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The Association of PON1192 Q/R Polymorphism with the Risk of Idiopathic Male Infertility in Northern Iran.

Introduction

Infertility is a disorder of the reproductive system defined by the failure to gain a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse'. In about half of the 15% of couples who cannot conceive, the cause is ascribed to male infertility (2). In spite of advances in clinical diagnostics, 50% of male infertility cases remain unclear which are referred to as idiopathic infertility (3). No identifiable cause is found in 37-58% of cases of male infertility (4). Most idiopathic cases are probable to be of genetic origin because many genes have been shown to be involved in human spermatogenesis (5). Moreover, idiopathic male infertility is assumed to be caused by several factors, including genetic and epigenetic abnormalities, Reactive Oxygen Species (ROS) or endocrine disruption as a result of environmental pollution (6).

ROS are generated as a by-product in mitochondria of normal mammalian cells. Low levels of ROS have physiological functions including activation and regulation of signal transduction pathways, modulation of activities of redox-sensitive transcription factors, and regulation of mitochondrial enzyme activities, but at high levels ROS are toxic to the cell (7). Moreover, ROS have a negative effect on sperm parameters (8). In 40% of infertile men, an increase in the seminal ROS level has been reported (9). Increased ROS levels can lead to damage with following sperm dysfunction or cell death (8).

Paraoxonase (PON) is a HDL-associated enzyme and a family of [Ca.sup.2+] depended hydrolase that inhibits low-density lipoprotein oxidation. It has antioxidant function and protects cells from oxidative stress (10). The PON gene family consists of 3 genes including PON1, PON2, and PON3 located on the long arm of chromosome 7. PON proteins localized in the seminiferous tubules and in spermatozoa have been shown to be implicated in the pathogenesis of male infertility (11). Furthermore, PONs can hydrolyze the hydrogen peroxide ([H.sub.2]O.sub.2]) (12). PON1 is the first member of the PON gene cluster to be discovered. Changes in the size and shape of HDL particles strongly influence the binding affinity and stability of PON] and result in a decreased antioxidative capacity (13). Inactivation of PON] reduces the ability of HDL to prevent both the oxidation of LDL and the interaction between macrophages and endothelium (12). The PONJ gene has more than seven polymorphisms in the coding region and five in promoter region. PON1 gene substitution of glutamine (Q) by arginine (R) at position 192 and leucine (L) by methionine (M) at position 55 of coding region has been shown (14). PON1 and PON3 are predominantly expressed in the liver and secreted into blood. PON2 is more widely expressed in a number of tissues including the brain, liver, kidney, and testis but not detectable in the blood (l5). PON has also been shown to play roles in lipid metabolism (13). The aim of this study was to analyze the PONJ (Glu/Arg 192) gene mutation in infertile men and men without infertility.

Materials and Methods

In the present study, 450 subjects including 220 men with idiopathic male infertility and 230 healthy men as the control group were assessed. Data on patient characteristics at the study entry for each subject were collected from the infertility clinic of Alzahra Educational and Remedial Hospital (Rasht, Iran). All patients underwent at least two semen analyses and those with a history of orchitis, obstruction of the vas deferens, hypogonadotroic hypogonadism, varicoceles, systemic diseases and sperm antibodies were excluded. Patients for at least two years had an infertility history with their spouses with confirmed normal gynecological assessment. Semen analysis results, age, smoking status, sperm motility and family history data were evaluated. A spermogram was made according to World Health Organizations (WHO) guidelines (16). Also, the healthy married male volunteers who had at least one child without assisted reproductive technologies were recruited as control group. Peripheral bloods (2 ml) were collected in the EDTA-coated tubes (Venoject, Belgium), which was used for DNA extraction. This project has been approved by the ethical committee of University of Guilan and informed consent was obtained from all subjects and has been performed according the Helsinki Declaration of 1975, as revised in 1983.

Genotyping

Genomic DNA was extracted from whole-blood samples using a DNA Extractor Gpp Solution Kit (Gen pajoohan, Iran) according to the manufacturer's instructions. The region of PON1 including the (192 Q/R) SNP site was amplified using primers:

(F: 5' CACGAAGGCTCCATCCCAC3' and R: 5' TC CTTCTGCCACCACTCGAAC3'). Each DNA sample was stored in TE buffer (5 m[micro] Tris-HCl, 0.1 m[micro] EDTA, pH=8.5) at -20 [degrees]C until analysis. Amplification of the PON] 192 Q/R polymorphism was accomplished with the use of polymerase chain reaction (PCR). The PCR was performed in 20 [micro]1. The PCR conditions for the PON] were as follows: 95 [degrees]C for 5 min, 34 cycles at 95 [degrees]C for 30 s, annealing at 60 [degrees]C for 40 s. Polymerase chain reaction products were subsequently digested with restriction enzyme AlwI. Enzyme digestion products were separated on 2% agarose gel electrophoresis and visualized by ethidium bromide staining.

