Analysis of the Functional Aspects of Sperm and Testicular Oxidative Stress in Individuals Undergoing Metabolic Surgery.

PURPOSE: Metabolic surgery is a recommended treatment for obese patients that results in BMI reduction; however, the observed impact of this therapy on male fertility is inconsistent. This research aimed to study the effects of BMI changes after metabolic surgery on seminal analysis, sex hormonal profile, sperm functional integrity, and the seminal plasma lipid peroxidation levels. MATERIALS AND METHODS: A prospective study was performed in 15 patients for whom metabolic surgery was recommended. The patients were evaluated by the techniques proposed in this study before and after the surgical procedure for 12 months. In each analysis, the male sex hormonal profile, semen analysis, sperm functional integrity, and seminal lipid peroxidation levels were assessed. RESULTS: The surgery resulted in BMI reduction and improvement in seminal characteristics and male sex hormone profile. The semen analysis showed increases in volume, sperm progressive motility, and in sperm morphology and a decrease in immotile sperms. Sperm mitochondrial activity and sperm DNA integrity were improved, and the levels of seminal lipid peroxidation were decreased. The hormonal profile showed lower levels of estradiol and highest levels of luteinizing hormone (LH), sex hormone-binding globulin (SHBG), and testosterone. CONCLUSION: BMI changes resulting from this treatment and its metabolic consequences can be associated with changes in the male fertile potential, leading to an improvement in the seminal quality, male sex hormone profile, sperm functional aspects, and levels of seminal lipid peroxidation, thus decreasing the testicular oxidative stress.

Evaluation of oxidative stress in seminal plasma of adolescents with varicocele.

Varicocele, defined by a dilation of efferent testicular veins, is the most commonly identifiable, surgically correctable lesion associated with male-factor infertility, starts at puberty and causes a progressive decline in fertility potential. The pathophysiology of infertility caused by this disease is still poorly understood, but it is suggested that the main mechanism is oxidative stress. Therefore, the aim of this study was to verify if the varicocele is associated with changes in enzymatic antioxidant mechanisms and seminal plasma lipid peroxidation levels in adolescents. We recruited 90 adolescents that were divided into control (C; n = 27); varicocele and normal semen (VNS; n =46); varicocele and altered semen (VAS; n =17). Seminal and serum levels of lipid peroxidation were quantified by thiobarbituric acid reactive substances (TBARS). Seminal plasma antioxidant profile was evaluated by the activities of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD). The VAS group had increased lipid peroxidation levels when compared to the other groups. The levels of serum lipid peroxidation and activities of the enzymes SOD and GPx did not differ between groups. CAT was undetectable by the method used. In conclusion, in adolescents with varicocele and altered semen analysis, there is an increase in seminal lipid peroxidation levels compared to adolescents with varicocele and without seminal change and adolescents without evident varicocele. However, the observed oxidative stress is not caused by a decrease in superoxide dismutase and glutathione peroxidase activities, which did not differ between adolescents with and without evident varicocele. LAY SUMMARY: Varicocele, defined by a dilation of efferent testicular veins, is the most commonly identifiable, surgically correctable lesion associated with male-factor infertility, starts at puberty and causes a progressive decline in fertile potential. There is still much that is not understood regarding how exactly it affects semen quality, but most studies agree that oxidative stress, which is defined as excessive amounts of free radicals in relation to antioxidant defense, is an important mechanism. In this study, we aimed to verify if the varicocele is associated with changes in antioxidant defense and semen oxidation in 90 adolescents with and without varicocele. In adolescents with varicocele and abnormal semen, there is an increase in semen oxidation compared to controls or to the group with varicocele and normal semen quality. Our results can help to understand how varicocele leads to infertility in adolescents, identifying changes in oxidative activity in semen, since the onset of varicocele and before damage to sperm production can be detected.

Restoration of the apoptosis pathways' proteins levels after orchiectomy in testicular tumour patients.

