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Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD14165

Decreased melatonin levels and increased levels of advanced oxidation protein products in the seminal plasma are related to male infertility Ewa Maria Kratz A,E, Agnieszka Piwowar B, Michal Zeman C, Katarı´na Stebelova´ C and Theresia Thalhammer D A

Department of Chemistry and Immunochemistry, Faculty of Medicine, Wrocław Medical University, O. Bujwida 44A, 50-345 Wrocław, Poland. B Department of Toxicology, Faculty of Pharmacy, Wrocław Medical University, Borowska 211, 50-556 Wrocław, Poland. C Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University, Mlynska dolina B2, 84215 Bratislava, Slovak Republic. D Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Waehringer Guertel 18-20 Vienna, Austria. E Corresponding author. Email: [email protected]

Abstract. Melatonin, an indolamine secreted by the pineal gland, is known as a powerful free-radical scavenger and wide-spectrum antioxidant. Therefore, the aim of this study was to correlate markers of oxidative protein damage (advanced oxidation protein products, AOPPs) and the total antioxidant capacity (TAC) with melatonin levels in the seminal plasma of men with azoospermia (n ¼ 37), theratozoospermia (n ¼ 29) and fertile controls (normozoospermia, n ¼ 37). Melatonin concentration was measured by radioimmunoassay. The levels of AOPP as well as TAC efficiency (determined by the ferric reducing antioxidant power, FRAP) were estimated by spectrophotometric methods. The concentration of melatonin and AOPP significantly differed in azoospermic (P , 0.0001) and theratozoospermic (P , 0.0001) patients versus fertile men, and correlated negatively (r ¼ 0.33, P ¼ 0.0016). The TAC levels were significantly higher in azoospermia than in theratozoospermia (P ¼ 0.0022) and the control group (P ¼ 0.00016). In azoospermia, the AOPP concentration was also significantly higher than that observed in theratozoospermia (P ¼ 0.00029). Decreased levels of melatonin together with elevated AOPP altered the oxidative–antioxidative balance in the ejaculate, thereby reducing fertility. Therefore, melatonin and AOPP levels may serve as additional diagnostic markers of semen quality and male reproductive potential. Additional keywords: azoospermia, male fertility, oxidative stress, theratozoospermia, total antioxidant capacity. Received 19 May 2014, accepted 23 July 2014, published online 12 September 2014

Introduction Oxidative stress is regarded as one potential cause of male infertility. For many infertile men increased levels of reactive oxygen species (ROS) in the ejaculate were detected. Under physiological conditions a well-controlled equilibrium exists between ROS production and antioxidant scavenging activities in the male reproductive tract and any disturbances of this balance can lead to damage of macromolecules and disruption of their function, e.g. inducing lipid peroxidation, modification of proteins and DNA damage (Wang et al. 2003; Agarwal et al. 2008; Makker et al. 2009). On the other hand, high levels of ROS in the seminal plasma are usually associated with an increased concentration of proteins known to protect against oxidative stress, such as antioxidative enzymes. However, the high Journal compilation Ó CSIRO 2014

concentration of ROS may lead to the production of oxidatively modified proteins that can contribute to male infertility (Sharma et al. 2013). Therefore, measurement of the concentration of advanced oxidation protein products (AOPPs) in human seminal plasma (Sharma et al. 2013) may provide a good measure for the contribution of oxidative stress to infertility. Severe urogenital diseases, infections and chronic inflammation, as well as exposure to toxic environmental agents (e.g. via smoking) are well known factors influencing the oxidative–antioxidative balance (Pasqualotto et al. 2000; Saleh et al. 2002). It is well documented that ROS are mediators of sperm dysfunction in both well-diagnosed and idiopathic cases of male infertility (Agarwal et al. 2008; Agarwal and Allamaneni 2011; Gharagozloo and Aitken 2011). Oxidative www.publish.csiro.au/journals/rfd

