Antisperm antibodies in infertile and homosexual men: relationship to serologic and clinical findings

The incidence and significance of antisperm antibodies in different groups of men were evaluated by a modified enzyme-linked immunosorbent assay. In serum, 4.0% of dermatologic patients (n = 223), 9.6% of andrologic patients (n = 178), and 28.6% of homosexual men (n = 42) were positive for IgG and/or IgM antibodies. In seminal fluids, 7.3% of the andrologic patients had IgA (and IgG) antibodies to spermatozoa. Only 1 of 29 positive men had antibodies both in serum and in seminal fluid. No correlation between antisperm antibodies and IgG/IgM concentrations was found in serum, whereas in seminal plasma men with antisperm antibodies showed higher IgG/IgA concentrations than men without (IgA, 3.2 versus 1.7 mg/dl; IgG, 9.8 versus 6.3 mg/dl). It is concluded that there is a high incidence of antisperm antibodies among homosexual men, probably because of contact of spermatozoa with the immune system by passive anal intercourse. There is little correlation between antisperm antibodies in serum and seminal plasma of infertile men because of a lack of relevant antibody transfer from the serum and the formation of local antibodies in seminal plasma. Antisperm antibodies in seminal fluid are associated with elevated local IgG and IgA concentrations.     

用ELISA法对不明原因不育夫妇血清和生殖道分泌液中抗精子抗体的研究

本文采用ELISA间接法对30对不明原因的不育夫妇血清及生殖道分泌液中ASA的阳性率、种类(IgG、IgM、IgA)及分布情况进行了研究。男性病人组血清中ASA阳性率为13.3%,精浆中为16.7%,和对照组相比无统计学意义。女性病人组血清中ASA阳性率为23.3%,宫颈粘液中为33.3%,和对照组相比均有显著差异。在不明原因的不育夫妇中,ASA主要发生在女方。检测结果表明,血清、精浆和宫颈粘液中均存在有IgG、IgM、IgA类ASA。但血清中主要为IgG、IgM类ASA,精浆及宫颈粘液中主要为IgA、IgG类ASA。ASA可同时存在于血清或生殖道分泌液中,也可单独存在于血清或生殖道分泌液中。     

精浆内抗精子抗体和免疫复合物的研究——对男性不育患者及输精管粘堵术者的观察

本文用ELISA法研究了174名男性不育患者及43名输精管粘堵术后精浆中的抗精子抗体及其种类。结果表明,精浆中抗精子抗体的检出率,在男性不育患者中为24.1%,输精管粘堵术后者为48.8%。在这些患者的精浆中可检出IgA、IgG和IgM型抗精子抗体,其中主要是IgA。同组人群血清和精浆中的抗精子抗体缺乏相关性。精浆中抗精子抗体的存在与精子活动率的下降有一定的关系。应用PEG法测定精浆中免疫复合物,在66例精子抗体阳性的患者中,阳性率为56.1%,表明局部抗精子的自身免疫反应,在一定条件下可导致免疫复合物的形成。     

Antisperm antibodies and fertility after vasovasostomy: a follow-up study of 216 men

A group of 216 vasovasostomized men were tested with the mixed antiglobulin reaction for immunoglobulin (Ig)G, IgA, and secretory IgA antisperm antibodies bound to the sperm membrane. Free antisperm antibodies in serum and seminal plasma were detected with the gelatin agglutination test and the tray agglutination test. The results were related to the conception rates. In a subgroup with a pure IgG response, the conception rate reached 85.7%, whereas only 42.9% of the men who also had IgA on the sperm induced pregnancy. When 100% of the spermatozoa were covered with IgA, the conception rate was reduced to 21.7%. The combination of IgA on all sperm and a strong immune response (titer in serum greater than or equal to 256) was associated with a conception rate of zero.     

