Immunology may be key to pregnancy loss (updated) by Carolyn Coulam and Nancy Hemenway

Graphis of words associated with immunology
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Graphis of words associated with immunologyUntil 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, has provided information on the causes of the heretofore unexplained pregnancy losses allowing more effective treatment enabling women to carry their babies 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 that 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 information is not available, 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 to obtaining genetic testing of the pregnancy is to test the chromosomes of the couple. This test is called a karyotype and 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. In addition to chromosomal problems, the pregnancies can have either abnormal genes or abnormal DNA contributing to their losses.  Gene abnormalities associated with recurrent pregnancy loss include mutations in HLAG genes contributed by either the father or the mother as well as gene deletions on the Y chromosome contributed by the father.  Fragmented DNA from the sperm has also been associated with early pregnancy loss.

The other major category of causes of RSA is a problem within the uterine environment that does not allow the pregnancy to grow properly. The most frequent environmental causes of pregnancy loss are attributable to immunologic factors followed by thrombophilic or blood clotting factors.  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 miscarriages is through immunologic and thrombophilic testing. To better understand the rationale for immunologic and thrombophilic testing, the roles of the immune and blood clotting systems in nature and reproduction will be reviewed.

 

Immune System

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 generating cells and molecules that are capable of identifying and eliminating potential  harmful “foreign” invaders.   The key to the function of the immune system is its ability to distinguish between the body’s own cells (self) and foreign cells (nonself). The body’s immune defenses normally coexist peacefully with cells that carry distinctive “self” marker molecules.  But when immune defenders encounter cells or organisms carrying markers that say “foreign”, they quickly launch an attack.  These cell markers as well as anything that can trigger an immune response is called an antigen.  Functionally, an immune response can be divided into two activities:  an innate immune response and an adaptive (or acquired) immune response. 
 
The innate immune system is an ancient mechanism of host defense found in essentially every multicellular organism from plants to humans.  It is the quick-to-respond, wired-in-the-genes immune system that serves as the body’s first line of defense that comes into play immediately or within hours of an antigen’s appearance in the body.  These actions are activated by chemical properties of the antigen and provide rapid, nonspecific and generalized defense mechanisms against a wide range of organisms.  The cells involved in innate immune responses include natural killer (NK) cells.
 

  • NK cells are a type of immune cells that are called lymphocytes.  NK cells secrete different proteins or cytokines depending on the signal they receive.  They also contain granules filled with potent chemicals that can destroy other cells NK cells recognize other cells that lack the so-called self molecules or antigens so they have the potential to attack many types of foreign cells.

 
If this first line of defense is not successful in neutralizing the potential harmful invader, the adaptive immune system is signaled.

The adaptive immune system is slower and more complex than the innate immune response.  The antigen must first be processed and recognized.  Once an antigen has been recognized, the adaptive system activates immune cells specifically designed to attack that antigen.  Adaptive immunity also includes a “memory” that makes future responses against that specific antigen faster.  The cells involved in the adaptive immune response include both T and B lymphocytes.
 

  • T cells are a subset of lymphocytes that play a large role in immune responses.  The abbreviation “T” stands for thymus. The organ in which T cells develop.  Most of the T cells in the body belong to three subsets:
    • Cytotoxic T cells express on their surface an antigen called CD8.  The role of cytotoxic T cell is to monitor all cells in the body ready to destroy and those express foreign antigens.  Destruction is mediated by molecules secreted CD8+ cells secrete molecules that destroy the cell to which they have bound
    • Helper T cells express on their surface CD 4 antigens and function as “middlemen” in immune responses.  When activated helper T cells proliferate and secrete proteins called cytokines that regulate or “help” other lymphocyte function.  There are two kinds of cytokines secreted by T helper cells:  pro-inflammatory cytokines that are largely involved in cell-mediated immunity (called Th1 responses) and anti-inflammatory cytokines that are involved in promoting B cells to secrete antibodies (called Th2 responses).
    • Regulatory T cells (also known as suppressor T cells) suppress activation of the immune system. Regulatory T cells express the cell surface antigens of CD8 and CD25.  Failure of regulatory T cells to function properly may result in autoimmune disease in which the immune cells attack healthy cells in the body
  • B cells when activated secrete proteins called antibodies Antibodies belong to a family of large proteins known as immunoglobulins. Antibodies inactivate antigens by several mechanisms:  1.complement fixation (proteins attach to antigen surface and cause holes to form, i.e. cell lysis), 2.neutralization (binding to specific sites to prevent attachment), 3.agglutination (clumping), and 4. precipitation (forcing insolubility and settling out of solution).  Thus B cells and antibody activity have been referred to as humeral immunity whereas T cells activity has been called cellular immunity.

