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Syst Biol Reprod Med, Early Online: 1–6! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/19396368.2014.960624
RESEARCH ARTICLE
Genotype and phenotype frequencies of paraoxonase 1 in fertile andinfertile men
Heidar Tavilani1*, Amir Fattahi2, Maryam Esfahani3, Iraj Khodadadi3, Jamshid Karimi3, Elham Bahrayni3,Akram Vatannejad4, Asad Vaisi-Raygani5, Marzyeh Ghorbani3, and Zeinab Latifi2
1Urology and Nephrology Research Center, Hamadan University of Medical Sciences, Hamadan, Iran, 2Department of Clinical Biochemistry,
Medical School, Tabriz University of Medical Science, Tabriz, Iran, 3Department of Biochemistry and Nutrition, Hamadan University of Medical
Sciences, Hamadan, Iran, 4Student’s Scientific Research Center, Tehran University of Medical Sciences, Tehran, Iran, and 5Fertility and Infertility
Research Center, Kermanshah University of Medical Science, Kermanshah, Iran
Abstract
Paraoxonase1 (PON1) is a glycoprotein associated with high density lipoprotein and hasantioxidant activity. The impact of PON1 in various stages of spermatogenesis has also beensuggested. This study was aimed to investigate frequencies of phenotypes and Q192Rgenotypes of PON1 in fertile and infertile males. Q192R variants of PON1 were determined in150 fertile and 150 infertile men using the polymerase chain reaction–restriction fragmentlength polymorphism (PCR-RFLP) technique. Plasma arylesterase and paraoxonase activitieswere detected by spectrophotometry and malondialdehyde (MDA) level was measured usingthiobarbituric acid. Our results showed no significant difference in the distribution of PON1genotypes and alleles between fertile and infertile groups. However morphology and motilityof sperm were associated with various genotypes of PON1. The number of fertile males with theBB phenotype (high activity) was significantly higher than that of infertile males, whereas thenumber of individuals with the AB phenotype (moderate activity) was statistically higher ininfertile men compared with the fertile group. Additionally, MDA and arylesterase activity levelswere significantly higher in infertile subjects compared with fertile men. We speculate that thelow activity of PON1 can be a risk factor for male infertility probably due to a decrease inantioxidant activity of PON1 and increase in lipid peroxidation.
Abbreviations: PON1: Paraoxonase1; PCR-RFLP: polymerase chain reaction–restrictionfragment length polymorphism; MDA: malondialdehyde; ROS: reactive oxygen species; HDL:high-density lipoprotein; LDL: low-density lipoprotein; HDL-C: HDL cholesterol; LDL-C: LDLcholesterol
Keywords
Genetic polymorphism, male infertility,paraoxonase 1, reproduction
History
Received 19 March 2014Revised 17 July 2014Accepted 18 July 2014Published online 24 September 2014
Introduction
Reactive oxygen species (ROS) are a necessity of aerobic
life. Nevertheless, inequality between ROS and antioxidant
systems causes oxidative stress which may result from both
extra production of ROS and/or a deficiency in the antioxi-
dant system [Sikka 2001]. Sperm damage by ROS was
shown in 30–80% of the cases of infertile males [Iwasaki and
Gagnon 1992; Shekarriz et al. 1995a,b; Zini et al. 1993].
Several studies have reported that subfertile patients have high
levels of seminal ROS and low levels of total antioxidant
capacity [Khosrowbeygi and Zarghami 2007; Lewis et al.
1995; Verit et al. 2009]. Furthermore, it has been demon-
strated that oxidative stress causes lipid peroxidation in sperm
plasma membranes and induces sperm DNA fragmentation
which influence sperm motility, morphology, capacitation,
and fertilization ability [Aitken and Krausz 2001].
Paraoxonase1 (PON1: EC 3.1.8.1) is a 43 kD glycoprotein
with paraoxonase, arylesterase, and lactonase activities which
are associated with high-density lipoprotein (HDL) [Camps
et al. 2009; Mackness et al. 1998a]. The PON1 gene is located
at 7q21–22 on the long arm of chromosome 7 [Primo-Parmo
et al. 1996]. It has been documented that PON1 has
antioxidant activity and inhibits oxidation of low-density
lipoprotein (LDL) and HDL particles and plays a key role in
preventing lipid peroxidation [Mackness et al. 1993]. The
exact role of PON1 in the male reproductive tract is unknown
but immunohistochemical analysis of human and mouse testis
has shown the existence of PON1 protein in spermatogonia,
spermatocytes, spermatids, Sertoli and Leydig cells which is
*Address correspondence to Heidar Tavilani, Urology and NephrologyResearch Center, Hamadan University of Medical Sciences, ShaheedFahmideh Ave., Hamadan, Iran 6517838678. Tel: 0098 8138381590.E-mail: [email protected]
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suggestive of a role for PON1 during various stages of
spermatogenesis [Marsillach et al. 2008; Marsillach et al.
