Skip to main content Accessibility help

Seminal cell-free DNA levels measured by PicoGreen fluorochrome are associated with sperm fertility criteria

  • F. Costa (a1), F. Barbisan (a1), C.E. Assmann (a2), N.K.F. Araújo (a3), A.R. de Oliveira (a3), J.P. Signori (a3), F. Rogalski (a3), B. Bonadiman (a1), M. S. Fernandes (a1) and I.B.M. da Cruz (a4) (a1) (a2)...


Previous investigations suggested that elevated cell-free DNA (cfDNA) can indicate non-healthy states. However, the potential association between cfDNA seminal plasma levels and fertility sperm parameters has not yet been determined. Therefore, the present study evaluated the association between seminal cfDNA levels and sperm fertility criteria to determine the use of seminal cfDNA quantification. An in vivo protocol quantified cfDNA levels of semen samples obtained from 163 male patients using fluorescent PicoGreen dye staining. To confirm if semen cfDNA quantification is realistic, an in vitro complementary test was performed using three or four semen samples. The fresh sperm samples were exposed to paraquat that generates high levels of superoxide anion causing oxidative stress and cell mortality. The results showed significant association between dsDNA levels and several sperm fertility parameters, such as low viability and alterations of motility and morphology. The in vitro analysis confirmed the association between dsDNA levels and sperm viability. Together, these results suggest that dsDNA levels could be an important biomarker to test sperm fertility.


Corresponding author

All correspondence to: Ivana Beatrice Mânica da Cruz. Laboratório Biogenômica. Universidade Federal de Santa Maria. Av Roraima 1000, Prédio 19, Santa Maria-RS 97105900, Brazil. E-mail:


Hide All
Bus, J.S. & Gibson, J.E. (1984). Paraquat: model for oxidant-initiated toxicity. Environ. Health Perspect. 55, 3746.
Cadona, F.C., Manica-Cattani, M.F., Machado, A.K., Oliveira, R.M., Flores, E.R.S., Assmann, C., Algarve, T.D. & Cruz, I.B.M. (2014). Genomodifier capacity assay: a non-cell test using dsDNA molecules to evaluate the genotoxic/genoprotective properties of chemical compounds. Anal. Methods 6, 8559–68.
Chou, J.S., Jacobson, J.D., Patton, W.C., King, A. & Chan, P.J. (2004). Modified isocratic capillary electrophoresis detection of cell-free DNA in semen. J. Assist. Reprod. Genet. 21, 397400.
Czamanski-Cohen, J., Sarid, O., Cwikel, J., Lunenfeld, E., Douvdevani, A., Levitas, E. & Har-Vardi, I. (2013). Increased plasma cell-free DNA is associated with low pregnancy rates among women undergoing IVF-embryo transfer. Reprod. Biomed. Online 1, 3641.
Halliwell, B. & Whiteman, M. (2004). Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br. J. Pharmacol. 2, 231–55.
Iommiello, V.M., Albani, E., Di Rosa, A., Marras, A., Menduni, F., Morreale, G., Levi, S.L., Pisano, B. & Levi-Setti, P.E. (2015). Ejaculate oxidative stress is related with sperm DNA fragmentation and round cells. Int. J. Endocrinol. 32, 1901–7.
Jentzsch, A.M., Bachmann, H., Fürst, P. & Biesalski, H.K. (1996). Improved analysis of malondialdehyde in human body fluids. Free Radic. Biol. Med. 2092, 251–6.
Levine, R.L. (1983). Oxidative modification of glutamine synthetase. I. Inactivation is due to loss of one histidine residue. J. Biol. Chem. 258, 11823–927.
Levy, M.S., Loptian, P., Kennedy, R.O., Lo-Yim, M.Y. & Shamlou, P.A. (2000). Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method. Nucleic Acids Res. 1, 28, 57.
Li, H.G., Huang, S.Y., Zhou, H., Liao, A.H. & Xiong, C.L. (2009). Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia: implications for non-invasive genetic utilities. Asian J. Androl. 11, 703–9.
Shiva, M., Gautam, A.K., Verma, Y., Shivgotra, V., Doshi, H. & Kumar, S. (2011). Association between sperm quality, oxidative stress, and seminal antioxidant activity. Clin. Biochem. 4, 319324.
Swarup, V. & Rajeswari, M.R. (2007). Circulating (cell-free) nucleic acids-a promising, non-invasive tool for early detection of several human diseases. FEBS Lett. 581,795–9.
Swarup, V., Srivastava, A.K., Padma, M.V. & Rajeswari, M.R. (2011). Quantification of circulating plasma DNA in Friedreich's ataxia and spinocerebellar ataxia types 2 and 12. DNA Cell Biol. 6, 389–94.
Uribe, P., Boguen, R., Treulen, F., Sánchez, R. & Villegas, J.V. (2015). Peroxynitrite-mediated nitrosative stress decreases motility and mitochondrial membrane potential in human spermatozoa. Mol. Hum. Reprod. 3, 237–43.
WHO (World Health Organization). (2010). WHO Laboratory Manual for the Examination of Human Semen and Sperm Cervical Mucus Interactions. Cambridge: Cambridge University Press 2.
Wu, T.L., Zhang, D., Chia, J.H., Tsao, K., Sun, C.F. & Wu, J.T. (2002). Cell-free DNA: measurement in various carcinomas and establishment of normal reference range. Clin. Chim. Acta 1, 7787.
Xie, G.S., Hou, A.R., Li, L.Y., Gao, Y.N. & Cheng, S.J. (2004). Quantification of plasma DNA as a screening tool for lung cancer. Chin. Med. J. (Engl.) 10, 1485–8.
Yan, L., Liu, J., Wu, S., Ji, G. & Gu, A. (2014). Seminal superoxide dismutase activity and its relationship with semen quality and SOD gene polymorphism. J. Assist. Reprod. Genet. 31, 549–54.
Yi, J., Zhang, Y., Zhang, Y., Ma, Y., Zhang, C., Li, Q., Liu, B., Liu, Z., Liu, J., Zhang, X., Zhuang, R. & Jin, B. (2014). Increased plasma cell-free DNA level during HTNV infection: correlation with disease severity and virus load. Viruses 7, 2723–34.
Zhang, W.D., Zhang, Z., Jia, L.T., Zhang, L.L., Fu, T., Li, Y.S., Wang, P., Sun, L., Shi, Y. & Zhang, H.Z. (2014). Oxygen free radicals and mitochondrial signaling in oligospermia and asthenospermia. Mol. Med. Rep. 10, 1875–80.
Zhao, X., Huang, S., Luo, H., Wan, X., Gui, Y., Li, J. & Wu, D. (2014). Evaluation of vesicular stomatitis virus mutant as an oncolytic agent against prostate cancer. Int. J. Clin. Exp. Med. 7, 1204–13.
Zhu, G., Ye, X., Dong, Z., Lu, Y.C., Sun, Y., Liu, Y., McCormack, R., Gu, Y. & Liu, X. (2015). Highly sensitive droplet digital PCR method for detection of EGFR-activating mutations in plasma cell-free DNA from patients with advanced non-small cell lung cancer. J. Mol. Diagn. 3, 265–72.


Seminal cell-free DNA levels measured by PicoGreen fluorochrome are associated with sperm fertility criteria

  • F. Costa (a1), F. Barbisan (a1), C.E. Assmann (a2), N.K.F. Araújo (a3), A.R. de Oliveira (a3), J.P. Signori (a3), F. Rogalski (a3), B. Bonadiman (a1), M. S. Fernandes (a1) and I.B.M. da Cruz (a4) (a1) (a2)...


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed