Immunological aspects of male infertility

Some immunological aspects of male infertility are discussed, including the mechanism of induction of auto-antibodies to sperm-specific antigens. Tests to determine antispermatozoal antibodies in serum are discussed. Since there is no direct relation with infertility, more attention is focused on the presence of antispermatozoal antibodies in semen. These antibodies affect male fertility by reducing the capacity of the spermatozoa to penetrate cervical mucus. This penetration inhibition is caused by autoagglutination of the spermatozoa in the ejaculate and by the shaking phenomenon. The sperm-cervical mucus contact test, based on the shaking phenomenon is described. The use of the Mixed Antiglobulin Reaction tests, to detect IgG and IgA antibodies on spermatozoa is discussed. Finally, the effect of antispermatozoal antibodies on the fertilization process is reviewed.     

Cervical Mucus Anti-Sperm Antibodies: Treatment With Intrauterine Insemination

PROBLEM: To determine (1) the incidence of cervical mucus anti-sperm antibodies in infertile women, and (2) the results of treatment by intrauterine insemination. METHOD: Cervical mucus was collected the morning after urinary LH surge occurred from 153 consecutive women being treated for unexplained infertility with intrauterine insemination. Immunobead testing for IgG, IgA, IgA1, and IgA2 was performed with only actively motile sperm being counted. RESULTS: Overall, 23/153 (15.0%) of cervical mucus samples were positive for anti-sperm antibodies: 9/23 (39.1%) were only IgA-positive (62% IgA1-positive, 38% IgA2-positive), 11/23 (47.8%) were only IgG-positive, and 3/23 (13.0%) were positive for both IgA and IgG. Insemination resulted in a pregnancy in 6/23 (26.1%) of women with cervical mucus anti-sperm antibodies after 1–3 cycles. CONCLUSIONS: Testing for cervical mucus anti-sperm antibodies should be performed in cases of “unexplained” infertility, and intrauterine insemination may be an effective treatment, resulting in pregnancies in over one-fourth of couples.     

Mucosal IgG and IgA responses to human papillomavirus type 16 capsid proteins in HPV16-infected women without visible pathology

Human papillomavirus type 16 (HPV16) may infect the cervical epithelium without producing pathological changes for a long time. To investigate if mucosal antibodies are induced in HPV16-infected women without visible pathology, cervical mucus from HPV16-infected patients with and without evident pathology, along with mucus from uninfected women were analyzed for the presence of mucosal IgG and secretory IgA (sIgA) antibodies to HPV16 capsid proteins by ELISA. sIgA and IgG antibodies were found in a significantly higher proportion of infected patients compared with uninfected women (p < 0.0001). sIgA antibodies were present in 13.1% of infected patients without visible pathology, the proportion of positivity increased to 27.0% in patients with visible pathology (p = 0.001). Mucosal IgG response was observed in 6.5% of patients without and 27.5% of patients with visible pathology (p = 0.00005). The antibody mean signal strength was significantly higher in patients with than in patients without pathological evidence (p < 0.005). In conclusion, both sIgA and IgG are found in patients without pathological signs of infection, however, the response increases significantly in patients with pathological evidence, suggesting that the appearance of these changes might be associated with a more vigorous antibody-mediated mucosal reaction.     

Immunoglobulin Class and Regional Specificity of Antispermatozoal Autoantibodies Blocking Cervical Mucus Penetration by Human Spermatozoa

ABSTRACT: The immunoglobulin class and regional specificity of sperm antibodies was analyzed in relation to in vitro cervical mucus penetration using a capillary test. There was a highly significant (X², P < 0.001) association of strong (≥ 95% sperm coated) immunobead test (IBT) results for sperm-bound antibodies of IgA immunoglobulin class with poor (< 3.0 cm/2 h) mucus penetration. There was also a significant (P < 0.01) association between poor penetration and the presence of antibodies on the sperm tail mainpiece. In contrast, IgG class antibodies did not show any significant association with poor penetration. These results confirm and extend previous work and imply that development of a male antispermatozoal vaccine that inhibits mucus penetration must involve stimulation of local IgA response rather than systemic antibody levels.     

