Effect of Selenium and Vitamin E on the Level of Sperm HSPA2+, Intracellular Superoxide Anion and Chromatin Integrity in Idiopathic Asthenoteratozoospermia: A Double-Blind, Randomized, Placebo- Controlled Trial Parvin Sabeti1, Soheila Pourmasumi 2, 3* , Niloofar Fagheirelahee 4 Purpose: Male infertility accounts for about half of all infertility cases. Asthenoteratozoospermia is a severe form of male infertility. Free radicals play an important role in infertility. In a previous study we found that asthenoter- atozoospermic men had a lower mean percentage of sperm HSPA2+ and higher intracellular anion superoxide than normozoospermia. Antioxidants are thought to be able to counteract the negative effects of free radicals. We explored the efficacy of vitamin E in combination with Se on the level of sperm HSPA2+, intracellular anion su- peroxide, and chromatin integrity in these patients. Materials and methods: 60 patients entered the study. They were randomized to the treatment group of oral Se (200 µg) in combination with vitamin E (400 units) for 3 months (n = 30) or placebo (n = 30). Semen samples were obtained and assessed for sperm parameters, intracellular O2-, protamine deficiency, sperm HSPA2+ and apoptotic spermatozoa at baseline and after the treatment phase. Results: There were no significant differences in baseline semen parameters, intracellular O2- protamine deficien- cy, sperm HSPA2+ and apoptotic spermatozoa between the treatment and placebo groups. There was a statistically significant decrease in sperm apoptosis and the level of anion superoxide (P = .001) and an increase in sperm motility and viability (P = .001) in the treated group, but no significant difference was found in the percentage of sperm HSPA2+ and sperm protamine deficiency compared with baseline. Moreover, no significant change was found in these parameters in the placebo group after 3 months. Conclusion: Our results showed that administration of vitamin E and selenium for three months may improve sperm motility and viability by decreasing intracellular anion superoxide and sperm apoptosis in asthenoterato- zoospermic infertile men. We suggest that consuming these supplements before assisted reproductive technology (ART) may improve outcomes in these patients. Keywords: infertility; male; selenium; vitamin E; HSPA2 protein INTRODUCTION Male factor infertility is a common cause of in-fertility and accounts for about 30% to 40% of infertility. Common causes of male infertility include gene mutations, aneuploidy, varicocele, radiation, chemotherapy, genital tract infections, and erectile dys- function(1). But sometimes other factors such as high levels of re- active oxygen species (ROS) can affect sperm as they pass through the ducts, causing subfertility or infertility. It has been reported that ROS may be a causative factor in 30–80% of infertile men(2). The cause of infertility in about 60%–75% of infertile men is unclear. This is called idiopathic infertility(3). Sperm are able to produce low levels of ROS, which are involved in their critical functions. As a result of an error in spermiogenesis, sperm with a large amount of extra cytoplasm are released. This ex- tra cytoplasm contains additional enzymes that promote ROS production by the redox system in the cytoplasmic membrane. Superoxide anion and hydroxyl radicals are 1Department of Anatomy, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran. 2Non-Communicable Diseases Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. 3Clinical Research Development Unit (CRDU), Moradi Hospital, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. 4Student Research Committee, Iran University of Medical Sciences, Tehran, Iran. *Correspondence: Shohada Street, Moradi Hospital, Rafsanjan University of Medical Science, Rafsanjan, Iran. Tel: +98 913391204. Email: Spourmasumi@yahoo.com. Received June 2020 & Accepted September 2020 the most important free radicals from oxygen deriva- tives. Oxidative stress (OS) occurs as a result of an im- balance between the produced ROS and antioxidant de- fense, neutralizing their toxicity and resulting in sperm DNA damage, reduced sperm motility, and fertilization potential(4,5). On the other hand, a study reported that the percentage of sperm HSPA2+ in infertile individuals was lower than in fertile individuals. HSPA2 plays a role in re- pairing DNA