UROL_V03_No3_001_Editorial.indd


Sexual Dysfunction and Infertility

154 Urology Journal   Vol 3   No 3   Summer 2006

Sperm Nuclear DNA in Ejaculates of Fertile and 

Infertile Men 
Correlation with Semen Parameters

Mohammad Ali Khalili,1 Fatima Aghaie-Maybodi,2 Morteza Anvari,2 Ali Reza Talebi2

Introduction: Our aim was to compare the nuclear DNA integrity of  the 

spermatozoa from infertile men with abnormal semen parameters with that from 

normospermic fertile men, and to evaluate the relationship between the sperm 

DNA integrity and semen parameters. 

Materials and Methods: Thirty ejaculate samples with abnormal semen 

analysis and 30 ejaculates with normal semen parameters were randomly collected 

from infertile and fertile men, respectively. The acridine orange test was used to 

assess the integrity of  sperm DNA. 

Results: The number of  ejaculates with a DNA Fragmentation Index (DFI) 

above 30% were 16 (53.3%) and 22 (73.3%) in fertile and infertile subjects, 

respectively (P = .10). The mean DFI was 37.7 ± 19.6% and 46.1 ± 16.7% in the 

fertile and infertile subjects, respectively (P = .24). The DFI of  the fertile men with 

normal sperm morphology (12 patients) ranged from 1% to 80%. In the samples 

with oligoasthenospermia, mean DFI was 52.7 ± 17.9%. There were 2 samples 

with severe teratospermia (normal morphology less than 4%) and DFIs of  70% 

and 87%. There were no significant correlations between the DNA integrity and 

the 3 parameters of  semen quality in our 60 subjects.  

Conclusion: Our results failed to show any significant difference in the DNA 

integrity of  the spermatozoa between infertile and fertile men. Also, no correlation 

was noticed between the DNA abnormality and the semen parameters in the 

studied samples. 

Urol J (Tehran). 2006;3:154-9. 

www.uj.unrc.ir

Keywords: DNA, spermatozoa, 

acridine orange, semen analysis

1Fertility and Infertility Research 

Center, Isfahan University of 

Medical Sciences, Isfahan, Iran
2Research and Clinical Center for 

Infertility, Yazd University of Medical 

Sciences, Yazd, Iran

Corresponding Author: 

Mohammad Ali Khalili, PhD

Fertility & Infertility Research Center, 

Dr Beheshti Hospital, Felezi Bridge, 

Isfahan, Iran

Tel: +98 913 311 3358 

E-mail: khalili59@hotmail.com

Received July 2005

Accepted May 2006

INTRODUCTION

Conventional semen analysis is still 

one of  the most popular tests for the 

evaluation of  male fertility. However, 

in many circumstances, semen 

analysis per se is unable to predict 

the fertilizing capacity of  ejaculated 

spermatozoa, because it does not 

assess some factors such as the 

integrity of  sperm nuclear DNA.(1-3)

It has been reported that some of  the 

infertile men are normospermic; while 

fertilization has occurred in others 

with an abnormal semen parameters.(4) 

Therefore, sperm function tests 

like sperm nuclear maturation assay 

have been developed to assess the 

fertility capacity of  individuals in 

assisted reproductive technologies 

(ART). It is now well understood 

that the integrity of  sperm DNA 

is an essential factor for normal 

transmission of  genetic materials 

during the process of  fertilization 

as well as embryo development.(1,3,4) 

Therefore, it is essential to develop 

accurate diagnostic tests that provide 

better prognostic capabilities than the 

conventional sperm assessments. At 

present, several assays are available for 



Sperm Nuclear DNA in Ejaculates of Infertile Men—Khalili et al

Urology Journal   Vol 3   No 3   Summer 2006 155

the evaluation of  the nuclear chromatin integrity or 

the maturity of  human spermatozoa.(4,5) One of  these 

tests which was first introduced by Evenson and 

colleagues in 1980 is acridine orange test (AOT).(6) 

