Multi-centre assessment of nitroblue tetrazolium reactivity in human semen as a potential marker of oxidative stress.

The nitroblue tetrazolium (NBT) reaction as a tracer of oxidative stress was examined in 707 ejaculates from seven clinics. Semen was initially surveyed by classifying the NBT reaction using a pre-established rank for the Oxisperm® test based on three colourimetric levels: L1, low (n = 141 [20%]); L2, medium (n = 538 [76%]) and L3, high (n = 28 [4%]). L3 was indicative of a high level of superoxide anions. Halosperm® chromatin dispersion assay was used to analyse samples of ejaculates 30 min after ejaculation; no difference was found in DNA fragmentation of L1 or L3; L3 category semen samples incubated for 24 h at 37oC showed a significantly faster rate (P < 0.001) of DNA damage than those in L1. The NBT reaction was further characterized in the ejaculates of 100 patients to determine the relative contribution of seminal plasma, spermatozoa, or both. Seminal plasma was the most significant fraction of •O2- localization, whereas sperm fractions generated detectable reactive oxygen species in only 32% of the ejaculates. Formazan precipitates were primarily associated with the sperm mid-piece and seminal leukocytes; however, not all spermatozoa stained positive to formazan and not all leukocytes presented with equivalent production of superoxide anions.

Rapid determination of colistin resistance in clinical strains of Acinetobacter baumannii by use of the micromax assay.

Colistin is an old antibiotic which has been used as a therapeutic option for carbapenem- and multidrug-resistant Gram-negative bacteria, like Acinetobacter baumannii. This pathogen produces life-threatening infections, mainly in patients admitted to intensive care units. Rapid detection of resistance to colistin may improve patient outcomes and prevent the spread of resistance. For this purpose, Micromax technology was evaluated in four isogenic A. baumannii strains with known mechanisms of resistance to colistin and in 66 isolates (50 susceptible and 16 resistant). Two parameters were determined, DNA fragmentation and cell wall damage. To assess DNA fragmentation, cells trapped in a microgel were incubated with a lysing solution to remove the cell wall, and the released nucleoids were visualized under fluorescence microscopy. Fragmented DNA was observed as spots that diffuse from the nucleoid. To assess cell wall integrity, cells were incubated with a lysis solution which removes only weakened cell walls, resulting in nucleoid release exclusively in affected cells. A dose-response relationship was demonstrated between colistin concentrations and the percentages of bacteria with DNA fragmentation and cell wall damage, antibiotic effects that were delayed and less frequent in resistant strains. Receiver operating characteristic (ROC) curves demonstrated that both DNA fragmentation and cell wall damage were excellent parameters for identifying resistant strains. Obtaining ≤11% of bacteria with cell wall damage after incubation with 0.5 µg/ml colistin identified resistant strains of A. baumannii with 100% sensitivity and 96% specificity. Results were obtained in 3 h 30 min. This is a simple, rapid, and accurate assay for detecting colistin resistance in A. baumannii, with strong potential value in critical clinical situations.

Fast assessment of resistance to carbapenems and ciprofloxacin of clinical strains of Acinetobacter baumannii.

Infections caused by multidrug-resistant Acinetobacter baumannii constitute a major life-threatening problem worldwide, and early adequate antibiotic therapy is decisive for success. For these reasons, rapid detection of antibiotic susceptibility in this pathogen is a clinical challenge. Two variants of the Micromax kit were evaluated for a rapid detection in situ of susceptibility or resistance to meropenem or ciprofloxacin, separately, in 322 clinical isolates. Release of the nucleoid is the criterion of susceptibility to the beta-lactams (carbapenems), whereas diffusion of DNA fragments emerging from the nucleoid characterizes the quinolone activity. All the susceptible and resistant strains were correctly categorized in 100 min according to the MIC results and CLSI criteria. Thus, our technology is a promising tool for rapid identification of carbapenem and quinolone resistance of A. baumannii strains in hospital settings.

Cell wall active antibiotics reduce chromosomal DNA fragmentation by peptidoglycan hydrolysis in Staphylococcus aureus.

Lysostaphin digestion of peptidoglycan (PG) from Staphylococcus aureus resulted in chromosomal DNA fragmentation by released DNase, as directly visualized in situ on isolated nucleoids. Nevertheless, DNA digestion was partially prevented by previous incubation with antibiotics that inhibit PG synthesis. This inhibitory effect was much more remarkable with glycopeptides vancomycin and mainly teicoplanin than with beta-lactams cloxacillin and ceftazidime. Therefore, inhibition of PG chain elongation has a more significant inhibition of DNA degradation than inhibition of PG cross-linking, possibly due to a reduction in DNase storage at the cell wall.