Seminal plasma proteins already demonstrated to reflect the testicular environment function and important regulatory mechanisms. However, it is crucial to understand which of these proteins participate in probable altered pathways in testicular germ cell tumours and after unilateral orchiectomy. In this study, we proposed to verify, by a multiplex approach, the levels of DNA damage and apoptosis pathways' proteins, in seminal plasma of men before and after unilateral orchiectomy, and also in control men. Comparing pre- and post-orchiectomy groups, just the apoptosis pathways' proteins presented different levels, in which Bad was lower and Bcl2, Akt, caspase-9, p53 and caspase-8 were higher after orchiectomy. When comparing pre- and post-orchiectomy groups with control, both presented lower levels of ChK1, Chk2, H2AX, p53 and p21, for DNA damage pathway. Regarding the apoptosis pathway, lower levels of JNK, Bcl2, Akt, caspase-9, p53 and caspase-8 and higher levels of Bad were observed before orchiectomy. The post-orchiectomy group did not differ from controls, demonstrating a probable restoration on its proteins levels. We can conclude that testicular tumours can alter both of the assessed pathways, and its removal is associated with a probable restoration of the apoptosis pathway.

One day is better than four days of ejaculatory abstinence for sperm function.

According to the World Health Organization guidelines, ejaculatory abstinence (EA) of 2-7 days is recommended for semen analysis. This study aimed to determine how seminal quality may be affected by two EA periods from the same man. Seminal samples from 65 men were evaluated by conventional semen analysis and qualitative characteristics after 1 and 4 days of EA (two samples/man). The semen was qualitatively analyzed by examining oxidative activity (intracellular and seminal plasma), sperm function (acrosome integrity, mitochondrial activity, and nuclear DNA integrity), and epididymal function. As expected, samples collected after 1 day of EA showed a decrease in volume and sperm total number compared to samples collected after 4 days of EA. The sperm motility of the samples collected after 1 day of EA was better compared to samples collected after 4 days of EA. Oxidative activity measured was lower after 1 day of EA compared with those measured after 4 days of EA. With regards to sperm function, samples collected after 1 day of EA showed an increase in acrosome integrity, mitochondrial activity, and nuclear DNA integrity compared with samples collected after 4 days of EA. Epididymal function showed no difference between the two-time points. Although samples collected after 4 days of EA showed better results for sperm quantity, samples collected after 1 day of EA showed better qualitative results, including motility, oxidative activity, and sperm function. Thus, it can be concluded that sperm storage at the epididymal tail may make spermatozoa more susceptible to oxidative damage. LAY SUMMARY: According to the World Health Organization guidelines, stopping ejaculation for 2 to 7 days is recommended before sperm collection for semen analysis. However, the evidence that supports these recommendations is limited. Our study aimed to compare how sperm quality was affected in samples collected after stopping ejaculation for 1 day and 4 days (two samples per man) in a total of 65 men. Although sample collection after stopping ejaculation for 4 days showed better semen quantity (volume and sperm concentration), sample collection after stopping ejaculation for 1 day showed better sperm motility and function. If not ejaculated, sperm are stored in the epididymis tail located in the scrotum beside the testicles and our study suggests that longer sperm storage may damage sperm quality. The results from this study may be used to inform guidance for sperm collection for use in assisted reproduction techniques, and lead to an improvement in both fertilization and implantation rates.

Effect of orchiectomy on sperm functional aspects and semen oxidative stress in men with testicular tumours.

To verify if quality of spermatozoa from men with testicular germ cell tumours is better before or after orchiectomy. This prospective study was carried out including 24 patients with testicular germ cell tumours, who provide one semen sample before they were submitted to unilateral orchiectomy and one other semen sample 30 days after the surgery. After collection by masturbation and liquefaction, an aliquot of the semen sample was used for semen analysis and another was used to evaluate sperm mitochondrial activity, DNA fragmentation and acrosome integrity. Seminal plasma was used to evaluate lipid peroxidation levels. Pre-orchiectomy sample and post-orchiectomy sample were compared using a paired Student's t test (normal distribution) or a paired Wilcoxon test, when appropriate (p Ë‚ 0.05). No significant difference was observed in semen analysis. A significant decrease in DNA fragmentation and lipid peroxidation and an increase in mitochondrial activity were observed after orchiectomy. Based on our findings, the semen quality from men with testicular germ cell tumours is better after orchiectomy.

