The effect of bacterial contamination of semen on sperm chromatin integrity and standard semen parameters in men from infertile couples.

A considerable proportion of male factor infertility cases are associated with inflammatory processes. The most common sexually transmissible agents causing sexually transmitted diseases in industrial countries are Chlamydia trachomatis, genital Ureaplasma and Mycoplasma spp. This study was undertaken to investigate whether these bacterial contaminants in semen affect sperm quality parameters and particularly DNA integrity (detected by sperm chromatin structure assay) in males from infertile couples (n = 293). The results showed that semen contaminations with the investigated bacterial species were not associated with sperm DNA fragmentation. However, contaminations with Mycoplasma spp. and C. trachomatis were associated with decreased sperm concentrations. Total sperm numbers in contaminated semen samples tended to be decreased, but not significantly. Mycoplasma had the highest adverse effect on sperm quality (concentration, motility, morphology and DNA condensation). Antibiotic therapy of the selected 47 men was successful in 55%, but semen quality parameters did not improve at least up to 3 months after the therapy. The presence of pathogenic bacteria in semen is primarily associated with low sperm production. Our data showed that Mycoplasma spp. contamination of semen had the highest adverse effect on sperm quality. Sperm chromatin integrity assessed by the presence of DNA breaks was not disturbed.

Fertile bull sperm aneuploidy and chromatin integrity in relationship to fertility.

Aneuploidy is associated with spontaneous abortions, perinatal mortality, mental retardation and with embryonic and foetal mortality. Most of these abnormalities originate as a result of meiosis errors during gametogenesis. The main purpose of the study was to analyse frequency of aneuploidies of sex chromosomes and chromosome 6 by three-colour fluorescence in situ hybridization (FISH) in 47 young bulls, candidates for artificial insemination programme with cryopreserved semen and to investigate the influence of aneuploidies and disturbed sperm chromatin integrity on non-return rates, the frequencies of abortions, perinatal mortality and stillbirths. The average frequencies of spermatozoa with disomy for chromosomes X, Y, XY and 6 were 0.032, 0.005, 0.003 and 0.039% respectively. The incidence of XX66, YY66 and XY66 diploidy was 0.017, 0.006 and 0.015% respectively. Frequencies of meiotic II errors were significantly higher than meiotic I errors (p < 0.01). More X bearing spermatozoa than Y bearing spermatozoa were detected (5151 vs. 5022; p < 0.01). Sperm chromatin damage expressed by DNA fragmentation index (DFI) was 5.3 +/- 3.7 and percentage of cells with defective chromatin condensation (HDS) was 1.4 +/- 0.8. No correlation was found between sperm aneuploidy and basic sperm analysis. The relationship was found between non-return rate and total aneuploidy (r = -0.310; p = 0.036). Significant correlation was found between sex disomy, total aneuploidy (disomy of chromosomes 6, X, Y and XY spermatozoa and diploidy) and stillbirths (r = 0.390; p = 0.013; and r = 0.331; p = 0.037). Chromosome 6 disomy correlated with perinatal mortality (r = 0.317; p = 0.047). HDS correlated significantly with total aneuploidy (r = 0.449; p = 0.002). Our study indicated that aneuploidy frequencies in young fertile bull spermatozoa are relatively low. Nevertheless, there exists a variability in aneuploidy frequencies amongst bulls, which appears to be able to have an influence on the fertility of these animals.

Genetic polymorphisms influence the susceptibility of men to sperm DNA damage associated with exposure to air pollution.

