Association of PON1-L55M Genetic Variation with Idiopathic Male Infertility in an Iranian Population: A Case-Control Study

doi:10.21203/rs.3.rs-3395305/v1

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Association of PON1-L55M Genetic Variation with Idiopathic Male Infertility in an Iranian Population: A Case-Control Study

https://doi.org/10.21203/rs.3.rs-3395305/v1

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The risk of male infertility is related to gene polymorphisms that reduce the activity of antioxidant enzymes. Serum paraoxonase 1 (PON1), is an antioxidant calcium-dependent enzyme has been implicated in the pathogenesis of male infertility. This study aimed to explore the relationship between PON1-L55M polymorphism with idiopathic male infertility. In this case–control study Semen samples were collected from 77 healthy men controls and 128 infertile donors. The paraoxonase activity of PON1 enzyme were measured by spectrophotometric methods. Genomic DNA was extracted from sperm and rs854560 (L55M) was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR sequencing. Statistical analysis indicated that significant difference in genotype frequencies of PON1 L55M polymorphism between patients and controls. Our results showed that, the men with a MM genotype (p= 0.021; OR= 0.091, CI=0.012-0.7) had a significant increased risk of idiopathic male infertility than those with a LM genotype (p= 0.013; OR=0.418, CI=0.27-0.855). In addition, PON1 activities in LL genotype were the highest than LM and MM genotype respectively (MM<LM<LL) in both patients and controls. So, the PON1-L55M is more likely to be a risk factor for idiopathic infertility in Iranian men. Based on the findings of this study, PON1 rs854560 (L55M) variation may be related to semen PON1 activity and the risk of idiopathic male infertility in Iranian population. Nevertheless, we suggested that, future studies focused on some factors such as, environmental and epigenetic factors in larger sample size are needed to reach accurate results.

PON1

Polymorphism

Male infertility

Oxidative stress

According to world health organization (WHO), male infertility as a global health issue refers to male's inability to cause pregnancy in a fertile female after 12 months of regular, unprotected sexual intercourse [1]. Approximately one in 20 of adult men in the reproductive age group are suffer from male factor infertility [2]. Nearly 25% of these cases of male infertility is unknown and is defined as idiopathic male infertility [3]. Many factors can impact male infertility including epigenetic, genetic, environmental, and lifestyle- related factors [4]. One of the main causes of Male infertility is stress oxidative (OS). Reactive oxygen species (ROS) is strongly related to impaired spermatogenesis and male infertility [5]. ROS is essential for signal transduction pathways, modulation of activities of redox-sensitive transcription factors, and regulation of mitochondrial enzyme activities in all living aerobic cells [6]. ROS includes free radical agents such as superoxide anions (O2•), hydrogen peroxide(H2O2), peroxyl (ROO•), and hydroxyl (OH•) radicals that are highly reactive [7, 8]. ROS in low levels is essential for sperm capacitation, acrosomal reaction, hyperactivation, and fertilization but over production of ROS deactive antioxidant in the seminal plasma and cause OS. Many studies have reported that infertile men have high levels of seminal ROS and low levels of the total antioxidant capacity [9]. Seminal plasma leukocytes and the mitochondria in the spermatozoa are the primary cellular sources of ROS production [1]. According to many studies, high levels of seminal oxidative stress have been associated with sperm dysfunction such as imperfect metabolism, morphology, motility and fertilization by lipid peroxidation in sperm cellular membranes and sperm DNA fragmentation and eventually leads to the cell death [10, 11]. ROS causes reproductive problems in men by damaging the balance of sex hormones [12]. Detoxification of ROS concentrations is conducted by well-organized antioxidative system which includes non-enzymatic and enzymatic antioxidants. The enzymatic antioxidants include superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), and paraxonase (PON). Most of these enzymes are encoded by SOD, glutathione peroxidase (GPx), CAT and PON genes [9, 13]. Many studies demonstrated that genetic mutations and genetic polymorphisms in these genes containing the single nucleotide polymorphisms (SNPs), can reduce the rate of fertilization [14, 15]. A wide range of genetic, epigenetic and lifestyle-related factors can break the balance between antioxidant defense system and ROS and lead to infertility [16]. Serum paraoxonase1 (PON1) is a 43kD extracellular HDL-associated antioxidant enzyme which is dependent on Ca²⁺ and prevents LDL and HDL oxidation to protect cells against OS [17, 18]. Infertility may be caused by hyperlipidemia therefore, PON1 prevents the occurrence of this disease by its key role in preventing lipid peroxidation [19–21]. Sperm parameters such as concentration, motility, and morphology can be influenced by paraoxonase levels and its activity in spermatozoa [22]. The long arm of chromosome 7 between q21.3 and q22.1 is belonging to multigene cluster which consists of PON1, PON2 and PON3 [23]. Liver is the predominant source of PON1 and PON3 expression which can be detected in the blood although, PON2 is extensively expressed in numerous tissues such as testis, brain, kidney and liver but not secreted into blood [24]. All three PON proteins localized in spermatozoa however only PON1 and PON3 were found in Sertoli and Leydig cells [25, 26]. PON1 mRNA has been detected in some tissues such as kidney. It has been documented that the ability of HDL to prevent both the oxidation of LDL and the interaction between macrophages and endothelium by inactivation of PON1 increases [27, 28]. Several researchers have reported that PON1 is present at various stages of spermatogenesis however the exact role of PON1 in male reproductive system is unknown [21, 25]. More than seven polymorphisms in the coding region and five in promoter region have been identified in the PON1 gene. It seems that substitution of glutamine (Q) by arginine (R) at position 192 (Q192R) and leucine (L) by methionine (M) at position 55 (L55M) of coding region are important functional genetic polymorphisms of PON1 protein [29, 30]. In fact, genetic analysis demonstrated that replacement of Leucine by Methionine at position 55 influences the paraoxonase and aryl esterase activities and the stability of the protein which can reduce the sperm motility [26, 31]. The L55M polymorphism shows three phenotypes (LL, LM and MM) which paraxonase activity in MM form is lower in compared with LL and LM [26]. Some report showed that L55M could be a risk factor in male infertility and influences the stability of the protein [18]. As of date, a little research has been conducted about the PON1 L55M (as known c.163T > A) gene polymorphism. The cytogenetic location (gene position), preferred name, rs number, human genome variation society (HGVS) of PON1 L55M showed in the Fig. 1a. In this present study, we determined the association of L55M and male infertility. In addition, PONase activities, the level of malondialdehyde (MDA) and total antioxidant capacity (TAC) in infertile men of Mazandaran population (North of Iran), were assessed.