fractures and replacing histones with pro- tamine during nuclear sperm compaction. Decreased expression of this protein is associated with increased aneuploidy, DNA fragmentation, apoptosis, and defect in histone - protamine translocation(6-8). In a previous study in this field, it was also found that infertile patients with asthenoteratozoospermia had a lower mean percentage of sperm HSPA2+ and higher intracellular anion superoxide than normozoospermic men(9). One of the most important antioxidant molecules against oxidative damage is vitamin E. This molecule is mainly located in the cell membrane and it prevents li- ANDROLOGY Urology Journal/Vol 18 No. 5/ September-October 2021/ pp. 549-555. [DOI: 10.22037/uj.v18i.6325] pid peroxidation and cell membrane damage by neutral- izing free radicals and enhancing other antioxidants(10). Selenium (Se) is an essential dietary micronutrient re- quired for reproductive functions such as testosterone metabolism and about 20-40% of infertile men who have deficiency in sperm production have been linked to selenium deficiency(11). Kaushal et al. found that var- iation in the amount of selenium can lead to OS and thereby affect reproductive potential(12). This demon- strates the importance of nutrition at the molecular lev- el. In this study, we investigated the effect of daily oral supplementation of Se and vitamin E on the level of intracellular superoxide anion, sperm HSPA2 +, pro- tamine deficiency, and sperm parameters in teratoas- thenozoospermia men. MATERIALS AND METHODS A double-blind, randomized, placebo-controlled trial study was conducted on 60 infertile men with asthe- noteratozoospermia (ATZ) (with normal morpholo- gy lower than 4% and total motility lower than 40%) at Royan Research and Clinical Center for Infertility (Tehran, Iran) from June 2014 to June 2016. All participants in this study were blinded to the inter- vention until the study was completed. Each participant randomly received two packs of pills in different colors from a doctor who was unaware of their contents and the record book remained in their hands until the end of the study. The treatment group received a daily supplement of vitamin E (400 IU) com- bined with selenium (200µg)(13) and the placebo group received two tablets of placebo for 3 months. Also, all patients were followed up by researchers dur- ing the study, and any adverse events were addressed. Out of 117 patients were included in this study, 54 pa- tients were excluded due to drug cessation and incom- plete consumption (n = 12) and/or declined to partici- pate (n = 42) (Figure 1). Semen samples were evaluated in two groups, treatment and placebo. Participants who met the study criteria, and who consented to participate were entered into the study. Inclusion criteria comprised history of infertility of at least one year despite regular unprotected inter- course; seminal analysis showing normal morphology lower than 4% and total motility lower than 40% as defined by WHO manual for semen analysis, (2010). Exclusion criteria comprised the cases with leukocy- tospermia (>1×106 WBC/mL), oligo and azoospermia, varicocele, cancer, endocrine disorders, genital tract in- fection, autoimmune disease, cryptorchidism, smoking or alcohol consumption which may impact the intracel- lular ROS, and patients who received chemotherapy, radiotherapy, and recent antioxidant intake. The study adhered to the local ethical protocol. Semen samples were obtained at baseline and after the treatment phase and were analyzed in accordance with WHO criteria. Semen collection and analyses Semen samples were obtained by masturbation after sexual abstinence of 2 to 4 days. After liquefaction in the lab at 37°C , samples were assessed for sperm pa- rameters, intracellular O2, protamine deficiency, sperm HSPA2+, and apoptotic spermatozoa. Semen Analysis Semen samples were collected into sterile containers in the laboratory by masturbation and after complete liquefaction at room temperature (22º C) for 30 min, they were assessed for macroscopic parameters such as color, pH, ejaculate volume, and viscosity. An aliquot of the sample was evaluated for sperm concentration, total motility, and morphology according to WHO cri- teria (WHO, 2010)(14). First, sperm concentration and total motility were as- sessed by CASA, then reanalyzed manually by a sin- gle experienced