Reproductive Immunology
Success After Multiple Miscarriages Is Possible
While some of our patients have a diagnosed inflammatory (this includes
endometriosis and
PCOS, which can cause multiple miscarriage and infertility) or autoimmune condition,
for many of our patients the only overt symptom of a potential underlying
inflammatory/autoimmune condition is their inability to become or stay
pregnant (and sometimes a family history of inflammatory and/or autoimmune
disorders). Scientific literature provides extensive evidence that derangement
of the immune system can occur many years (even decades) prior to the
onset of the classical clinical symptoms of an autoimmune disease (such
as inflamed synovial joints in rheumatoid arthritis).
Therefore, it is possible to have significant immune system dysfunction
for a long period of time without any noticeable clinical symptoms that
would precipitate a diagnosis. However, even in these preclinical stages
of autoimmunity the underlying immune system changes can lead to a failure
in the immune system to properly develop tolerance for an embryo which
can lead to implantation failure, miscarriage, multiple miscarriage, or
later pregnancy complications. Similarly, significant underlying immune
system derangement and inflammation can also exist in many patients with
endometriosis and/or PCOS with no or minimal overt clinical symptoms.
What Makes Our Approach to Multiple Miscarriage and Infertility Different
In patients with a history of idiopathic (unexplained) infertility, recurrent
miscarriage, or significant later pregnancy complications, it is critical
to undergo a thorough investigation of immune system function to identify
any potential immune system dysfunction and inflammation which may be
contributing to the obstetric problems. It is also critical to characterize
the nature of the underlying autoimmune/inflammatory condition as this
is crucial to designing an appropriate course of treatment. For example,
while lupus and rheumatoid arthritis are both autoimmune conditions, the
nature of the immune system derangements in these conditions are largely
separate and in some ways completely opposite. Therefore, effective immune
therapies for these conditions frequently do not overlap, and some treatments
used for one condition even have the potential to exacerbate the other.
Many centers claiming to have expertise in reproductive immunology only
test for a very small number of immunologic parameters which are wholly
inadequate in determining:
- whether an underlying autoimmune/inflammatory condition may be present, and
- anything about the nature of the autoimmune/inflammatory condition.
It is simply not possible to define the state of somebody’s immune
system by measuring 2 or 3 markers any more than you can look at 2 or
3 stars to identify a constellation. It is necessary to gather many pieces
of the immunological “puzzle” and determine how they all fit
together before the clinical picture that they form becomes clear. Looking
at 2-3 puzzle pieces is simply not performing diagnostics; it’s
guessing. Immune system responses in autoimmune and inflammatory conditions
are extremely complex and scientific advances in the understanding of
these responses move much too fast for most clinicians to keep up with.
Our team of scientists reviews this literature on a daily basis and attends
many national and international conferences so that we are constantly
on the leading edge of advancements in reproductive immunology. By using
the testing described below, we are able to detect and define the presence
of immune problems affecting pregnancy in an unprecedented and unmatched
way. It is the ability to precisely define the nature of underlying immune
issues affecting fertility/pregnancy that allows us to tailor treatment
protocols for optimal efficacy and safety in a highly individualistic way.
While the full set of testing listed below is critical for obtaining a
comprehensive picture of a patient’s immune system function (and
therefore design a highly tailored treatment regimen), a subset of the
testing listed below is also used to monitor the immune response to pregnancy.
The subset of testing performed during pregnancy has been carefully selected
based on our unmatched experience and extensive retrospective analysis
of our data. This combination of experience and extensive retrospective
data analysis has allowed us to select a panel of markers that is highly
sensitive and specific for identifying improper immune responses to an
embryo/fetus. Detection of these changes allows us to make highly informed
changes to treatment protocols to correct these immune responses and further
encourage immunological tolerance to the conceptus.
Killer Immunoglobulin Receptor (KIR) Genes
- KIR2DL1
- KIR2DL2
- KIR2DL3
- KIR2DL4
- KIR2DL5
- KIR3DL1
- KIR3DL2
- KIR3DL3
- KIR2DS1
- KIR2DS1
- KIR2DS2
- KIR2DS3
- KIR2DS4
- KIR2DS5
- KIR3DS1
Certain maternal KIR haplotypes (combinations of KIR genes) in combination
with maternal and paternal HLA-C genotypes can result in a failure in
embryo implantation or defective placentation leading to miscarriage or
later pregnancy complications including preeclampsia, intrauterine growth
restriction (IUGR), or stillbirth.
