The Link Between Immunologic Implantation Dysfunction (IID), unexplained Infertility, IVF failure and Recurrent Miscarriages
In 15-20% of women who have infertility or RPL, the cause will be immunologic implantation dysfunction (IID). Accordingly, all women who have predisposing factors such as endometriosis, unexplained infertility/repeated failed IVF, RPL, or have a personal/family history of primary autoimmune conditions, should be evaluated and treated appropriately. By doing so, we as physicians will not only be promoting a safer journey from “Infertility to family” but by promoting better implantation, will enhance placentation and prenatal development with the ultimate objective of optimizing the quality of life after birth.
Immunologic acceptance of the implanting embryo by the uterus of the mother is both highly complex and magnificent. Not only is it essential for pregnancy to occur, but it also sets the scene for our body’s own cells, tissues, and organs to be shielded from attack by our immune systems. For a moment, consider how, when confronted by foreign proteins (bacteria viruses, foreign tissue grafts/transplantation), the body’s immune system goes on the attack but yet an embryo that is partially derived from proteins that come from another individual (the sperm or paternal antigen), usually safely implants in the pre-pregnancy uterine lining and then grows into a healthy baby. This phenomenon has come to be referred to as the “immunologic riddle of pregnancy.”
For such a complex arrangement never to fail would be without precedent in human biology. To argue to the contrary is, in my opinion, an absurdity, bordering on arrogance. It can and does go wrong in about 15–20% of women with reproductive failure and when it does, it sometimes presents as failed implantation (presumed by the patient to be infertility), as miscarriage, or (much less frequently) as placental failure and compromised fetal development or intrauterine death. It all depends on the timing, nature, and severity of the immune assault.
It is well known that the reason the implanting normal embryo thrives in the womb is that unique immunologic adjustments convert the pre-pregnancy uterine lining (decidua) into a “privileged site” where the embryo and the fetus come to be regarded as “bodies own” (“self”) and as such are protected from immune attack. This initial acceptance of the embryo as “self” or “friend” rather than “non-self” or “foe” (despite of it being a semi-allograft) is one of the miraculous adaptations of nature and is in large part responsible for our survival as a species.
As soon as implantation begins, the paternal genetic contribution to the embryo (so called DQ alpha genes) initiates a signal to the pre-pregnancy decidual immune system which thereupon determines whether the embryonic allograft should be welcomed (i.e., be accepted as “friend”) or be regarded as “foe” and be rejected through immune attack. The process is referred to as “alloimmune recognition.” Given that with the exception of monozygotic twins, interpersonal differences in genotype are inevitable, it follows that maternal and paternal DQ alpha gene combinations will usually also differ in the vast majority of cases. Thus, preservation of the human species required that despite of such immunogenetic dissimilarities, the immune system of the pre-pregnancy endometrium (decidua) adapt and recognize the embryo as “self” or “friend” rather than as “non-self” or “foe.”
Upon reaching the uterine environment, the “genetically competent” embryo, hatches and thereupon, within 12–24 hours starts sending its root system (trophoblast) into the decidua. The trophoblast has both villous (root-like) and extravilous (diffuse) components. The extravillous trophoblast, which diffusely permeates the decidua expresses several so-called major histocompatibility complex (MHC) class 1 genes [e.g., histocompatibility leukocyte antigen (HLA-) C, E, and G]. These HLA genes, (primarily HLA-G) regulate primarily two types of lymphocytes present in the decidua. These are uterine natural killer (NK) cells and cytotoxic lymphocytes (CTL). NK cells comprise approximately 75% of decidual lymphocytes and CTL comprise about 10%. They both likely play a vital role in regulating the normal implantation process by control- ling the penetration and functioning of the trophoblast.
