Radiol Oncol 2021; 55(1): 97-105. doi: 10.2478/raon-2020-0062 97 research article Effect of the oral intake of astaxanthin on semen parameters in patients with oligo- astheno-teratozoospermia: a randomized double-blind placebo-controlled trial Senka Imamovic Kumalic1,2, Irma Virant Klun1,2, Eda Vrtacnik Bokal1,2, Bojana Pinter1,2 1 Division of Obstetrics and Gynecology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2021; 55(1): 97-105. Received 17 August 2020 Accepted 14 September 2020 Correspondence to: Assoc. Prof. Bojana Pinter, M.D., Ph.D., M.S. (Econ), Specialist in Obstetrics and Gynecology, University Medical Centre Ljubljana, Division of Obstetrics and Gynecology; Faculty of Medicine University of Ljubljana, Šlajmerjeva 3, 1000 Ljubljana, Slovenia. E-mail: bojana.pinter@guest.arnes.si Disclosure: No potential conflicts of interest were disclosed. Background. Higher concentrations of seminal reactive oxygen species may be related to male infertility. Astaxanthin with high antioxidant activity can have an impact on the prevention and treatment of various health conditions, including cancer. However, efficacy studies on astaxanthin in patients with oligospermia with/without astheno- or teratozoospermia (O±A±T) have not yet been reported. Our aim was to evaluate the effect of the oral intake of astaxanthin on semen parameters. Patients and methods. In a randomized double-blind trial, 80 men with O±A±T were allocated to intervention with 16 mg astaxanthin orally daily or placebo. At baseline and after three months basic semen parameters, sperm de- oxyribonucleic acid (DNA) fragmentation and mitochondrial membrane potential (MMP) of spermatozoa and serum follicle-stimulating hormone (FSH) value were measured. Results. Analysis of the results of 72 patients completing the study (37 in the study group, 35 in the placebo group) did not show any statistically significant change, in the astaxanthin group no improvements in the total number of spermatozoa, concentration of spermatozoa, total motility of spermatozoa, morphology of spermatozoa, DNA frag- mentation and mitochondrial membrane potential of spermatozoa or serum FSH were determined. In the placebo group, statistically significant changes in the total number and concentration of spermatozoa were determined. Conclusions. The oral intake of astaxanthin did not affect any semen parameters in patients with O±A±T. Key words: antioxidant; male infertility; oligo-astheno-teratozoospermia; semen quality; DNA fragmentation; cancer Introduction Currently, almost every seventh couple is infertile. The male factor as the single or additional reason for infertility is presented in nearly 50% of infertile couples.1 World Health Organization (WHO) de- fines oligo-astheno-teratozoospermia (OAT) as the concentration and the proportions of motile and morphologically normal spermatozoa below the reference values.2 One of the many pathophysiological factors of male infertility are higher concentrations of seminal reactive oxygen species (ROS).3 The un- controlled production of these molecules may be harmful to cells and different biomolecules such as carbohydrates, amino acids, proteins, lipids, and deoxyribonucleic acid (DNA), which can be damaged. ROS can have a negative influence on sperm function and quality4-7 due to the reduced motility of spermatozoa8, DNA damage9-11 and the Radiol Oncol 2021; 55(1): 97-105. Imamovic Kumalic S et al. / Oral intake of astaxanthin and semen parameters in infertile patients98 impaired integrity of the cellular membrane.7,12,13 It was revealed that infertile men with low semen concentration, poor semen motility, and a high pro- portion of morphologically abnormal spermatozoa were associated with an increased risk of testicular cancer.14,15 Several antioxidants are present within the ejaculate as protection against excessive ROS- induced lipid peroxidation.16 It has been confirmed that antioxidant capacity in semen is decreased in infertile men and men with testicular cancer with a high ROS proportion, compared to men with a normal proportion of ROS.17–19 Sperm function tests have arisen as a supplement to standard semen analysis.20 The DNA integrity of spermatozoa has a critical impact in determining sperm competence21,22 which is related to ROS lev- els9,10 and can be measured by sperm DNA frag- mentation (SDF) assays.21,22 A recent meta-analysis of 28 studies demonstrated that SDF is more ac- curate in determining the function of spermatozoa compared to conventional semen parameters.23 A study of apoptotic DNA fragmentation in human spermatozoa found a significant increase in apop- totic SDF in the semen of testicular cancer patients