Effects of Sperm-Reactive Monoclonal Antibodies on the Cervical Mucus Penetrating Ability of Human Spermatozoa

ABSTRACT: A group of monoclonal antibodies (Mabs) was tested by immunobead binding and indirect immunofluorescence to determine its reactivity with antigenic moieties present either on the surface of motile human spermatozoa or internally. Those Mabs reactive with the surface of motile spermatozoa impaired their ability to penetrate cervical mucus in vitro, while Mabs solely reactive with freeze-thawed, nonmotile sperm (presumptive subsurface epitopes) were without effect on sperm function.     

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.     

Characterization of Antibodies Generated Against a Conserved Portion of Oviductal Glycoprotein (OGP) and Endogenous Hamster OGP and Their Ability to Decrease Sperm Binding to the Zona Pellucida In Vitro

PROBLEM: The effect of antibodies generated against hamster oviductal glycoprotein (OGP) on sperm binding to the zona pellucida (ZP) was evaluated. METHOD OF STUDY: Antibodies against a 17-amino-acid sequence of the OGP core protein (amino acids 52–68) and the denatured hamster OGP protein were generated, characterized, and tested in an in vitro sperm binding assay. RESULTS: Sperm binding was significantly decreased (P < 0.05) when oviductal oocytes were incubated for 2 hr with 4 or 8 mg/ml of immune IgG of both antibodies when compared with normal rabbit IgG. A fluorescence assay showed binding of both antibodies to the endogenous OGP associated with the ZP of ovulated hamster oocytes. CONCLUSIONS: These results suggest that OGP may be a potential immunocontraceptive target because both antibodies significantly decreased sperm binding to the ZP of oviductal oocytes. Immunocontraception may be accomplished by attempting to generate active immunity to a recombinant OGP, to the region selected in this study (amino acids 52–68) or to some other region of the core protein.     

Identification of Epitopes of Monoclonal Antibodies to Porcine Zona Pellucida 3β Glycoprotein,a Homologue of the Mouse/Human Sperm Receptor

PROBLEM: Immunization with zona pellucida (ZP) glycoproteins leads to a block in fertility with a variable degree of ovarian dysfunction. To avoid autoimmmune oophoritis, synthetic peptides corresponding to B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. The main objective of the present study is to define the epitopes recognized by monoclonal antibodies (mAbs) generated against porcine ZP3β, a homologue of the designated primary sperm receptor in mice and humans. METHODS: A multipin synthetic peptides approach has been used to map the epitopes recognized by mAbs. Dodecapeptides with an overlap of 6 amino acids corresponding to a precursor pZP3β-deduced amino acid sequence (excluding the signal sequence) were synthesized on polypropylene pins and were tested for their reactivity with mAbs by enzyme-linked immunoadsorbent assay (ELISA). The ability of synthetic peptides corresponding to the identified epitopes to inhibit the binding of mAbs to pZP3β in a competitive inhibition ELISA was investigated to confirm the above findings. RESULTS: Reactivity of the mAbs with the pin-bound peptides in ELISA-identified epitopes for mAb-451 to EEKLVF (166–171) and mAb-462/470 to FKAPRP (250–255) amino acid residues. mAb-30 recognized QPVWQDEGQRLR (23–34) and VICRCC (316–321) amino acid residues. Competitive inhibition with synthetic peptides encompassing the motifs corresponding to 23–34 and 316–321 for binding of mAb-30 to pZP3β revealed the epitopic domain to be 23–34 amino acids. Synthesis of overlapping octapeptides further identified WQDE as the minimum motif for binding of mAb-30, and the replacement of one amino acid at a time with glycine revealed tryptophan as the critical residue. CONCLUSIONS: Collectively, these results describe peptide epitopes that will help in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZP3β or its homologues in other species.     