The purpose of the present study was to investigate the impact of carcinogenic polycyclic aromatic hydrocarbons and volatile organic compounds on sperm quality in a group of city policemen in Prague during a period of increased concentrations of ambient air-pollutants (winter season) compared to a period of low exposure (spring). Polymorphisms in metabolic genes (CYP1A1, EPHX1, GSTM1, GSTP1, GSTT1), folic acid metabolism genes (MTR, MTHFR) and DNA repair genes (XRCC1, XPD6, XPD23, hOGG1) were evaluated in these men as potential modifiers of associations between air pollution exposure and changes in sperm quality. The study population was a group of 47 policemen working in the center of the city. Seasonal differences in exposure were verified by ambient and personal monitoring. Markers of sperm injury included semen volume, sperm concentration, sperm morphology, sperm motility, and sperm DNA damage measured with the sperm chromatin structure assay The sperm chromatin structure assay (SCSA) includes a measure of DNA damage called DNA Fragmentation Index (DFI). The % of cells with detectable DFI (detDFI) by this assay includes sperm with either medium or high DNA damage; the term hDFI is used to define the % of sperm with only high DNA damage. The assay also detects immature sperm defined by high density staining (HDS). No significant differences were found in any of the standard semen parameters between the sampling periods except for vitality of sperms. Both DFI and HDS were significantly higher in winter than in spring samples for all men and for non-smokers. At the bivariate level, significant associations between hDFI or detDFI and polymorphisms of the repair genes XRCC1, XPD6 and XPD23 were observed. In multivariate models, polymorphisms of the genes XPD6, XPD23 and CYP1A1MspI were associated with hDFI and HDS. Moreover, HDS was significantly associated with polymorphisms in GSTM1 gene.

Sperm chromatin integrity in young men with no experiences of infertility and men from idiopathic infertility couples.

Damage to the genetic component of spermatozoa seems to play the main role in a majority of cases where current approaches fail to reveal the specific cause of male infertility. In this study, we compared semen quality in men assigned to two defined groups: men from couples with unexplained infertility - idiopathic infertility (A) and young men with no experiences of infertility (B). All samples were examined by standard ejaculate analysis and sperm chromatin structure assay (SCSA). Sperm chromatin damage was significantly higher in men from group A than in those from group B. Similar results were obtained by comparison of men from group A (all men were normozoospermic) with normozoospermic men from group B. According to these results, we can suppose that chromatin disorders may be the causal factor of subfertility or infertility in some of these men. No evidence for a strong association between chromatin disorders and standard parameters of ejaculates was found. We failed to confirm a relationship between smoking and sperm quality in men from any of the investigated groups. SCSA is a method that facilitates the identification of infertile men who otherwise show normal semen variables.

[Infection with Chlamydophila pneumoniae as a cause of female infertility of tubal origin?]. / Infekce Chlamydophila pneumoniae jako prícina neplodnosti zen tubárního puvodu?

OBJECTIVE: A contribution to the role of Chlamydophila pneumoniae in women from the IVF program. DESIGN: A serological study proving the antibodies against the antigens of Chlamydophila pneumoniae (C. pneumoniae) and Chlamydia trachomatis (C. trachomatis) in women from the IVF program suffering from different factor of infertility. SETTING: Veterinary Research Institute, Brno. METHODS: The complement fixation test with chlamydial antigen and ELISA tests proving IgA and IgG antibodies against genus-specific antigen (cLPS) and species-specific chlamydial major outer membrane protein (cMOMP) of C. trachomatis and C. pneumoniae in the blood serum of 32 females being in the fertilization program due the tubal factor infertility have been estimated and the results compared with those obtained in 26 females being in the fertilization program due the ovarian factor and in 42 female with andrological factor, respectively. RESULTS: The frequency of positive complement fixation test proved in the group of women with tubal factor infertility was significantly higher than in the groups with ovarial and andrological factor, respectively. The number of the strongly positive reactions (with high titres) in the group with tubal factor infertility was higher than in two remaining groups. The occurrence of species-specific IgG antibodies (anti-C. trachomatis) and chlamydia genus-specific IgA antibodies proved by means of ELISA in the group with tubal factor was statistically more frequent. The difference of the species-specific anti-C. pneumoniae antibodies in all three groups examined was not significant. The simultaneous occurrence of species-specific antibodies against anti-C. pneumoniae and C. trachomatis in the group with tubal factor was higher than in other groups (in comparing with the group with andrological factor statistically significant). CONCLUSION: The opinion of the authors being in agreement with the view of most specialists is that the most important agent in the pathogenesis of tubal factor fertility is C. trachomatis. C. pneumoniae is above all a respiratory pathogen with a high prevalence and its impact for fertility disorders may be in the synergismus with C. trachomatis. The serological examination of both chlamydial infections should be a standard part of diagnostic algorithm.

[Importance of regular screening for genital chlamydiosis in adolescents]. / Význam pravidelne opakovaného screeningu genitální chlamydiózy u dospívající mládeze.

