Dig Dis Sci (2010) 55:94–100 DOI 10.1007/s10620-008-0704-1
ORIGINAL ARTICLE
Infection by CagA-Positive Helicobacter pylori Strains may Contribute to Alter the Sperm Quality of Men with Fertility Disorders and Increase the Systemic Levels of TNF-a Giulia Collodel Æ Elena Moretti Æ Maria Stella Campagna Æ Serena Capitani Æ Cristina Lenzi Æ Natale Figura
Received: 3 September 2008 / Accepted: 30 December 2008 / Published online: 22 January 2009 Ó Springer Science+Business Media, LLC 2009
Abstract This study was aimed to address the possibility that Helicobacter pylori infection may play a detrimental role in semen quality of men with idiopathic infertility. Infection by H. pylori and by strains expressing CagA was determined in 80 male infertile patients by Western blotting and ELISA. Semen analysis was performed by light microscopy and transmission electron microscopy quantitatively elaborated (fertility index, immaturity, necrosis, and apoptosis percentages). Systemic levels of IL-6 and TNF-a were evaluated. Infertile patients infected with H. pylori showed a low sperm quality respective to uninfected patients. Particularly, in CagA-positive patients we observed a significant reduction in sperm motility and in the fertility index, while apoptosis and necrosis were increased. In these patients, the means of systemic TNF-a levels were higher than those of uninfected patients. The negative influence of CagA-positive H. pylori infection on sperm quality may help to understand the role of chronic infections in reproductive disorders.
G. Collodel E. Moretti (&) Department of Biomedical Sciences, Applied Biology Section, Policlinico Le Scotte, University of Siena, Viale Bracci, 14, 53100 Siena, Italy e-mail:
[email protected] G. Collodel E. Moretti S. Capitani Interdepartmental Centre for Research and Therapy of Male Infertility, University of Siena, Siena, Italy M. S. Campagna C. Lenzi N. Figura Department of Internal Medicine, Endocrine-Metabolic Sciences and Biochemistry, University of Siena, Siena, Italy S. Capitani Department of Physiopathology, Experimental Medicine and Public Health, University of Siena, Siena, Italy
123
Keywords H. pylori infection CagA Male idiopathic infertility Spermatozoa TEM TNF-a
Introduction Helicobacter pylori is a microaerophilic Gram-negative helical-shaped bacterium that has evolved together with man and has adapted to survive in the hostile gastric environment. This bacterium infects almost half the population worldwide and represents the major cause of gastroduodenal pathologies, such as duodenal and gastric ulcer, gastric cancer, B-cell lymphoma of mucosa associated lymphoid tissue (MALT), and autoimmune gastritis [1]. The possible clinical consequences of H. pylori infection are not restricted to the gastroduodenal tract. Helicobacter pylori infection is putatively associated with extra-digestive disorders [2–8] and may also play a role in the development of autoimmune diseases [9–11]. The list of proposed ‘‘extragastric’’ associations continues to grow despite the fact that H. pylori is a noninvasive organism and the infection is essentially confined at the surface of gastric-type mucosa. Helicobacter pylori infection can directly or indirectly cause extragastric manifestations using various mechanisms, including the release of inflammatory mediators, molecular mimicry, and systemic immune response [12]. Helicobacter pylori strains bearing the cytotoxin-associated gene A protein (CagA) were found to induce increased local and systemic levels of interleukin-8 (IL-8), IL-1 b, IL-6, tumor necrosis factor-a (TNF-a) and inflammation in the gastric mucosa compared to the levels of inflammatory mediators generated by infection by CagA-negative strains [13]. Molecular mimicry of host structures with proteins encoded by micro-organisms can have pathogenic consequences;
Dig Dis Sci (2010) 55:94–100
infection may induce antibodies and T cells to react against bacterial cell constituents that can also recognize self components and immunomediated damage may follow. Despite a vigorous humoral response against H. pylori antigens, most infected subjects fail to eliminate the pathogen spontaneously. Both the virulence properties of H. pylori strains and host factors strongly influence the clinical outcome of the infection. Concerning the potential repercussions of H. pylori infection for the whole organism, some of our group [14] have hypothesized the existence of an antigenic mimicry between bacterial flagella of H. pylori and the only flagellated human cells, the spermatozoa. A partial linear homology was observed between human tubulin (the main constituent of spermatozoon flagellum) and H. pylori proteins (flagellin, CagA, and VacA). This antigenic mimicry may commonly stimulate cross-reactive antibodies. In fact, an immune reaction was observed between H. pylori and human spermatozoa, especially in the first part of flagella at the centriolar region, which is particularly rich in tubulin, and also in the equatorial zone of sperm cells [14]. The present study was aimed at exploring the possibility that H. pylori infection, particularly in the presence of CagA-positive strains, could play a detrimental role on sperm quality, since the infection, which usually lasts for the whole life of the patients, may have systemic repercussions and may provoke autoimmune reactions, especially towards spermatozoa. In addition, IL-6 and TNF-a levels were evaluated in blood serum from the same patients in order to verify whether the H. pylori infection status could influence systemic indices of inflammation.
