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Immunology Testing
Introduction
The most common reason why IVF does not work is the failure of an embryo to implant. Also, up to two-thirds of early pregnancies miscarry. Although chromosomal abnormalities in embryos is a major factor in this, another significant cause of these problems may relate to abnormalities in the immune system which compromise Research has suggested that during a normal pregnancy, a unique type of immunity occurs that stops a woman rejecting an embryo. If this immunity does not exist embryos may not implant, early pregnancies may miscarry or later complications may occur to the mother or baby. Special tests may identify couples who are at risk Treatment that stimulates the proper immune response (immuno-modulation) in the mother may then improve the chances of a successful pregnancy. However, this research is stil in the experimental stages and some of the possible treatments (immuno-modulation) are not universal y accepted. Further information can be found the Royal Col ege of Obstetricians and Gynaecologists “Immunological Testing and Interventions for Reproductive Failure”.
Here at CRGW we are committed to reviewing the available evidence and to provide clear information about the treatments, their efficacy and safety to enable patients to It is important to note that immunological factors are only one of a number of issues which may contribute to recurrent treatment failure +/- miscarriage.
Recognised causes of recurrent failure/pregnancy loss include: Indications for Immune Testing
The fol owing couples may be at increased risk of immune problems and might wish  Women over the age of 35 who have had two miscarriages or two failed IVF or  Women under the age of 35 who have had three miscarriages or three failed IVF  Poor egg production from a stimulated IVF/GIFT cycle (less than 6 eggs);  Unexplained infertility of over 3 years;  Previous immune problems (Anti-nuclear antibody test {ANA} positive,  Previous pregnancies that have resulted in smal babies (fetal growth retardation)  One living child and repeated miscarriages while attempting to have a second Categories of Immune Problems
Listed below are four categories of immune problems that may be associated with pregnancy loss, IVF failures and infertility.
Class I
Women in this category have altered systemic inflammatory immune responses demonstrated by increased T helper 1 / T helper 2 balance [also cal ed TH1:TH2 ratio abnormalities, or Cytokine ratio abnormalities] and/or increased natural kil er cel numbers or activity. Possible treatment includes steroids, low dose aspirin, Heparin and Intralipid intravenous infusion. Intravenous immunoglobulin(IVIg) or Humira may be used in women al ergic to egg or soya.
Class II
Women in this category have blood clotting tendencies due to a either a genetic mutation or other reasons including the presence of antiphospholipid antibodies.
Treatment includes low dose aspirin and Heparin for most genetic clotting tendencies [the exception being that caused by the MTHFR mutation which requires treatment with activated folic acid – Metafolin]. Patients with antiphospholipid Class III
Women in this category have autoimmunity to fatty molecules, nuclear antigen, thyroid molecules or other tissues. Some of these patients have clinical manifestation of early mixed connective tissue disorder. Treatment may include steroids, low dose aspirin and Heparin. Patients with thyroid autoantibodies also require stabilisation and monitoring of thyroid function during treatment and Class IV
Women in this category have a lack of appropriate immune response to a pregnancy due to close genetic compatibility to their partner which can be identified through ‘DQ alpha matching’ of the couple. This may be treated with steroids, low dose aspirin, Heparin & Intralipid infusions. Some authorities recommend Lymphocyte Immune Therapy (LIT) for this. This is a very controversial treatment and we do not offer it at Tests Available for Immunology Testing
 Natural Kil er Cel (NK cel ) assay panel Other Tests which may be advised for Recurrent Treatment Failure/Pregnancy
General Blood tests
 Ful Blood Count, Urea &Electrolytes, Liver function tests, fasting glucose  Thyroid function tests (free T4, TSH) Genetic Blood tests
Thrombophilia & Immune Screen Blood tests
 Autoantibody screen (including Antinuclear and Thyroid  Coagulation profile, Antithrombin III, Factor V Leiden, Factor II Prothrombin Gene Mutation, Activated Protein C Resistance, Protein C & Protein S, Lupus Anatomical
 Ultrasound to exclude hydrosalpinx and major uterine anomaly, fibroids or  Consider Hysteroscopy if history of uterine surgery Sperm Tests
Treatments for Immune problems
It is important to understand that these treatments remain experimental and
that some of these drugs are not licensed for the immune treatment of
infertility or for use in pregnancy. There are potentially serious adverse
reactions to some of the medication, eg. IVIg or Humira (though these are no
longer used routinely) if not used with due care under strict supervision and
they can also be very expensive.
1. Aspirin Therapy
Low dose aspirin (75mg/day) is often used in women with antiphospholipid antibody syndrome, recurrent pregnancy losses or infertility caused by immunity. Low dose aspirin is prescribed alone or combined with heparin or steroid treatment. Aspirin 75mg tablets can be purchased over the counter and do not require a prescription.
