Association between the SIRT1 and SIRT3 levels and gene polymorphisms with infertility in war zones of Kermanshah province, Iran

doi:10.21203/rs.3.rs-1419037/v2

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Association between the SIRT1 and SIRT3 levels and gene polymorphisms with infertility in war zones of Kermanshah province, Iran

https://doi.org/10.21203/rs.3.rs-1419037/v2

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Background: War stresses and war toxin including mustard gas as mutagenic alkylating agent through inducing production of ROS and DNA mutagenesis, result in infertility in men. SIRT1 and SIRT3 are multifunctional enzymes involved in DNA repair, oxidative stress responses. The aim study is to assessed the correlation between serum levels of SIRT1, SIRT3 and both rs3758391T>C and rs185277566C>G gene polymorphisms with infertility in war zones of Kermanshah province, Iran.

Methods and Results: Based on spermogram analysis, samples were divided into two groups infertile (n=100) and fertile (n=100). HPLC method was used for determined MDA level and sperm chromatin dispersion (SCD) test was used for evaluated DNA fragmentation. Superoxide dismutase (SOD) activity was measured using the colorimetric assays. SIRT1 and SIRT3 protein levels were determined by using ELISA. The genetic variants of SIRT1 rs3758391T>C and SIRT3 rs185277566C>G were assessed by PCR-RFLP techniques.

MDA level and the percentage of DNA fragmentation were higher in infertile samples but serum levels of SIRT1 and SIRT3, and SOD activity was lower in infertile compared to fertile samples (p< 0.001). The TC + CC genotypes and the C allele from SIRT1 rs3758391T>C polymorphism, and CG + GG genotypes and the G allele from SIRT3 rs185277566C>G polymorphism could increase risk of infertility (p< 0.05).

Conclusions: The results of this study suggest that war toxins through the impact on genotypes, decreasing levels of SIRT1 and SIRT3 and increasing levels of oxidative stress, lead to defects in the concentration, motility and morphology of sperms and thus, infertility in men.

Infertility

Oxidative stress

SIRT1

SIRT3

War chemical toxin

About half of the all infertility is related to male factors (Agarwal et al. 2019). One of the most important causes of male infertility is an increase in levels of reactive oxygen species (ROS) compared to antioxidants. Evidence shows that ROS participates in the pathology of 30–80% of infertility in infertile men (Barati et al. 2020). ROS by lipid peroxidation, DNA damage, oxidation of proteins, and inactivation of enzymes in spermatozoa, eventually result in genetic mutations, reduced sperm volume and count, reduction of sperm motility, abnormal morphology of sperm (Alahmar 2019). Sources of ROS production are Various and are generally classified into two groups: endogenous and exogenous (Condorelli et al. 2017, Alahmar 2019). From endogenous sources can mention genital tract infection, metabolic syndromes and varicocele and from exogenous sources can mention, smoking, ionizing radiation, war chemical toxin and environmental pollution (Bolouki et al. 2020). Sulfur mustard (SM) is one of the most common war chemical agents, that widely used in several wars, such as the Iran-Iraq war (Bolouki and Zal 2020). Studies have shown that SM by inducing production of ROS, reducing seminal plasma antioxidants and Ultimately, imbalance in ROS level and antioxidant defense, result in reduces sperm quality and infertility in men (Safarinejad 2010).

Sirtuins (SIRTs) are NAD-dependent deacetylase enzymes that are involved in various function such as DNA repair, resistance to oxidative stress, apoptosis and control of metabolic enzymes. They play this key role by regulating the transcription factors downstream such as PGC1α, p53, FOXO, and NF-κB (Santos et al. 2016, Di Emidio et al. 2021). In mammals, seven SIRTs are known (SIRT 1-7), that to differ in intracellular position and role (!!! INVALID CITATION !!! ). Among SIRTs, SIRT1 and SIRT3 play the most important role in resistance to oxidative stress and DNA repair (Nasiri et al. 2021). humans, SIRT1 gene has 11 exons on chromosome 10 (10q21.3( and displaying expression in the cytoplasm and nucleus of all tissues, especially the adrenal and testes (Fagerberg et al. 2014).The SIRT3 gene has 10 exons on chromosome 11(11p15.5( and displaying expression in mitochondria testis, heart and brain and other tissues. SIRT1 polymorphisms, rs3758391 T/C and SIRT3 polymorphisms rs185277566 C/G, have been located in the promoter region, and may account for differential SIRT1,SIRT3 expression, function and susceptibility to certain disorders (Mohtavinejad et al. 2015).

