Leukocyte detection in human semen using flow cytometry

This study set out to establish a new method, using flow cytometry, to evaluate leukocytes in semen. Ejaculates of 59 males, asymptomatic for genitourinary infections, were examined. Routine semen analyses were carried out as well as peroxidase and polymorphonuclear granulocyte-elastase detection. Leukocytes were detected combining flow cytometry and monoclonal antibodies (anti-CD45, anti-CD53). This technique reliably assessed the total number of leukocytes and differentiated subpopulations even at low concentrations. The peroxidase test and elastase determination showed good specificity, but only moderate sensitivity versus flow cytometry combined with monoclonal antibodies. No significant association was observed between semen parameters and leukocytospermia whether evaluated by conventional methods or flow cytometry except for a moderate correlation between spermatozoa and CD53-positive cell concentrations. A first comparison of data from patients grouped on the basis of leukocytospermia (>10(6) white blood cells, WBC/ml) or non-leukocytospermia revealed no significant differences in semen parameters; lowering the threshold value for leukocytospermia to 2x10(5) WBC/ml, sperm concentration was reduced in the group with a low number of WBC identified by monoclonal antibodies. Flow cytometry using monoclonal antibodies was seen to be a simple, reproducible method that enables leukocytes in semen to be accurately detected and to identify WBC subpopulations without preliminary purification procedures.     

DNA flow cytometry of human semen.

The aim of this study was the evaluation of DNA flow cytometry for the analysis of male infertility. 171 ejaculates from 155 patients with fertility problems were analysed by flow cytometry and by conventional microscopical procedures. Using flow cytometry, it was possible to determine the relative proportions of the various cell populations: mature haploid and abnormal diploid mature spermatozoa, cellular fragments, immature germ cells (haploid round spermatids, diploid cells, S phase and 4C cells), and of leukocytes as indicators of infection. A linear association was observed between sperm concentration in semen as quantified by light microscopy and by flow cytometry, even with fewer than 20x10(6) spermatozoa/ml. Eight classes of histograms, each with differing fractions of spermatozoa and other particles, were obtained and correlated with the results of the spermiograms. During the 10 year follow-up, the two patient groups with a low sperm concentration or a high concentration of cellular debris exhibited significantly impaired fertility. The two patient groups with >/=5% diploid spermatozoa and with malcondensed sperm chromatin were also subfertile. No ovulatory disorders were revealed in the 155 female partners. DNA flow cytometry thus provides an additional dimension to semen analysis not easily gained by other methods and has the advantage of being rapidly performed and interpreted. We therefore recommend application of this technique in the diagnosis of male infertility.     

Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters

BACKGROUND: Detection of apoptosis in sperm samples may help evaluate sperm quality. Recently, it has been suggested that in some ejaculated sperm populations, apoptosis is caspase dependent. The aim of this study was to investigate the presence of activated caspases and examine possible correlations with apoptosis and sperm parameters in semen samples prepared for IVF. METHODS: To detect activated caspases, neat semen from infertile patients and sperm prepared by PureSperm gradient were stained with the fluorescein isothyocyanate-Val-Ala-Asp-fluoromethylketone (FITC-VAD-fmk) and analysed by flow cytometry. Cell death was determined by DNA fragmentation (TUNEL) and mitochondrial membrane potential. Sperm parameters were studied by conventional microscopy. RESULTS: FITC-VAD-fmk stained sperm cells in situ and the subcellular labeling pattern was compatible with the known localization of caspases. A significant correlation was found between the frequency of FITC-VAD-fmk stained cells and cell death markers. In both prepared sperm and neat semen a negative correlation was found between the percentage of FITC-VAD-fmk positive cells and standard parameters (concentration/motility). FITC-VAD-fmk positive cells negatively correlated with high fertilization rates after IVF. CONCLUSIONS: Labelling of sperm cells with the activated caspases-reacting fluorochrome provides a sensitive assay for detection of sperm apoptosis. This cytometric assay can be helpful to test sperm before IVF.     

