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The Miscarriage Manual: Coping with the Emotional Aspects of Pregnancy Loss by Elizabeth Carney

Miracles and Memories Family-Building Pins

Miracles and Memories Family-Building PinsFourteen years ago I gave birth to a baby girl. Four hours later she died because of an internal malformation that was undetectable during my pregnancy. During my short hospital stay, nurses and doctors seemed to avoid me and my questions. What they did say was about the same as what my friends and family were saying. "You're young. You'll have other babies. Try to forget." 

I didn't want any other baby; I wanted that one! Forget? How could I forget? Instead I was overwhelmed with crushing, breathtaking grief. I remember how empty I felt the day I left the hospital...an empty womb and empty arms. I never really knew her but I missed her and ached for her so desperately. 

Soon after I returned home, everyone acted as if they had already forgotten her, as if they expected me to also. Someone had removed all the baby items I had acquired before coming home, hoping to spare me the pain. Instead, it felt like a further denial of her existence. When I tried to talk about her everyone became very quiet, or changed the subject, or left the room. Friends were very careful not to say anything that might remind me of my experience. Baby shower invitations didn't come in the mail. Birth announcements didn't come in the mail. Many stayed away because they simply did not know what to say. My husband had three days to "get over it" before he was expected back at work. The world kept on spinning as if nothing had happened. I remember thinking that I must have lost my mind. I thought that if my baby had lived for a while, if people had gotten to know and love her, maybe then I would have been given the affirmation to grieve the way I needed to. But I was the only one with any memory of her, the only one who had the chance to love her. I had no one to share that with, not even my husband. Most of his grief was for me and for the dreams we had shared for this child. I felt all alone as I began my mourning. 

Over the years, after much healing, I have had the opportunity to speak with other parents who have had experiences which were similar to mine. As a result of that, and also as a result of my search for answers to all those unanswered questions, I have compiled a list of several "truths and non-truths" concerning the grieving process as it relates to perinatal bereavement. 

This is not intended to be the absolute word on the subject, but rather a gauge for the unexpected emotions felt by parents who have suffered this type of loss. Most of the parents I have spoken to agreed that the uncertainty of their grief was frightening and may have been alleviated had they known what to expect. 

Friends and family may also benefit from reading this over so they might understand the special kinds of pain and emotions involved in this type of loss and allow them to be expressed. 

The Truth Is...

1. The truth ISN'T that you will feel "all better" in a couple of days, or weeks, or even months. 

The truth IS that the days will be filled with an unending ache and the nights will feel one million sad years long for a while. Healing is attained only after the slow necessary progression through the stages of grief and mourning. 

2. The truth isn't that a new pregnancy will help you forget. 

The truth is that, while thoughts of a new pregnancy soon may provide hope, a lost infant deserves to be mourned just as you would have with anyone you loved. Grieving takes a lot of energy and can be both emotionally and physically draining. This could have an impact upon your health during another pregnancy. While the decision to try again is a very individualized one, being pregnant while still actively grieving is very difficult. 

3. The truth isn't that pills or alcohol will dull the pain. 

The truth is that they will merely postpone the reality you must eventually face in order to begin healing. However, if your doctor feels that medication is necessary to help maintain your health, use it intelligently and according to his/her instructions. 

4. The truth isn't that once this is over your life will be the same. 

The truth is that your upside-down world will slowly settle down, hopefully leaving you a more sensitive, compassionate person, better prepared to handle the hard times that everyone must deal with sooner or later. When you consider that you have just experienced one of the worst things that can happen to a family, as you heal you will become aware of how strong you are. 

5. The truth isn't that grieving is morbid, or a sign of weakness or mental instability. 

The truth is that grieving is work that must be done. Now is the appropriate time. Allow yourself the time. Feel it, flow with it. Try not to fight it too often. It will get easier if you expect that it is variable, that some days are better than others. Be patient with yourself. There are no short cuts to healing. The active grieving will be over when all the work is done. 

6. The truth isn't that grief is all-consuming. 

The truth is that in the midst of the most agonizing time of your life, there will be laughter. Don't feel guilty. Laugh if you want to. Just as you must allow yourself the time to grieve, you must also allow yourself the time to laugh. Viewing laughter as part of the healing process, just as overwhelming sadness is now, will make the pain more bearable. 

7. The truth isn't that one person can bear this alone. 

The truth is that while only you can make the choices necessary to return to the mainstream of life a healed person, others in your life are also grieving and are feeling very helpless. As unfair as it may seem, the burden of remaining in contact with family and friends often falls on you. They are afraid to "butt in," or they may be fearful of saying or doing the wrong thing. This makes them feel even more helpless. They need to be told honestly what they can do to help. They don't need to be told, "I'm doing fine" when you're really NOT doing fine. By allowing others to share in your pain and assist you with your needs, you will be comforted and they will feel less helpless. 

 

8. The truth isn't that God must be punishing you for something. 

The truth is that sometimes these things just happen. They have happened to many people before you, and they will happen to many people after you. This was not an act of any God; it was an act of Nature. It isn't fair to blame God, or yourself, or anyone else. Try to understand that it is human nature to look for a place to put the blame, especially when there are so few answers to the question, "Why?" Sometimes there are answers. Most times there are not. Believing that you are being punished will only get in the way of your healing. 

9. The truth isn't that you will be unable to make any choices or decisions during this time. 

The truth is that while major decisions, such as moving or changing jobs, are better off being postponed for now, life goes on. It will be difficult, but decisions dealing with the death of your baby (seeing and naming the baby, arranging and/or attending a religious ritual, taking care of the nursery items you have acquired) are all choices you can make for yourself. Well-meaning people will try to shelter you from the pain of this. However, many of us who have suffered similar losses agree that these first decisions are very important. They help to make the loss real. Our brains filter out much of the pain early on as a way to protect us. Very soon after that, we find ourselves reliving the events over and over, trying to remember everything. This is another way that we acknowledge the loss. Until the loss is real, grieving cannot begin. Being involved at this early time will be a painful experience, but it will help you deal with your grief better as you progress by providing comforting memories of having performed loving, caring acts for your baby. 

10. The truth isn't that you will be delighted to hear that a friend or other loved one has just given birth to a healthy baby. 

The truth is that you may find it very difficult to be around mothers with young babies. You may be hurt, or angry, or jealous. You may wonder why you couldn't have had that joy. You may be resentful, or refuse to see friends with new babies. You may even secretly wish that the same thing would happen to someone else. You want someone to understand how it feels. You may also feel very ashamed that you could wish such things on people you love or care about, or think that you must be a dreadful person. You aren't. You're human, and even the most loving people can react this way when they are actively grieving. If the situations were reversed, your friends would be feeling and thinking the same things you are. Forgive yourself. It's OK. These feelings will eventually go away. 

11. The truth isn't that all marriages survive this difficult time. 

The truth is that sometimes you might blame one another, resent one another, or dislike being with one another. If you find this happening, get help. There are self-help groups available or grief counselors who can help. Don't ignore it or tuck it away assuming it will get better. It won't. Actively grieving people cannot help one another. It is unrealistic, like having two people who were blinded at the same time teach each other Braille. Talking it out with others may help. It might even save your marriage. 

 

12. The truth isn't that eventually you will accept the loss of your baby and forget all about this awful time. 

The truth is that acceptance is a word reserved for the understanding you come to when you've successfully grieved the loss of a parent, or a grandparent, or a beloved older relative. When you lose a child, your whole future has been affected, not your past. No one can really accept that. But there is resolution in the form of healing and learning how to cope. You will survive. Many of us who have gone through this type of grief are afraid we might forget about our babies once we begin to heal. This won't happen. You will always remember your precious baby because successful grieving carves a place in your heart where he or she will live forever.

 

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Immunology May Be Key To Pregnancy Loss by Carolyn Coulam and Nancy Hemenway

African American couple listening to news on the telephone

Background
 
Introduction

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, 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 15-20% 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 11-13 %. In a pregnancy immediately following that loss, the risk of miscarriage is 13-17 %. But the risk to a third pregnancy after two successive losses nearly triples to 38 %.
 
Many doctors do not begin testing for the cause of pregnancy loss until after three successive miscarriages. However, because the risk of a third pregnancy loss 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 functions as the first line of defense against disease and is one of the most intricate and complex systems in the body. It works by generating cells and molecules that are capable of identifying and eliminating potentially 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.  Cell markers, as well as any other substance triggering an immune response, are called antigens.  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 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)
4.      Precipitation (forcing insolubility and settling out of solution). 
 
Thus B cells and antibody activity have been referred to as humoral 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 also 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 will not 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 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.

Antiphospholipid Antibodies

In pregnancy, phospholipids act like a sort of glue that holds the dividing cells together and is necessary for the 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 cause 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 kinds of antiphospholipid antibodies indicates the woman has the immune response that can cause 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 11-20%. 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 70-80%.
 

Antithyroid Antibodies

Women with thyroid antibodies face doubling 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% of women experiencing RSA are positive for one or both antibodies. Chances of a loss in the first trimester of pregnancy increase to 20%, and there is also an increased risk of postpartum 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 pro-inflammatory cytokines stimulate an inflammatory response, while others inhibit the inflammatory response of cells. The embryotoxicity 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 from 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 the response of ovaries to gonadotropin stimulation 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 4% of women with recurrent miscarriage test positive for lupus-like anticoagulant and 9% 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%, compared to 20% 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% 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 outcomes 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.
 

A successful pregnancy requires fibrin polymerization to stabilize the placental basal plate as well as to prevent excess fibrin deposition in placental vessels and intervillous spaces. Thus, a balance between coagulation and fibrinolysis is mandatory to ensure a successful pregnancy outcome as early as implantation.  The following bullets breakdown the complicated relations involved.

  • 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 the generation of thrombin.
  • Thrombin converts fibrinogen to fibrin. Fibrinogen is a protein with three 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. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the 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 the incidence of dizygotic twinning and an increase in fetal neural tube defects.

 

The Thrombophilia Panel of tests includes 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 sites in the proximal AZFc region of the Y chromosome have been shown to be microdeletion 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 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. Further information on each of the treatment options is presented below.
 

