WHY DO WOMEN HAVE SPORADIC MISCARRIAGE: A NEW CLINICAL RESEARCH STUDY IF YOU HAVE HAD ONE OR MORE UNEXPLAINED “SPORADIC” MISCARRIAGES, A SINGLE BLOOD SAMPLE WITH STATE-OF-THE-ART TESTING FOR BLOOD CLOTTING ABNORMALITIES MAY REVEAL INHERITED OR ACQUIRED CLOTTING DISORDERS WHICH CAUSE MISCARRIAGE. If these inherited or acquired clotting disorders are known before your next pregnancy, then safe, simple, anticoagulation during pregnancy with low molecular weight heparin can, to a large degree, prevent subsequent pregnancy loss. CALL (513-924-82500), FAX (513-924-8273), OR EMAIL ([email protected]) TO MAKE AN APPOINTMENT FOR A FREE EVALUATION AT THE JEWISH HOSPITAL CHOLESTEROL CENTER. This will take about 1 hour of your time. All results will be entirely confidential, and will be released only to yourself and to your physicians of record. Health Alliance, Jewish Hospital Cholesterol Center, Charles J. Glueck MD, Medical Director ABC Building, 3200 Burnet Ave, Cincinnati OH, 45229 Phone: 513-924-8250 Fax: 513-924-8273 E-mail: [email protected] or [email protected] web: /glueck/cholesterol.html HERITABLE HYPOFIBRINOLYSIS AND THROMBOPHILIA: PATHOETIOLOGIES OF FIRST TRIMESTER MISCARRIAGES, AND DISORDERS OF PREGNANCY BACKGROUND: Miscarriage occurs in approximately 13% to 20% of pregnancies.1 In recurrent spontaneous miscarriage, which is the most thoroughly studied, parental karyotyping reveals chromosomal abnormalities in < 5% of couples.1 Anatomic uterine cavity abnormalities can be found in up to 20% of women.1 Recent studies suggest that the great majority of other first trimester miscarriages are caused by inherited and/or acquired thrombophilia or hypofibrinolysis, or both.2-23 However, all published studies have focused on women with recurrent pregnancy loss ≥ 3 consecutive miscarriages before 20 weeks, and none have focused on women with a single first trimester miscarriage. Historically, known disease and environmental risk factors for clinically serious complications of pregnancy have included juvenile onset diabetes, gestational diabetes, cocaine-heroin-crack abuse, alcohol abuse, and cigarette smoking. Historically, women with otherwise unexplained recurrent first trimester miscarriages have usually been evaluated for disease and environmental risk factors (as above), and for the antiphospholipid antibody syndrome. Pre-conception knowledge of heritable thrombophilic or hypofibrinolytic risk factors2-23 in women and with recurrent miscarriage is important. We have shown that Lovenox thromboprophylaxis is effective in women with recurrent pregnancy loss.3 Bick et al21 have shown that preconception treatment with low dose aspirin and within-pregnancy treatment with low molecular weight heparin will protect against the pregnancy complications of thrombophilia. Fragmin (a low molecular weight heparin), 5000 units/day, is effective in thromboprophylaxis of pregnancy. It is also likely that preconception treatment with low dose aspirin and within pregnancy treatment with low molecular weight heparin (Fragmin, Lovenox) will protect against very early pregnancy loss (EPL). SPECIFIC AIM: We will assess thrombophilia and hypofibrinolysis to determine whether, and to what degree coagulation disorders are pathoetiologic for first trimester miscarriages. Two groups of women will be studied, as follows: 1. > 1First trimester miscarriage: Women having sustained > 1 first trimester miscarriage will be eligible for participation. Up to 100 women will be studied. 2. > 1 normal pregnancy without any complication of pregnancy: Women with > 1 naturally occurring pregnancy with no first trimester miscarriage and without complications of pregnancy will be matched by women with = 1 complication of pregnancy (as above), by are, race, and where possible, body mass index. Up to 100 women will be studied. PREVIOUS STUDIES FROM THE JEWISH HOSPITAL CHOLESTEROL CENTER: We have now documented that genetic thrombophilia, particularly the G1691A Factor V Leiden mutation is a major etiology of recurrent pregnancy loss, and that thromboprophylaxis with low molecular weight heparin will, to a large degree, prevent pregnancy