OBJECTIVE: Determination of prevalence of genital chlamydiosis in similar group of adolescents as in that examined two years ago. Comparison of the results with the previous ones to confirm positive impact of a repeated screening on drop of prevalence of the infection. Another objective is to make the epidemiological data on genital chlamydiosis in the countries of the "Eastern Block" more accurate, and at the same time to assess the most suitable screening method. DESIGN: Prospective epidemiological study aimed at finding whether the repeated screening examinations of genital chlamydiosis in adolescents will result in decreased incidence of the infection. SETTING: Veterinary Research Institute, Department of Clinical Microbiology of the Faculty Hospital, Regional Hygienic Station, Department of Gynecology and Obstetrics, Medical Faculty, Masaryk University, Brno. METHODS: The groups under testing comprised male and female students of two secondary medical schools and one private grammar school older than 18 (n = 232, of which 33 were boys and 199 girls). Direct detection of C. trachomatis was carried out from the urinary sediment of the sample of morning urine using ligase reaction, polymerase chain reaction, ELISA and direct immunofluorescence. RESULTS: Identical positive result of C. trachomatis detection by four methods was obtained 4 times, i.e. 1.7%. The result of LCR and PCR were positive identically (4 times), ELISA and IF were, besides the above results, positive in other 1 resp. 5 cases. CONCLUSION: Screening examinations of genital chlamydiosis were performed in adolescents in the town of Brno. Morning samples of urine from 232 students of secondary schools were examined on C. trachomatis using the following methods: LCR, PCR, ELISA, and IF. The LCR and PCR were positive 4 times, ELISA 5 times, IF 9 times. Positive results by LCR and PCR were identical. ELISA and IF were positive in other cases. Noninvasive methods are suitable for screening of latent genital chlamydiosis and for estimation of its prevalence. The authors of the study recommend the ligase chain reaction (LCR) as a most suitable screening method.

[Noninvasive screening for genital chlamydiosis in adolescents in Brno]. / Neinvazivní screeningové vysetrení genitální chlamydiózy adolescentu mesta Brna.

OBJECTIVE: To run a screening for genital chlamydiosis in adolescents living in the town Brno as the first action of this type in the Czech Republic and to use the results of the screening for the elaboration of recommendations for running similar actions on the national scale. DESIGN: Prospective epidemiological study. SETTING: Veterinary Research Institute, Brno; Institute of Clinical Biochemistry, 1st Faculty of Medicine, Charles University and General Faculty Hospital, Prague; Section of Public Health, Municipal Authorities of Brno; Bioplus Ltd., Brno; Regional Hygienic Services, Brno; Faculty Hospital Brno; with technical assistance of teachers and students of two Medical Assistant Schools in Brno. METHODS: Students (337 females and 15 males) of two Medical Assistant Schools, older than 18 years, were used as probands within the study. Sediments of the first portions of urine collected from the individual probands were tested for the presence of Chlamydia trachomatis using the direct fluorescent test, ELISA, and the ligase chain reaction. RESULTS: Positive reactions in any of the three tests were found in 31 of the 352 probands (8.8%). Positive and doubtful reactions in the direct fluorescent antibody test were obtained in 11 (35.5%) and 3 (9.6%) of the 31 reactors, respectively. The corresponding values for ELISA were 9 (23.0%) and 8 (25.8%), respectively, and those for the ligase chain reaction 3 (16.6%) and 3 (16.6%), respectively. The overall prevalence of 8.8% is higher than the European mean. CONCLUSIONS: The first limited screening for genital Chlamydia infections in the Czech Republic was run in Brno. Urinary samples were collected from 337 females and 15 males aged approximately 18 years. The presence of Chlamydia trachomatis in the urinary sediment was demonstrated by the direct fluorescent test, ELISA, and the ligase chain reaction. The established prevalence of 8.8% exceeded the European mean (3.9%).

Interaction of bull, stallion and boar seminal plasma proteins and sperms with acidic polysaccharides.

