An important cause of unexplained Infertility, unexplained IVF failure and Recurrent Pregnancy Loss (RPL).

Healthy embryo implantation is one of the key factors that determines successful procreation. About 15-20% of the time, infertility and miscarriage is in part or completely attributable to immunologic implantation dysfunction (IID). Women who have predisposing factors such as endometriosis, unexplained infertility/repeated failed IVF, recurrent pregnancy loss (RPL), and those with a personal/family history of primary autoimmune conditions such as Lupus erythematosus, rheumatoid arthritis, Hashimoto’s autoimmune hypothyroidism, scleroderma, dermatomyositis, etc. are most at risk. So too in certain circumstances where the male and female partners share certain genetic similarities involving certain HLA genes such as DQ alpha (alloimmune), IID also can result. Both autoimmune and alloimmune causes of IID should be thoroughly investigated and addressed.

Immunologic Implantation Dysfunction (IID)Immunologic acceptance of the implanting embryo is essential for pregnancy to occur. When confronted by foreign proteins (bacteria viruses, foreign tissue grafts/transplantation), the body’s immune system goes on the attack.

So why is it that embryo that are partially comprised of other than self-proteins from a different individual (sperm), usually will safely implant and go on to propagate a healthy baby, rather than being rejected? This phenomenon has come to be referred to as the “immunologic riddle of pregnancy.” Such acceptance of a foreign allograph is without precedent in human biology. For this to happen, requires unique immunologic adaptation. To believe that this cannot go wrong is to say the least, naïve. In fact it does go wrong in about 15–20% of women with reproductive failure. When this occurs prior to the pregnancy, it will present as “infertility”. If it arises later, it will often present as a miscarriage and should the pregnancy it will often manifest as placental insufficiency, intrauterine growth retardation (IUGR) and 2nd or 3rd trimester fetal demise. The presentation depends on the timing, nature, and severity of the IID.

In pregnancy, the uterine environment is transformed into a site where the conceptus (in spite of being an immunologically distinct allograph) comes to be regarded as “bodies own” (“self”) and is protected from immunologic rejection. This truly a miraculous transformation is in large part responsible for the survival of the species. With the very initiation of the implantation process, the male genetic contribution to the embryo (DQ alpha genes) sends a signal to immune cells in the pre-pregnancy endometrium (decidua), setting in motion a process that permits recognition of this “foreign” embryonic allograft to be accepted as “friend”, rather than “foe”, protecting it from being summarily rejected. Such “alloimmune recognition.” This paradox is remarkable because, with the exception of monozygotic twins, interpersonal differences in genotype are inevitable. As such, it follows that maternal and paternal DQ alpha gene-combinations usually also differ. The very survival of the species hinges on the absolute requirement that in spite of such alloimmune dissimilarities, the immune system of the decidua comes to accept the embryo as “self” or “friend” rather than as “non-self” or “foe.”

Within 6 hours of reaching the uterine cavity, the embryo will become wedged in between decidual glands. Within 12 hours it will” hatch”, breaking through its envelopment (the Zona Pellucida) and within 24 hours the implantation process will commence with the hatched embryo sending its root system (trophoblast) into the decidua. The trophoblast has both villous (root-like) and extravilous (diffuse) components. It expresses several major histocompatibility complex (MHC) class 1 HLA genes (e.g. histocompatibility leukocyte antigen (HLA- C, E, and G]. These, (primarily HLA-G) regulate certain uterine lymphocytes (primarily, natural killer -NK cells (that comprise >70% of the decidual lymphocyte population, and cytotoxic lymphocytes-CTL, account for about 10%). Both NK cells and CTLs promote orderly trophoblastic penetration of the decidua and thus, facilitate normal implantation.

Certain highly specialized decidual immune lymphocytes known as regulatory T-cells (Treg cells) can   recognize proteins as “self” (“friend”) or “non-self” (“foe”).  They are capable of “turning off” immune reactions even once these are in progress and play a vital role in preventing embryo rejection. Other immune cells known as dendritic cells, present antigenic proteins to Treg cells. If the presenting antigen is recognized as “self” the concentration of Treg cells increases and if it is recognized as “foe”, the concentration decreases. The interaction between MHC (primarily HLA-G) and Treg lymphocytes, in combination with other regulatory proteins, triggers and influences the production and release of growth factors known as cytokines by both NK and CTL cells. There are three (3) varieties of cytokines, two are vital for maintenance of implantation. They are: TH-2 cytokines, which promote trophoblastic growth and permeation as well as vascular proliferation (angiogenesis) and TH-1 cytokines that destroy trophoblastic cells by cytolysis. These are also procoagulants, causing blood to clot. Healthy implantation and placentation require that there a balance is established between decidual TH-1 and TH-2 cytokine activity. Over-activity (dominance) of TH-1 occurs with NK cell and CTL over- activation and can lead to, implantation dysfunction.

