is not necessary if they do not need reproductive assistance.
Conclusion
We believe that even if sexual transmission of HCV is very low, in subfertile or infertile couples, sperm-washing should be used to treat HCV-positive semen before ART. We suggest that it might not be necessary to perform nested PCR to detect HCV RNA in the final pellet, as suggested by Bourlet et al. and presently required by French legislation [11]. As the presence of HCV in semen implies a possible risk of nosocomial contamination, safety regulations must be strictly applied in assisted reproduction laboratories. As reported by Englert et al. [21] it seems to be “safer and more ethically acceptable to handle patients with the same levels of risk together, i.e. detected viral carriers in one laboratory, negatively screened patients in another, rather than to mix patients with clearly different risk levels”. Therefore, the laboratory used for ART should be a “viral risk” area separated from the laboratory used for couples negative for HIV, HBV, HCV. International guidelines should be developed through studies on larger populations of chronically HCV-infected individuals.
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