Evaluation of seminal oxidation–reduction potential in male infertility

The role of oxidative stress in male infertility has been broadly recognised, and the search for a new marker to determine the redox environment in semen has gained considerable interest. Oxidation–reduction potential (ORP) or redox potential, is a measure of the electron transfer from antioxidants to oxidants and provides information on the redox balance. In this review, the benefits of ORP as a new oxidative stress marker, the protocol for its evaluation and the importance of its measurement in the context of male infertility are discussed. In association with the standard semen analysis, seminal ORP has been analysed to evaluate semen quality and male fertility status. However, further studies are required to establish its use in assisted reproductive techniques (ART) practice.     

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.     

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.     

Effect of reactive oxygen species in semen on the pregnancy of infertile couples

Objective:   We intended retrospectively to investigate whether reactive oxygen species (ROS) levels, detected in whole semen, were correlated with the actual pregnancy rate.
Methods:   A total of 89 patients with data of ROS in semen, attending our male infertility clinics from April 1994 to June 2000, were evaluated. Semen parameters were determined with computer assisted semen analyzer (CASA) and ROS production levels were measured using a computer-driven LKB Wallac 1251 Luminometer after the addition of 40 µL of 4 mM luminol at the patients' first visits. All of the participants were inquired about their partners' pregnancies after the mean follow-up of 24.0 months (range 1.4 to 74.1). They were divided into two groups (pregnant group: n  = 41, non-pregnant group: n  = 48) and their characteristics, semen profiles and integrated ROS levels were analyzed.
Results:   There was no difference between the pregnancy rate of ROS detectable cases and negative cases. However, the mean integrated ROS level in detectable cases of the non-pregnant group was significantly higher than that in detectable cases of the pregnant group (115.61 ± 74.32 mV/30 min/10⁶ sperm versus 7.22 ± 4.69 mV/30 min/10⁶ sperm, P  = 0.0033). Then, by calculating the receiver operating characteristics curve with 95% confidence intervals, 4.35 mV/30 min/10⁶ sperm was considered as a cut-off value of ROS in semen for pregnancy.
Conclusion:   These results indicate that (i) highly detectable ROS in whole semen of infertile patients may have implications in their partners' pregnancies and that (ii) detection of ROS in whole semen has a prognostic value for idiopathic male infertility.     

Effect of levofloxacin treatment on semen hyperviscosity in chronic bacterial prostatitis patients

Changes in seminal fluid viscosity (SFV), reactive oxygen species (ROS) production, cytokines and seminal leucocyte concentration related to microbiological outcome in patients with chronic bacterial prostatitis (CBP) were studied. One hundred and ten infertile patients with CBP (positive sperm culture ≥10⁵ colony‐forming units [CFU] ml^?1, pathogens or Chlamydia in expressed prostatic secretions) were treated with levofloxacin 500 mg daily for 14 consecutive days per month for 3 months. In case of bacterial prostatitis, two conditions were examined: responders, eradication of 0 to <10³ CFU ml^?1 (n = 78) and poor responders, >10³ to <10⁵ CFU ml^?1 (n = 32). Compared with poor responders, responders showed a significant increase of sperm progressive motility and a significant decrease in seminal leucocyte count, SFV, liquefaction time, ROS production (in all fractions and conditions), seminal tumour necrosis factor‐α and interleukin 6. None of these variables showed significant differences compared with a control group of 37 fertile men. On the other hand, the poor responders showed significant changes in these variables compared with matched pretreatment values. In patients with CBP, antibiotic therapy alone leads to eradication in ≈71%, with improvement of sperm progressive motility, SFV and the framework of prooxidative factors. However, in the remaining ≈29% with poor antibiotic responsiveness, a deterioration of all variables is observed.     

CD 45/67 immunobead preparation of human semen activates granulocytes

Summary. In human semen reactive oxygen species (ROS) produced by spermatozoa or leukocytes can impair spermatozoa functions. The objective of this study was to assess the effects of CD 45- and/or CD 67 immunobead preparation on the chemiluminescence (CL) of seminal plasma free ejaculate cells (=original cell suspension), as well as of the spermatozoa and leukocyte fractions. The original cell suspensions of 68 infertile and 8 fertile men were incubated with CD 45 or CD 67 immunobeads. After separation in a magnetic field the luminol chemiluminescence of the original cell suspensions, the spermatozoa and the leukocyte fractions were recorded on a luminometer. Spermatozoa fractions did not contain any leukocytes as no increase in CL-counts occurred after addition of FMLP. The number of peroxidase-positive cells (per 10⁷ spermatozoa) correlated with the CL of the original cell suspensions (r = 0.7; P < 0.0001) as well as the CL of the spermatozoa and the leukocyte fractions after CD 45 or CD 67-preparation (r = 0.64; P < 0.0001). The CL of the spermatozoa and of the leukocyte fractions after CD 45 immunobead incubation were significantly correlated (r = 0.91; P < 0.0001). According to these data contaminating leukocytes could be eliminated by immunobead preparation. However, incubation of original cell suspensions with CD 45 or CD 67 immunobeads stimulated leukocytes to release soluble products resulting in elevated CL signals both in the leukocyte and the spermatozoa fractions. These effects have to be taken into account when using immunobeads for the preparation of human semen.     