Antispermatozoal Antibodies in Human Follicular Fluid

ABSTRACT: The possibility of antispermatozoal antibodies in women having significant effects in the higher regions of the female reproductive tract has been investigated. Follicular fluids (FF) and sera taken at the time of oocyte recovery from women undergoing in vitro fertilisation and embryo transfer (IVF-ET) were tested for the presence of antispermatozoal antibodies, and the concentrations of IgM, IgG, IgA, and complement C3 were determined. The concentrations of immunoglobulins and C3 in FF were consistent with transudation from serum inversely proportional to molecular weight. Titres of agglutinating and immobilising antibodies in FF were usually one or two dilution steps below those of serum except where immobilising activity was associated with IgM. IgG: IgA ratios were lower in FF from women with antispermatozoal antibodies, suggesting local production or enhanced transudation of IgA; however, a secretory component could not be detected in any of the follicular fluids in this study. Two women with antispermatozoal antibodies and infertility in excess of 5 years had successful IVF-ET and have delivered healthy infants.     

Interleukins-6 and −8 Levels in Sera and Cervical Mucus of Fertile,Idiopathic Infertile,and Immunoinfertile Women: Implication in Infertility

PROBLEM: The purpose of this study was to investigate the levels of interleukin-6 and ?8 (IL-6 and IL-8) in sera and cervical mucus of infertile (idiopathic and immunoinfertile) women and to compare the levels with those in age-matched normally cycling fertile women. METHODS: Levels of IL-6 and IL-8 were measured in the sera and cervical mucus of fertile and infertile women by the enzyme-linked immunosorbent assay (ELISA). A non-parametric Mann-Whitney test was used to evaluate significance between the means. Linear regression analysis of IL-6 and IL-8 concentrations in serum versus cervical mucus and with antisperm antibody titers was performed by condition as well as all groups together. P < 0.05 was considered significant. RESULTS: Both IL-6 and IL-8 were detected in sera and cervical mucus of fertile as well as infertile groups. Although serum levels of IL-6 and IL-8 were significantly different between fertile and infertile groups, the differences were more pronounced in the cervical mucus samples. Cervical mucus of idiopathic and immunoinfertile women demonstrated significantly (P = 0.002 to < 0.0001) greater concentrations of IL-6 and IL-8 compared to fertile controls. In general, there was no significant correlation between the serum and cervical mucus IL-6 and IL-8 levels, whether analyzed by condition or all groups together. CONCLUSIONS: Elevated levels of IL-6 and IL-8 in cervical mucus of infertile groups may play a role in etiology of idiopathic and immunologic infertility. These findings suggest that the measurements of cytokines (especially IL-6 and IL-8) in cervical mucus may provide a tool for specific diagnosis and possibly treatment of infertility, especially idiopathic infertility.     

Sperm penetration through cervical mucus in infertile couples in relation to selected cervical factors

One-hundred-and-fourteen women of infertile marriages with negativepost-coital test (PCT) results were studied. The women weredivided into three grou those with anatomical anomalies of thecervix, abnormalities of the cervical mucus and a negative PCIand normal parameters in cervical mucus. Patients were treatedwith mestranol when the physicochemical characteristics of thecervical mucus were impaired. Those with antispermatozoal antibodiesfound by means of the Katsh test were treated with desensitizingtherapy, condoms and homologous artificial insemination (AIH).Among all the patients therapy resulted in 23 pregnancies. Afterapplying these treatments to women, 49 of them (43%) had positivePCT results.     

The treatment of infertility by the high intrauterine insemination of husband's washed spermatozoa

A total of 345 couples with non-tubal infertility on an IVFwaiting list underwent 702 treatment cydes involving daily intrauterineinseminatlons of husband's washed spermatozoa (AIH) over 3 daysof the periovulatory period, following ovarian stimulation.Pregnancy rates achieved were depen dent upon the underlyingInfertility disorder, with similar rates noted in those witha negative post-coltal test (15.8%) or where antispermatozoalantibodies were present in either the male (18.5%) or female(17.1%) partner. These rates were significantly higher thanfor couples with poor cervical mucus (4.7%), asthenozoospermia(0%), endonietriosis (mild, 7.7%; severe, 4.1%) or unexplainedinfertility (8.5%), while discrete oligozoospermia showed mid-rangeresults (10.3%). Preg nancy outcome revealed a high level ofearly wastage (33.3%), mainly in the blighted ovum category,however congenital abnormalities (5.6%) were not significantlyincreased. It is concluded that the procedure of Affi shouldbe considered for infertility due to poor sperm-mucus interaction,antispermatozoal antibodies and simple oligozoospermia, priorto IVF-related treatments.     

Detection of Antispermatozoal Antibodies of IgG,IgA, and IgM Immunoglobulin Classes in Cervical Mucus

ABSTRACT: The immunobead test (IBT) was applied to bromelin-treated cervical mucus (CM) samples from 78 infertile women. Seven (8.9%) of the patients were positive for IgG and/or IgA class antibodies, whilst none (0/35) were found positive for IgM class antibodies. We found that 57% of the positive samples contained IgG and IgA classes, whilst the remaining samples contained IgA alone. The implications of these results were discussed.     

