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 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 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)

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 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 mA for 20 minutes at room temperature 30 minutes at 4 °C 2 Lysis for 1 hour at 4 °C followed by overnight incubation with Proteinase K solution at 37 °C 25 V at 300mA for 5 minutes at room temperature 3 Lysis at 4 °C for 1 hour followed by incubation with dithiothreitol solution for 30 minutes at 4 °C 25 V at 300mA for 10 minutes at room temperature 4 Lysis for 1 hour at 4 °C 20 V at 300mA for 20 minutes at room temperature

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

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

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

DISCUSSION… Electrophoresis Neutralization 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

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

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