A new multiparameter flow cytometric method for human semen analysis

BACKGROUND: The objectives of this study were (i) to evaluate whether the combined use of Syto 16 and 7-amino-actinomycin-D (7-AAD) allows the detection of sperm apoptosis and (ii) to describe a new multiparameter flow cytometric method to assess simultaneously sperm concentration (SC), viability and apoptosis as well as leukocyte concentration. METHODS: Semen samples from 68 patients were evaluated according to World Health Organization (WHO) criteria (normal, n=26; abnormal, n=42). The detection of activated caspases before and after betulinic acid (BA) incubation was carried out in 13 semen samples by flow cytometry using fluorescein-labelled inhibitors of caspases (FLICA). A multiparameter flow cytometric analysis was performed in 55 semen samples. Fluorescent microspheres were used to assess SC. Sperm apoptosis was detected by staining sperm with Syto 16 and 7-AAD. Leukocytes were counted using monoclonal anti-CD45. RESULTS: A significant correlation between the percentage of the spermatozoa with low Syto 16 fluorescence and the percentage of spermatozoa containing activated caspases was found (r=0.68, P=0.0106; n=13). After incubation with BA, an increase of the percentage of apoptotic cells was observed in all samples, using both the Syto 16/7-AAD and the caspase activation methods. There was a good correlation between flow cytometry and optical microscopy for sperm (r=0.98, P < 0.0001) and leukocyte counting (r=0.64, P <0.0001). The percentage of apoptotic sperm was inversely correlated with both SC (r=-0.303, P=0.0246) and morphology (r=-0.384, P=0.0050) but not with motility. CONCLUSIONS: The combination of Syto 16/7-AAD provides a sensitive assay to detect sperm apoptosis. The multiparameter flow cytometric method described offers the possibility of a simultaneous, simple, rapid and accurate assessment of several semen parameters.     

Sperm ubiquitination positively correlates to normal morphology in human semen

BACKGROUND: Global ubiquitination in human semen has been found to negatively correlate with standard semen parameters, indicating that ubiquitination can be considered a marker of poor semen quality. However, the inclusion of all semen components in the analysis may be misleading on the biological significance of ubiquitination of sperm cells. We have recently demonstrated the variable presence of bodies of different size, with the highest concentration in oligoasthenoteratozoospermia. The purpose of the present study was to evaluate the relationship between ubiquitination and standard semen parameters, after distinguishing between ubiquitinated sperm and bodies in each sample. METHODS: Ubiquitination was evaluated by flow cytometric sperm ubiquitin tag immunoassay (SUTI) in sperm samples from 45 subjects. Semen analysis was performed according to WHO (1999) guidelines. RESULTS: When only ubiquitinated sperm were considered, a positive correlation with number, motility and normal morphology was found. When correlation was evaluated considering the percentage of ubiquitinated bodies, a negative correlation was found with good semen quality. CONCLUSIONS: Results indicate that the negative correlations previously found between global semen ubiquitination and parameters of semen analysis are mainly driven by components other than sperm. The positive correlation between sperm ubiquitination and good quality parameters suggests a previously unrecognized role for sperm ubiquitination.     

Apoptosis in human sperm: its correlation with semen quality and the presence of leukocytes