Antisperm antibody production following intrauterine insemination.

Fifty-one infertile women were studied prospectively to assess whether intrauterine insemination (IUI) led to the development of antisperm antibodies in cervical mucus and/or serum. All women were tested for the presence of antisperm antibodies in cervical mucus and serum before and after IUI treatment. Each woman underwent between 1 and 9 cycles of IUI (mean: 4.0 cycles). Five women (9.8%) developed serum antisperm antibodies after IUI treatment. Three of 49 women (6.1%) developed cervical mucus antisperm antibodies and three other women demonstrated disappearance of antibodies following IUI treatment. IUI did not increase mucus or serum antibody titres in women who presented with antisperm antibodies. The number of IUI cycles did not correlate with the development of antisperm antibodies. We conclude that IUI increases the risk of the female partner developing systemic antisperm antibodies but the clinical significance of this finding in unclear.     

Does In Vitro Fertilization (IVF) Influence the Levels of Sperm and Zona Pellucida (ZP) Antibodies in Infertile Women?

The levels of sperm and zona pellucida antibodies in 250 women divided into four groups according to number of recurrent IVF failures (1–4) were analysed and compared with results of a control group of 211 unexplained infertile women never treated by IVF. Sperm antibodies in serum and in ovulatory cervical mucus were determined by mixed antiglobulin reaction (MAR) test, serum zona pellucida antibodies were detected using passive haemagglutination and ELISA. These tests showed increased occurrence of zona pellucida antibodies in women after repeated IVF. Zona pellucida antibodies were found in 20% after one unsuccessful IVF (similarly to 27% in the control group), but in 64% after two, in 91% after three and in 4 of 5 cases after four IVF failures. Sperm IgG, A, M and E antibodies in serum and in ovulatory cervical mucus do not seem to be influenced by IVF procedure. The results show evolution of autoimmune process due to repeated ovarial intervention during oocyte collections. Presence of zona pellucida antibodies, on the other hand, may become a cause of IVF failure.     

Immunological assessment of infertility by estimation of antisperm antibodies in infertile couples.

160 clinical samples were collected from 40 infertile couples with unexplained infertility. The samples collected included serum and seminal plasma of the male partners and serum and cervical mucus samples of the female partners. 25 fertile healthy couples were investigated as controls. All the samples collected were then tested for class-specific antisperm antibodies by an Enzyme linked immunosorbent assay (ELISA). Antisperm antibodies were detected in 30% of the infertile couples which included 25% female and 10% male partners. Amongst the cases positive for antisperm antibodies, antibodies were detected most frequently in female sera 58.4% followed by male sera 33% and 25% in cervical mucus. The isotyping of antisperm antibodies in various samples showed IgG to be the most frequent type specific antibody followed by IgM & IgA types of antibodies. ELISA has provided a relatively simple, reliable and highly reproducible method of detection of antisperm antibodies. Thus application of antisperm antibody testing especially in cervical mucus should become an integral part of the investigation of immunologic infertility.     

Chlamydial serology in 1303 asymptomatic subfertile couples

The clinical significance of antichlamydial antibodies (Chlam Ab) was determined in a total of 1303 subfertile couples consulting for infertility investigation and treatment. Median age of the women was 30 (range 22-44) years and of the men 33 (range 21-53) years. The median duration of infertility was 4 (range 1-21) years. All patients were asymptomatic for genital tract infection. A comprehensive infertility investigation included examination of the endocrine, cervical, and tubal factor, and semen analysis, antisperm antibody (ASA) testing, sperm-mucus interaction testing in vitro using a standardized protocol, and post-coital testing (PCT). Screening for Chlam IgG Ab was performed in serum of both partners, obtained at the same time. Simultaneous microbial cultures in genital secretions of both partners included a broad spectrum of potentially pathogenic bacteria. Elevated titres of Chlam IgG Ab as seromarker for previous infection were found in 20.8% of all women, and in 12.6% of men. Chlam Ab were significantly more frequent in partners of seropositive patients (in 51.8% of women with a Chlam Ab positive partner, compared to 15.8% of the other women). Microbial screening outcome was not significantly related to results of chlamydial serology in both partners. In women, elevated titres of Chlam Ab were significantly associated with a tubal factor, but were not related to reduced quality of the endocervical mucus (CM), including the in-vitro penetrability of the CM (using partners' or donors' spermatozoa). In males, Chlam Ab were not significantly related to the outcome of semen analysis, including screening for ASA (IgG and/or IgA) in semen, and several parameters of sperm functional capacity. After exclusion of couples with tubal disease, subsequent male fertility did not significantly differ in males with or without Chlam Ab. The results suggest that during basic infertility investigation, positive chlamydial serology as an easy screening procedure indicates a higher risk for a tubal infertility factor. However, in asymptomatic patients, Chlam IgG Ab in serum are not associated with a cervical factor or with the male factor, using several determinants for evaluation of semen quality including subsequent fertilizing capacity.      

