Protective effect of Propolfenol® on induced oxidative stress in human spermatozoa.

The propolis extract was shown to possess the capacity to protect sperm membrane from the deleterious action of oxidative attack. Oxidative stress can induce propagation of a lipid peroxidation (LPO) chain reaction because spermatozoa contain high concentration of unsaturated fatty acids. This study aimed at evaluating in vitro the possible toxicity and/or the antioxidant properties of Propolfenol® in ejaculated human spermatozoa. A colorimetric assay determined the total flavonoid content by spectrophotometry and a high-performance liquid chromatography-diode array detection analysis the quantity of galangin, pinocembrin and caffeic acid phenylethilic ester (CAPE). Sperm parameters such as motility, vitality and DNA integrity were assessed utilising optical microscopy. The antioxidant properties Propolfenol® against LPO induced by tert-Butyl Hydroperoxide were evaluated using the C11-BODIPY581/591 probe. Chemical analysis of Propolfenol® revealed low quantities of galangin, pinocembrin and CAPE; cyclic voltammetry experiments showed that Propolfenol® may exert an antioxidant activity. A protective action of Propolfenol® (20 and 100 µg/ml) on induced LPO in human spermatozoa was detected. Propolfenol® may be proposed as the supplement in media for sperm preparation techniques or cryopreservation to counteract the increased presence of reactive oxygen species generated by these methods.

Sperm with fibrous sheath dysplasia and anomalies in head-neck junction: focus on centriole and centrin 1.

Spermatozoa with a rare combination of two monomorphic sperm defects, dysplasia of the fibrous sheath (DFS) and alterations in head-mid-piece junction were analysed. The main focus was to explore the status of the centriole, a key organisation during fertilisation, using the centrin 1, a calcium-binding protein linked to this structure. The sperm quality was examined by light, scanning and transmission electron microscopy (SEM, TEM); immunocytochemistry was performed for tubulin, A-kinase anchor protein 4 (AKAP4) and centrin 1. Spermatozoa showed DFS defect associated with anomalies in head-tail attachment detected by SEM and TEM. Immunolocalisation of tubulin, AKAP4 and centrin 1 confirmed these alterations. Centrin 1 was visible in 67% of spermatozoa (in only 13% centrin localised in a normal position); in the majority of sperm centrin 1's location was altered, sometimes bent; often four spots, indicating the presence of two implantation fossae, were detected. At the centriolar level, immunoreactive fragments, frequently invading the entire short and thick tail, were observed. Centrin 1 is an essential component of the spermatozoa connecting piece and plays a role in centrosome dynamics during sperm morphogenesis and in zygotes and early embryos during spindle assembly. It is important to shed light on these rare conditions in order to better manage the patients during assisted reproductive technology.

Evaluation of enzymatic and non-enzymatic antioxidants in seminal plasma of men with genitourinary infections, varicocele and idiopathic infertility.

UNLABELLED: This study was aimed to assess the antioxidant enzymatic and non-enzymatic compounds in semen of infertile men. Seventy-four infertile patients were grouped according to their clinical diagnosis: genitourinary infection, varicocele, idiopathic infertility. Semen samples of fertile men represent the control. Semen characteristics were evaluated by light and transmission electron microscopy (TEM). TEM data was quantified with a mathematical formula, which provides numerical scores. Spectrophotometric and HPLC methods were used to measure the amount of reduced (GSH), oxidised glutathione (GSSG), ascorbic acid (AA) and malondialdehyde (MDA, marker of lipid peroxidation) and the activity of glutathione reductase, catalase (CAT), glutathione peroxidase. Infertile groups showed significantly decreased values of sperm parameters vs. CONTROLS: In infection and varicocele groups, the seminal MDA levels were significantly increased when compared to controls (p < 0.001), indicating an alteration of oxidative status and a peroxidative damage. In infection and varicocele groups, AA levels were reduced (p < 0.05) vs. control; in the varicocele group, the GSH levels were also decreased (p < 0.05). Significantly higher CAT activity was observed in infection and varicocele groups vs. fertile men (p < 0.001 and p < 0.05 respectively). The GSH/GSSG ratio was significantly decreased in varicocele and idiopathic infertility groups vs. control (p < 0.01). The study of the alteration of a single parameter of oxidative stress or of the antioxidant system may not have a relevant clinical value to estimate male fertilising potential and the background of infertility causes, since complex and multifactorial mechanisms are involved in different pathologies. In our study, each pathology is characterised by a definite pattern of markers such as MDA and enzymatic and non-enzymatic antioxidant compounds. In the different pathologies related to infertility, the identification of the complex of involved parameters could be useful in the diagnosis, prognosis and in the choice of a possible treatment such as specific antioxidant supplements.

