Accepted: 10 October 2016

DOI: 10.1111/and.12765

ORIGINAL ARTICLE

Assessment of human sperm DNA integrity using two

cytochemical tests: Acridine orange test and toluidine blue
assay

T. Ajina1 | O. Ammar1 | Z. Haouas1 | A. Sallem1,2 | L. Ezzi1 | I. Grissa1 | 

W. Sakly3 | A. Jlali1 | M. Mehdi1,2

1
Faculty of Medicine, Department
of Histology, Embryology and Summary
cytogenetics, University of Monastir, Primary infertility affects approximately 15% of couples, with male factor infertility
Monastir, Tunisia
2
accounting for 50% of cases. Semen samples from 41 patients with asthenoterato-
Laboratory of Cytogenetics and Reproductive
Biology, Fattouma Bourguiba University spermia and 28 men with proven fertility were analysed according to World Health
teaching hospital, Monastir, Tunisia
Organization guidelines. Abnormal sperm chromatin structure was assessed by tolui-
3
Faculty of Pharmacy, Laboratory of
dine blue assay (TBA), and DNA denaturation (DD) was detected by the acridine or-
Parasitology-Medical and Molecular
Mycology, Department of Clinical Biology ange test (AOT). The mean (±SEM) rates of DD and abnormal chromatin structure
B, University of Monastir, Monastir, Tunisia
were significantly higher in infertile subjects compared to fertile group respectively
Correspondence p = .003 and p < .001. A significant correlation was established between sperm DD
Tesnim Ajina, Faculty of Medicine, Department
and abnormal chromatin structure (R = .431, p < .001). Sperm DNA damage correlated
of Histology, Embryology and cytogenetics,
University of Monastir, Monastir, Tunisia. significantly with abnormal morphology, sperm motility and necrozoospermia. Our
Email: [email protected]
study shows that men with increased levels of abnormal sperm chromatin structure
Funding information have a high incidence of DNA denaturation and altered semen parameters. These find-
Research Unit of Histology and Genetic,
ings suggest that male infertility has been linked to sperm DNA damage.
Grant/Award Number: UR12ES10; Ministère
Tunisien de l’Enseingement Supérieur et de la
Recherche Scientifique. KEYWORDS
abnormal chromatin structure, acridine orange, denaturation, toluidine blue

1 |  INTRODUCTION may cause male infertility (Carrell & Liu, 2001; Oliva, 2006). The in-
ter-­ and intramolecular disulphide bonds between the protamine mol-
Semen analysis is traditionally used as the first step to evaluate the ecules are essential for sperm nuclear compaction and stabilisation.
male factor infertility, and it is believed that semen analysis is insuf- It is generally accepted that this type of nuclear compaction protects
ficient to determine the fertility in vivo or in vitro. Semen quality sperm genome from external damages including oxidative stress,
is determined according to motility, morphology and concentration temperature elevation and acid-­induced DNA denaturation (Chohan,
of the spermatozoa. However, a failure of the conventional semen Griffin, Lafromboise, DeJonge, & Carrell, 2006; Kosower, Katayose, &
parameters to predict fertilisation indicates that hidden anomalies Yanagimachi, 1992). Recent evidence shows that recurrent sponta-
lie at the sperm membrane level or at the chromatin level (Bianchi, neous abortion (RSA) may be the result of increased levels of sperm
Manicardi, Urner, Campana, & Sakkas, 1996; Iranpour, Nasr-­Esfahani, DNA denaturation (Bhattacharya, 2008; Nijs et al., 2009; Talebi et al.,
Valojerdi, & al-­Taraihi, 2000). In the process of spermiogenesis, the 2012). Evenson et al. (1999) and Spano et al. (2000) have demon-
degree of compaction of sperm chromatin changes considerably as strated that when denaturated DNA was above a threshold of 30%,
histones are replaced in a step manner by testis-­specific nuclear pro- ultimately fertile couples took longer time to conceive. Furthermore,
teins, transitional proteins and finally by protamines (Aoki & Carrell, it is reported that sperm cells with denatured DNA may result in
2003; Dadoune, 2003). Any abnormality in expression of each kind ­zygotes, but the occurrence of embryo fragmentation and retarded
of sperm-­specific nucleoprotein alters sperm chromatin structure and embryo growth with poor biological quality is very common (Tesarik,

