INFERTILITY AN OVERVIEW n Infertility affects 15 of

INFERTILITY – AN OVERVIEW n Infertility affects 15% of all couples trying to conceive (1 in every 6) n 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 § Most significant - free radicals § Species with unpaired electrons, highly reactive

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

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

ASSESSMENT OF DNA INTEGRITY ü High levels of DNA fragmentation, decline in spermoocyte fusion rates and motility - exposure to H 2 O 2 (Aitken et al. , 1998) ü Level of DSB’s high in infertile patients with abnormal semen parameters (Agarwal et al. , 2004) ü High proportion of sperm with DNA damage - may be a cause of infertility (Singh et al. , 2003) ü 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)

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

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

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

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

DIFFERENCE IN THE METHODOLOGIES ADOPTED FOR COMET ASSAY Protocol Lysis Electrophoresis Neutralisation 1 Lysis with proteinase K solution at 37 °C for 2 hours 12 V at 250 m. A for 20 minutes at room temperature 30 minutes at 4 °C 2 Lysis for 1 hour at 4 °C 25 V at 300 m. A for 5 followed by overnight minutes at room incubation with temperature Proteinase K solution at 37 °C 3 4 Lysis at 4 °C for 1 hour followed by incubation with dithiothreitol solution for 30 minutes at 4 °C Lysis for 1 hour at 4 °C 30 minutes at 4 °C 25 V at 300 m. A for 10 minutes at room temperature 30 minutes at 4 °C 20 V at 300 m. A for 20 minutes at room temperature 30 minutes at 4 °C

DISCUSSION n n 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

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

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

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

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

CONCLUSION…. n ? of the different protocols would be the most useful for description of clinically relevant sperm DNA damage is yet to be determined n 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