The interaction of seminal plasma proteins, sperms and detergent-released sperm proteins of three species with different types of acidic polysaccharides was studied. Heparin-binding activity of boar, bull and stallion seminal plasma proteins, sperms and sperm proteins was compared with their ability to interact with polysaccharides differing in the presence of the sulfate groups or in their saccharide moiety (chondroitin sulfate, dextran sulfate, fucoidan, hyaluronic acid). Bull seminal plasma proteins were characterized by higher affinity to heparin, fucoidan and dextran sulfate, while significant differences between different types of polysaccharides were detected in the case of boar proteins. Sperm protein interactions with acidic polysaccharides in bull and stallion were analogous to the binding of seminal plasma proteins. Bull and stallion seminal plasma proteins inhibited the interaction of corresponding sperm proteins with acidic polysaccharides.

[Direct and indirect detection of Chlamydia in infertile married couples in the assisted reproduction program]. / Prímý a neprímý prukaz chlamydií u neplodných manzelských páru zarazených do programu asistované reprodukce.

The authors pay attention to the relationship of pregnancy in women enlisted in the IVF programme and the detection of Chlamydia infection. A group of 74 infertile couples were investigated. A significant relationship was found between confirmed Chlamydia infection in women and men. Moreover a significant relationship was found between Chlamydia infection confirmed by immunofluorescence in men and impregnation in women in the IVF programme. This means that in female partners of men with acute Chlamydia infection the results of IVF are less successful. The authors recommend that all couples enlisted in the IVF programme should be examined for this infection and if it is positive given adequate treatment.

[Occurrence of antibodies to chlamydial group antigens in the population of the Czech Republic]. / Výskyt protilátek proti skupinovému chlamydiovému antigenu v lidské populaci CR.

A set of 4,178 human blood sera was tested including 3,093 samples collected from blood donors, 624 randomly collected samples. 124 samples collected from persons possibly exposed to Chlamydia infection, and 337 samples from patients of various hospitals in the districts of Brno, Olomouc and Rumburk. All the sera were examined using the complement fixation microtest and the antigen of Chlamydia psittaci for CFR (Bioveta, Ivanovice na Hané, CzR). Moreover, a subset of samples collected from selected groups (n = 209) was tested by CFR using the genus-specific Ornithose-Antigen (Behring, Germany) (n = 209), another subset of 116 samples by indirect immunofluorescence using the biological Chlamydia trachomatis Spot IF (Bio Merieux) (n = 116), and a subset of 224 samples by ELISA for the demonstration of IgG, IgM and IgA class of antibodies using the kit rELISA Medac. Complement fixing antibodies assayed with the Chlamydia psittaci antigen (Bioveta), reached titres of 1.5, 1:10 and 1:20 in 15.5%, 5.29% and 0.48% of the randomly collected samples, respectively. The corresponding values for the tests carried out with the Ornithose-Antigen were 1:10 in 10.9%, 1:20 in 8.9%, 1:40 in 2.9% and 1:80 in 1.4%. ELISA yielded 52.7%, 26.85% and 17.59% positive results in the globulin classes IgG, IgA and IgM, respectively. Antibody titres from 1:20 and from 1:10, determined with the Bioveta antigen, were found in 2.36% and 9.47% within a representative group of blood donors. The corresponding values for the group of the hospitalized patients were 14.54% and 24.93%, respectively, and for the group of persons possibly exposed to Chlamydia infection 2.42% and 8.87% respectively. The indirect immunofluorescence test yielded positive, negative ad doubtful results in 47.41%, 27.59% and 25.0% persons of the latter group. This group showed also a higher percentage (37.07%) of positive results in ELISA in the class IgA than the randomly selected persons.

Effects of oral intake of nitrates on reproductive functions of bulls.

