create the summary of the findings "Methods
Patients
Semen samples from 60 men referred to our research
and clinical center for infertility were divided into two
groups for analysis. Case group consisted of 30 infertile
men with any level of SCI who were injured at least 1
year before the start of the study. The majority of
patients had been injured on the battlefield or in vehicular accidents. Cases with azoospermia and semen
samples contaminated with blood were excluded from
the study. The control group consisted of 30 healthy
donors with proven fertility and normal spermograms
and whose partners had successful pregnancies within
the last 24 months. Written informed consent was
obtained from the patients in both groups. This prospective study was approved by the institutional review
board of Shahid Sadoughi University of Medical
Sciences, Yazd, Iran.
Semen analysis
Samples from all patients in case group were taken using
penile vibratory stimulation or using electroejaculator
(EEJ) (Seager model 14 electroejaculator system,
Dalzell, VA, USA) by the same urologist. Stimulation
was performed by using 2.5 mm amplitude and a frequency of 100 Hz in 2–3 minutes period of time until
ejaculation occurred. Regarding EEJ, 12–15 V stimulation rate was applied in short time intervals until ejaculation. All specimens in control group were collected
by masturbation following 2–4 days of abstinence. All
raw semen samples were analyzed according to World
Health Organization criteria.21 After semen liquefaction, to evaluate morphological abnormalities, Diff
quick staining was applied. To determine sperm motility
we used Makler chamber (Sefi Medical Co., Haifa,
Israel) and phase-contrast microscopy (Olympus Co.,
Tokyo, Japan) at ×200. All analyses were performed
by one experienced laboratory technician blinded to
the study.
Sperm chromatin and DNA study
For the evaluation of sperm DNA integrity and chromatin condensation, four different cytochemical tests
including aniline blue (AB), chromomycin A3
(CMA3), toluidine blue (TB), and acridine orange
(AO) test were used.
AB staining
Regarding AB staining, fresh sperm smear of each case
was air dried and then fixed in 3% buffered glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 30
minutes at room temperature. Each smear was treated
with 5% aqueous AB stain in 4% acetic acid (pH 3.5)
for 5 minutes. At least 200 spermatozoa were counted
in each slide by light microscopy (Olympus Co.,
Tokyo, Japan).
CMA3 staining
At first, smears were dried and then fixed in Carnoys
solution (methanol/glacial acetic acid, 3:1) at 4°C for
10 minutes. Each slide was stained with 100 μl of
CMA3 (Sigma, St Louis, MO, USA) (0.25 mg/ml in
McIlvaine buffer; 7 ml citric acid, 0.1 M + 32.9 ml
Na2HPO4 7H2O 0.2 M, pH 7.0 containing 10 mM
MgCl2) for 20 minutes. Finally, the slides were washed
in buffer and mounted with buffered glycerol (1:1).
TB staining
To do this staining, after air drying of smears, they were
fixed in fresh 96% ethanol-acetone (1:1) at 4°C for 30
minutes and then hydrolyzed in 0.1 NHCl at 4°C for 5
minutes. The slides were rinsed thrice in distilled water
for 2 minutes and finally stained with 0.05% TB in
50% McIlvaine buffer ( pH 3.5) for 10 minutes at
room temperature.22,23 The chromatin quality of
Talebi et al. Sperm chromatin condensation, DNA integrity, and apoptosis in men with SCI
The Journal of Spinal Cord Medicine 2013 VOL. 36 NO. 2 141
spermatozoa was determined according to metachromatic staining of sperm heads with the aid of light
microscopy at ×100 magnification.
AO test
In this staining, smears were fixed overnight in Carnoy’s
solution after primary air dried. Each sample was
stained for 10 minutes in freshly prepared AO
(0.19 mg/ml) in McIlvaine phosphate citrate buffer
( pH 4) for 5 minutes. Smears were assessed on the
same day using fluorescent microscopy (460 nm filter).
The duration of illumination was restricted to 40
seconds per field.
Terminal deoxynucleotidyl transferase-mediated dUTP
nick-end labeling assay
To determine the percentage of apoptotic spermatozoa
in each sample, we used the terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling assay
(TUNEL) staining by In Situ Cell Death Detection
Kit (Roche Diagnostics GmbH, Mannheim,
Germany). At first, air-dried smears were washed in
phosphate-buffered saline (PBS) and then fixed in 4%
paraformaldehyde for 15 minutes at room temperature.
Then the slides were permeabilized with 0.5% Triton X100 in 0.1% sodium citrate for 5 minutes on ice. The permeabilized spermatozoa were washed in PBS, and then
covered with TUNEL reaction mixture in a dark room
at 37°C for 1 hour. Smears were treated with enhanced
by peroxidase (POD) convertor at 37°C for 1 hour. We
did the second washing with PBS and then enhanced in
50–60 μl diaminobenzidine for 15 minutes in room temperature. Final washing with PBS was done before staining
with hematoxylin for 30 seconds and dehydration by
serial alcohols (70, 90, and 100°) for 3 minutes. To clear
the slides, the xylens I and II were used before mounting.
