Association between Helicobacter pylori infection and the presence of anti-sperm antibodies

ABSTRACT

The aim of this study was to investigate the possible association between serum anti-Helicobacter pylori antibodies and anti-sperm antibodies (ASA) in patients with gastroduodenal diseases caused by H. pylori, infertile patients positive for ASA and healthy fertile blood donors as controls. Serum ASA were studied by sperm agglutination tests, sperm immobilization test and ELISA against sperm antigens (ELISAsp). Serum IgG anti-H. pylori antibodies were detected by ELISA (ELISAHp). The tests revealed significantly higher incidence of ASA in patients with gastroduodenal diseases compared to the controls (P < 0.0001). The median levels of both types of antibodies were significantly higher in infertile patients than in patients with upper gastroduodenal diseases in all tests applied (sperm agglutination and immobilization tests P < 0.0001; ELISAsp P = 0.006; ELISAHp, P = 0.0008). Significant linear correlation was found between anti-H. pylori antibodies and ASA detected by sperm immobilization test (r = 0.613, P = 0.05) in the group of patients with gastroduodenal disease, as well as a weak linear correlation was established between anti-H. pylori antibodies and ASA in ELISAsp (r = 0.275, P = 0.0051) in the same group. These results suggest that H. pylori infection may play a role in the induction of ASA.

KEYWORDS:

• Helicobacter pylori
• anti-Helicobacter pylori antibodies
• immune infertility
• anti-sperm antibodies
• sperm antigens

Introduction

Anti-sperm antibodies (ASA) are a factor contributing to unexplained infertility, as have been shown by numerous human population studies [1–4]. Among the risk factors for ASA production, the chronic inflammatory diseases with different localization are believed to play an important role [5–8]. The inflammatory response is associated with production of antibodies to spermatozoal and microbial antigens. This may be due to cross-reactivity between antigens of spermatozoa and some exogenous antigens of bacteria, viruses, fungi, and allergens. Common antigenicity has been found between spermatozoa and: streptococci, Streptococcus viridans, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Candida albicans, Ureaplasma urealyticum, Chlamydia trachomatis, Escherichia coli and Salmonella typhi [9–16]. On the other hand, data concerning the presence of cross-reacting antigens of spermatozoa and Helicobacter pylori, as well as association between H. pylori infection and infertility are scarce. The spiral-shaped, Gram-negative bacterium H. pylori is an important human pathogen. Infection with it occurs worldwide and its prevalence is correlated with upper gastroduodenal diseases [17]. Perez-Perez et al. [18] investigated the prevalence of H. pylori infection in infertile couples by testing for H. pylori-specific IgG antibodies using ELISA. These authors, however, were more interested in the pathogen transmission and did not compare the infertile patients to healthy fertile controls. Figura et al. [19] hypothesized for the first time that H. pylori infection could be involved in the development of infertility and may increase the risk of reproductive disorders and aggravate their clinical expression. They also raised the possibility of cross-reaction between sperm and H. pylori antigens.

The aim of the present study was to investigate whether there is a correlation between anti-H. pylori antibodies (anti-Hp Abs) and ASA in patients with H. pylori infections and infertile patients, in view of the possibility for induction of human immune infertility, and whether there are cross-reacting sperm and H. pylori antigens.

Materials and methods

All procedures were performed at the Laboratory of Reproductive Immunology, Department of Biology, Medical University of Sofia after institutional ethical board approval and in agreement with the Declaration of Helsinki for Medical Research Involving Human Subjects.

Serum samples

A total of 314 infertile patients (157 couples) without apparent cause of infertility were tested for ASA by agglutination tests, immobilization test and ELISA. Sera from 106 of these patients (55 women and 51 men aged 23–46 years) gave a positive result in at least one of the tests and were included in the study as representing infertility with auto-/isoimmune response against sperm antigens.

