Recurrent Miscarriage - The Immunologic Causes

Immunologic causes of recurrent pregnancy loss are poorly understood. The theories proposed by authorities in this field appear to be constantly evolving and most of the theories that have been proposed to date have been proven to be either incorrect or largely incomplete. Two major categories of immunologic causes of recurrent pregnancy loss are Autoimmune, in which the woman's immune system attacks her own organs and tissues. 
Alloimmune, in which the immune system attacks tissues considered foreign. The immune system is designed to protect oneself against infectious organisms and their toxins. The system identifies, immobilizes and eliminates invaders. The two major mechanisms of surveillance are Nonadaptive immunity, in which cells respond nonspecifically to foreign molecules or material via either phagocytosis and lysis (by macrophages), lysozyme secretion (by lachrymal cells) or cell lysis (by natural killer cells). This type of response does not adapt and so its efficiency is not improved with further exposure . Adaptive immunity, in which action against specific foreign molecules (antigens) is enhanced by re-exposure. This is mediated by lymphocytes which produce highly specific antibodies that bind to the foreign molecules to further elicit (amplify) an immune response. The immune system is constantly operational (turned on) since it must synthesize an enormous catalog of different antibodies and cell surface receptors to deal with the wealth of foreign material that it is presented with.
An important feature of the immune system is its ability to distinguish foreign (unwanted) material from its own (desired) self. If this ability to distinguish non-self from self fails, then the system produces an immune response against itself (or its own tissues). This is called autoimmune disease.
Autoimmune disease or dysfunction may play a role in up to 20% of recurrent pregnancy loss. Phospholipids are molecular building blocks that help to make up a large portion of the walls around the cells of the body, including placental cells. Anti-phospholipid syndrome (APS) is the autoimmune dysfunction that is classically associated with recurrent pregnancy loss.
APS is associated with pregnancy loss in any trimester, placental thrombosis (blood clots), and small placentae. The interruption of the circulation to the fetus via these blood clots is a possible reason for the fetal losses. Identifying the mechanism behind the fetal losses would allow specific treatment to be developed. Clotting mechanisms are difficult to understand without a background in this area. Thrombosis may be caused by a relative deficiency in prostacyclin production within the cells that line the blood vessels (endothelial cells) since prostacyclin is a potent vasodilator and inhibitor of platelet aggregation. Thrombosis may also be caused by a relative insufficiency of the active form of the endogenous anticoagulant protein C, which normally degrades certain clotting factors to limit thrombosis, since phospholipids are required to activate protein C. At this time, the mechanism of thrombosis and fetal loss with APS is largely unknown.
Establishing the diagnosis of APS is important since most of the treatment options involve considerable expense and some added risk. Antiphospholipid antibodies are a large varied group of immunoglobulins directed against several different negatively charged cell surface phospholipids. Many of these phospholipids have been identified, with the best known being cardiolipin. Tests for APS can be divided into coagulation based tests and tests that detect the presence of the antibodies directly.
A group of phospholipid dependent coagulation tests are available (such as the kaolin clotting time, the plasma clotting time, dilute Russell viper venom time, and activated partial thromboplastin time) and serve as popular screening tests for antiphospholipid antibodies. Each of these coagulation tests relies on the activation of a prothrombin activator complex to allow for clot formation. Antiphospholipid antibodies block this activation to delay clot formation, such that in the presence of these antibodies there will be a prolongation of the time required for clotting and an abnormal result for these coagulation tests.
There are several available sensitive and specific assays for anti-cardiolipin antibodies, one of which should be obtained when there is a history of recurrent pregnancy loss. The classic assay for anti-cardiolipin antibodies is the Loizou ELISA, which has been modified over the years. Results that are negative or low positive are generally considered clinically irrelevant and do not require treatment. There are commercial assays for some of the other phospholipids such as phosphatidyl-serine, phosphatidyl-inositol, phosphatidyl-ethanolamine, phosphatidly-choline and phosphatidyl-glycerol. Rather than testing for each phospholipid individually, the more cost efficient test is one that detects a panel (usually all) of these phospholipids (such as an antiphospholipid antibody package). If the panel is positive then more specific detection of specific phospholipids can be considered. Clinically, it is not necessary to test for each of these specific antibodies since the treatment is the same for any of them. Specific testing is most appropriate in a research setting.