2011]. In addition, low PON1 activity in semen of subfertile
men has been reported by Verit et al. [2009]. It is suggested
that over production of ROS in subfertile men may be a result
of low PON1 activity in these patients [Verit et al. 2009].
Furthermore, a significant positive correlation between PON1
activity and semen parameters such as concentration, motility,
and morphology has been noted [Verit et al. 2009].
Several PON1 gene polymorphisms have been described
and substitution of glutamine to arginine at position 192
(Q192R) has been shown as a functional polymorphism
[Adkins et al. 1993; Humbert et al. 1993]. Studies have shown
that the 192R isoform has higher lactonase activity and higher
binding affinity to HDL and hydrolyzes paraoxon faster
compared to the 192Q isoform, whereas the 192Q isoform
exhibits lower stability and hydrolyzes diazoxon faster
[Bhattacharyya et al. 2008; Gaidukov et al. 2006; Gupta
et al. 2011; Veiga et al. 2011]. In comparison, the 192Q
isoform of PON1 is more efficient in reducing total lipid-
peroxide content and therefore shows higher antioxidant
effects [Mackness et al. 1998b].
The role of this PON1 gene polymorphism in the male
reproductive system has been well documented. Some studies
have reported that the Q192R polymorphism of PON1 which
modifies paraoxonase activity is associated with increased
risk of male infertility [Corbo et al. 2008; Lazaros et al. 2011;
Verit et al. 2009; Volk et al. 2011]. Lazaros et al. [2011] have
observed a significantly higher frequency of the QQ192
genotype in normozoospermic men compared with oligos-
permic patients. In addition, they have reported higher
motility of sperm in men with the 192Q allele and increased
sperm concentration in the PON2 311S allele. They
hypothesized that paraoxonase may affect spermatogenesis
and thus the quality of human sperm [Lazaros et al. 2011],
although Volk et al. [2011] did not observe significant
differences in the frequency of the PON1 Q192R genotypes
between fertile and infertile men. In another study, it was
reported that the PON1 QQ192 genotype was associated with
increased sperm DNA damage and thus risk of male infertility
[Ji et al. 2012].
There is a little information about the PON1Q192R
polymorphism gene. Thus the question of whether the
PON1 gene polymorphism distribution is different between
fertile and infertile men has yet to be addressed. In this study,
we determined the distribution of the PON1 Q192R poly-
morphism in fertile and infertile men. In addition, plasma
PON1 activity, PON1 phenotype, and the level of malondial-
dehyde (MDA) were investigated.
Results
The semen analysis profiles from infertile men are presented
in Table 1. For genotype determination of the PON1 Q192R
polymorphism, we used the polymerase chain reaction–
restriction fragment length polymorphism (PCR-RFLP) tech-
nique and the DNA fragments were visualized by silver
staining (Figure 1). The frequencies of the QQ, QR, and RR
genotypes from PON1 Q192R polymorphism, and odds ratio
of the two groups studied are described in detail in Table 2.
The distribution of the PON1 genotype of fertile and infertile
individuals was consistent with the predicted genotype by the
Hardy-Weinberg equilibrium. Our results indicated that the
distribution of PON1 genotypes and alleles was not signifi-
cantly different between fertile and infertile individuals. The
presence of PON1 Q192R genotypes (including QQ, QR, and
RR) and alleles are not a risk factor for male infertility
(Table 2).Association between semen parameters and PON1
genotypes in infertile men showed that sperm with normal
Table 1. Semen profile of infertile males.
Semen Parameters Infertile (n¼ 150)
Sperm concentration (million/ml) 39.11 ± 24.94Sperm normal morphology (%) 17.55 ± 14.30Progressive motility1 (%) 9.2 ± 7.9Non-progressive motility1 (%) 11.25 ± 7.90Immotility1 (%) 76.60 ± 20.9
Results are presented as mean ± SD. 1sperm movement according toWHO [2010] criteria: Progressive motility: spermatozoa movingactively, either linearly or in a large circle, regardless of speed;Non-progressive motility: all other patterns of motility with an absenceof progression; Immotility: no movement.
