"Personalized medicine" is a hot phrase in the health care community these days, used to describe the use of ever more highly precise methods for diagnosing and treating patients at an individual level. One critical component to practicing personalized medicine is the application of genetic approaches to diagnosis and selection of treatments – after all, it is small differences at the genetic level that truly distinguish each of us as individuals. Here at Braverman IVF & Reproductive Immunology we have long believed in the power of applying genetic approaches to the diagnosis of causes for infertility, implantation failure and recurrent pregnancy loss. Genetics are immutable – they are not affected by variables such as the level of psychological or physical stress a patient is experiencing, having a cold or bacterial infection, or even the time of day, the way that some other diagnostic tests can be, and they do not change over time – meaning that genetic tests only need to be done once and that the results are both reliable and stable over time. Genetic tests are also a particularly rich source of data that can provide insight into a large number of diverse biological processes.
One particularly useful genetic test that comprises an important component of our diagnostic tests is identification (typing) of the human leukocyte antigen (HLA) genes - also commonly referred to as the major histocompatibility complex (MHC) genes. The HLA genes are a family of genes located on the 6th chromosome that encode for proteins that play a key role in the regulation of immune responses (for a further description of the role of HLA genes in the immune system see our website
here). Each person has several HLA genes and each gene is highly variable in its composition amongst individuals in a population. Copies of HLA genes that vary amongst individuals are referred to as "alleles" and different alleles of the same HLA gene can encode for proteins that have slightly different functions. The presence of several HLA genes in each individual and many different possible alleles of each of these HLA genes leads to complex combinations of specific HLA alleles, called "haplotypes", amongst individuals in a population.
While genetic data, such as HLA haplotypes, are reliable and stable over time, the key to their diagnostic utility lies in possessing the necessary knowledge and experience to properly interpret and effectively apply the results. Because of this we are dedicated to maintaining a thorough and current knowledge of the vast scientific literature relating to infertility, implantation failure and recurrent pregnancy loss, and we recently enhanced our capabilities in this area by the addition of our new Director of Research. We routinely test our patients and their partners to determine their HLA haplotypes because we have determined from our extensive reviews of the literature and from our own data that they significantly impact fertility, implantation, and the ability to carry a pregnancy to term in several ways that are outlined below:
HLA Matching
The immune system constantly monitors the expression of HLA proteins on the surface of the body's cells. HLA proteins also bind to and present pieces (peptides) of the proteins from inside the cells to the immune system. These peptides may be pieces of HLA proteins themselves or of other proteins. When the immune system detects that these peptides presented by HLA proteins on a cell are normal "self" peptides, then the cell is ignored. When the immune system detects peptides from bacteria, viruses or other "non-self" proteins, then an immune reaction can be elicited. In most cases this immune reaction leads to the destruction of cells displaying these "non-self" peptides.
Upon transplantation of cells (such as a whole organ) from one individual to another, peptides of the HLA proteins from the donor cells are presented to the immune system of the recipient. If the donor and recipient have different HLA haplotypes (different combinations of alleles of HLA genes) then some of the donor HLA peptides will be seen as "non-self" peptides by the recipient's immune system. In most types of transplantation this leads to the generation of immune reactions that target and kill the donor cells. The strength of the immune reaction often depends on the number of different HLA alleles between the donor and recipient as well as exactly which different alleles are present. Thus, the goal in most types of transplantation is to identify donors that have HLA alleles that match the HLA alleles of the recipient as closely as possible. This kind of immune tolerance of the transplanted cells can be thought of as a "passive" tolerance, where the goal is to avoid recognition by the recipient's immune system as much as possible.
Pregnancy is similar to transplantation in many ways, although very different in others. An embryo/fetus contains a combination of the mother's and the father's genetic makeup, including HLA alleles from both the mother and father. In the great majority of cases, the mother's and father's HLA haplotypes will differ at least to some extent. Thus, the cells of the embryo express HLA proteins from the father's HLA alleles that are presented as peptides and recognized as "non-self" by the mother's immune system. By analogy to transplantation, the father can be thought of as the donor and the mother as the recipient. In pregnancy though, in stark contrast to transplantation, differences in HLA alleles between the father (donor) and mother (recipient) does not normally lead to rejection of the embryo (donor cells). Rather a certain amount of dissimilarity between the mother's and father's HLA alleles actually appears to be necessary for immune tolerance of the embryo. This difference between transplantation and pregnancy is because the type of immune tolerance that is generated to an embryo can be thought of as an "active" tolerance, as opposed to the "passive" tolerance that is the goal of most types of transplantation.
Active tolerance to an embryo is achieved through the generation of immune suppressive regulatory T cells (Tregs). These Tregs are initially generated in response to exposure to paternal HLA alleles differing from the mother's HLA alleles. The ability to spontaneously generate Tregs in response to dissimilar HLA alleles is a critical difference between most types of transplantation and pregnancy, and is most likely a result of the uterus being an "immune privileged" site where specific kinds of Tregs, called iTregs, can be generated. Thus, while HLA dissimilarity in transplantation (between donor and recipient) leads to generation of T cells that attack and kill donor cells, similar HLA dissimilarity in pregnancy (between mother and father) leads to the generation of immune suppressive Tregs that exert active, dominant tolerance to the embryo.