Cytokines release and oxidative status in semen samples from rabbits treated with bacterial lipopolysaccharide.

This study was aimed to evaluate the effects of a lipopolysaccharide- (LPS) induced inflammation on cytokines release and oxidative status of semen samples from buck rabbits at different times after treatment. Semen analysis was performed by optical microscopy and sperm motility evaluation by the computer-assisted sperm analyzer. The presence of activated macrophages and apoptotic/necrotic sperm was evaluated by fluorescent microscopy. A panel of cytokines, interleukin (IL)-6, IL-8, IL-1ß, and tumor necrosis factor-α, were detected and quantified in seminal plasma using the Bio-Plex Cytokine assay. Reactive oxygen metabolite and thiobarbituric acid-reactive substance determinations were carried out by spectrophotometry and tocopherol analysis by high performance liquid chromatography. The sperm motility and track speed were reduced in LPS-treated rabbits. The activated macrophages in LPS-treated buck rabbits significantly increased from 0.50 × 10(6)/mL (baseline) to 27 × 10(6)/mL on Day 21; successively, there was a progressive reduction. Apoptotic and necrotic sperm in LPS rabbits followed more or less the same trend. The reactive oxygen metabolite levels in semen from LPS-treated rabbits showed higher values compared with those evaluated in controls, particularly during the lag time, Days 1 to 3. The sperm thiobarbituric acid-reactive substances highlighted a peak in LPS-treated rabbits compared with those of controls on Day 1 after LPS treatment, and the different T isoforms (α, δ, and γ+ß) showed a similar trend with a significant decrease on Day 1 after injection and a recovery on Days 30 to 56. Until Days 3 to 21 from the treatment, higher levels of IL-1ß and tumor necrosis factor-α were detected in seminal plasma of LPS-treated rabbits. Interleukin-6 showed a peak on Day 3 after LPS treatment, and on Day 7, the value was similar to the control group. In conclusion, this study confirms that the buck rabbit is a good model for mimicking and understanding the inflammation mechanisms, which may induce male infertility, in particular that a systemic inflammatory status causes alterations to the sperm cells through a shift in the balance between the oxidant and antioxidant systems.

Semen characteristics and malondialdehyde levels in men with different reproductive problems.

