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Contents Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 μg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal. [ABSTRACT FROM AUTHOR]

Abnormal standard semen characteristics and reduced sperm chromatin maturity can appear with increasing male age. However, the influence of paternal age on semen parameters is still controversial. Therefore, this study was designed to estimate the influence of paternal age not only on conventional semen characteristics but also on sperm DNA integrity. This research was carried out on ejaculated sperm cells obtained from men (n = 1124) aged ≥40 y and <40 y. Our data revealed a decreased semen volume and an increased percentage of DFI (sperm DNA fragmentation index) in older men compared to younger men in the entire study cohort, in men with normozoospermia and in men with abnormal semen parameters. Moreover, there was a higher incidence of sperm DNA damage (>10% DFI, low fertility potential) in the groups of men aged ≥40 y than in the groups of men aged <40 y. Older men had over twice the odds ratio for high sperm DNA damage as younger men. Our findings suggest a detrimental effect of advanced paternal age on sperm chromatin integrity. The data show that the evaluation of sperm DNA has greater clinical utility than standard semen analysis in case of male fertility potential assessment.

Because sperm chromatin may play a key role in reproductive success, we verify the associations between sperm chromatin abnormalities, embryo development and the ability to achieve pregnancy. The evaluation of sperm chromatin maturity using aniline blue (AB), chromomycin A3 (CMA3) and toluidine blue (TB) staining were carried out in group of males from infertile couples that underwent ICSI. Low levels of sperm chromatin abnormalities (< 16%) were found in most subjects (> 50%). A higher percentage of TB-positive sperm cells were discovered in the men from couples who achieved ≤ 50% fertilized oocytes compared to men who achieved > 50%. No significant differences were discovered by the applied tests between the men from couples who achieved ≤ 50% and those who achieved > 50% high-quality embryos on the 3rd or 5th day after fertilization, nor between the men from couples who achieved pregnancy and those who failed. The sperm chromatin maturity did not correlate with the ICSI results. However, the ROC analysis revealed a significant predictive value of TB-positive spermatozoa only for fertilization. Therefore, the TB assay can be considered as a useful test for the prediction of fertilization. Our findings suggest that the level of sperm chromatin abnormalities of the examined men was not clinically significant. No found associations between sperm chromatin maturity and embryo development and the ability to achieve pregnancy. We could not exclude the effects of the repairing processes in the fertilized oocyte. The use of complementary tests that verify the status of the sperm chromatin seems justified.

Bacterial semen inflammation/infection is an important diagnostic and therapeutic problem in contemporary andrology. The molecular mechanism by which inflammatory mediators compromise the fertilizing potential of germ cells is complex and multifactorial, and it remains unclear. To improve the understanding of the pathophysiology of human subfertility/infertility caused or complicated by reproductive tract inflammation/infection, we simultaneously evaluated a set of conventional (standard semen analysis) and nonconventional sperm parameters, including subcellular changes in sperm membranes (phospholipid scrambling, peroxidative damage, and phosphatidylserine (PS) externalization), mitochondria (mitochondrial transmembrane potential, ΔYm, and oxidoreductive capability), and DNA fragmentation in healthy young normozoospermic males with asymptomatic bacteriospermia and leukocytospermia. Both bacteriospermia and leukocytospermia had a deleterious effect on standard sperm parameters, including sperm concentration, motility and morphology. Bacteriospermia was associated with a simultaneous decrease in mitochondrial transmembrane potential and an increase in PS externalization, and with DNA fragmentation in both live and dead sperm. The highest MDA concentrations in sperm lysates were observed in the presence of leukocytes. This study demonstrates for the first time that bacteriospermia and leukocytospermia compromise sperm quality in healthy young normozoospermic males. Bacteria mainly participate in intrinsic mitochondria-dependent apoptotic cell death mechanisms. Oxidative stress plays a relevant role in decreasing routine sperm parameters during leukocytospermia. The value of these observations may be significant and may support the development of a new diagnostic platform (biomarkers) for infertile males with infections in the reproductive tract., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Purpose: To evaluate whether ejaculated human spermatozoa undergo complete apoptosis or necrosis during experimental semen bacterial infection in vitro., Methods: Apoptotic markers, including mitochondrial transmembrane potential (ΔΨm), phosphatidylserine (PS) externalization, and DNA fragmentation, have been detected simultaneously in ejaculated human sperm after their incubation with a known pathogenic (Escherichia coli), as well as with conditionally pathogenic bacterial strains (Staphylococcus haemolyticus, Bacteroides ureolyticus) and/or leukocytes. The ΔΨm and translocation of PS was evaluated using the JC-1 and Annexin V binding tests, respectively. A modified TUNEL assay with additional staining for sperm viability was used to detect the DNA fragmentation level., Results: The exposure of ejaculated spermatozoa to bacterial strains was associated with a simultaneous decrease in the percentage of sperm with normal ΔΨm and an increase in the proportion of Annexin V-positive sperm. Additionally, in the presence of S. haemolyticus, B. ureolyticus and/or leukocytes, a significant increase in the percentage of live TUNEL-positive (apoptotic) as well as dead TUNEL-positive (necrotic) sperm cells was also observed., Conclusions: The cellular death observed in spermatozoa in the presence of inflammatory mediators may be due to both apoptosis and necrosis. Here, we demonstrate for the first time that direct contact of conditionally pathogenic bacteria with ejaculated human sperm may play an even greater role in the promotion of apoptosis than in case of some pathogenic bacterial strains. These findings suggest that significant bacteriospermia and leukocytospermia may be direct causes of subfertility or additional negative factors worsening the prognosis of fertility in natural and assisted procreation.

