Characteristics and Possible Role of Bovine Sperm Head-to-Head Agglutination.

Although sperm head-to-head agglutination has been reported in many mammalian species, the biological significance of this unique sperm-sperm interaction remains largely unknown. Here, we aimed to examine the functional characteristics of agglutinated bovine sperm to determine the possible role of sperm agglutination in the fertilization process. We initially examined temporal changes to the degree of head-to-head agglutination in culture, and found that bovine sperm agglutinated despite the lack of sperm agglutination inducers in medium. Sperm viability and motility were evaluated by SYBR14/PI and JC-1 staining, respectively, to identify the relationship between sperm agglutination and fertilizing ability. Agglutinated sperm had increased motility, viability, and intact mitochondrial function compared with unagglutinated sperm. Furthermore, we found that heparin significantly increased the percentage of unagglutinated sperm, but did not affect viability of both agglutinated and unagglutinated sperm, suggesting that sperm agglutination dictated the viability. In conclusion, agglutinated bovine sperm maintained viability and motility for a longer time than unagglutinated sperm. Thus, we propose that the head-to-head agglutination is a crucial sperm-sperm interaction to ensure the fertilizing ability of sperm.

Impairment of spermatogenesis and sperm motility by the high-fat diet-induced dysbiosis of gut microbes.

OBJECTIVE: High-fat diet (HFD)-induced metabolic disorders can lead to impaired sperm production. We aim to investigate if HFD-induced gut microbiota dysbiosis can functionally influence spermatogenesis and sperm motility. DESIGN: Faecal microbes derived from the HFD-fed or normal diet (ND)-fed male mice were transplanted to the mice maintained on ND. The gut microbes, sperm count and motility were analysed. Human faecal/semen/blood samples were collected to assess microbiota, sperm quality and endotoxin. RESULTS: Transplantation of the HFD gut microbes into the ND-maintained (HFD-FMT) mice resulted in a significant decrease in spermatogenesis and sperm motility, whereas similar transplantation with the microbes from the ND-fed mice failed to do so. Analysis of the microbiota showed a profound increase in genus Bacteroides and Prevotella, both of which likely contributed to the metabolic endotoxaemia in the HFD-FMT mice. Interestingly, the gut microbes from clinical subjects revealed a strong negative correlation between the abundance of Bacteroides-Prevotella and sperm motility, and a positive correlation between blood endotoxin and Bacteroides abundance. Transplantation with HFD microbes also led to intestinal infiltration of T cells and macrophages as well as a significant increase of pro-inflammatory cytokines in the epididymis, suggesting that epididymal inflammation have likely contributed to the impairment of sperm motility. RNA-sequencing revealed significant reduction in the expression of those genes involved in gamete meiosis and testicular mitochondrial functions in the HFD-FMT mice. CONCLUSION: We revealed an intimate linkage between HFD-induced microbiota dysbiosis and defect in spermatogenesis with elevated endotoxin, dysregulation of testicular gene expression and localised epididymal inflammation as the potential causes. TRIAL REGISTRATION NUMBER: NCT03634644.

Life-time exposure to waterborne copper IV: Sperm quality parameters are negatively affected in the killifish Poecilia vivipara.

In previous studies, we have shown that copper (Cu) is significantly accumulated in various tissues of killifish Poecilia vivipara following chronic exposure. Also, we showed that chronic metal exposure disrupted energy production and growth in this species. In the present study, we aimed to evaluate if chronic exposure to this metal could also affect reproductive parameters of P. vivipara males (sperm quality). In order to test that, newborn (<24 h-old) fish were exposed to two concentrations of waterborne Cu (5 and 9 µg/L) for 345 days. After exposure, fish were euthanized and the testes were collected for sperm analysis. We could observe that exposed animals had reduced sperm motility and period of motility. Also, the sperm of exposed fish had reduced plasma membrane integrity, mitochondrial functionality and DNA integrity when compared to sperm of control animals. It is suggested that the well-known association of Cu with elevated oxidative damage, endocrine disruption and energetic disturbance are involved with the observed outcomes. The results obtained in the present study show that chronic exposure to environmentally relevant concentrations of waterborne Cu caused reductions in all parameters used to evaluate sperm quality. Therefore, it is concluded that life-time exposure to this metal may disrupt fish reproduction and negatively affect the maintenance of its populations.