Figure 1. Genotype analysis of theparaoxonase 1 Q192R polymorphism usingthe polymerase chain reaction–restrictionfragment length polymorphism (PCR-RFLP)method. PCR products were digested byAlwI restriction enzyme, run on a 15%polyacrylamide gel, and visualized using thesilver-staining method. Lane 1 is DNAmolecular marker, Lane 2 and 3 arehomozygote QQ 99 bp, Lane 4 and 5are homozygote RR 63 and 36 bp, andLane 6 is heterozygote QR 99, 63, and 36 bp.
1 2 3 4 5 6125
100
75
50
Primer dimer
99 bp
63bp
36 bp
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morphology and sperm with progressive and non-progressive
motility were significantly different in various PON1 geno-
types (Table 3).
Three phenotypes for paraoxonase including AA
(low activity), AB (moderate activity), and BB (high activity)
are illustrated in Table 4. The distribution of AB and BB
phenotypes were significantly different between fertile and
infertile groups. In addition, the difference between the
paraoxonase phenotype in the two groups remained signifi-
cant also after adjustment for HDL-C, LDL-C, and
malondialdehyde levels (Table 5). The number of individuals
with the BB phenotype was significantly higher in fertile
males compared with infertile males (38.7% vs. 7.8%), while
the number of individuals with the AB phenotype was
statistically higher in infertile men than in fertile subjects
(37.3% vs. 21.3%).
The analysis of relationships between PON1 genotypes and
phenotypes showed that the frequency of the BB phenotype in
the fertile group with QQ and QR genotypes was higher than
in the infertile group (p50.01). Although the number of
individuals with BB phenotype was higher in the fertile
compared to the infertile individuals, the difference was not
significant in the individuals with the RR genotype.
The plasma levels of MDA as well as arylesterase and
paraoxonase activities of PON1were measured in fertile and
infertile men (Table 6). MDA and arylesterase activity levels
were significantly higher in infertile men compared with
fertile males (p50.05). In contrast, paraoxonase activity
levels were statistically higher in fertile subjects than in
infertile men (p50.05).
Discussion
Paraoxonase is an HDL-bound antioxidant enzyme which
is involved in to neutralizing of lipid peroxides
[Mackness et al. 1993]. Some studies have investigated the
possible roles of this enzyme and its polymorphisms in male
infertility [Corbo et al. 2008; Volk et al. 2011; Lazaros et al.
2011]. In this study, we have examined the PON1 Q192R
polymorphism (glutamine is replaced by arginine), plasma
Table 2. The frequency of paraoxonase 1 (Q192R) genotypes, alleles, and odds ratios in fertile (n¼ 150) and infertile (n¼ 150) males using �2 test andregression logistics analysis.
PON1 Genotypes Fertile n (%) Infertile n (%) p Value (�2, df) Odd ratio, 95% CI (Lower-Upper, p)
QQ 86 (57.3%) 94 (62.7%) 0.346 (�2¼ 0.889, df¼ 1) Reference groupQR 59 (39.3%) 48 (32%) 0.185 (�2¼ 1.758, df¼ 1) 1.344 (0.831–2.1720, p¼ 0.228)RR 5 (3.3%) 8 (5.3%) 0.395 (�2¼ 0.724, df¼ 1) 0.827 (0.464–1.473, p¼ 0.518)QR+RR 64 (42.7%) 56 (37.3%) 0.346 (�2¼ 0.889, df¼ 1) 1.249 (0.786–1.984, p¼ 0.346)
PON1 AllelesQ 231 (77%) 236 (78.7%) 0.623 (�2¼ 0.242, df¼ 1) Reference groupR 69 (23%) 64 (21.3%) 1.050 (0.866–1.273, p¼ 0.623)
df: degrees of freedom; CI: confidence interval
Table 3. Semen parameters according to the various genotypes of paraoxonase 1 in infertile group.
Sperm motility1 (%)Sperm concentration Sperm normal
Genotypes Progressive motility Non-progressive motility Immotility (million/ml) morphology (%)
QQ 10.8 ± 9.2 12.71 ± 8.73 75.42 ± 20.21 41.88 ± 27.44 20.40 ± 15.43QR 6.8 ± 4.4 8.72 ± 5.20 79.81 ± 20.13 33.18 ± 19.38 13.34 ± 11.32RR 6.4 ± 5.5 10.08 ± 8.31 71.78 ± 30.95 42.66 ± 19.40 11.28 ± 9.51p Value 0.028 0.034 0.459 0.197 0.018
Results are presented as mean ± SD. 1sperm movement according to WHO [2010] criteria: Progressive motility: spermatozoa moving actively, eitherlinearly or in a large circle, regardless of speed; Non-progressive motility: all other patterns of motility with an absence of progression; Immotility:no movement.