loss in women heterozygous for the Factor V Leiden mutation.3 As the placenta rapidly grows, it must quickly establish arterio-venous anastomoses with the maternal circulation in the endometrium and myometrium, which, of necessity must involve hemorrhage and thrombosis. It is our hypothesis, and that of Kupferminc et13 that thrombophilia and/or hypofibrinolysis, leading to increased placental thrombosis, produce placental insufficiency and complications of pregnancy. Abstracts 2003-2004 Glueck CJ, Wang P, Bornovali S, Sieve L. Pregnancy loss, polycystic ovary syndrome, thrombophilia, hypofibrinolysis, low molecular weight heparin. Abstract, J Invest Med, 2004;;52 (Suppl 2), S349 Presented, Chicago, IL, national Clinical Research Meetings, 2004. Bornovali S, Glueck CJ, Wang P, Goldenberg N, Sieve-Smith L. Recurrent pregnancy loss, thrombophilia, hypofibrinolysis, and polycystic ovary syndrome. J Invest Med 2003, suppl 2, S364., Presented, National Clinical Research Meetings, Baltimore MD, 3/14/-16/03. Publications 2003-2004 542. Glueck CJ, Wang P, Bornovali S, Goldenberg N, Sieve L. Polycystic ovary syndrome, the G1691A factor V Leiden mutation, and plasminogen activator inhibitor activity: Associations with recurrent pregnancy loss. Metabolism 2003;;Dec 52:1627-32. 543. Glueck CJ, Wang P, Goldenberg N, Sieve L. Pregnancy loss, polycystic ovary syndrome, thrombophilia, hypofibrinolysis, enoxaparin, metformin. Clinical and Applied Thrombosis/Hemostasis, In Press, 12/18/03 EXPERIMENTAL DESIGN AND METHODS: Subjects: Through our website (/glueck/cholesterol.html) and through obstetricians, gynecologists, and family practice physicians in the tristate area, we plan to recruit two groups of 100 women, one having had ≥ 1 unexplained miscarriage and 100 women (matched to the first group by age, race, and BMI) with ≥ 1 uncomplicated pregnancy. Inclusion criteria will be as follows, by group: 1. ≥ 1 unexplained 1st trimester pregnancy loss: Women with ≥1 unexplained pregnancy first trimester pregnancy loss. These will be women with no known uterine anatomical abnormalities or known karyotypic abnormalities. 2. Women with ≥1 uncomplicated pregnancy without any pregnancy loss. Exclusion criteria Alcoholism, juvenile onset diabetes, known cocaine, heroin, or crack use. Women with known anatomic defects (bicornuate uterus, etc) will also be excluded. Cigarette smoking will not be an exclusion criterion. PROTOCOL: Study Entry 1. Inclusionary criteria for ≥ 1 first trimester miscarriage in the absence of known uterine anatomic abnormality or karyotypic abnormality will be verified by review of hospital and physician records, by history, and by brief physical examination. Inclusionary criteria for women with ≥ 1 uncomplicated pregnancy without 1st trimester miscarriage will be verified by review of hospital and physician records, by history, and by brief physical examination 2. History: Information will be obtained on demographic details: age, race, height, weight, systolic and diastolic blood pressures and a detailed health history. 3. Laboratory measures: The following measures will be obtained in a blood sample: PCR measures: G1691A Factor V Leiden mutation, G20210A Prothrombin gene mutation, C677T and A1298C MTHFR mutations, 4G4G mutation of the PAI-1 gene: Serologic measures: Homocysteine, anticardiolipin antibodies, lupus anticoagulant, Factor VIII, Factor XI, resistance to activated protein C, protein C, protein S, plasminogen activator inhibitor activity (PAI- Fx). DATA ANALYSIS : Sample Size and power estimates: Given the prevalence of V Leiden heterozygosity of 5%, prothrombin gene heterozygosity of 6%, MTHFR C677T homozygosity or C677T-A1298C compound heterozygosity of 11%, and PAI-1 gene 4G4G homozygosity of 20%, in order to declare a significant difference against a 3 fold increase in gene mutations in the miscarriage group, at a p<.05, 123, 91, 29, and 13 patients would need to be studied. Hence, our patient group sizes of 100 should be sufficient to allow declaration of significant differences versus healthy normal controls. Data Management: The data management system for the study will