In human testis, the histone nucleoprotein is replaced 

by protamine during spermiogenesis. Protamine is 

rich in cysteine and also other basic amino acids. The 

structure of  the sperm nuclear chromatin becomes 

stable following transformation to DNA-protamine 

complex.(2,3,7) During the epididymal passage, the 

thiols of  cysteines in protamine are oxidized to 

disulfide bonds (S-S). This process will make the 

spermatozoa more stable than testicular sperm. At 

ejaculation, sperms are matured enough for the next 

step to occur—fertilization. The maturity of  sperm 

nuclei (rich in S-S) can be determined by the use of  

AOT. The acridine orange molecules are intercalated 

into double-stranded DNA and green fluorescence 

is emitted from the sperm nuclei. However, with 

immature nuclei (poor S-S), sperm DNA is easily 

denatured into single strands. Consequently, the 

acridine orange molecules aggregate in the nuclei and 

the color of  the fluorescence changes into orange-

red.(8)

It has previously been demonstrated that significant 

differences exist in the proportion of  the 

spermatozoa with impaired DNA integrity between 

fertile and infertile populations.(3,9) Also, it has 

been demonstrated that human spermatozoa with 

impaired chromatin structure, detected by AOT, are 

not able to penetrate the zona pellucida and fuse 

with the oolemma of  the hamster oocytes.(10) This 

indicates that a sperm chromatin abnormality seems 

more likely to affect the fertilization process. Also, 

in another clinical study, it has been shown that in 

individuals with immature sperm nuclei, DNA is 

easily denatured into single strands, thus reducing the 

chance of  a successful in vitro fertilization (IVF).(1) 

A correlation between the staining of  sperms by 

acridine orange and sperm morphology has also been 

found.(11) 

Although most studies have supported the significant 

correlation between the acridine orange fluorescence 

capability and the in vitro fertilizing ability of  human 

spermatozoa, a negative correlation has also been 

reported.(12) In addition, there have been controversial 

issues in terms of  the relationship between the 

integrity of  sperm DNA and semen parameters 

and whether measurements of  DNA integrity can 

differentiate the spermatozoa of  fertile and infertile 

men.(1,3) It is still unclear whether the widely used 

DNA integrity tests are sensitive enough to reveal 

the differences in baseline sperm DNA integrity 

between the semen collected from fertile and infertile 

populations. The purpose of  this cross-sectional 

study was first, to compare the nuclear DNA integrity 

of  spermatozoa using AOT in ejaculates of  infertile 

patients with abnormal semen parameters with 

that of  normospermic fertile people, and second, 

to evaluate the correlation between the integrity 

of  sperm DNA and semen parameters (sperm 

concentration, motility, and morphology) in both 

groups of  fertile and infertile men. 

MATERIALS AND METHODS

Patients 

This cross-sectional study included a randomly 

selected group of  30 Iranian men with a history 

of  infertility presented to our clinics. They were 

diagnosed with male factor infertility, and their wives 

were reported to have normal reproductive function 

following gynecological and endocrinological 

examinations. Infertile patients with normospermia 

were excluded. Also, 30 randomly selected married 

fertile men were served as controls. They had 

normal semen parameters and presented with other 

complaints. All subjects provided informed consent 

and the study was approved by our university’s 

research and ethics committees. Every single patient 

was asked to collect a fresh ejaculated semen sample 

into a sterile wide-mouth container. 

Semen Analysis

All semen analyses were performed after the 

liquefaction of  the semen samples within 15 to 20 

minutes. Using the WHO criteria (sperm count, 

> 20   106/mL; normal sperm motility, ≥ 50%; and 

normal sperm morphology, ≥ 30%), the samples 

were categorized into normospermic and those with 

single, double, and triple defects in spermatozoa.(13) 

Following macroscopic evaluation of  each 

specimen, sperm count and motility were evaluated 

using Makler Chamber and light microscopy at 

× 200 magnification. Motility was expressed as 

the percentage of  progressive and nonprogressive 

spermatozoa. Sperm morphology was assessed on 



Sperm Nuclear DNA in Ejaculates of Infertile Men—Khalili et al

156 Urology Journal   Vol 3   No 3   Summer 2006

smears with the Giemsa staining (Merck Chemical 

Co, Darmstadt, Germany). The percentage of  

spermatozoa with a normal morphology was 

determined by assessing 100 sperms under oil 

immersion with magnification of  × 1000 under 

bright-field illumination.(2,14) 