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 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.
  • 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 an 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 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 suggests 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% glycerine, 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 the 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 dose 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.  The 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 the 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 numbers of eggs retrieved and numbers 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 than 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 anti prostaglandin 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 rationale for using heparin is that it is a blood thinner and inhibits clot formation by a different pathway than 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 is noticed when prednisone is added to heparin and aspirin therapy for the 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 the 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 the “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 of 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 co-founder and serves as the INCIID Executive Director

 

 

Intralipid Treatment Vs. IVIg for Reproductive Failure

Intralipid Diagram
Intralipid diagram 2
Intralipid diagram 3
Intralipids instead of IVIg

By Carolyn Coulam, M.D. and Nancy Hemenway

Intralipids instead of IVIgFailure to reproduce is a physically and emotionally challenging ordeal. When reproductive failure is repetitive feelings of shame, of grief and loss and failure are magnified. Before embarking on an effective and successful treatment plan patients need to determine the causes of their inability to conceive and to carry a pregnancy to termThere are many reproductive variables that can delay, interrupt or disrupt a pregnancy. Some of these include:

  • Chromosomal Anomalies (i.e. Numerical chromosomal abnormalities or aneuploidies Extra Chromosomes or trisomy, Missing chromosome –monosomy)
  • Anatomic Abnormalities (i.e. Missing or blocked fallopian tubes, Misshaped uterus)
  • Hormonal Irregularities (i.e. Elevated FSH, Low Progesterone, Irregular  levels of gonadotropin releasing hormone - GnRH)
  • Immunological Issues (See article) and
  • Thrombophilic Abnormalities (Blood clotting disorders)

Patients should not use their obstetrical history alone to determine whether an immunological reproductive issue exists. This means women may experience reproductive failure even after one or more successful full term pregnancies. When implantation failure occurs or when there is a loss after successful attempts, immunotherapy will be useful. 

Be aware that only patients experiencing reproductive failure with an immunologic cause are expected to respond to immunotherapy. Patients who have unexplained infertility should consult a reproductive endocrinologist with reproductive immunological experience to determine if immunotherapy may be a consideration. Not all reproductive clinics have this physicians with reproductive immunological expertise or the correct lab assays to evaluate needs for immunotherapy.

The most frequently studied risk factors to identify an immunologic cause of reproductive failure have traditionally included the presence of antiphospholipid antibodies (APA) and elevated natural killer (NK) cells.

Elevated APAs are equally prevalent among women experiencing unexplained infertility, recurrent implantation failure, and recurrent pregnancy loss.  It is important to note that Heparin and aspirin are successful in the treatment of elevated APA among women with a history of recurrent miscarriage but not with recurrent implantation failure.  Intravenous immunoglobulin (IVIg) has been successful in the treatment of recurrent miscarriage and recurrent implantation failure among women with elevated APA and/or NK cell activity.  Because of the very high cost (as much as $125,000 for treatment throughout pregnancy) and potential risks of IVIg, a blood product, alternative were pursued. 

What is Intralipid? 

Lipid is a descriptive term. The lipids are a group of fats and sterols. The term “Lipid” is descriptive rather than a chemical name such as “protein” or “carbohydrate.” Lipids include true fats (esters of fatty acids and glycerol); lipoids (phospholipids, cerebrosides, waxes); and sterols (cholesterol, ergosterol found in plants).

Intralipid is a brand name for an IV solution that has been made suitable to give IV to patients. The lipid has been broken into small droplets and suspended in water. Intralipid is a combination of liquid, lipid, and an emulsifying system suitable for intravenous.  

Intralipid is the brand name for an IV medication used primarily to add calories to a patient.  However, a number of studies suggest that Intralipid modifies or controls some immune functions including suppression of NK cells ability to destroy and become toxic to the cells.

These studies also suggest that Intralipid suppresses the pro-inflammatory cytokine generations of cells. Cytokines are any of more than 100 proteins produced by the white blood cells. Pro-inflammatory cytokines are the circulating substances in the blood that deplete lean body mass during a critical illness. They include several types of cytokines - interleukins (IL-1, IL-6, and IL-8) and also tumor necrosis factor (TNF) secreted by NK cells. TNF provides the body’s defense against disease and gives the killer cells killing power. We all have NK cells.  Sometimes the TNF secretions destroy cells that are perceived to be foreign bodies, a virus or tumor when in fact they are destroying a fetus. (See Immunology may be key to pregnancy loss article.)

Recently, in an in vitro assay, Intralipid shows capacity to suppress NK cytotoxicity with equal effectiveness as IVIg

The first week after an Intralipid infusion, 78% of women experiencing reproductive failure who had elevated NK activity showed NK’s ability to destroy cells was suppressed to within normal range. 

For the remaining 22%, the NK activity was also suppressed to within normal range after 2 or 3 infusions of Intralipid, given at 2-3 week intervals.

The duration of the suppression of the NK cell activity after normalization of NK cells with Intralipid infusions lasted between 4 and 9 weeks with the majority lasting 6-9 weeks during the first trimester of pregnancy. 

The pregnancy outcomes of 200 women experiencing recurrent reproductive failure who had elevated NK cell activity and who were treated with Intralipids are shown in Figure1

Figure 1.  Successful pregnancy rates with Intralipid treatment

The pregnancy rate per cycle of treatment with Intralipid for women experiencing recurrent implantation failure with elevated NK cell activity was 52%. Among those women with a history of reproductive failure  who became pregnant, the pregnancy loss rate was 9% and live birth or ongoing pregnancy rate was 91%.

When the pregnancy outcomes of women with a history of reproductive failure and elevated NK cell cytotoxicity treated with intralipid were compared with age- and indication-matched women treated with IVIg, no significant differences were seen as shown in figure 2.

 Figure 2.  Comparison of live birth rates among women with a history of reproductive failure and elevated NK cytotoxicity treated with IVIg and Intralipid.

 

The overall live birth pregnancy rate per cycle of treatment was 61% for women treated with Intralipid and 56% with IVIg.

While the mechanism by which intralipids suppresses NK function is not known, effects of fatty acids have been demonstrated to be mediated through receptors such as peroxisome proliferator-activated receptors (PPARs), G-protein-coupled receptors,  and CD1 receptors as shown in figure 3.                                                  

                    

Figure 3.  Pathways involved in fatty acid actions

 

Intralipid 20& is a sterile fat emulsion prepared for intravenous administration.  It is made up of 20% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin and76.5% water.  The fatty acid components of the soybean oil include:

  • Linoleic acid 44-62%
  • Oleic acid 19-30%
  • Palmitic 7-14&
  • Linolenic 4-11%
  • Stearic 1.4-5.5%

The major fatty acid component is linoleic acid.  A number of studies have demonstrated conjugated linoleic acid improves reproduction in cows.  Furthermore, conjugated linoleic acids  have recently been shown to attenuate LPS-Induced proInflammatory gene expression by Inhibiting the NF-κB translocation through PPAR (see figure 3).   

 

Summary

Intralipid is effective in the treatment of women experiencing reproductive failure who display elevated NK cell activity. A controversy that has occurred involves answering the general question  ‘Does immunotherapy for treatment of reproductive failure enhance livebirths?’ The answer to the question is yes, BUT a treatment is more likely to work if it is given to those with physiological abnormality that the treatment can correct, and, if the treatment in fact corrects it.   Elevated levels of NK cell cytotoxicity have been linked to recurrent miscarriage and recurrent implantation failure.  Increased killing activity can be the result of elevated numbers of NK cells or increased cytotoxicity within each cell.  Detection  of elevation of circulating CD56+ (NK) cells and NK cell activity has been shown to be helpful in identifying individuals at risk for not implanting embryos  and for losing karyotypically normal pregnancies .  While both IVIg and intralipid are  effective in suppressing NK cell cytotoxicity and enhancing live births among women experiencing reproductive failure who display elevated NK cell activity, only IVIg has been shown to enhance live births in women displaying APAs.   The results of published studies suggest that intralipid can be used successfully as a therapeutic option to modulate abnormal NK activity in women with reproductive problems.  Patients carrying chromosomally abnormal pregnancies and patients with anatomic or thrombophilic risk factors including APAs  should not be treated with intralipid since they would not be expected to respond to intralipid therapy.

 

Carolyn Coulam MD will answer your questions here

IVIg for ART and Implantation Failure Controversy by Geoffrey Sher, M.D.

The controversy regarding immunologic implantation failure and failed IVF has become a political football. The individuals who criticize the use of heparin/IVIG in IVF implantation are the same ones who originally criticized immunologic factors as causative in non-chromosomal "recurrent miscarriages". Now that the roll of immunology in the latter has been established and IVIG/heparin therapy has been accepted as a standard of care in the treatment of "unexplained" recurrent miscarriages, the same critics have turned their attention to the IVF arena.

There is an ongoing and deliberate agenda (on the part of a few) to discredit immunologic testing and the use of heparin/IVIG in IVF. In support of this position, the critics argue neither a positive relationship between APAs (antiphospholipid antibodies) and poor IVF outcome, nor a benefit from selective immunotherapy has been proven through randomized and controlled studies. In this they are correct. No one in our field would deny that the "gold standard" for scientific validation, is proof through the performance of randomized and controlled prospective studies. Yet, virtually nothing we offer patients in IVF has been validated in this way, ...not the protocols we use for ovarian stimulation, not IVF versus GIFT or ZIFT or IUI, not Clomiphene vs. Pergonal/Gonal F /Folistim, not Lupron vs. Antagon/Cetrotide, not Assisted Hatching, not ICSI, not one type of culture media over another, not day 3 vs. Day 5 (blastocyst ) embryo transfers, not progesterone vs. hCG vs. no support of the luteal phase...and the list goes on and on. In fact if the IVF literature were to comprise only those things that have been proven by this "gold standard", it would probably be less than one page in length. There are so many variables associated with IVF outcome:

 

  • woman's age,
  • number of prior failed IVF attempts,
  • protocols used,
  • timing for hCG injection,
  • method and duration of ovarian stimulation,
  • the collective and/or individual prowess of the M.Ds, embryologist(s),who perform embryo transfer( ET)
  • the catheter used,
  • loading technique,
  • time taken,
  • ultasound guided vs non-ultrasound guided,
  • uterine lining,
  • immunologic factors,
  • type of luteal support and

 

It is virtually impossible to evaluate the influence of a single variable on outcome, by this approach. At best one would be making an educated guess. This explains why almost all advances in the IVF arena have been as a result of controlled longitudinal cohort studies rather than randomized controlled studies.
There appears to be a double standard when it comes to immunologic implantation failure. I believe the double standard is attributable to the introduction of financial considerations. Consider the following:

 

By and large IVF is paid for on a fee for service basis and the IVF medical arena is highly competitive, with more than 360 IVF programs in the US competing annually for about 80,000 cycles of treatment. In most States IVF constitutes an out-of pocket expense to the consumer. Since immunotherapy is expensive (IVIG costs about $60 per gram and full treatment can require 40-80 grams), it is very difficult for the RE to persuade couples to agree to IVIG treatment, and at the same time remain competitive in the IVF arena. At the same time, competitors, slam this treatment as being exploitatory.