Human Leukocyte Antigen (HLA) Haplotyping (Patient and Partner/Sperm Donor)
-
Class I
-
Class II – includes determination of whether class II HY-restricting
HLA (HYrHLA) alleles are present
- HLA-DQa1
- HLA-DQb1
- HLA-DRB1
- HLA-DRB3/4/5
- HLA-G 14 bp ins/del
Many HLA alleles and haplotypes (combinations of HLA alleles) predispose
to the development of conditions such as autoimmune conditions, endometriosis,
and PCOS that can affect the ability to become or stay pregnant. When
used in combination with our other immunological testing, HLA haplotyping
can provide important insight into the nature of any underlying immunological
conditions and help direct proper treatment.
A lack of certain kinds of mismatching between maternal and paternal HLA
alleles can also contribute to a failure in the maternal immune system
to tolerate the embryo. Proper analysis of HLA allele mismatching is far
more involved than simply looking at the DQA1 locus as other centers do.
Intracellular Cytokine Testing
-
CD4+ T cells
- TNFa (CD3+/4+/TNFa+)
- IFNg (CD3+/4+/IFNg+)
- IL-17 (CD3+/4+/IL-17+)
- IL-4 (CD3+/4+/IL-4+)
- IL-10 (CD3+/4+/IL-10+)
-
CD8+ T cells
- TNFa (CD3+/8+/TNFa+)
- IFNg (CD3+/8+/IFNg+)
- IL-17 (CD3+/8+/IL-17+)
- IL-4 (CD3+/8+/IL-4+)
- IL-10 (CD3+/8+/IL-10+)
-
NKT cells
- TNFa (CD3+/56+/TNFa+)
- IFNg (CD3+/56+/IFNg+)
- IL-17 (CD3+/56+/IL-17+)
- IL-4 (CD3+/56+/IL-4+)
- IL-10 (CD3+/56+/IL-10+)
-
NK cells
- TNFa (CD3+/56-/TNFa+)
- IFNg (CD3+/56-/IFNg+)
- IL-17 (CD3+/56-/IL-17+)
- IL-4 (CD3+/56-/IL-4+)
- IL-10 (CD3+/56-/IL-10+)
We determine percentages of cells for each cell type (CD4+ T cells, CD8+
T cells, NKT cells, NK cells) that are positive for each of the indicated
cytokine (TNFa, IFNg, IL-17, IL-4, IL-10), as well as ratios of these cells. Taken together
with genetic data (HLA haplotyping), patient/family history, and our other
immunologic testing, these data can be highly revealing regarding the
extent and nature of any underlying immunological conditions. The ability
to discern among various kinds of immunological abnormalities is critical
in the selection of appropriate treatments and doses. For example, lupus
is almost always associated with a Th2 bias (low ratio of CD4+ T cell IFNg
positive cells to CD4+ T cell IL-4 positive cells) whereas Hashimoto’s
thyroiditis is almost always associated with a Th1 bias (elevated ratio
of CD4+ T cell IFNg
positive cells to CD4+ T cell IL-4 positive cells) together with significant
CD8+ T cell activation (indicated by elevated ratios of TNFa, IFNg, and IL-17 positive CD8+ T cells and NKT cells to IL-4 and IL-10 positive
CD8+ T cells and NKT cells). Autoimmune conditions including rheumatoid
arthritis and psoriasis also are highly associated with CD8+ T cell and
NKT cell activation whereas endometriosis and PCOS are frequently associated
with NK cell activation. Many other centers only look at levels of one
cell type (CD4+ T cells) that are positive for 2 or 3 of these cytokines
(and frequently the wrong ratios of these cells are used to determine
Th1 and Th2 ratios). Testing of only one cell type is simply insufficient
to give a thorough picture of the state of the immune system.
Complete Blood Count (CBC) With Differential
- WBC
- RBC
- Hemoglobin
- Hematocrit
- MCV
- MCH
- MCHC
- RDW
- Platelet Count
- Absolute Neutrophils
- Absolute Monocytes
- Absolute Eosinophils
- Absolute Basophils
- Neutrophils
- Lymphocytes
- Monocytes
- Eosinophils
- Basophils
Abnormalities in a CBC can also provide important information regarding
the presence of underlying immune conditions. For example, a low neutrophil
count can indicate potential autoimmune neutropenia (which can be further
investigate by testing for anti-neutrophil antibodies).