Serum samples were also taken from 106 patients (52 women and 54 men aged 23–65 years) suffering from upper gastroduodenal diseases with clinical symptoms of infection caused by H. pylori (dyspeptic symptoms, gastritis, gastroduodenitis chronica or gastric ulcer) in the beginning of the active stage of the disease. In this group: 89 patients were with dyspeptic symptoms and preliminary diagnosis gastritis or gastroduodenitis (as 11 of them undergone endoscopy and were positive for the H. pylori infection after histological examination); 11 patients were with endoscopycal diagnosis gastroduodenitis chronica (as in 2 cases urease test on bioptat was positive and 1 had positive result in stool antigen test for H. pylori infection); 6 patients were with endoscopycal diagnosis gastric ulcers (as 1 patient was positive for the H. pylori infection after histological examination, 1 patient had positive urease test on bioptat and 1 had positive result in stool antigen test for H. pylori infection).

A negative control group comprising of 104 sera from healthy age-matched blood donors (BD) of proven fertility (46 females and 58 males) without clinical symptoms of upper gastroduodenal diseases was also tested.

Anti-sperm antibody tests

ASA were detected by gelatin agglutination test (GAT) of Kibrick, tray agglutination test (TAT) of Friberg, microsperm immobilization test (SIT) of Isojima and Koyama and enzyme-linked immunosorbent assay against sperm antigens (ELISAsp). All sera were heat-treated (56 °C, 30 min) before used and tested for ASA.

Human semen samples were collected from normal healthy donors of proven fertility according to the criteria of WHO [20]. Motile spermatozoa were obtained after ‘swim-up’ method [21] and used in TAT, SIT and ELISA.

GAT was applied according to Rose et al. [22] using total ejaculates. Sera were serially diluted in Baker's buffer, loaded in Kibrick 5/0.4 cm test tubes (100 µL per tube) and gently mixed with equal volumes of semen/gelatin suspension to a final concentration of 1 × 10⁷ spermatozoa/mL in a 2.5% gelatin. Tubes were incubated at 37 °C and read after 1 h. Sera with sperm agglutination titer ≥16 were considered positive. TAT was performed in 60 well low profile stacking trays (Robbins Scientific, Sunnyvale, CA, USA) according to the standard protocol as described in [22] They were loaded with serum samples in serial dilutions and 1 μL of ‘swim-up’ sperm suspension (1 × 10⁷ spermatozoa/mL) was added in each well. Trays were observed after incubation for 1 h at 37 °C. Sera with sperm agglutination titer ≥32 were considered positive.

Sperm immobilizing antibodies were detected by SIT – a semi-quantitative method as described in [23]. Serum samples diluted 1:2 in Baker's buffer were loaded in 60 well low profile stacking trays (Robbins Scientific), 10 µL/well. After that 1 μL/well of motile ‘swim-up’ spermatozoa (1 × 10⁷ spermatozoa/mL) and 2 μL/well of whole guinea-pig complement (National Center for Infectious and Parasite Diseases, Sofia, Bulgaria) were added. The trays were incubated for 1 h at 37 °C and then the percentage of motile spermatozoa in controls and tested sera was determined by counting 100 spermatozoa/well. For each serum, a sperm immobilization value (SIV) was calculated as $\text{SIV}=C/T$. Here, C is the mean percentage of the motile spermatozoa in the negative control and T is the mean percentage of the motile spermatozoa in the tested serum. Sera with SIV > 2.0 were considered positive.

ELISAsp was performed as described previously [24,25]. U-bottomed 96-well PVC plates (Becton Dickinson, Oxnard, CA, USA) were coated with 50 μL/well ‘swim-up’ sperm suspension (1 × 10⁷ spermatozoa/mL) and then incubated with blocking buffer of 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS). Serum samples diluted 1:20 in PBS were added to the plates. Peroxidase-conjugated anti-human IgG (National Center for Infectious and Parasite Diseases) diluted 1:1600 in blocking buffer was used as second antibody and o-phenylenediamine (Sigma-Aldrich) as chromogenic substrate. The optical density (OD) of the tested serum was measured at 492 nm on a Uniscan reader (Lab systems, Espoo, Finland), and mean value ($\overline{x}$) of the OD of each serum was determined. ELISA barrier value $I_b=\overline{x}+2\text{SD}$ was introduced with $\overline{x}$ as the mean value of OD_{492 nm} from the negative control group and SD as the standard deviation. Sera with $\overline{x}$> I_b were considered positive.

