History
Before the development of SCSA, diagnosis or prognosis of male infertility/subfertility was principally referenced the World Health Organisation (WHO) manual-based semen parameters, [3] including semen concentration, motility, and morphology. Yet, several reports of pregnancy failure had the parameters within normal range, suggesting that none of these measurements has drawn a reliable conclusion to reflect chance of fertility of a couple. [4] Furthermore, such parameters are often associated with high labour intensity and lack of statistical power.
In the late 1970s, Donald P. Evenson at Memorial Sloan Kettering Cancer Centre in the United States received an NIH Research Project Grant (RO1) for mammalian sperm chromatin structure study. [5] [6] Various techniques have since been adopted to gain access to sperm DNA integrity. In particular, transmission electron microscopy reflected a significant amount of sperm chromatin heterogeneity. [4] [7]
The heterogeneity was then confirmed through flow cytometry by contrasting AO staining results between human and mouse sperm nuclei. Homogeneous results were observed in the mouse sample while heterogeneous fluorescence intensity varied among the human sample. A hypothesis was proposed “single-stranded/double-stranded DNA breaks-induced sperm DNA fragmentation is correlated to male infertility.” [4] [5] In 1980, Evenson et al. published papers that synthesise this knowledge into clinical tests and found SCSA.
Initially, utilization of thermal energy in buffer (100 °C, 5 min) was proposed and used for denaturation of DNA at sites DNA damage. [8] However, the heated sperm protocol was time-consuming and induced random loss of sperm sample. Therefore, acid-induced denaturation has replaced heat-induced denaturation due to greater convenience of low pH technique and similarity in results. [5]
Principle
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. [9] SCSA defines sperm abnormality as an increased vulnerability of sperm DNA to in-situ heat/acid-induced denaturation. [4] Theoretically, a completely mature and healthy sperm nuclei, which is rich in disulfide bond (S-S), shall have its DNA preserved in double-stranded form. [5] 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. [4] [5] Undergoing flow cytometry (blue light), green (native DNA) and red (damaged DNA) fluorescence will be emitted from intact and defective sperms respectively. [2] [4] [10] 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.
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. [13] Such observation is implicative of deterioration of sperm selection, quality, and integrity. [20]
- Heat stress: High temperatures cause adverse effects to sperm DNA and male fertility. Excessive heat is related to impaired sperm chromatin integrity, [21] and testis overheating is associated with reduced fertility potential. [22]
- Smoking: Toxins in common tobaccos may increase the prevalence of fragmented DNA. [4] Smoking is associated with significantly escalated levels of seminal ROS and oxidative stress. [23] Increased ROS activity leads to apoptosis and increased fragmentation of DNA. [16]
Procedure
Currently, only the SCSA protocol developed by Evenson et al. has received trademark protection in achievement of clinical relevance between different laboratories. [4] The individual steps of SCSA are as follows:
- Freezing/Thawing: After ejaculation, human sperm samples are subjected to a 30-minute semen liquefaction at 37 °C, followed by cryopreservation in an ultra-low temperature freezer (–70 to –110 °C) or placed directly into liquefied nitrogen cryovial. [24] 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). [24]
- 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, [4] [24] 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. [4] [24] 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. [9] 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). [9] [4]
- 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; [9] 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). [4] [5]
Parameters
SCSA consists of a fixed flow cytometry protocol and a specific computing program, SCSAsoft ®. Measurements include DNA fragmentation index (DFI) and High DNA Stainable (HDS) fraction, which represent the percentage of sperm with DNA breaks/protamine defects and immature spermatozoa without full protamination respectively. [10]
DNA fragmentation index (DFI)
Also known as Cells Outside the Main Peak of αt (COMPαt), DFI can be further sub-classified into mean DFI (X DFI) and standard deviation DFI (SD DFI). [5] The index has been determined as the most sensitive criteria for fertility assessment in reflection of sperm DNA integrity. Normal DFI implies no measurable value; moderate DFI sample infers normal sperm morphology; and high DFI fractions exhibited elongated nuclei and signs of apoptosis. In general, the greater the DFI, the higher the chance of infertility or subfecundity.
Within DFI of 0-20%, the occurrence of spontaneous pregnancy remains consistent; [2] when DFI exceeds 20%, the rate of natural fertility gradually declines; [2] when DFI exceeds 30%, the odds ratio for natural or Intrauterine insemination (IUI) fertility is greatly reduced by 8-10 folds, suggesting a close-to-zero chance of pregnancy. [2]
High DNA Stainable (HDS) fraction
The HDS sperm population has a remarkably high degree of DNA staining by AO molecules due to the presence of unprocessed P2 protamines. [9] [25] Determination of HDS value reflects structural chromatin abnormalities. A high HDS value is indicative of immature sperm morphology and hence pregnancy failure. [25] [26]
Applications
Diagnosis of male infertility or subfertility
Since the SCSA can be performed to assess the sperm abnormality, it is a valid instrument to determine male infertility or subfertility.
Although the causes and events that actuate sperm DNA damage and fragmentation are not yet fathomed, Sperm DNA fragmentation has been shown to be closely correlated with fertility and subfertility in not only humans, but also bulls, boars, and stallions. [5] [27] [28] [29] Such finding asserts the DFI determined by SCSA to be a strong independent predictor of in vivo pregnancy and a clinically useful technique. [13] [23] [30] [31] [8]
Currently, 25% DFI is the established clinical threshold in classifying males into statistical probability of: 1) increased time for natural pregnancy, 2) lower chance of Intrauterine insemination (IUI) success, 3) more miscarriage, or 4) infertility. High HDS values are in positive correlation to pregnancy failures.
In such cases, other assisted reproductive technologies (ART) may be performed, including intracytoplasmic sperm injection (ICSI) (for sperm sample with DFI>25%) or testicular sperm extraction (TESE) (for sperm sample with DFI>50%). [9]
Evaluation of cool-stored semen
SCSA is also performed to assess the quality of laboratory sperm samples that have been stored for at least 24 hours. Semen samples that have been stored at appropriate conditions will have essentially no change, while greater change in DNA quality indicates an improper handling. [32]
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