Correlation Between Protamine-2 and miRNA-122 in Sperm from Heroin-addicted Men: A Case-Control Study Zohreh Nazmara1, Mohammad Najafi2,3, Mansoureh Movahedin4, Zahara Zandieh1,5, Peymaneh Shirinbayan6, Hamid reza Asgari1, Mohsen Roshanpajouh7, Chad B. Maki8, Zahra Bashiri1,2, Morteza Koruji1,2* Purpose: Recreational use of illicit drugs is one of the main factors affecting male fertility. However, the mech- anisms of heroin smoke-associated damage to mature spermatozoa are still completely unknown. The aim of this study was to concomitantly examine the levels of protamine-2 gene and protein concentrations, the amount of miRNA-122 in seminal plasma and semen analysis findings in heroin-addicted men. Materials and Methods: In a case control study, twenty-four fertile men that lacked any recreational drug abuse were considered as the healthy group, and 24 addicted men who used only heroin for at least four months were selected as the addicted group. Semen samples were gathered by masturbation after 2 - 5 days of sexual abstinence. Following the preparation of a semen analysis by computer-assisted sperm analysis according to WHO (2010), the level of protamine-2 gene expression in sperm and miRNA-122 in seminal plasma was measured using real-time sqPCR. Also, protamine-2 protein concentrations were quantified by nuclear protein extraction, SDS-Page and western blotting. Results: Among the studied variables, body mass index (27.75 ± 0.88 vs. 22.30 ± 0.36, p = 0.001), seminal pH (7.79 ± 0.06 vs. 7.58 ± 0.06, p = 0.003), white blood cell count in semen (1.69 ± 0.41 vs. 8.61 ± 1.73, p = 0.001), motility (65.51 ± 2.57 vs. 41.96 ± 3.58, p = 0.001) and survival rate (87.41 ± 1.00 vs. 71.50 ± 4.59, p = 0.002) of sperm cells was significantly different between the healthy and addicted groups. In addition, the levels of protamine-2 gene and protein expression in the addicted group (0.05 ± 0.02 and 0.10 ± 0.02, respectively) were significantly lower than the healthy group (3.59 ± 0.94 and 0.27 ± 0.06, respectively) (p = 0.002 and p = 0.017, respectively). Seminal miRNA-122 levels in addicted men (3.51 ± 0.73) were statistically higher than in healthy men (1.52 ± 0.54) (p = 0.034). Conclusion: This is one study on human infertility that evaluates the effects of heroin on protamine deficiency and seminal small RNAs expression levels. Heroin abuse may lead to male infertility by causing leukocytospermia, asthenozoospermia, protamine deficiency, and seminal plasma miRNA profile alteration. Keywords: protamine-2; miRNA-122; sperm; male infertility; heroin; illicit drugs; addiction INTRODUCTION Infertility is one of the major medical challenges that affects over 15% of couples worldwide. Approxi- mately 50% of the infertile cases are due to male fac- tors(1). Semen abnormalities are one of the most common types of infertility due to multiple potential causes including inheritance, hormonal defects, drug abuse, (especially illicit drugs), and infection(2,3), but alarmingly, 60 to 75% of causes are still unknown or idiopathic(4,5). It has been explored that drug abuse affects the hormonal 1Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. 2Iran University of Medical Sciences, Tehran, Iran. 3Department of Biochemistry, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. 4Department of Anatomical Sciences, School of Medicine, Tarbiat Modares University, Tehran, Iran. 5Shahid Akbar-Abadi IVF Center, Iran University of Medical Sciences, Tehran, Iran 6Pediatric Neuro-Rehabilitation Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran. 7Department, School of Behavioral Sciences and Mental Health(Tehran Institute of Psychiatry) Iran University of Medical Sciences, Tehran, Iran. 8VetCell Therapeutics, Santa Ana, California, USA. *Correspondence: Cellular and Molecular Research Center & Department of Anatomy, Iran University of Medical Sciences (IUMS), Hemmat Highway, P. O. Box 14155-5983, Tehran, Iran Tel & Fax: +98 21 88622689, Email: koruji.m@iums.ac.ir. Received November 2019 & Accepted June 2020 balance and quality of semen, which leads to increased DNA fragmentation in sperm cells(6). Although close scrutiny of the patient's history along with semen analysis to diagnose idiopathic infertility is necessary, it is not enough(7,8). In recent years, alongside with conventional sperm parameters, the attention to molecular details, especially the seminal