Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa

Reactive oxygen species (ROS) can be detected in the semen of 40% of infertile men, whereas none is detected in semen from normal men. The ROS detected in semen are a reflection of the imbalance between ROS production and degradation. The aim of the present study was to determine whether a lowered scavenging capacity or an increased production of ROS was responsible for the ROS detected in semen samples from infertile men. Two activities were investigated: (1) catalase-like activity, which is responsible for the degradation of H₂O₂, and (2) superoxide dismutase-like (SOD-like) activity which is responsible for the degradation of O₂-_-. Catalase-like and SOD-like activities were found in whole seminal plasma, in dialyzed seminal plasma (> 12 kD), in an ultrafiltrate of seminal plasma (< 5 kD) and in spermatozoa. There was no significant difference in the SOD-like activities measured in spermatozoa, or in seminal plasma (whole or fractionated) from samples that did or did not produce ROS. SOD-like activity originated mostly from the high molecular weight components of seminal plasma. However, the catalase-like activity of whole seminal plasma and of spermatozoa was significantly greater ( P = 0.01) in those samples that produced ROS as compared to those that did not. The catalase-like activity in dialyzed seminal plasma, and an ultrafiltrate of seminal plasma from semen samples that did or did not produce ROS were not statistically different. The catalase-like activity of the seminal plasma originated equally from high and low molecular weight components. In conclusion, the data suggest that the ROS detected in the semen of infertile patients are likely due to increased ROS production rather than to decreased ROS scavenging capacity.     

Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions

The contribution of leukocytes and of spermatozoa to reactive oxygen species (ROS) production in prepared sperm suspensions from donors and subfertility patients was compared. In both groups, more leukocytes/10(6) spermatozoa were counted in samples which produced detectable ROS than in those that did not: Donors-645 vs. 170 (medians, n = 7; p < 0.01, Kruskal-Wallis), Subfertile group-1785 (n = 18) vs. 11 (n = 8) (p < 0.005, Kruskal-Wallis), respectively. Leukocyte concentrations were correlated with basal (r = 0.826, p < 0.001) and with ROS production stimulated with 50 mumol N-formyl, met, leu, phe l-1 (N-FMLP) (r = 0.835, p < 0.001) and 100 nmol phorbol 12-myristate 13-acetate l-1 (PMA) (r = 0.835, p < 0.001) measured using a chemiluminescence assay. Leukocytes were removed from the sperm suspensions of 6 donors and from 96 ejaculates from 21 subfertility patients and ROS production was determined. Subsequently, in all 6 donors, N-FMLP did not stimulate ROS production indicating that leukocyte removal was complete, though in one case PMA stimulated low levels of ROS production. In 65 ejaculates from subfertile men the N-FMLP response was completely eliminated but in 7 of these samples PMA continued to stimulate ROS production. We conclude that infiltrating leukocytes are the predominant source of ROS production in unpurified sperm preparations. Some purified sperm suspensions could be stimulated to produce ROS by the addition of PMA indicating that spermatozoa themselves may produce ROS, albeit in much smaller amounts.     

Differentiation of ejaculates showing reactive oxygen species production by spermatozoa or leukocytes

Summary. Differences between subgroups and correlations between reactive oxygen species (ROS), sperm motility, concentration of leukocytes and viability in semen samples from 143 men were investigated. Patients with azoospermia or leuko-cytospermia were excluded from the study. Spermatozoa were separated by means of glass wool nitration. Reactive oxygen species were determined by means of luminol chemiluminescence before and after sperm separation; thereafter, normozoospermic and oligozoospermic patients were divided into three subgroups using the mean of all patients investigated (17462 count 10⁻⁷ viable spermatozoa) as cut-off value as follows: G1—high reactive oxygen species production in native semen and after glass wool nitration; G2—high production of reactive oxygen species only in native semen; G3—low levels of reactive oxygen species before and after glass wool filtration. In general, reactive oxygen species were significantly higher in oligozoospermic samples than in normozoospermic samples. In men with normal sperm count, ROS production correlated significantly with the number of leukocytes in the ejaculate before glass wool nitration, but not thereafter. Glass wool nitration is useful to distinguish between sperm samples in which reactive oxygen species are generated by leukocytes and those in which reactive oxygen species are excessively generated by the spermatozoa themselves.     