The aim of this study was to assess the level of malondialdehyde (MDA) in the seminal plasma of infertile men and to highlight a relationship between the level of MDA and semen parameters. Eighty-one infertile patients were divided into groups according to their clinical diagnosis: genitourinary infections, varicocele and idiopathic infertility. Semen quality was assessed by light and transmission electron microscopy (TEM). TEM data were quantified with a mathematical formula able to obtain a fertility index and the percentage of sperm apoptosis, immaturity, and necrosis. Seminal MDA levels were determined by spectrofluorometry. Scrotal Eco-color Doppler was used to detect the varicocele. Infected patients had a positive bacteriological semen analysis. A control group consisted of 14 normospermic fertile men. Fertile group showed significantly increased values of sperm concentration, motility, and fertility index compared to infertile groups. In the infertile groups, sperm motility, concentration, apoptosis, and fertility index were not significantly different. In infection group, the percentage of necrosis was significantly higher than that observed in fertile men, varicocele, and idiopathic infertility groups (p < 0.001). MDA levels increased significantly in infection group in comparison with varicocele group (p < 0.01), idiopathic infertility group, and fertile men (p < 0.001) and in varicocele group compared to idiopathic infertility group (p < 0.001). In infection group, MDA levels positively correlated with sperm concentration (p < 0.01), fertility index (p < 0.05), and necrosis (p < 0.001), whereas a negative correlation was found with motility (p < 0.01). In varicocele group MDA levels correlated positively with necrosis and negatively with immaturity (p < 0.05). In fertile men and idiopathic infertility group, they did not show any correlation. In conclusion, we suggest that the evaluation of seminal MDA may be a good marker for understanding pathologies responsible of a sperm motility reduction such as urogenital infections or inflammatory status.

Immunolocalisation of ghrelin and obestatin in human testis, seminal vesicles, prostate and spermatozoa.

The role of ghrelin and obestatin in male reproduction has not completely been clarified. We explored ghrelin and obestatin localisation in the male reproductive system. Polyclonal antibodies anti-ghrelin and anti-obestatin were used to detect the expression of these hormones in human testis, prostate and seminal vesicles by immunocytochemistry, while in ejaculated and swim up selected spermatozoa by immunofluorescence. Sertoli cells were positive for both peptides and Leydig cells for ghrelin; germ cells were negative for both hormones. Mild signals for ghrelin and obestatin were observed in rete testis; efferent ductules were the most immune reactive region for both peptides. Epididymis was moderately positive for ghrelin; vas deferens and seminal vesicles showed intense obestatin and moderate ghrelin labelling; prostate tissue expressed obestatin alone. Ejaculated and selected spermatozoa were positive for both peptides in different head and tail regions. This study confirms ghrelin localisation in Leydig and Sertoli cells; the finding that ghrelin is expressed in rete testis, epididymis, vas deferens and seminal vesicles is novel, as well as the localisation of obestatin in almost all tracts of the male reproductive system. This research could offer insights for stimulating other studies, particularly on the role of obestatin in sperm physiology, which is still obscure.

Thermal water of Vetriolo, Trentino, inhibits the negative effect of interleukin-1β on nitric oxide production and apoptosis in human osteoarthritic chondrocyte.

The thermal water of Vetriolo in Trentino, Italy (VW) has been known over 150 years for its therapeutic properties in the treatment of osteoarthritis (OA). This is a highly mineralized water, strongly acidic sulfate, rich in calcium, magnesium and iron and used for balneotherapy after dilution. The aim of our study was to investigate the possible in vitro effects of the VW in human OA chondrocytes cultivated in the presence or in the absence of Interleukin-1 beta (IL-1beta). OA chondrocytes were cultivated in Deionized Water (DW) (DW-DMEM, controls), or in one of three different VW-DMEM media, in which DW had been totally (100 percent) or in part (25 or 50 percent) substituted with VW. All samples were analyzed before and after treatment with IL-1beta at a concentration of 5 ng/ml. After 48 h, we evaluated the cell viability, the release of nitric oxide (NO) in culture medium, the inducible nitric oxide synthase (iNOS) expression, and the percentage of apoptosis and necrosis. Finally, we carried out a morphological assessment using a transmission electron microscope (TEM). Our data showed that VW alone at 25 or 50 percent concentration did not affect the viability of cultured OA chondrocytes, and determined a significant survival recovery rate in cultures stimulated with IL-1beta. On the contrary, the VW alone at 100 percent of concentration reduced, in a significant (P less than 0.05) manner, the cells viability. NO levels were low both in DW-DMEM cultures and in those reconstituted with 25 or 50 percent of VW, and were significantly (P less than 0.05) increased in cultures with 100 percent of VW. VW at 25 or 50 percent concentration significantly (P less than 0.001) reduced the NO production induced by IL-1beta. The data of the NO levels were confirmed by the immunocytochemistry assay for iNOS. Our experiments confirmed the pro-apoptotic effect of IL-1beta and demonstrated a protective effect of VW at 25 or 50 percent concentration. These findings were confirmed by TEM. In conclusion, our study demonstrated that VW alone at 25 or 50 percent concentration modifies neither morphology nor NO production and neither iNOS expression nor apoptosis, but it inhibits the negative effects of IL-1beta in chondrocytes cultures.