technician. The semen samples were mixed well; 10 µl of the sample was placed on a clean glass slide that had been stored at 37 °C and it was cov- ered with a coverslip. The samples were placed on the heating stage of a mi- croscope at 37 °C and were immediately observed at ×400 magnification. Sperm vitality and morphology in 200 spermatozoa per slide were evaluated by two experienced technicians us- ing the Eosin/Nigrosine and Papanicolaou staining, re- spectively. The same experienced technician performed all the semen analyses. Assessment of intracellular O2, HSPA2 and apop- totic spermatozoa by flowcytometry DHE (Dihydroethidium) is a specific probe for O2-. and a cell permeable stain. Sperm suspension was in- cubated with DHE (1.25µM; Sigma) at 25 °C for 25 Antioxidants and male infertility – Sabeti et al. Sperm Parameters Vit E+ selenium(n=30) Placebo(n=30) P- value Male age, year; mean ± SD (range) 31.90 ± 3.68 33.46 ± 3.72 0.107 Sperm volume. ml; mean ± SD (range) 3.35 ± 0.35 3.51 ± 0.27 0.125 Sperm PH; mean ± SD (range) 7.31 ± 0.13 7.27 ± 0.07 0.467 Vitality; mean ± SD (range) 55.16 ± 14.84 56.64 ± 14.24 .975 Sperm concentration ×106/ml; mean ± SD (range) 40.46 ± 18.51 43.86 ± 19.66 .893 Total motility, %; mean ± SD (range) 28.88 ± 6.66 29.47 ± 5.77 .987 Normal morphology, %; mean ± SD (range) 1.83 ± .74 2.03 ± .80 .799 CMA3+,%; mean ± SD (range) 37.06 ± 6.24 35.69 ± 5.05 .782 HSPA2+ ,%; mean ± SD (range) 22.26 ± 7.48 22.56 ± 6.37 .998 DHE+ ,%; mean ± SD (range) 35.40 ± 6.26 35.02 ± 5.58 .995 YO+ ,%; mean ± SD (range) 37.07 ± 8.40 40.97 ± 8.75 .345 Table 1. Demographic characteristics, sperm analysis and the DHE CMA3, HSPA2 and YO levels before treatment in two study groups Abbreviations: CMA3= Chromomycine A3 Staining, HSPA2= Heat-shock protein A2, DHE = Dihydroethidium, YO= Yo-pro-1 Iodide. Note: Values are presented by mean± SD. Paired sample t-test was used to compare dependent variables. P-value<0.05 was considered statistically significant. By comparing the hormonal levels in Sertraline and control group we founded that FSH, LH and testosterone levels all increased in the Sertraline group, but this increase was only significant for FSH (P < 0.05). There was no significant difference in LH, FSH and testoster- one levels between the 80th day and 170th day in the control group (P > 0.05) Andrology 550 min. DHE is oxidized by O2- and produces ethidium bromide which binds to the sperm DNA and emits red fluorescence which is then analyzed by a flowcytome- ter (FACS Caliber; BD Biosciences, USA) between 590 and 700 nm. Yo-pro-1 Iodide (Y3603- Life Technolo- gy) was used as a counterstain dye for DHE and exclud- ed the apoptotic spermatozoa(9,15). To measure the percentage of HSPA2+ spermatozoa, all samples were washed twice in cold phosphate – buffered saline (PBS, Gibco, USA), 4% paraformal- dehyde was added and samples were incubated for 20 min at room temperature, and then centrifuged for 5 min at 300g. Test fractures were permeable in 5% Tri- ton X-100 for 5 min and they were incubated overnight with the primary anti-HSPA2 antibody (Santa Cruz Co.) at a dilution of 1:100 in 3% bovine serum albu- min (BSA; Sigma Co.) at 4 °C; Control samples were incubated under the same conditions with 3% BSA. Two samples were washed and incubated with PE-con- jugated Donkey anti-goat IgG (1:200, Santa Cruz Co.) in 1.5% BSA at 4 ⁰C for 1 h. After washing, BD FACS Caliber flow-cytometry was used for further analysis(16). We assessed at least 10000 spermatozoa in each sample using the flowcytometry software (Flowjo 7.6.1) and expressed in percentage. Chromomycin A3 (CMA3) staining for Protamine deficiency assessment Chromomycin A3 (CMA3), is an indirect assessment for protamine content that competes with protamine to bind DNA. Using this procedure the semen sam- Sperm Parameters Vit E+ selenium(n=30) Placebo (n=30) P- value Sperm volume, ml; mean ± SD (range) 3.06 ± 0.21 3.32 ± 0.19 0.001 Sperm PH; mean ± SD (range) 7.25 ± 0.11 7.33 ± 0.08 0.02 Vitality; mean ± SD (range) 69.86 ± 12.56 46.75 ± 12.74 0.0001 Sperm concentration ×106/ml; mean ± SD (range) 40.66 ± 17.15 42.20 ± 18.93 .989 Total motility, %; mean ± SD (range) 44.39 ± 8.91 29.11 ± 5.09 0.0001 Normal morphology, %; mean ± SD (range) 2.16 ± 1.05 1.66 ± 0.75 .110 CMA3+,%; mean ± SD (range) 36.76 ± 5.96 37.13 ± 5.11 .994 HSPA2+,%; mean ± SD (range) 22.49 ± 