Alloimmune Implantation Dysfunction

All humans have 2 DQ alpha genes, located on p-arm of Chromosome #6.  When an embryo’s sperm-derived, paternal DQ-alpha gene matches one or both of the mother’s DQ alpha genes, it will come to be regarded by the early pregnancy uterine lining (decidua) as being a “foreign invader”. This will often activate decidual NK cells /CTLs leading to an IID. But the IID will usually not occur following a single exposure to a matching embryo. It will usually take repeated exposures to several matching embryos for NK cells and CTL to become activated. However, such activation can occur even in the absence of a biochemically or ultrasound confirmed implantation.  Such woman often presents with “presumed infertility” or with RPL. It is important to recognize that for paternal-maternal DQ alpha matching to result in IID there MUST also be of concurrent Nka. In the absence of NKa there will in my opinion be no risk of pregnancy loss.

This is how alloimmune implantation dysfunction happens:

Immunogenetically triggered HLA-G signaling on the part of the implanting embryo leads to a reduction in decidual Treg cells and ultimately to the activation of NK/CTLs with dominance of TH-1 over TH-2 activity. Depending on the severity of this response, there could be : a) total destruction of the early trophoblast with complete implantation failure with no evidence that implantation was attempted (“presumed infertility”),b) partial trophoblastic damage that would allow the pregnancy to limp along until reserve runs out and a miscarriage c)  later pregnancy loss (an infrequent presentation of IID)..

Autoimmune Implantation Dysfunction

With autoimmune implantation dysfunction, NK cell activation is already well established by the time the embryo reaches the uterus. Accordingly, in such cases the pregnancy is usually lost before its presence can be established by a blood pregnancy test or an early ultrasound examination (i.e., it presents as a negative pregnancy test or a chemical gestation).

So how is autoimmune implantation dysfunction established?

The initial recognition of the non-DQ alpha matching embryo as “friend” or “self” sets the stage for the cells/tissues of our tissues not coming under immune attack. However, under certain circumstances, genetic, infection, toxic, and degenerative influences can result in our own body’s proteins coming to be regarded as “non-self” (“foe”). When this happens, the immune system starts to produce antibodies that are directed against our body’s own proteins. These so-called auto-antibodies then start attacking the body’s own cells/tissues/organs creating pathological states (diseases) such as can be seen with certain (autoimmune) disease states—e.g., lupus erythematosus, autoimmune hypothyroidism (Hashimoto’s disease), and rheumatoid arthritis.

There are also certain reproductive diseases such as endometriosis, where cell membrane phospholipids are altered by the disease process and then combine with proteins to evoke the production of antiphospholipid antibodies (APA). Certain APAs can damage the trophoblast and lead to a reduction of Treg lymphocytes, culminating in activation of NK/CTLs. This reaction is referred to as autoimmune implantation dysfunction. Autoimmune implantation dysfunction is much more common than alloimmune implantation dysfunction. In fact, it is responsible for more than 80% of IID. When it comes to IID, the three most common auto antibodies involved are: antiphospholipid antibodies (APA), anti-thyroid antibodies (ATA), and possibly, antiovarian antibodies (AOA).

Management of Immunologic Implantation Dysfunction (IID)

In my opinion, effective treatment of NK/CTL activation ideally requires the timely intravenous administration of 100cc of 20% IL) Intralipid (IL) taken daily -to down-regulate NKa combined with oral corticosteroid (0.75mg Dexamethasone or Prednisone) to address CTL. The success of such treatment is in large part dependent on the cause of the IID. It is far more likely to be successful in the case of` autoimmune-induced NKa than alloimmune-NKa. The reason is that with the former, clearance of NKa is sustained while with the latter the embryo continues to activate NK cells through several months of gestation.

Treating a partial DQ alpha match + NKa/CTL: It is not yet possible to differentiate between maternal and paternal DQ alpha in the embryo. Thus, in cases where the paternal DQ alpha genes only match one of the mother’s two DQ alpha genes (i.e., a partial match) there is a 50% chance that a transferred embryo will match with result in an alloimmune implantation dysfunction.  This is why IL therapy will only be half as likely to result in a viable pregnancy with alloimmune dysfunction than with autoimmune-induced NK cell activation.  Treatment with intralipid/steroid will in my opinion NOT be helpful when there is NKa in association with a DQ alpha matching embryo. The only time IL/steroid therapy is likely to be effective in the treatment of alloimmune implantation dysfunction is in cases where there is a partial DQ alpha match and the embryo being transferred does NOT match. IL will, by down regulating local NKa will protect such a non-matching embryo from being rejected. Given this reality, I always recommend to patients who have a partial DQ alpha match (with NKa/CTL), that only a single embryo be transferred. If two are transferred an one “matches” the latter could activate NK and  “muddy the waters” for the non-DQ alpha matching which otherwise might have propagated a healthy baby.

What to do in cases of a total/complete DQ alpha match + NKa/CTL: A real problem arises where both paternal DQ alpha genes match one or both of the woman’s DQ alpha genes (i.e. a “complete/total match).  The reason is that in such cases, every embryo will express a paternal DQ alpha gene that matches that of the mother’s DQ alpha. IL/steroid therapy will rarely (<10% of cases) propagate a viable pregnancy in such circumstances.  In cases of a complete DQ alpha matching + associated NK/CTL activation, gestational surrogacy or the use of non-DQ alpha matching donor sperm, in my opinion, offers the only reasonable chance of a successful IVF.