Effect of sperm antibodies on pregnancy outcome in a subfertile population

The relationship between sperm antibodies, conception, and miscarriage was examined in 109 infertile couples. Antibodies present on the surface of husbands' ejaculated sperm and antibodies in husbands' or wives' sera that reacted with a purified population of the husbands' motile spermatozoa were detected by an enzyme-linked immunosorbent assay. During an 18-month period, conception occurred in 33 (30.3%) of the couples; 16 (14.7%) women subsequently suffered a spontaneous miscarriage during the first trimester, whereas 17 (15.6%) women maintained their pregnancies past this time period. Antisperm antibodies were present in sera from only two of 17 (11.8%) women with successful pregnancies, whereas seven of 16 (43.8%) women who miscarried and 29 of 76 (38.2%) who did not conceive had these antibodies in their sera. IgG (22.4%) and IgM (21.1%) antisperm antibodies predominated in sera of women who did not conceive, whereas IgA (37.5%) and IgG (37.5%) antibodies were most prevalent in sera of women with miscarriages. In men, the presence of antisperm antibodies in sera was unrelated to fertility. However, there was a correlation between sperm surface antibodies and an inability to conceive. IgG was identified on ejaculated spermatozoa from eight of 76 (10.5%) men whose wives failed to conceive and in none of 33 men whose wives conceived. Similarly, IgA was present on spermatozoa from 16 (21.1%) infertile and two (6.1%) fertile men. Thus antisperm antibodies in female sera and on ejaculated spermatozoa were associated with a failure to conceive and first-trimester miscarriage.     

Demonstration of antispermatozoal antibodies in varicocele-related infertility with an enzyme-linked immunosorbent assay (ELISA)

To assess the existence of a possible immunologic factor in varicocele-associated infertility, we searched for antispermatozoal antibodies in serum, seminal plasma, and bound to spermatozoa in 32 infertile men with varicocele and 22 infertile patients without palpable varicocele, with the use of an enzyme-linked immunosorbent assay. In addition, we performed morphologic and microbiologic analyses of the semen and urethral smears for isolation of Chlamydia trachomatis. Twenty-nine men from the varicocele group (90.6%) demonstrated antispermatozoal antibodies, compared with only 9 men (40.9%) in the control group. The antibodies in both groups, when present, were mainly serum and seminal plasma immunoglobulins IgA and IgM. A significant quantitative difference between the varicocele and control groups was also observed for serum IgA, seminal plasma IgA and IgM, and sperm-bound IgG, IgA, and IgM. Oligozoospermia and asthenozoospermia were significantly more prevalent in the varicocele men. An asymptomatic genital tract infection with C. trachomatis, Ureaplasma urealyticum, and Escherichia coli was traced in 40.6% of the varicocele men and in 45.5% of the control group. No interaction could be demonstrated between the infection and antispermatozoal antibody formation. These data suggest that an immunologic factor may play a role in varicocele-associated infertility; however, its impact on reproduction has yet to be assessed.     

A reverse (antibody capture) enzyme-linked immunosorbent assay for detection of antisperm antibodies in sera and genital tract secretions

A reverse (antibody capture) enzyme-linked immunosorbent assay (ELISA) for detection of antisperm antibodies has been developed. The assay enables detection of immunoglobulin (Ig) M, IgG, IgA, or IgM, IgG, and IgA--antisperm antibodies in serum, cervical mucus, and seminal plasma samples. The reverse ELISA is more specific and sensitive than conventional ELISA in detecting human antisperm antibodies of different isotypes. Using this assay, statistically significant differences in levels of antibodies between infertile and fertile individuals were demonstrated in sera and in genital tract secretions. Studies with 143 infertile couples revealed that the presence of antibodies in sera was not necessarily reflected in individual's genital tract secretion and vice versa. These data emphasize the importance of detecting antisperm antibodies in sera as well as in genital tract secretions for correct evaluation of sperm immunity.     

