Repromedix - Helping Doctors Help Couples

Amit, Ami MD

Beer, Alan MD

Branch, Ware MD, R.E.

Brown, David PhD

Cataldo, Nicholas MD, R.E.

Clark, Al MD, PhD, R.E.

Coulam, Carolyn MD, R.E.

Cowchick, Susan MD

Decherney, Alan MD, R.E.

Dunaif, A. MD

Fisher, Robin MD, R.E.

Friedman, Andrew MD, R.E.

Geva, E. MD, R.E.

Groome, N.P. PhD

Hill, Joseph MD, R.E.

Kent, First, MJ PhD

Kutteh, William MD, PhD, R.E.

Lambert, Messerlian, G PhD

Legro, R.S. MD

Lipschutz, Larry MD

Lockwood, G.M. MD

Luborsky, Judith PhD, R.E.

Matzner, William MD

Milunsky, Aubrey MD, PhD

Milunsky, Jeffrey MD

Nestler, J.E. MD

Ohashi, K. MD

Rivnay, Benjamin PhD

Scher, Geoffrey MD, R.E.

Seifer, David MD, R.E.

Stephenson, Mary MD, R.E.

Vought, P.H. MD

Zouves, C. MD, R.E.

Zuffardi, O. MD

Value Added Services

Repromedix is a single referral source for advanced reproductive disorder and infertility testing. We offer:

n Serving all of North America, Central America, Western Europe, and Parts of South America.

Commitment to Excellence

REPROMEDIX meets the following requirements to guarantee our commitment to excellence:

n Performs consistent procedures with a high level of confidence in test reproducibility on test results.

n Quality Control, Quality Assurance, and Proficiency Testing routinely incorporated in all aspects of the operation.

n Thorough laboratory record keeping and documentation indicating performance and consistency of each assay.

n All our personnel exceed the required qualifications and are staffed appropriately.

n Meet all necessary State, Federal, and Associated Society Regulation Requirements such as CLIA, CAP, AAB, and New York State Licensure.

Requirements for Quality Assurance

Clinical laboratories must comply with state, federal, and regulation agencies governing " the examination of human specimens for the diagnosis, prevention, or treatment of any disease or impairment of, or the assessment of health of, human beings," personnel safety and employment.

Under these requirements, clinical laboratories must show documentation verifying proper certifications, regular inspections, and active participation in a proficiency testing program, quality control, quality assurance, and proper record keeping. These activities must be proven through proper documentation. Written procedures and policies, active infectious control measures, required staffing requirements, inventory of supplies, sources of chemicals, general supplies and a listing of all suppliers must be available on site at all times and documentation of annual review must be available. Appropriate storage of patient samples, reagents, and controls are required.

n Continuous evaluation of corrective actions taken for quality control measures.

n Identify and evaluate patient test results that are inconsistent with clinical data.

n Documentation of any noted problems that occur due to communication breakdown between laboratory and provider ordering the test.

At Repromedix, we are active participant in the programs of the following regulatory agencies:

Comprehensive Panels

Guide to Infertility Testing

A. Ovarian Reserve / Premature Ovarian Failure (POF)

Inhibin B and FSH (day 3) have both been related to ovarian reserve - the potential to respond to hormonal stimulation with ovulation of multiple eggs with good quality. However, data suggest that the decrease in Inhibin B, characteristic of a reduced reserve, precedes the increase in FSH. AOA may be causative for these changes or may result in other ovarian/endocrine functional manifestations. It can also show up earlier than the drop in Inhibin B. The EFORT test measures the ability of the ovaries to respond to stimulation with an exogenous injected FSH dose on day 3. Premature Ovarian Failure may also result from Fragile X Syndrome, an inherited genetic mutation. Results from the complete panel may provide better information as to the temporal positioning of the patient in this long process of transition into menopause. It may also trigger a treatment.

This ELISA-based test detects autoimmunity to the ovary. Ovary-reactive antibodies may mask functional proteins on ovarian cells or interfere with intracellular protein functions, blocking ovarian functions and interfering with folliculogenesis and fertility. Women with elevated AOA may have reduced and impaired response to ovulation induction. Women with strong positive AOA may have Premature Ovarian Failure (POF, aka early menopause). In the course of reproductive function deterioration, AOA may precede the reduction in Inhibin B or cause it.

This new ELISA-based hormonal test measures the levels of Inhibin B in serum on day 3 of the menstrual cycle. Since only Follicular Granulosa cells secrete this hormone, its level reflects on the potential of the ovary to grow follicles, with subsequent successful ovulation of good quality eggs and embryo development (collectively related to "ovarian reserve"). Women with low levels of the hormone were found to have more impaired ovulation in the course of the IVF cycle, lower pregnancy rates, higher cancellation rates and higher abortion rates. While Inhibin B is secreted by the granulosa cells, estradiol (E2) involves other cell types in the ovary (theca). Thus, the Day 3 Inhibin B test provides the physician with a more direct test - monitoring a follicular component compared to a pituitary component (FSH). It may provide the couple some qualitative estimate of success in future cycles of artificial reproductive techniques.

Instead of measuring the status of ovarian reserve at an early cycle point (day 3) by a single Inhibin B measurement, the EFORT test measures the ability of the ovaries to respond to stimulation with an exogenous FSH dose. A blood sample is drawn on day 3 for measuring the basal Inhibin B level and a dose of 300 i.u. of FSH is injected I.m. on day 3. A second blood sample is drawn on day 4, 24 hours after the FSH injection for assessing the ovarain response. Inhibin B is measured in both (d3 and d4) serum samples and a change of "d4 Inhibin B - d3 Inhibin B" is determined.

