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Chapter 3

Alkaline comet assay reveals elevated sperm DNA

damage in idiopathic and oligozoospermic infertile

men

Introduction

The crucial importance of germ cell DNA integrity in the establishment and maintenance of a

viable pregnancy has been widely recognized (Sakkas and Alvarez, 2010; Barratt et al., 2010). It

is well known that sperm chromatin is structurally and functionally different from that of somatic

cells: it is not organized in nucleosomes (Ward, 2010). DNA in sperm is 6-fold more compacted

and has 40-fold less volume than somatic cell DNA (Ward and Coffey, 1991) and has lost most,

if not all, the repair capability (Aitken et al., 2004; Baumgartner et al., 2009). Despite the

compact packing and anti-oxidant defense provided by seminal fluid (Smith et al., 1996; Potts et

al., 2000), DNA damage does occur in both developing and mature sperm, high levels of which

has been reported in infertile men (Kodama et al., 1997; Sun et al., 1997; Evenson et al., 1999,

Spano et al., 2000; Zini et al., 2001; Singh et al., 2003).

The role that sperm DNA damage plays in reproductive outcome has become increasingly

debated and the subject of much research (Barratt and De Jonge, 2010; Sakkas and Alvarez,

2010; Schulte et al., 2010). Research over the last two decades has established that maintenance

of sperm genomic integrity is crucial for the health of future generations (Ahmadi and Ng,

1999a, 1999b; Fernandez-Gonzalez et al., 2008; Zini et al., 2008). The need for assessing the

integrity of the male gamete has been further intensified by growing concerns regarding

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transmission of genetic diseases through ICSI (Barroso et al., 2009). In this context, direct assays

on spermatozoa, which represent the functional end product of the whole intra-testicular

spermatogenesis process and its maturation during epididymal transit, have the main advantage

of being able to detect effects on terminally differentiated male gametes ready to undergo

fertilization (Villani et al., 2010).

The comet assay has gained wider acceptance across multiple fields as a quick and simple

method of analyzing DNA damage in single cells (McArt et al., 2009) and is emerging as a

promising tool in male reproductive toxicology and infertility evaluation (Baumgartner et al.,

2009). Although today we have a battery of tests to assess DNA damage in sperm, the alkaline

comet assay remains the most sensitive and simple method of evaluating DNA damage in

individual sperm cells (Singh et al., 2003). It is being increasingly used in the diagnosis of male

infertility, and also as a prognostic tool to determine reproductive outcomes (Lewis and Agbaje,

2008; Simon et al., 2011). The technique is rapid, non-invasive, sensitive and inexpensive

compared to other techniques and therefore been considered suitable for the assessment of DNA

damage in sperm (Collins et al., 2008).

Typically, single-cell gel electrophoresis is performed on gel-restrained, disrupted spermatozoa

whose nuclei display a comet like tail. This comet tail consists of the fragmented, fluorescently

labeled, ss and ds-DNA that migrates away from the core of the intact DNA in an electrophoretic

field. The slides are then examined microscopically and quantified as the percentage of cells with

comet tails; alternatively the length, width and area can be computed with relevant software

(Lovell and Omori, 2008).

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As a preliminary approach, to substantiate further work proposed in the thesis, it has been

planned to assess and compare sperm DNA damage in fertile controls and infertile subjects.

Owing to its distinct advantages, the alkaline comet assay has been employed in the present

study to assess genetic integrity of the sperm.

Materials and Methods

Subjects:

Patients visiting the infertility clinic of Kasturba Medical College for fertility evaluation and

treatment participated in this prospective study. Control group included fertile men, known to

have fathered a child within 12 months. Care was taken to ensure that the samples in the control

group and infertile group were age matched. Idiopathic infertility included infertile patients, with

normozoospermic semen parameters, who had a history of infertility of at least 2 years and

normal female partners i.e., normal reproductive history, normal day 2 follicular stimulating

hormone (FSH) and luteinizing hormone (LH) levels, normal ovulation (by follicular ultrasound

study), and tubal patency (by hysterosalpingogram). A total of 75 subjects participated in the

study, after provision of a written, informed consent. All subjects were asked to provide semen

samples after 3-5 days of ejaculatory abstinence. Semen specimens were produced by

masturbation directly into a sterile plastic container, in a room specially provided for this purpose

and located adjacent to the laboratory.

Semen Analysis:

After liquefaction, routine semen analysis was performed according to WHO criteria (WHO,

1999). Seminal volume was determined in a graduated tube and sperm concentration was

assessed by conventional method using Makler counting chamber (Sefi Medical Instruments,

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Israel) and expressed in millions/mL. The sperm motility was assessed in at least 100 sperm and

expressed as percent of motile sperm (sum of rapid progression plus slow progression sperm).