The precise mechanism of how war chemical toxin affects the reproductive systems and male infertility is not entirely understood (Bolouki and Zal 2020). Previous studies have shown that a decrease in SIRT1 and SIRT3 leads to defects in spermatogenesis and in general infertility in men (Niu et al. 2016, Liu et al. 2017, Nasiri et al. 2021), However, the mechanism of its effect on the reproductive system is unclear. Although polymorphic variants of SIRT1, SIRT3 have been associated with susceptibility to urinary bladder cancer, acute coronary syndrome and myocardial infarction (Yin et al. 2016, Razi et al. 2017, Shafieian et al. 2019), the study of the relationship between SIRTs variants and infertility is very little. Since oxidative stress and environmental factors such as war chemical toxin are one of the main causes of fragmentation and mutation DNA and infertility in men, and due to the important role that SIRT1, SIRT3 play in neutralizing oxidative stress and DNA repair, and since the prevalence of these two variants is more than 0/1 and the mechanism of its effect on the reproductive system is unclear. The main purpose of this study was to investigate the relationship between the levels of SIRT1, SIRT3 and SIRT1 (rs3758391) and SIRT3 (rs185277566) variants with oxidative stress, DNA fragmentation and infertility in war zones of Kermanshah province, Iran.

2.1. Study subjects

Study population consisted of 200 healthy men, aged between 30-55 years, from Kermanshah who have referred to Motazedi Hospital in Kermanshah for diagnosis of infertility. Based on place of residence and spermogram analysis (according to the WHO standards), samples were divided into two groups: 1) 100 men infertile patients (men who participated in the imposed Iran–Iraq war or live in war zones of Kermanshah) and 100 men fertile (men who did not participate in the imposed war and live in non-war zones of Kermanshah). The study was approved by the Research Ethical Committee of Baqiyatallah University of Medical Sciences (IR.BMSU.REC.1399.317). Informed consent was obtained from each participant before participation. The exclusion criteria were samples with an infection bacteria or viruses, subjects with a history of diseases (diabetes mellitus, chronic diseases, hypertension and varicocele) and smoking.

Semen samples were collected by masturbation into wide-mouthed sterile containers after 48 hours of abstinence. After liquefaction of semen samples in an incubator at 37 ° C, A portion of semen sample was used for routine analysis of semen parameters (macroscopic and microscopic parameters) according to World Health Organization (WHO-2010) (Organization 2010) and a portion of semen sample was used for assessment of sperm DNA fragmentation. The rest of the semen sample was centrifuged at 600 g for 10 minutes to separate the sperm cell from the seminal plasma, and stored at -20°C for subsequent measurements.

6 milliliters of fasting blood was received from each subject, 3 milliliters of which was transferred into tubes containing anticoagulants (EDTA) for DNA extraction and another 3 milliliters were poured into anticoagulant-free tubes to isolate serum samples for analysis of SIRT1 and SIRT3 in serum.

2.2. Assessment of antioxidants and oxidative stress biomarkers

The level ofmalondialdehyde (MDA) (Lipid peroxidation marker) was determined by reverse phase high-performance liquid chromatography (HPLC) (Agilent Technologies1200 Series). Device specifications included Nucleodur100–5 C18ec column (Macherey-Nagel, Duren, Germany), a fluorescence detector using EC 250/4.6, mobile phase 60:40 V/V of methanol: potassium monobasicphosphate buffer (50 Mm) and detection, Ex515-Em555 (Nasiri et al. 2021).

Superoxide dismutase (SOD) activity was measured by Kiazist SOD kit (Kiazist, Iran). This protocol employs the production of superoxide radicals by xanthine oxidase.

2.3. Determination of Sperm DNA fragmentation

Sperm chromatin dispersion (SCD) test (Halosperm kit INDAS) used for detecting DNA fragmentation in sperm. In SCD method, the spermatozoa with non-fragmented DNA shown a large halo dispersed DNA loops, whereas the spermatozoa containing fragmented DNA display very small or no halos (Medicine 2013).