The ovarian response to FSH stimulation could be useful in predicting the response in IVF cycle stimulation, especially for oocyte retrieval and thus, to identify the poor responders before IVF for better assessment and counseling. The significance in treatment outcome (good and poor response to hormonal treatment) related to this test was 10 times better that the basal d3 inhibin B test and 100 times better than the basal FSH test. EFORT is currently the best ovarian reserve response test available, It is argued to be more direct than the clomiphene citrate challenge test (CCCT) .

Fragile X syndrome is the most common cause of inherited mental retardation. Although affected females generally suffer less, about 20-33% of female carriers have pre-mature ovarian failure (POF). The gene defect is caused by instability of the FMRI gene region.

The FMRI gene normally has less than 45 tandem repeating tri-nucleotide (CGG) repeats. If an expansion occurs, 50-200 CGG repeats or > 200 CGG repeats, that defect can be passed on to their children.

Carriers of moderately expanded sequence are called pre-mutation (50-200 CGG repeats) and are mostly asymptomatic except for premature ovarian failure in carrier females.

B. Poly Cystic Ovary Syndrome

These panels cover both aspects of the PCOS syndrome: the endocrine (free testosterone and SHBG) and the metabolic (Insulin, Glucose and IGFBP-1). Thus, the complete panel confirms if hyperandrogenism is the underlying reason for hirsutism, polycystic appearance of the ovary in ultrasound and anovulation, and at the same time it also identifies the cause for these changes and indicates a treatment. This is a better alternative to sequential testing for hormones that may identify hyperandrogenism without linking the phenomenon to a metabolic disorder (IR). The panel is important as it indicates the only treatment for PCOS that is compatible with fertility (all other treatments are directed towards the hyperandrogenic manifestation from the cosmetic point of view). It also indicates a treatment that can reduce the probability of developing hypertension, cardiovascular disease and diabetes at later stages.

Poly Cystic Ovary Syndrome (PCOS) is a metabolic-endocrine-reproductive disorder that occurs in 5-10% of women in reproductive age and represents a major fertility problem. Infertility results from disturbances in follicle development, leading to oligo-ovulation. PCOS patients have high levels of LH and normal levels of FSH (high LH/FSH ratio), which in turn results in high levels of free testosterone in the circulation. {In ~80% of these patients, insulin resistance (IR) with elevated levels of insulin in circulation, seems to be the underlying mechanism for the development of this syndrome}. The classic manifestations of PCOS are hyper-androgenism (hirsutism and acne), menstrual irregularities due to anovulation (dysfunctional bleeding, absence of menses or prolonged menses intervals), and obesity. Long term, PCOS patients are at high risk of developing heart disease, hypertension, type-II diabetes and uterine cancer due to elevated levels of insulin and androgens. PCOS patients who have insulin resistance show no response or very poor response to treatment using the ovulation inducing drug clomiphene citrate. Once PCOS patients do respond to anovulation drugs, they tend to have multiple follicle development and be prone to ovarian hyperstimulation syndrome. The success of treatment is dramatically improved if insulin resistance is identified and treated with insulin sensitizing drugs such as metformin (Glucophage), roziglitazone (Avandia) or pioglitazone (Actos). Correction of insulin resistance also minimizes development of ovarian hyperstimulation syndrome.

This is an ELISA based test for the hormone levels in fasting plasma and in plasma collected two hrs after oral glucose load. This test must be done in conjunction with Glucose testing to evaluate the metabolic status of the patient. Insulin resistance due to reduced glucose utilization (e.g. impaired fasting insulin) can be treated. The combination of insulin and glucose testing would also rule out other causes for insulin elevation. Patients will be defined as normal or insulin resistant.

This is a highly sensitive colorimetric test. This test provides information on glucose metabolism. The test will reveal if the patient has normal glucose, impaired fasting glucose (IFG), impaired glucose tolerance (IGT) or if they are suspected for diabetes. The combination of glucose with insulin (G/I ratio) yields a more rigorous indication for Insulin Resistance.

This is an ELISA based test for the level of IGFBP-1 in plasma. IGFBP-1 synthesis is suppressed by high insulin, leading to a consequent increase in free IGF-1, which acts together with insulin to exacerbate hyper androgenism by enhancing testosterone production in the ovaries (stimulates P-450 c17aenzyme activity). Hence, IGFBP-1 is a more comprehensive probe for the levels/activities of both Insulin and IGF-1. IGF- BP1 is much more stable than insulin and hence sensitive to fasting, which renders the test and panel more accurate.

This is a competitive enzyme immune assay (EIA) for free testosterone. This is a measure of the end product of this hormonal system, resulting in hyperandrogenism. Elevation in Testosterone causes the increase in luteinizing hormone (LH), leading to the increase in the LH/FSH ratio which results in major perturbations in cycling and fertility functions. Unless high levels of this hormone originate from insulin resistance Metformin should not be prescribed, thus, this test must be done as part of the complete panel. This test by itself is not sufficient to identify the etiology of the disease.

This is an ELISA based test to measure plasma levels of this protein that are responsible for binding the majority of testosterone in the blood into a complex. Lower plasma levels of this protein suggest insulin resistance, and provide an explanation for the increase in Free Testosterone. This protein is much more stable than insulin in the blood, and thus increases the diagnostic value of the panel over insulin alone (insulin requires immediate separation of plasma from cells and immediate testing or freezing).

