Immunology May Be Key To Pregnancy Loss
Until the last decade, there was little a couple could do if they suffered from recurrent pregnancy losses. Miscarriages that couldn't be attributed to chromosomal defects, hormonal problems or abnormalities of the uterus were labeled "unexplained," and couples would continue to get pregnant, only to suffer time and again as they lost their babies. New research, however, indicates that as many as 80 percent of "unexplained" losses may the attributable to immunological factors-and some new therapies are enabling up to 80 percent of those affected to carry a baby to term.
About 15 to 20 percent of all pregnancies result in miscarriage, and the risk of pregnancy loss increases with each successive pregnancy loss. For example, in a first pregnancy the risk of miscarriage is 11 to 13 percent. In a pregnancy immediately following that loss, the risk of miscarriage is 13 to 17 percent. But the risk to a third pregnancy after two successive losses nearly triples to 38 percent.
Many doctors do not begin testing for the cause of pregnancy loss until after three successive miscarriages. However, because the risk of loss to a third pregnancy after two successive miscarriages is so high, the American College of Obstetrics and Gynecologists (ACOG) now recommends testing after a second loss-especially for women over the age of 35.
There are two major reasons for recurrent spontaneous abortion (RSA), or miscarriage. One is that there is something wrong with the pregnancy itself, such as a chromosomal abnormality, which curtails embryonic development. (A fertilized ovum is an embryo until 10 weeks gestation, and a fetus thereafter. Most miscarriages, though not all, occur between six and eight weeks, with expulsion taking place four weeks later, between 10 and 12 weeks.)
The best way to find out if the pregnancy itself is the problem is to test the chromosomes of the aborted embryo. While in many cases this is not an option, requesting genetic testing after a dilation and curettage (D&C) for a missed abortion can often give couples some definitive answers about what went wrong. An alternative is genetic testing for the couple, called "karyotyping." This involves a blood test for each partner so that both sets of chromosomes can be evaluated for abnormalities which may cause RSA, or which may be passed on to children.
The other cause of RSA, and the category into which immunological problems fall, is an environmental barrier to pregnancy-something wrong with he environment in which the pregnancy grows. In addition to immunological problems, other possible environmental causes of pregnancy loss are hormonal (not enough of necessary hormones to sustain the pregnancy) and anatomic (such as structural abnormalities of the uterus).
Anatomic problems may be detected with a hysterosalpinogram, hysteroscopy or hysterosonogram. Assessment of the hormonal environment looks at hormone levels and uterine response at the expected time of ovulation and implantation, usually through an endometrial biopsy or high level ultrasound examination.
The final way to determine an environmental cause of multiple miscarriage is through immunologic testing.
The immune system, one of the most intricate and complex systems in the body, functions as the first line of defense against disease. It works by identifying proteins as normal or foreign. The immune response to a foreign protein is to neutralize or destroy the antigen. An antigen is a protein marker on the surface of a cell that identifies the cell as "self" or "nonself" An antigen can cause the production of antibodies. Antibodies are complex compounds made by the white blood cells (WBCs) that combine with specific antigens to destroy or control bacterial infections. As bacteria enter the body, WBCs produce antibodies to provide protection against illness.
Immune Causes of Recurrent Pregnancy Loss
The immunologic causes for pregnancy loss and implantation failure are the result of abnormalities in antibody responses. These responses fall into two categories: autoimmune and alloimmune.
Contributions to a pregnancy represent the pairing of genes from both man and woman. Autoimmune represents the immunologic response of the mother to a pregnancy ("self-immune" problems). Autoimmune disorders that can cause rejection of a pregnancy mean the woman is rejecting her own proteins-in other words, treating them like they are an invading illness. Autoantibodies are antibodies which attack one's own antigens.
Alloimmune problems indicate a mother's response to the man's genetic contribution to the pregnancy ("other-immune" problems). Alloimmune disorders are the rejection of a protein from the man. Both kinds of immune disorders can be determined with blood tests.
There are four different autoimmune problems that can cause RSA. A woman may have one or more of these underlying problems: antiphospholipid antibodies; antithyroid antibodies, antinuclear antibodies and lupus-like anticoagulant. Thirty percent of women with "unexplained" RSA will test positive for an autoimmune problem.
In pregnancy, phospholipids act like a sort of glue that holds the dividing cells together, and are necessary for growth of the placenta into the wall of the uterus. Phospholipids also filternourishment from the mother's blood to the baby, and in turn, filter the baby's waste back through the placenta.
