Sperm-immobilizing Antibodies Research Articles

Anti-tubulin beta 4A (TBB4A) antibody immobilized sperm in a complement-dependent manner in humans

Sperm-immobilizing antibodies (SI-Abs) are detected in the sera of 3 % of infertile women. SI-Abs are occasionally produced as allogeneic antibodies against sperm, causing immune infertility. SI-Abs inhibit the passage of sperm through the female reproductive tract. Research on anti-sperm antibodies (ASA) remains of great importance for population control. We aimed to identify the antigens recognized by SI-Abs and elucidate the pathogenesis of immune infertility. Twelve sperm-immobilization test (SIT)-positive and fourteen SIT-negative sera were analyzed by two-dimensional electrophoresis and western blotting. Antigenic materials were extracted from well-motile sperm prepared using 0.1 % sodium dodecyl sulfate. In total, 22 different spots were detected in the 12 positive sera. Among these, three positive serum samples showed two positive signals with similar migration patterns. The significant positive spots were Mr: 49 K, pI: 5.1 and Mr: 51 K, pI: 5.6. All these positive spots were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS); tubulin beta-4A (TBB4A) was identified from the spot Mr: 49 K, pI: 5.1. TBB4A is a major component of tubulin and constitutes the axoneme in the sperm tail and the centrosome in the sperm neck; it is generally located inside the cell. An authentic antibody against TBB4A showed a positive reaction in the sperm neck and tail regions in an immunofluorescence study. This antibody also inhibited sperm motility in a complement-dependent manner. Sperm membrane permeability reportedly changes during swimming and capacitation. We identified TBB4A as an antigenic molecule recognized by SI-Abs, which may be relevant to immunological contraception in the future.

Advancements in the detection and implications of sperm-immobilizing antibodies in female infertility

This review highlights over five decades of research on sperm-immobilizing antibodies (SI-Abs), which are crucial for understanding female infertility due to their effects on sperm motility and fertilization. Since the 1960s, Isojima et al. have made significant strides, notably with the Sperm Immobilization Test (SIT), which revolutionized the quantification of SI-Abs and their roles in infertility. Drawing from a comprehensive PubMed search on "the sperm immobilization test" and "sperm immobilizing antibody," our review underscores the critical insights gained into SI-Abs' impact on reproductive functions. SI-Abs result from the body's response to sperm antigens, potentially leading to infertility by affecting post-intercourse sperm function. However, the presence of anti-sperm antibodies does not guarantee infertility, indicating a complex relationship between these antibodies and reproductive outcomes. Isojima et al.'s pioneering studies paved the way for SIT and sperm immobilization titer (SI50), tools that have clarified the link between SI-Abs and infertility, focusing on disrupted sperm mobility and fertilization as key infertility mechanisms. Clinically, interventions such as in-vitro fertilization (IVF), which bypasses or eliminates SI-Abs, have improved pregnancy rates, whereas Freund's complete adjuvant therapy has deepened our understanding of infertility mechanisms. The SI50 value is crucial for predicting fertility treatment success and guiding therapeutic decisions based on antibody levels. In summary, the evolution of SI-Abs research has provided new hope for addressing infertility, significantly enriching the field of reproductive immunology, and highlighting the need for ongoing investigation.

O8-2 - Detection of Sperm Antigens Corresponding to Sperm-Immobilizing Antibodies in Infertile Women

O8-2 - Detection of Sperm Antigens Corresponding to Sperm-Immobilizing Antibodies in Infertile Women