While the large diversity of possible HLA haplotypes makes it relatively rare that 2 unrelated individuals will match for a significant number of HLA alleles, it does indeed happen in many couples attempting to have a child. While these couples are relatively rare in the general population, they appear to comprise a significant portion of the population experiencing extended infertility, repeated implantation failure, and recurrent pregnancy loss. Literature and our own data support the concept that allele differences at only certain HLA genes are critical to initiate the development of Tregs that are then able to dominantly promote tolerance to the embryo despite allele differences at other HLA genes that would otherwise generate T cells that would attack the embryo. Thus, couples that share significantly matched alleles for these particular HLA genes fail to generate the necessary immune tolerance to the embryo, leading to infertility, repeated implantation failure, and recurrent pregnancy loss.
Understanding exactly which HLA genes are involved in reproductive failure due to matching between partners is, of course, critical to making a proper diagnosis on this basis. Our combined depth of knowledge of the both the basic science and clinical literature on this issue, and our own extensive data and experience lead us to strongly believe that the simplistic approach taken by most clinicians to this issue is grossly inadequate, and in many cases leads to an incorrect diagnosis and inappropriate recommendations for how a couple should pursue having children. Put more plainly, we believe that diagnosis of HLA matching as a significant factor in reproductive failure on the basis of allele matching for a single HLA gene (i.e., DQalpha) - or even worse, a partial match at a single HLA gene - is outdated, oversimplified, and ultimately, incorrect.
The scientific literature and our own extensive data simply do not support the assertion that complete allele matching at a single HLA gene (nevermind partial matching) is a significant barrier to successful pregnancy. The incorrect conclusion that matching at a single HLA gene is a significant impediment to successful pregnancy is further based on a completely unsupported belief that this matching somehow directly leads to activation of natural killer (NK) cells that will attack and kill the embryo. There are several things wrong with this: 1) there is not yet a single demonstration in the scientific literature that NK cells can directly attack embryos during pregnancy, 2) there is no evidence to support that NK cells have any means to directly detect expression of DQalpha (or any other class II HLA protein), on the surface of cells, much less be able to discern whether the DQalpha is "self" or "non-self", 3) NK cells that reside in the uterus – uterine NK (uNK) cells – are very different from peripheral blood NK (pbNK) cells, 4) while activation of pbNK cells primarily leads to cell killing (cytotoxic) function, activation of uNK cells in contrast leads to the production of cytokines and growth factors necessary to support blood vessel growth and transformation vital to support a pregnancy, and 5) the most recent and convincing literature on this subject supports the view that, in fact, adequate activation of uNK cells is necessary to prevent miscarriage, intrauterine growth restriction and preeclampsia. These issues are explored a little further below in the section entitled "HLA-C and KIR".
In summary, many practitioners cling to views of HLA matching in pregnancy that are outdated by at least a decade or more, and are for the most part based on old assumptions that have since been refuted by the scientific literature. Thus, while many practitioners believe that allele matching at a single HLA gene leads to reproductive failure through activation of NK cells, our more current and extensive knowledge of the literature and our experience teach us that a more sophisticated approach to evaluating HLA matching must be taken, and that when a true problem with HLA matching exists, it is inadequate generation of Tregs (and not activation of NK cells) that is directly affected. The difference in our approach to diagnosis of HLA matching issues in reproductive failure, and in our understanding of which cell types are directly impacted, the biological mechanisms involved, and the biological processes that are ultimately undermined, is not strictly an academic issue. This difference in approach and knowledge of the biological subject matter is critical both for accurate and specific diagnoses and for proper selection of therapeutics. For example, many practitioners are quick to advocate for patients to move on to the use of donor sperm or gestational surrogates when matches at DQalpha are identified. Again, we strongly disagree that matching alleles for a single HLA gene is a barrier to successful pregnancy, but
even when our more sophisticated approach to HLA matching analysis reveals an
actual HLA matching issue, our experience teaches us that in the majority of cases, this is readily treatable with
appropriate therapies – those therapies that address the
true cells and biological processes affected by the matching.
HY-Restricting HLA Alleles
In addition to the utility of HLA data for revealing potential problems due to matching between two partners, it can also independently reveal many other details about the function of the maternal immune system. Another important piece of information that we can extract from HLA haplotyping is whether the female's haplotype includes HY-restricting HLA alleles.
As noted in our earlier post, a recent study found that females with a history of a firstborn boy are susceptible to secondary recurrent miscarriage and to giving birth to boys with a low birth weight when they possess specific HYrHLA alleles. These HYrHLA alleles can specifically alert the mother's immune system to the presence of male-specific proteins (HY antigens) encoded by genes on the Y chromosome of a male fetus. In some women this can lead to the development of a dangerous immune reaction to the fetus mediated through T cells and HY antibodies produced by B cells.