BACKGROUND: Apoptosis plays an important role in regulating spermatogenesis. However, the biological significance of apoptosis in ejaculated sperm is not yet clear. This study set out to investigate how apoptosis correlates with semen quality and the presence of seminal leukocytes. METHODS: Fifty-seven semen samples from the male partners of infertile couples were classified as normal or abnormal according to World Health Organization guidelines. Flow cytometry was used to evaluate sperm populations and seminal leukocytes. Preliminary flow cytometry analysis using 6-carboxyfluoresceindiacetate (6-CFDA), which identifies live cells, and propidium iodide (PI), which stains only dead cells, was performed in order to pinpoint the sperm region accurately. Having thus gated the sperm population, bivariate Annexin V/PI analysis was then carried out in order to measure the percentage of apoptotic and necrotic sperm and the apoptotic index (the ratio between apoptotic:live sperm). Leukocytes were counted by the standard peroxidase test and by flow cytometry using monoclonal antibody (mAb) anti-CD45 or anti-CD53. RESULTS: No significant differences in the apoptotic index and the percentage of live and apoptotic sperm were detected between the subjects with normal and abnormal semen. A significant inverse correlation between sperm concentration and the apoptotic index was observed only in the normal sperm group. There was no correlation between the concentration of leukocytes, detected either by peroxidase or by mAb anti-CD45 or anti-CD53, either with the percentage of apoptotic sperm or with the apoptotic index. In contrast, the ratio between CD45 positive leukocytes and sperm showed a significant correlation with the apoptotic index. A weaker correlation was found when leukocytes were counted by peroxidase, while no correlation was observed using mAb anti-CD53. CONCLUSIONS: Sperm apoptosis did not seem to be correlated with semen quality. In the absence of genito-urinary infection, one of the main functions of seminal leukocytes is probably to provide for the removal of apoptotic sperm.     

Nuclear staining identifies two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters

BACKGROUND: Sperm DNA fragmentation is a possible predictive parameter formale fertility status. The occurrence of M540 bodies in semenof subfertile subjects affects flow cytometric investigationsin sperm. We set up a new method to evaluate DNA fragmentationexcluding M540 bodies. METHODS: DNA fragmentation was evaluated by flow cytometry in semen of75 subjects both by terminal deoxynucleotidyl transferase-mediatedfluorescein-dUTP nick end labeling (TUNEL, traditional method)and by double staining with TUNEL and propidium iodide (PI,new method). RESULTS: The use of the new method revealed that TUNEL underestimatessperm DNA fragmentation in flow cytometry and showed two spermpopulations stained with low (PI^dim) and high (PI^br) avidityfor PI. The PI^dim population is entirely composed of DNA fragmentedsperm and its incidence shows highly significant negative correlationswith morphology, motility, sperm count and concentration (respectively,r = –0.51, –0.52, –0.46 and –0.32, n= 75). DNA fragmentation in the PI^br sperm population is independentfrom semen quality. CONCLUSIONS: The correlations between sperm DNA breakage and semen qualitypreviously reported are mainly driven by the occurrence of thePI^dim population. DNA fragmented sperm in this population aremore likely to have poorer morphology, reduced motility andthus a reduced chance to fertilize an oocyte than DNA damagedsperm in PI^br population. Distinguishing between the two typesof sperm DNA fragmentation appears to be important in clinicalinvestigations.     

Density gradient centrifugation and glass wool filtration of semen remove spermatozoa with damaged chromatin structure.

The ability of double-layered density gradient centrifugation (DGC) or glass wool filtration (GWF) of semen to remove spermatozoa with damaged chromatin structure was assessed by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility to sperm nuclear denaturation in situ. Ejaculates from 26 men attending a university-affiliated assisted reproduction laboratory were processed by DGC and GWF. Unprocessed, DGC- and GWF-processed specimens were assessed by the SCSA and by conventional semen parameters. Changes in chromatin structure were compared with conventional semen parameters. Both sperm preparation techniques yielded sperm suspensions with improved sperm chromatin structure as well as motility (%), forward progression (1-4) and viability (%). DGC was superior to GWF in the efficiency of recovering motile, morphologically normal, mature sperm suspensions. However, GWF produced improved chromatin integrity (SDalpha(t)) and viability. Moderate correlations between SCSA and conventional sperm parameters were observed. Nevertheless, the SCSA provides additional information about the biochemical integrity of sperm DNA and may be used in future studies to provide insight into assisted reproduction technology outcomes not explained by conventional sperm parameters.     

Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm

BACKGROUND: Sperm cell death appears to be a cause of male infertility. The objective of this study was to determine the most reliable method for the evaluation of sperm quality in semen samples during sperm preparation for IVF. METHODS: Conventional analysis of semen samples was compared with several cytofluorometric methods detecting death-associated changes. Neat semen from infertile patients and sperm prepared by PureSperm gradient were studied by conventional microscopy and analysed for mitochondrial membrane potential (Delta Psi(m)), generation of reactive oxygen species, DNA fragmentation and cell viability. RESULTS: In neat semen, a positive correlation was found between the percentage of Delta Psi(m)(high) sperm cells and standard semen parameters (concentration/motility). Sperm cells depicting Delta Psi(m)(high) and cells with low DNA fragmentation displayed high fertilization rate after IVF. The only changes that could be detected in prepared sperm were changes in Delta Psi(m), with Delta Psi(m)(high) sperm positively correlated with forward motility and also with high fertilization rates after IVF. CONCLUSION: Analysis of mitochondrial membrane potential is the most sensitive test by which to determine sperm quality. These findings promise development of a test that may help to predict successful IVF.     

Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception

To evaluate and optimize the sperm separation efficiency of a novel silane-coated silica bead (Puresperm), serial studies were carried out to compare the various sperm parameters between: (i) three-layer (90%-70%-40%) Puresperm and three-layer (90%-70%-40%) conventional polyvinylpyrrolidone (PVP)-coated silica bead (Percoll) gradients; (ii) three-layer (90%-70%-40%) and two-layer (90%-45%) Puresperm gradients and separately the same for Percoll; and (iii) large (3.0 ml) and small (0.75 ml) semen loading volumes on three-layer Puresperm gradients. Normozoospermic semen samples were treated and analysed in 12 replicates for each experiment. Manual evaluation of concentration, percentage motility, percentage vitality, percentage normal morphology; computer-assisted semen analysis evaluation of concentration, percentage motility, grade of motility, motion characteristics (curvilinear velocity, linearity, amplitude of lateral head velocity, beat cross frequency, percentage hyperactivation); and yields from the initial semen samples were compared. Percoll was found to be superior to Puresperm in concentration, percentage motility, percentage vitality and yields after three-layer density gradient centrifugation. There were no significant differences in sperm parameters between two- and three-layer Percoll gradients, but three-layer Puresperm gradients behaved significantly better than two-layer gradients. Large semen volume loads on three-layer Puresperm gradients resulted in greater sperm concentrations, percentage motility, percentage vitality and percentage normal morphology, but small semen volume loads produced greater yields of good-quality spermatozoa. In the light of Percoll being withdrawn from the shelf for the use of assisted reproduction because of the presence of PVP, three-layer Puresperm gradients with large semen loading volumes appear to be an attractive alternative for sperm separation in medically assisted conception.     

Does the potential for selection bias in semen quality studies depend on study design? Experience from a study conducted within an infertility clinic

BACKGROUND: The low participation rates in human semen quality studies raises concern for the potential of differential participation based on semen quality (or a surrogate). To explore the potential for differential participation, we compared semen analysis results from study subjects with those of non-study subjects. METHODS: We obtained semen analysis results from 235 study subjects and retrospectively obtained results from a subset of 235 infertility clinic patients that were not study subjects but met the same eligibility criteria. The study was conducted at the Massachusetts General Hospital Infertility Clinic. All semen samples (study subjects and non-study subjects) were analysed for sperm concentration and motility by computer-aided semen analysis (CASA), and morphology was assessed using strict criteria. Semen analysis parameters for the non-study subjects were compared with the semen analysis results from study subjects. RESULTS: For all semen characteristics (sperm concentration, total sperm count, sperm motility and morphology), there were only marginal (non-significant) differences between study subjects and non-study subjects. CONCLUSIONS: Among men from an infertility clinic, we found no strong evidence of differential participation based on semen quality. This is reassuring since the potential for selection bias is of concern in semen quality studies. However, the potential for selection bias in other study designs remains unclear.     