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stress significantly impairs spermatogenesis and function of human spermatozoa by causing peroxidative damage of macromolecules and loss of DNA integrity. However, the overall role of ROS in normal and abnormal sperm function leading to male infertility is incompletely understood (Saalu 2010). On the other hand, spermatozoa produce a small amount of ROS in the respiratory chain, which, at physiological levels, are essential for sperm motility, hyperactivation and capacitation, acrosome reaction and fertilisation, as well as oocyte fusion (Agarwal et al. 2004, 2008; Saalu 2010). Other major sources of ROS in the human ejaculate are infiltrating leucocytes and damaged spermatozoa (Aitken et al. 2003; Fra˛ czek and Kurpisz 2007). Seminal plasma contains a variety of enzymatic and nonenzymatic antioxidants, which are induced under stress conditions for the neutralisation of the increased amount of ROS. A well-known powerful free-radical scavenger and widespectrum antioxidant is the pineal indolamine, melatonin (Poeggeler et al. 1994). It has been proven to be twice as active as vitamin E, believed to be the most effective natural lipophilic antioxidant (Pieri et al. 1994). It exerts pleiotropic actions in the human body and influences different functional systems. The best known physiological role of melatonin is to encode the daily light–dark cycle, but various important physiological processes related to growth, development and senescence of cells and organs in the human body are also modulated by melatonin (Bauer et al. 2013; Reiter et al. 2013). Melatonin can easily cross cell membranes (Hardeland 2005; Reiter et al. 2010; Pohanka 2011) and protect intracellular macromolecules and DNA by acting as a direct scavenger of radical oxygen and nitrogen species including OH, O2–, H2O2 and NO (Poeggeler et al. 1994; Hardeland 2013). In addition to scavenging several radicals and their derivatives, melatonin has been shown to stimulate the expression and function of antioxidative enzymes and glutathione, another important antioxidant (reviewed by Reiter et al. 2009, 2013). These potent antioxidative functions of melatonin may have implications for the optimal function of cells and organs in the male and female reproductive system (reviewed by Reiter et al. 2009) and may protect male spermatozoa from oxidative damage (reviewed by Reiter et al. 2009, 2013). Also, several melatonin metabolites are known free-radical scavengers e.g. N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK; Galano et al. 2011, 2013). It is suggested that melatonin treatment preserved mouse spermatogenesis that was interrupted due to toxin exposure (Sarabia et al. 2009). Under other conditions in which testicular free radicals are generated, melatonin significantly reduces the level of gonadal oxidative stress (Semercioz et al. 2003; Sarabia et al. 2009). Ji et al. (2012) reported that melatonin alleviates cadmium-induced cellular stress and germ cell apoptosis in testes and may be useful as a pharmacological agent to protect against cadmium-induced testicular toxicity. Also, Espino et al. (2011) indicated that stimulation with melatonin triggers a set of events culminating in cell-death prevention in ejaculated human spermatozoa. Furthermore, Fujinoki (2008) documented that melatonin may influence hyperactivation of hamster spermatozoa by stimulation of flagellar motility, directly influencing successful zona pellucida penetration and egg fertilisation.

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Indeed, incubation of ejaculated spermatozoa with melatonin improves their motility and prolongs their viability (Reiter et al. 2013). Additionally, melatonin was found to act with other antioxidants to improve the overall effectiveness of each antioxidant (Hardeland 2013). Therefore, it is important to determine the antioxidant power of biological fluids not only by quantification of an individual antioxidant but also by determination of their cumulative action and synergic effects. The second concept is known as total antioxidant capacity (TAC) and reflects complex interactions among antioxidants and their effects on the oxidative–antioxidative balance. It reflects the sum of low-molecular-weight antioxidants that possess the ability to break oxidation chain reactions, e.g. vitamin C, E, thiols and others (Lee et al. 2012). The aim of our study was to examine the antioxidative system efficiency in seminal plasma of azoospermic and theratozoospermic patients distinguished on the basis of standard semen analysis according to the World Health Organisation (WHO 2010). Since the level of seminal oxidative stress parameters together with melatonin concentration may contribute to further understanding of the mechanisms regulating oxidative– antioxidative balance in human ejaculate (Espino et al. 2010) and serve as additional diagnostic markers of semen quality and male reproductive potential, we measured melatonin concentration as well as the total antioxidant capacity for reduction of excessive free radicals together with markers of oxidative protein damage (AOPPs). Material and methods Patients’ samples Samples of ejaculate were collected during the daytime from infertile men living in childless couples who visited a fertility clinic for assisted reproduction, and samples from apparently fertile volunteers were used as a control. According to WHO (2010), male infertility was defined as the seminal plasma failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse (Zegers-Hochschild et al. 2009). The semen probes were collected by masturbation into sterile containers after 3–5 days of sexual abstinence and were allowed to stand at 378C until liquefaction (no longer than 1 h). Semen samples were centrifuged at 3000g for 10 min at room temperature to obtain plasma. Seminal plasma was divided into small aliquots and frozen at 768C until analysis. The samples were obtained from 66 infertile men and, based on standard semen analysis according to the criteria of WHO (2010), were assigned into two groups: (1) theratozoospermic, exhibiting abnormal sperm morphology with normal other ejaculate parameters (29 patients 26–47 years old) and (2) azoospermic, lacking spermatozoa in ejaculate (37 patients 26–64 years old). Samples from 37 normozoospermic fertile men aged 27–46 years were used as controls. The characteristics of the studied seminal groups are shown in Table 1; for lower reference limits for human semen characteristics see Table 2. The ejaculates were examined at the Department of Laboratory Diagnostics, Wrocław Medical University Academic Hospital for standard analysis (volume, pH, morphology, sperm concentration, motility and viability) performed in accordance with the