2.4. Assessment of SIRT1 and SIRT3 assay

To measurement of SIRT1 and SIRT3, 3 ml fasting blood sample was taken. Serum SIRT1 and SIRT3 were determined by Enzyme-Linked Immunosorbent Assay (ELISA) kit (Eastbiopharm, Hangzhou, USA).

2.5. DNA extraction and genotyping

The standard procedure of phenol-chloroform method was used to DNA extraction of whole blood leukocytes (Chacon Cortes et al. 2014, Mohammadi-Noori et al. 2020). The genotypes of SNPrs3758391 (SIRT1) and SNPrs185277566 (SIRT3) were determined by PCR–RFLP technique. Based on gene sequences in the GeneBank, From Primer 3 online website (https://primer3.ut.ee/) was used to design the pair of primers sequences of SIRT1 rs3758391 and SIRT3 rs185277566 SNPs.

The forward (F); 5′-TGGCCAGAACCCATACTAGG-3′, and reverse(R); 5′-AGCCCTTCCACTTTCCTCTC-3′, primers were used to determined SIRT1-3758391 SNP. PCR reaction was carried out in total volume of 25 µL (18 μL of ddH2O, 0.5 μL of dNTPs at a concentration of 10 mM, 0.75 μL of MgCl2 at a concentration of 50 mM, 2.5 μL of 10X PCR Bufer, 1 µL of each primer (F and R) at a concentration of 10 pmol and 0.2 μL of Taq DNA Polymerase at a concentration of 5 U/µL). The PCR protocol consisted of 1cycle initial denaturation (at 95 °C for 5 min), and 40 cycles amplification (at 95 °C for 30 seconds , 61.3 °C for 30 seconds, 72 °C for 30 seconds ), with a final extension at 72 °C for 10 min. Almost 20 µL of PCR product at 37 °C overnight was digested by StyI enzyme. The digested products were electrophoresed in 2% agarose gel. The homozygote mutant CC was not cleaved by the StyI enzyme and a fragment with a length of 205 bp was created, heterozygote mutant TC fragments were 205 bp, 120 bp, 85 bp and homozygote wild TT fragments were 120 bp, 85 bp as shown in Fig1A.

The forward (F); 5′-ACCCCAGAACGCCATGTT-3′, and reverse(R); 5′-TGAGACACCAGACTAGGGAACTT-3′, primers were used to determined SIRT3-185277566 SNP. PCR reaction was carried out in total volume of 25 µL (16 μL of ddH2O, 2 μL of DMSO, 0.5 μL of dNTPs at a concentration of 10 mM, 0.75 μL of MgCl2 at a concentration of 50 mM, 2.5 μL of 10X PCR Bufer, 1 µL of each primer (F and R) at a concentration of 10 pmol and 0.2 μL of Taq DNA Polymerase at a concentration of 5 U/µL). The PCR protocol consisted of 1cycle initial denaturation (at 95 °C for 5 min), and 40 cycles amplification (at 95 °C for 30 seconds , 55 °C for 30 seconds, 72 °C for 30 seconds ), with a final extension at 72 °C for 10 min. Almost 20 µL of PCR product at 37 °C overnight was digested by Cfr421 enzyme. The digested products were electrophoresed in 3% agarose gel. The homozygote wild CC was not cleaved by the Cfr421 enzyme and a fragment with a length of 246 bp was created, heterozygote mutant CG fragments were 246 bp, 186 bp, 60 bp and homozygote mutant GG fragments were 186 bp, 60 bp as shown in Fig1B.

2.6. Statistical analysis

The normality of distribution of quantitative data was evaluated by Kolmogrov-Smirnov (KS) test. Continuous and comparison data were performed using the Independent t-test or Mann–Whitney test. SPSS (version 16, Chicago, IL) was used for analyzing data and Statistical significance was assumed at the p ≤ 0.05. The frequencies of the genotypes and allele (SIRT1 and SIRT3) in patients and control groups were compared by Chi-square test. Odds ratios (OR), 95% confidence intervals (CI) and interaction between the two polymorphisms (SIRT1 T3758391C and SIRT3 C185277566G) were determined by SPSS logistic regression. Pearson correlation was used for Correlation between SIRT1 and SIRT3 levels with evaluated parameters.

