Novak 2003

2. Recurrent Pregnancy Loss Novak 2003

3. Introduction • Etiology • Evaluation • Treatment Contents

4. introduction

5. 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

6. 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.

7. etiology

8. Genetic = 3.5-5% • Anatomical = 12-16% • Endocrine = 17-20% • Infection = 0.5-5% • Immunological = 20-50% • Others = 10% 10-15% of RPL is due to APAS and chromosomal abnormalities.

9. Mostly due to balanced translocation: Reciprocal translocation normal gametes Robertsoniantranslocationbalanced 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 ). Genetic causes

10. 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

11. 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.

12. Parents with history of: • spontaneous abortion • ± SB • ± live birth ( ± anomalies ) are candidates for parental karyotype, but this may be insufficient because: • aneuploid sperm may be motile • chromosomal abnormality may be in 1 sperm

13. 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).

14. 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 Heritable thrombophilias

15. Homocysteine is derived from dietary methionine. It is metabolized in the presence of methyl-tetra-hydro-folatereductase 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. 1- hyperhomocycteinemia

16. 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. 2- Antithrombin iii mutation

17. 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. 3- protein-c and protein-s deficiency

18. Factor V prevents interaction with protein-C. 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. 4– factor v leiden mutation

19. It is linked to thrombosis and pregnancy loss. 5– prothrombin mutation

20. Increase in Japanese people and is linked to recurrent pregnancy loss. 6– factor xii deficiency

21. Both are associated with fetal loss and involve other complex mechanisms than simple thrombosis and infarction. 7- factor xiii and fibrinogen defects

22. 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 • Ashermansyndrom • cervical incompetence Ii-anatomical causes

23. Endocrine changes of menstrual cycle • Endocrine factors: • Luteal phase defect • Diabetes • Thyroid disease • Hyperprolactinemia • Ovarian reserve Iii- endocrine causes

24. 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 changes of menstrual cycle

25. 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 endocrine factors:1- lpd

26. DM type II • ↑ spontaneous abortion IDDM: - Pregestationalglycemic threshold above which  abortion ↑ - Hyperglycemia damage the embryo - Vascular changes in advanced IDDM ↓placental blood flow. 2- diabetes

27. Ovulatorydisfunction 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). 3 – thyroid disease

28. Affect reproduction by: - Direct effect on endometrium - Indirect immuno-mediator effect. 4 - hyperprolactinemia

29. Can be evaluated by: - Day 3 FSH - Day 3 estradiol - CC challenge test. 5 – diminished overian reserve

30. 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. 4 - infection

31. Basic immunology Cellular immunity Humoral immunity 5 – immune causes

32. 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. I - Basic immunology

33. 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. Antigen specificity

34. 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. Education and homing

35. 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. Mucosal and peripheral immune systems

36. Resident cells Immune cell education and homing to the reproductive tract Antigen presentation at the maternal-fetal interface Regulation of the decidual immune cells Ii - Cellular immune response

37. 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. 1- resident cells

38. Most peripheral immune system T-cells express TCR-αβ. In the reproductive system T-cells express in addition to this TCR-γδwhich ↑ 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.

39. 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. 2 – immune cell education and homing

40. 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. Extravillouscytotrophoblastcells 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. 3 – antigen presentation

41. 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.

42. Alterations in T- helper cell phenotype Reproductive hormones and immunosuppression Tryptophan metabolism 4– regulation of decidual immune cells

43. Ag-stimulated immune responses involving CD4+ cells can be divided into: T-helper cell subset 1 (TH1) T-helper cell subset 2 (TH2) I –alteration in t-helper cell phenotype 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.

44. 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.

45. 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 “.

46. 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 maternal-fetal interface may be vital. II- reproductive hormones

47. 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. progesterone

48. 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. estrogen

49. 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. Iii – tryptophan metabolism

50. Antiphospholipid antibody syndrome Antithyroid antibodies Blocking antibody deficiency theory Novel HLA-linked alloantigen Humeral immunity