Materials and Methods Patients Between September 2005 and November 2007, we enrolled 82 consecutive patients (aged 25–40 years) who were suffering from idiopathic infertility at the Interdepartmental Centre for Research and Therapy of Male Infertility. Idiopathic infertility was defined when patients did not show severe anatomical pathologies related to fertility, such as varicocele, cryptorchidism, hormonal imbalance, or sperm defects of supposed genetic origin. Moreover, genitourinary infections were excluded on the basis of negative results of culture for aerobic and anaerobic bacteria and genomic amplification by PCR using specific primers for Chlamydia trachomatis, Ureaplasma urealyticum, Trichomonas vaginalis and herpes simplex virus 2. All patients had failed to procreate during the two previous years of unprotected sexual intercourse and their partners had no fertility problems. Only patients with a normal 46, XY karyotype, evaluated with conventional cytogenetic analysis, were included in
95
this study. Moreover, these patients had no history of radiotherapy, chemotherapy, chronic illness, or medication and autoimmune disorders. In patients with a sperm concentration lower than 10 million/ml, PCR analysis on DNA extracted from blood lymphocytes was carried out to exclude the presence of Y chromosome microdeletions. Patients did not suffer from any dyspeptic symptoms and had not taken antibiotics potentially active against H. pylori over the previous 3 months, including proton pump inhibitors. Their H. pylori infection status was previously unknown. Patients provided written informed consent before inclusion in this study and the study approved by the local Ethics Committee. Determination of H. pylori Infection Helicobacter pylori infection status was determined serologically using a commercially available enzyme-linked immunosorbent assay with a sensitivity and specificity of around 96% (Helicobacter pylori IgG, Diesse, Monteriggioni, Siena, Italy). Helicobacter pylori infection status was confirmed by Western blotting (WB). WB was also used to detect antibodies to H. pylori CagA. Briefly, a whole cell suspension of H. pylori CCUG 17874 (a CagApositive and cytotoxic strain) was denatured in Laemmli’s buffer at 100° for 5 min and electrophoresed in a 10% polyacrylamide gel with sodium dodecylsulphate. The resolved proteins were transferred electrophoretically onto nitrocellulose membranes, and the free sites were saturated with 3% skim milk in phosphate buffered saline (PBS) pH 7.4 containing 0.1% Triton X (PMT). Afterwards, strips were cut and immunoblotted with serum samples diluted at 1:100 in PMT for immunoglobulin G (IgG). After overnight incubation at room temperature, the strips were washed three times with PMT, and a peroxidase-labeled antibody to human IgG, diluted in PMT 1:2,000 (Sigma Chem. Co., Milan), was added and incubated at room temperature for 90 min. The strips were then washed three times with PMT, once with PBS-Triton X, and twice with Tris buffer 0.05 mol/l pH 6.8. The reaction was visualized by addition of the substrate (H2O2 in a solution of 4-chloro- 1-naphthol in Tris buffer 0.05 M pH 6.8). The reaction was stopped with water. As positive controls, an anti-H. pylori whole cell suspension and anti-CagA rabbit polyclonal antibodies (kindly donated by R. Rappuoli, Novartis, Siena) were used. Semen Analysis Light Microscopy Semen samples were collected by patient masturbation after 4 days of sexual abstinence and examined after liquefaction for 30 min at 37°C. Volume, pH, sperm
