Seminal fibronectin-like antigen and transferrin concentrations in infertile and fertile men

Summary.  Fibronectin like antigen (Fn) and transferrin (Trs) levels were measured in the seminal plasma of 40 fertile and 102 infertile men. The concentrations of both proteins were significantly ( P <0.001) higher in the fertile controls compared to the infertile groups. The levels of Fn and Trs (mean value ± SEM) in the fertile men were 857.9 ± 9.8 μg ml^-1 and 164.0 ± 6.5 μg ml^-1, respectively; in the azoospermic men ( n = 17) 552.7 ± 24.65 μg ml^-1 and 20.7 ± 2.19 μg ml^-1, respectively; in the group of severe oligozoospermia ( n = 35) 568.34 ± 25.7 μg ml^-1 and 31.1 ± 4.18 μg ml^-1, respectively; in the moderate oligozoospermic group ( n = 8) 572.50 ± 47.9 μg ml^-1 and 43.4 ± 15.4 μg ml^-1 respectively, and in the asthenozoospermic group ( n = 26) 512.76 ± 40.4 μg ml^-1 and 47.0 ± 7.9 μg ml^-1, respectively. Of special interest was the finding from a group of 16 normospermic men (partners of couples with unexplained infertility) who showed significantly lower levels of Fn like antigen, 632.5 ± 26.9 μg ml^-1 ( P <0.001) and Trs 41.8 ± 6.94 μg ml^-1 ( P <0.0001) compared to normals. No correlation was found between Fn levels with either Trs or FSH levels or sperm count. In conclusion, our results indicate that male infertility is associated with changes in seminal plasma Fn like antigen concentrations and that it can be possibly used as an index of sperm fertilizing capacity.     

Mast cells in the seminal plasma of infertile men as detected by flow cytometry

Increased numbers of mast cells (MCs) in the testis have been associated with testicular dysfunction, where accumulation of MCs occurs. Furthermore, it has been reported that MCs might affect sperm function as it has been demonstrated that MC-derived tryptase in the seminal fluid might reduce sperm motility. Although MCs have been detected in rat epididymis, only little is known about the presence of MCs in human seminal plasma. Thus, we analysed MC numbers in the ejaculate of men during routine semen analysis of male patients suspected for infertility ( n  = 100). MCs were detected by c-kit (CD117) expression using flow cytometry. Thereby, we detected significant numbers of MCs in the ejaculate of most patients (559 ± 525 MCs ml⁻¹, mean ± SD). However, we could neither detect a correlation with respect to MCs and sperm count, motility or morphology nor to the seminal inflammatory markers like polymorphonuclear elastase. Nevertheless, a significant correlation of MCs to spermatozoa-bound IgA ( r  = 0.5; P  = 0.03; n  = 21) was observed. It is concluded that significant numbers of MCs can be detected in the human ejaculate without necessarily influencing sperm function. A potential role of MCs in seminal plasma as well as the association between MCs and IgA on spermatozoa remains to be elucidated.     

精浆游离L-肉毒碱水平及其与精子密度、活动率及活力的相关性研究

目的:研究正常生育及不育男性精浆中游离L-肉毒碱水平差异及其与精子密度、活动率(a+b+c级精子百分率)及活力(a+b级精子百分率)之间的相关性,探讨精浆中游离L-肉毒碱水平对男性生育力的影响及其在不育症检查和治疗中的作用。方法:分别采用高效液相色谱法和计算机辅助精液分析系统,测定了230例不育症患者(精子密度正常117例,少精子症81例,无精子症32例)和30例正常生育男性精浆中游离L-肉毒碱水平及精子密度、活动率、活力等参数。根据检查结果对不育症患者分组后,以SPSS12.0软件包进行统计学分析,比较各组间游离L-肉毒碱水平的差异以及游离L-肉毒碱水平与精子密度、活动率、活力之间的相关性。结果:正常生育组精浆游离L-肉毒碱水平明显高于不育组(P<0.01)。精液中精子密度越低、活力越弱,这种差异性越显著。相关性分析结果显示,精浆游离L-肉毒碱水平与精子密度呈显著正相关关系(r=0.521,P<0.01),与精子活动率和活力之间也具有正相关关系(r=0.319,P<0.01;r=0.251,P<0.01)。结论:精浆L-肉毒碱水平与精子密度、活动率和活力之间密切相关,其含量测定作为一项有用的生化指标,可为男性不育症检查及临床诊治和进行有关男性生殖功能机制研究提供参考。     

