Effect of vitamin E on Semen Quality Parameters: A Meta-Analysis of a Randomized Controlled Trial Rui Wang1#, Shangren Wang1#, Yuxuan Song2#, Hang Zhou1, Yang Pan1, Li Liu1, Shuai Niu1, Xiaoqiang Liu1* Purpose: To explore the effectiveness of vitamin E in male infertility, a systematic review and meta-analysis was conducted. Materials and Methods: The retrieval time was from January 1947 to May 2021, without language restriction. Stata 12.0 was used for the meta-analysis. Results: A total of 8 randomized controlled trials involving 459 patients were included. The results showed that af- ter vitamin E treatment, semen volume was reduced (95% CI: - 0.55 to - 0.06, SMD = - 0.30, p = 0.015), total sperm count was increased (95% CI: 0.02-0.45, SMD = 0.23, p = 0.035), and the differences were statistically significant. There were no statistically significant differences in increasing sperm concentration (95% CI: -0.21-0.29, SMD = 0.04, p = 0.769), total sperm motility (95% CI: -0.01-0.42, SMD = 0.20, p = 0.061) or sperm forward motility rate (95% CI: -0.06-0.65, SMD = 0.29, p = 0.106). Subgroup analysis showed that vitamin E treatment for six months could improve sperm forward motility (95% CI: 0.46-1.14, SMD = 0.80, p <0.001). Conclusion: Vitamin E could increase the total sperm count and reduce the volume of semen in male infertility patients, and long-term treatment could improve the forward motility rate of sperm. The decrease of semen volume may be the result of different abstinence time before and after the test. Keywords: vitamin E; sperm quality; male infertility; sperm ; meta-analysis. INTRODUCTION Worldwide, infertility is becoming increasingly serious. At present, male infertility has received widespread attention. As a common clinical disease of the male reproductive system, the incidence of male infertility is increasing year by year.(1,2)Infertility is de- fined clinically as having a normal sexual life without contraception and failing to achieve clinical pregnancy for 12 months or more.(3) The incidence of infertility is approximately 15%, and male factors account for at least 50% of all infertility cases.(4) The mechanism of male infertility is complex. One of the important causes of male infertility is the decline in semen quality, which leads to oligozoospermia or asthenospermia. These patients are prone to infertility. (5)There are many factors that can affect the quality of male semen, such as radiation, smoking, varicocele, uri- nary tract infection, oxidative stress, and other factors. (6) In recent years, people have gradually paid attention to the influence of oxidative stress on male infertility and how it affects the quality of semen. When oxidative stress occurs, reactive oxygen species (ROS) produced by organisms exceed the body's natural antioxidant de- fense. When the production of ROS is too high, it will exert toxic effects on many kinds of cells and tissues. Among them, male germ cell sperm are very sensitive 1Department of Urology, Tianjin Medical University General Hospital, Tianjin, 300052, China. 2Department of Urology, Peking University People's Hospital, No. 11 Xizhimen South Street Xicheng District, Beijing, 100044, China. #contributed equally to this work. Xiaoqiang Liu, PHD; Department of Urology, Tianjin Medical University General Hospital, 154 Anshan Road, Heping District, Tianjin 300052, China. Tel:+60361614 , E-mail: xiaoqiangliu1@163.com. Received January 2022 & Accepted July 2022 to ROS. Excessive ROS can cause changes in sperm structure and function and affect sperm motility. Evi- dence shows that the semen antioxidant capacity of in- fertile men is lower than that of fertile men, and the level of ROS in the body is higher.(7,8) The most common treatment for oxidative stress is an- tioxidants. Quite a few studies have started to explore whether the use of antioxidants improves semen quali- ty, and antioxidants mainly include vitamin C, vitamin E, vitamin B12. Among them, the most commonly used is vitamin E, which is a fat-soluble antioxidant that can neutralize free radicals, prevent lipid peroxidation, and inhibit the production of ROS in infertile men. Previous studies have shown that vitamin E deficiency might lead to impaired human fertility.(9) However, the therapeutic effect of vitamin E on male infertility is controversial. Some studies have suggested that vitamin E is ineffec- tive in the treatment of male infertility.