C. Hyper-androgenism Panel

The Hyper-androgenism panel includes tests for Total Testosterone, DHEA-S and 17-OH progesterone. It will establish a differential diagnosis of some of the important disease conditions that are associated with high androgen levels and its consequent symptoms in women (e.g. PCOS). Some adrenal disease conditions that have defects / disruptions in cortisol synthesis pathway could often contribute to increased testosterone production by producing high circulating levels of androgen precursors (e.g. 17-OH progesterone and DHEA-S) that are converted to testosterone in ovaries.

High total testosterone levels are indicative of the degree of androgen excess in women. Mild to moderately elevated total testosterone levels are associated with conditions such as PCOS, HAIRAN, or adrenal hyperplasia. Persistently elevated (very high) levels of total testosterone will suggest androgen producing tumors in ovaries.

The 17-OH Progesterone is an intermediate compound in the cortisol synthetic pathway and is the substrate for 21-hydroxylase enzyme which converts it to cortisol precursor, 11 Deoxy cortisol. This coristol synthesis is activated by the ACTH from pituitary. In non- classical congenital adrenal hyperplasia (NCAH) the patients have a severe deficiency of 21-Hydroxylase enzyme and thus will accumulate 17 -OH progesterone without being converted to 11-deoxy cortisol, thus high levels of 17-OH progesterone would be a specific diagnostic indicator for 21-hydroxylase deficient NCAH . As 17-OH Progesterone is also produced by corpus luteum, basal level of 17-OH progesterone should be measured in the follicular phase, preferably in the morning. If follicular phase 17-OH progesterone level is over 80-100 ng/dL (using the 17-OH Progesterone ELISA method) the patient should undergo an acute ACTH stimulation test. 250 µg of synthetic ACTH is injected intravenously to the patient and serum levels of 17-OH progesterone is measured after 60 minutes of ACTH administration. If post- ACTH levels of 17-OH progesterone are above 320 ng/dL (using the 17-OH progesterone ELISA method), the 21-hydroxylase deficient NCAH could be established.

DHEAS, a precursor of testosterone, is a useful clinical marker for androgen excess as well as adrenocortical hyperactivity or adrenal tumors. Moderately high DHEA-S serum levels (greater than 430 ng/dL) would indicate either to be of PCOS origin or adrenal hyperactivity (adult onset adrenal hyperplasia). Persistently high serum DHEA-S levels greater than 700 ng/dL will indicate adrenal tumors.

D. Ovarian Tumor Markers

Ovarian tumors may develop from different types of cells in the ovary. Testing for tumors must be done in addition and in conjunction with other functional tests and physical examination of the patient. Biochemical testing for ovarian tumors are performed on two types of markers: functional markers that simply quantify excessive activity of specific cells, that could be related to tumor development and specific tumor markers, i.e. markers whose secretion and expression is triggered or enhanced by the appearance of the tumor cells. This panel currently includes two tests: one for the function of granulose cells (Inhibin B), enabling the identification of granulose cell tumors, and the other (CA-125) for the currently most reliable ovarian tumor marker available. The panel should be useful in differentiating between benign and malignant tumors, and help reduce the false negative results.

This is an ELISA test that measures hormone levels in serum. Inhibin B is secreted from the ovarian follicular granulose cells, and thus is highly elevated (10-50 fold over the values normally found for Day 3 of the menstrual cycle) when a granulosa-cell tumor is present. The value remains elevated until the tumor is removed surgically. Due to the fast turnover of Inhibin B, values one-day post operation should provide an accurate monitor for the success of the surgery. Postoperative values should be as low as normal. Inhibin B can also be used to follow the progression or recurrence of such tumors. The combination with CA-125 increases the predictability of detecting all stages of ovarian tumors and reduces the potential of false negative results.

This is an ELISA test, which measures the serum level of the protein antigen CA-125 derived from epithelial cell surface. This protein is associated with ovarian malignancy however it is also elevated in multiple nonmalignant situations such as endometriosis or pregnancy. It is the most reliable marker of a dozen studied, for ovarian cancer, however it has a low positive predictability and low sensitivity. Thus, it should not be used alone. Likewise, it should not be used for prognostic (early detection) purposes, but rather for monitoring the progression of disease or the efficacy of treatment(s). It should be a useful adjunct to clinical examination and vaginal ultrasound with Doppler flow studies, and a good adjunct to Inhibin B testing.

E. Unexplained Infertility /Implantation Failure & Recurrent Pregnancy Loss

This panel encompasses multiple assays that assess factors related to unexplained infertility or recurrent pregnancy loss. These cover primarily immunological or coagulation disorders, as they had been correlated with reduced egg and embryo quality, and impaired mother-embryo cross talk in all stages of the early pregnancy. These tests need not be performed at specific days of the cycle, but should be done just prior to or during the first trimester of the current cycle. Sufficient time should be allowed for treatment before the next cycle if patient is positive.

This is an important pre-IVIG test, which consists of 3 tests, one each for levels of IgG, IgM, and IgA. IgG gives the physician a baseline for the patient. This will show how the patient was impacted after subsequent treatment. IgM will indicate other potential immunological issues. The most important test is IgA. Low IgA has been shown to be significant in predicting which patient will have a likelihood of complications from IVIG treatment.

Patients with low IgA should never receive IVIG treatments. It is recommended that the test be initially run with all tests that require IVIG treatment when positive, so that patient does not lose time before IVIG treatment is initiated. Patients with abnormally high IgM should be followed for autoimmune antibody responses, as IgM normally represent a fresh onset of humoral immune response.