Dietary boron supplementation enhances sperm quality and immunity through influencing the associated biochemical parameters and modulating the genes expression at testicular tissue.

INTRODUCTION: Dietary boron improves immune and antioxidant status and calcium metabolism in mammals. However, till date the effects of dietary boron supplementation on male reproduction, especially on sperm production and sperm quality in farm animals are not documented. OBJECTIVE: The present study was aimed to investigate the influence of dietary boron on semen production, semen quality, immunity and molecular changes in the testis, blood and seminal plasma and to assess the interrelationship with other minerals in male goats. METHODOLOGY: The study was conducted in 21 adult male goats divided into 3 groups (control, boron and selenium supplemented groups, n = 7 each). In boron group, boron was supplemented at 40 ppm and in selenium group, selenium was supplemented at 1 ppm over and above the basal level. In control group, only the basal diet was fed without supplementary boron or selenium. The feeding trial was carried out for 60 days. Selenium was taken as a positive control for the dietary boron supplementation experiment. Following feeding trials, the sperm concentration, kinematics and functional attributes, immunity and molecular level changes in the testis, biomolecular changes in the blood and seminal plasma and also interrelationship with other minerals were studied. RESULTS: The average sperm concentration (million/ml) and the total sperm production (million/ejaculate) were significantly (p < 0.05) increased in boron supplemented group when compared to selenium and control groups. The boron levels in blood plasma (r = 0.65) and seminal plasma (r = 0.54) showed a positive correlation with sperm progressive motility. Blood and seminal plasma metabolic biomarker namely, aspartate aminotransferase (AST) (p < 0.01) was significantly lower in the boron and selenium supplemented group than control, while alanine aminotransferase (ALT) (p < 0.05) was significantly lower in the boron supplemented group than selenium and control group. There was a significant increase in the mRNA expression of serine proteinase inhibitor (SERPIN) and interferon γ (IFNγ) in the testis of boron supplemented than the control group. Boron supplementation up-regulated the immune-regulatory gene, interleukin 2 (IL2) and antioxidant gene, catalase (CAT) in the peripheral blood mononuclear cells (PBMC). On contrary, toll-like receptor 2 (TLR2) mRNA expression was significantly (p < 0.05) down-regulated in boron and selenium supplemented groups. CONCLUSION: The study revealed that dietary boron supplementation increased the sperm output, sperm motility and enhanced the immune and antioxidant defense capacity in male goats. The improved semen quality can be attributed to enhanced expression of testicular SERPIN, a crucial protein for the regulation of spermatogenesis process.

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

PROBLEM: 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. METHOD OF STUDY: In this study, we developed a novel method using computer-aided sperm analysis. The results were compared with those obtained by the traditional method. RESULTS: 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. CONCLUSION: 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.

Human Papillomavirus Prophylactic Vaccination improves reproductive outcome in infertile patients with HPV semen infection: a retrospective study.

In this study we aimed to evaluate the effect on reproductive outcome of HPV vaccination in male subjects of infertile couples with HPV semen infection. In this single-center study, we retrospectively enrolled 151 infertile couples with detection of HPV in semen, attending our Hospital Unit of Andrology between January 2013 and June 2015, counseled to receive adjuvant HPV vaccination. Seventy-nine accepted vaccination (vaccine group) whilst 72 did not (control group). Our protocol of follow-up, aimed to evaluate HPV viral clearance, consisted in semen analysis, INNO-LiPA and FISH for HPV in semen cells after 6 and 12 months from basal evaluation. Spontaneous pregnancies, miscarriages and live births were recorded. Progressive sperm motility and anti-sperm antibodies were improved in the vaccine group at both time points (p < 0,05 vs control arm). Forty-one pregnancies, 11 in the control group and 30 in the vaccine group, were recorded (respectively 15% and 38,9%, p < 0,05) and resulted into 4 deliveries and 7 miscarriages (control group) and 29 deliveries and one miscarriage (vaccine group, p < 0,05 vs control group). HPV detection on sperms was predictive of negative pregnancy outcome. Adjuvant vaccination associated with enhanced HPV healing in semen cells and increased rate of natural pregnancies and live births.