Semen quality and infertility status can be identified through measures of oxidation–reduction potential

Standard analyses for evaluating semen quality require technical expertise and are interpretive in nature. Oxidative stress (OS) alters many of the semen parameters; thus, a measure of OS could be an indicator of semen quality. Static oxidation‐reduction potential (sORP) is a universal measure of OS traditionally used in environmental applications but is increasingly used in biomedical studies. sORP was measured to determine how well it associates with semen quality and if it differentiates semen from infertile patients and fertile donors. All study participants (Infertile, n = 365 and Fertile, n = 50) underwent standard semen analyses, and sORP was measured in unprocessed semen. In infertile patients, sORP increased with decreased total sperm number, motility and morphology. sORP values were higher in samples with abnormal quality (low number, motility and/or normal morphology) compared with those of normal quality. Infertile patients had higher sOPR values compared to fertile donors. A sORP cut‐off value of 1.38 mV/10⁶ sperm/ml can differentiate normal from abnormal semen samples, while a cut‐off value of 1.41 mV/10⁶ sperm/ml, can differentiate between infertile and fertile semen samples. In conclusion, sORP provides a quick and unbiased indicator of semen quality that can be a beneficial addition to semen analysis to determine semen quality and fertility status.     

Determination of seminal oxidation–reduction potential (ORP) as an easy and cost‐effective clinical marker of male infertility

Oxidative stress (OS) is an important contributing factor to male infertility. While previous methods to measure seminal OS are time‐consuming and limited to the use of freshly produced semen, oxidation reduction potential (ORP) is easier and quicker to perform and can also be used in frozen semen. Therefore, this study evaluated the clinical utility of ORP as a potential marker of male infertility. ORP was measured in semen samples from 293 patients and 15 fertile controls and categorised according to WHO criteria as normozoospermic, oligozoospermic, asthenozoospermic, teratozoospermic and oligoasthenoteratozoospermic. Receiver operating characteristic (ROC) curves were generated to differentiate these categories. Semen parameters were significantly different when subjects were grouped as control and patients or between the patient and normozoospermic group for concentration and morphology. ORP levels were significantly different between the control and normozoospermic group. When subjects were grouped based on concentration, motility, morphology or a combination of these, the area under the ROC curve, sensitivity, specificity, positive predictive value and cut‐off values were significantly different. These differences were significant when combined with ORP and grouped with any two sperm abnormalities. In conclusion, ORP is a quick, easy, cost‐effective and reliable marker of semen quality as well as oxidative stress for use in a clinical setting.     

Reassessment of the accuracy of traditional sperm characteristics and adenosine triphosphate (ATP) in estimating the fertilizing potential of human semen in vivo

Receiver operating characteristic curves and accuracy parameters were computed for traditional sperm characteristics (concentration, motility, morphology) and the number of peroxidase negative cells, and the concentration of adenosine triphosphate (ATP) in semen from populations of fertile and infertile men, and men who achieved a pregnancy after varicocele treatment. The percentage and concentration per millilitre of spermatozoa with rapid linear progressive motility, and the ATP concentration, provided the best discrimination between fertile and treated fertile from infertile men. The misclassification rate was higher for sperm morphology, total progressive motility and viability, whereas sperm concentration and the total sperm count per ejaculate had the worst discriminating power. The number of peroxidase negative cells per 100 spermatozoa was highly specific in identifying men who achieved pregnancy after varicocele treatment. The lower limit of normality of sperm characteristics was remarkably different between fertile men and men achieving pregnancy after treatment or during infertility work-up.     

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.     

Evaluation of reference values of standard semen parameters in fertile Egyptian men

The reference values of human semen, published in the WHO's latest edition in 2010, were lower than those previously reported. The objective of this study was to evaluate reference values of standard semen parameters in fertile Egyptian men. This cross‐sectional study included 240 fertile men. Men were considered fertile when their wives had recent spontaneous pregnancies with time to pregnancy (TTP) ≤12 months. The mean age of fertile men was 33.8 ± 0.5 years (range 20–55 years). The 5th percentiles (95% confidence interval) of macroscopic semen parameters were 1.5 ml for volume and 7.2 for pH. The 5th percentiles of microscopic parameters were 15 million/ml for sperm concentration, 30 million per ejaculate for total sperm count, 50% for total motility, 40% for progressive motility, 62% for vitality, 4% for normal sperm forms and 0.1 million/ml for seminal leucocyte counts. In conclusion , fertile Egyptian men had higher reference values of sperm total motility, progressive motility and vitality, and lower reference values for total sperm counts as compared to those determined by the latest edition of the WHO laboratory manual in 2010. Other semen parameters were identical to those defined by the WHO 2010 manual.     

