The document discusses the Sperm Chromatin Structure Assay (SCSA), which detects damaged sperm DNA and altered nuclear proteins. The SCSA relies on differences in DNA susceptibility to acid or heat-induced denaturation between normal and abnormal sperm. Samples are stained with acridine orange and analyzed by flow cytometry to measure denatured DNA levels. A high DNA Fragmentation Index (DFI) indicates a greater proportion of sperm with fragmented DNA. The SCSA provides an objective measure of sperm DNA integrity and is useful for evaluating male fertility potential and treatment outcomes.

1. SPERM CHROMATIN STRUCTURE ASSAY
SPERM CHROMATIN DISPERSION TEST

2. SPERM DNA
• Sperm DNA differs from the somatic DNA
• DNA completely occupy nucleus
• 9.9µm^3 -> twice
• Protamine's
• DNA domains loop – Donut shape toroid
• Protamine's and MAR fixed below
• Toroid -> Disulfide bond
• Histones

6. SPERM CHROMATIN DISPERSION
• Fenandez 2003
• When sperm is treated with an acid solution
prior to the lysis buffer, absence or minimal
halo is produced -> fragmented DNA
• Sperm DNA acid solution depolarized nuclei
lysis solution
Forms halo indicates Absent halo
DNA intact DNA damage

7. Principle
• When sperms are treated with an acid
solution prior to lysis buffer, the DNA
dispersion halos that are observed in sperm
nuclei with non-fragmented DNA due to
altered nuclear protein
• Which is minimal present or not present at
all in sperm nuclei with fragmented DNA
• Observed in fluorescent microscopy

9. PREPARATION
• Sperm conc with 5-10 million / ml prepared with PBS
• Mix with 1ml low M.P agarose at 37˚ C
• 50µl -> glass slide coated with 0.65% standard agarose dried at
80˚C put coverslip
• Solidify ( agarose) for 4 min at 4˚C in fridge
• Coverslip is removed
• Horizontally immersed in the tray of freshly prepared acid
denaturation solution (0.08N HCL) for 7 min in dark
• Transfer the slide to tray with neutralizing and lysing solution 1
for 10 min
• Neutralizing and lysing solution 2 incubate for 5 min
• Wash -> Tris – borate EDTA buffer for 2 min
• Dehydrated with 70%, 50%, 100% ethanol baths 2 min each
• Air-dried
• Stained with DAPI for florescence microscopy

11. DFI
• Sperm nucleoid has *central core * outer
halo
• SDFI = 100 ˣ Fragmented + degraded
Total cells

12. ADVANTAGE
• Minimal requirement
• Simple
• Cost effective
• Oxidative DNA damage can be determined
by adding 8- oxoguanine DNA probe

13. The Sperm Chromatin Structure Assay
• 1980s -> DNA fragmentation
• First test
• Evenson
• Detect the damaged sperm DNA and altered protein
in sperm nuclei, view flow cytometry, acridine
orange (AO) stained sperm
• 2 types : SCSA heat and SCSA acid
• SCSA acid is frequently used
• Abnormal protein / DNA complex/ immature
spermatozoa -> AO dye -> high HDS ( high level DNA
stainability)

14. Principle
• SCSA relies on the fact that abnormally
sperm chromatin has a greater susceptibility
to the physical induction of partial DNA
denaturation in situ
• Extent of DNA denaturation following heat
or acid treatment is determined by
measuring the metachromatic shift from
green fluorescents to red fluorescents
• By flow cytometry

15. • AO binds to DNA helix as an intercalate and
emits green fluorescence when bounded to
intact ds DNA
• Red fluorescence when bound to fragmented
DNA

17. Preparation

19. • Stained sample -> flow cytometry -> fluidics system
transport spermatozoa in a stream to the laser beam
of interrogation
• Optics system illuminates the particle and directs the
resulting light signals to the appropriate detector
• Equipment convers light signals to electronic system
-> processes by dedicated software

23. • % DFI = DNA stainability with red fluorescence
intensity / total red + green florescence
• Left panel -> Total DNA stainability v/s DFI
This reorients data into vertical / horizontal
pattern of dots
• Right panel -> frequency histogram of DFI
 Non – detectable DNA fragmentation
 Moderate level DNA fragmentation
 High level DNA fragmentation
• Total 5000 sperms are recorded on flow
cytometer

24. Advantages
• Abnormal sperm -> lack of protamine's
• Most precise
• Non-biased flow cytometer machine
• Repeatability
• cut-off point (25% DFI) to differentiate
between fertile and infertile samples
• successful assay ->ART -> fertilization and
implantation

25. Reference
• Assessment of sperm deoxyribose nucleic acid fragmentation
using sperm chromatin dispersion assay
• Textbook of assisted reproductive techniques vol1
• Clinical application of Sperm Chromatin Structure Assay in
diagnosis and treatment of infertility
• The Sperm Chromatin Structure Assay (SCSA®) and other sperm
DNA fragmentation tests for evaluation of sperm nuclear DNA
integrity as related to fertility
• Sperm chromatin structure assay (SCSA ® ) Article in Methods in
Molecular Biology
• Sperm chromatin assessment Ashok Agarwal, Tamer M Said
• The Sperm Chromatin Dispersion Test: A Simple Method for the
Determination of Sperm DNA Fragmentation
• Sperm chromatin dispersion test in the assessment of DNA
fragmentation and aneuploidy in human spermatozoa