Novel association between sperm reactive oxygen species production, sperm morphological defects, and the sperm deformity index.

OBJECTIVE: To examine the relationship between sperm reactive oxygen species (ROS) production and sperm morphology in a group of infertile men and healthy fertile donors. DESIGN: A prospective clinical study. SETTING: Male infertility clinic, Glickman Urological Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, and the Reproductive Medicine Unit, Liverpool Women's Hospital, United Kingdom PATIENT(S): Thirty-nine infertile men and 13 healthy fertile donors (control). INTERVENTION(S): Standard semen analysis, seminal leukocyte concentration, assessment of sperm morphology, and measurement of sperm ROS production. MAIN OUTCOME MEASURE(S): Levels of sperm ROS production, percentages of different sperm morphological abnormalities, and the sperm deformity index (SDI) scores. RESULT(S): A significant negative correlation was observed between sperm ROS production and the proportion of sperm with normal morphology and borderline morphology. Reactive oxygen species production was positively correlated with the proportion of sperm with amorphous heads, damaged acrosomes, midpiece defects, cytoplasmic droplets, tail defects, and SDI scores. Logistic regression analysis identified a two-variable model including SDI and percentage sperm motility, which correctly identified 84% of individuals with high seminal ROS and 85% of individuals with low seminal ROS. The model had an overall accuracy of 85%. CONCLUSION(S): The standard semen analysis to assess sperm motility, sperm morphology, and the SDI scores is a useful tool in identifying infertile men with high seminal ROS in infertility clinics where facilities for measuring levels of seminal ROS are not available.

Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele.

OBJECTIVE: To examine levels of sperm DNA damage and oxidative stress (OS) in infertile men with varicocele. DESIGN: Prospective controlled study. SETTING: Male infertility clinic, Glickman Urological Institute, Cleveland Clinic Foundation, Cleveland, Ohio. PATIENT(S): Thirty-one infertility patients and 16 fertile controls. INTERVENTION(S): Sperm DNA fragmentation index (DFI), levels of seminal reactive oxygen species (ROS), and total antioxidant capacity (TAC) were assessed using the sperm chromatin structure assay, chemiluminescence assay, and enhanced chemiluminescence assay, respectively. ROS-TAC score was calculated as a measure of OS. MAIN OUTCOME MEASURE(S): Median (interquartile range) DFI and ROS-TAC scores. RESULT(S): Sixteen of the 31 patients had left varicocele [grade I (n = 3), grade II (n = 10), and grade III(n = 3)], and the remaining 15 had normal genital examination. Patients with varicoceles had significantly higher percent DFI than controls (25%, range: 20%-35%; vs. 15%, range: 10%-22%). Patients with varicoceles had significantly lower ROS-TAC scores (21, range: 9.5-31) than the infertile patients with normal genital examination (34, range: 28-42) or the controls (40.3, range: 38-44). CONCLUSION(S): Infertile men with varicoceles showed significantly increased spermatozoal DNA damage that appears to be related to high levels of OS in semen.

Utility of the nitroblue tetrazolium reduction test for assessment of reactive oxygen species production by seminal leukocytes and spermatozoa.

The purpose of this study was to evaluate the ability of spermatozoa and leukocytes in semen to produce reactive oxygen species (ROS) by using nitroblue tetrazolium (NBT) staining and to examine the association between NBT staining and levels of ROS as measured by chemiluminescence. Twenty-one infertility patients (leukocytospermia; n = 8; nonleukocytospermia, n = 13) and 9 healthy donors were included. Standard semen analysis and density gradient centrifugation were performed to test NBT staining, ROS, and total antioxidant capacity. A ROS-total antioxidant capacity (ROS-TAC) score was calculated by using principal component analysis. In the leukocytospermic group, after separation on a density gradient, the percentage of NBT-positive staining was significantly higher in sperm suspensions contaminated with leukocytes (median [25th, 75th percentiles]; 70% [61%, 79%]) compared to the nonleukocytospermic group (14.5% [9%, 25.5%]; P =.03) and donors (7% [3%, 11%]; P =.02), respectively. A strong positive correlation was seen between levels of ROS in whole ejaculates and NBT-positive staining in leukocytes (r = 0.59; P <.0006) and in leukocyte fractions (r = 0.72; P <.0001) after density gradient separation. Similarly, ROS was positively correlated with excessive cytoplasmic retention in spermatozoa from whole ejaculates and abnormal spermatozoa after separation on density gradients (r = 0.72; P <.0001). The ROS-TAC score was inversely correlated with NBT staining in leukocytes in whole ejaculates (r = -0.960, P <.0007) and in both leukocyte fractions (r = -0.39; P <.04) and spermatozoa with cytoplasmic retention (r = -0.38; P <.04). Our results indicate that the NBT reduction test can be used to assess the contribution of seminal leukocytes and defective spermatozoa towards ROS generation in semen. Levels of ROS assessed by chemiluminescence assay are strongly correlated with the results of NBT staining.

Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility.

OBJECTIVE: To examine the effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Prospective study. SETTINGS: Infertility clinic at a tertiary care academic institution. PATIENT(S): Ninety-two infertile men with normal female partners. Sixteen fertile donors served as the control group. INTERVENTION(S): Standard semen analysis and assessment of levels of seminal oxidative stress. Assisted reproductive techniques in 33 of the 92 patients (IUI [n = 19], IVF [n = 10], and intracytoplasmic sperm injection [n = 4]). MAIN OUTCOME MEASURE(S): Sperm DNA damage by sperm chromatin structure assay. Results were expressed as DNA fragmentation index. RESULT(S): Patients were classified as having either idiopathic (n = 23) or male factor infertility (n = 69). Patients with idiopathic and male factor infertility had significantly higher DNA fragmentation index and oxidative stress compared with the case of fertile donors. A clinical pregnancy was achieved in 9 (27%) of 33 patients with assisted reproductive techniques. Significantly higher DNA fragmentation index and oxidative stress were found in men who failed to initiate a pregnancy after assisted reproductive techniques (n = 24), compared with the cases of those who succeeded and of the fertile donors. DNA fragmentation index was correlated positively with oxidative stress (r = 0.27), and negatively with fertilization (r = -0.70) and embryo quality (r = -0.70). CONCLUSION(S): Sperm DNA damage is significantly increased in men with idiopathic and male factor infertility and in men who failed to initiate a pregnancy after assisted reproductive techniques. Such an increase may be related to high levels of seminal oxidative stress.

Role of reactive oxygen species in the pathophysiology of human reproduction.

OBJECTIVE: To summarize the role of reactive oxygen species (ROS) in the pathophysiology of human reproduction. DESIGN: Review of literature. SETTING: Fertility research center and obstetrics and gynecology department in a tertiary care facility. RESULT(S): ROS plays an essential role in the pathogenesis of many reproductive processes. In male-factor infertility. oxidative stress attacks the fluidity of the sperm plasma membrane and the integrity of DNA in the sperm nucleus. Reactive oxygen species induced DNA damage may accelerate the process of germ cell apoptosis, leading to the decline in sperm counts associated with male infertility. ROS mediated female fertility disorders share many pathogenic similarities with the ones on the male side. These similarities include a potential role in the pathophysiology of endometriosis and unexplained infertility. High follicular fluid ROS levels are associated with negative IVF outcomes, particularly in smokers. Moreover, oxidative stress may be responsible in hydrosalpingeal fluid mediated embryotoxicity as well as poor in vitro embryonic development. CONCLUSION(S): High levels of ROS are detrimental to the fertility potential both in natural and assisted conception states.

Sexual dysfunction in men undergoing infertility evaluation: a cohort observational study.

OBJECTIVE: To study psychosexual problems in men undergoing infertility evaluation. DESIGN: A cohort observational study. SETTING: Male infertility diagnostic center. PATIENT(S): Four hundred twelve men undergoing infertility evaluation between 1999 and 2001. INTERVENTION(S): Baseline and follow-up data on sexual functions. Semen analysis for samples collected by masturbation. A second analysis was requested in 2 weeks upon finding an abnormality of semen parameters. Penile vibratory stimulation to help men who failed to collect semen on their second or subsequent appointments. MAIN OUTCOME MEASURE(S): Sexual functions (erection and orgasm) during the time of infertility evaluation. RESULT(S): Seven of 412 men were excluded from the analysis due to a past history of sexual dysfunction. Of the remaining 405 men, 46 (11%) failed to collect semen by masturbation for a second semen analysis after repeated (2-4 times) attempts at 2- to 3-day intervals. Nine of these men (20%) were able to collect semen using vibratory stimulation. All 46 men experienced problems with erection or orgasm and had severe anxiety during attempts to masturbate and during sexual contact with their partners. CONCLUSION(S): Our study indicates that some men may experience sexual dysfunction of a psychogenic nature in response to the diagnosis of infertility.

Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa.