HLA-G Linkage Disequilibrium
HLA-G is an HLA gene that has a very restricted tissue pattern of expression compared with most other HLA genes, and its significant expression on trophoblast cells of the embryo point to important roles in implantation and regulation of the maternal immune response. Many studies indeed support that certain alleles of HLA-G, both from the mother and father, make important contributions to pregnancy success or failure. Unfortunately, since most HLA haplotyping is performed by labs that support transplant clinics, and since HLA-G does not have a prominent role in most types of transplantation, genotyping of HLA-G is not usually performed as part of an HLA haplotype test. We are currently actively working on adding direct detection of HLA-G alleles to our panel of tests. While we work on adding this test we are fortunately still able to deduce, with a fairly high degree of confidence, the presence or absence of some of these alleles once the rest of the HLA haplotype is identified. This is possible because of recent large epidemiological studies that have demonstrated a positive or negative connection of certain HLA-G alleles to certain alleles of other HLA genes – a phenomenon referred to as linkage disequilibrium.
The precise role of HLA-G in regulating embryo implantation and modulation of the maternal immune response to the embryo – in particular how it regulates NK cell function – is currently undergoing reexamination in the literature and will be the topic of one of our future blog posts.
HLA-C and KIR
HLA haplotyping data can also make important contributions to the diagnosis of causes of infertility, implantation failure and recurrent miscarriage in combination with other genetic data.
As briefly discussed above, it is now well-understood that uterine NK (uNK) cells differ greatly from the NK cells that circulate in the peripheral blood (pbNK cells). The differences between these cell types and the role of uNK cells in embryo implantation and early steps in the establishment of pregnancy will be featured in more detail in an upcoming blog post. Briefly, for now, we will mention that while pbNK cells are highly cytotoxic once activated by certain stimuli, activation of uNK cells in contrast does not cause these cells to become cytotoxic, but rather to secrete a unique repertoire of cytokine and growth factors that regulate blood vessel growth and development. In fact it has been shown that sufficient activation of uNK cells and their release of these cytokines and growth factors is necessary to promote a transformation of the uterine spiral arteries from small, coiled tubes to the voluminous conduits for blood that are needed to support the growth of the embryo.
Activation of uNK cells to secrete cytokines and growth factors that transform the uterine spiral arteries is regulated by a number of interactions between the trophoblast cells of the embryo and the uNK cells. Studies have shown that one of these important interactions is between HLA-C on the trophoblasts and a family of proteins on uNK cells that bind to HLA-C called killer immunoglobulin-like receptors (KIRs). Like HLA genes, KIRs are a family of genes that are variable amongst individuals – different individuals can have different numbers and types of KIR genes. The numbers and types of KIR genes that a person possesses determines, in part, how strongly their NK cells react to another cell displaying HLA-C. Large epidemiological studies of humans have recently shown that the combination of the 1) KIR haplotype of the mother and of 2) the HLA-C genotype of both the father and the mother has a significant impact on the risk for defective placentation which can manifest as recurrent miscarriage, intrauterine growth restriction and/or preeclampsia.
Based on these studies, it is now clear that certain maternal KIR haplotypes in combination with certain maternal and paternal HLA haplotypes (in particular HLA-C alleles) can lead to reproductive failure and complications of pregnancy. Thus, in addition to HLA haplotyping, we recently added KIR haplotyping to our panel of diagnostic tests. A future blog post will explore how we analyze the combined HLA haplotype and KIR haplotype data to identify those patients at risk for reproductive failure associated with inadequate activation of uNK cells.
Identification of Anti-Paternal HLA Antibodies
HLA haplotyping data can also be very useful in combination with other non-genetic data to identify specific issues associated with infertility, implantation failure and recurrent pregnancy loss. Pregnancy is associated with the production of several types of antibodies in the mother. These antibodies can be directed at proteins made from genes contributed by the father, including HLA genes. Historically, the leukocyte antibody detection (LAD) assay has been used to detect antibodies in the mother specific for paternal proteins. However, as we have discussed in previous blog posts, the LAD test is not able to distinguish what paternal proteins the antibodies are binding to, nor between antibodies that could potentially harm the embryo/fetus and those that may play a protective role. Therefore, for any patients that first test positive on the
LAD assay, we previously started testing maternal blood using a more specific assay using the Luminex platform that detects the presence of antibodies specific for the entire range of HLA proteins. This assay can specifically detect if the patient is making antibodies for HLA proteins, and if so, exactly which ones. In combination with the paternal HLA haplotype data we can then determine if any of these anti-HLA antibodies are specific for HLA proteins produced by the father (and by inheritance potentially the embryo/fetus). In patients that contain anti-HLA antibodies specific for one or more paternal HLA proteins, we have also now started to test these antibodies to determine if they are the type that can potentially harm the embryo/fetus (complement-fixing) or if they are the protective type. Thus, we now use the LAD test simply as a screen to determine when further tests should be run to detect if anti-paternal HLA antibodies are present (in combination with HLA haplotyping data), and if so, what type of antibodies they are. A future blog post will address this topic in more detail.