Interaction between leucocytes and human spermatozoa influencing reactive oxygen intermediates release

The relationship between the presence of white blood cells (WBCs) and the fertilizing potential of human semen is still an open question. It is well known that the presence of leucocytes in human semen can be related to the production of reactive oxygen intermediates (ROI). Semen samples were obtained from 15 normozoospermic men and leucocytes were isolated from heparinized blood drawn from 15 volunteers. Lucigenin and luminol-mediated chemiluminescence assays were used to determine reactive oxygen species (ROS) generation by non-activated or activated leucocytes through 12-myristate-13-acetate or N-formyl-methionyl-leucyl-phenyalanine (FMLP) before the addition of spermatozoa isolated by swim-up or Percoll procedures. All spermatozoal fractions used in this study were characterized by defining their motility, morphology and viability. The levels of ROS formation by non-activated as well as stimulated leucocytes were significantly decreased after addition of swim-up separated spermatozoa (p < 0.01). The ability to inhibit the basal chemiluminescence was of lower degree for spermatozoa isolated from 90% Percoll fractions than for swim-up sperm. However, addition of sperm cells from 47% Percoll fraction was found to increase both lucigenin and luminol signals. Moreover, the determined ROI levels changed depending on the type of inducing factor used for oxidative burst. Then, spermatozoa selected by swim-up procedure although with only slightly higher viability and morphology than sperm obtained from 90% Percoll fraction clearly exhibited much higher capacity to inhibit ROI secretion by receptor-stimulated leucocytes (FMLP-activation) than Percoll fractionated sperm. Such results may indicate that within normal semen may exist sperm subpopulations with different biochemical mechanisms controlling the interaction between spermatozoa and contaminating leucocytes. When ROI levels contained in normozoospermic semen are dependent on the WBCs activation, it seems that spermatozoa with preserved normal functional competence are able to defend themselves against leucocytes-derived ROI. Also for normozoospermic ejaculates, swim-up sperm may improve semen antioxidant characteristics when comparing with Percoll (90%) separated sperm. It may help for optimal sperm preparation when assisting to infertility treatment.     

精子凋亡率与精子密度、活率、活力及活性氧关系探讨

目的 探讨精子凋亡率与精子密度、活率、活力与精浆活性氧关系.方法 分析85例男性不育患者精液样本,采用计算机辅助精液分析进行精液参数检测,采用Annexin V/PI染色法检测精子凋亡率,采用TBA法测定精浆活性氧.结果 精子凋亡率与精子密度之间有显著负相关性(P＜0.01);与精子活率之间无显著相关性(P＞0.05):与精子活力之间有显著负相关性(P＜0.05);与精浆活性氧之间有显著正相关性(P＜0.05).结论 精子捌亡率与精子密度和活力有相关性,精子凋亡率增加可能是少、弱精子症患者不育的原因之一,精浆活性氧升高可能是精子凋亡率增加的原因之一.     

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.     

Analysis of the relationship between reactive oxygen species production and leucocyte infiltration in fractions of human semen separated on Percoll gradients

We have shown previously that the reactive oxygen species generated by washed human ejaculates originate from cells which can be isolated in the low density region of discontinuous Percoll gradients. In this study we have used a simplified two-step (40/80%) Percoll gradient to separate human ejaculates (n = 109) into two populations of spermatozoa, exhibiting either a high or a low capacity for reactive oxygen species generation. We have then examined the relationships between this activity and other properties of the isolated fractions, with particular emphasis on the presence of leucocytes, which we have quantified using a monoclonal antibody directed against the common leucocyte antigen. The low-density cells recovered from the 40%/80% interface of the Percoll gradients, differed from the high-density fraction in exhibiting significantly reduced sperm motility, poorer sperm morphology, and a considerably enhanced capacity for reactive oxygen species production (P less than 0.001). In six cases the elevated levels of reactive oxygen species generation were associated with leucocyte concentrations in excess of 1 x 10(6) per 10(7) sperm, suggesting that leucocytes enter the seminal compartment in an activated, oxygen-radical generating, state. However, in the majority of cases exhibiting high levels of reactive oxygen species production, leucocyte numbers were low or absent and the semen profiles were unremarkable, except that seminal sperm concentrations tended to be low. These results suggest that the oxidative stress responsible for defective sperm function involves reactive oxygen species originating from two sources; the sperm and infiltrating leucocytes.     

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.     