DNA fragmentation dynamics allows the assessment of cryptic sperm damage in human: evaluation of exposure to ionizing radiation, hyperthermia, acidic pH and nitric oxide.

Sperm DNA fragmentation (SDF) is not a static seminal parameter, since the longevity of sperm DNA decreases progressively with time following ejaculation or thawing. While the dynamics of SDF is a species-specific characteristic, in the case of humans, there is still significant variation within patients. To evaluate the suitability of the dynamic SDF assay to assess the adverse effects of agents that cause genetic damage, fresh semen samples from different donors were exposed in vitro to (1) increasing acute doses of ionizing radiation, (2) elevated temperature (41 °C and 45 °C), (3) acidic pH (pH 4) and (4) the nitric oxide (NO) donor sodium nitroprusside (SNP). Sperm DNA fragmentation was analyzed after an incubation period of chronic (24h), or acute (1h) exposure to each treatment followed by incubation at 37 °C over a period of 24h. SDF was assessed using the sperm chromatin dispersion (SCD) test. Dynamic SDF for each treatment was analyzed using Kaplan-Meier survival curves. All agents, except for ionizing radiation, accelerated SDF kinetics following chronic exposure over a 24h period. Transient exposure to NO and heat but not acidic pH increased the basal (T0) level of SDF. Despite the removal of the three toxicants, the remaining sperm following acute exposure showed a decrease in their expected DNA longevity. It is concluded that the assessment of sperm DNA fragmentation dynamics is an effective methodological approach for revealing latent damage associated with toxicants that is not initially expressed following a single initial observation of SDF.

A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis.

BACKGROUND: Antibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. We have adapted a kit for rapid determination of bacterial DNA fragmentation, to assess cell wall integrity. RESULTS: Cells incubated with the antibiotic were embedded in an agarose microgel on a slide, incubated in an adapted lysis buffer, stained with a DNA fluorochrome, SYBR Gold and observed under fluorescence microscopy. The lysis affects the cells differentially, depending on the integrity of the wall. If the bacterium is susceptible to the antibiotic, the weakened cell wall is affected by the lysing solution so the nucleoid of DNA contained inside the bacterium is released and spread. Alternatively, if the bacterium is resistant to the antibiotic, it is practically unaffected by the lysis solution and does not liberate the nucleoid, retaining its normal morphological appearance. In an initial approach, the procedure accurately discriminates susceptible, intermediate and resistant strains of Escherichia coli to amoxicillin/clavulanic acid. When the bacteria came from an exponentially growing liquid culture, the effect on the cell wall of the ß-lactam was evident much earlier that when they came from an agar plate. A dose-response experiment with an E. coli strain susceptible to ampicillin demonstrated a weak effect before the MIC dose. The cell wall damage was not homogenous among the different cells, but the level of damage increased as dose increased with a predominant degree of effect for each dose. A microgranular-fibrilar extracellular background was evident in gram-negative susceptible strains after ß-lactam treatment. This material was digested by DNase I, hybridised with a specific whole genome probe, and so recognized as DNA fragments released by the bacteria. Finally, 46 clinical strains from eight gram-negative and four gram-positive species were evaluated blind for susceptibility or resistance to one of four different ß-lactams and vancomycin, confirming the applicability of the methodology. CONCLUSION: The technique to assess cell wall integrity appears to be a rapid and simple procedure to identify resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis.

Swim-up procedure selects spermatozoa with longer telomere length.

Telomere length and sperm DNA fragmentation were determined in sperm samples from 27 patients, using a quantitative PCR (Q-PCR) assay and the Sperm Chromatin Dispersion (SCD) test, respectively. Comparisons of the samples before and after swim-up processing demonstrated that this procedure selects a sperm population with longer average telomere size and lower frequency of sperm cells with fragmented DNA.

Rapid and simple determination of ciprofloxacin resistance in clinical strains of Escherichia coli.

We recently reported a simple new in situ diffusion assay, developed as a kit, to visualize DNA fragmentation in single bacterial cells. Use of this assay in a collection of 95 genetically unrelated Escherichia coli clinical strains resulted in correct identification of all of the isolates as resistant or susceptible to ciprofloxacin, consistent with the MIC results. This relevant information is obtained in 80 min.

Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay.

BACKGROUND: Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP)-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide. RESULTS: Exposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC) of 0.012 microg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 microg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 microg/ml but scarce after 10 microg/ml. The repair activity was not strictly related to viability. Four E. coli strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose. CONCLUSION: This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.

DNA fragmentation in microorganisms assessed in situ.

Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions.

Rapid Determination of Resistance to Antibiotic Inhibitors of Protein Synthesis in Staphylococcus aureus Through In Situ Evaluation of DNase Activity.

A rapid assay was designed for the detection of resistant strains of Staphylococcus aureus to antibiotic inhibitors of protein synthesis. The assay was based on the fact that a brief cell wall digestion with lysostaphin resulted in fragmentation of the chromosomal DNA by releasing the characteristic DNase stored in the cell wall. DNase activity was ascertained by visualization of the DNA fragments released from the isolated nucleoids. Lysostaphin-released DNase activity was found to be influenced by ribosomal protein synthesis. Inhibition of protein synthesis resulted in the prevention of lysostaphin-DNase induced DNA fragmentation when susceptible clinical strains were incubated with erythromycin, azithromycin, or doxycycline for 2 hr before enzymatic treatment. However, in nonsusceptible strains where protein synthesis was unsuccessfully inhibited, this suppression of lysostaphin-DNase was not, or only very slightly, evident. This assay was highly efficient, identifying resistance to erythromycin and azithromycin with 88-90.9% sensitivity and 100% specificity and with 100% sensitivity and specificity to gentamicin and doxycycline, within a 2 hr and 45 min period.

Rapid Detection of Bacterial Susceptibility or Resistance to Quinolones.

The emergence of multidrug resistant microorganisms together with the decline in discovery and development of new antibiotics is of great concern in health-care policy. In this alarming context, an early and well-tailored antibiotic therapy is a relevant strategy not only to improve clinical outcome but also to avoid appearance and spreading of perilous resistant strains. One of the most common antibiotic classes is fluoroquinolones. They trap the DNA girase and/or topoisomerase IV on the DNA, resulting in DNA fragmentation. We have developed the Micromax® assay to determine, in situ, the integrity of the chromosomal DNA-nucleoid from microorganisms. This was validated as a simple procedure for the rapid assessment of the susceptibility or resistance to quinolones in gram-negative bacteria. After incubating with the quinolone, cells are trapped in an agarose microgel on a slide and incubated with a specific lysing solution to remove the cell wall and visualize the nucleoids under fluorescence microscopy. If the strain is susceptible to the quinolone, the bacterial nucleoids show a halo of diffusing DNA spots of fragmented DNA, whereas they appear intact in the resistant strain. The technical processing is performed in 40 min with practically total sensitivity and specificity.

Rapid Assessment of Resistance to Antibiotic Inhibitors of Protein Synthesis in the Gram-Positive Pathogens, Enterococcus faecalis and Streptococcus pneumoniae, Based on Evaluation of the Lytic Response.

A novel assay for rapid determination of resistance to antibiotic inhibitors of protein synthesis was developed for the gram-positive pathogens, Enterococcus faecalis and Streptococcus pneumoniae. To this purpose, a lytic response was obtained by a brief incubation with lysozyme or a mixture of lysozyme, Triton X-100, and EDTA for E. faecalis (n = 82) and S. pneumoniae (n = 51), respectively. Lysis was quantified by visualizing the released nucleoids. Antibiotic-susceptible bacteria treated with Clinical and Laboratory Standards Institute (CLSI) breakpoint doses of erythromycin, azithromycin, or doxycycline that inhibited protein synthesis demonstrated a large reduction of lysed cells with respect to the control, that is, without antibiotics. However, cell lysis prevention was much lower in nonsusceptible strains, with unsuccessful inhibition of protein synthesis. ROC analysis showed that a reduction value of ≥35.6% and ≥40.4% discriminates susceptible and nonsusceptible strains for erythromycin and for doxycycline, respectively, in E. faecalis, whereas ≥20.0% is adequate for both macrolides and doxycycline in S. pneumoniae. Resistant stains were identified in 90-120 min with sensitivity and specificity between 91.7% and 100%. This is a proof of concept that evaluation of the lytic response may be a rapid and efficient test for determination of resistance to antibiotic inhibitors of protein synthesis.

Rapid Detection of Antibiotic Resistance in Gram-Negative Bacteria Through Assessment of Changes in Cellular Morphology.