post-orchiectomy and in OAT patients compared to a control group of healthy men.24 In addition to DNA integrity, mitochondria have an important impact on cell function25, and the sperm mitochon- drial membrane potential (MMP) contributes some benefit to male fertility potential and sperm qual- ity.26 Combined SDF and MMP may act as better predictors of natural conception than standard se- men parameters.27 Regarding the role of seminal antioxidant capac- ity in male infertility, several trials on the impact of antioxidants on semen characteristics have been performed. The review of randomized and place- bo-controlled trials affirmed the favorable impact of some types of antioxidants on different semen characteristics.28 Several studies have shown fa- vorable effects, especially on the motility of sper- matozoa.29-34 In some trials, antioxidant combina- tions (coenzyme Q10, vitamin C, vitamin E, sele- nium, zinc, L-carnitine, etc.) were assessed.35–40 The most recent Cohrane review shows that for couples attending fertility clinics, pregnancy rates and live births may be improved with antioxidant supple- mentation in subfertile males.41 As one of many antioxidants, ketocarotenoid astaxanthin has the potential to prevent and treat various diseases, including diabetes, liver and re- nal diseases, cancer, chronic inflammatory diseas- es, cardiovascular diseases, eye and skin diseases, metabolic syndrome, gastrointestinal diseases, and neurodegenerative diseases.42 Astaxanthin accumulates in the microalga Haematococcus plu- vialis, and compared to vitamin E, this compound has up to 100 times higher antioxidant activity.43 Astaxanthin is a food supplement and widely available over-the-counter. Until now, there were two “in vitro” studies that first showed the posi- tive effect of astaxanthin on sperm capacitation in 24 healthy, fertile men44; subsequently, the positive effect on sperm capacitation in 27 men from cou- ples who did not succeed to conceive after at least twelve months of regular unprotected intercourse was confirmed.45 On the other hand, there has been only one clinical trial investigating the influence of astaxanthin on male fertility published until now. The trial included a low number of patients with a history of male infertility – 11 patients in the study group and 19 patients in the placebo group; how- ever, these patients were enrolled without regard for semen characteristics.46 Favorable changes in inhibin B concentration, sperm linear velocity and ROS levels, and pregnancy rate were determined. However, the efficacy of astaxanthin oral intake on semen parameters in OAT patients treated for in- fertility has not yet been reported. The aim of this prospective randomized double- blind placebo-controlled clinical trial was to assess the effect of three-month intake of astaxanthin in infertile men with oligospermia with/without astheno- or teratozoospermia (O±A±T) on basic semen parameters, DNA fragmentation and mi- tochondrial membrane potential of spermatozoa, and serum level of follicle-stimulating hormone (FSH). Patients and methods The prospective randomized double-blind place- bo-controlled trial was performed from November 2014 to January 2019 at the outpatient infertility clin- ic, Department of Human Reproduction, Division of Obstetrics and Gynecology of the University Medical Centre Ljubljana, Slovenia. The study was approved by the Slovenian National Medical Ethics Committee (consent number 145/02/14) and was registered at ClinicalTrials.gov, NCT02310087. All participants were enrolled after they provided written informed consent to participate in the trial. Participants A total of 80 infertile men with O±A±T were en- rolled after they signed a written informed consent Radiol Oncol 2021; 55(1): 97-105. Imamovic Kumalic S et al. / Oral intake of astaxanthin and semen parameters in infertile patients 99 to participate in this trial. They were considered O±A±T after at least two previous semen analysis (seminogram) and andrological examination in the frame of their infertility treatment after their part- ner being unable to conceive for at least 12 months of unprotected sexual intercourse or after a failed assisted conception procedure. Semen quality was defined as O±A±T according to the WHO 2010 guidelines: oligospermia (O) − sperm concentra- tion < 15 million/ml; asthenozoospermia (A) − pro- gressive motility of spermatozoa < 32%; terato- zoospermia (T) − < 4% spermatozoa with normal morphology.20 The exclusion criteria were smoking more than 20 cigarettes per day, genetic causes of infertility, endocrinopathies, genital tract infec- tions, undescended testis, systemic diseases, his- tory