Protection of melatonin against damage of sperm mito-chondrial function induced by reactive oxygen species

Aim: To study the mitochondrial function damage of sperm in-duced by reactive oxygen species (ROS) and the protection of melatonin (MLT) against the damage. Methods: Normal function spermatozoa were selected from semen samples by Percoll gradi-ent centrifugation technique. The ROS generated by the hypoxan-thine xanthine oxidase system was incubated with the normal sper-matozoa in the presence or absence of MLT (6 retool/L) for 30 and 60 minutes.     

Effects of prilocaine and articaine on human leucocytes and reactive oxygen species in vitro

BACKGROUND: The aim of this study was to investigate the ability of local anaesthetics to inhibit reactive oxygen and nitrogen species generated by either stimulated human leucocytes or cell-free systems using luminol chemiluminescence (CL). METHODS: Free radical generation was stimulated in leucocyte assay by formyl-methionyl-leucyl-phenylalanine (FMLP, 2 microM). In cell-free experiments, hydrogen peroxide (H2O2) 3.5 mM, sodium hypochloride 5 microM, ferrous sulphate (FeSO4) 40 nM, peroxynitrite 50 nM and xanthine 0.1 mmol l(-1) plus xanthine oxidase 0.25 U ml(-1) were used to produce H2O2, hypochlorous acid (HOCl), hydroxyl radical, peroxynitrite and superoxide-induced CL, respectively. RESULTS: Prilocaine inhibited FMLP-induced CL in leucocytes (94+/-1%, at 1 mM), whereas articaine showed an activation (59+/-7%) at high concentration (1 mM) and inhibition (13+/-6%) at low concentration (0.1 mM). In cell-free experiments, prilocaine (22+/-6%, at 1 mM) and articaine (85+/-1%, at 1 mM) caused concentration-dependent inhibition in xanthine-xanthine oxidase-induced CL. Although articaine had no effect on H2O2-induced CL, prilocaine significantly attenuated the H2O2 signal (97+/-0.3%, at 1 mM). Prilocaine (99+/-0.04%, 1 mM) and articaine (70+/-6%, 1 mM) markedly inhibited HOCl-induced CL, whereas these drugs had no effect on FeSO4-induced CL. Articaine inhibited peroxynitrite CL (63+/-6%, 1 mM), but prilocaine did not produce any depression on this signal. CONCLUSION: Prilocaine interacted with superoxide, HOCl and H2O2, whereas articaine reacted with superoxide, HOCl, and peroxynitrite. The direct scavenging properties of these drugs might be involved in the inhibition observed in leucocyte assay and could provide experimental support for investigating the potential benefit of using these local anaesthetics in patients presenting pathologies associated with free radical reactions.     

精子优选后冷冻保存对活性氧生成的影响

目的观察优选技术在精液冷冻保存中对常规精液参数、线粒体膜电位(△Ψm)和活性氧(ROS)的影响。方法分别用计算机辅助精子分析仪(CASA)、流式细胞仪(FCM)检测优选或未优选的精子在冷冻前后常规精液参数、△Ψm和精子细胞内ROS的变化。结果各冻融组精子活率、a级精子、(a+b)级精子、正常形态率均比冷冻前有不同程度降低,有显著性差异;而优选冻融组和优选加精浆冻融组间常规精液参数无显著性差异(P>0.05),但都明显高于原始冻融组(P<0.01)。原始精液冷冻后比冷冻前△Ψm下降(P<0.05),ROS显著上升(P<0.01)。优选冻融组和优选加精浆冻融组与冷冻前相比,以及组间无显著性差异(P>0.05);但与原始冻融组分别比较,△Ψm均显著提高(P<0.05),ROS均显著减少(P<0.01)。各冻融组△Ψm和ROS都有明显相关性,相关系数(r)依次为-0.528、-0.537、-0.606(P<0.001)。结论精液优选后冷冻可以在保持良好的△Ψm同时,减少ROS生成,因此建议临床可应用该技术冷冻保存精子。     

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.     

Effect of vitamin E on human sperm motility and lipid peroxidation in vitro

Aim: To assess the protective efficacy of vitamin E to counteract the reactive oxygen species (ROS) mediated damage onsperm motility, viability and lipid peroxidation. Melhods: Human semen samplns were obtained from the local hospi-tal. The split seminal fractions freed of seminal plasma v, ere reeonstimted in Ringer-Tymde and subjected to varied vita-min E concentrations (0.1-2 mmol/L), Results: Dose-dependent improvement in both motility and viability accom-panied by concomitant decrease in malondialdehyde (MDA an end product of lipid peroxidation) following vitamin Esuppllementation was noticed. Conclusion: Vitamin E protects against the ROS mediated damage on spermatozoa.Vimmth E supplementation could be of clinical importance for prolonged spermatozoal storage whenever needed.     

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.     