Changes in sperm antibody test results when spermatozoa are subjected to capacitating conditions

Cytotoxic and immunobead-binding antibody assays were used to document possible changes in antigenicity after the spermatozoa were subjected in vitro to capacitating conditions. Serum and seminal plasma immunobead-binding immunoglobulin (Ig)A, sperm surface IgG, and IgA, and seminal plasma cytotoxic antibody titers in 26 autoimmune infertile men were decreased against autologous spermatozoa under capacitating conditions. Cytotoxic antibodies and immunobead-binding IgG, IgA, and IgM against husbands' sperm undergoing capacitation, in the cervical mucus of these women, were less than those against uncapacitated sperm. Conversely, sperm antibody levels were increased against the capacitated husbands' sperm in the husbands' serum and wives' cervical mucus, originally testing negative for cytotoxic antibodies against uncapacitated sperm. Antibody levels were unaltered against donors' sperm undergoing capacitation, except for a reduction in titers in the serum of isoimmune wives. The differences in sperm antibody assay results, depending on whether or not spermatozoa are incubated under conditions favoring capacitation, are specific to the husbands' and not to the donors' spermatozoal antigens.     

Detection of autoimmunity to sperm: mixed antiglobulin reaction (MAR) test or sperm agglutination? A study on 537 men from infertile couples

Two different ways of testing for antisperm antibodies were compared: the mixed antiglobulin reaction (MAR) test for demonstration of antibodies of the IgG and IgA classes bound in vivo to the sperm membrane antigens and the gelatin agglutination test for detection of nonbound antisperm antibodies in serum and seminal plasma. Samples from 537 men from infertile couples were investigated. Antibodies bound to the sperm membrane were detected in 49 men (9.1%), IgG in 44 (8.2%), and IgA in 38 cases (7.1%). Sperm agglutinins were recorded in seminal plasma from 30 men (5.6%) and in serum (titer greater than or equal to 16) from 43 men (8.0%). The investigation revealed a very close correlation between the results of MAR testing and the occurrence of sperm agglutinins in serum and seminal plasma. However, if one focuses on antisperm antibodies of the IgA class, which seem to play the major role in male immune infertility, the MAR test offered the advantage that a minor group of patients with pure IgG responses could be distinguished, and rare cases with mainly or exclusively locally produced IgA antibodies could be detected.     

Treatment of infertility caused by antisperm antibodies.

Twenty infertile couples with antisperm antibodies in the male or in the female partner were scheduled for treatment. In 15 couples the male and in 5 couples the female partner was the antisperm antibody carrier. In all the couples the result of the in vivo and in vitro sperm-penetration test was negative or poor. The SCMC-test was strongly positive in each of the couples. In all the couples IgG and IgA antisperm antibodies could be demonstrated on the spermatozoa or in the cervical mucus. It was postulated that antisperm IgA and not antisperm IgG is responsible for the penetration reduction of spermatozoa in cervical mucus and for the "shaking phenomenon" in the SCMC-test. Intrauterine inseminations, performed in 20 couples, resulted in four pregnancies. Condom therapy in three couples, for at lest 6 months, had no result. Two men were treated with 96 mg methylprednisolone per day for 7 days; this resulted in a slight decrease of the sperm-agglutination titre, but no pregnancy occurred.     

Comparison of mixed antiglobulin reaction and direct immunobead test for detection of sperm-bound antibodies in subfertile males.

OBJECTIVE: To compare two methods of detection of surface bound antibodies on spermatozoa from subfertile males. DESIGN: Prospective comparison of direct mixed antiglobulin reaction (MAR) for immunoglobulin (Ig)G with direct immunobead test (IgG and IgA) applied to spermatozoa from male partners of infertile couples. Circulating unbound antibody measured by tray agglutination test in serum and seminal plasma in a representative proportion. SETTING: Seminology laboratory. PATIENTS: One hundred nine male partners of infertile couples. RESULTS: Highly significant correlation between direct MAR (IgG) and direct immunobed test (IgG) and between both of these tests and serum unbound antibody measured by tray agglutination test. Highly significant correlation between direct immunobead test (IgA) and seminal plasma unbound antibody measured by tray agglutination test, but no correlation with MAR (IgG). CONCLUSIONS: Mixed antiglobulin reaction (IgG) is a cheap, quick, and sensitive screening test, but immunobead test (IgA) provides useful additional information on class of antibody on spermatozoa that may be clinically more important.     