Effect of hepatitis B virus infection on sperm quality and oxidative stress state of the semen of infertile males.

PROBLEM: The effects of hepatitis B virus (HBV) infection on sperm quality and oxidative stress state of the semen of infertile males remain undetermined. METHOD OF STUDY: Normal males and 60 semen samples from infertile males (with or without HBV infection) were subjected to semen analysis. RESULTS: Semen volume, semen pH, sperm density, percentage of forward, movement of sperm, sperm activation rate, sperm survival rate, rate of normal sperm morphology of infertile males with HBV infection were significantly lower than those of infertile males without genital infection and of normal males (P<.05), while interleukin (IL)-17, IL-18, and malondialdehyde (MDA) levels in subjects with HBV infection were significantly higher than those of infertile males without genital infection and of normal males (P<.05). In patients with HBV infection, MDA level was found to be negatively correlated with semen quality, but positively correlated with semen IL-17 and IL-18 concentrations. CONCLUSIONS: HBV infection increased MDA level, induced abnormal expression of IL-17 and IL-18, and negatively affected male reproductive capacity, resulting in male infertility.

Incidence of Sperm Surface Autoantibodies and Relationship with Routine Semen Parameters and Sperm Kinematics.

PROBLEM: Antisperm antibodies (ASA) are associated with male subfertility. However, results on sperm surface autoantibodies are controversial, the relationship between ASA and semen parameters (WHO, 2010) is unknown, and data on ASA and sperm kinematics are scarce. METHOD OF STUDY: A retrospective study carried out in men undergoing routine semen analysis (WHO 2010), ASA evaluation (direct SpermMAR(™) (IgG) test), and computer-assisted sperm analysis (CASA). RESULTS: A 2.6% and a 5.9% incidence of ASA-positive cases were found (cut-off 50% and 10%, respectively; n = 7492). ASA-positive samples had lower (P < 0.0001) sperm concentration, count, motility, and hypo-osmotic swelling (HOS) test score. HOS results did not correlate with sperm vitality in normozoospermic samples with high ASA levels. In unselected samples, ASA-positive samples (cut-off 50%) showed decreased sperm kinematics (VSL, VAP, LIN, ALH, STR, BCF, WOB), but in normozoospermic samples, ASA-positive and ASA-negative subgroups had similar CASA results. CONCLUSIONS: ASA evaluation is highly relevant in full semen assessment.

Reduction of fertility in male mice immunised with pSG.SS.C3d3.YL.Bin1b recombinant vaccine.

OBJECTIVES: To study the anti-fertility effect of a DNA vaccine using Bin1b as the target antigen in male mice. METHODS: A novel recombinant eukaryotic vector containing a fusion gene sequence of mouse Bin1b in tandem with three copies of C3d fragment (C3d3) was used to construct pSG.SS.C3d3.YL.Bin1b. The correct expression of the Bin1b-C3d3 protein was confirmed in transfected HEK293 cells by indirect immunofluorescence and western blot analysis. The fertility of immunised mice was determined by a mating experiment and sperm motility test. Anti-Bin1b antibody titres in sera were examined by ELISA assays. Binding activity of C3d3 fragment of the fusion protein was verified in C3d receptor-expressing Raji cells and flow cytometric analysis. RESULTS: Immunisation of pSG.SS.C3d3.YL.Bin1b recombinant DNA vaccine significantly decreased sperm motility and compromised fertility in male mice. ELISA results showed that the titres of anti-Bin1b IgG in sera of immunised mice increased markedly with the immunisation process. Further, the anti-fertility effect of pSG.SS.C3d3.YL.Bin1b was significantly better than that of pSG.SS.YL.Bin1b DNA vaccine and generated higher titres of anti-Bin1b antibody. CONCLUSIONS: Our results show that recombinant DNA vaccine targeting Bin1b can markedly reduce fertility in male mice, providing an alternative approach for birth control.

