Nuclear staining identifies two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters.
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 (PI(dim)) and high (PI(br)) avidity for PI. The PI(dim) 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 PI(br) 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 PI(dim) 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 PI(br) population. Distinguishing between the two types of sperm DNA fragmentation appears to be important in clinical investigations.
Muratori
M
M
Department of Clinical Physiopathology, Andrology Unit, Center for Research, Transfer and High Education DeNothe, University of Florence, Viale Pieraccini, 6, I-50139 Firenze, Italy. m.muratori@dfc.unifi.it
Marchiani
S
S
Tamburrino
L
L
Tocci
V
V
Failli
P
P
Forti
G
G
Baldi
E
E
D000328:Adult; D002467:Cell Nucleus / Q000648:ultrastructure*; D053938:DNA Fragmentation; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods; D007248:Infertility, Male / Q000175:diagnosis / Q000235:genetics; D008297:Male; D011419:Propidium; D012661:Semen / Q000502:physiology*; D013076:Sperm Count; D013081:Sperm Motility; D013094:Spermatozoa / Q000648:ultrastructure*
D011419:Propidium