Relationship between human sperm-hyaluronan binding assay and fertilization rate in conventional in vitro fertilization

BACKGROUND: Sperm-hyaluronan-binding assay (HBA) is one of the commercial kits being marketed for routine testing of sperm maturity and fertility. However, there is no report of whether the HBA can provide additional information over standard semen analysis for sperm-fertilizing ability. The objective of this study was to investigate the relationship between HBA and fertilization rate in conventional IVF. METHODS: A total of 175 IVF patients with > or = 3 mature oocytes inseminated were included in the study. Both the standard semen analysis and the HBA were performed on the same ejaculated sperm samples used for IVF treatments. Relationships between the semen analysis and the HBA results and fertilization rate were analysed by both the Spearman test and the multivariate logistic regression analysis. RESULTS: Both total and progressive sperm motility and normal morphology were highly correlated with HBA scores. While both normal sperm morphology and HBA scores were statistically significantly related to fertilization rates, the HBA was less significant than normal sperm morphology. The HBA does not provide additional information for identifying patients with a poor fertilization rate. CONCLUSION: HBA is highly significantly correlated with sperm motility and morphology but is less significant than sperm morphology in relation to the fertilization rate in IVF. Thus, the clinical predictive value of HBA for sperm-fertilizing ability in vitro is limited.     

Andrology: External quality assessment for semen analysis and sperm antibody detection: results of a pilot scheme

The need for quality assurance in the seminology laboratoryis clear, as the techniques of semen analysis and sperm antibodydetection are just as susceptible to variation as any otherroutine pathology test. Semen samples were distributed to 20laboratories on six occasions, four samples per distribution,for sperm concentration and morphology assessment under routineconditions, together with an equal number of serum samples forsperm antibody detection. The semen analysis results showeda wide range of values for any given sample, which did not seemto be related to the methodology used. However, this variationappears to be related to the presence of persistent errors,as most laboratories showed reasonable between-assay and within-assayvariation. Detection of sperm antibodies by the tray agglutination,gelatin agglutination or indirect immuno-bead test showed aconsistent discrimination between the intended positive andintended negative samples. However, the use of fluorescent microscopywas unable to do this. This study has shown the feasibilityof operating external quality assessment schemes for semen analysisand sperm antibody detection. These schemes provide the opportunityfor individual laboratories to fully evaluate their own methodsagainst those of others and to determine the stages at whichany errors occur. An increased number of participants will ultimatelyenable a systematic comparison of different methods.     

Hepatocyte growth factor in human semen and its association with semen parameters

Hepatocyte growth factor (HGF) is a structurally unique growth factor with potent motogenic (motility inducing) effects. Studies in the murine male genital tract have suggested important associations between HGF and the acquisition of sperm motility during epididymal maturation. The aim of this study was, therefore, to determine the concentration of HGF in human semen and assess its correlation, if any, with sperm motility and other semen parameters. Semen samples were collected by masturbation and analysed using standard procedures. HGF concentrations were measured in duplicate using an enzyme-linked immunoassay technique. Total protein estimations were also made in a subset of samples. The 95 subjects were divided into three groups for analysis: normozoospermic, subnormal semen and azoospermic. HGF was detected in all samples (median 0.456, 25th centile 0.388, 75th centile 0.556 ng/ml). No significant correlations were found between semen HGF concentrations and sperm concentration, motility, total sperm count or total motile count. There were no significant differences in mean HGF concentrations between the three subgroups. In conclusion HGF is present in human semen in significant quantities. The data do not suggest HGF concentrations are correlated with parameters of sperm motility.     