RNA content, has significantly grown. Micro RNAs (miRNA) are a class of non-coding small RNAs which exist in serum and plasma, semen and other body fluids. However, their production pat- Urology Journal/Vol 17 No. 6/ November-December 2020/ pp. 638-644. [DOI: 10.22037/uj.v16i7.5747] ANDROLOGY terns differ in different disease(9). The seminal plasma miRNAs are implicated in the regulation of spermat- ogenesis and gene expression in spermatozoa and the zygote during fertilization. These molecules were used as a novel, non-invasive biomarkers for the diagnosis of infertility and the classification of the types of them(10- 12). miRNA-122 is one of the high-expressing seminal miRNAs which alters in patients with azoospermia and asthenozoospermia(10,13). However, the role of miRNAs, and more specifically, miRNA-122 in infertility are not yet clear(14,15). Likely, recognizing this role will make miRNAs more reliable indicators for identifying and treating infertility. Recognizing the role of miRNAs in semen comes down to this: miRNAs are bound to be used as more reliable biomarkers for diagnosing different types of infertility, and subsequently dictating their treatment protocol (14). The health of the head and more specifically the nucle- us of the sperm depends on the correct expression of the proteins and protamine genes. One of the issues raised in idiopathic infertility is protamination abnormalities(16). In human, there are two types of protamine: protamine-1 (P1) and protamine-2 (P2) and they are expressed equal- ly in the sperm cells(17). The protamine mRNA (PRM) is expressed in round spermatids, whereas the translation is postponed until spermatid elongation(18,19). According to this time interval, protamines play an important role in post-transcription and epigenetics. In addition, a sub- set of protamine mRNAs are never translated and re- main in mature sperm cells, which is inherited in the zy- gote after fertilization (20). Previous studies have shown that miRNA-122a, through binding to its complemen- tary sequences in the 3’ untranslated regions (UTRs) of the transition protein 2 (Tnp2) mRNA, decreases the target transcription(21). P1, P2 and Tnps have the same promoter thus, their transcription occurs simultaneous- ly(22,23). On the other hand, miRNA-122 is likely to have similar effects on the protamine genes based on miRNA base data. Although the effect of the drug on infertility has been reported, its mechanism and molecular chang- es remain unclear. In this study, the correlation among miRNA-122 and protamine gene and protein expres- sion levels were studied alongside semen analysis, and miRNA-122’s association with protamine and seminal parameters were investigated. PATIENTS AND METHODS Study population The medical ethics committee of Iran University of Medical Science approved this study (code: IR.IUMS. rec.1394.9211313202). In case control study, 24 men with normal semen analysis [according to World Health Organization (WHO) 2010 criteria] and 24 her- oin-addicted men whose addiction were confirmed by a psychiatrist and experiments, were considered as the control and addiction groups, respectively. All volun- teers executed the written informed consent which was conducted according to the Declaration of Helsinki and a questionnaire containing exact demographic informa- tion. Inclusion and exclusion criteria All participants were 20-50-year-old men with normal body mass index (BMI). Other criteria included normo- zoospermia, in the control group, and only heroin con- sumption for at least four months and collecting sam- ples before initiating drug-tapering treatment protocols, in the addiction ones. Subjects with infertility, AIDS and hepatitis, and alcohol consumption were excluded from the study. Also, if addicted men consumed other narcotics in the last four months, they would be exclud- ed from the study. Sampling and preparation Semen samples were collected by masturbation in ster- ile containers after 2 to 5 days of sexual abstinence and immediately sent off for processing according to WHO (2010) guidelines. A part of the samples was cryopre- served for nuclear protein evaluation. The rest of the samples were incubated to liquefy at 37 °C for 30 min- utes. Gradient-swim up technique was used to precisely separate spermatozoa from seminal plasma. The pellet Protamine-2 and miRNA-122 in sperm-Nazmara et al. Andrology 