Sperm morphology was assessed by Shorr staining and sperm viability by Eosin-Nigrosin stain.

The reference values of semen variables and nomenclature for some semen variables used

throughout the thesis has been presented in the subsequent section.

Comet Assay:

A part of the sample was used for assessment of DNA damage by alkaline comet assay (Singh et

al., 1988). It was performed as adapted for sperm, with modifications. Normal melting agarose

(NMA) (1.0%) was prepared by heating agarose in phosphate buffered saline (PBS, 137 mM

NaCl, 12 mM phosphate, 2.7 mM KCl). Following successive cleaning of the slide with soap

water and methanol, 200 µl of 1% NMA was spread uniformly and the slide was allowed to dry

at room temperature. The slides were stored in a dry place at room temperature until further use.

The cell suspension containing sperm (approximately 5 µl) was thoroughly mixed with low

melting point agarose (LMPA) (0.5%), prepared in PBS and maintained at 37°C. 200 µl of the

cell suspension with LMPA was layered onto the slide. Care was taken to ensure that the number

of cells was appropriate to facilitate easy image acquisition and analysis. A cover slip was placed

over the cell suspension and slide was placed on ice until the agarose layer hardened.

Subsequently, the cover slip was gently slided off and a third agarose layer (150 µl LMPA)

added, to the slide. Followed by hardening of agarose on ice and removal of the cover slip, the

slides were slowly lowered into cold, freshly made lysing solution (2.5 M sodium chloride, 100

mM EDTA, 10 mM Tris, 1% Triton X-100, 10% dimethylsulphoxide, pH:10). Following

overnight incubation, 15 mM dithiothreitol was added to the lysing solution and the slides were

incubated for additional 4 hours. The slides were then carefully removed from the lysing solution

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and transferred to an electrophoretic chamber and placed side by side. The buffer reservoirs were

filled with freshly made electrophoresis buffer (1 mM EDTA and 300 mM NaOH buffer,

pH>13) and left to stand for 20 minutes to allow for alkaline unwinding of DNA. Electrophoresis

was carried out at 25V (~0.74 V/cm) and 300 mA for 30 minutes. The slides were then

neutralized with the neutralization buffer (0.4 M Tris, pH 7.5) at 4°C for 10 minutes. The slides

were then dehydrated in absolute ethanol for 2 hours and immersed for 5 minutes in 70%

ethanol. Slides were air-dried at room temperature. Immediately before scoring, slides were

rehydrated and stained with 100 µl of ethidium bromide stain (EtBr, 20 μg/ml), rinsed once in

PBS, coverslipped and analysed within 3 hours. Slides were examined at 40X under a fluorescent

microscope, equipped with a 490 nm excitation filter (Imager-A1, Zeiss, Germany). While

scoring, each slide was imaginarily divided into 8 parts and from each part, random images of 6-

7 spermatozoa were obtained. Approximately fifty cells were scored from each specimen. A

computerized image analysis system (Komet 6.0, Kinetic Imaging, Nottingham, UK) was used to

measure the different comet parameters.

Statistical Analysis

Basic descriptive statistics were calculated for standard semen parameters and different comet

assay parameters using Statistical Package for Social Sciences (SPSS 16.0). The value represents

Mean ± SEM. Statistical analysis of the means between different study groups was performed

using one way analysis of variance (ANOVA). A P value < 0.05 was considered statistically

significant.

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Reagents

Sigma Aldrich, St. Louis, MO, USA

Normal melting agarose (NMA) Cat No A9539

Low melting point agarose (LMPA) Cat No A9414

Potassium chloride Cat No P5405

Tris base Cat No T4661

Dithiothreitol Cat No D9760

Triton X-100 Cat No T8532

Dimethylsulphoxide (DMSO) Cat No D5879

Nigrosin Cat No N4754

Ethidium Bromide Cat No E8754

Himedia, India

Sodium chloride Cat No MB023

Disodium Ethylene diamine tetra acetic acid Cat No MB011

Disodium hydrogen phosphate Cat No RM1154

Potassium dihydrogen phosphate Cat No RM249

Tris Hydrochloride Cat No RM613

Sodium hydroxide Cat No RM467

Ethidium bromide Cat No E8754

Merck & Co, USA

Shorr stain Cat No UN1993

Xylene Cat No 1330-20-7

Ammonia solution (about 25% pure) Cat No 1336-21-6

Thermo Fisher Scientific, UK

Methanol Cat No 32407

Hayman, UK

Absolute Alcohol (Ethanol)

