Seasonal variation of peptidase activities in the reproductive tract of Crotalus durissus terrificus

Seasonal quantitative patterns of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolylimino (PIP) aminopeptidases and prolyl oligopeptidase (POP) activities in soluble (SF) and solubilized membrane-bound (MF) fractions from ductus deferens, vagina and uterus were studied to evaluate their relationships with the reproductive cycle and the extensive long-term spermatozoa storage (LTSS) of the Neotropical rattlesnake Crotalus durissus terrificus. APB, PIP and POP were detected only in SF, while other peptidases were detected in SF and MF. APB, APN-PI and APN-PS were predominant in most tissues in all seasons. Peptidase activities had a common pattern of increment during the dry season (winter/autumn), which coincides with the mating period (autumn) and LTSS in the female (winter), as well as the reduction of spermatozoa motility and maintenance of fertilization capacity of spermatozoa. The high CAP activity in the soluble fraction of the vagina during winter, compared to summer (time of parturition) and spring, coincides with the relaxation of this tissue. In the soluble fraction, the low PAP-1 activity of the ductus deferens coincided with its high activity in the vagina during the winter; and the inverse occurred in summer, which is consistent with the physiological process of preserving spermatozoon viability. In conclusion, the studied peptidase activities had seasonal and tissue-specific characteristics, which suggest a relevant role in the reproductive physiology of C. d. terrificus. (C) 2008 Elsevier Inc. All rights reserved.

Inactive free : total prostate specific antigen ratios in ejaculate from men with suspected and known prostate cancer, compared with young control men

Objective To measure free:total prostate specific antigen (PSA) ratios in ejaculate from men with suspected and known prostate cancer, and in young control men, to determine if this ratio might be useful in discriminating benign from malignant prostatic conditions. Patients, subjects and methods Forty-seven men with prostate cancer (positive biopsies), 52 men with suspected prostate cancer but who had negative biopsies and 28 young men (< 30 years old) and with no family history of cancer, provided either a single ejaculate specimen (total 59) or multiple specimens (total 193) on subsequent occasions. Free and total PSA were measured using appropriate assays. All specimens were diluted in a PSA-negative female serum pool. Results The median free:total PSA ratios were 0.76-0.81 among the patient groups and control men, and there was no statistical difference between the groups. These data presumably only reflect the inactive component of free PSA, given that any alpha(2)-macroglobulin or alpha(1)-antichymotrypsin in the assay serum diluent was likely to have bound the active free PSA component in these samples. Similar results were obtained from those providing single and multiple samples, suggesting that a single specimen is sufficient to reflect the seminal plasma free:total PSA ratio over that period. There was no relationship between seminal plasma free:total PSA ratio and age for the controls or the positive biopsy group, although there was a negative relationship (i.e. a decline with age) that almost reached significance in those with negative biopsies (P = 0.058, R-2 = 0.07). Conclusions This is the first report of free:total PSA ratios in the ejaculate of men with suspected and known prostate cancer compared with young control men. Although no significant changes were detected in the free:total PSA ratios in ejaculate, these results may be confounded by differences in ratios with age, as is the case for serum PSA or different molecular forms of PSA. Indeed, these data suggest that a large proportion of free PSA in seminal plasma may be inactive. Further studies are needed to determine the potential utility of measuring free:total PSA, or other candidate markers, in ejaculate to better discriminate benign from malignant prostate disease.

Measuring male infertility: epidemiological aspects

Evidence suggests that human semen quality may have been deteriorating in recent years. Most of the evidence is retrospective, based on analysis of data sets collected for other purposes. Measures of male infertility are needed if we want to monitor the biological capacity for males to reproduce over time or between different populations. We also need these measures in analytical epidemiology if we want to identify risk indicators, risk factors, or even causes of an impaired male fecundity-that is, the male component in the biological ability to reproduce. The most direct evaluation of fecundity is to measure the time it takes to conceive. Since the time of conception may be missed in the case of an early abortion, time to get pregnant is often measured as the time it takes to obtain a conception that survives until a clinically recognized pregnancy or even a pregnancy that ends with a live born child occurs. A prolonged time required to produce pregnancy may therefore be due to a failure to conceive or a failure to maintain a pregnancy until clinical recognition. Studies that focus on quantitative changes in fecundity (that does not cause sterility) should in principle be possible in a pregnancy sample. The most important limitation in fertility studies is that the design requires equal persistency in trying to become pregnant and rather similar fertility desires and family planning methods in the groups to be compared. This design is probably achievable in exposure studies that make comparisons with reasonable comparable groups concerning social conditions and use of contraceptive methods.

