Derlin-dependent retrograde transport from endosomes to the Golgi apparatus.

Cells have to maintain stable plasma membrane protein and lipid compositions under normal conditions and to remodel their plasma membranes in response to stimuli. This maintenance and remodeling require that integral membrane proteins at the plasma membrane that become misfolded, because of the relatively harsher extracellular milieu or carbohydrate and amino acid sequence changes, are degraded. We had previously shown that Derlin proteins, required for quality control mechanisms in the endoplasmic reticulum, also localize to endosomes and function in the degradation of misfolded integral membrane proteins at the plasma membrane. In this study, we show that Derlin proteins physically associate with sorting nexins that function in retrograde membrane transport from endosomes to the Golgi apparatus. Using genetic studies in Caenorhabditis elegans and ricin pulse-chase analyses in murine RAW264.7 macrophages, we show that the Derlin-sorting nexin interaction is physiologically relevant. Our studies suggest that at least some integral membrane proteins that are misfolded at the plasma membrane are retrogradely transported to the Golgi apparatus and ultimately to the endoplasmic reticulum for degradation via resident quality control mechanisms.

Evaluation of acrosomal status and sperm viability in fresh and cryopreserved specimens by the use of fluorescent peanut agglutinin lectin in conjunction with hypo-osmotic swelling test.

OBJECTIVE: In this study, we evaluated whether the hypo-osmotic swelling test (HOST) can be used as a vital marker in combination with peanut agglutinin (PNA) - labeling in fresh and cryopreserved spermatozoa. MATERIALS AND METHODS: Human sperm populations were exposed to a hypo-osmotic medium for 60 minutes, and then incubated in a 1 microg/mL solution of the fluorescent dye Hoescht 33258 (H33258) for 10 minutes. Excess stain was removed by washing in phosphate-buffered saline (PBS) solution, and the pellet was resuspended in 100 microL of culture medium. Twenty microliters of this solution were subsequently smeared on a microscope slide, and fixed in ice-cold methanol to permeabilize the sperm membranes. The fixed smears were finally incubated in a 40-microg/mL FITC-PNA solution for 20 minutes. Simultaneous assessment of acrosome and viability scores was done in a fluorescent microscope equipped with appropriate filters and phase contrast illumination. The same slide was examined for FITC-PNA labeling, tail swelling, and for Hoechst-33258 staining by interchanging the filters and phase contrast optics. RESULTS: In fresh specimens, HOST was found to provide viability assessments comparable to those obtained using the H33258 method (r = 0.95). However, the results of HOST and H33258 were not correlated in cryopreserved specimens (r = 0.22). There was no alteration of PNA-labeling due to the HOST or H33258. CONCLUSIONS: FITC-PNA labeling in conjunction with the visualization of the morphological change induced by exposure to hypo-osmotic solution provides a simple but effective method for establishing the state of acrosomal membrane and viability in fresh human spermatozoa, but this technique is not reliable for cryopreserved ones.

Reassessing the value of varicocelectomy as a treatment for male subfertility with a new meta-analysis.

OBJECTIVE: To determine the efficacy of varicocelectomy as a treatment for male factor infertility by improving the chance of spontaneous pregnancy. DESIGN: Meta-analysis. SETTING: Cleveland Clinic's Glickman Urological Institute. PATIENT(S): Infertile men with abnormal results on semen analyses and a palpable varicocele. INTERVENTION(S): Surgical varicocelectomy. MAIN OUTCOME MEASURE(S): Spontaneous pregnancy outcome. RESULT(S): The odds of spontaneous pregnancy after surgical varicocelectomy, compared with no or medical treatment for palpable varicocele, were 2.87 (95% confidence interval [CI], 1.33-6.20) with use of a random-effects model or 2.63 (95% CI, 1.60-4.33) with use of a fixed-effects model. The number needed to treat was 5.7 (95% CI, 4.4-9.5). CONCLUSION(S): Surgical varicocelectomy in infertile men with palpable lesions and at least one abnormal semen parameter improves the odds of spontaneous pregnancy in their female partners. Five studies were included (two randomized, three observational). All were scored for bias. Our study suggests that varicocelectomy in selected patients does indeed have beneficial effects on fertility status.

Cryosurvival of testicular spermatozoa from obstructive azoospermic patients: The Cleveland Clinic Experience.

We analyzed the testicular spermatozoa of 23 infertile men after cryopreservation to evaluate their cryosurvival and to scrutinize the effectiveness of freezing protocol used in our institution. An excellent postthaw motility was observed indicating an effective cryosurvival protocol.