Effects of oral intake of nitrates on selected biochemical and endocrinological indices and its impact on reproductive functions were investigated in five feeder bulls aged 16-18 months. The bulls were tested prior to (30 days), during (30 days) and after (35 days) the period of the nitrate administration. The initial dose of 100 g potassium nitrate per day was increased at weekly intervals by 50 g up to 250 g per day. The administration of nitrates resulted in a highly significant (P < 0.01) increase in methaemoglobin concentration and a non-significant decrease in the concentration of beta-carotene and a highly significant (P < 0.01) decrease in the concentration of E vitamin in blood serum. A significant (P < 0.01) increase in blood serum concentration of bile acids and prolonged biological half-life of progesterone were suggestive of an impairment of liver metabolism. Prolonged intake of excessive doses of nitrates resulted in a significant (P < 0.05) increase in cortisol concentration during and after the administration period, while depressed thyroid gland activity was evident from a significant (P < 0.05) decrease in thyroxin concentration during the administration period. A suppression of hypothalamic functions after the administration period was documented by non-detectable levels (< 0.001 microgram/ml) of thyrotropin in TRH test. Depressive effects of nitrates on the function of Leydig cells during and particularly after the administration period were apparent from weakening testicular responses to a treatment with GnRH. Biochemical analyses of seminal plasma revealed a highly significant (P < 0.01) increase in total acid phosphatase activity and a significant (P < 0.05) decrease in the concentration of fructose. No other significant changes in seminal plasma components were observed. Adverse effects of excessive intake of nitrates were also evident from reduced sperm motility in the 120-min thermal test. While no difference was found in the frequency of primary morphological abnormalities, the number of secondary abnormalities rose by 115% in the post-administration period and was suggestive of damaged membrane integrity. Histological examinations revealed degenerative lesions in cells of the spermiocyte and spermatid layers.

[Detection of Chlamydia in inflammation of the cervix and uterus in cows]. / Nálezy chlamydií u zánetu delozního krcku a pochvy krav.

Clinical findings in cows of seven farms with a frequent occurrence of birth canal inflammations were matched against results of serological and direct immunofluorescence tests for Chlamydia sp. infection. Normal clinical picture, weak to moderate and serious inflammations were found in 16.1%, 46.7% and 37.2% of the 205 examined cows, respectively. The results of micro CFT were negative in 32.7% and titres 1:5, 1:10, 1:20, and 1: > or = 40 were found in 8.7%, 38.0%, 12.3%, and 8.3%, respectively. Direct immunofluorescence test of cervical swabs, using the Progen Biotechnic GmbH set, was negative in 29.1% and clearly positive in 34.7% of the tested cows. The rest was animals in which sporadic elementary bodies were seen. The significance of the findings is discussed and the necessity to test Chlamydia infections in herds affected by frequent and/or chronic inflammations of genital organs is stressed. Evidence of the transmission of Chlamydia infection by contaminated semen is presented.

[Detection of chlamydia in the intestines and lungs in pigeons and humans]. / Prukaz chlamydií ve streve a plicích holubu a lidí.

Attempts were made to detect Chlamydia sp. by direct immunofluorescence (IF) in intestinal and lung tissues collected from wild town pigeons, young reared pigeons, and human corpses. Chlamydia in any organ were detected in 86.6% of the 427 adult pigeons and 27.3% of 16 young pigeons. Positive IF results were in intestinal and lung samples, collected from 427 adult pigeons, in 34.3% and 40.05% of the birds, respectively. Human (n = 100) intestinal and lung tissues were positive in 22% and 20% of the corpses, respectively. Titres 1:20 and higher were demonstrated by CF in 53.86% of the 427 adult pigeons. The highest titre found was 1:5 120.

[Detection of chlamydiae in animal and human semen using direct immunofluorescence]. / Prímý prukaz chlamydií v semeni zvírat a lidí imunofluorescenci.

Frequency of elementary and reticular chlamydial bodies was investigated by direct immunofluorescence tests in ejaculates collected from 52 men, 60 stallions, 42 bulls, and 66 boars using the kits of Progen Biotechnic GmbH and the microscope Labophot-2 Nikon. At the same time, qualitative semen tests, including ejaculate volume, sperm motility, percentage of live and dead sperms and morphological' analyses (Vezník and Svecová, 1992) were done. Repeatability of the findings was checked in a group of nine bulls housed at the institute and sampled at weekly intervals for 3 to 4 months (Tab. 1). Chlamydiae were demonstrated in 3.8%, 14.3%, 3.4% and 9.1% of the human, bull, stallion and boar ejaculates, respectively (Fig. 1). A relation between the presence of Chlamydiac and impaired functional and morphological quality of ejaculates was found in contaminated human (only two samples) and bull ejaculates (Fig. 2) and in one of the two positive stallion ejaculates (Tab. II, Fig. 3). Direct tests for Chlamydiae should be included into semen health and quality checks as one of the major tasks of assisted reproduction in human and veterinary medicine.

Average testosterone levels in bulls after GnRH stimulation at breeder bull rearing stations and A. I. stations.