Vasectomy: consequences of autoimmunity to sperm antigens.

Data from studies examining the effects of vasectomy in a large number of nonhuman primates vasectomized for periods ranging up to 14 years are summarized, and these findings and speculations are used as a framework with which to review the subject of autoimmunity and vasectomy. Attention is directed to autoimmunity to sperm antigens following vasectomy (factors affecting antisperm antibody levels, characteristics of circulating antisperm antibodies, antisperm antibodies in seminal plasma, and cellular immunity following vasectomy), and immunopathology of antisperm autoimmunity (local effects on the male reproductive tract and systemic effects on the male reproductive tract). The 6 hypotheses that have been advanced to explain individual variations in dynamics and types of antisperm antibodies produced following vasectomy are reviewed. 3 tests are commonly used to detect free antisperm antibodies after vasectomy: 1) the spermagglutination test; 2) the sperm immobilization test; and 3) the immunofluorescence test. Spermagglutinating (SA) antibodies, the most common type of antibody produced after vasectomy, occur in approximately 2/3 of vasectomized men and in a majority of vasectomized rhesus monkeys. Sperm-immobilizing (SI) antibodies are also produced in a large percentage (40%) of vasectomized men and rhesus monkeys. About 30% of vasectomized men also have antiprotamine antibodies.     

A radiolabeled antiglobulin assay to identify human cervical mucus immunoglobulin (Ig) A and IgG antisperm antibodies

Antisperm immunoglobulin (Ig) A and IgG antibodies in human cervical mucus (CM) were identified by a radiolabeled antiglobulin assay. Cervical mucus samples from fertile and infertile women were exposed to a 1:3,200 dilution of 2-mercaptoethanol (2-ME), and 5 micrograms of the solubilized CM protein were assayed for the presence of IgA and IgG antisperm and anti-Candida activity by the radiolabeled antiglobulin assay. Purified human secretory IgA and IgG exposed to 2-ME retained the molecular integrity and functional activity of the untreated antibody molecules. CM aliquots collected after high-performance liquid chromatography (HPLC) fractionation were assessed for antisperm antibody activity; antisperm antibody activity was retained in the appropriate IgA or IgG CM fractions. The incidence of CM antisperm antibodies was minimally affected when the radiolabeled antiglobulin assay was performed with a motile sperm population. Approximately 70% of the CM IgA antisperm antibodies were of the IgA1 subclass; CM IgG was primarily of the IgG4 subclass. When Candida antigen was substituted for sperm in the radiolabeled antiglobulin assay, the CM antisperm antibodies were found to be exclusively sperm-specific. These data indicate that the radiolabeled antiglobulin assay using 2-ME to extract CM antibodies is a specific method for the assay of antisperm antibodies in CM.     

ELISA法检测不育男子精浆中抗精子IgG和IgA

将精子经Tritonx－100处理，冷冻高速离心后，经抗人全血清-SephadexG－75亲和柱层析分离，提取分子量为59KD人精子膜蛋白作为抗原，经ELISA间接法对20例生育男子和50例不育男子精浆中抗精子IgG和IgA进行了测定。结果显示：生育男子精浆中抗精子IgG为阴性；抗精子IgA阳性率为5％。不育男子精浆中抗精子IgG和IgA阳性率分别为10％、30％；抗精子IgG和IgA均为阳性者3例，均为阴性者33例；抗精子IgG阳性而IgA阴性者2例；抗精子IgA阳性而IgG阴性者12例。不育组与生育组间抗精子IgG阳性率无显著性差异（P＞0．05）；而IgA阳性率间则有显著性差异（P＜0．05）。不育组抗精子IgG和IgA阳性率间有极显著性差异（P＜0．01）。本文利用ELISA法对精浆中抗精子抗体的分类及可能来源进行了讨论。     

Assessment by fluorescence-activated cell sorting of whether sperm-associated immunoglobulin (Ig)G and IgA occur on the same sperm population.

