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
30 minutes at 4 °C
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
30 minutes at 4 °C
4 Lysis for 1 hour at 4 °C 20 V at 300mA for 20 minutes at room temperature
30 minutes at 4 °C
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 parametersConcentration 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
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
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