Showing papers in "Journal of Andrology in 2003"

The Sperm Chromatin Dispersion Test: A Simple Method for the Determination of Sperm DNA Fragmentation

Abstract: Sperm DNA fragmentation is being increasingly rec- ognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non- fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hy- bridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (49,6-diamidino-2-phenylindole) and/ or the Diff-Quik reagent, and the percentages of sperm with nondis- persed and dispersed chromatin loops were monitored by fluores- cence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmen- tation, as measured by DBD-FISH. Conversely, all sperm with dis- persed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had par- ticipated in a donor insemination program. The coefficient of varia- tion obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly repro- ducible, and inexpensive method for the analysis of sperm DNA frag- mentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test for the screening of sperm DNA fragmentation in the andrology laboratory.

Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa.

Abstract: The objective of this study was to examine the effect of reactive oxygen species (ROS) and cryopreservation on DNA fragmentation of equine spermatozoa. In experiment 1, equine spermatozoa were incubated (1 hour, 38 degrees C) according to the following treatments: 1) sperm alone; 2) sperm + xanthine (X, 0.3 mM)-xanthine oxidase (XO, 0.025 U/mL); 3) sperm + X (0.6 mM)-XO (0.05 U/mL); and 4) sperm + X (1 mM)-XO (0.1 U/mL). In experiment 2, spermatozoa were incubated (1 hour, 38 degrees C) with X (1 mM)-XO (0.1 U/mL) and either catalase (200 U/mL), superoxide dismutase (SOD, 200 U/mL), or reduced glutathione (GSH, 10 mM). Following incubation, DNA fragmentation was determined by the single cell gel electrophoresis (comet) assay. In experiment 3, equine spermatozoa were cryopreserved, and DNA fragmentation was determined in fresh, processed, and postthaw sperm samples. In experiment 1, incubation of equine spermatozoa in the presence of ROS, generated by the X-XO system, increased DNA fragmentation (P <.005). In Experiment 2, the increase in DNA fragmentation associated with X-XO treatment was counteracted by the addition of catalase and GSH but not by SOD, suggesting that hydrogen peroxide and not superoxide appears to be the ROS responsible for such damage. In experiment 3, cryopreservation of equine spermatozoa was associated with an increase (P <.01) in DNA fragmentation when compared with fresh or processed samples. This study indicates that ROS and cryopreservation promote DNA fragmentation in equine spermatozoa; the involvement of ROS in cryopreservation-induced DNA damage remains to be determined.

Cavernous neurotomy causes hypoxia and fibrosis in rat corpus cavernosum.

Abstract: The etiologies of erectile dysfunction (ED) after nerve-sparing radical prostatectomy have not been clearly elucidated. The aim of this study was to evaluate the effects of cavernous nerve injury on cavernous fibrosis, and to consider measures to prevent irreversible damage to the cavernous tissues. Twenty male Sprague-Dawley rats constituted the study population. The animals were divided into 2 groups; group 1 consisted of sham-operated rats (n = 10), and group 2 consisted of rats that underwent incision of both cavernous nerves (n = 10). Three months later, all rats underwent intracavernous papaverine injection (300 and 600 mg), and intracorporal pressures were recorded. Transforming growth factor-beta(1) (TGF-beta(1)) messenger RNA (mRNA) expression from rat penile tissue was measured using reverse transcriptase-polymerase chain reaction. Hypoxia-inducible factor-1alpha (HIF-1alpha), TGF-beta(1), and collagen I and III protein expressions were determined by Western blot analysis and immunohistochemical staining. Erectile function as studied with intracavernosal papaverine injection and histological analysis of penile cross-sections at 3 months was similar in both groups. TGF-beta(1) mRNA expression, HIF-1alpha, TGF-beta(1), and collagen I and III protein expressions were significantly greater in the neurotomy group. Immunohistochemical staining for TGF-beta(1), HIF-1alpha, and collagen III were qualitatively more positive in the neurotomy group, whereas collagen I staining was similar. This study demonstrates an increase in TGF-beta(1), HIF-1alpha, and collagen III synthesis in rat cavernosal smooth musculature after cavernous neurotomies. In theory, cavernous fibrosis may be reduced by employing various vasoactive agents or interventions that increase oxygenation to the corporal tissues during the postoperative period.

Endothelial dysfunction in erectile dysfunction: role of the endothelium in erectile physiology and disease.