123
96
Dig Dis Sci (2010) 55:94–100
mathematical formula by Baccetti et al. [16], which considers 16 selected submicroscopic characteristics of sperm organelles able to define sperm function and calculates the number of spermatozoa probably free of structural defects (fertility index) and the percentages of three main phenotypic sperm pathologies: immaturity, necrosis, and apoptosis [17], each one being characterized by typical alterations of organelles. Reduced acrosomes, round or elliptical nuclei with uncondensed chromatin and the presence of cytoplasmic droplets were the examined characteristic for immaturity. Marginated chromatin and altered shaped nuclei were considered as the main ultrastructural markers of apoptosis, whereas spermatozoa with broken plasma membranes, reacted acrosomes, and disrupted chromatins were affected by necrosis. Moreover, in Table 1 we report the sperm characteristics from 25 men of proven fertility as reference values, already published by Collodel and Moretti [17].
concentration, and motility were evaluated according to WHO guidelines [15]. Sperm concentration was determined by use of a Bu¨rker counting chamber. We diluted the sample in natrium chloride 0.9% in distilled water prewarmed at 37°C and counted the spermatozoa in 20 square fields under a microscope. During scoring, we categorized the sperm that exhibited rapid progressive motility (grade a), slow progressive motility (grade b), non-progressive motility (grade c) and those that were immotile (grade d). Reference value for motility indicated by WHO (1999) is expressed by the percentage of spermatozoa that were 50% or more motile (grades a ? b) or 25% or more with progressive motility (grade a). Transmission Electron Microscopy For electron microscopy, sperm samples were fixed in cold Karnovsky fixative and maintained at 4°C for 2 h. Fixed semen was washed in 0.1 mol/l cacodylate buffer (pH 7.2) for 12 h, postfixed in 1% buffered osmium tetroxide for 1 h at 4°C, and then dehydrated and embedded in Epon Araldite. Ultra-thin sections were cut with a Supernova ultramicrotome (Reickert Jung, Vienna, Austria), mounted on copper grids, stained with uranyl acetate and lead citrate, and then observed and photographed with a Philips CM10 transmission electron microscope (TEM; Philips Scientifics, Eindhoven, The Netherlands). For each patient, three hundred ultra-thin sperm sections were analyzed. Major submicroscopic characteristics were recorded by two trained examiners who were blind to the experiment. TEM data were evaluated using the statistical
Detection of Cytokines Levels of IL-6 and TNF-a were measured in serum samples using enzyme-linked immunosorbent assays (ELISA) with ‘‘Human IL-6’’ and ‘‘Human TNF-a’’ kits provided by Bender MedSystems (Vienna, Austria). The results are expressed in pg/ml. Statistical Analysis Statistical analysis was performed using the StatGraphicsPlus (vers. 5.0) statistical package. P \ 0.05 was considered
Table 1 Sperm concentration, motility, and TEM data in patients with idiopathic infertility infected with H. pylori and uninfected Groups and number () of examined patients and controls
Variables
Concentration 9106
Motility (%)
Fertility index
Immaturity (%)
Apoptosis (%)
Necrosis (%)
H. pylori positive patients (36)
Median
24.5
22
14270?
