Immunology testing

PI- Immunology V1 Feb 2010 V1 AOL Page 1

Immunology Testing

IntroductionThe most common reason why IVF does not work is the failure of an embryo toimplant. Also, up to two-thirds of early pregnancies miscarry. Although chromosomalabnormalities in embryos is a major factor in this, another significant cause of theseproblems may relate to abnormalities in the immune system which compromisesuccessful embryo implantation.

Research has suggested that during a normal pregnancy, a unique type of immunityoccurs that stops a woman rejecting an embryo. If this immunity does not existembryos may not implant, early pregnancies may miscarry or later complicationsmay occur to the mother or baby. Special tests may identify couples who are at riskof these problems.

Treatment that stimulates the proper immune response (immuno-modulation) in themother may then improve the chances of a successful pregnancy. However, thisresearch is still in the experimental stages and some of the possible treatments(immuno-modulation) are not universally accepted. Further information can be foundthe Royal College of Obstetricians and Gynaecologists “Immunological Testing andInterventions for Reproductive Failure”.

Here at CRGW we are committed to reviewing the available evidence and to provideclear information about the treatments, their efficacy and safety to enable patients toreach informed decisions.

It is important to note that immunological factors are only one of a number of issueswhich may contribute to recurrent treatment failure +/- miscarriage.

Recognised causes of recurrent failure/pregnancy loss include:

Genetic causes Endocrine (hormonal) causes Anatomical causes Immunological causes Miscellaneous causes


Indications for Immune TestingThe following couples may be at increased risk of immune problems and might wishto consider immune testing: -

Women over the age of 35 who have had two miscarriages or two failed IVF orGIFT cycles;

Women under the age of 35 who have had three miscarriages or three failed IVFor GIFT

cycles; Poor egg production from a stimulated IVF/GIFT cycle (less than 6 eggs); One blighted ovum (missed abortion); Unexplained infertility of over 3 years; Previous immune problems (Anti-nuclear antibody test {ANA} positive,

rheumatoid arthritis, and/or lupus) Previous pregnancies that have resulted in small babies (fetal growth retardation) One living child and repeated miscarriages while attempting to have a second

child.

Categories of Immune ProblemsListed below are four categories of immune problems that may be associated withpregnancy loss, IVF failures and infertility.

Class IWomen in this category have altered systemic inflammatory immune responsesdemonstrated by increased T helper 1 / T helper 2 balance [also called TH1:TH2ratio abnormalities, or Cytokine ratio abnormalities] and/or increased natural killercell numbers or activity. Possible treatment includes steroids, low dose aspirin,Heparin and Intralipid intravenous infusion. Intravenous immunoglobulin(IVIg) orHumira may be used in women allergic to egg or soya.

Class IIWomen in this category have blood clotting tendencies due to a either a geneticmutation or other reasons including the presence of antiphospholipid antibodies.Treatment includes low dose aspirin and Heparin for most genetic clottingtendencies [the exception being that caused by the MTHFR mutation which requirestreatment with activated folic acid – Metafolin]. Patients with antiphospholipidantibodies also require steroids.

Class IIIWomen in this category have autoimmunity to fatty molecules, nuclear antigen,thyroid molecules or other tissues. Some of these patients have clinicalmanifestation of early mixed connective tissue disorder. Treatment may include


steroids, low dose aspirin and Heparin. Patients with thyroid autoantibodies alsorequire stabilisation and monitoring of thyroid function during treatment andpregnancy.

Class IVWomen in this category have a lack of appropriate immune response to a pregnancydue to close genetic compatibility to their partner which can be identified through ‘DQalpha matching’ of the couple. This may be treated with steroids, low dose aspirin,Heparin & Intralipid infusions. Some authorities recommend Lymphocyte ImmuneTherapy (LIT) for this. This is a very controversial treatment and we do not offer it atCRGW.

Tests Available for Immunology Testing

TH1:TH2- cytokine ratio Natural Killer Cell (NK cell) assay panel Leucocyte antibody Detection (LAD)

DQ alpha and beta antigen

Endometrial Biopsy for NK cells

Other Tests which may be advised for Recurrent Treatment Failure/PregnancyLoss

General Blood tests Full Blood Count, Urea &Electrolytes, Liver function tests, fasting glucose Thyroid function tests (free T4, TSH)

Genetic Blood tests Karyotype

Thrombophilia & Immune Screen Blood tests Cardiolipin Antibodies (IgG, IgM) Autoantibody screen (including Antinuclear and Thyroid

Peroxidase Antibodies) Coagulation profile, Antithrombin III, Factor V Leiden, Factor II Prothrombin

Gene Mutation, Activated Protein C Resistance, Protein C & Protein S, Lupusanticoagulant,

MTHFR gene, PAI – 1 mutation

Anatomical Ultrasound to exclude hydrosalpinx and major uterine anomaly, fibroids or

polyps Consider Hysteroscopy if history of uterine surgery


Sperm Tests Sperm DNA Fragmentation Index (DFI)

Genetic Karyotype Blood test Consider Sperm Aneuploidy

Treatments for Immune problems

It is important to understand that these treatments remain experimental andthat some of these drugs are not licensed for the immune treatment ofinfertility or for use in pregnancy. There are potentially serious adversereactions to some of the medication, eg. IVIg or Humira (though these are nolonger used routinely) if not used with due care under strict supervision andthey can also be very expensive.