Andrologia. 2017;49:e12765. wileyonlinelibrary.com/journal/and © 2017 Blackwell Verlag GmbH  |  1 of 6

https://doi.org/10.1111/and.12765
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Mendoza, & Greco, 2002 Virant-­Klun, Tomazevic, & Meden-­Vrtovec, with normal values of sperm concentration (>15 × 106 sperm/ml) and
2002). normal values of sperm viability (≥58%). In addition, 28 healthy men
Recently, much attention has been given to methods evaluating with normal semen profiles and proven fertility were recruited as con-
the DNA integrity of spermatozoa, hoping that it would add a new trols. Men with a leucocyte concentration more than 106/mL of ejacu-
dimension to the diagnosis of male infertility. Many of the more late were excluded from the study.
popular tests available require flow cytometry, which is expensive Semen samples were collected by masturbation in to sterile cups
and often times difficult to implement (De Jonge, 2002). In this re- following 3 days of sexual abstinence. After liquefaction of the sper-
spect, cytochemical assays for assessment of sperm DNA integrity matozoa for 30 min at 37°, total motility, concentration and viability
or evaluating chromatin structure, based on different staining meth- were evaluated according to the World Health Organization guidelines
ods or fluorochromes, have been used. Acridine orange test (AOT) (2010). Sperm viability was assessed using the eosin test, and sperm
is an established cytochemical method for determining sperm DNA concentration was determined with Thoma cell counting chamber.
integrity which allows differentiation between normal double-­ Sperm morphology was evaluated using the Spermoscan kit. At least
stranded and abnormal denatured/single-­stranded DNA using the 100 spermatozoa per patient were examined at ×100 magnification
metachromatic properties of the stain (Tejada, Mitchell, Norman, according to David’s modified classification (Auger & Eustache, 2000).
Marik, & Friedman, 1984). In the case of completely matured sperm All subjects in either group had a normal 46, XY karyotype and
nuclei, which are rich in S-­S, DNA is kept in double-­stranded form testicular volume within the normal range, and they had no history of
even if it suffers stresses such as exposure to acid or heat. After radiotherapy, chemotherapy, chronic illness, medication or varicocele.
being treated with acridine orange staining, acridine orange mole- The mean age of our study participants was 34.31 ± 7.07 years.
cules are intercalated into ds-­DNA. Green fluorescence is emitted This protocol was approved by the local ethics committee, and all
from the sperm nuclei under blue light with wavelengths of 450– patients and controls had previously given informed consent for the
490 nm. In the case of sperm nuclei poor in S-­S, on the other hand, study.
DNA could be denatured to a single strand, and the aggregation
of acridine orange molecules occurs within the nuclei. Red flores-
2.2 | Semen preparation
cence can then be observed under the same wavelength (Acheson,
1973). Other assays for sperm chromatin integrity assessment in- After performing a semen analysis, an aliquot of unprocessed semen
clude toluidine blue assay (TBA). TBA has been reported to be a (about 25–100 μl, containing two million spermatozoa) was sus-
sensitive test for incomplete DNA structure and packaging (Talebi pended in ice-­cold buffer composed of Tris-­HCl, NaCl and EDTA
et al., 2012). It has been used for visualisation of sperm chroma- (TNE) (0.01 mol/L Tris-­HCl, 0.15 mol/L NaCl and 1 mmol/L EDTA, pH
tin abnormalities (Hee-­Sun et al., 2013). Toluidine blue (TB) is a 7.4) and immediately stored at −80°C until later assessment of sperm
cationic dye that binds to negatively charged phosphate residues. DNA denaturation with AOT. Another aliquot of 200 μl of semen was
Therefore, lightly or orthochromatically stained cells have few TB freshly used for TBA.
binding sites and indicate tightly bound DNA. Conversely, dark or
metachromatically stained cells reveal high incidence of coopera-
2.3 | Toluidine blue assay
tive binding between the TB stain and available phosphate resi-
dues, indicating poor DNA integrity (Dennis, Hannah, Scott, Avner, The TBA was performed as described earlier (Erenpreiss, Bars,
& Huai, 2010). Lipatnikova, Erenpreisa, & Zalkalns, 2001; Erenpreiss et al., 2004).
The goal of this study was to determine, through toluidine blue Briefly, 200 μl of fresh semen was washed in phosphate-­buffered sa-
assay and the acridine orange test, the levels of sperm chromatin line (PBS, pH 7.4) (Sigma, St. Louis, MO, USA) by centrifugation at
abnormalities and sperm DNA denaturation, and to examine the 500 g for 10 min. Smears were prepared from the pellet and air-­dried.
relationship among DD, abnormal chromatin structure and semen The air-­dried smears were then fixed in freshly prepared 96% ethanol-­
parameters. acetone (1:1) at 4°C for 1 hr and air-­dried, and then hydrolysed in
0.1 mol/L HCl at 4°C for 5 min. Thereafter, the slides were rinsed
three times in distilled water for 2 min and stained with 0.05% TB for
2 |  SUBJECTS AND METHODS
17 min at room temperature. The component of staining buffer was
50% citrate phosphate (McIlvain buffer, pH 3.5). The slides were fi-
2.1 | Collection and processing of semen samples
nally dehydrated in successive baths of ethanol (70%, 96% and 100%).
A prospective controlled study was employed, involving 41 patients Under light microscopic evaluation, a total of 300 spermatozoa were
presenting for infertility evaluation at our Department of Cytogenetic counted in different areas of each slide using oil immersion with ×
and Reproductive Biology, Fattouma Bourguiba Teaching Hospital, 100 magnifications. Sperm cell heads with good chromatin integrity
Monastir, Tunisia. This study group consists of asthenoteratozoosper- were light blue; however, those of diminished integrity were deep vio-
mic men, whose subinfertility is determined mainly by less than normal let (dark). TB-­dark cells were considered to be abnormal (Figure 1),
levels of morphologically normal spermatozoa (≤15%) and less than and the percentage of spermatozoa with a deep violet colour was
normal levels of progressively motile spermatozoa (a+b+c ≤ 40%), determined.
AJINA et al. |
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F I G U R E   1   Sperm chromatin structure

