Sperm Chromatin Structure Assay

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Sperm Chromatin Structure Assay (SCSA) is a diagnostic approach that detects sperm abnormality with a large extent of DNA fragmentation. [1] First described by Evenson in 1980, the assay is a flow cytometric test that detects the vulnerability of sperm DNA to acid-induced denaturation DNA in situ. [2] SCSA measures sperm DNA fragmentation attributed to intrinsic and extrinsic factors and reports the degree of fragmentation in terms of DNA Fragmentation Index (DFI). The use of SCSA expands from evaluation of male infertility and subfertility, toxicology studies and evaluation of quality of laboratory semen samples. Notably, SCSA outcompetes other convention sperm DNA fragmentation (sDF) assays such as TUNEL and COMET in terms of efficiency, objectivity, and repeatability.

Contents

Schematic representation of the SCSA. Use of flow cytometry in differentiating intact sperms from defected sperms. Laser beam strikes each sperm cell. Photomultiplier tube identifies and quantifies the results. Schematic representation of the SCSA.jpg
Schematic representation of the SCSA. Use of flow cytometry in differentiating intact sperms from defected sperms. Laser beam strikes each sperm cell. Photomultiplier tube identifies and quantifies the results.

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.

Intrinsic factors

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:

  1. 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]
  2. 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.
  3. 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.
  4. 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]
  5. 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]

Toxicology studies

Sperm DNA damage can be attributed to exposure chemotherapy, radiotherapy, or other environmental toxicants. SCSA is highly dose-responsive to sperm DNA fragmentation induced by chemical toxicants. [13] Therefore, SDαt is the most important variable for toxicology studies.

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]

Advantages

SCSA has numerous advantages when compared to other sperm DNA fragmentation (sDF) assays [ TUNEL assay, COMET assay, and Sperm Chromatin Dispersion (SCD)], which include:

Limitations

Despite the objective data and advantages offered, the efficacy of SCSA in fertility assessment remains doubted clinically. Suggested limitations include:

Related Research Articles

<span class="mw-page-title-main">Spermatozoon</span> Motile sperm cell

A spermatozoon is a motile sperm cell produced by male animals relying on internal fertilization. A spermatozoon is a moving form of the haploid cell that is the male gamete that joins with an ovum to form a zygote.

<span class="mw-page-title-main">Intracytoplasmic sperm injection</span> In vitro fertilization procedure

Intracytoplasmic sperm injection is an in vitro fertilization (IVF) procedure in which a single sperm cell is injected directly into the cytoplasm of an egg. This technique is used in order to prepare the gametes for the obtention of embryos that may be transferred to a maternal uterus. With this method, the acrosome reaction is skipped.

<span class="mw-page-title-main">Infertility</span> Inability to reproduce by natural means

Infertility is the inability of a couple to reproduce by natural means. It is usually not the natural state of a healthy adult. Exceptions include children who have not undergone puberty, which is the body's start of reproductive capacity. It is also a normal state in women after menopause.

<span class="mw-page-title-main">Pyknosis</span> Irreversible condensation of chromatin in the nucleus of a dying cell

Pyknosis, or karyopyknosis, is the irreversible condensation of chromatin in the nucleus of a cell undergoing necrosis or apoptosis. It is followed by karyorrhexis, or fragmentation of the nucleus.

DNA fragmentation is the separation or breaking of DNA strands into pieces. It can be done intentionally by laboratory personnel or by cells, or can occur spontaneously. Spontaneous or accidental DNA fragmentation is fragmentation that gradually accumulates in a cell. It can be measured by e.g. the Comet assay or by the TUNEL assay.

<span class="mw-page-title-main">Spermatogonium</span> Undifferentiated male germ cell

A spermatogonium is an undifferentiated male germ cell. Spermatogonia undergo spermatogenesis to form mature spermatozoa in the seminiferous tubules of the testicles.

Terms oligospermia, oligozoospermia, and low sperm count refer to semen with a low concentration of sperm and is a common finding in male infertility. Often semen with a decreased sperm concentration may also show significant abnormalities in sperm morphology and motility. There has been interest in replacing the descriptive terms used in semen analysis with more quantitative information.

<span class="mw-page-title-main">TUNEL assay</span> Molecular biology method used to detect DNA fragmentation

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the 3′- hydroxyl termini in the double-strand DNA breaks generated during apoptosis.

Male infertility refers to a sexually mature male's inability to impregnate a fertile female. In humans, it accounts for 40–50% of infertility. It affects approximately 7% of all men. Male infertility is commonly due to deficiencies in the semen, and semen quality is used as a surrogate measure of male fecundity. More recently, advance sperm analyses that examine intracellular sperm components are being developed.

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<span class="mw-page-title-main">Acridine orange</span> Organic dye used in biochemistry

Acridine orange is an organic compound that serves as a nucleic acid-selective fluorescent dye with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows the dye to interact with DNA by intercalation, or RNA via electrostatic attractions. When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein. Acridine orange and fluorescein have a maximum excitation at 502nm and 525 nm (green). When acridine orange associates with RNA, the fluorescent dye experiences a maximum excitation shift from 525 nm (green) to 460 nm (blue). The shift in maximum excitation also produces a maximum emission of 650 nm (red). Acridine orange is able to withstand low pH environments, allowing the fluorescent dye to penetrate acidic organelles such as lysosomes and phagolysosomes that are membrane-bound organelles essential for acid hydrolysis or for producing products of phagocytosis of apoptotic cells. Acridine orange is used in epifluorescence microscopy and flow cytometry. The ability to penetrate the cell membranes of acidic organelles and cationic properties of acridine orange allows the dye to differentiate between various types of cells. The shift in maximum excitation and emission wavelengths provides a foundation to predict the wavelength at which the cells will stain.