1. Aspirin TherapyLow dose aspirin (75mg/day) is often used in women with antiphospholipid antibodysyndrome, recurrent pregnancy losses or infertility caused by immunity. Low doseaspirin is prescribed alone or combined with heparin or steroid treatment. Aspirin75mg tablets can be purchased over the counter and do not require a prescription.

Side EffectsThe possible side effects of full dose aspirin are not often seen with low dose aspirintreatment. These side effects are nausea, heartburn, upset stomach, decreasedappetite and microscopic amounts of blood in stools. On very rare occasions, allergicreactions have been observed following aspirin ingestion. If you have any history ofaspirin sensitivity, please inform your nurse and your doctor. The above-mentionedside effects are mainly experienced in patients taking a normal adult dose or highdose of aspirin therapy. Low dose aspirin treatment is reported to have minimal, ifany, side effects.

Aspirin intolerance manifested by exacerbation of asthma (bronchospasm) andrhinitis may occur in a patient with a history of nasal polyps, asthma, allergic skinreactions or rhinitis. If you have any past history of any of the above, please notify usbefore starting aspirin.Enteric coated aspirin is also available for women with a history of gastrointestinalside effect of plain aspirin or condition requiring chronic or long-termaspirin therapy

InteractionWhen you start to take low dose aspirin, moderation in taking the following food isrecommended.Curry powder, paprika, liquorice, prunes, raisins, gherkins, tea and other than theoccasional use of antacids. Phenobarbitone decreases aspirin efficacy.

Usage During Pregnancy


The use of aspirin during pregnancy, especially chronic or intermittent high doses,may affect the maternal and baby’s blood clotting mechanisms, leading to anincreased risk of bleeding. High dose aspirin may be related to increased perinatalmortality, intrauterine growth retardation, and congenital defects. Recent evidencesuggests that aspirin may increase the relative risk of early miscarriage and for thisreason patients are now advised to stop low dose aspirin just before embryo transferand not to take in early pregnancy. Patients on short term Aspirin having an eggcollection should discontinue Aspirin 2 days before egg collection. Patients on longterm Aspirin therapy for other medical reasons who are having an egg collectionshould discontinue Aspirin at least 3 days before egg collection as significantbleeding has been reported if Aspirin is not discontinued.

2. Heparin TherapyLow molecular weight heparin is often used in treatment for women with inheritedthrombophilia –clotting problems - with the presence of factor V (Leiden) mutation orabnormalities in Protein C or S. It is also used empirically as a “suppressor” of theimmune and clotting systems. Several commercial preparations are available, eg.Fragmin and Clexane. It is known that regular heparin (or high molecular weightheparin) does not cross the placenta in pregnant women. This seems to be the samefor low molecular heparin.

a) ClexaneClexane is a low molecular weight heparin. Clexane will usually be prescribed as 20mg or 40mg, subcutaneously, once daily.

b) FragminFragmin is a low molecular weight heparin. Fragmin is often prescribed as 2500 IUor 5000 IU,subcutaneously daily. Dosage and injection frequency can be changed based on thepatients’ need.

Contraindication to low molecular weight heparinPatients with following concerns should not use Clexane/Fragmin injections: Known hypersensitivity Active bleeding Thrombocytopenia (decrease in the number of platelets) Hypersensitivity to heparin or pork products Severe hypertension Osteoporosis

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

molecular weight heparin 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 neuraxialhematoma reported with the concurrent use of Clexane and spinal/epiduralanaesthesia resulting in paralysis

Common Side EffectsMild local irritation, pain, bruising, ecchymoses (small purple skin patch) anderythema (redness, flush, of skin) may occur at the injection site. Osteoporosis afterprolonged use, hair loss (very rarely).

Calcium supplementationTo minimise the bone thinning effect of heparin, we advise patients takingFragmin/Clexane to use a calcium supplement (500mg tablet, twice a day). Thesecan be purchased over the counter and do not require a prescription.

MonitoringPeriodic full blood count and platelet count should be considered for long-termusage.