Induction of the Shaking Phenomenon by IgA Class Antispermatozoal Antibodies From Serum

ABSTRACT: Serum was obtained from a woman with a high (> 1000) titre of antispermatozoal antibody of IgA immunoglobulin class determined by the indirect immunobead test (IBT). It was found that the serum could sensitize normal sperm to give a strong shaking reaction in the semen-cervical mucus contact test (SCMCT). The capacity to induce shaking was retained after absorption of the serum with protein A-sepharose, but lost after absorption with rabbit antihuman IgA. The results therefore suggest that antispermatozoal antibodies of IgA class from serum can induce shaking, and are consistent with the possibility that the Fc fragment (rather than secretory component or J-chain) may be important for induction of shaking in the conventional SCMCT.     

Allergic infertility: laboratory techniques to detect antispermatozoal antibodies

Twenty sera from fertile and infertile women were examined for antibodies to spermatozoa or to seminal plasma, to determine which of the five techniques tested provided reliable evidence of allergic infertility. Ten sera agglutinated seminal plasma coated tanned red cells, six agglutinated spermatozoa, ten gave mixed cell antiglobulin agglutination and fourteen gave immunofluorescent reactions for IgG or igM antispermatozoal antibodies, and three were cytotoxic to sperm. The results of tests for seminal plasma agglutinins were unrelated to those of any of the other tests, and it is believed that factors other than antibody also clump these antigen-coated cells. There was good agreement in the results obtained by direct sperm agglutination, mixed cell antiglobulin agglutination and strong immunofluorescent staining of the sperm head. The antiglobulin and immunofluorescent techniques also detect nonagglutinating (‘incomplete’ or ‘univalent’) antibody. One of the three sera cytotoxic to sperm had no antibody detected by the other tests. The sperm may have been killed by an inflammatory substance or a systemically absorbed contraceptive. This case illustrates the necessity of combining tests for cytoxicity with at least one technique for antispermatozoal antibody in order to detect allergic infertility.     

The prevalence of abnormal cervical cytology in women with infertility

The aim of this study was to compare the frequency of abnormal cervical cytology in women with infertility problems with that of fertile women by using ThinPrep® liquid‐based Pap Tests™. A retrospective case–control study for over 2 years was conducted. The cases included all women with infertility problems who had Pap tests during their infertility treatment period. The cases were further subdivided into primary and secondary infertility groups. The control group included all women without infertility problems who had routine Pap tests in the same period. The age and demographic features were adjusted and matched for both groups. Statistical analysis included chi‐square test and Fischer exact test. The infertility group (n = 490) showed significantly (P < 0.05) more abnormal cervical squamous epithelial abnormalities (48 positive cases, 9.8%) than the controls (n = 7,150, 216 positive cases, 3%). Women with secondary infertility had more epithelial abnormalities and more high‐grade lesions than women with primary infertility. Women with infertility had statistically significant higher frequency of squamous intraepithelial lesions than women without infertility problems of similar age and demographic background. The data suggest that women with infertility might benefit from more frequent cervical cytology screening. Diagn. Cytopathol. 2010;38:791–794. © 2009 Wiley‐Liss, Inc.     

Immunoglobulin Class of Sperm Antibodies in Cervical Mucus From Infertile Women

ABSTRACT: By immunoaffinity chromatography using anti-IgG, anti-IgM, and anti-IgA coupled to CNBr-activated sepharose 4B, the immunoglobulin class of sperm-agglutinating antibodies was investigated in cervical mucus from four infertile women. In all patients, it was found that the sperm antibodies in cervical mucus belonged to the IgA class, whereas in serum, which was studied in two of the patients, IgG sperm antibodies were demonstrated. Absorption of the four cervical mucus samples with anti-secretory component sepharose 4B revealed that the IgA antibodies in at least two of the samples were SC-IgA antibodies. Investigation of a third sample by sucrose gradient ultracentrifugation revealed that the IgA sperm antibodies were characterized by a sedimentation coefficient between 9S and 13S, strongly suggesting the presence of SC-IgA antibodies. Accordingly, the sperm-agglutinating antibodies were SC-IgA antibodies in at least three of the four samples studied.     