If a woman tests positive for any one of variety of antiphospholipid antibodies (APA), it indicates the presence of an underlying process that can cause recurrent pregnancy loss. The antibodies themselves do not cause miscarriage, but their presence indicates that an abnormal autoimmune process will likely interrupt the ability of the phospholipids to do their job, putting the woman at risk for miscarriage, second trimester loss, intrauterine growth retardation (IUGR) and pre-eclampsia.
While testing for anticardiolipins (cardiolipins are a kind of phospholipid) is standard in some infertility clinics, this test alone cannot identify the presence of all underlying autoimmune processes that causes RSA. A panel of tests for antibodies to six additional phospholipids is recommended to determine the presence of APA. Testing positive for one or more kind of antiphospholipid antibodies indicates the woman has the immune response that can causes RSA. (See the full range of APA tests in the accompanying chart.)
The markers tested for each of seven phospholipids include IgM, IgG and IgA. These are circulating immunoglobulins (proteins that ward off potential harmful invaders). In some patients, measuring these 21 markers can identify elevations of immunoglobulins to unknown proteins, and signal some as-yet-unidentified process exists that can trigger RSA.
About four percent of women with recurrent miscarriage test positive for lupus-like anticoagulant, and nine percent of individuals diagnosed with SLE have a positive lupus anticoagulant test, or activated partial thromboplastin time (APTT). APTT is an adequate screening test for lupus-like anticoagulant antibodies, but there is a high incidence of false positives. Women who have a positive APTT should also have more specific tests, such as Kaolin clotting time, Russel viper venom assay and the platelet neutralization assay a to confirm the presence of lupus anticoagulant antibody activity. And, since some women do not test positive until they are pregnant or have suffered a pregnancy loss, repeat testing during early pregnancy is highly recommended when there is a history of RSA.
Because some circumstances can cause false positives for these tests, it is important to determine persistent positive levels by repeating the tests in six to eight weeks.
The live birth rate for a patient with untreated APA ranges from 11 percent to 20 percent. Individuals with recurrent pregnancy loss and/or implantation failure, venous or arterial, thrombosis, thrombocytopenia, elevated APTT or a circulating lupus-like anticoagulant are among those at risk for development of APA. Also at risk may be women experiencing infertility associated with endometriosis, premature ovarian failure, multiple failed in-vitro fertilization, and unexplained infertility. With treatment, the live birth rate for women with APA increases to 70 to 80 percent.
Antinuclear antibodies react against normal components of the cell nucleus. They can be present in a number of immunologic diseases, including: systemic lupus erythematosus (SLE or Lupus), progressive systemic sclerosis, Sjorgen's syndrome, scleroderma polymyositis, dermatomyositis and in persons taking hydralazine and procainamide or isoniazid. In addition, ANA is present in some normal individuals or those who have collagen vascular diseases. The presence of ANA indicates there may be an underlying autoimmune process that affects the development of the placenta and can lead to early pregnancy loss.
Histones are proteins which combine with the DNA of the cell nucleus to govern the development of tissues. Histones are the smallest building blocks of DNA. Antibodies to these histones mean the mother is developing an immunity to histone components of DNA. The mechanism by which ANA cause pregnancy loss is not known. (See the accompanying chart for specific ANA tests.)
Women with thyroid antibodies face double the risk of miscarriage as women without them. Increased levels of thyroglobulin and thyroid microsomal (thyroid peroxidase) autoantibodies show a relationship in an increased miscarriage rate, and as many as 31 percent of women experiencing RSA are positive for one or both antibodies. Chances of a loss in the first trimester of pregnancy increase to 20 percent, and there is also an increased risk of post-partum thyroid dysfunction. Therefore, antithyroid antibody testing should be routine in women with a history of two or more losses or thyroid irregularities.
It is important to note that when only the hemagglutination blood test is used, one out of five women with thyroid antibodies will not be correctly screened. More sensitive tests, enzyme linked immunosorbant assays (ELISAs), or gel agglutination tests, have become the standard for thyroid antibodies associated with recurrent pregnancy loss.