Seminal leucocyte subpopulations and sperm function in fertile and infertile Chinese men

The level of seminal leucocytes and the prevalence of leucocytospermia was determined in a group of fertile and infertile southern Chinese men in Hong Kong. Sixteen normal fertile semen donors and 49 men with male factor infertility were studied prospectively. None had antisperm antibodies and past or present evidence of genital tract infection. Seminal leucocytes and their subsets were analysed using monoclonal antibodies and an immunocytochemical alkaline phosphatase-anti-alkaline phosphatase conjugate technique. Seminal leucocytes were detectable in 94% and 86% of the fertile and infertile men respectively, with the predominant subset being granulocytes. Leucocytospermia (> 1 × 10⁶ leucocytes/ml) was found in only one of the 49 (2%) infertile men without clinical evidence of genito-urinary infection. Inverse correlations were observed between (1) the percentage of spermatozoa with normal morphology and the number of T-helper/inducer cells, (2) the linearity of sperm movement and the number of T-lymphocytes. In conclusion, the level of seminal leucocytes and the prevalence of leucocytospermia is low in infertile Chinese subjects. The effect of seminal leucocytes on sperm function in these subjects needs further evaluation.     

Assessment of seminal plasma laminin in fertile and infertile men

Aim： To assess laminin levels in the seminal plasma of infertile and fertile men, and to analyze the correlation of laminin levels with sperm count, age, sperm motility and semen volume. Methods： One hundred and twenty-five recruited men were equally divided into five groups according to their sperm concentration and clinical examination： fertile normozoospermia, oligoasthenozoospermia, non-obstructive azoospermia （NOA）, obstructive azoospermia （OA） and congenital bilateral absent vas deferens （CBAVD）. The patients＇ medical history was investigated and patients underwent clinical examination, conventional semen analysis and estimation of seminal plasma laminin by radioimmunoassay. Results： Seminal plasma laminin levels of successive groups were： 2.82 ± 0.62, 2.49 ± 0.44, 1.77 ± 0.56, 1.72 ± 0.76, 1.35 ± 0.63 U/mL, respectively. The fertile normozoospermic group showed the highest concentration compared to all infertile groups with significant differences compared to azoospermic groups （P 〈 0.05）. Testicular contribution was estimated to be approximately one-third of the seminal laminin. Seminal plasma laminin demonstrated significant correlation with sperm concentration （r = 0.460, P 〈 0.001） and nonsignificant correlation with age （r = 0.021, P = 0.940）, sperm motility percentage （r = 0.142, P = 0.615） and semen volume （r = 0.035, P = 0.087）. Conelusion： Seminal plasma laminin is derived mostly from prostatic and testicular portions and minimally from the seminal vesicle and vas deferens. Estimating seminal laminin alone is not conclusive in diagnosing different cases of male infertility.     

Total anti-oxidant status: a biochemical predictor of human male fertility

The objective of the study was to assess whether seminal plasma total antioxidant status (TAS) can be used as a biochemical predictor of male fertility and its variation in different categories of infertile male subjects in our population. The study population consisted of 28 fertile and 127 infertile [teratozoospermic (30), asthenoteratozoospermic (30), azoospermic (21), oligoastheno-teratozoospermic (20), polyzoospermic (15) and oligozoospermic (11)] male subjects. Seminal plasma was separated by centrifugation and stored at minus 80 degree Celsius. Semen was analysed using computer-assisted semen analysis according to WHO criteria. Seminal plasma TAS was estimated by colorimetric method using Randox total antioxidant status kit. TAS of fertile male subjects was significantly (P < 0.001) higher than that of infertile patients. In whole studied population, seminal plasma TAS showed a significant positive correlation with sperm concentration (P < 0.001), sperm motility (P < 0.0001), and spermatozoa with normal morphology (P < 0.0001). In conclusion, this study suggests that TAS of seminal plasma is one of the important factors contributing to male infertility, and it can be used as a biochemical predictor for male fertility.     