To summarize, all couples with recurrent pregnancy loss should be screened for APS. The tests that we routinely order include one of the coagulation tests (aPTT) that relies on the activation of the prothrombin activator complex and which will be appropriately diluted with normal plasma when abnormal,the anti-cardiolipin antibody test (positive in 2-3% of the general population, 7-45% of women with recurrent pregnancy loss- depending on what level is considered abnormal) 
& the lupus anticoagulant test (positive in 1-2% of the general population, 10% of women with recurrent pregnancy loss) We do not routinely order specific anti-phospholipid antibody tests since ourmy management is not altered by the results. Some research centers may order these tests to determine experimental treatment protocols.
APS is classically defined as a triad of recurrent pregnancy loss, thrombosis and autoimmune thrombocytopenia (decreased platelet concentration). For those couples with recurrent pregnancy loss, the positive finding (on 2 separate occasions) of either an appropriately performed coagulation based test or a direct antibody test is generally all that is required to propose treatment. Without treatment, couples with APS have a poor chance of carrying a fetus to term. The worst prognosis appears to occur when there is a prior fetal loss and high anti-cardiolipin antibodies. Treatment options for APS include 1)Low dose aspirin (75-81 mg per day) starting prior to pregnancy. Rationale for this treatment is based on the theory that a relative decrease in prostacyclin is the cause for thrombosis. Aspirin at these low doses has the effect of increasing the prostacyclin to thromboxane (its natural competitor) ratio to enhance the effect of prostacyclin. 
2)Prednisone (30-60 mg per day) to suppress the immune system. This corticosteroid can have several potentially serious complications. When given during pregnancy for this indication, prednisone has been associated with preterm premature rupture of the fetal membranes, preterm delivery and pregnancy-induced hypertension. This medication should only be given by physicians experienced in its use for this indication and typically in a research setting. 
We avoid this approach completely.3)Low molecular weight Heparin (2750 IU s/c per day in the first trimester after fetal viability is seen on ultrasound, continued through to the second trimester). Typically the aPTT test is used to monitor LMW heparin dosing but these test results are abnormal in APS so cannot be used. We generally go by the platelet count (If less than 100,000, we give LMWH on every second or third day). Use of LMW heparin is based on the theory that decreased levels of activated protein C may be responsible for the thrombosis seen, and acts as an anti-coagulant. We have treated over 300 patients using this approach over the last 10 years. 4)Immunoglobulin (Ig) therapy, with intravenous injections of Igs, has been used for several decades in the treatment of immunodeficiency conditions and more recently in the treatment of autoimmune disorders. The mechanism of action is not known, the dosing is not standardized for recurrent pregnancy loss, and this treatment is very expensive. I believe that it is important for this treatment to be administered in a research facility until it is better understood.
Alloimmune dysfunction resulting in recurrent pregnancy loss has also been proposed. Allogeneic antigens are molecular structures that occur in different members of the same species and have the ability to elicit an immune response. Normally, a person will reject dissimilar (non-self) tissues or structures from the body using the immune system. In pregnancy, the placenta and growing embryo are not entirely self but rather are a result of both the maternal and paternal genetic heritages (referred to as a semi-allograft). The placenta (and pregnancy) has a privileged relationship with the pregnant woman that allows it to escape rejection. The mechanism for this privilege is not known.There have been several interesting and complex theories attempting to describe how the normal pregnancy achieves its privileged status in the maternal uterus. Thus far, none of these theories has been generally accepted and proven. Some of the theories are based on
Increased sharing of HLA types (genes encoding antigens that distinguish and mark tissue as self) within the maternal and paternal chromosomes. With increased sharing the placenta may not trigger the production of special blocking antibodies which confer privilege . Decreased numbers of blocking factors that normally allow the placenta to be retained as a privileged site, either due to increased HLA sharing or other factors such as decreased numbers of natural suppresser cells in the uterus, which may control the activity of the natural killer cells and allow for placental survival within the uterus. The diagnosis of alloimmune recurrent pregnancy loss is one of exclusion. That is, when all other tests have been performed and the findings have come back normal then some of those with unexplained losses are thought to fall into this category. Several physicians refuse to treat alloimmune recurrent pregnancy loss since there are no direct diagnostic tests, treatment options are expensive and their benefits are largely unproven, and treatment options potentially involve risk. I think that it is prudent to limit treatment to a research facility with expertise in these therapies. Also, you must consider that there is reportedly up to a 60-70% chance of carrying a pregnancy to term even after 3 spontaneous abortions without treatment. The two main treatment options include 1)Unified leukocyte (white blood cell, WBC) immunization with paternal or donor blood cells, using 200-300 million mononuclear cells from the isolated buffy coat of blood, once the woman is pregnant and prior to 6 weeks gestation on one occasion only . 2)Immunoglobulin (Ig) therapy, with intravenous injections of Igs. The mechanism of action is not known, the dosing is not standardized for recurrent pregnancy loss, and this treatment is very expensive. With treatment, viable pregnancy rates of 70-80% have been reported in uncontrolled studies. In my experience, better candidates for this treatment are couples who have no other treatment options available and are willing to commit themselves to the time, energy (especially emotional) and money required to pursue experimental techniques.

