Qualitative differences in sperm antibody responses in mice of different inbred strains and sexes

To better understand the immunogenetic basis and potential pathological consequences of anti-sperm humoral immunity, age-matched female mice of 9 different inbred strains were immunized with syngeneic sperm and were tested for qualitative (specificity) and quantitative (titer) antibody differences by radioimmunoassay, immunofluorescence and immunoblot techniques. All mice developed antisperm antibodies, although titers varied considerably between inbred strains. Antisperm antibodies produced in this study did not cross-react with membrane antigens on thymocytes, brain or immature testicular germ cells. Immunoblot tests identified 17 major sperm antigen bands; this approach also revealed considerable inter- and intra-strain variation in antisperm antibody specificities among female mice. In a parallel study C57BL/6 male mice demonstrated significantly lower antisperm antibody titers and an absence of response to certain sperm antigens in immunoblot tests when compared to age-matched females of the same inbred strain. These findings provide evidence that genetic factors (including sex) interact with environmental (nongenetic) factors in the control of immune responses to sperm antigens.     

Reconstruction of humoral response to sperm antigens in scid mice

OBJECTIVE AND DESIGN: Production of specific human antisperm antibodies by using human-SCID mice model with deposited peripheral blood lymphocytes. MATERIALS AND METHODS: Human peripheral blood lymphocytes (PBL's; CD8(+)-negative cell fraction) were grafted to the peritoneal cavity of severely-combined immunodeficient (SCID) mice at concentration of 20-35 x 10(6) cells per mouse. Lymphocytes were obtained from non-sensitized individual (to sperm antigen) and from in vivo primed males (vasectomized). Two sets of experiments were carried out, with 'native' (glycosylated) and enzymatically deglycosylated sperm antigenic extracts. In all applied variants, sperm antigens were administered with Complete and then with Incomplete Freund adjuvant to improve an immune response. RESULTS AND CONCLUSION: This approach allowed us to obtain better pronounced humoral antisperm response, specific to sperm deglycosylated antigens when PBL's were obtained from individuals in vivo sensitized to sperm (after vasectomy).     

Immunobiological consequence of immunization of female mice with homologous spermatozoa: induction of infertility.

Female Swiss Webster mice were immunized intraperitoneally with mouse epididymal spermatozoa or with phosphate buffered saline (PBS) and their fertility was compared by (1) incidence and size of litters, (2) number of uterine implantation sites, and (3) incidence and number of fertilized eggs in the oviducts. Statistically significant reduction in fertility was noted following two courses of injections of spermatozoa; 12% of mice injected with spermatozoa had litters compared with 80% of mice injected with PBS. The infertility did not seem to be related to a failure in fertilization since the two groups of mice had a similar incidence and number of fertilized eggs in the oviducts. All female mice were found to have a "natural' anti-acrosomal antibody. Following immunization with spermatozoa, antibodies to "postacrosomal' region, the main piece and the midpiece of the tail, as well as cytotoxic antisperm antibodies, appeared. Anti-LDH-X antibody was not detected. However, correlation was not found between infertility and antisperm antibodies or sperm granulomata that developed in the peritoneal cavities. It is concluded that female mice receiving repeated i.p, injections of mouse spermatozoa become infertile and that the infertility is related to interference with events after fertilization.     

Infertility in mice after unilateral vasectomy

The effects of unilateral vasectomy upon fertility and antisperm antibody production were studied using DBA/1J mice. Thirty-six males underwent either unilateral vasectomy, unilateral orchiectomy, or sham surgery. In vivo effects upon fertility were monitored by matings. Antisperm antibody titers were measured monthly. In vitro fertilization was performed in the presence of serum obtained 4 months postoperatively, and serum testosterone levels were also determined. After 3 months, only 1 male in the vasectomy group induced a pregnancy (1 of 12), while all but 1 of the males in the two control groups induced a pregnancy (20 of 21). The geometric mean antisperm antibody titer was 1:169 in the vasectomy group, while the orchiectomy and sham surgery groups had titers of 1:4 and 1:14, respectively (P less than 0.0001). The percentage of eggs fertilized in vitro in the presence of serum from experimental mice fell from 80% in the two control groups to 40% in the unilateral vasectomy group. Unilateral vasectomy induced infertility in DBA/1J mice and an antisperm antibody response. Sera containing these antibodies inhibited in vitro fertilization. This suggests that infertility after unilateral vasectomy may be immunologically mediated by antisperm antibodies.     

