SPERM CELL

Correlation of spermiogram profiles with DNA damage in sperm cells ofinfertile men: a comet assay study

NIYAZI TUG1,2, SULEYMAN SANDAL3, BERNA OZELGUN1, & BAYRAM YILMAZ1

1Yeditepe University, Faculty of Medicine, Department of Physiology, 34755 Istanbul, Turkey, 2Zeynep Kamil Maternity and

Children’s Hospital, Department of Obstetrics and Gynecology, Istanbul, Turkey, and 3Inonu University, Faculty of Medicine,

Department of Physiology, Malatya, Turkey

(Received 31 October 2009; revised 31 January 2010; accepted 16 March 2010)

AbstractWe have investigated a relationship between DNA damage in sperm and spermiogram profiles in the infertile men. Twenty-one non-smoking infertile men540 years of age with no systemic or genetic disease were randomly selected from the pool ofinfertile couples applied to our clinic. Sperm samples were collected and evaluated according to WHO guidelines. DNAdamage of sperm cells was assessed using neutral comet assay. Fifty cells per slide and two slides per sample were scored toevaluate DNA damage. The cells were visually classified into four categories based on DNA migration such as undamaged(UD), little damage (LD), moderate damage (MD) and significant damage (SD). Total comet scores (TCS) were calculatedas: 16UDþ 26LDþ 36MDþ 46SD. There was a negative correlation between the percentage of slow- and in situ-motion sperm cells in spermiograms and TCS (p5 0.001 and p5 0.05, respectively). The relationship between thepercentage of non-motile sperm cells and TCS was negative (p5 0.001). This study provides new evidence that DNAdamage in spermatozoa and sperm motility parameters are negatively correlated. We suggest that evaluation of sperm DNAby the neutral comet assay may be valuable to use in fertility research.

Keywords: Sperm, DNA damage, infertile men, comet assay, spermiogram

Introduction

Sperm chromatin is a highly organised compact

structure consisting of DNA and proteins and

relatively more resistant to damage compared to the

other cell types of the body [1]. DNA fragmentation is

a sign of poor quality chromatin of sperm cells, which

has been suggested to be closely associated with, and

highly indicative of, some fertility problems [2].

Indeed, a recent survey has shown that sperm DNA

damage is more common in the infertile men and may

contribute to poor reproductive outcome [3].

Morphology, motility and concentration of sper-

matozoa are among the conventional parameters of

semen analysis [4]. The morphological analysis of

spermatozoa gives important prognostic clues about

the success of fertilisation, abortus rate and congenital

anomalies [5]. However, conventional spermiogram

has been reported to be insufficient for the diagnosis

of male infertility since it lacks data about the genomic

status of the sperm cells [6]. Recent studies have

indicated that nearly 20% cases of idiopathic infertility

cases have high levels of sperm DNA fragmentation,

which is a negative influence for infertility [6,7]. It has

been suggested that analysis of the DNA integrity may

be a better diagnostic and prognostic marker as

compared to the routine semen analysis, especially

in assisted reproduction [8].

There are several methods of genetic analysis being

used to measure DNA defects in the human

spermatozoa, but the conventional spermiogram is

still the only test that has been accepted as the routine

sperm qualification method [4]. Among these meth-

ods, the comet assay (single-cell gel electrophoresis) is

a simple, fast and informative test for investigation of

DNA damage of the sperm cells. Its sensitivity for

detecting low levels of DNA damage and requirement

of small numbers of cells per sample increased its

popularity in genotoxicity testing [9,10]. The comet

Correspondence: Bayram Yilmaz, Yeditepe University, Faculty of Medicine, Department of Physiology, Kayisdagi Cad., Istanbul 34755, Turkey. Tel: þ90-216-

5780675. Fax: þ90-216-5780575. E-mail: bayram2353@yahoo.com

Gynecological Endocrinology, January 2011; 27(1): 49–54

ISSN 0951-3590 print/ISSN 1473-0766 online ª 2011 Informa UK, Ltd.

DOI: 10.3109/09513590.2010.487598

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assay can be conducted in neutral or alkaline

conditions depending on the type of DNA damage

to be investigated [4]. The neutral comet assay detects

double strand DNA breaks whereas the alkaline

version identifies single strand DNA breaks and alkali

labile sites. These alkali fragments are abundant in

spermatozoa and thus decrease the sensitivity of the

assay to DNA fragments resulting from double strand

DNA breaks that are indicative of DNA damage and

infertility [11,12].