Melatonin and AOPP in male fertility

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Table 1. Characteristics of groups studied Phenotypes are related to semen quality, determined according to criteria recommended by the WHO (2010). PRþNP, total motility; PR, progressive motility; NP, non-progressive motility Phenotype

WHO criteria

Normozoospermia

$15  106 spermatozoa mL1, $39  106 total sperm count, $1.5 mL semen volume, $40% of (PRþNP) or $32% (PR) motility, high percentage of normal forms ($4%) No spermatozoa in the ejaculate ,4% of spermatozoa with normal morphology

Azoospermia Theratozoospermia

Table 2. Lower reference limits for human semen characteristics Presented lower reference limits (5th percentiles and their 95% confidence intervals) for human semen characteristics are recommended by the WHO (2010). The reference values given here have been generated from the results of several prospective, cross-sectional studies of semen quality and fertility. They were obtained by direct, retrospective selection of fertile men, defined as men whose partner conceived within 12 months after stopping use of contraception (Cooper et al. 2010). Only complete semen samples – one per man (the first where several were given), obtained following 2–7 days of abstinence – were included in this analysis. Semen volume was measured using methods recommended by the WHO (2010), total sperm number was calculated from concentrations measured by haemocytometer on fixed, diluted samples. Total motility (PRþNP), progressive motility (PR), non-progressive motility (NP) and immotile spermatozoa were measured at room temperature or at 378C. Vitality was determined by exclusion of vital dye (eosin) from spermhead membranes Parameter

Lower reference limit

Semen volume (mL) Total sperm number ( 106 per ejaculate) Sperm concentration ( 106 mL1) Total motility (PR þ NP, %) Progressive motility (PR, %) Vitality (live spermatozoa, %) Sperm morphology (normal forms, %) Other consensus threshold values pH Peroxidase-positive leucocytes ( 106 per mL) MAR test (motile spermatozoa with bound particles, %) Immunobead test (motile spermatozoa with bound beads, %) Seminal zinc (mmol per ejaculate) Seminal fructose (mmol per ejaculate) Seminal neutral glucosidase (mU per ejaculate)

criteria of WHO (2010). All samples were collected between 2:00 and 7:00 p.m. Informed consent was obtained from all patients and the study was approved by the Wrocław Medical University Bioethics Committee (KB-631/2012). Melatonin determination Melatonin concentrations were measured by radioimmunoassay directly in seminal plasma validated in our laboratory (Vician et al. 1999). We used melatonin antiserum prepared towards N-acetyl-5-methoxy-tryptamine conjugated on bovine thyreoglobuline (Stockgrand Ltd, University of Surrey, Guildford, UK) and tritium-labelled melatonin (O-methyl-3H melatonin, TRK 798, specific activity: 3.07 TBq mmol1 (83.0 Ci mmol1); Perkin Elmer, Waltham, MA, USA). Sample radioactivity was measured in the scintillating b-counter for liquid samples (Packard Tri-Carb 2900 TR; Packard Instruments,

1.5 (1.4–1.7) 39 (33–46) 15 (12–16) 40 (38–42) 32 (31–34) 58 (55–63) 4 (3.0–4.0) $7.2 ,1.0 ,50 ,50 $2.4 $13 $20