Subjects and sample collection.

In this case-control study, semen samples were collected from 128 idiopathic male infertility and 77 fertile men without any history of infertility in their first-degree family as a healthy control group. All semen samples prepared from Fatemeh Al-Zahra IVF and Pastor Laboratory (Babol, Mazandaran, Iran), and stored at -20°C for other use. The infertile men who referred to Fatemeh Al-Zahra IVF and Pastor Laboratory had no history of cryptorchidism, orchitis, infectious disease, diabetes mellitus, drug abuse, obstruction of the vas deferens, varicocele, abnormal profiles of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone, abnormal karyotype as well as Y-chromosome microdeletion. In addition, the infertile men’s women had no problem in their reproductive systems. Also, there was no sign of coronary heart disease (CHD), atherosclerosis, liver diseases, hypertension, diabetes and cancer in all fertile and subfertile cases which are paraxonase-affecting diseases. The semen analysis was evaluated according to the world health organization [32] guidelines with respect to sperm motility (normal ≥ 25%) concentration (normal ≥ 20× 10⁶ spermatozoa/ml), and normal morphology (normal ≥ 14%).

This study was confirmed by the principles outlined in the Declaration of Helsinki and approved by the ethics committees of University of Mazandaran (#IR.UMZ.REC.1399.033).

DNA extraction and PON1 L55M genotyping.

Genomic DNA was extracted from semen samples by a conventional salting-out method, which described by Mwer et al. [33] and then stored in -20°C until to use. The PON1 L55M (NM_000446.7: c.163T > A) gene polymorphisms were genotyped using polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP).