This study was undertaken to investigate any

relationship between DNA damage in spermatozoa

determined by the neutral comet assay and conven-

tional spermiogram profiles in the infertile men.

Efficacy of the routine sperm analysis to reflect

DNA defects of the spermatozoa was also sought.

Patients and methods

Patients

Twenty-one non-smoking infertile men540 years of

age with no systemic or genetic disease were

randomly selected from the pool of infertile couples

applied to our clinic.

Spermiogram

Sperm sample collection procedure was approved by

the ethics committee of Zeynep Kamil Hospital.

Samples were collected by masturbation into sterile

specimen cups following an abstinence period of

2–5 days. After liquefaction at 378C, a small amount

was removed from each specimen and then the

sperm parameters were examined under Makler

Chamber according to World Health Organisation

(WHO) guidelines [13].

The density gradient centrifugation method was

used for all specimens. Discontinuous density

gradients were prepared with two layers consisting

of 45% and 90% working density solutions. These

solutions had been prepared by using commercial

sperm separation medium concentrate (Isolate,

Irvine Scientific) and sperm washing medium (Pure

Sperm Wash, Nidacon Int, Sweden). Fresh liquefied

semen (1 ml) was gently layered on top of two-

layered gradient in a centrifuge tube using transfer

pipette. It was centrifuged at 2000 rpm for 20 min.

Then, the density gradient and seminal fluid were

removed without disturbing the sperm pellet. The

pellet was washed twice by following procedure: 2 ml

fresh medium was added gently on the tube. Samples

were centrifuged at 1300 rpm for 10 min to wash

away residual silica. Washing medium was gently

removed from the centrifuge tube by a transfer

pipette. 0.5 ml fluid was left at the bottom of the

tube. After the washing procedure, sperm quality

assessment was done by the same technician. A total

of 21 semen samples were collected with a progres-

sive motile sperm count of �56106/ml.

Comet assay

Neutral comet assay was performed as previously

described [14,15] with slight modifications. Briefly,

the fully frosted microscope slides were covered with

100 ml of 0.7% normal melting-point agarose in PBS

and then dried at room temperature. Approximately

105 sperm cells (10 ml) were mixed with 0.7% (w/v)

low melting agarose (80 ml) to form a cell suspension,

which was pipetted onto the precoated slides. The

slides were immediately covered with a cover slip and

kept at 48C for 5 min to allow the agarose to solidify.

After the cover slips were removed, the slides were

immersed in ice cold, freshly prepared neutral lysing

solution (2.5 M NaCl, 100 mM EDTA, 10 mM

Tris, adjusting pH to 10 and adding 1% Triton

X-100 and 40 mM DL-Dithiothreitol before using)

at 48C for 1 h.

Electrophoresis

After lysis, the slides were placed in a horizontal gel

electrophoresis tank positioned close to the anode

and covered with fresh alkaline electrophoresis buffer

solution containing 100 mM Tris, 300 mM sodium

acetate (pH¼ 9) for 20 min at 48C. Electrophoresis

was conducted at 25 V (0.83 V/cm) and 100–

120 mA for 15 min. After electrophoresis, the slides

were washed three times with neutralising buffer

(0.4 M Tris, pH¼ 7.5) at 48C for 5 min for each.

Then the slides were dried and stained with 20 mg/ml

ethidium bromide and covered with a cover slip. The

slides were incubated at 48C in a refrigerator for

20 min before scoring. The entire procedure was

conducted in the dark to prevent additional DNA

damage.

Analysis of DNA damage

Fifty cells per slide and two slides per sample were

scored to evaluate DNA damage. The cells were

visually classified into four categories on the basis of

extent of migration (Figure 1) such as undamaged

(UD), little damage (LD), moderate damage (MD)

and significant damage (SD). Total comet scores

(TCS) were calculated as: 16UDþ 26LDþ 36MDþ 46SD. Comet Assay IV (Perspective

Instruments, Suffolk, UK) imaging system was

attached to a fluorescent inverted microscope in our

laboratory. Sperm cells were initially analysed using

this software. However, some highly damaged sper-

matozoa leading to highly scattered comet could not

be evaluated effectively. Therefore, visual classifica-

tion by an expert (and blind to the study design) was

performed as we previously reported [16].