Antioxidant enzyme profile and lipid peroxidation products in semen samples of testicular germ cell tumor patients submitted to orchiectomy

ABSTRACT Purpose To determine enzymatic antioxidant and lipid peroxidation levels in seminal plasma of patients orchiectomized for testicular tumors. Materials and Methods The study included 52 patients: 26 control men and 26 orchiectomized patients for testicular tumor, of which 12 men had seminoma tumor and 14 men non-seminoma tumor. After semen analysis performed according to the WHO guidelines, an aliquot of semen was centrifuged and the seminal plasma was collected. Lipid peroxidation was performed by thiobarbituric acid reactive substances (TBARS) assay and antioxidant profile was assessed by analyzing catalase, glutathione peroxidase (GPx) and superoxide anion (SOD) activities using colorimetric assays with a standard spectrophotometer. Data were tested for normality and compared using one-way ANOVA (p<0.05). Results Seminoma and non-seminoma groups presented lower sperm concentration and morphology when compared to control group (p=0.0001). Both study groups (seminoma and non-seminoma) presented higher TBARS levels when compared to control group (p=0.0000013). No differences were observed for SOD (p=0.646) andGPx (p=0.328). It was not possible to access the enzymatic activity of catalase in any group. Conclusion Patients with testicular tumor present increased semen oxidative stress, but no differences were observed in antioxidant levels, even after orchiectomy. This indicates that most likely an increased generation of oxidative products takes place in these patients.

Antioxidant enzyme profile and lipid peroxidation products in semen samples of testicular germ cell tumor patients submitted to orchiectomy.

PURPOSE: To determine enzymatic antioxidant and lipid peroxidation levels in seminal plasma of patients orchiectomized for testicular tumors. MATERIALS AND METHODS: The study included 52 patients: 26 control men and 26 orchiectomized patients for testicular tumor, of which 12 men had seminoma tumor and 14 men non-seminoma tumor. After semen analysis performed according to the WHO guidelines, an aliquot of semen was centrifuged and the seminal plasma was collected. Lipid peroxidation was performed by thiobarbituric acid reactive substances(TBARS) assay and antioxidant profile was assessed by analyzing catalase, glutathione per-oxidase (GPx) and superoxide anion (SOD) activities using colorimetric assays with a standard spectrophotometer. Data were tested for normality and compared using one-way ANOVA (p<0.05). RESULTS: Seminoma and non-seminoma groups presented lower sperm concentration and morphology when compared to control group (p=0.0001). Both study groups (seminoma and non-seminoma) presented higher TBARS levels when compared to control group (p=0.0000013). No differences were observed for SOD (p=0.646) and GPx (p=0.328). It was not possible to access the enzymatic activity of catalase in any group. CONCLUSION: Patients with testicular tumor present increased semen oxidative stress, but no differences were observed in antioxidant levels, even after orchiectomy. This indicates that most likely an increased generation of oxidative products takes place in these patients.

Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation.

PURPOSE: Sperm DNA fragmentation has been suggested as a marker for infertility diagnosis and prognosis. Hence, understanding its impact on male physiology and post-genomic pathways would be clinically important. We performed the proteomics and functional enrichment analyses of viable spermatozoa from ejaculates with low and high sperm DNA fragmentation to identify protein expression and pathways altered in association with sperm DNA fragmentation. METHODS: Sperm DNA fragmentation using the Comet assay and the Komet 6.0.1 software was assessed in raw samples from 89 subjects from a human reproduction service. The Low and High sperm DNA fragmentation groups were formed according to the Olive Tail Moment variable. Spermatozoa proteins from these groups were pooled and analyzed by a shotgun proteomic approach (2D nanoUPLC-ESI-MS(E)). Differentially expressed proteins were used for a functional enrichment study. RESULTS: Two hundred and fifty-seven proteins were identified or quantified in sperm from the Low and High sperm DNA fragmentation groups. Of these, seventy-one proteins were exclusively or overexpressed in the Low group, whereas twenty-three proteins were exclusively or overexpressed in the High group. One hundred and sixty-three proteins were conserved between these groups. We also functionally related the differentially expressed proteins in viable spermatozoa from the groups. Processes such as triacylglycerol metabolism, energy production, protein folding, response to unfolded proteins, and cellular detoxification were found to be altered in these cells. CONCLUSIONS: Sperm DNA fragmentation is associated with differential protein expression in viable spermatozoa. These proteins may potentially be used as biomarkers for sperm DNA integrity.