as assessed by toluidine blue staining in
fertile (a) and infertile (b) men. Sperm cell
heads with good chromatin structure were
light blue (TB-­light cells); those of abnormal
chromatin structure were deep violet
(TB-­dark cells) (a) (b)

F I G U R E   2   Sperm DNA denaturation as

assessed by acridine orange test in fertile
(a) and infertile (b) men. Sperm cell heads
with double stranded DNA were green
(AO-­green cells); those of single-­stranded
(denaturated) DNA were red (AO-­red cells) (a) (b)

and semen parameters. A significant statistical difference was ac-

2.4 | Acridine orange test
cepted when p ≤ .05.
The acridine orange test was performed as described in detail by
Tejada et al. (1984) with slight modifications. Briefly, a suspension
3 | RESULTS
of 200 μl of diluted spermatozoa was treated with an acid detergent
solution (pH 1.2) containing 0.1% Triton X-­100 (Sigma), 0.15 mol/L
3.1 | Conventional semen analysis
NaCl and 0.08 mol/L HCl. After 30 s of acid treatment, 1.2 ml of AO
solution containing 50 μg/ml purified AO (Sigma) in staining buffer The conventional semen parameters of patients and controls are re-
(0.037 mol/L citric acid, 0.126 mol/L Na2HPO4.7H2O, 0.011 mol/L ported in Table 1. The percentage of total sperm motility, sperm mor-
EDTA and 0.15 mol/L NaCl, pH 6.0) was added to the sperm suspen- phology and sperm viability was significantly lower in infertile group
sion and incubated for 3 min. The tubes were centrifuged at 500 g for compared with controls.
10 min, and the liquid was decanted. The spermatozoa were resus-
pended in 100 μl of TNE buffer and transferred to a glass slide. The
3.2 | Sperm DNA assessment
percentage of spermatozoa with denaturated DNA was determined
by counting at least 300 spermatozoa under a fluorescent microscope As shown in Figure 3, the proportion of spermatozoa with DNA
in 40 ×  magnification, with excitation at 450–490 nm. Spermatozoa denaturation as assessed by AOT was significantly higher in
with normal, intact double-­stranded DNA stained green and those
with denatured ones showed red or orange fluorescence (Figure. 2). T A B L E   1   Standard semen parameters in fertile and infertile men
AO-­red cells were considered to be abnormal, and the percentage of
Fertile Infertile
spermatozoa with red colour was determined.
Sperm parameters (n = 28) (n = 41) p-­value