(10,11) Therefore, in order to resolve these disputes, we conducted a me- ta-analysis to evaluate the effectiveness of oral vitamin E in improving semen quality . MATERIALS AND METHODS Search strategy To formulate search strategies according to the require- ments of the Cochrane Collaboration, we retrieved Pu- Urology Journal/Vol 19 No. 5/ September-October 2022/ pp. 343-351. [DOI: 10.22037/uj.v19i.7160] REVIEW bMed, the Cochrane Library, Web of Science , Scopus , the VIP database, the Wanfang Database, and the China National Knowledge Infrastructure (CNKI). The used search strategy included the following :(((((((((((male fertility) OR (sterility, male)) OR (male sterility)) OR (subfertility, male)) OR (male subfertility)) OR (sub- fertility, male)) OR (male subfertility)) OR (subfertil- ity, male)) OR (infertility)) AND (semen quality)) OR (sperm)) AND (vitamin E) .The time of retrieval was from database building to May 2021, without language restriction. In addition, a manual search of relevant cita- tions of the included studies was performed. Literature selection criteria We used PICO/PECO statement (population—inter- vention/exposure—comparator—outcomes statement) to set the inclusion criteria and exclusion criteria for- further selecting eligible studies. The detailed criteria for selecting studies in the systematic review and me- ta-analysis are outlined in Table 1. Data extraction General information was extracted by two reviewers independently, including basic information of partici- pants, interventions, the period of outcomes observed, the results and other information. Disagreements be- tween the two authors were resolved by a third senior person. Quality assessment All included literature was evaluated for quality by 2 in- vestigators independently referencing the modified Ja- dad scoring criteria and resolved after discussion with a third party if a disagreement arose. The scoring criteria included (1) random sequence generation; (2) alloca- tion hiding; (3) blinding; and (4) follow-up. The total score is 1 to 7, of which 1 to 3 are low-quality research and 4 to 7 are high-quality research.(12) Statistical analysis Stata software (version 12.0) was used for statistical analysis. Standard statistical tables and charts were used to describe the characteristics of the respondents. The standardized mean difference (SMD) and 95% con- fidence interval (CI) were used to analyze the continu- ous data. The Q test and I2 statistics were used to test the heterogeneity of the included results. When I2 > 50% or p < 0.05, there was significant heterogeneity. If the heterogeneity between studies was obvious, the reasons for heterogeneity were analyzed, and subgroup analysis and sensitivity analysis were used. A p value of less than 0.05 for the difference was statistically significant. When I2 < 50% or p > 0.05, the fixed effects model was used for meta-analysis. In this study, Begg’s test and Egger’s test were used to evaluate publication bias. If the p value of the above test was less than 0.05, it indicated that there was significant publication bias.(13) RESULTS Literature search results A total of 545 studies were identified, including 265 from PubMed, 67 from Web of Science, 32 from Scop- us, 26 from the Cochrane Library, 27 from CNKI, 118 from the Wanfang Database, and 23 from VIP. After reading titles and abstracts, 491 articles were excluded for animal experimentation, review, duplicate articles, and those that clearly did not meet the inclusion criteria. Then, the remaining 54 articles were searched for full- text reading, 46 articles were excluded that did not meet the inclusion criteria, and 8 articles(10,11,14-19) were final- ly included, with a total of 459 patients, including 238 cases in the experimental group and 221 cases in the control group. The literature screening flow is shown in Figure 1, and the basic information of the included studies is shown in Table 2. Methodological quality assessment results The modified Jadad score was used to evaluate the methodological quality of the included studies. Four articles(10,11,14,17) were high-quality literature, and four (15,16,18,19) were low-quality literature, as detailed in Ta- ble 3. Meta-analysis results 1 Ejaculate volume Five studies(10,14,16,17,19) reported the relationship between vitamin E treatment of male infertility and semen vol- ume, for a total of 263 patients. There was no statisti- cal heterogeneity among studies (p = 0.697, I2 = 0%), and the pooled effect size of the fixed-effect model was used for analysis. The results of the meta-analy- sis showed that patients in the test group had less se- men volume than those in the control group, and the difference was statistically significant (95% CI: -0.55 to -0.06, SMD = -0.30, p = 0.015), as detailed in Figure 2. The sensitivity analysis showed that the results of the meta-analysis were robust, and the results of the funnel plot and Egger's test (p = 0.422) indicated the absence of publication bias, as shown in Figure S1. 