CagA-positive Helicobacter pylori infection and reduced sperm motility, vitality, and normal morphology.

Helicobacter pylori (HP) infection, particularly when caused by strains expressing CagA, may be considered a concomitant cause of male and female reduced fertility. This study explored, in 87 HP-infected males, the relationship between infection by CagA-positive HP strains and sperm parameters. HP infection and CagA status were determined by ELISA and Western blotting; semen analysis was performed following WHO guidelines. The amino acid sequence of human enzymes involved in glycolysis and oxidative metabolism were "blasted" with peptides expressed by HP J99. Thirty-seven patients (42.5%) were seropositive for CagA. Sperm motility (18% versus 32%; P < 0.01), sperm vitality (35% versus 48%; P < 0.01) and the percentage of sperm with normal forms (18% versus 22%; P < 0.05) in the CagA-positive group were significantly reduced versus those in the CagA-negative group. All the considered enzymes showed partial linear homology with HP peptides, but four enzymes aligned with four different segments of the same cag island protein. We hypothesize a relationship between infection by strains expressing CagA and decreased sperm quality. Potentially increased systemic levels of inflammatory cytokines that occur in infection by CagA-positive strains and autoimmune phenomena that involve molecular mimicry could explain the pathogenetic mechanism of alterations observed.

Alterations of the FSH and LH receptor genes and evaluation of sperm ultrastructure in men with idiopathic hypergonadotropic hypogonadism.

PURPOSE: Gonadotropins, interacting with their gonadal receptors, play a key role in sexual development, reproductive functions and metabolism. In this study we performed the genetic analysis of FSHR and LHR and semen investigation in 14 infertile men with normal level of T and elevated levels of FSH and/or LH in the absence of other causes of infertility. METHODS: Sperm parameters were analysed following WHO (2010) guidelines and sperm morphology by Transmission Electron Microscopy (TEM) analysis mathematically elaborated. FSHR and LHR gene mutations have been searched by PCR technique, followed by DHPLC analysis and direct sequencing. RESULTS: In FSHR, we found no difference in the frequency between Ala or Thr at position 307, Ser was at codon 680 in all subjects. Three patients had an heterozygous mutation at codon 419. Three intronic polymorphisms (rs2091787, rs6708637, rs1922464) were significantly found compared to controls; the single allele frequency and the odds ratio were calculated. Two new variants: the Cys338Arg and the Gln123Glu were detected in two different patients. Regarding LHR, three patients were heterozygous for the known variant Glu354Lys and two for Ile374Thr. Intronic polymorphisms were not identified. A new variant, the Val144Ile was found. By the routine semen analysis, variable seminal conditions in this group of patients was observed, on the contrary TEM data mathematically elaborated showed a homogeneous decrease in fertility index and increase in sperm pathologies such as apoptosis and immaturity. CONCLUSIONS: The obtained results suggest that a deeper examination of spermatozoa, achieved by the use of more powerful tools such as TEM or molecular analysis, are advisable in patients with hypergonadotropic hypogonadism.

In vitro effect of gold and silver nanoparticles on human spermatozoa.