 

Central to the controversy regarding immunologic implantation failure and selective immunotherapy is the association between the presence of circulating antiphospholipid antibodies (APA) and activated Natural killer Cells (NKa). When it comes to the role of APAs, there is published data supporting both sides of the argument. The problem with regard to interpreting APA data is that there is no commercial Kit available that tests for all 21 APA's. So each laboratory has to develop its own reagent process. There is also currently no uniform agreement on the exact cut-off values to use in deciding on a "+ve vs. a -ve" result. The truth is that most RE's still rely on non-specific and relatively insensitive tests such as PTT, lupus anticoagulant and anticardiolipin antibodies. Relatively MD's in the IVF arena measure an entire panel of 18-21 different types of APAs and as stated above, most immunology laboratories vary in the methodology they use and disagree on the exact cut-off points beyond which reported results should be regarded as "positive" or "negative".

 

The origin of most of the contrary information regarding APA testing stems primarily from an article by Denis and Scott, et.al. which appeared in "Fertility and Sterility" in 1997. This article reported on an absence of any correlation between APA+ and IVF outcome and was, in my opinion, seriously flawed in design and methodology, sufficient to evoke a flurry of strong criticism by way of letters to the editor in a subsequent edition of the journal . The critics cited the following significant problems with this study :

  • First, the IVF patients reported on were not confined to those with female causes of infertility. (We have never claimed that APA positivity has any significance in cases where IVF is done for an isolated male factor). In fact about one half of the cases reported by Denis and Scott had male infertility as the primary indication for IVF
  • Secondly, the methodology for APA testing in this study was shown to be seriously flawed and lacking both in sensitivity and specificity. About a year ago we reported in "Human Reproduction" on a positive association between the presence of certain types of APAs and increased NKa in the blood.

 

More recently we have shown that when IVIG is given to APA+/NKa+ women (>200 cases) the pregnancy rate is comparable to negative testing IVF patients . AND subsequently out of 21 women NKa+ women who refused IVIG/heparin, only one(1). conceived. After reconsidering the situation, 8 of those who did not take IVIG and failed to conceive, came back and did IVF again, this time using IVIG/heparin. About half of these women conceived straight away. We believe that the evidence shows that many APA+ women who undergo IVF for female factors, will not achieve viable pregnancies that continue to delivery without minidose heparin therapy and if in addition, they test NKa+ less than 10% will have IVF babies unless IVIG is added to the heparin regime.

Against this background we recently initiated a long awaited randomized and controlled study that will, hopefully, settle this controversy. This IRB approved study addresses, the use of IVIG/heparin therapy in APA+/NKa+ women undergoing IVF and confirm what our prospective longitudinal cohort studies have already shown , namely, that selective immunotherapy significantly enhances IVF outcome. In an attempt to reduce the number of confounding variables, we intend making the qualifying criteria tight, performing all IVF at a single SIRM center , and confining the performance of IVF to the same two(2) treating physicians and the same embryologists, throughout.We anticipate that it will take about eighteen (18)months to complete this study. Enrolment has already started.

 

If you are interested in participating and would like to determine whether you qualify, please contact Linda Danner R.N at "ldanner@sherinstitute.com" or call 800-780-7437 ASAP . SIRM is accepting enrollees on a first come first serve basis.

Intravenous Immunoglobulin for Treatment of Recurrent Pregnancy Loss

Abstract

 

Intravenous Immunoglobulin for the Treatment of Recurrent Pregnancy Loss , C.B. Coulam, L. Kyrsa, J. J. Stern, M. Bustillo, Genetics & IVF Institute, Fairfax VA.

Objective: To evaluate the efficacy of intravenous immunoglobulin for treatment of individuals experiencing unexplained recurrent pregnancy loss.

Design: Prospective randomized, placebo-controlled clinical trial.

Materials and Methods: 95 women experiencing 2 or more consecutive spontaneous abortions (SA) with no known cause were randomized and received either intravenous immunoglobulin (IVIG) 500/mg/kg/mo or placebo (albumin)

Results: Of 95 women participating in he study, 47 received IVIG and 48 received placebo. Medication was discontinued in 34 women who failed to conceive within 4 cycles. The remaining 61 women achieved pregnancy. Pregnancy outcomes included 29 delivery and 32 recurrent SA. Among women delivering live births 18 (62%) received IVIG and 11 (33%) received placebo. By contrast 21 (67%) women experiencing SAs received placebo and 11 (33%) received IVIG. Among 61 women who conceived, 29 received IVIG and 32 received placebo. Of the 29 women who conceived and received IVIG 18 (62%) delivered live births and 11 (33%) experienced recurrent SA. Of the 33 women who conceived and received placebo, 11 (33%) delivered live births and 22 (67%) had recurrent SA. The difference in live birth rates between women receiving IVIG and placebo was significant
(P = 0.01, odd ratio 0.2)

Conclusion: IVIG is effective in enhancing the percentage of live births among women experiencing unexplained recurrent SA.

 

 

INTRODUCTION

Recurrent spontaneous abortion is a common complication of pregnancy for which there is no known cure.1 An immunologic cause has been suggested for more than 80% of otherwise unexplained recurrent spontaneous abortion and various immunotherapies have been proposed as treatment for these couples.2 White blood cell immunization has been the most widely used immunotherapy.3 The efficacy of this treatment is low, with an absolute reduction of risk of another abortion between 8% and 10%. Thus, the number of cases needing to be treated in order to prevent a single pregnancy failure was between 9 and 13.4 Because of the low treatment effect, alternative treatments for recurrent spontaneous abortion have been sought. Among alternative treatments reported to result in successful pregnancies is intravenous immunoglobulin.7,15 None of the studies reporting successful pregnancies after treatment with intravenous immunoglobulin included control subjects. We therefore undertook a prospective randomized placebo-controlled double-blinded clinical trial to define the efficacy of intravenous immunoglobulin in the treatment of recurrent spontaneous abortion. We now report the results of this randomized placebo-controlled trial.

 

Materials and Methods

Patients

Women experiencing two or more consecutive spontaneous abortions with the same partner were offered the opportunity of participating in an Institutional Review Board (IRB) approved randomized placebo controlled trial using intravenous gammaglobulin (IVIg) or albumin (placebo) from January 1991 to March 1994. The obstretrical histories of each of the women were obtained and the number of total pregnancies, live births stillbirths, abortions, ectopic pregnancies, and hydatidiform moles and the number of partners for each pregnancy were recorded. All couples were investigated with chromosome analysis, hysterosalpinography, and hysteroscopy, luteal phase endometrial biopsy, and serum progesterone timed with ovulation documented by ultasonic monitoring of folliculogenesis, anticardiolipin antibody (ACA) and activated partial thromboplastin time. All couples with a diagnosis of chromosomal, anatomic, endocrinologic and autoimmunologic etiology of recurrent pregnancy loss were excluded from the study. Also excluded were women less than 18 years or greater than 45 years of age, and women with a history of IgA deficiency or hypersensitivity to immunoglobulin. Each woman had blood screened for the presence of HIV antibodies and the Hepatitis B antigen.

 

Sample-Size Consideration

The major determinant of sample size for this type of study is the expected proportion of subsequent pregnancies to end in a spontaneous abortion among the nonintervention group. Estimates provided in the literature suggest 40% as a reasonable expected proportion of third to sixth spontaneous abortions.1,14,16 The next major consideration is the level of reduction to be achieved by the intervention (therapy). If the causes of recurrent spontaneous abortion differ from the causes of isolated spontaneous abortions and can be eliminated by the intervention, the base line risk for subsequent abortion would be about 12%.16Thus the maximum effect of the treatment would be a decrease in risk of abortion from 0.40 to 0.12. According to traditional parameters for sample size computations, including type 1 error of 0.05, type 2 of 0.2 and a one sided test, 25 pregnancies would be required in the treated and untreated groups. This sample size requires that all of the patients conceive. Previous data indicate 73% of women with three spontaneous abortions and no viable pregnancies have a subsequent pregnancy.16Therefore both the treated and untreated groups should consist of a minimum of 43 patients. Since the reduction of the excess risk of spontaneous abortion due to "recurrent causes" might not be complete due to heterogeneity of the causes of spontaneous abortion and /or effectiveness of the intervention, a sample size of 45 in each group would ensure reliable data (Graph PAD, InStat Version 1.12a, Graph PAD Software, 1990.)

 

Protocol

A total of 95 women were randomized using computer-generated random number, on half receiving intravenous immunoglobulin (IVIg) and the remaining one half albumin infusions. Each patient received an intravenous infusion of the follicular phase of the cycle when pregnancy was desired. Patients were randomized in a double-blinded fashion to receive either (IVIg 500 mg/kg per month of albumin 0.5% in an intravenous infusion. The patient received the infusion every 28 days until pregnant or for 4 months. If the patient was not pregnant in 4 months, she was dropped from the study and replaced with another patient. Once conception occurred, the patient received an infusion every 28 days until delivery or until 28-32 weeks gestation.

 

Results

Patients

Ninety-five women participated in the study. Their mean age was 35 years (range 27-44) and gravidity 5.7 (range 2-18). Forty-six women experienced recurrent spontaneous abortion after a previous live birth (secondary recurrent spontaneous abortion), and 49 women lost two or more pregnancies with no pregnancy progressing beyond 20 weeks of gestation (primary recurrent spontaneous abortion). Of 95 women participating in the study 47 received IVIG and 48 received placebo. The mean age of women receiving IVIg was 35 years (range 27-44), mean gravidity was 5 (range 2-11), 27 women were primary aborters, and 20 were secondary aborters. No differences in age, gravidity, parity, or proportion of primary and secondary aborters existed between the group receiving IVIg and placebo. Medication was discontinued in 34 women (18 receiving IVIg and 16 placebo) because of lack of conception.