 

Role of the Immune System in Pregnancy

Since the pregnancy contains antigens contributed by the father, they will be foreign to the mother.  Thus the mother must adapt her immune response so as not to reject or destroy the pregnancy.  At the same time the maternal immune system has to tolerate the contribution of paternal antigens, it must maintain anti-infectious immune responsiveness to protect both the mother and the pregnancy.  Pregnancy has therefore been thought to be a state of immunologic tolerance.   This tolerance is thought to result from signals given by the pregnancy to the mother’s immune cells.  Such signals include secretion of a protein called soluble HLA G.  HLA G turns off the innate immune response by inactivating NK cells. After the innate immune response has been suppressed, the adaptive immune response directed toward the foreign antigens of the pregnancy must be curtailed.  Recent research suggests that regulatory T cells are increased during normal pregnancy and decreased in pregnancies complicated by loss.  Regulatory T cells are known to suppress T cell activation and provide tolerance.  In addition, T cells during normal pregnancy predominantly secrete anti-inflammatory cytokines (Th2 response) compared with increase pro-inflammatory cytokines (Th1 response) observed in patients with recurrent miscarriage.   Pro-inflammatory (Th1 type) cytokines can induce blood clotting.  Clotting off of placental vessels leads to pregnancy complications and failures.                                                           

                                                               

Immune Causes of Recurrent Pregnancy Loss

Incomplete tolerance results in pregnancy loss.  Thus, immunologic causes for pregnancy loss include a problem within the embryo such that the signals to the maternal immune cells are inappropriate or a problem within the maternal immune cells such that they don’t respond properly to normal embryo signals.
 

Problems with embryo signaling

Antigens on the surface of the invading embryo or secreted by the embryo must signal the maternal immune cells that it is “self” rather than “nonself” or foreign so that the mother wont mount an immune response to reject the embryo.  Soluble HLA G is an antigen secreted by the embryo that signals the mother’s immune cells that it is “self” and should not be rejected.  Abnormalities in HLA G signaling as a cause of recurrent pregnancy loss can be detected by looking at HLA G gene in the mother and the father or by measuring soluble HLA G protein in culture media of in vitro fertilized embryos.  The most frequent HLA G gene mutation found in couples experiencing recurrent miscarriage is HLA G-725C/G.
 

Problems with Maternal Immune Response
 
 When the mother’s immune system cannot or does not respond appropriately to embryonic signals, pregnancy loss can occur.  How can we tell if the maternal immune cells cannot respond appropriately?  There are blood tests that can identify inappropriately functioning immune cells:

  • NK cells can be tested with the Reproductive Immunophenotype (RIP) and the NK activation (NKa) assays.
  • T cells can be assessed by measuring the activated RIP and regulatory T cells (CD4+25+).  In addition T cell function has been associated with the presence of Anti-thyroid Antibodies as well as the presence of circulating embryotoxins in the Embryotoxicity Assay (ETA).
  • B cells function is evaluated by their production of autoantibodies including antiphospholipid antibodies, antinuclear antibodies, antithyroid antibodies and lupus-like anticoagulant

 