Differences in immunogenicity indicating polymorphism of sperm antigens from mice of different inbred strains

Polymorphism of mouse sperm was investigated by analysis of immune sera generated in BALB/c female mice against sperm from 6 inbred strains. The immune sera were analyzed by immunofluorescence and Western blot techniques against sperm antigens from the 6 immunizing strains. Immunofluorescence revealed no differences in reactivity patterns or titers. However, several different reaction patterns were detected by Western blot technique which indicated that both the sperm extracts and the antisperm immune sera contained different components. Syngeneic (anti-BALB/c sperm) antisera showed far fewer reactive antibody species than allogeneic immune sera. The anti-BALB/c sera recognized an antigen of 23 kDa in sperm extracts from DBA/2J and C57BL/6 mice, and failed to react with an antigen of the same molecular weight when applied to sperm from A/J and 129/J mice, indicating antigenic differences between sperm from these inbred strains. Anti-C57BL/6 sera contained a unique antibody which reacted with an antigen of 80 kDa in all of the 6 sperm extracts, whereas others antisera did not detect this antigen. These findings indicate antigenic and immunogenic polymorphism in sperm from different inbred strains of mice.     

Detection and titration of class-specific antisperm antibodies in serum using an enzyme-linked immunosorbent assay

An antisperm antibody enzyme-linked immunosorbent assay (ELISA) that uses whole unfixed sperm and detects immunoglobulin G (IgG) and IgA antibodies in serum was developed. Donor sperm were washed and plated on poly-L-lysine-treated microtiter plates. The patient's sera were diluted to concentrations of 1:4 to 1:256 and incubated with sperm. Positive and negative sera had been previously tested for IgG antisperm antibody activity with a radiolabeled antiglobulin assay. Samples were considered positive when the mean absorbance of triplicate wells was greater than 2 SD above the pooled negative mean. Intra-assay variation was 7.9 and 9.6% for pooled negative and positive controls, respectively. Identical titers of control positive serum were consistently detected. A correlation of 0.83 was observed between ELISA IgG serum titers and radiolabeled antiglobulin results (N = 12). All negative samples tested negative in both assays (N = 21). Some serum samples showed IgA antisperm antibodies. Determination and titration of class-specific antibodies in serum should facilitate initial screening and follow-up of patients at risk for antisperm antibodies.     

Monoclonal antibodies to rabbit sperm autoantigens

Rabbits were immunized with homologous spermatozoa to investigate their autoimmunogenic nature. Major sperm autoantigens that elicit antisperm antibodies were analyzed molecularly by the sodium dodecylsulfate (SDS) gel/protein blot radioimmunobinding method. IgG fractions of the autoimmune sera were purified by a protein A-Sepharose column, immobilized on Sepharose as affinity ligands, and utilized to purify major sperm autoantigens from rabbit testes. The autoimmunogenicity of the purified autoantigens was verified by reimmunizations in rabbits. BALB/c mice were immunized with the affinity-purified autoantigens to raise monoclonal antibodies by modified hybridoma techniques. Following fusions and clonings, we have established more than 100 hybrid cell lines that were shown to secrete antibodies to purified autoantigens and to rabbit sperm. A variety of techniques was employed to characterize these monoclonal antibodies. By the SDS gel/protein blot radioimmunobinding method, some were found to react with unique proteins of rabbit spermatozoa. By indirect immunofluorescent assay, about one third were shown to bind different cytologic regions of spermatozoa from rabbit, man, and/or mouse. Six were found to inhibit rabbit sperm binding to rat ova in vitro. In addition, agglutinating and immobilizing activities of these antibodies on live sperm were observed.     

Oral immunization with sperm antigens: possible therapy for sperm antibodies?

