The role of the intrinsic pathway of apoptosis in human ejaculated sperm damage under a state of scrotal heat stress.

PURPOSE: The study aimed to determine the associations among standard sperm characteristics and oxidative/apoptotic markers in ejaculated sperm of men exposed to prolonged scrotal hyperthermia of either environmental or clinical origin. METHODS: The original study design included four research groups: professional drivers (n = 54), infertile men with varicocele (n = 78), infertile men not exposed to prolonged genital heat stress (n = 37), and fertile individuals serving as the control group (n = 29). Standard semen analysis was performed according to the 5th WHO laboratory manual. The following oxidative and apoptotic parameters of sperm were investigated: mitochondrial superoxide anion generation (MitoSOX Red dye), phosphatidylserine externalization (Annexin V binding assay), mitochondrial membrane potential (JC-1 dye), DNA fragmentation (TUNEL/PI assay), and membrane fluidity (merocyanine 540 dye). RESULTS: All the studied groups presented a strong deterioration in routine sperm parameters and a strongly apoptotic phenotype in sperm, characterized by both decreased mitochondrial membrane potential and enhanced DNA fragmentation, regardless of the thermal insult. Significant induction of mitochondrial superoxide anion generation was noted only in the groups exposed to genital heat stress. A positive correlation between the production of superoxide anion in the mitochondrial chain and the level of DNA fragmentation in drivers was also noted. CONCLUSION: Long-term exposure to scrotal hyperthermia in real-life situations is sufficient to reduce sperm quality in humans. The thermal stress directly induces the oxidative stress cascade in ejaculated sperm, affecting the plasma membrane fluidity, mitochondrial homeostasis, and sperm DNA integrity.

Male Infertility Coexists with Decreased Sperm Genomic Integrity and Oxidative Stress in Semen Irrespective of Leukocytospermia.

Our research was designed to verify the relationship between male infertility, basic semen characteristics (with respect to detailed sperm morphology), sperm DNA fragmentation (SDF), oxidation-reduction potential in semen (ORP), and leukocytospermia. The obtained results showed that infertile groups (with or without leukocytospermia) had significantly lower basic semen characteristics and higher SDF, raw ORP, and static ORP (sORP) than fertile controls. The thresholds of 13% SDF (AUC = 0.733) and 1.40 sORP (AUC = 0.857) were predictive values for discriminating infertile from fertile men. In infertile groups, a higher prevalence and risk for >13% SDF and >1.40 sORP were revealed. Unexpectedly, leukocytospermic subjects had lower sORP, prevalence, and risk for >1.40 sORP than leukocytospermic-negative men. These groups did not differ in SDF and raw ORP. Both SDF and sORP negatively correlated with basic semen parameters but positively correlated with sperm head and midpiece defects. sORP positively correlated with sperm tail defects, immature sperm cells with excess residual cytoplasm, and SDF. In turn, raw ORP negatively correlated with sperm count but positively correlated with SDF and sORP. These findings indicate that (1) there is a relationship between male infertility, SDF, and OS in semen; (2) in infertile men, there is a clinically significant risk of SDF and OS irrespective of leukocytospermia; and (3) the assessment of SDF and oxidative stress should be independent of leukocytospermia.

Global 5mC and 5hmC DNA Levels in Human Sperm Subpopulations with Differentially Protaminated Chromatin in Normo- and Oligoasthenozoospermic Males.

Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm chromatin protamination in three subpopulations of fertile normozoospermic controls and infertile patients with oligo-/oligoasthenozoospermia. For the first time, a sequential staining protocol was applied, which allowed researchers to analyse 5mC/5hmC levels by immunofluorescence staining, with a previously determined chromatin protamination status (aniline blue staining), using the same spermatozoa. TUNEL assay determined the sperm DNA fragmentation level. The 5mC/5hmC levels were diversified with respect to chromatin protamination status in both studied groups of males, with the highest values observed in protaminated spermatozoa. The linkage between chromatin protamination and 5mC/5hmC levels in control males disappeared in patients with deteriorated semen parameters. A relationship between 5mC/5hmC and sperm motility/morphology was identified in the patient group. Measuring the 5mC/5hmC status of sperm DNA according to sperm chromatin integrity provides evidence of correct spermatogenesis, and its disruption may represent a prognostic marker for reproductive failure.