Meriden, CT, USA). All samples were measured in a single assay; intra-assay coefficient of variation was 3.46% and the sensitivity of the assay 1.3 pg per tube. Measurement of advanced oxidation protein products The concentration of AOPPs was measured by a spectrophotometric assay according to Witko-Sarsat et al. (1996). The method is based on the colour reaction of AOPP with a potassium iodide solution in an acidic environment. Briefly, 1 mL of 10-fold diluted seminal plasma in 0.9% NaCl was mixed with 50 mL potassium iodide (1.16 M; CHEMPUR, Piekary S´la˛ skie, Poland) and 100 mL 10% (v/v) acetic acid. The absorbances were measured immediately at 340 nm using a STATFAX 1904þ Chemistry Analyzer (Awareness Technology Inc., Palm City, FL, USA) against blank samples without seminal plasma, but containing all other reagents. The results were expressed in

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Table 3. The concentrations of melatonin, AOPP and TAC in studied groups of men AOPP, advanced oxidation protein products; TAC, total antioxidant capacity expressed as ferric reducing antioxidant power (FRAP). # median; significant differences versus: 1normozoospermic fertile men, 2theratozoospermic patients. A two-tailed P-value of less than 0.05 was considered significant Group

n

Melatonin (ng L1) Mean  s.e.m.

Median

AOPP (mmol L1)

P value vs normo

Mean  s.e.m.

Median

Normozoospermic fertile Azoospermic

37 37

23.7  10.9 14.3  9.1

20.8 11.9

,0.0001

83.1  93.6 273.9  123.7

41.8 268.4

Theratozoospermic

29

14.5  7.1

12.1

,0.0001

195.4  91.1

176.3

chloramine T units. A calibration curve was made for chloramine T concentrations ranging from 10 mM up to 100 mM. Blood serum and seminal plasma samples with known AOPP concentration were used as controls. All determinations were done in duplicate. The concentration of AOPP in seminal plasma was expressed in mmol L1. Determination of total antioxidant capacity In seminal plasma samples, the TAC values for reduction of excessive free radicals were determined by measuring the ferric reducing antioxidant power (FRAP) with ferric tripyridyltriazine (Benzie and Strain 1996). Reduction of Fe3þ to Fe2þ at low pH creates a coloured Fe2þ-tripyridyltriazine complex. For this reaction, 1.3 mL of FRAP reagent (freshly prepared by mixing 25 mL 300 mM acetate buffer (pH 3.6), 2.5 mL 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ; Sigma-Aldrich, St Louis, MO, USA) in 40 mM HCl and 2.5 mL 20 mM FeCl3  6H2O (CHEMPUR, Piekary S´la˛ skie, Poland) in distilled water) and 300 mL of a sixty-fold diluted sample in 0.9% NaCl were mixed. The reaction mixture was incubated at 378C for 5 min and absorbance was then immediately recorded at 594 nm using a GENESYS 10S Series UV-VIS Spectrophotometer (Thermo Fisher Scientific Inc., Madison, WI, USA). The absorbance of the samples was read against a blank sample containing the FRAP reagent without seminal plasma. A calibration curve was made for known amounts of Fe2þ in solutions (ranging from 0.025 mM up to 0.05 mM Fe2þ). Blood serum and seminal plasma samples with a known FRAP concentration were used as controls. All measurements were done in duplicate. The concentration of TAC in seminal plasma was expressed in mmol L1. Statistical analysis For statistical analysis STATISTICA 10.0 computer software was used (StatSoft Inc., Tulsa, OK, USA). Results obtained are presented as the mean value  s.e.m. and median with the interquartile range (25th percentile, 75th percentile) as the parameters more accurately describing the data spread. The concentrations of melatonin, AOPP and FRAP were not normally distributed as calculated according to the Shapiro–Wilk W test, thus the nonparametric Mann–Whitney U-test was used to determine differences between studied groups. The correlation with 95% confidence interval was estimated according to the

TAC (mmol L1) P value vs normo, therato ,0.0001, ¼0.00029 ,0.0001

Mean  s.e.m.