Thrombophilic Causes of Recurrent Pregnancy Loss                                     

Once tolerance has been established and implantation completed, the mechanism of other immunologic causes of pregnancy loss involve blood clotting or thrombophilia.  Vessels of the placenta that take blood and nutrients to the fetus clot off and the pregnancy “withers on the vine.” Cytokines, especially Th1 cytokines can cause the placental vessels to clot.  Th1 type of cytokines can be secreted by either activated NK or T cells. Other reasons for clotting of the placental vessels include both acquired and inherited thrombophilia.  The most common cause of acquired thrombophilia is antiphospholipid antibodies. Inherited thrombophilias can result from gene mutations involved in coagulation (Factor V von Leiden, Factor II Prothrombin, Fibrinogen, Factor XIII), fibrinolysis (PAI-1) and thrombosis (Human Platelet Antigen-1, Methylenetetrahydrofolate reductase).

 

TESTS AVAILABLE TO DIAGNOSE IMMUNOLOGIC CAUSES OF PREGNANCY LOSS

There are a number of tests mentioned in the above description of immunologic causes of pregnancy loss available to diagnose immunologic causes of pregnancy failure.  These are listed below in alphabetical order.
 

Activated Reproductive Immunophenotype

Identification of the type of relative concentrations of various white blood cell populations in blood is valuable in determining risk factors for pregnancy loss. The Reproductive immunophenotype has been shown to be useful in identifying individuals at risk for not implanting embryos and for loosing karyotypically normal pregnancies due to elevated circulating Natural Killer (CD56+) cells. The Activated Reproductive Immunophenotype measures not only the percentage of circulating lymphocytes as the Reproductive Immunophenotype does, but also activated NK and T cells. Women experiencing implantation failure after IVF/ET have significantly higher expression of NK cell activation marker of CD69+ and of T cell activation marker of HLA-DR.
 

Antinuclear Antibodies

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 that 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 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.)
 

Antiphospholipid Antibodies

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 filter nourishment 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. 
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.
 

Antithyroid Antibodies

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.
 

Embryotoxicity Assay

Cells make proteins called cytokines. Different cytokines do different things. Some stimulate growth of cells while others inhibit growth. The proinflammatory cytokines stimulate inflammatory response, while others inhibit inflammatory response of cells. The embryo toxicity assay (ETA) is looking for cytokines that 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.
 

HLA G Testing

The major histocompatability complex (MHC), well known for its role in the regulation of cell-cell interaction in the immune response, also influences reproductive success. The MHC affects a variety of reproductive parameters including spontaneous abortion, protection of fetus from attack by the maternal immune system and regulation of preimplantation embryo growth and survival.
One gene in the MHC has that has had special attention with respect to reproduction is the class I gene HLA-G because it is important in establishing immunotolerance of the pregnancy. Mutations in the HLA gene could interfere with this vital process, resulting in pregnancy loss.
 

Immunoglobulin Panel

Patients with autoimmune diseases characteristically exhibit significant abnormalities in total immunoglobulin isotypes. A very high incidence of such gammopathies is also seen in women experiencing endometriosis, recurrent pregnancy loss, infertility and failure of implantation after in vitro fertilization. The occurrence of hypergammaglobulinemias has been reported to decrease the clinical pregnancy rate with IVF. Hypogammaglobulinemia of IgA needs to be further evaluated to rule out IgA antibodies before treatment with intravenous immunoglobulin is considered.