To investigate the role of increased seminal leukocytes in enhancing reactive oxygen species (ROS) production by human spermatozoa.A prospective study. Male infertility clinic.Forty-eight infertile men. Standard semen analysis. Assessment of sperm nuclear DNA damage by sperm chromatin structure assay. Incubation of spermatozoa from nonleukocytospermic samples with blood neutrophils. Spontaneous and phorbol 12-myristate 13-acetate (PMA)-induced ROS production in pure-sperm suspensions (after removal of leukocytes) as measured by a chemiluminescence assay. Levels of spontaneous and PMA-induced ROS production in pure-sperm suspensions from the infertile men with a diagnosis of leukocytospermia (n = 16) were significantly higher compared with the case of infertile men without leukocytospermia (n = 32) and with the case of a control group of healthy volunteers (n = 13). A similar pattern of increased ROS was observed when spermatozoa were incubated with blood neutrophils. Leukocytospermia was associated with a significant decrease in sperm motility and increase in DNA damage. Increased seminal leukocytes may play a role in stimulating ROS production by human spermatozoa. Such stimulation may be mediated via direct cell-cell contact or by soluble products released by leukocytes. Poor sperm quality in leukocytospermic samples may be due to leukocyte-mediated oxidative stress.

Effects of temperature on sperm motion characteristics and reactive oxygen species.

OBJECTIVE: To evaluate the effect of temperature on sperm motility, sperm motion characteristics, and production of reactive oxygen species (ROS) in semen. METHODS: We collected semen samples from 12 infertile patients and 12 healthy donors and divided each sample into four aliquots just after liquefaction. One aliquot was analyzed immediately after liquefaction and the remaining three were analyzed after one hour of incubation at 4 degrees C, 25 degrees C, and 37 degrees C. Sperm motility and motion characteristics were measured by computer-assisted sperm analysis (CASA). Levels of ROS were determined in washed sperm suspensions using a chemiluminescence assay and results were expressed as x10(6) counted photons per minute (cpm)/20 million sperm/mL. RESULTS: Motility, curvilinear velocity, straight-line velocity, average path velocity, and amplitude of lateral head displacement decreased significantly from baseline values in both the patients and donors after incubation at 4 degrees C. Percentage motility was highest and ROS levels were lowest in the samples that were incubated at 37 degrees C. CONCLUSION: Based on our results, we suggest that semen samples be stored at 37 degrees C after collection and during transportation and processing.

Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a prospective study.

OBJECTIVE: To investigate levels of seminal oxidative stress (OS) and sperm quality in a group of infertile men with a history of cigarette smoking. DESIGN: A prospective clinical study. SETTING: Male infertility clinic, Urological Institute, the Cleveland Clinic Foundation, Cleveland, Ohio. PATIENT(S): Infertile men who smoked cigarettes (n = 20), infertile men who were nonsmokers (n = 32), and healthy nonsmoking donors (n = 13). INTERVENTION(S): Genital examination, standard semen analysis, sperm DNA damage. MAIN OUTCOME MEASURE(S): Levels of seminal reactive oxygen species (ROS) and total antioxidant capacity (TAC) measured by a chemiluminescence assay and seminal OS assessed by calculating a ROS-TAC score. Sperm DNA damage was measured by sperm chromatin structure assay. RESULT(S): Smoking was associated with a 48% increase in seminal leukocyte concentrations (P<.0001), a 107% increase in ROS levels (P=.001), and a 10-point decrease in ROS-TAC scores (P=.003). Differences in standard sperm variables and DNA damage indices between the infertile smokers and infertile nonsmokers were not statistically significant. CONCLUSION(S): Infertile men who smoke cigarettes have higher levels of seminal OS than infertile nonsmokers. Given the potential adverse effects of seminal OS on fertility, physicians should advise infertile men who smoke cigarettes to quit.

Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study.

OBJECTIVE: To evaluate levels of sperm nuclear DNA damage in infertile men with normal and abnormal standard semen parameters. DESIGN: Prospective study. SETTING: Male infertility clinic. PATIENT(S): Ninety-two men seeking infertility treatment and 16 fertile volunteers. INTERVENTION(S): Standard semen analysis was performed according to the World Health Organization guidelines. MAIN OUTCOME MEASURE(S): Sperm DNA damage was assessed by sperm chromatin structure assay and the results expressed as %DFI. RESULT(S): Of the 92 patients, 21 (23%) had normal standard sperm parameters (concentration, motility, and normal sperm forms), while 71 (77%) had an abnormality in one or more of these parameters. The %DFI [median (25th and 75th percentiles)] in infertile men with normal sperm parameters [23 (15, 32)] was significantly higher than fertile donors [15 (11, 20)] (P=.02), but not significantly different from infertile men with abnormal sperm parameters [28 (18, 41)] (P=.27). CONCLUSION(S): The results of this study indicate that a significant increase in SCSA-defined DNA damage can be found in sperm from infertile men with normal standard sperm parameters. Therefore, sperm DNA damage analysis may reveal a hidden abnormality of sperm DNA in infertile men classified as idiopathic based on apparently normal standard sperm parameters.