Among 61 women who conceived, 29 received IVIg and 32 received placebo. Of the 29 women who conceived and received IVIg, 18 (62%) delivered live births and 11 (38%) experienced recurrent spontaneous abortion. Of 32 women who conceived and received placebo, 11 (34%) delivered live births and 21 (66%) had recurrent spontaneous abortion. The difference in live birth rates between women receiving IVIg and placebo was significant (P=0.04, odds ratio 3.1)

Pregnancy Outcome

Pregnancy outcomes included 29 deliveries and 32 spontaneous abortions (Table 1). Among women delivering live births, 18 (62%) received IVIg and 11 (38%) received placebo. By contrast, 21 (66%) women experiencing recurrent spontaneous abortions received placebo and 11 (34%) received IVIg.
TABLE I. Out come of 61 pregnancies randomized to receiving intravenous immunoglobulin (IVIg) or placebo (albumin)

Pregnancy Outcome    n    IVIg        Placebo          P value
                                                   # (%)          # (%)  

 
Delivery                            29   18 (62)        11 (38)            0.04
Abortion                           32   11 (34)        21 (66)            0.04
Blighted Ovum                15    8  (53)          7 (47)            NS
Intrauterine Death           17    3  (18)        14 (82)         0.004
Total                                    61   29 (48)        32 (52)            NS

 

Thirty-two women experienced recurrent spontaneous abortion (Table I.) Ultrasonographic finding of the 32 pregnancy losses included 15 (47%) empty embryonic gestational sacs or blighted ova and 17 (53%) intrauterine deaths after establishment of cardiac activity in the first trimester of pregnancy. Eight of the blighted ova occurred in women receiving IVIg and 17 in those receiving placebo. Of 17 intrauterine embryonic deaths, 3 (18%) occurred in women receiving IVIg and 14 (82%) in women receiving placebo. Among the 11 pregnancy losses occurring in women receiving IVIg, 8 (73%) were blighted ova, and 3 (27%) were intrauterine embryonic deaths. Twenty-one pregnancy losses occurred in women receiving placebo; 7 (33%) were blighted ova, and 14 (67%) were intrauterine embryonic deaths. The difference in intrauterine embryonic deaths between women receiving IVIg and placebo was significant (P<0.004, odds ratio 0.1)

 

Complications

No reactions to study medications occurred. One infant was born with Mosaic Down syndrome and one pregnancy was complicated by an umbilical cord accident at 30 weeks gestation. Both women received IVIg.

 

DISCUSSION

Analysis of results from this randomized, double blinded placebo-controlled clinical trial suggests that IVIg us efficacious in the treatment of recurrent spontaneous abortion. Another randomized, placebo-controlled trial has been performed in Germany as a multicenter study.17 A significant specific effect of IVIg on live birth rate could not be demonstrated. However , success rates for both IVIg and albumin were in the same range as allogeneic leukocytes.17 The difference in interpretation of results in the current study and the German experience 17 has at least three explanations. The first explanation involves patient selection. The women included in the studies could represent different populations with different risk factors for pregnancy loss. More sensitive and specific markers are needed to identify individuals most likely to respond to immunotherapy before differences in study populations can be prepared. The second explantation involves the differences in the study design between the two studies. In the current study, therapy was begun before conception, whereas the German study instituted all treatment after a positive pregnancy test was obtained.17 Preconception treatment for recurrent spontaneous abortion using various forms of immunotherapy has been shown to be more effective than postconception treatment.17 The third exception for differences in success rate is co-intervention by the control treatment. The reason the German study showed no treatment effect of IVIg is that the effect seen was the same as that of albumin. Both effects were the same as the treatment effect seen by IVIg in the current study and by leukocyte immunization in the worldwide prospective collaborative study.4 The concentration of albumin in the German study17 was a 5% solution in contrast to the current study in which 0.5% albumin was used. Little is known about the immunomodulating effects of albumin. Recently, soluble HLA molecules have been detected, not only in IVIg19 but also in smaller amounts, in albumin preparations.20

Intravenous immunoglobulin therapy has been previously reported to be effective in prevention of recurrent spontaneous abortion (5-13). The mechanism of this antiabortive effect is not known. Immune modulation by IVIg has been speculated to result from passively transferred blocking or antiidiotypic antibodies,22 blockage of Fc receptors,23 enhancement of suppressor T-cell function,24 down regulation of B-cell function,25 and/or reduction of activation of complement components, 13,26,27 natural killer cell function, and cytokine production.28

Whatever the mode of action, the mechanism does not maintain pregnancies associated with blighted ova29 but does maintain pregnancies that are lost as a result of intrauterine demise after the establishment of embryonic cardiac activity (Table I.).

The majority of pregnancies (73%) lost after treatment with intravenous immunoglobulin are blighted ova (Table I.). Limited data in the literature suggest that ultrasonographic demonstration of an empty sac is associated with an abnormal analysis of the chorionic villus sampling .30,31 If these observations can be confirmed in women experiencing recurrent pregnancy loss, then pregnancies lost after treatment with intravenous immunoglobulin will be those with abnormal chromosome complements. IVIg is effective in enhancing the percentage of live births among women experiencing unexplained recurrent spontaneous abortion. Since IVIg preparations are free of cells and can be quarantined for prolonged periods, IVIg provides a safer alternative for treatment of recurrent pregnancy loss than white blood cell immunization.

 

Acknowledgments

The authors should like to thank the following physicians who provided patients who participated in the study: S. Alexander, D. Fein, J. Langley, A. Toofanian, G. Janneck, M. Liptak, D. Koepping, G. Shuster-Haynes, M. Hinton, K.Duprey, A. Haney, J. Eberhardy, A. Hough, R. Zold, R. Nehls, D. Mullaney, R. Suarez, M. Maloney, M. Aiken, M. Jones, M. Freedman, J. Davidson, P. Taylor, C. Whitworth, D. Ross, S. Marynick, B. Wassell, L. Beard, M. Utley, S. Reager, R. Chopyck, S. Collins, C. Calvello, M. Turner, T. Markus, A. West, A. Gonzales, J. Thompson, L Underwood, J. Jones, K. Fischer, R. Reinsch, D. Bewall, A. Peters, and R. Lloyd.

 

  1. Coulam, CB. Unification of immunotherapy protocols. Am J Reprod Immunol 1991: 25:1-6
  2. McIntyre JA, Coulam CB, Faulk WP. Recurrent spontaneous abortion. Am J Reprod Immunol 1989; 21:100-104
  3. Mowbray JF, Lidlee H, Underwood JL, et al. Controlled trial of treatment of recurrent spontaneous abortionby immunization with paternal cells. Lancet 1985; 1:941-949
  4. Fraser EJ, Grimes DA, SchultzKF, Immunization as therapy for recurrent spontaneous abortion; a review and meta-analysis. Obstet Gynecol 1993; 82:854-859
  5. Coulam CB, Peters AJ, McIntyre JA, Faulk WP. The use of intravenous immunoglobulin for the treatment of recurrent spontaneous abortion. Am J Reprod Immunol 1990; 22:78.
  6. Mueller-Eckhardt G, Heine O, Neppert J, Kunzel W, Mueller-Eckhardt C. Prevention of recurrent spontaneous abortion by intravenous immunoglobulin. Vox Sang 1989:56:151-154
  7. Mueller-Eckhardt G, Huni O, Poltrin B. IVIg to prevent recurrent spontaneous abortion. Lancet; 1991; 1:424
  8. Berstein RM, Crawford RJ. Intravenous IgG therapy for anticardiolipin syndrome: A case report (abstract). Clin Exp Rheumatol 1988; 6:198
  9. Scott JR, Branch DW, Kochenour NK, Ward K. Intravenous immunoglobulin treatment for pregnant patients with recurrent pregnancy loss caused by antiphospholipid antibodies and Rh immunization. Am J Obstet Gynecol 1988; 159:1055-1056
  10. Carreras I.O., Perez GN. Vega HR. Casavilla F. Lupus anticoagulant and recurrent fetal loss: Successful treatment with gammaglobulin. Lancet 1988; 2:393-394
  11. Francois A. Freund M. Daffos F. Remy P. Risch M. Jacquor C. Repeated fetal losses and lupus anticoagnulant. Ann Intern Med 1988: 109:993-994
  12. Parks A. Maer D. Wilson D. Andreoli J. Ballow M. Intravenous gamm-globulin, anti-phospholipid antibodies and pregnancy. Ann Intern Med 1989: 110:495-496
  13. Christriansen OB. Mathiesen O. Lauristen JG. Grunner N. Intravenous immunoglobulin treatment of women with multiple miscarriages. Human Reprod 1992; 7:718-722.
  14. Parazzini F.Acais B. Ricciardeiello O. Fedele L. Liata P. Candiani GB. Short-term reproductive diagnosis when no cause can be found for recurrent miscarriage. Br J Obstet Gynaecol 1988; 95:654.
  15. Risch HA. Weiss NB. Clarke EA. Miller AB. Risk factors for spontaneous abortion and its recurrence. Am J Epidemiol 1988; 128:420.
  16. Poland BJ. Miller JR. Jones DC. Trimble BK. Reproductive counseling in patients who have had spontaneous abortion. Am J Obstet Gyencol 1977; 127:685.
  17. The German RSA/IVIG Group. Intravenous immunoglobulin in the prevention of recurrent miscarriage. Br J Obstet Gynecol 1994; In press.
  18. Kwak JYH. Gilman-Sachs A. Beamen KD. Beer AE. Reproductive outcome in women with recurrent spontaneous abortions of alloimmune and autoimmune causes: preconception vs. postconception treatment. Am J Obstet Gyencol 1992; 166:1787-1795.
  19. Gross-Wilde II. Blasczyk R. Westhoff U. Soluble HLA class I and II concentrations in commercial immunoglobulin preparations. Tissue Antigens 1992; 39:74-77.
  20. Sancoso S. Kiefel V. Voiz H. Mueller-Echardt C.Quantitation of soluble HLA class I antigen in human albumin and immunoglobulin preparations for intravenous use by solid-phase immunoassay. Vox Sang 1993; 62:29-33.
  21. Hay CRM. The effect of chronic exposure to clotting factor concentrates on the immune system. In Coagulation and blood transfusion. Smit Sibings (T. Das PC. Mannucci PH(eds). Dordrecht. Boston. London; Kluwer Academic Publisher. 1991:227-240
  22. Brand A. Wirvliet M. Claas FHJ. et.al. Benificial effect of intravenous gammaglobulin in a patient with complement-mediated autoimmune thromboeytopenia due to IgM-anti-platelet antibodies. Br J Haemarol 1988; 69:507-511.
  23. Kimberly RP. Salmon JE. Bussell JB. et al. Modulation of mononuclear phagocte function by intravenous gammaglobulin. J. Immunol 1987; 132:745-750.
  24. Delfraissy JF, Tchernia G. Laurian Y. et al. Suppressor cel function after intravenous gammaglobulin treatment in adult chronic idiopathic thrombocytopenic purpura. Br J Haematol 1985; 60:315-322.
  25. Nydegger UE. Hypotheric and established action mechanisms of therapy with immunoglobulin G. In immunotherapy with intravenous immunoglobulin. Imbach P (ed). Academic Press. London. 1991. pp 27-36.
  26. Kulies J. Rajnavolgya E. Fust G. Gergely J. Interaction of C3 and C3h with immunoglobulin and complement concentration. Nephron 1985; 40:253-254.
  27. Zielinski CC. Pries P. Eibl MM. Effect of immunoglobulin and complement concentration. Nephron 1985; 40:253-254.
  28. Newland AC. The use and mechanisms of action of intravenous immunoglobulin: An update. Br J Haematol 1989; 72:301-305.
  29. Coulam CB. Stern JJ, Bustillo M. Ultrasonographic findings of pregnancy losses after treatment for recurrent pregnancy loss: intravenous immunoglobulin versus placebo. Fertil Steril 1994; 61:248-251.

