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The risk of hepatitis C virus (HCV) transmission in medically assisted procreation (MAP) is debated and some researchers have proposed to exclude MAP for HCV-positive infertile patients. The objectives of this study were to assess the presence of viral RNA in the final preparation of density gradient semen fractions collected from men with chronic HCV and HIV co-infection participating in a MAP program, and to assess whether HIV co-infection influences the rate of the presence of HCV RNA in the semen.The study was based on a cohort of 170 HCV-infected male patients (93 HIV co-infected) participating in a MAP program in a French center. Semen samples were subjected to standard MAP sperm preparation, using density-gradient centrifugation with 40 and 90% layers. All aliquots were tested with a commercially available HCV RNA assay (Roche Monitor), adapted for use with semen after a nucleic HCV RNA extraction modification (Organon Technika).Seminal plasma samples from 19 (11%) patients were HCV RNA positive. The positive HCV viral load in semen was less than 600 IU/ml. None of the 90% fractions from HCV-infected patients were HCV RNA positive. Among the 93 co-infected patients, 10 were positive for HCV RNA in semen and three were HIV/HCV RNA positive in semen.Although HCV RNA was found in the semen of 11% of patients, no purified sperm fraction, or spermatozoa used in MAP were HCV RNA positive. The 90% purified sperm fraction discards the virus and must be used with care in MAP.