Here in our 11th case, we discuss a couple who had experienced eight (8) prior 1st trimester miscarriages due to a hitherto undiagnosed immunologic implantation dysfunction (IID). We identified the underlying cause as a shared, “partial DQalpha match” with uterine natural killer cell activation (NKa). Treatment involvedIntralipid (IL) and prednisone, down-regulation of NKa followed by IVF using CGH embryo selection to identify genetically “competent” embryos. Once the CGH results were available, a frozen embryo was transfer performed (Staggered IVF), in which a single blastocyst was transferred (SBT). Upon establishing successful implantation, IL/steroid therapy was continued until the 24th week. She gave birth to a full-term healthy baby girl. This discussion centers on the importance of establishing an early diagnosis of IID in all cases of RPL and why IVF with a single blastocyst transfer of a CGH-selected chromosomally “competent” embryo was the favored approach in this case.

Background
Monica and her partner Andrew were 34 years and 37 years of age, respectively, when they first presented to me with a history of eight (8)recurrent first trimester miscarriages over a period of four (4) years. Fetal cardiac activity had been observed prior to the miscarriage in all but two cases. D&C’s were conducted to evacuate the uterus after 5 of the 8 miscarriages, and products of conception were submitted for chromosomal analyses (karyotyping). Pregnancies number 1 (male), 7 (female), and 8 (male) yielded products of conception with normal chromosomal configurations or karyotypes (i.e.”euploid”).

In 3 cases, the spontaneous miscarriages were deemed (through ultrasound examination) to have been “complete”, thus no D&C’s were done and no products were available for karyotyping. The 1st three miscarriages occurred prior to 8 weeks gestation while the last two occurred between 10 and 12 weeks gestation. The first 4 pregnancies occurred spontaneously, the next three followedintrauterine inseminations (IUI) with clomiphene citrateovulation inductions, and the last pregnancy occurred with a second IVF attempt.

Both Monica and Andrew had their blood tested for structural chromosomal abnormalities known as translocations….None were present. Monica underwent hormonal evaluation to exclude subtle causes of ovulation dysfunction (FSH, LH, luteal phase estradiol, and progesterone/prolactin) and had tests for thyroid function, all of which were normal. A hysterosonogram (HSN), (also known as a saline sonogram) done prior to her initial IVF attempt confirmed the absence of surface lesions involving the uterine cavity, and an ultrasound examination of the uterine lining (done prior to ovulation/egg retrieval) consistently revealed an adequate endometrium (trilaminar configuration and a thickness of >9mm).

A thrombocytopenia panel done on Monica’s blood, aside from a heterozygous single mutation, showed no major problems in this regard. Both Monica and Andrew tested negative for reproductive ureaplasma infection and Andrew had normal semen analysis andSCSA tests…

Clearly, while Monica and Andrew had undergone all standard tests for causes of RPL, no testing had been done for analloimmune or autoimmune cause for immunologic implantation dysfunction. Accordingly I had the following immunologic tests performed:

1. Monica’s blood : Antiphospholipid antibodies, Antinuclear antibodies, Immunophenotype, and antithyroid (antithyroglobulin and antimicrosomal) antibodies, Natural Killer Cell activation (NKa) – using the K-562 Target Cell Test
2. Both Monica and Andrew’s blood: DQalpha and HLA matching

The results of these tests revealed that Monica was NKa positive and that she and Andrew shared a “partial” DQalpha match (one of two DQalpha genes matched).

I strongly recommended that they proceed with a cycle of Staggered IVF with CGH embryo selection (see below). Monica’s COS cycle was launched with her coming off a birth control pill (BCP) and using a modest agonist-antagonist Conversion (stimulation) Protocol (A/ACP). I treated her with prednisone from the 1st day of stimulation. This was continued throughout the cycle. On cycle day 7, Monica received an infusion of 20% intralipid. She produced 11 mature (MII) eggs at ER. Following ICSI, these propagated 8, 5-9 cell, day-3 embryos.