consist of four major components;; i. a baseline data entry system at the clinic;; ii. a laboratory data entry system for the urine assays;; iii. a computer mainframe data merging and management system;; iv. a statistical analysis and summarization system. Data Collection: The baseline data entry system will be facilitated on a Pentium-PC using display screens, which resemble paper forms. These data screens will be windows-menu driven and will prompt data entry in an orderly sequence. Baseline data will be cumulated weekly on diskette and transferred to the mainframe computer. Duplicate data files will be maintained at the clinic and at the mainframe computer. The laboratory data system will be maintained on a Pentium-PC using a spreadsheet program. Data will be cumulated on a weekly basis and transferred to the mainframe computer for merging with the baseline data. Duplicate data files will be maintained at both locations. Data Editing and Documentation: The task of editing the baseline data will occur at three levels. First, a validity check will be performed by the computer program for each data item as it is entered. Inappropriate characters or out-of-range values will be challenged. Second, when the entry of the baseline interview data has been completed, a consistency check will be performed by the computer, and prompt notification of any problems will be made. Third, when the merging of the baseline and laboratory data is made in the mainframe computer, further consistency reviews will be made. The laboratory values will also be scrutinized at entry into the spreadsheet by the computer for validity and consistency. Any corrections to the data base which are made subsequent to the initial entry will be noted and kept in a separate data file to provide an "audit trail" of data corrections. Quality Control: Duplicate assays will be prepared on a random basis and compared for consistency with their counterpart assays. Serious discrepancies will be noted and appropriate remedial action will be taken. Data Confidentiality and Security Several steps will be taken to ensure the privacy and security of the study data. Each user of the data entry system will be issued a password by the supervisor, or Principal Investigator. Data diskettes and duplicate diskettes will be kept in a locked file or other secure location when not in use. Merged data files at the mainframe computer will be password protected, and duplicate files will be maintained in a secure fireproof vault. Data Analysis Summary statistics will be prepared quarterly (monthly) and patient accrual will be monitored on a weekly basis. The rates of occurrence of the several polymorphism's of interest in the study will be noted both for maternal and fetal patients. At a convenient intermediate time point in the study a more complete analysis of the genetic factors and their influence will be made using maximum likelihood methods and Bayesian and likelihood ratio tests. Poisson regression analysis will also be used so that ancillary baseline data can be incorporated into the analysis. To ensure the absence of bias in the final significance levels for the study, the method described in Pocock will be used for the intermediate analysis. First: The frequency of abnormalities in the measurements of thrombophilia and hypofibrinolysis between women having at least one 1st trimester miscarriage those with ≥1 pregnancy and no miscarriage will be compared using non-parametric tests of difference and X2 analyses. Comparisons will also be made against the population distribution of wild type normal genes and thrombophilic-hypofibrinolytic gene mutations in 234 healthy normals previously studied by our group. Second: simple correlations between the measurements of coagulation and pregnancy outcomes will be done in the 2 groups of women whose pregnancy outcomes are known. Third: stepwise logistic regression will be done for each pregnancy outcome with the dependent variable being outcome yes (for example first trimester miscarriage) vs outcome no (no first trimester miscarriage), and explanatory variables including age, sex, number of pregnancies, Quetelet