Beneficial effect of microsurgical varicocelectomy on human sperm DNA integrity

BACKGROUND: Human sperm DNA damage may adversely affect reproductive outcomes, and the spermatozoa of infertile men possess substantially more DNA damage than that of fertile men. To date, there is no available treatment for men with high levels of sperm DNA damage. The objective of this study was to examine the effect of varicocelectomy on sperm DNA denaturation (DD, an index of sperm DNA damage) in infertile men with a clinical varicocele. METHODS. We reviewed the reports of 37 men who underwent microsurgical varicocelectomy at our institution from September 2001 to July 2002. Standard semen parameters and the percentage of spermatozoa with DD (monitored by flow cytometry analysis of acridine orange-treated spermatozoa) were assessed before and 6 months after varicocelectomy. RESULTS. The percentage of spermatozoa with DD decreased following varicocelectomy compared with pre-operatively (27.7 versus 24.6%, respectively, P < 0.05). Sperm concentration and the percentages of motile sperm and normal forms (WHO criteria) increased following varicocelectomy, but the difference did not reach statistical significance. CONCLUSIONS. Our data suggest that varicocelectomy can improve human sperm DNA integrity in infertile men with varicocele. These data represent the first report of improved sperm DNA integrity after therapy and further support the beneficial effect of varicocelectomy on human spermatogenesis.     

Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters

BACKGROUND: Standard semen parameters have proven poor at predicting the outcomes of IVF treatment cycles. As recent studies suggest that the male genome may play an important role in early embryogenesis, this study attempts to correlate the level of sperm DNA damage in fresh semen and prepared sperm with the outcomes of conventional IVF treatment cycles. METHODS: Forty patients embarking on IVF treatment were recruited into this prospective observational study. Both fresh semen and PureSperm-prepared sperm were processed using a modified comet assay 3-6 months prior to the patients' IVF treatment cycles. Comet head DNA (mean and integrated head density) and tail DNA parameters (length and moment) were measured separately. RESULTS: Significant correlations between total sperm concentration and between comet length, moment, mean head density with embryo quality were detected in fresh semen and prepared sperm. Surprisingly, no significant correlations between head and tail parameters were detected. CONCLUSIONS: Comet head and tail DNA parameters appear to be potentially useful as predictors of embryo quality and IVF outcomes, especially in couples with unexplained subfertility. The lack of correlation between head and tail parameters may be due to a different mechanism of DNA damage within these two compartments.     

Increased levels of sperm ubiquitin correlate with semen quality in men from an andrology laboratory clinic population

BACKGROUND: Ubiquitin, a house-keeping protein that marks other proteins for proteasomal degradation, tags defective sperm during epididymal passage. To establish ubiquitin as a biomarker of human infertility, the present study examines the relationships between sperm ubiquitin content and clinical semen parameters among men from an infertility clinic population with varied aetiologies. METHODS: Anti-ubiquitin immunoreactivity was measured by flow cytometric sperm-ubiquitin tag immunoassay (SUTI) in sperm samples of 28 infertility patients and 15 fertile donors. Semen analyses were performed by computer-assisted semen analysis and World Health Organization morphology. RESULTS: Median values of ubiquitin-induced fluorescence had a strong negative correlation with sperm count (r = -0.63, P = 0.0003) and a positive correlation with % abnormal morphology (r = 0.55, P = 0.01). Infertility patients (n = 28) had significantly higher levels of sperm ubiquitin. Out of 28 patients, six reported possible occupational exposures to solvents, three were current smokers and six were ex-smokers. Within the patient group, men with known male factor infertility, those with self-reported occupational exposure to solvents and current smokers had the highest sperm ubiquitin levels. When men with jobs involving potential occupational exposure to solvents were combined with current smokers, the highest correlations were found between sperm ubiquitin and motility (r = -0.74), count (r = -0.82) and % sperm abnormalities (r = 0.73). CONCLUSIONS: Increased sperm ubiquitin was inversely associated with sperm count, motility and % normal morphology, supporting the use of ubiquitin as a biomarker of human semen quality. SUTI assay confirmed poor semen quality in all men with poor clinical semen parameters, but also was high in some patients with seemingly good clinical semen parameters. Occupational exposure to solvents and smoking may have contributed to high levels of sperm ubiquitin in some of these patients.     

Intra- and inter-laboratory variability in the assessment of sperm morphology by strict criteria: impact of semen preparation, staining techniques and manual versus computerized analysis.

