Stesura Seveso Archivio Italiano di Urologia e Andrologia 2025; 97(2):13554 1 ORIGINAL PAPER further 20%. Idiopathic male subfertility is the common- est cause in most cases (1). D-aspartic acid (D-Asp) is an endogenous amino acid occurring in several tissues and cells of both invertebrates and vertebrates (2). It was first detected in the brain and optic lobes of the cephalopod mollusy Octopus vulgaris and later in the nervous and endocrine systems of various animal phyla such as crus- taceans, amphibians, reptiles, fish, chicken, rat, and man (3-14). Topo et al. conducted a clinical trial on 23 healthy male volunteers who consumed 3.12 gram of sodium D- Asp for 12 consecutive days (15). They found significant increase in LH and testosterone levels after 12 days by 33% and 42%, respectively (15). Thus, Topo et al. postu- lated that consumed D-Asp may also be remained in the testis and it continued to stimulate the testosterone pro- duction in the testis (15). In the aforementioned study the mean baseline testosterone were within 25% of the lower clinical range (3-10 ng/mL) and D-Asp supplemen- tation elevated testosterone levels to approximately 50% of the clinical range (4.5-6.4 ng/mL) (15). These findings can be explained by the fact that D-Asp acts directly on the pituitary gland inducing an increase of LH releasing (15-16). Furthermore, two in vitro studies had demon- strated a beneficial effect of Zn, D-Asp and Co-enzyme Q10 (Co-Q10) on sperm motility, recovery of spermato- zoa by swim-up and lipid peroxidation (17, 18). On the contrary, Willoughby et al. who conducted a study on twenty apparent healthy and heavy resistance-trained men (19). The participants trained 4 times/week while orally taking either 3 gram of placebo or D-Asp in the morning upon waking (19). There was no effect on mus- cle strength, body mass and serum hormones after 28 days of D-Asp supplementation (19). To the best of our knowledge, the current study is one of the first to evaluate the in vivo supplementation of D-Asp in male infertility. Thus, we aimed in the current study to evaluate the in vivo effect of D-Asp, zinc and Co-Q10 supplementation on different semen parameters and serum testosterone level in idiopathic male infertility. Introduction: About 20-30% of cases of infertility are attributed to male factor and males are also contributing to infertility in a further 20%. Idiopathic male subfertility is the commonest cause in most cases. D-aspartic acid (D-Asp) is an endogenous amino acid occurring in several tissues and cells of both invertebrates and vertebrates. The current study is one of the first to evaluate the in vivo sup- plementation of D-Asp in idiopathic male infertility. Thus, we aimed in the current study to evaluate the in vivo effect of D-Asp, zinc and Co-enzyme Q10 (Co-Q10) supplementation on different semen parameters and serum testosterone level in idiopathic male infertility. Methods: A total of 75 infertile patients were recruited from the outpatient andrology clinic from March 2023 to June 2024. The current study was registered at the UMIN clinical registry trials prior to initiating the study (UMIN000050023). Group (A) included 24 infertile patients who received 2660 mg D-Asp plus 200 mg of ubiquinol plus 10 mg zinc once daily for 3 months. Group (B) included 24 infertile patients who received placebo (starch granules) daily for 3 months. Results: Interestingly, patients in group (A) who received 2660 mg D-Asp plus 200 mg of ubiquinol plus 10 mg zinc once daily for 3 months showed significant improvement in progres- sive sperm motility after 3 months (10.63 ± 8.64 vs 15.21 ± 12.11, p = 0.047). Also, they showed highly significant increase in total testosterone level (5.06 ± 1.74 vs 5.89 ± 1.62, p = 0.009). Conclusions: D-Asp plus ubiquinol plus zinc are promising ingredients that showed good results when administrated once daily to infertile males. KEY WORDS: Idiopathic male infertiliy; Total testosterone; Progressive sperm motility; D-aspartic acid; Ubiquinol; Zinc. Submitted 30 December 2024; Accepted 17 January 2025 INTRODUCTION About 20-30% of cases of infertility are attributed to male factor and males are also contributing to infertility in a Evaluation of in vivo supplementation of 2660 mg D-aspartic acid and 200 mg ubiquinol and 10 mg zinc on different semen parameters in idiopathic male infertility: A randomized double blind placebo controlled study Sameh Fayek GamalEl Din 1, Elnashar A.M. 1, Yasser Elkhiat 1, Tarek Hussein 2, Mohamed Ahmed AbdElSalam 1, Ayman Alam 1, David Ramzy 1, Islam Moatamed 1, Ashraf Zeidan 1, Amr Elahwany 1, Mohamed Wael Ragab 1, Omar Zahran 1, Hany Saad 3 1 Department of Andrology, Sexology, and STDs, Faculty of Medicine, Cairo University; 2 Department of Andrology, Sexology, and STDs, Faculty of Medicine, Alexandria University; 3 Department of Andrology, Sexology, and STDs, Faculty of Medicine, Suez Canal University. DOI: 10.4081/aiua.2025.13554 Summary Archivio Italiano di Urologia e Andrologia 2025; 97(2):13554 S. Fayek GamalEl Din, Elnashar A.M., Y. Elkhiat, et al. 2 MATERIALS AND METHODS A total of 75 infertile patients were recruited from the outpatient andrology clinic from March 2023 to June 2024. The recruited patients were assessed for eligibility to join the study (Figure 1). The institutional ethical com- mittee of Alexandria University approved the study on 23/1/2023 that conforms to Helsinki declaration 2013 (20). Also, the current study was registered at the UMIN clinical registry trials prior to initiating the study (UMIN000050023). Twenty-three patients were exclud- ed from the study. The remaining 52 patients were equal- ly randomized by simple numbering method into 2 groups. Unfortunately, 4 patients dropped out of the study, 2 patients were from group (A) and 2 patients were Figure 1. Study flow chart. Archivio Italiano di Urologia e Andrologia 2025; 97(2):13554 3 In vivo supplementation of D-aspartic acid and zinc and ubiquinol on different semen parameters from group (B) (Figure 1). Group (A) included 24 infer- tile patients who received Spertility® that consists of 2660 mg D-Asp plus 200 mg of ubiquinol (active form of Co- Q10) plus 10 mg zinc once daily for 3 months. Group (B) included 24 infertile patients who received placebo (starch granules) daily for 3 months. Five cc blood was withdrawn for total testosterone evaluation at the begin- ning of the study and after 3 months. All participants brought 2 semen samples at the beginning and at the end of the study. Semen analysis was processed according to the 5th guidelines of the WHO (21). Inclusion criteria of the patients Any infertile male aged 25 to 40 years old suffering from idiopathic infertility was included. Exclusion criteria of the patients Any idiopathic infertile male suffering from varicocele or leukocytospermia or immunological infertility was excluded. Also, any infertile patient with hormonal imbalance was excluded. Finally, any infertile patient with small testicular volume < 8 ml was also excluded. RESULTS The current study did not show any significant difference in the mean age of the participants of both groups (31.25 years, ± 7.86; 33.96 years, ± 8.11, respec- tively, p = 0.062). Interestingly, patients in group (A) who received Spertility® showed significant improvement in progressive sperm motility after 3 months of daily sup- plementation of D-Asp (10.63 %, ± 8.64, 15.21 %, ± 12.11, p = 0.047, respectively) (Table 1). They also showed significant increase in total