Methods for the detection of male genital tract inflammation

Summary. Male genital tract inflammation is reflected by increased numbers of white blood cells (WBC) in semen. An ejaculate containing more than 10⁶ WBC ml⁻¹ semen is termed leukocytospermic. Among male infertility patients, the frequency of leukocytospermia is between 10% and 20%. By conventional light microscopy or sperm staining techniques, it is not possible to reliably differentiate WBC from immature germ cells in semen. In contrast, the cytochemical peroxidase method reliably identifies granulocytes, the most prevalent WBC type in semen. The method is cheap, fast and easy to perform. The gold standard for the detection of all WBC populations in semen is immunocytology using monoclonal antibodies. However, it is expensive and time-consuming, thus remaining a research tool at present. The measurement of granulocyte elastase in semen provides information on the number of granulocytes and their inflammatory activation. However, commercial granulocyte elastase enzyme immunoassays are expensive and due to logistical reasons often delay the results for more than 1 week. Leukocyte esterase dipstick tests lack both sensitivity and specificity for the detection of inflammatory changes in semen. For clinical purposes, the peroxidase method is ideally suited to detect inflammatory changes in semen.     

Nonsperm cells in human semen and their relationship with semen parameters

The prevalence and clinical significance of leukocytes (WBC) and immature germ cells in semen is currently a matter of controversy. The aim of this work was to assess the prevalence of leukocytospermia in semen samples from Venezuelan men and its possible effects on sperm parameters. The concentration of WBC and round cells (RC) was evaluated in 118 semen samples from 19 fertile subjects (group 1), 62 infertile patients (group II), and 37 men with varicocele (group III). Semen WBC concentration was assessed by peroxidase assay. Twenty-six (22%) of the total samples had more than 10 WBC/mL semen. Twenty of the infertile men had leukocytospermia (32%) compared with 16% in the fertile group and 8% in the varicocele group. Semen RC concentration was lower than 5 x 10(6)/mL in all groups but, in groups II and III was significantly higher compared with group I. Infertile men had the highest WBC concentration. WBC concentration was negatively correlated with progressive motility, percentage of morphologically normal sperm, and hypoosmotic swelling test in infertile men but not in the varicocele group. In this group a negative correlation was obtained between immature germ cells and normal sperm morphology. The data show that leukcytospermia occurs frequently in infertile patients and is associated with poor semen quality parameters. In contrast, in men with varicocele, the increased number of immature germ cells might play a pivotal role in the pathogenesis of abnormal spermatozoa.     

Sperm morphology and male urogenital infections

Summary. The aim of this study was to investigate the influence of urogenital infections as indicated by leukocytospermia on human sperm morphology, diagnosed cytologically and by means of a leukocyte peroxidase test. A basic semen analysis, including a leukocyte peroxidase test, was prospectively performed on 150 consecutive semen samples. Cytology smears were microscopically investigated for the presence of WBC and the results expressed on a 4 point scale as ± to + + + WBCs/high power field (HPF). ROC curve analysis indicated that for cases with more than ± WBC/HPF the peroxidase determined WBC count cut-off value was >0.25 times 10⁶ WBC ml⁻¹ with a sensitivity of 75% and specificity of 90%. The presence of more than ±WBC/HPF was negatively correlated with sperm morphology characteristics studied. The mean (±SD) percentage of morphological normal spermatozoa was 7.0% (SD 4.4) in the WBC negative group ( n =134) compared to 4.3% (SD 3.5) in the WBC positive ( n =16) group ( P <0.0001). There was also an associated increase, 15.3% (SD 13.3) to 23.6% (SD 13.8), in the percentage of spermatozoa with elongated head forms in the WBC positive group ( P =0.0218). No other effect on sperm and acrosome morphology could be found. With the peroxidase determinations there was also a tendency in the WBC positive group ( n =10) towards poorer sperm morphology characteristics, but these changes were not statistically significant. The presence of urogenital infections as diagnosed cytologically was associated with statistically poorer sperm morphology characteristics. This statistical relationship was not found in the peroxidase diagnosed leukocytospermia positive groups.     