Volume (ml) 2.84 ± 0.25 3.64 ± 0.52 .01*

2.5 | Statistical analysis pH 8.10 ± 0.04 8.06 ± 0.03 .49

Statistical analysis was performed using the Statistical Package for Sperm concentration 94.67 ± 9.54 83.08 ± 11.91 .45
(106/ml)
Social Sciences, version 21 (SPSS, Chicago, IL). Data are represented
as Mean ± SEM (standard error of the mean). The comparisons be- Total motility (%) 50 ± 2.28 29 ± 1.44 <.001*

tween fertile group and infertile group were made using one-­way Normal morphology 19 ± 1.15 6 ± 0.55 <.001*
(%)
analysis of variance (ANOVA) following by Tukey’s post hoc compari-
sons. Pearson’s correlation was performed to examine the relation- Viability (%) 84 ± 1.86 72 ± 2.24 <.001*

ship among abnormal chromatin structure, sperm DNA denaturation Values are mean ± SEM (standard error of the mean), *p < .05.
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4 | DISCUSSION

During spermiogenesis, nucleoprotein histones are gradually replaced

with protamines, which are placed in the minor groove of double-­
stranded DNA to form a DNA–protamine complex. Any abnormal-
ity in expression of each kind of sperm-­specific nucleoprotein alters
sperm chromatin structure and may cause male infertility (Carrell &
Liu, 2001; Oliva, 2006).In the present work, we have shown a sig-
nificant increase in chromatin abnormalities level in infertile patients
compared with controls. Our data are in agreement with previous ex-
perimental results showing that light blue sperm cell heads prevailed
in normal samples, whereas dark and blue sperm cell heads dominated
in abnormal samples (Erenpreisa et al., 2003). The proportion of TB-­
dark cells in normal samples did not exceed 35% which is in accord-
ance with our data. The 35% threshold is within the range from 27% to
F I G U R E   3   Levels of abnormal chromatin structure as assessed 30% (Evenson et al., 1999; Larson, De Jonge, Barnes, Jost, & Evenson,
by toluidine blue (TB) assay and DNA denaturation as evaluated
2000) to 40% (Spano et al., 2000). Using TB image cytometry, Tsarev
by acridine orange (AO) test in patients and controls. *: significant
difference between patients and controls et al., (2009) reported that the threshold for this staining is 45% and
a significant difference in the percentage of TB-dark and TB-light cells
between fertile and infertile men was shown. (Tsarev et al., 2009).
infertile patients compared with fertile group (31.06% ± 3.26 vs Moreover, the percentage of spermatozoa with abnormal sperm chro-
17.12% ± 3.15; p = .003). The analysis of sperm chromatin structure matin structure was significantly higher in infertile group compared
by TBA revealed that infertile patients had higher percentage of sperm with controls (Talebi, Moein, Tabibnejad, & Ghasemzadeh, 2008).
chromatin abnormalities compared with fertile men (61.37% ± 3.81 vs Using the same staining test on recurrent spontaneous abortion pa-
30.88% ± 3.41; p < .001). tients, it is demonstrated that higher percentage of sperm chromatin
abnormalities in RSA patients was found in comparison with control
group (Talebi et al., 2012). As well as, BT staining on rabbit spermato-
3.3 | Correlation between sperm DNA and
zoa suggested that this test, contrary to the Feulgen reaction, another
semen parameters
cytochemical assay, allowed better distinction between spermatozoa
The results of correlation analysis between sperm parameters and with normal and altered chromatin because of the difference of col-
the two markers of sperm DNA assessment are listed in Table 2. our between these two sperm populations (Belettia & Mello, 2004). In
Significant correlations between the percentage of TB-­dark cells keeping with these observations, TBA may be regarded as a potential
and normal morphology (r = −.449, p < .001), motility (r = −.244, alternative that allows better measure of DNA integrity using only
p = .044) and viability (r = −.374, p = .002) were found. In addi- a light microscope which can be found in most laboratories (Dennis
tion, the percentage of AO-­red cells correlated significantly with et al., 2010). It is a cheaper and logistically easier test than the more
both normal morphology (r = −.25, p = .038) and viability (r = −.261, popular SCSA (sperm chromatin structure assay) and TUNEL (terminal
p = .030). deoxynucleotidyl transferase-­mediated dUTP nick-­end labelling) as-
A significant and positive correlation between the proportion of says, with some even suggesting that TBA may measure DNA–pro-
spermatozoa with DNA denaturation (AO-­red cells) and those with tein interactions better than the latter two (Schlegel & Paduch, 2005).
abnormal chromatin structure (TB-­dark cells) was shown (r = .431, In contrast with our results, it has been reported that the difference
p < .001). in the proportion of spermatozoa with abnormal, easily denaturable

