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Serum Levels of Lycopene, Beta-Carotene, and Retinol and Their Correlation with Sperm DNA Damage in Normospermic and Infertile Men

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Serum Levels of Lycopene, Beta-Carotene, and Retinol and Their Correlation with Sperm DNA Damage in Normospermic and Infertile Men Int J Reprod BioMed Vol. 13. No. 12. pp: 787-792, December 2015 Original article Serum levels of lycopene, beta-carotene, and retinol and their correlation with sperm DNA damage in normospermic and infertile men Taiebeh Ghyasvand1 M.Sc., Mohammad Taghi Goodarzi2 Ph.D., Iraj Amiri3 Ph.D., Jamshid Karimi1 Ph.D., Marzieh Ghorbani3 M.Sc. 1. Department of Clinical Abstract Biochemistry, Hamadan University of Medical Sciences, Background: Oxidative stress in reproductive system leads to sperm DNA damage Hamadan, Iran. and sperm membrane lipid peroxidation and may play an important role in the 2. Research Center for Molecular pathogenesis of male infertility, especially in idiopathic cases. Antioxidants such as Medicine, Hamadan University carotenoids function against free radical damages. of Medical Sciences, Hamadan, Iran. Objective: The aim of this study was to determine the levels of lycopene, beta- 3. Endometer and Endometriosis carotene and retinol in serum and their relationship with sperm DNA damage and Centre, Fatemieh Hospital, lipid peroxidation in infertile and normospermic males. Hamadan University of Medical Materials and Methods: Sixty two infertile men and 71 normospermic men Sciences, Hamadan, Iran. participated in this study. Blood and semen samples were collected from all subjects. Sperm DNA damage was measured using TUNEL method. Carotenoids, retinol, and malonedildehyde in serum were also determined. Results: DNA fragmentation was higher in infertile group comparing to control group. Serum levels of lycopene, beta-carotene and, vitamin A in infertile men were significantly lower than normospermic men (p< 0.001, =0.005, and =0.003 respectively). While serum MDA was not significantly different between two groups, MDA in seminal plasma of infertile men was significantly higher than Corresponding Author: Mohammad Taghi Goodarzi, control group (p< 0.001). Research Center for Molecular Conclusion: We concluded that lycopene, beta-carotene, and retinol can reduce Medicine, Hamadan University of sperm DNA fragmentation and lipid peroxidation through their antioxidant effect. Medical Sciences, Sh Fahmideh St, Therefore the DNA fragmentation assay and determination of antioxidants factors 65178, Hamadan, Iran. Email: [email protected] such as lycopene, beta-carotene and retinol, along with sperm analysis can be useful Tel: (+98) 81 38380462 in diagnosis and treatment of men with idiopathic infertility. Received: 15 August 2015 Revised: 12 September 2015 Key words: Infertility, Lycopene, Beta- carotene, Retinol, DNA damage, Lipid peroxidation, Accepted: 18 November 2015 Malondialdehyde. Introduction glutathione reductase, and catalase, and also non-enzyme antioxidants, such as ascorbate, nfertility has a high incidence in young alpha-tocopherol, urate, coenzyme Q10, couples and more than 50% of the cases albumin, N-acetyl-L-cysteine, pyruvate, I are related to the male factors. taurine and hypurate (3, 4). Approximately 25% of the male infertility ROS attack polyunsaturated fatty acids causes are idiopathic (1). (PUFA) in sperm membrane and lead to Reactive oxygen species (ROS) are accumulation of lipid peroxidation products released by leukocytes and abnormal sperms and destruction and deterioration of PUFA. (2). Increased ROS production in sperms and This process causes reduction in mobility, reduction in its scavengering capability leads fluidity, function and fertility of sperms (5). to oxidative stress and can cause infertility in Maintaining sperm DNA integrity is men (3). A set of antioxidants with important for the transfer of genetic scavengering properties, maintains a steady information and fetal development (6). state of ROS in seminal plasma. Damage to sperm DNA causes DNA Seminal plasma contains a number of fragmentation, cross-reaction of protein, and antioxidant enzymes, including superoxide low ability of oocyte fertility. ROS can cause dismutase, glutathione peroxidase, genetic mutations including point mutations, Ghyasvand et al and polymorphisms resulting in a decrease in chemotherapy, and smokers were excluded semen quality (7). DNA is modified and from the study. changed by mutagens such as ROS, Blood and semen samples were collected ultraviolet, gamma, X-rays, and also alkali from all participants 3 to 4 days after the last elements (6, 8). sexual intercourse using self- arousal. As Nearly 90% of dietary carotenoids include leukocytes can produce ROS (16), the alpha- and beta-carotene, beta-krypto- samples containing white blood cells and gezantyne, lutein and lycopene having high immature sperms were excluded from the double bonds