Reactive oxygen species as an independent marker of male factor infertility.

OBJECTIVE: To determine the abnormal patterns of reactive oxygen species (ROS) production in male factor infertility (MFI) patients and to define the ROS reference values in such patients. DESIGN: A retrospective study. SETTING: Male infertility clinic at a tertiary healthcare center. PATIENT(S): We examined 132 MFI patients (all normal sperm parameters, n = 24, and all abnormal sperm parameters, n = 38) and 34 healthy donors. INTERVENTION(S): Routine semen analysis, measurement of ROS. MAIN OUTCOME MEASURE(S): Sperm parameters, ROS levels (10(4) cpm/20 x 10(6) sperm). RESULT(S): Normal, healthy donors had significantly higher (P<.0001) sperm concentration, motility, and morphology compared with all MFI patients. Univariate analysis indicated a significant association between MFI and log (ROS + 1) (odds ratio [OR] = 3.84), besides sperm parameters and age. A multivariate model using logistic regression analysis also indicated an independent association of log ROS with MFI (OR = 4.25). The ROS cutoff values of 1.2-1.4 had a sensitivity of 0.70-0.78 with a corresponding specificity of 0.82. However, at a cutoff point of 1.2, the OR was 68.6, which increased with an increase in the cutoff. CONCLUSION(S): High ROS is an independent marker of MFI, irrespective of whether these patients have normal or abnormal semen parameters. We suggest the inclusion of ROS measurement as part of idiopathic infertility evaluation. Treatment with antioxidants may be beneficial in such patients.

Arteriovenous malformation of scrotum causing virtual azoospermia.

We report a case of surgical correction of a scrotal arteriovenous malformation (AVM) that reversed severe oligospermia. A 31-year-old man was found to have virtual azoospermia and left scrotal swelling. The left scrotal mass was diagnosed as a varicocele, but the mass did not resolve after bilateral microsurgical varicocelectomies. Postoperative pelvic arteriography revealed a scrotal AVM that was subsequently embolized. With a microsurgical approach, the mass was excised. Surgical correction of the scrotal AVM resulted in marked improvement in his semen parameters. The effect of the AVM on spermatogenesis was most likely mediated by an elevation in scrotal temperature.

Sperm harvesting and cryopreservation during vasectomy reversal is not cost effective.

OBJECTIVE: To determine whether sperm harvesting and cryopreservation at the time of vasectomy reversal is cost-effective. DESIGN: Model of actual costs and results at five institutions. SETTING: Multicenter study comprising five centers, including university hospitals and private practices. PATIENT(S): Men undergoing vasectomy reversal. INTERVENTION(S): We established two models for vasectomy reversal. The first model was sperm harvesting and cryopreservation at the time of vasectomy reversal. The second model was sperm harvesting at the time of IVF only if the patient remained azoospermic after vasectomy reversal. Vasectomy reversal procedures modeled included bilateral vasovasostomy and bilateral epididymovasostomy. The costs for each procedure at the five institutions were collated and median costs determined. MAIN OUTCOME MEASURE(S): Median cost of procedure and calculated financial comparisons. RESULT(S): The median cost of testicular sperm extraction/cryopreservation performed at the time of bilateral vasovasostomy was $1,765 (range, $1,025-$2,800). The median cost of microsurgical epididymal sperm aspiration or testicular sperm extraction with cryopreservation performed at the time of epididymovasostomy was $1,209 (range, $905-$2,488). The average of the median costs for percutaneous sperm aspiration or testicular sperm aspiration for those patients with a failed vasectomy reversal was $725 (range, $400-$1,455). CONCLUSION(S): Sperm retrieval with cryopreservation at the time of vasectomy reversal is not a cost-effective management strategy.

Relationship between epidemiological features and aetiology of male infertility as diagnosed by a comprehensive infertility service provider.

This study examined the relationship between demographic features and aetiological causes of male infertility. Primary infertility was the presentation in 78% of patients. The incidence of varicocele was the highest (31%), whereas only 4.6% had vasectomy reversal and 7.4% of men were diagnosed with idiopathic infertility. Using the chi-squared test, there was no significant difference in the incidence of different causes of infertility among different ethnic groups (White, African-American, Asian, Hispanic, and other). Furthermore, there was no increased incidence of infertility aetiology with any particular occupation, race, religion, smoking or alcohol intake. In this study population, there was no association between the various risk factors (occupation, smoking, alcohol intake, and race) and the aetiologies of infertility. The proportion of patients diagnosed with idiopathic infertility was significantly less than reported in the literature.

Multi-institutional validation of vasectomy reversal predictor.