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Do We Really Need To Use Immunosuppressant Therapy For Antiovarian Antibodies?

Over three decades ago researchers observed a high rate of premature ovarian failure associated with poly-endocrine autoimmune disease and suggested that there might be an autoimmune disease of the ovary. At about the same time IVF workers observed auto-antibodies to eggs in the ovary in women with infertility and also suggested there might be an autoimmune disease of the ovary. Since that time, the concept of ovarian autoimmune disease has become more widely recognized but significant advances in our understanding are only just beginning to occur.
Such antibodies would bind to important functional sites in the ovary and granulosa cells and impair the normal response. The anti-ovarian antibodies were found fairly recently, and their complete function is not well known yet. It is believed that these antibodies cause disturbances that are a cause of ovarian failure/non-ovulation or poor ovulation. They are believed also to be a cause of less-than-expected response to various medications to stimulate proper ovarian function, and possibly even formation of less-than-excellent and normal eggs. The treatment of this condition is more or less experimental. Very infrequently, this condition can be helped through in vitro fertilization and if everything else fails an egg donation from a fertile donor is the evidence based-medicine’s answer to this disorder. When such a pregnancy is properly supported by administration of exogenous hormones – progesterone and sometimes even estrogen – it has an excellent chance to lead to normal delivery.
Ovarian autoimmune disease is principally associated with Premature Ovarian Failure (POF) and with unexplained infertility. It is possible that autoimmune unexplained infertility is an early stage of autoimmune POF but this remains to be demonstrated conclusively. POF is the onset of menopause before age 40 and occurs in one to two percent, or about one to two million women. Like natural menopause around age 50, premature menopause is identified by cessation of menstrual cycles for one year, accompanied by elevated follicle stimulating hormone (FSH) and reduced estradiol levels in blood . While some cases of POF have a genetic cause (such as Turner syndrome), or are due to chemotherapy, recent studies suggest that about half the cases of premature menopause are due to an autoimmune attack on ovarian follicles and the eggs (oocytes).
There is still a lack of information on the specific features of autoimmune POF. Some women with POF may have a family history of POF but most do not. However, women with POF have a higher risk for other autoimmune endocrine diseases, such as thyroid disease, Addison disease, type 1 diabetes, and autoimmune poly-glandular syndromes.
Women with infertility and ovarian auto-antibodies tend to have lower than expected estradiol responses to gonadotropin hormone stimulation (i.e. poor responders) and lower pregnancy rates following infertility treatment. Poor responders with ovarian antibodies were younger than poor responders without ovarian antibodies. This suggests that although some poor responses are associated with early stages of the menopause progression and reduced number of functional follicles, others are associated with an autoimmune process.