不同人群血清中抗透明带抗体的检测

为了探讨抗透明带抗体的临床意义 ,本研究用纯化的猪卵透明带ZP3抗原制备抗卵透明带抗体酶联免疫检测试剂盒 ,采用ELISA方法检测不同人群血中抗透明带抗体 ,并用阳性血清与人卵巢组织切片进行免疫组化分析。结果显示不同年龄组妇女抗透明带抗体阳性率差异不显著 (P >0 0 5 ) ,不孕组阳性率 15 % ,与其他组相比有显著性差异 (P <0 0 5 )。ELISA检测到的透明带抗体阳性血清不与人卵巢组织切片中的透明带产生肉眼可见的沉淀反应。除了早孕组和老龄妇女 ,任何年龄阶段的女性均可出现抗透明带自身抗体 ,鉴于这些抗体不引起周期紊乱现象 ,却可能阻止受精 ,这为透明带作为避孕疫苗候选抗原的安全提供理论依据     

Characterization of Monoclonal Antibodies Against Human Sperm Antigens by Immunoassays Including Sperm Function Assays and Epitope Evaluation

ABSTRACT: Fifteen monoclonal antibodies (MAbs) raised against human sperm cells were evaluated for reactions against human sperm by indirect immunofluorescence, immunocytochemistry, agglutination, complement-dependent immobilization, cervical mucus penetration, and hamster egg penetration assays. The MAbs were analyzed for specificity by immunofluorescent reactions with peripheral blood lymphocytes and sperm and classified into three main groups based on regional staining, ie, acrosome, plasma membrane, or tail. One MAb (218) bound to the sperm neck. Three MAbs, (80, 85, and HS-126) were found to react with lymphocytes. Three of five acrosome-reactive MAbs (11, 63, 106), two of five tail-staining MAbs (97, HS-30), and the neck reactor (218) showed significant to highly significant inhibition of sperm penetration of eggs but without significant effects on sperm agglutination, immobilization, or the mucus penetration assay. The three nonspecific MAbs gave strong plasma membrane reactions in the agglutination and immobilization assays and also caused highly significant inhibition of sperm penetration of both cervical mucus and zona-free ova. Preliminary analysis of the complementary antigens suggested that epitopes reacting with MAbs 33 (acrosome) and 85 (plasma membrane) were carbohydrate chains on glycoproteins. Three of five MAbs recognizing tail antigen, the neck-staining MAb, and the nonspecific MAb (HS-126) appeared to be reactive against glycolipid moieties. Seven of the 12 specific MAbs also reacted in indirect immunofluorescence with mouse and rabbit sperm in patterns similar to those observed with human sperm.     

Anti-ZP3 Antibodies Binding to the Human Zona Pellucida: Effect of Oocyte-Storage Conditions

PROBLEM: The zona pellucida protein 3 (ZP3) is a zona pellucida (ZP) glycoprotein crucially involved in fertilization. ZP3 plays a major role in sperm binding and induction of the acrosome reaction. In different species, ZP3 proteins differ in their primary structure as derived from cDNA clones. The hemizona assay (HZA) is a bioassay that evaluates binding of human sperm to human ZP and is highly predictive of fertilization outcome under in vitro conditions. METHOD: In these studies, we used antisera generated against synthetic ZP3 peptides to compare antibody binding patterns to ZP with sperm-ZP binding capacity under different HZA conditions. RESULTS: Analysis of antibody binding to hemizonae derived from metaphase II human oocytes that were used either after refrigeration at 4°C or stored in a hyperosmotic salt solution revealed a strong reaction with human ZP3. However, treatment of human oocytes using a protocol to freeze embryos with the addition of 1,2 propanediol drastically reduced binding of ZP3 antibodies to the hemizonae. Nevertheless, no significant difference of sperm binding occurred under HZA conditions when oocytes were refrigerated, salt-stored, or frozen with 1,2 propanediol. CONCLUSIONS: Our results indicate that the ZP3 protein backbone might be altered by 1,2 propanediol-treatment while the glycoprotein-receptor remains intact. We conclude that antisera against ZP3 peptides can be used as markers for the ZP3 protein backbone in human oocytes and might be useful tools for the evaluation of ZP3 protein integrity.     