Cadmium, lead, selenium, and zinc in semen of occupationally unexposed men

Summary. Concentrations of cadmium, lead, selenium, and zinc were determined in semen and seminal plasma of 22 volunteers by atomic absorption spectrometry. Additionally conventional semen parameters and, by means of computer videomicrography, motion parameters of spermatozoa were evaluated. Concentrations of Cd, Pb, and Zn determined in semen were not significantly different from those measured in seminal plasma. However, selenium levels were significantly higher in semen (53.8 ± 22.9 μg 1⁻¹) than in seminal plasma (40.4 ± 15.5 μg 1⁻¹, P <0.01). The investigated semen samples on average contained low levels of Cd (0.4 ± 0.23 μg 1⁻¹) and Pb (9.8 ± 6.5 μg 1⁻¹). Studies on the intra-individual variability revealed the following average coefficients of variation (%) for element concentrations: Pb (70), Cd (53), Se (27), and Zn (23); and for semen parameters: total sperm count (46), sperm concentration (37), motility (22), ejaculate volume (21), linearity (19), linear velocity (11), curvilinear velocity (10), and percentage of normally formed sperm (9). Significant positive correlations were detected between semen selenium levels and sperm concentration ( r =0.51, P <0.05), and percentage of normally formed sperm ( r =0.46, P <0.05), respectively. Sperm motility ( r =0.53, P <0.02), linear ( r = 0.76, P <0.001) and curvilinear velocity ( r = 0.64, P < 0.002) were significantly correlated with semen cadmium levels.     

不育男性精浆总抗氧化能力与精子运动功能的关系

目的:研究不育男性精浆总抗氧化能力(TAC)与精子运动能力和方式之间的关系,探讨精浆TAC水平在男性生育中的临床意义。方法:113例精子密度正常的不育男性,28例正常生育男性作为对照组。精液于37℃液化后采用计算机辅助精液分析(CASA)系统进行精液常规分析,采用比色法进行精浆TAC分析。结果:正常生育组精浆TAC为(19.82±6.33)U,不育男性精子密度正常组精浆TAC为(14.37±8.45)U,不育男性精子密度正常组与正常生育组比较存在显著性差异(P<0.01)。精浆TAC与a级精子百分率(r=0.208,P<0.05)和(a+b)级精子百分率(r=0.231,P<0.05)呈显著正相关,精浆TAC与精子运动参数中的前向性(r=0.200,P<0.05)、直线性(r=0.208,P<0.05)、曲线速度(r=0.189,P<0.05)、直线速度(r=0.210,P<0.05)、平均移动速度(r=0.215,P<0.05)及鞭打频率(r=-0.248,P<0.01)之间有显著的相关性,其中前向性、直线性、直线速度、曲线速度、平均移动速度与TAC呈正相关(P<0.05),而鞭打频率与TAC呈负相关(P<0.01)。精浆TAC与摆动性、侧摆幅度、平均移动角度之间无显著相关。结论:精浆中TAC水平与精子运动能力和运动方式密切相关,适宜的精浆TAC为精子运动提供了良好的外部环境,精浆中过低的TAC水平与精子运动能力下降和运动方式改变有关,可能是引起男性不育的病因之一。精浆中TAC分析可为探讨男性不育的发病机制以及临床用药提供依据。     

Relationship between sperm cell ubiquinone and seminal parameters in subjects with and without varicocele

Summary. In a previous paper it was demonstrated that Coenzyme Q10, a lipidic molecule with important antioxidant properties, is present at remarkable levels in human seminal fluid, and shows a direct correlation with seminal parameters (sperm count and motility). In patients with varicocele, on the contrary, correlation with sperm motility was lacking and a higher proportion of Coenzyme Q10 was found in seminal plasma. In the present study, the levels of Coenzyme Q10 in the cell pellet of spermatozoa, obtained after centrifugation of semen, were evaluated. In nonvaricocele subjects it was observed that a higher concentration of Coenzyme Q 10 (expressed as ng of the molecule per million of cells) was present in the spermatozoa of oligospermic and asthenospermic patients (sperm count <20*10⁶ spermatozoa ml⁻¹, sperm motility <40%). This relationship was not observed in varicocele subjects, who also showed slightly lower intracellular absolute values of the coenzyme.
Since Coenzyme Q10 is an antioxidant molecule involved in the defence of the cell from free radical damage, higher intracellular concentrations may represent a mechanism of protection of the spermatozoa. In varicocele patients, this mechanism could be deficient, leading to higher sensitivity to oxidative damage.     