64.80
9.29
54.72
Group 1
I–III quartile 8.75–57.25
12.25–32 3183–407247
56.63–74.76 4.38–13.56 46–62.97
H. pylori-negative patients (44)
Median
28.5
43329
64.79
Group 2
I–III quartile 7–47.75
12.5–35
1829–309917
56.39–69.83 5.94–12.42 39.86–59.41
Infected CagA-positive patients (17)
Median
18*
16056*
62.69
Group 3
I–III quartile 23.25–37
2–31
3721–444161
56.35–67.24 5.34–14.85 54.37–80.40
29 17.5–32
32041 3469–820019
65.19 7.76 51.00 57.95–75.78 4.50–10.46 43.90–62.97
Median
3807391
47.29
I–III quartile
2057544–8308132 38.59–58.21 3.58–4.67
23.5 25.5
Infected CagA-negative patients (19) Median 23 Group 4 I–III quartile 5–57 [20 9 106
WHO (1999) Reference values of 25 fertile controls
Fertility index: number of spermatozoa devoid of ultrastructural defects ?Group 1 vs. group 2 P \ 0.05 * Group 3 vs. group 4 P \ 0.05
123
11.19*
51.26 59.11*
[50%
The number of patients in each group is reported in parentheses Fertile controls as reported in Collodel and Moretti [17]
9.26
4.06
34.63 24.74–40.10
Dig Dis Sci (2010) 55:94–100
97
significant. All groups of quantitative variables were checked for normal distribution by the Kolmogorov–Smirnov test and the F-test was used to compare variances between the groups. The Mann–Whitney test, rather than the t-test, was performed to evaluate the statistical differences between the variables when the conditions of normality of distribution and of homogeneity of variance were not satisfied. The variables were expressed as mean ± standard deviation (SD) in the case of normal distribution or as median and interquartile ranges in cases of non-normal distribution.
Sperm Motility and Concentration
Results
Motility grade a and b (rapid a ? slow b) was reduced in both groups of patients, infected and uninfected, according to WHO parameters [14] (normal value: rapid ? slow progressive motility [ 50%). In the group of H. pylori-infected patients, the mean percentage of motile spermatozoa was lower than that obtained in uninfected ones (Table 1), but the difference was not statistically significant. However, infected patients seropositive for CagA had a significant reduction (about 50%) in sperm motility, with a P-value of \0.05 (Table 1). The medians of sperm concentration were 20 9 106 sperm/ml, a value considered ‘‘normal’’ by the WHO [14]. No significant difference was found in the number of sperm/ml among the analyzed groups (Table 1).
Prevalence of H. pylori Infection and Type of Infecting Organism
Number of Spermatozoa Devoid of Ultrastructural Defect: Fertility Index
Out of the 82 patients examined, ELISA and WB results were concordant in 80 cases. Two patients were seronegative by ELISA for whole H. pylori antigens, although they had serum antibodies to CagA detected by WB. The interpretation of this data was dubious and we excluded these patients from the study. Thus, the final number of patients studied was 80. Thirty-six patients (45.0%) were seropositive for H. pylori infection (group 1), while 44 patients (55.0%) were seronegative and therefore were considered uninfected (group 2). Among the infected patients, 17 men (47.2%) were seropositive for CagA (group 3), indicating that they were colonized by strains expressing this marker of increased pathogenicity; 19 infected patients (52.7%) did not have anti-CagA antibodies, hence they were considered as infected by ‘‘less virulent’’ H. pylori strains (group 4).
The results concerning this subject are reported in Table 1. The mean number of spermatozoa devoid of defects was significantly lower in the group of infected patients at a P level of \0.05. A similar level of significance was detected when we compared the mean of fertility index numbers in CagA-positive infected patients with that of CagA-negative ones, i.e., the fertility index was lower in the former group (Table 1). Sperm Pathologies and Organelles To better understand the presumptive effect of H. pylori infection on sperm morphology, in particular by strains expressing CagA, we considered the incidence of sperm pathologies (Table 1) and the status of the most important organelles involved in each sperm pathology (Table 2).
