helicobacter pylori ( hp ) , a gram - negative bacterium that colonizes the stomach , is the main etiologic factor in the development of gastritis , peptic ulcer disease , and malignant gastric lesions [ 1 , 2 ] . the clinical consequences of hp infection are not limited to the gastroduodenal tract since many studies reported that it could contribute to cause extradigestive disorders   and to play a role in the development of autoimmune diseases . recently , rising evidence has indicated a relationship between hp infection and reduced fertility , in both men and women . the first observation on this topic was made by some of our group   who reported that the infection is significantly more common in both men and women with fertility problems ; in semen , follicular fluid , and vaginal secretions of infected individuals , we detected specific antibodies , which cross - reacted in vitro with spermatozoa , suggesting the existence of an autoimmune phenomenon . the amino acid alignment of the human tubulin , a constituent of sperm axoneme , with hp peptides indicated a possible antigenic mimicry with the cytotoxin - associated gene a protein ( caga ) and other hp antigens . caga is expressed by hp strains endowed with enhanced inflammatory potential and the infection by caga - positive strains is significantly associated with gastric and extragastric diseases such as cardiovascular disorders and autoimmune thyroid diseases . caga is expressed by one of the 30 genes enclosed in the pathogenicity island cag and it is a marker of the presence of such an insertion in the bacterial chromosome . most strains harboring cag also express the vacuolating toxin vaca , which contributes to the aggressiveness of the strains . later on , in a japanese study , hp infection was reported to be twice as prevalent in women with idiopathic procreation problems , compared to women with known causes of fertility disorders . lastly , an italian team have recently found that in vitro sperm motility through cervical mucus of infected women was significantly reduced , concluding that anti - hp igg antibodies could possibly hamper the meeting of sperm with the oocyte . the previous observation , made in a small number of patients , that hp infection was associated with reduced sperm motility [ 9 , 10 ] prompted us to carry out the present study , in which we explored the role of caga in decreasing sperm motility in a large male population . to substantiate whether antigenic mimicry mechanisms may influence the sperm motility , we aligned the aminoacid sequence of some enzymes involved in energy production , with peptides expressed by helicobacter pylori j99 ( taxid : 85963 ) strain possessing the pathogenicity island cag . should a homology exist , the immune response against hp infection could also react with human cells with possible harmful consequences . from january 2010 to may 2013 , we enrolled 97 males ( aged 1845 years ) with hp infection attending the department of medical and surgical sciences and neurosciences for dyspepsia . patients did not take antibiotics potentially active against hp , including proton pump inhibitors , in the past three months . kg / m , no history of diabetes , radiotherapy , chemotherapy , chronic illnesses , medication , or autoimmune disorders . the subjects showed normal follicle stimulating hormone ( fsh ) , luteinizing hormone ( lh ) , and testosterone ( t ) evaluated in serum by chemiluminescence using commercial kit ( beckman coulter access for fsh , lh , and testosterone , beckman coulter s.p.a . , milano , italy ) . normal ranges were 0.711.00  mui / ml for fsh ( sensitivity 0.2  mui / ml , intra- and interassay coefficient of variation < 10% ) , 0.88.0  mui / l for lh ( sensitivity 0.2  mui / ml , intra- and interassay coefficient of variation < 10% and 2.710.9  ng / ml for t ( sensitivity 0.1  ng / ml , intra- and inter - assay coefficient of variation < 10% ) . clinical and physical examinations and scrotal eco - color doppler were performed on all patients to exclude the possible presence of varicocele . all subjects gave their permission to collect blood samples in order to investigate their hp infectious status and semen samples for determination of semen parameters and for microbiological investigations ; they signed a written informed consent for this research , and the local ethical committee has approved the study . after semen analysis , 10 individuals were excluded from the study : 3 subjects ( 2 caga negative and 1 caga positive ) resulted azoospermic ; 6 other patients ( 2 caga negative and 4 caga positive ) had semen culture positive for escherichia coli ( 3 men ) , enterococcus faecalis ( 2 men ) , and staphylococcus epidermidis ( 1 man ) ; in addition , one caga - negative patient showed the sperm defect of possible genetic origin named  dysplasia of the fibrous sheath ,  characterized by short , thick tails with a peculiar dysplastic fibrous sheath , as detected by transmission electron microscopy , which severely compromises sperm motility . the azoospermic subjects and the patient with the sperm showing  dysplasia of the fibrous sheath  were excluded from the study after three different semen analyses which confirmed the first diagnosis . hp infectious status was determined in serum using a commercially available enzyme - linked immunosorbent assay with a sensitivity and specificity of around 96% ( helicobacter pylori igg , hpg screen elisa kit , genesis diagnostics ltd , littleport , uk ) . briefly , a whole cell suspension of h. pylori ccug 17874 ( a caga - positive and cytotoxic strain ) was denatured in laemmli 's buffer at 100c for 5  min and electrophoresed in 10% polyacrylamide gel with sodium dodecylsulphate . resolved proteins were transferred electrophoretically onto nitrocellulose membranes , and free sites were saturated with 3% skim milk in phosphate buffered saline ( pbs ) ph 7.4 containing 0.1% triton