3.1. Semen analysis

Table 1 presents Age, body mass index (BMI) and semen parameter of the study subjects. There was no significant difference in the mean of age, BMI and semen volume among the study groups. The mean concentration, motility and morphology of sperm in the infertile group were significantly lower than the fertile group.

3.2. Levels of oxidative stress biomarkers and Sperm DNA fragmentation

The seminal plasma levels of MDA and sperm DFI were significantly (P<0.001) higher in the infertile groups compared to fertile group. SOD activity in infertile group was remarkably (P<0.001) lower than in the fertile groups (Table 2).

3.3. Quantification of SIRT1 and SIRT3 proteins

A significantly lower (P < 0.001) proteins level of SIRT1 and SIRT3 were detected by ELISA in the serum of infertile groups compared with the fertile group. The amount of SIRT1 protein in fertile samples was 6.98±0.63 ng/ml and in infertile samples is 5.14±0.62 ng/ml (Fig2A). The amount of SIRT3 protein in fertile samples was 4.20±0.34 ng/ml and in infertile samples is 3.46±0.66 ng/ml (Fig2B).

3.4. Genotypes

The frequency of SIRT1 rs3758391T>C and SIRT3 185277566C>G genotypes and alleles in infertile and fertile group is demonstrated in Table 3 and Table 4. The distribution of SIRT1 rs3758391T>C genotypes (χ2 =14.03, df =2, p= 0.001) and alleles (χ2 =15.22, df =1, p= 0.014) in infertile men were significantly different compared to fertile men (Table 3). The OR for C allele and TC + CC vs. T/T genotype (dominant model) from SIRT1 rs3758391T>C was found to be 2.20(1.47–3.28, p =0.014) and 2.61(1.37–4.96, p =0.003) in the infertile group, that significantly increased the risk of infertility in the subject studied. Interestingly, distribution of SIRT3 185277566C>G genotypes (χ2 =6.34, df=2, p= 0.04) and alleles (χ2 =8.23, df =1, p= 0.004) evidence a signifcant diference in the infertile group in compare to the fertile group (Table 4). The presence of dominant (C/G+ G/G vs. C/C) genotypes [2.14(1.07–4.24, p= 0.02)] and the G allele [1.81(1.20–2.71, p=0.004)] of SIRT3 185277566C>G SNP significantly increased the risk of infertility in the subject studied.

The correlation of rs3758391 and 185277566 genotypes with semen levels of volume, concentration, motility, morphology, MDA, SOD, DFI, and serum levels of SIRT1, SIRT3 between the infertile and fertile groups is shown in Tables 5 and 6. For SIRT1, the presence of TT and TC+CC genotype in infertile men was strongly related with lower of motility , morphology , SOD, SIRT1, SIRT3 and related with higher of MDA (no significant only in the TC + CC genotype), DFI compared to fertile men (Table 5). Similar results were observed for SIRT3 genotypes rs185277566 CC and CG+GG (Table 6).

The correlation of SIRT1 concentration with the levels of semen and serum parameters and the severity of disease in the infertile men showed that there was a positive correlation between the levels of motility (r = 0.58, p < 0.001), morphology (r = 0.66, p < 0.001), SOD (r = 0.72, p < 0.001), SIRT3 (r = 0.26, p < 0.001), as well as a negative correlation between the levels of MDA(r = -0.09, p = 0.20), DFI (r = -0.74, p < 0.001), with SIRT1 level in serum (Table 7). A positive correlation has been found between morphology (r = 0.28, p < 0.001), SOD (r = 0.26, p < 0.001) and SIRT1 (r = 0.26, p < 0.001) as well as a negative correlation between DFI(r = - 0.25, p < 0.001), MDA (r = - 0.02, p = 0.76), concentration (r = - 0.04, p = 0.56) with SIRT3 level in serum (Table 7).

In this study, we detect that oxidative stress biomarker (like MDA), DNA fragmentation were higher in infertile specimen but SIRT1 and SIRT3 levels, and SOD activity was lower in infertile compared to fertile specimen. We observed that the distribution of rs3758391T>C and rs185277566C>G genotypes and alleles in infertile men were significantly different compared to fertile men.