We designed prospective studies to compare manual and computerized analysis of sperm morphology by strict criteria using different semen processing and staining techniques. A total of 54 semen samples were studied; slides were prepared from each subject from liquefied semen and after washing, and stained with Diff-Quik or Papanicolaou. An intra-laboratory, blind assessment was performed manually (two observers) and using a computerized analyser (two readings). This demonstrated a very good correlation between manual analysis of liquefied and washed samples with both staining techniques [intraclass coefficient (ICC) = 0.93 and 0.83]. Greater agreement was observed between computerized readings (washed samples) of Diff-Quik (ICC = 0.93) than of Papanicolaou-stained slides (ICC = 0.66). An excellent intra-laboratory correlation was observed for within-computer readings (ICC = 0.93). There was moderate agreement between inter-laboratory computer readings (two centres, ICC = 0.72). Although there was lower inter-laboratory agreement for manual and manual versus computer readings, overall results of all manual and computer analyses showed good agreement (ICC = 0.73). Diff-Quik staining is reliable for both manual (liquefied) and computer (washed) analysis of strict sperm morphology. Intra- and inter-computer analyses using this method reached satisfactory levels of agreement. There is still high inter-laboratory variability for the manual method.     

Quantitative study of caspase-3 activity in semen and after swim-up preparation in relation to sperm quality

BACKGROUND: There are no studies relating the apoptotic marker caspase-3 in human sperm to different degrees of abnormal sperm concentration, morphology and rapid progressive motility. METHODS: Semen from 67 males with abnormal semen analyses (n = 61) and normozoospermia (n = 6) were used. In each case, sperm from the neat semen (semen fraction) and after gradient centrifugation and swim-up (swim-up fraction) were incubated with a caspase-3 profluorogenic substrate. Caspase-3 activity was quantified in 119 850 sperm, 67 488 from semen and 52 362 from swim-up fractions. Logistic regression was used to estimate odds ratio (with 99% confidence intervals) for the presence of caspase-3-positive sperm. RESULTS: In semen fractions, no relationship was found between abnormal semen analysis subgroups and sperm caspase-3 activity. On the contrary, a significantly increased number of sperm with caspase-3 activity was found in the swim-up fractions from samples with poor sperm morphology. When analysis was restricted to single semen analysis defects, a significant increase of caspase-3-positive sperm was found in the semen fractions of cases with asthenozoospermia, and in the swim-up fractions of cases with teratozoospermia. CONCLUSIONS: Sperm caspase-3 activity seems to be associated with teratozoospermia and asthenozoospermia, thus suggesting that nuclear, mitochondrial and cytoskeletal abnormalities induce caspase-3 activation during spermiogenesis or sperm maturation.     

Antioxidant treatment of patients with asthenozoospermia or moderate oligoasthenozoospermia with high-dose vitamin C and vitamin E: a randomized, placebo-controlled, double-blind study

In a randomized, placebo-controlled, double-blind study we investigated whether high-dose oral treatment with vitamins C and E for 56 days was able to improve semen parameters of infertile men. Ejaculate parameters included semen volume, sperm concentration and motility, and sperm count and viability. Thirty-one patients without genital infection but with asthenozoospermia (< 50% motile spermatozoa) and normal or only moderately reduced sperm concentration (> 7 x 10(6) spermatozoa/ml) (according to WHO criteria) were examined. To investigate the influence of the epididymal storage period on semen parameters, the patients were asked to deliver two semen samples with abstinence times of 2 and 7 days both before and at the end of vitamin treatment. After randomization, the patients received either 1000 mg vitamin C and 800 mg vitamin E (n = 15) or identical placebo capsules (n = 16). No changes in semen parameters were observed during treatment, and no pregnancies were initiated during the treatment period. Combined high-dose antioxidative treatment with vitamins C and E did not improve conventional semen parameters or the 24-h sperm survival rate. Prolonged abstinence time increased ejaculate volume (P < 0.05), sperm count (P < 0.05), sperm concentration (P < 0.05) and the total number of motile spermatozoa (P < 0.05).