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:

  1. whether an underlying autoimmune/inflammatory condition may be present, and
  2. 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
    • HLA-A
    • HLA-B
    • HLA-C
  • 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.


  • 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.