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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: tavilani@gmail.com

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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.

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