testosterone level after 3 months of daily supplementation of D-Asp (5.06 ng/dl, ± 1.74; 5.89 ng/dl, ± 1.62, respective- ly, p = 0.009) (Table 1). Conversely, total sperm concentration and sperm motility and abnormal forms did not show any improvement after daily supple- mentation of D-Asp in patients of group (A) (Table 1). Furthermore, patients in group (B) who received placebo did not show any improvement in total sperm concentration, sperm motility, progressive sperm motility, abnormal forms and total testosterone (Table 2). Despite, significant improvement in pro- gressive sperm motility and significant increase in total testosterone in patients in group (A), yet, pregnancy rate was 8.3% only. Owing to the fact that only 2 patients out of 24 patients in group (A) succeeded to impregnate their wives. Statistical methods Data management and statistical analysis were performed using the Statistical Package for Social Sciences (SPSS) version 25. Numerical data were summarized using means and stan- dard deviations or medians and ranges. Data were explored for normality using Kolmogrov-Smirnov test and Shapiro- Wilk test. Mann-Whitney U and Wilcoxon Signed Ranks Tests were used for the comparison between groups. All p- values are two-sided. P-values ≤ 0.05 were considered sig- nificant. DISCUSSION The current study had demonstrated significant improve- ment in progressive sperm motility in group (A) patients who received Spertility® daily for 3 months. Similarly, two previous studies demonstrated beneficial effects of in vitro supplementation of D-Asp on progressive sperm motility (17, 18). Moreover, an animal study revealed an improve- ment in sperm function of rabbits after being adminstered D-Asp (22). In the same context, D’Aniello et al. (2005) was one of the first to demonstrate lower levels of this amino acid in the semen of patients with oligoastenotera- tozoospermia than in fertile men (23). The significant improvement in sperm motility in the current study can also be explained by the presence of zinc and Co-Q10 in the ingredients of Spertility®. Several studies had shown the beneficial effects of zinc in male inferility (18, 24-26). In contrats, Foresta et al. (2014) failed to demonstrate any correlation between zinc and sperm motility (27). Table 1. Changes in semen parameters and total testosterone in group (A) before and after Spertility®. Mean Std deviation Minimum Maximum P-value Sperm concentration (106/ml) baseline 35.15 ± 23.74 9.30 110.20 0. 989 After 3 months 36.14 ± 24.33 6.30 92.80 Sperm motility % baseline 45.83 ± 10.18 25.00 65.00 0. 441 After 3 months 44.79 ± 8.91 30.00 65.00 Sperm progressive motility % baseline 10.63 ± 8.64 .00 25.00 0.047 After 3 months 15.21 ± 12.11 .00 60.00 Abnormal forms % baseline 61.46 ± 11.75 20.00 80.00 0.242 After 3 months 63.96 ± 13.67 20.00 80.00 Total testosterone (ng/dl) baseline 5.06 ± 1.74 2.56 8.20 0.009 After 3 months 5.89 ± 1.62 3.80 9.50 Table 2. Changes in semen parameters and total testosterone in group (B) before and after placebo. Mean Std deviation Minimum Maximum P-value Sperm concentration (106/ml) baseline 32.75 ± 40.22 5.00 201.00 0.255 After 3 months 31.58 ± 39.44 0.00 180.00 Sperm motility (%) baseline 37.21 ± 23.86 0.00 88.00 0.269 After 3 months 35.00 ± 20.54 0.00 70.00 Sperm progressive motility (%) baseline 6.25 ± 6.63 0.00 25.00 0.331 After 3 months 7.08 ± 6.24 0.00 20.00 Abnormal forms (%) baseline 54.38 ± 16.17 20.00 80.00 0.982 After 3 months 53.96 ± 18.33 20.00 98.00 Total testosterone (ng/dl) baseline 5.75 ± 1.67 3.00 8.80 0.884 After 3 months 5.82 ± 1.77 2.70 9.70 Archivio Italiano di Urologia e Andrologia 2025; 97(2):13554 S. Fayek GamalEl Din, Elnashar A.M., Y. Elkhiat, et al. 4 Additionally, Co-Q10 has a well-established