NK Cell Cytotoxic Activity
- E:T 50:1 Native State
- E:T 25:1 Native State
- E:T 12.5:1 Native State
- E:T 25:1 + IL-2 Stimulation
- E:T 25:1 + Intralipid
- E:T 25:1 + 12.5 mg/dl IgG
- E:T 25:1 + 6.25 mg/dl IgG
NK cell cytotoxic activity (NKa) is one of the most commonly tested immune
variables in clinical reproductive immunology and is frequently used entirely
by itself to make a diagnosis. While this test can be useful together
with all of the other immune testing we perform as a piece of the diagnostic
puzzle, by itself it is completely inadequate to make any determinations
at all. In fact, many centers interpret this testing in a completely incorrect
biological context leading to misinterpretation of the results.
Reproductive Immunophenotype
- Total T cells (CD3+)
- Total NKT cells (CD3+/56+)
- Total NK cells (CD3-/56+)
- CD16+ NK cells (CD3-/56+/16+)
- CD16- NK cells (CD3-/56+/16-)
- CD4+ NKT cells (CD3+/4+/8-/56+)
- CD8+ NKT cells (CD3+/4-/8+/56+)
- CD4-8- NKT cells (CD3+/4-/8-/56+)
- CD8+ T cells (CD3+/8+)
- CD4+ T cells (CD3+/4+)
- Activated CD4+ T cells (CD3+/4+/25+)
- Activated T cells (CD3+/25+)
- CD28+ T cells (CD3+/28+)
- Total B cells (CD19+)
- B1a B cells (CD19+/5+)
- Activated T cells (CD3+/HLA-DR+)
- Regulatory T cells (Treg cells) (CD3+/4+/25hi/127lo/FoxP3+)
This testing is useful particularly in conjunction with the intracellular
cytokine testing to further define the extent and nature of the underlying
immune dysfunction.
Anti-HLA Antibodies
- HLA Class I Antibodies (HLA-A, -B, -C)
- HLA Class II Antibodies (HLA-DQA1, -DQB1, -DRB1, -DRB3, -DRB-4, -DRB5)
- Complement (C1q) fixation
HLA antigens are expressed by the conceptus at various stages of development
and binding of antibodies to these antigens can variably affect fertility
and pregnancy outcome. Detection of anti-HLA antibodies using a single
antigen bead (SAB) assay allows for highly sensitive and specific determination
of the full array of anti-HLA antibodies that may be present, their levels,
and whether they are specific for partner HLA antigens. Together with
testing to determine f any partner-specific anti-HLA antibodies fix complement
(C1q), this information allows for a full evaluation regarding the significance
of antibodies that are present. Many centers perform a leukocyte antigen
detection (LAD) test which is meant to detect HLA antibodies. However,
this test is very non-specific and results of this test are often misinterpreted
based on outdated science relating to the concept of “blocking antibodies”.
Unfortunately, this misinterpretation is often used to encourage patients
to undergo a procedure that can actually significantly negatively impact
future attempts at pregnancy.
Serum Cytokines
- TNFa
- IL-6
- IL-8
- IL-17
- TGFb-1/2/3
Serum levels of cytokines reflect levels of systemic inflammation and their
profile can provide valuable information regarding the extent and nature
of immune system activation that is present.
Autoantibody Testing
- Antinuclear antibodies
- Antiphospholipid antibodies
-
Thyroid autoantibodies
- Anti-TPO
- Anti-thyroglobulin
- Anti-TSH receptor antibody
- Rheumatoid factor
- Anti-CCP
We test for a diverse array of antibodies that bind to self antigens (autoantibodies)
and can detect the presence of various autoimmune conditions, including
antiphospholipid syndrome (APS), lupus, scleroderma, Hashimoto’s
thyroiditis, Graves’ disease, rheumatoid arthritis, and many others.
Other
- C3 complement activity
- C4 complement activity
- Total IgM
- Total IgA
- Total IgG
- Total IgE
- Vitamin D level
- Homocysteine level
- MTHFR polymorphisms (C677T and A1298C)
- TSH
Various other analytes are tested which can reveal underlying immune/inflammatory
disorders, as well as deficiencies in levels of vitamin D (a major immunoregulatory
hormone) and polymorphisms in the MTHFR gene which affect folic acid metabolism.