639 Table 1. Gene primers. Gene Forward Primer Reverse Primer PRM-2 5'-CACGCACGAGGTGTACAGG-3' 5'-CAGTGTCTGCGCCTAAAGTGA-3' Β-actin 5′-TCCCTGGAGAAGAGCTACG-3′ 5′-GTAGTTTCGTGGATGCCACA-3′ miRNA-122 5′- TGGAGTGTGACAATGG-3′ Parameter Control group Mean ± SD (n) Addicted group Mean ± SD (n) p-value Demographic data Age (year) 34.41 ± 1.25 34.87±1.80 0.836 BMI (kg/m2) 27.75 ± 0.88 22.30 ± 0.36 0.001 Seminal parameters Semen physical parameters Volume (ml) 3.90 ± 0.35 3.22±0.35 0.136 Semen pH 7.79 ± 0.06 7.58±0.06 0.003 WBC 1.69 ± 0.41 8.61±1.73 0.001 Sperm concentration (×106/ml) 136.18 ± 25.23 146.22±37.32 0.823 Sperm motility (%) 65.51±2.57 41.96 ± 3.58 0.001 Sperm viability (%) 87.41±1.00 71.50 ± 4.59 0.002 Normal morphology (%) 16.98±3.97 12.48±1.49 0.300 Molecular data Protamine-2 expression 3.59 ± 0.94 0.05 ± 0.02 0.002 level (2-ΔΔCT) Relative density of protamine-2 protein 0.27 ± 0.06 0.10 ± 0.02 0.017 miR-122 expression levels (2-ΔΔCT) 1.52 ± 0.54 3.51 ± 0.73 0.034 Table 2. Demographic, semen analysis, and molecular data in the participants. and supernatant of each sample were also subsequently used for total RNA extraction and miRNA assessment. RNA extraction and cDNA synthesis The spermatozoa RNA was prepared using RNeasy Mini Kit (Qiagen, Germany). RNA 260/280 OD ratio and concentration were evaluated by nanodrop. cDNA was synthesized with RT primers according to the man- ufacturer's instructions (QuantiTect Reverse Transcrip- tion Kit, Qiagen, Germany). Real-time semi-quantitative PCR technique The PRM-2 (NM_001286356.1) expression level was determined by QuantiNova SYBR Green PCR Kit (Qia- gen, Germany) and were normalized with Beta-actin gene. The primers for target genes are presented in Ta- ble 1. The temperature cycles (n = 45) were generally performed at 95 °C for 5 s and 59 °C for 30 s. Total RNA isolation from seminal plasma After thawing, seminal plasma aliquots were centri- fuged twice (1,600 g for 10 min, then 16,000 g for 10 min) at 4 °C to harvest cell-free seminal plasma. The supernatant was carefully collected for subsequent as- says. For purification of cell-free total RNA, (primarily miR- NAs and other small RNAs), from seminal plasma, we used miRNeasy Serum/Plasma Kit (Qiagen, Germany) according to the manufacturer’s recommendations. The purity and integrity of RNA were checked by a 260/280 nm ratio measurement. cDNA synthesis and real-time semi-quantitative PCR for miRNA The miScript PCR System (Qiagen, Germany) was used for cDNA synthesis and determination of miRNA-122 expression levels. This system consists of the miScript II RT Kit for cDNA synthesis with oligo-dT primers, Ce_miR-39_1 miScript Primer Assay to monitor miR- NA purification and amplification, and miScript SYBR Green PCR Kit to enable quantification of miRNA-122 by real-time sqPCR. Expression values were normal- ized with C. elegans miR-39 mimic as a reference gene. Nuclear protein extraction 10 million spermatozoa from each sample were individ- ually centrifuged at 3000 g for 5 minutes at room tem- perature (RT), and the pellet was washed three times with PBS (500 µl each time). The pellet was finally re- suspended in 300 µl PBS and was sonicated for 5 min- utes under these circumstances: 80 % strength, pulse on 15 seconds and pulse off 5 seconds. Then the samples were boiled for 30 minutes at 95 °C. After centrifuga- tion at 15294 ×g for 20 minutes at 4 °C, and discarding of supernatant, 200 µl lysis buffer (0.2 M Tris-HCl, pH 7.5, containing 1 % SDS, and 10 % glycerol) was added and pipetted. Finally, after freezing and thawing of the specimens in liquid nitrogen five times, samples were centrifuged at 15294 ×g for 20 minutes at 4 ºC and the Table 3. Partial correlation between study variables. Addiction Seminal pH Seminal WBC Sperm motility Sperm viability PRM-2 P2 miRNA-122 - -0.362 0.391 -0.416 -0.315 -0.562 -0.382 0.440 Addiction Correlation 0.020 0.011 0.007 0.045 0.002 0.144 0.019 p value Seminal Correlation - - -0.350 0.234 0.043 0.190 0.008 -0.302 pH p value 0.025 0.141 0.790 0.343 0.975 0.118 Seminal Correlation - -0.467 -0.097 -0.267 -0.225 0.471 WBC p value 0.012 0.547 0.179 0.402 0.011 Sperm Correlation - 0.832 0.437 0.457 -0.404 motility p value 0.001 0.023 0.075 0.033 Sperm Correlation - 0.288 0.378 -0.533 viability p value 0.145 0.149 0.004 PRM-2 Correlation - 0.772 -0.391 p value 0.001 0.050 P2 Correlation - -0.309 p value 0.244 