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Recurrent Pregnancy Loss NOVAK 2003 CONTENTS Introduction Etiology Evaluation Treatment INTRODUCTION 70% of pregnancies are lost<viability 50% “”””””””””””””””””””””””<1st missed period 31% “””””””””””””””””””” >implantation(by β-HCG) 15% ””””””””””””””””””””””’ < 20 weeks Definition: ≥ 3 spontaneous abortions < 20 weeks % = 1:300 Risk of recurrence : > 2 abortions = 24% > 3 “””””””””” = 30% > 4 “””””””””” = 40-50% Evaluate RPL after 2 spontaneous abortions if: age > 35 years subfertility/infertility FHMs are seen in both = 1% of pregnancies require evaluation. ETIOLOGY 1) 2) 3) 4) 5) 6) Genetic Anatomical Endocrine Infection Immunological Others = 3.5-5% = 12-16% = 17-20% = 0.5-5% = 20-50% = 10% 10-15% of RPL is due to APAS and chromosomal abnormalities. GENETIC CAUSES Mostly due to balanced translocation: Reciprocal translocation normal gametes Robertsonian translocationbalanced gametes or unbalanced gametes Most embryos with chromosomal abnormalities do not survive. Live offspring are either carriers of: Balanced translocation Monosomy/trisomy ( in robertsonian T ). Monosomic fetuses: may be mosaic X gametes can only survive ↑ in IVF Trisomic fetuses: may also be mosaic. include trisomies 13,16,18,and 21 survive more than monosomic fetuses We can't exclude chromosomal abnormalities by: negative family history history of a live term birth The frequency of chromosomal abnormalities is inversely proportional to the number of previous abortions. Couples with genetic abnormalities are usually with no children. Parents with history of: spontaneous abortion ± SB ± live birth ( ± anomalies ) are candidates for parental karyotype, but this may be insufficient because: o aneuploid sperm may be motile o chromosomal abnormality may be in 1 sperm Other structural chromosomal anomalies include: • Inversion • Insertion • Chromosomal mosaicism • X-linked anomalies (males do not survive) • Single gene anomalies such as cystic fibrosis (detected by detailed family history). HERITABLE THROMBOPHILIAS 1. 2. 3. 4. 5. 6. 7. Hyperhomocysteinemia mutation Antithrombin III mutation Protein-C and protein-S deficiency Factor V Leiden mutation Prothombin mutation Factor XII deficiency Factor XIII and fibrinogen defects 1- HYPERHOMOCYCTEINEMIA Homocysteine is derived from dietary methionine. It is metabolized in the presence of methyl-tetra-hydro-folate reductase enzyme (MTHFR) + dietary folic acid, vit B6, vit B12 into: Cystathione or back into methionine. Hyperhomocysteinemia may be either: - Congenital due to point mutation in MTHFR (common ) - Acquired due to dietary deficiency of folic acid, vit B6, or vit B12 Hyperhomocysteinemia is linked to thrombosis and RPL. 2- ANTITHROMBIN III MUTATION Antithrombin III inhibits: - thrombin - factor IXa,Xa,XIa,XIIa Antithrombin III mutation is rare but it is the worst prognostic factor of all heritable thrombophilias. It is associated with RPL and thrombosis. 3- PROTEIN-C AND PROTEIN-S DEFICIENCY Protein-C and protein S affect: - X Xa reaction - prothrombin thrombin reaction Protein-C and protein S deficiency is linked to placental thrombosis and pregnancy complications. 4– FACTOR V LEIDEN MUTATION Factor V prevents interaction with proteinC. Multiple point mutation in the cleavage site of factor V Leiden resistance to protein-C. Factor V Leiden is associated with RPL. It is the most common heritable thrombophilia among white people. 5– PROTHROMBIN MUTATION It is linked to thrombosis and pregnancy loss. 6– FACTOR XII DEFICIENCY Increase in Japanese people and is linked to recurrent pregnancy loss. 7- FACTOR XIII AND FIBRINOGEN DEFECTS Both are associated with fetal loss and involve other complex mechanisms than simple thrombosis and infarction. II-ANATOMICAL CAUSES 1- Congenital causes: incomplete fusion of Mϋllerian ducts incomplete septum resorption (septate, subseptate) cervical anomalies The most commonest anomalies are septate 60% of spontaneous abortion in the 2nd trimester and sometimes the 1st trimester. Inutero exposure to DES may cause congenital anomalies. The most common lesion in DES is hypopleasia abortion in the 1st or 2nd trimesters. 