The recognition of proteins as “self” (“friend”) or “non-self” (“foe”) is propagated by highly specialized immune lymphocytes known as regulatory T-cells. These so-called Treg cells can “turn off” immune reactions even once they have been started by conventional immune cells. They play a pivotal role in the immune system’s ability to prevent rejection of an embryo whether due to an autoimmune or alloimmune response. Other immune cells known as dendritic cells, introduce antigenic proteins to these Treg cells, whose concentration increases when the antigen is recognized as “self” and decreases when recognized as “foe.” MHC (primarily HLA-G) signaling, through the Treg lymphocyte mechanism working in combination with other regulatory proteins, influences the production and release of so-called cytokines by the NK and CTL cells. There are three varieties of cytokines, two of which play defining roles in the maintenance of implantation: The first is TH-2 cytokines, which encourage growth and expansion of the trophoblast and promote proliferation of blood vessels (angiogenesis). The second, TH-1 cytokines, promote destruction (cytolysis) of trophoblastic cells and also cause blood to clot (procoagulant effect). A balance between TH-1 and TH-2 cytokines is essential for normal implantation and development of the placenta (placentation).
Over-activity (dominance) of TH-1, the hallmark of NK cell and CTL activation, leads to damage of the trophoblast, implantation dysfunction, and reproductive failure.
Alloimmune Implantation Dysfunction
Every human being has two DQ alpha genes. One is contributed by the father and the other by the mother. When (albeit in a small percentage of patients undergoing IVF) paternal-maternal DQ-alpha gene similarities occur, it will, following repeated exposures to such genetically matching embryos, provoke activation of the decidual immune system. Usually, this will, through the mechanisms described above, ultimately lead to NK/CTL activation and reproductive failure (i.e., infertility, and pregnancy loss) in most cases. We refer to this phenomenon as alloimmune implantation dysfunction.
This is how alloimmune implantation dysfunction happens: Immunogenetically triggered HLA-G signaling on the part of the implanting embryo leads to a reduction in Treg cells and eventually to a destabilization of NK/CTLs with domination of TH-1 over TH-2 activity. The severity with which this occurs is an important determinant of whether total implantation failure will occur or whether there would remain enough residual trophoblastic activity that would allow the pregnancy to limp along until the nutritional supply can no longer meet the demands of the pregnancy, at which point miscarriage or pregnancy loss occurs.
With paternal-maternal DQ alpha matching it will often take the passage of several pregnancies for NK cell activation to build to the point that woman with alloimmune implantation dysfunction will present with clinical evidence of implantation dysfunction. Sometimes it starts off with one or two pregnancies surviving to birth of a baby, whereupon NK/CTL cell activity starts to build, leading to one or more early miscarriages. Eventually the NK /CTL activity is so high that subsequent pregnancies can be lost before the woman is even aware that she was pregnant at all. At this point she is often diagnosed with secondary, “unexplained” infertility and/or “unexplained” IVF failure. The case report below illustrates the interplay of factors involved in Alloimmune 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, infective, 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 autoantibodies then start attacking the body’s own cells/tissues/organs creating pathologic 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 often altered by the disease process and then combine with proteins to evoke the production of so-called antiphospholipid antibodies (APA). Certain types of APAs can both directly damage the trophoblast and can also lead to a reduction of Treg lymphocytes, culminating in activation of NK/CTLs. This type of reaction—albeit due to a predisposition to auto-immune diseases such as lupus erythematosus, Hashimoto’s disease, or reproductive conditions such as endometriosis—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 85% of reproductive failure due to immunologic implantation dysfunction. The three most common types of autoantibodies involved are antiphospholipid antibodies (APA), anti-thyroid antibodies (ATA), and possibly, antiovarian antibodies (AOA).
Management of Immunologic Implantation Dysfunction (IID)
In the United States, effective treatment of NK/CTL activation associated with either alloimmune or autoimmune implantation dysfunction requires the administration of primarily Intralipid (IL). Such treatment is much more likely to be successful in the case of` autoimmune implantation dysfunction where the NK/CTL activation is present in advance of the uterus being exposed to the embryo. It is not nearly as effective for the treatment of alloimmune implantation dysfunction where a DQ alpha-matching embryo will exert a sustained activation of NK/CTLs over several months of gestation.
