Intermittent detection of hepatitis C virus (HCV) in semen from men with human immunodeficiency virus type 1 (HIV-1) and HCV

HCV is usually transmitted via the blood, but HCV RNA has been detected recently in seminal fluid. This study was done to study HCV seminal shedding and factors that could influence the presence of HCV in the seminal fluid of men coinfected with HCV and HIV-1. HCV and HIV-1 genomes were assayed in multiple paired blood and semen samples obtained from 35 men enrolled in an assisted medical procreation protocol. HCV RNA was found intermittently in semen samples from 9 patients (25.7%). Samples from 9 men with HCV RNA in their semen and 26 men without were compared to further analyze these parameters. No correlation was found between HCV RNA in the seminal fluid and age, HCV virus load, the duration of HIV-1 infection, HIV treatment, the CD4(+) cell count, HIV-1 virus load or HIV-1 detection in the semen. The intermittent detection of HCV RNA in semen samples support the systematic search for HCV RNA in semen and the use of processed spermatozoa in assisted medical procreation of infertile HCV serodiscordant couples.     

Hepatitis C virus infection and assisted reproduction

BACKGROUND: In assisted reproduction, hepatitis C virus (HCV) transmission may pose a risk for the baby, technicians, and gametes or embryos from non-contaminated parents. This study aimed at determining the prevalence and risk factors for HCV infection in a group of infertile couples. METHODS: HCV infection was investigated in 409 patients attending the infertility clinic at Hospital de Clínicas de Porto Alegre, Brazil, between 1997 and 1998. Serum was screened for anti-HCV using ELISA and for hepatitis B surface antigen (HBsAg) using an enzyme-linked fluorescent assay (ELFA). HCV infection and semen viraemia was also investigated using HCV RNA detection. RESULTS: The overall prevalence of anti-HCV was 3.2% (8/248) among women and 3.7% (6/161) among men. All subjects were negative for hepatitis B virus (HBV) and human immunodeficiency virus (HIV). From the 14 HCV-positive patients, two were lost, and serum was collected from the remaining 12 patients for assessment of HCV RNA, resulting in five HCV-positive cases (one woman and four men). Only one of the HCV-positive men had viraemia levels >500 000 RNA copies/ml. There was a significant risk associated with being HCV-positive in women with HCV-positive male partners (P < 0.001). In male patients, the correlation between use of intravenous drugs and HCV-positivity was also significant (P < 0.001). CONCLUSIONS: Since the risk for vertical and laboratory HCV infection is not well determined, and HCV prevalence is not negligible in this group, we recommend that infertile patients be screened before assisted reproductive techniques.     

A modified RT-PCR technique to screen for viral RNA in the semen of hepatitis C virus-positive men

BACKGROUND: Our objective was to use an adapted RT-PCR technique to assess the presence of hepatitis C virus (HCV) in semen and also in different density gradient semen fractions collected from men with chronic viral hepatitis participating in an assisted reproduction programme. METHODS: This study included 50 semen samples from 35 HCV(+) men, with active viral replication assessed by RT-PCR, collected the day of oocyte retrieval and used for assisted reproduction. These samples were subjected to standard assisted reproduction sperm preparation conditions, using density-gradient centrifugation with 45 and 90% layers. Aliquots of semen, 45 and 90% fractions, and embryo culture media were frozen at -80 degrees C for subsequent virological analyses. All aliquots were tested with a commercially available HCV RNA assay, adapted for use with semen after a number of technical changes. This assay yielded a sensitivity of 50-100 HCV RNA copies/ml and strongly diminished the effect of seminal amplification inhibitors. RESULTS: HCV RNA was detected in 7/50 (14%) semen samples tested, 5/35 (14.3%) men. HCV RNA was found in only 1/50 45% fractions but never in the 90% fraction or embryo culture media. Sera from 3/5 men contained 3.19-7.40 x 10(5) IU/ml, while the two others had 4.5 and 11.7 x 10(6) IU/ml. However, HCV RNA was quantified at <600 IU/ml in the HCV(+) semen of these five patients. The ongoing pregnancy rate was of 20% (10/50) with one delivery at the time of the present report. No anti-HCV antibody was found in any of the women or the newborn. CONCLUSIONS: Although HCV is present at low concentrations in the semen of a few HCV(+) patients, no purified sperm fraction (i.e. 90% fraction) used in assisted reproduction was HCV(+) and no seroconversion was observed in the women and the newborn, thereby suggesting a very low risk of virus transmission. Nevertheless, because the presence of HCV in semen implies a possible risk of nosocomial contamination, safety regulations must be strictly applied in assisted reproduction laboratories.     

Transmission risk of hepatitis C virus via semen during assisted reproduction: how real is it?