Ascorbic acid reduces redox potential in human spermatozoa subjected to heat‐induced oxidative stress

Oxidation–reduction potential (ORP) is a new measure of oxidative stress. It is a balance between the total available oxidants and reductants. This study measures the efficiency of ascorbic acid (AA) against oxidative stress induced by either heat alone or heat and hydrogen peroxide in sperm suspensions using the MiOXSYS System. Two concentrations of ascorbic acid (400 and 600 μmol/L) were tested against heat‐ and heat plus hydrogen peroxide‐induced oxidative stress in sperm suspensions after 2 and 4 hr of incubation. Sperm motility and static oxidation reduction potential (sORP) were measured at 2 and 4 hr of incubation at three different temperatures. A significant decrease in sORP was observed as a function of AA concentration. The 600 μmol/L AA had more pronounced reduction in sORP compared to 400 μmol/L AA (p = .001). Significant decreases in sperm motility ranging from 4.89% to 14.02% were observed both as a function of incubation time and addition of H₂O₂ (p < .001). Ascorbic acid is efficacious to reduce heat‐induced oxidative stress in sperm preparations in vitro. The supplementation of ascorbic acid may be advantageous for semen preparations in IUI, IVF and ICSI.     

Effect of reactive oxygen species and the activity of antioxidant systems on human semen; association with male infertility

A range of compounds with a role in oxidative stress were measured in ejaculates from 40 normozoospermic individuals and 93 infertile males. Ejaculates were classified according to WHO criteria. Seminal plasma and the sperm cell fraction were assessed separately for superoxide dismutase (SOD), catalase, xanthine oxidase, capability for singlet oxygen trapping and content of thiobarbituric acid-reactive substances (TBARS). Pathological cases defined as oligozoospermia, asthenozoospermia, or teratozoospermia revealed different backgrounds of oxidative stress as reflected by different levels of tested substances in every type of sperm pathology. In the majority of abnormal ejaculates, a significant increase in intracellular activity of SOD, decreased intracellular levels of catalase, elevated levels of xanthine oxidase and TBARS, and severely impaired singlet oxygen trapping were observed when compared to normozoospermic ejaculates. Interrelationships between SOD and TBARS, and between xanthine oxidase and catalase, appeared to be of key importance when analysed separately in seminal plasma and in spermatozoa or in a combination of both. Elevated xanthine oxidase levels and low capacity for singlet oxygen trapping are statistically significant factors for the evaluation of male infertility which can develop as a result of persistent oxidative stress.     

Pentoxifylline increases the level of nitric oxide produced by human spermatozoa

Pentoxifylline (PF) is a xanthine derivative drug primarily used to treat peripheral vascular disorders. It is currently used in assisted reproductive technologies to enhance human sperm motility. However, the mechanism by which this enhancement occurs is not fully understood. Given that nitric oxide has been identified as a trigger to sperm motion, we asked whether nitric oxide modulates the stimulatory effect of PF on sperm motility. A total of 41 semen samples from infertile males were studied. Nitric oxide production in the presence of 5 mm PF was tested using different bio‐analytical methods (spectrophotometry, fluorometry and fluorescence microscopy). The spectrophotometric determination showed higher levels of nitrite, an indirect measure for nitric oxide, in sperm samples supplemented with PF compared to controls. The fluorometric experiment showed higher 4, 5‐diaminofluorescein triazole, a product from the reaction between nitric oxide and 4, 5‐diaminofluorescein diacetate, after adding PF to spermatozoa. The fluorescence microscopy images of the spermatozoa supplemented with PF showed higher green fluorescence, indicating higher 4, 5‐diaminofluorescein triazole levels, compared to controls. It is concluded that PF enhances nitric oxide production in human spermatozoa, which explains, at least in part, the mechanism by which PF stimulates human sperm motility.     