Several studies have been investigated on the short-term and long-term effects of war on infertility (Amirzargar et al. 2009, Bolouki and Zal 2020). Evidence suggests that patients who have been exposed to war chemical toxin such as SM have reduced sperm count and sperm motility, increased abnormal morphology and lead to oligozospermia, asthenozospermia and teretozospermia compared to nonexposed individuals (Shakeri et al. 2007, Bolouki and Zal 2020). These results are consistent with our data and show that various reproductive toxicants through impacts on sperm quality lead to infertility. Many studies have shown, war chemical toxin, especially SM, through increase inflammatory reactions, inducing production of ROS in the testicular tissue, reducing seminal plasma antioxidants and Ultimately, imbalance in ROS level and antioxidant defense, result in reduces sperm quality and infertility in men (Jafari et al. 2010, Agarwal et al. 2019). In our study, there was a direct relationship between increased biomarkers of oxidative stress (MDA) and decreased antioxidants (SOD) with infertility. Increase the level of MDA in seminal plasma consistent with oxidation of sperm membrane lipids, resulting in abnormal morphology and reduced motility in sperm. SOD plays an important role in improving sperm quality (Macanovic et al. 2015). In this study we have shown that decreased SOD activity in seminal plasma is directly related to poor morphology and motility of sperms. However, in some studies, no significant correlation between the level of MDA and SOD activity with the quality of sperms (Suleiman et al. 1996, Shiva et al. 2011).

Increased DNA fragmentation rate in sperms can affect negatively on sperm viability and sperm fertility. In our study, there was a direct relationship between increased DNA fragmentation with decrease concentration, motility and abnormal morphology of sperm in infertile samples. Connection between DNA fragmentation and war chemical toxin was investigated. Safarinejad showed the direct correlation between DNA fragmentation rate and the amount of exposure to mustard gas in sperm sample. The frequency of DNA fragmentation was higher in the group with severe mustard gas injury (2010). These data are consistent with our results and indicates that the rate of DNA fragmentation in people exposure to war chemical toxin is high. Many studies have shown that elevation of ROS in seminal fluid is the most important factors in DNA fragmentation (Atig et al. 2017, Agarwal et al. 2019). Our study also shows that there is a direct relationship between increased ROS (increase in MDA level and decrease of SOD activity) and DNA fragmentation. These results in combination with our findings, suggest that the war chemical toxin by increasing oxidative stress lead to DNA fragmentation.

In our previous study, we found that SIRT1 and SIRT3 have an important role in improves sperm quality and it is a major regulators of ROS (Nasiri et al. 2021). According to our findings it can be guessed that the correlation between low levels of SIRT1 and SIRT3 protein in serum with high ROS and DFI of sperm in infertile samples. Loganathan et al. have shown the possible role of SIRT1 and SIRT3 in spermatogenesis and stated that SIRT1 and SIRT3 play an important role in spermatids differentiation and reducing oxidative stress and absence of this SIRTs leads to DNA damage (2021). In SIRT1 knockout mice in their reproductive system, this lead to suppression of germ cell differentiation (Niu et al. 2016) defective acrosome formation (Liu et al. 2017), increased ROS production (Nasiri et al. 2021), DNA fragmentation, poor genomic integrity in sperm (Atig et al. 2017) and consequently infertility in male. SIRT3 is a mitochondrial enzyme that plays a role in neutralizing oxygen radicals in the reproductive system. Studies have shown that SIRT3 increases the quality of sperm parameters by increasing the activity of antioxidant enzymes(Di Emidio et al. 2021), and reduces the production of ROS (Loganathan et al. 2021). These result are consistent with our findings and suggest that war chemical toxin through reducing the levels of SIRT1 and SIRT3 leads to increase the production of oxidative stress, DNA fragmentation and infertility in men.

Our data detected a significant differences between the genotypes and alleles of SIRT1 rs3758391T>C and SIRT3 rs185277566C>G polymorphism, between the two groups of infertile men and fertile men. The C allele from SIRT1 rs3758391T>C and The G allele from SIRT3 rs185277566C>G polymorphism could increase 2.20 and 1.80 times the risk of infertility in comparison with T and C alleles, respectively. The presence of TC+CC genotype compared to TT genotype of SIRT1 rs3758391T>C 2.61 times and the presence of CG+GG genotype compared to CC genotype of SIRT3 rs185277566C>G 2.14 times increases the risk of infertility.