Index, fasting blood sugar, and coagulation disorders. This logistic regression will be done separately for each coagulation disorder and categorically for women having ≥1 coagulation disorder to try to dissect out the independent contributions of coagulation disorders to complications of pregnancy. Setting: The study will be carried out in the outpatient Alliance Cholesterol Center on the fifth floor of Burnet Ave pavilion, ABC building. Laboratory methods and facilities The cDNA PCR assays will be carried out in the coagulation laboratory of the Health Alliance or at the Coagulation Laboratory of Children’s Hospital Medical Center. Similarly, all serologic assays will be carried out at these laboratories. RISKS AND BENEFITS: Documentation of coagulation disorders of a heritable nature might, were they known to medical insurance companies, be identified as a pre-existing risk for thrombosis. However, the data for this study will be processed following strict confidentiality rules and will be released only with signed patient consent. Benefits include ability to preventatively treat severe coagulation disorders with targeted anticoagulant therapy. Payment: There will be no financial remuneration. Parking will be free in the Alliance ABC garage. Subject costs: There are no anticipated costs. The study is entirely free to participants LITERATURE CITED: 1. Tulppala M, Palousuo T, Ramsay T, et al: A prospective study of 63 couples with a history of recurrent spontaneous abortion: contributing factors and outcome of subsequent pregnancies. Hum Rep 1993;;8:764-70. 2. Glueck CJ, Wang P, Bornovali S, Goldenberg N, Sieve L. Polycystic ovary syndrome, the G1691A factor V Leiden mutation, and plasminogen activator inhibitor activity: Associations with recurrent pregnancy loss. Metabolism 2003;;Dec 52:1627-32. 3. Glueck CJ, Wang P, Goldenberg N, Sieve L. Pregnancy loss, polycystic ovary syndrome, thrombophilia, hypofibrinolysis, enoxaparin, metformin. Clinical and Applied Thrombosis/Hemostasis, In Press, 12/18/03 4. Glueck CJ, Wang P, Fontaine RN, et al. Plasminogen activator inhibitor activity: an independent risk factor for the high miscarriage rate during pregnancy in women with polycystic ovary syndrome. Metabolism 1999;;48:1589-95 5. Glueck CJ, Phillips H, Cameron D, et al. The 4G/4G polymorphism of the hypofibrinolytic plasminogen activator inhibitor type I gene: an independent risk factor for serious pregnancy complications. Metabolism 2000;;49:845-52. 6. Glueck CJ, Awadalla SG, Phillips H, et al. Polycystic ovary syndrome, infertility, familial thrombophilia, familial hypofibrinolysis, recurrent loss of in vitro fertilized embryos, and miscarriage. Fertil Steril 2000;;74:394-7. 7. Rey E, Kahn, David M, et al: Thrombophilic disorders and fetal loss: a meta-analysis. Lancet 2003;;361:901-8 8. Rai R. Is miscarriage a coagulopathy. Curr Opin Obstet Gynecol 2003;;15:265-8 9. Loomenthal D, Von Dadelszen P, Liston P, et al: The effect of factor V Leiden carriage on maternal and fetal health. CMAJ 2002;;167:48-54. 10. Balasch J, Creus M, Fabregues F, et al. Antiphospholipid antibodies and human reproductive failure. Human Reprod 1996;;11:2310-5. 11. Kutteh WH, Rote NS, Silver R. Antiphospholipid antibodies and reproduction: the antiphospholipid antibody syndrome. Am J Reprod Immunol 1999;;41:133-152. 12. Cubillos J, Lucena A, Lucena C, et al: Incidence of autoantibodies in the infertile population. Early Pregnancy 1997;;3:119-124. 13. O'Connor JF, Ellish N, Kakuma T, et al. Differential urinary gonadotrophin profiles in early pregnancy and early pregnancy loss. Prenat Diagn 1998;;18:1232-40. 14. Kupferminc MJ, Eldor A, Steinman N, et al: Increased frequency of genetic thrombophilia in women with complications of pregnancy. New Eng J Med 240:9-13, 1999. 15. Rand JH, Wu XX, Andree HA, et al: Pregnancy loss in the antiphospholipid-antibody syndrome--a possible thrombogenic mechanism. N Engl J Med 337:154-160, 1997. 16. Younis JS, Ohel G, Brenner B, Ben-Ami M: Familial thrombophilia--the scientific rationale for thrombophylaxis in recurrent pregnancy loss? Hum Reprod 12:1389-90, 1997. 