Sperm nuclear DNA fragmentation rate is associated with differential protein expression and enriched functions in human seminal plasma.

OBJECTIVE: To analyse the proteomic profile of seminal plasma with the aim of identifying the proteins and post-genomic pathways associated with sperm DNA fragmentation. MATERIALS AND METHODS: A cross-sectional study including 89 subjects from a human reproduction service was carried out. All semen samples were assessed for sperm DNA fragmentation using a comet assay. Results from 60 sperm were analysed using Komet 6.0.1 software and the 'Olive tail moment' variable was used to stratify these into low and high sperm DNA fragmentation groups. Seminal plasma proteins from the two groups were pooled and used for proteomic analysis. Quantitative data were used for functional enrichment studies. RESULTS: Seventy-two proteins were identified or quantified in seminal plasma. Of these, nine were differentially expressed in the low group and 21 in the high group. Forty-two proteins were conserved between these groups. Functional enrichment analysis indicated that sperm DNA fragmentation was related to functions such as lipoprotein particle remodelling and regulation, fatty acid binding and immune response. Proteins found exclusively in the low group may be involved in correcting spermatogenesis and/or improving sperm function. Proteins in the high group were associated with increased innate immune response, sperm motility and/or maturation and inhibition of mitochondrial apoptosis. CONCLUSION: Protein expression and post-genomic pathways of seminal plasma differ according to the rate of sperm DNA integrity.

Differential seminal plasma proteome according to semen retrieval in men with spinal cord injury.

OBJECTIVE: To evaluate protein expression profile and to quantify proteins present in seminal plasma from men with spinal cord injury (SCI) and healthy men without SCI. DESIGN: Experimental study. SETTING: University hospital. PATIENT(S): Twelve SCI patients divided into two groups, six who underwent electroejaculation (EEJ) and six who underwent penile vibratory stimulation (PVS); and ten control subjects presenting normal sperm motility and concentration. INTERVENTION(S): EEJ and PVS. MAIN OUTCOME MEASURE(S): The seminal plasma protein profile was analyzed by two proteomic strategies: data-independent label-free quantitative proteomics (MS(E)) and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). RESULT(S): A total of 638 different proteins were identified by MS(E) and 18 by 2D SDS-PAGE followed by tandem mass spectrometry. Interactome analysis showed key reproductive biologic processes-insemination, sperm and oocyte fusion, and acrosome reaction-related to all groups, as were triglyceride stimuli. Processes related to actin and muscle function and to iron oxidation, transportation, and homeostasis were found only in the EEJ and PVS groups; response to hydrogen peroxide and increased immune response was found only in the PVS group. CONCLUSION(S): This study was able to demonstrate differential protein expression among control, PVS, and EEJ groups; SCI is responsible for alterations in seminal plasma protein profile leading to a deviation from homeostasis; proteins reported in both PVS and EEJ groups correlate with the pathophysiology of SCI-related infertility.

Changes in the seminal plasma proteome of adolescents before and after varicocelectomy.