2- Acquired causes: fibroid polyps adenomyosis Asherman syndrom cervical incompetence III- ENDOCRINE CAUSES Endocrine changes of menstrual cycle Endocrine factors: 1. Luteal phase defect 2. Diabetes 3. Thyroid disease 4. Hyperprolactinemia 5. Ovarian reserve ENDOCRINE CHANGES OF MENSTRUAL CYCLE Follicular phase and ovulation: - abnormal blastocyst transport - alteration of uterine receptivity - improper function of CL From ovulation until 7-9 weeks - failure of CL to produce enough P - impaired delivery of P to the uterus - inappropriate use of P by the decidua Near the time of luteal-placental shift - trophoblast unable to produce biologically active P ENDOCRINE FACTORS: 1- LPD It is inadequate or inappropriately timed endometrial maturation due to: - Premature aging of the oocyte - Dyssynchronous maturation of the endometrium - PCO: . PCO is present in 80%of RPL . It is associated with: , obesity , ↑ androgen level affect markers of uterine receptivity , ↑ insulin resistance 2- DIABETES DM type II ↑ spontaneous abortion IDDM: - Pregestational glycemic threshold above which abortion ↑ - Hyperglycemia damage the embryo - Vascular changes in advanced IDDM ↓placental blood flow. 3 – THYROID DISEASE Ovulatory disfunction LPD Pregnancy is associated with ↑ requirement to T4. Hypothyroidism is associated with ↑ spontaneous abortion. Even when T4 is normal, ↑ antithyroid antibodies may cause ↑ RPL. (Antithyroid antibodies could be markers of a generalized autoimmune disease). 4 - HYPERPROLACTINEMIA Affect reproduction by: - Direct effect on endometrium - Indirect immuno-mediator effect. 5 – DIMINISHED OVERIAN RESERVE Can be evaluated by: - Day 3 FSH - Day 3 estradiol - CC challenge test. 4 - INFECTION Mycoplasma Ureaplasma Chlamydia β-streptococcus: BV is linked to ↑ RPL and PTL in a large study . Viral infection as: - cyromegalovirus - herpes simplex Mechanisms of action: - villitis - mechanisms protecting the fetus from autoimmune rejection↑ infection - activation of immune reactions PTL, PROM, IUGR. 5 – IMMUNE CAUSES I. II. III. Basic immunology Cellular immunity Humoral immunity I - BASIC IMMUNOLOGY 1- Innate responses: 1st line defense. None antigen specific. Mechanisms are: - phagocytosis - complement fixation, - lysis by NK cells. 2- Acquired responses: Antigen-specific. Modulated by T and B cells. Divided into: - Primary immune response: on 1st contact with the Ag - Secondary immune response: on subsequent contact with the Ag. Rapid and strong. ANTIGEN SPECIFICITY Regulated by 2 sets of genes present at major histocompatability complex (MHC) located on chromosome 6: Class I MHC molecules: HLA A, B, C Present at the surface of nearly all cells for intracellular pathogens. Major ligand for: TCR on CD 8+ cytotoxic and suppressor cells many receptors on NK cells. Class II MHC molecules: HLA DR, DP, DQ Present on the surface of antigen-presenting cells as: Dendritic cells, macrophages- monocytes, B-cells, and tissue specific cells. Protect against extracellular pathogens. Major ligand for: TCR on CD-4+ T- helper cells. EDUCATION AND HOMING Fetal bone-marrow derived T-cells move with the blood until they reach the thymus. In the thymus they are divided into: - Those who will express CD4 co-receptor - Those who will express CD8 co-receptor And auto-reactivity is eliminated; that is the cells can recognize non-self but not react against self. MUCOSAL AND PERIPHERAL IMMUNE SYSTEMS Peripheral immune system: Located in the blood and spleen. Responsible for protection against blood-born pathogens. Mucosal immune system: Located in GIT, RT, GUT, lacrimal and mammary glands. Responsible for protection against exogenous pathogens. II - CELLULAR IMMUNE RESPONSE 1. 2. 3. 