Sperm Chromatin Structure Assay (SCSA) is a diagnostic approach that detects

sperm Sperm is the male reproductive cell, or gamete, in anisogamous forms of sexual reproduction (forms in which there is a larger, female reproductive cell and a smaller, male one). Animals produce motile sperm with a tail known as a flagellum, whi ...

abnormality with a large extent of

DNA fragmentation 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 a ...

. 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. SCSA measures sperm

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 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 subfertility, toxicology studies and evaluation of quality of laboratory semen samples. Notably, SCSA outcompetes other convention sperm DNA fragmentation (sDF) assays such as

TUNEL Tunel or Tünel may refer to: * TUNEL assay (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling assay), in genetics, a method for detecting DNA fragmentation * Tunel (band), Yugoslav rock band * Tunel (railroad station), railroa ...

and

COMET A comet is an icy, small Solar System body that, when passing close to the Sun, warms and begins to release gases, a process that is called outgassing. This produces a visible atmosphere or coma, and sometimes also a tail. These phenomena ar ...

in terms of efficiency, objectivity, and repeatability. Schematic representation of the SCSA

History

Before the development of SCSA,

diagnosis Diagnosis is the identification of the nature and cause of a certain phenomenon. Diagnosis is used in many different disciplines, with variations in the use of logic, analytics, and experience, to determine " cause and effect". In systems engin ...

prognosis Prognosis (Greek: πρόγνωσις "fore-knowing, foreseeing") is a medical term for predicting the likely or expected development of a disease, including whether the signs and symptoms will improve or worsen (and how quickly) or remain stabl ...

/ subfertility was principally referenced the World Health Organisation (WHO) manual-based

semen Semen, also known as seminal fluid, is an organic bodily fluid created to contain spermatozoa. It is secreted by the gonads (sexual glands) and other sexual organs of male or hermaphroditic animals and can fertilize the female ovum. Semen i ...

parameters, including semen

concentration In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished: '' mass concentration'', ''molar concentration'', ''number concentration'', an ...

motility Motility is the ability of an organism to move independently, using metabolic energy. Definitions Motility, the ability of an organism to move independently, using metabolic energy, can be contrasted with sessility, the state of organisms th ...

, and

morphology Morphology, from the Greek and meaning "study of shape", may refer to: Disciplines *Morphology (archaeology), study of the shapes or forms of artifacts *Morphology (astronomy), study of the shape of astronomical objects such as nebulae, galaxies, ...

. 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. Furthermore, such parameters are often associated with high labour intensity and lack of

statistical power In statistics, the power of a binary hypothesis test is the probability that the test correctly rejects the null hypothesis (H_0) when a specific alternative hypothesis (H_1) is true. It is commonly denoted by 1-\beta, and represents the chances ...

. In the late 1970s, Donald P. Evenson at Memorial Sloan Kettering Cancer Centre in the

United States The United States of America (U.S.A. or USA), commonly known as the United States (U.S. or US) or America, is a country primarily located in North America. It consists of 50 states, a federal district, five major unincorporated territorie ...

received an NIH Research Project Grant (RO1) for

mammal Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur or ...

ian sperm chromatin structure study. Various techniques have since been adopted to gain access to sperm DNA integrity. In particular,

transmission electron microscopy Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a g ...

reflected a significant amount of sperm chromatin heterogeneity. The heterogeneity was then confirmed through

flow cytometry Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flo ...

by contrasting AO staining results between

human Humans (''Homo sapiens'') are the most abundant and widespread species of primate, characterized by bipedalism and exceptional cognitive skills due to a large and complex brain. This has enabled the development of advanced tools, culture, ...

and

mouse A mouse ( : mice) is a small rodent. Characteristically, mice are known to have a pointed snout, small rounded ears, a body-length scaly tail, and a high breeding rate. The best known mouse species is the common house mouse (''Mus musculus' ...

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

.” 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. 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.

Principle

SCSA is a widespread diagnostic tool in detection of

samples with a high degree of

and absence of

histone In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn are wr ...