50 N. Tug et al.

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Statistics

Results are expressed as mean+ standard deviation

(STD). Statistical analysis was performed by using

two-tailed Pearson correlation test (SPSS 13.5 for

Windows). p5 0.05 was considered to be statistically

significant.

Results

Mean age and the spermiogram profiles of the

patients are summarised in Table I. Mean age of

the 21 patients in the study was 30.29+ 5.44 years.

Mean total motile sperm count of the ejaculates was

31.9+ 11.66106/ml. Total sperm count of the

washed samples was 13.5+ 3.596106, the total

motility rate of which was 69.86%+ 18.87%. Of

these, 59.11%+ 19.46% were progressively motile

and 12.28%+ 7.16% in situ motile. 27.31%+18.87% of the samples were non-motile. Mean total

comet score of the same samples was 132.81+32.75. Percentage score of UD and LD sperm cells

was 81.29+ 12.40 and 2.45+ 1.91, respectively.

Moderately damaged and significantly damaged

scores of the samples were 3.75+ 3.43 and 9.18+9.06, respectively.

Correlation of spermiogram parameters with TCS

of individual patients is illustrated in Table II. A

positive correlation was determined between total

comet score (TCS) and non-motile sperm count

(n¼ 21, r¼ 0.830, p5 0.001). TCS and slow-

progressive motility rates were found to be negatively

correlated (n¼ 21, r¼70.707, p5 0.001).

However, no significant correlation was observed

between TCS and in-situ motion rate of the

spermatozoa (n¼ 21, r¼ 421, p¼ 0.053).

Discussion

In this study, spermiogram parameters according to

the WHO guidelines and degree of DNA damage of

sperm cells as determined by the neutral comet assay

were evaluated in 21 infertile male patients. There

was a negative correlation between TCS and the

percentage of slow-motility and the total motility

scores of spermiograms. It has been reported that the

relationship between DNA damage and standard

semen parameters is still controversial [7]. In a recent

study, it was shown that no DNA fragmentation was

found in spermatozoa with normal morphology

collected from a fertile group of donors [17].

Figure 1. Appearance of sperm cells stained with ethidium bromide in neutral comet assay experiments. Undamaged (A) low damaged (B),

moderate damaged (C) and high damaged (D).

Table I. Age and sperm motility scores of the 21 infertile patients

evaluated in this study according to the WHO guidelines.

Parameters Mean+STD

Age 31.2+1.41

Total motile sperm (million/ml) 31.9+11.6

Sperm count after wash (million/ml) 13.5+3.59

Total motility (%) 72.3+4.12

Slow-progressive motility (%) 61.0+4.25*

In situ motile (%) 12.5+1.56

Non-motile (%) 27.7+4.12*

Total comet score 137.6+7.2

There was a significant (*p50.001 using two-tailed Pearson

correlation test) correlation with total comet scores.

DNA damage in spermatozoa 51

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However, a high proportion of DNA damage in

spermatozoa was observed in samples from the

infertile group in the same study. Our study provides

new evidence that DNA damage in spermatozoa and

conventional spermiogram parameters are signifi-

cantly correlated.

Most of the comet assay studies with human

sperm have been performed in the context of male

infertility and assisted fertilisation [1]. This is a

useful method for investigating pre-existing DNA

damage of germ cells, particularly spermatozoa.

Because of the highly condensed chromatin structure

of the sperm DNA, additional steps for chromatin

decondensation is needed to detect DNA breaks

(either single or double stranded). There are various

modifications of the comet assay protocols with

sperm and there is no general agreement on

translating the results of these studies [1,4]. Alkaline

version (pH4 13) is the most sensitive among those

protocols which detects a broad spectrum of primary

DNA lesions with high sensitivity [9,18]. However,

an increased background level of DNA migration has

been observed in alkaline comet assay protocols that

results a high background noise [11]. Therefore,

alkali comet assay experiments with sperm cells are

to be interpreted with caution as it might result an

overestimation of the DNA damage [1]. Background

DNA migration decreases as the pH of the solutions

decrease to neutral in the comet assay protocols

which also reflect single stranded DNA breaks like

the alkali version, but with a lesser sensitivity [19–

21]. Additional steps such as use of dithiothreitol

(DTT) have been recommended to decondense

sperm chromatin to allow DNA migration

[14,15,22]. In our experiments, DTT was also added

to lysis solution to optimise the protocol. We suggest

that the comet assay protocol used in this study is

well standardised and can be used as a part of the

male infertility workup.