Symposium 1-4 - Immunological infertility caused by gamete immunity

1. Infertility caused by anti-sperm antibodies (ASA) 1) Female infertility caused by sperm-immobilizing antibodies in the sera In some women, ASA are produced as allogenic immunity, which causes infertility. Various methods for the antibody detection have been developed, but sperm-immobilization antibodies (SI-Ab) detected by the sperm immobilization test (SIT) have the greatest influence on the development of infertility. Sperm-immobilizing antibodies are present in the female genital tract, and also in the follicle fluid.SI-Ab cause a post-coital test (PCT) negative or impaired sperm transport from the uterine cavity to the Fallopian tubes, and fertilization disorders. There is a method to quantitatively measure SI-Ab titer, SI50 unit, and treatment methods are decided based on this SI 50 value. In cases with a SI50 value of 10 or more, it is difficult to conceive with AIH, and IVF is effective after preventing antibody contamination into the culture medium. As a mechanism for producing sperm-immobilizing antibodies, it has been speculated that women with disease-specific genes may be exposed to enough amount of sperm through sexual intercourse and develop symptoms triggered by such as chlamydia infection. CD52 is considered to be a promising antigen against sperm-immobilization antibodies, and is expected to be applied for the development of contraceptive vaccines. 2) Male infertility caused by ASA that bind to ejaculated sperm In some men, ASA are produced as autoantibodies, causing infertility. In men, it is not useful for measuring serum antibodies, therefore antibodies that bind to ejaculated sperm (sperm-binding antibodies) should be detected with ImmunoSpheres○R. However, since there are antibodies that are not related to the development of infertility, the treatment strategy can be determined by confirming whether the antibody has a biological effect. Inhibitory effect on cervical sperm penetration is confirmed by PCT and fertilization disorder is examined by the hemizona assay (HZA). If HZA cannot be performed, ICSI is recommended because it is likely to be accompanied by fertilization disorder in cases where the ratio of ASA-bound sperm to total motile sperm is 80% or more. 2. Female infertility caused by anti-zona pellucida (ZP) antibodies The ZP plays an important role in terms of egg formation, fertilization, protection of the embryo. These include the recognition of sperm species of animals, the induction of acrosome reaction, and the prevention of polysperm fertilization. On the other hand, since there is a strong antibody production ability as an antigen, if an anti-ZP antibodies are produced, the function of the ZP is impaired by the antibody, and there is a possibility of developing infertility. It was reported that anti-ZP antibodies were detected at a high rate in the sera of infertile women of unknown cause by the anti-ZP antibody detection method using porcine ZP, followed by controversial reports by another investigators. Therefore, we developed a detection method using human ZP. Anti-ZP antibodies disturb the follicular development, inhibit sperm-ZP binding and hatching. For the purpose of appropriate treatments, it was shown that ICSI is effective for fertilization disorder and assisted hatching (AHA) is effective for hatching disorder, respectively.

Sperm immobilization test and quantitative sperm immobilization test using frozen-thawed sperm preparation applied with computer-aided sperm analysis.

PurposeIn a previous study, a new method was described using the sperm immobilization test (SIT) with computer‐aided sperm analysis (CASA). However, obtaining high‐quality sperm as needed was a known issue. Here, we compared the results of using frozen‐thawed sperm and fresh sperm for the SIT using the CASA method.MethodsFor the frozen‐thawed preparation, 500 μL of condensed semen and 500 μL of Sperm Freeze were mixed in a cryovial and cryopreserved in liquid nitrogen. Density gradient centrifugation was used for the collection of motile sperm in both the fresh and frozen‐thawed sperm preparations. A total of 50 serum samples were prepared for both the fresh and frozen‐thawed sperm with each sample tested containing 10 μL of serum, 1 μL of either fresh or frozen motile sperm suspension, and 2 μL of complement. Sperm motilities were measured using CASA after a 1‐hour incubation period for both fresh and frozen‐thawed sperm.ResultsBoth fresh and frozen‐thawed sperm reacted similarly when exposed to serum containing sperm‐immobilizing antibodies asserting the use of frozen‐thawed sperm for the diagnosis of immunological infertility.ConclusionThese results suggest the possibility of using cryopreserved sperm for the SIT when fresh sperm is unavailable.

Anti-sperm antibodies and reproductive failures.