Rapid antimicrobial susceptibility testing has the potential to improve patient outcomes and reduce healthcare-associated costs. In this study, a novel assay based on bacterial cell elongation after exposure to an antibiotic (ceftazidime) was evaluated for its ability to rapidly detect resistance in Gram-negative bacteria. The assay was used to detect resistance in a large collection of strains containing 320 clinical isolates of Acinetobacter baumannii, 171 clinical isolates of Klebsiella pneumoniae, and 212 clinical isolates of Pseudomonas aeruginosa, and the results were compared to those obtained using standard antimicrobial susceptibility testing methods. The assay identified ceftazidime-resistant strains with 100% sensitivity and 100% specificity for A. baumannii, 100% sensitivity and 97.2% specificity for K. pneumoniae, and with 82.3% sensitivity and 100% specificity for P. aeruginosa. Importantly, results were obtained in 1 hour 15 minutes from exponentially growing cultures. This study demonstrates that changes in cell length are highly correlated with phenotypic antibiotic susceptibility determined using standard susceptibility testing methods. This study therefore provides proof-of-concept that changes in cell morphology can be used as the basis for rapid detection of antibiotic resistance and provides the basis for the development of novel rapid diagnostics for the detection of antibiotic resistance.

Assessment of Chromosomal DNA Fragmentation by Quinolones in an Isogenic Collection of Escherichia coli with Defined Resistance Mechanisms.

The aim of this study was to investigate the potential usefulness of DNA fragmentation as a quick and simple procedure for detecting resistance to fluoroquinolones (FQ) in isogenic Escherichia coli strains harboring defined and multiple quinolone resistance mechanisms, including low-level quinolone resistance (LLQR) phenotypes. DNA fragmentation assay (Micromax(®)) was evaluated for detecting resistance to FQ in 71 isogenic strains of E. coli harboring specific quinolone resistance mechanisms frequently found in clinical isolates. These isogenic strains represent a consistent and reliable model of increasing minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP), ranging from 0.004 to 16 mg/L. According to CLSI criteria, the assay correctly identified all CIP-resistant strains (MIC ≥4 mg/L). As regards susceptible strains, 96% of bacterial strains were correctly assigned as susceptible to CIP. Moreover, the procedure enabled LLQR phenotypes to be efficiently identified; this subset may show different levels of DNA damage depending on the strain, even with similar MIC. Interestingly, despite increasing the dose according to the MIC, a lower response to quinolones occurs in strains with higher MIC values. This is a simple, rapid, and reliable test for evaluating susceptibility to FQ of E. coli, including the detection of strains harboring LLQR mechanisms.

Effect of sperm DNA fragmentation on pregnancy outcome depends on oocyte quality.

OBJECTIVE: To quantify the effect of sperm DNA fragmentation (SDF) on reproductive outcome by evaluating the most statistically significant bias factors using logistic regression. DESIGN: Prospective blind observational cohort study. SETTING: University affiliated private IVF unit. PATIENT(S): Two hundred ten male partners of couples undergoing in vitro fertilization (IVF) or first intracytoplasmic sperm injection (ICSI) cycles with fresh or thawed sperm with the women's own or donated oocytes. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): SDF determined before and after swim-up (n=420), odds ratio calculated of the effect of an increase of one unit of SDF on pregnancy, and stratified regression analysis performed to evaluate the confusion effect of oocyte quality, sperm origin, and the fertilization procedure. RESULT(S): The effect of SDF on pregnancy was not affected by sperm origin (fresh or thawed) or fertilization procedure when measured both before and after swim-up. When oocytes from infertile patients were employed, SDF had a statistically significant negative impact on chance of pregnancy. For every 10% increase in SDF, the probability of not achieving pregnancy increased by 1.31. When donated oocytes were employed, SDF did not have a statistically significant effect. CONCLUSION(S): The effect of SDF on the probability of pregnancy can be calculated independent of the fertilization procedure or sperm origin. Oocyte quality conditions the extent of the negative impact of SDF on pregnancy; this can be overcome when good quality oocytes are employed.

Simultaneous determination in situ of DNA fragmentation and 8-oxoguanine in human sperm.

Deoxyribonucleic acid fragmentation and oxidative DNA damage were simultaneously determined in the same sperm cell, incubating with an 8-oxoguanine DNA probe on human spermatozoa processed by the sperm chromatin dispersion test. The assay was validated by incubation with agents that induce DNA fragmentation with or without oxidative base damage. In all samples examined, increased levels of 8-oxoguanine were present only in those spermatozoa with fragmented DNA, suggesting a link between both DNA damage types.