Important Points To Remember 1 Preferred site of injection is the abdominal area. Injections must be given 2

inches away from umbilicus (belly button). If you need another area to give yourheparin, you may use your thighs or buttocks, but this is only if there is no otherplace in your abdominal area.

2 Rotate your sites of injections. Never inject in the same place as a previousinjection or in a bruised area.

3 Some bruising at the site of injection is normal (less than 2p size). If increasedbruising occurs, you may use ice before you clean the area for injection and/orafter you have given yourself the injection.

4 Notify your doctors that you are taking heparin before any medication orsurgical procedure.

5 Contact your doctor if any of the following symptoms occur: Nose Bleeds Blood in the urine or stool Excessive bleeding lasting greater than 15 minutes and not controlled by direct

pressure. Unusual bruising not at the site of injection.

Possible Side Effects Bleeding Local irritation – redness, mild pain, and itching at site of injection Nausea and vomiting, chill and fever (rare)

3. Steroid Treatment

Indications:


a) Immune suppressionPrednisolone and dexamethasone are two commonly prescribed steroids.Prednisolone is prescribed to suppress abnormal autoimmunity such as ANA andautoantibodies to DNA and/or histones.Prednisolone treatment can be combined with aspirin or heparin, or both.

b) Poor respondersDexamethasone is also used in the stimulation phase of an IVF cycle to try toincrease egg numbers.

Possible Side EffectsThe principal complications resulting from prolonged therapy with steroids are fluidand electrolyte disturbances, hyperglycemia, glycosuria (abnormal amounts ofglucose 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, buffalopads, central obesity, ecchymosis, acne, and hirsuitism (hair growth) can occur.Your features will return to normal following cessation of steroids.

Diet RestrictionAverage and large doses of prednisolone can cause elevation of blood pressure, saltand water retention and increased excretion of potassium and calcium. Dietary saltrestriction, potassium supplementation and regular blood pressure monitoring isadvised is steroids are used in high dose for long periods. Only moderate doses are,however, used in reproductive immune therapy.

DiabetesPrednisolone can induce diabetic tendency. If prednisolone is indicated, your bloodsugar level will need to be monitored, especially during pregnancy.

Other CautionsPrednisolone should be used with caution if you have ulcerative colitis, abscess orother pyogenic (pus forming) infection, diverticulitis, peptic ulcer, hypertension,congestive heart failure, history of blood clots, osteoporosis, Cushing syndrome orconvulsive disorder. Osteoporosis can be prevented by calcium supplementation,which is reported to suppress bone resorption without detectable suppression ofindices of bone formation in steroid treated patients.Notify your doctor that you are taking steroids before taking any medication or havinga surgical procedure and carry an identification card in your wallet stating that youare on steroids.

WithdrawalToo rapid withdrawal of prednisolone during the weaning process may causenausea, fatigue, anorexia, dyspnea (laboured breathing), hypotension,hypoglycemia, myalgia, fever, malaise, arthralgia, dizziness, sloughing off of skinand fainting. If you have these problems, contact us at CRGW immediately. Forpatients on prednisolone we advise a gradual step-wise reduction by 5mg every 3days, ending with 5mg every other day for 3 doses.


There are a number of studies in which pregnant patients received prednisolone andhave shown little, if any effect on the developing foetus. The drug does not cross theplacenta to the foetus.

4. Intravenous Intralipid 20% Solution Infusion TreatmentEvidence from both animal and human studies suggests that intravenous Intralipidadministration may enhance embryo implantation. Although the exact mechanism ofthis beneficial action has not been completely elucidated, it has been suggested thatIntralipid stimulates the immune system to remove “danger signals” that can lead topregnancy loss. Also, recent evidence has confirmed the ability of Intralipid tosuppress Natural Killer (NK) cytotoxicity for a sufficient duration of time to enhanceimplantation and maintain pregnancy. It has also been shown to be the mosteffective treatment to correct Th1/TH2 abnormalities.

Clinical studies using Intralipid have shown improved pregnancy and live birth ratesin recurrent embryo implantation failure\miscarriage after IVF in women with elevatedNK activity and Th1/Th2 abnormalities. Studies comparing IVIg, Intralipid and sHLA-G confirmed they all suppressed NK cell cytotoxicity with equal efficacy.However, Intralipid has the advantage that it is relatively inexpensive and it is not ablood product.Intralipid treatment is not licensed for use in reproductive failure or pregnancy and isconsidered investigational.

Nature and Duration of ProcedureTreatment is given intravenously and dosage and infusion protocols are determinedbased on laboratory testing and clinical responses. Treatment is usually given whileyou are attempting to achieve a pregnancy and during a pregnancy. The infusion isgiven as a drip in the arm and is administered at CRGW. The procedure takes aminimum of two hours.