At least 200 sperm cells in each smear were counted using
light microscopy at ×100 magnifications.
Statistical analysis
According to the results of a pilot investigation which
was done on a few patients, we considered 30 cases in
each group to find a difference by 80% power with a
5% significance (a = 0.05, b = 0.2). The Statistical
Package for the Social Sciences (SPSS) 15.0 software
was used to analyze the data. Data were expressed in
mean ± SD. The baseline characteristics of the two
groups of patients were compared using the Student
t-test for normal distributed variables and
Mann–Whitney U test for abnormal distributed variables. A P value of <0.05 was considered statistically
significant.
Results
The mean age was similar between case and control
groups (35.62 ± 5.65 and 36.43 ± 5.44, respectively).
The mean post injury years in case group was 13.37 ±
7.03 ranging from 3 to 28 years and the most frequent
level of injury was at lumbar region with frequency of
66.6%. The other injury levels were high thoracic
(20.8%), low thoracic (8.3%), and cervical (4.2%). The
results of sperm parameters analysis of both groups
are listed in Table 1. It should be noted that the duration
and level of injury have no effect on sperm parameters
and DNA chromatin status.
There was a significant difference between case and
control groups regarding sperm concentration and morphology (P < 0.05). In addition, the total motility
(grades “a” and “b” and “c”), the progressive (grades
“a” and “b”) and non-progressive motility (grade “c”)
were significantly higher in control men than patients
with SCI (P < 0.05). In addition, 75% of samples of
SCI patients have contaminated with bacteria and
white blood cells (≥106
) that specified the
leukocytospermia.
The sperm nuclear integrity of patients and control
group was shown in Table 2.
In sperm chromatin study, the rates of spermatozoa
that reacted with AB (AB+) were significantly different
in both groups (P < 0.05). The spermatozoa with
unstained or pale-blue nuclei were recorded as normal
and those with dark-blue nuclei were regarded as abnormal ones.11,22 Furthermore, 87.5% of patients with SCI
Table 1 Mean and standard deviation of sperm parameters of two groups under investigation
Sperm variables SCI (n = 30) Control (n = 30) P value*
Concentration (×106
/ml) 70.16 ± 46.32 105.70 ± 59.24 0.020
Total motility (%) (grade “a” + “b” + “c”) 19.58 ± 18.30 71.36 ± 8.38 0.000
Progressive motility (%) (grade “a” + “b”) 11.58 ± 13.94 58.30 ± 10.93 0.000
Non-progressive motility (%) (grade “c”) 8 ± 5.92 13.06 ± 4.63 0.001
Normal morphology (%) 13.04 ± 7.05 37.43 ± 15.66 0.000
SCI, spinal cord injury.
Data are presented as mean ± SD.
*SCI vs. control.
Talebi et al. Sperm chromatin condensation, DNA integrity, and apoptosis in men with SCI
142 The Journal of Spinal Cord Medicine 2013 VOL. 36 NO. 2
showed higher AB-reacted spermatozoa than “cut-off”
value (30%) (Fig. 1).24
Regarding CMA3 staining, there was a statistically
difference between the rates of CMA3-reacted spermatozoa in both groups. In other words, the percentage
of men with SCI who had higher CMA3+ sperm cells
than the “cut-off” value (30%)25 was 91.7% (Fig. 1).
The CMA3-reacted spermatozoa (CMA3+) showed
bright yellow stain, and non-reacted ones (CMA3−)
were yellowish green when viewed under axiplane fluorescent microscope with a 460 nm filter (Zeiss Co., Jena,
Germany) at ×100 magnification.
The rate of orange-red fluorescence spermatozoa was
significantly higher in patients with SCI in comparison
with control group (62.66 ± 18.14 vs. 30.06 ± 10.78;
P < 0.05). The percentage of green fluorescence spermatozoa (normal double-stranded DNA) and orange/red
fluorescence spermatozoa (abnormally denatured
DNA) per sample were calculated.11,22,26 Only 29.2%
of SCI men showed AO-reacted sperms below 50%
which considered as “cut-off” value for this test.27
In TB staining, we also found a significant difference
between control and case groups (P < 0.05). The percentage of men with SCI who had samples with more than
45% TB-reacted spermatozoa,28 was notably higher
than controls (P < 0.05) (Fig. 1). The scores were as followed: score 0 = light blue (good chromatin), score 1 =
dark blue (mild abnormal chromatin), and score 2 =
violet and purple (severe chromatin abnormality).29
So, the sum of spermatozoa with scores 1 and 2
(TB+) were assumed as abnormal chromatin, whereas
score 0 (TB−) as sperm cells with normal chromatin.
In TUNEL assay, the results showed that the rate of
sperm apoptosis was higher in patients with SCI when
compared with controls (32.91 ± 12.55 and 9.03 ±
2.94), respectively, (P < 0.05). The 79.2% of the
samples of patients with SCI had above 20% apoptotic
sperm cells (Fig. 1).30 Spermatozoa with light-bluestained nuclei were considered as alive and those with
dark brown pigments in their heads were counted as
apoptotic ones."