Side Effects
The possible side effects of ful dose aspirin are not often seen with low dose aspirin treatment. These side effects are nausea, heartburn, upset stomach, decreased appetite and microscopic amounts of blood in stools. On very rare occasions, al ergic reactions have been observed fol owing aspirin ingestion. If you have any history of aspirin sensitivity, please inform your nurse and your doctor. The above-mentioned side effects are mainly experienced in patients taking a normal adult dose or high dose of aspirin therapy. Low dose aspirin treatment is reported to have minimal, if Aspirin intolerance manifested by exacerbation of asthma (bronchospasm) and rhinitis may occur in a patient with a history of nasal polyps, asthma, al ergic skin reactions or rhinitis. If you have any past history of any of the above, please notify us Enteric coated aspirin is also available for women with a history of gastrointestinal side effect of plain aspirin or condition requiring chronic or long-termaspirin therapy Interaction
When you start to take low dose aspirin, moderation in taking the fol owing food is Curry powder, paprika, liquorice, prunes, raisins, gherkins, tea and other than the occasional use of antacids. Phenobarbitone decreases aspirin efficacy.
Usage During Pregnancy
The use of aspirin during pregnancy, especial y chronic or intermittent high doses, may affect the maternal and baby’s blood clotting mechanisms, leading to an increased risk of bleeding. High dose aspirin may be related to increased perinatal mortality, intrauterine growth retardation, and congenital defects. Recent evidence suggests that aspirin may increase the relative risk of early miscarriage and for this reason patients are now advised to stop low dose aspirin just before embryo transfer and not to take in early pregnancy. Patients on short term Aspirin having an egg col ection should discontinue Aspirin 2 days before egg col ection. Patients on long term Aspirin therapy for other medical reasons who are having an egg col ection should discontinue Aspirin at least 3 days before egg col ection as significant bleeding has been reported if Aspirin is not discontinued.
2. Heparin Therapy
Low molecular weight heparin is often used in treatment for women with inherited thrombophilia –clotting problems - with the presence of factor V (Leiden) mutation or abnormalities in Protein C or S. It is also used empirical y as a “suppressor” of the immune and clotting systems. Several commercial preparations are available, eg.
Fragmin and Clexane. It is known that regular heparin (or high molecular weight heparin) does not cross the placenta in pregnant women. This seems to be the same a) Clexane
Clexane is a low molecular weight heparin. Clexane wil usual y be prescribed as 20 mg or 40mg, subcutaneously, once daily.
b) Fragmin
Fragmin is a low molecular weight heparin. Fragmin is often prescribed as 2500 IU subcutaneously daily. Dosage and injection frequency can be changed based on the Contraindication to low molecular weight heparin
Patients with fol owing concerns should not use Clexane/Fragmin injections:  Thrombocytopenia (decrease in the number of platelets)  Hypersensitivity to heparin or pork products Warnings for low molecular weight heparin
 Clexane and Fragmin are not intended for intramuscular administration  Clexane and Fragmin cannot be exchangeable with heparin or other low  In patients with a history of low platelet count either induced by heparin or other reason it should be used with extreme caution  As with other anticoagulants, there have been rare cases of neuraxial hematoma reported with the concurrent use of Clexane and spinal/epidural Common Side Effects
Mild local irritation, pain, bruising, ecchymoses (smal purple skin patch) and erythema (redness, flush, of skin) may occur at the injection site. Osteoporosis after prolonged use, hair loss (very rarely).
Calcium supplementation
To minimise the bone thinning effect of heparin, we advise patients taking Fragmin/Clexane to use a calcium supplement (500mg tablet, twice a day). These can be purchased over the counter and do not require a prescription.
Monitoring
Periodic ful blood count and platelet count should be considered for long-term Important Points To Remember
1 Preferred site of injection is the abdominal area. Injections must be given 2
inches away from umbilicus (bel y button). If you need another area to give your heparin, you may use your thighs or buttocks, but this is only if there is no other  2 Rotate your sites of injections. Never inject in the same place as a previous
3 Some bruising at the site of injection is normal (less than 2p size). If increased
bruising occurs, you may use ice before you clean the area for injection and/or after you have given yourself the injection.
4 Notify your doctors that you are taking heparin before any medication or
5 Contact your doctor if any of the fol owing symptoms occur:
 Excessive bleeding lasting greater than 15 minutes and not control ed by direct  Unusual bruising not at the site of injection.
Possible Side Effects
 Local irritation – redness, mild pain, and itching at site of injection  Nausea and vomiting, chil and fever (rare) 3. Steroid Treatment
Indications:
a) Immune suppression
Prednisolone and dexamethasone are two commonly prescribed steroids.