Infection and reactive oxygen species

Summary. In previous years the physiologic and pathophysiologic significance of reactive oxygen species (ROS) on sperm function has been recognized. The impact of ROS during the invasion, adhesion and multiplication of microorganisms in the male genital tract are largely unknown. However, it is known that the resulting activation of leukocytes leads to an increased generation of ROS. There is growing evidence that spermatozoa are protected from detrimental ROS effects by the powerful antioxidants in seminal plasma since disturbances of sperm function by ROS were demonstrated in the absence of seminal plasma, i.e., during epididymitis or after semen preparation. If seminal plasma is present, ROS generated by physiologic numbers of granulocytes (< 1 times 10⁶ ml⁻¹) apparently do not damage spermatozoa. Interestingly, ROS generated by leukocytes during male genital tract infections are critical for the techniques of semen preparation for assisted reproduction. These ROS impair sperm function if the protective effects of seminal plasma are not present. The relevance of ROS production by higher leukocyte numbers in human semen is presently unknown as is the relevance of ROS generated in the female reproductive tract.
In previous years the ambiguous role of reactive oxygen species (ROS) generation in human semen has been recognized. On one hand, ROS appear to be involved in physiologic reactions such as capacitation (De Lamirande & Gagnon, 1995; Griveau et al. , 1995a), while on the other hand, there is ample evidence that an increased ROS production impairs sperm functions and the fertilizing capacity of spermatozoa (Ochsendorf & Fuchs, 1993; Aitken, 1994; Griveau et al. , 1995b). The objective of this review is to summarize the relevance of reactive oxygen species during infections of the male genital tract.     

Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection

High seminal reactive oxygen species (ROS) are related to poor semen quality and impaired fertilization. We aimed at finding whether there is an association between ROS and fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). In prepared semen of 147 male partners of infertile couples, ROS were assessed with luminol chemiluminescence. Spermiogram was assessed in native semen. ROS were negatively correlated with standard sperm characteristics and testicular volume, and positively with abnormal sperm head morphology. Fertilization rate and embryo morphology on day 2 and on day 4 were assessed in 41 IVF and 106 ICSI cycles. The influence of maternal (female age and number of oocytes) and paternal (sperm motility, morphology and ROS) factors on fertilization and embryo quality were assessed by means of regression analyses. After IVF, fertilization and pregnancy rates were negatively associated with ROS level (p = 0.031 and 0.041, respectively). In case of higher ROS, significantly fewer ICSI-derived embryos (p = 0.036) reached the morula-blastocyst stage on day 4. High seminal ROS levels are associated with impaired sperm fertilizing ability and lower pregnancy rates after IVF. In ICSI, a negative association of ROS with embryo development to the blastocyst stage has been observed.     

Scavenging effect of N-acetyl-L-cysteine against reactive oxygen species in human semen: a possible therapeutic modality for male factor infertility?

Summary. A new approach to reduce the level of reactive oxygen species (ROS) in human semen by using N-acetyl-L-cysteine (NAG) was evaluated. Semen samples were incubated with or without NAC (1.0 nig ml⁻¹) at room temperature. The chemiluminescent signal of the oxidation of luminol was detected by means of an MTP reader after 0, 20, 40, 60 and 120 min, respectively, using 200 μM luminol. In addition, the dose-dependent action of NAC (0.1, 1.0 and 5.0 mg ml⁻¹) and the influence of NAC on functional sperm parameters (motility and acrosome reaction) were studied.
ROS levels decreased significantly after 20 min incubation with NAG. This reduction was greater in the high ROS group (>30 000 counts/10⁷ viable sperm at t = 0) than in the low ROS group (<30 000). In addition, a marked dose-dependence of NAC was observed. Concerning sperm function, total sperm motility improved after incubation with NAC, but no significant change was observed with respect to the acrosome reaction.
NAC (at concentrations of 1.0 mg ml⁻¹) significantly reduced ROS in human semen and showed the possibility of improving impaired sperm function. After further testing NAC might be useful for the treatment of male infertility patients.     