2 Sperm concentration Four studies(10,11,14,16) reported the relationship between vitamin E treatment of male infertility and sperm con- centration in a total of 247 patients, as shown in Figure 3. After testing, there was no significant heterogeneity between the studies (p = 0.604, I2 = 0%). The fixed-ef- fect model analysis showed that the difference between the treatment group and the control group was not sta- Review 344 Vitamin E and male infertility-Wang et al. Table 1. Criteria for Selecting Studies Based on PICO/PECO Statement. Inclusion Criteria Exclusion Criteria Population patients with male infertility who were diagnosed according to the WHO and other diagnostic criteria Studies performed on cells or animals Intervention/exposure the test group received vitamin E as the main treatment The test group was mainly supplemented with other supplements, such as coenzyme Q10, folic acid, etc Comparator/comparison placebo Antioxidants or drugs Outcomes sperm parameters and pregnancy rates serum vitamin E levels in initial Study design Randomized controlled trial No-control studies including single arm studies, case reports, meta-analyses, reviews, and comments. Study was not a randomized controlled trial. Abbreviations: PICO/PECO,population-intervention/exposure-comparator-outcomes statement. tistically significant (95% CI: -0.21-0.29, SMD = 0.04, p = 0.769). 3 Total sperm count Five studies(10,14,15,17,19) reported the association between male infertility treated with vitamin E and total sperm count in 246 patients, as shown in Figure 4. After test- ing, there was no significant heterogeneity among the studies (I2 = 39.0%, p = 0.132), and the fixed-effects model was used to combine the effect size. The results Vol 19 No 5 September-October 2022 345 Figure 1. Flow chart of included studies Abbreviations: IU,international unit; T/C, treatment group/control group; RCT, randomized controlled trial. Table2. Characteristics of included studies. Author Country Age Patients Case of Intervention Duration of Outcomes (year) (mean ± sd) recruited infertility male study (T/C) (T/C) C.Rolf (1999) Germany 36.1±5.0/35.2 ± 4.8 15/16 asthenospermia 800 mg vitamin E+1000 56 days sperm parameters mg vitamin C daily/placebo Hussein Ghanem Egypt 31.8±8.1 30/30 Oligoasthenozo-ospermia 400 mg vitamin E+25 6 months sperm parameters (2010) mg clomiphene daily/ pregnancy rates placebo Ermanno Greco European unclear 32/32 men consulting for 500 mg vitamin E+500 mg 2 months sperm parameters (2005) infertility vitamin C twice daily/placebo sperm DNA fragmentation Suleiman SA Saudi Arabia unclear 52/35 Asthenozoospe-rmia 100 mg vitamin E three 6 months sperm parameters pregnancy rates (1996) times a day/placebo Ghazaleh Eslamian Iran 32.80±4.13/33.04±4.08 45/45 idiopathic 600IU vitamin E+ placebo/ 12 weeks sperm parameters (2020) asthenozoosper-mia placebo+ placebo K. Ener Turkey 25.8±4.6 22/23 varicocele vitamin E capsules 12months sperm parameters (2016) (300 mg×2)daily/nothing pregnancy rates Rezvan Bahmyari Iran 37.23±7.09/36.65±6.41 30/32 Oligo, astheno, vitamin E capsule 3 months sperm parameters (2021) teratozoospermia or (400 IU/day) + oligoasthenozo-ospermia selenium tablet (200 µg/day) + folic acid tablet (5 mg/day) L. KESKES- Tunisie unclear 12/8 unclear 400 mg vitamin E daily 3months sperm parameters AMMAR (2003) (Ephynal 100 mg, 2 tablets, twice daily) or 225 mg selenium (Bioselenium 35 mg, 2 capsules×3/day)/vitamin B Vitamin E and male infertility-Wang et al. showed that the total sperm count was significantly im- proved in the treatment group compared with the con- trol group (95% CI: 0.02-0.45, SMD = 0.23, p = 0.035), as shown in Figure 4. Sensitivity analysis showed that the results of the meta-analysis were relatively robust, and no obvious publication bias was detected (Egger test: p = 0.553), as shown in Figure S2. Funnel plot found no significant publication bias in Figure S6. 