of testicular cancer and treatment with other drugs and food supplements, such as antioxidants, during the last three months before enrolling in this study. Intervention and methods Eighty eligible infertile men with O±A±T were al- located at random to daily intake of 16 mg of astax- anthin capsules (Astasan) or placebo capsules, both produced by the same manufacturer (Sensilab). Astaxanthin capsules contained astaxanthin, vita- min E as a stabilizer, safflower oil, gelatin, water and glycerine. Placebo capsules were identical both in appearance and taste, containing gelatine, water and glycerine only. A computerized randomiza- tion table was used for the purpose of randomi- zation. A random allocation sequence was gener- ated and participants were enrolled and assigned to interventions by a third party, thus ensuring that both the enrolled participants and researchers were blinded. At baseline and after three months of treatment, all participants answered a question- naire about their age, height, weight, smoking sta- tus and the history of current occupational expo- sure to high temperatures, ultraviolet or radiology radiation, chemicals, or a predominant sedentary job in order to exclude the potential effect of these factors on semen quality. The semen samples were obtained by masturbation after 2−5 days of sexual abstinence. Semen quality was assessed according to the WHO guidelines.20 The total number, con- centration, motility and morphology were deter- mined by international standard laboratory micro- scopic analyses.47 Sperm DNA fragmentation in our study was evaluated using a TUNEL assay – measuring DNA fragmentation in spermatozoa using terminal de- oxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labelling.48 The calculated threshold value for the TUNEL assay to distinguish between normal and abnormal semen samples in men was 20%.49 MMP was measured by means of 3,3’-dihex- yloxacarbocyanine iodide (DiOC6(3)) staining as an indicator of mitochondrial membrane integrity and the mitochondrial potential capacity to gener- ate ATP by oxidative phosphorylation.50 Propidium iodide (PI) was used as a supravital fluorescent dye and stained spermatozoa were analyzed by flow cytometry. MMP was considered to be normal in our laboratory setting when the proportion of spermatozoa with normal MMP (attributed to cells with high fluorescence signals) was higher than 60%.27 The serum level of FSH was measured by a solid-phase, two-site chemiluminescent immuno- metric assay. The normal FSH values for males in our laboratory setting were 0.7−11.1 IU/L. Sample size calculation To estimate the sample size for the study, we an- ticipated an increase in sperm concentration in the astaxanthin group, as reported in the only clinical study until now.46 With the assumption of a 34% increase as reported, with alpha 0.05 and power 80%, 32 cases were needed in each group. To ac- count for drop-outs, we aimed to recruit 40 cases in each group. Statistical analysis Statistical analysis was carried out using the Statistical Package for the Social Science (SPSS), version 25, IBM Corp, Armonk, NY, USA. An in- dependent Student’s t-test was used to analyze normally distributed data, and the Mann-Whitney test was used to analyze data that were not distrib- uted normally. A paired t-test or the Wilcoxon test were applied to analyze the differences in the se- men variables within groups. The chi-square test was applied to compare the exposure to occupa- tional factors between groups. The results of con- tinuous variables were expressed as the mean ± SD. P-values < 0.05 were considered significant. Results A group of 256 men was assessed for eligibility; half (126 men, 49.2%) declined to participate, and 50 did not meet the inclusion criteria; 80 men were eligible and willing to participate and these indi- Radiol Oncol 2021; 55(1): 97-105. Imamovic Kumalic S et al. / Oral intake of astaxanthin and semen parameters in infertile patients100 viduals were randomized. Eight patients in both groups (10%) dropped out for personal reasons during the treatment, and thus, 72 patients com- pleted the trial. No adverse effects during the treat- ment were reported. Finally, we analyzed the effect of treatment in 37 patients in the astaxanthin and 35 patients in the placebo group (Figure 1). Five patients were not included in the statisti- cal analyses on changes in sperm total number and concentration and 27 patients were not included in the statistical analyses on the motility of sper- matozoa as in these patients only a few mobile or immobile spermatozoa in the semen sample were present. At baseline, all patients in the astaxanthin group were