Sarumaru et al. reported that among Japanese population the distribution of rs3758391T>C polymorphism genotypes had a significant correlation between autoimmune thyroid patients and control group and the C allele increased the risk of autoimmune thyroid disease (2016). Shafieian et al. stated that the frequency distribution of TT genotype of SIRT1 rs3758391 was significantly different among urinary bladder cancer and the control group. In this study, the wild TT genotype increases the risk of urinary bladder cancer (2019). While in our study the mutant CC genotype is associated with an increased risk of infertility. Inconsistent results between the SIRT1 rs3758391 polymorphism and the incidence of a disease may be due to differences in the type of damaged tissue, lifestyle, environmental factors and ethnicity. The results of this study show that a significant correlation between TC + CC, TT genotype from rs3758391T>C polymorphism with the serum SIRT1 levels, in which the patient (infertile men) with TC + CC, TT genotype, the level of SIRT1 is lower than in control subjects (fertile men) with the same genotype. Consistent with our results, there is a significant relationship between SIRT1 gene genotypes with the serum levels of this protein. Hu et al. tested the correlation of SIRT1 gene genotypes with the serum levels of this protein in acute coronary syndrome patients and their results showed that the C allele could reduce gene expression and serum SIRT1 levels between the acute coronary syndrome (2014). Consistently, in the study of Peng et al., the distribution of rs3758391T>C polymorphism genotypes associated with a decrease in serum SIRT1 levels, However, lowering the level of this protein reduces the susceptibility to diabetic foot ulcers (2018). These results suggest that serum levels of SIRT1 are associated with the SIRT1 rs3758391T>C genotypes.

Few studies have examined the genotype of SIRT3 rs185277566C>G with the incidence of a disease. Yin et al. examined the correlation of SIRT3 rs185277566C>G genotypes with myocardial infarction in the Chinese population. Their results show that a significant correlation between myocardial infarction patients and control group and the G allele increased the risk of myocardial infarction (2016). In addition, in our study, there was a significant relationship between SIRT3 rs185277566C>G polymorphisms and its serum level between patients and the control group. In patient (infertile men) with CG + GG, CC genotype, the level of SIRT3 is lower than in control subjects (fertile men) with the same genotype. These results suggest that serum levels of SIRT3 are associated with the SIRT3 rs185277566C>G genotypes.

The results of this study demonstrated that carriers of one or two copies of the C (TC+CC of SIRT1) or G (CG+GG of SIRT3) allele had significantly lower levels of sperm motility, sperm morphology, SOD, serum SIRT1 level, serum SIRT3 level, and higher levels of MDA (only in CG + GG genotype of SIRT3) and DFI in comparison with the control group with the same genotype. Furthermore, a significant positive correlation between serums SIRT1, SIRT3 levels with sperm motility, sperm morphology and SOD as well as a negative relationship between MDA, DFI with the level of these proteins. In the study of Mostafa et al. a negative correlation was observed between SIRT1 level with concentration, motility and morphology of sperm in varicocoele men (2018). Nasiri et al. stated that there is a positive relationship between SIRT1, SIRT3 level with antioxidants and a negative relationship between these proteins with oxidative stress in plasma seminal (2021). Loganathan et al. showed that a negative correlation between SIRT1 and SIRT3 level with DNA fragmentation and infertility (2021). According to these results, it can be guessed that the correlation between low levels of SIRT1 and SIRT3 protein in serum with high ROS and DFI of sperm in infertile samples and decreased levels of SIRT1 and SIRT3 protein may have led to increased ROS, DNA fragmentation, and infertility in men.

The results of know study demonstrated a correlation between SIRT1 rs3758391 and SIRT3 rs185277566
polymorphisms with infertility in the war zones of Kermanshah province, Iran. The genetic variants of SIRT1 rs3758391 and SIRT3 rs185277566 were correlated with diminish serum levels of SIRT1 and SIRT3 in infertile men. The levels of ROS and DNA fragmentation in people exposure to war chemical toxin are high. A significant positive correlation between serums SIRT1, SIRT3 levels with sperm motility, sperm morphology and SOD as well as a negative relationship between MDA, DFI with the level of these proteins. In summary, the results of present study showed that war chemical toxin through the impact on genotypes, decreasing levels of SIRT1 and SIRT3 and increasing levels of oxidative stress, lead to defects in the concentration, motility and morphology of sperms and thus, infertility in men.