4. Resident cells Immune cell education and homing to the reproductive tract Antigen presentation at the maternal-fetal interface Regulation of the decidual immune cells 1- RESIDENT CELLS The endometrium is populated by: T-cells NK-like cell Macrophages Very few B-cells At implantation, there is a dramatic change in the decidual cells and 70-80% of the cells are called: Decidual granular lymphocytes, or Large granular, or Decidual NK If these cells are NK, then implantation site represent the largest accumulation of NK in humans. The function of these cells is unclear. In animals, peripheral NKT cells (which have the characteristics of both T cells and NK cells) ↑ spontaneous abortion. Most peripheral immune system T-cells express TCR-αβ. In the reproductive system T-cells express in addition to this TCR-γδ which ↑ early in pregnancy and are responsible for direct, none MHC restricted immune recognition of Ag within tissues = more protection. A subset of macrophage called suppressor cells promot anti-inflammatory properties and may be implicated in maintenance of pregnancy. To sum up: Human decidua is populated by characteristic immune cells. Alteration of these cells may pregnancy loss. Alteration of these cells occur in RPL and not isolated abortion. 2 – IMMUNE CELL EDUCATION AND HOMING How these cells are selected, educated, and how they home to the reproductive tract is still unknown, but animal studies showed that: These cells may be educated outside the thymus They may have different mechanisms other than MHC They may differ from mucosal and peripheral immune systems. In the mucosal immune system, cells select these sites through interaction between: Cell surface molecules on the immune cell= integrins “”””””””””””””””””””””””””” endothelial cell= selectins or vascular wall adhesive molecule (VCAM) == homing. Animal studies showed that: Integrins are expressed in the reproductive tract Knowing the mechanism of selection and homing may aid in ttt of RPL. 3 – ANTIGEN PRESENTATION In the past trophoblast escape recognition by ↓ expression of MHC Ag. Now obsolete, although trophopblast cannot express class II MHC molecules and class I MHC HLA: A and B Ags. Extravillous cytotrophoblast cells express class I: C, E and D Ags. These cells are characterized by excessive invasive properties: They move from the tip of villi deep into the decidua, invade decidual blood vessels, and replace endothelial cells of decidual spiral arteries. Their behavior reflect non-MHC mechanisms as integrin switching and expose the fetus to recognition. Any cell not expressing Ag is killed by NK. In addition to protection from killing by NK cells, expression of MHC HLA: C, E, and G by the trophoblast may modulate cytokines expression, aid in invasion, and aid in maternal acceptance. G Ag was linked to disorders of placental invasion. MHC polymorphism was not linked to RPL. Interferon-γ was believed to ↑ spontaneous abortion by ↑ expresion of class I and class II MHC which ↑ cytotoxic attack of T cells, but aborted tissues were not shown to express MHC Ags. Class II genotypes were linked to adverse pregnancy outcome, RPA, and ↑ susceptibility to disease as autoimmune disease and DM. 4– REGULATION OF DECIDUAL IMMUNE CELLS I. II. III. Alterations in T- helper cell phenotype Reproductive hormones and immunosuppression Tryptophan metabolism I –ALTERATION IN T-HELPER CELL PHENOTYPE Ag-stimulated immune responses involving CD4+ cells can be divided into: T-helper cell subset 1 (TH1) T-helper cell subset 2 (TH2) based on the character of CD4+ cell and associated cytokine. Undifferentiated T- helper cells are differentiated into: TH 1 in the presence of interferon γ (INF-γ). TH 1 is associated with inflammatory responses and: INF-γ, IL 2, IL 12. TH 2 in the presence of IL 4. TH2 is associated with antibody responses and IL 4, 5, 6, 10, TNF-β. TNF-α is expressed by both T-helper cells. There is a reciprocal relationship between TH 1, TH 2, and cytokines. Most researches agree that: TH 1 responses are harmful to the embryo TH 2 responses are present in most normal pregnancies