 
Inhibin B

Inhibin-B serum concentration provides a new measure of ovarian reserve. Ovarian reserve describes the ovary's capacity to respond to gonadotropin stimulation by producing a sufficient number of good quality eggs capable of generating normal embryos. Granulosa cells of the ovarian follicle secrete Inhibin-B. Most of the serum Inhibin-B concentration originates from large or dominant follicles since these follicles secrete ten fold higher concentrations follicles measuring 4mm. Inhibin-B controls FSH secretion form the pituitary gland. Thus, Inhibin-B is a more direct measurement of assessing ovarian function
than FSH. Inhibin-B serum concentrations drawn on cycle day 3 have been shown to predict response of ovaries to gonadotropin stimulation in in vitro fertilization (IVF) cycles. Women who had less than 45pg/ml Inhibin-B on cycle day 3, required 50% more ampoules of the day of hCG, 33% reduction in the number of oocytes retrieved, less embryos transferred per cycle and 70% reduction in pregnancy rate, than women with day 3 Inhibin levels greater than 45pg/ml. The women with day 3 Inhibin levels less than 45pg/ ml that did get pregnant had an 11 fold increase in spontaneous abortions compared with greater than 45pg/ml.
 

Lupus-like Anticoagulant

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.
 

Natural Killer Activity

Natural Killer cell activity or activation assay (NKa) measures the killing activity (cytotoxicity) within each cell.  Increased killing activity is associated with implantation failure and pregnancy loss.  A value of greater than 105 killing with a target to effector ratio of 1:50 is considered abnormal.  The NKa also measures the ability of IVIg to suppress the killing activity.  Patients with high NK cell activity that suppress with IVIg in the NKa will respond very well to intravenous immunoglobulin (IVIg) therapy. In fact, the live birth rate with preconception IVIg is more than 80 percent, compared to 20 percent without treatment.
 

Reproductive Immunophenotype

White blood cells that belong to the innate or primitive immune system  kill anything perceived as foreign .  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 immunophenotype (RIP) test, which looks for cells that have the CD56+ marker. An NK (CD56+) cell range above 12 percent is abnormal.
 

Sperm DNA Integrity assay

Results of recent research indicate that sperm influences not only rates of fertilization of eggs but also subsequent embryo development. The markers of sperm quality used to predict pregnancy outcome are not the parameters included in the standard semen analysis (sperm concentration, motility or morphology) but rather the results of the Sperm DNA Integrity assay, which measures the amount of sperm DNA that is fragmented.  A sperm DNA fragmentation index of greater than 30% is associated with poor fertility potential.
 

Thrombophilia Panel

Thrombophilia is defined as a predisposition for thrombosis. Increased thrombosis can result from defects in coagulation, fibrinolysis, platelet aggregation and endothelial damage. About 40% of patients with thrombosis are inherited.
Inherited thrombophilias have been associated with early and late recurrent pregnancy loss as a result of uteroplacental microvascular thrombosis and hypoperfusion. Obstetrical complications such as intrauterine growth retardation, placental abruption as well as preeclampsia have also been related to abnormal placental vasculature. Genetic thrombophilia are suspected to account for about 30% of these obstetrical complications. Poor pregnancy outcomes are associated with maternal thrombophilia but may also be associated with fetal thrombophilia by inheritance of maternal and paternal thrombophilic genes.
 
Successful pregnancy requires fibrin polymerization to stabilize the placental basal plate as well as to prevent excess fibrin deposition in placental vessels and intravillous spaces. Thus, a balance between coagulation and fibrinolysis is mandatory to ensure successful pregnancy outcome as early as implantation. Coagulation factors linked to reproductive disorders include mutations of Factor V, Factor II and Factor XIII. Factor V mutations associated with reproductive problems have included G1691A (von Leiden), H1299R (R2) and
Y1702C. Factor V von Leiden and Factor II prothrombin mutation G20210A are twice as common among women experiencing recurrent first trimester pregnancy loss and are suspected of tripling the risk of late fetal loss. The mechanism of loss is through generation of thrombin. Thrombin converts fibrinogen to fibrin. Fibrinogen is a protein with 3 polypeptide chains. A mutation in the b chain (-455G1A) has been associated with thrombosis. Fibrin is stabilized by cross-linking polymers under the influence of Factor XIII. One of the variations in the Factor XIII A gene, the Val34Leu polymorphism, has been correlated with thrombosis. Women who are homozygous for Factor XIII mutations also have a high risk for recurrent spontaneous abortion. Increased thrombosis can result from a defect in fibrinolysis as well as coagulation.