These embryos were biopsied for metaphase CGH analysis (full karyotyping) and were then allowed to continue in culture to the blastocyst stage. At that time, the four embryos that made it to this stage were vitrified and cryobanked for future dispensation in a later cycle after the CGH results were returned and reviewed (Staggered IVF ). Two of the four blastocysts were found to be CGH-normal (euploid) and thus were classified as “competent”.

In a subsequent hormone replacement cycle, one was transferred to her uterus (the others three were kept in storage for future use). She conceived and subsequently gave birth to a healthy baby girl at term. Steroid/IL therapy was continued through the 24th week of pregnancy.

A Rational Approach to Managing Recurrent Pregnancy Loss
In more than 75% of cases, early miscarriage is caused by achromosomally defective embryo (aneuploidy). In the remaining 25% of cases, pregnancy loss is due to uterine (endometrial) rejection of the embryo (i.e. implantation dysfunction). The latter can be due to an anatomical defect involving the uterine cavity (polyps, fibroids and scarring), a thin (inadequate) endometrial lining, or an immunologic implantation dysfunction (IID). However, since embryo aneuploidy occurs sporadically, it rarely repeats over and over in the same person. When this happens (i.e. RPL), the cause is more likely to be due to implantation dysfunction – of which there are two varieties:

1. Alloimmune Implantation Dysfunction – This is the most common immunologic cause of recurrent miscarriages. here is how it works: With fertilization, the sperm contributes genetic material to the fertilized egg or embryo. That is why an implanting embryo, is in about 90% of cases immunogenetically different from the mother’s genotype…Yet, in spite of this difference the embryo is accepted and allowed to continue to develop into a viable pregnancy. This so called “paradox of pregnancy” is due to magnificent immunologic adaptations in the uterus, brought about by interaction (“communication) between an immunologically distinct embryo and the predominating immune lymphocytes in the uterine lining known as natural killer (NK) cells. Sometimes (in <10% of cases) the embryo and the mother might share certain similarities involving genes known as DQalpha and HLA. When this happens, after repeated exposures of the uterus to consecutive matching pregnancies, uterine NK cells (as well as and other immune cells known as cytotoxic lymphocytes) begin to release excessive amounts of cytotoxic cytokines that attack the embryo’s trophoblast (root system) , thereby initiating a rejection process that often precipitates a first trimester miscarriage.Such alloimmune implantation dysfunction is thus a relatively common immunologic cause of recurrent first trimester miscarriages , making testing for alloimmune similarities in both partners an important part of the evaluation of non-chromosomal recurrent pregnancy loss. This requires comparing the mother’s and father’s HLA and DQ alpha genetic status for “matching similarities”.When the sperm provider and the embryo recipient share one or both DQ alpha genes and/or have several other HLA genes in common, implantation failure can occur, usually manifesting as early RPL (and sometimes, albeit less common, as “unexplained” infertility or IVF failure). However, for such alloimmune matching to lead to implantation dysfunction (e.g. miscarriage) there must be associated activation of uterine NK cells (NKa) as evidenced by the K-562 Target cell test and/or increased endometrial TH-1 cytokine activity determined through sampling. In the absence of such NKa, even the severest form of alloimmune matching will not prevent a perfectly normal pregnancy from occurring.
2. Autoimmune Implantation Dysfunction is the most common cause of immunologic failed implantation. This usually presents as “unexplained infertility” and/or “unexplained IVF failure” rather than miscarriage. Autoimmunity is an immunologic reaction produced by the individual to his/her body’s own cellular components. The most common autoantibodies that form in such situations are: a) Antiphospholipid antibodies (APA); b) Antithyroid antibodies (ATA); c) Antiovarian antibodies.But as with alloimmune implantation dysfunction, it is only when uterine NK cells become activated (NKa) and start to release excessive amounts of TH-1 cytokines, which attack the root system of the embryo, that implantation potential is jeopardized. Since autoimmune implantation dysfunction is often genetically transmitted, it is not surprising that it is more likely to exist in women who have a family (or personal) history of primary autoimmune diseases such as lupus erythematosus (LE), scleroderma, clinical or subclinical hypothyroidism, and rheumatoid arthritis.Reactionary (secondary) autoimmunity can occur in conjunction with any medical condition associated with widespread tissue damage. One such gynecologic condition is endometriosis (where 1/3 of cases have associated NKa). Autoimmune implantation dysfunction is usually immediately lethal to the implanting embryo. This is because it destroys most of the embryo’s root system from the get-go. That is why it most commonly presents as “unexplained infertility” or “unexplained (often repeated) IVF failure” rather than as a miscarriage.