-to- protamine

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

s exchange in sperm nuclei. SCSA defines sperm abnormality as an increased vulnerability of sperm DNA to in-situ heat/acid-induced denaturation. Theoretically, a completely mature and healthy sperm nuclei, which is rich in disulfide bond (S-S), shall have its DNA preserved in

double-stranded A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DN ...

form. 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. Undergoing

(blue light), green (native DNA) and red (damaged DNA)

fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...

will be emitted from intact and defective sperms respectively. Signals will be analysed with

software programming Computer programming is the process of performing a particular computation (or more generally, accomplishing a specific computing result), usually by designing and building an executable computer program. Programming involves tasks such as anal ...

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 An oocyte (, ), oöcyte, or ovocyte is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in a female fetus in the ovary during female gametogenesis. The female ...

during

fertilisation Fertilisation or fertilization (see spelling differences), also known as generative fertilisation, syngamy and impregnation, is the fusion of gametes to give rise to a new individual organism or offspring and initiate its development. Proce ...

. The etiology of sperm DNA damage can be subdivided into intrinsic and extrinsic factors. The former is attributed to a series of

pathophysiological Pathophysiology ( physiopathology) – a convergence of pathology with physiology – is the study of the disordered physiological processes that cause, result from, or are otherwise associated with a disease or injury. Pathology is th ...

phenomena during

spermatogenesis Spermatogenesis is the process by which haploid spermatozoa develop from germ cells in the seminiferous tubules of the testis. This process starts with the mitotic division of the stem cells located close to the basement membrane of the tubule ...

; the latter is caused by postnatal exposure to endogenous sources of DNA breaks.

Intrinsic factors

* ''Abnormality in recombination and

chromatin Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...

restructuring'': During

, crossing-over of

chromatid A chromatid (Greek ''khrōmat-'' 'color' + ''-id'') is one half of a duplicated chromosome. Before replication, one chromosome is composed of one DNA molecule. In replication, the DNA molecule is copied, and the two molecules are known as chro ...

segments between homologous chromosomes may have occurred abnormally. Specific

nuclease A nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their ta ...

s 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 Meiosis (; , since it is a reductional division) is a special type of cell division of germ cells in sexually-reproducing organisms that produces the gametes, such as sperm or egg cells. It involves two rounds of division that ultimately resu ...

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 Ejaculation is the discharge of semen (the ''ejaculate''; normally containing sperm) from the male reproductory tract as a result of an orgasm. It is the final stage and natural objective of male sexual stimulation, and an essential componen ...

spermatozoa A spermatozoon (; also spelled spermatozoön; ; ) is a motile sperm cell, or moving form of the haploid cell that is the male gamete. A spermatozoon joins an ovum to form a zygote. (A zygote is a single cell, with a complete set of chromosomes, ...

. However, such a conclusion is theoretically speculative and currently there is no direct confirmation of this

hypothesis A hypothesis (plural hypotheses) is a proposed explanation for a phenomenon. For a hypothesis to be a scientific hypothesis, the scientific method requires that one can test it. Scientists generally base scientific hypotheses on previous obse ...

in humans. During

spermiogenesis Spermiogenesis is the final stage of spermatogenesis, during which the spermatids develop into mature spermatozoa. At the beginning of the stage, the spermatid is a more or less circular cell containing a nucleus, Golgi apparatus, centriole and ...

, DNA double-strand breaks are introduced to relieve torsional stress and to enable the substitution of

nucleosome A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone proteins and resembles thread wrapped around a spool. The nucleosome is the fundamen ...

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 cell Germ or germs may refer to: Science * Germ (microorganism), an informal word for a pathogen * Germ cell, cell that gives rise to the gametes of an organism that reproduces sexually * Germ layer, a primary layer of cells that forms during embry ...

s or escape of defective germ cells will lead to sperm DNA damage. * '' Oxidative stress:'' Oxidative stress denotes the imbalance of activity between

reactive oxygen species (ROS) In chemistry, reactive oxygen species (ROS) are highly reactive chemicals formed from diatomic oxygen (). Examples of ROS include peroxides, superoxide, hydroxyl radical, singlet oxygen, and alpha-oxygen. The reduction of molecular oxygen () pr ...

and endogenous antioxidant agents. High levels of ROS cause DNA damage in terms of single-stranded breaks and double-stranded breaks frequently recorded in

infertile Infertility is the inability of a person, animal or plant to reproduce by natural means. It is usually not the natural state of a healthy adult, except notably among certain eusocial species (mostly haplodiploid insects). It is the normal state ...

men's sperms.