Acridine Orange Staining

For assessment of  sperm DNA integrity, the smears 

were air-dried for 1 hour and then fixed overnight 

in freshly made Carnoy’s solution (one part glacial 

acetic acid, three parts methanol) at 4ºC. The slides 

were rinsed 2 times with distilled water and dipped 

in McIlvaine phosphate-citrate buffer (pH = 4) 

for 5 minutes. Each sample was then stained with 

freshly prepared acridine orange (0.19 mg/mL; Sigma 

Chemical Co, St Louis, USA) in MciIvaine phosphate-

citrate buffer for 10 minutes in the darkness.(15) 

The preparations were washed with distilled water, 

covered with glass cover slips, and assessed on 

the same day using fluorescent microscope (Zeiss, 

Oberkochen, Germany) with a 460-nm filter. The 

duration of  illumination was limited to 40 seconds 

per field. The percentage of  green (normal DNA 

integrity) and orange-red (abnormal DNA integrity) 

spermatozoa per 100 spermatozoa in each sample 

was calculated by a same single person. An abnormal 

integrity of  sperm nuclear DNA was considered 

as more than 30% denaturation (orange-red 

spermatozoa on acridine orange staining).(16) The 

DNA Fragmentation Index (DFI), which is the ratio 

of  the orange-red to the total (orange-red + green) 

fluorescence intensities of  spermatozoa, was also 

calculated for the samples.

Statistical Analyses

For statistical analyses, SPSS software (Statistical 

Package for the Social Sciences, version 9, SPSS Inc, 

Chicago, Ill, USA) was used. The chi-square test 

and the Fisher exact test were used to compare the 

frequencies and Student t test to compare quantitative 

values. The Pearson correlation test was used to 

evaluate the relationship between sperm DNA 

integrity and sperm parameters. A P value less than 

.05 was considered significant.

RESULTS

Overall, semen samples of  30 infertile and 30 fertile 

men were assessed. The mean sperm concentration, 

motility, and normal morphology were significantly 

higher in the fertile subjects. The mean values of  

semen parameters are presented in Table 1. Also, the 

viscosity of  the seminal specimens were abnormally 

high in 4 (13.3%) and 2 (6.7%) patients in the 

fertile and infertile groups, respectively (P = .67). 

Nineteen men (63.3%) in the infertile group had 

asthenoteratospermia, 7 (23.3%) had asthenospermia, 

and 4 (13.4%) had oligoasthenoteratospermia. 

Therefore, the majority of  our patients suffered from 

abnormal progressive motility.

The number of  ejaculates with denatured sperm 

DNA (DFI) above the normal value of  30% were 

16 (53.3%) and 22 (73.3%) in fertile and infertile 

subjects, respectively (P = .10). The mean DFI was 

37.7 ± 19.6% and 46.1 ± 16.7% in the fertile and 

infertile subjects, respectively (P = .24). The mean 

percentage of  green fluorescent sperm in AOT was 

62.4 ± 28.8% and 53.8 ± 24.3% in the fertile and 

infertile subjects, respectively (P = .22). A total of  

4 (13.3%) and 1 (3.3%) ejaculates were presented 

with over 97% double-stranded-DNA sperms 

(green staining) in the fertile and infertile groups, 

respectively (P = .35). Interestingly, the mean DFI of  

the fertile men with a normal sperm morphology (12 

patients) was 32.2 ± 8.3% (range, 1% to 80%), and 6 

of  them had a DFI greater than 30%. 

Table 1. Semen Parameters and Sperm DNA Integrity for 60 Ejaculates of Fertile and Infertile Men*

*Values are demonstrated as means ± standard deviations (ranges).

Semen Parameters Fertile Group Infertile Group P

Volume, mL 3.8 ± 1.6 (1 to 8)   3.5 ± 1.8 (1 to 7) .46 

Sperm concentration, × 10
6
   81.5 ± 36.0 (38 to 180)   55.1 ± 36.3 (3 to 170)   .006 

Progressive motility, %   60.3 ± 7.5 (50 to 73) 19.3 ± 16.4 (2 to 47) < .001 

Nonprogressive motility, % 10.3 ± 2.6 (5 to 17)   16.3 ± 7.1 (7 to 38) < .001 

Normal morphology, % 52.5 ± 11.7 (38 to 78) 22.6 ± 12.4 (2 to 48) < .001 

Round cells, × 10
6
    0.3 ± 0.1 (0 to 0.4)    0.52 ± 0.9 (0 to 0.3)  .36 

Normal DNA integrity, %   62.4 ± 28.0 (19 to 100)   53.8 ± 24.3 (12 to 98)  .22 



Sperm Nuclear DNA in Ejaculates of Infertile Men—Khalili et al

Urology Journal   Vol 3   No 3   Summer 2006 157

In the samples with oligoasthenospermia, the mean 

percentage of  green sperms was 48.25 ± 6.8% 

(range, 28% to 71%) and the mean DFI was 52.7 ± 

17.9%. In addition, there were 2 samples with severe 

teratospermia (normal morphology less than 4%) and 

DFIs of  70% and 87%. 