17. Gris JC, Ripart-Neveu S, Maugard C, et al: Respective evaluation of the prevalence of haemostasis abnormalities in unexplained primary early recurrent miscarriages. The Nimes Obstetricians and Haematologists (NOHA) Study. Thromb Haemost 77:1096-1103, 1997. 18. Rai RS, Regan L, Chitolie A, Donald JG, Cohen H: Placental thrombosis and second trimester miscarriage in association with activated protein C resistance. Br J Obstet Gynaecol 103:842-4, 1996. 19. Younis JS: Factor V Leiden--a novel etiology of the long-standing thrombosis theory for recurrent pregnancy loss. Am J Obstet Gynecol 178:1107-8, 1998. 20. Richards EM, Makris M, Preston FE: The successful use of protein C concentrate during pregnancy in a patient with type 1 protein C deficiency, previous thrombosis and recurrent fetal loss. Br J Haematol 98:660-1, 1997. 21. Brenner B, Maudel H, Lanir N, et al: Activated protein C resistance can be associated with recurrent fetal loss. Br J Haematol 97:551-4, 1997. 22. Bick RL. Recurrent miscarriage syndrome and infertility caused by blood coagulation protein or platelet defects. Hematol Oncol Clin North Am 2000;;14:1117-31 23. Blomback M, Bremme K, Hellgren M, Siegbahn A, Lindberg H: Thromboprophylaxis with low molecular mass heparin 'Fragmin' (dalteparin) during pregnancy-a longitudinal safety study. Blood Coag and Fibrinolysis 9:1- 9, 1998 24. Clifford K, Rai R, Watson H, Regan L: An informative protocol for the investigation of recurrent miscarriage: preliminary experience of 500 consecutive cases. Human Reproduct 9:1328-32, 1994. STATEMENT OF INFORMED CONSENT: First trimester miscarriage 1. Introduction: Before agreeing to participate in this study, it is important that the following explanation of the proposed procedures be read and understood. It describes the purpose, procedures, benefits, risks and discomforts, and precautions of the study. It also describes alternative procedures available and the right to withdraw from the study at any time. It is important to understand that no guarantee or assurance can be made as to the results. 2. Objectives: We will carry out measures of thrombophilia and hypofibrinolysis to determine whether, and to what degree these coagulation disorders cause 1st trimester miscarriage. 3. Procedures: Through our website (http://www.health-alliance.com/glueck.html), and through obstetricians, gynecologists, and family practice physicians, we plan to recruit the following groups of women, as follows: 1) those having sustained at least 1 first trimester miscarriage;; 2) those with > 1 naturally occurring pregnancy without complications of pregnancy. In these 2 groups of women, we will obtain detailed measures of blood clotting. 4. All measures of blood coagulation are entirely free. The results will be provided to each subject and to the obstetrician. No data will be released without written subject consent. This research study does not, however, cover the usual and customary costs of the subjects’ regular visit to their obstetrician. Six teaspoons of blood (30 cc) will be obtained by a single blood drawing for measurement of clotting factors known to cause complications of pregnancy. These clotting factors include those which increase the risk of blood clotting, called "thrombophilic" and those which reduce the ability to dissolve blood clots, called "hypofibrinolytic" When and if laboratory tests for coagulation disorders which can cause 1st trimester miscarriage are found to be present, if approved by the subject, discussions will be initated with the subject's doctors to review medical interventions which might promote conception and prevent future miscarriages. 5. Exclusion Criteria: Alcoholism, juvenile onset diabetes, uterine abnormalities known to complicate pregnancy, crack, heroin, or cocaine addiction will exclude subjects from participation, because these are known "risk factors" which can predispose to complications of pregnancy, separate from the coagulation risk factors being studied. 