Median

2120  400 2913  900

2120 2720

2269  570

2210

P value vs normo, therato ¼0.00016, ¼0.0022

Spearman test. A two-tailed P value of less than 0.05 was considered to be statistically significant. Results In the normozoospermic fertile group, the sperm number was higher than 15  106 mL1 and more than 4% of them expressed regular sperm morphology with a total motility of $40% or progressive motility $32% at 1 h after ejaculation, fulfilling the requirement for normal sperm physiological parameters according to the criteria of WHO (2010). No spermatozoa were found in the azoospermic samples. In the theratozoospermic group, normal sperm morphology was below 4% while their vitality and motility were comparable to that of normal controls (Table 1). None of the seminal plasma samples were leucocytospermic, excluding the possibility that inflammation may contribute to infertility. Also, other parameters (pH, semen volume, level of zinc, fructose and neutral glucosidase) were within the normal range in all samples (see Table 2). The concentration of melatonin as determined by a highly specific and sensitive radioimmunoassay revealed that seminal plasma melatonin levels were approx. 30% higher in normozoospermic fertile men (23.7  10.9 ng L1, median 20.8 ng L1) than in samples from infertile men (P , 0.0001). There were no differences in melatonin levels in samples from the azoospermic group (14.3  9.1 ng L1, median 11.9 ng L1) as compared with theratozoospermic patients (14.5  7.1 ng L1, median 12.1 ng L1) underlining the importance of the indolamine for normal sperm development and quality (Table 3, Fig. 1a). In line with the proposed role of oxidative stress in infertility, we found 2.0–3.5-fold higher levels of AOPP in the seminal plasma of infertile subjects. In azoospermia, AOPP levels were 273.9  123.7 mmol L1 (median 268.4 mmol L1, P , 0.0001) and in theratozoospermia 195.4  91.1 mmol L1 (median 176.3 mmol L1, P , 0.0001) while in the normozoospermic samples of fertile volunteers only 83.1  93.6 mmol L1 (median 41.8 mmol L1) AOPPs were determined (Table 3, Fig. 1b). The data indicate that AOPPs in azoospermic samples were 1.5-fold higher than in the theratozoospermic group (P ¼ 0.00029; Table 3, Fig. 1b). Using the Spearman test (Fig. 2) we further demonstrated that high AOPP levels significantly correlated with low melatonin levels (r ¼ 0.33, P ¼ 0.0016; Fig. 2a). On the other hand, high AOPP levels were associated with high TAC levels

Melatonin and AOPP in male fertility

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Fig. 1. Concentrations of melatonin, AOPP and TAC in human seminal plasma. (a) Seminal plasma melatonin concentrations were estimated by radioimmunoassay and the (b) AOPP and (c) TAC levels by spectrophotometric assays. Subjects: NF, normozoospermic fertile; Az, azoospermic; T, theratozoospermic. AOPP, advanced oxidation protein products; TAC, total antioxidant capacity. Significant differences versus: 1normozoospermic fertile group, 2theratozoospermic group. A two-tailed P value of less than 0.05 was considered to be statistically significant.

(r ¼ 0.34, P , 0.0011; Fig. 2c), but TAC levels did not correlate with the levels of melatonin (r ¼ 0.11, P ¼ 0.29; Fig. 2b). In contrast to the decreased levels of the antioxidant melatonin in samples from infertile patients, the TAC levels (measured as FRAP) in seminal plasma samples followed a different

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600 500 400 300 200 100 0 1000

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TAC (µmol L1) Fig. 2. The association between melatonin, AOPP and TAC in human seminal plasma. The figures show the association between concentrations of determined seminal plasma parameters: (a) melatonin and AOPP, (b) melatonin and TAC and (c) AOPP and TAC. Melatonin concentrations were estimated by radioimmunoassay and AOPP and TAC by spectrophotometric assays. The correlations were estimated according to the Spearman test and a two-tailed P value of less than 0.05 was considered to be statistically significant. The dashed line shows 95% confidence interval. AOPP, advanced oxidation protein products; TAC, total antioxidant capacity.