7.10 22.08 ± 5.58 .995 DHE+,%; mean ± SD (range) 26.61 ± 7.66 38.96 ± 5.13 0.0001 YO+,%; mean ± SD (range) 28.34 ± 10.58 44.02 ± 8.36 0.0001 Table 2. Comparison of the levels of LH, FSH and Testosterone between 80th and 170th day in Sertraline and control group. Abbreviations: CMA3= Chromomycine A3 Staining, HSPA2= Heat-shock protein A2, DHE = Dihydroethidium, YO= Yo-pro-1 Iodide Note: Values are presented by mean ± SD. Independent sample t-test was used to compare dependent variables. P-value < 0.05 was con- sidered statistically significant. Figure 1. Study design, and distribution of patients into treatment Antioxidants and male infertility – Sabeti et al. Vol 18 No 5 September-October 2021 551 ples were washed with PBS, smears of them were prepared and dried. Then, they were fixed in Carnoy,s solution (Methanol/Glacial acetic: 3:1) at 4 ⁰C for 10 min, stained with 100µl of CMA3 (0.25 mg/mL) (Sig- ma Co.) for 25 min in dark at room temperature and mounted with buffered glycerol. We counted 200 sper- matozoa under a fluorescence microscope at 1000x magnification in all samples. Spermatozoa with normal protamine content (CMA3 – negative) and spermatozoa with protamine deficiency are stained dull green and bright yellow, respectively(9). This study was approved by Institutional Review Board of Yazd Research and Clinical Center for Infertility and informed consent forms were signed by all participants. Ethical committee registration: Yazd Research and Clinical Center for Infertility, Ethical code: 342/26/693 Clinical trial registration: IRCT20140409017210N2 Statistical analysis Statistical analysis was performed using SPSS soft- ware, (version 16.0, SPSS Inc., Chicago, IL, USA). The data distribution was normalized with K-S test. Inde- pendent sample t-test was used to compare two study groups. Paired sample T test was used to compare pre and post-treatment parameters. One-way analysis of variance (ANOVA) was used for comparison of param- eters between groups. Two tailed p-value less than 0.05 was considered as statistically significant outcome for the measured cases. All data was presented as mean ± standard deviation. RESULTS Demographic characteristics in two study groups before treatment (Vit E+ Selenium and Placebo) are summa- rized Table 1. Demographic characteristics and sperm parameters were similar between the two groups. Also, comparison of post-test seminal parameters, the lev- el of DHE, CMA3+, HSPA2+ and YO+ between two groups are shown in Table 2. Comparison of pre and post treatment seminal parameters, the level of DHE, CMA3+, HSPA2+ and YO+ in two study groups are summarized in Table 3. Sperm concentration There were no differences between the two groups be- fore treatment (p = .893) and post treatment (p = .989) (Tables 1 and 2 respectively). There were no statically significant changes in sperm concentration in place- bo (p = .986) and Vit E+ selenium group (p = 1.000) groups after 3 months (Table 3). Sperm total motility There was no difference between two groups before treatment (p = .987), but it was significant post treat- ment (p = .0001) (Table 1 and Table 2).While for the placebo group no change was observed (p = .99), the increase in the Vit E+ selenium group was highly sig- nificant (p = .0001) (Table 3). Normal morphology There were also no differences between the two groups before treatment (p = .799) and post treatment (p = .110). In placebo (p =.345) and treatment group (p =. 43, no significant changes were observed after 3 months. Sperm vitality There was no difference for the comparison between two groups at the baseline (p = .975) but it increased in the treatment group significantly. (p = .0001) (Ta- ble 1 and Table 2, respectively). While sperm vitali- ty decreased in the placebo group significantly after 3 months (p =.030), and it increased in Vit E+ selenium group significantly (p = .001) (Table 3). Semen volume There was no statistically significant difference be- tween the two groups before treatment (Table 1). Com- parison between post-treatment groups showed changes were significant and in the placebo group volume in- creased significantly (Table 2). Pre and post-treatment comparison showed a significant decrease in volume in VIT E+ selenium group (Table 3). Sperm pH There was no statistically significant difference be- tween the two groups before treatment (Table 