Studies on Sperm Survival and Motility in the Presence of Cytotoxic Sperm Antibodies

ABSTRACT: The effect of cytotoxic sperm antibodies and native complement in the serum and secretions from 40 fertile and 93 infertile couples on in vitro sperm survival and motion characteristics was studied. Sperm survival in vitro was unaffected by sera from fertile and infertile subjects without cytotoxic sperm antibodies and from infertile men with antibodies to control but not to autologous sperm. Sperm survival was reduced (P < .001) by sera from infertile men with antibodies to autologous sperm or to antologous and control sperm and from women with cytotoxic antibodies to sperm from both. Sera from fertile couples without sperm antibodies enhanced sperm swimming speed and motility index (P < .0001). Sera from infertile women with or without cytotoxic sperm antibodies did not affect sperm motility. Sperm survival and motility were reduced by seminal plasma from infertile men with cytotoxic antibodies to autologous and/or control sperm. Seminal plasma from fertile men enhanced sperm survival. Cervical mucus from infertile women with antibodies to autoimmune husbands' sperm or to husbands' and control sperm inhibited sperm motion, whereas cervical mucus from infertile women without sperm antibodies and women with antibodies to control sperm failed to have any effect. It is concluded that cytotoxic sperm antibodies developed through exposure to sperm antigens in autoimmune infertile men decrease in vitro sperm survival and/or motility.     

Occurrence of the Interference of Sperm-Associated Antibodies on Sperm Fertilizing Ability as Evaluated by the Sperm-Zona Pellucida Binding Test and by the TEST-Yolk Buffer Enhanced Sperm Penetration Assay

PROBLEM: This study was performed to evaluate the occurrence as well as the level of the interference of sperm-associated antibodies on fertilization process. METHOD: Motile sperm suspensions from 28 infertile patients with high degree of autoimmunization against the sperm head were tested with the zona pellucida (ZP) binding test and with the sperm penetration assay (SPA) enhanced with TEST-yolk buffer. Both tests were also performed using donor sperm exposed and non-exposed to the patients' circulating sperm antibodies. RESULTS: A low ZP-binding was exhibited by sperm from 50% of patients with normal semen profile. All normozoospermic patients with low ZP-binding showed circulating sperm-antibodies with inhibitory effect on ZP-binding, while no patient with normal ZP-binding showed circulating sperm-antibodies with inhibitory effect. No normozoospermic patient exhibited a negative SPA result, and only in 16% of cases the penetration index was slightly less than 2 (the lowest value exhibited by fertile controls). Circulating antisperm-antibodies did not significantly affect the hamster egg penetration. CONCLUSION: Even in the presence of high degree of autoimmunization against the sperm-head, sperm fusion with oolemma is not impaired after sperm preincubation with TEST-yolk buffer, while an impairment of the ZP-binding is demonstrable in half cases, when non-immunologic factors are excluded. A substantial role in this interference is likely exerted by IgG antibodies transuded from the blood into the genital tract.     

Monoclonal Sperm Antibodies: Their Potential for Investigation of Sperms as Target of Immunological Contraception

ABSTRACT: We generated 149 hybridoma cell lines secreting antibodies against human spermatozoal antigens of which antibodies from 136 hybridoma lines also reacted with seminal plasma constituents. The occurrence of common antigeneic determinants on spermatozoa and seminal plasma was further demonstrated by competitive inhibition ELISA tests. We found that seven hybridoma clones secreted antibodies reactive with spermatozoa but nonreactive with seminal plasma. Antibodies from 5 clones were sperm-agglutinating and from 15 clones sperm-immobilizing. Localization of sperm antigens reactive with the monoclonal antibodies was demonstrated by indirect immunoperoxidase staining. A synthetic decapeptide, earlier shown to be reactive with naturally occurring human iso- and autosperm antibodies, was shown to be reactive with the monoclonal antibody VII-5 in ELISA tests.     

