This review summarizes the advancements over a decade of research on antigens of anti-sperm antibodies (ASAs), which are key to male immune infertility. Despite the progress in assisted reproductive technologies, understanding the roles and mechanisms of ASAs and their antigens remains vital for immune infertility management. We conducted a comprehensive literature search on PubMed from January 2013 to December 2023 using the following keywords: \"anti-sperm antibody,\" \"sperm antigen,\" and \"immune infertility.\" In this review, we focus on the discoveries in sperm antigen identification and characterization through proteomics, gene disruption technology, and immunoinformatics, along with the development of fertility biomarkers. Here, we discuss the clinical applications of improved ASA detection methods and the progress in the development of immunocontraceptive vaccines. The intersection of advanced diagnostic techniques and vaccine development represents a promising frontier in reproductive health. The findings also highlight the need for standardized ASA detection methods and a comprehensive molecular-level approach to understanding ASA-related infertility. These insights underscore the significance of ongoing reproductive immunology research in enhancing clinical fertility outcomes and contraceptive vaccine development.

CatSper is a voltage dependent calcium ion channel present in the principal piece of sperm tail. It plays a crucial role in sperm hyperactivated motility and so in fertilization. Extracellular loops of mouse sperm CatSper were used to develop a vaccine to achieve protection from pregnancy. These loops were inserted at one of the three hypervariable regions of Human Papilloma Virus (HPV) capsid protein (L1). Recombinant vaccines were expressed in E.coli as inclusion body (IB), purified, refolded and assembled into virus-like particles (VLP) in vitro, and adsorbed on alum. Four vaccine candidates were tested in Balb/C mice. All the constructs proved immunogenic, one showed contraceptive efficacy. This recombinant contraceptive vaccine is a non-hormonal intervention and is expected to give long-acting protection from undesired pregnancies.

Rodent population control through contraception requires species-specific oral contraceptive vaccines. Therefore, in this study, we produced putative mouse-specific contraceptive peptides, mZP2 (from oocyte) and mIzumo1 (from sperm), in plants using Agrobacterium-mediated transient expression. Peptides were produced separately in Nicotiana benthamiana using constructs encoding antigens containing three copies of each peptide. We also determined the immunogenicity and contraceptive effects of the plant-produced antigens in female BALB/c mice. Mice immunized subcutaneously with a relatively low amount of antigen (5 µg/dose of each peptide in a mixture) showed systemic immune responses against mZP2-3 and mIzumo1-3 antigens. Moreover, the mean litter size of mice treated with the plant-produced antigens was reduced by 39% compared to that of the control mice. Notably, there was a significant negative correlation between the number of pups born and individual antibody levels against both antigens. Immunofluorescence assays demonstrated the binding of induced antibodies to the oocytes of BALB/c and wild-type mice in vivo and in vitro, respectively. Our study demonstrate the feasibility of producing small contraceptive peptides in plants that can be further used to develop oral contraceptive vaccines against mouse populations.

A short mouse-specific peptide from zona pellucida 3 (mZP3, amino acids 328-342) has been shown to be associated with antibody-mediated contraception. In this study, we investigated the production of mZP3 in the plant, as an orally applicable host, and examined the immunogenicity of this small peptide in the BALB/c mouse model. The mZP3 peptide was inserted into the major immunodominant region of the hepatitis B core antigen and was produced in Nicotiana benthamiana plants via Agrobacterium-mediated transient expression. Soluble HBcAg-mZP3 accumulated at levels up to 2.63 mg/g leaf dry weight (LDW) containing ~172 µg/mg LDW mZP3 peptide. Sucrose gradient analysis and electron microscopy indicated the assembly of the HBcAg-mZP3 virus-like particles (VLPs) in the soluble protein fraction. Subcutaneously administered mZP3 peptide displayed on HBcAg VLPs was immunogenic in BALB/c mice at a relatively low dosage (5.5 µg mZP3 per dose) and led to the generation of mZP3-specific antibodies that bound to the native zona pellucida of wild mice. Oral delivery of dried leaves expressing HBcAg-mZP3 also elicited mZP3-specific serum IgG and mucosal IgA that cross-reacted with the zona pellucida of wild mice. According to these results, it is worthwhile to investigate the efficiency of plants producing HBcAg-mZP3 VLPs as immunogenic edible baits in reducing the fertility of wild mice through inducing antibodies that cross-react to the zona pellucida.

To manage population of dogs (Canis familiaris), the efficacy of recombinant proteins-based contraceptive vaccines to inhibit fertility has been evaluated in female beagle dogs.
Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli-expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT-KK-pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase-KK-pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3-GnRH2 ); control animals received only alum, the adjuvant. The immunized animals were followed-up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs.
Active immunization of female dogs following a three injections schedule at 4-week intervals with a physical mixture of TT-KK-pZP3 + bRNase-KK-pZP4 as well as dZP3-GnRH2 , led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3-GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT-KK-pZP3 + bRNase-KK-pZP4 or dZP3-GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3-GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3-GnRH2 immunized group was associated with antibody titres against dZP3-GnRH2 . However, immunization with recombinant TT-KK-pZP3 + bRNase-KK-pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control.
These studies revealed the potential of recombinant dZP3-GnRH2 -based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted.