1). Com- parison between post-treatment groups showed changes were significant and in the placebo group pH increased significantly (Table 2). Pre and post-treatment com- parison showed there was no statistically significant difference between pre and post-treatment with Vit E+ selenium (Table 3). Percentage of sperm CMA3+ There was significant difference between the two groups pre-treatment (p = .782) and post-treatment (p = .994) (Table 1 and Table 2). No changes occurred in percentage of sperm CMA3+ in placebo Vit E+ sele- nium group, (p =.756) (p =.997), after 3 months (Table 3). Percentage of sperm HSPA2+ There were also no differences between the two groups pre-treatment (p = .998) and post-treatment (p = .995) (Table 1 and Table 2). Percentage of sperm HSPA2+ +, Andrology 337 Sperm Parameters Vit E+ selenium(n=30) P- value Placebo (n=30) P- value Pre-treatment Post-treatment Pre-treatment Post-treatment Sperm volume. ml; mean ± SD (range) 3.35 ± 0.35 3.06 ± 0.21 0.0001 3.51 ± 0.27 3.32 ± 0.19 0.045 Sperm PH; mean ± SD (range) 7.31 ± 0.13 7.25 ± 0.11 0.171 7.27 ± 0.07 7.33 ± 0.08 0.102 Vitality; mean ± SD (range) 55.16 ± 14.84 69.86 ± 12.56 .0001 56.64 ± 14.24 46.75 ± 12.74 .030 Sperm concentration ×106/ml; mean ± SD (range) 40.46 ± 18.51 40.66 ± 17.15 1.000 43.86 ± 19.66 42.20 ± 18.93 0.986 Total motility, %; mean ± SD (range) 28.88 ± 6.66 44.39 ± 8.91 0.0001 29.47 ± 5.77 29.11 ± 5.090 0 .997 Normal morphology, %; mean ± SD (range) 1.83 ± .746 2.16 ± 1.05 .430 2.03 ± .80 1.66 ± .75 0.345 CMA3+, %; mean ± SD (range) 37.06 ± 6.24 36.76 ± 5.96 .997 35.69 ± 5.05 37.13 ± 5.14 0.756 HSPA2+, %; mean ± SD (range) 22.26 ± 7.48 22.49 ± 7.10 .999 22.56 ± 6.37 22.08 ± 5.58 0.992 DHE+ , %; mean ± SD (range) 35.40 ± 6.26 26.61 ± 7.66 .0001 35.02 ± 5.58 38.96 ± 5.13 0.074 YO+ , %; mean ± SD (range) 37.07 ± 8.40 28.34 ± 10.58 .002 40.97 ± 8.75 44.02 ± 8.36 0.563 Table 3: Comparison of the pre and post treatment seminal parameters, the level of DHE CMA3, HSPA2 and YO in two study groups. Abbreviations: CMA3= Chromomycine A3 Staining, HSPA2= Heat-shock protein A2, DHE = Dihydroethidium, YO= Yo-pro-1 Iodide Note: Values are presented by mean± SD. Paired sample t-test was used to compare dependent variables. P-value<0.05 was considered statistically significant. Antioxidants and male infertility – Sabeti et al. Andrology 552 in both groups, showed no significant changes after 3 months (Table 3). Intracellular O2- There was no difference between the two groups before treatment (p = .995) but it was decreased in post-treat- ment significantly (p = .0001) (Table 1 and Table 2). Pre and post-treatment placebo groups showed no change (p = .074) in the level of intracellular O2- , though in the VitE+ selenium group it decreased signif- icantly after 3 months(p =.0001) (Table 3). Percentage of apoptotic spermatozoa There was no difference between the two groups be- fore treatment (p = .345) but a significant decrease was observed in VitE+ selenium group (p = .0001) (Table 1 and Table 2). In the placebo group, no change was observed (p = .563), but in the Vit E+ selenium group, the percentage of apoptotic spermatozoa decreased sig- nificantly after 3 months (p =.002) (Table 3). DISCUSSION Infertility is a common medical and social problem that affects about one out of eight couples and approximate- ly 40–50% is due to “male factor” (16, 17). Asthenoterato- zoospermia is a severe form of male infertility and our findings in the previous study showed that they have a higher level of intracellular superoxide anion compared to normosospermic men(9). Antioxidants are molecules that are able to reduce or in- hibit oxidative stress by scavenging free radicals. When the concentration of free radicals in the body increases, the endogenous antioxidant system is compromised and unable to fully protect the body. In this situation, the use of exogenous antioxidants in dietary supplements or medications can be helpful(18). There are several antioxidants in seminal plasma which improve sperm quality. Some of them are vitamins E and C, along with selenium and zinc, which are constit- uents of the antioxidant system(19). Selenium is an essential element in the biosynthesis of the hormone testosterone and sperm formation and is the constituent of different selenoproteins. At least 25 selenoproteins in human and animals are known and are involved in maintaining the normal structure of sperm (20). 