It is presently not yet possible to recognize paternal DQ alpha in the embryo. Accordingly, in cases where the paternal DQ alpha genes only match with one of the mother’s DQ alpha’s (i.e., a partial match) there is a one out of two chance that a transferred embryo will inadvertently be a match with at least one of the mother’s DQ alpha genes. Thus, IL and IVIg therapy will only prove half as likely to propagate a viable pregnancy in cases of partial DQ alpha matching as it can achieve in the treatment of NK/CTL activation associated with autoimmune implantation dysfunction. Thus, we prefer to transfer only one embryo (rather than multiples) at a time in such cases, for fear of there being one DQ alpha matching embryo in the mix and so “muddying the waters” for the non-DQ alpha matching that otherwise might have propagated a healthy baby.
A real problem arises in cases of a complete match, where both paternal DQ alpha genes match with at least one of the mother’s DQ alphas. Here, every embryo will express a paternal DQ alpha gene that matches that of the mothers. In such cases, IL therapy will rarely work. The reason is that such treatment cannot match the sustained provocation of NK/CTL activity brought about by an ever-present DQ alpha “clash.” In cases of a complete DQ alpha matching (with associated NK/CTL activation), where all the embryos will inevitably carry one or both paternal DQ alpha that match(es) the mother, there is in my opinion little hope of success, even with Intralipid/steroid therapy. In such cases, gestational surrogacy or the use of non-DQ alpha matching donor sperm may offer the only reasonable chance of a successful IVF outcome.
Some patients ask whether using an egg donor might not offer another solution in such cases. The answer is no! The matchup is between the paternal DQ alpha contribution (in the sperm) and the mother’s uterus. It is not between the sperm and the egg.
IL therapy should be administered in combination (with corticosteroids) at an adequate dosage, 7–14 days prior to planned embryo transfer, and with alloimmune implantation dysfunction it should (ideally) be maintained, at least through the 1st half of pregnancy. The goal is to down-regulate activated NK/CTL and thereby reinstate a healthy TH-1: TH-2 cytokine balance in advance of a “competent” non-DQ alpha matching embryo reaching the uterus. Treatment of autoimmune implantation dysfunction requires that IL (with corticosteroids) be administered only twice, once 7–14 days prior to embryo transfer and then one more time when the beta hCG blood level has shown evidence of an appropriate rise, thereby suggesting that healthy implantation could be in progress. Supplementation with heparinoid is indicated when there is evidence of concomitant antiphospholipid antibodies or certain types of hereditary clotting defects (thrombophilias) such as a homozygous MTHFR mutation.
The Role of PGS (Full Embryo Chromosomal Karyotyping) in the Treatment of Alloimmune Implantation Dysfunction
Intralipid (IL)/Prednisone therapy only addresses the implantation issue, not embryo competency (which resides in the chromosomal integrity of the embryo transferred. Moreover, as previously alluded to, with a partial DQ alpha match/NK cell activation each blastocyst transferred has a 50:50 chance of matching. Consider the fact that the transfer of a single expanded blastocyst to a young woman (who did not have a DQ alpha match) would yield at best about a 35% chance of propagating a healthy pregnancy. Now, if the woman had a partial DQ alpha match with her partner, given that each of her embryo’s embryo would have a 50:50 chance of matching (and there is currently no way to identify the DQ alpha genotype of an embryo), the chance of a viable pregnancy would be one half of the otherwise anticipated 35% (i.e. about 17%). If on the other hand the woman’s transferred embryo had been tested and found through PGS Next Generation Gene Sequencing – NGS) to have a full component of 46 chromosomes (i.e. euploid) then the chance of a viable pregnancy would be about 32% (half of an otherwise 65% chance had she not had a partial DQ alpha match with her partner. Now add to this equation the fact that with a partial DQ alpha match it is probably best to transfer only one embryo at a time in order to reduce the risk that the inadvertent delivery of a DQ alpha matching embryo could potentially cause activation of local uterine NK cell activation that might prejudice the implantation of all embryos being transferred.