Two-color fluorescence-activated cell sorting of antisperm antibody-positive sperm was used to detect simultaneously the presence of immunoglobulin (Ig)A and IgG antisperm antibodies associated in vivo on a man's sperm. Sperm positive for sperm-associated Ig were analyzed using phycoerythrin-conjugated antihuman IgA and fluorescein isothiocyanate-conjugated antihuman IgG; up to 87% of the same spermatozoa were stained with both labels. Sperm positive for only one of the antisperm antibody isotypes stained up to 90% of a man's sperm with only one fluorochrome. Immunocytochemistry studies revealed similar patterns of sperm binding for sperm-associated IgG and IgA. These results suggest that the sperm antigenic determinants reacting with antisperm IgA and IgG are present on the same sperm population at similar locations on the sperm surface.     

Occurrence of serum antisperm antibodies in patients with cystic fibrosis

OBJECTIVE: To determine if acquired obstruction of the vas deferens in men with cystic fibrosis (CF) induced the development of antisperm antibodies with genital tract obstruction similar to other men. DESIGN: Serum antisperm antibodies were assayed by an indirect immunobead test and an indirect immunofluorescence assay. Both homologous (human sperm/human zona) and heterologous (human sperm/zona-free hamster ova) sperm/egg interactions were evaluated in the presence of serum antisperm antibodies from patients with CF. SETTING: Cystic Fibrosis Clinic at the University of Oklahoma Health Sciences Center, a tertiary care referral center. PATIENTS: Fifteen CF patients (10 male and 5 female), 3 non-CF antisperm antibody-positive infertile patients (2 male and 1 female), 20 fertile controls (7 males and 13 females), and 9 fertile sperm donors were used. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Serum antisperm antibody levels in patients with CF. In those patients with antisperm antibodies, determine effect of these sperm antibodies on sperm/egg interactions and complement-mediated events. RESULTS: Sera from 3 (30%) of 10 men with CF demonstrated immunoglobulin (Ig)G, IgA, and/or IgM antisperm antibodies, whereas sera from all 5 CF women and the 20 control sera were negative for antisperm antibodies. The maximal titers for IgG, IgA, and IgM antisperm antibody were 1:8, 192, 1:256, and 1:64, respectively. The immunobead binding, which was restricted to the sperm head and tail-tip or the midpiece and tail-tip, correlated with the indirect immunofluorescence pattern. Antisperm antibody-positive sera from men with CF impaired both the binding and penetration of human zonae and the penetration of hamster ova by human sperm. CONCLUSIONS: Similar to other men with congenital or acquired obstruction of their genital tract, antisperm antibodies may occur in some men with CF. Antisperm antibodies may contribute to immune sperm dysfunction in some men with CF by activated complement-mediated events and interfering with sperm/egg interactions.     

Effect of antisperm antibodies in males on in vitro fertilization

To investigate the effect of antisperm autoantibodies on the process of in vitro fertilization, sperm immobilization test and sperm agglutination test with serum, and direct immunobead test with semen were conducted on the male partners of 101 infertile couples. Fourteen patients were positive in at least one of these three antisperm antibody tests. Six of them were associated with a low fertilization rate (= LFR group, less than 50% of mature oocytes were fertilized), while the other 8 patients had a normal fertilization rate (= NFR group). The spermatozoa in the LFR group were bound to immunobeads of both IgG and IgA classes. In contrast, the spermatozoa in the NFR group were not bound to IgA. The results of the tests with serum were not related to the outcome of fertilization. By rapid dilution and washing of the semen containing antisperm autoantibodies, the percentage of spermatozoa bound to IgG decreased significantly. However, the rate of fertilization was not improved. Six pregnancies were achieved after 13 embryo transfers in the 14 patients. We conclude that the fertilization rate is reduced when spermatozoa are bound to both IgG and IgA antisperm autoantibodies, or to IgA alone. Antisperm autoantibodies do not seem to hamper embryonal development and implantation.     