Prednisolone is prescribed to suppress abnormal autoimmunity such as ANA and autoantibodies to DNA and/or histones.
Prednisolone treatment can be combined with aspirin or heparin, or both.
b) Poor responders
Dexamethasone is also used in the stimulation phase of an IVF cycle to try to Possible Side Effects
The principal complications resulting from prolonged therapy with steroids are fluid and electrolyte disturbances, hyperglycemia, glycosuria (abnormal amounts of glucose in urine), increased susceptibility to infection, peptic ulcer, osteoporosis, behavioural disturbance, e.g. nervousness, insomnia, changes in mood, cataracts, and striae (skin stretch marks). Cushingoid features consisting of moon face, buffalo pads, central obesity, ecchymosis, acne, and hirsuitism (hair growth) can occur.
Your features wil return to normal fol owing cessation of steroids.
Diet Restriction
Average and large doses of prednisolone can cause elevation of blood pressure, salt and water retention and increased excretion of potassium and calcium. Dietary salt restriction, potassium supplementation and regular blood pressure monitoring is advised is steroids are used in high dose for long periods. Only moderate doses are, however, used in reproductive immune therapy.
Diabetes
Prednisolone can induce diabetic tendency. If prednisolone is indicated, your blood sugar level wil need to be monitored, especial y during pregnancy.
Other Cautions
Prednisolone should be used with caution if you have ulcerative colitis, abscess or other pyogenic (pus forming) infection, diverticulitis, peptic ulcer, hypertension, congestive heart failure, history of blood clots, osteoporosis, Cushing syndrome or convulsive disorder. Osteoporosis can be prevented by calcium supplementation, which is reported to suppress bone resorption without detectable suppression of indices of bone formation in steroid treated patients.
Notify your doctor that you are taking steroids before taking any medication or having a surgical procedure and carry an identification card in your wal et stating that you Withdrawal
Too rapid withdrawal of prednisolone during the weaning process may cause anorexia, dyspnea (laboured breathing), hypotension, hypoglycemia, myalgia, fever, malaise, arthralgia, dizziness, sloughing off of skin and fainting. If you have these problems, contact us at CRGW immediately. For patients on prednisolone we advise a gradual step-wise reduction by 5mg every 3 days, ending with 5mg every other day for 3 doses.
There are a number of studies in which pregnant patients received prednisolone and have shown little, if any effect on the developing foetus. The drug does not cross the 4. Intravenous Intralipid 20% Solution Infusion Treatment
Evidence from both animal and human studies suggests that intravenous Intralipid administration may enhance embryo implantation. Although the exact mechanism of this beneficial action has not been completely elucidated, it has been suggested that Intralipid stimulates the immune system to remove “danger signals” that can lead to pregnancy loss. Also, recent evidence has confirmed the ability of Intralipid to suppress Natural Kil er (NK) cytotoxicity for a sufficient duration of time to enhance implantation and maintain pregnancy. It has also been shown to be the most effective treatment to correct Th1/TH2 abnormalities.
Clinical studies using Intralipid have shown improved pregnancy and live birth rates in recurrent embryo implantation failure\miscarriage after IVF in women with elevated NK activity and Th1/Th2 abnormalities. Studies comparing IVIg, Intralipid and sHLA- G confirmed they al suppressed NK cel cytotoxicity with equal efficacy.
However, Intralipid has the advantage that it is relatively inexpensive and it is not a Intralipid treatment is not licensed for use in reproductive failure or pregnancy and is Nature and Duration of Procedure
Treatment is given intravenously and dosage and infusion protocols are determined based on laboratory testing and clinical responses. Treatment is usual y given while you are attempting to achieve a pregnancy and during a pregnancy. The infusion is given as a drip in the arm and is administered at CRGW. The procedure takes a Potential Risks - Patients al ergic to eggs or soya bean oil MUST NEVER have
Intralipid.
Side effects are otherwise rare and include febrile episodes (fever) and less frequently shivering, chil s and nausea/vomiting (less than 1%). The infusion should be discontinued in such cases. Other adverse effects are extremely rare, occurring in less than 1 in 1 mil ion infusions e.g. hypersensitivity reactions (anaphylaxis, skin rash & urticaria), respiratory symptoms (rapid breathing) circulatory effects (high or low blood pressure), abdominal pain, headache and tiredness. These side effects are mainly seen in patients having Intralipid daily for intravenous feeding. The safety profile for Intralipid is regarded as extremely good.
5 Immunoglobulin G (IVIg) Infusion Treatment – NOT AVAILABLE AT CRGW
IVIg infusion treatment alone or in conjunction with conventional immuno-modulation such as anticoagulation or immunosuppression may be indicated in some women with recurrent spontaneous abortions or infertility caused by immune problems who are al ergic to egg or soya and, therefore cannot have Intralipid.