The Anti-cardiolipin Antibody ELISA test measures 3 different species of antibodies to the phospholipid cardiolipin. This test is essentially an anti-phospholipid antibody test, with all features similar to those of the APA. Its stand-alone status is solely due to historical reasons. It is clear today that the phospholipid testing must cover as many as possible different phospholipids since serum samples found negative for ACA may be positive for other phospholipids. Such samples would be diagnosed falsely negative if ACA was performed without APA.

This is an ELISA-based test, which evaluates and quantifies the presence of any one of 12 different species of antibodies to 4 different phospholipids in patient's serum. These antibodies bind primarily to negatively charged phospholipids and interfere with functions related to the exposure of these moieties or with functions of phospholipid binding proteins. Elevated concentrations of these antibodies are thought to interfere with feto-maternal blood flow due to enhanced blood clotting-related functions. Anti-Phospholipid Antibodies may also interfere with the formation of the syncithiotrophoblast formation, which is an important function of implantation. These impairments may lead to retardation of fetal development, first trimester abortions or in extreme cases interference with the embryo implantation in the uterus. The test is usually ordered for women with failed IVF cycles and recurrent pregnancy loss (natural or following ART procedures).

Some patients with systematic lupus erythematous (SLE) produce auto-antibodies against components of the blood clotting system. This antibody is called "Lupus Anti-Coagulant (LAC)" and is often grouped as a heterogeneous anti-phospholipid antibody. LAC test (also referred to as Dilute Russell Viper Venum Test) is a sensitive coagulation test used to specifically detect the defect. LAC is associated with more thrombotic events (venous thrombosis) and often correlated with fetal loss and recurrent spontaneous abortion (during first 4 month of gestation) in women with lupus (SLE).

Note: Although LAC antibodies are associated with thrombotic events in vivo, presence of lupus anti-coagulant in patients plasma is detected by a prolonged clotting time in the in-vitro testing system (modified Russell Viper Venum Test or LAC test).

APTT is a general screening clotting time test used primarily to evaluate abnormalities in the enzymes and co-factors of the intrinsic coagulation pathway necessary for prothrombin activation. APTT is especially sensitive to functional deficiencies of factor VIII, IX, XI, and XII, Fletcher factor or Fitzgerald factor. APTT will be prolonged in coagulation defects associated with these clotting factors deficiencies. Coagulation disorders associated with these intrinsic factor deficiencies have been related to failed implantations and recurrent pregnancy loss.

APTT may be shortened, normal or slightly prolonged in lupus patients. More sensitive modified Russell Viper Venom Test (LAC Test) is required to detect the presence of lupus anti-coagulant antibodies (See LAC test above).

The cell test is a white blood cell typing analysis, using fluorescence-tagged antibodies to specific markers on the surface of those cells (flow cytometry). When activated, NK cells function to fight, kill and destroy their targets. Excessive NK cells in blood (particularly CD56⁺16⁺) , and very likely in the uterine issue were correlated with pregnancy loss and reduced success in IVF cycle utcome. The test is recommended within the first trimester of pregnancy but elevated levels may be detectable earlier. Positive test results may indicate a few types of therapeutic treatments all aimed at suppressing the immune response for the early parts of the pregnancy, thus alleviating the woman of this fetus-rejecting activity and allowing a successful implantation and normal development of the pregnancy.

This is a FACS (Fluorescence Activated Cell Scanning) technique employing a panel of fluorescent antibody mixtures to stain/tag specific surface markers on peripheral blood white cells for analyzing lymphocyte subpopulations. The panel includes 2 markers for Natural Killer (NK) cell subpopulations, 3 markers for T-cells subpopulations and 2 markers for B cells. Increases in NK and T or B cytotoxic cells is indicative of increased risk of (recurrent) pregnancy loss as these types of cells may be involved in cellular activity against the developing embryo through a direct attack or by secreting deleterious cytokines such as TNF aor Interferon g (TH1 cytokines).

These are ELISA-based tests used to identify high levels of these autoantibodies. Women with elevated levels were found to have higher risk of RPL (Recurrent Pregnancy Loss) and were associated with IVF failures. These autoantibodies interfere with thyroid functions and thus, with normal metabolism, which is higher in pregnancy than in normal non-pregnant women and thus sub clinical thyroid metabolism may impair metabolism in pregnancy. They are also indicators for a predisposition of the patient to autoimmunity, which may involve additional autoantibodies that interfere with the reproductive process (e.g., AOA, APA, ACA, ASA).

If the general screening test is positive, expand to the ANA PANEL (Anti-Nuclear Antibodies).

This is a test for an autoimmune response (woman's immune system attacking her own cell nucleus). ANA's are antibodies against one or more elements within a biological cell, involved in the machinery of translating gnomic message into proteins. These antibodies can destroy cells, and their effect usually leads to the disease Systemic Lupus Erythematosus (SLE). Women with 'Lupus' or positive ANA test results are at a higher risk of miscarriage and IVF implantation failure due to inflammatory effects on the placenta and or the fetus. The first test is usually a screen, which identifies up to 100 of this type of antibodies as a group. If the ANA Screen is positive, the ANA Panel is automatically reflexed to determine if any of the 10 most clinically important antigens in the group is involved.