Extrinsic factors

* ''Age:'' Although males produce sperm throughout their

adult An adult is a human or other animal that has reached full growth. In human context, the term ''adult'' has meanings associated with social and legal concepts. In contrast to a " minor", a legal adult is a person who has attained the age of major ...

hood, older age is associated with increased number of DNA double-stranded breaks and decreased frequency of sperm

apoptosis Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...

. Such observation is implicative of deterioration of sperm selection, quality, and integrity. * '' Heat stress:'' High temperatures cause adverse effects to sperm DNA and male fertility. Excessive heat is related to impaired sperm

integrity, and testis overheating is associated with reduced fertility potential. * ''

Smoking Smoking is a practice in which a substance is burned and the resulting smoke is typically breathed in to be tasted and absorbed into the bloodstream. Most commonly, the substance used is the dried leaves of the tobacco plant, which have bee ...

:'' 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.

Procedure

Currently, only the SCSA protocol developed by Evenson ''et al.'' has received

trademark A trademark (also written trade mark or trade-mark) is a type of intellectual property consisting of a recognizable sign, design, or expression that identifies products or services from a particular source and distinguishes them from others ...

protection in achievement of clinical relevance between different laboratories. The individual steps of SCSA are as follows: # ''

Freezing Freezing is a phase transition where a liquid turns into a solid when its temperature is lowered below its freezing point. In accordance with the internationally established definition, freezing means the solidification phase change of a liquid o ...

/ Thawing:'' After

ejaculation Ejaculation is the discharge of semen (the ''ejaculate''; normally containing sperm) from the male reproductory tract as a result of an orgasm. It is the final stage and natural objective of male sexual stimulation, and an essential componen ...

, human sperm samples are subjected to a 30-minute semen

liquefaction In materials science, liquefaction is a process that generates a liquid from a solid or a gas or that generates a non-liquid phase which behaves in accordance with fluid dynamics. It occurs both naturally and artificially. As an example of the ...

at 37 °C, followed by cryopreservation in an ultra-low temperature freezer (–70 to –110 °C) or placed directly into liquefied nitrogen cryovial. Frozen or fresh sperm samples are thawed in a 37 °C water bath and diluted with a TNE buffer to obtain 200

μl The litre (international spelling) or liter (American English spelling) (SI symbols L and l, other symbol used: ℓ) is a metric unit of volume. It is equal to 1 cubic decimetre (dm3), 1000 cubic centimetres (cm3) or 0.001 cubic metre (m3). ...

suspension (sperm concentration: 1-2 x 10^6 mL). # ''Acid-induced denaturation:'' 400

acidic solution ( pH 1.2) containing 0.15M

sodium chloride Sodium chloride , commonly known as salt (although sea salt also contains other chemical salts), is an ionic compound with the chemical formula NaCl, representing a 1:1 ratio of sodium and chloride ions. With molar masses of 22.99 and 35.45 g ...

solution, 0.08M Tris

hydrochloric acid Hydrochloric acid, also known as muriatic acid, is an aqueous solution of hydrogen chloride. It is a colorless solution with a distinctive pungent smell. It is classified as a strong acid Acid strength is the tendency of an acid, symbol ...

, 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 DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA da ...

. # '' 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 Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of Cell (biology), cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and inject ...

, 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

wavelength In physics, the wavelength is the spatial period of a periodic wave—the distance over which the wave's shape repeats. It is the distance between consecutive corresponding points of the same phase on the wave, such as two adjacent crests, tro ...

s of 450-490 nm, double-stranded DNA from intact sperms emit green

(488 nm) while aggregation of AO molecules single-stranded DNA from defective sperms leads to metachromatic shift to red

(>630 nm). # ''

Data analysis Data analysis is a process of inspecting, cleansing, transforming, and modeling data with the goal of discovering useful information, informing conclusions, and supporting decision-making. Data analysis has multiple facets and approaches, enco ...

:'' A

scattergram A scatter plot (also called a scatterplot, scatter graph, scatter chart, scattergram, or scatter diagram) is a type of plot or mathematical diagram using Cartesian coordinates to display values for typically two variables for a set of data ...

(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

SCSA consists of a fixed

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

without full protamination respectively.