Determination of antibodies to H. pylori-specific antigens

Serum anti-H. pylori IgG antibodies (anti-Hp Abs) were detected by indirect sandwich ELISA with recombinant antigens (ELISAHp) using a commercially available kit (recomWell Helicobacter IgG, Mikrogen GmBH, Martinsried, Germany) and a protocol provided by the manufacturer. Positive, cut-off and negative controls included in the kits were always included. These controls and patient sera were diluted 1:100 before application. OD was measured on Uniscan reader at 450 nm, as required by the chromogenic substrate (tetra-methylbenzidine). The average OD_{450 nm} of the two cut-off controls at the beginning and end of the series of the testing was designated as $\overline{x}$_cut-off. The barrier value (I_b) was introduced as I_{b =} $\overline{x}$_cut-off + 20%, and sera with $\overline{x}$> I_b were considered positive. The anti-Hp Ab activity levels in units per ml were calculated using the following formula:$\text{Sample (U}/\text{ml)}=(\text{OD}{}_{\text{sample}}/\text{OD}{}_{\text{cut-off}})\times 20$

Absorption experiments with human spermatozoa

To study the cross-reaction between sperm cell surface and H. pylori antigens, we absorbed with ‘swim-up’ spermatozoa: (1) all sera from infertile patients (n = 25) positive simultaneously in SIT, ELISAsp and ELISAHp; (2) all sera from patients with gastroduodenal diseases (n = 20) positive both in ELISAsp and ELISAHp. Absorption was performed by incubating the serum with an equal volume of sperm suspension (1 × 10⁷ spermatozoa/mL) for 2 h at room temperature. After that, the samples were centrifuged and the supernatant was tested. The activity of absorbed and unabsorbed serum of the same individual was compared and its reduction was calculated.

Statistical analysis

Incidence of ASA and anti-Hp Abs was found in each tested group based on the above criteria for clinical relevance. The magnitude of positive response for each group (presented as median value – titer, SIV or OD depending on the method) was determined. The data were processed with the GraphPad software package (GraphPad, San Diego, CA, USA).

Statistical evaluation of the results from the experiments was done by non-parametric analysis [26]. Nonparametric method of Pearson (criterion χ²) was applied to evaluate differences in antibody incidence, and Mann–Whitney test was applied to compare the median antibody levels between tested groups. The activity of each absorbed serum and an unabsorbed sample of the same individual was also compared.

The association between serum anti-Hp Abs and ASA in tested groups was investigated by correlation analysis, with Pearson's correlation coefficient r_xy used as measure for linear dependence between the accidental variables x (anti-Hp Abs) and y (ASA). The following linear regression model can be constructed as described before [8,27]:$y=A+Bx$where y (ASA) is the mean value of measured quantity, x (anti-Hp Abs) is the mean value of the independent variable and A and B are parameters in the model. The level of significance was set as P < 0.05 where P is level of significance of the null hypothesis.

Results and discussion

After testing 106 infertile patients with humoral immune response against sperm antigens (InfP), 106 patients suffering from upper gastroduodenal diseases with clinical symptoms of H. pylori infection (HpP) and 104 healthy age-matched BD of proven fertility without clinical symptoms of gastroduodenal diseases for ASA incidence by agglutination tests (GAT, TAT), SIT and ELISAsp as well as for anti-Hp Abs incidence by ELISAHp, the results are summarized in Table 1.

Table 1. Clinically relevant anti-sperm and anti-H. pylori antibody incidence among infertile patients, patients with H. pylori infection and controls.