One pair of primers were designed using with oligo primer analysis software ver. 7.0 and were listed as follows: forward primer: 5´-GAAGAGTGATGTATAGCCCCAGTT, reverse primer: 5´-AGTGGGCATGGGTATACAGAAA. The amplification reactions were carried out in a total volume of 25 µl consisting: 100 ng (2 µl) genomic DNA, 10 pmol (1 µl) of each primer, 2.5 µl of 10X PCR buffer, 0.5 µl of four mixed dNTPs (10 mM, Cinnagen Inc, Iran), 1µl of MgCl₂ (50 mM, Cinnagen Inc, Iran), 0.25 µl of 5U/µl Taq DNA polymerase (Cinnagene, Co., Iran). PCR program was used for the amplification: 5 min at 94°C, 32 cycles of 30s at 94°C, 30s at 56°C, 30s at 72°C and finally extension step 5 min at 72°C. PON1 gene rs854560 polymorphism was amplified by PCR and then genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. For the PCR RFLP analysis, PCR products were digested with Hin1II restriction enzyme (Thermo Fisher Scientific, USA). The restriction reactions were carried out in a total volume of 10 µl containing: 5µl PCR product (~ 100ng); 3µl DNAase free H₂O; 1.5 µl 10X fast digest green buffer and 0.5µl (~ 3 Units) of the restriction enzymes. These components were incubated 5min at 37°C. Restriction fragments were separated by 1.5% agarose gel electrophoresis and visualized by UV-transilluminators after staining with 1 µg/ml ethidium bromide [34]. Function of Hin1II restriction enzyme, PCR amplification product sizes and genotyping map are summarized in Figure. 1b.

TAC, MDA and PON1 activities.

Total antioxidant capacity (TAC) level was measured by its ability to reduce Fe³⁺ to Fe²⁺ using the ferric reducing ability of plasma (FRAP) method [35]. Malondialdehyde (MDA) level determined by its reaction with thiobarbituric acid (TBA) at 100°C [36]. Procedure these methods in semen fluid and described by Fallah et al [37].

The PONase activity was assessed spectrophotometrically using paraoxon (Sigma Chem, USA) as substrate and the absorbance was recorded at 412 nm, because of 4 -nitrophenol formation [38]. Briefly, the paraxonase activity was measured at 25°C during 3 min after 10 µl of seminal plasma were added to each well containing 150 µl of Tris-Cl (100 mM, pH 8.5) buffer including 2 mM CaCl₂ and 6 mM of paraoxon. All results are expressed in U/ml which is defined as 1 nmol of 4- nitrophenol formed per minute. The PON1 enzymatic activity was calculated by using the molar extinction coefficient 18 053 M⁻¹ cm⁻¹.

Statistical analysis.

Statistical analysis of difference in allele and genotype frequencies between controls and infertility groups were performed by using SPSS ver. 26.0 (SPSS, Inc., Chicago, IL, USA) software. After assessing the normality of the constant variables, using the Shapiro-Wilk test, quantitative data were presented as mean ± SD (normally distributed data) however qualitative variables were represented as a number or percentage. The Hardy–Weinberg equilibrium (HWE) test was used to estimate genotype frequencies. Results were revealed by odds ratios (ORs) and 95% confidence intervals (CI). Both clinical and laboratory data were checked for their correlation with PON1 polymorphism. A p-value less than 0.05 (typically ≤ 0.05) were accepted to be statistically significant.

in silico analysis.

L55M is one of the most studied polymorphisms has been associated with PON1 levels and activity. In this study, some bioinformatics tools were used to investigate the molecular effects of this substitution. To prediction the effects of L55M substitution on the structure and function on PON1 protein, the PolyPhen-2 in silico prediction server (http://genetics.bwh.harvard.edu/pph2/index.shtml) was used. SIFT server is an online tool to predict if an amino acid substitution effects on protein function (https://sift.bii.a-star.edu.sg/). The MutPred server which is based on SIFT, was developed to classify amino acid substitutions (AAS) as benign or disease-associated (http://mutpred.mutdb.org/).To determine the effect of mutation on protein stability and structure according to free energy change value (ΔΔG), I-Mutant 2.0 online server was used (https://folding.biofold.org/i-mutant/i-mutant2.0.html). SNP & GO is an online server which predict human disease-related Mutations in proteins by determination of Reliability Index (RI) (https://snps-and-go.biocomp.unibo.it/snps-and-go/). To determine the effect of this polymorphism on m-RNA secondary structure SNAP software was used. For the purpose of finding PON1 expression in different tissues and the relationship between rs854560 polymorphism and PON1 expression levels in testis, GTEx portal was used. Kyte-Doolittle scale was used for detection protein hydrophobic and hydrophilic tendencies before and after mutation.