TH 1 responses are present in some patients of RPL 60-70% of none pregnant patients with history of RPL show abnormal T-cell responses invitro compared to <3% in controls. The type of CD4+ cellular response to the implanting fetus is controlled not only by the types of cells in the decidua but also by the cytokine enviroment at the maternal –fetal inteface. Cytokines may affect reproduction by regulating: Type of cytokine expressed Concentration of cytokine expressed Differential stage of effector cell. Cytokine alteration can be detected in: Endometrium Decidual immune cells Peripheral blood lymphocytes Animal studies showed that the only factor absolutely essential for implantation is: ” Leukemia inhibitory factor “. II- REPRODUCTIVE HORMONES Many mechanisms are present to avoid fetal recognition by the mother. Maternal immune responses to the fetus can be detected and if these mechanisms are regulated, this may help to treat RPL. Increased reproductive hormones during pregnancy is known to suppress maternal immunity, but the overall immune responses during pregnancy appears to change little, while local immune suppression at the maternalfetal interface may be vital. PROGESTERONE Progesterone is partially responsible for pregnancy maintenance. Progestrone suppress T-cell activity by altering K-channels in cell depolarization which affect: gene expression intracellular Ca And both may be none receptor mediated, resulting in: TH 2 responses LIF expression Both will help in maintenance. Recently it was found that progesterone can inhibit CD 8+ T cell proliferation and cytokine secretion. ESTROGEN Estrogen modulate immune reactions. In male animals: Estrogen improve immune responses after hemorrhage, trauma and thermal injury. Estrogen protect against chronic renal allograft rejection In humans: Estrogen ↓ delayed-type hypersensitivity Estrogen ↑ TH 2 responses. III – TRYPTOPHAN METABOLISM Tryptophan is important for T-cell activity and proliferation. Local alterations in tryptophan metabolism may either: activate or fail to suppress maternal immune response. In animals: - Tryptophan rich diet ↑ spontaneous abortion - Inhibition of indolamine 2,3 dioxygenase enzyme (IDO) loss of allogeneic fetuses but not syngeneic. In humans: - IDO is expressed in the decidua. - As GA ↑ alteration in maternal serum tryptophan level Both= potential local immune regulation. HUMERAL IMMUNITY I. II. III. IV. Antiphospholipid antibody syndrome Antithyroid antibodies Blocking antibody deficiency theory Novel HLA-linked alloantigen ANTIPHOSPHOLIPID ANTIBODY SYNDROME In the past , it was thought that these antibodies were against: cardiolipin and phosphatidylserine. Now it is believed to be against a protein cofactor : β2 glycoprotein-1 Which assist antibody association with phospholipid. % 3-5 CHARACTERISTICS OF APAS: Prolonged aPTT Thrombosis Obstetric complications: Abortion Stillbirth PTL PROM IUGR Preeclampsia SAPPORO CRITERIA FOR APAS DIAGNOSIS(1998) clinical and ≥ laboratory criteria must be present to diagnose APAS: clinical criteria:≥ ≥ 1 episode of thrombosis in artery, vein, or small vessel obstetric complication: ≥ 3 spontaneous abortions ≥ 10 weeks ≥ 1 fetal death ≥ 10 weeks ≥ 1 preterm labor ≤ 34 weeks for preeclampsia or placental insufficiency laboratory criteria: 2 results must be (+)ve on ≥ 2 occasions with ≥ 6 weeks in between +ve ACA (Ig G or Ig M medium to high level) +ve LA History of systemic lupus erythromatosus=less favorable outcome. MECHANISMS OF ACTION ↓ prostacyclin, ↑ thromboxane thrombosis placental infarction II. Inhibition of syncytiotrophoblast formation III. Increase β2 glycoprotein 1 rapid atherosclerosis in decidual artries inhibit trophoblast adhesion to endothelial cells ↓ annexin V = placental antithrombotic molecule To summarize: Pathological evidence of APAS are often equivocal Characteristic criteria “””””””””””””””””” lacking “”””””””””””””””””””””””””””””””””””””””” present in other lesions I. ANTITHYROID ANTIBODIES