IMMUNOLOGICAL BASIS OF INFERTILITY IN ANIMALS Introduction Infertility, the inability of a couple to conceive after a period of 12 months of intercourse without

t the use of contraception, is a worldwide problem. Pathologic immune response is one of its aetiologies even if beyond doubt proofs have not been so far established. The objective of our review on immunologic infertility is to synthesize data on auto antigens tolerance, its loss, the pathogenesis of immune induced infertility, the laboratory support for diagnosis and the treatment. Literature search was performed on Medline. It is admitted that the cellular and the humoral components of immune system interact in a balanced manner. Once tipped in favour of pathologic influence, the antibodies occur. The intimate mechanism of infertility is unclear, the results of laboratory tests are less sharp and the treatment outcome is conflicting. Medically assisted procreation is promising but its success should not hide possible deleterious effects. More investigations are required to clarify the pathogenesis of immune induced infertility and thus, the improvement of the treatment.(Smith et al 1999)

DEFINTION Infertility is defined as the inability of a couple to conceive after a period of 12 months of intercourse without the use of contraception . This public health problem involves all regions of the World. Its prevalence varies from 10 to 30 % . Immune disease is a condition resulting from a conflict between antigens and antibodies. An autoimmune disease occurs when the immune system attacks self molecules as a result of a breakdown of immunologic tolerance to auto reactive immune cells . It is admitted today that unexplained infertility is often associated to immunologic factors . This condition may be a result of autoimmunity (in man and woman) or of isoimmunity (in woman).

The purpose of summarise the current knowledge in this field for the following points: 1. The factors of antigens tolerance 2. How this tolerance is broken-down 3. The pathogenesis of the immunologic infertility 4. The laboratories tools for the detection of the antibodies 5. The therapeutic means available. Data for the 2 first points are quite established. The 3 last points are debating so far; they will constitute the main subjects of our discussion. The review will be closed by the key features of the future. A. The self antigens tolerance The cellular and humoral components of the immune system interact with the testis and the ovary in a well orchestrated manner . ( Risvanli, et. al, 2004). Testis and sperm At the puberty, in testis, the differentiating germ cells begin to express new antigens that the immune system has not seen before. These antigens are considered as foreign according to the Burnets clonal selection theory. In the interstitial spaces between seminiferous tubules exists an immunocompetent tissue (macrophages and lymphocytes) .Many theories are evoked to explain the tolerance between sperm as auto antigen and the neighbour immunocompetent cells.The concept of blood testis barrier: this barrier includes the myoid cells lining the outer surface of the seminiferous tubules, Sertoli cell tight junctions, various immunosuppressive agents and genetic influences. This theory is not sufficient because some molecules may cross the myoid cells and the tight junctions of the Sertoli cells are less dense at the level of the rete testis, afferent ductules and epididymis, rendering these areas unprotected. The following theory suggests the existence of a down regulation of cell mediated immune system at this level by possible four mechanisms : the vascular endothelium confines the orchitogenic T cells and reduce their number, the T Suppressor cells suppress any immune reaction, the environmental defect hinders the proper presentation of sperm antigens to lymphocytes and a small, constant antigen leak through these weaker areas serves to desensitise the immune system to sperm antigens. The 3rd theory: cytokins

and other humoral mediators of the immune response may contribute to tolerance within the testis. Ovary In mice, the data indicate that the potentially pathogenic T cells (pathogenic self-reactive T cells = oophoritogenic T cells) are present in adult spleens. The Suppressor (Regulatory) T cells, present in adult thymus and adult spleen have the capacity to render pathogenic selfreactive T cells non functional in the normal individual. This is thought that the same mechanism protects the animal ovary. Autoimmunity and antigenic targets in ovarian pathology The involvement of autoimmune mechanisms in premature ovarian failure has been put forward by numerous investigators. In various other ovarian pathologies, such as idiopathic infertility, polycystic ovary syndrome, or endometriosis, similar mechanisms have been suggested. However, the exact role of autoimmunity in the patho physiology of these diseases still remains controversial. The diagnosis of autoimmune ovarian disease relies on several clinical, biological and histological findings, but special interest has been focused on antiovarian autoantibodies. The search for these antibodies has been undertaken by several authors and yielded somewhat conflicting results which might be conditioned by methodological differences and by the multiplicity of potential immune targets. These targets, which comprise various steroidogenic enzymes, gonadotrophins and their receptors, the corpus luteum, zona pellucida and oocyte, are reviewed. Further investigation of these targets is required to improve the diagnostic tools that will lead to a precocious and reliable diagnosis of autoimmune ovarian disease, an appropriate clinical surveillance as well as the selection of patients who may benefit from immunemodulating therapy and possibly recover ovarian function and fertility B. Female tolerance to sperm antigens The female genital tract is endowed with immunological competent cells which phagocytose sperm and process their antigens for immune recognition. This operation may be influenced by the following factors : From the large number of sperm introduced into the upper vagina, it is possible that only a minority, immunologically different to the residual majority, gains access to the Fallopian tube.