Abstract: Erectile dysfunction (ED) is defined as the consistent inability to obtain or maintain an erection for satisfactory sexual intercourse. Basic science research on erectile physiology has been devoted to investigating the pathogenesis of ED and has led to the conclusion that ED is predominately a disease of vascular origin. The incidence of ED dramatically increases in men with diabetes mellitus, hypercholesterolemia, and cardiovascular disease. Loss of the functional integrity of the endothelium and subsequent endothelial dysfunction plays an integral role in the occurrence of ED in this cohort of men. This communication reviews the role of the vascular endothelium in erectile physiology and the influence of endothelial dysfunction in the pathogenesis of ED. Future pharmacological and gene therapy interventions to restore endothelial function may represent exciting new therapeutic strategies for the treatment of ED. Penile erection is a neurovascular phenomenon that depends upon neural integrity, a functional vascular system, and healthy cavernosal tissues (Giuliano et al, 1995). Normal erectile function involves 3 synergistic and simultaneous processes: 1) neurologically mediated increase in penile arterial inflow, 2) relaxation of cavernosal smooth muscle, and 3) restriction of venous outflow from the penis. The corpus cavernosum of the penis is composed of a meshwork of interconnected smooth muscle cells lined by vascular endothelium. Of note, endothelial cells and underlying smooth muscle also line the small resistance helicine arteries that supply blood to the corpus cavernosum during penile tumescence. Pathological alteration in the anatomy of the penile vasculature or impairment of any combination of neurovascular processes can result

Epididymosomes and Prostasomes: Their Roles in Posttesticular Maturation of the Sperm Cells

Abstract: The occurrence of membrane vesicles along the male reproductive tract and in the ejaculated semen appears as a common feature among different species, including humans. Indeed, prostasomes (prostate-derived vesicles) were first described in human semen in 1978 (Ronquist et al, 1978), and vesicular structures similar to prostasomes were also found in the seminal plasma of rabbit (Davis, 1978), ram (Breitbart and Rubinstein, 1982), and stallion (Arienti et al, 1998; Minelli et al, 1998). Furthermore, ‘‘prostasome-like’’ particles are present in the epididymal fluid of rat (Fornes and De Rosas, 1991), hamster (Yanagimachi et al, 1985), and bull (Frenette and Sullivan, 2001) and are also secreted by the bull seminal vesicles (Agrawal and Vanha-Perttula, 1987). The biochemical composition of prostasomes purified from human semen has been well documented. These are multilamellar lipoprotein membrane particles with a diameter of 50 to 500 nm. Their cholesterol-phospholipid ratio reaches 2, sphingomyelin being the major phospholipid. Many proteins are associated with prostasomes, some of them having a catalytic activity (Review: Kravets et al, 2000; Ronquist and Nilsson, 2002). This review will focus on the physiological implication of these membranous structures with regard to the posttesticular sperm maturation.

Marked differences in protamine content and P1/P2 ratios in sperm cells from percoll fractions between patients and controls.

Abstract: The various sperm cell types present in a simple ejaculate differ in their motility and morphology. However, little is known about the nuclear maturity of these sperm cells and their relationship with morphological and motile characteristics. Protamines are considered a good marker of sperm nuclear maturity since they are added to the DNA in the last stage of spermatogenesis. We have analyzed the P1/P2 ratio and the protamine content of subpopulations of human spermatozoa at different stages of maturation, isolated by density gradient centrifugation of ejaculated spermatozoa obtained from 3 groups of patients from our Assisted Reproduction Unit: 10 men of proven fertility, 12 oligozoospermic men, and 13 asthenozoospermic men. Four different fractions (F2-F5) were collected from the top to the bottom of the Percoll gradient. Differences in the motion and morphology were found between the fractions in each of the groups studied, with fraction F5 being the one with the best morphology and motility. However, no significant differences in the P1/P2 ratio were found between fractions within the same group of samples, indicating that the P1/P2 ratio and the amount of protamines are relatively independent of the morphology and motility of sperm cells. In contrast, statistically significant differences were found in the P1/P2 ratio and in the relative amount of protamines between the 3 groups.

Effects of medical or surgical castration on erectile function in an animal model.