Selective Immunotherapy for RPL: Both alloimmune and autoimmune implantation dysfunction are amenable to timely and appropriately administered selective immunotherapy with steroids (Prednisone/dexamethasone) and intralipid (IL) therapy. Achievement of optimal success with IL/corticosteroid therapy requires that the treatment be initiated well before ovulation takes place (about 7-14 days prior to anticipated implantation).

Reaching a diagnosis and developing a treatment plan for Monica and Andrew: The fact that at least three of Monica’s prior miscarriages were associated with chromosomally normal (euploid) pregnancies strongly suggested to me that her RPL was not due to embryo aneuploidy. By exclusion it had to be due to an implantation dysfunction. Since prior ultrasound examinations had shown that she generated a well-developed endometrium, it was apparent that Immunologic Implantation Dysfunction lay at the root of her recurrent miscarriages. This prompted immunologic testing that confirmed that she had NKa – apparently due to alloimmune dysfunction resulting from a partial DQa match with Andrew. This is what formed the basis for my proposing intralipid and steroid therapy that, in the event of pregnancy, would be continued through the 24th week of pregnancy.

An Argument for Staggered IVF with CGH Testing
Any exposure of Monica’s endometrial NK cells to an embryo with a matching DQa (derived from partner’s sperm) would evoke a local response in the uterus that would cause the release of TH-1 cytokines and the subsequent rejection of any/all embryos in the uterus at the time. Therefore, with the couple’s partial DQalpha match, each embryo propagated by Andrew’s sperm would have a 1:2 chance of matching Monica’s DQalpha type (there is currently no reliable way to determine which embryo(s) would “clash”). It follows that if more than one single embryo was to be transferred and one of these “matched”, it could lead to the rejection of both embryos and a failed embryo transfer. That is why I strongly advocate for the transfer of only one embryo at a time in cases of partial DQalpha matching.

Moreover, because of the partial match in his case, each embryo transferred would have a 1-in-2 chance of being accepted by the uterine immune system. This meant that each embryo transferred would have half the chance of propagating a pregnancy as compared to what would otherwise have been the case in the absence of such an alloimmune match with accompanying NK cell activation.

Since Intralipid therapy will only protect the non-matching embryo from rejection, the chance of Monica having a baby when one embryo is transferred was reduced from about 25% to about 12% using traditional embryo selection methods. I thus favored increasing those odds through CGH embryo selection. In this way, the chance of such a “competent” embryo propagating a baby would be about 35% in her case (half of 70%)…much better odds, don’t you think? Implementation of this approach mandated that IVF be used.

Conclusion: Immunotherapy in IVF still represents a “much debated” issue, with many RE’s (albeit a declining number) still stubbornly refusing to even consider its relevance. However, Monica and Andrew’s case represents one of many examples where failure to evaluate IID in cases of “unexplained” infertility, “unexplained” IVF failure and RPL results in a couple “spinning their wheels” with significant emotional, financial and even physical consequences.. Patients with such issues should demand of their RE’s that they investigate and treat such issues.

Addendum: IVF is an ART-Science blend and not all practitioners agree on the same strategies. Thus, in the final analysis, it is important, after discussion with your personal doctor, to follow his/her advice to the letter.