The Role of Seminal Oxidative Stress Scavenging System in the Pathogenesis of Sperm DNA Damage in Men Exposed and Not Exposed to Genital Heat Stress.

Responding to the need for the verification of some experimental animal studies showing the involvement of oxidative stress in germ cell damage in the heat-induced testis, we investigated the possibility of a direct relationship between seminal oxidative stress markers (total antioxidant capacity, catalase activity, superoxide dismutase activity, and malondialdehyde concentration) and ejaculated sperm chromatin/DNA integrity (DNA fragmentation and chromatin condensation abnormalities) in distinct groups of men exposed and not exposed to prolonged scrotal hyperthermia. A statistical increase in the proportion of sperm with DNA fragmentation was observed in all the studied subgroups compared to the fertile men. In turn, the groups subjected to heat stress as professional drivers or infertile men with varicocele presented greater disturbances in the oxidative stress scavenging system than men not exposed to genital heat stress. Based on the comparative analysis of the studied parameters, we can conclude that alterations in the seminal oxidative stress scavenging system are directly engaged in the pathogenesis of ejaculated sperm DNA damage regardless of the intensity of the impact of thermal insult. To the best of our knowledge, this study, for the first time, revealed the co-existence of oxidative stress and sperm DNA damage in the semen of professional drivers.

The Negative Impact of Varicocele on Basic Semen Parameters, Sperm Nuclear DNA Dispersion and Oxidation-Reduction Potential in Semen.

Since varicocele is so common in infertile men, this study intends to analyse the relationships between varicocele and conventional semen characteristics, sperm nuclear DNA dispersion and oxidation-reduction potential (ORP) in semen. Varicocele-positive and varicocele-negative infertile men (study groups) showed significantly lower standard sperm parameters and higher sperm DNA fragmentation (SDF) and ORP in semen than healthy volunteers and subjects with proven fertility (control groups). A lower proportion of low SDF levels (0-15% SDF) and higher incidence of high SDF levels (>30% SDF), as well as a higher prevalence of high ORP values (>1.37 mV/106 sperm/mL), were found in the study groups vs. the control groups. Moreover, infertile men had significantly lower odds ratios (ORs) for low SDF levels and significantly higher ORs for high SDF levels and high ORP. SDF and ORP were negatively correlated with sperm number, morphology, motility and vitality. Furthermore, a significant positive correlation was found between SDF and ORP. The obtained results suggest that disorders of spermatogenesis may occur in varicocele-related infertility. These abnormalities are manifested not only by reduced standard semen parameters but also by decreased sperm DNA integrity and simultaneously increased oxidative stress in semen.

Seminal Plasma Analysis of Oxidative Stress in Different Genitourinary Topographical Regions Involved in Reproductive Tract Disorders Associated with Genital Heat Stress.

The pathophysiological mechanisms responsible for male subfertility/infertility caused by or complicated by genital heat stress remains unclear in many respects. Because seminal plasma creates the environment for the proper functioning of spermatozoa, in this study, we verified the associations among standard spermiograms, seminal biochemical parameters (neutral alpha-glucosidase, fructose, and citric acid) and oxidative stress markers (total antioxidant capacity, catalase activity, superoxide dismutase activity, and malondialdehyde concentration) in distinct entities associated with male infertility with and without long-time exposure to local hyperthermia. We demonstrated that men exposed to prolonged environmental or clinically recognized local heat stress in adulthood may suffer from dysregulation of seminal antioxidant components, which can be directly associated with epididymal and prostate function. The comparative analysis of the studied parameters showed numerous correlations among all biochemical parameters (particularly neutral alpha-glucosidase) with low standard semen quality in almost all the investigated infertile groups. In light of the data obtained in this originally designed study, we conclude that more attention should be paid to the epididymis and accessory gland function in subfertile and infertile men exposed to genital heat stress, especially in the context of novel treatment algorithms (targeted therapies).

Age-related changes in human sperm DNA integrity.