The main cause of defective fibrinolysis is an increase in plasmin activator inhibitor (PAI 1) concentrations. PAI 1 is induced by insulin and is increased in patients with polycystic ovary syndrome (PCOS) associated with insulin resistance. Clotting problems associated with increased PAI 1 may cause abnormal uterine artery blood flow, thus contributing to miscarriage associated with PCOS.  Thrombosis can also result from increased platelet aggregation and endothelial cell damage. Human platelet activator 1 (HPA-1) is part of the thrombosis system involved in platelet aggregation. It is a member of the integrin family. The integrin b3 gene encodes glycoprotein IIIa (GP IIIa) which is part of GP IIb/IIIa complex when activated interacts with fibrinogen to cross-link platelets to one another and causes platelet aggregation. Two allelic forms of GPIIIa have been identified (PLA1 and PLA2). The A2 form has been associated with increased thrombosis. Endothelial damage leading to thrombosis can be caused by hyperhomocysteinemia or antiphospholipid antibodies. Methylenetetrahydro-folate reductase (MTHFR) catalyzes remethylation of homocysteine to methionine.

Several mutations in the MTHFR gene, C677T and A1298C, leads to hyperhomocysteinemia via decreased enzyme activity. Hyperhomocysteinemia is a major risk factor for both arterial and venous thrombolic disease. Individuals homozygous for the MTHFR gene are at increased risk for thrombosis and pregnancy related disorders. The risk of embryonic and fetal loss is increased if the MTHFR gene mutation is combined with additional thrombophilic factors. Disturbance of maternal and fetal homocysteine metabolism has also been implicated in a decrease in incidence of dizygotic twinning and an increase in fetal neural tube defects.
 
The Thrombophilia Panel include testing for the following gene mutations:

  • Factor V Y1702C mutation
  • Factor V G1691A (Leiden)
  • Factor V H1299R (R2)
  • Factor II Prothrombin G20210A
  • b-Fibrinogen –455 G>A
  • Factor XIII V34L
  • PAI 1 4G/5G
  • HPA1 a/b Human Platelet Glycoprotein (PLA1/PLA2)
  • MTHFR C677T
  • MTHFR A1298C

Results are reported as normal, heterozygous or homozygous.
 

Y Chromosome Microdeletion Assay Related to Recurrent Pregnancy Loss (MYC/RPL)

While Y chromosome deletions were initially reported to be associated with infertility due to oligo-azospermia, more recently sequence tagged site in the proximal AZFc region of the Y chromosome have been shown to be microdeleted among men whose partners experienced recurrent pregnancy loss.  The four sites analyzed for deletions are:

  • DYS262
  • DYS220
  • DYF85S1
  • DYF86F1

 

TREATMENT FOR IMMUNOLOGIC CAUSES OF RECURRENT PREGNANCY LOSS

Effective treatment depends on the cause of the pregnancy loss.  If the cause of the pregnancy loss is a problem within the embryo itself, elimination of the problem involves treatments including donor egg, donor sperm or IVF with preimplantation genetic diagnosis (PGD).  If, however, the cause  is related to activated immune cells and their cytokines, treatments include:  Intravenous Immunoglobulin (IVIg), Intralipid, and Phosphodiesterase Inhibitors.  If either acquired or inherited thrombophilia is causing clotting of the placental vessel and subsequent pregnancy loss, then heparin and aspirin is the treatment of choice.  If the blood clotting is the result of an immune process, then steroids and/or IVIg can be used.
 