 Sperm flushing from the genital tract reduces the number of those gaining access to the immune system. Degradation of sperm antigens by extracellular enzymes ,especially in the uterus. The phagocytosis of sperm by somatic cells and macrophages, the chemotactic attraction of sperm for macrophages and neutrophils in the uterine cervix and in the peritoneal cavity. The nonspecific binding of immunoglobilins or of non-pathogenic naturally occurring antibodies, onto senescent sperm as a preliminary to their phagocytosis. This mechanism may, in the opposite, enhance phagocytosis and immunogenicity. The presence of immunosuppressive factors in semen and follicular fluid. Individual specific characteristics of semen : sperm cells appear to be inherently immunosuppressive as a function of their physiological cell surface characteristics. Integrity of mucosal epithelium of the reproductive tract. Genetic influences The occurrence of immune disease

A. Autoimmune disease In both, testis and ovary, the autoimmune disease (orchitis and oophoritis) occurs when the balance is tipped in favour of pathogenic self reaction T cells, either by 1. Depletion of Suppressor (Regulator)T cells 2. Activation of pathogenic self-reactive T cells. Cellular and humoral response are both involved (Moncayo, et.al, 1992).

Testis Immunogenic testicular antigens or auto antigens can be classified as one or both the 2 types : testis specific and aspermatogenic. Testis specific antigens consist of any antigens in the testis that can induce orchitis. Aspermatogenic auto antigens are subset of testis specific antigens that induce an immune response resulting in destruction of germ cells and decreased sperm production. Only germ cells express aspermatogenic autoantigens . The autoimmune orchitis may lead ultimately to testicular atrophy and necrosis. (Fijak, et.al, 2004)

Ovary Ovarian structures recognised by the auto antibodies include Oocyte of developing follicles, The zona pellucida, Granulosa cells, Theca cells, Luteal cells and Receptors for human chorionic gonadotropin. Immune oophoritis occurs when animals are immunised with the crude ovarian extracts. It is characterised by massive atresia of the follicles followed by perivascular mononuclear cell inflammation. This leads to premature ovarian failure. The zona pellucida appears to be the most relevant and immunogenic component of these extracts. If its antigens are used as immunogens, oophoritis doesnt occur but permanent interruption of follicular development does . The pathology of autoimmune ovarian failure in female human is similar to that seen in experimental autoimmune oophoritis in neonatally thymectomised female mice .A possible role of the ovarian hyperstimulation before invitro fertilisation (IVF) in ovarian auto antibodies production is claimed by some authors and also the role of the ovarian repeated trauma for oocyte retrieval before IVF . (Agarwal, et. al, 2007). B . Antisperm antibodies (ASA) The fact that sperm can induce antibodies is known since 1899.ASA are detected in 7 to 15 % of men and in 13 to 80 % in women with unexplained infertility . They can be found in serum, seminal plasma, bound to sperm, cervical mucus. They are of the immunoglobulin (Ig)G, IgM and IgA class. Systemic inoculation of sperm antigen thought to stimulate IgG production, whereas local reproductive tract antigens stimulate IgA production . IgM is generally the 1st synthetised in response to a novel antigen. Because of their role in antibody mediated infertility presumably is limited, IgM antibodies are not routinely measured .( Bronson et al) The development of ASA in the male depends on the sequestration of antigens on germ cells by the presence of the bloodtestis barrier. During maturation of spermatogenesis, new antigens are expressed on developing spermatocytes and spermatids. When these antigens come into contact with immunocompetent cells, ASA formation occurs. Develop mental abnormalities of the formation of the blood testis barrier, traumatic disruption or unilateral focal cryptic obstructions could lead to