Base average testosterone levels and testosterone levels in 60 and 90 min after GnRH administration were determined in bulls at five breeder bull rearing stations at their age of 13 months and at nine A. I. stations at their age of 17-34 months. Significant increases in testosterone levels in 60 and 90 min after GnRH administration were the responses of all groups of bulls. There were differences between the average testosterone levels typical of the various BRSs and AISs. Highly significant correlation coefficients 0.344 for base levels and 0.583 for induced levels were calculated from a comparison of the testosterone levels determined at BRSs at the bull's age of 13 months and of those recorded after a six-month stay of the same bulls at AISs. A decrease in testosterone levels was also observed.

[Testosterone production after in vitro stimulation of testicular cells with gonadotropins in bulls]. / Produkce testosteronu po stimulaci varletních bunek býku gonadotropinem in vitro.

The response of bulls' testicular cells to the stimulation by serum gonadotropin ad us. vet. (Bioveta, Ivanovice in Haná) was investigated in in vitro tests. The testicular tissue was sliced, collagenase-split and the loosened cells were stimulated by gonadotropin concentrations of 7.8 to 250 I U per l; control samples without gonadotropin treatment were used for comparisons. The testicular cells responded to the increasing stimulation by higher production of testosterone.

Significance of progesterone determination in milk.

The determination of progesterone in milk sampled on the day of insemination is used for fertilization check in dairy cattle. Thus, the corpus luteum function confirms insemination failures due to the false estrus. By this way it is possible to detect 10 to 20 per cent of cows which cannot be successfully inseminated. Further estimation is performed 3 weeks after the insemination when low concentration of progesterone reflects an insemination failure. By this way it is possible to follow up cows inseminated without success and without any clinical symptoms of the next estrus. The repeated estimation of progesterone can objectively monitor the corpus luteum function. This is of great importance for the confirmation of puerperal anestrus, diagnosis of fertility disorders and/or assessment of therapy of sexual cycle disorders.

[The effect of the light schedule on biochemical indicators in the blood, serum and urine of heifers during their estrus cycle]. / Vliv svetelného rezimu na biochemické ukazatele krve, séra a moce jalovic v prubehu ríjového cyklu.

The dynamics of 38 biochemical parameters of blood, serum and urine was studied in ten heifers during their oestrous cycle in a light house (80-340 lux) and a dark small house (10-40 lux). In the light stable a significant influence (P less than 0.05) was found to be exerted on five parameters and in the dark stable nine parameters, four being influenced in the same way in both houses: haematocrit value and haemoglobin in blood and cholesterol and progesterone in serum; the relationship of progesterone content in serum to the days of oestrous cycle was particularly high in the light house. The comparison of the parameters between the groups demonstrated a significant (P less than 0.05) influence of light regime, exerted on some days of the oestrous cycle upon the levels of inorganic phosphorus, total protein and aspartate aminotransferase activity (AST) in serum and upon haematocrit, haemoglobin and ketone bodies in the blood of heifers. However, the light regime did not influence the levels of progesterone which were somewhat higher in the light house but with no statistically significant difference from the dark house. A significant difference between the groups was obtained in the levels of sodium and phosphorus in urine (P less than 0.05) between the first and tenth days of the oestrous cycle. No differences in the oestrous cycle between the heifers in the light and dark stables were recorded. Neither was the duration of heat influenced significantly; it was only less manifest in the dark stable.

[Determination of the activity of luteinizing hormone in PMSG preparations using testosterone synthesis in vitro]. / Stanovení aktivity luteinizacního hormonu v preparátech PMSG podle syntézy testosteronu in vitro.

The activity of luteinizing hormone (LH) in serum gonadotropin (PMSG) was studied by a bioassay in vitro, using the cells of mouse testes prepared by the combination of enzymatic and mechanical diffociation. The activity of individual preparations was calculated according to the amount of produced testosterone measured by radioimmunoassay. Comparison with the 2nd international standard of PMSG (WHO) showed that in the PMSG substandard (Dessau) the activity of LH was twice lower, and in three charges of the commercial PMSG preparation (Bioveta, Ivanovice in Haná) four-and-a-half times lower than declared. No great variability of LH activity was observed among the charges of the commercial PMSG preparation.