Intravenous Immunoglobulin (IVIg)

IVIg has been used to treat both pre-implantation and post-implantation recurrent pregnancy loss associated with elevated levels of antiphospholipid antibodies, antithyroid antibodies, circulating NK cells and NK cell killing activity and embryotoxins.  It has also been used for treatment of unexplained recurrent implantation failure and pregnancy loss.   The mechanisms by which IVIg works include:

  • IVIg provides antibodies to antibodies (anti-idiotypic antibodies)
  • IVIg suppresses B cells production of autoantibodies
  • IVIg enhances regulatory T cell activity
  • IVIg suppresses NK cell killing activity

Originally, IVIg therapy was used to treat women who had not been successful in pregnancies previously treated with aspirin and prednisone or heparin. The rationale for the use of IVIG in the original studies was the suppression of the lupus anticoagulant in a woman being treated for severe thrombocytopenia. IVIg was often given with prednisone or heparin plus aspirin. The estimated success rate of 71% for women at very high risk for failure with a history of previous treatment failures suggested IVIg treatment was effective. More recently, IVIg therapy alone has been used to successfully treat women with antiphospholipid antibodies as well as women who become refractory to conventional autoimmune treatment with heparin or prednisone and aspirin.

Proinflammatory cytokines at the maternal-fetal surface can cause clotting of the placental vessels and subsequent pregnancy loss. One source of these cytokines is the NK cell. Biopsies of the lining of the uterus from women experiencing repeat pregnancy loss reveal an increase in activated NK cells. Peripheral blood NK cells are also elevated in women with repeat pregnancy loss compared with women without a history of pregnancy loss. Measurement of NK cells in peripheral blood of women with a history of recurrent miscarriage and a repeated failing pregnancy has shown a significant elevation associated with loss of a normal karyotypic pregnancy and a normal level associated with loss of embryos that are karyotypically abnormal. Furthermore, increased NK activity in the blood of nonpregnant women is predictive of recurrence of pregnancy loss. Suppressor T cells with are required for protection against NK cytokine-dependent pregnancy loss. IVIg has been shown to decrease NK killing activity and enhance Suppressor T cell activity. Both of these events are necessary for pregnancy to be successful. IVIg has been used to successfully treat women with elevated circulating levels of NK cells, NK cell killing activity and embryotoxins with live birth rates between 70% and 80%.

IVIg has also been used to treat women with unexplained repeat pregnancy loss. Four randomized, controlled trials of IVIg for treatment of repeat pregnancy loss have been published. A European-based study showed a positive trend but did not achieve statistical significance due to too few patients for adequate statistical power given the magnitude of the effect. However, a US-based trial did show a significant benefit, the difference in live birth rates being 62% among women receiving IVIg and 33% among women receiving placebo. The greater magnitude of effect in the US-based study than the European-based trial could have arisen from the use of a different study design. Patients began IVIg treatment before conception in the US-based trial, but after implantation in the European-based trial. By waiting until 5-8 weeks of pregnancy to begin treatment, women with NK cell-related pathology occurring earlier would have been excluded and those pregnancies destined to succeed would be included, providing an opportunity for selection bias. Indeed, a negative correlation with delay in treatment was significant in this study. A third trial treated only women who had a previous live birth, a group that showed no significant benefit of treatment using leukocyte immunization, but significant benefit from IVIg. The fourth Canadian-based trial had too few patients for adequate statistical power to give significant results but did show a trend toward benefit in women with a history of previous live birth followed by recurrent miscarriage. When the results of all of these trials were combined in a meta-analysis the conclusion showed IVIg to be effective treatment for repeat pregnancy loss. None of the studies took into account the pregnancies lost as a result of chromosomal abnormalities except the US-based trial.