The Effect of Immunoglobulin Occurring on Human Sperm In Vivo on the Human Sperm/Hamster Ova Penetration Assay

ABSTRACT: We selected 91 infertile men who were tested for increased sperm-associated immunoglobulin and also tested in the human sperm/hamster ova penetration assay. There was a statistically significant association between the presence of increased sperm-associated IgG alone (p = 0.0218) and both sperm-associated IgG and A (p = 0.0187) when correlated with the failure to penetrate any hamster ova. There was a trend but no statistical significance when sperm-associated immunoglobulin A alone was present. There was a trend but no statistical relationship between the presence of sperm-associated immunoglobulin and the sperm penetration assay when the criteria for normality of the sperm penetration assay was a 15% or greater ovum penetration rate.     

EBV Transformation and Microfusion as the Potential Source of Human Monoclonal Antisperm Antibodies

ABSTRACT: Peripheral blood lymphocytes were obtained from vasectomized men with high serum titers of anti-sperm antibodies. An Epstein-Barr virus (EBV) transformation was performed either with B cells or mononuclear leukocytes. The effect of feeder cells (irradiated umbilical cord blood lymphocytes), cyclosporin A, and in vitro stimulation of lymphocytes with sperm extract on EBV transformation was evaluated. Antibody-producing cells were screened for specificity against human sperm by an enzyme-linked immunosorption assay (ELISA) one to six weeks after transformation. Using B cells or leukocyte mononuclear cells, we found that the percentage of wells containing antibody reactive against human sperm was greatest two weeks after transformation (range 3% to 7.5% positive wells). To increase and maintain antibody synthesis by these transformed cells, microfusions were performed in those wells positive for antisperm antibody using the UC 729-6 lymphoblastoid cell partner. Then resultant hybridomas were expanded, subcloned, and preliminarily characterized.     

The Interfering Effect of Human IgG Antisperm Antibodies on Human Sperm Penetration of Zona-Free Hamster Eggs*

ABSTRACT: A liquid-phase radioimmunoassay was used to quantitate sperm-associated IgG in a population of patients with unexplained infertility. Plasma IgG antisperm antibody activity was identified in a subpopulation of these patients. Using the human sperm/hamster egg assay, a unique functional test of human sperm penetrating capacity, we identified a subset of these positive patients whose plasma significantly and reproducibly inhibited penetration. This study implies that antigen-antibody reactions may prevent sperm responses that are critical to sperm-egg fusion.     

A new test for the assessment of sperm- zona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro

The spermatozoa of some patients attending for in-vitro fertilization(IVF) fail to penetrate the zona pellucida in vitro. A testhas been devised to identify these cases. It is based on thenumber of spermatozoa penetrating into the zona pellucida, whichwere counted after removing spermatozoa bound to the zona surfaceby vigorous aspiration of each oocyte through a narrow gauge(120 µm) glass pipette. The oocytes were collected from197 patients undergoing IVF treatment with their own gametes;79 with no oocytes fertilized and 118 with some oocytes fertilized.Sperm motility, morphology and DNA normality (acridine orangestain) were also measured. The relationships between sperm testresults and IVF rate were examined by logistic regression. Theproportions of penetrated zonae, normal sperm morphology andnormal DNA were the most significant factors related to IVFrate in the whole group. Also, in patients with 30 spermatozoabound per zona pellucida or with normal sperm morphology 30%,the proportion of penetrated zonae and normal DNA were mostsignificant. Oocytes from 42 patients who had zero fertilizationand low sperm-zona binding (average, 2.2 spermatozoa/zona pellucida)were re-incubated with normal donor spermatozoa: large numbersof spermatozoa bound (average, 88 spermatozoa/zona pellucida)and each zona was penetrated by at least one spermatozoon. Inconclusion, the percentage of zonae penetrated was the variablemost significantly correlated with IVF rate. Penetration ofthe zona was also strongly related to fertilization rates inpatients without defects of sperm morphology and sperm-zonabinding. In patients where all zonae were penetrated, poor fertilizationmay be due to sperm morphology and DNA abnormalities. Failureof sperm-zona binding and penetration in vitro in patients withfailure of fertilization was mainly due to sperm defects andnot oocyte defects     