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BDH Chemicals, UK

Eosin Cat No 3419720

Sisco Research Laboratories, India

DPX Mountant for histology Cat No 42848

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Reference values of semen variables (World Health Organization, 1999)

Volume 2.0 ml or more

pH 7.2 or more

Sperm concentration 20 x 106 spermatozoa/ml or more

Total sperm number 40 x 106

spermatozoa per ejaculate or more

Motility 50% or more motile (grades a + b) or 25% or more with

progressive motility (grade a) within 60 minutes of

ejaculation

(a - rapid progressive motility; b - slow progressive motility)

Morphology *

Vitality 50% or more live

White blood cells Fewer than 1 x 106

/ ml

Immunobead test Fewer than 50% motile spermatozoa with beads bound

MAR† test Fewer than 50% motile spermatozoa with adherent particles

† Mixed antiglobulin reaction test

* The reference value for normal morphology in the present study, using the methods and

definitions described in the WHO 4th

ed. (1999) manual is 30%.

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Nomenclature for some semen variables (Based on World Health Organization, 1999)

Normozoospermia - Normal ejaculate as defined by the reference values

Oligozoospermia – Sperm concentration less than the reference value

Asthenozoospermia – Less than the reference value for motility

Teratozoospermia – Less than the reference value for morphology

Oligoasthenoteratozoospermia – Signifies disturbance of all three variables – count, motility and

normal morphology below the reference range

Asthenoteratozoospermia – Signifies disturbance of motility and morphology below the

reference range

Azoospermia – No spermatozoa in the ejaculate

Severe oligozoospermia – Although literature supports the use of 5 x 106 spermatozoa/ml to be

the most commonly used definition, in the present study, this term is used to represent a sperm

concentration of 1 x 106 spermatozoa/ml or less

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Results

Assessment of sperm DNA damage by alkaline comet assay

Semen characteristics of different categories of infertile subjects and fertile controls are

presented in Table 3.1. The values represent Mean ± SEM. Komet 6, comet analysis system by

Kinetic Imaging (Nottingham, UK), is a simple and most advanced and powerful software

solution for analysis, data management and presentation of comet assay samples. 24 parameters

are computed from the comet image based on intensity and migration patterns for comparison

between control and experimental groups. Among the various parameters available, percent of

tail DNA, olive tail moment and tail length were analysed as a measure to compare the extent of

DNA damage and is presented in Table 3.2.

The difference between the amount of total DNA and the percent DNA in the head region yields

percent tail DNA. The mean percent tail DNA in fertile controls was approximately 9.4

(Table 3.2). The percent tail DNA was significantly higher (P<0.001) in subjects with idiopathic

infertility and oligozoospermia (Table 3.2). Representative images of alkaline comet assay in

control, oligozoospermic and idiopathic infertile men is presented in Figure 3.1. The increase in

percent tail DNA was approximately 1.7, 1.4 fold higher in the idiopathic and oligozoospermic

group. However, in patient‟s asthenozoospermia and teratozoospermia, a non-significant increase

in percent tail DNA compared to the control group was observed.

The Olive tail moment (OTM), which is expressed in arbitrary units, represents the product of

amount of DNA in the tail and mean distance of migration in the tail. The mean OTM in control

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group was approximately 1.1 while in those with idiopathic infertility and oligozoospermia, it

was 2.22 and 1.78 respectively, which was significantly higher (P<0.001). Those with

asthenozoospermia and teratozoospermia also had a marginal, non-significant increase in OTM

compared to the fertile, control group (Table 3.2).

The extent of migration of DNA in the comet tail is provided by the tail length, expressed in

micrometer. The mean tail length observed in control group was 10.77 µm. The tail length

observed in asthenozoospermic and teratozoospermic groups was not significantly different from

the control group. However, in idiopathic and oligozoospermic category, the tail length was

significantly higher (P<0.001) in comparison to the control group (Table 3.2).

Therefore, from the data, in comparison to the control group, it can be observed that patients

with idiopathic infertility (infertile subjects with normozoospermic semen parameters) and

oligozoospermia had significantly higher DNA damage compared to the control group. Although

the DNA damage was high in the other groups of infertile subjects studied (asthenozoospermia

and teratozoospermia), this difference was statistically insignificant (Figure 3.2). The idiopathic

group had the highest amount of DNA damage, to be followed by oligozoospermic,

asthenozoospermic and teratozoospermic samples (Table 3.2; Figure 3.2).