The Role of Embryo Banking in Cases of Alloimmune Implantation Dysfunction with a Partial DQ alpha Match
Bear in mind that less than 1:2 embryos are chromosomally normal even in young women, and this decreases further with advancing age. Furthermore, where there is a partial DQ alpha match between partners, only 50% of the embryos will be non-matching, reducing the chances of successful implantation again by half. It is advisable to only transfer one embryo at a time in such cases. Indeed, a strong case could be made for full embryo karyotyping (using PGS) to allow for the selective transfer (one at a time) of only those embryos that are chromosomally normal (euploid). In most cases, this will require biopsying the fresh embryos for PGS testing, allowing them to progress to blastocysts and then cryopreserving these for subsequent single embryo transfer. This would allow for more competent blastocysts to be available and for a much higher success rate per blastocyst transferred and accordingly, improved IVF outcomes.
Use of a Gestational Surrogate for Alloimmune Implantation Dysfunction
A gestational surrogate is used when there is a complete DQ alpha match with NK cell activation between the patient and the sperm provider. It has no real merit when there is only a partial match. Ordinarily, provided that an embryo recipient is NK negative, a DQa match between recipient and sperm provider should theoretically not preclude an ensuing pregnancy. Notwithstanding this, there should in our opinion be reluctance to accept NK negative Gestational Surrogates (GS) who share a DQa match with the sperm provider……An exception could be made only if following full disclosure of this concern to both parties in advance of treatment that although unlikely, a pregnancy with a matching DQa, NK negative pair could (although unlikely) suddenly cause the newly pregnant embryo recipient to convert to NK+, placing the pregnancy (as well as all future pregnancies) in jeopardy.
Use of a Sperm Donor in Cases of Alloimmune Implantation Dysfunction
This is an acceptable option in cases of a partial or complete DQ alpha match, provided that the sperm donor and the embryo recipient do not match, and any coexisting NK cell/CTL activation is treated concurrently with IL/steroids.
Use of Medications in the Treatment of IID
- Intralipid (IL) Therapy:
About a decade ago, a Sher-IVF Reproductive Endocrinologist, along with a geneticist in an affiliated Reproductive immunology Laboratory in Chicago, IL, were the first to report on the potential advantage of supplanting IVIg therapy.
Intralipid (IL) is a solution of small lipid droplets suspended in water. When administered intravenously, IL 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 a modulating effect on certain immune cellular mechanisms largely by down-regulating cytotoxic /activated natural killer cells (NKa). This effect is enhanced through the concomitant administration of corticosteroids such as dexamethasone, prednisone, and prednisolone, by suppressing cytotoxic/activated T-lymphocytes. 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. In this regard it is just as effective as IVIg but at a fraction of the cost and with a far lower incidence of side-effects. Its effect 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 (or IVIg) therapy can take several weeks to become detectable, it follows that there is really no benefit in trying to assess the potential efficacy of such treatment by retesting NKa in the laboratory after adding IL (or IVIg) to the sample.
Treatment of Autoimmune NKa Using Intralipid: When it comes to NKa in IVF cases complicated by autoimmune implantation dysfunction, the combination of daily oral dexamethasone commencing with the onset of ovarian stimulation and continuing until the 10th week of pregnancy, combined with an initial infusion of IL (100ml, 20% IL dissolved in 500cc of saline solution, 10-14 days prior to embryo transfer and repeated once more (only), as soon as the blood pregnancy test is positive), the anticipated chance of a viable pregnancy occurring within 2 completed IVF attempts (including fresh + frozen ET’s) in women under 40Y (who have normal ovarian reserve) is above 80%.
Treatment of Alloimmune NKa Using Intralipid:
Partial DQ alpha Match: IVF patients who have NKa associated with a partial alloimmune implantation dysfunction (DQ alpha match between partners) we use the same IL, infusion as with autoimmune-NKa, only here we prescribe oral prednisone rather than dexamethasone until the 10th week of pregnancy and IL infusions are repeated every 2-4 weeks following the chemical diagnosis of pregnancy until the 24th week. Additionally, (as alluded to elsewhere) in such cases we transfer only a single embryo at a time. This is because in such cases, the likelihood is that one out of two embryos will “match” and we are fearful that if we transfer >1 embryo, and one of the transferred embryos “matches” it could cause further activation of uterine NK cells and so prejudice the implantation of all transferred embryos. Since we presently have no way of determining which embryo carries the matching paternal DQ alpha gene and thus would transfer only one embryo at a time, it follows that the anticipated viable pregnancy rate per cycle will be much lower than with autoimmune implantation dysfunction. It also follows that the only way to improve success with a single embryo being transferred would be to perform PGS on the embryos in advance of ET and then selectively transfer a “chromosomally normal-euploid (“competent”) embryos.