miRNA- Correlation - 122 p value This correlation was adjusted for cigarette smoking in the addicted group. parameters Unstandardized Coefficients Standardized Coefficients p-value B Std. error Beta Age (year) 0.137 0.395 0.052 0.731 BMI (kg/m2) -0.772 0.756 -0.175 0.314 Cigarette smoking -5.929 6.954 -0.158 0.400 Heroin addiction (yes or no) -24.794 11.148 -0.672 0.033 Duration of opioid dependence (year) -0.478 0.410 -0.224 0.252 Duration of heroin dependence (year) 0.259 0.662 0.070 0.699 Amounts of heroin consumed (mg/day) 4.168 4.394 0.185 0.349 Table 4. Simple linear regression analysis for sperm motility. Protamine-2 and miRNA-122 in sperm-Nazmara et al. Vol 17 No 06 November-December 2020 640 supernatant was collected. Extracted protein was con- centrated by a concentrator machine and protein con- tent of each sample was calculated by the BCA method (Thermo Scientific™ Pierce™ BCA™ Protein Assay Kit, USA). Western blotting An extracted nuclear protein from each sample was separated by 15 % polyacrylamide SDS-PAGE. After electrotransfer onto a PVDF membrane, blocking was performed in buffer containing 5 % non-fat dry milk in 4 ºC overnight and the membranes were washed with 0.05 % Tween 20 in PBS. Proteins were detected using anti-protamine 2 antibody (ERP15738, Abcam; MV= 17 kDa) and anti-beta-ac- tin antibody (ab8227, Abcam, MW=41.7 kDa) as pri- mary antibodies, and goat anti-rabbit IgG H&L (HRP) (ab6721, Abcam) as the secondary antibody and were visualized with the ECL reagent (Amersham, Canada) according to the manufacturer’s instructions. The pri- mary antibodies were diluted 1:1,000 in 2 % non-fat dry milk (Biolife) in PBS. The level of protamine was quantified via densitometry and normalized to beta-ac- tin protein levels. Statistical analysis Statistical analysis was performed using a statistical software package SPSS (Ver. 16.0, Chicago, SPSS Inc.). The parametric distribution was evaluated with Kolmogorov- Smirnov test. The differences between groups were statistical- ly determined by Chi-square, independent t-test, and Mann-Whitney test. The binary and multiple regression analyses were performed among gene and protein ex- pression levels and other parameters. 2-ΔΔCT values were used to compare the target gene expression levels. p-value < 0.05 was proposed to be significant. The al- phaEasseFC software was also used to determine the protamine-2 density versus β-actin. RESULTS Demographic and semen analysis information of all participants is shown in Table 2. There was no signif- icant difference in the mean age between the 2 groups (34.41 ± 1.25 vs. 34.87 ± 1.80). Although BMI was in the normal range in both groups, this parameter in the addicted men (22.30 ± 0.36 kg/m2) was significantly lower than the healthy men (27.75 ± 0.88 kg/m2) (p ≤ 0.01). All subjects in the addicted group smoked ciga- rettes, so this factor was statistically different between the two groups (p ≤ 0.01). There was no significant difference in semen volume (3.90 ± 0.35 vs. 3.22 ± 0.35), sperm concentration (136.18 ± 25.23 vs. 146.22 ± 37.32) or normal morphology (16.98 ± 3.97 vs. 12.48 ± 1.49) between healthy and addicted men. However, seminal pH (7.79 ± 0.06 vs. 7.58 ± 0.06), white blood cells (WBCs) in semen (1.69 ± 0.41 vs. 8.61 ± 1.73), and sperm viability (87.41 ± 1.00 vs. 71.50 ± 4.59) and motility (65.51 ± 2.57 vs. 41.96 ± 3.58) were signifi- cantly altered in the heroin consumption group. Protamine-2 gene and protein content and miR- NA-122 expression PRM-2 concentrations that was given by real-time sqP- CR were significantly decreased in addicted men versus healthy ones (3.59±0.94 and 0.05±0.02, respectively; p ≤ 0.01). In western blots of sperm nuclear proteins sep- arated by SDS–PAGE (Fig 1 A, B), increased density in P2 content was also observed in the addicted group (0.27 ± 0.06) as compared to the healthy group (0.10 ± 0.02) (p ≤ 0.05) (Table 2). In addition, the real-time sqPCR analysis revealed that miRNA-122 expression levels were increased in addicted men as compared to the healthy group (3.51 ± 0.73 versus 1.52 ± 0.54) (p ≤ 0.05). Correlation analysis Figure 1. Analysis of protamine 2 (P2), beta actin (β-act). (A) Western blot, using an antibody specific for P2 and an antibody specific for (β-act). (B) Nuclear proteins extracted from spermatozoa, separated on a polyacrylamide gel and stained with Coomassie Blue. Protamine-2 