用免疫珠试验检测不育者抗精子抗体

应用免疫珠试验(Immunobead test,IBT)检测251例不育者血清抗精子抗体及51例不孕妇女宫颈粘液抗精子抗体,将IBT与明胶凝集试验(Gelatin agglutination test,GAT)试管玻片凝集试验(Tube-slide agglutination test,TSAT)及精子制动试验(Spermimmobilization test,SIT)比较。结果表明,抗精子抗体是不育原因之一,宫颈粘液与血清中抗精子抗体的存在不一致,IBT的敏感性、特异性及重复性较好,且能作抗体分类和定位,对临床诊治免疫不育有重要指导意义。     

Influence of antisperm antibodies on the acrosome reaction as determined by flow cytometry

OBJECTIVE: To evaluate the influence of antisperm antibodies on the acrosome reaction (AR). DESIGN: Clinical study. SETTING: University of Marburg, Department of Andrology, Clinical Training Center of the European Academy of Andrology. PATIENT(S): Spermatozoa from a pool of healthy donors were incubated with 30 seminal plasma samples from infertile men containing antisperm antibodies; they were compared to a control group of 10 samples without antisperm antibodies and five samples with buffer only. INTERVENTION(S): The spontaneous acrosome reaction (SAR) and the induced acrosome reaction (IAR) by calcium ionophore A23187 were observed and determined by means of a flow cytometer. Flow cytometric double-staining estimates of acrosomal integrity were determined by using a monoclonal antibody (TUS 19), marked with a secondary fluorescein isothiocyanate-labeled antibody. Cell viability was determined by counterstaining with propidium iodide (PI). MAIN OUTCOME MEASURE(S): Number of acrosome reacted spermatozoa. RESULT(S): The spermatozoa treated with antisperm antibodies showed significantly higher SAR and IAR responses than the control group. CONCLUSION(S): Some of the antisperm antibodies from individual patients are able to enhance the acrosome reaction in donor sperm, but none of them appeared to inhibit acrosome reaction.     

In vitro immune absorption of antisperm antibodies with immunobead-rise, immunomagnetic, and immunocolumn separation techniques

Fourteen men with a mean duration of infertility greater than 3 years who had significant sperm immobilizing or sperm-agglutinating antibodies were studied. All patients had greater than 20% IgG or IgA immunobinding to sperm in their seminal plasma and 7 had immunobinding levels of greater than 50%. Sperm from these men were less able to penetrate an overlaying buffer layer than sperm from a fertile control. Addition of immunobeads to the specimen was of little use, because few motile sperm could swim into the overlaying buffer; retained immunobeads were noted in the buffer layer of 18-hour capacitated specimens. Magnetic isolation of antibody-coated sperm from antibody-free sperm avoids potential damage to fragile sperm through centrifugation. Viable spermatozoa were isolated from magnetite-complexed spermatozoa, but the motility of the isolated spermatozoa deteriorated rapidly during the subsequent capacitation period. Passage of diluted ejaculate through a column of dextran beads for antisperm antibody processing (ASAP) was associated with superior sperm quality and fertilizing potential. The use of ASAP resulted in good sperm velocity and linearity and improved sperm function, as measured with the hamster egg penetration test. Sperm from men with immunologically mediated infertility can be processed through the ASAP and used for artificial insemination of their partners or in an in vitro fertilization program.