OBJECTIVE: To compare seminal plasma protein profiles before and after varicocele correction to assess if surgical intervention alters the protein profile. DESIGN: Prospective study. SETTING: Academic research environment. PATIENT(S): Nineteen adolescent boys with varicocele grades II or III. INTERVENTION(S): Two semen samples were collected before bilateral subinguinal microsurgical varicocelectomy, and two semen samples were collected 3 months after surgery. Seminal plasma protein profiles were determined with the use of two-dimensional gel electrophoresis. Proteins were separated in 18-cm 3-10 pH strips and 10%-17.5% gradient gels. Gels were stained, scanned, and compared with the use of Imagemaster 2D platinum 7.0. Spots of interest were removed from gels, and protein digestion was performed with the use of trypsin. Digests were identified with the use of electrospray ionization-quadrupole/time-of-flight tandem mass spectrometry (ESI-QTOF MS/MS), and spectra were analyzed with the use of the Mascot software. MAIN OUTCOME MEASURE(S): Proteins uniquely or overexpressed in each period (before or after varicocelectomy). RESULT(S): Nineteen spots were differentially expressed between pre- and postsurgery samples. Identified proteins were albumin, proteasome subunit alpha type 6, alpha-1-antitrypsin, fibronectin, CD177, prostatic acid phosphatase, specific prostatic antigen, alpha-2-antiplasmin, vitamin D-binding protein, gastricsin, clusterin, semenogelin-1, semenogelin-2, superoxide dismutase, protein-glutamine gamma glutamyltransferase-4, and prolactin-inducing protein. CONCLUSION(S): Varicocelectomy is associated with changes in the seminal plasma protein profile. Understanding specific pathways leading to male infertility may further assist physicians in demonstrating deviation from homeostasis in male infertility. In addition, it may be possible to observe if surgical intervention does indeed revert altered pathways toward a homeostatic state.

Proteomic analysis of seminal plasma in adolescents with and without varicocele.

OBJECTIVE: To compare proteomic profiles of seminal plasma from adolescents with varicocele and changes in semen quality with the plasma from adolescents with varicocele without seminal changes and from adolescents without varicocele. DESIGN: Observational study. SETTING: Patients in an academic research environment. PATIENT(S): Adolescents without varicocele (control group), adolescents with varicocele and normal semen quality (VNS group), adolescents with varicocele and abnormal semen quality (VAS group). INTERVENTION(S): Two semen collections at 1-week interval. Protein separation by two-dimensional protein electrophoresis, analysis by gel densitometry, and identification by mass spectrometry. MAIN OUTCOME MEASURE(S): Overexpressed proteins in each group, observed by increased densitometric signal in gels, and exclusively identified proteins in each group. RESULT(S): No differences were observed among the three groups regarding clinical parameters. In semen analysis, the VAS group presented lower sperm concentration, motility, and morphology compared with the VNS and control groups. Forty-seven protein spots of interest were submitted to mass spectrometry identification. Apoptosis regulation proteins were overexpressed in the VAS group, whereas spermatogenesis proteins were overexpressed in the VNS group. Controls presented proteins related to homeostasis. CONCLUSION(S): Changes in the proteomic profile of adolescents with varicocele and normal semen parameters (VNS group) indicate that normal semen analysis may not reflect alterations in proteins in seminal plasma. Implementation of proteomics will help characterize proteins identified in seminal plasma and will facilitate detection of new proteins associated with spermatogenesis and sperm function.

Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele.

STUDY QUESTION: What are the effects of smoking on the functional aspects of the sperm, the levels of lipid peroxidation and the protein profile of seminal plasma in patients with varicocele? SUMMARY ANSWER: In men with varicocele, smoking is associated with altered semen quality, decreased sperm functional integrity and seminal oxidative stress. Alterations in seminal plasma protein profiles are also present and may explain the altered semen phenotype. WHAT IS KNOWN ALREADY: Varicocele is a major cause of male infertility. It reduces testicular blood renewal with a consequent accumulation of toxic substances. Thus, it can potentiate the toxic effects of environmental exposure to genotoxic substances such as those found in cigarette smoke. STUDY DESIGN, SIZE AND DURATION: A cross-sectional study was performed in 110 patients presenting with variococele to the Human Reproduction Section of the Sao Paulo Federal University (2006-2010). The patients were divided into a control group of non-smokers, a moderate smokers group and a heavy smokers group. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Semen parameters were analysed by standard methods. Sperm DNA integrity and mitochondrial activity were assessed by Comet assays and by 3,3'-diaminobenzidine deposition, respectively. The level of lipid peroxidation in semen was determined by malondialdehyde quantification. Proteomic studies were performed by 2D-electrophoresis and mass spectrometry. MAIN RESULTS AND THE ROLE OF CHANCE: Both groups of smokers showed reduced semen quality in comparison with the control group. In the groups of smokers, sperm DNA integrity and mitochondrial activity were also decreased and lipid peroxidation levels were increased. Proteomic analyses revealed 20 proteins differentially expressed between the study groups. LIMITATIONS AND REASONS FOR CAUTION: A study including smokers without varicocele is still warranted as these results apply only to smokers who present varicocele. WIDER IMPLICATIONS OF THE FINDINGS: Patients with varicocele who are exposed to tobacco smoking present more important alterations to semen quality and sperm functional integrity and show changes in the seminal plasma proteome. This suggests testicular, and possibly systemic, adverse effects of smoking. STUDY FUNDING/COMPETING INTEREST(S): Funding for the study was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) (2007/59423-7) and by the Division of Urology, Human Reproduction Section at the São Paulo Federal University.