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Association between different comet parameters assessed

To examine the possible association between the different comet parameters- percent tail DNA,

olive tail moment and tail length, a regression analysis was carried out. A positive association

was observed between all the measured parameters mentioned above. Percent tail DNA versus

Olive tail moment and tail length exhibited a good correlation with R=0.89 and R=0.67

respectively (Figure 3.3; Figure 3.4). Olive tail moment versus tail length also exhibited a good

positive correlation with R=0.72 (Figure 3.5).

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Table 3.1: Mean semen characteristics of fertile controls and infertile subjects who underwent Comet assay

Group N Age (Yrs) Seminal volume

(ml)

Sperm count

(Millions/ml)

Total sperm

motility (%)

Sperm with

normal

morphology

(%)

Control 6 36.17 ± 1.60 2.67 ± 0.40 64.50 ± 9.94 71.50 ± 5.74 35.5 ± 3.01

Idiopathic infertility 17 36.24 ± 0.99 2.12 ± 0.18 60.41 ± 5.37 66 ± 1.97 35.41 ± 1.77

Oligozoospermia 18 35 ± 1.26 3.97 ± 0.72 9.57 ± 1.07 40.11 ± 4.49 16.56 ± 1.91

Asthenozoospermia 17 37.53 ± 1.05 3.26 ± 0.26 48.06 ± 5.71 43 ± 3.73 20.82 ± 2.01

Teratozoospermia 17 38.94 ± 1.53 3.76 ± 0.35 57.29 ± 6.03 52.41 ± 4.74 17.35 ± 1

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Table 3.2: Comet assay parameters in different groups of infertile subjects and fertile controls

Group N Cells analysed Head DNA (%) Tail DNA (%) Tail length (µm) Olive tail moment

Control 6 343 90.51 ± 0.54 9.48 ± 0.54 10.77 ± 0.38 1.10 ± 0.08

Idiopathic infertility 17 931 83.52 ± 0.57* 16.47 ± 0.57* 11.99 ± 0.32* 2.22 ± 0.10*

Oligozoospermia 18 978 86.60 ± 0.52* 13.39 ± 0.52* 11.65 ± 0.31* 1.78 ± 0.10*

Asthenozoospermia 17 923 89.44 ± 0.38 10.55 ± 0.38 11.45 ± 0.29 1.25 ± 0.07

Teratozoospermia 17 929 89.90 ± 0.41 10.09 ± 0.41 7.83 ± 0.25 1.16 ± 0.08

* p<0.001 compared to the control group

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Discussion

The technique of micro-gel electrophoresis of immobilized cells lysed at high salt concentrations

was first introduced in 1984 (Ostling and Johanson, 1984). The use of alkaline conditions for

DNA unwinding and electrophoresis (pH≥ 13) were incorporated later (Singh et al.,1988),

allowing the detection of double, and single-strand breaks, in addition to expression of alkali-

labile sites (ALS). Widespread acceptance of this technique and extensive application of the

same in various disciplines subsequently led to the establishment of guidelines for its use (Tice et

al., 2000). Today, the comet assay is a genotoxicity testing method widely applied both in vivo

and in vitro, to different organs and tissues. This method can be applied to non-proliferating cells

and for this reason it is one of the few cytogenetic assays applicable to detect DNA damage in

vitro on mature spermatozoa (Villani et al., 2010).

The alkaline comet assay can be applied to measure DNA damage at the single cell level since it

requires only a relatively small cell population. Further, the cells require no prelabelling with

radioactivity and can be non-invasive (Collins, 2004). As data are collected at an individual cell

level, the comet assay also gives a measure of the heterogeneity in a sample (Lovell and Omori,

2008). In addition, it is a simple, robust technique, capable of detecting low levels of DNA

damage (Tice et al., 2000, Collins, 2002, 2004). It can detect DNA damage equivalent to as few

as 50 single-strand breaks per cell and varying number of double strand breaks (Singh et al.,

1988; Tice and Strauss, 1995; Olive et al., 1998). The assay is able to measure both single- and

double-strand breaks with versatility, sensitivity (Leroy et al., 1996) and speed (Fairbairn et al.,

1995). It has therefore been considered to be suitable for the assessment of DNA damage in

sperm (Collins et al., 2008; Lewis and Agbaje, 2008) and relatively few numbers of cells ~50,

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has been found to be sufficient for the reproducibility of the assay (Hughes et al., 1997). Owing

to the above-mentioned advantages, it has been used extensively to detect sperm DNA damage in

human biomonitoring, in the context of ART and in in vivo and ex vivo genotoxicity studies

(Lewis and Agbaje, 2008; Baumgartner et al., 2009; Speit et al., 2009).