This is a combination of two procedures. The first involves a maternal cell (lymphocyte) culture in which stimulation by trophoblast antigen (derived from a cell line) takes place. The second procedure involves mouse embryo culture. These procedures are set in order to determine if the patient's lymphocytes had been sensitized to secrete anything toxic to the embryo (test utilizes 2-cell stage mouse embryos) e.g. TH1 type cytokines. The production of the cytokine gamma Interferon IFN gin response to the trophoblast antigen is also examined in this test. This cytokine may interfere with pregnancy but is not the sole factor responsible for such activities. Women, who have been sensitized in the course of their earlier pregnancies or in any other mode, could amass an immune response against their own fetus in the following pregnancy, and end up losing the pregnancy (in the implantation process or later in the first trimester). The toxicity, if established by this test, may result from multiple factors. It may exist in the cells prior to stimulation in the culture or be induced by the exposure to the trophoblast components. This test is not a serum test, and therefore is not affected by anti mouse antibodies. It is more sensitive than serum testing as it targets immune memory which may not be triggered at the time of serum sampling.

Human Lymphocyte Antigen DQ (HLA DQ) is a test for both the male and female genotypes that indicate a predisposition to alloimmune response function between the 2 partners. In order to be of value, the HLADQα must be ordered for both male and female partners. According to theory, if the male and female genotypes overlap significantly, there is a predictable adverse effect on the early pregnancy due to lack of immune protection. Also, if specific HLA genotype values are found, they can be indicative of an abnormal immune response. A male value of 2.0 will automatically reflex the HLA DQβ test for the male only.

This immuno-bead-based test measures antibodies to the sperm cells in the woman's serum (IgG, IgM, IgA) or cervical mucus. Such antibodies would interfere with the ability of normal sperm to move up the female reproductive tract and fertilize.

The LAD test is another measure of immuno-campatibility between husband and wife. According to theory, in order for the maternal immune system to down regulate during pregnancy, and in order for her to produce blocking antibodies that would prevent her system from attcking the embryo/fetus, she must respond to her husband's alloantigens. LAD tests the presence of blocking allo-antibodies in maternal blood. A positive test result is desired.

It is widely known that the majority of recurrent first trimester spontaneous abortions (Recurrent Pregnancy Loss) are caused by an abnormal karyotype (abnormal number and shape of chromosomes in the cell nucleus). In many cases this is due to one of the parents being the carrier of an abnormal chromosome, and the incidence of this increases with the maternal age. The test involves a 3-day stimulation of the peripheral blood cells, spreading of the chromosomes on a microscope slide, and karyotyping (analysis of chromosome number and shape). Several specific karyotypes have been associated with reproductive health and infertility.

F. Thrombophilia (Coagulation Disorders)

Blood Coagulation is a vital physiological process that is paramount to prevent excessive bleeding and blood loss when a blood vessel gets injured/damaged. A series (cascade) of events happens at the site of vessel injury, ultimately resulting in formation of a platelet plug and a mesh of cross-linked fibrin referred to as a blood clot or thrombus. After healing occurs at the site of the injury, the blood clot is broken down and removed by a process called fibrinolysis, and the normal vessel structure is restored. Mechanisms that restrict excessive or prolonged clotting processes at the site of vessel injury are necessary to maintain the normal fluidity of the blood and prevent hyper-coagulatory events. Excessive activities of pro-coagulant factors and deficiencies in anti-coagulant factor activities or fibrinolytic mechanisms will lead to thrombotic conditions/diseases. Thrombosis is a pathologic process in which platelet aggregation and fibrin clot formation occur in intact blood vessels rteries or veins) and is often associated with heart disease (myocardial infarction), neuro-vascular complications (ischemic stroke), pulmonary embolism and edema, inflammation and necrosis. In pregnancy, excessive coagulatory events lead to the disruption of the blood supply to the fetus or thrombosis in the placenta resulting in many complications: implantation failures, recurrent pregnancy loss, fetal death, fetal growth retardation (IUGR) , preeclampsia or stillbirths

Antithrombin is a potent inhibitor of the multiple reactions in the coagulation cascade (factor XIa, IXa, Xa and thrombin) and is one of the primary natural anticoagulant proteins. In type 1 antithrombin III deficiency , the patients have inadequate amount of antithrombin III present in their circulation. In type 2 deficiency, the amount of antithrombin III present is normal, but it does not function properly. The occurrence of antithrombin III deficiency in the population is low (0.02 - ~1%) but it is seen in ~4% of patients who have had venous thrombosis events. Antithrombin III (Activity) Test is available to diagnose the condition

APTT is a general screening clotting time test used primarily to aluate abnormalities in the enzymes and co-factors of the intrinsic coagulation pathway necessary for prothrombin activation. APTT is a especially sensitive to functional deficiencies of factor VIII, IX, XI, and XII, Fletcher factor or Fitzgerald factor. APTT will be prolonged in coagulation defects associated with these clotting factors deficiencies. Coagulation disorders associated with these intrinsic factor deficiencies have been related to failed implantations and recurrent pregnancy loss.

NOTE: Prothrombin Time (PT) test is used to determine the deficiencies of factors involved in down stream events (extrinsic factors) of the coagulation cascade, such as factor VII, X, V, or II. Thrombin time (TT) is used to detect Fibrinogen deficiencies (the most proximal event in clotting). An Algorithm of a combination of these tests are used to differentiate individual or combined clotting factor abnormalities. More detailed hematological evaluation is recommended for patients suspected of severe clotting factor abnormalities and prolonged bleeding.