PURPOSE: Some urologists who perform vasectomy reversals are not experienced with performing VE. A model to preoperatively identify patients who may require referral to an experienced VE surgeon was created (). We tested the model at multiple institutions. MATERIALS AND METHODS: The model had previously been designed in 483 patients who underwent vasectomy reversal at 1 institution (100% sensitive and 59% specific for predicting the need for VE). It was based on time since vasectomy and patient age. We tested it prospectively in 33 patients and retrospectively in a total of 312 at 6 other institutions. The predictive accuracy of the model was compared to using a simple duration from vasectomy cutoff alone, as is used in clinical practice. RESULTS: The model had 84% sensitivity and 58% specificity for detecting the need for VE in a total of 345 patients at 7 institutions. If using only a duration from vasectomy cutoff of 10 years to predict the need for VE, sensitivity was only 69%. At a cutoff of 4 years sensitivity was 99% but specificity was only 23%. Thus, the model performed better than any specific duration cutoff alone. CONCLUSIONS: The predictive model provides 84% sensitivity for detecting patients who may require VE during vasectomy reversal across 7 institutions (58% specificity). The model more accurately predicts the need for VE than using a specific duration from vasectomy cutoff alone.

Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model.

Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.

Novel association between sperm deformity index and oxidative stress-induced DNA damage in infertile male patients.

AIM: To investigate the impact of abnormal sperm morphology using the sperm deformity index (SDI) on reactive oxygen species (ROS) production and its correlation with sperm DNA damage. METHODS: Semen samples were collected from men undergoing infertility screening (n = 7) and healthy donors (n = 6). Mature spermatozoa were isolated and incubated with 5 mmol/L beta-nicotinamide adenine dinucleotide phosphate (NADPH) for up to 24 h to induce ROS. Sperm morphology was evaluated using strict Tygerberg's criteria and the SDI. ROS levels and DNA damage were assessed using chemiluminescence and terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) assays, respectively. RESULTS: SDI values (median [interquartiles]) were higher in patients than donors (2 [1.8, 2.1] vs. 1.53 [1.52, 1.58], P = 0.008). Aliquots treated with NADPH showed higher ROS levels (1.22 [0.30, 1.87] vs. 0.39 [0.10, 0.57], P = 0.03) and higher incidence of DNA damage than those not treated (10 [4.69, 24.85] vs. 3.85 [2.58, 5.10], P = 0.008). Higher DNA damage was also seen following 24 h of incubation in patients compared to donors. SDI correlated with the percentage increase in sperm DNA damage following incubation for 24 h in samples treated with NADPH (r = 0.7, P = 0.008) and controls (r = 0.58, P = 0.04). CONCLUSION: SDI may be a useful tool in identifying potential infertile males with abnormal prevalence of oxidative stress (OS)-induced DNA damage. NADPH plays a role in ROS-mediated sperm DNA damage, which appears to be more evident in infertile patients with semen samples containing a high incidence of morphologically abnormal spermatozoa.

Model to predict if a vasoepididymostomy will be required for vasectomy reversal.

PURPOSE: We devised a model to predict, preoperatively, the need for a vasoepididymostomy (VE) when performing a vasectomy reversal. Urologists could use it to identify those patients who need a referral to an experienced VE surgeon. MATERIALS AND METHODS: We performed a retrospective review of 483 patients who underwent vasectomy reversal by a single surgeon (AJT) including 393 vasovasostomies and 90 vasoepididymostomies. Selection was based on chart availability. Established criteria were used in deciding the type of reversal (eg gross appearance and microscopic examination of vasal fluid). Type of reversal, patient age and time since vasectomy were recorded. Univariate analysis revealed that patient age (p <0.001) and time since vasectomy (p <0.001) were significant predictors of reversal type. On multivariate logistic regression analysis, time since vasectomy (p <0.001) was the only significant independent predictor. We designed a linear regression algorithm based on time since vasectomy and patient age to predict if a VE would be performed. The model was designed using 433 patients and then tested on a separate randomly selected 50 patient group. The model was designed to be 100% sensitive in detecting patients requiring VE. RESULTS: In the test group the model was 100% sensitive in predicting VE with a specificity of 58.8%. The area under the ROC curves for the design and test groups was 0.8. Palm (PalmSource Inc., Sunnyvale, California) and Windows (Microsoft Corporation, Redmond, Washington) versions are available as free shareware from www.uroengineering.com. CONCLUSIONS: The model is 100% sensitivity in detecting those patients who may require a VE during vasectomy reversal (specificity of 58.8%). It may allow urologists to preoperatively identify these patients.