dual action as electron/proton carrier in mitochondrial bioenergetic chain and antioxidant agent (18). In the same context, Li et al. (2006) found significant different concentrations of this antioxidant in the seminal plasma of fertile men and infertile patients (28). Conversely, Nadjarzadeh et al. (2014) had revealed that this molecule had a potent antioxidant effect (29). Furthermore, Spertility® contains ubiquinol which is the active form of Co-Q10 that facili- tates its gastro-intestinal absorption. Similarly, Garrido- Maraver et al. (2014) demonstrated the effect of different formulations on gastro-intestinal absorption (30). Another interesting finding of the current study was the highly significant increase in total testosterone level in group (A) patients who received Sperility once daily for 3 months. Consistently, a study had reported a steroido- genic role of D-Asp in humans and rats (15). Quite the reverse, Willoughby et al. (2014) failed to demonstrate any change in serum testosterone level in resistance-trained men after being resistance trained 4 times weekly for 1 month and after ingesting 3 g daily of D-Asp (19). It should be mentioned that the dose of D-Asp in the aforementioned study was higher than the concentration of D-Asp in Spertility®. Notably, the role of testosterone in male infertility is well established by two important reviews showing the pivotal role of androgen in spermatogensis (31, 32). In the same context, Carvalho et al. (2022) had revealed that eugenol admin- istration to Wistar rats reduced serum testosterone and sperm viability (33). On the contrary, a previous study had stated that serum testosterone levels demonstrate no relationship to sperm concentration (34). To wrap up, Spertility® administration in the form of once daily sachet for 3 months had shown significant improvement in pro- gressive sperm motility and highly significant increase in serum total testosterone. To the best of our knowledge, the current study is one of the first to demonstrate the impact of in vivo supplementation of D-Asp plus ubiquinol plus zinc to infertile males. Despite such achievements that were shown in the current study, yet, the pregnancy rate was very low as two patients only from group (A) succeeded to conceive naturally. This disappointing finding could be attributed to the small sample size and the short period of follow up. Admittedly, there are several limitations of the current study. Firstly, the small sample size and short duration of follow up are seen as major limitations of the current study. Nevertheless, the proper and consistent study design and being a prospective one added strength to the current findings. Also, we were not able to measure luteinizing hormone and sperm DNA fragmentation index adding further limitations of the current study. Finally, we were not able to utilize the 6th edition of the WHO for semen analysis processing (35). CONCLUSIONS D-Asp plus ubiquinol plus zinc are promising ingredients that showed good results when administrated once daily to infertile males. Future cohort studies that evaluate these ingredients versus L-carnitine are needed to affirm these findings. REFERENCES 1. Agarwal A, Baskaran S, Parekh N, et al. Male infertility. The Lancet. 2021; 397:319-333. 2. D'Aniello A. D-Aspartic acid: an endogenous amino acid with an important neuroendocrine role. Brain Res Rev 2007; 53:215-234. 3. D'Aniello A, Giuditta A. Identification of D-aspartic acid in the brain of Octopus vulgaris lam. J Neurochem. 1977; 29:1053-1057. 4. Okuma E, Fujita E, Amano H, et al. Distribution of Free D-Amino Acids in the Tissues of Crustaceans. Fisher Sci. 1995; 61:157-160. 