Effects of white blood cells on the in vitro penetration of zona-free hamster eggs by human spermatozoa

The presence of white blood cells in semen has been associated with male infertility. Previous studies indicate that pyospermia occurs in conjunction with decreases in sperm motility, number of normal sperm forms, and penetration rates in the zona-free hamster egg sperm penetration assay. We have evaluated the relationship of seminal white blood cells and sperm function, as reflected in the zona-free hamster egg penetration assay, and have investigated the possible mode of action of the white cells. Egg penetration rates decreased when white blood cells from fertile or potentially fertile donors were added to their sperm suspensions prior to preincubation and at insemination in the in vitro assay. Zona-free hamster egg penetration assay results were also inhibited when the supernatant from white blood cells incubated in Biggers, Whitten, and Whittingham (BWW) medium overnight were introduced to sperm-oocyte suspensions at insemination. Conversely, egg penetration rates were enhanced in samples from hypofertile individuals when white blood cell concentrations in the semen or WBC/sperm ratios were reduced, either by physical removal or as a result of antibiotic therapy. The physical presence of leukocytes, and possibly, the extracellular release of lysosomal enzymes may be responsible for the inhibitory effects in vitro. Although the mechanism(s) by which white blood cells interfere with the fertilizing capacity of spermatozoa are not clear, it is quite obvious that their presence in the in vitro environment is undesirable and can mask an individual's actual fertilizing potential.     

Increased levels of interferon-gamma in seminal plasma of infertile men

Summary. The role of the cell-mediated immunity in male infertility is still far from clear. Interferon-gamma (INF-γ), a secretory product of activated T cells and natural killer cells, has been hypothesized to have a toxic effect on sperm function. The presence of INF-γ was investigated in seminal plasma of fertile and infertile subjects, using a specific enzyme-linked immunosorbent assay, in order to study its role in male infertility. Forty-one subjects were studied; 20 had proven fertility and normal semen quality (fertile group) and 21 showed male infertility for at least 2 years and poor semen quality (infertile group). INF-γ was present in significantly higher levels in the seminal plasma of infertile subjects (6.36±0.72 fmol ml⁻¹) compared to fertile subjects (3.68±0.30 fmol ml⁻¹). Moreover, a significant negative correlation between INF-γ levels and sperm count, motility and morphology was detected, whereas no correlation between INF-γ levels and leukocyte count was found. These findings (i) confirm INF-γ to be present in seminal plasma; (ii) show increased INF-γ secretion in the infertile group; (iii) demonstrate negative correlations of INF-γ with the main spermiogram parameters and (iv) no correlation with leukocyte count. INF-γ may therefore play an important role in male infertility.
Seminal plasma—     

Progress in leukocytospermia research]

Leukocytospermia is a most common cause of male infertility, but the distribution, origin and role of leukocytes in semen are still controversial. Some reports on leukocytospermia have indicated its negative effects on semen parameters and even in vitro fertilization (IVF). Recent literature has made it clear that the most deleterious effect of leukocytospermia is that the increased reactive oxygen species (ROS) may cause sperm damage, leading to significantly increased male infertility. The treatment and prevention of leukocytospermia have been proven of help for improving semen parameters.     

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。     

Relationship between seminal white blood cell counts and oxidative stress in men treated at an infertility clinic.

In semen, granulocytes are major producers of reactive oxygen species (ROS), which can damage sperm. The diagnosis of leukocytospermia is usually based on the World Health Organization (WHO) definition of 1 x 10(6) white blood cells per milliliter, but controversy remains over the minimum leukocyte level that impairs fertility. The goals of this study were to clarity the relationship between leukocyte count and oxidative stress and to establish the minimum leukocyte count associated with oxidative stress. To do so, we compared oxidative stress in semen samples with different leukocyte counts (by the Endtz test) after a simple wash-and-resuspend procedure and determined the correlation between leukocyte counts and oxidative stress (expressed as ROS-TAC score, a composite score calculated from ROS levels and total antioxidant capacity (TAC), both measured with chemiluminescence assays). ROS-TAC decreases as oxidative stress rises. We compared specimens from 271 men attending an infertility clinic and 28 healthy controls. About 9% of patients had WHO-defined leukocytospermia and an additional 16% had some leukocytes. Samples with no seminal leukocytes had significantly lower ROS levels and significantly higher ROS-TAC scores than samples with any seminal leukocytes, even very low levels. Oxidative stress was correlated with rising white blood cell (WBC) count (r = .39; P < .001). Receiver operating characteristics curves showed that ROS-TAC score would be fairly accurate at distinguishing between patients with any leukocytes and those with no leukocytes (area under the curve, 75%). In conclusion, oxidative stress occurs even in patients with very low seminal WBC counts (between 0 and 1 x 10(6)/mL) and rises with an increase in WBC count. Therefore, we are unable to determine a safe minimum WBC count; the presence of any WBCs is associated with oxidative stress and may therefore impair fertility. Complete removal of WBCs from semen samples used for assisted reproduction may help reduce oxidative stress.     