20 to 40% of infertile men whose infertility is re- lated to decreased sperm production is due to seleni- um deficiency(21). It is involved in the structure of the enzyme glutathione peroxidase, which is an important antioxidant and a marker for oxidative stress(22). Oral supplementation of selenium (50 microgram) has been reported to significantly increase in sperm param- eters, serum testosterone and glutathione levels. Also, serum MDA significantly decreased in patients after treatment(11). In a study, selenium levels were assessed in idiopathic infertile men. In this study, selenium con- centration in the seminal plasm and sera of 60 infertile men with oligospermia and azoospermia was measured (case group) along with 40 fertile men with normozoo- spermia (control group). They concluded that the mean serum selenium level in infertile men with oligospermia was significantly higher than in infertile men with azo- ospermia and a significant inverse relationship between selenium levels and sperm count was found. They also found that there was a relationship between selenium levels in the plasma seminal and other sperm parame- ters(23). Hamza et al. investigated the protective and antioxidant effects of selenium nanoparticles (Se NP) on testicular structure changes in male mice treated with monosodi- um glutamate (MSG). SeNP is known as a flavor en- hancer that has toxic effects on the male reproductive system. They found that, SeNP inhibit testicular injury and improve the antioxidant state in male mice treated with MSG(24). In a study by Scott et al., 96 infertile men were treated with selenium or selenium in combination with vitamin E, A and C and they showed significant improvements in sperm motility(25). In another study, selenium and N-acetyl-cysteine ad- ministration in infertile men with idiopathic oligo-as- thenoteratospermia for 30 weeks improved all sperm parameters(26). Vitamin E is a fat-soluble vitamin that is able to neu- tralize free radicals and protects cell membranes against the ROS by preventing lipid peroxidation as well as en- hancing the function of other antioxidants(27). In sum- mary, the mechanism by which vitamin E protects cells from oxidative stress includes maintaining normal glutathione levels as an intracellular scavenger of free radicals, protecting cell membranes by inhibiting per- oxidation, and clearing cells of ROS(28,29), and reduced apoptosis(30). On the effect of vitamin E, Keshtgar et al. showed one hour invitro incubation of semen samples from tera- tozoospermia patients with vitamin E significantly in- creased sperm motility and viability, but sperm DNA fragmentation and acrosome reaction did not change (31). Kemal Ener et al. found that oral administration of vitamin E increased sperm parameters after varicoce- lectomy but it was not statistically significant(32). Inhi- bition of ROS production in infertile men with vitamin E administration has also been reported(33). Abad and colleagues designed a study in which 20 infertile asthe- noteratozoospermic men were treated with a multi an- tioxidants combined with vitamin E and selenium for 3 months. The results showed a significant improvement of DNA integrity and significant increase in concentra- tion, motility, vitality, and normal morphology(34). In 2020, Matorras R. et al. examined the effect of vi- tamin E administration on men from infertile couples on sperm parameters and ART results in a double-blind randomized study. Vitamin E improved sperm param- eters but they did not find a significant difference be- tween the vitamin E treatment and the placebo groups. However, administration of vitamin E significantly in- creased live birth rates compared with placebo, and a tendency to achieve better results was seen in other IVF parameters in the treatment group(35). In this study, the effect of Se as a component of an- tioxidant system and Vitamin E on different parame- ters, intracellular anion superoxide, sperm apoptosis, and sperm chromatin deficiency was studied. Abad and colleagues designed a study in which 20 infertile asthe- noteratozoospermic men were treated with multi anti- oxidants combined with vitamin E and selenium for 3 months. The results showed a significant improvement of DNA integrity and a significant increase in concen- tration, motility, vitality, and