Abstract: The goal of this study was to investigate the effects of medical castration (luteinizing hormone-receptor hormone [LH-RH] agonist treatment) or surgical castration on erectile function in an animal model. New Zealand White male rabbits were either kept intact (control); surgically orchiectomized; or treated for 2, 4, or 8 weeks with the LH-RH agonist leuprolide acetate (107 microg/kg/mo). At 2 weeks, plasma testosterone levels of orchiectomized and leuprolide acetate-treated animals were 12.8% and 57.4% of intact control animals, respectively. Erectile function was assessed by continuously recording systemic arterial pressure (SAP) and intracavernosal blood pressure (ICP) and determining the ICP:SAP ratios in response to electrical stimulation of the pelvic nerve at varying frequencies (2.5-32 Hz). Androgen deprivation by surgical (orchiectomy) or medical (leuprolide acetate) castration reduced ICP at all frequencies tested but did not alter SAP. Administration of the phosphodiesterase type 5 inhibitor vardenafil (10 microg/kg) did not enhance ICP in surgically orchiectomized or leuprolide acetate-treated animals. Nitric oxide synthase and arginase activities in the corpus cavernosum were not significantly altered by surgical or medical castration. Further, Masson trichrome staining of erectile tissue from androgen-ablated animals showed a reduction in smooth muscle content. These data demonstrate that androgen deprivation achieved by surgical or medical castration adversely affects penile hemodynamics and erectile function without producing significant changes in the activities of nitric oxide synthase or arginase. We conclude that androgen deprivation produces structural alterations in the corpus cavernosum leading to corporal veno-occlusive dysfunction.

Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation.

Abstract: The origin and the meaning of DNA fragmentation in ejaculated human spermatozoa are not yet clear, although some hypotheses have been proposed. In the present study, we used investigated sperm DNA fragmentation by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL)-coupled flow cytometry to investigate DNA fragmentation in spermatozoa that were selected by the swim-up procedure and incubated long-term. In addition, using flow cytometry we detected annexin V binding assay and propidium iodide staining, and we also studied membrane phosphatidylserine translocation and the loss of membrane integrity in the same sperm populations that we used in the TUNEL investigation. We found that in vitro sperm DNA fragmentation 1) occurs after ejaculation under experimental conditions without the involvement of any external factor, 2) is not affected by treatment with the nuclease inhibitor aurintricarboxylic acid, 3) is increased by treatment with the glutathione peroxidase inhibitor mercaptosuccinate, 4) correlates with basal values (ie, just after swim-up selection) of DNA fragmentation in teratozoospermic but not in normospermic semen samples, 5) develops in a sharply associated manner with the in vitro occurrence of sperm necrosis, and 6) is predicted by the basal value of annexin V binding in viable spermatozoa. These findings suggest an involvement of endogenously produced reactive oxygen species as the possible cause of in vitro sperm DNA fragmentation.

Deterioration of plasma membrane is associated with activated caspases in human spermatozoa.

Abstract: Spermatozoa with deteriorated plasma membranes can be separated by magnetic-activated cell sorting (MACS) after binding superparamagnetic annexin V-conjugated microbeads (ANMBs) to membrane phosphatidylserine (PS). Semen samples from 15 donors and 25 infertile patients were divided into 2 spermatozoal fractions by annexin V-MACS. Activated caspases (aCPs), which mediate degradations of cell quality, were determined by CaspaTag in the 2 subpopulations. Spermatozoa from donors showed lower levels of bound annexin V (3.6% +/- 0.5% vs 11.9% +/- 1.1%; P <.01) and aCPs (21.8% +/- 2.6% vs 43.2% +/- 2.1%; P <.01) than did spermatozoa from infertile patients. MACS resulted in a decrease of spermatozoa with aCPs from 21.8% +/- 2.6% (before separation) to 9.2% +/- 1.4% (in the ANMB-negative fraction) in donors and from 43.2% +/- 2.1% to 18.8% +/- 2.6% in infertile patients (mean +/- SEM; P <.01). Separation effects of the MACS technique were confirmed with flow cytometry using anti-annexin V antibodies and with electron microscopy. ANMB-MACS removes spermatozoa with PS-bound annexin V and produces a higher quality spermatozoal fraction. Spermatozoa with a deteriorated membrane are characterized by an increase in aCPs. A higher percentage of spermatozoa with ANMBs bound to PS and with aCPs were found in infertile patients.

Clinical and diagnostic features of patients with suspected Klinefelter syndrome.