IVIg consists of concentrated human immunoglobulins, primarily IgG (Immunoglobulin G), prepared from pooled human donors (8,000-13,000 donors per lot), screened free of blood-borne disorders.
Dosage and infusion protocol wil be determined based on laboratory testing and Before IVIg can be given, all patients must have a blood test to check an
Immunoglobulin Panel to rule out deficiency in Immunoglobulin A. This is
because in such women there is a risk of severe reaction.
Side Effects
Side effects to IVIg infusion tend to be related to the rate of infusion. Possible side effects include malaise, a feeling of faintness, fever, chil s, headache, nausea, and vomiting. Shortness of breath, chest tightness, thrombosis and joint pains have also Viral Safety
No cases of human immunodeficiency virus transmission have been related to the administration of IVIg. Hepatitis B and C virus transmission have been reported in However, there are stil concerns about possible viral transmission. It is recommended to use only IVIg products that have been prepared with an additional Hypersensitivity
Anaphylactic reactions may occur during IVIg treatment in patients with IgA (Immunoglobulin A)deficiency. Before IVIg infusion, serum IgA level should be checked. Patients with IgA deficiency need further workup before IVIg infusion.
6. Adalimumab (Humira)- NOT AVAILABLE AT CRGW
Humira belongs to a new class of drugs that block the effects of the products of NK cel s (tumour necrosis factor-alpha – TNF-a). It is only used in women with immune abnormalities causing recurrent IVF failure or miscarriage, who are al ergic to egg or soya and, therefore, cannot have Intralipids. It is licensed for the treatment of patients with rheumatoid arthritis or Crohn’s disease. Recent work done at the Chicago Medical School indicates that it may be useful in the treatment of recurrent miscarriage and recurrent embryo implantation failure. Although it is not licensed for this purpose, it can be used after proper investigation if there is a clear indication for use. This drug wil be used preceding fertility treatment or pregnancy cycle only. The aim is simply to bring TNF – α levels back to normal prior to treatment or pregnancy, thereby reducing any potential adverse effects from the raised levels.
We do not advise its use in pregnancy. Treatment with anti TNFa drugs should not be initiated in patients with active infections, including tuberculosis, chronic or localised infections until infections are control ed.
Before starting Humira patients must rule out Tuberculosis by TD Gold test.
TNF-α may be important in immunological defence against cancer. There is, however, no clinical evidence that anti TNF-a drugs increase the risk of cancer.
Administration of Humira (Adalimumab)
Humira comes as a single dose (40mg), pre-fil ed syringe and is given as a subcutaneous injection once every two weeks. Complete elimination of Humira may take up to five months. Patients usual yneed 2 to 4 doses, dependent on the severity Side Effects
In placebo-control ed studies 20% of patients treated with Humira developed injection site reactions (redness and/or itching, bleeding, pain or swel ing) compared to 14% who received placebo. The incidence of serious infections was extremely low. The infections were primarily upper respiratory tract infections, bronchitis and urinary tract infections. In clinical trials with Humira for up to 53 months no increase in rates and incidences of malignancies were observed. After 24 weeks of Humira 12.6% of patients with negative anti-nuclear antibodies tested positive compared with 7.3% of placebo-treated patients. The significance of this is unclear. A drug- induced lupus-like syndrome may occur and the drug must be discontinued in that case. Although developmental toxicity study in monkeys showed no maternal toxicity, no toxicity to embryos and no developmental abnormalities, we do not advise the use of Humira in pregnancy.
In up to 20% of patients, elevated TNF alpha levels may not be successfully
reduced with Humira.
Infections
Severe infections, sepsis and re-activation of tuberculosis have been reported with TNF-α blockers. These drugs should not be started and should be discontinued if the patient develops serious infection. These drugs are used cautiously in patients with a history of recurrent infections or medical condition that predisposes to infections. Live vaccines should not be given concurrently with these drugs. The recommendation is that al patients should be screened for tuberculosis before starting the drugs. If symptoms develop suggesting tuberculosis, e.g. persistent cough, weight loss and low-grade fever, medical advice should be sought. Also, because of this slow elimination of the drugs, infections should be reported while on the drugs and for at least 6 months afterwards. Severe leukopenia (reduction of white blood cel s), pancytopenia (deficiency of al blood cel s) and aplastic anaemia and onset of demyelinating diseases of the central nervous system including multiple sclerosis and optic neuritis have been rarely associated with some anti TNF alpha drugs. The drug should be used with great caution in patients with a history of these disorders or discontinued if the problems occur.

Source: http://crgw.co.uk/userfiles/file/PI%201_42%20Immunology%20Testing.pdf

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