Variations in creatine kinase activity and reactive oxygen species levels are involved in capacitation of bovine spermatozoa

The generation of reactive oxygen species (ROS) is associated with some factors such as oxidative substrate sources, mitochondrial function and NAD(P)H oxidase activity. In bovine spermatozoa, heparin capacitation produces a respiratory burst sensitive to diphenyleneiodonium (DPI). Creatine kinase (CK) is related to extramitochondrial ATP disponibility. Our purpose was to determine the variation in ROS level and its relation with NAD(P)H oxidase sensitive to DPI and CK participation, as factors involved in redox state and energy generation in capacitation. The chlortetracycline technique was used to evaluate capacitation. CK activity and ROS level were measured by spectrophotometry and spectrofluorometry respectively. The capacitation percentage was increased by heparin or quercetin treatment (P < 0.05) and no significant differences in sperm viability were observed. Samples treated with heparin or quercetin maintained the same ROS level as control (238.62 ± 23.47 arbitrary units per 10⁸ spermatozoa) (P > 0.05). CK activity decreased by 50% with heparin or quercetin (P < 0.05). In DPI presence, capacitation was inhibited and differential CK activities and ROS level variations were observed in heparin- or quercetin-treated samples (P < 0.05). In cryopreserved bovine spermatozoa, capacitation requires equilibrium between oxidative damage susceptibility and ROS levels. CK activity is associated with redox state variation and energy sources. In conclusion, capacitation induction depends on NADPH oxidase and the shuttle creatine–creatine phosphate, both sensitive to DPI.     

Separation of human spermatozoa with hyaluronic acid induces, and Percoll® inhibits, the acrosome reaction

The effect on the acrosome reaction of human spermatozoa of two widely used sperm separation media, hyaluronic acid (Sperm Select®) and Percoll®, was studied. Viable and highly motile fractions of human spermatozoa were separated from seminal plasma using self-migration on a Percoll® gradient. After translocation of separated spermatozoa from the Percoll® solution to a culture medium, serum, Percoll® or hyaluronic acid (Sperm Select®) was added to aliquots of the spermatozoa containing culture medium. At increasing time intervals, the influx of ⁴⁵Ca²⁺ into spermatozoa was measured and the concentration of viable spermatozoa that had undergone the acrosome reaction was analysed using the triple stain technique. Serum was found to be necessary to support sperm motility and viability. Compared to culture medium with serum only, addition of hyaluronic acid induced influx of ⁴⁵Ca²⁺ and the acrosome reaction, whilst Percoll® inhibited both of these actions. Hyaluronic acid (Sperm Select®) added to spermatozoa separated by a 'swim-up' method induced, and the addition of Percoll® inhibited, influx of ⁴⁵Ca²⁺ when compared to the addition of culture medium with serum only. This study demonstrates that both hyaluronic acid (Sperm Select®) and Percoll® affect the acrosome reaction and the prerequisite for Ca²⁺ influx in human spermatozoa. These effects should be taken into consideration when using these media for preparation of spermatozoa for insemination or for fertilization in vitro.     

Monitoring the reactive oxygen species in spermatozoa during liquid storage of boar semen and its correlation with sperm motility,free thiol content and seasonality

Oxidative stress is an important factor affecting the quality of spermatozoa during liquid storage of boar semen; however, monitoring of reactive oxygen species (ROS) that provides direct insight into the oxidative status is not yet attempted. This study aimed to monitor ROS in boar sperm during liquid semen storage to determine its correlation with sperm motility and free thiol (SH) content, and seasonality. Ejaculate was collected from mature Duroc boars in a commercial farm in autumn and spring, diluted in Mulberry III extender, stored at 15°C, and examined daily for sperm ROS level, SH content and motility. The ROS levels in spermatozoa prepared during autumn and spring were constantly low until days 4 and 5 of storage, respectively, which thereafter progressively increased in association with the loss of sperm motility. The increased sperm ROS level correlated with the higher SH level and lower motility, which was accentuated from day 4 of storage and was higher in September, or early autumn. This study indicates that increased sperm ROS levels during liquid storage results in oxidative damage, causing loss of sperm motility, presumably through decreased sperm viability, suggesting that sperm ROS monitoring effectively evaluates the quality of boar semen.     