Association between obesity and alteration of sperm DNA integrity and mitochondrial activity.

UNLABELLED: What's known on the subject? and What does the study add? The relationship between high levels of BMI and changes in altered standard semen analysis parameters are described in the literature. However, the functional characteristics of the sperm are essential to complete the evaluation of male infertility. Thus, this study provides important information about the functionality of the sperm of men with different levels of BMI. OBJECTIVE: To assess the effect of obesity on semen analysis, sperm mitochondrial activity and DNA fragmentation. MATERIALS AND METHODS: A transversal study of 305 male patients, presenting for clinical evaluation, was carried out. The patients were divided into three groups according to body mass index (BMI) as follows: eutrophic (BMI < 25 kg/m(2), n = 82), overweight (BMI ≥ 25 kg/m(2) and <30, n = 187) and obese (BMI ≥ 30 kg/m(2), n = 36). The variables analysed were semen analysis, rate of sperm DNA fragmentation and sperm mitochondrial activity. Groups were compared using one-way analysis of variance followed by a least significant difference post-hoc test. A P-value of <0.05 was considered to indicate statistical significance. RESULTS: No differences were observed in age, ejaculatory abstinence, ejaculate volume, sperm vitality, morphology or round cell and neutrophil count among the groups. The eutrophic group had a higher percentage of sperm with progressive motility (P = 0.001). Mitochondrial activity was lower in the obese group (P = 0.037) when compared to the eutrophic, and the percentage of sperm with DNA damage was higher in the obese group (P = 0.004) than the other two groups. CONCLUSION: Increased BMI values are associated with decreased mitochondrial activity and progressive motility and increased DNA fragmentation.

Individual and seminal characteristics of patients with testicular germ cell tumors.

OBJECTIVE: To analyze the characteristics of patients with testicular germ cell cancer and compare patients' sperm quality according to histologic type (seminomatous and nonseminomatous tumors). DESIGN: Prospective study. SETTING: Sperm bank at a university. PATIENT(S): One hundred consecutive patients with testicular tumor who had been referred to our infertility center for cryopreservation, between 2004 and 2006. INTERVENTION(S): A questionnaire, through personal interview, was given to all patients and collection of seminal data before cryopreservation was performed. MAIN OUTCOME MEASURE(S): Patient characteristics, including age, time between diagnosis and orchiectomy, history of cryptorchidism, histologic type, and seminal analysis were taken into consideration. RESULT(S): The mean age of the patients at the time of diagnosis was 26.9 years. The mean time between cancer suspicion and the diagnosis of neoplasm was 58.9 days, and 19.4 more days were necessary until orchiectomy was performed. Eleven patients had a history of cryptorchidism. Thirty-seven patients had seminomatous tumors. Men with a seminoma present a higher number of motile and morphologically normal sperm in the ejaculate than men with a nonseminoma, although individual semen variables are not different. CONCLUSION(S): The majority of the patients with testicular cancer, referred to our infertility center, are very young, single, do not have children, and are unaware of their fertility potential status by the time diagnosis is made. Men with a nonseminoma present semen of lower quality.

Is sperm cryopreservation an option for fertility preservation in patients with spinal cord injury-induced anejaculation?