Table 4. Distribution of paraoxonase 1 phenotypes according to dividingsalt-stimulated paraoxonase by arylesterase activity in fertile and infertilegroups (using �2 test).
Phenotype* Fertile n (%) Infertile n (%) p Value (�2, df)
AA 60 (40%) 81 (54%) 0.075 (�2¼ 3.177, df¼ 1)AB 32 (21.3%) 56 (37.3%) 0.023 (�2¼ 5.165, df¼ 1)BB 58 (38.7%) 13 (8.7%) 0.001 (�2¼ 18.363, df¼ 1)
df: degrees of freedom; *AA: low activity, AB: moderate activity, BB:high activity.
Overall PON1 phenotypes between fertile with infertile males aresignificantly different (p50.01).
Table 5. Distribution of PON1 phenotypes according to dividing salt-stimulated paraoxonase by arylesterase activity in fertile and infertilegroups (using �2 test) after adjusted for multiple comparisons (levels ofHDL-C, LDL-C, and MDA in plasma).
Phenotype
Fertile(n¼ 131)
n (%)
Infertile(n¼ 116)
n (%) p Value (�2, df)
AA 53 (40.4%) 63 (54.3%) 0.075 (�2¼ 3.177, df¼ 1)AB 28 (21.3%) 45 (38.8%) 0.023 (�2¼ 5.165, df¼ 1)BB 50 (38%) 8 (6.8%) 0.001 (�2¼ 18.363, df¼ 1)
HDL-C: high-density lipoprotein cholesterol; LDL-C: low-densitylipoprotein cholesterol; MDA: malondialdehyde; df: degrees offreedom; AA: low activity; AB: moderate activity; BB: high activity.
Overall PON1 phenotypes between fertile with infertile males aresignificantly different (p50.01).
DOI: 10.3109/19396368.2014.960624 Paraoxonase 1 and male infertility 3
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activity and phenotype of PON1, and level of MDA in fertile
and infertile males.
Comparing the PON1 genotype and the allele distribution
showed no significant difference between fertile and infertile
males. Previous studies have reported conflicting results on
the distribution of the PON1 Q192R polymorphism. While
Volk et al. [2011] reported no significant differences in the
PON1 Q192R polymorphism between fertile and infertile
men, Lazaros et al. [2011] have reported a statistically higher
frequency of the QQ genotype in normozoospermic compared
with oligospermic Greek men, although they have not
clarified whether normozoospermic men were fertile or
infertile. Corbo et al. [2008] have reported no association
between QQ, QR, and RR genotypes of men and the number
of their children in an Italian population. Similarly, our results
suggested that the presence of different PON1 genotypes and
alleles is probably not a risk factor for male infertility.
Inconsistent results about the distribution of PON1 genotypes
between fertile and infertile individuals may originate from
different ethnic populations and different sample sizes used in
these studies.
We found that men with the QQ genotype had higher
sperm progressive motility and normal morphology compared
with the QR and RR genotypes. In accordance with this
finding, it has been reported that normozoospermic men with
the PON1 192Q allele have higher sperm motility than
carriers of the R allele [Lazaros et al. 2011]. It is reported that
the PON1 192 R allele carriers have lower PON1 activity and
higher lipid peroxidation compared with Q allele carriers,
which may explain the effect of various PON1 genotypes on
sperm parameters [Costa et al. 2003; Mackness et al. 1998b].
It can be concluded that, because the QQ genotype has high
antioxidant activity it improves motility and morphology of
sperm by reducing lipid oxidation. Although our results
indicated a significant difference in the percent of motile
sperm in various PON1 genotypes, the difference in the
percent of immotile sperm in the QQ, QR, and RR genotypes
were not significant. The reason for no significant difference
can be that immotile sperm have other defects such as mito-
chondrial dysfunction, increased level of ROS, and structural
problems in the sperm tail. For this reason, movement of
immotile sperm may not be affected by the PON1 genotype.
