Cigarette smoking is a widely recognized health
hazard and a major case of mortality. Previous
studies have shown that one-third of the world’s
populations over the age of 15 years are smokers
Creatine kinase (CK) is an enzyme (EC 184.108.40.206)
expressed by various tissues and cell types that
require high energy. This enzyme reversibly catalyzes
conversion of creatine and adenosine triphosphate
(ATP) to phosphocreatine and adenosine
diphosphate (ADP). Its biological role is to
provide an ATP buffering system for tissues that require large amounts of energy (
Numerous researches have been undertaken regarding
the effect of cigarette smoking on male
reproductive function (
ADP, adenosine monophosphate (AMP), nicotinamide adenine dinucleotide phosphate (NADP), glucose 6-phosphate dehydrogenase, hexokinase and Triton x-100 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The highest grade and purity of reagents were used in this research.
This was a case-control study that performed in
Ahwaz, Iran. Study population was selected from
males who attended Razi Laboratory in Ahwaz,
Iran for routine semen analyses. Prior to the collection
of semen samples, information was obtained
from subjects regarding their ages, occupation,
smoking habits, alcohol consumption, and use
of other substances and drugs. Exclusion criteria
included alcohol use in the three months before
study entry, recent fever, exposure to gonad toxins
and heavy metals. A total of 147 men, ages 17 to
41 years were included in the study. There were 64
smokers (20-39 years) and 83 nonsmokers (17-41
years) enrolled. This study was conducted in 2009.
According to the number of cigarettes smoked per
day and duration of cigarettes smoked in a year, we
categorized participants as either light, moderate
or heavy smokers (
Smoking status of study participants
|Smoking status||Number of cigarettes/day||Number of cases (n)|
Smoking duration of study participants
|Smoking status||Duration of smoking (Y)||Number of cases (n)|
Semen samples were collected by masturbation
into sterile containers after sexual abstinence for 2
to 3 days. Semen samples were kept at 37°C and
processed immediately after complete liquefaction.
All semen samples were analyzed for appearance,
volume, pH, sperm motility, sperm count,
and sperm morphology according to World Health
Organization guidelines (
CK of each semen sample was partially isolated
as previously described by Miyaji et al. (
CK activity within seminal plasma, sperm cells and
total semen of each sample were separately measured
by the Rosalki method (
Results are presented as mean ± standard deviation (mean ± SD). All assays were performed in triplicate, and the mean was used for the calculation. Semen analysis and CK activity were compared using the independent samples t test in both smokers and nonsmokers. The t test was employed for comparisons between different subgroups. Coefficients of correlation were analyzed with linear (Pearson) analysis. P≤0.05 was considered statistically significant.
The study population consisted of 147 male participants.
Smoking status was classified as follows:
56.5% (83/147) were nonsmokers, 29.2% (43/147)
were short-term smokers, and 14.3% (21/147) were
long-term smokers. There were 17.7% (26/147)
light smokers, 20.4% (30/147) moderate smokers,
and 5.4% (8/147) who were heavy smokers.
Semen characteristics and CK activity in smokers
and nonsmokers are given in table 3. Semen
volume, concentration, motility, normal sperm
morphology, and CK activity in semen, seminal
plasma, and sperm in smokers were significantly
lower than nonsmokers (
There were statistically significant associations observed
between sperm motility, CK activity in seminal
plasma, sperm, and total semen in some of the
subgroups of male smokers compared to nonsmokers
The same results were obtained for CK activity
of sperm in short-term smokers (
Semen characteristics in non-smokers and smokers
|Characteristics||Non-smokers (n = 83)||Smokers (n = 64)||P value|
|4.1 ± 1.4||3.2 ± 1.06||<0.001|
|8.01 ± 0.22||8.03 ± 0.32||0.659|
|547.31 ± 193.8||377.83 ± 187.2||<0.001|
|764.24 ± 259.7||510.03 ± 205.98||<0.001|
|65.5 ± 22.9||52.8 ± 20.7||0.001|
|46.5 ± 13.8||38.6 ± 17.5||0.003|
|47.5 ± 18.9||37 ± 21.2||0.002|
|0.22 ± 0.07||0.16 ± 0.05||<0.001|
Sperm motility and CK activity of seminal plasma, sperm cells, total semen in smokers
|41.09 ± 18.4||31.9 ± 14.79*||41.38 ± 16.26||38.7 ± 19.02**||29.12 ± 13.05*||46.48 ± 13.84|
|418.18 ± 198*||289.05 ± 189.99*||432.35 ± 33.02***||358.13 ± 185.51*||274.5 ± 216*||599.23 ± 246.89|
|0.2 ± 0.01||0.12 ± 0.05*||0.17 ± .04*||0.16 ± 0.05*||0.12 ± 0.05*||0.22 ± 0.07|
|555.98 ± 186.4*||403.2 ± 214.4||577.27 ± 192*||490.57 ± 193.58*||356.12 ± 228.12*||788.48 ± 309.2|
*;p<0.001, **; p=0.02 and***; p=0.002 vs. the corresponding values for non-smokers.