Antibody assay		Infertile patients (InfP)	H. pylori infection patients (HpP)	Blood donors (BD)
GAT	No. of tested/No. positive (%), titer ≥ 16	106/38 (35.85%)	106/8 (7.55%)	104/1 (0.96%)
	InfP/BD	P < 0.0001 (χ^2 = 1369.11)^a
	HpP/BD		P < 0.0001 (χ^2 = 49.02)^a
	InfP/HpP	P = 0.0002 (χ^2 = 14.156)^b
TAT	No. of tested/No. positive (%), titer ≥ 32	106/79 (74.53%)	106/19 (17.92%)	104/2 (1.92%)
	InfP/BD	P < 0.0001 (χ^2 = 1482.79)^a
	HpP/BD		P < 0.0001 (χ^2 = 72.25)^a
	InfP/HpP	P = 0.0012 (χ^2 = 10.448)^b
SIT	No. of tested/No. positive (%), SIV > 2	106/68 (64.15%)	106/16 (15.10%)	104/5 (4.81%)
	InfP/BD	P < 0.0001 (χ^2 = 159.14 0)^a
	HpP/BD		P = 0.0277 (χ^2 = 4.848)^c
	InfP/HpP	P < 0.001 (χ^2 = 10.896)^b
ELISAsp	No. of tested/No. positive (%), cut-off > Ib	106/56 (52.83%)	106/27 (25.47%)	104/3 (2.88%)
	InfP/BD	P < 0.0001 (χ^2 = 312.36)^a
	HpP/BD		P < 0.0001 (χ^2 = 64.047)^a
	InfP/HpP	P > 0.05 (P = 0.256,χ^2 = 1.288)^d
ELISAHp	No. of tested/No. positive (%), cut-off > Ib	106/79 (74.53%)	106/86 (81.13%)	104/73 (70.19%)
	InfP/BD	P > 0.05 (P = 0.878,χ^2 = 0.023)^d
	HpP/BD	P > 0.05 (P = 0.719,χ^2 = 013)^d	P > 0.05 (P = 0.691,χ^2 = 1.158)^d
	InfP/HpP

Note: GAT, gelatin agglutination test; TAT, tray agglutination test; SIT, sperm immobilization test; ELISAsp; ELISAHp = enzyme-linked immunosorbent assay for anti-sperm antibodies (ASA); for anti-H. pylori antibodies; barrier value, I_b; method of Pearson (criterion χ²): significantly higher incidence of ASA in InfP or HpP than the BD.

^aP < 0.0001; ^bP ≤ 0.0012; ^cP = 0.0277. Not significant differences in the antibody incidence ^dP > 0.05.

In the group of infertile patients, clinically relevant ASA levels were demonstrated by GAT in 35.85% of the sera, TAT in 74.53%, SIT in 64.15% and ELISAsp in 52.83%. After testing for anti-Hp Abs by ELISAHp, 74.53% of the sera proved positive. Relevant ASA levels in the group of patients with upper gastroduodenal diseases were demonstrated in 7.55% of the sera by GAT, in 17.92% by TAT, in 15.10% by SIT and in 25.47% by ELISAsp. When tested by ELISAHp, 81.13% of these sera were positive for anti-Hp Abs. ASA incidence among negative controls (BD group) was much lower: 0.96% in GAT, 1.92% in TAT, 4.81% in SIT and 2.88% in ELISAsp. 70.19% of tested sera from BD were positive for anti-Hp Abs detected by ELISAHp.

The differences in antibody incidence among the tested groups of subjects were evaluated by nonparametric method of Pearson (criterion χ²) (Table 1). ASA incidence was extremely higher (P < 0.0001) in infertile patients than in BD (in all applied tests) and significantly higher (P < 0.001) in InfP than in patients with H. pylori infection (in GAT, TAT and SIT). No significant difference in ASA incidence was found between infertile patients and patients with H. pylori infection in ELISAsp (P > 0.05). An extremely higher (P < 0.0001) ASA incidence was noted in patients with H. pylori infection compared to BD in GAT, TAT and ELISAsp, and significantly higher (P = 0.0277) ASA incidence in SIT.

No significant differences (P > 0.05) were observed in the incidence of anti-Hp Abs between tested groups of InfP and HpP and between either of them and BD (Table 1). There were no statistically significant differences in reactivity of male and female sera in GAT, TAT, SIT, ELISAsp and ELISAHp in all tested groups.