Potential Risks - Patients allergic to eggs or soya bean oil MUST NEVER haveIntralipid.

Side effects are otherwise rare and include febrile episodes (fever) and lessfrequently shivering, chills and nausea/vomiting (less than 1%). The infusion shouldbe discontinued in such cases. Other adverse effects are extremely rare, occurring inless than 1 in 1 million infusions e.g. hypersensitivity reactions (anaphylaxis, skinrash & urticaria), respiratory symptoms (rapid breathing) circulatory effects (high orlow blood pressure), abdominal pain, headache and tiredness. These side effectsare mainly seen in patients having Intralipid daily for intravenous feeding. The safetyprofile 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-modulationsuch as anticoagulation or immunosuppression may be indicated in some women


with recurrent spontaneous abortions or infertility caused by immune problems whoare allergic 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 perlot), screened free of blood-borne disorders.Dosage and infusion protocol will be determined based on laboratory testing andclinical responses.

Before IVIg can be given, all patients must have a blood test to check anImmunoglobulin Panel to rule out deficiency in Immunoglobulin A. This isbecause in such women there is a risk of severe reaction.

Side EffectsSide effects to IVIg infusion tend to be related to the rate of infusion. Possible sideeffects include malaise, a feeling of faintness, fever, chills, headache, nausea, andvomiting. Shortness of breath, chest tightness, thrombosis and joint pains have alsorarely been reported.

Viral SafetyNo cases of human immunodeficiency virus transmission have been related to theadministration of IVIg. Hepatitis B and C virus transmission have been reported inIVIg improperly prepared.However, there are still concerns about possible viral transmission. It isrecommended to use only IVIg products that have been prepared with an additionalviral inactivation procedure.

HypersensitivityAnaphylactic reactions may occur during IVIg treatment in patients with IgA(Immunoglobulin A)deficiency. Before IVIg infusion, serum IgA level should bechecked. 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 NKcells (tumour necrosis factor-alpha – TNF-a). It is only used in women with immuneabnormalities causing recurrent IVF failure or miscarriage, who are allergic to egg orsoya and, therefore, cannot have Intralipids. It is licensed for the treatment ofpatients with rheumatoid arthritis or Crohn’s disease. Recent work done at theChicago Medical School indicates that it may be useful in the treatment of recurrentmiscarriage and recurrent embryo implantation failure. Although it is not licensed forthis purpose, it can be used after proper investigation if there is a clear indication foruse. This drug will be used preceding fertility treatment or pregnancy cycle only. Theaim 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 notbe initiated in patients with active infections, including tuberculosis, chronic orlocalised infections until infections are controlled.

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-filled syringe and is given as asubcutaneous injection once every two weeks. Complete elimination of Humira maytake up to five months. Patients usuallyneed 2 to 4 doses, dependent on the severityof immune abnormality.

Side EffectsIn placebo-controlled studies 20% of patients treated with Humira developedinjection site reactions (redness and/or itching, bleeding, pain or swelling) comparedto 14% who received placebo. The incidence of serious infections was extremelylow. The infections were primarily upper respiratory tract infections, bronchitis andurinary tract infections. In clinical trials with Humira for up to 53 months no increasein rates and incidences of malignancies were observed. After 24 weeks of Humira12.6% of patients with negative anti-nuclear antibodies tested positive comparedwith 7.3% of placebo-treated patients. The significance of this is unclear. A drug-induced lupus-like syndrome may occur and thedrug must be discontinued in that case. Although developmental toxicity study inmonkeys showed no maternal toxicity, no toxicity to embryos and no developmentalabnormalities, we do not advise the use of Humira in pregnancy.

In up to 20% of patients, elevated TNF alpha levels may not be successfullyreduced with Humira.

InfectionsSevere infections, sepsis and re-activation of tuberculosis have been reported withTNF-α blockers. These drugs should not be started and should be discontinued ifthe patient develops serious infection. These drugs are used cautiously in patientswith a history of recurrent infections or medical condition that predisposes toinfections. Live vaccines should not be given concurrently with these drugs. Therecommendation is that all patients should be screened for tuberculosis beforestarting the drugs. If symptoms develop suggesting tuberculosis, e.g. persistentcough, 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 onthe drugs and for at least 6 months afterwards. Severe leukopenia (reduction ofwhite blood cells), pancytopenia (deficiency of all blood cells) and aplastic anaemiaand onset of demyelinating diseases of the central nervous system including multiplesclerosis and optic neuritis have been rarely associated with some anti TNF alphadrugs. The drug should be used with great caution in patients with a history of thesedisorders or discontinued if the problems occur.


Health & Medicine

Immunology testing