IVIg Safety

Safety of Therapeutic Immune Globulin Preparations with Respect to Transmission of Human T-Lymphotropic Virus Type III/ Lymphadenopathy-Associated Virus Infection

Publication date: 04/11/1986

 


Table of Contents

Article
Editorial Note
References
POINT OF CONTACT FOR THIS DOCUMENT:

 


Article

Immune globulins produced by plasma fractionation methods approved for use in the United States have not been implicated in the transmission of infectious agents. Nevertheless, because immune globulins manufactured before 1985 were derived from plasma of human donors who were not screened for antibody to human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), CDC and the U.S. Food and Drug Administration (FDA) have received inquiries concerning the safety of immune globulin (IG), hepatitis B immune globulin (HBIG), and intravenous immune globulin (IVIG). Current epidemiologic and laboratory evidence shows that these preparations carry no discernable risk of transmitting HTLV-III/LAV infection and that current indications for their clinical use should not be changed based on such concerns.

 

BACKGROUND

The IG, HBIG, IVIG, and other special immune globulins used in the United States are produced by several manufacturers using the Cohn-Oncley fractionation process (1,2). This process involves a series of precipitation steps performed in the cold with addition of varying concentrations of ethanol. Production lots of IG and IVIG are made from plasma pools from at least 1,000 donors; HBIG and other specific immune globulins (e.g., varicella-zoster IG) may be prepared from plasma pools from fewer donors.
Before 1985, donors were screened only for hepatitis B surface antigen but not by other tests for specific diagnosis of viral infections. Since April 1985, all donor units also have been screened for antibodies to HTLV-III/LAV, and all repeatedly reactive units have been discarded. Tests conducted at FDA and CDC have shown that as many as two-thirds of HBIG lots, as well as some lots of IG and IVIG, produced between 1982 and 1985 may have been positive for HTLV-III/LAV antibody. The question of safety arises out of concern that some immune globulins currently available were prepared from plasma pools that included units from donors who may have had HTLV-III/LAV viremia.

 

EPIDEMIOLOGIC STUDIES

Several studies have shown that recipients of HBIG and IG, including recipients of lots known to be positive for antibody to HTLV-III/LAV, did not seroconvert to antibody to HTLV-III/LAV-positivity and have not developed signs and symptoms of acquired immunodeficiency syndrome (AIDS) or other illnesses suggesting HTLV-III/LAV infection.

Since August 1983, CDC has enrolled 938 individuals who have had parenteral or mucous-membrane exposures to blood or body fluids of AIDS patients in a prospective surveillance study. To date, 451 entrants have been followed and tested for HTLV-III/LAV antibody. Of these, 183 persons received IG and/or HBIG as prophylaxis against hepatitis B infection; 100 (55%) received only IG; 65 (36%) received only HBIG; and 18 (10%) received both. One of the 183 HBIG recipients is now positive for HTLV-III/LAV antibody, but no preexposure serum was available for this individual, and seropositivity may have predated the needlestick exposure and IG prophylaxis. Further, heterosexual transmission of HTLV-III/LAV infection in this individual cannot be ruled out. No documented seroconversions have occurred in any of the 183 health-care workers who received IG or HBIG. Studies have been reported of 16 subjects who received HBIG that was strongly positive for HTLV-III/LAV antibody (3). Each patient had been given one to five ampules. A total of 31 doses were administered to 16 individuals. Low levels of passively acquired HTLV-III/LAV antibody were detected shortly after injection, but reactivity did not persist. Six months after the last HBIG injection, none of the 16 individuals had antibody to HTLV-III/LAV.

In a study of prophylaxis against cytomegalovirus (CMV) infections among kidney-transplant patients, 16 patients received CMV-specific IVIG preparations subsequently found to contain HTLV-III/LAV antibody. After 10 months or longer of follow-up, none of the 16 recipients developed antibody or other evidence of HTLV-III/LAV infection.
In studies of a group of IVIG recipients, most of whom had idiopathic thrombocytopenia, none of 134 patients developed antibodies or other evidence of HTLV-III/LAV infection.
Information regarding past therapy with immune globulins is available from 10,227 of 17,115 AIDS patients reported to CDC. Three hundred fifty-eight (4%) reported receipt of an IG preparation. All but seven of these patients also were members of groups known to be at high risk for developing AIDS. The percentage of patients with no recognized risk factors for AIDS was not significantly different among those who received immune globulins (7/358 (2%)) than among those who did not (358/9,869 (4%)).

 

LABORATORY STUDIES

Scientists at FDA recently evaluated the basic fractionation processes (1,2) used for production of immune globulins to determine effectiveness of those procedures in eliminating HTLV-III/LAV infectivity (4). Six sequential steps in a typical process were evaluated. The study was designed so that efficiency of eliminating HTLV-III/LAV at each step was measured. The degree to which HTLV-III/LAV was reduced by partitioning or inactivation at individual steps ranged from 10))-1)) to more than 10))-4)) of in vitro infectious units (IVIU)/ml. The effectiveness of virus removal in the entire process by partitioning and inactivation was calculated to be greater than 1 x 10((15)) IVIU/ml.

Concentrations of infectious HTLV-III/LAV in plasma of infected persons have been estimated to be less than 100 IVIU/ml. Further, FDA scientists have shown that the geometric mean infectivity titer of plasma from 43 HTLV-III/LAV infected persons was 0.02 IVIU/ml (4). Thus, the margin of safety based on the removal of infectivity by the fractionation process is extremely high.

Scientists at CDC and FDA also cultured 38 lots of HBIG, IVIG, and IG, most of which contained HTLV-III/LAV antibody. HTLV-III/LAV was not recovered from any lot tested.

Reported by J Bossell, MD, Cornell University, New York City; Central Laboratories Swiss Red Cross Blood Transfusion Svc, Berne, Switzerland; Immuno A.G., Vienna, Austria; KabiVitrum AB, Stockholm, Sweden; Massachusetts Public Health Biologics Laboratories, Boston, Massachusetts; Miles Laboratories, Inc., Berkeley, Travenol Laboratories, Inc., Glendale, California; Center for Drugs and Biologics, U.S. Food and Drug Administration; Center for Infectious Diseases, CDC.

 


Editorial Note

Editorial Note:The laboratory and epidemiologic studies referred to have shown that concern about HTLV-III/LAV infection associated with the use of immune globulins available in the United States is not warranted. Strategies for using immune globulins recommended by the Immunization Practices Advisory Committee should be followed (5). Recently, concern has been expressed that patients who received IG prepared from plasma of donors not screened for HTLV-III/LAV antibody may have a passively acquired false-positive reaction for antibody (6). Passively acquired HTLV-III/LAV antibody from HBIG known to contain high levels of antibody has been reported (3). Based on the estimated half-life of globulins in plasma, it can be calculated that passively acquired antibodies might be detected in sera of recipients for as long as 6 months after administration of immune globulins. It is important to recognize this possibility when attempting to determine the significance of HTLV-III/LAV antibody in a person who has recently received immune globulins, especially HBIG.

 


References

  1. Cohn EJ, Strong LE, Hughes WI Jr, et al. Preparation and properties of serum and plasma proteins. IV: A system for the separation into fractions of protein and lipoprotein components of biological tissues and fluids. J Am Chem Soc 1946;68:459-75.
  2. Oncley JL, Melin M, Richert DA, Cameron JW, Gross PM Jr. The separation of the antibodies isoagglutinins, prothrombin, plasmonogen and beta-lipoprotein into subfractions of human plasma. J Am Chem Soc 1949;71:541-50.
  3. Tedder RS, Uttley A, Cheingsong-Popov R. Safety of immunoglobulin preparation containing anti-HTLV-III (Letter). Lancet 1985;I:815.
  4. Wells, MA, Wittex AE, Epstein, JS, et al. Chemical and physical inactivation of human T lymphotropic virus, Type III (HTLV-III). Transfusion 1986;26:110-30.
  5. ACIP. Recommendations for protection against viral hepatitis. MMWR 1985;34:313-24, 329-35.
  6. Steele DR. HTLV-III antibodies in human immune g-globulin (Letter). JAMA 1986;255:609.

IVIg Trial

Intravenous Immunoglobulin for Treatment of Recurrent Pregnancy Loss

 

Abstract

Intravenous Immunoglobulin for the Treatment of Recurrent Pregnancy Loss , C.B. Coulam, L. Kyrsa, J. J. Stern, M. Bustillo, Genetics & IVF Institute, Fairfax VA.

Objective: To evaluate the efficacy of intravenous immunoglobulin for treatment of individuals experiencing unexplained recurrent pregnancy loss.

Design: Prospective randomized, placebo-controlled clinical trial.