DNA fragmentation index (DFI)

Also known as Cells Outside the Main Peak of αt (COMPαt), DFI can be further sub-classified into

mean There are several kinds of mean in mathematics, especially in statistics. Each mean serves to summarize a given group of data, often to better understand the overall value (magnitude and sign) of a given data set. For a data set, the ''arithme ...

DFI (X DFI) and

standard deviation In statistics, the standard deviation is a measure of the amount of variation or dispersion of a set of values. A low standard deviation indicates that the values tend to be close to the mean (also called the expected value) of the set, while ...

DFI (SD DFI). 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

; and high DFI fractions exhibited elongated nuclei and signs of

. In general, the greater the DFI, the higher the chance of

infertility Infertility is the inability of a person, animal or plant to reproduce by natural means. It is usually not the natural state of a healthy adult, except notably among certain eusocial species (mostly haplodiploid insects). It is the normal state ...

or subfecundity. Within DFI of 0-20%, the occurrence of spontaneous pregnancy remains consistent; when DFI exceeds 20%, the rate of natural fertility gradually declines; when DFI exceeds 30%, the

odds ratio An odds ratio (OR) is a statistic that quantifies the strength of the association between two events, A and B. The odds ratio is defined as the ratio of the odds of A in the presence of B and the odds of A in the absence of B, or equivalently (due ...

for natural or Intrauterine insemination (IUI) fertility is greatly reduced by 8-10 folds, suggesting a close-to-zero chance of

pregnancy Pregnancy is the time during which one or more offspring develops ( gestates) inside a woman's uterus (womb). A multiple pregnancy involves more than one offspring, such as with twins. Pregnancy usually occurs by sexual intercourse, but ca ...

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. Determination of HDS value reflects structural chromatin abnormalities. A high HDS value is indicative of immature sperm morphology and hence pregnancy failure.

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

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. Such finding asserts the DFI determined by SCSA to be a strong independent predictor of

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...

and a clinically useful technique. Currently, 25% DFI is the established clinical threshold in classifying males into statistical

probability Probability is the branch of mathematics concerning numerical descriptions of how likely an Event (probability theory), event is to occur, or how likely it is that a proposition is true. The probability of an event is a number between 0 and ...

of: 1) increased time for natural pregnancy, 2) lower chance of Intrauterine insemination (IUI) success, 3) more

miscarriage Miscarriage, also known in medical terms as a spontaneous abortion and pregnancy loss, is the death of an embryo or fetus before it is able to survive independently. Miscarriage before 6 weeks of gestation is defined by ESHRE as biochemical lo ...

, or 4)

. High HDS values are in positive

correlation In statistics, correlation or dependence is any statistical relationship, whether causal or not, between two random variables or bivariate data. Although in the broadest sense, "correlation" may indicate any type of association, in statistics ...

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%).

Toxicology studies

Sperm DNA damage can be attributed to exposure

chemotherapy Chemotherapy (often abbreviated to chemo and sometimes CTX or CTx) is a type of cancer treatment that uses one or more anti-cancer drugs (chemotherapeutic agents or alkylating agents) as part of a standardized chemotherapy regimen. Chemotherap ...

radiotherapy Radiation therapy or radiotherapy, often abbreviated RT, RTx, or XRT, is a therapy using ionizing radiation, generally provided as part of cancer treatment to control or kill malignant cells and normally delivered by a linear accelerator. Radia ...

, or other environmental toxicants. SCSA is highly dose-responsive to sperm DNA fragmentation induced by chemical toxicants. 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 Semen, also known as seminal fluid, is an organic bodily fluid created to contain spermatozoa. It is secreted by the gonads (sexual glands) and other sexual organs of male or hermaphroditic animals and can fertilize the female ovum. Semen i ...

samples that have been stored at appropriate conditions will have essentially no change, while greater change in DNA quality indicates an improper handling.

Advantages

SCSA has numerous advantages when compared to other sperm DNA fragmentation (sDF) assays nowiki/>TUNEL assay, Comet assay">COMET assay A comet is an icy, small Solar System body that, when passing close to the Sun, warms and begins to release gases, a process that is called outgassing. This produces a visible atmosphere or coma, and sometimes also a tail. These phenomena are ...