Semen decontamination for the elimination of seminal pathogens

Abstract

The presence of pathogens in semen can compromise the outcome of assisted reproductive treatment, together with the possibility of the female partner or offspring becoming infected. This is cause for concern, especially in South Africa with a high prevalence of HIV-1. Most of these infected individuals are in their reproductive years with the desire to have their own genetically related children. Therefore, assisted reproductive treatment with effective risk reduction procedures, such as semen processing for the elimination of these pathogens is crucial. However, during sperm preparation by standard discontinuous density gradient centrifugation, the supernatant is aspirated to allow access to the purified sperm pellet. Pathogens from the upper layers can adhere to the inside surface of the test tube and flow down to re-infect the purified sperm sample. The use of a centrifuge tube insert may prevent the re-contamination of sperm samples after discontinuous density gradient centrifugation. Furthermore, seminal pathogens can bind specifically or non-specifically to spermatozoa, rendering semen decontamination procedures ineffective. Serine proteases, such as trypsin, have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could have a negative impact on sperm parameters. This research was therefore aimed towards the determination of: i) The effect of semen processing with trypsin and trypsin inhibitor on sperm parameters. ii) The prevalence of various bacteria in semen samples from men attending the Reproductive and Endocrine Unit at Steve Biko Academic Hospital. iii) The effectiveness of semen processing by discontinuous density gradient centrifugation with a centrifuge tube insert, for the elimination of some of the most prevalent bacteria, white blood cells and in vivo derived HIV-1. Evaluation of sperm parameters after semen processing indicated that trypsin and trypsin inhibitor did not have an impact on sperm mitochondrial membrane potential, vitality, motility and zona binding potential, or acrosin activity, respectively. Seminal bacteria were highly prevalent in patients wishing to participate in the Unit’s assisted reproductive program, with 49.5% of semen samples presenting with positive bacterial cultures. Semen processing by means of discontinuous density gradient centrifugation with the tube insert, eliminated significantly more in vitro derived (spiked) bacteria and white blood cells from semen compared to processing without the insert. Furthermore, the semen decontamination procedure was effective in removing HIV-1 RNA from 100% of samples and proviral DNA from 98.1% of semen samples from HIV-1 sero-positive patients. The effectiveness of discontinuous density gradient centrifugation for the elimination of seminal pathogens could, therefore, be improved by the addition of trypsin to the upper density layer, without supplementing the bottom layer with trypsin inhibitor. Additionally, semen decontamination efficiency could also be improved by the prevention of re-contamination of processed sperm samples by the utilization of a tube insert during discontinuous density gradient centrifugation.