T A B L E   2   Correlation analysis between

Abnormal chromatin structure Sperm DNA denaturation
sperm parameters and sperm DNA
Correlation Correlation assessment
Sperm parameters coefficient (r) p-­value coefficient (r) p-­value

Sperm −.42 .73 −.84 .49

concentration
Total sperm motility −.244 .044* −.12 .324
Normal morphology −.449 <.001* −.25 .038*
Viability −.374 .002* −.261 .030*

*p < .05.
AJINA et al. |
      5 of 6

DNA is not statistically significant between infertile and fertile men protamine deposition or aberrant protamine subtypes may also in-
(Hoshi, Katayose, Yanagida, Kimura, & Sato, 1996; Tejada et al., 1984). duce sperm DNA denaturation (Bianchi, Manicardi, Bizarro, Bianchi,
In addition to the high levels of chromatin abnormalities, our patients & Sakkas, 1993; Lolis et al., 1996) that is significantly correlated with
showed significantly higher levels of DNA denaturation compared high levels of free sperm SH groups, suggesting that DD may result
with controls. This observation is in agreement with the reports of from incomplete oxidation of sperm protamine SH groups during sper-
several studies which showed an increase in the percentage of AO-­ migenesis (Zini, Kamal, & Phang, 2001). The possible role of topoisom-
red cells in infertile group compared with fertile donors (Talebi et al., erase II in the introduction and relegation of such strandbreaks has also
2008). Using SCSA, infertile men have higher percentage of sperma- been shown (Laberge & Boissonneault, 2005). The final step in sperm
tozoa with denaturated DNA than fertile donors, noting that male chromatin packaging during nuclear maturation includes formation of
infertility is associated with poor sperm DNA integrity.(Zini, Bielecki, covalent disulphide bonds between cysteine residues of protamines.
Phang, & Zenzes, 2001). Similarly, AOT found in fertile population ex- If chromatin remodelling is perturbed, then most of the chromatin re-
hibits least amount of DNA abnormalities versus subfertile population mains in the less compact and more hydrated nucleosomic form with
(Varghese et al., 2009). In addition to the importance of DNA integrity the increased affinity for cationic dyes, including TB and AO.
assessment, it is believed that sperm chromatin condensation has also In conclusion, our study shows that patients with abnormal
a critical role in male fertility, early embryonic development and preg- sperm chromatin structure had a significantly greater incidence of
nancy outcome (Bhattacharya, 2008; Nijs et al., 2009; Tesarik et al., sperm DNA denaturation. Moreover, significant correlations among
2002). The effect of altered sperm chromatin integrity on postimplan- the levels of sperm chromatin abnormalities, DNA denaturation and
tation embryonic development and also on recurrent pregnancy loss is altered semen parameters were found. Our results suggest that acri-
still a matter of debate, and there are insufficient cytochemical-­based dine orange test (AOT) and toluidine blue assay (TBA) may be re-
studies indicating the effects of sperm chromatin abnormalities on garded as useful tools for the evaluation of sperm DNA integrity in
unexplained spontaneous recurrent abortion (Kazerooni et al., 2009; infertile men.
Saxena et al., 2008). A higher percentage of abnormal spermatozoa
in patients with RSA compared with fertile controls suggesting that
AC KNOW L ED G EM ENTS
AOT is a useful tool in evaluation of sperm nuclear chromatin integrity
which is essential for normal embryonic development (Talebi et al., This study was supported by the funds allocated to the Research Unit
2012). However, Kazerooni et al. (2009), using acridine orange, did of Histology and Genetic UR12ES10 by the “Ministère Tunisien de