Table 2 Mean ± standard deviation (SD) of ultrastructural sperm defects affecting organelles in different pathologies in H. pylori-infected patients (group 1), uninfected patients (group 2), patients infected with CagA-positive (group 3) and CagA-negative strains (group 4) Parameters
Sperm organelles
Groups 1 vs. 2
Groups 3 vs. 4
Apoptosis
Marginated chromatin
2.17 ± 2.35 vs. 1.89 ± 2.49
5.8 (3.5–6.6) vs. 3.7 (3–6.7)*
Immaturity
Altered-shape nucleus Uncondensed chromatin
42.41 ± 12.41 vs. 42.75 ± 17.20 35.25 ± 14.81 vs. 33.82 ± 12.30
40 (36.6–44.11) vs. 44 (35.5–50.1) 26.6 (26.6–41.9) vs. 36.1(25.7–40.7)
Reduced acrosome
45.94 ± 17.39 vs. 41.44 ± 15.94
40 (33.3–50) vs. 46.4 (30.3–59.4)
Necrosis
Cytoplasmic droplets
8.07 ± 3.56 vs. 7.89 ± 2.68
7.89 ± 2.55 vs. 8.34 ± 3.67
Elliptical/Round nucleus
8.27 ± 5.8 vs. 10.74 ± 5.84
6.6 (5.4–7.1) vs. 7.9 (6.1–12.2)
Broken plasma membrane
40.57 ± 12.51 vs. 34.16 ± 7.68
42 (38–46) vs. 37 (32–47.5)*
Reacted acrosome
24.22 ± 15.82 vs. 17.92 ± 12.21
23.5 (12–23.8) vs. 13.5 (8.6–20)*
Disrupted chromatin
18.32 ± 15.29 vs. 13.04 ± 6.21
21.4 (20.6–30) vs. 7.8 (4–21)*
Means ± standard deviation (SD) were considered when group 1 was compared with group 2 Medians were considered when group 3 was compared with group 4 * Difference was significant at a P level of \0.05
123
98
Dig Dis Sci (2010) 55:94–100
TEM data highlighted that sperm pathologies were higher than the reference values, related to individuals of proven fertility, reported in Table 1. Apoptosis As reported in Table 1, apoptosis significantly increased (P \ 0.05) in CagA-positive patients compared to CagAnegative patients. Apoptotic sperm are characterized by marginated chromatin and an altered shape of the nucleus. We found similar ratios of these alterations in the two groups of infected and uninfected individuals (Table 2); nevertheless, marginated chromatin was found significantly more present (Table 2) in CagA-positive infected patients (Fig. 1) compared to those who did not have anti-CagA serum antibodies (Fig. 2). Necrosis As reported in Table 1, necrosis significantly increased (P \ 0.05) in CagA-positive patients compared to CagAnegative patients. The sperm characteristics typical of necrosis are a broken plasma membrane, an absent or reacted acrosome, and
Fig. 2 TEM micrograph of longitudinal sections of spermatozoa from a CagA-negative infected patient. The figure shows normal sperm with well-shaped acrosomes (A) and nuclei (N) and necrotic sperm with absent acrosomes (aA) and altered nuclei with disrupted chromatin (dCh). A necrotic sperm also shows a disintegrated axoneme (aAX) coiled into a cytoplasmic droplet (RC). Bar 1 lm
disrupted chromatin. The percentages of these defects were higher in infected patients, but the difference reached statistical significance (Table 2) only when we compared their prevalence in CagA-positive (Fig. 1) and CagA-negative patients (Fig. 2). Immaturity Finally, the percentage of immaturity and the related defective organelles (reduced acrosome, round/elliptical nucleus, uncondensed chromatin) and cytoplasm residues did not seem to be influenced by the presence of infection (Table 2). Cytokine Levels
Fig. 1 TEM micrograph of longitudinal sections of spermatozoa from a CagA-positive infected patient. The figure shows a necrotic sperm characterized by the absence of acrosome (aA) and the disrupted chromatin (dCh), swollen mitochondria (sM) and broken plasma membrane (arrows); apoptotic sperm with marginated chromatin (mCh); a sperm with a normal, but empty acrosome (eA) surrounding an almost well-shaped nucleus (N) with condensed chromatin and a coiled altered axoneme (aAx) embedded in a large cytoplasmic residue (RC). Bar 1 lm
123
The mean concentrations of IL-6 and TNF-a in infected patients were similar to those measured in uninfected ones (Table 3). However, the mean level of TNF-a was significantly higher in patients infected by CagA-positive strains compared to the mean level measured in uninfected ones (Table 3). Mean concentrations of TNF-a in infected CagA-negative patients were similar to those found in uninfected patients. Nevertheless, the difference in the mean level in CagA-seropositive patients was not statistically significant (P = 0.125) between these two groups, which is probably due to the small number of patients. Helicobacter pylori infection status did not seem to influence the amounts of IL-6 secreted in the bloodstream (Table 3).