The risk of viral transmissibility in assisted reproduction is still a much-debated issue, especially for hepatitis C virus (HCV). HCV is a common causative agent for parenterally transmitted viral hepatitis. In addition, it has been incriminated in other routes of transmission, including sexual transmission and nosocomial infections. The management of infertility, in association with HCV, has sparked debates about the potential risk of spread of infection to virus-free individuals, embryos and/or semen. The lack of worldwide-accepted screening policies has helped to fuel this debate. Today, it is evident that there is a potential risk of spread of HCV through biological fluids, including semen, to other individuals. This risk can only be marginalized by the use of well-established criteria for safety in infertility centres, and by the use of proper initial detection and segregation of potentially hazardous materials. Techniques and protocols have been established to help the andrologist and embryologist to safeguard patients against such dangers, and should be imposed in all centres, allowing HCV-positive males to enter their assisted reproduction programmes.     

Pregnancy after safe IVF with hepatitis C virus RNA-positive sperm

In France, assisted reproductive technology (ART) for hepatitis C virus (HCV)-infected patients is now subject to strict control after the publication of recent guidelines. Infertile serodiscordant couples (HCV-viraemic men and their seronegative female partners) require special care to carried out in designated 'viral risk' laboratories. Twelve sequential semen samples taken from an HCV chronically infected patient were analysed within 22 months. HCV RNA was detected in all the seminal plasma sampled before antiviral treatment with relatively high viral loads, and in two of the corresponding fractions of motile sperm obtained after a gradient selection, suggesting that a contamination risk by HCV through ART cannot be excluded. When the selection of sperm on a discontinuous gradient was followed by an additional swim-up step, HCV RNA was never detected in the motile sperm suspension that was frozen in highly secure straws. IVF was performed using cryopreserved sperm that tested negative for HCV RNA, resulting in a pregnancy. One month after embryo transfer, testing for HCV RNA and antibodies in the woman gave negative results.     

Multicenter quality control of the detection of HIV-1 genome in semen before medically assisted procreation

Couples in whom the man is HIV-1-positive may use medically assisted procreation in order to conceive a child without contaminating the female partner. But, before medically assisted procreation, the semen has to be processed to exclude HIV and tested for HIV nucleic acid before and after processing. The performance was evaluated of the technical protocols used to detect and quantify HIV-1 in 11 centers providing medically assisted procreation for couples with HIV-1 infected men by testing panels of seminal plasma and cells containing HIV-1 RNA and/or DNA. The performance of these tests varied due to the different assays used. False positive results were obtained in 14-19% of cases. The sensitivity for RNA detection in seminal plasma was 500-1,000 RNA copies/ml, over 500 RNA copies/10(6) cells in semen cells, and for DNA detection in semen cells 50-500 DNA copies/10(6) cells. The use of silica-based extraction seemed to increase the assay performance, whereas the use of internal controls to detect PCR inhibitor did not. This first quality control highlights the need for technical improvements of the assays to detect and quantify HIV in semen fractions and for regular evaluation of their performance.     

Validation of safety procedures for the cryopreservation of semen contaminated with hepatitis C virus in assisted reproductive technology

BACKGROUND: In France, assisted reproductive technologies involving a hepatitis C virus (HCV)-infected man requires the cryopreservation of potentially infected semen (in order to establish the presence of HCV), hence the need for a safe and secure storage system. We evaluated the safety of high-security straws for the conservation of semen containing HCV RNA under routine conditions. METHODS: Ionomeric resin (IR) straws were filled with seminal plasma spiked with different concentrations of HCV RNA and sealed using a thermo-solder. After a 4% sodium hypochlorite treatment and/or cryopreservation for 7 days in liquid nitrogen, the outside ends of each straw were rinsed with RNAse-free water. RESULTS: No HCV RNA could be detected in any of the water samples. Additional samples included the rinsing water from straws sealed by thermo-solder and from the heating wire used to cut the end of straws containing HCV-positive semen. The latter samples were found positive for both HCV RNA and the protamine-2 gene expressed by spermatozoa. CONCLUSIONS: These results demonstrate the safety of IR straws, the filling system and the thermo-solder for cryopreservation of semen containing HCV in liquid nitrogen. Decontamination of the straw after sealing and the use of disposable scissors to open the straws are strongly recommended.     

Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation

Cryopreservation of human spermatozoa is extensively used in artificial insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post-thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. This aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation (95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared spermatozoa from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze-thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared samples from fertile and infertile men.     