and miRNA-122 in sperm-Nazmara et al. Andrology 641 Cigarettes were smoked by all participants in the ad- dicted group, but not by participants in the healthy group, so partial correlation was used to eliminate that variable. The correlation results have been presented in Table 3. WBC count in seminal plasma was negative- ly correlated with seminal acidity and sperm motility. However, there was a positive correlation between the presence of WBC and miRNA-122 expression levels in seminal fluid. The increase in miRNA-122 is associated with a decrease in motility and survival rate of sperma- tozoa and a reduction in the amount of PRM-2. Among the groups, a positive significant relationship between sperm cell motility and spermatozoa survival rate, and PRM-2 expression levels was demonstrated. The number of copies of PRM-2 was directly correlated with the P2 content. Simultaneous analysis of demographic variables on sperm motility by simple linear regression Among the age, BMI, cigarette smoking, heroin ad- diction, duration of opioid and heroin dependence, and amounts of heroin consumed, only heroin addiction pa- rameter affects sperm motility (p < 0.05, Table 4). DISCUSSION To the best of our knowledge, no study has been con- ducted to addiction science that simultaneously evalu- ates the conventional and molecular parameters asso- ciated with infertility. Main obstacles on the way of human studies can be legislation and ethical considera- tions as well as simultaneous polydrug abuse. Our find- ings can contribute to increase our knowledge about the seminal molecular changes in the addicted men. Although BMI of heroin-addicted men was in the nor- mal range, the mean of this variable in that group was statistically lower than the healthy one. Based on demo- graphic data, the economic and educational condition of heroin consumers were different from healthy men. Based on the previous many human and animal studies, we conclude that a decrease in BMI in addiction cases (3,5,24,25). Yilmaz et al. (1999) determined morphine re- duced body weight with decreased metabolism caused by inhibition of androgen production along with the reduction of gastrointestinal activity(25). Illicit drugs can compete with food for brain reward sites, and de- crease appetite(26,27). Based on these results, it would be expected BMI is a heroin-dependent variable and is considered as a physiological change in the drug users. It seems that one of the possible pathways for heroin effects on gametogenesis is with BMI reduction. PH and WBC count are some of the seminal quality cri- teria, which decreased and increased in the addiction group, respectively. The results of partial correlations and previous studies suggests that the presence of WBC changes seminal pH leads to acidification of seminal fluid(28) and reduced survival rate and motility of sperm cells(29). Similar to the link between heroin addiction and BMI reduction, leukocytospermia, which refers to the presence of leukocytes in semen, is one of the inherent heroin-related clinical manifestations. Leuko- cytes produce reactive oxygen species (ROS) in semen and contribute to male infertility (7,8,30). Few studies have investigated sperm microenvironment in addicted cas- es. However, our finding is in line with Agrawal et al. (2014) and Nazmara et al. (2019) who reported leuko- cytospermia in addicted cases (5,8). In this study, protamine-2 content (gene and protein) in ejaculated spermatozoa and miRNA-122 levels in sem- inal fluid showed that addiction could lead to protamine deficiency and alter the functionality of cell-free sem- inal RNAs. It seems that insufficiency of protamine-2 in the addicted group is dependent on epigenetic regu- lators. Data on the copy number of protamine-2 mRNA confirms this conclusion in three ways. First: the signif- icant positive correlation between protamine-2 protein and mRNA levels could support the idea that dimin- ished P2 content was due to the low levels of PRM2. Second: the negative correlation between PRM2 and miRNA-122 levels suggests that abnormally high lev- els of miRNA-122 may lead to protamine-2 transcripts would be inaccessible or non-functional ones. Third: an abnormally high level of miRNA-122 and its associa- tion with seminal acidification and leukocytospermia, along with the correlation of these factors with addic- tion, suggests that the increase of