Controversial One study link it to RPL Other study no link BLOCKING- ANTIBODY DEFICIENCY THEORY Studies using mixed lymphocyte culture with paternal stimulator cells suggest that HLA-sharing between the two parents may cause blocking-antibody deficiency. Larger studies showed that HLA heterogeneity is not necessary for successful pregnancy, although complete sharing of the whole genome (occur in isolated groups of people and is very rare) is associated with ↑ spontaneous abortion. Thus, HLAtyping is rarely required. NOVEL HLA-LINKED ALLOANTIGEN SYSTEM Studies showed that this substance is actually CD 46 which is a placental complementary receptor responsible for protecting the placenta from complement -mediated attack. Conclusion: Successful pregnancy may not require an intact maternal immune system. Evidence: Successful pregnancy can occur in: Agamma-globulinemic women Severe immune deficiency Animals lacking T and C cells Animals with congenital absence of the thymus OTHER FACTORS VEGF Apoptosis Uterine receptivity Endometrial mucin Cellular and extracellular matrix adhesion properties Soluble intercellular adhesive molecules. Environmental factors: o Medications: Antiprogestogens Antineoplastic agents Anhalation anesthetics Ionized radiation prolonged organic solvents o Heavy metals o Alcohol o Coitus cause abortion only if the cervix is abnormal or incompetent EVALUATION PRECONCEPTION EVALUATION History Physical examination Laboratory investigations POSTCONCEPTIONAL EVALUATION Β-hCG U/S Α-fetoprotein HISTORY History of previous pathologic studies or karyotyping of the abortus tissues. 60% of abortions < 8 weeks are due to chromosomal abnormalities. Most common anomalies are trisomy 16 or monosomy 45X. Documentation of aneuploidy in the abortus tissues does not affect future fertility. GA of previous abortion: Most RPL occur at the same GA every time. Menstrual history may detect oligomenorrhia or endocrine abnormalities. The timing of coitus in relation to fertilization may detect dyssynchronous fertilization or old ovum. Subfertility or infertility= 33% in RPL couples. Abortion may occur before the 1st missed period and is diagnosed as subfertility. Personal or family history of thrombosis or renal abnormalities. Family history of pregnancy loss or obstetric complications. Drug intake or environmental exposure. PHYSICAL EXAMINATION General examination: Obesity Hirsuitism and acanthosis Brest examination and glactorrhea Thyroid Pelvic examination: Anatomy: uterus size, shape, DES cervix vagina Trauma Infection Estrogenization LABORATORY ASSESSMENT Standard investigations: I. Parental peripheral blood Karyotyping II. Hysteroscopy / HSG III. Luteal-phase endometrial biopsy at day 24 IV. Thyroid function tests ± prolactin V. Platelet/ complete blood picture VI. ACA+LA (aPTT or Russel viper venom) LABORATORY ASSESSMENT UNDER INVESTIGATION • • • • • • • • • Ovarian reserve: day 3 FSH, CC challenge test PCO: LH, A ATA Autoantibodies : antiphospholipid, antiphosphatidylserine, anti-β2 glycoprotein 1 Nk cells activity TH 1, TH 2 Hypercoagulability; aPTT Hyperhomocysteinemia: fasting and after methionine loading (dangerous to the fetus). Cervical cultures: mycoplasma, ureaplasma, chlamydia EXPERIMENTAL STUDIES Mixed lymphocyte culture Parental HLA-typing Serum or site-specific autoantibodies & alloantibodies Others: - Suppressor cell - Cytokines - GF - Embryotoxic factor - Oncogenes POSTCONCEPTIONAL EVALUATION If become pregnant psychological support Confirm: - intrauterine pregnancy - viability Because of ↑ % of ectopic and molar pregnancy Pregnancy complications Only ↑ in APAS and intrauterine infections. Monitoring: Β-hCG Not always ↓ before abortion If missed period Β-hCG until= 1500mIU/ml U/S Every 2 weeks GA of abortion If no FH activity until 6-7 weeks terminate and karyotype Maternal serum α-fetoprotein At 16-18 weeks. Karyotyping of the abortus tissues: - Suggest chromosomal abnormalities in the parent - Prevent other costly