<span class="mw-page-title-main">Comet assay</span> Test for damage to DNA

The single cell gel electrophoresis assay is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It was first developed by Östling & Johansson in 1984 and later modified by Singh et al. in 1988. It has since increased in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. The term "comet" refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet.

<span class="mw-page-title-main">Semen analysis</span> Scientific analysis of semen

A semen analysis, also called seminogram or spermiogram, evaluates certain characteristics of a male's semen and the sperm contained therein. It is done to help evaluate male fertility, whether for those seeking pregnancy or verifying the success of vasectomy. Depending on the measurement method, just a few characteristics may be evaluated or many characteristics may be evaluated. Collection techniques and precise measurement method may influence results. The assay is also referred to as ejaculate analysis, human sperm assay (HSA), sperm function test, and sperm assay.

<span class="mw-page-title-main">Semen quality</span> Measure of male fertility

Semen quality is a measure of male fertility, a measure of the ability of sperm in semen to accomplish fertilization. Semen quality involves both sperm quantity and quality. Semen quality is a major factor for fertility.

<span class="mw-page-title-main">Ashok Agarwal</span> Medical Scientist

Ashok Agarwal is the Director of the Andrology Center, and also the Director of Research at the American Center for Reproductive Medicine at Cleveland Clinic, Cleveland, USA. He is Professor at the Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, USA. Ashok is a Senior Staff in the Cleveland Clinic's Glickman Urological and Kidney Institute. He has published extensive translational research in human infertility and assisted reproduction.

Marc Goldstein is an American urologist who is the Matthew P. Hardy Distinguished Professor of Reproductive Medicine, and Urology at Weill Cornell Medical College; Surgeon-in-Chief, Male Reproductive Medicine and Surgery; and Director of the Center of Male Reproductive Medicine and Microsurgery at New York Presbyterian Hospital. He is Adjunct Senior Scientist with the Population Council's Center for Biomedical Research, located on the campus of Rockefeller University.

Antisperm antibodies (ASA) are antibodies produced against sperm antigens.

Robert John Aitken is a British reproductive biologist, widely known for identifying oxidative stress as a significant contribution to infertility and its actions on human sperm function. He also made substantial contributions to clinical practice translation in male reproductive health, notably the development of new contraceptive vaccine.

The male infertility crisis is an increase in male infertility since the mid-1970s. The issue attracted media attention after a 2017 meta-analysis found that sperm counts in Western countries had declined by 52.4 percent between 1973 and 2011. The decline is particularly prevalent in Western countries such as New Zealand, Australia, Europe, and North America. A 2022 meta-analysis reported that this decline extends to non-Western countries, namely those in Asia, Africa, Central America, and South America. This meta-analysis also suggests that the decline in sperm counts may be accelerating.

<span class="mw-page-title-main">PRM2</span> Protein-coding gene in the species Homo sapiens

Protamine 2 is a protein that in humans is encoded by the PRM2 gene.

References

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  3. ↑ "Aktuelles". Praxis. 92 (20): 978. 2003. doi:10.1024/0369-8394.92.20.978 (inactive 14 December 2024). ISSN   0369-8394.{{cite journal}}: CS1 maint: DOI inactive as of December 2024 (link)
  4. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 EVENSON, DONALD P.; LARSON, KJERSTEN L.; JOST, LORNA K. (2002-01-02). "Sperm Chromatin Structure Assay: Its Clinical Use for Detecting Sperm DNA Fragmentation in Male Infertility and Comparisons With Other Techniques". Journal of Andrology. 23 (1): 25–43. doi: 10.1002/j.1939-4640.2002.tb02599.x . ISSN   0196-3635. PMID   11780920.
  5. 1 2 3 4 5 6 7 8 9 Bungum, Mona; Bungum, Leif; Giwercman, Aleksander (2011). "Sperm chromatin structure assay (SCSA): a tool in diagnosis and treatment of infertility". Asian Journal of Andrology. 13 (1): 69–75. doi:10.1038/aja.2010.73. ISSN   1008-682X. PMC   3739398 . PMID   21057512.
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  29. ↑ Castilla, J.A.; Zamora, S.; Gonzalvo, M.C.; Luna del Castillo, J.D.; Roldan-Nofuentes, J.A.; Clavero, A.; Björndahl, L.; Martínez, L. (2010). "Sperm chromatin structure assay and classical semen parameters: systematic review". Reproductive BioMedicine Online. 20 (1): 114–124. doi: 10.1016/j.rbmo.2009.10.024 . ISSN   1472-6483. PMID   20158996.
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  33. ↑ Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert (2016-11-10). Drevet, Joël R (ed.). "Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis". PLOS ONE. 11 (11): e0165125. Bibcode:2016PLoSO..1165125C. doi: 10.1371/journal.pone.0165125 . ISSN   1932-6203. PMC   5104467 . PMID   27832085.
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