A critical method of evaluating tests for male infertility

Considerable uncertainty surrounds the selection of test values that separate infertile from fertile men in the evaluation of male infertility. We herein describe an objective method of determining these values, referred to as threshold values, for different infertility tests. Using test results from fertile men threshold values were chosen such that 96 per cent of the semen samples from the fertile men were scored as fertile. These threshold values then were used to evaluate 100 semen samples from 74 men presenting for evaluation of infertility. Using this method we constructed infertility profiles on each of the 100 semen samples presented for infertility evaluation and found that the zona pellucida-free hamster egg penetration test (a measure of a spermatozoon's ability to undergo capacitation and penetrate an egg) identified 66 per cent of these samples as infertile, while multiple exposure photomicrography (a quantitative measure of sperm motility) identified 54 per cent of these samples as infertile. This compares with results from routine semen analyses using the same method, which identified none of the samples as infertile by sperm motility grade, 1 per cent by semen pH, 4 per cent by the percentage of motile sperm, 7 per cent by the total count of motile sperm, 10 per cent by the total sperm count, 11 per cent by the semen leukocyte concentration, 12 per cent by the concentration of motile sperm, 13 per cent by ejaculate volume, 16 per cent by sperm concentration and 27 per cent by sperm morphology. This method of analyzing infertility test results provides insight into the potential causes of male infertility and offers a critical approach towards understanding the complex problem of male fertility dysfunction.     

Melatonin hormone profile in infertile males

Melatonin is a hormone produced by the pineal gland. There is much controversy about its relationship to the male reproductive process. In this study, seminal plasma as well as the serum melatonin levels were studied in different infertile male groups and were correlated with their semen parameters and hormonal levels. One hundred twenty male cases subdivided into six equal groups were consecutively included; fertile normozoospermic men, oligoasthenozoospermia (OA), OA with leucocytospermia, OA with varicocele, non-obstructive azoospermia (NOA) with high serum follicle stimulating hormone (FSH) and NOA with normal FSH. Semen analysis, estimation of melatonin, FSH, testosterone (T) and prolactin (PRL) hormone was carried out. Mean level of serum melatonin was higher than its corresponding seminal concentrations in all investigated groups with a positive correlation between their levels (r = 0.532, p = 0.01). Serum and seminal plasma melatonin levels in all infertile groups were reduced significantly compared with their levels in the fertile group. The lowest concentrations were in OA with leucocytospermia group. Melatonin in both serum and semen demonstrated significant correlation with sperm motility (r = 607, 0.623 respectively, p = 0.01). Serum melatonin correlated positively with serum PRL (r = 0.611, p = 0.01). It may be concluded that melatonin may be involved in the modulation of reproductive neuroendocrine axis in male infertility. Also, low levels of melatonin in semen were observed in infertile groups having reduced sperm motility, leucocytospermia, varicocele and NOA.     

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.     

Modulation of insulin-like growth factor-1 in the seminal plasma of infertile men

It has been suggested that insulin-like growth factor (IGF)-1 plays an important role in the regulation of spermatogenesis in the testes. In the present study, concentrations of total IGF-1 were determined in the seminal plasma of fertile (n = 44), male-factor infertile (n = 34), and immunoinfertile (n = 10) men in order to investigate the role of IGF-1 in male infertility. Levels of IGF-1 were expressed both as nanograms per milliliter and as nanograms per milligram of protein. IGF-1 was detected in the seminal plasma of both fertile and infertile men. IGF-1 levels differed significantly between fertile and immunoinfertile groups (P < 0.035 to P < 0.0001), whether expressed as nanograms per milliliter or as nanograms per milligram of protein. The immunoinfertile group showed a 31.3-37.9% increase in the mean IGF-1 concentration over the fertile group. There was no statistical difference in the mean or median levels of IGF-1 between the fertile and male-factor infertile groups, whether expressed as nanograms per milliliter or as nanograms per milligram of protein. However, when the male-factor infertile subjects were divided into four subgroups based on which seminal parameter was defective, the subgroup having a low sperm count had IGF-1 levels that were significantly different from the fertile group, the immunoinfertile subgroup, and the other male-factor infertile subgroups. The low-sperm-count subgroup had the lowest mean and median IGF-1 levels of all the groups and subgroups tested. IGF-1 levels linearly correlated (r = 0.30-0.499) significantly (P = 0.023-0.027) with the total sperm count in the semen, whether analyzed with all groups together or in subgroups by condition. These findings suggest that IGF-1 has a role in fertility and that its derangement may be involved in male infertility, especially when mediated through low sperm count and immunologic factors.     