Materials and Methods: 95 women experiencing 2 or more consecutive spontaneous abortions (SA) with no known cause were randomized and received either intravenous immunoglobulin (IVIG) 500/mg/kg/mo or placebo (albumin)

Results: Of 95 women participating in he study, 47 received IVIG and 48 received placebo. Medication was discontinued in 34 women who failed to conceive within 4 cycles. The remaining 61 women achieved pregnancy. Pregnancy outcomes included 29 delivery and 32 recurrent SA. Among women delivering live births 18 (62%) received IVIG and 11 (33%) received placebo. By contrast 21 (67%) women experiencing SAs received placebo and 11 (33%) received IVIG. Among 61 women who conceived, 29 received IVIG and 32 received placebo. Of the 29 women who conceived and received IVIG 18 (62%) delivered live births and 11 (33%) experienced recurrent SA. Of the 33 women who conceived and received placebo, 11 (33%) delivered live births and 22 (67%) had recurrent SA. The difference in live birth rates between women receiving IVIG and placebo was significant
(P = 0.01, odd ratio 0.2)

Conclusion: IVIG is effective in enhancing the percentage of live births among women experiencing unexplained recurrent SA.

 

INTRODUCTION

Recurrent spontaneous abortion is a common complication of pregnancy for which there is no known cure.1 An immunologic cause has been suggested for more than 80% of otherwise unexplained recurrent spontaneous abortion and various immunotherapies have been proposed as treatment for these couples.2 White blood cell immunization has been the most widely used immunotherapy.3 The efficacy of this treatment is low, with an absolute reduction of risk of another abortion between 8% and 10%. Thus, the number of cases needing to be treated in order to prevent a single pregnancy failure was between 9 and 13.4 Because of the low treatment effect, alternative treatments for recurrent spontaneous abortion have been sought. Among alternative treatments reported to result in successful pregnancies is intravenous immunoglobulin.7,15 None of the studies reporting successful pregnancies after treatment with intravenous immunoglobulin included control subjects. We therefore undertook a prospective randomized placebo-controlled double-blinded clinical trial to define the efficacy of intravenous immunoglobulin in the treatment of recurrent spontaneous abortion. We now report the results of this randomized placebo-controlled trial.

 

Materials and Methods

Patients

Women experiencing two or more consecutive spontaneous abortions with the same partner were offered the opportunity of participating in an Institutional Review Board (IRB) approved randomized placebo controlled trial using intravenous gammaglobulin (IVIg) or albumin (placebo) from January 1991 to March 1994. The obstretrical histories of each of the women were obtained and the number of total pregnancies, live births stillbirths, abortions, ectopic pregnancies, and hydatidiform moles and the number of partners for each pregnancy were recorded. All couples were investigated with chromosome analysis, hysterosalpinography, and hysteroscopy, luteal phase endometrial biopsy, and serum progesterone timed with ovulation documented by ultasonic monitoring of folliculogenesis, anticardiolipin antibody (ACA) and activated partial thromboplastin time. All couples with a diagnosis of chromosomal, anatomic, endocrinologic and autoimmunologic etiology of recurrent pregnancy loss were excluded from the study. Also excluded were women less than 18 years or greater than 45 years of age, and women with a history of IgA deficiency or hypersensitivity to immunoglobulin. Each woman had blood screened for the presence of HIV antibodies and the Hepatitis B antigen.

 

Sample-Size Consideration

The major determinant of sample size for this type of study is the expected proportion of subsequent pregnancies to end in a spontaneous abortion among the nonintervention group. Estimates provided in the literature suggest 40% as a reasonable expected proportion of third to sixth spontaneous abortions.1,14,16 The next major consideration is the level of reduction to be achieved by the intervention (therapy). If the causes of recurrent spontaneous abortion differ from the causes of isolated spontaneous abortions and can be eliminated by the intervention, the base line risk for subsequent abortion would be about 12%.16 Thus the maximum effect of the treatment would be a decrease in risk of abortion from 0.40 to 0.12. According to traditional parameters for sample size computations, including type 1 error of 0.05, type 2 of 0.2 and a one sided test, 25 pregnancies would be required in the treated and untreated groups. This sample size requires that all of the patients conceive. Previous data indicate 73% of women with three spontaneous abortions and no viable pregnancies have a subsequent pregnancy.16Therefore both the treated and untreated groups should consist of a minium of 43 patients. Since the reduction of the excess risk of spontaneous abortion due to "recurrent causes" might not be complete due to heterogeneity of the causes of spontaneous abortion and /or effectiveness of the intervention, a sample size of 45 in each group would ensure reliable data (Graph PAD, InStat Version 1.12a, Graph PAD Software, 1990.)

 

Protocol

A total of 95 women were randomized using computer-generated random number, on half receiving intravenous immunoglobulin (IVIg) and the remaining one half albumin infusions. Each patient received an intravenous infusion of the follicular phase of the cycle when pregnancy was desired. Patients were randomized in a double-blinded fashion to receive either (IVIg 500 mg/kg per month of albumin 0.5% in an intravenous infusion. The patient received the infusion every 28 days until pregnant or for 4 months. If the patient was not pregnant in 4 months, she was dropped from the study and replaced with another patient. Once conception occurred, the patient received an infusion every 28 days until delivery or until 28-32 weeks gestation.

 

Results

Patients

Ninety-five women participated in the study. Their mean age was 35 years (range 27-44) and gravidity 5.7 (range 2-18). Forty-six women experienced recurrent spontaneous abortion after a previous live birth (secondary recurrent spontaneous abortion), and

49 women lost two or more pregnancies with no pregnancy progressing beyond 20 weeks of gestation (primary recurrent spontaneous abortion). Of 95 women participating in the study 47 received IVIG and 48 received placebo. The mean age of women receiving IVIg was 35 years (range 27-44), mean gravidity was 5 (range 2-11), 27 women were primary aborters, and 20 were secondary aborters. No differences in age, gravidity, parity, or proportion of primary and secondary aborters existed between the group receiving IVIg and placebo. Medication was discontinued in 34 women (18 receiving IVIg and 16 placebo) because of lack of conception.

Among 61 women who conceived, 29 received IVIg and 32 received placebo. Of the 29 women who conceived and received IVIg, 18 (62%) delivered live births and 11 (38%) experienced recurrent spontaneous abortion. Of 32 women who conceived and received placebo, 11 (34%) delivered live births and 21 (66%) had recurrent spontaneous abortion. The difference in live birth rates between women receiving IVIg and placebo was significant (P=0.04, odds ratio 3.1)

 

Pregnancy Outcome

Pregnancy outcomes included 29 deliveries and 32 spontaneous abortions (Table 1). Among women delivering live births, 18 (62%) received IVIg and 11 (38%) received placebo. By contrast, 21 (66%) women experiencing recurrent spontaneous abortions received placebo and 11 (34%) received IVIg.

TABLE I. Out come of 61 pregnancies randomized to receiving intravenous immunoglobulin (IVIg) or placebo (albumin)

Pregnancy Outcome

n

IVIg

Placebo

P
value

 

#

(%)

#

(%)

 

Delivery

29

18

(62)

11

(38)

0.04

Abortion

32

11

(34)

21

(66)

0.04

Blighted Ovum

15

8

(53)

7

(47)

NS

Intrauterine Death

17

3

(18)

14

(82)

0.004

Total

61

29

(48)

32

(52)

NS

Thirty-two women experienced recurrent spontaneous abortion (Table I.) Ultrasonographic finding of the 32 pregnancy losses included 15 (47%) empty embryonic gestational sacs or blighted ova and 17 (53%) intrauterine deaths after establishment of cardiac activity in the first trimester of pregnancy. Eight of the blighted ova occurred in women receiving IVIg and 17 in those receiving placebo. Of 17 intrauterine embryonic deaths, 3 (18%) occurred in women receiving IVIg and 14 (82%) in women receiving placebo. Among the 11 pregnancy losses occurring in women receiving IVIg, 8 (73%) were blighted ova, and 3 (27%) were intrauterine embryonic deaths. Twenty-one pregnancy losses occurred in women receiving placebo; 7 (33%) were blighted ova, and 14 (67%) were intrauterine embryonic deaths. The difference in intrauterine embryonic deaths between women receiving IVIg and placebo was significant (P<0.004, odds ratio 0.1)

 

Complications

No reactions to study medications occurred. One infant was born with Mosaic Down syndrome and one pregnancy was complicated by an umbilical cord accident at 30 weeks gestation. Both women received IVIg.

 

DISCUSSION

Analysis of results from this randomized, double blinded placebo-controlled clinical trial suggests that IVIg us efficacious in the treatment of recurrent spontaneous abortion. Another randomized, placebo-controlled trial has been performed in Germany as a multicenter study.17 A significant specific effect of IVIg on live birth rate could not be demonstrated. However , success rates for both IVIg and albumin were in the same range as allogeneic leukocytes.17 The difference in interpretation of results in the current study and the German experience 17 has at least three explanations. The first explanation involves patient selection. The women included in the studies could represent different populations with different risk factors for pregnancy loss. More sensitive and specific markers are needed to identify individuals most likely to respond to immunotherapy before differences in study populations can be prepared. The second explantation involves the differences in the study design between the two studies. In the current study, therapy was begun before conception, whereas the German study instituted all treatment after a positive pregnancy test was obtained.17 Preconception treatment for recurrent spontaneous abortion using various forms of immunotherapy has been shown to be more effective than postconception treatment.17 The third exception for differences in success rate is co-intervention by the control treatment. The reason the German study showed no treatment effect of IVIg is that the effect seen was the same as that of albumin. Both effects were the same as the treatment effect seen by IVIg in the current study and by leukocyte immunization in the worldwide prospective collaborative study.4 The concentration of albumin in the German study17 was a 5% solution in contrast to the current study in which 0.5% albumin was used. Little is known about the immunomodulating effects of albumin. Recently, soluble HLA molecules have been detected, not only in IVIg19 but also in smaller amounts, in albumin preparations.20

Intravenous immunoglobulin therapy has been previously reported to be effective in prevention of recurrent spontaneous abortion (5-13). The mechanism of this antiabortive effect is not known. Immune modulation by IVIg has been speculated to result from passively transferred blocking or antiidiotypic antibodies,22 blockage of Fc receptors,23 enhancement of suppressor T-cell function,24 down regulation of B-cell function,25 and/or reduction of activation of complement components, 13,26,27 natural killer cell function, and cytokine production.28

Whatever the mode of action, the mechanism does not maintain pregnancies associated with blighted ova29 but does maintain pregnancies that are lost as a result of intrauterine demise after the establishment of embryonic cardiac activity (Table I.).