Effects of clinical stage and immunological status on semen analysis results in human immunodeficiency virus type 1-seropositive men

Summary. Complete semen analyses including computer-assisted sperm motility and morphology assessments were performed to determine if semen and sperm differed between HIV-seropositive men and fertile controls, or differed with symptoms, or CD4+ peripheral cell count categories. Previous studies included small numbers of men and presented conflicting conclusions. Two hundred and fifty non-vasectomized HIV-seropositive men and 38 fertile controls each provided one semen sample. Non-parametric statistics were used to analyse both continuous and nominal data. Fertile men had significantly greater semen volume, sperm concentration, percent motility, percent rapid and linear motility and total strictly normal spermatozoa than HIV seropositive men. Neither total number nor subtypes of leukocytes in semen differed between the two groups. Among the HIV seropositive men, significant differences in semen analyses were found between CD4+ cell count, clinical, and AIDS categories. Lower CD4+ cell counts (<200 mm⁻³) were associated with significantly lower percent motility, percent normal sperm morphology by strict criteria, significantly more spermatids in semen, and higher percentages of teratozoospermia, oligoasthenoteratozoospermia and leukocytospermia. Healthier men, based on clinical categories, had significantly more normal shaped spermatozoa and fewer had azoospermia, oligoasthenoteratozoospermia or leukocytospermia. Many HIV-seropositive men have normal semen analyses, but as the disease progresses more defects are found, particularly in strict criteria sperm morphology.     

精液粘度与精液分析参数、解脲支原体感染和抗精子抗体关系分析

目的探讨精液粘度增高导致男性不育的机理。方法共4337例不育门诊就诊者,分为精液粘度增高和正常组,观察粘度与主要精液常规参数、UU感染率和AsAb阳性率关系。结果精液粘度增高率为65.02%。粘度增高组精子活动率、a,b级活力精子率显著低于粘度正常组(P<0.05,P<0.001);畸形精子率、液化时间均明显高于粘度正常组(P<0.001);两组精液量、精子密度和精液pH比较无显著性差异(P>0.05)。精液白细胞>5个/HP组粘度增高率明显高于白细胞<5个/HP组(P<0.001)。粘度增高组精浆AsAb阳性率和精液UU阳性率均明显高于粘度正常组(P<0.001)。结论精液粘度可影响精液参数,并与精液白细胞数、精浆AsAb和UU感染有关。     

Use of methenamine hippurate in male infertility.

In sperm-freezing for infertility patients with low sperm counts, we found that semen containing high numbers of white blood cells, greater than 15 to 20 per high-power field, was both poor in quality and difficult to freeze. In examining these patients carefully, we found that many had smoldering prostatitis and some an overt history of prostatitis. We treated them with methenamine hippurate using various methods and found that by decreasing the white blood cell count in their semen, we could frequently improve the semen quality, particularly sperm motility, also achieve pregnancies in some cases.     

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.     

Scrotal temperature and semen quality in men with and without varicocele

The exact role of varicocele in human male infertility remains controversial. Fifty-five male partners of infertile couples randomly selected and 17 fertile semen donors were evaluated for semen quality, scrotal temperature, and presence of varicocele using clinical palpation and Doppler ultrasound. The incidence of varicocele was 42% in male partners of infertile couples and 41% in fertile semen donors. Left scrotal temperature was significantly (p less than .001) higher in infertile males with varicocele as compared to all groups. No significant differences were observed in the percentage of morphologically normal sperm in semen of males with and without varicocele. However, the incidence of tapered, elongated, and immature sperm was significantly higher in the infertile patient population with a varicocele. Measurement of scrotal temperature and assessment of sperm morphology may be used as predictors of the presence and deleterious effect of varicocele.     