Abnormal standard semen characteristics and reduced sperm chromatin maturity can appear with increasing male age. However, the influence of paternal age on semen parameters is still controversial. Therefore, this study was designed to estimate the influence of paternal age not only on conventional semen characteristics but also on sperm DNA integrity. This research was carried out on ejaculated sperm cells obtained from men (n = 1124) aged ≥40 y and <40 y. Our data revealed a decreased semen volume and an increased percentage of DFI (sperm DNA fragmentation index) in older men compared to younger men in the entire study cohort, in men with normozoospermia and in men with abnormal semen parameters. Moreover, there was a higher incidence of sperm DNA damage (>10% DFI, low fertility potential) in the groups of men aged ≥40 y than in the groups of men aged <40 y. Older men had over twice the odds ratio for high sperm DNA damage as younger men. Our findings suggest a detrimental effect of advanced paternal age on sperm chromatin integrity. The data show that the evaluation of sperm DNA has greater clinical utility than standard semen analysis in case of male fertility potential assessment.

Utility and Predictive Value of Human Standard Semen Parameters and Sperm DNA Dispersion for Fertility Potential.

Because the assessment of sperm DNA fragmentation (SDF) plays a key role in male fertility, our study was designed to find the relationships between SDF and standard semen parameters. The receiver operating characteristic (ROC) curve showed that 18% SDF is a prognostic parameter for discriminating between men with normal and abnormal standard semen parameters (n = 667). Men with > 18% SDF had significantly lower quality semen, a higher prevalence of abnormal semen characteristics, and a higher odds ratio for abnormal semen parameters compared to men with ≤ 18% SDF. An ROC analysis provided predictive values for age and semen parameters to distinguish between men with SDF > 18% and men with ≤ 18% SDF. SDF was positively correlated with male age and teratozoospermia index but negatively with sperm concentration, total number of spermatozoa, sperm morphology, progressive motility, and vitality. Our study shows that 18% SDF has a predictive value for distinguishing between men with normal and abnormal semen characteristics. Men with >18% SDF have a higher risk for abnormal semen parameters, while age and obtained semen parameters have a predictive value for SDF. There is a relationship between SDF and conventional sperm characteristics, and thus, SDF can be incorporated into male fertility assessment.

The impact of sedentary work on sperm nuclear DNA integrity.

INTRODUCTION: Contemporary professional jobs that often enforce a sedentary lifestyle and are frequently associated with testicular overheat, deserve special attention with respect to male fertility potential. Interestingly, the harmful effect of testicular heat stress on sperm characteristics including nuclear DNA integrity was well characterized; however, the influence of sedentary work on sperm chromatin has not yet been documented. Therefore, our research was designed to examine the potential effects of sedentary work not only on conventional semen features but also on sperm nuclear DNA status. MATERIALS AND METHODS: The study was carried out on ejaculated sperm cells obtained from men who spent ≥ 50% of their time at work (≥ 17.5 h per week) in a sedentary position (n = 152) and from men who spent < 50% of their time at work in a sedentary position (n = 102). Standard semen characteristics were assessed according to the WHO 2010 recommendations, while sperm nuclear DNA fragmentation (SDF) was evaluated using the Halosperm test. RESULTS: There were no significant differences in the standard semen parameters between the study groups. The groups differed only in SDF parameter. The men who spent at least 50% of their work time in a sedentary position had a higher proportion of SDF than the men who spent < 50% of their time at work in a sedentary position (median value 21.00% vs. 16.50%, respectively). The incidence of low SDF levels (related to 0-15% sperm cells with abnormal DNA dispersion) was significantly lower (27.63% vs. 45.10%), the percentage of men with high SDF levels (related to > 30%) was significantly higher (30.92% vs. 16.67%) in group of men who spent at least 50% of their work time in a sedentary positon. Furthermore, these men were more than twice as likely to have not a low SDF level (OR: 0.4648) and had more than twice the risk of having a high SDF level (OR: 2.2381) than the men in less sedentary occupations. CONCLUSIONS: Despite lack of association between sedentary work and conventional semen characteristics our study revealed detrimental effect of seated work on sperm nuclear DNA integrity. A sedentary job doubled the risk of high levels of sperm DNA damage. The pathomechanism could be related to testicular heat stress resulting in sperm chromatin remodelling failure during spermiogenesis. Therefore, it seems reasonable to simultaneously carry out routine seminological analyses and tests assessing sperm chromatin status while diagnosing male infertility.