Antibodies to Spermatozoa: XI. The Use of Immunobeads for the Detection of Sperm Antibodies in Serum

ABSTRACT: Two procedures were developed and evaluated that used either larger or smaller volumes for the detection of sperm antibodies in serum by means of an indirect immunobead test (IBT). The immunobeads, coated with rabbit antibody to each of the major human immunoglobulins (IgG, IgA, or IgM), were mixed with preparations of donor sperm, onto which antibody had been coated by passive transfer from various serum samples. The results of the IBT could be evaluated in various ways: (1) positive or negative; (2) if positive, whether binding is to the tail, the head, or the head and tail of the sperm cells; (3) if positive, whether binding is by IgG, IgA, or IgM. The diverse IBT results were obtained from a group of 50 serum samples; these sera were also tested by two sperm agglutination methods; the gelatin agglutination test (GAT) and the tube-slide agglutination test (TSAT). There was an excellent agreement between the IBT and the GAT; it was not as good between the IBT and the TSAT. However, considering both agglutination methods together, 90% of the IBT-positive sera were agglutination-positive. In terms of morphological sites, tail binding occurred in 27 of 31 sera, head binding in 12 of 31 sera, and head-tail binding in 15 of 31 sera. The number with tail binding was very close to the number that were GAT-positive (26). As for the immunoglobulins, the most frequent class was IgG. IgA was 83% as frequent and IgM was only 25% as frequent as IgG. In a larger group with only IgG and IgA, of 31 IBT-positive sera, 26 showed IgG and 23 showed IgA; 18 showed both. Hence, only eight showed IgG exclusively, and only five showed IgA exclusively. One final point is that several sera with GAT titers of only 4 were IBT-positive, adding strength to the concept that such a low GAT titer does have antibody significance.     

A digital method of sperm immobilization test: comparison to the conventional method

Antisperm antibodies have been found in infertile patients and those causing immobilization of sperm are considered to be closely related to unexplained infertility. These antibodies are usually identified by a sperm immobilization test which involves counting motile sperm under microscope. This test is subjective as it relies on the judgement of the examiner with respect to sperm motility. In this study, we analyzed motile sperm by a digital method using Sperm Quality Analyzer. The results were compared with those obtained by the conventional method. We found that the two methods yielded identical results, with 14 of 66 samples tested being positive and 52 negative for sperm immobilizing antibodies. These results show that the digital method is objective and of value in the measurement of motile sperm in determination of sperm immobilizing antibodies.     

精子形态与活率相关性的研究及其在男性不育中的应用

目的探讨精子形态与活率的相关性,以便为男性不育的诊断提供理论指导。方法分别利用改良的巴氏染色法和伊红染色法,对精子形态和精子活率进行分析。结果与精子形态正常组相比,畸形精子组中精子活率异常的比例明显上升,差异具有统计学意义(P<0.05)。结论精子活率与精子形态呈正相关。精子形态可影响精子活率,进而影响男性的生育。     

抗大肠杆菌_(506)株36kD蛋白单抗的制备及其在人精子上的免疫定位

用纯化的36kD蛋白免疫Balb/c小鼠。取免疫脾细胞与小鼠骨髓瘤细胞SP2/0以PEG进行细胞融合,融合率为86％。采用ELISA筛选出杂交瘤细胞阳性孔。经过4～7次有限稀释克隆化,共获得7株能稳定分泌36kD相应MCAb的杂交瘤细胞。将杂交瘤细胞分别注射小鼠腹腔,测定腹水McAb的效价为1:5×10~3～1:1×10~5,抗体亚型均为IgG1。用荧光显微镜观察,结果发现EC2作用的人精子头颈部出现明显荧光反应,提示共同抗原存在于人精子头颈部。这些结果有助于进一步探讨细菌感染引起男性不育的机制以及研制细菌避孕疫苗等工作。