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Article
Paternal age and sperm DNA decay: discrepancy
between chromomycin and aniline blue staining
Stéphanie Belloc, graduate of medicine and reproductive biology, Paris VI Tenon Hospital,
has trained in reproductive biology since 2002. She is currently working as the head of
the male infertility department in Eylau Laboratories, Paris. She researches actively in the
area of assisted reproduction technology within the Eylau team, particularly male predictive
factors, for example DNA fragmentation, sperm chromatin decondensation and intra
morphologically selected sperm injection (IMSI) tests.

Dr Stéphanie Belloc

Stéphanie Belloc1, Moncef Benkhalifa1,2, Anne Marie Junca1, Martine Dumont1, Paul Cohen Bacrie1, Yves Ménézo1,2,3
1
UNILABS, Centre d’AMP Eylau, Clinique Pierre Cherest et Clinique de la Muette, 55 Rue Saint Didier, 75116 Paris,
France; 2ATL, R and D, 78320 La Verriere, France
3
Correspondence: e-mail: yves.menezo@club-internet.fr

Abstract
The effect of paternal age on sperm DNA fragmentation and decondensation was determined in a retrospective study involving
1769 patients. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay was used to
assess fragmentation, and DNA decondensation was measured with either chromomycin or aniline blue staining. The impact of
atypical forms was also analysed. DNA fragmentation increases with age, but is independent of the percentage of atypical forms.
Both staining techniques revealed a negative correlation between the quality of sperm packaging and the percentage of atypical
forms. Decondensation increases with increasing age and fragmentation when measured with chromomycin; however, an inverse
relationship is observed when testing is performed using aniline blue. These observations are discussed in relation to the specificity
of the dyes, the deposition of protamines and the impact of age and reactive oxygen species on protamine cross-linking.

Keywords: aniline blue, chromomycin, paternal age, sperm decay

Introduction
For many years, with the advent of IVF, male fertility has
been defined in terms of the ability to produce an amount
of spermatozoa that is able to fertilize an oocyte. In human
IVF, the development of a 2- to 4-cell embryo was regarded
as a test for sperm-fertilizing ability. However, the impact of
sperm morphology on early embryonic development has now
been clearly demonstrated, even in the presence of acceptable
fertilization rates (Ron-el et al., 1991; Janny and Ménézo, 1994).
The consensus of opinion now is that male fertility is not defined
by the capacity of a spermatozoon to penetrate the oocyte, but
instead by the ability of a spermatozoon to establish a full-term
pregnancy (Lewis and Aitken, 2005; Ménézo, 2006; Wyrobek
et al., 2006). Intracytoplasmic sperm injection (ICSI), which
bypasses the problem of sperm penetration, does not achieve
full-term pregnancies for an important part of the infertile
population. Although this technique has shown a high efficiency,
its complete safety is far from demonstrated, and negative real
or potential aspects have been highlighted (Aitken and Krausz,
2001; Aitken et al., 2004; Morozumi and Yanagimachi, 2005).
264 Basic parameters such as concentration, motility and morphology
are of limited value in determining the embryotrophic potential of
spermatozoa. Only motility appears to have the highest correlated
values in relation to sperm DNA fragmentation (Evenson and
Wixon, 2006a). Progress has been made with the analysis of
DNA integrity, including secondary and tertiary structure (i.e.
DNA fragmentation and decondensation). However, formation of
DNA adducts (Badouard et al., 2008) and aberrant methylation
of imprinted loci in spermatozoa (Kobayashi et al., 2007) that
modify the primary structure are also sources of further anomalies.
Multiple technologies have been used to detect chromatin
structure anomalies: sperm chromatin structure assay (Evenson
et al., 2002) for both fragmentation and decondensation, TdT
(terminal deoxynucleotidyl transferase)-mediated dUDP nick-
end labelling (TUNEL) for fragmentation. Aniline or toluidine
blue (Dadoune et al., 1988; Auger et al., 1990; Hammadeh et
al., 2001), which selectively stain lysine-rich histone proteins,
and chromomycin A3 (CMA3), a guanine–cytosine-specific
fluorochrome that competes with protamines for access to DNA
(Sakkas et al., 1996), have been used to detect anomalies in
protamine packaging.