Vacuolar-ATPase isoform a2 regulates macrophages and cytokine profile necessary for normal spermatogenesis in testis.

a2V is required for maturation of sperm. The decreased expression of a2V at the feto-maternal interphase causes poor pregnancy outcome. The present study examined the role of a2V in spermatogenesis and inflammatory network in the testis. A single dose of anti-a2V mouse IgG or mouse IgG isotype (3 µg/animal) was injected i.p. into male mice on alternate days for 10 days. Anti-a2V-treated males exhibit severe deficiencies of spermatogenesis, which is indicated by the presence of less numbers of postmeiotic cells. Sperm counts and sperm motility were reduced significantly in anti-a2V-treated males. The release of the cleaved a2NTD was significantly lower in anti-a2V-treated testes. The TMs were identified as M2-like macrophages, and this population and the expression of various cytokines/chemokines (Tgf-ß, Il-6, Nos2, Tnf, Lif, Mcp1, Ccl5) were decreased significantly in anti-a2V-treated testis compared with control testis. Moreover, the cleaved a2NTD acts as a key mediator of TMs and significantly up-regulates the secretion of testicular cytokines/chemokines, which are associated with normal spermatogenesis. When these anti-a2V-treated males were used for mating with normal females, the number of implantation sites was decreased significantly in the females mated with anti-a2V-treated males than the females mated with control males. These observations suggest that a2V plays a crucial role in spermatogenesis by regulating testicular immune responses, and its inhibition in males leads to poor pregnancy outcome in females.

The effect of 17α-ethynylestradiol on steroidogenesis and gonadal cytokine gene expression is related to the reproductive stage in marine hermaphrodite fish.

Pollutants have been reported to disrupt the endocrine system of marine animals, which may be exposed through contaminated seawater or through the food chain. Although 17α-ethynylestradiol (EE2), a drug used in hormone therapies, is widely present in the aquatic environment, current knowledge on the sensitivity of marine fish to estrogenic pollutants is limited. We report the effect of the dietary intake of 5 µg EE2/g food on different processes of testicular physiology, ranging from steroidogenesis to pathogen recognition, at both pre-spermatogenesis (pre-SG) and spermatogenesis (SG) reproductive stages, of gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost. A differential effect between pre-SG and SG specimens was detected in the sex steroid serum levels and in the expression profile of some steroidogenic-relevant molecules, vitellogenin, double sex- and mab3-related transcription factor 1 and some hormone receptors. Interestingly, EE2 modified the expression pattern of some immune molecules involved in testicular physiology. These differences probably reflect a developmental adjustment of the sensitivity to EE2 in the gilthead seabream gonad.

Methods for direct and indirect antisperm antibody testing.

Antisperm antibodies (ASA) are one well-known cause of refractory infertility in both males and females. In females, a sperm immobilization test, which detects sperm-immobilizing antibodies indirectly in the patient's serum, requires complement for the reaction and thus seems to be a more specific immunological reaction. In males, an immunobead test or a mixed antiglobulin reaction test, which detects ASA directly on the sperm surface, is a screening test because of the nonspecific reaction.

Evaluation of contraceptive potential of a novel epididymal sperm protein SFP2 in a mouse model.

PROBLEM: Sperm flagellar protein 2 (SFP2), which was earlier identified using a novel combinatorial approach, was evaluated for its contraceptive potential in mice. METHOD OF STUDY: Male mice were actively immunized with two synthetic peptides of SFP2. Antipeptide antibody was characterized by Western blot and indirect immunofluorescence. Immune response was monitored, and mating studies were performed 6 and 22 weeks post-immunization. RESULT: Antibodies to the SFP2 peptide 1 recognized a doublet at 220- to 230-kDa region only in the epididymal protein extract. Peptide 1 antibody recognized the cognate protein on spermatozoa from mouse, rat, and human. Histological analysis of testis and epididymis of the immunized mice indicated no deleterious effect. Incubation of sperm with the immune sera of peptide 1 caused significant reduction in motility and viability but did not agglutinate sperm. Only synthetic peptide 1 gave rise to high-level antibodies in all the immunized mice, which on mating resulted in reduced fertility rate (20%) when compared with PBS control animals (100%). The antibody levels in the immunized males declined by 22 weeks post-immunization, resulting in 100% reinstatement of fertility. CONCLUSION: These data provide an experimental basis for the development of effective contraceptive vaccine based on new epididymal target.

Anti-Helicobacter pylori antibodies in cervical mucus: a new cause of infertility.