Ovarian autoimmunity is identified by the presence of anti-ovarian antibodies. There are several different types of tests and consequently different names for these antibodies. Antibodies specifically to eggs and to the zona pellucida (an area surrounding the eggs), or to ovarian cells have been described (9). Some women have antibodies to both ovarian cells and to eggs and others have only one of these antibodies. Ovarian autoimmunity is not identified by traditional tests for ovarian function, such as elevated pituitary follicle stimulating hormone in POF. Although the cause may differ, the symptoms of autoimmune and non-autoimmune POF are the same as in a normal menopause, including hot flashes, dry vaginal tissues, painful sex, infertility, bone loss, and an increased risk of cardiovascular disease. Likewise, autoimmune infertility can only be distinguished from non-autoimmune infertility by specific antibody tests.
As with other endocrine disorders, POF is treated by replacing the lost hormone, in this case hormone replacement therapy (HRT) with estrogen and progesterone to protect the heart, bones, genital and urinary tract tissues, and the nervous system. However, if a woman with infertility or POF wants to become pregnant, treatment with hormones that stimulate ovarian follicles to grow and produce eggs can be tried. If hormone stimulation alone does not result in a pregnancy, women may be treated by more aggressive methods such as in vitro fertilization (IVF). There have been reports of success in combination with low dose immunosuppression. However, success rates are relatively low and there are concerns about the side effects of immunosuppressant therapy. We, at Rotunda do not believe in Immunosuppression with steroids. This sort of approach has not been helpful or useful for any patients in our experience. In some rare cases of POF, follicular function may spontaneously resume, and a pregnancy can occur.
The next significant advance in characterizing ovarian autoimmune disease will be identification of the specific ovarian proteins recognized by the auto antibodies. Once we have tests that detect specific auto-antigens, we will be moving closer to some evidence-based medicine answers. This will permit development of tests based on use of specific antigens, which will improve clinical diagnosis. An understanding of how these proteins become targets of an autoimmune attack would be a significant step toward designing therapies for reversing the effects of this disorder.
As of now, the only known treatment which gives results for this disorder is Donor Egg IVF.

Is Co-culture The Magic Recipe For Improving IVF Implantation Rates?

Although I am writing about co-culture in IVF, we are not currently doing any co-culture. With recent improvements in IVF culture media and techniques, our IVF pregnancy rates (without co-culture) have improved dramatically. Therefore, we no longer see a need for co-culture. In vitro fertilization with co-culture has been utilized in animal in vitro embryo culture systems for over 30 years and more recently in some clinical human in vitro fertilization laboratories as well. The basic concept involves growing embryos in a culture medium on top of a proliferating monolayer of cells such as fallopian tube cells or cells from the lining of the uterus called endometrial cells. The idea is that these cells, which are sometimes referred to as "feeder" cells or "helper" cells, will stimulate development of the embryos by removing toxins from the medium, adding growth factors, or some other beneficial effect. Some studies have demonstrated improved pregnancy rates and delivery rates with utilization of co-culture for human in vitro fertilization.
Why don't all IVF centers use co-culture? There are several reasons that co-culture is not currently more widely used for human IVF:
1. Co-culture involves a lot of tedious work in the laboratory which leads to additional expense.
2. Most IVF labs are not experienced with culture of cells other than eggs, sperm, and embryos. Although culturing cells from the endometrium or fallopian tube is not extremely difficult, it does involve learning some new techniques.
3. There is no universal agreement that co-culture is necessary to provide optimal pregnancy rates from human in vitro fertilization.
4. Another issue is that depending on the source of cells used for the co-culture there may be concerns about transmission of infectious diseases such as viruses from the cell line to the developing embryos. To date there have been no reported cases of viral transmission to a human fetus. Non-autologous cell lines should be screened for infectious diseases prior to use in human embryo coculture.
Coculture is usually not applied universally to all cases in an IVF program. It is usually reserved for use in the "poor prognosis" patients. Studies have suggested that these patients can benefit the most from IVF with co-culture. Examples of poor prognosis patients include women over 40, women with previous IVF failures, women with elevated FSH (follicle stimulating hormone) levels, and women who respond poorly to ovarian stimulation with gonadotropins.
In general there are two schools of thought in this area. One school says that co-culture can be of a benefit for some patients undergoing in vitro fertilization. The philosophy here is that we do not need to know the exact mechanism of the benefit of co-culture, or exactly how standard in vitro culture systems are deficient - what we want is to help the couple get their baby. More like experienced hunches & claims rather than solid evidence based medicine. The other school says co-culture is a crutch that masks the real problem which is sub-optimal in vitro embryo culture systems. These people would prefer to use very pure and carefully defined media in order to maximize the culture environment. They believe that this can yield an equally high pregnancy rate without the use of co-culture. This is what we strongly believe in. I believe that the heart of an IVF clinic are good solid culture systems.