Reproductive failures include infertility and recurrent pregnancy loss (RPL). Although the relative importance of immunological factors in human reproduction remains unclear, there may be immune-mediated reproductive failures in a portion of unexplained infertility and RPL. As a cause of immunological factor, anti-sperm antibodies (ASA) are produced both in men and women. There have been reported several antigens in the surface of sperm that are especially foreign to women. The presence of ASA, especially sperm-immobilizing antibodies, in the sera of infertile women has been shown to inhibit sperm migration in the female genital tract. Therefore, the effectiveness of the treatments for infertile women with sperm-immobilizing antibodies by timed intercourse or intra-uterine insemination is limited. Such antibodies can also exert inhibitory effects on various stages of sperm-egg interaction and subsequent embryo development in vitro. It is suggested that ASA testing for infertile women should be performed before proceeding IVF. The manipulation of gametes and embryos from patients having sperm-immobilizing antibodies should be carefully carried out especially to avoid contaminating patient's serum and follicular fluid in the culture medium in order to overcome the immunological causes of female infertility by ASA, and satisfactory results under suitable conditions for gametes and embryos have been obtained. The relationship between ASA and RPL was controversially reported. Increased miscarriage rates in women with ASA were demonstrated by some authors. In contrast, lack of association between ASA and RPL was reported. In this manuscript, we are focusing the roles of ASA in women with reproductive failures.

Application of computer-aided sperm analysis (CASA) for detecting sperm-immobilizing antibody.

Since the 1970s, anti-sperm antibodies have been studied as a pathogenic factor contributing to infertility. The complement-dependent sperm-immobilization test (SIT) and quantitative SIT have been used as effective tools for detecting anti-sperm antibodies in clinical settings. These tests have been carried out traditionally by manually counting the number of motile sperm through eye estimation. In this study, we developed a novel method using computer-aided sperm analysis. The results were compared with those obtained by the traditional method. The results were identical and 25 of 78 samples tested were positive and 53 samples were negative for sperm-immobilizing (SI) antibodies based on both methods. For SI-positive samples, the values of SI50 obtained using the two methods correlated closely with high co-efficiency. Using the novel method, manually counting the number of motile spermatozoa becomes unnecessary. The novel method presented here will increase the objectivity and convenience of using the SIT as a clinical indicator.

The status of sperm immobilizing-antibody (SI-Ab) detected in infertile females (therapy and pathogenic mechanisms)

Chronic prostatitis is a risk factor for impaired male fertility potential, and anti-sperm antibodies (ASAs) cause the autoimmune disease immune infertility, which has a negative effect on semen parameters. Current studies have investigated the ASA-positive relationship between chronic prostatitis versus normal controls, but have shown inconsistent results. Hence, we systematic searched the PubMed, EMBASE, Science Direct/Elsevier, Medline, and the Cochrane Library up to October 2015 for case-control studies that involved the ASA-positive relationship between chronic prostatitis patients versus normal controls. The meta-analysis was performed with Review Manager and Stata software. After literature search, six studies were identified, including 721 cases of chronic prostatitis and 160 normal controls. Our results illustrated a significant correlation of the ASA-positive relationship between chronic prostatitis patients versus normal controls. The combined odds ratio of the ASA-positive rate in chronic prostatitis patients and normal controls was 3.26 (1.86–5.71). There was also a significant correlation of the ASA-positive relationship between National Institutes of Health (NIH) III versus normal controls, and the combined OR was 2.46 (1.10–5.51). However, there was no significant correlation of the ASA-positive relationship between National Institutes of Health (NIH) II versus normal controls. The present study illustrates that the positive rate of ASAs in chronic prostatitis patients was significantly higher than in the control group, suggesting that chronic prostatitis has a negative effect on male reproductive function. However, studies with larger samples are needed to better illuminate the correlation between ASAs and chronic prostatitis.

The Status of Sperm Immobilizing-Antibody (SI-Ab) detected in Infertile Females (Therapy and Pathogenic Mechanisms)

The Status of Sperm Immobilizing-Antibody (SI-Ab) detected in Infertile Females (Therapy and Pathogenic Mechanisms)

Incidence of Sperm-immobilizing Antibodies in Infertile Women with Past Chlamydia trachomatis Infection

Among the risk factors for antisperm antibody production, inflammatory diseases of the genital tract are believed to play an important role. Chlamydia trachomatis infection is one of the most common sexually transmitted diseases. There are some reports suggesting that human sperm have antigens that cross-react immunologically with certain microbial antigens, such as C. trachomatis. However, this is still controversial. We performed a retrospective study to investigate the correlation between anti-chlamydial antibodies and sperm-immobilizing antibodies in infertile women. Between January 2007 and March 2009, the presence of sperm-immobilizing antibodies was examined by the sperm immobilization test using sera from 273 infertile women. Anti-chlamydial antibodies (IgG and IgA) were examined to prove past C. trachomatis infection by ELISA using the same sera from infertile women. The overall incidence of sperm-immobilizing antibodies was 2.9% (8/273) in infertile women. The incidences of sperm-immobilizing antibodies were 6.4% (5/78) in cases with past C. trachomatis infection and 1.5% (3/195) in cases without past C. trachomatis infection. There was a significant difference between the two groups (P = 0.031). A significantly higher incidence of sperm-immobilizing antibodies was noted in infertile women with past C. trachomatis infection compared with that of those without past C. trachomatis infection. This is the first demonstration that C. trachomatis infection could play a role in the production of sperm-immobilizing antibodies in infertile women.

