infertility – an overview infertility affects 15% of all couples trying to conceive (1 in every 6)...

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