Abstract: Klinefelter syndrome, with an incidence of 1:600 male newborns, is the most frequent form of male hypogonadism. However, despite its relatively high frequency, the syndrome is often overlooked. To prevent such oversights, the clinical features should be better characterized, and simple screening tests should be used more frequently. In a cohort of 309 patients suspected of having Klinefelter syndrome, we evaluated the clinical symptoms as well as the diagnostic value of the Barr body test for screening procedures. On the basis of chromosome analysis, 85 patients (group I) were diagnosed as having Klinefelter syndrome, and 224 patients had a 46,XY karyotype (group II). Barr body analysis revealed a specificity of 95% and a sensitivity of 82% for the diagnosis of Klinefelter syndrome. General features (eg, reason for admission, age, age of the parents, body weight, and frequency of maldescended testes) were not different between the groups, except that group I had a higher proportion of patients with a lower educational background. Compared to group II, patients with Klinefelter syndrome were taller (P <.001); had smaller testis volumes (P <.0001), higher follicle-stimulating hormone (FSH) and luteinizing hormone (LH) values; and carried a tendency for less androgenic phenotype and secondary hair distribution. Testosterone, estradiol, sex hormone-binding globulin (SHBG), and prostate-specific antigen (PSA) serum levels as well as prostate volume were not significantly different between the groups. In patients who provided an ejaculate, azoospermia was found in 54% of the patients in group II and in 93% of the patients with Klinefelter syndrome. Although not exclusively characteristic for Klinefelter syndrome, the combination of low testicular volume and azoospermia, together with elevated gonadotropins, is highly indicative for a Klinefelter syndrome and should stimulate further clinical investigations. Barr body analysis provides a quick and reliable screening test, which, however, must be confirmed by karyotyping.

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

Abstract: 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.

Seasonal variation and age-related changes in human semen parameters

Abstract: Although semen quality has been discussed extensively with regard to age and season in the andrology literature, the results vary and firm conclusions are still outstanding. To investigate seasonal and age-related variations in human semen parameters, we analyzed data that were collected from an andrology clinic population. We performed a retrospective review of 551 semen analysis records collected from 1989 to 2000 from the Vincent Memorial Andrology Laboratory at Massachusetts General Hospital. Semen volume, sperm concentration, total sperm count, motility, total motile sperm, and morphology significantly decreased as age increased. In addition, as age increased, the percentage of sperm with tail defects increased. Sperm concentration was significantly higher in winter (mean 157.9 million/mL) than in fall (mean 119.1 million/mL) (P <.05). The mean percentage of sperm with normal morphology was significantly higher in winter (9.2%) than in summer and spring (7.0% and 7.5%, respectively; P <.05). The mean percentage of sperm with head defects was significantly higher in fall and summer (74.0% and 72.3%, respectively) than in winter (68.6%; P <.05). Seasonal variations were found in sperm concentration and morphology, with higher sperm concentrations in winter than in fall, and a greater percentage of sperm with normal morphology in winter than in spring and summer. Sperm concentration was lowest in the fall, whereas the percentage of sperm with normal morphology was lowest in summer. Semen volume, sperm concentration, total sperm count, motility, total motile sperm, and morphology decreased as age increased.

Male genital tract inflammation: The role of selected interleukins in regulation of pro-oxidant and antioxidant enzymatic substances in seminal plasma.

Abstract: Human semen contains spermatozoa as well as populations of round nonspermatozoal cells primarily consisting of leukocytes. Activation of white blood cells present in the seminal plasma during genital tract inflammation and cellular reactions against microbial agents may provoke a release of a variety of products such as cytokines and reactive oxygen species. The aim of this study was to evaluate whether a panel of selected cytokines (interleukin [IL]-1 beta, IL-6, IL-8, and tumor necrosis factor-alpha [TNF alpha]) detectable in seminal plasma during male genital tract inflammation could be considered as mediators between altered semen parameters and changed levels of pro-oxidant and antioxidant substances. Studies using chemiluminometric, spectrophotometric, and enzyme-linked immunosorbent assay methods indicate that proinflammatory cytokines such as IL-1 beta, IL-6, IL-8, and TNF alpha may modulate pro-oxidant and antioxidant activities in the male genital tract. The data also suggest that the function of pro-oxidant and antioxidant systems in semen may directly influence basic semen parameters. The elevated numbers of leukocytes present in semen during male genital tract inflammation without an associated contribution of cytokines and semen antioxidant capacity appear to be of little prognostic value in evaluating male fertilization potential.

Cognitive changes associated with supplementation of testosterone or dihydrotestosterone in mildly hypogonadal men: a preliminary report.

Abstract: This study prospectively examined changes in cognition in hypogonadal men given testosterone (T) or older hypogonadal men given dihydrotestosterone (DHT) gel. A battery of cognitive tests assessing verbal and spatial memory, language, and attention was administered at baseline (prior to medication) and again at days 90 and 180 of treatment for men receiving T gel and at baseline and days 30 and 90 of treatment for men receiving DHT gel. For men receiving T gel, circulating total T and estradiol (E(2)) were significantly raised compared with baseline, and a significant improvement in verbal memory was observed. For men receiving DHT gel, serum DHT levels increased and T levels decreased significantly compared with baseline, and a significant improvement in spatial memory was observed. The results suggest that beneficial changes in cognition can occur in hypogonadal men using T replacement levels and DHT treatment, and these changes in cognition can be reliably measured during a relative steady-state dose level. Further, our results suggest that aromatization of T to E(2) may regulate verbal memory in men, whereas nonaromatizable androgens may regulate spatial memory.