ASA formation. Also gastrointestinal exposure to sperm has been associated with the development of ASA both in animal experiments as well as in homosexual men. There are also results suggesting active local immunoregulatory mechanisms being operative within the testis (Bronson et al1999). The direct proof is easy to achieve for ASA, because they can be transferred to the sperm of healthy animals. This is a great advantage in comparison with auto antibodies in diseases of other organs. The impairment of functions (e.g. decrease of motility) can be demonstrated in the receiving cells. Since the direct proof of ASA specificity is easy to achieve, experimental models giving indirect proof, in particular in animals, are rare. Also circumstantial evidence is rare, in particular the favourable response to immunosuppressant was not demonstrated in ASA. This may be because it is impossible to treat patients seeking fertility with cytotoxic drugs, but not because of the ineffectiveness of the treatment. The observation that the expression of ASA is associated with certain HLA classes supports the suggestion that immune infertility represents an autoimmune disease.

In male, the following conditions are involved in generation of ASA : Testicular trauma (torsion, biopsy, obstruction vasa or seminal vesicle agenesis vasectomy) Infection (sexually transmitted infections, prostatitis, orchitis) Cancer Cryptochidism Varicocele. (Agarwal, et. al, 2007) Female possible contributing factors to the sperm isoimmunity are : Mechanical such as uterine cervix surgery chemical disruption of the mucosal layer of the genital tract. The possible immunopotentiating effects of other foreign antigens gaining access to the female genital tract. Lymphocytes in semen induce in female the sensitisation to histocompatibility antigens of the male partner. Sperm with surface bound antibodies are lymphocytes activators and gamma interferon inducers and hence, enhance ASA response in the female. Abnormal, senescent or damaged sperm increase sperm immunogenicity. Presumably, gastrointestinal exposure to sperm. Sperm within the peritoneal cavity after transtubal passage .

Pathogenesis of immunologic infertility The pathogenesis of infertility is quite clear in case of testis or ovary autoimmune disease. Indeed, infertility is due to the gonadal failure which leads to azoospermia in man and in premature ovarian failure.Infertility induced by ASA is more complex and is to date matter of debate. What seems to be widely admitted is the fact that local genital tract antibodies are more relevant in terms of fertility impairment than systemic antibodies and the fact that antibodies may have multiple biological functions and sites of action. iven the interaction between partners factors, we present for both sex simultaneously, the mechanisms evoked in the pathogenesis of infertility in the presence of ASA : Disordered spermatogenesis resulting in Oligozoospermia Azoospermia Cytotoxic antibodies Cellmediated immunity are involved. Antibodies binding to posttesticular sperm inhibit effective transport of sperm in the male reproductive tract. Auto agglutination of ejaculated sperm impairs sperm motility. Sperm cytotoxicity may be induced in both, male and female reproductive tract, by sperm antibodies or by a local cell mediated immune attack. In the female, one of the result is the activation of cytokines which may mimic the activity of intra uterine device. In the female tract, sperm antibodies arm macrophages and enhance phagocytose clearance of sperm within the uterine cavity or may immobilize sperm. ASA prevent sperm from adequately traversing of cervical mucus (due to agglutination or sperm shaking phenomenon) and may inhibit sperm transport and function within the tube and alters the percentage of normal sperm. By

interfering with sperm capacitation, inactivating sperm enzymes or disturbing acrosome reaction (premature lysis of the acrosome or premature acrosomal reaction). By blocking spermovum interaction, thus preventing fertilization or by inhibiting penetration of the oocyte. The ASA in sperm may induce sperm immunity in the female. Evidences from experimental animal models suggest that sperm antibodies can cause post fertilization reproductive failure and occult abortion by arresting embryo development and/or preventing implantation. The destruction of cells in maturing follicles before ovarian failure occurs.