There were no significant correlations between the 

DNA integrity (demonstrated by the green staining of  

the sperm nucleus in AOT) and the 3 parameters of  

semen quality in our 60 subjects (Table 2). 

DISCUSSION

In clinical practice, the conventional subjective 

semen analysis using light microscopy still plays the 

central role in the assessment of  fertility in men. 

However, a definitive diagnosis of  infertility cannot 

often be made solely on the basis of  semen analysis 

results. Although in many circumstances the sperm 

parameters are impaired in infertile population, 

there is a significant overlap between the semen 

parameters of  infertile and fertile men.(3,16) Thus, 

to upgrade the prognostic and diagnostic ability 

of  semen analysis, an sperm function assay should 

be included. Nowadays, there are several assays 

available to assess the sperm nuclear integrity in 

clinical setting. However, the efficacy or prognostic 

value of  each assay for prediction of  fertilization or 

pregnancy outcome following ART is still a matter 

of  controversy. One of  the standard assays is AOT, 

which is quick and easy to be performed at clinical 

andrology laboratories. Application of  acridine 

orange fluorescent staining allows the nuclear 

chromatin integrity of  the human spermatozoa to 

be analyzed under either fluorescent microscope 

or by flowcytometry. This can be used for testing 

the sperm maturity and predicting the fertilization 

capacity of  ejaculated spermatozoa.(16) Hoshi and 

colleagues showed that men who had ejaculates 

with more than 50% green sperms in AOT (double-

stranded DNA) had a significantly higher fertilization 

capacity for IVF compared with men with immature 

sperms.(17) Therefore, it seems that sperm chromatin 

abnormality affects the fertilization process in clinical 

setting.

It has been previously reported that a significant 

difference exists between the proportions of  the 

sperms with impaired DNA integrity in the fertile 

and the infertile populations.(9,16,18) Zini and coworkers 

studied the prevalence of  sperm DNA denaturation 

by AOT among 13 fertile and 88 infertile men. 

They observed that the rate of  DNA denaturation 

was significantly lower among infertile men with 

normospermia compared with infertile patients with 

abnormal spermatozoal parameters (11.1% versus 

23.1%, respectively). Thus, a negative correlation 

was reported between the DNA integrity and semen 

parameters.(16) However, our results disagree with 

the aforementioned studies; the proportion of  

sperms with a normal DNA integrity in our fertile 

(normospermic) subjects was slightly higher than that 

in infertile subjects with impaired semen parameters, 

but the difference was not significant. Neither 

could we find any correlation between the status 

of  sperm DNA integrity and the sperm parameters 

in our patients. The rate of  sperm DNA integrity 

was not consistent among the fertile men with 

normal semen parameters. For example, 54% of  the 

fertile men had a sperm DFI over 30%. In addition, 

samples with normal sperm morphology revealed 

a 32.2% DFI which is above the normal range. 

Evaluation of  sperm nuclear integrity with AOT 

and fluorescent microscopy, as used in our study, 

is a routine procedure in andrology units; however, 

usage of  flowcytometry is more efficient as stated by 

Erenpreiss and coworkers.(9) But, flowcytometry is an 

expensive technique and also time consuming, thus, 

less practical for daily evaluation of  sperm DNA in 

ART. 

In our previous studies, we noticed that the 

sperm parameters were more defective in samples 

extracted from the testis comparing with the ones 

extracted from the ejaculates. This is due to the 

fact that sperms are immature in the testis and 

should reach the epididymis to achieve the maturity. 

Also, the fertilization rates were lower with sperms 

retrieved from testis than either epididymal or 

Table 2. Correlations Between DFI and Sperm Parameters in 

Fertile and Infertile Men* 

* Values are correlation coefficient (P). DFI indicates DNA 

Fragmentation Index.

