Sperm DNA fragmentation

Androlog Mail

Androlog has previously focused on the fascinating topic of sperm DNA
fragmentation (reviewed in Makhlouf and Niederberger, J Androl, 2006).
Our research group comes from genetics-mutagenesis field and not from
clinical andrology or fertility. The SCD (Sperm Chromatin Dispersion)
test, the original technique which produced the Halosperm (bright-field
microscopy) and Halomax (fluorescence microscopy) kits, has been
developed in our laboratories. Due to the recent commentaries, we would
like to provide our viewpoint about some of the questions presented.

Should sperm DNA fragmentation be assessed?. It is obvious that any cell
with broken DNA or with evident DNA damage is not a good one. If DNA
breaks (as well as base damages and other DNA lesions) are not repaired
or misrepaired, they can result in cell arrest, mutations, or chromosome
aberrations that may compromise cell viability. This basic fact makes it
relevant to assess DNA integrity in spermatozoa. The available data
indicate that the frequency of sperm cells with broken DNA is increased
in infertile patients, in those with varicocele, local infections,
cancer, and after exposure to certain genotoxic agents or environments.
This is also demonstrated in animals. Then, sperm DNA fragmentation must
be a parameter of sperm quality, complementary to the conventional
evaluations.

Has it a value in fertility evaluation?. From studies performed to date,
a correlation is always found with one or several parameters of
fertility: oocyte fertilization, embryo development, embryo quality,
blastocyst rate, implantation rate or pregnancy. The surprising fact is
that correlations are diverse in the different studies, even using the
same tests, evidencing the complexity of the topics. This will therefore
be an interesting research area for many years. The fact that no
specific treatment for DNA fragmentation exists for some conditions does
not mean it should not be an important topic for study. Moreover, this
evaluation should not only be focused in fertility. Sperm DNA
fragmentation may also help to assess treatment effectiveness in those
clinical conditions where it is elevated and a therapy may exist, like
infections, cancer, varicocele, as well as exposure to genotoxic agents.

What technique should be used?. Each researcher defends the procedure
used in its own lab. All (ISNT, TUNEL, Comet, SCSA, Halosperm) seem
optimal because all essentially determine spermatozoa with fragmented
DNA, and with excellent correlations among them. This is because the
presence of DNA fragmentation a) makes DNA more susceptible to
electrophoresic migration, as in the comet assay; b) makes DNA more
susceptible to acid denaturation, as in the SCSA or Halosperm kit, and
c) makes DNA more susceptible to incorporate modified nucleotides by
certain enzymes, though only in accessible and 'clean' 3'-OH groups, as
in the ISNT or TUNEL assays.

We believe all are indirect ways to evidence DNA breaks. In any case,
whether direct or indirect, does not provide any advantage. At this
point, it must be stressed that the acid incubation performed in the
SCSA or Halosperm, does not create new breaks in DNA, in susceptible
sites or 'potential DNA breaks', as repeatedly cited. The only concept
in the mutagenesis field, that could resemble 'potential DNA breaks', is
that of alkali-labile sites. These are 'real'-mutagenic DNA lesions
(abasic sites and some deoxyribose damages) that the alkaline treatment
may transform into single-strand breaks. This does not happen for the
short acid unwinding treatment. From early research, in the seventies,
on unwinding assays for determination of radiation-induced DNA breaks,
it is suggested that DNA breaks behave as starting points for DNA
denaturation. Moreover, acid is much less a denaturant than alkali, so
only extensive DNA breaks result in significant DNA denaturation after a
short incubation. Strong and prolonged acid incubation may induce some
loss of purines, which does not correspond to any previous 'potential'
lesion. If it happens, this is irrelevant for the test result since
apurinic sites are not transformed into new DNA breaks.

Since all tests detect fragmented DNA, with possible minor peculiarities
among them, the important advantages or disadvantages of each procedure,
are perhaps more dependent on logistic factors, e.g. time, costs,
technical equipment and personnel. These can be important facts in a
decision to investigate sperm DNA fragmentation in your own patients.
Obviously, we use and support the Halosperm kit. It is an easy, simple
procedure, which only needs the conventional bright-field microscope,
slides being permanent. An even faster protocol is being implemented, so
sperm preparation, incubations, staining and scoring is completed in one
hour. This is a very versatile procedure and allows for sequential FISH,
incubation with anti-DNA antibodies (e.g. against 5-methylcytosine), or
with an 8-oxoG probe (in development), or residual protein staining, so
sperm DNA fragmentation can be related in the same sperm cell with
chromosomal aberrations, DNA methylation level or oxidative DNA damage.
More applications in advanced research depend on imagination. This may
be a quite practical procedure, and the subject of interest for many
laboratories interested in the assessment of sperm DNA integrity.

Jose Luis Fern=E1ndez, M.D., Ph.D.
Secci=F3n de Gen=E9tica y Unidad de Investigaci=F3n,
Hospital materno infantil 'Teresa Herrera',
Complejo Hospitalario Universitario Juan Canalejo
As Xubias, 84,
15006-A Coruna, Spain
JLFernandez@canalejo.org ; genetica@cog.es

Jaime Gosalvez, Ph.D.
Unidad de Gen=E9tica, Facultad de Biolog=EDa,
Universidad Aut=F3noma de Madrid,
Cuidad Universitaria de Cantoblanco
28049-Madrid, Spain
jaime.gosalvez@uam.es
Received on Fri Jan 18 16:28:33 2008