Young adult male CD-1 mice were given intraperitoneal injections (IP) of saline (controls) and pooled sperm or seminal plasma of two autoimmune infertile men and two nonautoimmune fertile men (n = 40 per treatment). Other mice received only an oral challenge with the same antigens (oral controls; n = 20 per treatment). Three weeks after the booster challenge (day 36), 20 mice in each group were orally immunized with the antigens, whereas the other 20 were not (IP controls). Cytotoxic antibody titers (immunoglobulin M) to human sperm were significantly higher in mice IP immunized with sperm or seminal plasma from autoimmune infertile men or orally immunized with autoimmune men's sperm, in contrast to the controls. Oral challenge with sperm or seminal plasma of autoimmune infertile men after the IP immunization with the same resulted in significantly decreased cytotoxic sperm antibody titers (P less than 0.001 versus oral or IP controls in sperm immunization; P less than 0.001 versus IP controls in seminal plasma immunization). Fertility was unaffected by any mode of immunization. It is concluded that, in mice, sperm and seminal plasma antigens from autoimmune infertile men are more immunogenic than those from nonautoimmune fertile men, and oral challenge with the former after an IP establishment of cytotoxic sperm immunity desensitizes the immune mice. These findings may have practical implications in the diagnosis and immunotherapy of infertile men with cytotoxic sperm antibodies.     

A comparison of specific antibody responses in mouse vaginal fluid after immunization by several routes

Mice were immunized with a protein antigen, horse ferritin, by eight different routes and the immune responses in the reproductive tract were compared by measuring specific IgA and IgG in vaginal fluid and by localizing anti-ferritin plasma cells in uterine horns, cervix and vagina. The eight routes of immunization were: subcutaneous with Freund's adjuvant (s.c.), intragastric (i.g.), intravaginal (i.v.), s.c.-i.g., s.c.-i.v., i.g.-i.v., i.v.-i.v. and s.c.-i.g.-i.v. The largest overall response, considering both IgA and IgG antibodies, was obtained by s.c. priming with ferritin in adjuvant followed by i.v. boosting. Intravaginal immunization also boosted priming by the i.g., s.c.-i.g. and i.v. routes, but the response to i.v. immunization alone was weak. All i.v. immunizations stimulated mainly IgA antibody responses in vaginal fluid. Specific plasma cells, mostly of the IgG isotype, were present in the vaginal fornix of several mice in the s.c.-i.v. and s.c.-i.g.-i.v. groups, but none were detected there in any other group and they were only rarely observed in the uterine horns. The results provide data on the relative effectiveness of different routes of immunization in producing a humoral immune response in vaginal fluid against a non-replicating antigen.     

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.     

Relationship between circulating antisperm antibodies in women and autoantibodies on the ejaculated sperm of their partners

The relationship between antibodies on the surface of ejaculated sperm and circulating antibodies in female partners was evaluated. Of 616 couples examined by the immunobead binding test, there was a 12.4% incidence of sperm-surface antibodies in men whose wives had antisperm antibodies in their sera, but only a 6.5% incidence in partners of women who lacked these antibodies (p less than 0.025). Sperm-bound immunoglobulin G and immunoglobulin A both occurred at a significantly higher frequency (p less than 0.05) in partners of women with serum antisperm antibodies. Increased incidence of both immunoglobulin G (p less than 0.01) and immunoglobulin M (p less than 0.005) circulating antisperm antibodies in females were observed when the male partners had antibody-bound sperm. Antibody-coated sperm may activate lymphocytes in the female partners after coitus, thus leading to the production of antisperm antibodies. This may be an additional mechanism that leads to female isoimmunity to sperm and infertility.     

影响不孕妇女IUI治疗成功率的因素分析

目的:探讨影响IUI治疗成功的各种临床因素。方法:回顾性分析260例接受IUI治疗妇女的促排卵方案、子宫内膜厚度、优势卵泡的数目和直径、IUI的时机、输卵管壶腹部的直径、输卵管伞端距宫角的距离、洗涤后精子的密度和精子的动力、IUI的周期数、AsAb与妊娠结局的关系。结果:IUI治疗结局与子宫内膜的厚度、IUI治疗的时机、输卵管壶腹部的直径、输卵管伞端距宫角的距离、洗涤后精子的密度和动力有关(P<0.01,P<0.05)。与促排卵方案、优势卵泡的数目和直径、抗精子抗体无关(P>0.05)。1-3个周期IUI治疗的妊娠率明显高于3个以上治疗周期的妊娠率(P<0.05)。结论:影响IUI结局的主要因素是:子宫内膜的厚度、IUI治疗的时机、输卵管壶腹部的直径、输卵管伞端距宫角的距离、洗涤后精子的密度和动力。延长IUI治疗的周期数,并不能提高病人的成功率。     

Lymphocyte proliferative response to fertilization antigen in patients with antisperm antibodies

The fertilization antigen, immunopurified from human testes, activated lymphocytes from three of the six men and women with antisperm antibodies. Lymphocytes from none of the six men and women without antibodies were activated with fertilization antigen. Another sperm surface antigen, the germ cell antigen, immunopurified from murine testes, did not activate lymphocytes from any of the individuals with or without antisperm antibodies. These results indicate that the men and women with antisperm antibodies are sensitized against fertilization antigen and not germ cell antigen. These results reinforce our previous findings on the involvement of fertilization antigen in immunoinfertility in humans.     