not found a significant difference between fertile group and patients l’Enseingement Supérieur et de la Recherche Scientifique”. We thank
with RSA. Another work has indicated that sperm cells with denatured all the staff in the Histology-­Embryology Laboratory and all the indi-
DNA as detected by AOT may result in zygotes, but the resulting em- viduals who volunteered to participate in this study.
bryo cannot complete its normal growth and leads to early pregnancy
loss (Virant-­Klun et al., 2002).
REFERENCES
In the present study, the proportion of AO-­red cells correlated
significantly with the proportion of TB-­dark cells, suggesting that the Acheson, R. M. (1973). ed. Acridines. New York, NY, USA: Interscience
Publishers.
susceptibility of sperm DNA to denaturation may be in part the result
Aoki, V. W., & Carrell, D. T. (2003). Human protamines and the developing
of incomplete chromatin compaction and stabilisation. Other authors spermatid: Their structure, function, expression and relationship with
supported our findings, noting that spermatozoa with loosely packaged male infertility. Asian Journal of Andrology, 5, 315–324.
chromatin is sensitive and become single stranded when exposed to Aoki, V. W., Emery, B. R., Liu, L., & Carrell, D. T. (2006). Protamine levels vary
between individual sperm cells of infertile human male and correlate
acid or heat denaturing conditions (Chohan, Griffin, & Carrell, 2004).
with viability and DNA integrity. Journal of Andrology, 6, 890–898.
Furthermore, DNA could be denatured to a single strand, in the case Auger, J., & Eustache, F. (2000). Standardisation de la classification mor-
of immature sperm nuclei with histone or S-­S-­poor protamine (Haruo, phologique des spermatozoïdes humains selon la méthode de David
Kaoru, Shinako, Hidekazu, & Akira, 2003). Such a correlation has been modifiée. Andrologie, 10, 358–373.
Belettia, M. E., & Mello, M. L. S. (2004). Comparison between the tolui-
also reported by several studies using the same tests (Erenpreisa et al.,
dine blue stain and the Feulgen reaction for evaluation of rabbit sperm
2003; Erenpreiss et al., 2001 and Erenpreiss et al., 2004).The strength
chromatin condensation and their relationship with sperm morphology.
relationship between sperm DNA denaturation and abnormal chroma- Theriogenology, 62, 398–402.
tin structure with higher levels of sperm DNA damage in infertile com- Bhattacharya, S. M. (2008). Association of various sperm parameters with
pared with fertile men indicates that male infertility may be associated unexplained repeated early pregnancy loss— which is most important?
International Urology and Nephrology, 40, 391–395.
with defects in sperm chromatin conformation or compaction. In fact,
Bianchi, P. G., Manicardi, D., Bizarro, D., Bianchi, U., & Sakkas, D. (1993).
during spermiogenesis, protamines facilitate strong intermolecular at- Effect of deoxyribonucleic acid protamination on fluorochrome stain-
traction between the positively charged protamine and the negatively ing and in situ nick-­translation of murine and human spermatozoa.
charged DNA to facilitate a compact DNA–protamine structure (Aoki Biology of Reproduction, 49, 1083–1088.
Bianchi, P. G., Manicardi, G. C., Urner, F., Campana, A., & Sakkas, D. (1996).
& Carrell, 2003). Hence, it is believed that abnormalities in protamine/
Chromatin packaging and morphology in ejaculated human spermato-
DNA content can increase sperm DNA vulnerability to assault (Aoki, zoa: Evidence of hidden anomalies in normal spermatozoa. Molecular
Emery, Liu, & Carrell, 2006). Other mechanisms such as deficient Human Reproduction, 3, 139–144.
 |
6 of 6       AJINA et al.