Mann–Whitney test was applied for the comparison of the median anti-sperm and anti-Hp Ablevels (represented the magnitude of positive response) between all tested groups (Table 2). Median anti-sperm and anti-Hp Ablevels were significantly higher among infertile patients than in BD in all tests applied (GAT, TAT and SIT P < 0.0001; ELISAsp P = 0.0463; ELISAHp P = 0.0059). A similar finding was observed between infertile patients and patients with gastroduodenal diseases. Median anti-sperm and anti-Hp Ab levels were significantly higher in infertile patients than in patients with upper gastroduodenal diseases for all tests applied (GAT, TAT and SIT P < 0.0001; ELISAsp P = 0.006; ELISAHp P = 0.0008). Median ASA levels were extremely significantly higher (P < 0.0001) in patients with upper gastroduodenal diseases compared to BD in TAT and ELISAsp (Table 2).

Table 2. Median anti-sperm and anti-H. pylori antibody levels among infertile patients, patients with H. pylori infection and controls.

Methods	GAT*	TAT	SIT	ELISAsp	ELISAHp
Group	Median log2titer (range)	Median log2titer (range)	Median SIV (range)	Median OD values (range)	Median OD values (range)
Infertile patients	1 (0–7)^c,i	6 (0–19)^c,i	2.429 (0.727–42.0)^c,i	0.8212 (0.2690–1.445)^a,g	1.891 (0.149–3.165)^b,h
H. pylori infection patients	0 (0–4)^f	2 (0–9)^c	1.296 (0.775–4.529)^e	0.7407 (0.3065–1.114)^c	0.6227 (0.109–3.212)^d
Controls (blood donors)	0 (0–4)	0 (0–6)	1.252 (0.8276–2.529)	0.8953 (0.4639–2.016)	1.212 (0.032–5.749)

*Abbreviations as in Table 1.

Mann–Whitney test. Significantly higher value in infertile patients or in patients with H. pylori infection as compared with controls: ^aP = 0.0463; ^bP = 0.0059; ^cP < 0.0001; statistically not significant values; ^dP = 0.051; ^eP = 0.6277; ^fP = 0.5525.

Significantly higher value in infertile patients as compared with patients with H. pylori infection: ^gP = 0.006; ^hP = 0.0008; ⁱP < 0.0001.

Statistical analysis of anti-Hp and ASA activity of the sera (Table 3) showed that there was no significant correlation between anti-Hp Abs and ASA for infertile patients in all ASA tests applied. The same was observed for patients with gastroduodenal diseases when tested by GAT and TAT. There was, however, a significant positive linear correlation between anti-Hp Abs and ASA by SIT (r_xy = 0.613, P = 0.05) and a weak linear correlation between anti-Hp Abs and ASA by ELISAsp (r_xy = 0.275, P = 0.0051), where r_xy is the correlation coefficient, P is the level of significance of the null hypothesis, for the patients with H. pylori infection.

Table 3. Correlation between anti-H. pylori and anti-sperm antibody in the groups of the infertile patients and patients with H. pylori infection.

Antibody assay	Infertile patients	H. pylori infection patients
GAT*	rxy = 0.05843, P = 0.5519	rxy = 0.01491, P = 0.8794
TAT	rxy = 0.1703; P = 0.081^c	rxy = 0.04707, P = 0.6318
SIT	rxy = 0.145, P = 0.687	rxy = 0.613, P = 0.05^a
ELISAsp	rxy = 0.5, P = 0.141	rxy = 0.275, P = 0.0051^b

*Abbreviations as in Table 1.

Correlation analysis. r_xy, Pearson's correlation coefficient; significant positive linear correlation between anti-Hp Abs and ASA by SIT (r_xy = 0.613, ^aP = 0.05) and weak linear correlation between anti-Hp Abs and ASA by ELISAsp (r_xy = 0.275, ^bP = 0.0051) in the group of the patients with H. pylori infection.