The relationship between smoking duration and sperm motility (A), CK activity in seminal plasma (B), sperm (C), and total semen (D).
The relationship between numbers of cigarettes smoked daily and sperm motility (A), CK activity in seminal plasma (B), sperm (C), and total semen (D).
The relationship between sperm motility and CK activity in seminal plasma (A), sperm (B), and total semen (C) in smokers.
The relationship between sperm CK activity and CK activity in seminal plasma (A) and total semen (B) in smokers
There was a significant (r = 0.88, p<0.001) positive association among all the analyzed samples in smokers with respect to CK activity (Fig 4).
Smoking presents with a lifestyle hazard for those
who smoke. Although the lungs are known to be a
primary target for carcinogens in tobacco, numerous
studies have suggested that smoking is associated
with altered semen quality (
This study demonstrated that smoking duration
and number of cigarettes smoked per day affected
sperm motility. Thus, we found that males who
smoked for a duration of ≤10 years (short-term)
had 12% decreased sperm motility whereas those
who smoked for a duration of >10 and <20 years
had 31% lower sperm motility than nonsmokers.
Males who smoked ≤10 cigarettes per day (light)
had an approximately 11% lower sperm motility,
those who were moderate smokers (>10 and
≤20 cigarettes per day) had approximately 17%
less motility and heavy (>20 cigarettes per day)
smokers had approximately 37% lower sperm
motility compared to nonsmokers. Pasqualotto et
The findings of the present study support those
of numerous other studies, which show a significant
relation between smoking duration, sperm
concentration, and motility as well as between the
number of cigarette smoked daily, sperm concentration
and motility (
CK has two distinct isomeric forms, brain CK
(B-CK) and muscle CK (M-CK) which are present
in the midpiece region and the sperm tail, respectively
According to Pearson's linear analysis, we found a significant negative relation between duration of cigarette smoking and sperm motility in smokers. A statistically significant negative correlation was also observed for CK activity in seminal plasma, sperm cells and total semen in smokers. In addition, our data showed that the daily number of cigarettes smoked had a significant negative effect on both sperm motility and CK activity in seminal plasma, sperm cells, and total semen.
The present study showed an insignificant positive relation between sperm motility and with CK activity of seminal plasma, sperm cells, and total semen samples in smokers. This investigation demonstrated a significant positive relationship between CK activity of sperm cells and activity of this enzyme in seminal plasma and total semen in smokers. The positive correlation between CK activity and sperm motility in smokers, has indicated that exposure to tobacco smoke can diminish sperm motility via inhibition of sperm CK activity. The insignificant correlation found in this study suggests the possibility of other causes that may be involved in this process.
These results show that maintaining of normal CK
activity at physiological levels may provide an important
contribution to sperm motility and fertility in
males. According to previous research, it has been
demonstrated that mammalian sperm must remain
motile in the female tract and free energy released
from the hydrolysis of ATP is required for this movement.
Additionally, ADP can primarily be rephosphorylated
by phosphocreatine. The rate of ATP synthesis
via CK activity is generally faster than its rate of
synthesis through oxidative phosphorylation (
We found that cigarette smoking in adult males impaired sperm quality. This negative effect was dosedependent, as increased duration and quantity of cigarettes smoked had a positive effect on decreased sperm motility and CK activity. Therefore, we have suggested cigarette components that diminish sperm CK activity may potentially impair sperm energy homeostasis and thus have an association with damaged sperm motility. As a consequence, this effect can be one of the several important mechanisms that possibly cause infertility in male smokers. However, further research is required to elucidate the underlying mechanism of sperm motility damage caused by cigarette smoking.