Sperm DNA fragmentation levels in testicular sperm samples from azoospermic males as assessed by the sperm chromatin dispersion (SCD) test.

OBJECTIVE: To analyze sperm DNA fragmentation (SDF) in testicular sperm samples from patients with azoospermia either from spermatogenic failure or from duct obstruction. Several technologies can be applied in the evaluation of SDF, but given the ease and low costs, the sperm chromatin dispersion test (SCD) has emerged as a promising standard. DESIGN: Prospective blind observational cohort study. SETTING: University-affiliated private IVF setting. PATIENT(S): Azoospermic patients from couples undergoing intracytoplasmic sperm injection cycles. INTERVENTION(S): Testicular sperm extraction (TESE). MAIN OUTCOME MEASUREMENT(S): We determined testicular SDF, and a basic comparison between nonobstructive (n = 22) and obstructive azoospermia (n = 40) was performed. We also correlated SDF with embryo quality and pregnancy outcome. RESULT(S): SDF in the testicular sperm of patients with nonobstructive azoospermia was significantly higher, 46.92% (SEM = 4.47), than that of patients with obstructive azoospermia, 35.96% (SEM = 2.63). A moderate relationship between embryo morphology and testicular SDF was detected. Logistic regression analysis of the effect of testicular SDF on pregnancy outcome revealed no significant effect (odds ratio = 1.015). CONCLUSION(S): Ours is the first report of SDF analysis in testicular sperm by using SCD in azoospermia. This result suggests that spermatogenesis failure may result in a severe affectation of sperm DNA integrity. The degree of DNA fragmentation using the SCD test is not reflected in pregnancy chances, and the explanation could be that embryos have been selected.

The effect of cancer on sperm DNA fragmentation as measured by the sperm chromatin dispersion test.

The percentage of spermatozoa with fragmented DNA from cancer patients before surgery, chemotherapy, or radiotherapy treatments was compared with infertile male patients in an assisted reproduction program and with sperm donors of proven fertility. The percentages of DNA fragmentation were 34.3% in cancer patients, 30.9% in infertile men whose partners did not become pregnant, 28.8% in men who partners became pregnant, and 10.8% in fertile sperm donors. The DNA fragmentation of sperm donors was statistically significantly lower compared the other groups. No statistically significant differences were found in the levels of DNA fragmentation when comparing cancer types, including those of testicular origin.

Sperm DNA fragmentation in infertile men with genitourinary infection by Chlamydia trachomatis and Mycoplasma.

OBJECTIVE: To determine the frequency of sperm cells with fragmented DNA in semen samples from men with genitourinary infection by Chlamydia trachomatis and Mycoplasma and the influence of antibiotic therapy, using the sperm chromatin dispersion test with the Halosperm kit. DESIGN: Prospective study. SETTING: University-affiliated reproductive medicine center, medical genetics laboratory, and academic biology center. PATIENT(S): One hundred forty-three male member of couples attending the andrology infertility center and a group of 50 fertile subjects. The effect of antibiotic treatment was evaluated in 95 male patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Classical semen analysis (concentration, motility, morphology, and vitality), sperm DNA fragmentation, and clinical outcome. RESULT(S): The mean percentage of sperm cells with fragmented DNA was 35.2% +/- 13.5%, 3.2 times higher than in the control fertile group (10.8% +/- 5.6%). Concentration, morphology, and motility were also significantly affected but to a much lower degree. Sperm vitality was not significantly affected. After 3.8 +/- 2.2 months of antibiotic treatment, the mean frequency of spermatozoa with fragmented DNA decreased from 37.7% +/- 13.6% to 24.2% +/- 11.2%. Sperm concentration and motility were not significantly improved. In a group of 16 couples who attempted pregnancy during antibiotic treatment course, only 12.5% achieved pregnancy. However, in a group of 14 couples who attempted pregnancy after finishing the antibiotic treatment, 85.7% achieved it. The only significant differences found between groups was the rate of sperm DNA fragmentation and morphology. CONCLUSION(S): Patients with genitourinary infection by Chlamydia trachomatis and Mycoplasma have increased sperm DNA fragmentation in comparison with fertile controls. This increase is proportionally greater than the influence on classical semen parameters and could result in a decreased fertility potential. Antibiotic therapy appears to be important in providing a remedy for infection-induced high DNA fragmentation levels.