Approximately 60% of the pregnancies lost in the clinical trial would be expected to have chromosomal abnormalities that would not be corrected by IVIg.
The usual dosage of IVIg for treatment of repeat implantation failure is 40 gm and repeat post-implantation pregnancy loss is 25 grams but successful pregnancies have been reported using dosages from 20 to 60 grams. The half-life in circulation is 28 days so infusions are usually given every 28days. Depending on the obstetric history, IVIg is continued every 28 days until the end of the first trimester (women with a history of first trimester pregnancy losses) or until 28-32 weeks gestation (women with a history of late pregnancy losses). Pregnancies are monitored with immunologic blood tests and treatment can be modified based on the results of the blood tests.
Side effects of treatment with IVIg include nausea, vomiting, headaches, chills, chest pain, difficulty breathing—all comfort side effects which usually occur during the infusion of IVIg and are related to the rate of infusion. If these side effects occur, the rate of the infusion of IVIg is slowed. Other side effects that have been reported much less frequently are migraine-type headaches and sore or stiff neck occurring from one to four days after the infusion. Last, but not least, while IVIg is a purified protein particulate that is reconstituted in fluid and infused in veins, the protein is extracted from human plasma. Therefore, it runs the same theoretic risks for transmittable disease as other blood products. However, IVIg has been available on the American market under FDA and CDC surveillance since 1981, with no reported instance of HIV transmission. There were reports of cases of hepatitis C after IVIg treatment reported in 1992 and the first part of 1993 for which some manufactures changed the method of extraction and added a detergent solubilization step. Thus the theoretic risk at this time is an unknown risk of transmission of presently unidentified infectious particles. Because of the rigorous screening it must undergo, the cost of IVIg is high. The high cost of IVIg therapy can be a deterrent to treatment for some individuals.
 

Intralipid

Evidence from both animal and human studies suggest that intralipid administered intravenously may enhance implantation and maintenance of pregnancy. Intralipid is a 20% intravenous fat emulsion used routinely as a source of fat and calories for patients requiring parental nutrition. It is composed of 10% soybean oil, 1.2% egg yolk phospholipids, 2.25% gylcerine and water. Intralipid stimulated the immune system to remove “danger signals” that can lead to pregnancy loss.  The appeal of Intralipid lies in the fact that it is relatively inexpensive and is not a blood product. Its likely benefit to IVF patients with immunologic dysfunction is under evaluation.
 

Phosphodiesterase Inhibitors

The phosphodiesterases are responsible for enzymatic degradation of molecules within the cells involved in generating energy for the cell to function.  They have anti-inflammatory effects.  Two phosphodiesterase inhibitors—Sildenfil (Viagra) and Pentoxiphylline (Trental) have been shown to increase blood flow to the uterus.  Viagra in the form of vaginal suppositories given in the dosage of 25 mg four times a day has been shown to increase uterine blood flow as well as thickness of the uterine lining. Significant improvement of the thickness of the uterine lining in about 70% of women treated. Successful pregnancy resulted in 42% of women who had previously experienced repeated IVF failures and who responded to the Viagra. Similar results were obtained when Trental was used in 400mg twice a day doses alone with vitamin E to treat women experiencing implantation failure associated with thin endometrium and elevated uterine NK cells.   Animal studies have demonstrated that pentoxifylline prevents miscarriages in abortion-prone mice.  Efficacy of pentoxifylline for treatment of recurrent pregnancy loss in human beings remains to be established.
 

 

Aspirin

Low-dose aspirin (80mg or 1 baby aspirin) alone has used for treatment of both repeat implantation failures and post-implantation pregnancy losses.  Aspirin therapy has been reported to enhance implantation rates in women undergoing IVF/ET.  In these studies the number of eggs retrieved and number of embryos generated were higher in the aspirin treated group than in the non-treated group making it unclear whether the enhancement in implantation rate was the result of better embryo selection or a direct effect on the lining of the uterus.  Among women with increased resistance of blood flow through their uterine arteries who were treated with aspirin for a minimum of two weeks, the pregnancy rate was increased from 17% to 47% and the miscarriage rate decreased from 60% to 15%.  As a prostaglandin inhibitor, aspirin would be expected to increase blood flow to the ovary prior to implantation, to the endometrium during implantation and to prevent clotting of the placental vessels following implantation.  However, in studies of women experiencing recurrent post-implantation pregnancy loss/miscarriage associated with antiphospholipid antibodies, results of clinical trials have shown aspirin alone to be half as effective as other treatments including heparin and steroids. In two studies women receiving aspirin alone or heparin plus aspirin for treatment of repeat pregnancy loss associated with antiphospholipid antibodies, heparin plus aspirin provided a significantly better outcome that aspirin alone (live birth rate of 80% vs 44%).
A rationale for the use of low-dose aspirin therapy during pregnancy for women with antiphospholipid antibodies is to decrease blood clots from forming in the placental vessels. The mechanisms by which aspirin prevents blood clots are through its antiprostaglandin and antiprostacyclin effects and inhibition of platelet adhesiveness and aggregation.

