Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia: implications for non-invasive genetic utilities

Abstract

We established a quick and reliable method for recovering cell-free seminal DNA (cfsDNA), by using the binding-washing-elution procedure on the DNA purification column. Low variations (below 15%) among the triplicate values of cfsDNA quantity verified the reproducibility of our cfsDNA recovery method. Similar cfsDNA yield and size distribution between seminal plasma acquired by filtration and centrifugation confirmed the presence of cfsDNA. To investigate the general characterization of cfsDNA, the quantitation and size distribution of cfsDNA from normozoospermic and azoospermic semen were analyzed by real-time PCR and electrophoresis, respectively. CfsDNA concentration in semen with normozoospermia (n = 11) was 1.34 +/- 0.65 microg mL(-1), whereas a higher cfsDNA concentration was observed in azoospermia (2.56 +/- 1.43 microg mL(-1), n = 9). The continuous distribution of DNA fragments ranging from approximately 1 kb to 15 kb and a spectrum of multiples of 180-bp fragments were observed in each normozoospermic and azoospermic sample. Distinct characteristic DNA ladder fragmentations in some azoospermic samples implicated that cfsDNA originate partly from apoptotic cells. CfsDNAs of 36 selected azoospermic patients with known information of Y chromosome microdeletion were subjected to the same microdeletion analysis by multiplex PCR and PCR amplification of sY114 (1450 bp). All multiplex PCR reactions with cfsDNA amplified successfully and provided the same result as leukocyte DNA. PCR amplification of sY114 gave a 1450-bp amplicon as expected. Our data suggested the potential use of cfsDNA in search of biomarker or diagnostic procedures.