Laboratories assays for immunologic infertility To date, very few cell mediated immunity assays have been introduced. This is due to the complexity of the cellular immune system and to the fact that the cellular components mediating infertility are still being defined and elaborated .When infertility caused by ASA is suspected (unexplained infertility, clinical status mentioned above, poor post coital test or shaking phenomenon), one may use one of the assays available. They can be divided into 5 groups :

1.Agglutination tests Macro agglutination tests Gel agglutination test (GAT) Mixed anti globulin reaction (MAR) Direct or indirect immunobead test (IBT)

Micro agglutination tests tray slide agglutination test (TSAT) microtray agglutination test(TAT or MAT)

2.Immobilization test Sperm immobilizing test (SIT)

3.Antibody fluorescence Immuno fluorescence (IF) Flow cytometry

4. Colorimetry Enzyme linked immunosorbent assay (ELISA)

5. Radio immuno assay Radiolabelled antiglobulin test

Each of these assays has advantages and drawbacks. The best assay may be a proper combination of tests that could accurately offer localisation, isotype and quantification of antibody binding . Treatment of immunologic infertility Immune gonadal failure In case of immune testis or ovarian failure, the possible treatment is gametes gift or adoption. Antisperm antibodies induced infertility Prevention : Animal data suggest that ASA formation may be prevented by prompt antibiotic treatment in case of suspected genital tract infection in men. Treatment : proposed therapies for ASA induced infertility include : Methods to reduce the production of ASA: Use of Processing sperm to decrease the effect of ASA: washing, enzymatic treatment, Split ejaculate, supplemented proteins medium, density gradient, swimup pentoxilline use Separation of ASA bound sperm from non bound sperm Immune depletion by coating magnetic micro beads with human antiimmunoglobulins mixing them with sperm Suppression of antibodies production : corticosteroids.

Overcoming possible ASA interference by Assisted Reproductive Technology (ART), 1. Intracervical insemination (ICI), 2. In vitro fertilization (IVF), 3. Gametes intra fallopian transfert (GIFT), 4. Subzonal sperm insertion (SUZI) 5. Intra cytoplasmic sperm injection (ICSI). 6. Intra uterine Insemination

Discussion We focused our discussion of three points : the pathogenesis of the immunologic infertility, the assays to detect ASA and the proposed therapies because they are the most conflicting matters in this field of medicine.Meanwhile, we will begin by a discussion on the possible effect of ovarian hyperstimulation (OHS) on ovarian auto-antibodies and the effect of uterine cervix surgery on the ASA production. Ovarian hyperstimulation,oocyte retrieval and anti-ovarian antibodies As we will see later, assisted reproductive technology-mainly IVF and ICSI are the most used therapies for the treatment of the immunologic infertility. The oocyte retrieval is preceded by OHS. Some studies found a relationship between OHS and ovarian autoantibodies. This relationship is supported by hormonal theory and clinical observations . Dynamic interrelationship among the hypothalamus and the pituitary, the ovary and the immune system might be disturbed by the addition of exogenous gonadotropin and Cutolo M.et al. ( mentioned by Smith et al. reported that oestrogen and oestrogen like chemical may alter the immune response. Clinically, a significant incidence of seroconversion (ASA negative to ASA positive) during hormonal stimulation is reported and also an association between ovarian auto antibodies and the development of occult POF in patients who fail to conceive after IVF.Many author found associated factors to cell-mediated immunity (Il6, T8 cells...) in follicular fluid ( .Their conclusions are as unclear as conflicting. Some studies found a relation between oocyte retrieval for IVF and the occurrence of antiovarian antibodies. For these authors, antiovarian antibodies are induced by repeated follicular puncture rather than hormonal stimulation. A great success of IVF and ICSI seems to obviate this question. Most of the aforementioned studies are old. We encourage current studies, scientifically valid, to elucidate this problem. B. Uterine cervix surgery and ASA The data are conflicting : Nicholson in 1996 found that uterine cervix surgery induces the production of ASA but recently. We think that the disruption of mucosal layer, putting in contact sperm and female blood, may really induce ASA formation. In waiting for the results of most numerous studies, we share the opinion of Nicholson who advises the use of the postponement of intercourse until the total healing occurs.