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pattern, with higher concentrations in azoospermic samples (2913  900 mmol L1, median 2720 mmol L1, P ¼ 0.00016) as compared with normozoospermic samples (2120  400 mmol L1, median 2120 mmol L1; Table 3, Fig. 1c). In seminal plasma of theratozoospermic patients, the TAC levels of 2269  570 mmol L1 (median 2210 mmol L1) were similar to controls. Although high TAC levels were associated with low melatonin levels in the azoospermic and normozoospermic samples, there was no significant correlation between these two parameters (r ¼ 0.11, P ¼ 0.29; Fig. 2b). No association between melatonin, AOPP and TAC concentrations versus sperm number or total motility was observed in the normozoospermic fertile or theratozoospermic groups. Discussion We have shown that high melatonin levels corresponded to decreased AOPP concentrations in the seminal plasma of fertile men. Moreover, in the seminal plasma of infertile patients diagnosed with azoospermia or theratozoospermia, significantly reduced levels of melatonin were associated with elevated levels of AOPPs. The regulation of oxidative–antioxidative balance in human ejaculate is only one of the various mechanisms influencing male reproductive potential. Seminal plasma serves as a natural reservoir of antioxidants. It helps to remove excessive formation of ROS and consequently reduce oxidative stress. Although, spermatozoa generate ROS themselves, which is important to assure fertilisation, the amount of ROS needs to be kept at a physiological level because developing and mature spermatozoa are extremely sensitive to oxidative stress. Therefore, extensive amounts of ROS delivered from damaged spermatozoa and infiltrating leucocytes together with insufficient antioxidant protection will increase the ROS levels. This may lead to infertility resulting from a lack of normal fertilisation by ROS-induced peroxidative damage in the sperm membrane and defects in spermiogenesis caused by ROS-induced DNA fragmentation (Aitken et al. 2003; Aitken and Curry 2011). The potent antioxidant melatonin is capable of scavenging free radicals and induction of antioxidative enzymes (discussed by Reiter et al. 2013) and has been proven to restore normal testis function and sperm quality (Ortiz et al. 2011) even under pathological conditions e.g. hyperlipidaemia (Zhang et al. 2012). Ortiz et al. (2011) documented that exposure of spermatozoa to melatonin improved the percentage of motile and progressively motile cells and decreased the number of static cells, thereby promoting the proportion of rapid spermatozoa. Therefore, insufficient levels of melatonin may prevent efficient sperm development and formation of normal morphology in azoospermic and theratozoospermic patients, respectively (Cruz et al. 2014). Melatonin concentrations determined in our study by using a specific and highly sensitive radioimmunoassay (23.7  10.9 ng L1 in fertile men vs 14.3  9.1 ng L1 and 14.5  7.1 ng L1 in azoospermic and theratozoospermic samples, respectively, measured between 2:00 and 7:00 p.m.) are in good agreement with the data of Awad et al. (2006), where seminal melatonin levels were determined by enzymelinked immunosorbent assay (ELISA; 44.8  11.0 ng L1 in the

E. M. Kratz et al.

seminal plasma of fertile and 20.3–36.2 ng L1 in infertile men, measured between 8:00 and 10:00 a.m.). The seminal melatonin levels were much lower than those observed in blood serum (111.5  35.3 ng L1 for fertile men and 50.7–66.7 ng L1 for infertile subjects, respectively), measured at the same time of the day, as reported by Awad et al. (2006). Melatonin is readily soluble in both lipid and aqueous environments and can readily cross the blood–testes barrier to protect the germinal epithelium (Awad et al. 2006). This suggests that seminal melatonin is derived from the pineal gland and is transferred to semen from the blood. Extrapineal melatonin production by seminal vesicles or the prostate gland has not been proven so far, although high concentrations of melatonin found in some organs suggest an extrapineal production of the hormone (Acun˜a-Castroviejo et al. 2014). A local synthesis of melatonin in the testis was suggested (Tijmes et al. 1996) and expression of mRNA for both melatonin-synthesising enzymes (N-acetyltransferase and hydroxyindole-O-methyltransferase) was detected in testes (Stefulj et al. 2001). Testicular concentrations of melatonin, measured in biopsies of patients suffering from idiopathic infertility, negatively correlated with the macrophage number and expression of pro-inflammatory cytokines such as tumour necrosis factor a (TNFa) and interleukin 1b (Rossi et al. 2014). Melatonin concentrations in the testis are in the same range as in other organs examined, such as the kidney, heart and pancreas (Stebelova´ et al. 2007). Otherwise, local seminal plasma melatonin concentrations may reflect systemic pineal-derived production influenced by the life-style and especially environmental lighting conditions. It is also possible that melatonin is stored locally in some cells and can be liberated ‘on demand’ to work as an antioxidant; however, this hypothesis is still not documented. We used the levels of advanced oxidation protein products in human seminal plasma as a good measure for the contribution of oxidative stress to infertility via protein modification. In blood plasma AOPPs are characterised as dityrosine-containing protein cross-linked products formed due to oxidation of amino acids by ROS. They represent predominantly aggregates of albumin damaged by oxidative stress but there is a possibility that they may be derived from other plasma proteins as well (Witko-Sarsat et al. 1996). A similar mechanism for AOPP formation in seminal plasma is proposed and AOPPs are thought to influence normal development of spermatozoa leading to infertility. In accordance with this hypothesis we observed an even higher AOPP concentration in the seminal plasma of patients with azoospermia than in the theratozoospermic group (P ¼ 0.00029; Table 3, Fig. 1b). We also measured the total antioxidant capacity, which reflects the complex interactions among antioxidants and their effect on the redox balance. While TAC reflects the effect of low-molecular-weight antioxidants, which possess the ability to break oxidation chain reactions, it does not reflect the activity of endogenous antioxidant enzymes (e.g. catalase etc.) or metalbinding proteins. These limitations may at least partly explain why we did not see a relationship between TAC values in seminal plasma samples and the fertility of patients. TAC values were increased in azoospermic infertile patients and remained unchanged in the infertile theratozoospermic group. Therefore,