Effects of H2O2 exposure on human sperm motility parameters,reactive oxygen species levels and nitric oxide levels

Research has revealed that reactive oxygen species (ROS) negatively affect sperm function, both in vivo and in vitro. Sperm preparation techniques for assisted reproductive technologies (ART) are potential causes for additional ROS production. This study aimed to correlate the concentration of exogenous H₂O₂ with sperm motility parameters and intracellular ROS and nitric oxide (NO) levels to reiterate the importance of minimising ROS levels in ART. Human spermatozoa from 10 donors were incubated and exposed to different exogenous H₂O₂ concentrations (0, 2.5, 7.5 and 15 μm ). Subsequently, motility was determined using computer‐aided semen analysis, while ROS (2,7‐dichlorofluorescin diacetate) and NO (diaminofluorescein‐2/diacetate) were analysed using fluorescence‐activated cell sorting. Results showed that H₂O₂ did affect the sperm parameters. Exogenous H₂O₂ was detrimental to motility and resulted in a significant increase in overall ROS and NO levels. A significant increase in static cells was seen as well. It is important to elucidate the mechanisms between intracellular ROS levels with sperm motility parameters. While this experiment demonstrated a need to reduce exogenous ROS levels during ART, it did not illustrate the cause and effect relationship of intracellular ROS and NO levels with sperm motility. Further research needs to be conducted to define a pathological level of ROS.     

In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation

The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham′s F10 medium plus bovine albumin at 37° and 5% CO₂ for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen‐free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.     

The limits for detection of activated caspases of spermatozoa by western blot in human semen

Detection of activated caspases of spermatozoa could be helpful to evaluate male infertility. Although western blot is validated as a highly specific method to detect the proteins extracted from cells, the ability of this technique to detect activated sperm caspases in human semen may be limited. Indeed, round cells, which potentially contain some activated caspases, may be present in semen and interfere with the detection of activated sperm caspases. Moreover, it is necessary to evaluate the minimum amount of spermatozoa necessary to optimise the detection of activated caspases in semen samples. Our results showed that interference due to round cells contained in semen with activated caspase-3 requires separation of spermatozoa by density migration. This sperm preparation selects a mature sperm population that does not reflect the whole sperm population, and in infertile men with oligoasthenoteratozoospermia, the amount of spermatozoa thus selected is usually low. Moreover, the western blot technique's low detection sensitivity and the low level of caspase enzyme activity in human spermatozoa for activated caspase-3, -8 and -9 mean that large quantities of spermatozoa are needed to detect the expression of the activated caspases. These limitations prevent this method being used for routine analysis in clinical practice.     

Proteomic analysis of sperm proteins in infertile men with high levels of reactive oxygen species

Oxidative stress is a significant risk factor for male infertility. A pro‐oxidant testicular environment may alter the expression profile of functional sperm proteins and result in poor sperm quality. Patients and donors were divided into ROS (‐) and ROS (+) groups. Using computational studies, and data mining of available literature on spermatozoa, oxidative stress and proteomics, we identified three core regulatory proteins angiotensin‐converting enzyme (ACE), heat‐shock protein (Hsp70) family A member 2 (HSPA2) and ribosomal protein subunit 27A (RPS27A) and seven interlink proteins NOS2, SUMO2, UBL4A, FBXO25, MAP3K3, APP and UBC. HSPA2 was validated by Western Blot, while the localisation of ACE, RPS27A, MAP3K3 and APP was identified by immunocytochemistry. The obtained results showed that HSPA2 was 1.2 (ROS+) and 2.1 (ROS?) fold downregulated in spermatozoa from patients with high levels of reactive oxygen species (ROS). ACE and APP were localised in the post‐acrosomal region of spermatozoa, whereas RPS27A and MAP3K3 were localised either in the tail or sperm neck area. Our data show that these proteins may play a role in ROS‐induced male infertility.     

Tissue inhibitors of metalloproteinases 1 and 2 in human seminal plasma and their association with spermatozoa

A 24-kDa heparin binding protein recently identified as tissue inhibitor of matrix metalloproteinases 2 (TIMP-2) in bovine seminal fluid was suggested to play an important role in bull fertility. As no data are present for men, the concentrations of tissue inhibitors of metalloproteinases 1 (TIMP-1) and TIMP-2 were quantified in human seminal plasma of normozoospermic and azoospermic men using enzyme-linked immunosorbent assay methods. TIMP-1 and 2 were not significantly different in both groups and there were no relationships between the concentrations of both TIMPs and other sperm characteristics. It is assumed that TIMPs are released from accessory sex glands.     