To assess the possibility for cross-reaction between sperm cell surface and H. pylori antigens, we absorbed with human spermatozoa two groups of sera: (1) from infertile patients simultaneously positive in SIT, ELISAsp and ELISAHp; (2) from patients with gastroduodenal diseases positive both in ELISAsp and ELISAHp. After the absorption we tested them by SIT (the first group), ELISAsp (both groups) and ELISAHp (both groups) and evaluated the reduction of serum activity. The absorption with human spermatozoa reduced completely (P < 0.001) the SIVs of all SIT-positive sera from the first group; after absorption, SIVs of all these sera were <2.0 and were considered negative. However, absorption did not decrease significantly (P > 0.05) the ASA activity in ELISAsp and anti-Hp Ab activity in ELISAHp of either the first or the second group of sera.

In the present study, we focused on the possibility that H. pylori infection may be associated with ASA production in affected individuals and could be involved in the development of infertility. To investigate this, we compared the incidence and levels of ASA and anti-Hp Abs between infertile patients, patients with gastroduodenal diseases and controls, and addressed the hypothesis of cross-reacting sperm and H. pylori antigens. According to the WHO recommendation based on the fact that ASA differ in their isotype, effects and target antigens, in our study we applied four different techniques for their detection.

The observed extremely significantly higher (P < 0.0001) ASA incidence in infertile patients than in BD in all applied tests and the significantly higher (P < 0.001) ASA incidence in InfP than in patients with H. pylori infection in GAT, TAT and SIT can be explained by the fact that ASA positivity of InfP was used as their inclusion criterion. It is more noteworthy that the difference in ASA incidence between infertile patients and patients with H. pylori infection in ELISAsp was not significant (P > 0.05). This means that gastroduodenal disease caused by H. pylori is associated with incidence of anti-sperm auto/isoimmunity comparable to that found in patients with unexplained infertility pre-selected for such auto/isoimmunity. This was confirmed by the finding of an extremely higher (P < 0.0001) ASA incidence in patients with H. pylori infection compared to BD in GAT, TAT and ELISAsp, and significantly higher (P = 0.0277) ASA incidence in SIT test. Hence, our results reveal enhanced humoral immunity against both surface and total sperm antigens in patients with H. pylori infection. No significant differences (P > 0.05) were observed in the anti-Hp Ab incidence between tested groups. This may be due to the high general prevalence of anti-Hp Abs in the studied population, reported also in other studies [28,29].

The median anti-sperm and anti-Hp Ab levels were significantly higher among infertile patients than in BD and patients with upper gastroduodenal diseases in all tests applied. The median anti-sperm antibody level was extremely higher (P < 0.0001) in patients with upper gastroduodenal diseases compared to BD in TAT and ELISAsp. This suggests a propensity for intensive humoral immune response in both groups of patients.

In this study, we confirmed and enriched the data that H. pylori infection may be associated with the production of ASA [18,19]. Perez-Perez et al. [18] investigated the prevalence of H. pylori infection in infertile couples. The authors found that 17.3% of the tested infertile patients (n = 554) were positive for H. pylori-specific antibodies by IgG ELISA. Figura et al. [19] discussed the possibility that H. pylori infection could be involved in the development of infertility and studied the prevalence of H. pylori infection in male and female patients with various reproductive disorders. The authors found that seropositivity for H. pylori is significantly higher in infertile patients than in controls (49.1% v. 33.5%, P < 0.001), as the prevalence of infection is 66.6% in patients with undetermined infertility. Hajishafiha et al. suggested [30] that infection with CagA-positive H. pylori strains is linked to an increase in women's potential to abort early. Infertile male patients infected with H. pylori showed a low sperm quality compared to uninfected males [31].

The antibodies detected by sperm agglutination and immobilization tests bind to cell surface antigens of the spermatozoa and can directly damage them; in fact, SIT measures the sperm immobilization and the cytotoxic potential of these antibodies in the presence of complement [3,32]. It is known that human ejaculates contain a certain proportion of dead sperm cells, which in some infertile patients is elevated to ‘necrozoospermia’[33]. It can be hypothesized that such antibodies in patients with H. pylori infection have an impact on their fertility, but this possibility requires further investigation.