5. Di Fiore M, Assisi L, Botte V, D'Aniello A. D-Aspartic acid is implicated in the control of testosterone production by the vertebrate gonad. Studies on the female green frog, Rana esculenta. J Endocrinol. 1998; 157:199-207. 6. Raucci F, Assisi L, D'Aniello S, et al. Testicular endocrine activity is upregulated by D-aspartic acid in the green frog, Rana esculenta. J Endocrinol. 2004; 182:365-376. 7. Assisi L, Botte V, D'Aniello A, Di Fiore M. Enhancement of aro- matase activity by D-aspartic acid in the ovary of the lizard Podarcis s. sicula. Reproduction. 2001; 121:803-808. 8. Raucci F, D’Aniello S, Di Fiore M. Endocrine roles of D-aspartic acid in the testis of lizard Podarcis s. sicula. J Endocrinol. 2005; 187:347-359. 9. D’Aniello A, Di Fiore M, Fisher G. Occurrence of Daspartic acid in animal tissues and its role in the nervous and endocrine systems. Trends Comp Biochem Physiol. 1998; 4:1-24. 10. Neidle A, Dunlop DS. Developmental changes in free D-aspartic acid in the chicken embryo and in the neonatal rat. Life Sci. 1990; 46:1517-1522. 11. Dunlop DS, Neidle A, McHale D, et al. The presence of free D- aspartic acid in rodents and man. Biochem Biophys Res Com. 1986; 141:27-32. DECLARATIONS Ethical approval: The study was prospectively registered at UMIN under the following serial number 000050023. Availability of data and material: All inquiries can be directed to the corresponding author. Competing interests: The authors declare no competing interests. Funding: This study did not receive any fund. Authors' contributions: SFG, AEN, TH and YE developed the conception and study design; MAA, IM, AZ, MWR, AA, DR and OZ recruited the cases; HS performed the statistics of the study; SFG intellectually drafted the manuscript and critically revised the data. All authors approved the final draft. SFG drafted the initial manuscript; SFG revised the article crit- ically; SFG reviewed and edited the article; NN designed the study; SAA performed the lab work of the study; AO, AE, AZ, AAS collected and analyzed the data. All authors reviewed the manuscript. Acknowledgments: We would like to thank Parkville Pharmaceutical Company for their provision of the drug and placebo once daily for 3 months. We would thank the surgeons who performed the sub inguinal micro-varicocelectomy. Archivio Italiano di Urologia e Andrologia 2025; 97(2):13554 5 In vivo supplementation of D-aspartic acid and zinc and ubiquinol on different semen parameters 12. Hashimoto A, Nishikawa T, Oka T, et al. Widespread distribu- tion of free Daspartate in rat periphery. FEBS Lett. 1993; 331:4-8. 13. Hashimoto A, Kumashiro S, Nishikawa T, et al. Embryonic development and postnatal changes in free D-aspartate and D-serine in the human prefrontal cortex. J Neurochem. 1993; 61:348-351. 14. Fisher GH, D'Aniello A, Vetere A, Padula L, Cusano GP, Man EH. Free D-aspartate and D-alanine in normal and Alzheimer brain. Brain Res Bulletin. 1991; 26:983-985. 15. Topo E, Soricelli A, D’Aniello A, et al. The role and molecular mechanism of D-aspartic acid in the release and synthesis of LH and testosterone in humans and rats. Reprod Biol Endocrinol. 2009; 7:1482-1488. 16. D’Aniello A, Di Fiore MM, Fisher GH, et al. Occurrence of D- aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. FASEB J. 2000; 14:699-714. 17. Talevi R, Barbato V, Fiorentino I, et al. Protective effects of in vitro treatment with zinc, d-aspartate and coenzyme q10 on human sperm motility, lipid peroxidation and DNA fragmentation. Reprod Biol Endocrinol. 2013;11:81. 18. Giacone F, Condorelli RA, Mongioì LM, et al. In vitro effects of zinc, D-aspartic acid, and coenzyme-Q10 on sperm function. Endocrine. 2017; 56:408-415. 