x ( pmt ) . strips were then cut and immunoblotted with serum samples diluted at 1  :  100 in pmt for immunoglobulin g ( igg ) . after overnight incubation at room temperature , strips were washed three times with pmt , and a peroxidase - labeled antibody to human igg , diluted in pmt 1  :  2000 ( sigma che . co. , milan ) , was added and incubated at room temperature for 90  min . strips were washed three times with pmt , once with pbs - triton x and twice with 0.05  mol / l ph 6.8 tris buffer . the reaction was visualized by addition of the substrate ( h2o2 in a solution of 4-chloro-1-naphthol in 0.05  m ph 6.8 tris buffer ) . anti - hp whole cell suspension and anti - caga rabbit polyclonal antibodies ( kindly donated by r. rappuoli , novartis , siena ) were used as positive controls . semen samples were collected by masturbation after 4 days of sexual abstinence and examined after liquefaction for 30  min at 37c . volume , ph , and sperm concentration and motility were evaluated according to who guidelines . sperm morphology was assessed by the papanicolaou ( pap ) staining modified for spermatozoa following the who guidelines . sperm vitality was assessed in semen samples showing a progressive motility < 40% : 37 specimens of caga - positive group , 41 of caga - negative one . the specimens were stained with 0.5% eosin y ( ci 45380 ) in a 0.9% aqueous sodium chloride solution . a few minutes after staining , the samples were observed by light microscope , and the stained ( dead ) cells and unstained ( living ) cells were scored . the amino acid sequence of some of human enzymes involved in the energy production was  blasted  in the protein databases at the national center for biotechnology information ( ncbi ; bethesda , md , usa ; http://www.ncbi.nlm.nih.gov/ ) , with proteins of helicobacter pylori j99 ( taxid : 85963 ) . sequences longer than five amino acids were also included , even if the alignment was interrupted by one or two nonmatching amino acids . five amino acids in a row is the minimum number to form an epitope , also called antigenic determinant , that is , the specific region on an antigen that an antibody recognizes and binds to . we consider also sequences longer than five amino acids because the longer the amino acid sequence , the higher the probability that two proteins are similar and antibodies may also be able to bind the native protein . hexokinase , phosphoglucose isomerase , phosphofructokinase , aldolase , triose phosphate isomerase , glyceraldehyde 3-phosphate dehydrogenase , phosphoglycerate kinase , phosphoglycerate mutase , enolase , and pyruvate kinase . citrate synthase , aconitase , isocitrate dehydrogenase , -ketoglutarate dehydrogenase , succinyl - coa synthetase , succinate dehydrogenase , fumarase , and malate dehydrogenase . nadh - coenzyme q oxidoreductase , succinate - q oxidoreductase , electron transfer flavoprotein - q oxidoreductase , q - cytochrome c oxidoreductase , cytochrome c oxidase , and atp synthase . 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 . since the conditions of normality of distribution and of homogeneity of variance were not satisfied , comparisons between the variables from hp caga - positive and hp caga - negative groups were performed using the mann - whitney u test . to investigate the role of caga in reduced semen quality , we have analyzed semen samples from a group of men with hp - infection , establishing their caga status . the characteristics of the 87 hp infected subjects , grouped following caga status , are shown in table 1 . thirty - seven hp - infected patients ( 42.53% ) were positive for caga . medians of semen volume and sperm concentrations were comprised between 25th centile and 50th centile in both groups , the median of progressive sperm motility percentage was under 2.5th centile in the group of caga - positive patients and at 5th centile in those negative for caga , the median of normal forms was between 50th centile and 75th centile in both groups , and finally the medians of vitality were < 2.5th centile . applying statistics , we obtained that sperm motility and vitality in the caga - positive group were significantly lower than those observed in the caga - negative group ( motility : 18% versus 32% ; vitality : 35% versus 48% ; p < 0.01 ) . using such a numerous group of infected patients , we confirmed that the infection by hp strains expressing caga may exert a negative effect on human sperm motility and vitality . these results are concordant with previous observations that in infertile male seropositive for caga , the frequency of sperm death , expressed as apoptosis and necrosis , was increased with respect to that detected in infected men seronegative for caga or uninfected . another interesting result emerged from this analysis : the percentage of sperm with normal forms appeared to be significantly lower in the caga - positive group with respect to that observed in caga - negative one ( 18% versus 22% ; p < 0.05 ) . this result can be explained by the increase of unviable sperm with broken plasma membrane ( eosin - stained ) in caga - positive group : the acrosome , the most fragile organelle of the spermatozoon , is reacted or absent in necrotic sperm . in addition , the manchette , the structure able to model the sperm shape during spermatogenesis , is constituted by microtubules , and it is known that human tubulin , the major constituent of this structure , shows homologies with some hp proteins , suggesting the phenomenon of antigenic mimicry . the other analyzed variables such as ph , volume , and sperm concentration were similar in both groups ( ph : p = 0.875 , volume p = 0.594 and sperm concentration p = 0.718 ) . also similar , between the two groups , were the levels of analyzed hormones , such as fsh ( caga positive : 4.30  mui / ml ( 1.80  mui / ml10.10  mui / ml ) ; caga negative : 3.80  mui / ml ( 1.70  mui / ml11  mui / ml ) ) , lh ( caga positive : 2.50  mui / ml ( 1.20  mui / ml6.50  mui / ml ) ; caga negative : 2.60  mui / ml ( 1  mui / ml6.70  mui / ml ) ) and t ( caga positive : 5.60  ng / ml ( 2.93  ng / ml9.86  ng / ml ) ; caga negative : 5.47  ng / ml ( 2.97  ng / ml9.98  ng / ml ) . to the best of our knowledge no clues on this topic are present in the literature ; a recent research reported that infection may regulate the seminal levels of ghrelin , a hormone endowed with anti - inflammatory properties   and involved in reproduction , suggesting that the infection may influence the semen concentration of peptides involved in the regulation of testicular function . the exact mechanism by which hp may influence sperm quality is still unknown , although some hypotheses involving antigenic mimicry have been proposed [ 5 , 9 ] . for instance , infected individuals may mount a humoral and cellular immune response to bacterial constituents and products that might cross - react with epithelial cells of various organs . ( for a comprehensive review on hp infection and autoimmunity , consult . ) in a previous study , we showed that antibodies raised in rabbit against hp , as well as serum samples from hp - infected patients , recognized immunologically human sperm , suggesting the existence of an antigenic mimicry between hp antigens and sperm peptides . as far as the autoimmune cell response is concerned , there are lines of evidences of a cross - reactivity of cd4 + th1 cells , infiltrating the gastric epithelium in response to the infection , with epitopes of the gastric k+/h+ atpase , the proton pump , with deleterious consequences for the gastric mucosa . these observations prompted us to align aminoacidic sequence of hp with some enzymes involved in glycolysis and oxidative metabolism processes , since both pathways are involved in providing energy for sperm movement [ 20 , 21 ] . all the considered enzymes showed partial linear homology with hp peptides ; four enzymes aligned with four different segments of the same cag protein ( table 2 , figure 1 ) . in addition , nadh dehydrogenase ( ubiquinone ) fe - s protein 1 showed partial linear homology with a different cag island protein , sequence i d : ref . finally , aldolase partially aligned with vacuolating cytotoxin ( vaca ) , sequence i d : ref . the observation that four different enzymes ( table 1 ) showed partial structural homology with different segments of the same cag island protein could suggest the existence of sperm damage mediated by antigenic mimicry mechanisms . of course , the considered enzymes are part of ubiquitous processes shared by all kinds of cells , so the damage caused by autoimmune reaction can not be severe . on the other hand we have to consider that the hp infection lasts for the patient 's entire life ( if not treated ) and that the immune reactions occurring in patients infected by caga positive hp strains , which are endowed with enhanced inflammatory potential , could concur to determine a more severe damage . another plausible concomitant explanation for the observed reduced sperm quality , in terms of vitality , motility , and normal morphology , could consist in the inflammatory response to the infection . hp strains bearing caga were found to induce increased local and systemic levels of interleukin-8 ( il-8 ) , il-1 , il-6 , tumor necrosis factor- ( tnf- ) , and inflammation in the gastric mucosa , compared to the levels of inflammatory mediators generated by infection by caga - negative strains [ 2224 ] ; to this purpose we demonstrated that hp infected men , especially those with serum antibodies to the caga protein , have increased systemic levels of tnf- , a proinflammatory cytokine that may cause sperm damage . we are working on measuring cytokines levels directly in seminal plasma of men infected by hp expressing / nonexpressing caga . since we hypothesized mechanisms such as antigenic mimicry and inflammation the blood - testis barrier ( btb ) protects haploid germ cells against immune attack ; however , it is possible that in case of increased level of interleukins ( as in case of infection by caga - positive hp ) the btb might be damaged ; it has been recently reported that il-6 might be involved in the downregulation of expression of occludin , a protein found in the tight junctions constituting the btb , and in the modulation of btb permeability that occurs in rats undergoing autoimmune orchitis . indeed , it is not excluded that the damage could occur in the epididymis , where the tight junctions are much less effective than in the testis , or directly in the semen . we definitely demonstrated , in a quite large population of hp - infected males , a relationship between the presence of strains expressing caga and decreased sperm quality , especially concerning motility and viability . we propose two possible explanations , not mutually exclusive , for the obtained results : the existence of an autoimmune phenomenon that involves molecular mimicry , an enhanced inflammatory response causing an increased cytokine production . the existence of an autoimmune phenomenon that involves molecular mimicry , an enhanced inflammatory response causing an increased cytokine production . in our opinion , there are enough indications that the chronic infection by hp expressing caga could contribute to the damage of organs other than the gastroduodenal tract and to negatively influence many functions , including reproduction . if other studies will confirm our findings , treatment of h. pylori infection is advisable . we tend , however , to prescribe the appropriate eradication therapy independently of the putative impact of infection upon the reproductive sphere ; our opinion is that there is no advantage for infected people to harbour an organism that causes chronic gastritis in all , peptic ulceration in 20% ca . and
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 .