In normal individuals, once clotting occurs, thrombin binds to thrombomodulin and activates a number of anti-coagulant proteins, including Protein C, in order to control/terminate excessive clotting. Activated Protein C (APC) binds Protein S and inactivates and degrades (by cleavage) coagulation Factor V, limiting and keeping further coagulation in check. In patients with a point mutation at the APC cleavage site in the Factor V gene (Factor V Leiden), the APC can no longer cleave and inactivate the Factor V, resulting in prolonged coagulation events (thrombophilic state). Excessive thrombotic events due to the Factor V Leiden mutation have been associated with recurrent spontaneous abortions, late abortions, fetal death and IUGR and pregnancy complications such as preeclampsia in affected women.

This laboratory test determines the APC-Resistance (APC-R) by using a coagulation-based assay to determine the APC-R ratio. The Factor V Leiden coagulation test is performed by activating the endogenous Protein C in patient's plasma by Southern Copperhead venom (activator) followed by a dilute Russell viper venom time (DRVVT) clotting test. In normal plasma, activation of Protein C will prolong the clotting time on DRVVT 2-3 fold compared to saline. In Factor V mutation (defective Factor V), no or only marginal change in the clotting time will be observed. The numerical values for APC-R are interpreted as APC-R ratios (i.e. DRVVT with activator divided by DRVVT with saline). APC-R ratios < 1.5 are classified as Factor V Leiden APC-R and ratios > 2.1 are the normal range. Ratios between 1.5 and 2.1 are automatically reflected to a DNA-based PCR assay for confirmation.

The Factor V Leiden APC-R is a single nucleotide point mutation at the site 1691 in the Factor V gene, which results in an amino acid replacement of arginine at position 506 with a glutamine in the Factor V protein. This is the exact site where APC cleaves Factor V and inactivates it in order to slow down and stop excessive coagulation. The Leiden variant Factor V protein becomes resistant to cleavage by APC due to the replacement of the amino acid arginine. The genomic DNA is isolated from patient whole blood and the target gene fragment is amplified by PCR. The PCR product is then analyzed by gel electrophoresis. This method determines whether the patient is wild-type (normal), heterozygous (+/-), or homozygous (+/+) for this mutation. Both (+/-) and (+/+) are considered abnormal. This molecular test detects the Factor V Leiden mutation with extremely high sensitivity and specificity and is used as a confirmation test for the coagulation-screening test.

Some patients with systemic lupus erythematous (SLE) produce auto-antibodies against components of the blood clotting system. This antibody is called 'lupus anti coagulant' (LAC) and is often grouped as a heterogeneous anti-phospholipid antibody. LAC test (also called as Dilute Russell Viper Venom Test) is a sensitive coagulation test used to specifically detect the presence of lupus anti-coagulant in patient's plasma. Although the name anti-coagulant usually refers to an agent that interferes with normal clotting resulting in abnormal bleeding, lupus anti-coagulant is never associated with abnormal bleeding. On the contrary, LAC is associated with more thrombotic events (venous thrombosis) and is often correlated with fetal loss and recurrent spontaneous abortions (during first four months of gestation) in women with lupus (SLE).

Note: Although LAC antibodies are associated with thrombotic events in vivo, presence of lupus anti-coagulant in patient's plasma is detected by a prolonged clotting time in the in vitro testing system (modified Russell viper venom test or LAC test).

Homocysteine is a compound involved in the pathways related to Methionine and Cysteine amino acid metabolism. In the absence or shortage of vitamin B6 and B12 , which are cofactors in these reactions, or in the presence of a specific mutation in the enzyme MTHFR involved in the synthesis of Folic acid, homocysteine accumulates to create hyper-homocysteinemia.

High levels of this chemical in the blood are associated in multiple ways with the hyper-coagulation status of the patient, as well as with reduced elasticity of blood vessels, which indirectly exacerbates thrombophilic events. As such, it was shown to constitute a thrombophilic risk factor that, like other components of thrombophilia, may cause recurrent pregnancy loss and other pregnancy risks. This test measures the amount of homocysteine in the patient's serum utilizing HPLC. The test is performed on serum, and specimens must be obtained while fasting, and separated and spun immediately.

Increased levels of homocysteine (hyper-homocysteinemia) is a risk factor for vascular thrombotic diseases. High levels of homocysteine in circulation inhibit prostacyclin synthesis, activate factor V, inhibit activation of protein C, down regulate thrombomodulin expression and block tPA binding to endothelial cells - all of which are procoagulant activities that can lead to excess thrombotic events. In addition, defects in folate metabolism have been correlated with neural tube defects (pregnancies complicated with neural tube birth defects). One of the most common genetic defects of homocysteine metabolism is a mutation in the enzyme methylene-tetrahydrofolate reductase (MTHFR). The MTHFR enzyme is responsible for conversion of 5,10-methyl tetrahydrofolate to 5- tetrahydrofolate for the synthesis of folic acid (folate). Folate is a cofactor for conversion (remethylation) of homocysteine to methionine. MTHFR gene mutation thus leads to reduced folate enzyme activity and accumulation of high levels of homocysteine. One of the most common MTHFR mutations is a nucleotide transition from C-to-T at position 677 that results in amino acid transition from valine to alanine.

The C677T MTHFR mutation is common in Caucasians with a homozygous frequency of ~5% (Dutch and Finnish) to 20% (in French Canadians and other European descents). The genomic DNA is isolated from patient whole blood and the target gene fragment is amplified by PCR. The PCR product is then analyzed by OLA using oligonucleotide probes. This method determines whether the patient is wild-type (normal), Heterozygous (+/-), or homozygous (+/+) for this mutation. Only homozygous mutations are considered abnormal .