in accordance with WHO criteria for OAT. In the placebo group, all patients were in ac- cordance with the WHO criteria for oligo-teratozo- ospermia and two-thirds for asthenozoospermia. The epidemiological and semen characteristics of the included patients are summarized in Table 1. There were no statistically significant differences between all parameters of both groups of patients upon enrollment. In addition, exposure of patients to chemicals, ultraviolet rays, X-ray radiation, high temperature, and a sedentary job according to their history (questionnaire) have not been related to the semen parameters in the studied population of men. FIGURE 1. CONSORT flow chart of study presenting the inclusion criteria, randomization and follow-up of participants. Radiol Oncol 2021; 55(1): 97-105. Imamovic Kumalic S et al. / Oral intake of astaxanthin and semen parameters in infertile patients 101 The pre- and post-treatment semen parameters and hormonal levels of both groups of patients are displayed in Table 2. The three-month treatment of patients with astaxanthin did not affect the se- men parameters or serum FSH levels in any way. Interestingly, the significant change after the three- month period was the increased total number (16.0 ± 21.1 vs. 38.4 ± 69.2 million/ejaculate, p = 0.002) and concentration (5.7 ± 5.9 vs. 10.2 ± 15.7 million/ml, p = 0.024) of spermatozoa in the placebo group, but not in the astaxanthin group (24.6 ± 28.6 vs. 31.7 ± 33.4 million/ejaculate, p = 0.186, and 7.0 ± 5.6 vs. 9.2 ± 7.9 million/ml, p = 0.100). To address the increase in the concentration of spermatozoa in the placebo group, we separately analyzed the seasonal inclusion of patients into the trial to review the potential seasonal changes of se- men quality. Nineteen patients in the astaxanthin group and 18 patients in the placebo group were enrolled in the study during the winter/spring pe- riod, and 18 patients in the astaxanthin group and 17 patients in the placebo group were enrolled dur- ing the summer/fall seasons. There was no signifi- cant difference in the seasonal inclusion of patients in the trial (p = 0.995). Discussion The present study is the first study of astaxanthin efficacy on semen parameters in oligo-astheno- teratozoospermic patients who were treated for infertility. Our results did not show any significant improvements in the basic semen parameters or at the cellular level (SDF, MMP) or serum FSH val- ues after oral intake of astaxanthin. In previously published studies on the effects of antioxidants in semen quality28,32,38,39, the semen quality was de- fined according to the WHO 1999 guidelines. On the other hand, our patients were included based on their semen quality in line with the WHO 2010 guidelines, which have lower threshold values. Therefore, our patients were enrolled in the study with considerably poorer semen quality compared to patients in previous studies, which followed old- er WHO guidelines. Considering cancer patients, both chemotherapy and radiation have measur- able effects on the sperm number, motility, mor- phology, and DNA integrity.51 Although treatment usually represents the greatest threat to the fertil- ity of male survivors, the underlying malignancy may additionally affect semen production and quality. In cases of testicular cancer, the rates of oligospermia and azoospermia at presentation are 50% and 10%, respectively.52–55 Similarly, Hodgkin Lymphoma is associated with oligospermia and azoospermia before the treatment, and depending on the treatment regimen, also after.56–58 According to poor starting values of basic sperm parameters in our patients, in addition to their low MMP and very high DNA fragmentation, it can be concluded that our patients, in general, had poorer semen quality as subjects in the previous studies due to the lower threshold of the new WHO guidelines. TABLE 1. Comparison of baseline characteristics of patients in astaxanthin and placebo groups Characteristic Astaxanthin (n = 37) Placebo (n = 35) p-value Age (years) 35.0 ± 5.2 36.4 ± 5.5 0.276 BMI (kg/m2) 25.9 ± 3.8 27.1 ± 4.6 0.162 Smoking (no. of cigarettes smoked / day) 4.8 ± 6.8 3.6 ± 6.8 0.264 Sperm concentration (million /ml)* 6.9 ± 5.7 5.4 ± 5.8 0.297 Total sperm number (million/ejaculate)* 23.9 ± 28.4 15.6 ± 21.0 0.164 Total sperm motility (%)** 29.9 ± 14.8 38.2 ± 14.8 0.063 Progressive sperm motility (%)** 25.1 ± 14.5 33.3 ± 14.6 0.063 Sperm morphology (%) 1.8 ± 2.1 1.9 ± 2.0 0.663 Sperm head defect (%) 95.1 ± 16.2 97.5 ± 3.3 0.936 Sperm neck + midpiece defect (%) 2.9 ± 16.4 0.2 ± 0.8 0.981 Sperm tail defect (%) 0.2 ± 0.6 0.5 ± 1.0 0.424 Leukocytes with peroxidase (million/ml) 0.3 ± 0.3 0.5 ± 0.8 