There is a general belief that alkaline conditions are required to reveal single-strand (SS) breaks,

and that therefore using a near-neutral pH ensures that only double-strand (DS) breaks are picked

up (Van Kooij et al., 2004; McArt et al, 2009). This theory has been revealed to be a

misconception as alkaline conditions are not specific to detecting single-strand breaks (Collins et

al., 2008). Alkaline conditions, for DNA unwinding and electrophoresis (Singh et al., 1988) is

known to assess actual DNA strand breaks and alkali labile sites (Baumgartner et al., 2009).

Moreover, as most protocols for the comet assay in sperm involve high salt extraction in the

presence of a reducing reagent which removes both protamines and histones (Tomsu et al., 2002;

McVicar et al., 2004), the comet assay possibly detects chromatin breaks in both types of

chromatin with equal efficiency (Shaman and Ward, 2006).

Of the three measures of DNA migration commonly used - tail length, tail moment and percent

tail DNA, there is an increasing emphasis on the use of the percent tail DNA as the preferred

metric or the primary end point (Kumaravel and Jha, 2006). While the percent tail DNA is a

measure of the relative fluorescent intensity in the head and tail (Collins, 2004), the tail moment

is an index that takes into consideration both the migration of the genetic material and the

relative amount of DNA in the tail. The Olive tail moment (OTM) is the product of the tail

length and the percent tail DNA (Lovell and Omori, 2008). Tail length is considered

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unsatisfactory as a measure because the length only increases at relatively low damage levels and

is sensitive to the background intensity of the image analysis system which affects the criteria for

determining the end of the tail (Collins, 2004). Tail length and moment, although consistent

within a study, may not be comparable across studies. In contrast, the percent tail DNA has the

distinct advantage that it can be „standardized‟ over studies (Lovell and Omori, 2008). It has

been recognized as the most suitable primary end point at the International Workshop on

Genotoxicity Test Procedures (Burlinson et al., 2007). Additionally, percent tail DNA has been

viewed as the most useful measure because it covers a wide range of damage (from 0 to 100%),

and is known to be independent of the threshold settings of the image analysis program (Collins,

2004). In view of good positive correlation obtained between percent tail DNA, Olive tail

moment and tail length in the present study, percent tail DNA is put forth as a most reliable

indicator of DNA damage in sperm.

Consistent with previously reported literature, in this study, the comet assay has been able to

detect DNA damage among the different groups of infertile men (Saleh et al., 2003a; Ahmad et

al., 2007; Erenpreiss et al., 2008). A high risk of infertility has been observed in men with sperm

DNA fragmentation at more than a diagnostic threshold of 25% by alkaline comet assay. The

risk of failure to achieve a pregnancy has also been reported to increase when sperm DNA

fragmentation exceeded a prognostic threshold value of 52% for semen and 42% for sperm

prepared by density gradient method (Simon et al., 2011). The results of the present study agree

with that of a recently published study where high degree of DNA damage has been reported in

idiopathic infertile men with normal semen profile as compared to fertile controls and variably

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higher DNA damage in subjects with oligospermia, asthenospermia and teratozoospermia in

comparison to fertile controls has been observed (Shamsi et al., 2010).

Although today, many different modifications of the alkaline and the neutral comet assay are in

use, a standard protocol has not been established yet. In addition, high and variable background

levels of DNA effects have been reported and there is still need for standardization and

validation of the comet assay with sperm (Speit et al., 2009). Therefore, before it can gain wider

acceptance, in the infertility clinic, the assay is yet to undergo appropriate multilaboratory,

international validation studies to demonstrate its interlab and intralab reproducibility, reliability

and adequacy of its performance against the currently adopted methods. Additionally, studies

that can potentially lead to a better understanding of the underlying mechanisms of comet

formation and how strongly the comet shape reflects the DNA damage that has occurred is

clearly warranted (McArt et al., 2009). Nevertheless, an in vitro assay with human spermatozoa

is considered to be valuable, as a sensitive system to assess potential transgenerational effects in

the subject of reproduction and development.

To conclude, in the present study, employing alkaline comet assay, it has been demonstrated that

DNA damage is higher in idiopathic and oligozoospermic infertile men in comparison to fertile

controls. In addition to providing a rationale for further continuation of the work proposed in the

thesis, the above finding, coupled with concomitant concerns of increased sperm DNA damage

with failed/delayed fertilization and aberrant embryo development clearly warrants larger,

prospective studies to refine this one measure of sperm quality.