© 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

Although paternal age has been implicated in an increase in rates
of spontaneous abortion (de la Rochebrochard and Thonneau,
2002; Klonoff-Cohen and Natarajan, 2004), the possibility of
other negative effects related to paternal age has only recently
been considered. Progress in the analysis of sperm DNA structure
has clearly demonstrated that sperm DNA fragmentation increases
with age, with or without oxidative stress as an additional
factor (Evenson et al., 2002; Aitken et al., 2003; Guérin et al.,
2005; Evenson and Wixon, 2006b). There is growing evidence
that these defects in sperm DNA may have a negative impact
on offspring (Aitken and Baker, 2006; Wyrobek et al., 2006;
Fernández-Gonzalez et al., 2008). There is now clear evidence
that increasing male age increases the frequency and the amount
of fragmentation, thus leading to impaired fertility (Aitken, 2007;
Vagnini et al., 2007; Belloc et al., 2008). The impact of age also
affects the quality of the sperm genome (Wyrobek et al., 2006).
However, no clear correlation has been established between DNA
fragmentation and packaging, i.e. tertiary structure in relation
to age. Wyrobek et al. (2006) found no correlation between
fragmentation and quality of packaging; in contrast, Plastira et
al. (2007) claimed that, for a short cohort of oligoasthenospermic
patients, fragmentation is linked to decondensation as measured
with chromomycin, and both are correlated with age. Sperm
packaging is of major importance, and it is surprising that this
parameter is often neglected in assisted reproduction treatment.
The tertiary structure of DNA carries epigenetic messages to the
embryo, affects post-fertilization genome reprogramming, and
then has an impact on early embryonic development (Carrell,
2008; Rousseaux et al., 2008). This retrospective study, involving
more than 1769 patients, studied sperm DNA fragmentation
using a TUNEL assay, and its correlation with decondensation,
measured either with chromomycin or with aniline blue. These
parameters were analysed in relation to paternal age and the
percentage of typical forms.
Materials and methods
All of the patients (n = 1769) were under clinical management
in a fertility centre that consisted of two clinics and one common
laboratory. The work was approved by the ethical committee
of the clinics and did not require consent from the Government
Agence de BioMedecine, which governs all assisted reproduction
procedures in France. Patients were informed and signed a
consent form.
Semen analyses were carried out according to World Health
Organization guidelines (1999) (see also Cohen Bacrie et al.,
2009). Sperm morphology analysis was performed after Harris–
Schorr staining. In order to avoid bias, abstinence length was
recorded; as strictly recommended, only samples obtained after
less than 5 days of abstinence were analysed.
DNA fragmentation was performed using the classical TUNEL
test. A fluorescein isothiocyanate-labelled deoxyuridine
triphosphate kit (Roche Diagnostics Corporation, Mannheim,
Germany) was used according to the instructions of the
manufacturer for in-situ or flow cytometry techniques. Briefly,
after centrifugation and washing (10 min at 750 g) to remove
seminal plasma, spermatozoa were treated for 20 min with
trypsin EDTA at 37oC, followed by 20 min of treatment at 37oC
with 1% of trisodium citrate for hypotonic shock to facilitate the
fragmented DNA TUNEL procedure, after which spermatozoa
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Article - Sperm DNA staining - S Belloc et al.
were fixed with 3:1 methanol:ethanol solution. Flow cytometry
was used to detect TUNEL staining of spermatozoa from patients
with >1 s 106 spermatozoa/ml. A Beckman Coulter (Villepinte,
France) cytometer at 488 nm, with a 15-mW argon-ion laser as
the light source, detected a minimum of 5000 events. Debris
were eliminated by establishing a region around the population
of interest. The percentage of labelled spermatozoa was
characterized by identifying a region that included >90% of
events in the frequency histogram of the positive control (Sergerie
et al., 2005). The data were processed on a computer using LYSIS
software (Becton-Dickenson, Mountain View, CA). For patients
with <1 s 106 spermatozoa/ml, an in-situ TUNEL technique
was used and a minimum of 200 spermatozoa were analysed by
epifluorescence microscopy. Positive and negative controls were
used daily. For a positive control, spermatozoa were treated with
0.2 IU of deoxyribonuclease (DNase I, RNase-free; Roche) for
20 min at room temperature, whereas the enzyme was omitted
from the reaction mixture and replaced with distilled water for
the negative control. To assess the efficiency and concordance of
the flow cytometry and the in-situ techniques, 68 semen samples
were analysed simultaneously with both techniques.
In a first group (from June 2007 to February 2008), chromatin
condensation was assessed by aniline blue staining as previously
described (Dadoune et al., 1988; Hammadeh et al., 1996). Briefly,
after sperm preparation, the pellet was incubated with sodium
dodecylsulphate (SDS; 1%) in sodium citrate (permeabilization).
Processed spermatozoa (5 Ml) were then spread onto a glass
slide, and allowed to dry. The smears were fixed in 3% buffered
glutaraldehyde in 0.2 mol/l phosphate-buffered saline (pH 7.2) for
30 min. The slides were then stained with 5% aqueous aniline
blue mixed with 4% acetic acid (pH 3.5) for 15 min at ambient
temperature. A total of 200 sperm cells were evaluated and the
percentage of blue-stained sperm heads was calculated. Two
classes of staining intensities were distinguished: unstained and
completely or partially stained sperm heads.
In a second group (from March 2008 onwards), chromatin
condensation was assessed with chromomycin. After sperm
processing, permeabilization and fixation were performed in
the same way as for aniline blue. Staining was then performed
first with chromomycin in NaCl/HEPES for 10 min at room
temperature. After washing with NaCl/HEPES for 30 min, the
samples were incubated with methyl green for 10 min at room
temperature; after washing in HEPES buffer, labelling was
assessed by fluorescence.
Statistical analysis
Age was ranked in four categories: <34, 35–39, 40–49 and 50
years and over. Four classes of fragmentation were used: <20,
20–30, 30–40 and over 40%, the limits being defined according
to experience and also to literature indicating a threshold at 30%
for fragmentation. The statistical analysis used was the Kruskall–
Wallis for these non-Gaussian populations. For the correlations,
Pearson, Spearman and Kendall coefficients were calculated.
The data were corrected for abstinence (strictly less than 5 days), as
the preliminary results showed a strong increase in fragmentation
with increasing abstinence, (P < 0.007, Kruskall–Wallis in a
subgroup with abstinence between 5 and 7 days).
265
 Article - Sperm DNA staining - S Belloc et al.