The cytotoxicity of Au/Ag nanoparticles (NPs) on human spermatozoa was investigated in vitro. Semen from donors were incubated (37 °C, 60'-120') with 30, 60, 125, 250 and 500 µM Au/Ag-NPs. Sperm motility was evaluated following WHO guidelines; sperm viability was assessed with eosin Y test. Au-NPs were characterised and localised with field emission gun-based scanning transmission electron microscope/energy dispersive spectroscopy and transmission electron microscopy. Both tested NPs exerted a significant dose-dependent effect on motility and viability of human spermatozoa (P < 0.001). Ag-NPs seem to show a slightly elevated toxicity although not significant (P > 0.05). Au-NPs were localised in spermatozoa, whereas Ag-NPs were undetectable. In conclusion, Au-NPs and Ag-NPs do not appear to be harmful for human spermatozoa up to high concentrations (250-500 µM) that are probably difficult to reach in vivo. It is mandatory to explore the genotoxic effect of NPs in germ cells.

Effect of quercetin, rutin, naringenin and epicatechin on lipid peroxidation induced in human sperm.

Quercetin, rutin, naringenin, epicatechin are flavonoids with diverse properties, including antioxidant potential. We evaluated, in vitro, the cytotoxicity of these flavonoids (20, 30, 50, 100, 200, 400 µM) in swim-up selected human sperm. Antioxidant activity was tested against tert-butylhydroperoxide induced lipid peroxidation using a C11-BODIPY(581/591) probe and transmission electron microscopy. A significant concentration-dependent effect on sperm viability (P<0.001) and motility (P<0.001) was observed. Lipid peroxidation was decreased in samples treated with 30 µM quercetin (P<0.01) and 30 µM rutin (P<0.05) versus samples incubated with tert-butylhydroperoxide alone. Naringenin (50-100 µM) showed a low protective effect and epicatechin (200 µM) was not efficacious. Transmission electron microscopy analysis confirmed the protective action of rutin and in particular quercetin on damages induced by lipid peroxidation. These results underlined the antioxidant properties of quercetin and rutin. A possible role of these compounds in the supplementation of media used during semen handling warrants attention and further studies.

Secretion patterns and effect of prostate-derived granules on the sperm acrosome reaction of rabbit buck.

There is increasing evidence that the particulate fraction of seminal plasma plays an important role in reproduction of several mammalian species. However, the origin and role of these granules in the physiology of rabbit spermatozoa is partially unknown. The aim of this study was to investigate the implication of prostate gland in the production and secretion of granules into the rabbit semen and the role of prostate-derived granules in the sperm acrosome reaction. Light and electron microscopy of the prostate gland showed that the anterior and middle tracts of the prostate (namely the proprostate and prostate, respectively) are chiefly implicated in the secretion of granules of different size: smaller granules (SG; 0.5 µm) and large granules (LG; 4 µm). Two major patterns of secretion were identified, based on electron microscope views: storage granules (large granules) seem to empty inner smaller granules directly into the duct by exocytosis, or the storage vesicle itself is released in toto into the ducts (diacytosis). In vitro experiments using granules from vasectomized rabbits, to exclude testicular origin of granules, showed that granules reduce the acrosome reaction of Percoll-selected spermatozoa, independently of the size. Interestingly, spermatozoa incubated with heat-treated granules showed a higher sperm acrosome reaction rate, suggesting a potential role of granule-derived proteins in this process. Inhibition of the acrosome reaction is a crucial event in rabbit reproduction; ejaculated spermatozoa have to wait for a long time (8-16 h) for egg availability in the female tract after mating. Taking together, our results demonstrate that prostate granules secreted either by exocytosis or diacytosis can preserve spermatozoa fertilizing ability, by preventing sperm acrosome reaction. The type of granule-derived proteins or other macromolecules implicated in this process should be further investigated.

Effect of a bacterial lipopolysaccharide treatment on rabbit testis and ejaculated sperm.