ROS Reactive oxygen species

SM Sulfur mustard

SIRTs Sirtuins

DFI DNA fragmentation index

WHO World Health Organization

MDA Malondialdehyde

SOD Superoxide dismutase

SCD Sperm chromatin dispersion

HPLC Reverse phase high-performance liquid chromatography

ELISA Enzyme-Linked Immunosorbent Assay

PCR Polymerase chain reaction

RFLP Restriction fragment length polymorphism

SNP Single nucleotide polymorphism

This work was supported by Human Genetics Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran (Grant number: 98000337) and Kermanshah University of Medical Sciences. We express our gratitude to the Motazedi and Baqiyatallah Hospitals for providing the samples. The authors declare that they have no conflicts of interest. A.N., S.M.-H., A.V.-R.; Participated in the study design, data collection, performed statistical analyses, and wrote the manuscript. H.M.; Participated in perform the PCR-RFLP tests and interpreted the data. M.R. Was involved in both writing and editing the manuscript. All authors have read and approved the final version of the manuscript. The study was approved by the Research Ethical Committee of Baqiyatallah University of Medical Sciences (IR.BMSU.REC.1399.317). Informed consent was obtained from each participant before participation.

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Table1. Age, BMI and semen parameters data in the fertile and infertile groups

Parameters

Groups

P-value

Fertile

(n=100)

Infertile

(n=100)

Age(y)

BMI(Kg M^-2)

Volume (ml)

Concentration (10⁶ per ml)

Motility (%)

Morphology (%)

40.93 ± 3.91

27.85 ± 3.11

3.36 ± 0.8

40.74 ± 10.19

23.45 ± 3.23

6.97 ± 0.95

40.73 ± 3.67

27.10 ± 3.20

3.60 ± 0.66

37.21 ± 13.62

16.75 ± 3.51

3.94 ± 1.1

0.714

0.095

0.920

0.041*

0.001*

a)^*P-value<0.05, significant difference among the groups

b) BMI: body mass index

Table2. MDA concentration, SOD activity and percentage of DNA fragmentation in the fertile and infertile groups

Parameters

Groups

P-value

Fertile

(n=100)

Infertile

(n=100)

MDA (µmol/L)

SOD (U/ml)

DFI (%)

0.91 (0.52-1.12)

23.55 (19.83-27.01)

20.59 (16-23)

1.72(1.51-3.01)

12.17 (8.64-15.01)

35.94 (33-39.21)

0.001*

Data are presented as median and interquartile range (IQR (
^*P-value<0.05, was considered statistically significant.
MDA; Malondialdehyde, SOD; Superoxide dismutase, DFI; DNA fragmentation index.

Table3. Genotype distribution and alleles frequency SIRT1 (rs3758391) gene polymorphisms in the studied subjects

SNPrs3758391 Genotype	Fertile n (%)	Infertile n (%)	OR (95%CI, p value)
TT	38 (38%)	19 (19%)
TC	46 (46%)	45 (45%)
CC	16 (38%)	36 (38%)
	χ2 =14.03, df =2,p= 0.001
TT	38 (38%)	19 (19%)	2.61(1.37–4.96, 0.003)
CT+CC	62 (62%)	81 (19%)
	χ2 =8.85, df =1,p= 0.003
Alleles
T	122 (61%)	83 (41.5%)	2.20(1.47–3.28, 0.014)
C	78 (39%)	117 (58.5%)
	χ2 =15.22, df =1,p= 0.014

P value < 0.05 was considered significant
Odd ratio (OR) was calculated 95% confidence interval

Table4. Genotype distribution and alleles frequency SIRT3 (rs185277566) gene polymorphisms in the studied subjects

SNPrs185277566 Genotype	Fertile n (%)	Infertile n (%)	OR (95%CI, p value)
CC	29 (29%)	16 (16%)
CG	34 (34%)	32 (32%)
GG	37 (37%)	52 (52%)
	χ2 =6.34, df =2,p= 0.04
CC	29 (29%)	16 (16%)	2.14(1.07–4.24,0.02)
CG+GG	71 (71%)	84 (84%)
	χ2 =4.84, df =1,p= 0.02
Alleles
C	92 (46%)	64 (32%)	1.81(1.20–2.71,0.004)
G	108 (52%)	136 (68%)
	χ2 =8.23, df =1,p= 0.004