normal morphology(34). Moreover, Moslemi et al. confirmed the protective and beneficial effects of Selenium–vitamin E supplementa- tion on semen parameters and pregnancy rate. In this study, idiopathic asthenoteratospermia men received daily supplements of vitamin E in combination with selenium for 100 days and they showed an increase in Antioxidants and male infertility – Sabeti et al. Vol 18 No 5 September-October 2021 553 sperm motility and morphology(13). Our investigation showed that Se (200 µg) in combi- nation with vitamin E (400 units) for 3 months has a potential effect on the reduction of sperm apoptosis and level of anion superoxide and also increases sperm mo- tility and viability (Table 3). In addition, we found that in comparison with posttest in the treatment group, the level of intracellular O2.- and apoptotic spermatozoa were lower than placebo group significantly in the final visit (p = .0001) (Table 2); also, sperm total motility and vitality were significantly higher (p = .0001) (Ta- ble 2). High levels of intracellular superoxide anion in men with asthenoteratozoospermia(9) can increase sperm ap- optosis. On the other hand, the high presence of poly- unsaturated phospholipids in sperm membrane, makes it very sensitive to high levels of ROS and increases the processes of lipid peroxidation(36), reducing sperm motility and vitality. Vitamin E is able to directly neutralize the anion super- oxide(37). In addition, a significant positive correlation has been found between serum selenium concentra- tion and glutathione peroxidase (GSH-PX)(38). GSH is a major antioxidant enzyme and a selenoprotein which protects the organism from oxidative stress by reducing reactive oxygen species(39). Therefore, according to the results of the current study, we may be able to say that, vitamin E and selenium in these patients play a role in free radicals removal and reduction of ROS and this reduction can lead to improv- ing sperm motility, viability, and apoptosis. Also, we observed no change in the level of sperm pro- tamine deficiency and HSPA2 after treatment. HSPA2 is involved in histone-protamine translocation. There- fore, no change in sperm protein deficiency after treat- ment may be attributed to the lack of change in HSPA2 level. According to the inclusion and exclusion criteria, the selection of patients was one of the major limitations of this study. Because, many patients with asthenoter- atozoospermia consumed alcohol or drugs in varying amounts, and some took a variety of antioxidants and medications, and this makes it very difficult to follow these patients. Another problem was the measurement of intracellular superoxide anion by flow cytometry in this trial study. Because it is a compound that must be quickly coor- dinated and measured with the flow cytometry depart- ment. Based on the results of this study, we propose other studies in higher populations in which pregnancy rates are also measured. CONCLUSIONS Our results showed that the administration of vitamin E and Selenium for three months decreased the level of intracellular anion superoxide in asthenoteratozoo- spermic men which may lead to improved motility, re- duced apoptosis, and increased sperm viability in these patients. However, it does not affect the sperm HSPA2 + as well as sperm protamine deficiency level in them. In conclusion, administration of vitamin E and seleni- um may have a positive effect as a low-cost supplement to improve sperm parameters in infertile asthenotera- tozoospermic men and prescribing them before using assisted reproductive techniques (ART) may improve outcomes. We also believe that the limitation of chang- es in sperm parameters to the period of administration may be due to the lack of effect of these supplements on factors such as HSPA2 in these patients. ACKNOWLEDGMENT This paper, derived from the Ph.D. thesis in reproduc- tive biology, has been funded by the Shahid Sadoughi University of Medical Sciences, Yazd, Iran. REFERENCES 1. Jungwirth A, Diemer T, Dohle G, Giwercman A, Kopa Z, Krausz C, et al. Guidelines on male infertility. Eur Urol. 2015;62:324-32. 2. Wagner H, Cheng JW, Ko EY. Role of reactive oxygen species in male infertility: An updated review of literature. Arab J Urol. 2018;16:35- 43. 3. Wu W, Shen O, Qin Y, Niu X, Lu C, Xia Y, et al. 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