Sperm Mitochondrial Mutations as a Cause of Low Sperm Motility

Abstract: We report the unique case of a 28-year-old man who, in spite of having a varicocele and a sperm concentration of 5 million/mL, of which 10% were motile and 20% had normal forms (oligoasthenoteratozoospermia [OAT]), was fertile. This was confirmed by paternity testing using 16 autosomal and 6 Y-chromosomal short tandem repeat (STR) loci. An analysis of mitochondrial genes that included cytochrome oxidase I (COI), cytochrome oxidase II (COII), adenosine triphosphate synthase6 (ATPase6), ATPase8, transfer ribonucleic acid (tRNA) serine I, tRNA lysine, and NADH dehydrogenase3 (ND3) revealed, for the first time, 9 missense and 27 silent mutations in the sperm's mitochondrial DNA (mtDNA) but not in the DNA from the blood cells. There was a 2-nucleotide deletion in the mitochondrial COII genes, introducing a stop codon, which might be responsible for low sperm motility.

Balance of apoptosis and proliferation of germ cells related to spermatogenesis in aged men.

Abstract: To clarify whether germ cell apoptosis is related to a decrease of germ cells in the aged testis with impaired spermatogenesis, we investigated the apoptotic rate of each germ cell type. Testicular specimens were obtained by orchiectomy from 36 men with advanced prostate cancer and by testicular biopsy from 21 men with obstructive azoospermia, which served as controls. The terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique was used to identify apoptosis. As a marker of cell proliferation activity, the expression of Ki-67 was immunohistochemically evaluated. Expression of Bcl-xl, which regulates apoptosis of germ cells, was also immunohistochemically examined. Histologically, except for spermatogonia, the ratios of primary spermatocytes, round spermatids, and elongated spermatids to Sertoli cells were significantly decreased in aged testes. The apoptotic rate in spermatogonia was significantly lower in aged men than it was in controls (0.11% +/- 0.06% vs 0.34% +/- 0.21%). Expression of Ki-67 in spermatogonia was decreased in aged men (18.6% +/- 6.0%) compared with that of controls (24.9% +/- 3.3%), suggesting that germ cell proliferation diminished with aging. Consequently, the balance of spermatogonial proliferation and apoptosis showed no difference between the two groups. This was believed to be one of reasons why spermatogonial numbers in aged testes was similar to those of controls. The apoptotic rate of primary spermatocytes in aged men was significantly elevated compared with that of controls (0.60% +/- 0.54% vs 0.22% +/-0.12%), resulting in a decrease of the number of primary spermatocytes per Sertoli cell. The expression of Bcl-xl was inversely correlated with the apoptotic rate in primary spermatocytes, suggesting that Bcl-xl may be related to the regulation of primary spermatocyte apoptosis. Based on these findings, we conclude that accelerated apoptosis of primary spermatocytes might account for a part of the mechanism of germ cell loss in aging men.

Enhanced chemiluminescence assay vs colorimetric assay for measurement of the total antioxidant capacity of human seminal plasma

Abstract: Although the enhanced chemiluminescence assay is commonly used to measure the nonenzymatic total antioxidant capacity (TAC) of the human seminal plasma, it is cumbersome, expensive, and time-consuming. We describe herein an alternate method to measure TAC that is based on the ability of antioxidants in seminal plasma to interfere with a reaction between 2,2'-azino-di-[3-ethylbenzthiazoline sulphonate] and metmyoglobin with H(2)O(2). This reaction produces a relatively stable blue-green color with absorbance maxima at 600 nm. We compared this colorimetric assay with our established chemiluminescence method and assessed quality control parameters (ie, intra-assay and interassay variabilities) in addition to intraobserver and interobserver differences. Our results show that the colorimetric assay was fairly predictive of antioxidant capacity similar to the chemiluminescence assay (P <.001). Furthermore, there was a high level of agreement between the duplicate measures by the same observer (intraobserver) and intra-assay variability, with a concordance correlation coefficient of 0.99. The interassay coefficient of variation was 4.7% (overall). The mean +/- SD of the difference between the 2 observers was 2.98% +/- 4.1%. In conclusion, we found that the colorimetric assay is a reliable and accurate method to evaluate seminal TAC, and it could be used as a simpler, rapid, and cheaper alternative to the chemiluminescence assay.