Chromosome (re)positioning in spermatozoa of fathers and sons - carriers of reciprocal chromosome translocation (RCT).

BACKGROUND: Non-random chromosome positioning has been observed in the nuclei of several different tissue types, including human spermatozoa. The nuclear arrangement of chromosomes can be altered in men with decreased semen parameters or increased DNA fragmentation and in males with chromosomal numerical or structural aberrations. An aim of this study was to determine whether and how the positioning of nine chromosome centromeres was (re)arranged in the spermatozoa of fathers and sons - carriers of the same reciprocal chromosome translocation (RCT). METHODS: Fluorescence in situ hybridization (FISH) was applied to analyse the positioning of sperm chromosomes in a group of 13 carriers of 11 RCTs, including two familial RCT cases: t(4;5) and t(7;10), followed by analysis of eight control individuals. Additionally, sperm chromatin integrity was evaluated using TUNEL and Aniline Blue techniques. RESULTS: In the analysed familial RCT cases, repositioning of the chromosomes occurred in a similar way when compared to the data generated in healthy controls, even if some differences between father and son were further observed. These differences might have arisen from various statuses of sperm chromatin disintegration. CONCLUSIONS: Nuclear topology appears as another aspect of epigenetic genomic regulation that may influence DNA functioning. We have re-documented that chromosomal positioning is defined in control males and that a particular RCT is reflected in the individual pattern of chromosomal topology. The present study examining the collected RCT group, including two familial cases, additionally showed that chromosomal factors (karyotype and hyperhaploidy) have superior effects, strongly influencing the chromosomal topology, when confronted with sperm chromatin integrity components (DNA fragmentation or chromatin deprotamination).

Global methylation status of sperm DNA in carriers of chromosome structural aberrations.

Male infertility might be clearly associated with aberrant DNA methylation patterns in human spermatozoa. An association between oxidative stress and the global methylation status of the sperm genome has also been suggested. The aim of the present study was to determine whether the global sperm DNA methylation status was affected in the spermatozoa of carriers of chromosome structural aberrations. The relationships between the 5-methylcytosine (m 5 C) levels in spermatozoa and chromatin integrity status were evaluated. The study patients comprised male carriers of chromosome structural aberrations with reproductive failure (n = 24), and the controls comprised normozoospermic sperm volunteers (n = 23). The global m 5 C level was measured using thin-layer chromatography (TLC) and immunofluorescence (IF) techniques. The sperm chromatin integrity was assessed using aniline blue (AB) staining and TUNEL assay. The mean m 5 C levels were similar between the investigated chromosome structural aberrations carriers (P) and controls (K). However, sperm chromatin integrity tests revealed significantly higher values in chromosomal rearrangement carriers than in controls (P < 0.05). Although the potential relationship between sperm chromatin integrity status and sperm DNA fragmentation and the m 5 C level juxtaposed in both analyzed groups (P vs K) was represented in a clearly opposite manner, the low chromatin integrity might be associated with the high hypomethylation status of the sperm DNA observed in carriers of chromosome structural aberrations.

The effect of bacteriospermia and leukocytospermia on conventional and nonconventional semen parameters in healthy young normozoospermic males.

Bacterial semen inflammation/infection is an important diagnostic and therapeutic problem in contemporary andrology. The molecular mechanism by which inflammatory mediators compromise the fertilizing potential of germ cells is complex and multifactorial, and it remains unclear. To improve the understanding of the pathophysiology of human subfertility/infertility caused or complicated by reproductive tract inflammation/infection, we simultaneously evaluated a set of conventional (standard semen analysis) and nonconventional sperm parameters, including subcellular changes in sperm membranes (phospholipid scrambling, peroxidative damage, and phosphatidylserine (PS) externalization), mitochondria (mitochondrial transmembrane potential, ΔYm, and oxidoreductive capability), and DNA fragmentation in healthy young normozoospermic males with asymptomatic bacteriospermia and leukocytospermia. Both bacteriospermia and leukocytospermia had a deleterious effect on standard sperm parameters, including sperm concentration, motility and morphology. Bacteriospermia was associated with a simultaneous decrease in mitochondrial transmembrane potential and an increase in PS externalization, and with DNA fragmentation in both live and dead sperm. The highest MDA concentrations in sperm lysates were observed in the presence of leukocytes. This study demonstrates for the first time that bacteriospermia and leukocytospermia compromise sperm quality in healthy young normozoospermic males. Bacteria mainly participate in intrinsic mitochondria-dependent apoptotic cell death mechanisms. Oxidative stress plays a relevant role in decreasing routine sperm parameters during leukocytospermia. The value of these observations may be significant and may support the development of a new diagnostic platform (biomarkers) for infertile males with infections in the reproductive tract.