Problem: Biological significance of increased number of leukocytes in ejaculate remains a subject of controversy. The aim of this research was to describe the morphological characteristics of the sperm elimination by leukocytes in in vivo and in vitro conditions using natural stimulator of the immune system--uropathogenic Escherichia coli, O75:HNT, isolated from semen., Method of Study: The study was performed on ejaculated spermatozoa from normozoospermic men with leukocytospermia (n=10, in vivo experiment) or without leukocytospermia (n=15, in vitro experiment). Morphological observations were performed using light and scanning electron microscopy., Results: Sperm removal by active leukocytes mediated by traditional phagocytosis and generation of extracellular traps were observed in in vivo and in vitro experiments., Conclusion: Our morphological data suggest that human germ cells activate leukocytes triggering both traditional phagocytosis and a novel trapping mechanism, followed by extensive sperm elimination., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To assess the in vitro effect of three bacterial isolates (Escherichia coli, serotype O75:HNT, Staphylococcus haemolyticus, and Bacteroides ureolyticus) and/or leukocytes on sperm motility, subcellular changes in sperm plasma membranes, and sperm fertilizing potential., Design: An in vitro model of semen bacterial infection., Setting: Basic research laboratory., Patient(s): Healthy normozoospermic volunteers and healthy blood donors., Intervention(s): None., Main Outcome Measure(s): Sperm plasma membrane stability was evaluated with a LIVE/DEAD Sperm Viability Kit and with the merocyanine 540 (M540) test both performed using flow cytometry. An oxiSelect TBARS Assay Kit was used for quantitative measurement of malondialdehyde content. Functional ability of spermatozoa was assessed by hypo-osmotic swelling (HOS) test and sperm penetration assay (SPA)., Result(s): The incubation of sperm with bacteria and/or leukocytes was associated with the reduction of their fertilizing potential demonstrated in both the HOS test and SPA, and this effect can be considered as a natural consequence of diminished motility and sperm membrane injury of lipid bilayers. Bacteroides ureolyticus demonstrated the most significant detrimental effect on sperm structure and function., Conclusion(s): Sperm motility and lipid sperm membrane status might be the earliest and the most sensitive indicators of sperm damage with negative consequences for male factor fertility, which can be attributed to both bacteria and leukocytes action., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to determine serum selenium concentrations in Polish Konik horses residing in the Odra Delta Nature Park (Poland) and to evaluate the activity of glutathione peroxidase and Se content in testes of this horse breed. In over 95% of cases, serum Se concentration was below the optimal range, and none of the horses examined was deficient in this trace element. The lack of Se deficiency in the animals examined suggests however, that the Polish Konik horses have a natural ability to the optimal use of nutrients available in their life area. Testicular content of Se and GSHPx activity in the colts was higher than those found in stallions, and a positive relationship between these antioxidants was demonstrated. The differences in Se contents and GSHPx activities in testes between colts and stallions suggest that selenoenzymes play important roles during the puberty of male horses.