show the highest antioxidant study. activity. Lycopene a member of this group has good antioxidant activity includes singlet Sample preparations and analysis oxygen and free radical scavenging (9). The semen analysis, according to World Based on epidemiological studies, a high Health Organization protocol (17) was carried uptake of dietary carotenoids on tissue is out manually and using computer analysis by positively correlated with carotenoid CASA (Camputer Aided Sperm Analysis) via concentrations and negatively with high risk of video test sperm 2.1 software. A serum chronic diseases (9-11). The inverse sample was also prepared from all relationship between lycopene and beta- participants for carotenoid analysis. Prepared carotene with risk of cardiovascular disease serum and semenial plasma samples were and certain cancers has been identified (9, 12, maintained at -70oC until analysis. 13). Unlike the other carotenoids, heat causes Sperm DNA fragmentation assay was the increase of lycopene absorption by carried out using TUNEL (Terminal converting trans- to cis-isomer; also cis-isomer deoxynucleotidyl transferas-mediated dUTP is more soluble in bile micelles and can be nick-end labeling) according to the kit absorbed better (14). manufacturer protocol (Roche, Cat. Retinol is a major vitamer of vitamin A that No.11684795910, Germany). DNA plays a role in the growth and differentiation of fragmentation was determined using a the cells. Also it is involved in differentiation of fluorescence microscope (Ziess, Germany) at germ cells in the gonads and in 488 nm. spermatogenesis (15). The aim of this study Malondialdehyde (MDA) as a lipid was to determine the levels of lycopene, beta- peroxidation index was determined both in carotene and retinol in serum and their serum and seminal plasma according to the relationship with sperm DNA damage and lipid thiobarbitoric fluorimetric assay (16). The peroxidation in infertile and normospermic fluorescence intensity was measured at males. excitation wavelength of 515 nm and emission wavelength of 553 nm (Fluorimete Jasco FP- Materials and methods 6200). Lycopen, beta carotene and viamin A were extracted from prepared samples This case control study was performed in according to the previously reported methods Research Center for Molecular Medicine and (18, 19). Endometer and Endometriosis Center, Hamadan University of Medical Sciences, Chromatographic conditions Hamadan, Iran, during 2012-2013. The Chromato-graphic analysis was carried out protocol of this study was approved by the using Waters HPLC device (isocratic pump, Ethics Committee of Hamadan University of column C18, 150 x 3.9mm, particle size 5µm, Medical Sciences, and informed consent was Novopack). Mobile phase was consisted of obtained from all studied participants. HPLC- grade methanol: acetonitrile: Two age matched groups (20-40 years) tetrahydrofuran (Merck, 5:50:45 v/v) containing 71 normospermic men and 61 men containing butyl hydroxy toluene 0.01%; flow with idiopathic infertility were participated in rate was 0.8 ml/min. Standards for lycopene this study. Infertile men were diagnosed (9879 Sigma), beta-carotene (Fluka 22040) according to world health organization criteria and retinol (7632 Sigma R) were prepared; that were referred by an urologist. Infertile also retinyl acetate (Sigma) was used as men with varicocele, cryptorchidism, renal and internal standard. Concentrations of lycopene urinary tract infections, trauma, and beta- carotene and vitamin A in serum 788 International Journal of Reproductive BioMedicine Vol. 13. No. 12. pp: 787-792, December 2015 Lycopen and sperm DNA damage in infertile men were calculated by the application of higher in infertile men compare to mellenium 1000 software according to the normospermic men (18.94 ± 2.68 vs. 13.42 ± standard curves. 2.14 %, p< 0.001). The results of MDA assay are presented in Statistical analysis table I. The findings showed while the level of Data analysis was performed using serum MDA was not significantly different Pearson and t-test in Statistical Package for between the two groups; in seminal plasma it the Social Sciences, version11, SPSS Inc, was significantly higher in infertile men Chicago, Illinois, USA (SPSS software). comparing to control group (p< 0.001). p<0.05 was considered significant. Lycopene, beta-carotene, and vitamin A levels in infertile men were significantly lower Results than those of normospermic men (Table I). Figure 2 shows a typical chromatogram of the The mean (SD) age of the participants in HPLC analysis. Relationship between serum normospermic and infertile groups was 32.40 lycopene and MDA of seminal plasma with (3.9) and 34.9 (4.5) years respectively, sperm mobility and TUNEL test results were showing no significant difference (p= 0.120). determined in both groups. Also the Results of sperm analysis are shown in table relationship of vitamin A and beta-carotene
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