Plasminogen Activator Inhibitor (PAI)-1 disease is a relatively less common genetic defect caused by defects in the PAI-1 gene transcription promoter region. It's a 4G/5G type polymorphism in the promoter region and certain genotypes (4G/4G and 4G/5G) will be associated with increased plasma levels of PAI-1 protein. PAI-1 enzyme inactivates the plasminogen activator (tPA) enzyme, which is responsible for the activation of plasminogen to plasmin that digests the fibrin/blood clots. Thus, elevated levels of PAI-1 will lead to a hypo-fibrinolytic and hyper-thrombotic status. Affected genotypes are associated with thrombotic diseases/complications in pregnancy. PAI-1 defects are associated with many conditions in infertility/pregnancy (e.g. RSA, preeclampsia, fetal growth retardation, fetal death etc). PAI-1 antigen and activity tests are available for the detection of plasma PAI-1 to aid in diagnosis.

Protein C deficiency is a genetic condition that predisposes a person to hyper-coagulation disorders and thrombosis. High levels of thrombin that was generated during the coagulation process binds to thrombomodulin and activates a number of anti-coagulant proteins including Protein C, in order to control/terminate excessive clotting. Activated Protein C (APC) binds Protein S and inactivates and degrades (by cleavage) coagulation Factor V, limiting further coagulation. Protein C deficiency will lead to inadequate/lack of degradation of factor V and progression of a hyper-coagulatory status. Protein C deficiency results from inadequate amounts of protein C synthesis (type I) or synthesis of defective protein C molecules (type II). Numerous defects in protein C molecules have been described that alter the interaction with Protein S, thrombomodulin, phospholipids, factor Va and factor VIIIa. Individuals who are heterozygous for Protein C deficiency are 7-fold more prone to thrombotic diseases/complications. Homozygotes may have severe thromboses even at birth. We offer two tests to detect a deficiency in Protein C. The first is a functional clotting assay that measures the activity of Protein C in the patient's plasma. The second is an ELISA assay that measures the amount of this anti-coagulatory protein. The results of the ELISA assay, in conjunction with a documented decrease in Protein C activity, will determine the type of deficiency the patient has.

Protein S binds to activated protein C and acts as a co-factor to cleave and inactivate factor V. Similar to protein C deficiency, protein S deficiency will thus lead to inadequate/lack of degradation of factor V and progression of hyper-coagulatory status. Protein S deficiency is an acquired or inherited condition resulting from inadequate amounts of protein S synthesis (type I), synthesis of defective protein S molecules (type II), or inadequate amounts of the free form of protein S to bind to APC but normal total levels of protein S (type III).

Protein S exists in two forms - only the free form can bind to APC and function as co-factor, the other is bound to an additional protein and thus cannot bind to APC. Individuals who are heterozygous for protein S deficiency have a 5-fold higher risk of thrombotic diseases/complications. Homozygotes may have severe thrombosis at birth. We offer two tests to detect a deficiency in Protein S. The first test is a clotting assay that measures the activity of Protein S in the patient's plasma. The second test is an ELISA assay that measures the amount of Protein S in the plasma. The measurement of Free Protein S (standard) or both Free and Total Protein S will confirm the functional assay and identify the type of deficiency the patient has.

Prothrombin G20210A is a mutation in the prothrombin gene and is associated with an increased risk of venous thrombosis and thrombophilia. Patients carrying prothrombin G20210A mutation have greater risk of thrombosis associated pregnancy complications such as recurrent spontaneous abortions, placental abruption, fetal death and preeclampsia.

The genomic DNA is isolated from patient whole blood and the target gene fragment is amplified by PCR. The PCR product is then analyzed by gel electrophoresis. This method determines whether the patient is wild-type (normal), heterozygous (+/-), or homozygous (+/+) for this mutation. Both +/- and +/+ are considered abnormal. This molecular test detects the G20210A mutation with extremely high sensitivity and specificity.

Please note sample integrity is vitally important for the Thrombophilia Panel.

G. High Risk Pregnancy Monitoring

This panel includes the immunological tests that are ordered for unexplained infertility / recurrent pregnancy loss. Unfavorable immune environment is responsible for recurrent pregnancy loss, but also for abnormal function in later stages of the pregnancy, and may be involved in disorders such as pre-eclampsia and other pregnancy complications.

This ELISA based test measures the levels of Inhibin-A, a peptide hormone secreted by corpus luteum and placenta, in maternal serum. Feto-placenta is a major source of Inhibin A, thus, its levels around 4 weeks of gestation reflect the status of the implanted embryo and is used as a diagnostic test for detection of early pregnancy problems such as loss of pregnancy (embryo death), singleton or multiple pregnancies. Inhibin A testing (4th week of pregnancy or 13 days after ET) could be useful for IVF patients for early diagnosis of pregnancy loss or multiple pregnancies.

H. Andrology (Male Factor)

This group covers immunological, cellular, endocrine and genetic tests. The tests may be used to assess the immediate sperm function, the hormonal status of the patient (as related to spermatogenesis) as well as the genetic predisposition and associated prognosis of risks for a successful fertilization, for the pregnancy and for the newborn.

This test examines the ability of the sperm to perform one of the functions involved in fertilization, namely the attachment to the egg and the actual penetration into the egg. The test is performed on the male partner's sperm using a Chinese hamster egg, which is generally suitable for this test and emulates a human egg. Reduced penetration reflects on the quality of the sperm and its ability to fertilize, assuming other parameters such as sperm motility and sperm morphology are normal.

