SCSA is a widespread diagnostic tool in detection of
sperm samples with a high degree of
DNA fragmentation and absence of
histone-to-
protamine proteins exchange in
sperm nuclei. SCSA defines sperm abnormality as an increased vulnerability of sperm DNA to in-situ heat/acid-induced
denaturation. A low
pH treatment opens up defective sperm DNA at the sites of damage. Through
acridine orange (AO) staining, AO molecules are intercalated into
double-stranded DNA in intact sperms while aggregation of AO molecules occurs at
single-stranded DNA in defective sperms. Signals will be analysed with
software programming in examination of both sperm DNA fragmentation (sDF) and atypical chromatin structure.
Causes for sperm DNA damage The integrity of sperm DNA is in close correlation with the transfer of paternal DNA into the
oocyte during
fertilisation. The etiology of
sperm DNA damage can be subdivided into intrinsic and extrinsic factors. The former is attributed to a series of
pathophysiological phenomena during
spermatogenesis; the latter is caused by postnatal exposure to endogenous sources of DNA breaks.
Intrinsic factors •
Abnormality in recombination and chromatin restructuring: During
spermatogenesis,
crossing-over of
chromatid segments between
homologous chromosomes may have occurred abnormally. Specific
nucleases are programmed to introduce
DNA double-stranded breaks for the progress of crossing-over. To prevent undesirable alterations,
DNA damage checkpoint is activated and progress of
meiosis will be suspended when DNA is damaged. Incorrect activation or inactivation of the checkpoint is suspected to be the cause of
fragmented DNA in
ejaculated spermatozoa. However, such a conclusion is theoretically speculative and currently there is no direct confirmation of this
hypothesis in humans. During
spermiogenesis,
DNA double-strand breaks are introduced to relieve torsional stress and to enable the substitution of
nucleosome histone cores by transitional proteins. Alteration to such processes may be detrimental to the chromosomal integrity of sperm. •
Abortive apoptosis: Apoptosis refers to a
programmed cell death that removes abnormal cells and to prevent their over-proliferation. If apoptosis is not activated efficiently, overpopulation of
germ cells or escape of defective germ cells will lead to
sperm DNA damage.
Extrinsic factors •
Age: Although males produce sperm throughout their
adulthood, older age is associated with increased number of DNA double-stranded breaks and decreased frequency of sperm
apoptosis. •
Heat stress: High temperatures cause adverse effects to sperm DNA and
male fertility. Excessive heat is related to impaired sperm
chromatin integrity, and
testis overheating is associated with reduced fertility potential. •
Smoking: Toxins in common tobaccos may increase the prevalence of
fragmented DNA. Smoking is associated with significantly escalated levels of seminal
ROS and
oxidative stress. Increased ROS activity leads to apoptosis and increased fragmentation of DNA. Frozen or fresh sperm samples are thawed in a 37 °C water bath and
diluted with a
TNE buffer to obtain 200
μL suspension (sperm concentration: 1-2 x 10^6 mL). •
Acid-induced denaturation: 400
μL acidic solution (
pH 1.2) containing 0.15M
sodium chloride solution, 0.08M
Tris hydrochloric acid, and 0.1%
Triton-X 100 are added into 200 μL sperm suspension, and the solution is mixed strictly for 30 seconds. Such a process enables
denaturation of
sperm nuclei with
DNA damage. •
Acridine orange (AO) staining: Next, 1.20 ml of AO staining solution with 6 μg AO/ml staining buffer is added into the mixture. The small AO molecules penetrate through the
sperm chromatin in access to double-stranded DNA and single-stranded DNA in intact and defective sperm nuclei respectively. •
Flow cytometry (FCM): Using a
flow cytometer, 500-1000 sperms can be examined within minutes on a 1024 x 1024 gradation scale through a dual parameter. Visualized under blue light at the
wavelengths of 450-490 nm, double-stranded DNA from intact sperms emit green
fluorescence (488 nm) while aggregation of AO molecules single-stranded DNA from defective sperms leads to metachromatic shift to red
fluorescence (>630 nm). •
Data analysis: A
scattergram (cytogram) will be generated from the flow cytometer reflecting DNA stainability from red (X-axis) and green (Y-axis) dots to single out the
heterogeneity; With SCSAsoft® software, data from the scattergram will be converted into
frequency histograms in calculation of DNA fragmentation index (DFI) / Cells Outside the Main Peak of αt (COMPαt), alpha t (αt), and High DNA Stainable fraction (HDS). == Parameters ==