We have evaluated the phenotypic AA (low activity),
AB (moderate activity), and BB (high activity) distribution of
PON1 in fertile and infertile men by the double substrate
method using paraoxon and phenylacetate, based on a
previously described method [Sepahvand et al. 2007]. The
results showed significant differences in frequencies of AB
and BB phenotypes between the study groups. The frequency
of the BB phenotype in fertile men was higher than infertile
men (38.7% vs. 8.7%), while infertile men had a higher
frequency of the AB phenotype compared with the fertile
group (37.3% vs. 21.3%). In other words, most fertile men are
categorized in a phenotype with moderate and high activity of
paraoxonase. In addition, we observed remarkably higher
plasma paraoxonase activity (according to hydrolysis of
paraoxon as a substrate) in fertile males compared with
infertile subjects. In similar studies, lower activity of seminal
PON1 in infertile men has been reported compared with
fertile subjects [Marsillach et al. 2011; Verit et al. 2009].
Our results showed that the frequency of the BB phenotype
in the fertile group with QQ, QR, and RR genotypes was
higher than the infertile group. This enzyme has two distinct
activities, i.e., paraoxonase and arylesterase and each of these
activities reflects a unique genotype [Furlong et al. 1988;
Gaidukov et al. 2006; Phuntuwate et al. 2005]. In accordance
with the data above, we can conclude that the BB phenotype
is not affected by the QQ, QR, or RR genotypes.
We have previously shown that semen lipid peroxidation
differs in fertile and infertile males [Tavilani et al. 2005;
Tavilani et al. 2008]. In the present study again we showed an
increased level of plasma MDA (a stable product of lipid
peroxidation) in the infertile group compared with fertile men,
while a lower activity of plasma paraoxonase has been
observed in infertile men as compared to fertile individuals.
This is in agreement with a study by Serhatlioglu et al. [2003]
who reported a negative correlation between paraoxonase
activity and MDA content in plasma of subjects exposed to
ionizing radiation. A similar inverse relationship between
seminal paraoxonase activity and total oxidant status in fertile
and infertile subjects has also been previously reported by
Verit et al. [2009].
Collectively, based on previous studies together with our
results, we speculate that low activity of PON1 can be a risk
factor for male infertility probably due to a decrease in
antioxidant activity of PON1 and the accumulation of lipid
peroxidation. Since the PON1 activity is mainly determined
by polymorphisms in the PON1 gene [Sepahvand et al. 2007]
and our results showed that the activity varies among fertile
and infertile men, it seems the measurement of PON1 activity
instead of determining PON1 Q192R genotypes would be a
useful tool to evaluate male infertility.
In conclusion, our findings showed that the distribution of
PON1 genotypes and alleles did not differ in fertile and
infertile individuals. In addition, higher paraoxonase activity
and lower MDA content were detected in fertile males
compared with infertile subjects. Therefore, we suggest that
decreased activity of PON1 may be a risk factor for male
infertility.
Materials and Methods
Study population
Three hundred Iranian males including 150 fertile men
(having at least one child) and 150 infertile men were
enrolled in this study. The infertile individuals were selected
Table 6. Arylesterase and paraoxonase activities of paraoxonase 1 andlevels of malondialdehyde (MDA), high-density lipoprotein cholesterol(HDL-C), and low-density lipoprotein cholesterol (LDL-C) in plasma offertile and infertile men.
Fertile n¼ 150 Infertile n¼ 150 p Value
Arylesteraseactivity (U/mL)
46.81 ± 10.89 50.15 ± 9.12 0.043
Paraoxonaseactivity (U/L)
181.87 ± 91.56 150.7 ± 82.83 0.022
MDA (nmol/mL) 1.04 ± 0.56 1.20 ± 0.59 0.016HDL-C (mg/dL) 33.07 ± 7.33 42.22 ± 8.06 0.01LDL-C (mg/dL) 128.45 ± 35.84 108.21 ± 48.86 0.001
Results are presented as mean ± SD
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from patients who were admitted to the Fattemieh Fertility
Clinic of Hamadan University of Medical Sciences and had
no child after at least a year of unprotected intercourse and
had an abnormal semen analysis. Inclusion criteria for the
participants were: having no known cause of infertility such
as varicocele or abnormal karyotype, having no history of
using lipid lowering drugs for a year ended up to the study,
having no history of diabetes, cardiovascular, and thyroid
diseases. Written informed consent was obtained from all of
the participants and the procedures described were approved
by the Ethics Committees of Hamadan University of Medical
Sciences.
Sampling
Blood samples were collected in tubes containing heparin,
plasma was separated and plasma aliquots stored at �20�C.
In addition, blood leukocytes were used for DNA extraction.