 

Heparin

Heparin has also been used in conjunction with aspirin to prevent blood clotting.  The rational for using heparin is that it is a blood thinner and inhibits clot formation by a different pathway that the aspirin.  While the effectiveness of heparin and aspirin for treatment of women with elevated circulating antiphospholipid antibodies and a history of recurrent miscarriage is well accepted, the use of heparin with or without aspirin to enhance implantation rates has been controversial. Most clinical trials of women with elevated antiphospholipid antibodies and a history of implantation failure undergoing IVF/ET show no enhancement of implantation rates with heparin and aspirin compared with no treatment.  This observation is not surprising since the action of heparin is on the cells lining the blood vessels and pre- and peri- implantation pregnancy loss occurs before placental blood vessels appear. The combination of both heparin and aspirin given to women experiencing repeat pregnancy loss who had antiphospholipid antibodies are associated with a live birth rate of 80% compared with a live birth rate of 44% in women receiving aspirin alone. Live birth rates with heparin, aspirin and a steroid called prednisone are 74%. Thus no enhancement of live birth rates are noticed when prednisone is added to heparin and aspirin therapy for treatment of recurrent miscarriage.
Heparin is usually administered at a dose of 5,000-10,000 units subcutaneous twice a day along with aspirin 80mg each day. In women with a circulating lupus-like anticoagulant, more heparin may be required. The side effects of heparin therapy include bleeding, decreased platelet count and osteoporosis or thinning of the bones. Calcium supplementation (two tablets of Tums a day) is recommended while taking heparin.  Low molecular weight heparins such as Lovenox and Fragmin have also been used to treat recurrent pregnancy loss associated with thrombophilias, either acquired or inherited.

 

Steroids

Steroid therapy in the forms of prednisone, prednisolone and dexamethasone has been used to prevent both pre-implantation pregnancy failure and post-implantation pregnancy loss.  Steroids are routinely administered in many IVF programs.  These medications are started prior to initiating ovarian stimulation with gonadotropins and continued until the diagnosis of pregnancy.  If the pregnancy test is negative, the dosage is tapered off over the next week and then discontinued.  If the pregnancy test is positive, treatment is continued until 8 to 12 weeks of gestation.  Steroids are believed to act by inhibiting the cellular immune response.  The exact mechanism and the degree to which implantation is enhanced by the use of steroids are not known.  Dosages of steroids for treatment of pre-implantation failure vary depending on the preparation.  A typical regimen is dexamethasone 0.5mg a day. 
Historically, repeat pregnancy loss associated with antiphospholipid antibodies was treated with combinations of prednisone and aspirin. The rationale for prednisone therapy is suppression of autoantibodies such as antiphospholipid and antinuclear antibodies. A study comparing live birth rates in women treated with heparin and aspirin with prednisone and aspirin showed 75% live births in both groups. However, both maternal complications and preterm delivery with premature rupture of membranes and toxemia of pregnancy were significantly higher in pregnant women treated with prednisone and aspirin compared with heparin and aspirin. Other side effects of steroid medication include fluid retention, weight gain, and mood changes. Therefore, the current recommendation for “first attempt” treatment for repeat pregnancy loss associated with antiphospholipid antibodies is heparin and aspirin.

 

 

SUMMARY

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 years. 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.
 

Carolyn B. Coulam, M.D. is Director of Millenova Immunology Laboratories and a physician at the Rinehart/Coulam Center for Reproductive Medicine 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 the INCIID Executive Director

 

 

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