4 Total sperm motility Four studies with a total of 257 patients(14-17) reported the association between male infertility and total sperm motility with vitamin E treatment, as shown in Figure 5. After testing, there was no significant heterogeneity among the studies (I2=49.9%, p = 0.076), and a fixed-ef- fects model was used to combine the effect sizes. We identified a trend toward better total activity of sperm in treatment group than in control group. 5 Forward motility Six studies(11,14,16-19) reported the association between male infertility treated with vitamin E and sperm for- ward motility rate in a total of 383 patients. After test- ing the heterogeneity between the studies (I2 = 64.8%, p = 0.014), the random-effects model was used to com- bine the effect size. There was no statistically signif- icant difference between the treatment group and the control group in terms of the sperm forward motility rate (95% CI: -0.06-0.65, SMD=0.29, p = 0.106), as shown in Figure 6. Subgroup analysis was performed according to the duration of vitamin E administration in the treatment group, as shown in Figure S3. The re- sults showed that there was no statistical significance in terms of treatment duration of three months (95% CI: -0.31-0.29), SMD = -0.01, p = 0.925). The treatment duration was statistically significant at six months (95% CI: 0.46-1.14, SMD = 0.80, p = <0.001). Sensitivity analysis and publication bias Compared with the results after excluding one by one and rerunning the meta-analysis, the statistical results did not change significantly, as shown in Figure S4 and S5. These results confirmed that the results of the me- ta-analysis of vitamin E treatment for male infertility were reliable, as shown in Figure S4 and Figure S5. Re- sults of sensitivity analyses and publication bias Begg’s test and Egger's test illustrated that no evidence of bias existed in this meta-analysis and the outcomes proved to be firm. DISCUSSION The aim of this meta-analysis was to assess whether oral vitamin E is effective in improving male infertility. Our meta-analysis found that oral vitamin E treatment could Review 346 Figure 2. Forest plot of Meta-analysis of semen volume. Table 3. Results of methodological quality evaluations of included studies (score). Author (year) Design Random sequence generation Concealment of randomization Blinding Sign out Score Ghazaleh Eslamian (2020) RCTa 2 2 2 0 6 K. Ener (2016) RCT 1 1 0 0 2 Rezvan Bahmyari (2021) RCT 2 0 2 0 4 C.Rolf (1999) RCT 2 2 2 1 7 Hussein Ghanem (2010) RCT 1 2 2 0 5 Ermanno Greco (2015) RCT 1 2 2 0 5 Suleiman SA (1996) RCT 0 0 1 0 1 L. KESKES-AMMAR (2003) RCT 2 1 0 0 3 Abbreviations: RCT:,randomized controlled trial. Vitamin E and male infertility-Wang et al. significantly increase the total sperm count and reduce the volume of semen. It was further found that oral vi- tamin E treatment for up to 6 months could improve the forward motility of sperm but not for 3 months. Eslamian et al. explored the separate effects of doco- sahexaenoic acid and vitamin E and showed that the effect of vitamin E alone on sperm concentration, total motility, and forward motility was significant, and the association was stronger than that of docosahexaeno- ic acid alone.(14)The study by Suleiman et al. reported that a 6-month vitamin E treatment can improve sperm motility.(18) A randomized double-blind controlled tri- al showed that oral administration of vitamin E could increase the levels of vitamin E in seminal plasma, and the levels of vitamin E in seminal plasma in the treatment group were significantly different from those in the control group.(20) Treatment with vitamin E was based on the fact that it could increase body vitamin E levels and decrease sperm ROS levels, thus protecting the function of sperm. Figure 3. Forest plot of Meta-analysis of sperm concentration Figure 4. Forest plot of Meta-analysis of total sperm count Vitamin E and male infertility-Wang et al. Vol 19 No 5 September-October 2022 347 Furthermore, it was found in many preclinical studies that vitamin E supplementation could indeed improve semen quality. As an antioxidant, vitamin E has a spe- cial affinity for membrane phospholipids, and it can prevent lipid peroxidation and protect the integrity of the sperm membrane. The in vivo study showed that vitamin E can increase the total sperm count and sperm concentration of boars; the in vitro study was carried out with a nonenzymatic method for lipid peroxida- tion of boar seminal plasma using a vitamin E analog that could inhibit seminal plasma lipid peroxidation to control levels.(21) Another animal trial showed that oral administration of vitamin E improved sperm concen- tration, total sperm count, and percentage of normal sperm in infertile sires, and the plasma levels of vitamin E were also significantly increased.