Semen characteristics in human immunodeficiency virus (HIV)- and hepatitis C (HCV)-seropositive males: predictors of the success of viral removal after sperm washing

BACKGROUND: Human immunodeficiency virus (HIV)/hepatitis C virus (HCV)-seropositive males can now father children safely, avoiding transmission risks to the mother and the children using sperm washing and nested PCR (nPCR) techniques. Nevertheless, we still lack enough data to determine the reasons why approximately 10% of the performed sperm washes remain positive, thus forcing the repetition of the treatment. Semen quality in infected males is also essential to these procedures. We aimed to determine the predictive value of the semen parameters, sperm washing procedure and the infection status for the post-wash viral positivity, as well as the correlation between the semen and the disease features. METHODS: Semen characteristics were evaluated in 136 samples provided from 125 males. We also included a control group of 125 males matched by age and length of sexual abstinence. At the time of semen retrieval, 70 of them were infected with HIV (45 also with HCV, 64.3%), and 55 of them with HCV alone. nPCR for viral detection was performed in each sample. RESULTS: Thirteen out of 136 (9.5%) of the samples were positive for one or more viral detections (HIV RNA, HIV DNA and HCV RNA, when needed). From a total of 240 nPCR viral analyses, 16 were positive (6.6%). None of the seminal parameters were adequate to predict post-wash results, nor was a positive result dependent on the volumes used in the semen wash. A positive correlation was found between post-wash progressive motility and CD4 blood levels, as well as a negative correlation between progressive motility and time of evolution of the disease in HIV-infected males. CONCLUSIONS: Semen analysis, according to the World Health Organization criteria, of HIV- and HCV-affected patients showed no differences from that of non-infected males. Moreover, low CD4 blood levels, and a long evolution of the disease do not negatively affect sperm motility.     

Virus et sperme : implications pour l'assistance médicale à la procréation (AMP)

In this article, we reviewed published data on the presence of HIV, HCV and HBV in semen. Means to achieve reduction in the risk of viral transmission during medically assisted reproduction are described. French national guidelines for management of couples infected with HIV, HCV and HBV during medically assisted reproduction are commented.     

Detection of human immunodeficiency virus-1 RNA and DNA by extractive and in situ PCR in unprocessed semen and seminal fractions isolated by semen-washing procedure

BACKGROUND: To determine the presence of human immunodeficiency virus-1 (HIV-1) viral RNA/DNA in whole semen, in properly isolated seminal fractions and in spermatozoa after swim-up, by extractive nested PCR and to compare the detection of HIV DNA by in situ PCR (IS-PCR) with the results of nested PCR. METHODS: We tested HIV-1 RNA and DNA by nested PCR in semen and in seminal fractions from 55 patients. Non-spermatic cells and spermatozoa pellet fractions from 10 HIV-1-positive and five HIV-1-negative men were tested for proviral DNA by IS-PCR. RESULTS: All samples of spermatozoa recovered after sperm washing were free of HIV RNA. HIV RNA tested positive in seven (13%) seminal plasma samples and only in two (4.2%) whole semen of these same samples. Of the seven seminal plasma samples testing positive for HIV RNA, four men had elevated blood viral load and three an undetectable viraemia. HIV DNA by IS-PCR turned positive in three of five samples in semen of HIV-noninfected men. CONCLUSION: HIV RNA/DNA detection in the semen of HIV-infected men proves the efficacy of sperm washing with swim-up of spermatozoa. It is recommended that nested PCR be conducted on purified seminal compartments. IS-PCR is inadequate for detecting HIV in semen.     

Density differences between spermatozoa with antisperm autoantibodies and spermatozoa covered with antisperm antibodies from serum.

Autoantibody-carrying spermatozoa from infertile men were processed using a discontinuous Percoll gradient. The treatment yielded a sperm fraction with significantly reduced antibody loading on the sperm head but did not affect antibody binding to the sperm tail. The immunodepleted sperm preparation demonstrated fertilizing ability when used in in-vitro fertilization. Normal, antibody-free semen samples were coated in vitro with antisperm antibodies from serum, followed by Percoll gradient centrifugation. No significant separation of antibody-free spermatozoa was obtained, regardless of the antibody binding site.     

Detection of HCV RNA in serum and seminal fluid from HIV-1 co-infected intravenous drug addicts

The presence of hepatitis C virus (HCV) RNA in serum and seminal fluid was investigated in eleven drug addicts coinfected with HIV-1 and HCV. Serum and seminal fluid were taken from each patient at the same time point. HCV RNA was found in ten of the eleven serum samples tested, but only in one of the semen samples. No relationship was observed between CD4 cell counts, the stage of HIV infection, extent of liver damage and the presence of HCV RNA in serum and semen. The results indicate that HCV is not usually present in the semen and provide further evidence against sexual transmission as an important mode of transmission of HCV infection. © 1995 Wiley-Liss, Inc.     

