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Hum. Reprod. Advance Access published online on March 6, 2008
Human Reproduction, doi:10.1093/humrep/den058
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© The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Nuclear staining identifies two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters

M. Muratori1, S. Marchiani1, L. Tamburrino1, V. Tocci1, P. Failli2, G. Forti1 and E. Baldi1,3

1 Department of Clinical Physiopathology, Andrology Unit, Center for Research, Transfer and High Education DeNothe, University of Florence, Viale Pieraccini, 6, I-50139 Firenze, Italy 2 Department of Pharmacology, University of Florence, Florence, Italy

3 Correspondence address. Tel: +39-0 554271486/7; Fax: +39-0 554271371; E-mail: m.muratori{at}dfc.unifi.it

BACKGROUND: Sperm DNA fragmentation is a possible predictive parameter for male fertility status. The occurrence of M540 bodies in semen of subfertile subjects affects flow cytometric investigations in sperm. We set up a new method to evaluate DNA fragmentation excluding M540 bodies.

METHODS: DNA fragmentation was evaluated by flow cytometry in semen of 75 subjects both by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL, traditional method) and by double staining with TUNEL and propidium iodide (PI, new method).

RESULTS: The use of the new method revealed that TUNEL underestimates sperm DNA fragmentation in flow cytometry and showed two sperm populations stained with low (PIdim) and high (PIbr) avidity for PI. The PIdim population is entirely composed of DNA fragmented sperm and its incidence shows highly significant negative correlations with morphology, motility, sperm count and concentration (respectively, r = –0.51, –0.52, –0.46 and –0.32, n = 75). DNA fragmentation in the PIbr sperm population is independent from semen quality.

CONCLUSIONS: The correlations between sperm DNA breakage and semen quality previously reported are mainly driven by the occurrence of the PIdim population. DNA fragmented sperm in this population are more likely to have poorer morphology, reduced motility and thus a reduced chance to fertilize an oocyte than DNA damaged sperm in PIbr population. Distinguishing between the two types of sperm DNA fragmentation appears to be important in clinical investigations.

Key words: sperm/DNA fragmentation/terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling/nuclear staining/M540 bodies

Submitted on September 13, 2007; resubmitted on December 6, 2007; accepted on February 7, 2008.


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