Sensitization to wasp venom does not induce autoantibodies leading to infertility

Antigenic cross-reactivity has been described between the venom allergen (antigen 5) and mammalian testis proteins. Based on an allergen database we have previously shown that allergens can be represented by allergen motifs. A motif group was found containing venom antigen 5 sequences from different vespids. Using an optimized amino acid profile based on antigen 5 sequences for searching cross-reactive proteins, three human semen proteins belonging to the family of cysteine-rich secretory proteins (hCRISP) were found in the Swiss Protein database. To analyze antigenic cross-reactivity between antigen 5 and hCRISPs, antigen 5 from yellow jacket venom (Ves v 5) and two hCRISPs (CRISP-2 and -3) were chosen and produced as recombinant proteins in E. coli. A correlation was found between antibodies reacting with rVes v 5 and rhCRISP-2, -3 in a small human sera population indicating the presence of cross-reactive antibodies in human serum. Using intravenous immunoglobulin (IVIg), a therapeutic multidonor IgG preparation, cross-reactive antibodies were isolated that recognize rVes v 5, hCRISP-2 and -3 suggesting the presence of common epitopes between Ves v 5 and hCRISPs. However this cross-reactivity seems not to be linked to allergy to wasp venom as we could show no correlation between increasing CAP-class IgE level to wasp venom and IgG to sperm extract and hCRISPs. These data suggest that higher sensitization to wasp venom does not induce more antibodies against autoantigens and might not represent a higher risk to develop autoantibodies leading to infertility.

The use of quantitative polymerase chain reaction amplification method to determine sex ratio of human spermatozoa in semen and to determine maternal contamination of amniotic fluid samples

We have modified a technique which uses a single pair of primer sets directed against homologous but distinct genes on the X and Y chromosomes, all of which are coamplified in the same reaction tube with trace amounts of radioactivity. The resulting bands are equal in length, yet distinguishable by restriction enzyme sites generating two independent bands, a 364 bp X-specific band and a 280 bp Y-specific band. A standard curve was generated to show the linear relationship between X/Y ratio average vs. %Y or %X chromosomal content. Of the 51 purified amniocyte DNA samples analyzed, 16 samples showed evidence of high % X contamination while 2 samples demonstrated higher % Y than the expected 50% X and 50% Y chromosomal content. With regards to the 25 processed sperm samples analyzed, X-sperm enrichment was evident when compared to the primary sex ratio whereas Y-sperm was enriched when we compared before and after selection samples.

Effects of semen storage and separation techniques on sperm DNA fragmentation

Objective: To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Design: Controlled clinical study. Setting: An assisted reproductive technology laboratory. Patient(s): Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. Intervention(s): One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD. Main Outcome Measure(s): DNA fragmentation as measured by SCD. Result(s): There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels. Conclusion(s): The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use. (Fertil Steril (R) 2010;94:2626-30. (C) 2010 by American Society for Reproductive Medicine.)

Age-related increase of reactive oxygen species in neat semen in healthy fertile men

OBJECTIVES The effects of advancing paternal age on the male reproductive system are well known, but its effects on fecundity remain controversial. Although oxidative stress is associated with poor semen quality and function, a relationship with advancing male age has not been established. The objective of this study was to analyze the relationship between male age and seminal reactive oxygen species (ROS) levels in men presenting for voluntary sterilization. METHODS We prospectively evaluated 98 fertile men who were candidates for vasectomy. These were divided into 2 age groups: less than 40 years (n = 78) and 40 or more years (n = 20). We used 46 infertile patients as positive controls. Standard semen analysis, seminal leukocyte count and ROS levels were measured in all samples. Fertile men with leukocytospermia were excluded. RESULTS The mean age of the men was 35.1 +/- 5.6 years. Men 40 years and older had significantly higher ROS levels compared with younger men (P < 0.001). We observed a positive correlation between seminal ROS levels and age (r = 0.20; P = 0.040). In addition, ROS was negatively correlated with sperm concentration (r = -0-48; P < 0.001) and motility (r = -0.21; P = 0.030). CONCLUSIONS Reactive oxygen species levels are significant higher in seminal ejaculates of healthy fertile men older than 40 years. ROS levels in whole ejaculate are significantly correlated to age among fertile men. Because ROS are clearly implicated in the pathogenesis of male infertility, these data suggest that delayed fatherhood may reduce the chances of pregnancy as men become progressively less fertile with age.

