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Sperm DNA fragmentation assessment: Is it really helpful?
Prof. Basil C. Tarlatzis and Dimitrios G. Goulis
Reprinted with permission of the
International Society of Gynecological Endocrinology
Fertilization, both natural and by assisted reproduction techniques (ART), needs
the dual support of the sperm not only for the messages it carries but for being
as a messenger cell the and carrier of these messages as well. Nevertheless, In
in the era of Intra-Cytoplasmic Sperm Injection (ICSI) though, only the sperm
messages are needed. These messages include the haploid genome, the centrosome,
necessary for the division of the cell, and important factors for the
development of the placenta. Sperm DNA contributes half of the offspring’s
genetic material and abnormal DNA in the form of fragmented DNA - when(i.e.
excessive strand breaks are present -) may lead to derangements in the
reproductive process. In this perspective, it seems plausible that strand breaks
in the sperm DNA may affect fertilization and delivery of a healthy child. In
mammals sperm chromatin differs from that in somatic cells in structure and
composition (…). In fact sperm chromatin forms the most compact structure among
all eukaryotic cells. This compact structure is important for the protection of
genetic integrity during transport of the paternal genome through the male and
female reproductive tracts (…). DNA fragmentation is characterized by single- as
well as double double-strand DNA breaks. During spermiogenesis, the maturing
sperm gradually loses its ability to repair possible DNA damage. Therefore, DNA
breaks occurring during or after DNA packaging may escape the corrective
mechanisms and be delivered to the mature sperm. The exact mechanisms leading to
DNA strand breaks are still widely elusive. The theories proposed involve
defective sperm chromatin packaging, abortive apoptosis and oxidative stress.
There is evidence that the mechanisms leading to DNA fragmentation are
inter-related, as an abnormal chromatin packaging due to defective protaminosis
makes sperm more vulnerable to environmental insults like excessive ROS. Some
external conditions factors have been associated with an increase in the
percentage of ejaculated spermatozoa with DNA damage, although the underlying
mechanisms still remain unclear. Among them, the most important are cigarette
smoking, genital tract infection, testicular cancer and Hodgkin’s disease,
iatrogenic damage, like such as in sperm preparation protocols for ART as well
as hyperthermia, exposure to pesticides, and air pollution. The assays used for
the assessment of sperm DNA fragmentation can be distinguished into direct and
indirect. Direct assays try to detect the actual DNA breaks, while indirect
assays quantify the susceptibility of sperm DNA to break after an external
insult, such as acid treatment. The most commonly used direct assays are;
Terminal Deoxynucleotidyl Transferase-mediated Nick End Labeling (TUNEL), Single
Cell Gel Electrophoresis (COMET) and In-Situ Nick Translation (NT) assay. The
most common indirect assays are; Flow flow cytometric acridine orange assay,
Acridine Orange test (AO), DNA Break Detection-Fluorescence In Situ
Hybridization (DBD-FISH) and Sperm Chromatin Dispertion test (SCD). Despite the
method of assessment, all assays attempt to determine the total amount of DNA
fragmentation, irrespectively of the region of the genome that occurs. It is
reasonable that breaks affecting certain genes are more detrimental than others
in “silent” areas of the genome, and although no assay can evaluate this factor
yet. Thus, for the present, there is no differentiation between clinically
significant and insignificant fragmentation. A recent meta-analysis showed a
significant odds ratio (OR) of 6.54 [(95% confidence interval (CI; ) 1.71, -
24.91]) and 7.58 (95% CI; 2.54, - 22.67), when couples with DNA Fragmentation
Index (DFI) of < 30% and < 40%, respectively, compared to couples with higher
DFIs. Many studies using a variety of assays have shown statistically
significant differences in sperm DNA fragmentation between fertile and infertile
men. Despite these differences - referring to the mean or median –, there is an
extensive overlap between the values found in fertile and infertile men. In
addition, clear reference values to distinguish the two groups have not yet been
obtained established or can not be widely accepted without concern. It is
important to note that values obtained using different assays can not be
compared. Sometimes in addition, it is inappropriate to compare values obtained
from different laboratories, even using the same technique, as many lab factors
and protocol variations can significantly affect the results. IVF and ICSI
techniques represent a very important treatment approach, even in cases of
severe male factor infertility. So far, conventional semen parameters were
proven disappointing at predicting the outcome of IVF; and during the last
years, sperm DNA fragmentation has been promoted as a promising alternative
predictive factor. Numerous studies of varying quality have examined the
association between sperm DNA fragmentation and pregnancy rates after standard
IVF and ICSI with conflicting results. In a recent meta-analysis of thirteen
studies, sperm DNA fragmentation was significantly, albeit weakly, associated
with pregnancy (OR= 1.44, 95% CI; 1.03, - 2.03). This weak association can not
be considered clinically important neither enough adequate evidence to
discriminate between couples who will conceive or will not conceive. Thus, there
is not enough evidence to support the routine use of DNA fragmentation as a
prognostic factor of pregnancy after IVF or ICSI procedures. In conclusion, DNA
fragmentation is a new parameter for the evaluation of male factor infertility
and a possible predictor of the outcome of ART. Although promising, there are
still concerns about regarding not only its utility usefulness as a part of the
fertility evaluation predictive factor, but also about what it actually
measures. Protocol variations among laboratories worldwide, lack of reference
values and absence of conclusive evidence about its actual value as an
independent or complementary factor of male infertility still prevent its
establishment in the routine diagnostic investigation of the infertile man.
© International Society of Gynecological Endocrinology - n. 34/ June 2009