OBJECTIVE: The aims of our study were to determine on the one hand a correlation between the presence of anti-Helicobacter pylori (anti-H. pylori) IgG antibodies in serum and cervical mucus of women with idiopathic infertility, and on the other hand the effect of these antibodies on cervical mucus quality, in particular related to the ability of spermatozoa to penetrate it. STUDY DESIGN: We analysed anti-H. pylori IgG antibodies in the serum and cervical mucus of 67 patients diagnosed with idiopathic infertility using the Quanta Lite H. pylori IgG test. The penetration of normal sperm, in 15 cervical mucus samples positive for anti-H. pylori antibodies and in 15 negative samples, was assessed using the simplified slide test. RESULTS: A significant positive correlation emerged between anti-H. pylori IgG antibody concentrations in the serum and in the cervical mucus (r=0.9275; p<0.00001). In the 15 anti-H. pylori IgG mucus-positive samples the slide test showed abnormal penetration by the spermatozoa. CONCLUSIONS: Our study demonstrated that the presence of anti-H. pylori antibody in the cervical mucus can be involved in female infertility, interfering with sperm progression. Considering the close correlation found between serum and cervical mucus anti-H. pylori antibody titres, measuring serum antibodies could become an additional test, in particular in couples with unexplained infertility.

Effect of vaccination of boars against porcine circovirus type 2 on ejaculate characteristics, serum antibody titers, viremia, and semen virus shedding.

The objective of this research was to determine the effect of vaccination against porcine circovirus type 2 (PCV2) on ejaculate characteristics, PCV2-specific antibody titers in serum, viremia, and viral shedding in the semen of PCV2-positive boars. Before vaccination, all boars were confirmed by PCR to be naturally infected with PCV2. The boars were vaccinated with a commercial killed vaccine against PCV2 (n = 5) or served as controls and received 2 mL of 0.9% saline (n = 5). Semen and blood samples were collected before vaccination at wk 0 and at 7-d intervals thereafter until wk 8. Sperm concentration and characteristics of sperm motility were assessed using a computer-assisted sperm analysis system, and sperm morphology was evaluated using light microscopy after staining. The PCV2 antibody titers were determined in serum using an ELISA, and the genomic copy numbers of PCV2 DNA in serum and semen were determined by real-time PCR. In general, there were no effects of treatment or treatment × week on semen or sperm characteristics (P > 0.10). An effect of treatment × week was detected for serum antibody titers (P < 0.01). Compared with controls, PCV2 antibody titers in vaccinated boars were less (P < 0.01) at wk 7 (1.01 ± 0.05 titer/mL vs. 1.23 ± 0.05 titer/mL) and tended (P = 0.07) to be less at wk 8 (1.05 ± 0.05 titer/mL vs. 1.17 ± 0.05 titer/mL). There were no effects of treatment or treatment × week for serum and semen genomic copy numbers of PCV2 DNA (P > 0.10). There was a tendency (P = 0.09) for an effect of week on serum viral load. It was evident that during this experiment, boars experienced reoccurring PCV2 infection, and the detection of an increased PCV2 DNA load in serum preceded that in semen; the duration of reoccurring infection appeared to be less in vaccinated boars compared with controls. In summary, vaccination against PCV2 can reduce antibody titers when given postinfection and has no dramatic effect on indicators of semen quality. Vaccination against PCV2 in naturally infected boars can also decrease the length of reoccurring infection and decrease the duration of viral shedding in semen.

Anti-sperm antibodies are not associated with sperm DNA damage: a prospective study of infertile men.