Semen analysis： a new manual and its application to the understanding of semen and its pathology

This article reviews the latest edition of the World Health Organization＇s manual on seme instructional guide. The methodology used in the assessment of the usual variables in semen many of the less common, but very valuable, sperm function tests. Seminal fluid preparatio such as in vitro fertilization and intrauterine insemination are also outlined in the manual. useful techniques for the assessment of seminal fluid. It will be a very useful manual for an analyses of seminal fluid.     

Seminal oxidation–reduction potential levels are not influenced by the presence of leucocytospermia

Oxidative stress (OS) is characterised by an excessive amount of reactive oxygen species (ROS) which negatively affect sperm functions. In this study, the influence of leucocytes on seminal oxidation–reduction potential (ORP) and sperm DNA fragmentation (SDF) was investigated in 1,068 men. Seminal leucocyte concentration did not correlate with SDF, unadjusted ORP, ORP normalised for sperm concentration (sORP), ORP normalised for total motile sperm concentration (motORP) or total motile sperm count (TMSC-ORP). Although receiver operator characteristic (ROC) curve analyses show that leucocytospermia does not predict high sORP values (>1.34 mV/10⁶ spermatozoa/ml), the motORP (AUC: 0.666) and TMSC-ORP (AUC: 0.683) predict the rate of leucocytospermia significantly (p = .0195 and p = .0085 respectively). Moreover, SDF can significantly predict leucocytospermia (AUC: 0.679; p = .011) and vice versa (AUC: 0.657, p = .0298). Our data confirm the association between OS and SDF. In conclusion, motORP and TMSC-ORP may be better predictive factors of leucocytospermia, probably because sperm motility, included in motORP and TMSC-ORP calculation, is the first seminal parameter to be affected by OS. Although all these parameters are indicative of OS, ORP values, SDF and leucocytospermia should be considered independently for the evaluation of redox seminal status, as they probe distinct seminal features.     

Diagnostic value of routine semen analysis in clinical andrology

Infertility is a major health issue affecting over 48.5 million couples around the world, with the male factor accounting for about 50% of the cases. The conventional semen analysis recommended by the World Health Organization (WHO) is the cornerstone in the evaluation of male fertility status. It includes macroscopic and microscopic evaluation of the ejaculate, which reflects the production of spermatozoa in the testes, the patency of the duct system and the glandular secretory activity. Evaluation of seminal fructose, sperm vitality and leucocytes (Endtz test) are useful adjuncts to semen analysis that provide information on specific clinical conditions. Though several computer-assisted sperm analysis (CASA) systems have been developed, conventional methods for semen analysis are still widely accepted in clinical practice. This review summarises the conventional techniques used in routine semen analysis and their diagnostic value in clinical andrology.     

Free radical scavenger effect of rebamipide in sperm processing and cryopreservation

Aim: To study the effect of rebamipide added to semen samples and cryoprotectant on reactive oxygen species (ROS) production. Methods: Semen samples from 30 fertile and healthy volunteers were collected by masturbation after 2 days - 3 days of abstinence. After liquefaction, the specimens were diluted with sperm wash media to a uniform density of 20×106/mL. Rebamipide was added to semen samples and cryoprotectant to a final concentration of 10 μmol/L, 30 μmol/L, 100 μmol/L or 300 μmol/L. Specimens were incubated at 37 ℃ in a 0.5 % CO2 incubator for 1 h or cryopreserved at -196 ℃ LN2 for 3 days. The sperm motility and viability and the levels of ROS and lipid peroxidation of sperm membrance were assessed before and after incubation and cryopreservation by means of computer assisted semen analyzer, eosin-nigrosin stain, chemiluminescence and thiobarbituric acid assay, respectively. Results: The sperm motility was significantly increased after incubation with 100 μmol/L and 300 μmol/L rebamipide (P     