C. Pathogenesis of immunologic infertility The occurrence or infertility in case of testis or ovarian autoimmune disease is due to the destruction of the tissue from which gametes are normally produced. Despite what we wrote above, the precise effect of ASA which result in impairment of fertility is less clear . The widely accepted impairment of sperm migration through cervical mucus by IgA ASA is hardly supported by clinical experiences . Why this impairment is not consistent or need high titres is not clear .The existence of many sperm antigens may explain it. We think that there are also many subsets of IgA, the basis of our hypothesis. The interference of ASA with sperm-egg interaction, with zygote development an implantation is debatable. Indeed, we think that ASA should no longer play a role after pro-nuclei fusion since this entity is a new structure which theoritically should be immunologically different from the gametes. The occult abortion might be a precocious expression of severe chromosomal abnormalities as observed during the 1st trimester spontaneous abortion . D. Assays to detect ASA There is a wide range of tests used for antispermes antibodies detection .The choice is based upon the sample under testing . The training of clinician and /or technologist, the conviction of clinician difficulties of processing. The conflicting data reported may be the result of differences between ASA testing modalities which vary in sensibility and specificity , specimen preparation, test interpretation, internal and between laboratories variability . Furthermore, sperm undergoes maturational changes (capacitation, acrosomal reaction) which results in changing ASA epitopes . World Health Organization (WHO) published last year a revised edition of its manual for examination of human semen and sperm cervical mucus interaction with the intention to reduce differences between laboratories as underlined by De Juge .An other effort to furnish is the elaboration of most specific and most sensitive tests. Treatment of immunologic infertility Several treatment have been and are still used. The outcome are very conflicting. That is the consequence of a lack of actual pathogenesis of the ASA induced infertility and the absence of standard means of diagnosis .The Chinese are experimenting a drug which possesses inhibitory effect on antisperm cytotoxic antibody .Appropriate treatment will be the

available when we will reach the exact diagnosis by improving our investigations tools. Meanwhile, therapy, specially for the majority of cases of sperm-bound ASA- associated infertility remains empiric and largely unproven.The ART, mainly IVF and ICSI, is largely used today . Its overwhelming success should not hinder the risk of adverse effects. If it is proven that the ovarian hyperstimulation induces POF, we will be in front of a dilemma : give just a child to a couple or help a couple to achieve the desired number of children ; one of the aspect of the reproductive health. CONCLUSION The immunologic infertility is a real clinical feature. Its pathogenesis is not yet elucidated. The mastery of the pathogenesis will allow the elaboration of most specific tests, the improvement of therapy. Our hypothesis: spermatozoa undergo change of its antigens through the migration. Male and female organisms are endowed with immunocompetent tissue able to react with the different sperm antigens. In normal conditions, this does not happen. The imbalance results in ASA production. We think that there are many subgroups of IgA and IgG ; each subgroup is able to react specifically and at each level with the new antigen presents by the spermatozoa. Some of them can than impair fertility. REFERENCES 1. Agarwal, S and Naik, S. (2007). Fundamentals of Immunogenetics: Principles and Practices. International Book Distributing Co (Publishing Division), Lucknow. Pp.No:217-228. 2. Bronson R. (1999). Antisperm antibodies: A critical evaluation and clinical guidelines. J Reprod Immunol., 45: 15983. 3. Fijak M, Meinhardt A. (2006). The testis in immune privilege. Immunological Reviews., 213: 6681. 4. Haribabu,Y.(2003). Veterinary Immunology: Basic concepts and applications. Jaypee Brothers, New Delhi. Pp.No: 135-160. 5. Lander, M.F., Hansen, P.J and Droset, M. (1990). Antisperm antibodies in cows after subcutaneous and intrauterine immunization. Vet.Rec., 126: 461-462. 6. Risvanli, A. and Kaygusuzoglu, E. (2004). Immunological infertility in domestic animals. J. Turk. Vet. Med., 3(4): 52-55. 7. Smith D.A. and Germolec D R . Introduction to immunology and auto-immunity. Environ Health Perspect 1999 Oct ; 107 Suppl. 5 : 661-5.