Determination of Free DNA (cfDNA) by RT-qPCR in Individuals in Sperm Alterations

Abstract

Previous studies have suggested that the presence of circulating nucleic acids (cell-free DNA) in seminal plasma may indicate disease states. However, the potential association between cell-free DNA (cfDNA) levels in seminal plasma and sperm fertility parameters has not yet been definitively determined. In this study, we will compare seminal free DNA levels between normozoospermic samples and those from different pathologies related to characteristic parameters of sperm quality (asthenozoospermia, azoospermia, teratozoospermia, oligozoospermia and a few samples with a high fragmentation index) in order to detect a potential association between free DNA levels in seminal plasma and these different pathologies of male fertility. The recovery of free DNA from our different samples was done with the MACHEREY-NAGEL NucleoSpin® kit. This kit allows isolation of DNA from cell-free biological fluids using rapid silica column procedures. The quantification of free DNA in our samples was performed by quantitative PCR (RT-qPCR). Our results showed a significant difference in the level of free seminal DNA between normozoospermic samples and oligozoosperimic, teratozoosperimic, azoosperimic samples and those with a high DNA fragmentation index. On the other hand, no significant difference in the level of seminal free DNA was noted between normozoospermic and asthenozoospermic samples. These results suggest that seminal free DNA may be an important biomarker in the assessment of human sperm fertility.