Infertile men have poorer sperm DNA integrity than do fertile men, and this damage may contribute to reducing male reproductive potential. However, the etiology of this damage has not been fully characterized. We sought to examine the relationship, if any, between anti-sperm antibodies and sperm DNA damage in a consecutive series of non-azoospermic, infertile men in order to determine whether anti-sperm antibodies (ASAs) may contribute to sperm DNA damage. We conducted a prospective study on consecutive semen samples obtained from men (n=75) presenting for infertility evaluation. Sperm concentration, motility, strict morphology, ASA levels (by direct mixed agglutination reaction, expressed as the percentage of spermatozoa with IgG or IgA antibodies) and sperm DNA damage (by sperm chromatin structure assay) were evaluated. Mean (+/-SD) sperm concentration and progressive motility were significantly lower in ASA-positive (>40% of sperm coated with ASAs) compared to ASA-negative samples (23.4+/-13.1 x 10(6)/mL and 27+/-15% vs. 74.6+/-61.2 x 10(6)/mL and 46+/-18%, respectively, P<0.05). Sperm progressive motility was inversely correlated with the percentage of IgG-bound (r=-0.33) and IgA-bound spermatozoa (r=-0.25). In contrast, sperm %DNA fragmentation index and percent normal forms were not significantly different in ASA-positive compared to ASA-negative samples (17.5+/-17.9% and 7.5+/-3.0% vs. 17.4+/-13.5% and 6.5+/-2.6%, respectively). The data indicate that ASAs are not associated with sperm DNA damage and suggest that ASAs are unlikely to have a significant direct or indirect effect on sperm DNA integrity.

Effect of lycopene on semen quality, fertility and native immunity of broiler breeder.

1. The effect of drinking water supplementation with lycopene on the semen quality, fertility and immunity of broiler breeders was evaluated. 2. Broiler breeder males were individually caged from 25 to 42 weeks old and divided into two group: L group, treated birds (lycopene 0.5 g/l) and C group, control birds. Laying hens were divided into two groups and artificially inseminated. 3. Semen variables were evaluated and daily fertility recorded. Serum bactericidal activity was tested. 4. Semen production and viability were affected by lycopene supplementation. Serum bactericidal activity was better in L than in C group. The fertility rate curve of the L group displayed a positive trend.

Immunization with Bin1b decreases sperm motility with compromised fertility in rats.

OBJECTIVE: To evaluate the contraceptive ability of a synthetic Bin1b peptide in vivo in the rat. DESIGN: Basic research. SETTING: University laboratory animal service center. ANIMAL(S): A peptide-based immunization model was developed; rats were injected with the Bin1b specific peptide. INTERVENTION(S): A synthetic peptide segment, MCRSGERKGDICSDP-conjugated with KLH (Bin1b), was used to immunize male wistar rats. Freund's complete adjuvant was used as a control. MAIN OUTCOME MEASURE(S): Anti-Bin1b levels in sera were evaluated by enzyme-linked immunosorbent assay (ELISA). Anti-Bin1b and control antisera were used to evaluate sperm function inhibition in vitro. The fertility of immunized rats was determined by mating experiment. The testis and epididymides were analyzed by histology. RESULT(S): Histological studies showed no evidence of orchitis or epididymitis in Bin1b-immunized animals. ELISA results revealed that the titers of anti-Bin1b antibodies in serum increased with the immunization process in immunized rats. Sperm recovered from the corpus epididymidis of the Bin1b-immunized animals exhibited a significant decrease in motility. Immunization of Bin1b also caused a reduction (25%) in fertility after the mating experiment. CONCLUSION(S): The present study has demonstrated that immunization with Bin1b peptide specifically interferes with sperm motility, resulting in a compromised fertilizing capacity of sperm.

Immunocontraceptive potential of the Ig-like domain of Izumo.

To investigate whether the Ig-like domain of sperm protein Izumo or the other part of the protein could be used as an immunocontraceptive antigen, three partially overlapping cDNA fragments (PA, PB, and PC), together covering entire mouse Izumo, were cloned, expressed, and purified. PB contains the whole Ig-like domain of mouse Izumo. The anti-PB antibody significantly inhibited the fusion of sperm with zona-free mouse eggs with no effect on sperm motility, while anti-PA and anti-PC antibodies virtually had no effect on sperm-egg fusion at the same concentration. Furthermore, in the presence of anti-PB antibody, the anti-sperm reactivity could be competitively inhibited by recombinant PB protein. The PB-specific antibody staining was restricted to the acrosome region in acrosome-reacted mouse spermatozoa by indirect immunofluorescence. Active immunization with the PB antigen sharply raised the antibody titers in mouse that were enough to cause a significant reduction in fertility compared to the PA and PC immunized groups. In conclusion, our data indicate that the Ig-like domain of Izumo plays an important role in the fertilization process, as verified by the dose-dependent reduction in fertilization rates in mouse IVF trials and mouse mating assay. These results indicate that the Ig-like domain of Izumo might be a new candidate for the development of a contraceptive vaccine.