First evaluation of human sperm quality in various geographic regions of Switzerland

A decline in human sperm quality and quantity has been reported in numerous Western countries. This observation was also accompanied by an increase in urogenital malformations. The need for epidemiological studies dealing with unbiased populations in order to understand the causes of these observations is obvious. In Switzerland, the large majority of young men are asked to attend a military camp to be drafted into the army. A few weeks before this camp, conscripts were contacted and invited to participate in a large national study on semen quality. The participation was totally voluntary and anonymous. From September 2005 to June 2007, 770 volunteers filled out a questionnaire, underwent a clinical examination and provided sperm, blood and urine samples. Using self-rated health assessments, the observed cohort could be considered as healthy and no testicular cancer was found. Moreover, the testicular volumes, measured using Prader's orchidometry and ultrasonography, were comparable to those already published for young male populations. The median sperm concentration was 47 x 10(6)/ml, which is close to the concentration reported in Denmark, known to have the highest incidence of testicular cancer in Europe. Statistically significant differences were observed between regions with a lower sperm concentration for men residing in the Alps (43 x 10(6)/ml) and in the Zurich area (36 x 10(6)/ml) compared to men from West Plateau (54 x 10(6)/ml) and from the Jura (54 x 10(6)/ml). Such a regional discrepancy could be related to environmental factors, including endocrine disruptors. In order to confirm such regional differences more volunteers from the already studied regions should be studied and other parts of the country should be investigated. The rather low sperm concentration of Swiss young volunteers should be considered as a national health issue and investigated further.

Effects of pentoxifylline treatment before freezing on motility, viability and acrosome status of poor quality human spermatozoa cryopreserved by the liquid nitrogen vapor method

The objective of the present study was to investigate the effects of the direct addition of pentoxifylline (PF) to the ejaculates of men with poor sperm quality before freezing on post-thaw sperm motility, viability, acrosome integrity, and agonist-induced acrosome reaction. Semen specimens from 16 infertile men with impaired sperm count and motility (oligoasthenozoospermia) were divided into two equal aliquots: one received no treatment (control) while the other was incubated with 5 mM PF (treated). Both aliquots were cryopreserved by the liquid nitrogen vapor method. Motility was assessed according to WHO criteria. Acrosome integrity and spontaneous and calcium ionophore-induced acrosome reactions were assessed with fluorescein isothiocyanate-conjugated peanut agglutinin combined with a supra-vital dye (Hoechst-33258). Cryopreservation impaired sperm motility (percentage reduction: 87.4 (interquartile range, IQ: 70.3-92.9) vs 89.1 (IQ: 72.7-96.0%)), viability (25.9 (IQ: 22.2-29.7) vs 25.6 (IQ: 19.7-40.3%)) and acrosome integrity (18.9 (IQ: 5.4-38.9) vs 26.8 (IQ: 0.0-45.2%)) to the same extent in both treated and control aliquots. However, PF treatment before freezing improved the acrosome reaction to ionophore challenge test scores in cryopreserved spermatozoa (9.7 (IQ: 6.6-19.7) vs 4.8 (IQ: 0.5-6.8%); P = 0.002). These data show that pre-freeze treatment of poor quality human sperm with pentoxifylline did not improve post-thaw motility or viability nor did it prevent acrosomal loss during the freeze-thaw process. However, PF, as used, improved the ability of thawed spermatozoa to undergo the acrosome reaction in response to calcium ionophore. The present data indicate that treatment of poor quality human sperm with PF may enhance post-thaw sperm fertilizing ability.

Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa

The aim of this investigation was to determine the presence of abnormal sperm chromatin packaging in spermatozoa with large nuclear vacuoles (LNV) selected via high magnification by analysing the pattern of chromomycin A3 (CMA3) staining. A prospective observational study was designed to analyse semen samples obtained from 66 men undergoing infertility diagnosis and treatment. The numbers of cells with normal (dull yellow staining of the sperm head/CMA3-negative) and abnormal (bright yellow fluorescence of the sperm head/CMA3-positive) chromatin packaging were determined on slides with normal and LNV spermatozoa. The presence of bright yellow fluorescence (CMA3-positive) was significantly higher (p < 0.0001) in spermatozoa with LNV than in normal spermatozoa (719/1351; 53.2% vs. 337/835; 40.3%, respectively), reflecting a higher percentage of abnormal chromatin packaging in spermatozoa with large LNV. Our data support the hypothesis that the presence of LNV reflects the presence of abnormal chromatin packaging, which may facilitate sperm DNA damage. As sperm nuclear vacuoles are evaluated more precisely at high magnifications using motile sperm organelle morphology examination (MSOME), the present results support the use of high-magnification sperm selection for intracytoplasmic sperm injection (ICSI).

Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-stranded or normal double-stranded DNA in spermatozoa with large nuclear vacuoles (LNV) selected by high magnification. Fresh semen samples from 30 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and LNV were selected at x8400 magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation in spermatozoa with LNV (29.1%) was significantly higher (P < 0.001) than in spermatozoa with NN (15.9%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono- and oligonucleosomes), and single-strand breaks (nicks) in high molecular weight DNA occur more frequently in spermatozoa with LNV. Similarly, the percentage of denatured-stranded DNA in spermatozoa with LNV (67.9%) was significantly higher (P < 0.0001) than in spermatozoa with NN (33.1%). The high level of denatured DNA in spermatozoa with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibres. The results show an association between LNV and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high magnification for ICSI.

Significance of extruded nuclear chromatin (regional nuclear shape malformation) in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-strand or normal double-strand DNA in spermatozoa with extruded nuclear chromatin (ENC) selected by high magnification. Fresh semen samples from 55 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and ENC were selected at 8400x magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation was not significantly different (p = 0.86) between spermatozoa with ENC (19.6%) and those with NN (20%). However, the percentage of spermatozoa with detectable denatured-stranded DNA in the ENC spermatozoon group (59.1%) was significantly higher (p < 0.0001) than in the NN group (44.9%). The high level of denatured DNA in spermatozoa with ENC suggests premature decondensation and disaggregation of sperm chromatin fibres. The results show an association between ENC and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high-magnification intracytoplasmic sperm injection.

Use of corticosteroid therapy on the modulation of uterine inflammatory response in mares after artificial insemination with frozen semen

In order to modulate uterine inflammatory response and evaluate the effect of corticosteroid therapy on fertility, 90 cycles of 45 mares were used for artificial insemination with frozen semen, using three different protocols: G1 - inseminated with frozen semen (800 x 10(6) viable spermatozoa pre-freezing) + 20 mL of seminal plasma; G2 - inseminated with frozen semen (800 x 10(6) viable spermatozoa pre-freezing) + corticosteroid therapy; G3 - inseminated with frozen semen (800 x 10(6) viable spermatozoa pre-freezing) + 20 mL of seminal plasma + corticosteroid therapy. Corticosteroid therapy consisted on one administration of prednisolone acetate (0.1 mg/Kg - Predef (R)) when mares presented 35mm follicles and uterine edema, concomitantly with the unique dose of hCG (human chorionic gonadotropin), then repeated each 12 hours until ovulation. on first fertility trial, with normal mares, there was no difference between control and treated groups (p>0.05), using seminal plasma associated with corticosteroid therapy (40 vs. 38%, respectively) or corticosteroid therapy alone (40 vs. 45% respectively). The second fertility trial, performed with mares with previous history of post-insemination endometritis, demonstrated a significant increase of pregnancy rate when mares were submitted to corticosteroid therapy (0.0 vs. 64.5%, respectively; p<0.05). Corticosteroid therapy was shown to be safe, with no physical or reproductive alterations on treated mares, demonstrating to be an adequate option to those animals with history of post-breeding or post-insemination endometritis. Further clinical research is necessary to confirm these results and contribute to the establishment of preventive therapy for cases of post-insemination endometritis.

What's the signification of nuclear vacuoles in human sperm?

The aim of this study was to determine the extent of DNA fragmentation and the presence of single/denatured or double stranded of DNA in sperm with large nuclear vacuoles (LNV) selected by high-magnification. A total of 30 patients had fresh semen samples prepared by discontinuous concentration gradient. Sperm with normal nucleus (NN) and LNV were selected at 8400x magnification and placed in different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double stranded DNA was identified by acridine orange fluorescence method. The percentage of DNA fragmentation in LNV sperm (29%) was significantly higher (P<0.001) than NN sperm (15.8%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono and oligonucleosomes), and single strand breaks (nicks) in high molecular weight DNA occur more frequently in LNV. Identically, the percentage denatured stranded DNA in sperm with LNV (67.9%) was significantly higher (P <0.0001) than NN sperm (33%). The high level of denatured DNA in sperm with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibers. Our results support an association between LNV sperm and DNA damage, and the routine selection and injection of morphological motile sperm at high magnification for ICSI. The adverse effect (DNA fragmentation or denaturation) leads to concern particularly about the possibility of iatrogenic transmission of genetic abnormalities. Copyright - SBRA - Sociedade Brasileira de Reprodução Assistida.