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The semen analysis is a time tested method of evaluating male fertility and has been utilized for over sixty years. However, it is limited in its ability as a consequence of what it examines. The routine semen analysis includes an assessment of the number of sperm, their shape (morphology) and motility. A new focus is being directed to other male fertility tests. One type of test that has been growing in popularity is an assessment of Sperm DNA “integrity” as a possible additional metric for fertility potential. However, the clinical utility of such tests is in question as is which of the available DNA integrity tests is the most accurate.
The four tests most commonly used for assessment of DNA fragmentation are SCSA, TUNEL, SCD and Comet. They are described as follows:
Sperm are exposed first to a denaturing acid solution and then exposed to Dye that stains sperm that have DNA damage which is then measured by a flow cytometer. SCSA is limited to assessing the proportion of sperm with DNA fragmentation in a sample, and several thousand sperm are typically necessary for assessment. This limits its use in men with cryptozoospermia (severe low sperm count) and or men who require testicular sperm extraction.
labels the 3’hydroxyl free ends of DNA and DNA “nicks” or damage is measured by a technique called epiflourescence microscopy or flow cytometry.
Assesses the capacity of sperm chromatin to disperse under hydrochloric acid used to denature the chromatin. It is \ measured using immunoflourescence or optical microscopy The amount of dispersion is correlates to the level of DNA Damage.
A single cell gel electrophoresis test. It applies electric current to sperm chromatin from a single cell and examines the amount of fragmented DNA that migrates away. The resulting image resembles a comet with the fragmented DNA in the comet’s tail and intact DNA in the head.
TUNEL, Comet and SCD tests are performed on individual cells making these tests applicable to patients with cryptozoospermia and surgically retrieved specimens.
Understanding the concept and the test: DNA fragmentation (loss of integrity) is commonly due to a phenomenon called Apoptosis. Apoptosis is a normal process of regulated cell death and serves to remove damaged cells from the body. Sperm cells have DNA which is condensed or wrapped differently from other cells in the body, enabling the head of a sperm to be fairly small and the DNA within it inherently more stable. Apoptosis in the body’s cells begins with generation of “reactive oxygen species (ROS) ” and ends with fragmentation of DNA and cell death. In the sperm, ROS generation occurs in the midpiece (which contains mitochondria) and causes impairment of sperm motility. DNA fragmentation may then occur, followed by destruction of the cell.
DNA breakage can also come from other intrinsic and extrinsic sources. DNA breaks may occur with DNA compaction as a normal step in sperm maturation. The breaks are typically repaired.
DNA damage can therefore come from unrepaired DNA breaks, from sperm with DNA damage undergoing incomplete apopotosis, or from sperm with DNA breaks which are the result of oxidative stress. The main cause of such DNA damage is ROS. ROS may be part of apoptosis or generated from post-testicular environmental causes such as increased abstinence, and the presence of a varicocele.
Is the test useful? Integrity of the Sperm DNA is important for normal embryo development. Several studies have correlated abnormalities in measurements of sperm DNA integrity decreased egg fertilization, and reduced pregnancy rates. A figure of greater than 30% DNA fragmentation has been correlated with reduced fertility. Interestingly, several subsequent studies have failed to make the same correlation, with some studies actually showing improved fertility correlated with more DNA damage. This was particularly apparent in studies of sperm DNA fragmentation and cycles of IVF with ICSI.
One confounder may be sperm motility which has been correlated with DFI (DNA Fragmentation index). Non motile sperm are much more likely to have DNA damage and increased measurement of DFI, yet may not affect fertility at all. In fact, the lack of correlation with IVF-ICSI success rates may be partly due to the fact that motile sperm are preferentially selected for ICSI.
In conclusion: The American Society of Reproductive Medicine currently considers such tests of DNA integrity to be for research purposes only and that their clinical utility is unclear. Refinements in the test, such as testing specimens after processing and removal of non motile sperm are currently under investigation and may hold potential in the future. However, currently, semen analysis remains the gold standard.
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