0.976 Mitochondrial membrane potential (%)*** 27.5 ± 18.5 26.9 ± 15.3 0.888 DNA fragmentation (%) 50.5 ± 23.9 45.8 ± 21.8 0.424 FSH (IU/l) 9.7 ± 7.8 9.2 ± 5.8 0.919 Chemicals at work (% exposed) 5.4 14.3 0.204 Ultraviolet rays at work (% exposed) 2.7 0 0.327 X-ray at work (% exposed) 0 11.4 0.051 High temperature at work (% exposed) 13.5 14.3 0.925 Sedentary job (% exposed) 45.9 40.0 0.611 Results are presented as average (± SD) or percentage (%). Differences at baseline assessed with independent Student t-test, Mann-Whitney U-test or Chi-square test, as appropriate. * = the analysis for total sperm number and sperm concentration was made for 34/37 patients from the astaxanthin group and 35/35 patients from the placebo group, since the excluded patients only had a few mobile or immobile spermatozoa in the semen sample; ** = the analysis for total and progressive sperm motility was made for 25/37 patients from the astaxanthin group and 20/35 patients from placebo group, since the excluded patients only had a few mobile or immobile spermatozoa in the semen sample; ***= the proportion of spermatozoa with normal MMP; BMI = body mass index; DNA = deoxyribonucleic acid; FSH = follicle-stimulating hormone Radiol Oncol 2021; 55(1): 97-105. Imamovic Kumalic S et al. / Oral intake of astaxanthin and semen parameters in infertile patients102 Under these conditions, our study revealed that antioxidant astaxanthin could not improve semen quality in cases of very poor semen quality, which could also be of great importance for cancer pa- tients. Our results are in contrast to a previous study by Comhaire, which determined favorable changes in the inhibin B concentration, sperm linear veloc- ity and ROS levels, and pregnancy rate after astax- anthin treatment in the same dosage of astaxanthin as in our study. However, the study enrolled a low number of infertile males (11 in the study group and 19 in the placebo group) and was not based on semen analysis but rather on a more than twelve months’ infertility history of female partners hav- ing no verifiable cause of infertility.46 Interestingly, another recent study, similar to our study, showed that different antioxidants did not affect the se- men quality in a larger group of infertile patients.59 Similar results of no effect of antioxidants were found in some other studies.38,60–63 These data show that antioxidants are interesting, but the clinical data should be comprehended with prudence. The improved total number and concentrations of spermatozoa after three months of treatment with a placebo could reflect the beneficial psycho- logical effect of placebo on patients. In the litera- ture, the strength of expectations is connected to the degree of the placebo effect.64,65 The appearance of a medication, past experience, verbal informa- tion, and relationship between a patient and phy- sician may influence the patient’s expectations.66 However, previously published reviews on the effects of oral antioxidants on semen quality did not show any positive effect of the placebo in the placebo-controlled studies.28,67 On average, the FSH value in the studied popu- lation was relatively high thus indicating the im- balance of the pituitary-gonadal endocrine axis and hypogonadism with a serious damage of sper- matogenesis. In infertile men, a higher concentra- tion of FSH is considered to be a reliable indicator of germinal epithelial damage, and was shown to be associated with azoospermia and severe oligo- zoospermia.68 This indicates that the studied popu- lation of men in our study was facing severe male infertility (severe OAT). TABLE 2. Comparison of basic semen parameters, sperm MMP, DNA fragmentation, serum FSH values in astaxanthin and placebo groups after three-month treatment period Parameter Astaxanthin (n = 37) Placebo (n = 35) Baseline After 3 months p-value Baseline After 3 months p-value Sperm concentration (million /ml)* 7.0 ± 5.6 9.2 ± 7.9 0.100 5.7 ± 5.9 10.2 ± 15.7 0.024 Total sperm number (million/ejaculate)* 24.6 ± 28.6 31.7 ± 33.4 0.186 16.0 ± 21.1 38.4 ± 69.2 0.002 Total sperm motility (%)** 32.3 ± 14.0 37.9 ± 14.7 0.172 38.0 ± 15.0 43.1 ± 12.8 0.164 Progressive sperm motility (%)** 27.3 ± 14.0 33.0 ± 14.7 0.171 33.1 ± 14.7 38.1 ± 12.8 0.176 Sperm morphology (%) 1.8 ± 2.1 1.6 ± 1.4 0.471 1.9 ± 2.0 2.0 ± 1.7 0.913 Sperm head defect (%) 95.1 ± 16.2 97.8 ± 2.2 0.785 97.5 ± 3.3 97.2 ± 2.7 0.836 Sperm neck + midpiece defect (%) 2.9 ± 16.4 0.2 ± 0.7 0.916 0.2 ± 0.8 0.3 ± 0.8 0.472 Sperm tail defect (%) 0.2 ± 0.6 0.4 ± 0.9 0.253 0.5 ± 1.0 0.5 ± 0.9 0.872 Leukocytes with peroxidase (million/ml) 0.3 ± 0.3 0.5 ± 0.7 0.155 0.5 ± 0.8 0.6 ± 0.9 0.365 Mitochondrial membrane