In a previous study, we reported the short- and long-term effects of bacterial lipopolysaccharide (LPS)-induced inflammation on rabbit sperm quality. This study was aimed at exploring the spermatogenesis of the rabbit model focussing on the possible damages occurring to the testis and ejaculated sperm. Twenty New Zealand White rabbit bucks were divided into two groups. One group was inoculated intra-peritoneally with LPS, the other group, considered as control, was treated under the same conditions with saline only. Semen samples were collected before LPS injection, the 7th, 14th, 21st, 30th, 45th, 60th and 90th day after LPS treatment. Semen parameters were evaluated following international guidelines. The kinetic characteristics of ejaculated sperm were analysed using a computer-assisted sperm analyzer and the ultrastructural characteristics were explored by transmission electron microscopy (TEM). On the 7th, 14th and 30th day, testis from treated rabbits and controls were obtained. Testis samples were analysed by light microscopy and TEM. The induced LPS lesions in the testis became evident the 7th day after treatment, with a decrease in germinal cells and with an increase in structurally altered Sertoli cells; normal spermatogenesis was restored on the 30th day. The testicular damages observed on day 7 were probably responsible for the reduction in sperm concentration and motility and the ultrastructural alterations that were detected in the ejaculated sperm on the 14th through the 30th days after treatment. In conclusion, rabbit buck treated with LPS could be a useful model for studying the effect of an induced systemic inflammation on spermatogenesis.

Effect of trans-resveratrol on induced oxidative stress in human sperm and in rat germinal cells.

Resveratrol is a phytoalexin with antioxidant properties. We evaluated resveratrol toxicity in swim-up selected human sperm and in rat spermatocytes and spermatids separated by the STAPUT method. Resveratrol antioxidant activity was tested against lipid peroxidation (LPO) induced by tert-butyl hydroperoxide. LPO was evaluated using the C11-BODIPY(581/591) probe and transmission electron microscopy in samples incubated with and without resveratrol. LD50 for human sperm and rat spermatids was 50 µM; spermatocytes were more sensitive to resveratrol cytotoxicity. Sperm motility increased progressively at 30 µM, 15 µM and 6 µM. 15 µM resveratrol acts against LPO, preserving sperm chromatin and plasma membranes. LPO were more marked in spermatocytes than in spermatids and the effect of resveratrol was more evident in spermatocytes. In this study, the scavenger properties of resveratrol were demonstrated in vitro in human sperm and rat germ cells, thus resveratrol could be added to the media used in assisted reproduction techniques and cryopreservation when oxidative stress is exacerbated.

Semen quality of male idiopathic infertile smokers and nonsmokers: an ultrastructural study.

This retrospective study was aimed at evaluating the effects of cigarette consumption on semen parameters in a group of men with idiopathic infertility. The semen quality of 2 groups of men with idiopathic infertility, smokers (n = 118) and nonsmokers (n = 153), were compared. Conventional semen analysis was performed and sperm morphology was assessed by transmission electron microscopy (TEM). TEM data were elaborated by means of a mathematical formula based on a Bayesian technique able to furnish a fertility index (FI), and the percentages of sperm apoptosis, necrosis, and immaturity. Values of normality recommended by World Health Organization guidelines were used as a control for conventional semen analysis, and values from sperm of 25 men of proven fertility were used for TEM indices. Infertile smoker and nonsmoker patients showed similar sperm parameters, although sperm motility and TEM analysis values in both groups were significantly impaired compared with controls. Smoker patients were then classified as mild (>or=1 and 10 and <20 cigarettes/day), or heavy smokers (>or=20 cigarettes/d). Sperm concentration and FI were significantly (P < .05) different among the 3 considered smoker classes. Comparing the pairs of smoker classes, sperm concentration and FI in heavy smokers were significantly lower (P < .05) than that observed in mild smoker and nonsmoker groups. Although semen quality in males with idiopathic infertility seems not to be dramatically affected by cigarette consumption, heavy smokers show significantly lower sperm concentration and FI: another strong reason to stop smoking.