6. Risks and Precautions: The only known risks of the study involve those of taking blood. These include commonly, the occurrence of discomfort and/or bruise at the site of puncture;; and less commonly, the formation of a small blood clot or swelling of the vein and surrounding tissue, and bleeding from the puncture site. 7. Benefits: In women with one or more miscarriages, identification of risk factors for future miscarriages should help promote successful conception and should help to prevent future miscarriages, by allowing preventive measures such as anticoagulation with low molecular weight heparin very early during subsequent pregnancies. This has been shown to sharply reduce risk of miscarriage in women with inherited coagulation disorders. In normal women, without infertility and with > 1 pregnancy without complications, benefit will be obtained by a broad assessment of coagulation disorders, which, in aggregate affect about 12% of "healthy normal" women. Knowledge of such disorders is important for future use of estrogen replacement therapy or oral contraceptives with estrogen, neither of which should be used in women with the inherited V Leiden or Prothrombin gene mutations. 8. Alternative Procedures Available: The laboratory measures for thrombophilia and hypofibrinolysis are commercially available in the community at very specialized centers which study blood clotting. 9. Right of Refusal: Participation in the study is voluntary. If you refuse to participate, there will be no penalty or loss of any benefit to which you are entitled. If you volunteer to participate in the study, you may withdraw from the study at any time without a penalty or loss of any benefit to which you are entitled. The patient will notify the study physician in the event of withdrawal and return to the center for a termination visit. At this visit, all unused study drug should be returned. 10. Confidentiality: Your medical records will be treated as confidential. Only authorized personnel will have access to the records. It is possible that an authorized person from the Department of Health and Human Services, Food and Drug Administration (FDA) or other federal agency will inspect the records. In all instances except the FDA, your name will remain confidential;; only the scientific information will be disclosed. In the case of new drug investigation, the FDA does have a right to know your name and this may be revealed to the proper authorities from the FDA if requested to do so. Unauthorized persons will not be permitted to examine your records without your written consent. 11. Availability of Information: Any question that you have concerning any aspect of this study or your rights as a subject in the study will be answered by: Charles J. Glueck, M.D., or my Associate, James E. Lang, M.D. at the Jewish Hospital Cholesterol Center, 569-2297. The Jewish Hospital of Cincinnati, Inc. follows a policy of making all decisions concerning financial compensation or medical care for injuries occurring during or caused by participation in biomedical or behavioral research on a case-by-case basis. If you believe you have been injured as a result of any procedure of research, contact Dr. Steven Goldberg, Chairperson, IRB, 513/686-5446. C O N S E N T I,__________________________, have read and understand the preceding explanation, and I consent to participate in the study as described above in section 2 captioned "Objectives." I understand that I am free to withdraw without penalty or loss of benefit from this investigation at any time. Should I wish to withdraw I have been assured that standard therapy for my condition will remain available to me. I have been informed of the probably consequences of my withdrawal from the study. SUBJECT:___________________________________________ DATE:_______________ INVESTIGATOR_______________________________________ DATE:_______________ WITNESS:___________________________________________ DATE:_______________ In the case of a minor or guardianship, the person signing the consent form must indicate the authority under which he/she is signing: ON BEHALF OF:______________________________________DATE:___________________ SIGNATURE:_________________________________________ PARENT GUARDIAN E-mail: [email protected] or [email protected] Fax: 513-924-8273