2343例男科就诊患者精子动态参数分析

目的探讨精子动态参数在评价男性生育能力方面的应用价值。方法严格按照WHO技术规范，用计算机辅助精液分析（CASA）系统对本院2343例男科门诊就诊的患者精液进行常规指标和精子动态参数分析。结果①正常精液组和异常精液组各项精子动态参数比较，均存在统计学差异；②随着精子存活率的下降，曲线速度（VCL）、直线速度（VSL）、平均路径速度（VAP）、平均移动角度（MAD）、侧摆幅度（ALH）、前向性（STR）下降明显，鞭打频率（BCF）呈上升趋势，而直线性（LIN）、摆动性（WOB）差异无显著性；③与密度活力正常组比较：少精组除LIN、STR有所增加，MAD、BCF有所下降，差别有统计学意义外，其余五项参数差别无统计学意义；弱精组除MAD无统计学差异外，其余八项参数均有统计学差异；少弱精组除MAD和ALH无统计学差异外，其余七项参数均有统计学差异。结论精子动态参数是评估精液质量的重要指标，为男性不育的诊断、治疗和研究提供了充分的依据。     

The effectiveness of Korean herbal medicine in infertile men with poor semen quality: A prospective observational pilot study

We examined the effects and safety of Korean herbal medicine (MYOMI‐14) to treat infertile men with low semen quality. This study included 17 patients who received MYOMI‐14 for 10 weeks. The primary outcomes were the mean differences between sperm test values. The secondary outcomes were changes in the Fertility Quality of Life and the difference in skin temperature between the thigh and scrotum. Adverse events were also monitored. The average values of sperm concentration, sperm progressive motility and total motile sperm count significantly improved after MYOMI‐14 treatment (36.2%, 51.7% and 55.5%, respectively; p < 0.05). The core Fertility Quality of Life and the difference in skin temperature between the thigh and scrotum did not change significantly. No adverse events were observed. MYOMI‐14 improved the semen quality of infertile men without adverse effects. Additional studies in a larger population and longer prospective randomised clinical trials are needed to confirm these results.     

Sperm separation in patients with urogenital infections

Summary. A total of 196 patients attending the Center of Dermatology and Andrology, Giessen, Germany, were examined for fertility problems. Polymorphonuclear elastase, reactive oxygen species (ROS) and the number of round and peroxidase-positive cells were investigated in addition to routine semen analysis. The ejaculates were also analysed before and after sperm separation by means of swim-up or glass wool filtration. In 20 cases of leukocytospermia, sperm concentration, motility, viability, production of reactive oxygen species, and the number of peroxidase-positive cells were evaluated before and after glass wool filtration. The results show that ROS production by viable spermatozoa is highly correlated with the concentration of PMN elastase and the number of both peroxidase-positive and round cells. Multiple regression analysis with motility as dependent parameter showed the number of round cells ( n = 91; r = -0.332; P = 0.0030) to be the most important parameter affecting motility, while ROS mainly affects the viability of spermatozoa ( n = 69; r = 0.250; P = 0.0107). In the case of leukocytospermia, glass wool filtration significantly reduced the number of peroxidase-positive cells and ROS production ( P = 0.0098 and P = 0.0005, respectively). Receiver operating characteristic (ROC) curve analysis for ROS production in the ejaculate using a concentration of 1.000 ng ml⁻¹ PMN elastase as decisive parameter resulted in a cut-off value of 49,489.9 counts 10⁻⁷ viable spermatozoa. The statistical parameters were: Sensitivity: 63.2%, specificity: 100%, positive predictive value: 100%, negative predictive value: 36.1%.     

Detection of DNA fragmentation in human spermatozoa: correlation with semen parameters

To determine the prevalence of high levels of sperm DNA damage among infertile men with normal and abnormal semen parameters, 90 patients were subdivided into the following three groups. Group A ( n  = 30): men with normal semen parameters who acted as the controls. Group B ( n  = 30): asthenozoospermic men and group C ( n  = 30): teratozoospermic men, suffering from male infertility. DNA damage was evaluated by the rate of DNA fragmentation index (DFI) as assessed by the terminal desoxynucleotidyl transferase-mediated dUTP nick-end labelling assay. It was found that the difference was not significant between the percentage of DFI in patients with asthenozoospermia and the normospermic men (9.46% ± 8.68 and 8.19 ± 6.84 respectively, P- value not significant). The patients with teratozoospermia showed a significantly higher percentage of DNA fragmentation compared with the controls (respectively 21.37 ± 17.26% and 8.19 ± 6.84%, P  < 0.001). There was a positive correlation between abnormal sperm morphology and the DFI ( r  = 0.44, P  < 0.01) in group C. It is concluded that the impairments of sperm parameters were associated with an increase of DNA fragmentation; this association was strictly related to atypical forms.     

白细胞精子症的研究进展

白细胞精子症是男性不育的一个重要原因,目前对精液白细胞的分布、来源和作用尚完全不清楚。精液中增多的白细胞及其产物可对精子功能和精液质量产生影响,其中活性氧(ROS)的增加与男性不育关系密切。白细胞精子症的治疗和预防对改善精液质量有一定作用。