Robotic-assisted vasovasostomy: a two-layer technique in an animal model.

OBJECTIVES: To assess the feasibility of a multilayered robotic-assisted vasovasostomy (RAVV) in a rabbit model. Microscope-assisted vasovasostomy (MAVV) is a technically challenging procedure. Robotics may be a surgical adjunct that helps overcome the microsurgical challenges, which include fine suture, delicate instruments, and tremor. A recent survey revealed that most urologists use a multilayered technique for vasovasostomies. METHODS: A surgeon performed eight vasovasostomies with 10-0 suture and a two-layer technique using an in vivo rabbit model-four were MAVV using conventional microsurgical instrumentation and four were RAVV using the da Vinci robot. Performance measures and adverse haptic events were recorded. Patency was evaluated by passing a 2-0 Prolene suture through the anastomoses. RESULTS: The mean operating time for the total procedure and for the mucosal layer only was longer for RAVV than for MAVV (75 versus 42 minutes, P = 0.03 and 38 versus 23 minutes, P = 0.03, respectively). The needle passes required for the mucosal layer and the number of mucosal and muscularis sutures were similar in both groups (9.5 versus 8.8 passes, P = 0.34; 4 versus 4, P >0.99; and 7 versus 6.3, P = 0.2, respectively). Unlike MAVV, no tremor was appreciated during RAVV. No adverse haptic events were observed in either group. All anastomoses were patent, and all rabbits were free of any crush injury. CONCLUSIONS: A multilayered RAVV can be performed in an in vivo rabbit model. Although it was associated with increased operative times, the absence of adverse haptic events and comparable patency rates continue to suggest a role for robotics in microsurgery.

Characterization of oxidative stress status by evaluation of reactive oxygen species levels in whole semen and isolated spermatozoa.

We defined the basal levels of reactive oxygen species (ROS) in normal donors in neat (whole unprocessed) semen specimens, and in mature and immature spermatozoa isolated by a double-density gradient technique. In addition, we demonstrated that the ROS levels were significantly lower in neat semen compared with washed spermatozoa. The reference values of ROS in neat semen and mature spermatozoa can be used to define the pathologic levels of ROS in infertile men and may guide in therapeutic interventions.

Impact of sperm morphology on DNA damage caused by oxidative stress induced by beta-nicotinamide adenine dinucleotide phosphate.

OBJECTIVE: To investigate the role of DNA damage induced by beta-nicotinamide adenine dinucleotide phosphate (NADPH) in human spermatozoa. DESIGN: Prospective controlled study. SETTING: Male infertility clinic at the Glickman Urological Institute, Cleveland Clinic Foundation, Cleveland, Ohio. PATIENT(S): Twenty-eight men undergoing infertility screening. INTERVENTION(S): Chemiluminescence assay and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay coupled flow cytometry after incubating mature and immature sperm separated by density gradient with 5 mM NADPH for 0, 3, and 24 hours. MAIN OUTCOME MEASURE(S): Reactive oxygen species (ROS) generation (10(6) counted photons per minute/10(6) sperm) and percentage of spermatozoa with fragmented DNA. RESULT(S): Immature sperm from teratozoospermic semen samples were characterized by a statistically significant presence of cytoplasmic residues in the mid-piece when compared with mature normozoospermic samples. Increased ROS production was observed in spermatozoa rich in cytoplasmic residues that showed a statistically significant positive correlation with sperm DNA damage in a time-dependent manner. CONCLUSION(S): Immature sperm contain high nicotinamide adenine dinucleotide phosphate (NADPH) in cytoplasmic droplets, but it has not yet been clear whether abnormal sperm morphology plays any role in sperm DNA damage induced by oxidative stress. Our data support the role of NAPDH in ROS-mediated sperm DNA damage and suggest that abnormal sperm morphology combined with elevated ROS production may serve as a useful indicator of potential damage to sperm DNA.

Increased seminal reactive oxygen species levels in patients with varicoceles correlate with varicocele grade but not with testis size.

Increased levels of reactive oxygen species (ROS) are associated with clinical varicoceles; however, its correlation with varicocele grade and testis size is unknown. In our study, seminal ROS levels showed significant correlation with left varicocele grade and significantly elevated seminal ROS levels were seen in men with left varicocele grade 2 and 3 compared to grade 1.

Relationship of interleukin-6 with semen characteristics and oxidative stress in patients with varicocele.