Short- and long-term effects of lipopolysaccharide-induced inflammation on rabbit sperm quality.

Infections and resulting inflammation are widely known to cause transient or permanent male infertility. The objectives of this study were (1) to provide a suitable animal model of a sub-acute inflammatory state by intraperitoneally inoculating bacterial lipopolysaccharides (LPS) and (2) to define the short- and long-term effects of this state on the sperm quality of rabbit bucks. Two series of experiments were performed to accomplish these objectives. In experiment 1, 15 healthy New Zealand White rabbit bucks were divided into five homogeneous groups, receiving 25, 50, 100 and 150 microg/kg body weight (b.w.) of E. coli LPS dissolved in 2ml of sterile saline or only saline (control), respectively. White blood cells (WBC), rectal temperature, feed intake and mating ability were observed for 3 consecutive days following inoculation. Inoculation of 50 microg/kg b.w. produces a reversible inflammation-like state that lasts for about 3 days, with minimal distress to the animals, and therefore it was used in our experiment. The major symptoms were fever and anorexia. Changes in WBC count and a moderate reduction in reproductive activity also occurred. In experiment 2, two groups of five rabbit bucks each were treated with 50 microg/kg b.w. E. coli LPS diluted in 2ml of saline or only saline (controls), respectively. Semen samples were collected weekly up to 56 days after inoculation and the changes in semen characteristics were examined. During the first 3 days, semen volume and concentration decreased in both experimental groups, probably due to the high collection frequency. Sperm membrane integrity and the number of necrotic sperm were seriously affected 30 days after the LPS challenge, reaching a maximum at the end of the spermatogenic cycle (56 days). These results suggest that a sub-acute inflammation may cause infertility by compromising sperm membrane integrity which decreases a month after LPS-treatment. In addition, the rabbit could be a useful LPS animal model for further study of the effects of inflammation and the underlying mechanisms on sperm characteristics.

TEM and FISH studies in sperm from men of couples with recurrent pregnancy loss.

The role of the male partner in recurrent pregnancy loss (RPL) is not clear. In this study, semen characteristics of 22 men whose partners had experienced RPL were examined by light microscopy. Sperm morphology was analysed by transmission electron microscopy (TEM) and the data were mathematically elaborated to obtain numerical indices expressing the status of an ejaculate: the fertility index and the percentage of apoptosis, necrosis and immaturity. Sperm apoptosis and necrosis were also evaluated by annexin V/propidium iodide assay. To explore the status of meiotic segregation, fluorescence in situ hybridisation (FISH) with probes for chromosomes 18, X and Y, was applied directly on sperm nuclei. Sperm characteristics from a group of men of proven fertility were used as controls. Among the considered sperm characteristics, apoptosis (P < 0.01), 1818YY diploidy (P < 0.05) and 18YY disomy (P < 0.01) scores were significantly higher in men with RPL compared with controls. Our study showed that some patients with normal semen parameters can present a slight increase in aneuploidy compared with controls, indicating a possible involvement of sperm in some cases of RPL. Chromosomal FISH analysis and chromatin tests of sperm could be included in RPL work-ups when no other cause has been detected.

Retrospective assessment of potential negative synergistic effects of varicocele and tobacco use on ultrastructural sperm morphology.