Melatonin and AOPP in male fertility

any increase in the antioxidative capacity of male semen may not necessarily lead to improved sperm parameters or structuredependent functionality of some proteins crucial in the fertilisation process. Furthermore, although increased AOPP values were associated with increased TAC values, this may not be sufficient to counteract the negative effect of AOPP. Our data are in contrast to some other studies on human seminal plasma TAC values (Pasqualotto et al. 2000; Benedetti et al. 2012; Pahune et al. 2013), in which the lower TAC values were found in infertile as compared with fertile patients. However, Pahune et al. (2013) showed that the TAC concentrations in azoospermic seminal samples were lower than in normozoospermic samples; in contrast to our normozoospermic fertile collective, they had normozoospermic samples from male partners from infertile couples. These data show that TAC values in the seminal plasma of infertile men are inconsistent and this might be caused by different study conditions applied. It has to be added that data on the antioxidant enzymes superoxide dismutase (SOD) and catalase in infertile patients published by various authors are controversial as well. For example, Siciliano et al. (2001) reported that seminal plasma non-enzymatic (TAC) and enzymatic (SOD, catalase) antioxidant capacities do not alter in asthenozoospermic subjects, whereas SOD activity was lower in oligoasthenozoospermic samples than normozoospermic males. Sanocka et al. (1996) documented statistically significant differences in SOD activity in infertile men compared with normozoospermic samples. They observed that only in oligozoospermic seminal plasma, SOD activity exceeds that of normozoospermic samples. In a later paper the same group of investigators (Sanocka et al. 1997) compared SOD and catalase activity in men with asthenozoospermia, theratozoospermia and oligozoospermia to that of normozoospermic samples, showing a significant increase in intracellular SOD and a decrease in catalase activity in infertile patients. Also Zini et al. (2000) showed higher seminal plasma SOD activity in infertile men but found no differences in catalase activity. Hsieh et al. (2002), however, did not find a significant difference in seminal plasma or sperm SOD activity between normozoospermic and oligo- or asthenozoospermic patients, while Tkaczuk-Włach et al. (2002) concluded that whole semen SOD activity is higher in men with oligozoospermia as compared with normozoospermia. Information on a positive impact of healthy lifestyle and healthy diet on male fertility may contribute to the fact that patients with diagnosed reduced fertility may try to change their lifestyle, dietary habits and eliminate addictions. Also, antioxidant supplementation (e.g. readily available vitamin A, C, E, GSH, coenzyme Q10) may contribute to improving the antioxidative potential of men with decreased fertility caused by oxidative stress as described by e.g. Makker et al. (2009), Showell et al. (2011), Reiter et al. (2013) and Mora-Esteves and Shin (2013). These circumstances might explain the contradictory data on TAC levels in seminal plasma of infertile patients. No information is available on the particular type of antioxidant, if any, used by these patients. Taken together, our data suggest that measurement of seminal plasma melatonin may be more conclusive than TAC in determination of the antioxidative capacity of human semen. Although melatonin is not usually tested in the first-level

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evaluation of infertile male patients, it seems to be a better and more accurate marker reflecting the antioxidant capacity of male ejaculate than TAC and should be tested together with AOPP in a larger group of patients. Data from these studies could provide further information on aspects of antioxidant supplementation in infertile male patients prepared for in vitro fertilisation, which may be extended to melatonin. Acknowledgements During the study implementation (melatonin determination) E. M. Kratz was supported by Wrocław Medical University Foundation (ISO 9001:2009) and the project was partially supported by APVV 0291–2012. The authors declare no conflict of interest.

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