Diagnosis and treatment of immunologically infertile women with sperm-immobilizing antibodies in their sera

Detection of sperm-immobilizing antibodies in women may have relevance for diagnosis of immunological infertility. Infertile women in whom sperm-immobilizing antibodies are detected can be refractory to conventional treatments such as timed intercourse or intrauterine insemination (IUI) because the antibodies secreted in the female reproductive tract might impair sperm passage, inhibit fertilization, and prevent normal post-fertilization processes. Hence, manipulation of gametes and embryos from patients with sperm-immobilizing antibodies should be carried out with additional care to avoid fertilization failure resulting from the presence of antibodies during in vitro fertilization (IVF). Moreover, the reasons for the why majority of women do not develop sperm-immobilizing antibodies on exposure to sperm is not clear. The production of sperm-immobilizing antibodies is likely to occur in women with particular HLA haplotypes after repeated exposure to sperm. Characterization of sperm-immobilizing antibodies may help in the identification and characterization of sperm specific antigens that can be used as candidate antigens for the development of sperm based contraceptive vaccines.

Functional aspects of CD52 in reproduction

CD52 is a GPI-anchored protein present in lymphocytes and male reproductive tissues (mrt) including mature sperm and seminal plasma. It has been shown that mrt-CD52 is synthesized in epithelial cells of the epididymis and vas deferens, but not in the testis. The mrt-CD52 is transported to mature sperm during sperm transition in the male reproductive tract. Lymphocyte CD52 functions to stimulate suppressor T cell induction, while mrt-CD52 is associated with seminogelin and involved in clot formation and liquefaction of semen. In a landmark study, a monoclonal antibody (Mab H6-3C4) from peripheral B lymphocytes of a patient with complement-dependent sperm-immobilizing antibody in the serum has been generated. Using Mab H6-3C4, the carbohydrate moiety of CD52 as an epitope for infertility-related antigen was identified. Mab H6-3C4 also shows strong complement-dependent sperm-immobilizing activity. This suggests CD52 may have a function in protecting sperm from complement activation. Indeed, purified mrt-CD52 from human sperm interferes with the classical pathway, but not lectin-binding or alternative pathways, of the complement systems. Recently, we found CD52 in ovulated cumulus cells from the female reproductive tissues (frt). The frt-CD52 is not recognized by Mab H6-3C4, suggesting that it harbors distinct carbohydrate antigenicity. Further studies are necessary to determine the molecular features and biological functions of CD52 in male and female reproductive tissues.

Andrological and hormonal findings in subjects with ductus deferens agenesia.

The aim of our study has been the complete andrological and endocrinological evaluation of seven cases of bilateral ductus deferens agenesia. In all cases testicular biopsy demonstrated a normal spermatogenesis. Urographic examination showed, in four cases, the existence of congenital anomalies of the urinary tract. The presence of spermioagglutinating and spermimmobilizing antibodies in blood and in seminal plasma was excluded in all cases. Basal and stimulated levels of FSH, LH, PRL and testosterone were within normal limits. Surgical exploration of seminal tracts and bilateral collection of sperm at the caudal portion of the epididymis appear to be mandatory in order to select the cases for surgical therapeutic approach.