Treatments for autoimmune risk factors include preconception administration of low-dose heparin (an anticoagulant produced naturally by the body), aspirin and prednisone (a steroid to decrease inflammation ). Heparin is administered (at 5,000 10,000 units) every 12 hours, subcutaneously, and is used to treat women with APA syndrome and to combat possible clotting problems. Prednisone (40-60 mg. per day) is given to decrease autoantibody levels, provide blood-thinning and anti-inflammatory reactions, and reduce the risk of clotting. Aspirin is a prostaglandin inhibitor which decreases agglutination of the platelets (clotting), and has some anti-inflammatory action.
Aspirin therapy, sometimes in concert with heparin and prednisone in severe cases), can increase blood flow to the placenta by inhibiting the tendency for clotting in women with abnormal levels of autoantibodies. Because of complications of pregnancy are significantly higher with prednisone, however, it is usually recommended for women who do not respond to aspirin and heparin therapy.
Another successful method for treatment of autoimmune factors is intravenous immunoglobulin (IVIg), a process which infuses the mother with antibodies from thousands of donors in the general population. The basic effect of IVIg is like neutralizing a large military force (the mother's dangerous antibodies) armed with weapons. The army is still present after administering the IVIg, but it is disarmed. The donor immunoglobulin keeps the attacking antibodies busy and away from the developing fetus.
Among women with the combined problems of APA and elevated NK cells who achieve pregnancy with preconception treatment, the subsequent live birth rate is about 70 percent. The initial treatment of choice is usually low-dose heparin and aspirin therapy because obstetrical complications, such as preterm birth, premature rupture of the membrane and gestational diabetes, are more common with prednisone. IVIg, while very effective, is also quite costly --- roughly $10-30,000 for treatments throughout pregnancy ($39 to $145 per gram, depending on the distributor). However, among women for whom pregnancy loss occurred even with preconception use of heparin and aspirin, IVIg remains an alternative that may allow them to carry a pregnancy to term.
There are two possible reasons that women with alloimmune problems lose their pregnancies in miscarriage: Either her immune system does not recognize the pregnancy, or she develops an abnormal immunologic response to the pregnancy.
Successful pregnancy has been associated with the presence of circulating "blocking antibodies." These are antibodies that are formed by a woman's immune system when she is pregnant, and they "mask," or disguise the pregnancy so it is not recognized as "foreign." Pregnancies that end with RSA have been associated with the absence of these blocking antibodies.
Recently, an antigen identified as R80K has been identified on the surface of syncytiotrophoblasts, the outer layer of cells covering of the chorionic villi of the placenta. These cells are in contact with maternal blood. R80K is a kind of protein marker to which the blocking antibodies respond during a successful pregnancy. The antibodies to this antigen react in a specific way to the antigens from the father's genetic material in the developing embryo, and thus create the protective, blocking antibodies.
For some women who lack the blocking antibodies, immunization with their husband's white blood cells may be an effective treatment. However, a leukocyte antibody detection assay (LAD) should be performed prior to initiating this treatment.
Also, IVIg treatment may be effective for some women who lack blocking antibodies because the immunoglobulin, which comes from thousands of donors, appear to contain small amounts of antibodies to R80K.
Leukocyte Antibody Detection Assay
Performed after a series of suspected losses when a woman is not pregnant, the Leukocyte antibody detection (LAD) test indicates a woman's physiologic response to pregnancy. Women who test for high levels of leukocyte antibodies have a history of carrying pregnancies longer than women who exhibit low levels. Women who have low levels of leukocyte antibodies generally had pregnancies that ended by week 12, or their immune systems did not respond to the stimulus of pregnancy by creating blocking antibodies. Only women with low levels of LAD are candidates for immunization with their husbands' white blood cells (leukocytes), so it is recommended that this assay be done prior to initiating an immunization protocol.
Natural Killer Cells
WBCs that belong to the innate or primitive group of cells that kill anything perceived as foreign . They kill abnormal invaders, including virally-affected cells. Some types of NK cells produce a substance called tumor necrosis factor (TNF), which might be described as your body's version of chemotherapy, and is toxic to a developing fetus. Patients who have high levels of these cells are at risk for implantation failure and miscarriage.
The proportion of NK cells is determined by a reproductive immunophynotype (RIP) test, which looks for cells that have the CD56+ marker. An NK (CD56+) cell range above 12 percent is abnormal. A patient with high NK cell activity will respond very well to intravenous immunoglobulin (IVIg) therapy. In fact, the live birth rate with preconception IVIg is more than 90 percent, compared to 20 percent without treatment.