Carrell, D. T., & Liu, L. (2001). Altered protamine 2 expression is uncommon Lolis, D., Georgiou, I., Syrrou, M., Zikopoulos, K., Konstantelli, M., &
in donors of known fertility, but common among men with poor fertil- Messinis, I. (1996). Chromomycin A3-­staining as an indicator of prota-
izing capacity, and may reflect other abnormalities of spermiogenesis. mine deficiency and fertilization. International Journal of Andrology, 19,
Journal of Andrology, 22, 604–610. 23–27.
Chohan, K. R., Griffin, J. T., & Carrell, D. T. (2004). Evaluation of chromatin Nijs, M., Creemers, E., Cox, A., Franssen, K., Janssen, M., Vanheusden, E., , …
integrity in human sperm using acridine orange staining with different Ombelet, W. (2009). Chromomycin A3 staining, sperm chromatin struc-
fixatives and after Cryopreservation. Andrologia, 36, 321–326. ture assay and hyaluronic acid binding assay as predictors for assisted
Chohan, K. R., Griffin, J. T., Lafromboise, M., DeJonge, C. J., & Carrell, D. T. reproductive outcome. Reproductive Biomedicine Online, 19, 671–684.
(2006). Comparison of chromatin assays for DNA fragmentation evalu- Oliva, R. (2006). Protamines and male infertility. Human Reproduction
ation in human sperm. Journal of Andrology, 27, 53–59. Update, 12, 417–435.
Dadoune, J. P. (2003). Expression of mammalian spermatozoa nucleopro- Saxena, P., Misro, M. M., Chaki, S. P., Chorpa, K., Roy, S., & Nandan, D.
teins. Microscopy Research and Technique, 61, 56–75. (2008). Is abnormal sperm function an indicator among couples with
De Jonge, C. (2002). Clinical value of sperm nuclear DNA assessment. recurrent pregnancy loss? Fertility and Sterility, 90, 1854–1858.
Human Fertility, 5, 51–53. Schlegel, P. N., & Paduch, D. A. (2005). Yet another test of sperm chromatin
Dennis, E. M., Hannah, B., Scott, F., Avner, H., & Huai, L. F. (2010). The ef- structure. Fertility and Sterility, 84, 854–859.
fect of semen processing on sperm DNA integrity: Comparison of two Spano, M., Bonde, J. P., Hjollund, H. I., Kolstad, H. A., Cordelli, E., & Leter,
techniques using the novel Toluidine Blue Assay. European Journal of G. (2000). Sperm chromatin damage impairs human fertility. The
Obstetrics & Gynecology and Reproductive Biology, 151, 176–180. Danish First Pregnancy Planner Study Team. Fertility and Sterility, 73,
Erenpreisa, J., Erenpreiss, J., Freivalds, T., Slaidina, M., Krampe, R., Butikova, 43–50.
J., … Pjanova, D. (2003). Toluidine blue test for sperm DNA integrity Talebi, A. R., Moein, M. R., Tabibnejad, N., & Ghasemzadeh, J. (2008). Effect
and elaboration of image cytometry algorithm. Cytometry, 52, 19–27. of varicocele on chromatin condensation and DNA integrity of ejacu-
Erenpreiss, J., Bars, J., Lipatnikova, V., Erenpreisa, J., & Zalkalns, J. (2001). lated spermatozoa using cytochemical tests. Andrologia, 40, 245–251.
Comparative study of cytochemical tests for sperm chromatin integrity. Talebi, A. R., Vahidi, S., Aflatoonian, A., Ghasemi, N., Ghasemzadeh, J.,
Journal of Andrology, 22, 5–53. Firoozabadi, R. D., & Moein, M. R. (2012). Cytochemical evaluation of
Erenpreiss, J., Jepson, K., Giwercman, A., Tsarev, I., Erenpreisa, J., & Spano, sperm chromatin and DNA integrity in couples with unexplained recur-
M. (2004). Toluidine blue cytometry test for sperm DNA conformation: rent spontaneous abortions. Andrologia, 44, 462–470.
Comparison with the flow cytometric sperm chromatin structure and Tejada, R. I., Mitchell, J. C., Norman, A., Marik, J. J., & Friedman, S. (1984). A
TUNEL assays. Human Reproduction, 19, 2277–2282. test for the practical evaluation of male fertility by acridine orange (ao)
Evenson, D. P., Jost, L. K., Marshall, D., Zinaman, M. J., Clegg, E., Purvis, K., fluorescence. Fertility and Sterility, 42, 87–91.