DFI

Semen Parameters Fertile Group Infertile Group 

Sperm concentration  -0.23 (.21) -0.29 (.12) 

Progressive motility  -0.02 (.91) -0.05 (.80) 

Normal morphology  -0.06 (.74) -0.15 (.44) 



Sperm Nuclear DNA in Ejaculates of Infertile Men—Khalili et al

158 Urology Journal   Vol 3   No 3   Summer 2006

seminal spermatozoa. In addition, the fertilization 

and pregnancy rates were higher in samples with 

normospermia compared with the abnormal samples 

with 2 or 3 defects in the sperm parameters.(3,19) 

Therefore, it can be concluded that mature sperms 

from ejaculates of  infertile patients are capable of  

fertilizing oocytes at higher rates comparing with 

the immature spermatozoa that are retrieved from 

the testis of  infertile subjects, and also, the seminal 

samples with normal sperm parameters have higher 

levels of  fertilizing potentials than abnormal samples 

regardless of  the source of  collection. 

It should be emphasized that normal DNA integrity 

of  spermatozoa is very important in intracytoplasmic 

sperm injection (ICSI), because the sperm selection 

is performed by the embryologist on the basis of  

the motility and morphology status. Bungum and 

colleagues observed significantly higher DFI levels 

in successful ICSI cases compared with those in 

successful IVF or intrauterine insemination cases.(20) 

Therefore, the highest level of  sperm DNA damage 

was detected in men with the poorest semen 

quality (ICSI candidates). The authors concerned 

the safety of  the ICSI procedure since damaged 

DNA is often observed more frequently among 

ICSI candidates. Developing assays for detecting 

and selecting spermatozoa with intact DNA for 

microinjection during ICSI procedure will be the 

future challenge. Our results showed that none of  

the sperm parameters correlated with DNA integrity 

of  the spermatozoa;(18) therefore, we cannot predict 

the outcome of  the ICSI cycles solely on the basis 

of  sperm parameters such as sperm motility and 

nonstained sperm morphology.

Acridine orange test has been successfully used by 

some laboratories in an attempt to improve the male 

fertility evaluations; however, the predictive value of  

AOT is still controversial.(1,12,16,20-22) Most investigators 

have noticed a significant relationship between the 

AOT and the sperm parameters or sperm fertilizing 

ability, while a negative correlation has also been 

reported.(12) As our results showed, AOT may be 

associated with severe teratospermia or oligoastheno

teratospermia. Therefore, we do not recommend the 

application of  AOT as a part of  the routine fertility 

workup. On the other hand, it should only be used 

in selected infertile men with severely abnormal 

sperm parameters. It should be emphasized that the 

application of  other chromatin integrity assays may 

reveal the precise quality of  the spermatozoa.(23) 

Thus, it may be necessary to apply several chromatin 

or DNA integrity assays for the elucidation of  the 

precise pathophysiology of  male infertility. In 

addition, as Katayose and associates stated, acridine 

orange fluorescent staining is very useful in cases 

with unexplained infertility.(11) We, however, have not 

investigated the DNA integrity of  such cases so far.

CONCLUSION

Our study showed that the relationship between 

DNA integrity status and the sperm quality is 

a rather complex issue. Although more DNA 

abnormalities were detected among infertile cases 

with abnormal sperm parameters, the difference 

was not significant when compared with ejaculates 

of  fertile controls. However, defects in sperm DNA 

integrity were associated with severe cases such 

as oligoasthenospermia or severe teratospermia. 

Additional large-scale trials are needed to confirm the 

predictive value of  sperm chromatin integrity using 

AOT for the outcome of  ART cycles.

ACKNOWLEDGEMENT

The authors would like to thank Mr Hossain Fazli 

and Mrs Habibeh Ghaisari for their technical 

assistance during the course of  this research study. 

Authors are also grateful to Mr Ahmedieh, MSc, for 

the statistical analysis.

CONFLICT OF INTEREST

None declared.

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2. Aitken RJ. Sperm function tests and fertility. Int J 

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3. Khalili MA, Vahidi S, Aflatonian A, Amir-Arjmand M. 

Intracytoplasmic sperm injection for the treatment 

of male factor infertility. Med J Islam Repub Iran. 

1997;11:181-5.

4. Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, 

Carrell DT. Comparison of chromatin assays for DNA 

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