Topology of chromosome centromeres in human sperm nuclei with high levels of DNA damage.

Several studies have shown that the 'poor' sperm DNA quality appears to be an important factor affecting male reproductive ability. In the case of sperm cells from males with the correct somatic karyotype but with deficient spermatogenesis, resulting in a high degree of sperm DNA fragmentation, we observed changes in the preferential topology of the chromosome 7, 9, 15, 18, X and Y centromeres. The changes occurred in radial localization and may have been directly linked to the sperm chromatin damage. This conclusion is mainly based on a comparison of FISH signals that were observed simultaneously in the TUNEL-positive and TUNEL-negative sperm cells. The analyzed cells originated from the same ejaculated sample and FISH was performed on the same slides, after in situ TUNEL reaction. Based on the observed changes and previous data, it appears that the sperm nucleus architecture can be disrupted by a variety of factors and has a negative influence on spermatogenesis at the same time. Often, these factors coexist (e.g. chromosomal translocations, aneuploidies, a higher DNA fragmentation, abnormal seminology), but no direct correlations between the factors were observed.

Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis.

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.

Semen Quality, Hormonal Levels, and Androgen Receptor Gene Polymorphisms in a Population of Young Male Volunteers from Two Different Regions of Poland.

BACKGROUND: The population of healthy Polish men has not been frequently and systematically investigated for fertility status. The aim of this study was to assess the quality of semen in a randomly recruited population of young males. The most important task was to find a relationship between semen parameters, sex hormones, and AR gene polymorphism. MATERIAL AND METHODS: Semen and blood samples from young men from the Poznan (n=113) and Lublin regions (n=89) were collected for semen analysis, assessment of hormonal concentrations, and calculation of the CAG and GGN repeats of the AR gene. RESULTS: Statistical comparisons of the hormones and circulating proteins and the seminological parameters revealed significant differences between the regional groups of males studied. Among the correlations found, we emphasize the positive relationship between inhibin B levels and both the number of spermatozoa per ml (R=0.37; p=0.0001) and the total sperm concentration (R=0.40; p=0.00003). Positive correlations between IGF1 and sperm morphology was also found (R=0.40; p=0.000004). The mean number of CAG repeats in our tested groups was 21.93±2.79, in a range from 16 to 31. The mean number of GGN repeats was 23.2±1.66 and ranged from 16 to 29. Numerous significant correlations were found between CAG or GGN repeats and blood hormones or circulating proteins and semen parameters; however, Spearman's rank correlations revealed rather weak coefficients. CONCLUSIONS: This report attempted to determine the quality of semen samples and sex hormones in a population of Polish young men. The results were found to be similar to data obtained in Scandinavia. The calculated means and range of CAG or GGN repeats of the AR gene in Polish males were similar to West European epidemiological data.

Mechanisms of the harmful effects of bacterial semen infection on ejaculated human spermatozoa: potential inflammatory markers in semen.

The invasion of the male reproductive tract by microorganisms, and its subsequent consequences for sperm fertilizing potential, has been intensely discussed. The role of the bacteria that are responsible for the colonization and contamination of the male urogenital tract, rather than its infection, in diminished sperm parameters raises the most controversy. There are numerous premises suggesting that bacterial semen infection is associated with male infertility. However, the molecular mechanism by which the fertility is affected is complex and multifactorial, and still presents a puzzle. Some authors have suggested that direct interactions between bacteria and human spermatozoa facilitate sperm immobilization, affect sperm morphology, and thus weaken the ability of sperm to fertilize. On the other hand, the massive infiltration of activated leukocytes into the inflammatory site may be associated with impairment of sperm fertilizing potential, due to oxidative, apoptotic, and immune processes. This review presents current research trends and aims to summarize the present knowledge of semen inflammation and causative bacterial agents in the male urogenital tract, with its consequence on seminological parameters, and male fertility status.