(22) Another animal experiment showed that vitamin E and vitamin C treat- ments can significantly improve the sperm quality of rabbits. After treatment with vitamins, rabbits signifi- cantly reduced the number of abnormally dead sperm, especially in the vitamin E supplement group.(23) The results also indicated that vitamin E was more effec- Review 348 Figure 5. Forest plot of Meta-analysis of total sperm motility. Figure 6. Forest plot of Meta-analysis of sperm forward motility Vitamin E and male infertility-Wang et al. tive than vitamin C in inhibiting lipid peroxidation, and the results of Massaeli et al. supported this conclusion. (24) In contrast, Rolf et al.'s study found that no changes in any of the conventional ejaculatory parameters were observed during short- or long-term vitamin treatment when compared with the parameters before treatment and with the placebo group.(10) Several trials reported similar results to those of Rolf et al .(20,25,26) The duration of treatment in the study by Rolf et al. was 8 weeks, which is relatively short, perhaps explaining why it is in contrast to the results of Suleiman et al. The same finding was reported by Ermanno Greco et al. After 2 months of treatment, there was no significant change in concentration, vitality and morphology in either the treatment group or the placebo group(11). However, the percentage of sperm with DNA fragmentation was sig- nificantly lower in patients treated for 2 months than in those treated with placebo. Only one of the studies we included reported that after vitamin E treatment, the proportion of sperm DNA fragments was significantly lower than that of the control group. Due to the lack of research data, no meta-analysis of sperm DNA frag- ments was performed. Vitamin E can inhibit the formation of free radicals and prevent their induced membrane lipid peroxidation, thereby maintaining the integrity of the sperm acroso- mal membrane and reducing the rate of sperm malfor- mation. High levels of reactive oxygen species reduced the activity of acrosin(27) and impaired the binding of sperm and oocytes.(28) A prospective study showed that men with high levels of reactive oxygen species pro- duction had a sevenfold lower chance of conception than men with low levels of production.(7) Trials have shown that fertility is significantly improved by anti- oxidant treatment.(29) Vitamin E had a positive effect on testicular and sperm fertility, and the prescription of a supplement containing vitamin E could improve sperm function in vivo and in vitro by reducing oxi- dative stress damage.(23) The issue of pregnancy rate was addressed in two of our included articles. Suleiman et al. mentioned in a placebo-controlled double-blind study that vitamin E produced good effects in reducing the concentration of malondialdehyde and improving sperm motility; eleven of the 52 spouses in the treat- ment group became pregnant during the 6-month treat- ment period, and none became pregnant in the place- bo group.(18) However, reasons for withdrawal or loss to follow-up were not described in this study, causing some degree of bias. Hussein Ghanem et al. showed four pregnancies in the placebo group and 11 pregnancies in the treatment group, with a significant increase in the cumulative number of pregnancies after treatment.(17) Kessopoulou et al. designed a double-blind randomized controlled crossover trial to treat male infertility caused by ROS using antioxidant vitamin E. The findings were not significant for semen parameters, but there was an improvement in sperm performance in the zona bind- ing assay. Thirty patients completed the 2-year trial. As a result, 108 of the 120 semen samples recovered vitality, and there were 3 successful pregnancies.(25) It was concluded that oral administration of vitamin E had beneficial effects on sperm fecundity and improved IVF rates.(25,30) Because the study by Kessopoulou et al. was not an RCT trial, this article was not included in our review. Therefore, only two studies in the included lit- erature mentioned pregnancy rates, with fewer data, so we did not perform a meta-analysis of pregnancy rates. We also included a study of vitamin E supplementation after varicocele surgery. The reason is that varicocele cause reflux of blood and other impaired microcircu- lation, and an increase in ROS levels will reduce the quality of semen and sperm function.