P value < 0.05 was considered significant
Odd ratio (OR) was calculated 95% confidence interval

Table5. Relationship of SIRT1 (rs3758391) genotypes with serum and semen parameters in fertile and infertile groups

Parameters	Fertile group TT	Infertile group TT	p values	Fertile group TC+CC	Infertile group TC+CC	p values
N (%)	38(38%)	19 (19%)		62 (62%)	81 (81%)
BMI(kg/m²)	27.60	27.84	0.80	28.00	26.93	0.04
Volume (ml)	3.40	3.07	0.10	3.33	3.80	0.30
Concentration (10⁶ per ml)	41.02	37.36	0.18	40.56	39.90	0.70
Motility (%)	23.55	16.84	0.001	23.38	16.72	0.001
Morphology (%)	6.60	3.78	0.001	7.19	3.97	0.001
MDA (µmol/L)	0.42	1.80	0.001	1.19	1.68	0.53
SOD (U/ml)	23.56	11.70	0.001	23.55	12.27	0.001
DFI (%)	19.63	36.15	0.001	21.17	35.88	0.001
SIRT1 (ng/ml)	7.05	5.19	0.001	6.93	5.12	0.002
SIRT3 (ng/ml)	4.30	3.46	0.001	4.24	3.50	0.001

BMI; Body mass index, MDA; Malondialdehyde, SOD; Superoxide dismutase and DFI; DNA fragmentation index.

Table6. Relationship of SIRT3 (rs185277566) genotypes with serum and semen parameters in fertile and infertile groups

Parameters	Fertile group CC	Infertile group CC	p values	Fertile group CG+GG	Infertile group CG+GG	p values
N (%)	29(29%)	16 (16%)		71 (62%)	84 (81%)
BMI(kg/m²)	27.63	27.75	0.88	27.95	26.18	0.07
Volume (ml)	3.18	5.40	0.13	3.43	3.33	0.40
Concentration (10⁶ per ml)	39.66	39.00	0.83	41.20	39.50	0.30
Motility (%)	22.66	17.18	0.001	23.78	16.66	0.001
Morphology (%)	6.68	3.68	0.001	7.01	3.98	0.001
MDA (µmol/L)	2.00	1.59	0.80	0.42	1.72	0.001
SOD (U/ml)	23.48	12.27	0.001	23.58	12.14	0.001
DFI (%)	20.50	35.56	0.001	20.62	36.01	0.001
SIRT1 (ng/ml)	6.94	5.30	0.001	6.99	5.10	0.001
SIRT3 (ng/ml)	4.28	3.29	0.001	4.26	3.49	0.001

BMI; Body mass index, MDA; Malondialdehyde, SOD; Superoxide dismutase and DFI; DNA fragmentation index.

Table7. Correlation of SIRT1 and SIRT3 levels with serum and semen parameters in infertile groups.

Parameters	Pearson Correlation for SIRT1.Conc	P value	Pearson Correlation for SIRT3.Conc	P value
BMI(kg/m²)	0.10	0.13	0.002	0.97
Volume (ml)	0.013	0.85	0.01	0.87
Concentration (10⁶ per ml)	0.06	0.39	-0.04	0.56
Motility (%)	0.58	0.001	0.121	0.88
Morphology (%)	0.66	0.001	0.282	0.001
MDA (µmol/L)	-0.09	0.20	-0.02	0.76
SOD (U/ml)	0.72	0.001	0.26	0.001
DFI (%)	-0.74	0.001	-0.25	0.001
SIRT1 (ng/ml)	-	-	0.26	0.001
SIRT3 (ng/ml)	0.26	0.001	-	-

BMI; Body mass index, MDA; Malondialdehyde, SOD; Superoxide dismutase and DFI; DNA fragmentation index.

No competing interests reported.

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Association between the SIRT1 and SIRT3 levels and gene polymorphisms with infertility in war zones of Kermanshah province, Iran

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Abstract

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1. Introduction

2. Materials And Methods

3. Results

4. Discussion

Abbreviations

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