potential (%)*** 27.5 ± 18.5 26.3 ± 18.5 0.375 26.9 ± 15.3 27.1 ± 15.9 0.925 DNA fragmentation (%) 50.5 ± 23.9 51.2 ± 17.9 0.958 45.8 ± 21.8 49.8 ± 16.9 0.116 FSH IU/l) 9.7 ± 7.8 10.0 ± 8.3 0.200 9.2 ± 5.8 9.7 ± 6.5 0.345 Results presented as average (±SD) or percentage (%). Differences within groups between baseline and after 3 months assessed with paired t-test and Wilcoxon test, as appropriate. * = the analysis for total sperm number and sperm concentration was made for 33/37 patients from the astaxanthin group and 34/35 patients from the placebo group, since the excluded patients only had a few mobile or immobile spermatozoa in the semen sample; ** = the analysis for total and progressive sperm motility was made for 22/37 patients from the astaxanthin group and 16/35 patients from the placebo group, since the excluded patients only had a few mobile or immobile spermatozoa in the semen sample; ***: the proportion of spermatozoa with normal sperm mitochondrial membrane potential (MMP); DNA = deoxyribonucleic acid; FSH = follicle-stimulating hormone Radiol Oncol 2021; 55(1): 97-105. Imamovic Kumalic S et al. / Oral intake of astaxanthin and semen parameters in infertile patients 103 The advantage of our prospective, double-blind study includes the good randomization of patients for astaxanthin or placebo treatment. At baseline, both groups were comparable by their characteris- tics. We included patients based on their previous seminograms, and they were all clinically exam- ined by an experienced andrologist. Additionally, strict exclusion criteria were used. Besides, this is the first clinical study of the efficacy of astaxan- thin oral intake on semen parameters in severe oligo-astheno-teratozoospermic patients treated for infertility. The number of patients included is higher compared to similar placebo-controlled studies of other antioxidants.32,39,69,70 Astaxanthin group was homogenous because all patients ac- corded with WHO 2010 criteria for O+A+T. In the placebo group, two-thirds of patients also met the criteria for O+A+T; the average progressive motil- ity of spermatozoa was just above the reference value (33.1 ± 14.7%), but the average total motil- ity of spermatozoa was below the reference value. Therefore, the placebo group was also considered to be homogenous. Nevertheless, our study had some limitations, since one-third of patients had only a few mobile or immobile spermatozoa dur- ing the semen analysis; therefore, the measure- ments and statistical analysis for the total number, concentration and motility of spermatozoa in these patients were not possible. In addition, a relatively high proportion of patients declined to participate in this study. As our study was performed with humans who gave consent to participate, it is our ethical obliga- tion to also report the results with no evidence of an effect.71,72 Also, our study delivers an important fact that the clinical study on the astaxanthin effect in oligo-astheno-teratozoospermia was carried out. Unreported or unpublished negative findings in- troduce a bias in meta-analysis.73 Last but not least, the results could be an important part of future sci- entifically fair and flawless meta-analyses and re- views of the effects of antioxidants in infertile men. Conclusions In conclusion, the oral intake of 16 mg astaxan- thin daily for three months did not significantly improve the semen parameters in patients with oligo-astheno-teratozoospermia compared to pla- cebo. Our findings are important for the treatment and counselling on possible improvements in se- men quality and fertilizing potential in infertile, and also post-testicular cancer patients. As a wide range of different antioxidant supplements is avail- able, including astaxanthin, these findings are also significant concerning the efficacy of supplements in the treatment of male infertility. Acknowledgments The authors would like to thank Branko Zorn, Saso Drobnic, Mojca Kolbezen Simoniti, Ivan Verdenik, Natasa Petkovsek, Sara Hajdarevic, and registered nurses of the Division of Obstetrics and Gynecology, University Medical Centre Ljubljana, Slovenia, and Andreja Natasa Kopitar from the Institute for Microbiology and Immunology, Faculty of Medicine, University of Ljubljana for their assistance in the study. The authors are grate- ful to manufacturer Sensilab, Ljubljana, Slovenia, for the donation of astaxanthin and placebo cap- sules. References 1. Sharlip ID, Jarow JP, Belker AM, Lipshultz LI, Sigman M, Thomas AJ, et al. Best practice policies for male infertility. Fertil Steril 2002; 77: 873-82. doi: 10.1016/s0015-0282(02)03105-9 2. 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