miRNA-122 may be affected by heroin-dependent seminal changes. Protamine-2 is the most important protein involved in spermatozoa chromosome condensation(31). It is said that a defect in protamine gene expression is not due to gene mutations and may reflect new transcription regulations or incomplete post-translational processes (32). As previously mentioned, protamine transcription and translation is temporally uncoupled during sper- miogenesis(33). Although this time-separation is critical to the sperm development and is transcriptionally gene silent, it can make protamine mRNAs sensitive to in- tra- and extra-cellular alterations which result in pro- tamine deficiency. The most important hypothesis for reducing protein in a condition where the level of gene expression is low, (as in the present study), or normal is summarized: (a)Abnormalities in the post-translational process of P2: Defective protein kinases and their acti- vating pathways were reported in patients with dimin- ished P2 concentration by Aoki and Carrell (2003)(34) and Wu et al. (2000)(35). (b) Reduction in PRM2 levels: in mice, miRNA-469 binds to the coding regions of Tnp2 and PRM2, and hence represses those protein ex- pressions at the translation level with minor effects on mRNA degradation(23). Furthermore, inhibition of Tnp2 transcription, (one of the most widely studied genes in humans), was achieved by binding miRNA-122 to 3'-UTR of Tnp2 and its endonucleolytic cleavage ac- tivity in sperm-like cells(22). These are two examples of post-transcriptional regulation by miRNAs and our findings were consistent with the reports by the afore- mentioned investigators. The presence of protamine-2 transcripts in mature sper- matozoa RNA profiles and its transfer to the oocyte during fertilization(36) indicates the importance of the protamine-2 mRNA as a gene expression regulator in those cells. In other words, PRM2 not only contributes to toroidal structure, but also can be considered as one of the biomarkers which guarantees successful fertili- zation. Motility was another modified parameter in the ad- diction group which was measured and reported by computer-assisted sperm analysis (CASA). Consider- ing that PRM2 and miRNA-122 levels had significant correlations with sperm motility, it is legitimate to speculate that heroin abuse affects chromatin packag- ing, expression of motility-related genes, and sperm viability, resulting in asthenospermia. Up-regulation of Protamine-2 and miRNA-122 in sperm-Nazmara et al. Vol 17 No 06 November-December 2020 642 miRNA-122 in patients with asthenozoospermia(14) de- creased motility with increased % DFI (DNA Fragmen- tation Index) in opiate users, especially heroin-addicted men(37), depression of sperm motility along with move- ment-related gene impairment, including Catsperes, in mice that were addicted to Iranian kerack(24), are part of the studies which have similarities with our study. Semen and sperm cells are a major source of endorphins and enkephalins(38), and their defined activity levels are required to regulate sperm motility(39). However, heroin as a µ-agonist decreased sperm motility(40). In view of cigarette smoking is the most confounding factor affect- ing sperm mobility(41), and all participants in addicted group smoking cigarettes, simple linear regression was used to explore the most important demographic factors affecting mobility. Our findings showed that among the studied demographic variables, heroin addiction has the most deleterious effect on sperm motility. Our recommendations for further research are: Evalu- ation of other factors associated with nuclear conden- sation; In vitro assessment of the impact of heroin on ejaculated semen; The study of sperm surface receptors which are necessary in sperm-oocyte attachment. ACKNOWLEDGEMENTS The research team would like to thank the individuals who participated in this study. This study was supported by Iran University of Medical Sciences (IUMS), (Num- ber: 94-02-30-25973) and INSF (number: 94017358). FUNDING This study was funded by a grant from Iran Universi- ty of Medical Sciences (IUMS), (Number: 94-02-30- 25973) and INSF (number: 94017358) for a Ph.D. stu- dent thesis. CONFLICT OF INTEREST No potential conflict of interest was reported by the au- thors. REFERENCES 1. Amini F, Mashayekhi Z, Rahimi H, Morad G. 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