(31) K. Ener et al. reported that vitamin E supplementation after varico- cele surgery resulted in an improvement in sperm con- centration and motility in both groups after varicocele resection compared to preoperative parameters, and the factor contributing to this improvement was merely var- icocele surgery.(15) The findings from the data obtained above were controversial. This may be explained by the fact that antioxidant treatment may have been ineffec- tive in some studies in which infertility was not a result of oxidative stress. The effects of vitamin E treatment depended strictly on its dosage. In the studies we includ- ed, the doses of vitamin E were different, but they were less than 1000 mg/d. The Food and Nutrition Commit- tee of the Institute of Medicine has determined that the tolerable upper intake of vitamin E for adults is 1000 mg.(32) Common side effects of vitamin E supplementa- tion include nausea, headache, visual changes, gastroin- testinal discomfort, risk of hemorrhagic stroke, slightly elevated urinary creatinine, and necrotizing enterocolit- is. There is a potential risk of increased bleeding during surgery because vitamin E is known to inhibit platelet aggregation and may increase the risk of anterior ade- nocarcinoma.(33,34)The dose of vitamin E used in several studies we included is within the safe range, which can show that our results are reliable. However, further re- search is needed to determine the optimal dose of oral vitamin E for infertile men. The treatment duration of these studies was 2 to 6 months. The data of our study showed that semen volume decreased after oral vitamin E treatment. This may be attributed to the difference in the number of days of abstinence before and after treatment and between groups. Long periods of absti- nence can lead to increased semen volume. Similarly, prolonged abstinence resulted in a higher total sperm count, so we need more trials to confirm the above re- sults. The advantages of our meta-analysis included: 1) five of the included studies used the double-blind method, the results were robust, and 2) the subgroup analysis found more accurate and meaningful results. Despite these advantages, we acknowledged that this meta-anal- ysis also had several limitations. We included only 8 articles, half of which were low-quality articles, and the number of patients was small. Due to the paucity of the included literature, there are certain limitations to the evaluation of publication bias. In several studies we included, vitamin E was combined with other drugs, which may confuse the results. The combined use of some antioxidants and vitamin E may increase or mask its own effects. The combination of drugs and vitamin E is used to treat semen quality. Drugs will interfere with the results of vitamin E treatment. It is impossible to determine whether the improvement of semen quality comes from the effect of vitamin E. Therefore, our con- clusion needs to be verified by larger RCTs with strict designs and long-term follow-up to further evaluate the effectiveness of oral vitamin E treatment for male in- fertility. Vol 19 No 4 July-August 2022 252 Vitamin E and male infertility-Wang et al. Vol 19 No 5 September-October 2022 349 CONCLUSIONS Vitamin E could increase the total sperm count and re- duce the volume of semen in male infertility patients. The decrease of semen volume may be the result of different abstinence time before and after the test. And long-term treatment could improve the forward motility rate of sperm. SUMMARY This article show that oral vitamin E can increase the to- tal number of sperm in male infertility patients, reduce semen volume, and long-term treatment can improve the positive motility of sperm. In other word long-term oral vitamin E and dosage within a safe range can im- prove men's semen quality. ACKNOWLEDGEMENT This study was financially supported by National Nat- ural Science Funds of China (No. 82171594) and Zhao Yi-Cheng Medical Science Foundation (No. ZYY- FY2018031). CONFLICTS OF INTEREST All authors have no relevant affiliations or financial involvement with any organization or entity with a fi- nancial interest in, or financial conflict with, the subject matter or materials discussed in the manuscript. APPENDIX https://journals.sbmu.ac.ir/urolj/index.php/uj/libraryFiles/downloadPublic/40 REFERENCES 1. Stahl P J, Schlegel P N.Genetic evaluation of the azoospermic or severely oligozoospermic male. Curr Opin Obstet Gynecolurnal.2012;24:221. 2. 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