Results decondensation as measured by chromomycin increased with age

Sperm DNA fragmentation measured with TUNEL increased
with age (Table 1): Kruskall–Wallis for non-Gaussian variables:
P < 0.001 (Figure 1). A weak but significant correlation was
found: Pearson: R = 0.221 (P < 0.001); Spearman: R = 0.162
(P < 0.001); Kendall: R = 0.112 (P < 0.001). TUNEL-linked
fragmentation did not increase with the percentage of atypical
forms (data not shown).
There was a clear discrepancy between the results obtained
for the analysis of sperm DNA decondensation (Tables 2 and
3) with aniline blue and chromomycin staining. First of all,
(P = 0.002, Kruskall–Wallis for non-Gaussian variables; Figure
2); all of the correlation tests yielded a significant but weak
correlation: Pearson: R = 0.115 (P = 0.003); Spearman: R = 0.099
(P = 0.011); Kendall: R = 0.072 (P = 0.01). However, this increase
was obviously linked to a strong jump observed at ages 50 years
or more. Decondensation measured with chromomycin increased
with DNA fragmentation (P < 0.001).
In contrast, the values obtained for sperm DNA decondensation
with aniline blue staining decreased, but not significantly, with
increased age (Figure 3). No significant correlation could be
found between DNA fragmentation and decondensation in this
group, although decondensation did decrease.

Table 1. Sperm DNA fragmentation according to age, as measured by TUNEL assay.

Age (years)
b34 35–39 40–49 r50

Mean DNA fragmentation (%) 21.1 23.2 24.7 31.9

Standard deviation ± 10.3 ± 10.8 ± 12.6 ± 17.7
95% confidence interval 20.2–21.9 22.3–24.0 23.6–25.8 28.7–35.1

Kruskall–Wallis for non-Gaussian variables: P < 0.001.

Figure 1. Sperm DNA fragmentation (FRG) according to age,

measured with TUNEL (P < 0.001, Kruskall–Wallis).

Table 2. Sperm DNA decondensation according to age, measured either with aniline
blue or chromomycin staining.