Seminal fibronectin in fertile and infertile males

This study aimed to assess seminal plasma fibronectin in fertile and infertile males. Ninety infertile males were investigated; asthenozoospermia (n = 27), asthenoteratozoospermia (n = 30), oligoasthenoteratozoospermia (n = 33) compared with 20 healthy fertile controls. They were subjected to semen analysis, seminal plasma fibronectin estimation by radial immune diffusion, serum testosterone (T) and follicle stimulating hormone (FSH) estimation by ELISA. There was significant increase of seminal plasma fibronectin among different infertile groups compared with the controls. Significant negative correlation was elicited between seminal fibronectin and sperm count, sperm motility grades A, B, A + B, sperm velocity, linear velocity, linearity index, sperm normal forms and serum T. Seminal fibronectin showed significant positive correlation with grade D sperm motility and serum FSH. ROC curve analysis discriminating controls and other infertile groups demonstrated criteria value of < 674 mg l(-1) (sensitivity 100% and specificity 96.4%). It is concluded that increased seminal fibronectin is associated with decreased sperm count and sperm motility.     

Leukocytospermia in male infertility patients in China

Summary. Recent studies have revealed a high prevalence of leukocytospermia (> 1 × 10⁶ white blood cells ml⁻¹ semen) in male infertility patients in the USA and certain European countries, and have implicated white blood cells as a cause of infertility. Since leukocytospermia may often be attributed to male genital-tract infections, its prevalence could vary widely in different populations depending on factors such as sexual practices and the prevalence of sexually transmitted pathogens. In the study described here the incidence of leukocytospermia was determined in a group of 101 male infertility patients and a small reference group of normal fertile men in Beijing, China. Seminal white blood cells (WBC) and WBC sub-populations were enumerated by peroxidase staining and immunohistological assay. Eight out of 101 (7.9%) samples from infertility patients and 0/10 samples from fertile donors were leukocytospermic. The incidence of leukocytospermia in the Chinese infertility patients was considerably lower than the 23% incidence observed in a recent study of infertility patients in the USA using a similar technique. All but one of the patients with leukocytospermia had a poor sperm count and/or poor sperm motility. However, due to the low incidence of leukocytospermia and the small number of patients in this group, a statistically significant association between leukocytospermia and poor semen quality was not attained. The simple peroxidase test correlated well with the more expensive and technically demanding immunohistological assay for detection of white blood cells in semen.     

METALLOTHIONEIN IN HUMAN SEMINAL PLASMA

Metallothionein (MT) concentrations were measured in the seminal plasma of 4 fertile and 35 infertile mbn and in the hydrocele and spermatocele fluids. The relationship between MT content and sperm density, total number of sperm per ejaculate, sperm motility and abnormal form rates, leukocyte count and zinc levels in seminal plasma, as well as the relationship between MT and serum follicle-stimulating hormone, luteinizing hormone, testosterone, and prolactin were examined. MT was not detected in the hydrocele and spermatocele fluids. MT levels were related to zinc levels and to the leukocyte count in seminal plasma, but there was no correlation between MT and the other factors examined. This study supported previous findings that MT was secreted predominantly from the prostate and induced by inflammation of the prostate gland or seminal vesicles; the findings suggest that MT binds mainly to zinc and is one of the zinc-binding proteins in seminal plasma.     