Dig Dis Sci (2010) 55:94–100
99
Table 3 Mean ± standard deviation of levels of circulating cytokines in patients infected with H. pylori (CagA-positive and CagA-negative) and uninfected Groups and number () of patients and controls examined
IL-6
TNF-a
H. pylori-positive patients (36)
2.57 ± 2.86
9.79 ± 7.01
2.90 ± 4.87
8.53 ± 4.17
2.43 ± 2.73
12.14 ± 9.22*
2.46 ± 2.72
8.56 ± 3.47
Group 1 H. pylori-negative patients (44) Group 2 CagA-positive infected patients (17) Group 3 CagA-negative infected patients (19) Group 4 The number of patients in each group is reported in parentheses * Group 3 vs. group 2 P = 0.039
Discussion In a recent study [14], some of our group have observed an association between H. pylori infection and infertility, in men and in women. Infected patients also have antiH. pylori antibodies in their biological fluids, including semen, which cross-reacted with spermatozoa, suggesting that this infection may concur to stimulate the production of anti-spermatozoa autoantibodies. Finally, a linear homology was found between beta-tubulin, a sperm flagellar protein, and some bacterial antigens, including CagA. To further investigate the pathogenic mechanisms through which H. pylori infection may increase the risk of male infertility, we have analyzed the semen quality of a group of patients with idiopathic infertility, either infected or uninfected with H. pylori. We found that the group with H. pylori infection showed a decreased fertility index compared to the uninfected infertile patients. In particular, our results showed that the sperm quality of patients infected by CagA-positive strains was significantly reduced compared to that of CagA-negative patients: motility and the fertility index were diminished, while the frequency of apoptosis and necrosis was increased. Two possible explanations could account for the observed findings: the inflammatory response to the infection and the existence of cross-mimicry phenomena between H. pylori and sperm antigens. The humoral and cellular immune responses to the invading organisms increase the local and systemic indices of inflammation, such as the levels of polymorphs and monocytes [18]. The colonized gastric epithelium and immunocytes stimulated by the bacterial determinants of pathogenicity secrete vasoactive substances and cellular mediators, such as cytokines. Certain clones of H. pylori are particularly effective in stimulating the production of these substances: increased amounts of TNF-a and IL-6 were detected in the gastric mucosa
colonized by CagA-positive H. pylori strains [19, 20]. TNF-a is a predominant cytokine in the gastric mucosa of patients with H. pylori infection; it induces apoptosis in a variety of cells, including spermatozoa, and also exerting a negative influence on sperm motility [21, 22]. The amounts of TNF-a may also increase systemically along infections. Some researchers [23] showed augmented concentrations of this cytokine in serum samples of H. pylori-infected dyspeptic patients; others discovered that the concentrations of TNF-a in the bloodstream were further increased when patients were colonized by strains expressing CagA [24]. In agreement with such observations, in the present study we found that the mean systemic levels of TNF-a were significantly increased in patients infected by CagApositive strains. We therefore hypothesize that the observed augmented apoptosis and necrosis of spermatozoa from patients seropositive for CagA may be due, at least in part, to the high pathogenic potential of such strains, which enhance the inflammatory response with an increased cytokine production, either by the gastric mucous cells and by the stimulated immunocytes present in the semen. To corroborate this supposition, we began a study aiming to explore the role of H. pylori genotypes and other agents of chronic infection in determining the levels of proinflammatory cytokines in semen samples. Male infertility could also be due to immunological factors. It is known that the alterations