Studies were performed on boar semen routinely used at the local artificial insemination (AI) centre. The semen was stored in a Safe Cell Plus commercial extender at 17 degrees C for nine days. The aim of our research was focused on changes in sperm plasma membrane integrity. The integrity of the sperm plasma membrane and acrosome as well as sperm motility decreased after dilution and during storage of the semen. The highest percentage of live sperm was identified by the eosin-nigrosin method, a lower percentage by the SYBR-14/PI test, and the lowest percentage of live cells was discovered by the hypoosmotic swelling (HOS) test (P < 0.01). There were significant differences between the results of staining methods and sperm motility (P < 0.01). No significant differences were found between the HOS test results and sperm motility. The plasma membrane integrity parameters positively correlated (P < 0.001) with each other and with sperm motility but negatively with aspartate aminotransferase activity. Our findings confirmed that the boar sperm aging changes, which increased during liquid semen preservation, were connected with the loss of function and integrity of the sperm plasma membrane. The employed complementary tests are comprehensive indicators of sperm membrane integrity during long-term semen preservation, and they can help establish the actual number of 'healthy' cells. The assays may be used in AI laboratories and should be incorporated into the routine of semen analysis.

The objective of the current study was to evaluate whether blood plasma progesterone (P(4)) measurements with a time-resolved fluorescent antibody test (TR-FAT) kit designed for humans was applicable for goats. The first experiment was designed to verify whether the concentrations of P(4) measured by TR-FAT can be used to monitor the estrous and ovarian activity in goats (n = 14). Blood samples (322) were collected, and the ovaries were scanned using ultrasonography. The second experiment was carried out on 4 goats (60 samples) and designed to compare the TR-FAT with radioimmunoassay (RIA). The time interval between the lowest concentrations of P(4) assayed by TR-FAT was 21 +/- 0.3 days and did not differ significantly from the length of the interestrous interval. The highest concentrations of P(4) were confirmed by detection of corpus luteum. During estrus, the mean concentration did not differ significantly between both methods. Significant differences were present during the luteal phases; however, the profiles of P(4) assayed by both methods followed a similar pattern. Regression analysis showed a correlation between the 2 methods (r = 0.98; r(2) = 0.96; P < 0.0001). The Bland-Altman plot showed that all averages were within the 95% limits of agreement; however, the differences between both methods tend to be greater as the average increases. The results demonstrated that the TR-FAT method can be applied to monitor estrous cycles in goats through measurements of plasma P(4) concentrations. Moreover, not only does the TR-FAT meet the requirements for safety, but it is also a method of high throughput, rapidity, and simplicity.

In the prostate the enzyme 5alpha-reductase converts testosterone into more active form--dihydrotestosterone (DHT). This hormone strongly influences prostate function and takes part in its pathology Since the connection between androgens and pathology of this gland has been proved, pharmacological inhibition of conversion became one of the therapy strategies both for benign prostate hyperplasia and prostate cancer. Last decade has brought numerous, long term clinical trials which involved numerous men being administered finasteride and other 5alpha-reductase inhibitors in bening prostate hypertrophy (BPH) treatment and prostate cancer (PCa) prevention. The results confirmed main assumptions, however revealed complex androgens and other factors' activity overlapping with the effect of therapy on prostate disease.

Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

The objective of our study was to evaluate the incidence of spermatozoa with nuclear DNA strand breaks in patients with normal routine sperm parameters (26 subjects). Sperm DNA fragmentation was measured using TUNEL test assessed in flow cytometer. Variable percentages of sperm with damaged DNA (9.42 +/- 7.68%; range: 2-36) were found. Two categories of patients were distinguished: (1) patients (8 out of 26 subjects) with < or = 4% of TUNEL-positive sperm and (2) patients (18 out of 26 subjects) with > 4% of TUNEL-positive sperm. A significantly lower percentage of normal sperm forms was found in patients with > 4% of TUNEL-positive sperm than in patients with < or = 4% of TUNEL-positive sperm. Moreover, a significant negative correlation (r(s) = -0.50) was noted only between a proportion of normal sperm forms and a proportion of TUNEL-positive spermatozoa. In electron microscope, a large number of spermatozoa with immature chromatin was observed more frequently in subjects with > 4% of TUNEL-positive cells (11 out of 18 subjects). Our results suggest that in some patients with normal routine sperm parameters, DNA fragmentation may be associated with poor sperm morphology. The diminished sperm genomic integrity may result from molecular disturbances in nuclear remodeling process during spermiogenesis. TUNEL assay is a screening tool that may help to discriminate between fertile and infertile men and may help to predict successful in vitro fertilization.

In the present paper, morphological and functional features of human sperm midpiece, contributing to the assessment of sperm fertility potential, have been described. The NADH-dependent NBT screening assay was used to identify and visualise: 1/ morphological defects of sperm midpiece, 2/ immature sperm forms with extensive cytoplasmic retention, reflecting developmental failure in spermatogenic remodelling process, 3/ cytoplasmic sperm conglomerates, related to apoptotic bodies and 4/ sperm NADH-dependent oxidoreductase system at the mitochondrial level, related to the reaction intensity. The used assay is an adequate marker of sperm mitochondrial activity and sperm maturity. It can also help discover sperm defects that result in asthenozoospermia and can be used as an additional indicator in the evaluation of the sperm midpiece, as well as in routine morphological examination of spermatozoa, having a considerable predictive value for in vivo and in vitro fertilization.

In several cases of asthenoteratozoospermia, electron microscopic investigation displayed immature sperm forms, morphological apoptotic patterns of spermatozoa and many cytoplasmic conglomerates with fragments of the sperm. In these patients, TUNEL assay showed a high percentage of spermatozoa with nuclear DNA fragmentation. Moreover, thickened and deformed midpieces were observed which contained supernumerary and redundant mitochondria with normal oxidoreductive capability and normal membrane potential. In these cases a high percentage of spermatozoa with normal DeltaPsim was detected. Nevertheless, a subpopulation of patients was found with an abnormal ultrastructure of sperm mitochondria and with a low percentage of spermatozoa with normal DeltaPsim. These findings indicate that low motility of spermatozoa may be related to abnormal morphogenesis of the midpiece containing functional mitochondria and that this may be a possible consequence of an apoptotic mechanism. Furthermore, our results show that asthenoteratozoospermia may result from dysfunction of sperm mitochondria and/or with alternations of the structures involved in sperm motility, i.e. the dense outer fibres, the fibrous sheath and the axoneme.

The purpose of our study was to determine the effect of dilution and liquid-preservation of boar sperm on oxidoreductive capability of their mitochondria. The semen was diluted with BTS extender produced from water purified by destillation or by reverse osmosis. The spermatozoa were stored over a four-day period at 16-18 degrees C. The function of sperm mitochondria was assessed using the screening cytochemical test for NADH-dependent oxidoreductases (diaphorase/NADH, related to flavoprotein). Morphological assessment of cytochemical reaction was carried out using a light microscope. The intensity of the reaction was evaluated by means of a computer image analysing system (Quantimet 600S), measuring the integrated optical density (IOD) and mean optical density (MOD) of the reaction product (formazans) occurring in the sperm midpieces. In the non-diluted semen, intensive cytochemical reaction throughout the length of the sperm midpiece was observed. Furthermore, spermatozoa with the intensive reaction displayed the high optical density values. After dilution the semen with two variants of experimental extender, and as the conservation time expired, the cytochemical reaction was less intensive. Moreover, the absence of formazan deposits in various parts of the sperm midpiece was also noted. These morphological features corresponded to low values of optical density. These findings suggest that the dilution of semen and the time of sperm preservation may be critical factors that handicap energy metabolism of sperm mitochondria. The type of water used in preparing BTS extender does not have any significant effect on the oxidoreductive capability of sperm boar mitochondria.