Can apoptosis and necrosis coexist in ejaculated human spermatozoa during in vitro semen bacterial infection?

PURPOSE: To evaluate whether ejaculated human spermatozoa undergo complete apoptosis or necrosis during experimental semen bacterial infection in vitro. METHODS: Apoptotic markers, including mitochondrial transmembrane potential (ΔΨm), phosphatidylserine (PS) externalization, and DNA fragmentation, have been detected simultaneously in ejaculated human sperm after their incubation with a known pathogenic (Escherichia coli), as well as with conditionally pathogenic bacterial strains (Staphylococcus haemolyticus, Bacteroides ureolyticus) and/or leukocytes. The ΔΨm and translocation of PS was evaluated using the JC-1 and Annexin V binding tests, respectively. A modified TUNEL assay with additional staining for sperm viability was used to detect the DNA fragmentation level. RESULTS: The exposure of ejaculated spermatozoa to bacterial strains was associated with a simultaneous decrease in the percentage of sperm with normal ΔΨm and an increase in the proportion of Annexin V-positive sperm. Additionally, in the presence of S. haemolyticus, B. ureolyticus and/or leukocytes, a significant increase in the percentage of live TUNEL-positive (apoptotic) as well as dead TUNEL-positive (necrotic) sperm cells was also observed. CONCLUSIONS: The cellular death observed in spermatozoa in the presence of inflammatory mediators may be due to both apoptosis and necrosis. Here, we demonstrate for the first time that direct contact of conditionally pathogenic bacteria with ejaculated human sperm may play an even greater role in the promotion of apoptosis than in case of some pathogenic bacterial strains. These findings suggest that significant bacteriospermia and leukocytospermia may be direct causes of subfertility or additional negative factors worsening the prognosis of fertility in natural and assisted procreation.

Cytokines in the male reproductive tract and their role in infertility disorders.

Cell integration between the immune and reproductive systems is the basis for normal male reproductive physiology. Cytokines are a part of the autocrine/paracrine network operating in the male reproductive tract. At the same time, immunological reactions occurring via cytokines appear to be both beneficial and/or risk factors for male fertility. As the cytokines are produced by a whole spectrum of cells in all compartments of the male genital tract, they can also be involved in a variety of andrological disorders. The monitoring of cytokines and other immune factors in seminal plasma may offer a chance to better understand the mechanisms leading to sub-/infertility. In this review, we present insights into cytokine interplay in some of the pathological conditions associated with male reproduction.

Fertilizing potential of ejaculated human spermatozoa during in vitro semen bacterial infection.

OBJECTIVE: To assess the in vitro effect of three bacterial isolates (Escherichia coli, serotype O75:HNT, Staphylococcus haemolyticus, and Bacteroides ureolyticus) and/or leukocytes on sperm motility, subcellular changes in sperm plasma membranes, and sperm fertilizing potential. DESIGN: An in vitro model of semen bacterial infection. SETTING: Basic research laboratory. PATIENT(S): Healthy normozoospermic volunteers and healthy blood donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm plasma membrane stability was evaluated with a LIVE/DEAD Sperm Viability Kit and with the merocyanine 540 (M540) test both performed using flow cytometry. An oxiSelect TBARS Assay Kit was used for quantitative measurement of malondialdehyde content. Functional ability of spermatozoa was assessed by hypo-osmotic swelling (HOS) test and sperm penetration assay (SPA). RESULT(S): The incubation of sperm with bacteria and/or leukocytes was associated with the reduction of their fertilizing potential demonstrated in both the HOS test and SPA, and this effect can be considered as a natural consequence of diminished motility and sperm membrane injury of lipid bilayers. Bacteroides ureolyticus demonstrated the most significant detrimental effect on sperm structure and function. CONCLUSION(S): Sperm motility and lipid sperm membrane status might be the earliest and the most sensitive indicators of sperm damage with negative consequences for male factor fertility, which can be attributed to both bacteria and leukocytes action.