Clinical in vitro fertilization programs that are utilizing co-culture for their human IVF generally use one of three cell types. However, there are numerous other cell lines that have been successfully utilized as well. The cell lines most often used are fallopian tube cells which can be from either human or animal origin, endometrial cells from the lining of the uterus, or Vero cells which are from an immortalized cell line derived from African Green Monkey kidney cells. Cumulus cells from around the egg with or without granulosa cells from the walls of the ovarian follicles where the eggs develop can also be used for co-culture.
Most commonly the eggs and sperm are mixed together on the day of egg retrieval without the co-culture cells. The next morning, after identification of the fertilized eggs (called zygotes), these embryos are then transferred on to the co-culture cells which have been prepared several days in advance. The embryos are then cultured with the helper cells until the time of embryo transfer. This is usually two more days of culture.
Another potential application of co-culture for human in vitro fertilization programs is that of culturing embryos to the blastocyst stage and then performing blastocyst transfer. This allows selection of embryos that have been able to survive through the early cleavage stages of the first five days after fertilization. It is generally very difficult to get good numbers of high quality blastocysts when culturing in defined medium (no co-culture). This technique can allow transfer of fewer embryos while still maintaining an excellent pregnancy rate. For example, some programs have cultured embryos to blastocyst stage and had very good pregnancy rates resulting from transfer of only two blastocysts. This would greatly reduce the risk of high order multiple pregnancy that is seen in some programs transferring higher numbers of embryos.
Further research is needed in order to define exactly which patients would be benefited by co-culture. Also, the co-culture technique itself may be able to be further modified such that in vitro embryonic development is even better than what can be achieved with current technology. For example, many aspects of the co-culture technique could be altered, such as using a different cell line, a different medium, smaller droplets for culture, changing the medium more frequently, or other changes. By varying the usual co-culture techniques, we might obtain a further improvement in embryonic development over what is currently possible.
A practical problem with research in this area is that studies using variations on standard techniques are relatively easy to perform using animal embryos, but studies using human embryos are problematic to set up and implement. Results from co-culture studies done with animal embryos will not necessarily be applicable to IVF with human embryos.
Much has been learned about co-culture both for animal in vitro embryo culture and for in vitro culture in the human as well. Studies continue to attempt to discover exactly how co-culture improves embryonic development. If the cells make certain products that stimulate development of healthier embryos, these products might be able to be produced commercially and added to conventional culture media.
It is possible that pure and exactly defined chemical media might someday be so improved as compared to what is now in use that co-culture would not offer any increase in pregnancy rates, even for poor prognosis patients. However, we do not appear to be at that point today. Further co-culture research is needed before we make tall claims about this being a panacea & a potential Nobel prize winner.

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The First Steps of an In-captivity born pre-term Panda

When the ancestors of human beings had only just learnt how to walk upright, the footprints of the Giant Panda were already all over East and South East Asia. But the peak population of the family of giant pandas has diminished greatly since then. Today, the total population of Pandas wavers at around one thousand, including some 100 animals in captivity. More seriously, in captivity few seem to have the natural desire to mate.
This year, a record number of 16 pandas have been born in captivity, according to China's Panda Breeding Programme. Because pandas in the wild number less than 1000, these sixteen babies are a significant addition to this endangered species population.This year the Wolong Chinese Giant Panda Protection Center has been successful in breeding two infants in total. It may seem like a small number, but around the world only 50 pandas are born every year and out of this number only 20 have a chance to grow up. Scientists are now investigating ways to get pandas bred in captivity back to where they belong - the wild. It is this that will complete the work of the researchers at the Wolong Chinese Panda Protection Center. Work which is ensuring the survival of the giant panda.
I recently came across these series of pictures of a pre-term Panda baby which would have made a baby-album proud! Pictures speak louder than a 1000 words & I will let you enjoy these from the pre-term delivery to 120 days of life...