3533例男性不育症患者精液标本的支原体检测与耐药性分析

目的了解我院近三年来男性不育症患者精液标本衣原体、支原体的感染情况和支原体对抗生素的耐药性,为临床治疗用药提供依据。方法对2004～2006年间来我院生殖中心就诊的不同病因的3533例男性不育症患者的精液标本进行支原体和衣原体的检测,同时对支原体检测阳性的标本进行了菌落计数和抗生素敏感性检测,分析其耐药情况。结果2004、2005、2006年的413、1303、1817份男性不育症患者的精液标本中分别有18、124、66份精液标本衣原体金标试验阳性,分别有119、304、408份精液标本检到支原体,检到的支原体以解脲支原体为主,解脲支原体对氧氟沙星耐药率最高、交沙霉素耐药率最低。结论我院近三年男性不育症患者的精液标本中支原体和衣原体是主要的病原,检测支原体的耐药性对合理用药、指导临床治疗和提高不育症患者的生育率具有重要意义。     

Medically assisted reproduction in the presence of chronic viral diseases

Teams practising medically assisted reproduction techniques try to avoid viruses as much as possible. Attitudes towards chronic carriers of viruses are rapidly changing, especially for human immunodeficiency virus (HIV) patients. We focus our attention on the legitimacy of systematic screening before assisted reproductive techniques and the need for specialized approaches including an adapted laboratory for viral hazards as well as the need for a multidisciplinary team. Specificities of HIV, hepatitis C virus (HCV), hepatitis B virus (HBV) carriers and the hypothesis of a reduced fertility potential are discussed. Are male HIV carriers a new indication for assisted reproductive techniques in order to prevent virus transmission? It is largely proven that sperm gradient preparation techniques efficiently decrease viral loads and therefore have a protective effect on contamination risk during assisted reproductive techniques. Although a few thousand assisted reproductive technique cycles were performed in the world for this indication without contamination, it is still too early to demonstrate that this technology is fully safe. Two examples of contaminations during insemination are examined. Many questions remain unresolved, such as the lack of standardized techniques for semen preparation or virus detection or the relative merits of intrauterine insemination or ICSI to prevent HIV contamination during assisted reproductive techniques. The authors plead for well-structured, separate programmes of care linked to research objectives.     

Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception

To evaluate and optimize the sperm separation efficiency of a novel silane-coated silica bead (Puresperm), serial studies were carried out to compare the various sperm parameters between: (i) three-layer (90%-70%-40%) Puresperm and three-layer (90%-70%-40%) conventional polyvinylpyrrolidone (PVP)-coated silica bead (Percoll) gradients; (ii) three-layer (90%-70%-40%) and two-layer (90%-45%) Puresperm gradients and separately the same for Percoll; and (iii) large (3.0 ml) and small (0.75 ml) semen loading volumes on three-layer Puresperm gradients. Normozoospermic semen samples were treated and analysed in 12 replicates for each experiment. Manual evaluation of concentration, percentage motility, percentage vitality, percentage normal morphology; computer-assisted semen analysis evaluation of concentration, percentage motility, grade of motility, motion characteristics (curvilinear velocity, linearity, amplitude of lateral head velocity, beat cross frequency, percentage hyperactivation); and yields from the initial semen samples were compared. Percoll was found to be superior to Puresperm in concentration, percentage motility, percentage vitality and yields after three-layer density gradient centrifugation. There were no significant differences in sperm parameters between two- and three-layer Percoll gradients, but three-layer Puresperm gradients behaved significantly better than two-layer gradients. Large semen volume loads on three-layer Puresperm gradients resulted in greater sperm concentrations, percentage motility, percentage vitality and percentage normal morphology, but small semen volume loads produced greater yields of good-quality spermatozoa. In the light of Percoll being withdrawn from the shelf for the use of assisted reproduction because of the presence of PVP, three-layer Puresperm gradients with large semen loading volumes appear to be an attractive alternative for sperm separation in medically assisted conception.     

Transmissions of hepatitis C virus during the ancillary procedures for assisted conception

Since mother to child transmissions of hepatitis C virus (HCV) have been reported to be low, teams involved in assisted reproductive technologies have accepted HCV positive patients into their programmes. We report in the present paper two cases of undoubted patient to patient HCV transmission while patients were attending for assisted conception. In both cases, HCV genotyping and sequencing of the first hypervariable region of the HCV genome provided molecular evidence for nosocomial transmission. Investigations made to elucidate the route of contamination have shown that the most likely route of contamination is through healthcare workers. Such nosocomial HCV infection has been reported in other healthcare situations, mainly in dialysis units, and physical proximity was also suspected to be at the origin of the infection. We conclude that assisted reproduction teams must be very prudent when including such patients in their programmes.