Cells make proteins called cytokines. Different cytokines do different things. Some stimulate growth of cells, some inhibit growth. The proinflammatory cytokines stimulate inflammatory response, while others inhibit inflammatory response of cells. The embryo toxicity assay (ETA) is looking for cytokines which kill embryos.
Embryotoxic factors have been identified in as many as 60 percent of women with recurrent, unexplained miscarriage, and also reported among women endometriosis-associated infertility.
For the ETA, blood serum from the woman is incubated with mouse embryos. If the embryos die, a toxin (to the embryo) cytokine is present. IVIg therapy controls these cytokines and allows a pregnancy to progress.
For a women who exhibits low levels of LAD, immunization with her husband's white blood cells results in about a 10 percent increase in the chance of live birth (to 60 percent) over the live birth rate without treatment. And risk of complications for these women, such as intrauterine growth retardation (IUGR), preterm birth and birth defects, are generally diminished with treatment.
Immunization can also be performed with seminal plasma vaginal capsules, inserted twice weekly from preconception to the 28th week of pregnancy. There is about a 15 percent increase in the live birth rate with this treatment compared to no treatment.
IVIg is also an effective, though more costly, treatment for women with low LAD levels. Research shows that there is a 28 percent increased live birth rate among women in this category who received IVIg, compared to women given a placebo.
For women with elevated LAD levels, IVIg is the recommended treatment. The dosage is 500 mg. per kg (2.2 lbs.) of weight per month. When treatment is started prior to conception and continued through 28 weeks of gestation, the overall success rate of IVIg is 70 percent. IVIg is also recommended for treatment of elevated circulating natural killer (CD56+) cells, circulating embryotoxins and unexplained recurrent miscarriage.
As much as 40 percent of unexplained infertility may be the result of immune problems, as are as many as 80 percent of "unexplained" pregnancy losses. Unfortunately for couples with immunological problems, their chances of recurrent loss increase with each successive pregnancy.
Certainly, couples with RSA (two or more) would benefit from the full range of available immunological testing, especially if a woman is older than 35. And, because immune problems are often the cause implantation failure, couples with good embryos that fail to implant during IVF procedures are also good candidates for immunological screening.
Medical researchers have begun to pay attention to the problems of recurrent pregnancy loss, and ongoing genetic and immunologic research will continue to improve the diagnosis and treatment of this heartbreaking problem.
The following chart includes the full range of Autoimmune and Alloimmune Risk Tests and their "normal" ranges as conducted by Dr. Coulam at the Center for Human Reproduction in Chicago, Ill. Other labs and doctors may use different norms. The full range of tests is about $1,300.
Autoimmune Risk Tests
Antiphospholipid Antibodies (APA) Each of three markers, IgM, IgG and IgA, are tested for the following phospholipids, for a total of 21 different markers.
Anti-Phospholipid Antibodies (APA) IgM IgG IgA
Anticardiolipin (ACA) .131 - .173 .209 - .254 .192-.212
Phosphoethanolamine .362 - .478 088 - .222 .046-.073
Phosphoinositol .136 - .178 .175 - .236 .093-.122
Phosphatidic Acid .137 - .214 .104 - .132 .131-.155
Phosphogylcerol .168 - .242 .143 - .185 .102-.139
Phosphoserine .101 - .134 .082 - .188 .123-.143
Phosphocholine .152 - .198 .131 - .170 .092-.219
Natural Killer Cells
Reproductive Immunophynotype (RIP) 3-12%
Leukocyte Antibody Detection (LAD) >10%
Embryo Toxicity Assay (ETA). <37% Artesia
Lupus - LikeAntibodies
Lupus-like Anticoagulant Antibodies (APA) 33.5 - 44.5 seconds
Kaolin ClottingTime 33.5 - 44.5 seconds
Platelet Neutralization Assay 33.5 - 44.5 seconds
Dilute Russel viper Venom Time 33.5 - 44.5 seconds
Thyroglobulin <1:7ssDNA 99
Sci-70 32Thyroid microsomal (thyroid peroxidase) autoantibodies <1:72
ELISAs Gel agglutination tests <1:72
Carolyn B. Coulam, M.D.
is the Medical Director, SIRM-Chicago and Millenova Laboratory in Chicago, IL. She has served as a member of INCIID's advisory board since the organization's inception.
Nancy P. Hemenway
is an INCIID cofounder and serves as INCIID's Executive Director.