DNA fragmentation Test
Sperm DNA fragmentation refers to deletion of bases or a break or separation in one or both DNA strands contained within sperm. Each tiny sperm has an extensive quantity of DNA, which is wrapped tightly around protein structures to preserve it. Chromatin is made up of DNA and protein structures. Even with protection, sperm chromatin is vulnerable to damage at several points in the sperm’s life cycle, while it’s being produced within the testicles by a series of cell divisions known as spermatogenesis, while it’s in “storage” in the epididymis prior to ejaculation, and possible even after ejaculation.
The genetic material in sperm freezes and wraps around particular proteins during spermatogenesis (sperm synthesis) to maintain the sperm head responsive. This permits enormous amounts of DNA to fit into a tiny amount of space, but it also has the potential to cause damage. To begin with, a vast amount of genetic material must be squeezed tightly in the sperm nucleus; this twisting is likely to induce physical breaks in the DNA. Furthermore, if there are issues with the mechanisms that pack this genetic material, such as a lack of proteins required for the condensation process.
Sperm DNA damage can be caused by many factors:
- Environmental pollution
- Excessive heat exposure
- Infections & certain cancers
- Diet & lifestyle factors
- Smoking & Alcohol consumption
Defective apoptosis, increased reactive oxygen species (ROS) generation, and reduced seminal antioxidants are all possible reasons of sperm DNA fragmentation. Increased sperm DNA damage has also been associated with toxic effects of drugs, cigarette smoking, pollution, and factors such as xenobiotics, high testicular temperature (fever, varicocele), and advanced age.
Sperm motility impairments are caused by sperm DNA damage or fragmentation, which has been linked to lower male fertility. Extensive DNA fragmentation has been associated to infertility and miscarriage, making natural pregnancy and IUI conception more challenging.
Advantage of the sperm DNA fragmentation test is, it provides a reliable analysis of sperm DNA integrity that may help to identify men who are at risk of subfertility, infertile.
Sperm DNA fragmentation test appears to be favored in couples presenting with recurrent conventional IVF failure and pregnancy loss following IVF and ICSI.
Several testing methods are available to determine sperm DNA integrity including:
- TUNEL assay
- COMET assay
- SCD or sperm chromatin dispersion test
- SCSA – sperm chromatin structure analysis
TUNEL assay: The TdT dUTP Nick-End Labeling (TUNEL) assay was designed to detect apoptotic cells that undergo substantial DNA degradation during the late stages of apoptosis. The method is based on the ability of TdT to label blunt ends of double-stranded DNA breaks independent of a template.
COMET assay: The Comet assay, also known as single-cell gel electrophoresis, is a quick and accurate method for detecting strand breakage in individual sperm DNA. This procedure removes protamines and histones from the nucleus, allowing it to form a nucleoid-like structure with supercoiled DNA loops. After gel electrophoresis, the Comet assay measures the shape of single cell nuclei. In an electrophoretic field, little fragmented DNA migrates quicker towards the anode (tail region) than bigger non-fragmented DNA (head region), resulting in a classic comet shape.
SCD: The principle behind the sperm chromatin dispersion (SCD) test is that spermatozoa with fragmented DNA do not produce the characteristic “halo” of dispersed DNA loops in the agarose gel that sperm without fragmented DNA do after being treated with acid Denaturation solution and having the nuclear proteins removed with lysis buffer. This methodology is routinely used in most of the laboratories, because of ease of the procedure.
SCSA: SCSA is a flow cytometric technique that evaluates sperm DNA breaks indirectly via DNA denaturability. The technique assesses sperm DNA’s susceptibility to acid-induced DNA denaturation in situ before staining with the fluorescent dye acridine orange.