Age (years)
b34 35–39 40–49 r50

Aniline blue staining

n (total = 1111) 371 359 315 66
Mean decondensation (%) 16.8 16.2 15.9 13.7
Standard deviation ± 10.7 ± 10.1 ± 10.2 ± 9.7
95% confidence interval 15.8–17.9 15.1–17.2 14.8–17.1 11.3–16.1
CMA3 staining
n (total = 658) 178 234 194 52
Mean decondensation (%) 12.1 11.1 12.4 16.4
Standard deviation ± 8.5 ± 8.4 ± 8.4 ± 10.5
95% confidence interval 10.9–13.4 10.1–12.2 11.2–13.6 13.1–19

CMA3 = chromomycin.
The decrease observed with aniline blue was not statistically significant, but the increase observed for CMA3
266 was significant (P = 0.002), especially in relation to the increase observed in the r50 years age group.

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Table 3. Relationship between sperm DNA fragmentation measured with

TUNEL assay and decondensation measured with aniline blue or chromomycin.

DNA fragmentation (%)

<20 20–<30 30–<40 r40

Aniline blue staining

n (total = 1111) 483 331 194 103
Mean decondensation (%) 15.5 16.8 16.6 16.8
Standard deviation ± 9.7 ± 10.6 ± 11.1 ± 10.7
95% confidence interval 14.6–16.5 15.6–17.9 15–18.1 14.7–18.9
CMA3 staining
n (total = 658) 306 194 89 69
Mean decondensation (%) 11.9 11.3 12.8 15.1
Standard deviation ± 9.2 ± 7.4 ± 8.6 ± ß9.3
95% confidence interval 10.9–12.9 10.2–12.3 11–14.6 12.9–17.4

CMA3 = chromomycin.
Decondensation measured with aniline blue was not statistically significantly different between any of the
DNA fragmentation groups, whereas CMA3 was positively correlated with DNA fragmentation (P < 0.001).

Figure 2. Sperm DNA decondensation (DCS) according to age, Figure 3. Sperm DNA decondensation (DCS) according to age,
measured with chromomycin (P = 0.002, Kruskall–Wallis). measured with aniline blue (non significant, Kruskall–Wallis).

The absolute decondensation values observed were higher in
the aniline blue group for all age groups, except for the patients
aged 50 and over where it was similar (chromomycin 16.4%
versus aniline blue 13.7%).
A significant correlation was found between DNA packaging
and the percentage of atypical forms for both aniline blue
and CMA3 (P < 0.006, Kruskall–Wallis for each test).
Chromomycin: Pearson: R = 0.261 (P = 0.013); Spearman: R
= 0.249 (P = 0.017); Kendall: R = 0.174 (P = 0.022). Aniline
blue: Pearson: R = 0.147 (P = 0.046); Spearman: R = 0.241 (P
< 0.001); Kendall: R = 0.172 (P < 0.001).
However, the fact that the R values are low must be addressed
and this limits the biological meaning of the correlations.
Yet, this is consistent with the fact that fragmentation may be
strongly linked to defective apoptosis and increased (local)
production of reactive oxygen species (ROS) (Kodama et al.,
1997; Lopes et al., 1998). This level of DNA damage affects
the outcome of assisted reproduction procedures, especially
in artificial insemination programmes with the male partner’s
spermatozoa (Belloc et al., 2008). In this study, atypical forms
and DNA fragmentation are not linked. Abnormal forms and
sperm decondensation are strongly linked on the basis of both
aniline blue and chromomycin staining, in agreement with the
work of Franken et al. (1999).

Discussion The analysis of tertiary DNA structure, i.e. condensation

These data confirm, in a large population, several observations
described in the literature. Sperm DNA fragmentation increases
with age (Aitken et al., 2003; Wyrobek et al., 2006; Vagnini et al.,
2007). The age of 40 years can be considered as a turning point
(Evenson et al., 2002; Guérin et al., 2005; Belloc et al., 2008).
of DNA, is far less studied and has been rather neglected,
especially as it does not impair fertilization; condensation is
usually considered to be a sign of gamete maturity. In addition
to sperm chromatin structure assay, two further tools can be
used to check DNA tertiary structure: guanine–cytosine-
specific fluorochrome, chromomycin A3, which competes 267

