SPERM DNA DAMAGE/SPERM DNA FRAGMENTATION.pptx

SPERM DFI, SPERM DNA DAMAGE,
TEST FOR DFI, CLINICAL IMPLICATIONS OF DFI

1. Sperm DNA Damage Dr. DEEPTHI REPALLE LAB DIRECTOR MOHAK IVF

2. Sperm Structure

3. Sperm DNA

4. DNA Damage

5. Definition Break in one or two strands of the DNA in sperm

6. Mechanisms Apoptosis Chromatin remodeling-Spermiogenesis ROS- migration from seminiferous tubules to epididymis Caspases and endonucleases Chemotherapy and radiotherapy Environmental toxins

7. Mechanisms

8. Methods Different methods for detection of sperm DNA damage Acridine orange Aniline blue Toulidine blue Tunnel assay SCSA SCD COMET

9. Types of methods

10. Acridine orange Microscopy Flow cytometry

11. SCSA

12. Aniline blue During spermatogenesis, sperm chromatin undergoes structural changes and results in a high condensation. Smears were fixed with a solution of 3% buffered glutaraldehyde in 0.2 M phosphate buffer for 30 minutes. Slides were then stained with 5% aqueous aniline blue solution mixed with 4% acetic acid for 5 minutes.

13. Toulidine blue Toluidine blue (TB) staining to evaluate sperm chromatin decondensation status and the exposure of phosphate groups. Pale blue spermatozoa are considered to be normal (TB−), and dark blue or violet/purple spermatozoa are categorized as abnormal cells (TB+) (fluorescent microscopy, ×100 eyepiece magnification).

14. CHROMAMYCIN A3 Differentiates Protamine deficiency and normal Protamine content

15. TUNEL The percentage of apoptotic spermatozoa with DNA fragmentation in each sample was determined by the TUNEL assay using an  in situ  cell death detection kit (Roche Diagnostics, Mannheim, Germany) using fluorescent microscopy, in which normal DNA was shown as light green and damaged DNA was seen as bright green.

16. TUNEL

17. SCD

18. Comet Test

19. Clinical Implications

20. Indications for DFI Unexplained or persistent infertility Failure to conceive after 5-6 IUI cycles despite good count and motility Low fertilization rates or poor embryo quality in IVF cycles Implantation failure after IVF Recurrent miscarriage Prolonged stay in an reproductive hazardous environment Advanced paternal age Abnormal semen analysis

21. Recent studies Increased miscarriage rates Spontaneous abortions Pregnancy rates ? Live birth rates reduced Female factors-oocyte quality-repairing capacity

22. Treatment options Life style modification Antioxidant treatments Sperm sorting devices MACS Zeta sperm selection Testicular Biopsy

23. Zeta sperm selection It refers to the electrical charge potentials between the surrounding medium and the negatively charged sperm membrane. This charge is influenced by epididymal proteins present on the sperm surface and is lower in sperm cells with damaged DNA. Mature sperm typically exhibit zeta potentials ranging from -16 to -120 mV. Moreover, the Zeta Potential method is cost-effective and simple to perform, allowing for the separation of sperm with intact DNA, superior motility, and normal morphology.

24. Zeta sperm selection

25. THANKS