OBJECTIVE: To evaluate the effects of cryopreservation on sperm mitochondrial activity and nuclear DNA integrity in men with spinal cord injury. DESIGN: Prospective controlled study. SETTING: Patients in an academic research environment. PATIENT(S): Men with and without spinal cord injury-induced anejaculation. INTERVENTION(S): Electroejaculation or penile vibrating stimulation semen cryopreservation using a commercial TEST-yolk-buffer technique. MAIN OUTCOME MEASURE(S): Rate of sperm DNA fragmentation as assessed by the comet assay, graded in Classes I (high DNA integrity) to IV (high DNA fragmentation). Mitochondrial activity as assessed by a method in which active mitochondria precipitate 3,3'-diaminobenzidine. Cells were classified as I (all active) to IV (all inactive). Semen was cryopreserved in a Test-yolk buffer, and motility, DNA fragmentation, and mitochondrial activity were analyzed precryopreservation and postthaw. RESULT(S): Before cryopreservation, when the study (SCI) and control groups were compared, no statistically significant differences were found with respect to concentration or total sperm count; however, the SCI group presented significantly lower ejaculate volume, decreased sperm morphology, and an increase in the round cell and neutrophils counts. In both groups, cryopreservation was associated with an increase in DNA fragmentation, a decrease in mitochondrial activity, and a decrease in motility, of which the latter was of greater importance in the control group. CONCLUSION(S): Cryopreservation causes a decrease in conventional seminal variables as well as in mitochondrial activity and DNA fragmentation. However, these were no more detrimental to sperm from men with SCI than to sperm from the control group.

Effect of leukocytospermia and processing by discontinuous density gradient on sperm nuclear DNA fragmentation and mitochondrial activity.

PURPOSE: To assess the effect of leukocytospermia and semen processing on sperm DNA and mitochondria. METHODS: Twenty-two patients with and 41 without leukocytospermia were included. Sperm DNA fragmentation was assessed by the Comet assay, and mitochondrial activity by a colorimetric method for active mitochondria. Semen was processed using Percoll, and motility, DNA fragmentation, and mitochondrial activity were analyzed pre- and post-processing. RESULTS: No differences were observed in age, abstinence, volume, sperm morphology, progressive motility, concentration, and vitality (p>0.10). Variables were grouped according to time (pre- vs post-processing) and group (leukocytospermia vs non-leukocytospermia) because no interactions could be observed. Leukocytospermia was associated to increased DNA fragmentation, while semen processing led to a decrease in DNA fragmentation and to increased mitochondrial activity. CONCLUSION: While semen processing selects sperm with higher rates of DNA integrity independent of the presence or absence of leukocytes in semen, samples without leukocytospermia present more sperm without DNA fragmentation. Semen processing also selects sperm with higher mitochondrial activity.

Quality and functional aspects of sperm retrieved through assisted ejaculation in men with spinal cord injury.

OBJECTIVE: To assess semen quality, sperm DNA fragmentation, and mitochondrial activity in fertile men as well as in men with spinal cord injury who were collecting semen through different methods. DESIGN: Prospective controlled study. SETTING: Academic research environment. PATIENT(S): Men with spinal cord injury who achieved ejaculation through electroejaculation (n = 12) and penile vibratory stimulation (n = 10); 30 fertile control men without spinal cord injury. INTERVENTION(S): Electroejaculation or penile vibratory stimulation, semen analysis according to World Health Organization guidelines, morphology by Kruger's strict criteria. MAIN OUTCOME MEASURE(S): Semen was analyzed according to World Health Organization guidelines; morphology was analyzed according to Kruger's strict criteria. Sperm DNA fragmentation, as assessed by the TUNEL technique, was classified as percentage positive. Mitochondrial activity was assessed by incorporation of diaminobenzidine by mitochondria. Cells were classified as I (all active) to IV (all inactive). RESULT(S): The control group presented a statistically significantly higher percentage of sperm with active mitochondria and a statistically significantly lower percentage of sperm with inactive mitochondria. Although sperm DNA fragmentation was not significantly different when considering collection method (electroejaculation: 30; 8.4; penile vibratory stimulation: 31.2; 8), both groups presented statistically significantly higher DNA fragmentation than did controls (11.8; 4.5). A strong inverse correlation was observed between sperm DNA fragmentation (assessed by in situ DNA nick end labeling) and mitochondrial activity in the case of electroejaculation (r = -0.714), but not in the case of penile vibratory stimulation (r = 0.060). CONCLUSION(S): Spinal cord injury led to a decrease in sperm mitochondrial activity and an increase in sperm DNA fragmentation, and the latter is a sign of testicular alterations. Studies should focus on improving the testicular environment in these men.