5-aza-2'-deoxycytidine induces alterations in murine spermatogenesis and pregnancy outcome.

Abstract: Because of the ability of cytidine analogues, such as 5-aza-2'-deoxycytidine, to incorporate into DNA and lead to decreases in DNA methylation, there has recently been renewed interest in using these drugs in anticancer therapy. To determine the effects of paternal 5-aza-2'-deoxycytidine treatment on spermatogenesis and progeny outcome in the mouse and whether effects are modulated by decreased levels of the predominant DNA methyltransferase, DNMT1, adult Dnmt1(+/+) and Dnmt1-deficient (Dnmt1(c/+)) male mice were treated with 5-aza-2'-deoxycytidine for 7 weeks, which resulted in dose-dependent decreases in testicular weight, an increase in histological abnormalities, and a decline in sperm counts, with no apparent effect on androgen status. Testes of Dnmt1(c/+) mice, however, were less severely affected by 5-aza-2'-deoxycytidine than were those of wild-type mice. The exposure of Dnmt1(+/+) male mice to even low doses of 5-aza-2'-deoxycytidine followed by mating elicited significantly reduced pregnancy rates and elevated preimplantation loss in females. Dnmt1 deficiency, however, protected against such drug-induced decreases in pregnancy rate but not preimplantation loss. Altered DNA methylation or DNMT1 activity may explain such adverse effects, because treatment resulted in dose-dependent decreases in the global methylation of sperm DNA. Thus, in the mouse, paternal administration of 5-aza-2'-deoxycytidine interferes with normal male germ cell development and results in reduced fertility, whereas lowering DNMT1 levels appears to partially protect the seminiferous epithelium from deleterious drug effects.

Induction of spermatogenesis by recombinant follicle-stimulating hormone (puregon) in hypogonadotropic azoospermic men who failed to respond to human chorionic gonadotropin alone.

Abstract: A multicenter, open-label, randomized efficacy and safety study was performed with combined human chorionic gonadotropin (hCG) and recombinant follicle-stimulating hormone (recFSH) (Puregon(R)) treatment to induce spermatogenesis in hypogonadotropic hypogonadal male patients. Patients were pretreated for 16 weeks with hCG to normalize testosterone levels. A total of 30 of 49 (61%) subjects had normalized testosterone levels but were still azoospermic after the hCG-alone phase. These patients were randomized into 2 treatment schemes with recFSH (2 x 225 IU recFSH per week [group A] or 3 x 150 IU recFSH per week [group B]), in combination with hCG for a period of 48 weeks. Total testosterone increased during the hCG-alone period from 1.08 and 1.22 ng/mL to 6.26 and 4.52 ng/mL for groups A and B, respectively. Combined gonadotropin treatment was effective in inducing spermatogenesis (sperm count >/=1 x 10(6)/mL) in 14 of 30 subjects (47%) and this was achieved after a median duration of treatment of approximately 5.5 months. Treatment time necessary for first sperm cells to appear in the ejaculate was related to the initial testicular volume. Subjects with a history of maldescended testes (11 of 30 subjects, 37%) showed a lower mean response to treatment as indicated by the relatively lower number of subjects reaching levels of at least 1 x 10(6) sperm cells per milliliter. Combined testicular volume increased during combined gonadotropin treatment from 11.4 to 24.0 mL. Although subjects with a history of maldescended testes had a lower starting testicular volume, subjects with and without a history of maldescended testes showed approximately the same relative increase in testicular volume. Total testosterone levels showed only a minor further increase during the combined gonadotropin treatment period. In conclusion, a weekly dose of 450 IU (3 x 150 IU or 2 x 225 IU) recFSH, in addition to hCG, was able to induce spermatogenesis in many hypogonadotropic azoospermic men who failed to respond to treatment with hCG alone.

New semen quality scores developed by principal component analysis of semen characteristics.