This immuno-bead based test checks the presence of auto-antibodies on the male partner's sperm (IgG, IgM). If excessive amounts of antibodies are bound to the surface of the sperm cell, they may mask receptors or other functionally important proteins, interfere with the sperm-egg interaction, thereby reducing the potential for successful fertilization. A positive test result may be indicative of past exposure of the male reproductive system to his own immune system in the course of injury (e.g.. Vasectomy procedure) or inflammation.

This is an immuno-bead-based assay for testing sperm function. In the process of fertilization, just prior to penetration into the egg, the sperm is activated to go through a phenomenon called the Acrosome Reaction, in which the apical membranes of the sperm cell ruptures, releasing various enzymes. This function exposes various surface antigens which were hidden inside the acrosome prior to this stage. The test examines the ability of the sperm to perform the Acrosome reaction by testing the exposure of a CD46 following artificial induction of this reaction.

An ELISA test for the Inhibin B levels in serum. This hormone is a gonadal product (primarily of the Sertoli cells) involved in spermatogenesis, and thus is a more direct measure of testicular disorders than the classical FSH. This hormone can distinguish between spermatogenetic vs. obstructive causes for azoospermia. It has been shown to be reduced in azoospermia, idiopathic hypogonadotropic hypogonadism (IHH), infertility with high FSH, and Kleinfelter Syndrome, but not in vasectomy or asteno- or teratozoospermia. It is practically absent following orchidectomy. Inhibin B, in males, can also be used to assess abstinence time, and to monitor the response to r-hFSH therapy.

This test should be offered as a male infertility test. Y-chromosome micro-deletions have been found in DNA from patients with azoospermia (lack of sperm in the semen), oligozoospermia (low titers of sperm in semen) or normo-spermic individuals. It utilizes 5 groups of primer pairs in a multiplex PCR amplification technology, to identify micro-deletions (small segments of DNA missing from the sequence of specific genes) in any one of 20 specific loci on the euchromatin of the long arm of the Y chromosome. All 20 amplified sites reside within 4 azoospermia factor (AZF) regions. Genes in these sites have been associated to male infertility, and thus, deletions therein do provide a genetic basis for the infertile male phenotype.

Assessment of abnormal sperm morphology using strict criteria has become a valuable tool in male fertility evaluation. Proportion (%) of sperm with abnormal forms in head, acrosome, midpiece, or tail carefully identified on thin stained sperm smears under a microscope and % of normal sperm are scored. Clinical studies have shown that cases with >4 % morphologically normal sperms have very poor fertilization and pregnancy rates , while cases with normal forms >14% shown high fertilization and pregnancy rates. Abnormal morphological features of sperms in ejaculates with relatively normal sperm counts and motility have been well correlated with low fertilization and pregnancy rates, and thus provide a very useful parameter in predicting success in infertility treatments, IVF or GIFT procedures.

DNA decondensation in the sperm nucleus is a prerequisite for the formation of the nucleus and the subsequent DNA synthesis and chromatin recondensation in the course of fertilization. Since in all the studies to date, sperm from males with either unexplained infertility showed 100 % correlation between positive (abnormal) SDD^TMtest result, and failure in pregnancy by any ART approach, this test should be offered to male infertility patients (to better define treatment solutions), men with unexplained infertility (to identify etiology) , men working in an environment where they may have been exposed to reproductive toxicant (to monitor employment related reproductive health hazards) and men with bad sperm scores following any treatment (to quantify improvement in semen quality) . The test involves sperm cell permeabilization by lysolecithin, followed by chemical reduction and exposure of the chromatin to Xenopus laevis frog extract, mimicking the human egg cytoplasm environment. This procedure results in DNA decondensation within 15 minutes of mixing, which is then visualized and quantitatively scored using a phase contrast microscopy. If less than 80% of the cells decondense, the sperm is incapable of fertilization at that time.

Physician's Guide to Reproductive Disorder Tests
- 2nd Edition

Table of contents

Contributors And References

Value Added Services

Commitment to Excellence

Requirements for Quality Assurance

Comprehensive Panels

Guide to Infertility Testing

A. Ovarian Reserve / Premature Ovarian Failure (POF)

B. Poly Cystic Ovary Syndrome

C. Hyper-androgenism Panel

D. Ovarian Tumor Markers

E. Unexplained Infertility /Implantation Failure & Recurrent Pregnancy Loss

F. Thrombophilia (Coagulation Disorders)

G. High Risk Pregnancy Monitoring

H. Andrology (Male Factor)

Specimen and Shipping Instructions Chart

Physician's Guide to Reproductive Disorder Tests - 2nd Edition

Table of contents

Contributors And References

Value Added Services

Commitment to Excellence

Requirements for Quality Assurance

Comprehensive Panels

Guide to Infertility Testing

A. Ovarian Reserve / Premature Ovarian Failure (POF)

B. Poly Cystic Ovary Syndrome

C. Hyper-androgenism Panel

D. Ovarian Tumor Markers

E. Unexplained Infertility /Implantation Failure & Recurrent Pregnancy Loss

F. Thrombophilia (Coagulation Disorders)

G. High Risk Pregnancy Monitoring

H. Andrology (Male Factor)

Specimen and Shipping Instructions Chart

Physician's Guide to Reproductive Disorder Tests
- 2nd Edition