of the blood-testis barrier may expose spermatozoa antigens to the immune system [25] and that inflammation of the genital tract may alter the composition of spermatozoa antigens by inducing a secondary autoimmune response that could cause sperm damage. The presence of anti-H. pylori and anti-CagA antibodies of both classes, IgG and IgA, in the seminal fluid of infected individuals and the existence of crossmimicry between H. pylori and spermatozoon epitopes [14] lead us to hypothesize that immune reaction phenomena could take place in semen specimens, with the consequent possible injury of spermatozoa. In conclusion, there is growing evidence of an unfavorable influence of H. pylori infection on organs and apparati different and distant from the gastroduodenal tracts, also involving the reproductive system. In this study we demonstrated a negative effect of CagA-positive H. pylori infection on the sperm quality of patients with idiopathic infertility. We therefore suggest testing all patients with hypofertility or infertility for H. pylori infection and the presence of anti-CagA serum antibodies, as the definition of the H. pylori infection status alone may not be conclusive enough. A possible follow-up study could be first to eradicate H. pylori from infected individuals and then to check the possible improvement of semen parameters and the reduction of TNF-a levels. In this study we determined the
123
100
cytokine level in the bloodstream, and our purpose will be to measure the cytokine concentration directly in the semen samples. Acknowledgments This work was supported in part by funds from the University of Siena P.A.R. 2006, ‘‘Role of Helicobacter pylori infection in the development of some autoimmune diseases, such as Hashimoto’s thyroiditis, Sjo¨gren’s syndrome and systemic sclerosis’’ and P.A.R. 2005, ‘‘Morphological, biochemical and molecular studies of human spermatogenesis in a flogistic environment.’’
References 1. Parsonnet J, Hansen S, Rodriguez L, et al. Helicobacter pylori infection and gastric lymphoma. N Engl J Med. 1994;330:1267– 1271. doi:10.1056/NEJM199405053301803. 2. Dufour C, Brisigotti M, Fabretti G, Luxardo P, Mori PG, Barabino A. Helicobacter pylori gastric infection and sideropenic refractory anemia. J Pediatr Gastroenterol Nutr. 1993;17:225– 227. doi:10.1097/00005176-199308000-00018. 3. Mendall MA, Goggin PM, Molineaux N, et al. Relation of Helicobacter pylori infection and coronary heart disease. Br Heart J. 1994;71:437–439. doi:10.1136/hrt.71.5.437. 4. Tebbe B, Geilen CC, Schulzke JD, Bojarski C, Radenhausen M, Orfanos CE. Helicobacter pylori infection and chronic urticaria. J Am Acad Dermatol. 1996;34:685–686. doi:10.1016/S0190-9622 (96)80086-7. 5. Gasbarrini A, Gabrielli M, Fiore G, et al. Association between Helicobacter pylori cytotoxic type I CagA-positive strains and migraine with aura. Cephalalgia. 2000;20:561–565. doi:10.1046/ j.1468-2982.2000.00077.x. 6. Franceschi F, Leo D, Fini L, et al. Helicobacter pylori infection and ischaemic heart disease: an overview of the general literature. Dig Liver Dis. 2005;37:301–308. doi:10.1016/j.dld.2004.10.015. 7. Franceschi F, Gasbarrini A. Helicobacter pylori and extragastric diseases. Best Pract Res Clin Gastroenterol. 2007;21:325–334. doi:10.1016/j.bpg.2006.10.003. 8. Manolakis A, Kapsoritakis AN, Potamianos SP. A review of the postulated mechanisms concerning the association of Helicobacter pylori with ischemic heart disease. Helicobacter. 2007;12:287–297. doi:10.1111/j.1523-5378.2007.00511.x. 9. Negrini R, Lisato L, Zanella I, et al. Helicobacter pylori infection induces antibodies cross-reacting with human gastric mucosa. Gastroenterology. 1991;101:437–445. 10. Appelmelk BJ, Simoons-Smit I, Negrini R, et al. Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity. Infect Immun. 1996;64:2031–2040. 11. D’Elios MM, Bergman MP, Amedei A, Appelmelk BJ, Del Prete G. Helicobacter pylori and gastric autoimmunity. Microbes Infect. 2004;6:1395–1401. doi:10.1016/j.micinf.2004.10.001.