Comparison of zinc concentrations in blood and seminal plasma and the various sperm parameters between fertile and infertile men

The aim of the study was to examine the relationships between concentrations of zinc in blood and seminal plasma and sperm quality among infertile and fertile men. One hundred seven male (infertile group) partners of couples who were undergoing investigation for infertility with no known cause for the infertility and 103 men (fertile group) whose wives were pregnant at the time of the study were recruited. The subjects' blood and seminal plasma concentration of zinc were determined by atomic absorption spectroscopy. Except for semen volume, all the other semen parameters for the infertile men were significantly lower than those for the fertile group. The geometric means of the seminal plasma zinc concentration were significantly lower in the infertile group compared with those in the fertile group; 183.6 mg/L (range, 63-499) versus 274.6 mg/L (range, 55-420). There were no significant differences in the geometric means of the blood zinc concentration between the 2 groups. Seminal plasma zinc concentration was significantly correlated with sperm density (r = 0.341, P < .0001), motility (r = 0.253, P < .0001), and viability (r = 0.286, P < .0001). On the basis of the findings of this study and those of other reports, zinc may contribute to fertility through its positive effect on spermatogenesis.     

Correlation between neutral alpha-glucosidase activity and sperm DNA fragmentation

To evaluate the association between neutral α-glucosidase (NAG) activity and sperm DNA fragmentation (DFI), ejaculates from 24 men undergoing evaluation for sperm DNA damage as a part of infertility assessment were analysed. The mean ± SD and range for the semen quality of the 24 ejaculates are as follows: volume (3.1 ± 1.3, 1.8–6.0 ml); sperm concentration (45.6 ± 41.1, 1.3–151.2 × 10⁶ ml⁻¹); sperm motility (52.8 ± 28.8, 1–95%); sperm with fragmented DNA (17.6 ± 15.4, 1.7–56.0%); sperm with immature chromatin (9.6 ± 3.8, 2.5–19.1%); NAG activity (37.9 ± 18.3, 4.4–75.3 mU ml⁻¹). The only sperm parameter significantly correlated with neutral α-glucosidase is the percentage of sperm DFI [correlation coefficient ( r ) = 0.4376, P  = 0.03].     

Are oxidative stress markers associated with unexplained male infertility?

Male infertility can be responsible for up to 20% of the cases attending fertility consultation facilities; nonetheless, the underlying molecular mechanisms that could explain it are still elusive. Therefore, we aimed to evaluate conventional and functional parameters of semen samples from patients who presented with male infertility of unknown origin. Conventional semen parameters and functional parameters (i.e. intracellular reactive oxygen species production, mitochondrial membrane potential, sperm chromatin structure assay, sperm membrane lipid peroxidation and antioxidant capacity of seminal plasma) were evaluated on semen samples from 54 healthy donors, 23 patients with idiopathic infertility and 34 fertile controls. No significant differences were observed in the conventional seminal parameters between the fertile and infertile men. However, increased intracellular reactive oxygen species (ROS) production and DNA fragmentation were observed in the infertile patients compared to the fertile group. Alterations in intracellular ROS production and DNA fragmentation could be associated with male idiopathic infertility. These parameters could eventually distinguish both groups more accurately than the conventional parameters. Our current results are encouraging, and the efficacy of these parameters in the clinical settings needs to be further assessed to establish their predictive potential as a marker of unexplained male infertility.     

Development of the NBT assay as a marker of sperm oxidative stress

Oxidative stress is a well-established cause of male infertility, with reactive oxygen species (ROS) causing infertility principally by impairing sperm motility and DNA integrity. Currently, most clinics do not test their infertile patients for the presence of oxidative stress because the available tests are expensive or difficult to perform. As antioxidant therapy may improve sperm DNA integrity and pregnancy outcomes, it has become apparent that there is an unmet clinical need for an inexpensive and easy-to-perform assay to identify sperm oxidative stress. The aim of this study was to develop a standardized protocol for performing a photometric nitro blue tetrazolium (NBT) assay for the measurement of seminal ROS production via production of coloured formazan, whilst correlating these results with impaired sperm function (motility and DNA integrity). Semen samples from 21 fertile and 36 male aetiology infertile men were assessed for ROS production (NBT assay), sperm DNA integrity (TUNEL), apoptosis (Annexin V) and sperm motility. Infertile men's semen contained on average fourfold higher levels of ROS than fertile men. The production of ROS by sperm was positively correlated with sperm DNA fragmentation and apoptosis, whilst being negatively correlated with sperm motility. Receiver-operating characteristic plot analysis established a cut-off point of 24 μg formazan/10⁷ sperm having a sensitivity of 91.7% and a specificity of 81% for determining the fertility status of an individual. This study has been successful in establishing a standardized protocol for performing a photometric seminal NBT assay that has significant clinical utility in identifying men with impaired fertility because of oxidative stress.