Urogenital inflammation: changes of leucocytes and ROS

The presence of excess leucocytes in the semen has been associated with male infertility. According to the WHO, concentrations of more than 106 leucocytes ml-1 are considered as leucocytospermia, indicating genital tract infections. Up to now, no consensus has been achieved on how leucocytes should be quantified in semen. Using the peroxidase staining and monoclonal antibodies to CD15, CD45 and CD68, we found significant differences between the detection methods. Only 47.4% of the semen samples that were assessed as leucocytospermic by CD45 were identified as such by peroxidase staining. The concentration of peroxidase-positive cells was significantly correlated with polymorphonuclear granulocyte (PMN) elastase (P < 0.0001). However, a negative correlation of peroxidase-positive cells with the sperm concentration was only found in oligozoospermic patients (P < 0.0001). Moreover, the slightly positive correlation with normal sperm morphology seems to be applicable only in cases of oligozoospermia. Significant negative correlation of the number of peroxidase-positive cells were found for both maximal inducible acrosome reaction (P = 0.0219) and the inducibility of acrosome reaction (P = 0.0370), indicating a rather deleterious effect of leucocytes on this important sperm function. Concerning the result in the in vitro fertilization programme, none of the examined parameters (PMN elastase, concentration of round cells and peroxidase-positive cells) showed a correlation with either fertilization or pregnancy. This result seems to be reasonable as severely damaged spermatozoa and leucocytes are eliminated from the ejaculate by different sperm separation methods. Interestingly, a significant negative correlation of the TUNEL assay as a measure of sperm DNA fragmentation was found only with pregnancy (P = 0.006) but not with fertilization. As DNA fragmentation can also be caused by ROS that are generated by leucocytes, this causality should not be neglected.     

皮肤光老化、活性氧簇与抗氧化剂

皮肤长期反复暴露于日光紫外线（UV）下可导致皮肤光老化.近年来研究表明活性氧簇（ROS）在UV致皮肤光老化过程中起重要作用.UV可在皮肤中诱生高浓度ROS,这些具有一个或多个未配对电子的原子或分子如不能被皮肤抗氧化系统及时清除则会和皮肤中的核酸、脂质、蛋白质等生物大分子发生化学反应,并影响相关细胞信号转导途径和基因表达,导致皮肤光老化发生,使用抗氧化剂清除ROS可能成为一种重要的光老化防治策略.     

The efficacy of antioxidants in sperm parameters and production of reactive oxygen species levels during the freeze-thaw process: A systematic review and meta-analysis

To investigate the impact of antioxidants in sperm parameters and reduction in reactive oxygen species production during the freeze-thaw process. PubMed, Scopus, Web of Science, Embase and Cochrane central library were systematically searched. Of the 1583 articles, 23 studies were selected for data extraction. Our results show that antioxidants improved sperm progressive motility (standardised mean difference (SMD) = 1; 95% CI: 0.62, 1.38; p < .001) and viability (SMD = 1.20; 95% CI: 0.50, 1.91; p = .001) and reduced sperm DNA fragmentation (SDF) and hydrogen peroxide (H₂O₂) production, but there was no significant improvement in total sperm motility after thawing. Acetyl-l-carnitine/l-carnitine, melatonin and catalase had a significant positive impact on progressive motility. The role of tempol and melatonin in improving viability was significant compared to other antioxidants. Moreover, a significant reduction in SDF was observed after addition of butylated hydroxytoluene, tempol and vitamin E. However, the prevention of H₂O₂ production was significant only after the addition of tempol. Our overall results displayed the positive impact of antioxidants on progressive sperm motility, viability and reduction in SDF and H₂O₂ production, but no significant impact of antioxidants on total sperm motility was seen during the freeze-thaw process.     

血小板内源性活性氧对血小板高敏性的调控机制

血小板高敏性对疾病的重要性越来越被学者们重视.本文着重讨论了血小板内源性活性氧对血小板高敏性的调控机制,以及该研究对相关药物的作用靶点、相关疾病的易感性及病因防治的参考作用.     

Clinical significance of reactive oxygen species in semen of infertile Indian men

Reactive oxygen species (ROS) levels in semen are believed to play both physiological and pathological roles in male fertility. The study was aimed to find the clinical significance of ROS levels in infertile Indian men. This pilot study included 33 idiopathic infertile men and 18 proven fertile controls. ROS levels in the washed sperm were measured using chemiluminescence assay and expressed as 10⁶ cpm per 20 million spermatozoa. Sperm count, percent sperm motility, and percent normal sperm morphology were found to be significantly ( P  < 0.0001) reduced in infertile men compared with the controls. Median (minimum, maximum range) ROS levels of the infertile group [24.90 (6.89, 44.71)] were found to be significantly ( P  < 0.0001) elevated compared with the fertile controls [0.167(0.15, 2.78)]. No significant correlation was seen between ROS levels and semen parameters. Elevated ROS levels in the idiopathic Indian infertile men may be one of the underlying reasons for impaired fertility. Therefore measurement of seminal ROS levels may be used in Indian infertile men for better understanding of the aetiology and selection of antioxidant regimen in the treatment of male infertility. However, large studies may be urgently warranted to find out the role of antioxidants in ROS elevated Indian infertile men through randomised, controlled clinical study.