Our study revealed a significant linear correlation between ASA (detected by sperm immobilization test and ELISAsp) and anti-H. pylori antibodies in the group of patients with gastroduodenal disease, but a correlation not found with agglutination tests. These our results are in accordance with the results of Figura et al. indicating also that rabbit hyperimmune sera against whole-cell H. pylori suspension reacted strongly with the rich in tubulin tail, the centrioles and the part of equatorial zone of human spermatozoa. In the study of Figura et al., a linear homology between human β-tubulin and H. pylori protein flagellin was found. Spermatozoa are flagellated human cells and proteins that compose structures with the similar functions with bacterial flagella are highly conserved in the evolution [19].

Unlike agglutination and immobilization tests for ASA, ELISAsp detects antibodies against total sperm antigens, including intracellular components. The spermatozoon is a highly differentiated cell containing numerous specific cytoskeletal and other intracellular proteins [34] which can serve as auto- or isoantigens. The fact that absorption of all sera from infertile patients, simultaneously positive in SIT, ELISAsp and ELISAHp, with human spermatozoa reduced completely their SIVs, but could not decrease significantly their activity in ELISAsp and ELISAHp confirmed the hypothesis that these antibodies were directed primarily against intracellular antigens. Humoral response against such antigens, while may be of little direct significance for development of infertility, seemed to be highly indicative for the predisposition of the patient's immune system to auto-/isoimmune antibody-mediated hypersensitivity reactions. The results from our study support the hypothesis that H. pylori infection is associated with generation of ASA. Helicobacter pylori infection is known to induce a vigorous systemic and mucosal humoral response [35]. This antibody production does not lead to eradication of the infection and may instead induce damage. Some H. pylori infected patients have an autoantibody response [36,37]. According to the data of Appelmelk et al. [38] and Ko et al. [39], H. pylori infection may be implicated in extra-digestive diseases and particularly in autoimmune response against epithelial cells of various organs. Antigenic mimicry is a hypothetical mechanism explaining the cross-reaction between antigens of H. pylori and cells of various host tissues such as kidney tubular cells, ductal cells of the salivary glands, follicles of the thyroid gland, etc. The same hypothesis may apply to underlying factors that trigger polycystic ovary syndrome in women with H. pylori seropositivity [40,41]. A number of investigators have demonstrated the presence of cross-reactivity between antigens of spermatozoa and some micro-organisms by different techniques. Common antigenicity has been found between spermatozoa and: E. coli, streptococcal antigens, T. vaginalis, M. hominis, U. urealyticum [9–16,42]. Of special interest is the cross-reactivity between sperm antigens and carbohydrate antigens on bacterial walls revealed by Kurpisz et al. [11,12]. All these reports show that chronic H. pylori infection may have systemic influence and be associated with various autoimmune and other hypersensitivity reactions, including anti-sperm immunity. The inflammatory response implicated in H. pylori extra-digestive diseases can result in production of antibodies to spermatozoal and microbial antigens by activated B-lymphocytes.

Conclusion

In conclusion, the literature data concerning the human populations showed sufficient evidence that ASA impair fertility in couples with unexplained infertility by different pathogenic mechanisms. Among the risk factors for ASA production, the inflammatory diseases with different localization are believed to play an important role. On the other hand, the data concerning the presence of cross-reacting antigens of spermatozoa and Helicobacter pylori, as well as association between H. pylori infection and infertility are rather dim. In the present investigation, high prevalence of serum ASA in patients with H. pylori gastroduodenal diseases compared to healthy controls was detected, and a significant linear correlation between ASA and anti-Hp Abs was found in the group of patients with gastroduodenal disease, suggesting that H. pylori infection may play a role in the induction of ASA. The good reproducibility of the results and the low cost of the methods for the ASA (tray agglutination test of Friberg, microsperm immobilization test of Isojima and Koyama and ELISA against sperm antigens), as well as indirect sandwich ELISA with recombinant antigens for the serum anti–H. pylori IgG antibodies, confirm their appropriate use for the diagnosis of sperm antibodies and anti-Hp Abs in patients with unexplained infertility and with H. pylori gastroduodenal diseases.

Acknowledgments

This study was partly based on ideas originated by the late Prof. Lyudmil S. Nakov, to whom the authors are immensely grateful for his long-standing support and guidance.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Medical University of Sofia [grant number 8/2004]; [grant number 36/2012].