OBJECTIVES: To examine levels of interleukin-6 (IL-6) in fertile semen donors and patients with varicocele and examine its association with semen characteristics and levels of reactive oxygen species (ROS). METHODS: We conducted a prospective study consisting of 15 fertile donors (controls) and 35 infertile patients with varicocele. Semen analysis was performed according to the World Health Organization guidelines. IL-6 levels were measured using the enzyme-linked immunosorbent assay. ROS (x10(4) counted photons per minute per 20 x 10(6) sperm) and total antioxidant capacity (molar trolox equivalents) were measured using a chemiluminescence assay. RESULTS: The sperm concentration and motility were significantly greater in the donors compared with the infertile patients with varicocele (P <0.0001 and P = 0.01, respectively). The IL-6 (log10 [IL-6 +1]) and ROS (log10 [ROS +1]) levels were significantly greater in infertile patients with varicocele than in the donors (IL-6: 2.1 [1.7, 2.4] versus 0.7 [0, 1.9], P = 0.003; ROS: 1.8 [1.2, 2.6] versus 1.0 [0.7, 1.6], P = 0.04). The total antioxidant capacity levels were significantly lower in the varicocele patients (1166.7 +/- 366.2) than in the donors (1556.4 +/- 468.1; P = 0.003). The IL-6 levels correlated significantly with the ROS levels in the infertile patients with varicocele (r = -0.39; P = 0.01). CONCLUSIONS: Infertile patients with varicocele exhibited elevated levels of IL-6 and ROS and decreased levels of total antioxidant capacity. Pro-inflammatory cytokine IL-6 and oxidative stress may play a role in the pathophysiology of infertility in these patients.

Human sperm superoxide anion generation and correlation with semen quality in patients with male infertility.

OBJECTIVE: To measure levels of reactive oxygen species (ROS) including H(2)O(2) and O(2)(.-) generation in infertile men and determine whether sperm quality is correlated with levels of ROS triggered by the exogenous reduced form of beta nicotinamide adenine dinucleotide phosphate (NADPH). DESIGN: Prospective study. SETTING: Male infertility clinic at a tertiary healthcare center. PATIENT(S): Eleven infertile men and six healthy donors. INTERVENTION(S): Chemiluminescence assay using luminol and lucigenin as probes before and after incubating sperm samples with 5 mM and 10 mM of NADPH. MAIN OUTCOME MEASURE(S): The ROS generation (10(6) counted photons per minute/10(6) sperm). RESULT(S): Baseline levels of O(2)(.-) generation were significantly higher in the infertile patients than in the healthy donors (r = 0.73, 95% confidence interval [median (25th, 75th percentiles): 0.73 (0.5, 5.5) vs. 0.2 (0.0, 0.5)] when lucigenin was used as the probe. Compared with basal levels, O(2)(.-) generation was significantly higher after coincubation with NADPH (5 mM and 10 mM) in the entire combined study population, and patients only but not donors. The O(2)(.-) generation was negatively correlated with sperm concentration (r = -0.75, 95% CI 0.38-1), motility (r = -0.69, 95% CI 0.28-1), and percentage of normal morphology (r = -0.78, 95% CI 0.36-1). CONCLUSION(S): Spermatozoa from infertile men produce higher levels of O(2)(.-) in the presence of exogenous NADPH compared to healthy donors. The ability of spermatozoa to generate O(2)(.-) increases as the semen quality declines.

Novel associations between specific sperm morphological defects and leukocytospermia.

OBJECTIVE: To examine the relationship between leukocyte concentrations in semen and sperm morphology in a group of infertile men and healthy fertile donors. DESIGN: A prospective clinical study. SETTING: Male infertility clinic at a tertiary care teaching hospital and a reproductive medicine unit at a Women's Hospital in the United Kingdom. PATIENT(S): Fifty-six infertile men and 13 healthy fertile sperm donors (control). INTERVENTION(S): Standard semen analysis, seminal leukocyte concentration, and the assessment of sperm morphology and sperm deformity index (SDI), applying Tygerberg's strict criteria. MAIN OUTCOME MEASURE(S): Granulocyte concentrations in semen, percentages of different sperm morphological abnormalities, and SDI scores. RESULT(S): Leukocyte concentrations were statistically significantly and negatively correlated with the proportion of sperm with damaged acrosomes, cytoplasmic droplet, tail defects, and SDI scores with normal and borderline morphology. The percentage sperm motility was significantly and negatively correlated with leukocytic concentration in semen. However, the leukocytic concentration was not significantly correlated with sperm concentration. CONCLUSION(S): This is the first study to report a significant positive correlation between leukocytospermia and sperm tail defects, acrosomal damage, and high SDI scores. These observations suggest that leukocytospermia is associated with compromised sperm structural integrity.