The sperm agglutination antigen-1 (SAGA-1) glycoforms of CD52 are O-glycosylated

CD52 is composed of a 12 amino acid peptide with N-linked glycans bound to the single potential glycosylation site at position 3, and a glycosylphosphatidylinositol-anchor attached at the C-terminus. Some glycoforms of this molecule expressed in the male reproductive tract are recognized by complement-dependent sperm-immobilizing antibodies in infertile patients making this antigen an important target for immunocontraception and fertility studies. Although the amount of posttranslational modification is already remarkable for such a small polypeptide, O-glycosylation of CD52 has additionally been implicated by several studies, but never rigorously characterized. In this report, we show clear evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 monoclonal antibody generated against sperm agglutination antigen-1 (SAGA-1), a male reproductive tract specific form of CD52. The O-glycans have been characterized by MALDI-TOF and tandem mass spectrometry after reductive elimination and permethylation. The data indicate that the major SAGA-1 O-glycans are core 1 and 2 mucin-type structures, with and without sialic acid (NeuAc(0-2)Hex(1-3)HexNAc(1-2)HexNAcitol). Minor fucosy- lated O-glycans are also present including some struc- tures with putative Le(y) epitopes (NeuAc(0-1)Fuc(1-3)Hex(1-2) HexNAc(0-1)HexNAcitol). Analysis of O-glycopeptides by tandem mass spectrometry provided an additional level of support for the O-glycosylation of SAGA-1. Elucidation of the O-glycosylation of SAGA-1 adds to the complexity of this molecule and may help to explain its biological activity.

Effects of invivo exposure to eggs with sperm-immobilizing antibodies in follicular fluid on subsequent fertilization and embryo development invitro.

Aims: It has been shown that supplementation of patients' sera that contains sperm-immobilizing antibodies results in failure of fertilization and embryo development invitro. The present study was carried out to investigate if exposing retrieved eggs to a high number of sperm-immobilizing antibodies in the follicular fluid (FF) invivo affected subsequent fertilization and embryo development invitro, even if they were washed with an antibody-free culture medium. Methods: Patients' sera and their FF were collected in 15 invitro fertilization-embryo transfer (IVF-ET) or intracytoplasmic sperm injection-embryo transfer (ICSI-ET) treatment cycles from 11 infertile women with sperm-immobilizing antibodies in their sera. Quantitative sperm-immobilizing antibody titers (SI50 titers) in the sera and FF were evaluated. The fertilization rate, good-quality embryo rate and implantation rate by IVF-ET were compared between infertile patients having higher (10≤) SI50 titers and lower (<10) SI50 titers in their FF. Results: There was a significant correlation in the SI50 titers between the patients' sera and their FF (P<0.0001). After thoroughly washing the collected eggs in culture medium without the patient's serum before IVF, there was no difference in the fertilization rate in the patients with high (10≤) and low (<10) SI50 titers in their FF (P=0.62). However, the good-quality embryo rate in the patients with a high SI50 titer was significantly lower than patients with a low antibody titer (P < 0.05). There was no significant difference in the implantation rate between the two groups (P=0.33). Conclusions: Similar amounts of sperm-immobilizing antibodies existed in the patients' FF and in their sera. ICSI did not seem to be necessary in patients having the antibodies if their sera were not supplemented in the culture media. Even with careful manipulation of eggs, it might be suggested that the harmful effects of sperm-immobilizing antibodies on embryo development cannot be completely avoided, especially in patients with high SI50 titers in the FF. (Reprod Med Biol 2006; 5: 137-143).

Antigenic epitope for sperm-immobilizing antibody detected in infertile women

CD52 is a glycosylphosphatidylinositol (GPI) anchor protein occurring in lymphocytes, the epididymis, seminal plasma and on ejaculated sperm surface. The molecular structure of male reproductive tract CD52 (mrtCD52) is quite different from that of lymphocyte CD52 except for a peptide sequence comprising 12 amino acids. The carbohydrate molecule may thus provide antigenic epitopes to females. The repeated N-acetyllactosamine units of the N-linked carbohydrate present in the mrtCD52 were identified as a pathogenic antigen for infertility using a monoclonal antibody (MAb H6-3C4) generated from an infertile patient's peripheral blood lymphocyte. We suggested also the possible presence of O-linked carbohydrate in human mrtCD52, although it is not known whether this carbohydrate is immunogenic or not. This article gives an overview of the structure and function of mrtCD52 as well as the fertilization-blocking mechanism of the antibody against this molecule.