Statistical analysis

Statistical analyses were performed using MedCalc (version 12.1, Mariakerke, Belgium). Analysis of difference in allele and genotype frequencies between cases and controls were compared by the [chi square] test. To estimate the association between the PON1 192 Q/R variant and the risk of idiopathic male infertility, odds ratios with 95% confidence intervals (95% CI) were evaluated by logistic regression. A value of p<0.05 was considered statistically s ignificant, Also , analyses for cas es and control s were perform ed by age (two gro ups: cases and controls were performed by age (two groups: [less than or equal to]30 years and >30 years), smoking status, family history of infertility and semen parameters. The homozygosity with the more frequent allele among controls was set as the reference group. A value of p<0.05 was considered statistically significant.

Results

The current study included a total of 220 patients with idiopathic male infertility and 230 disease-free control subjects. The mean age of study subjects was (36.2[+ or -]2.1 years) and for controls (34.5[+ or -]2.3 years), that was not significantly different between infertile patients and controls (p>0.05). Genotyping of 192 Q/R was done by PCR-RFLP method (Figure 1). The main characteristics of the patients are presented in table 1. Analysis suggested that age, smoking status and family history of infertility were not significantly different between cases and controls. The prevalence of genotype frequencies for QQ, QR and RR were 40.8, 54.7 and 4.3% in controls, and 57.2, 38.7, 4.5% in infertile subjects, respectively. Statistical analysis showed that there was significant difference between two groups (p=0.004).

The results indicated that the subgroup with QR genotypes was associated with decreased risk of idiopathic male infertility (OR=0.49, 95%CI=0.33-0.73, p= 0.0004). Moreover, analyses showed that R allele may have a protective effect on susceptibility of idiopathic male infertility (OR=0.31, 95%CI=0.21-0.47, p=0.0001). All information about allele and genotype frequencies and associated ORs (95%CI) for infertile cases and controls are summarized in table 2.

Discussion

In this case-control study, the role of PON I 192 Q/R polymorphism in 220 infertile patients and 230 controls was evaluated. Our results suggest that there is a significant association in genotype distribution between cases and controls (p= 0.001). The individuals with QR genotypes were associated with decreased risk of idiopathic male infertility (OR=0.49, 95%CI=0.33-0.73, p=0.0004). It has been shown that R allele may have a protective effect on susceptibility of idiopathic male infertility (OR=0.31, 95%CI=0.21-0.47, p=0.0001).

In spite of enormous progress in discovering the reproductive biology, the underlying mechanism of male infertility remains unclear in about 50% of cases referred to as idiopathic male infertile patients (17). Para-xonase is a High Density Lipoprotein (HDL)-associated enzyme that prevents Low-Density Lipoprotein (LDL) oxidative modification (l8). In humans, PON1 gene is expressed basically in the liver and kidney (19). PON1 is a calcium-depended esterase that circulates in plasma associated with HDL and contributes to the protective effect of this lipoprotein on LDL oxidation (20). Several studies have presented increased susceptibility of LDL to oxidation (21). It has been demonstrated that PON1 genetic variations have a crucial role in increasing the risk in many kinds of diseases. Bhatta-charyya et al reported that the PON gene may contribute to genetic susceptibility of cardiovascular risk (22). Moreover, Aydin et al found that PON1 192 Q/R polymorphism was significantly associated with stroke severity (23). Recently, Erlich et al revealed that PONS 192 Q/R polymorphisms are associated with increased Alzheimer risk (24).

According to the role of PON in oxidative stress and also the effect of genetic variations of PON which might be effective in male germ line cells and fertility, in the present study, the association between 192 Q/R PON1 SNP and susceptibility to idiopathic male infertility was investigated. In Iranian population, the PON1 gene polymorphism was associated with systemic lupus erythematosus (14), High LDL/HDL ratios (25) and psoriasis (26). The finding of the current study is inconsistent with those of Marsillach who found that the PON I 192 Q/R polymorphism was not associated with male infertility risk (27). However, our results indicate that PON1 192 Q/R polymorphism had a significant decreased risk of idiopathic male infertility and the protective effect of QR was more predominant among other subgroups (OR=0.49,95%CI=0.33-0.73, p=0.0004). The contradictory results of these reports may be due to the differences in sample sizes, gene pool and the impact of other genetic and environmental factors.