Sperm nuclear apoptotic DNA fragmentation in men with testicular cancer.

OBJECTIVE: To verify whether sperm from patients with a seminoma and patients with a non-seminoma present with an increased rate of apoptotic DNA fragmentation, when compared with men without testicular cancer and who had fathered a child in the 2 years preceding the study. DESIGN: Controlled prospective study. SETTING: Patients referred to a sperm bank in an academic research environment. PATIENT(S): Men with a diagnosed seminoma, men with a diagnosed non-seminoma, both after orchiectomy and before adjuvant therapy, and men with proven paternity in the 2 previous years. MAIN OUTCOME MEASURE(S): Rate of nuclear apoptotic sperm DNA fragmentation as assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay, classified as positive (with DNA fragmentation) or negative (without DNA fragmentation). RESULT(S): Of the 48 men with testicular cancer included in the study, 29 (60.4%) presented a non-seminoma and 19 (39.6%) a seminoma. Patients with non-seminoma presented with lower progressive sperm motility than the control group (57.4% and 66.3%, respectively), but both were still within normal ranges. Sperm concentration was lower in seminoma (31.2 x 10(6)/mL) and in non-seminoma (20.6 x 10(6)/mL) when compared with the control group (78.1 x 10(6)/mL), but values did not differ between the two testicular cancer groups. Sperm morphology was lower in patients with non-seminoma than in the control group (10% and 13.1%, respectively). Results for sperm nuclear apoptotic DNA fragmentation (mean; standard deviation) were 12.6%; 4.5% for the control group, 12.2%; 5.5% for the non-seminoma group, and 12.5%; 6.4% for the seminoma group. No differences were found between the three groups. CONCLUSION(S): Our results demonstrate that the presence of a seminoma or a non-seminoma is not associated with an increase in sperm apoptotic DNA fragmentation.

Sperm nuclear DNA fragmentation and mitochondrial activity in men with varicocele.

OBJECTIVE: To verify the impact of varicocele on semen quality and sperm function (DNA integrity and mitochondrial activity). DESIGN: Prospective study. SETTING: Patients in an academic research environment. PATIENT(S): Seventeen patients with a clinical diagnosed varicocele of grade II or III and 20 men without a varicocele. MAIN OUTCOME MEASURE(S): Rate of sperm DNA fragmentation as assessed by the Comet assay and categorized as classes I (no DNA fragmentation), II (little DNA fragmentation), III (meaningful DNA fragmentation), and IV (high DNA fragmentation). Rate of mitochondrial activity as assessed by the diaminobenzidine (DAB) assay and categorized as grades I (all mitochondria active), II (most mitochondria active), III (most mitochondria inactive), and IV (all mitochondria inactive). RESULT(S): No statistically significant differences were found between the study and control groups with respect to age, ejaculatory abstinence, and round cell count. Men with varicocele had significantly higher ejaculate volume, concentration of immotile sperm, and neutrophil count and lower mean percentage of sperm concentration, progressive motility, and morphology than men in the control group. The study group presented a lower percentage of sperm with little DNA fragmentation (class II) and a higher percentage of sperm with DNA fragmentation (class IV). In addition, the study group presented a greater percentage of sperm with inactive mitochondria (class III). CONCLUSION(S): Compared with men without varicocele, men with varicocele had a higher percentage of cells with DNA fragmentation and sperm with inactive mitochondria. Indeed, varicocele causes a decrease in motility, concentration, and morphology and an increase in volume and concentration of immotile sperm and neutrophils. The sperm functional evaluation (DNA fragmentation and mitochondrial activity) could be important factors in deciding treatment options for men with varicocele.