A total of 205 individuals were participated in this present study which include 128 patients with idiopathic male infertility and 77 fertile men as control group. The average age of the healthy donors was 35.25 ± 7.19. The case group included 128 infertile men which the average of age was 37.62 ± 9.24 Seminal factors such as, motility, sperm count, morphology and volume in both infertile and fertile groups were are summarized in Table 1.

Table 1

Sperm parameters and biochemical parameters of fertile and infertile men.
Sperm parameters	Fertile (n = 77)	Infertile (n = 128)	p-value
Age (year)	35.25 ± 7.19	9.24 ± 37.62	p = 0.056
Morphology (percentage of normal sperm)	24.49 ± 8.35	9.24 ± 11.08	p < 0.0001
Motility (motile sperm percentage)	62.18 ± 16.07	38.18 ± 8.69	p < 0.0001
Seminal volume (ml)	2.43 ± 0.889	1.77 ± 3.75	p < 0.0001
Sperm count (×10⁶/ml)	93.31 ± 32.26	32.80 ± 33.47	p < 0.0001
Biochemical parameters
MDA µM. L^− 1	1949.33 ± 229.56	1513.43 ± 412.65	p < 0.0001
TCA µM. L^− 1	7.61 ± 2.53	9.65 ± 5.49	p < 0.0001

The levels of MDA and TAC (as biochemical parameters) in seminal fluid was assayed by TBA and FRAP methods, respectively and results reported in Tables 1. The concentration of MDA in the seminal fluid of the infertile men (9.65 ± 5.49) and fertile subjects (7.61 ± 2.53) was evaluated. Statistically, the plasma levels of MDA significantly (p < 0.001) increased in infertile men compare with fertile subjects. In contrast, seminal TAC levels were considerably (p < 0.001) higher in fertile donors (1949.33 ± 229.56) than in men with idiopathic infertility (1513.43 ± 412.65). In general, seminal MDA levels were significantly higher and seminal TAC levels were considerably lower in men with idiopathic infertility than in fertile (Table 1).

Genotyping results

The amplified 259 bp fragment of PON1 L55M, which flanked the PON1 L55M (c.163T > A) loci, were used for SNP genotyping. After digestion of PCR products by Hin1 II and subjected to electrophoresis on an 1.5% agarose gel, gene variants have been displayed in Fig. 1b-c. In this figure, an undigested single band of 259 bp was detected in LL homozygotes, digested fragments of 125 and 134 bp were identified in MM genotypes and three different fragments (134, 125 and 259 bp) were obtained for heterozygous genotypes. The direct DNA sequencing revealed that PCR-RFLP results for the three mentioned SNPs are reliable Fig. 1d.

Distribution of genotypic and allelic frequency

The analysis of T163A polymorphism correlation with sperm abnormality and sperm motility showed that these clinical data of infertile patients were associated with T163A polymorphism genotypes. Statistical analysis showed that PON1 L55M allele frequencies and genotype distribution in the fertile and infertile subjects were agreed with Hardy-Weinberg equilibrium (p > 0.05). Statistical analysis showed that PON1 L55M allele frequencies and genotype distribution in the fertile and infertile subjects were agreed with Hardy-Weinberg equilibrium (p > 0.05). The frequencies of the alleles and genotypes of the L55M polymorphism in infertile and fertile men are shown in Table 2. As shown in Table 2, T163A SNP, included frequent heterozygous genotype TA (p = 0.013; OR = 0.418; 95% CI = 0.27–0.855), TA + AA genotypes (p < 0.001; OR = 0.139; 95% CI = 0.124–0.299), and A allele (p < 0.001; OR = 0.193; 95% CI = 0.124–0.299), revealed a significant with male infertility. And, there was no significant difference in these frequencies between infertile and healthy control groups. Investigation of allele and genotype frequencies for the PON1 T163A polymorphism showed that the frequency of 163A allele in the infertile was higher than the control group ((p < 0.001; OR = 0.193; 95% CI = 0.124–0.299). Additionally, subjects who receive the A allele (TA and AA genotypes) have an increased risk of infertility (p < 0.001; OR = 0.139; 95% CI = 0.124–0.299).