It has been suggested that standard sperm para-

meters have a limited power for prediction of the

chance of natural conception [23]. Therefore, re-

search for a better diagnostic and screening method to

evaluate genetic material in spermatozoa is ongoing.

In a recent study, 30 normozoospermic infertile men

seeking infertility treatment and 20 fertile donors

were studied, and no relationship was found between

the DNA damage scores and idiopathic infertility

[24]. In their study, the sperm DNA damage was

assessed by alkaline comet assay. It is possible that

high background noise (fluorescence) due to alkaline

conditions overshadowed differences between the

infertile men and fertile donors. DNA fragmentation

index findings as studied using sperm chromatin

structure assay have been reported to be associated

with semen analysis results and fertility in vivo [23].

Duru et al. [25] have shown a significant and negative

relationship between sperm motility and percentages

of sperm with DNA fragmentation determined

using terminal deoxynucleotidyl transferase-

mediated deoxyuridine triphosphate nick end label-

ling (TUNEL) procedure in 10 infertile men.

Similarly, significant inverse correlations were

detected between percentage of TUNEL-positive

cells and both concentration and motility of sperma-

tozoa in 37 patients applied for treatment [26].

However, in a different study conducted in France,

Cohen-Bacrie et al. [27] have shown that semen

Table II. Percentages of motility, slow progressive motility, in-situ motility, non-motile sperm counts and total comet scores (TCS) for each

patient.

Patient Age (years) Motile (%) Slow prog. (%) In situ mot. (%) Non-motile (%) Total comet scores

1 31 91 83 8 9 100

2 29 96 81 15 4 108

3 34 92 89 34 8 109

4 32 87 72 12 13 109

5 24 88 75 13 12 111

6 34 72 56 16 28 113

7 28 90 83 5 10 115

8 25 80 65 15 20 116

9 34 70 51 19 30 118

10 35 81 70 11 19 118

11 34 80 70 10 20 120

12 25 83 70 13 17 126

13 30 79 68 11 21 135

14 26 57 36 21 43 136

15 33 84 74 10 16 157

16 34 64 57 7 36 167

17 29 29 15 14 71 172

18 48 50 47 3 50 176

19 29 56 38 18 44 180

20 39 39 31 8 61 193

21 28 50 50 – 50 210

52 N. Tug et al.

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parameters were correlated with high TUNEL rates

in only one-third of the samples collected from

infertile patients. They suggested that the WHO

sperm parameters and DNA damage are comple-

mentary, rather than strongly linked. In our study,

non-motile spermatozoa samples were shown to have

significantly more DNA damage. We suggest that

evaluation of the sperm DNA may significantly add to

the value of semen analysis in prediction of the chance

of natural conception. Results of this study also

confirm that the neutral comet assay is a sensitive and

valuable method to use in fertility research.

Mechanisms by which damage in the genetic

material is triggered are still largely unresolved and

the susceptibility of each individual appears to

depend on their genetic background, lifestyle and

exposure to various insults [28]. Environmental

pollutants have been linked to high incidence of

damage in the genetic material of sperm cells [29].

Increased sperm DNA damage as determined by the

neutral comet assay has been correlated with

presence of low levels of a non-persistent pesticide

in men [30]. However, the same group also

previously reported lack of any correlation between

DNA fragmentation and organochlorinated chemi-

cals in sub-fertile patients applied to Massachusetts

General Hospital Andrology Laboratory [31]. Thus,

there may be various endogenous and exogenous

causes for DNA fragmentation in human sperm. In

addition, the sperm processing procedure itself has

been suggested to carry a slight genotoxic potential,

which most probably occurs secondary to reactive

oxygen species formation during sperm preparation

[32]. This may account for the basal comet noise

observed in our study, besides the actual DNA

damage of the sperm cells.

In conclusion, results of this study provide new

evidence that DNA damage in the spermatozoa and

sperm motility parameters are negatively correlated. We

suggest that evaluation of sperm DNA may significantly

add to the value of semen analysis. Our results also

confirm that the neutral comet assay is a sensitive and

valuable method to use in fertility research.

Acknowledgements

This study is supported by The Scientific and

Technological Research Council of Turkey (TUBI-

TAK project No. 104-T-240). We thank Dr. Gulhan

Aktas of the Zeynep Kamil Maternity and Children’s

Hospital in Istanbul for her technical assistance in the

Andrology Laboratory.

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