Sperm-immobilizing antibodies suppress an increase in the plasma membrane fluidity of human spermatozoa

To clarify the mechanism by which capacitation is blocked by sperm-immobilizing antibodies, changes in the plasma membrane fluidity of human spermatozoa exposed to sperm-immobilizing antibodies were evaluated. In vitro cell culture study using human spermatozoa. Department of Obstetrics and Gynecology, School of Medicine, The University of Tokushima. Semen samples were obtained from four healthy, fertile volunteers. The internalization of [3H]lyso-platelet activating factor (lyso-PAF) across the plasma membranes of human spermatozoa, which were exposed to sperm-immobilizing antibodies (antisperm group) or not exposed (control group), was measured at 20 and 60 minutes after the addition of a phospholipid probe using the modified albumin-back extraction method. The percentage of internalization of [3H]lyso-PAF across the plasma membrane of human spermatozoa. Although the percentages of internalization of [3H]lyso-PAF (mean +/- SE) in the antisperm and control groups 20 minutes after addition of [3H]lyso-PAF were not significantly different (6.6% +/- 1.5% and 9.2% +/- 2.1%, respectively), at 60 minutes after the addition, the percentage in the antisperm group (9.0% +/- 1.3%) was significantly lower than that in the control group (13.4% +/- 1.3%). This inhibitory effect was diminished when spermatozoa exposed to sperm-immobilizing antibodies were incubated in an antibody-free medium. Sperm-immobilizing antibodies suppress the increase in internalization of an alkyl ester lysophospholipid probe in plasma membranes of human spermatozoa, and this inhibitory effect is reversible. Therefore, sperm-immobilizing antibodies suppress the fluidity of the plasma membranes of human spermatozoa, thus blocking capacitation.

Infertile women without sensitization to an appropriate amount of sperm do not produce sperm-immobilizing antibodies in their sera.

Background and Aim: The factors that affect sperm immobilizing antibody production in some women are not fully understood. This study was performed to investigate if production of sperm immobilizing antibodies in women is associated with their husbands' sperm count. Methods: The sperm immobilization test (SIT) was performed on 221 infertile women whose husbands had normal semen characteristics according to the criteria by WHO; 160 patients were treated by intracytoplasmic sperm injection (ICSI) because of poor semen characteristics, and 1013 virgin female children acted as the controls. Results: A significant difference of the incidence in SIT was observed between the virgin female children and the women whose husbands had a normal sperm count (P<0.0001). There was also a significant difference of the incidence in SIT between the women treated by intracytoplasmic sperm injection (ICSI) because of a severe male factor and those whose husbands had a normal sperm count (P<0.05). Conclusions: These results indicate that production of sperm immobilizing antibodies in women begins after they have been exposed to a large enough amount of sperm. However, the precise amount of sperm required to produce the antibodies is not clarified by the present study. (Reprod Med Biol 2003; 2: 105-108).

Epitope analysis for human sperm-immobilizing monoclonal antibodies, MAb H6-3C4, 1G12 and campath-1.

Human monoclonal antibody, MAb H6-3C4, possesses strong sperm immobilizing activity. MAb H6-3C4 has been suggested by several research groups to react with a carbohydrate moiety of male reproductive tract CD52 (mrtCD52). In the present study, we analysed the epitope on mrtCD52 for MAb H6-3C4 and found that it was polymorphic in Western blot analysis and disappeared after enzymatic removal of the N-linked carbohydrate moiety. Two other monoclonal antibodies (1G12, campath-1) with sperm-immobilizing activity recognized mrtCD52 in a polymorphic manner similar to MAb H6-3C4. Further analysis showed that 1G12 recognized a structure formed by the peptide and/or a glycosylphosphatidylinositol (GPI) anchor portion as does campath-1. Results of a lectin binding assay suggested the presence of O-linked carbohydrates on mrtCD52. Our results also indicated that the peptide portion of CD52 could serve as an epitope for sperm-immobilizing antibodies. It was concluded that the epitope of MAb H6-3C4 is similar to, but distinct from, those of 1G12 and campath-1, and that mrtCD52 contains different antigenic epitopes.

Effect of sperm-immobilizing antibodies bound to the surface of ejaculated human spermatozoa on sperm motility in immunologically infertile men

Effect of sperm-immobilizing antibodies bound to the surface of ejaculated human spermatozoa on sperm motility in immunologically infertile men