Abstract: The purpose of this study was to determine whether semen characteristics can be reduced to 2 semen quality (SQ) scores and whether these new scores can help the clinician in assessing the reproductive outcome. A cross-sectional sample of 250 patients seeking infertility treatment were analyzed for semen characteristics. In addition, 177 male-factor patients (prostatitis with infection, n = 40; varicocele, n = 77; varicocele with infections, n = 11; and vasectomy reversal, n = 43) were also assessed. Sperm motion kinetics were measured by computer-assisted semen analysis (CASA) (concentration, percent motility, curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], and amplitude of lateral head displacement [ALH]). Sperm morphology was assessed by both World Health Organization (WHO) guidelines and Tygerberg strict criteria. The principal component analysis model was used to construct an SQ score and a relative semen quality (RQ) score. A separate set of 25 normal donors was included as controls to determine normal ranges of the semen scores. Among the patient samples, SQ and RQ scores (median and 25% and 75% interquartile values) were 89.9, 25.1, and 130.4 and 106.1, 45.2, and 165.9, respectively. The SQ score for the varicocele and varicocele with infection groups was comparable (78.6 +/- 17.4 and 84.8 +/- 20.6) but significantly different from the control (100 +/- 10, P <.001 and.03). Vasectomy reversal patients had an SQ score of 78.2 plus or minus 16.8 that was significantly lower than controls (P <.001). The correlation among semen characteristics allows for the efficient combining of semen measures. The composite scores can summarize overall SQ and quantity. Both SQ and RQ scores provide meaningful information on the quality of semen specimens for the clinician.

Y chromosome deletions in azoospermic men in India.

Abstract: Genetic factors cause about 10% of male infertility. Azoospermia factors (AZFa, AZFb, AZFc) are considered to be the most important for spermatogenesis. We therefore made an attempt to evaluate the genetic cause of azoospermia, Y chromosome deletion in particular, in Indian men. We have analyzed a total of 570 men, including 340 azoospermic men and 230 normal control subjects. DNA samples were initially screened with 30 sequence-tagged site (STS) markers representing AZF regions (AZFa, AZFb, AZFc). Samples, with deletion in the above regions were mapped by STS walking. Further, the deletions were confirmed by Southern hybridization using the probes from both euchromatic and heterochromatic regions. Of the total 340 azoospermic men analyzed, 29 individuals (8.5%) showed Y chromosome deletion, of which deletion in AZFc region was the most common (82.8%) followed by AZFb (55.2%) and AZFa (24.1%). Microdeletions were observed in AZFa, whereas macrodeletions were observed in AZFb and AZFc regions. Deletion of heterochromatic and azoospermic regions was detected in 20.7% of the azoospermic men. In 7 azoospermic men, deletion was found in more than 8.0 Mb spanning AZFb and AZFc regions. Sequence analysis at the break points on the Y chromosome revealed the presence of L1, ERV, and other retroviral repeat elements. We also identified a approximately 240-kb region consisting of 125 bp tandem repeats predominantly comprised of ERV elements in the AZFb region. Histological study of the testicular tissue of the azoospermic men, who showed Y chromosome deletion, revealed complete absence of germ cells and presence of only Sertoli cells.

Testicular pain following vasectomy: a review of postvasectomy pain syndrome.

Abstract: Since the late 1970s the definition and etiology of chronic testicular pain following vasectomy has been evolving, as have the names for this syndrome, including postvasectomy orchalgia (Shapiro and Silber, 1979), late postvasectomy syndrome (Selikowitz and Schned, 1985), congestive epididymitis (Schmidt and Free, 1978), chronic testicular pain (McMahon et al, 1992), and postvasectomy pain syndrome (McCormack and LaPointe, 1988). Today, the syndrome is generally recognized by the term postvasectomy pain syndrome (PVPS). Although the mechanism has not been proven, a number of studies have shown changes in the histology of the epididymis and the testis following vasectomy. Patients with PVPS generally present with orchalgia; pain with intercourse, ejaculation, or both; pain with physical exertion; and tender or full epididymides (Nangia et al, 2000). The theory that these symptoms are caused by infection has long since been discarded, due to the lack of response to antibiotics in these patients and absence of acute infection when epididymal and vasal sections are removed during surgery and analyzed histologically (Selikowitz and Schned, 1985; Chen and Ball, 1991). Instead, histologic studies of samples collected during vasovasostomy or epididymectomy have demonstrated a chronic inflammatory process that begins to explain the development of PVPS.

