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INFERTILITY – AN OVERVIEW

INFERTILITY – AN OVERVIEW. Infertility affects 15% of all couples trying to conceive (1 in every 6) Male factor held responsible in roughly half of all cases of infertility. INTRODUCTION. Spermatogenesis : Complex, diverse, 74 days Sperms prone for disruption by potential targets

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INFERTILITY – AN OVERVIEW

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  1. INFERTILITY – AN OVERVIEW • Infertility affects 15% of all couples trying to conceive (1 in every 6) • Male factor held responsible in roughly half of all cases of infertility

  2. INTRODUCTION • Spermatogenesis : Complex, diverse, 74 days • Sperms prone for disruption by potential targets • Most significant - free radicals • Species with unpaired electrons, highly reactive

  3. OXIDATIVE STRESS ? • Overproduction of free radicals (ROS) • Decreased clearance of ROS by scavenging mechanisms • Imbalance – results in oxidative stress • Oxidative stress – disruption of functional competence of human spermatozoa

  4. HYDROGEN PEROXIDE • Sperm plasma membrane • Loss of membrane fluidity and integrity • Sperm DNA • Strand breaks & oxidative base damage • Loss of competence to participate in membrane fusion events - fertilization

  5. ASSESSMENT OF DNA INTEGRITY • High levels of DNA fragmentation, decline in sperm-oocyte fusion rates and motility - exposure to H2 O 2 (Aitken et al., 1998) • Level of DSB’s high in infertile patients with abnormal semen parameters (Agarwal et al., 2004) • High proportion of sperm with DNA damage - may be a cause of infertility (Singh et al., 2003) • Studies - DNA fragmentation - sperms used for ART. Evidence is accumulating on the importance of sperm DNA integrity during both fertilization and embryogenesis(Miller et al., 2002)

  6. COMET ASSAY • Routine examination of sperm & need for novel techniques • Advantages • Simple, non invasive • Fast, relatively inexpensive, highly sensitive • Applied to any eukaryotic cell • Less amount of sample required • Software facilitated analysis

  7. OBJECTIVE • To standardize a protocol for the evaluation of genomic integrity of the human spermatozoa exposed to hydrogen peroxide treatment using comet assay

  8. TECHNIQUE • MICROGEL PREPARATION • SAMPLE PREPARATION - SWIM UP METHOD • EMBEDDING OF CELLS IN MICROGELS • LYSIS - USING HIGH SALT & DETERGENTS • EXPOSURE TO HYDROGEN PEROXIDE • ALKALINE ELECTROPHORESIS • NEUTRALISATION AND STAINING • IMAGE ANALYSIS & AND INTERPRETATION

  9. RESULTS • 4 different protocols followed which differed in • Composition of buffers • Duration and temperature of lysis • Level of genotoxic insult • Electrophoretic conditions • Each of the experiment done for at least 3 times and Protocol 2 – best results • ? Due to longer duration of lysis and high levels of ROS induction

  10. DIFFERENCE IN THE METHODOLOGIES ADOPTED FOR COMET ASSAY

  11. DISCUSSION • Reproducible and Reliable results • Strict quality control • All steps equally important • No single correct method -critical steps • Slide preparation Goal - uniform gels with stability & easy visualization • Important parameters Concentration of cells in agarose & agarose concentration itself

  12. DISCUSSION… • 5ml of 1% agarose at 70-80°C for 2 hrs –best results • Single layer procedure suited the study • Cell density • Optimal number of cells • Not > few per visual field • Higher cell densities • Overlapping comets at higher levels of DNA migration

  13. DISCUSSION… • Lysis - parameters - highly variable • Detergent & Reagent requirements • Duration and temperature of lysis • Minimal time required • Incubation of slides in a solution of Proteinase K at 37°C for 8 hours • Genotoxic agent • Nature, concentration, sequence of steps in the assay

  14. DISCUSSION… • Electrophoresis • Length of time for unwinding and expression • Electrophoretic conditions • Ideal - 25 V, 300mA for 5 minutes at room temperature • Neutralization • Optimal time - 30 minutes at 4°C • Use of chemical Spermine - enhanced the clarity

  15. CONCLUSION • An in vitro assay with human spermatozoa - valuable - sensitive system - assess potential genetic effects - various factors in human reproduction • Need for assessment further intensified - genetic disorders - transmitted through ART’s • The comet assay - promising tool - evaluation - genetic aspects of male infertility

  16. CONCLUSION…. • ? of the different protocols would be the most useful for description of clinically relevant sperm DNA damage is yet to be determined • The comet assay - yet to undergo - appropriate multilaboratory, international validation studies - demonstrate - interlab and intralab reproducibility, reliability and adequacy of it’s performance against the currently adopted methods

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