123
Dig Dis Sci (2010) 55:94–100 12. De Korwin JD. Does Helicobacter pylori infection play a role in extragastric diseases? Presse Med. 2008;37:525–534. doi: 10.1016/j.lpm.2007.07.029. 13. Bodger K, Crabtree JE. Helicobacter pylori and gastric inflammation. Br Med Bull. 1998;54:139–150. 14. Figura N, Piomboni P, Ponzetto A, et al. Helicobacter pylori infection and infertility. Eur J Gastroenterol Hepatol. 2002;14: 663–669. doi:10.1097/00042737-200206000-00012. 15. World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interactions. 4th ed. Cambridge, United Kingdom: Cambridge University Press; 1999. 16. Baccetti B, Bernieri G, Burrini AG, et al. Notulae seminologicae. 5. Mathematical evaluation of interdependent submicroscopic sperm alterations. J Androl. 1995;16:356–371. 17. Collodel G, Moretti E. Morphology and meiotic segregation in spermatozoa from men of proven fertility. J Androl. 2008; 29:106–114. doi:10.2164/jandrol.107.002998. 18. Graham DY, Osato MS, Olson CA, Zhang J, Figura N. Effect of H. pylori infection and CagA status on leukocyte counts and liver function tests: extra-gastric manifestation of H. pylori infection. Helicobacter. 1998;3:174–178. doi:10.1046/j.1523-5378.1998.08 018.x. 19. Yamaoka Y, Kita M, Kodama T, Sawai N, Kashima K, Imanishi J. Induction of various cytokines and development of severe mucosal inflammation by CagA gene-positive Helicobacter pylori strains. Gut. 1997;41:442–451. 20. Kowalski M, Konturek PC, Pieniazek P, et al. Prevalence of Helicobacter pylori infection in coronary artery disease and effect of its eradication on coronary lumen reduction after percutaneous coronary angioplasty. Dig Liver Dis. 2001;33: 222–229. doi:10.1016/S1590-8658(01)80711-8. 21. Perdichizzi A, Nicoletti F, La Vignera S, et al. Effects of tumour necrosis factor-alpha on human sperm motility and apoptosis. J Clin Immunol. 2007;27:152–162. doi:10.1007/s10875-0079071-5. 22. Shibata J, Goto H, Arisawa T, et al. Regulation of tumor necrosis factor (TNF) induced apoptosis by soluble TNF receptors in Helicobacter pylori infection. Gut. 1999;45:24–31. 23. Russo F, Jirillo E, Clemente C, et al. Circulating cytokines and gastrin levels in asymptomatic subjects infected by Helicobacter pylori (H. pylori). Immunopharmacol Immunotoxicol. 2001;23: 13–24. doi:10.1081/IPH-100102563. 24. Perri F, Clemante R, Festa V, et al. Serum tumour necrosis factoralfa is increased in patients with Helicobacter pylori infection and CagA antibodies. Ital J Gastroenterol Hepatol. 1999;31:290– 294. 25. Heidenreich A, Bonfig R, Wilbert DM, Strohmaier WL, Engelmann UH. Risk factors for antisperm antibodies in infertile men. Am J Reprod Immunol. 1994;31:69–76.