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with protamines for access to DNA, and aniline blue, a dye
that selectively stains lysine-rich histone proteins, leaving
the arginine-rich protamine proteins unstained. A positive
correlation was found between age and CMA3 staining, but
in contrast with aniline blue, DNA staining has a tendency to
decrease with age (not significant). In the same manner, no
relationship could be found between fragmentation and aniline
blue staining whereas, using CMA3 staining, decondensation
increased as DNA fragmentation increased. Manicardi et al.
(1995) and Aoki et al. (2005) found a correlation between
defective tertiary structure and DNA strand breaks as measured
by nicks, but with wide individual variations. In contrast, as
suggested by Wyrobek et al. (2006) using sperm chromatin
structure assay, the increases in DNA fragmentation index and
decondensation arise from different mechanisms. Using the
same technique, the same result was observed: fragmentation
and decondensation may vary independently at an individual
level (Ménézo et al., 2007a). This fits with the data obtained
using sperm chromatin structure assay and aniline blue, but not
CMA3 as it is less specific as DNA breaks may simply allow
increased access for CMA3 to DNA.
Decondensation is mainly a feature of sperm maturity in
relation to protamine deposition, starting from the deposition
of transition proteins to replacement of histones by protamines
(Foresta et al., 1992). A defective ratio of protamine 1:protamine
2 seems to be associated with reduced condensation and
fertility (Balhorn et al., 1988; Corzett et al., 2002). Aoki et
al. (2005), more surprisingly, found that DNA fragmentation
increases when the concentration of either protamine 1 or
protamine 2 decreases. This probably renders sperm cells more
susceptible to DNA defects. However, it could be speculated
that this also allows extended access to DNA repair capacity
during spermiogenesis, when this is still available. These
observations are not necessarily in favour of a correlation
between decondensation and DNA fragmentation.
The second common aspect is that defective cross-linking
between cysteine residues impedes complete packaging of the
nucleo-protamine complex. This fits with these data and suggests
that a high intake of antioxidants can lead to decompaction
of the nucleus, independently of sperm maturity (Ménézo et
al., 2007b). This faulty cross-linking is not consistent with a
positive correlation between CMA3 staining and age, as ROS
increase and defence against them decreases with age, thus
decreasing CMA3 access to DNA.
The third point concerns other epigenetic aspects of nuclear
remodelling. Before being replaced by protamines, histones are
methylated and massively acetylated in elongating spermatids,
starting the reprogramming process; 15% of these histones
remain in the nucleus. Methylation of DNA and proteins also
affects the tertiary structure of the DNA, even if the essential
role is probably linked to imprinting of sex-specific epigenetic
information. Global methylation patterns in spermatozoa
are not different in men with abnormal protamine expression
(Aoki et al., 2006). The protective function of tight protamine
packaging on sperm DNA is one aspect. However, the specific
epigenetic remodelling that modifies the tertiary structure of
DNA is important in the correct presentation of male DNA, a
prerequisite for further correct decondensation of the paternal
genetic material at the time of fertilization (Carrell, 2008;
268 Rousseaux et al., 2008), and then during early embryonic
development. This study found a strong correlation between
faulty packaging and abnormal forms, and this correlation was
higher with the use of aniline blue. This is in agreement with
Sati et al. (2008) who moreover found, using double probing,
that aniline blue is an excellent marker of maturity.
In conclusion, DNA fragmentation and decondensation are
not necessarily processes that are linked: aniline blue and
chromomycin do not assess exactly the same parameters of
sperm DNA tertiary structure. Due to its better specificity,
aniline blue appears to be more informative: fragmentation,
but not decondensation, increases with age. However, this does
not mean that this parameter should be neglected irrespective
of the age of the male partner: in the authors’ clinical units,
carrying out more than 4000 IVF/ICSI cycles per year, ongoing
pregnancies/deliveries have never been achieved after the use of
sperm samples showing decondensation of over 40%, measured
with either aniline blue or using sperm chromatin structure
assay. Anomalies in the tertiary structure of sperm DNA do
affect, sooner or later, normal embryonic development, even if
fertilization is not impaired (Rousseaux et al., 2008). However,
the age of the female partner can sometimes compensate, as the
oocyte is weakly able to repair some tertiary structure anomalies
(Ménézo et al., 2007a).
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Declaration: The authors report no financial or commercial
conflicts of interest.
Received 9 October 2008; refereed 17 November 2008;
accepted 20 March 2009.
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