What about using eggs derived from a non-DQ alpha matching egg donor to overcome the problem of a complete DQ alpha match (+NKa/CTL)? This will not work because with alloimmune implantation dysfunction, the problem relates to a match-up between the paternal DQ alpha contribution to the embryos and the mother’s uterus. It is not between the sperm and the egg.

IL/corticosteroid therapy should be administered by slow infusion (over 2-3 hours), 10–14 days prior embryo transfer. With autoimmune-NKa, a second infusion is advisable at the first indication of a positive blood pregnancy test. With alloimmune implantation dysfunction repeated infusions every 2-4weeks should be maintained, at least through the 1st half of pregnancy. In both autoimmune and alloimmune NKa/CT cases, steroid therapy is tailed off between the 8th and 10th week of pregnancy.  The goal ofIL/steroid therapy is to down-regulate activated NK/CTL and reestablish TH-1: TH-2 cytokine balance in advance of embryo transfer. Supplementation with heparinoid such as Lovenox or Clexane is in my opinion only indicated in cases where there is concomitant antiphospholipid antibodies (APA) or with certain types of thrombophilia such as a homologous MTHFR mutation.

The Role of PGT (Full Embryo Chromosomal Karyotyping) in the Treatment of Alloimmune Implantation Dysfunction

Since with a partial DQ alpha match/NK cell activation each blastocyst transferred has a 50:50 chance of matching and Intralipid (IL)/Prednisone therapy only addresses the implantation issue, the success rate after transferring a single embryo could be markedly improved by selectively transferring a chromosomally normal (euploid) embryo. ,

Medications in the Treatment of IID

  1. Intralipid (IL) Therapy:

Intralipid (IL) is a solution of small lipid droplets suspended in water. When administered intravenously it provides essential fatty acids, linoleic acid (LA), an omega-6 fatty acid, alpha-linolenic acid (ALA), an omega-3 fatty acid. IL is made up of 20% soybean oil/fatty acids (comprising linoleic acid, oleic acid, palmitic acid, linolenic acid and stearic acid), 1.2% egg yolk phospholipids (1.2%), glycerin (2.25%) and water (76.5%).

IL exerts regulates certain immune cellular mechanisms involved in NK cell activation, but not necessarily CTL activation (the latter requires the addition of steroids as described). This effect of IL might be due to its ability to suppress pro-inflammatory cellular (Type-1) cytokines such as interferon gamma and TNF-alpha. In-vitro testing has shown that IL successfully and completely down-regulates activated natural killer cells (NKa) within 2-3 weeks in 78% of women experiencing immunologic implantation dysfunction. This effect of IL lasts for 4-9 weeks when administered in early pregnancy.

Can in-vitro tests done in the laboratory assess for an immediate benefit of Intralipid on NKa?

Since the down-regulation of NKa through IL therapy can take several weeks to achieve, there is in reality no benefit in trying to assess the potential efficacy of such treatment by retesting NKa in the laboratory after adding IL to the sample.

  • Corticosteroid Therapy (Prednisone, Prednisolone, and Dexamethasone)

Corticosteroid therapy has become the mainstay in the treatment for many women undergoing IVF. It is believed by most to enhance implantation due to an overall immunomodulatory effect. Some IVF programs prescribe daily oral methyl prednisolone (Medrol) while others prefer prednisone or dexamethasone, commencing 10-14 days prior to egg retrieval and continuing until pregnancy is discounted or until the 10th week of pregnancy.

  • Heparinoid Therapy

There is evidence to show that the subcutaneous administration of heparin twice daily or low molecular heparin (Clexane, Lovenox) once daily, (starting with the onset of ovarian stimulation) can improve IVF birthrate in women who test positive for certain antiphospholipid antibodies (APA) and with certain  thrombophilias (e.g. homozygous MTHFR mutation)

  • What About Baby Aspirin?

In my opinion, aspirin therapy has little (if any) benefit when it comes to IID and in fact  might even reduce the chance of success because it thins the blood and increases the potential to bleed, thereby complicating  an egg retrieval procedure or causing  intrauterine bleeding at the time of embryo transfer. I do not prescribe it during IVF cycles.

  • TH-1 Cytokine Blockers (Enbrel, Humira)

I do not advocate the use of TH-1 cytokine blockers such as Enbrel and Humira. I believe this to be relatively ineffective in the IVF setting.

  • Leukocyte Immunization Therapy (LIT)

This treatment is illegal in the U.SA. Some couples travel to Mexico or elsewhere abroad to get LIT. There is evidence to show that LIT enhances the ability for the mother’s decidua (uterus) to recognize the DQ alpha matching embryo as “self” or “friend” and thereby avert its rejection. However, the same benefit can be achieved through the use of IL plus corticosteroids.