The majority of pregnancies (73%) lost after treatment with intravenous immunoglobulin are blighted ova (Table I.). Limited data in the literature suggest that ultrasonographic demonstration of an empty sac is associated with an abnormal analysis of the chorionic villus sampling .30,31 If these observations can be confirmed in women experiencing recurrent pregnancy loss, then pregnancies lost after treatment with intravenous immunoglobulin will be those with abnormal chromosome complements. IVIg is effective in enhancing the percentage of live births among women experiencing unexplained recurrent spontaneous abortion. Since IVIg preparations are free of cells and can be quarantined for prolonged periods, IVIg provides a safer alternative for treatment of recurrent pregnancy loss than white blood cell immunization.

 

Acknowledgments

The authors should like to thank the following physicians who provided patients who participated in the study: S. Alexander, D. Fein, J. Langley, A. Toofanian, G. Janneck, M. Liptak, D. Koepping, G. Shuster-Haynes, M. Hinton, K.Duprey, A. Haney, J. Eberhardy, A. Hough, R. Zold, R. Nehls, D. Mullaney, R. Suarez, M. Maloney, M. Aiken, M. Jones, M. Freedman, J. Davidson, P. Taylor, C. Whitworth, D. Ross, S. Marynick, B. Wassell, L. Beard, M. Utley, S. Reager, R. Chopyck, S. Collins, C. Calvello, M. Turner, T. Markus, A. West, A. Gonzales, J. Thompson, L Underwood, J. Jones, K. Fischer, R. Reinsch, D. Bewall, A. Peters, and R. Lloyd.

 

Coulam, CB. Unification of immunotherapy protocols. Am J Reprod Immunol 1991: 25:1-6

McIntyre JA, Coulam CB, Faulk WP. Recurrent spontaneous abortion. Am J Reprod Immunol 1989; 21:100-104

Mowbray JF, Lidlee H, Underwood JL, et al. Controlled trial of treatment of recurrent spontaneous abortionby immunization with paternal cells. Lancet 1985; 1:941-949

Fraser EJ, Grimes DA, SchultzKF, Immunization as therapy for recurrent spontaneous abortion; a review and meta-analysis. Obstet Gynecol 1993; 82:854-859

Coulam CB, Peters AJ, McIntyre JA, Faulk WP. The use of intravenous immunoglobulin for the treatment of recurrent spontaneous abortion. Am J Reprod Immunol 1990; 22:78.

Mueller-Eckhardt G, Heine O, Neppert J, Kunzel W, Mueller-Eckhardt C. Prevention of recurrent spontaneous abortion by intravenous immunoglobulin. Vox Sang 1989:56:151-154

Mueller-Eckhardt G, Huni O, Poltrin B. IVIg to prevent recurrent spontaneous abortion. Lancet; 1991; 1:424

Berstein RM, Crawford RJ. Intravenous IgG therapy for anticardiolipin syndrome: A case report (abstract). Clin Exp Rheumatol 1988; 6:198

Scott JR, Branch DW, Kochenour NK, Ward K. Intravenous immunoglobulin treatment for pregnant patients with recurrent pregnancy loss caused by antiphospholipid antibodies and Rh immunization. Am J Obstet Gynecol 1988; 159:1055-1056

Carreras I.O., Perez GN. Vega HR. Casavilla F. Lupus anticoagulant and recurrent fetal loss: Successful treatment with gammaglobulin. Lancet 1988; 2:393-394

Francois A. Freund M. Daffos F. Remy P. Risch M. Jacquor C. Repeated fetal losses and lupus anticoagnulant. Ann Intern Med 1988: 109:993-994

Parks A. Maer D. Wilson D. Andreoli J. Ballow M. Intravenous gamm-globulin, anti-phospholipid antibodies and pregnancy. Ann Intern Med 1989: 110:495-496

Christriansen OB. Mathiesen O. Lauristen JG. Grunner N. Intravenous immunoglobulin treatment of women with multiple miscarriages. Human Reprod 1992; 7:718-722.

Parazzini F.Acais B. Ricciardeiello O. Fedele L. Liata P. Candiani GB. Short-term reproductive diagnosis when no cause can be found for recurrent miscarriage. Br J Obstet Gynaecol 1988; 95:654.

Risch HA. Weiss NB. Clarke EA. Miller AB. Risk factors for spontaneous abortion and its recurrence. Am J Epidemiol 1988; 128:420.

Poland BJ. Miller JR. Jones DC. Trimble BK. Reproductive counseling in patients who have had spontaneous abortion. Am J Obstet Gyencol 1977; 127:685.

The German RSA/IVIG Group. Intravenous immunoglobulin in the prevention of recurrent miscarriage. Br J Obstet Gynecol 1994; In press.

Kwak JYH. Gilman-Sachs A. Beamen KD. Beer AE. Reproductive outcome in women with recurrent spontaneous abortions of alloimmune and autoimmune causes: preconception vs. postconception treatment. Am J Obstet Gyencol 1992; 166:1787-1795.

Gross-Wilde II. Blasczyk R. Westhoff U. Soluble HLA class I and II concentrations in commercial immunoglobulin preparations. Tissue Antigens 1992; 39:74-77.

Sancoso S. Kiefel V. Voiz H. Mueller-Echardt C.Quantitation of soluble HLA class I antigen in human albumin and immunoglobulin preparations for intravenous use by solid-phase immunoassay. Vox Sang 1993; 62:29-33.

Hay CRM. The effect of chronic exposure to clotting factor concentrates on the immune system. In Coagulation and blood transfusion. Smit Sibings (T. Das PC. Mannucci PH(eds). Dordrecht. Boston. London; Kluwer Academic Publisher. 1991:227-240

Brand A. Wirvliet M. Claas FHJ. et.al. Benificial effect of intravenous gammaglobulin in a patient with complement-mediated autoimmune thromboeytopenia due to IgM-anti-platelet antibodies. Br J Haemarol 1988; 69:507-511.

Kimberly RP. Salmon JE. Bussell JB. et al. Modulation of mononuclear phagocte function by intravenous gammaglobulin. J. Immunol 1987; 132:745-750.

Delfraissy JF, Tchernia G. Laurian Y. et al. Suppressor cel function after intravenous gammaglobulin treatment in adult chronic idiopathic thrombocytopenic purpura. Br J Haematol 1985; 60:315-322.

Nydegger UE. Hypotheric and established action mechanisms of therapy with immunoglobulin G. In immunotherapy with intravenous immunoglobulin. Imbach P (ed). Academic Press. London. 1991. pp 27-36.

Kulies J. Rajnavolgya E. Fust G. Gergely J. Interaction of C3 and C3h with immunoglobulin and complement concentration. Nephron 1985; 40:253-254.

Zielinski CC. Pries P. Eibl MM. Effect of immunoglobulin and complement concentration. Nephron 1985; 40:253-254.

Newland AC. The use and mechanisms of action of intravenous immunoglobulin: An update. Br J Haematol 1989; 72:301-305.

Coulam CB. Stern JJ, Bustillo M. Ultrasonographic findings of pregnancy losses after treatment for recurrent pregnancy loss: intravenous immunoglobulin versus placebo. Fertil Steril 1994; 61:248-251.

Embryo Interaction May Be Key to Diagnosis and Treatment of Immunological Implantation Failure by Geoffrey Sher, M.D.

What is the basis for the diagnosing and treating of immunologic implantation failure with IVIG and corticosteroids?

There have recently been several significant developments in the reproductive immunology arena that promise to soon resolve the raging controversy regarding the issue of immunologic implantation failure with IVF and the role of immunotherapy. Recent recognition of the fact that the implanting embryo engages in a "cross talk" with immune cells in the endometrium (lymphocytes; Natural Killer [NK] cells and T-cells) through the interaction of growth factors known as cytokines (i.e. the cytokine network) to reach a "negotiated settlement" with the embryo (a semi-allograph) allowing it to develop unhindered in the uterus rather than be rejected.

The embryo, upon arrival in the uterus, relays its desire to enter into a "dialogue" with the endometrial immune system, through releasing specific genes known as Human Lymphocyte Antigens (HLA). If the signal is appropriate and is well received by the endometrial NK and T-cells, the cytokine network is established and the "dialogue" is both initiated and propagated thereby.

Cytokines produced by both the embryo and the immune cells of the endometrium, fall in to two categories. TH-1 cytokines tend to destroy the cells of the trophoblast (the root system of the early embryo and of the placenta), and TH-2 cytokines propagate implantation and placentation. A harmonious interaction of TH-1 and TH-2 cytokines is necessary for healthy implantation and for the developing conceptus to survive and thrive. It is perhaps not surprising that clear evidence has recently emerged of TH1 dominance in cases of "unexplained" recurrent pregnancy loss, pregnancy-induced complications (such as pre-eclampsia) that threaten fetal survival and in "unexplained (often repeated) IVF failure. Cases at risk of the latter are women who exhibit local (endometrial) and peripheral (blood) immunologic and immunophenotypic evidence of NK cell hyper activation (NKa) and or cytotoxic T-cells (CTL), prior to undergoing IVF. These findings will hopefully pave the way to a rational discourse. Since THI cytokine dominance in large part originates from increased NKa and CTL activity in the endometrium, it follows that rational treatment must focus upon immunoregulation/modulation of the culpable cells, prior to the embryo’s arrival in the uterus. Currently, immunoglobulin-G (IVIG) and steroid therapy offers the best method for down-regulating NKa and CTL activity in the uterine lining.

It has long been our position at SIRM that the unwillingness on the part of ART practitioners to recognize the entity of immunologic implantation failure and the use of NKa and CTL regulators such as IVIG and corticosteroids has been motivated by an agenda propagated by a few individuals who were desirous aimed at "killing the messengers". Now, emerging clinical/scientific evidence offers renewed hope that will force a healthy debate on this highly charged, hitherto controversial, but nevertheless very important cause of IVF failure.

 

Why do we measure HLA-G to determine embryo implantation potential?