Long-term culture and transplantation of murine testicular germ cells

Abstract: The objectives of this study were to develop an in vitro culture system to optimize germ cell proliferation and to measure the potential of the cultured germ cells to produce mature spermatozoa after transplantation into a recipient. Donor germ cells isolated from ROSA26 male mice were cultured with a STO feeder cell layer in Dulbecco's minimal essential medium (DMEM) supplemented with fetal bovine serum (FBS), stem cell factor, leukemia inhibitory factor, basic fibroblast growth factor, insulin-like growth factor 1, interleukin-11, L-glutamine, sodium pyruvate, 2-mercaptoethanol, murine oncostatin M, and platelet-derived growth factor. Donor germ cells formed colonies in the primary cultures after 8-21 days. These cultured colonies were maintained for 4 weeks or longer without subculture and proliferated for up to 8 passages over a period of 3 months. These colonies had alkaline phosphatase activity and incorporated 5-bromo-2'-deoxyuridine. These colonies were positive partially when screened with antibody for germ cell nuclear antigen and c-kit. Germ cells cultured with this supplemented medium showed enhanced colonization vs controls cultured with DMEM and FBS. Cultured germ cells from Rosa26 donors were transplanted into testes and were identified by X-gal staining and histological screening. The cells cultured in the supplemented medium colonized the tubules and initiated spermatogenesis in the recipient mice. This is an improved method for culturing germ cells and may be useful in gene therapy and the production of transgenic animals.

Effect of transient embryonic in vivo exposure to the endocrine disruptor methoxychlor on embryonic and postnatal testis development.

Abstract: The current study was designed to examine the effects of a transient embryonic exposure to the pesticide methoxychlor, an endocrine disruptor, on in vivo rat testis development and function. Gestating female rats were transiently administered methoxychlor (MXC) from embryonic day 7 (E7; EO = plug date) through E15. Embryonic testes were collected at E16 and postnatal (PO = day of birth) testes at P4, P10, P17-20, and P60. Seminiferous cords formed in testes from MXC exposed males. However, at E16, there was a decrease in the area of cords and an increase in interstitial area in MXC exposed testes when compared with controls. At all postnatal ages collected, there did not appear to be differences in seminiferous cord/tubule area, interstitial area, or number of seminiferous cords/tubules between untreated controls and males exposed to MXC. Exposure to the endocrine disruptor also had no effect on the postnatal organ weights of a variety of different organs, nor were testosterone levels altered. Interestingly, there were reductions in the number of germ cells in testes from MXC-exposed males at P17-P20 when compared with untreated controls. Furthermore, there was a twofold increase in apoptotic cells in tubules from pubertal P17-P20-MXC exposed males when compared with untreated controls. Testes were collected from adult P60 males to determine if early embryonic and postnatal alterations in germ cell numbers or testis cellular composition had compromised spermatogenesis. In adult P60 MXC exposed testes there were no gross morphological changes in testis structure or cellular composition over that of controls. However, there was an increase in apoptotic cell number in elongating spermatids in MXC exposed testes. Four P60 males that were exposed to MXC during gestation and 4 control males were bred with unexposed females to determine their ability to produce offspring. All MXC exposed males were capable of impregnating females and had normal litter size and pup weights. Combined observations demonstrated that exposure to MXC during gestation at a critical stage of testis development (ie, sex determination) affects embryonic testis cellular composition, germ cell numbers, and germ cell survival. While alterations in these parameters does not affect the ability of males to produce offspring, there appears to be a reduced spermatogenic capacity associated with MXC treatment. Therefore, transient embryonic exposure to an endocrine disruptor (methoxychlor) during gestation can influence the germline and fertility in adult males.

A simple self-report diary for assessing psychosexual function in hypogonadal men

Abstract: To examine the performance of a self-report diary to assess psychosexual function in hypogonadal men, 2 groups of eugonadal men and 2 groups of hypogonadal men were asked to record and score parameters for sexual desire, sexual enjoyment, sexual performance, sexual activity, and positive and negative moods daily for 7 days before a clinic visit (data set 1 and 2). The hypogonadal men were also assessed after testosterone replacement and some of the eugonadal men were studied while they were on placebo treatment. In this retrospective analysis, sexual function parameters (sexual desire, performance, and activity score) in the diary discriminated between the hypogonadal and eugonadal men with all measures significantly lower in hypogonadal men (all parameters P <.0001). Significant improvements in sexual desire and performance as well as sexual activity scores (P <.0001 for all parameters) in hypogonadal men after testosterone treatment were readily detected within 30 days. Mood and functional parameters did not show any change over time in eugonadal men on placebo treatment. The mood parameters assessed by the diary showed an excellent correlation with those assessed by the Profile of Mood States. Mood parameters were not clearly different between eugonadal and hypogonadal men at baseline. With testosterone treatment positive mood parameters were significantly increased (P <.0028 and.0001 set in data 1 and 2, respectively), and negative mood parameters improved in hypogonadal men (P <.0003 in data set 2). We conclude that this simple self-report diary is useful in assessing the sexual function and mood profile of hypogonadal subjects in clinical research.