Mammalian zona pellucida (ZP) is composed of three to four glycoproteins, which plays an important role during fertilization. Mutations in the genes encoding zona proteins are reported in women with empty follicle syndrome, degenerated oocytes and those with an abnormal or no ZP further emphasizing their relevance during fertility. Immunization with either native or recombinant ZP glycoproteins/proteins leads to curtailment of fertility in various animal species. Observed infertility is frequently associated with ovarian pathology characterized by follicular atresia and degenerative changes in ZP, which may be due to oophoritogenic T cell epitope(s) within ZP glycoproteins. To avoid ovarian dystrophy, B cell epitopes of ZP glycoproteins have been mapped by using bio-effective monoclonal antibodies. Immunization with the immunogens encompassing the mapped B cell epitopes by and large led to amelioration of follicular atresia. However, their use for human application will require more rigorous research to establish their safety and reversibility of the contraceptive effect. Nonetheless, to minimize human-animal conflicts, ZP-based contraceptive vaccines have been used successfully in the population management of free-ranging animal species such as feral horses, white-tailed deer and elephants. To control zoonotic diseases, attempts are also underway to control the population of other animal species including stray dogs, which acts as one of the major vectors for the rabies virus.

The cyclical proliferation of the wild fossorial rodent Arvicola terrestris scherman (ATS) is critical in mid-mountain ecosystems of several European countries. Our goal is to develop an immunocontraceptive vaccine to control their fertility, as a sustainable alternative to chemical poisons currently used. Indeed, these chemicals cause the death of ATS predators and animals sharing their ecosystem, and current laws progressively limit their use, making the development of a targeted vaccination strategy an interesting and efficient alternative. In order to identify species-specific sperm antigens, male and female ATS received subcutaneous injections of whole ATS spermatozoa to elicit an immune response. The analysis of the immune sera led to the identification of 120 immunogenic proteins of sperm cells. Of these, 15 were strictly sperm-specific and located in different regions of the male gamete. Some of these antigens are proteins involved in molecular events essential to the reproductive process, such as sperm-egg interaction, acrosomal reaction, or sperm motility. This approach not only identified a panel of immunogenic proteins from ATS sperm cells, but also demonstrated that some of these proteins trigger an immune response in both male and female ATS. These spermatic antigens are good candidates for the development of a contraceptive vaccine.

BACKGROUND: The management of stray dog population has been of utmost importance due to their overpopulation, increase in dog bites incidence, and rabies. Contraceptive vaccines, a non-surgical alternative to spaying and neutering are viewed as a valuable option for the management of dog population. In this study, the contraceptive potential of a recombinant fusion protein containing the three genes GnRH, GnRH receptor, and ZP3 was explored.
RESULTS: The gene fragment encoding GnRH, GnRHR, and ZP3 along with the antigenic epitopes of canine distemper virus and tetanus toxoid was assembled, synthesized, and cloned into pET28a expression vector. The resulting construct GVAC08 was successfully transformed into BL21DE3 strain of E. coli and confirmed by colony PCR. The recombinant GVAC08 protein was expressed and purified using Ni-NTA and was confirmed to be a 50-KDa protein by SDS PAGE and Western blot. Mice were immunized with the GVAC08 protein using Freund\'s complete adjuvant followed by a booster using Freund\'s incomplete adjuvant. This induced a high antibody titer against GnRH, GnRH receptor, and ZP3 which was determined by ELISA.
CONCLUSIONS: Mating studies showed that the GVAC08 recombinant protein was able to reduce the litter size in immunized mice showing improved efficacy. However, the vaccine candidate with further improvements will be a viable contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 μg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

The functional competence of leukemia inhibitory factor (LIF), as immunocontraceptive vaccine in mice, was investigated. Balb/c mice were divided into two groups of vaccinated and controls. The recombinant human LIF (rhLIF) protein and phosphate buffer saline was emulsified with Freund\'s adjuvant and injected into vaccinated and control groups, respectively. Theinhibition of implantation was evaluated in mice uterine. The concentration of secreted interferon-γ (IFN-γ) and interleukin (IL)-4 were measured in cultured splenocyte of mice stimulated by rhLIF. The expressions of immune responsive gene 1 (IRG-1), cochlin (COCH), amphiregulin(Ar), and heparin-binding EGF-like growth factor (HB-EGF) genes were determined. Mice were assessed for inhibition of fertility after delivery, reversibility of immune response against rhLIF, and survival rate. Active immunization of mice with rhLIF resulted in reduction of the implantation and fertility rate up to 80.49% and 75%, respectively. All mice produced a high titer of anti-rhLIF antibodies in serums and vaginal fluids washes after 16 weeks; however, these antibodies were cleared from vaginal fluid washes after six months. A significant down-regulation in mRNA levels of IRG-1, Ar and HB-EGF was observed in vaccinated group compared to controls; however, no significant change in the expression profile of cochlin gene was detected. The results showed that rhLIF prevented pregnancy in a high percentage of female mice. Although the immunization of female Balb/c mice with rhLIF inhibited fertility and expression of genes associated with this molecule, further studies are needed to support this protein as a suitable candidate for contraceptive vaccine in mammals.