OBJECTIVES: To investigate, in a retrospective study, whether smoking cigarettes increases the effect of varicocele on sperm morphology. METHODS: The semen quality of 2 groups of patients with varicocele were compared, those who smoked (n = 121) and those who did not (n = 158). The semen parameters were evaluated, and sperm morphology was assessed using transmission electron microscopy and quantitatively elaborated (fertility index, immaturity, necrosis, and apoptosis percentages). RESULTS: In the smoker and nonsmoker varicocele-associated cases, sperm motility and the results from transmission electron microscopy analysis were significantly impaired compared with controls. However, a nonsignificant difference was detected when the semen parameters were compared. Subsequently, we divided the patients into 4 groups: mild (> or = 1 but < or = 10 cigarettes/d), moderate (>10 but <20 cigarettes/d), and heavy (> or = 20 cigarettes/d) smokers and a group of randomly chosen nonsmoker patients with varicocele. The sperm motility, sperm concentration, and fertility index decreased and the percentage of sperm pathologic features increased as the number of cigarettes smoked daily increased. CONCLUSIONS: A detrimental effect of cigarette smoking (>10 cigarettes/d) associated with varicocele on sperm motility and morphology was observed. Because much of reduced fecundity associated with smoking may be reversed within 1 year of cessation, as reported in published studies, effective interventions targeted at helping patients quit smoking should be addressed for the benefit of general health and fertility.

Semen characteristics and inflammatory mediators in infertile men with different clinical diagnoses.

This study was aimed at investigating whether semen characteristics in different clinical diagnoses of infertility are associated with PMN elastase, IL-6, IL-8, IL-1beta and TNFalpha levels detected in seminal plasma. Sixty-eight patients were divided into groups according to their clinical diagnosis: idiopathic infertility (group I), varicocele with infections (group II), varicocele (group III), infections (group IV), controls (group V). Physical examination and scrotal Eco-color Doppler was used to detect the varicocele. Patients with positive bacteriological semen analysis were considered as having an infection of the male reproductive tract. Samples were examined by light microscopy and transmission electron microscopy (TEM). TEM data were quantified with a mathematical formula furnishing a fertility index and the percentage of sperm apoptosis, immaturity and necrosis. PMN elastase/alpha1-PI complex levels were determined by ELISA and IL-6, IL-8, IL-1beta, TNFalpha by Bio-Plex Cytokine assay. Sperm concentration (I-II: p < 0.005; III-IV: p < 0.0001), motility (I-IV: p < 0.0001) and the fertility index (I: p < 0.005; II-IV: p < 0.0001) were significantly lower in the groups vs. controls, whereas sperm pathologies, except for apoptosis, were significantly higher in group I and apoptosis and necrosis were higher in group III. An increase in immaturity (p < 0.005) with a decrease in necrosis (p < 0.005) were observed in group III vs. group IV. Significantly higher levels of inflammatory mediators were detected in groups III and IV vs. controls. Despite a broad relationship among different inflammatory mediators, no correlation was found among them and the semen parameters, including indices from TEM analysis. In conclusion, patients with idiopathic infertility showed altered semen quality and normal levels of inflammatory mediators. Genitourinary infection and varicocele induced an inflammatory effect which could play a detrimental role in spermatogenesis, revealed by a decrease in sperm motility and the fertility index, concomitant with an increase in immaturity mainly in varicocele and necrosis in infection.

Sperm ultrastructure and meiotic segregation in a group of patients with chronic hepatitis B and C.

Little is known about the effect of chronic hepatitis B and hepatitis C on sperm quality. In this study, we analysed sperm quality from selected patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections. Semen samples were examined by light and transmission electron microscopy (TEM). TEM data were elaborated with a mathematical formula able to indicate a fertility index and the presence of the three main sperm pathologies: apoptosis, immaturity and necrosis. Meiotic chromosome segregation was investigated by fluorescence in situ hybridisation carried out on sperm nuclei, using probes for chromosomes 18, X and Y. Despite normal sperm concentration, we observed reduced motility. TEM analysis highlighted that 35.7% of patients showed generally good semen quality. However, significantly higher values of apoptosis and necrosis, compared with controls, were observed, demonstrating spermatogenetic alterations. Regarding meiotic segregation, we found an incidence of disomies similar to that observed in control samples, whereas diploidy resulted higher in HCV patients, without reaching statistical significance. In conclusion, sperm quality in the studied group was not impaired; however, apoptosis and necrosis resulted out of normal range and the fertility index was significantly lower in HCV- and HBV-infected patients versus controls.