Total (Complete) DQ alpha Match: In cases where the partners have a total alloimmune (DQ alpha) match with accompanying NKa the chance of a viable pregnancy occurring or (if it does) resulting in a live birth at term, is so small as to be an indication for using a non-matching sperm donor or resorting to gestational surrogacy would in our opinion be preferable by far.
Contraindications and Cautions with Intralipid Infusion: IL is only contraindicated in conditions associated with severely disordered fat metabolism (e.g. severe liver damage, acute myocardial infarction and shock,
Rarely, hypersensitivity has been observed in patients allergic to soybean protein, egg yolk and egg whites and where fat metabolism may be disturbed (e.g. renal insufficiency, uncontrolled diabetes, certain metabolic disorders and in cases of severe infection (sepsis).
Adverse Reactions during Infusions of IL (Rare): These include transient fever, chills, nausea, vomiting, headache, and back or chest pain with shortness of breath and cyanosis.
Composition and Storage of IL: IL should be stored at a controlled room temperature below 25°C. It should not be frozen.
IVIg versus Intralipid Therapy: Until about a decade ago, the only effective and available way (in the US) to down-regulate activated NK cells was through the intravenous administration of a blood product known as immunoglobulin-G (IVIg). The fear (albeit unfounded) that the administration of this product might lead to the transmission of viral infections such as HIV and hepatitis C, plus the high cost of IVIG along with the fact that significant side effects occurred about 20% of the time, led to bad press and bad publicity for the entire field of reproductive immunology. It was easier for RE’s to simply say “I don’t believe IVIg works” and thereby avoid risk and bad publicity. But the thousands of women who had babies because of NK cell activity being down-regulated through its use, attests to IVIg’s efficacy. But those of us who felt morally obligated to many desperate patients who would not conceive without receiving IVIG were facing an uphill battle. The bad press caused by fear mongering took its toll and spawned a malicious controversy. It was only through the introduction of IL less (than a decade ago), that the tide began to turn in favor of those patients who required low cost, safe and effective immunotherapy to resolve their IID.
- Corticosteroid Therapy (Prednisone, Prednisolone, and Dexamethasone)
Corticosteroid therapy has become a mainstay in the treatment of most 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 compelling evidence 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 APAs and can prevent later pregnancy loss when certain thrombophilias (e.g. homozygous MTHFR mutation)
- What About Baby Aspirin?
In our opinion, aspirin has little (if any) value when it comes to IID, and besides, could even reduce the chance of success. The reason for this is that aspirin thins the blood and increases the potential to bleed. This effect can last for up to a week and could complicate an egg retrieval procedure or result in “concealed” intrauterine bleeding at the time of embryo transfer, thereby potentially compromising IVF success.
- TH-1 Cytokine Blockers (Enbrel, Humira)
TH-1 cytokine blockers, (Enbrel and Humira) are in our opinion relatively ineffective in the IVF setting. There has to date been no convincing data to support their use. Conversely, these blockers could have a role in the treatment of a threatened miscarriage thought to be due to CTL/NK activation, but not for IVF. The reason is that the very initial phase of implantation requires a cellular response involving TH-1 cytokines. To block them completely (rather than simply restore a TH-1: TH-2 balance as occurs with IL therapy) so very early on could compromise rather than benefit implantation.
- Leukocyte Immunization Therapy (LIT)
The subcutaneous injection of the male partner’s lymphocytes to the mother is thought to enhance the ability for the mother’s decidua (uterus) to recognize the DQ alpha matching embryo as “self” or “friend” and thereby avert its rejection. LIT has been shown to up-regulate Treg cells and thus down-regulate NK cell activation and thereby improve decidual TH-1: TH-2 balance. Thus, there could be a therapeutic benefit from such therapy. However, the same benefit can be achieved through the use of IL plus corticosteroids. Besides, IL is much less expensive, and the use of LIT is prohibited by law in the U.S.A.