19. Willoughby DS, Leutholtz B. D-Aspartic acid supplementation combined with 28 days of heavy resistance training has no effect on body composition, muscle strength ,and serum hormones associated with the hypothalamo-pituitary-gonadal axis in resistance-trained men. Nutr Res. 2013; 33:803-810. 20. World Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013; 310:2191-2194. 21. World Health Organization (WHO). WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva. WHO. 2010; 271 p. 22. Macchia G, Topo E, Mangano N, et al. DLAspartic acid admin- istration improves semen quality in rabbit bucks. Anim Reprod Sci. 2010; 118:337-343. 23. D’Aniello G, Ronsini S, Guida F, et al. Occurrence of D-aspartic acid in human seminal plasma and spermatozoa: possible role in reproduction. Fertil Steril. 2005; 84:1444-1449. 24. Hadwan MH, Almashhedy LA, Alsalman AR. Oral zinc supple- mentation restores high molecular weight seminal zinc binding pro- tein to normal value in Iraqi infertile men. BMC Urol. 2012; 13:12- 32. 25. Mocchegiani E, Costarelli L, Giacconi R, et al. Zinc-binding pro- teins (metallothionein and alpha-2 macroglobulin) and immunose- nescence. Exp Gerontol. 2006; 41:1094-107. 26. Macanovic B, Vucetic M, Jankovic A, et al. Correlation between sperm parameters and protein expression of antioxidative defense enzymes in seminal plasma: a pilot study. Dis Markers. 2015; 2015:436236. 27. Foresta C, Garolla A, Cosci I, et al. Role of zinc trafficking in male fertility: from germ to sperm. Hum Reprod. 2014; 29:1134-45. 28. Li K, Shi Y, Chen S, et al. Determination of coenzyme Q10 in human seminal plasma by high-performance liquid chromatography and its clinical application. Biomed Chromatogr. 2006; 20:1082-6. 29. Nadjarzadeh A, Shidfar F, Amirjannati N, et al. Effect of Coenzyme Q10 supplementation on antioxidant enzymes activity and oxidative stress of seminal plasma: a double-blind randomised clinical trial. Andrologia. 2014; 46:177-83. 30. Garrido-Maraver J, Cordero MD, Oropesa-Avila M, et al. Clinical applications of coenzyme Q10. Front Biosci (Landmark Ed). 2014; 19:619-33. 31. Holdcraft RW, Braun RE. Hormonal regulation of spermatogen- esis. Int J Androl. 2004; 27:335-42. 32. Smith LB, Walker WH. The regulation of spermatogenesis by androgens. Semin Cell Dev Biol. 2014; 30:2-13. 33. Carvalho RPR, Lima GDA, Ribeiro FCD, et al. Eugenol reduces serum testosterone levels and sperm viability in adult Wistar rats. Reprod Toxicol. 2022; 113:110-119. 34. Jackaman R, Ghanadian R, Ansell ID, et al. Relationships between spermatogenesis and serum hormone levels in subfertile men. Br J Obstet Gynaecol. 1977; 84:692-6. 35. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen, 6th ed.; WHO Press: Geneva, Switzerland, 2021. Available online: https://www.who.int/pub- lications/i/item/9789240030787 (accessed on 3 December 2021). Correspondence Sameh Fayek GamalEl Din, MD (Corresponding Author) samehfayek@kasralainy.edu.eg Department of Andrology and STDs Kasr Al-Ainy, Faculty of Medicine Cairo University, Al-Saray Street, El Manial, Cairo, 11956, Egypt Elnashar A.M., MD abdelrahmanelnashar@kasralainy.edu.eg Yasser Elkhiat, MD elkhiat@hotmail.com Mohamed Ahmed AbdElSalam, MD moh_756@cu.edu.eg Ayman Allam, M.M.B.c.H aymanallam085@gmail.com David Ramzy, M.M.B.c.H davidramzy1994@gmail.com Eslam Meatmed, M.M.B.c.H eslammeatmed@gmail.com Ashraf Zeidan, MD zidana2000@gmail.com Amr Alahwany, MD amralahwani@hotmail.com Mohamed Ragab, MD m.w.ragab@kasralainy.edu.eg Omar Zahran, M.M.B.c.H omar5b2007@gmail.com Department of Andrology, Sexology, and STDs, Faculty of Medicine, Cairo University, Cairo, Egypt Tarek Hussein, MD tarekmhaa@hotmail.com Department of Andrology, Sexology, and STDs, Faculty of Medicine, Alexandria University, Alexandria, Egypt Hany Saad, MD hanysaad@med.suez.edu.eg Department of Andrology, Sexology, and STDs, Faculty of Medicine, Suez Canal, Egypt