Conclusion

In conclusion, our results indicated that the PON1 192 Q/R polymorphism is associated with decreased risk of idiopathic male infertility. Further studies with larger numbers of patients and controls are needed to confirm our results.

Acknowledgement

We are especially grateful to all the experts who were integral partner in the preparation of facilities.

References

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(2.) Avenarius MR, Hildebrand MS, Zhang Y, Meyer NC, Smith LL, Kahrizi K, et al. Human male infertility caused by mutationsin the CATSPER1 channel protein. Am J Hum Genet 2009;84(4): 505-510.

(3.) Hildebrand MS, Avenarius MR, Fellous M, Zhang Y, Meyer NC, Auer J, et al. Genetic male infertility and mutation of CATSPER ion channels. Eur J Hum Genet 2010;18(11): 1178-1184.

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(5.) Plaseska-Karanfilska D, Noveski P, Plaseski T, Maleva I, Madjunkova S, Moneva Z. Genetic causes of male infertility. Balkan J Med Genet 2012; 15(Suppl):31-34.

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(7.) Oberley TD. Oxidative damage and cancer. Am J Pathol. 2000;160(2):403-408.

(8.) Olayemi FO. A review on some causes of male infertility. Afr J Biotechnol 2010;9(20):2834-2842.

(9.) Lewis SE, Sterling ES, Young IS, Thompson W. Comparison of individual antioxidants of sperm and seminal plasma in fertile and infertile men. Fertil Steril 1997:67 (1):142-147.

(10.) Lazaros L, Markoula S, Kkyritsis A, Georgiou I. Paraox-onase gene polymorphisms and stroke severity. Eu J Neurol 2010;17(5):757-759.

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(12.) Kim DS, Burt AA, Ranchalis JE, Richter RJ, Marshall JK, Eintracht JF, et al. Additional common polymorphisms in the PON gene cluster predict PON1 activity but not vascular disease. J Lipids 2012;2012: 476316.

(13.) She ZG, Chen HZ, Yan Y, Li H, Liu DP. The human paraoxonase gene cluster as a targetin the treatment of atherosclerosis. Antioxid Redox Signal 2012;16(6):597-632.

(14.) Bahrehmand F, Vaisi-Raygani A, Ahmadi R, Kiani A, Rahimi Z, Tavilani H, et al. Paraoxonase (PON1) 55 polymorphism and association with systemic lupus erythematosus. Iran J Allergy Asthma Immunol 2013; 12(3): 211-219.

(15.) Eom SY, Kim YS, Lee CJ, Lee CH, Kim YD, Kim H. Effects of intronic and exonic polymorphisms of paraoxonase l(PONl) gene on serum PON1 activity in a Korean population. J Korean Med Sci 2011;26(6): 720-725.

(16.) World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Geneva: World Health Organization. 1999. 271 p.

(17.) Ferlin A, Arredi B, Foresta C. Genetic causes of male infertility. Reprod Toxicol 2006;22(2): 133-141.

(18.) Mackness MI, Arrol S, Durrington PN. Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein. FEBS Lett 1991 ;286( 1-2): 152-154.

(19.) Reddy ST, Wadleigh DJ, Grijalva V, Ng C, Hama S, Gangopadhyay A, et al. Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids. Arterioscler Thromb Vase Biol 2001;21(4):542-547.

(20.) Mahadesh Prasad AJ, Kemparaju K, Elizabeth AF, Jaich-ander P, Arun A, Cletus JM, et al. Lack of correlation of paraoxonase (PON1) activity with smoking among the south Indians and risk of cardiovascular disease. World Appl Sci J 2010;9:194-198.

(21.) Haj Mouhamed D, Ezzaher A, Mechri A, Neffati F, Omezzine A, Bouslama A, et al. Effect of cigarette smoking on paraoxonase 1 activity according to PON1 L55M and PON1 Q192R gene polymorphisms. Environ Health Prev Med 2012;17:316-321.

(22.) Bhattacharyya T, Nicholls S.I, Topol EJ, Zhang R, Yang X, Schmitt D, et al. Relationship of paraoxonase I (PON1) gene polymorphisms and functional activity with systemic oxidative stress and cardiovascular risk. JAMA 2008;299(11): 1265-1276.

