immunological basis of infertility in animals
TRANSCRIPT
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IMMUNOLOGICAL BASIS OF INFERTILITY IN ANIMALS
Introduction
Infertility, the inability of a couple to conceive after a period of 12 months of
intercourse without 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).
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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 Burnet‟s 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
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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 self-
reactive 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 immune‐modulating 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.
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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)
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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 doesn‟t 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 blood–testis 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
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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 immuno‐regulatory 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 .
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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.
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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
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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
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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 auto-
antibodies. 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.
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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 the
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
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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.
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