fertility test (male)

1. SPERM NUCLEAR INTEGRITY
ASSESSMENT

2. OBJECTIVE OF STUDY
SPERM NUCLEAR INTEGIITY ASSESSMENT
• IMPORTANCE OF NUCLEAR DNA IN SPERM
• CAUSE OF ABNORMALITY IN SPERM NUCLAR DNA
• ASSESSMENT METHOD TO DETACT THE ABNORMALITY

3. IMPORTANCE OF NUCLEAR DNA IN SPERM
• IN MOST OF THE WESTERN COUNTRIES AND INDIA
ONE FOURTH COUPLES IN REPRODUCTIVE AGE ARE
SEEKING MEDICAL HELP FOR INVOLUNTARY
CHILDLESSNESS.
• BOTH MALE GAMET AND FEAMALE GAMET HAD
EQUAL WATAGE FOR A PERFECT DEVELOPMENT OF
EMBRYO AND ITS FATE.
• WHEN WE CAME TO THE PERSPECTIVE MALE, IN
1994 WHO DEVELOP STANDARD SEMEN PARAMETER
ASSAY WHEATHER A MALE CONSIDER TO BE
FERTILE OR INFERTILE.
• BESIDE THIS PARAMETER SPERM DNA QUALITY IS
IMPORTANT IN MAINTAINING THE REPROTIVE
POTENTIAL OF MEN.

4. • THE FERTILITY POTENTIAL OF SPERM
DEPENDS NOT ONLY UPON THE FUNCTIONAL
COMPENTANCE OF SPERMATOZOA BUT ALSO
ON SPERM DNA INTIGRITY.
• MEN WITH NORMAL SPERMATOZOA MAY STILL
INFERTILE THE CAUSE COULD BE RELATED TO
ABNORMAL SPERM DNA.
• IF SOME HOW THE FERTILIZATION OCCURE
THE ZYGOTE WILL NOT DELVELOP NORMALLY.
• THUS THE NUCLEAR SPERM INTIGRITY
ASSESSEMNT METHODS IS DONE TO DETACT
THE ABNORMALITY OF SPERM AT NUCLEAR
AND GENETIC LEVEL.

5. CAUSE OF ABNORMALITY IN SPERM NUCLAR
DNA

6. ASSESSMENT METHOD TO DETACT THE
ABNORMALITY
• MANY NEW TECHNIQUES HAVE BEEN DEVELOPED DURING THE PAST TWO DECADES TO
ASSESS THE SPERM NUCLEAR INTEGRITY.
• SUCH TECHNIQUES, USING BOTH MICROSCOPY- AND FLOW CYTOMETRY-BASED
ANALYSES, EVALUATE SPERM DNA INTEGRITY AND SPERM CHROMATIN QUALITY.
• CURRENTLY, FOUR MAJOR TESTS OF SPERM DNA FRAGMENTATION ARE MOST
FREQUENTLY USED, INCLUDING THE COMET ASSAY (SINGLE CELL GEL
ELECTROPHORESIS) , THE TUNEL (TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE-
MEDIATED DUDP NICK END LABELLING) ASSAY , THE SPERM CHROMATIN DISPERSION
(SCD) TEST AND THE SPERM CHROMATIN STRUCTURE ASSAY (SCSA).
• THEY ALL LABEL SINGLE- OR DOUBLE-STRANDED DNA BREAKS; HOWEVER
UNFORTUNATELY, MOST OF THE AVAILABLE TECHNIQUES FOR DETECTION OF SPERM
DNA DAMAGE PROVIDE LIMITED INFORMATION ON THE NATURE OF THE DNA LESIONS
DETECTED AND NONE OF THEM ENABLES US TO DEPICT THE EXACT AETIOLOGY AND

7. COMET ASSAY
• THE COMET ASSAY IS A SIMPLE METHOD
FOR MEASURING DNA STRAND BREAKS.
• IN COMET ASSAY SPERM CELLS ARE MIXED
WITH MELTED AGAROSE AND THEN PLACED
ON A GLASS SLIDE. THE CELLS ARE LYSED
AND THEN SUBJECTED TO HORIZONTAL
ELECTROPHORESIS.
• DNA IS VISUALIZED WITH THE HELP OF A
DNA SPECIFIC FLUORESCENT DYE AND DNA
DAMAGE IS QUANTIFIED BY MEASURING
THE DISPLACEMENT BETWEEN THE
GENETIC MATERIAL OF THE NUCLEUS
COMET HEAD AND THE RESULTING TAIL.

8. TUNEL ASSAY
• TUNEL ASSAY IS A METHOD FOR
DETECTING DNA FRAGMENTATION BY
LABELING THE 3’-HYDROXYL TERMINAL
IN THE DOUBLE STRAND DNA BREAKS
GENERATE DURING APOPTOSIS.
• THE FLUORESCENCE INTENSITY OF
EACH ANALYZED SPERM IS DETERMINED
AS A “POSITIVE” OR “NEGATIVE” FOR
SPERM ON A MICROSCOPE SLIDE.

9. SCSA TEST
• SCSA IS A FLOW CYTOMETRY TEST WHERE
SPERM DNA BREAKS ARE EVALUATED
INDIRECTLY THROUGH THE DNA
DENATURABILITY. THE ASSAY MEASURES THE
SUSCEPTIBILITY OF SPERM DNA TO ACID
INDUCED DNA DENATURATION IN SITU,
FOLLOWED BY STAINING WITH THE
FLUORESCENCE DYE ACRIDINE ORANGE.
• BY USING A FLOW CYTOMETER 5.000–10.000
SPERM CAN BE ANALYZED WITHIN FEW
SECONDS AND THUS PROVIDE A LESS
SUBJECTIVE RESULT COMPARED TO THE WHO
ANALYSIS WHERE ONLY 1–300 CELLS ARE
ANALYZED.
• THROUGH A SPECIFIC SCSA-SOFTWARE (SCSA-
SOFT) A SCATTER PLOT IS CREATED, SHOWING
THE RATIO OF GREEN AND RED SPERM. THE
PERCENTAGE OF RED SPERM IS CALLED DNA

10. SCD TEST

11. THANK YOU