(23.) Aydin M, Gencer M, Cetinkaya Y, Ozkok E, Ozbek Z, Kilic G, et al. PON1 55/192 polymorphism, oxidative stress, type, prognosis and severity of stroke. IUBMB Life 2006;58(3):165-172.

(24.) Erlich PM, Lunetta KL, Cupples LA, Huyck M, Green RC, Baldwin CT, at al. Polymorphisms in the PON gene cluster are associated with Alzheimer disease. Hum Mol Genet 2006;15(l):77-85.

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(26.) Asefi M, Vaisi-Raygani A, Bahrehmand F, Kiani A, Rahimi Z, Nomani H, et al. Paraoxonase 1 (PON1) 55 polymorphism, lipid profiles and psoriasis. Br J Dermatol 2012;167(6): 1279-1286.

(27.) Marsillach J, Lafuente R, Checa MA, Maestre-Martinez C, Fabian E, Brassesco M, et al. Paraoxonase-1 is only present in traceable amounts in seminal fluid and does not show any relationship with male subfertility. BJU Int 2010;108(4):566-570.

Setareh Behrouzi (1*), Farhad Mashayekhi (1), and Mohammad Hadi Bahadori (2)

(1.) Department of Biology, Faculty of Sciences, University of Cuilan, Rasht, Iran

(2.) Cellular and Molecular Research Center, Faculty of Medical Sciences, Cuilan University of Medical Sciences, Rasht, Iran

(*) Corresponding author:

Setareh Behrouzi, M.Sc.,

Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran

Tel: +98 937 964 5020

Fax: +98 131 3233647

E-mail:

sara.behrozi7@gmail.com

Received: 11 Oct 2017

Accepted: 19 Dec 2017
Table 1. Characteristics of idiopathic male infertility patients and
controls enrolled in the study

                                    Controls (n=230)
Variable                            No. (%)

Age (mean[+ or -]SD)                 34.5[+ or -]2.3
<30                                  44 (19.3)
>30                                 186 (80.8)
Smoking status
Never                               114 (49.5)
Former                               42 (18.2)
Current                              74 (32.2)
Family history of infertility
No                                  171 (74.3)
Yes                                  59 (25.6)
Semen parameters (mean[+ or -]SEM)
Concentration (x[10.sup.6]/m/)      100[+ or -]2.4
Motility (%)                         63.5[+ or -]1.21
Volume (ml)                           2.9[+ or -]0.05

                                    Cases (n=220)
Variable                            No. (%)             P

Age (mean[+ or -]SD)                 36.Z[+ or -]2.1     0.365
<30                                  34 (15.4)
>30                                 186 (84.5)
Smoking status                                           0.181
Never                                90 (41.0)
Former                               48 (21.8)
Current                              82 (37.2)
Family history of infertility                            0.121
No                                  148 (67.2)
Yes                                  72 (32.8)
Semen parameters (mean[+ or -]SEM)
Concentration (x[10.sup.6]/m/)       80.7[+ or -]4.21   <0.001
Motility (%)                         35.7[+ or -]0.63   <0.001
Volume (ml)                           2.85[+ or -]0.06  <0.001

Table 2. Allele and genotype frequencies of PON1 192 Q/R polymorphism
among cases and controls and the associations with risk of idiopathic
male infertility

              Controls                   infertile cases
              (n=230)                    (n=220)
              n (%)         n (%)        OR (95% CI)       P (a)   P (b)

Alleles (192  Q/R)
Q              460 (68.0)   336 (76.0)   1.00 (reference)  0.0001  -
R              146 (32.0)    34 (24.0)   0.31 (0.21-0.47)  0.0001
Genotypes     (192 Q/R)
QQ              94 (40.86)  126 (57.27)  1.00 (reference)  0.001   -
QR             126 (54.78)   84 (38.18)  0.49 (0.33-0.73)  0.0004
RR              10 (4.34)    10 (4.54)   0.74 (0.29-1.86)  0.530

a) Allele and genotype frequencies in cases and controls were compared
using [chi square] test.
b) Significance level for allele and genotype frequencies in cases and
controls.
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Title Annotation:Original Article; Paraoxonase1 192
Author:Behrouzi, Setareh; Mashayekhi, Farhad; Bahadori, Mohammad Hadi
Publication:Avicenna Journal of Medical Biotechnology (AJMB)
Article Type:Report
Geographic Code:7IRAN
Date:Oct 1, 2018
Words:2925
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