An investigation of allogeneic pregnancy in multiparous mice subjected to in vivo depletion of CD8 (Ly2)-positive lymphocytes by monoclonal antibody treatment

Adult thymectomized C57/Bl (H-2b) and DBA/1 (H-2q) female mice were subjected to treatment with rat anti-mouse CD8 and mouse anti-rat Ig (kappa) prior to entering their third pregnancy with CBA/Ca (H-2k) males. The treatment protocol drastically reduced the number of CD8 (Ly2)-carrying lymphocytes (T-cytotoxic/suppressor phenotype) in the spleen and para-aortic lymph nodes, as assessed by immuno-staining. All mice were investigated on day 18 of their third gestation. The following data were collected from experimental and control groups: (1) resorption frequency, (2) weight of the placenta, fetuses, spleen and para-aortic lymph nodes, (3) immunohistochemical analysis of maternal lymphoid tissues, (4) level of anti-paternal IgG serum antibodies, (5) content of "background" IgM and IgG-secreting cells in spleen and para-aortic lymph nodes. Neither the resorption frequency nor placental/fetal weight was affected by anti-CD8 treatment. However, the formation of anti-paternal antibodies was enhanced in anti-CD8 treated C57/Bl mice.     

Interference of antisperm antibodies with the induction of the acrosome reaction by zona pellucida (ZP) and its relationship with the inhibition of ZP binding

Objective: To determine whether antisperm antibodies can interfere with the induction of the acrosome reaction (AR) by the zona pellucida (ZP) and whether this interference also can occur in the absence of an inhibitory effect on ZP binding.

Design: Prospective in vitro study.

Setting: A tertiary care center, the Andrologic Clinic, University of L'Aquila.

Patient(s): Sera from 12 infertile patients with high titers of circulating antibodies directed against the sperm head were studied.

Intervention: None

Main Outcome Measure(s): The effect of antisperm antibodies on ZP binding was evaluated by matching antibody-exposed and nonexposed donor sperm suspensions labeled with fluorescein or rhodamine, respectively, and incubated with the same salt-stored human ZPs. The effect of antibodies on ZP-induced AR was determined by challenging antibody-exposed and nonexposed donor sperm suspensions with human ZPs disaggregated with acidic NaH₂PO₄. Acrosomal status was evaluated using fluorescein-labeled Pisum sativum agglutinin and supravital stain Hoechst 33258. In some selected cases, the effect of antisperm antibodies on the acrosomal status of sperm bound to intact ZP also was evaluated using transmission electron microscopy.

Result(s): Five of 12 sera exhibited an inhibitory effect on ZP binding. An inhibition of AR induction by disaggregated ZPs (ranging from 64% to 98%) was produced by all 5 sera with an inhibitory effect on ZP binding and by 2 of 7 sera without an inhibitory effect on ZP binding. The different effects of antisperm antibodies on AR induction by disaggregated ZP were confirmed by comparing with ultrastructural evaluations on the acrosomal status of sperm bound to intact ZP.

Conclusion(s): Antisperm antibodies can interfere with the induction of AR by ZP. This inhibition can occur even in the absence of an inhibitory effect on ZP binding. Neither effect may occur.     

Circulating antisperm antibodies in recurrently aborting women

One hundred seventy-three women with a history of three or more recurrent consecutive abortions were analyzed for circulating antisperm antibodies with a radiolabeled antiglobulin assay (RAA), a modified enzyme-linked immunosorbent assay (ELISA), a tray agglutination test (TAT), and a sperm immobilization test (SIT). No pregnancies were subsequently gestated to term in women who were antisperm antibody-positive unless they were inoculated with their husband's leukocytes as treatment for an immune basis (not related to antisperm antibodies) for their recurrent abortions. In women with an immune basis for their recurrent abortions, immunization with leukocytes from their male partners increased the ability of these women previously aborting their fetuses to carry their fetuses to term, even if they had positive results in the ELISA, TAT, and SIT; women with positive results in the RAA continued to abort subsequent pregnancies, despite leukocyte immunization. Immunization of antisperm antibody-positive women with their partner's leukocytes did not incite or increase the antisperm antibody titer, with any of the assay techniques.     