Effect of leukocytospermia on sperm DNA integrity: a negative effect in abnormal semen samples

Controversy exists over levels of DNA integrity in the sperm of fertile and infertile men. In addition, the effect of leukocytospermia on sperm DNA in these 2 groups is unclear. We decided to address these questions by collecting semen samples from men known or presumed to be fertile and men from infertile couples. Samples were analyzed and assessed for sperm concentration, motility, and morphology. Samples failing to meet World Health Organization (WHO) standards in one or more of these parameters were judged abnormal. Samples were then arbitrarily assigned normalized scores in each of the above parameters, and scores were summed to give a normalized value for overall sperm quality. DNA abnormality was determined by an in situ DNA denaturation test with acridine orange and expressed as a percentage of cells with abnormal DNA integrity (ADI). Assessment of 187 samples revealed a moderate inverse correlation between ADI and sperm quality (r =.58), although a large degree of ADI dispersion was observed in abnormal semen samples. The average ADI for normal and abnormal semen samples was 18% +/- 2.8% and 36% +/- 5.8%, respectively, with the threshold of 95% probability set at 30%. When sorted for leukocytospermia, the difference in ADI between normal and abnormal semen groups without leukocytospermia was much smaller (17% +/- 2.2% and 22% +/- 4.6%; P =.023). Leukocytospermia had no significant effect on ADI in the normal semen group (P = .46); however, ADI was more than double the ADI in the abnormal semen group (18% +/- 2.4% and 50% +/- 11%; P < .001). The results of our analysis show that at least 3 factors affect net DNA integrity in leukocytospermic samples that fail to meet WHO standards: 1) primary DNA damage, which is moderately inverse to sperm quality, in particular to sperm concentration; 2) effect of leukocytes increasing primary or provoking potential DNA damage in a cascade-like manner, particularly in sperm with poor morphology and motility; and 3) a decreasing proportion of cells with damaged DNA in semen with the worst quality.     

Apoptosis and kinematics of ejaculated spermatozoa in patients with varicocele

Increased DNA fragmentation is found in sperm from infertile men. Varicocele is an important cause of male infertility, even though it is present in 15% of men who father children. Semen analysis does not always identify infertility in these patients. Sperm motility is strongly correlated with male fertility potential. The goal of this study was to determine the correlation between apoptosis and kinematics in the ejaculated spermatozoa of patients affected by varicocele. Fresh semen samples were obtained from 30 patients with varicocele and 15 fertile controls. These samples were compared using computer-assisted semen analysis and were assayed to determine the degree of sperm apoptosis. The apoptotic index (AI) was calculated by dividing the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate nick end labeling (TUNEL) stained spermatozoa by the total number of Hoechst 33258-stained sperm cells for 300 sperm. Five microscopic fields were analyzed to obtain 5 AIs for each individual. Results demonstrated no significant difference in semen quality and sperm motion characteristics; however, a significantly higher AI (23.05% +/- 4.07%: mean difference +/- SE, 95% CI, 15.06%-31.03%, P <.0001) was identified in the varicocele group than in the fertile controls. We concluded that sperm apoptosis does not seem to correlate with semen quality and sperm kinematics and that apoptosis is increased in ejaculated spermatozoa in patients with varicocele compared to normal fertile men.     

Relationship between acrosin activity of human spermatozoa and oxidative stress

Aim: To study the association between seminal oxidative stress and human sperm acrosin activity. Methods: It is a prospective study consisting of 30 infertile men and 12 fertile normozoospermic volunteers. A full history, clinical examination and scrotal ultrasound were done to exclude other related factors such as smoking and varicocele. Presence of white blood cells (WBCs) in semen samples was evaluated by peroxidase staining. Lipid peroxidation in spermatozoa was induced after incubating with ferrous sulphate (4 mmol/L) and sodium ascorbate (20 mmol/L). Induced peroxidation of spermatozoa was assessed by determining the production of thiobarbituric acid reactive substances (TBARS). Acrosin activity was measured using the gelatinolysis technique. The halo diameters around the sperm heads and the percentages of spermatozoa showing halo formation were evaluated. An acrosin activity index was calculated by multiplying the halo diameter by the halo formation rate. Results: A significant difference was obse     

Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests

Varicocele occurs in approximately 15% to 20% of the general male population and it is the most common cause of poor semen production and decreased semen quality. It has been demonstrated that patients with varicocele have a significantly higher DNA fragmentation index (DFI) and spermatozoa with nuclear anomalies than healthy fertile men. Therefore, the aim of this study was to evaluate sperm chromatin integrity in these patients. Sixty men referring to the andrology laboratory were categorised into three different groups: 20 infertile men with varicocele, 20 infertile men with abnormal semen parameters and 20 fertile men who had normal spermatogram were considered as control group. Semen analysis was performed according to WHO criteria. To evaluate sperm chromatin quality and DNA integrity, after fixation of sperm smears, aniline blue, toluidine blue, chromomycin A₃ and acridine orange staining were applied in three groups. The slides were analysed by light and fluorescent microscopy and to determine the percentage of mature or immature spermatozoa, 200 spermatozoa were counted in each slide. The results showed that the rates of aniline blue-reacted spermatozoa were significantly higher in infertile and varicocele patients than in the normal group ( P  < 0.001). In addition, with regard to chromomycin A₃, acridine orange and toluidine blue staining, there was a significant difference between the three groups ( P  < 0.001). The results showed that the varicocele samples contain a higher proportion of spermatozoa with abnormal DNA and immature chromatin than those from fertile men as well as infertile men without varicocele. Therefore, varicocele results in the production of spermatozoa with less condensed chromatin and this is one of the possible causes of infertility due to varicocele.     

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.     

Male immunity to the chlamydial 60 kDa heat shock protein (HSP 60) – associated with semen quality?

The role of Chlamydia trachomatis for male infertility is a matter of constant debate. It is assumed that in its persistent form this pathogen may produce high levels of 60 kD heat shock protein (Chlam HSP60). Cross‐reactivity between epitopes of the bacterial and human HSPs, involved in many steps of the reproductive process, might induce an autoimmune response with potential impairment of semen quality and sperm fertilising capacity. This prospective study included asymptomatic males of a total of 128 unselected subfertile couples (median duration of infertility 3 years) to determine the clinical relevance of male immunity to Chlam HSP60 during infertility investigation. After medical history and clinical examination of both partners, serum antibodies (Ab) to Chlam HSP60 were determined. Same day semen quality evaluation included microscopical standard sperm analysis, determination of seminal white blood cells (WBC) and of antisperm Ab (ASA) of the Ig G‐ and Ig‐A class (mixed antiglobulin reaction, MAR), microbial screening and examination of sperm functional capacity. Sperm/mucus interaction was tested in vitro and in vivo. Simultaneously, patients′ female partners were tested for Chlam HSP60 Ab and results were compared with a standard serology evaluation for antichlamydial IgG Ab. The presence of ChlamHSP60 Ab (positive in 24% of males) was not significantly associated with semen quality, seminal WBC and antisperm AB of the IgG‐ or Ig A‐class, the outcome of the microbial screening nor with sperm functional capacity and results of sperm/mucus interaction testing in vitro and in vivo. Chlam HSP60 Ab were significantly more frequent in female partners of Chlam HSP60 Ab‐positive men, and results correlated with the outcome of standard chlamydial serology evaluation. In conclusion, when serum Chlam HSP60 Ab are used as marker, male immunity to the chlamydial 60 kD heat shock protein is not associated with semen quality, sperm functional capacity and other clinically relevant parameters of male fertility.     

Evaluation of the Sysmex UF‐1000i system as an alternative for the screening of genital tract inflammation in male infertility patients

The number of white blood cell (WBC) in semen is an important indicator of genital tract inflammation in male infertility. The peroxidase assay is the recommended reference method for seminal WBC counting. However, it is time‐consuming and may cause relatively heavy workload in daily routine. Meanwhile, the main component in the reagent of peroxidase test is harmful to human and the environment. In this study, we evaluated the analytical performance of the Sysmex UF‐1000i that is a urine flow cytometer as a screening tool for genital tract infection in male infertility patients through the counting of seminal WBC. We examined 143 semen samples and compared the results of UF‐1000i and manual microscopy. The intra‐assay variability, stability and linearity studies were performed. The intravariability (CV %) of seminal WBC count by Sysmex UF‐1000i was 2.34%–9.65%. The method of UF‐1000i displayed a good agreement with the reference assay of manual microscopy, and the r value for correlation of seminal WBC count between UF‐1000i and manual microscopy was over 0.999 (p < 0.001). The Sysmex UF‐1000i is capable of producing reliable seminal WBC count consistent with that obtained by manual microscopy. It is a suitable alternative to the manual microscopy, thus reduces the workload.