… Claussen, O. P. (1999). Utility of the sperm chromatin structure assay Tesarik, J., Mendoza, C., & Greco, E. (2002). Paternal effects acting during
as a diagnostic and prognostic tool in human fertility clinic. Human the first cell cycle of human preimplantation development after ICSI.
Reproduction, 14, 1039–1049. Human Reproduction, 17, 184–189.
Haruo, K., Kaoru, Y., Shinako, H., Hidekazu, Y., & Akira, S. (2003). Use of Tsarev, I., Bungum, M., Giwercman, A., Erenpreisa, J., Ebessen, T., Ernst,
diamide–acridine orange fluorescence staining to detect aberrant pro- E., & Erenpreiss, J. (2009). Evaluation of male fertility potential by
tamination of human-­ejaculated sperm nuclei. Fertility and Sterility, 79, Toluidine Blue test for sperm chromatin structure assessment. Human
670–676. Reproduction, 1, 1–6.
Hee-Sun, K., Moon Joo, K., Sung Ah, K, Sun Kyung, O., Hoon, K., Seung-Yup, Varghese, A. C., Bragais, F. M., Mukhopadhyay, D., Kundu, S., Pal, M.,
K., … Young Min, C. (2013). The utility of sperm DNA damage assay Bhattacharyya, A. K., & Agarwal, A. (2009). Human sperm DNA integ-
using toluidine blue and aniline blue staining in routine semen analysis. rity in normal and abnormal semen samples and its correlation with
Clinical and Experimental Reproductive Medicine, 1, 23–28. sperm characteristics. Andrologia, 41, 207–215.
Hoshi, K., Katayose, H., Yanagida, K., Kimura, Y., & Sato, A. (1996). The rela- Virant-Klun, I., Tomazevic, T., & Meden-Vrtovec, H. (2002). Sperm sin-
tionship between acridine orange fluorescence of sperm nuclei and the gle stranded DNA, detected by acridine orange staining, reduces
fertilizing ability of human sperm. Fertility and Sterility, 66, 634–639. fertilization and quality of ICSI-­derived embryos. Journal of Assisted
Iranpour, F. G., Nasr-Esfahani, M. H., Valojerdi, M. R., & al-Taraihi, T. M. Reproduction and Genetics, 19, 319–328.
(2000). Chromomycin A3 staining as a useful tool for evaluation of male World Health Organization (2010). Laboratory Manual for the Examination
fertility. Journal of Assisted Reproduction and Genetics, 1, 60–66. and Processing of Human Semen, 5th ed.. New York: Cambridge
Kazerooni, T., Asadi, N., Jadid, L., Kazerooni, M., Ghanadi, A., Ghaffarpasand, University Press.
F., … Zolghadr, J. (2009). Evaluation of sperm’s chromatin quality Zini, A., Bielecki, R., Phang, D., & Zenzes, M. T. (2001). Correlations be-
with acridine orange test, chromomycin A3 and aniline blue staining tween two markers of sperm DNA integrity, DNA denaturation and
in couples with unexplained recurrent abortion. Journal of Assisted DNA fragmentation, in fertile and infertile men. Fertility and Sterility,
Reproduction and Genetics, 26, 591–596. 75, 674–677.
Kosower, N. S., Katayose, H., & Yanagimachi, R. (1992). Thiol-­disulfide Zini, A., Kamal, K. M., & Phang, D. (2001). Free thiols in human spermato-
status and acridine orange fluorescence of mammalian sperm nuclei. zoa: Correlation with sperm DNA integrity. Urology, 58, 80–84.
Journal of Andrology, 13, 342–348.
Laberge, R., & Boissonneault, G. (2005). On the nature and origin of DNA
strand breaks in elongating spermatids. Biology of Reproduction, 73,
How to cite this article: Ajina T, Ammar O, Haouas Z, et al.
289–296.
Assessment of human sperm DNA integrity using two
Larson, K. L., De Jonge, C. J., Barnes, A. M., Jost, L. K., & Evenson, D. P.
(2000). Sperm chromatin structure assay parameters as predictors of cytochemical tests: Acridine orange test and toluidine blue assay.
failed pregnancy following assisted reproductive techniques. Human Andrologia. 2017;49:e12765. https://doi.org/10.1111/and.12765
Reproduction, 15, 1717–1722.