Interrelationships among semen characteristics, antisperm antibodies, and cervical mucus penetration assays in infertile human couples

Semen characteristics, antisperm antibodies, and cervical mucus penetration studies were analyzed in 754 couples and 95 men undergoing infertility evaluation. The means for the different semen/sperm variables were within ranges published for fertile men. Ages of the men ranged from 22 to 55 years and accounted for a small amount of variation. Sperm counts were lowest in September, December, and January, and highest in April, May, October, and November. Of the sperm characteristics, morphology appeared to be associated with the most other variables. Specimens with more than 50% abnormal sperm forms were overall of significantly poorer quality in terms of sperm counts, motility, forward progression, and ability to penetrate cervical mucus. Antisperm antibodies (agglutinating and immobilizing) were detected in the serum samples of 19.0% of the men, 20.4% of the women, and 32.8% of the couples where one or both partners were positive. Agglutinating antibody titers were significantly correlated between partners. Serum titers of antisperm antibodies were associated with decreased sperm counts, motility, forward progression, and normal forms (immobilizing antibodies). Multiple correlation analysis indicated significant independent effects of sperm concentration, motility, forward progression, and antibodies on sperm-cervical mucus penetration scores of the men. In women, cervical mucus penetration was adversely affected by the presence in the serum of sperm agglutinating antibodies and of immobilizing activity in the cervical mucus.     

Suspected ectopic pregnancy: ultrasound findings and hCG levels assessed by an immunofluorometric assay

One hundred suspected ectopic pregnancies were assessed by ultrasound on the basis of the following criteria: (A) viable intrauterine fetus, intrauterine pregnancy is certain; (B) intrauterine double sac or eccentric ring, intrauterine pregnancy is probable; (C) empty uterus or central ring but no adnexal mass or cul-de-sac fluid, ectopic pregnancy is possible; (D) empty uterus or central ring and an adnexal mass or cul-de-sac fluid, ectopic pregnancy is probable; (E) viable ectopic fetus, ectopic pregnancy is certain. Serum human chorionic gonadotrophin (S-hCG) was detected by an immunofluorometric assay (sensitivity 0.2 i.u./l, cut-off level 10 i.u./l). All the 51 patients in groups A and B had an intrauterine pregnancy. Normal gestational sacs were found also at S-hCG levels of less than 3600 i.u./l, the lowest level being 894 i.u./l. Ectopic pregnancy was confirmed in 29 of the 30 women in groups D and E. In the 19 women categorized into group C serial hCG assay and repeated sonography diagnosed ectopic pregnancy in 12 and miscarriage of an intrauterine pregnancy in the other seven. Ectopic pregnancy was always found when no gestational sac was seen by sonography and the hCG level was greater than 1000 i.u./l.     

Sperm washing and intrauterine insemination for cervical factor, oligospermia, immunologic infertility and unexplained infertility

The cumulative pregnancy rate was evaluated for 56 couples undergoing 227 intrauterine inseminations (IUIs) after sperm washing. The indications for IUI were cervical factor, oligospermia, immunologic infertility and unexplained infertility. The overall pregnancy rate was 21.4%, with a 0.289 cumulative probability of conception after six cycles. Within each category the pregnancy rates were 25% for cervical factor, 60% for women with antisperm antibodies, 20% for men with antisperm antibodies and 7% for oligospermia.     

不育或流产患者抗精子及抗附睾的人单克隆抗体制备

我们用ＥＢ病毒转化法从免疫性不育或流产患者的淋巴细胞中制备了一批抗精子和抗附睾的人单克隆抗体。抗精子抗体与全精子、精子头、精子尾、赤道部或核后部反应；抗附睾抗体与附睾基细胞或纤毛区反应。这些来源于不育患者淋巴细胞的抗体具有抗生育效应的可能性较大，对人体具有副作用的可能性相对较小。其中抗附睾基细胞抗体的作用区段与精子在附睾内成熟的区段相一致，这种抗体对基细胞的功能及其在生殖过程中的作用均有特殊的研究价值。