The effects of pentoxifylline on the generation of reactive oxygen species and lipid peroxidation in human spermatozoa

Summary. The aims of this study were to compare the in vitro effects of 3.6 mM and 7.2 mM pentoxifylline on the ability of spermatozoa to generate reactive oxygen species (ROS) and on lipid peroxidation (LPO). Semen samples were obtained from 10 asthenozoospermic men who had been previously identified as producing ROS after addition of Phorbol 12-myristate 13-acetate (PMA) during the screening of patients attending with male factor infertility. Spermatozoa were prepared by a swim-up technique from unprocessed semen and divided into 3 aliquots. To the control aliquot [A] an equal volume of BWW medium was added. To aliquots B and C an equal volume of BWW medium containing pentoxifylline was added to obtain final concentrations of 3.6 and 7.2 mM, respectively. ROS production was measured from peak luminescence (mV 10⁻⁷ sperm) using a lucigenin chemiluminescent probe. LPO was also measured in the medium surrounding the spermatozoa after 30 min exposure to pentoxifylline using the thiobarbituric acid (TBA) assay for malondialdehyde (MDA). The reduction in ROS production was significantly greater in the samples exposed to 7.2 mM pentoxifylline as compared with the control and 3.6 mM pentoxifylline samples. There was no significant difference in peak luminescence between control and 3.6 mM pentoxifylline specimens. Both concentrations of pentoxifylline caused comparable reductions in MDA concentration in the medium ( P <0.05) surrounding the spermatozoa compared with control after 30 min exposure. Extracellular ROS generation may damage surrounding healthy spermatozoa. These findings suggest that higher concentrations of pentoxifylline are protective against ROS release in susceptible spermatozoa and may also reduce collateral LPO.     

Effects of semen processing on the generation of reactive oxygen species and mitochondrial membrane potential of human spermatozoa

This study was designed to evaluate the effects of semen processing on the generation of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in spermatozoa, and to develop reliable indexes for the evaluation of sperm quality during sperm preparation. Swim-up and density gradient centrifugation methods were used to separate semen in oligoasthenoteratozoospermia (OAT), leucocytospermia (LC) and normozoospermia groups. Levels of ROS and MMP were measured by flow cytometry. Before preparation, the patients with abnormal semen parameters had a lower MMP and higher ROS, and there was a negative correlation between MMP and ROS. The levels of MMP and ROS increased significantly, especially ROS produced by swim-up. A significant difference was found between the correlation of MMP and total normal motile sperm count after preparation in the OAT group. The level of ROS was associated with the amount of white blood cells in the LC group. The MMP can be used as an objective index to evaluate the sperm quality of OAT patients, and the combination of MMP and ROS can be used to assess the efficiency of sperm preparation in LC patients. These findings can guide selection of the ideal sperm separation technique for different sperm samples.     

Association of ureaplasma urealyticum with abnormal reactive oxygen species levels and absence of leukocytospermia

PURPOSE: Ureaplasma urealyticum is a commensal of the lower genitourinary tract of many sexually active adults. The organism is more common in partners of infertile than fertile marriages. We conducted a prospective study at our tertiary care center to confirm a possible association between U. urealyticum and abnormal sperm function parameters. MATERIALS AND METHODS: A total of 50 consecutive male patients seeking general urology consultation for lower urinary tract symptoms characteristic of chronic prostatitis were evaluated. Urine and semen localization cultures were performed with additional semen cultures for U. urealyticum, Chlamydia trachomatis and Mycoplasma hominis. Specimens from 21 healthy men were used as controls. Specimens were analyzed by a computer assisted semen analyzer, and verified manually for concentration, percent motility and morphology. Leukocytospermia was measured by the Endtz test. Semen specimens were also analyzed for reactive oxygen species (ROS), acrosome reaction and mannose binding assay. RESULTS: Of the patients 17 had positive U. urealyticum cultures and the other cultures were negative. Patients with U. urealyticum had significantly higher ROS levels (log [ROS + 1] = 2.52 +/- 0.25) than those without U. urealyticum (1.49 +/- 0.20, p = 0.002) or controls (1.31 +/- 0.19, p = 0.002). Leukocytospermia was detected in only 1 of the 17 (6%) positive specimens and 4 (12%) negative specimens. CONCLUSIONS: Seminal ROS levels are elevated among patients with U. urealyticum. ROS induces lipid peroxidation, which reduces membrane fluidity and sperm fertilization capability, and may be the mechanism by which U. urealyticum impairs sperm function. Absence of leukocytospermia does not exclude U. urealyticum.