Table 2

Analysis of PON1 L55M (c.163T > A) gene variants with the risk of male infertility.
Genotype/ Allele	No. and Percentage		OR (95% CI)	p-value	HWE χ2 (p value)
Genotype/ Allele	Control (77)	Case (n = 128)	OR (95% CI)	p-value	HWE χ2 (p value)
Genotype (Phenotype)
TT (LL)	38 (49.35%)	28 (21.9%)	-	-	0.0042**
TA (LM)	39 (50.64%)	84 (65.63%)	0.418(0.27–0.855)	0.013*	0.283
AA (MM)	1 (1.3%)	16 (12.5%)	0.091(0.012-0.7)	0.021*	0.0085**
TA (LL) + AA (MM)	40 (51.95%)	100 (78.13%)	0.139(0.124–0.299)	< 0.001**	0.0832
Allele
T	115 (74.67%)	140(54.68)	-	-	0.054*
A	41(26.62%)	116(45.31)	0.193(0.124–0.299)	< 0.001**	0.010**
OR = odds ratio; CI = confidence interval; HWE = Hardy-Weinberg equilibrium; P value < 0.05; P Value < 0.01*

Analysis of paraxonase activity in seminal plasma

Our findings revealed plasma PON activity had significant difference between infertile and control groups (p < 0.001) so that, infertile patients had considerably lower PON activity (0.046 ± 0.002) in semen plasma compared to healthy men (0.063 ± 0.002) (Fig. 2a). Collectively, PON activities in MM phenotype was the lowest than LM and LL phenotypes respectively (MM < LM < LL) in both patients and controls (Fig. 2b).

in silico results.

According to PolyPhen-2 online tool, L55M was found as benign. L55M polymorphism which submitted to PolyPhen was also submitted to the SIFT server. Therefore, a strong correlation between the results obtained from the PolyPhen and SIFT server was found. There was a remarkable relationship between the results obtained from the PolyPhen and MutPred servers. Due to, I-Mutant 2.0 online server, negative ΔΔG value induces that the protein being mutated is of lower stability and vice versa is also true. Our results show that L55M substitution can reduce the protein stability by negative ΔG (ΔΔG=-0.64). According to SNP& GO server, rs 854560 with Reliability Index value greater than 5 (RI = 7) can have disease causing abilities although the cell continues to live despite it. Each amino acid has a numeric value according to how hydrophobic or hydrophilic it is. Then a change in an amino acid in protein sequence can has an effect on its hydrophilicity and hydrophobicity. PON1 analysis by Kyte scale (Kyte-Doolittle Hydropathy Plot), showed that the peak of hydrophobic pattern in wild type in position 55 for Leucine − 0.44 (Fig. 3b) is higher than Methionine for mutant variant (-0.82) (Fig. 3a). As a result, the Leucine to Methionine substitution can decrease the tendency of PON1 protein hydrophobicity (Fig. 3).

By using GTEx, PON1 gene expression in different tissues such as Adrenal glands, Breast, Fallopian Tube, Liver, Nerve, Ovary, Prostate, Testis, Thyroid and vagina shown in Fig. 4. According to GTEx portal out comes, rs854560 variant is remarkably related to different levels of PON1 expression in liver, testis and ovary (Fig. 4).

The data in SNAP software revealed that Leucine substitution by Methionine didn’t make a significant change on m-RNA secondary structure p = 0.8427; the p-value < 0.2 is a fundamental structural change), (Fig. 5).

Infertility is one of the most social problems which known as a multifactorial disease. Epigenetic modifications, genetic variants and environmental factors, play a key role in the risk of Male infertility [2, 39]. Male reproductive system may be affected by environmental factors and pollution. Cigarette smoking is one of the main risk factors for male infertility. Acrosome reaction and capacitation which are two necessary processes for fertilization can be impairs by smoking also smoking can causes sperm ultra-structural abnormalities. According to American Society of Reproductive Medicine researches Cigarette smoking is associated with poorer semen parameters in smokers than in non-smokers [40]. Sperm creatine kinase activity can reduce by using cigarettes. Cigarette smoking modify the PON1 thiol groups, then inhibit hydrolytic activity of this enzyme [41]. In pregnant women who are exposed to smoking, during the first trimester of pregnancy, PON1 activity can be reduce [42].