investigations ↓ cost Difficulties: - Difficult culturing of inflamed or necrotic cells - Contamination with maternal cells Recent advances: Genomic hybridization technology: Used on archived and paraffin-embedded tissues Karyoyping of nucleated fetal RBCs in maternal blood TREATMENT TREATMENT Most ttt are experimental, use only with informed consent. Most patients conceive irrespective of ttt. Genetic causes Anatomical causes Endocrine causes Infection Immunological ttt immune-stimulation immune-suppression Antithrombotic ttt TREATMENT OF GENETIC CAUSES Antithrombotic ttt for inherited thrombophilias Preimplantation genetic diagnosis in known heritable disorders Donor oocyte or sperm in robertsonian translocations Recently: PGD+antithrombotic ttt+ ART for all unexplained RPL TREATMENT OF ANATOMICAL CAUSES Hysteroscopic resection/metroplasty ± laparoscopy U/S guided transcervical metroplasty Fluoroscopic guided septum resection Laparoscopic excision of endometriotic lesions Cervical cerclage. TREATMENT OF ENDOCRINLOLGICAL CAUSES Ovulation induction Thyroid hormone replacement Insulin-sensitizing agents for overt DM Bromocriptine TREATMENT OF INFECTION Empiric antibiotics for immunosuppressive patients Specific antibiotic ttt for known infections to both partners with posttreatment culture before attempting conception. TREATMENT OF IMMUNOLOGICAL CAUSES Immunostimulating ttt leukocytes others Immunosuppressive ttt I.V. immunoglobulin progesterone others cyclosporine nifedipine corticosteroids LDA LEUKOCYTES Large studies useless Complications: Graft versus host reaction Severe IUGR Fatal fetal thrombocytopenia Autoimmune/isoimmune complications May benefit 1 : 11 patients Routine use unjustified OTHERS I. I.V. injection of syncytiotrophoblast microvillous plasma membrane vesicles. II. 3rd party seminal plasma suppositories. INTRAVENOUS IMMUNOGLOBULIN Indicated in: APAS Inappropriate cellular immunity Possible Mechanisms of action: ↓ autoantibodies ↑autoantibodies clearance T-cell regulation Complement inactivation ↑ T-cell suppressor function ↓ T-cell adhesion to extracellular matrix ↓ TH1 cytokine production Side effects: nausea myalagias hypotension headache anaphylaxis Disadvantages: expensive invasive time consuming require multiple I.V.D. PROGESTERONE Partially responsible for maintenance. Inhibits TH1 responses shift to TH2 responses Vaginal progesterone: Is better than systemic treatment ↑ local suppression ↑ intrauterine concentrations ↓ adverse effects. OTHERS Cyclosporine Nifedipine Plasmaphoreses Corticosteroids: Prednisone + low dose Aspirin same pregnancy outcome as controls, but: ↑ HTN ↑ DM ↑ PTL ANTITHROMBOTIC TREATMENT For APAS and thrombophilic disorders Treat hypercoagulability Start before conception continue although pregnancy Unfractionated heparin 5000 IU S.C. twice/day + low dose Aspirin 75-80mg/day. Monitoring by aPTT/week. Risks: PTL PROM IUGR Preeclampsia Abruptioplacenta Stillbirth Gastric bleeding Ostiopenia Recently: low molecular weight heparin(LMWH) Indications: APAS Non-APAS patients with thrombophilia Patients without thrombophilia =unexplained RPL Advantages: Less bleeding Less osteopenia More antithrombotic ratio More patient compliance: - less frequent doses - less frequent monitoring The role of LDA alone: Studies useful for APAS and RPL Studies useful in unexplained RPL Routine use alone cannot be justified. Other antithrombotic treatment: Vit B6, B12, folate Protein C concentrate Both for hyperhomocysteinemia whether congenital or aquired. PROGNOSIS Depends on cause and number of abortions. Even if >4 abortions, successful birth rate may reach 60% Genetic causes 20-80% live birth Anatomical causes 60-90% “””””””””” Endocrine causes > 90% “””””””””” Infection 70-90% “””””””””” APAS 70-90% “””””””””” FHM documentation: It has a prognostic value depending on the cause: FHM documentation in 77% of live birth “”””””””””””””””””””””” in 86% of APAS before abortion “”””””””””””””””””””””” in 3% of unexplained RPL