In-vitro cervical mucus -- sperm penetration tests and outcome of infertility treatments in couples with repeatedly negative post-coital tests

The results of in-vitro cervical mucus—sperm penetrationtests and cross-hostility tests in 178 couples with repeatedlynegative post-coital tests were recorded. Using a protocol ofthree cycles with intra-uterine inseminations (IUI) followedby three cycles with ovulation induction + IUI, the associationbetween the cause of infertility, results of the in-vitro testsand the outcome of infertility treatment was investigated. Wefound that repeatedly negative post-coital tests are a goodindicator of a cervical mucus — sperm penetration problem.The crosshostility test clearly differentiates the abnormalfactor in this interaction, and a good performance of the donors'spermatozoa in the cervical mucus correlates with increasedpregnancy rate. In male factor infertility, failure of the husbands'spermatozoa to penetrate cervical mucus was not indicative ofa deficient fertilization potential in vivo. In these patientsa serious attempt should therefore be made to reverse the infertilityby treatment with IUI or ovulation induction + IUI before attemptingassisted reproduction. Women with polycystic ovaries and repeatedlynegative post-coital tests should be investigated for spermreceptivity of the cervical mucus. Low receptivity of the cervicalmucus may imply that endometrial receptivity and oocyte qualityare also low. Ovulation induction and not IUI alone should thereforebe used as the preferred mode of treatment to improve pregnancyrate.     

Diagnosis and follow-up of genital chlamydial infection by direct methods and by detection of serum IgG, IgA and secretory IgA

Purpose: To determine the prevalence of Chlamydia trachomatis infection in a high-risk population by direct and indirect methods and to evaluate the diagnosis of secretory immunoglobulin A (sIgA). Patients and Methods: Urethral or endocervical specimens from 78 patients (48 females and 30 males) were examined by cell culture, direct fluorescence assay, PCR Cobas Amplicor (Roche Molecular Diagnostics), and sIgA was detected by the recombinant lipopolysaccharide (LPS)-enzyme-linked immunoassay (rELISA). Serum from each patient was also obtained and analysed for the presence of IgG and IgA antibody by in-house microimmunofluorescence (MIF) and by the rELISA method (Medac, Hamburg, Germany). Results: The overall C. trachomatis prevalence determined by direct methods was 28%. The detection of sIgA antibodies was significantly higher in the group of patients with a positive direct detection (50%) than in the group of negative direct detection (10.7%). The Chlamydia-specific IgA antibodies were detected by the rELISA in 40.9 and 53.6% of group I (positive direct detection) and group II patients (negative direct detection), respectively. The species-specific IgA antibodies were detected by the MIF method in 18.2 and 16.1% of group I and II patients, respectively. Chlamydia genus-specific IgG antibodies were detected by the rELISA in 86.4 and 83.9% of group I and group II patients and, C. trachomatis specific IgG were present in 81.8 and 73.2% of group I and group II patients, respectively, as assessed by the MIF test. Conclusion: Combining the positive direct methods and/or positive sIgA antibody results from cervical or urethral specimens had an indication of current C. trachomatis infection.     

Viral proteins VP2, VP6, and NSP2 are strongly precipitated by serum and fecal antibodies from children with rotavirus symptomatic infection

Rotavirus-specific IgA has been correlated with immune protection against rotavirus reinfection and symptomatic disease. Systemic and mucosal antibody responses were determined by an enzyme-linked immunosorbent assay in 11 infants with severe rotavirus gastroenteritis. Geometric mean titers of antirotavirus serum IgG and IgA antibodies were significantly higher during the convalescence of the disease (P < 0.001 vs. acute-phase titers). Rotavirus-specific fecal sIgA antibodies increased 4 times during the convalescence in 9 (81.8%) children (P < 0.001). The serum IgG and IgA antibody and fecal sIgA antibody responses to individual rotavirus polypeptides were characterized by radioimmunoprecipitation assay (RIPA) using Staphylococcus aureus protein A and the lectin jacalin to precipitate IgG- and IgA-immune complexes, respectively. The main IgG response was directed toward the structural viral proteins VP2, VP4, and VP6 and toward the nonstructural protein NSP2. Serum IgA reactivity was detected by RIPA in all serum samples, with major responses to VP2, VP6, and NSP2. Interestingly, fecal sIgA in convalescent samples reacted strongly toward NSP2 and VP6. These data reinforce the antigenic importance of rotaviral proteins other than VP4 and VP7, such as VP2, VP6, and NSP2, as main targets in the immune response to rotavirus. J. Med. Virol. 56:58–65, 1998. © 1998 Wiley-Liss, Inc.