Given the pivotal role of HLA-signaling (predominantly HLA-G) in establishing the ground work for a healthy TH-1:TH-2 balance by initiating the cytokine network (and thereby, facilitating normal implantation), it occurred to us and to other researchers that sHLA-G secretion by individual early embryos in culture, might provide insight into embryos subsequent potential for implantation and viability. This led us to measure sHLA-G expression in the media surrounding each embryo, 46 hours following forced fertilization by intracytoplasmic sperm injection (ICSI). Since at SIRM (unlike almost all other IVF programs) we culture each embryo in a separate aliquot of culture medium and inseminate all mature eggs by ICSI, we were well positioned to accurately time and measure early embryo sHLA-G expression. We soon came to recognize (and recently published) our observation that the embryo’s sHLA-G expression provides a reliable measure (unavailable through microscopic assessment anywhere) of its subsequent implantation and pregnancy-generating potential. We have since validated these findings through sHLA-G expression of embryos
transferred to more than 500 women. As a result, we currently offer sHLA-G embryo testing (performed bi-coastally) to all SIRM IVF patients.

 

What are the barriers to widespread use of sHLA-G embryo testing?

Since in order to evaluate sHLA-G expression of each embryo, one must of necessity culture each fertilized egg separately, the vast majority of IVF programs who culture embryos in groups or batches would not be able to use this method unless they completely change embryo labs. Moreover, the precise timing for the collection of media surrounding each developing embryo (46 hours post-fertilization), an important step in the testing methodology, is hard to effect unless all oocytes are inseminated by ICSI (which provides for precise timing of fertilization). Since most IVF programs do not perform ICSI uniformly on all oocytes, this would be difficult to do, adding a significant additional impediment to the widespread dissemination of this methodology.

What promise does sHLA-G embryo testing hold for the future?

  • The emergence of an era where a rational basis can be established for the transfer of few, and in many cases of a single "competent" embryo at a time, while maintaining optimal IVF success rates. (We are soon to launch such a study at SIRM where only 1 sHLA-G +ve embryo will be transferred)
  • Measurement of embryo-derived markers such as sHLA-G could help us to better understand and perhaps "interpret" what the embryo is trying to "relate" to the endometrial immune cells.... in the hope of gaining insight in to the subsequent quality of the pregnancy that follows, and
  • An opportunity to test and develop new formulations of fertility drugs as well as individualize the protocols of stimulation.... based on the potential to produce "competent "embryos.

 

Geoffrey Sher, M.D. is the executive medical director for the Sher Institutes for Reproductive Medicine and an executive board member for INCIID. Dr. Sher, founder of SIRM, has been influential in assisting more than 140,000 women have babies following the treatment of infertility in general, and for the births of more than 4,000 IVF babies in specific.

Phone: (702) 892-9696 in Nevada and New York City
Toll Free 800-780-7437
Email: gsher@sherinstitute.com
Website: http://www.haveababy.com

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

Graphis of words associated with immunology

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

 

 

IVIg THERAPY TEMPLATE LETTER

PRE-TREATMENT LETTER OF MEDICAL NECESSITY/REQUEST
AND/OR POST TREATMENT APPEAL

 
Date:
 
To:       Medical Director Name
            Insurance Company Name
            Address

 
RE:       LETTER OF MEDICAL NECESSITY AND REQUEST FOR PRE-AUTHORIZATION
            FOR INTRAVENOUS IMMUNOGLOBULIN THERAPY
 
            Patient Name:
            Patient Policy & Group Numbers:
            Policy Holder Name:

 
Dear ______________________________,
 
           
            The ability to successfully host a pregnancy is largely dependent upon complex immunologic interactions designed to promote orderly accommodation of the invading trophoblast (developing embryo).  Peer-reviewed studies provide compelling evidence that functional failure of these intricate immunologic interactions during implantation lead to infertility, failure of IVF, recurrent miscarriage, and late pregnancy fetal loss, and pirimarily involve  immunologic factors including anti-phospholipid antibodies (APA), anti-thyroid antibodies (ATA), and activated Natural Killer Cells (NKa).  These immunologic markers are measured via blood tests analyzed by specialized reproductive immunology laboratories.
 
            An increased incidence of detectable APA’s has been reported in women with pelvic endometriosis, unexplained infertility and repeated IVF failure (1-5).  Recent evidence strongly suggests that the presence of APA’s in cases of non-male factor infertility resulting in immunologically-associated implantation failure is likely mediated by activation of a sub-population of lymphocytes known as Natural Killer (NK) cells, in particular, CD 56 lymphocytes, that comprise more than 80% of the lymphocyte population in the late secretory and early pregnancy endometrium (6).   NK cells contain / produce a variety of TH-1 cytokines [tumor necrosis factor alpha (TNFa), interferon gamma and interleukins (IL) 1&2] and TH-2 cytokines (IL 3,4,6,7,8,11,12). Excessive release of TH-1 cytokines, particularly TNFa, is cytotoxic to trophoblast and endometrial glandular cells, causing unregulated apoptosis and subsequent failed implantation. Orderly, controlled release of TH-1 cytokines, occurring in association with an appropriate production of TH-2 cytokines, is vital to proper placentation.  This TH1/TH2 homeostasis creates an environment fostering implantation and optimal intrauterine development.
 
Endometrial NK cells are normally predominantly of the CD56+ and CD16- variety. However, in some situations NK cells may become sensitized and express the cell surface marker CD16+. These include:  (1) inappropriate HLA signaling (possibly due to allogeneic compatibility between the conceptus and the maternal organism) and (2) occult or overt organic pelvic disease where the female tests positive for APA, particularly for anti-phosphoethanolamine (PE)/anti-phosposerine (PS) (e.g., endometriosis).  CD56+ and CD16+ NK cells are highly susceptible to activation by TH-1 cytokines such as IL2, transforming them into lymphokine activated killer cells (LAK) which in turn, release large amounts of TH-1 cytokines that threaten implantation.  Because these activated NK cells (NKa) can migrate into the peripheral blood their cytotoxicity can readily be assayed.
 
IVIG is thought to offset or counter the anti-implantation effects associated with APA positivity and NKa because:
 
(1) IVIG is a potent suppressor of NKa;
(2) IVIG contains anti-idiotypic antibodies which counter the effects of harmful APA’s; and
(3) IVIG also suppresses activated T-cells and polyclonal B-cells. Studies suggest that this may explain why IVIG therapy improves reproductive performance in females who test positive for antithyroid antibodies (ATA) (7).
 
IVIG has a profound ability to down-regulate and deactivate endometrial/decidual LAK cells over a period of one to two weeks, therefore, the assay used to measure NK cell activity--the administration of titrated dosages of IVIG to NK cells--determines the amount of IVIG necessary to neutralize NK cell activation.  It has been recently demonstrated that IgG or IgM antibodies to PE or PS in non-male factor infertility cases is often accompanied by increased peripheral NK activity and that IVIG therapy selectively benefits this group of patients (6,8) as well. This suggests that APA’s, rather than being causally related to IVF implantation failure, may act as markers of an underlying abnormality of cellular immunity and shows that appropriate IVIG dosing improves outcome in this patient population. Because the immunologic expression of the fetoplacental unit converts from an atypical Class I (i.e. HLAG) expression to a typical Class I type, it becomes much less susceptible to immunologic injury. This change in HLA antigenicity confers improved immunologic protection to the trophoblast.  Therefore, it is probably unnecessary to continue IVIG therapy beyond the 6th week of gestation, the time at which this conversion occurs.
 
In conclusion, patients undergoing assisted reproduction may experience failed IVF cycles, implantation failure, clinical miscarriages, or other pregnancy wastage on the basis of pathologic immune processes. Clear evidence now exists to support the fact that patients with serologically demonstrable levels of APA’s and NK’s may benefit from immunotherapy in selected cases.  Further, other perturbations of the immune system, including activation of T cells and polyclonal B cells, and ATA’s, if associated with NKa, may represent an additional indication for IVIG treatment (7).
 
            In pursuit of optimizing the outcome of IVF, we have a profound responsibility to make every effort to enhance implantation, and hence the chance of pregnancy.  We believe that it is appropriate for you, as the insurer, to recognize that IVIG therapy for this patient is medically prudent and cost-effective in light of the potential alternative need for repeat treatment and/or third-party assisted reproduction in the event of IVF failure, and to authorize benefits for this patient accordingly.
 

           

Sincerely,
 
 
 
Physician and/or Patient

 

 
REFERENCES:
 
1.   Fisch B., Rikover Y., Shohat L., Zurgil N., Tadir Y., Ovadia J., Wik I., and Yron I.: The relationship between in vitro fertilization and naturally occurring antibodies; evidence for increased production of antiphospholipid antibodies. Fertil. Steril. 56(4), 718-724, 1991.
 
2.   Gleicher N., Liu H.L., Dudkievicz A., Rosenwaks Z., Kaberlien G., Pratt D., et al.: Autoantibody profiles and immunoglobulin levels as predictors of in vitro fertilization success. Am J Obstet. Gynecol. 170:1145-1149, 1994.
 
3.   Birkenfeld A., Mukaida T., Minichiello L.., Jackson M., Kase N.G., Yemini M.: Incidence of      autoimmune antibodies in failed embryo transfer cycles. Am. J Reprod. Immunol.  31:65-68, 1994
 
4.   Kaider B.D., Price D.E., Roussev R.G., Coulam C.B.: Antiphospholipid antibody prevalence in patients with IVF failure. Am. J Reprod. Immunol. 35:383-393, 1996.
 
5.   Bustillo M. Goodman C.: Assisted reproductive technologies and immune infertility. Am. J.      Reprod. Immunol., 35:205-289, 1996.
 
6.   Matzner W. Presentation at Pacific Coast Infertility Meeting, 1998.
 
7.   Sher G., Maassarani G., Zouves C., Feinman M., Sohn S., Matzner W., Chong P., Ching W. The Use of Combined Heparin/Aspirin and Immunoglobulin-G Therapy in the Treatment of In Vitro Fertilization Patients with Antithyroid Antibodies. Amer. J. of Reprod. Immunol. 39: 223-225, 1998.
 
8.   Sher G., Matzner W., Feinman M., Maassarani G., Zouves C., Chong P., Ching W.: The selective use of heparin/aspirin therapy, alone or in combination with intravenous immunoglobulin G, in the management of antiphospholipid antibody-positive women undergoing in vitro fertilization. Am. J of Reprod. Immunol., 40:74-82, 1998.

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