Stress oxidative (OS) and reactive oxygen species (ROS) are strongly related to impaired spermatogenesis and male infertility [5]. ROS has both favorable and disadvantageous effects on spermatozoa as low levels of ROS have been shown to be necessary for sperm fertilization, motility, capacitation and acrosome reaction, but high levels of ROS may cause male infertility by ROS-induced sperm morphology, membrane and DNA damage [10, 11]. The human body has several antioxidant enzymes to protects itself against ROS damage [9, 43]. Antioxidant enzymes play a key role in maintain the oxidant and antioxidant balance [22]. PON1 is an antioxidant calcium-dependent enzyme that appears to play an important role in the development of a large variety of diseases. This enzyme possesses three enzymatic activities: lactonase, aryl esterase, and paraoxonase activity. PON1 is mainly synthesized in the liver, then transported from the liver to several tissues and released into the blood circulation in which it binds to cell membranes and protects lipids against peroxidation [44]. PON1 received its name from its ability to hydrolyze paraoxon (diethyl p-nitrophenyl phosphate). Several studies have reported that PON1 is present at various stages of spermatogenesis [21, 25] Sperm plasma membrane has a high level of polyunsaturated fatty acids. The PON1 gene encodes an enzyme which protects sperm plasma membrane against lipid peroxidation [22]. There is enough evidence that show genetic risk factors play an important role in increasing the risk of male infertility [45, 46]. Leu 55 Met is one of the most common types of genetic variation in coding region which is identified in PON1 gene and has effect on PON1 serum concentration and activity [47]. PON1 L55M polymorphism has been reported in various diseases, including cardiovascular diseases [48], rheumatoid arthritis [49], Breast cancer and Parkinson [28]. In this present study, we found significant association between the substitution of Leucine (TTG) at position 55 by Methionine (ATG) at third exon (L55M) and the risk of male infertility. L55M polymorphism (rs854560-T163A) displays three phenotypes (LL, LM and MM) which paraxonase phosphodiesterase activity in MM form is lower than LL and LM [26]. Although serum PON1 aryl esterase activity wasn’t linked to this polymorphism but L55M can regulate PON1 aryl esterase activity in the blood [42]. M allele carriers have significantly lower PON1 mRNA and protein levels. Some researches represented those men with MM genotype might be predisposed to infertility. On the other hand, it was shown that the T163A AT genotype is associated with less sperm motility. As a keynote, L55M could be a risk factor in male infertility and influences the stability of the PON1 protein [18].

The present study establishes an association L55M polymorphism can be a genetic marker to male infertility in Iranian population. Limitations of this study was the sample size. Nevertheless, we suggested that, future studies focused on some factors such as, environmental and epigenetic factors in larger sample size to significant of these findings.

Acknowledgments: Thanks to Mr. Hossein Moeini from Islamic Azad University (Qaemshahr, Iran) for preparing of samples. We are grateful to our colleagues for critical reviews of the manuscript.

Authorship Contribution Statement: All authors contributed to the writing of this article: AHC was involved in the conceptualization, methodology, and software sections. HM and FF contributed to the material preparation, data curation and analysis, and preparation of the original draft. AT and GJ contributed to the data analysis and editing of the manuscript.

Funding: This study was supported by a grant from the University of Mazandaran, dedicated to the MSc thesis of Hanie Mortezapour (#IranDoc 1550260)

Availability of Data and Materials: The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval: Informed consent was obtained from all subjects, and the study protocol was approved by the ethics committee of the University of Mazandaran and conducted in accordance with Iran National Committee for Ethics in Biomedical Researches (#IR.UMZ.REC.1399.033).

Consent to Participate.As corresponding author, I confirm that the manuscript has been read and approved for submission by all the named authors.

Consent to Publish. All authors would like to submit this manuscript, to be considered for publication as a Research Article, in the ESPR journal. We declare that it is original, has not been published before and is not currently being considered for publication elsewhere.

Conflict of Interest. There is no conflict of interest to declare.

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Association of PON1-L55M Genetic Variation with Idiopathic Male Infertility in an Iranian Population: A Case-Control Study

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Abstract

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Introduction

Materials and methods

Statistical analysis.

Results

Genotyping results

Distribution of genotypic and allelic frequency

Analysis of paraxonase activity in seminal plasma

Discussion

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