Semen samples were collected from infertile men by mastur-
bation after 3 days of abstinence and semen parameters were
determined based on World Health Organization standard
procedures [WHO 2010].
DNA extraction and genotyping of PON1
The Phenol-Chloroform extraction method was used for
DNA extraction from peripheral blood. Variants of
paraoxonase-1 Q192R polymorphism were determined by
amplification of a 99 bp fragment (PCR PreMix; BIONEER
kit, Bioneer, Daejeon, Korea) using a pair of forward
(50-TATTGTTGCTGTGGGACCTGAG-3
0) and reverse (5
0-
CACGCTAAACCCAAATACATCTC-30) primers, as previ-
ously described [Serrato and Marian 1995]. PCR products were
digested by AlwI restriction enzyme (Fermentas, USA), run on
15% polyacrylamide gel, and visualized using silver-staining
method.
Biochemical measurements
PON1 activity:
Plasma arylesterase activity of PON1 was measured by
spectrophotometric method as previously described [Eckerson
et al. 1983; Furlong et al. 1988]. Phenylacetate was used as
a substrate and phenol product of substrate hydrolysis was
measured at 270 nm. Briefly 5 ml of the plasma sample was
added to 3 ml of the reaction mixture (1 mmol/L phenylace-
tate, 1 mmol/L CaCl2 and 20 mmol/L Tris-HCl buffer pH 8).
The absorbance at 270 nm and 37�C was measured per min
and PON1 activity was calculated using the molar absorption
coefficient 1310 mol�1 L cm�1. Finally, results were
expressed as U/mL.
Paraoxonase activity of plasma was determined using
paraoxon as a substrate and monitoring of the formation of
p-nitrophenol (diethyl-p-nitrophenyl phosphate) by recording
absorbance at 405 nm. Briefly 25 mL of plasma was added to
an assay tube containing 535 mL buffer (0.132 M Tris-HCl,
pH 8.5 and 1.32 mM CaCl2) and 140 mL freshly prepared
paraoxon. The molar absorption coefficient for calculating the
formation of p-nitrophenol was 18,050 M�1 cm�1 and the
activity was expressed as U/L [Eckerson et al. 1983; Furlong
et al. 1988].
PON1 phenotype:
For determining the distribution of PON1 phenotypes, double
substrate method was used by using paraoxon and phenyla-
cetate as substrates. The ratio of salt-stimulated paraoxonase/
arylesterase activities for each sample was used for the
determination of PON1 phenotypes. Therefore, the ratio was
divided into three phenotypes; AA (low activity), AB
(moderate activity), and BB (high activity) [Eckerson et al.
1983].
Malondialdehyde:
The content of lipid peroxidation in plasma was measured by
the reaction of thiobarbituric acid (TBA) with MDA accord-
ing to the Yagi method [Yagi 1984]. The MDA level was
measured spectrofluorometrically via excitation at 515 nm
and emission at 553 nm and tetraethoxypropane was used as
standard.
Plasma lipoproteins:
Levels of plasma lipoproteins including high-density lipopro-
tein cholesterol (HDL-C) and low-density lipoprotein choles-
terol (LDL-C) were measured by the commercially available
kit (Pars Azmoon, Tehran, Iran).
Statistical analysis
Data were analyzed using SPSS V.16 software and the results
were expressed as mean ± SD. According to One-Sample
Kolmogorov-Smirnov test results the variables were normally
distributed. Differences between plasma MDA concentration
and enzyme activities of fertile and infertile men were tested
using Independent sample t-test. Comparison of allele,
genotype, and phenotype frequencies and semen parameters
between groups were determined by Chi-Square test. One-
way ANOVA test was used to compare the means of
quantitative parameters in different genotypes and statistical
significance was assumed at the p50.05 level.
Acknowledgments
The authors appreciate the Fattemieh Fertility Clinic of
Hamadan for providing the samples. This study was finan-
cially supported by Hamadan University of Medical Sciences.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper. The
authors are not directly employed by the Government of Iran.
Heidar Tavilani and Iraj Khodadadi are academic members of
Hamadan University of Medical Sciences. Asad Vaisi-
Raygani is an academic member of Kermanshah University
of Medical Sciences. Other authors are students in Clinical
Biochemistry.
Author contributions
Designed the study and wrote the revised paper: HT, IK;
Wrote the paper: AF; Statistical analysis and interpretation of
data: AV-R; Performed experiments: JK, EB, AV, ZL;
Provision of patients: MG, ME.
DOI: 10.3109/19396368.2014.960624 Paraoxonase 1 and male infertility 5
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