Increased DNA damage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay.

OBJECTIVE: To determine DNA damage as measured by the sperm chromatin structure assay (SCSA) in subsets of human spermatozoa at different stages of maturation in patients who are undergoing infertility evaluation. DESIGN: Prospective study. SETTING: Andrology laboratory at a tertiary care hospital. PATIENT(S): Fifty-six patients undergoing infertility evaluation. Patients with normal semen parameters (n = 17), abnormal semen parameters (n = 29), leukocytospermia (n = 10), and a group of healthy fertile men (n = 18) were included in the study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The shift of green (native DNA) to red (denatured, single-stranded DNA) fluorescence was measured and quantified using the expression alpha(t) (red fluorescence/[red + green fluorescence] per cell). Sperm DNA damage was examined in subsets of spermatozoa isolated by a three-step density gradient. The DNA damage was correlated with classic semen characteristics. RESULT(S): Leukocyte concentration in semen was directly correlated with chromatin alterations in immature and mature sperm. Leukocyte concentration in semen was also directly correlated with immature germ cell concentration and the percentage of abnormal forms in semen. CONCLUSION(S): The increase in chromatin alterations and DNA damage in sperm, as defined by the sperm chromatin structure assay from leukocytospermic samples may be related to alterations in the regulation of spermatogenesis.

Positive bacterial culture of semen from infertile men with asymptomatic leukocytospermia.

OBJECTIVE: To examine results of semen culture in a population of infertile men with asymptomatic leukocytospermia, and to determine the correlation between culture results and sperm characteristics in these patients. METHODS: Semen samples were collected from a group of infertile men (n = 80) after 2-3 days of sexual abstinence. Standard semen analysis was performed according to World Health Organization (WHO) guidelines. Seminal leukocyte concentrations were determined by a myeloperoxidase staining technique. Culture of semen was performed on enriched and specific culture media. RESULTS: Based on the results of semen culture, the samples were classified into three groups: group 1 (enteric gram-negative bacilli [Enterobacteriaceae], n = 13), group 2 (bacteria other than Enterobacteriaceae, n = 15), and group 3 (negative cultures, n = 52). No correlation was found between positive semen culture and sperm characteristics (concentration, motility, and morphology). However, seminal leukocyte concentrations were negatively correlated with percentages of motile sperm and normal sperm morphology. CONCLUSION: Our data indicate that semen samples from infertile men with asymptomatic leukocytospermia may have bacterial contamination, evidenced by positive bacterial cultures. Lack of correlation between positive semen cultures and sperm characteristics may be indicative of early or mild (subclinical) infection.

Role of oxidants in male infertility: rationale, significance, and treatment.

Interest in the physiologic and pathologic effects of ROS on male fertility is growing. The controlled generation of very low amounts of ROS seems to regulate the acquisition of sperm-fertilizing ability. High levels of ROS endanger sperm function and viability. Oxidative stress can arise as a consequence of excessive production of ROS or impaired antioxidant defense mechanisms in semen. Oxidative stress precipitates a range of pathologies that are thought to affect the male reproductive system. ROS-mediated peroxidative damage to the sperm plasma membrane may account for the defective sperm functions observed in a high proportion of infertility patients. High levels of ROS in semen have been correlated with reduced sperm motility and damage to sperm nuclear DNA. Determining the levels and the sources of excessive ROS production in human semen and precise evaluation of the scavenger system may be useful tools to develop therapeutic strategies for male infertility. Therapeutic interventions directed toward the isolation of mature spermatozoa by in vitro separation techniques may benefit infertile men in whom oxidative stress has a significant role. Nevertheless, this approach may be limited by the fact that the prolonged centrifugation involved in these procedures may enhance ROS production by spermatozoa. Clinical trials using antioxidants in vivo and in vitro have resulted in a major debate, and further research is required before one can be optimistic about a role for antioxidants in the treatment of infertile men. A double-blind, placebo-controlled study underway at the authors' center is evaluating the effects of treating infertile men with a combination of vitamins E and C after adjusting for all causes of controversy.