1. بسم الله الرحمن الرحیم

2. Reproductive Immunology
Samira Rajaei, MD, PhD
Assistant Professor of Immunology

3. Fetus
Autograft or
Allograft
Xenograft ?

4. Is there any contact between mother’s immune system and blastocyst?

5. Progesterone and estradiol
Decidualization & Implantation
Endometrial stromal cells
Progesterone and estradiol
Decidual stromal cells
Inner cell mass:Embryo
Distinct trophoblast subsets
Blastocyst
~ 6 days

6. Implantation
6 days after fertilization, the embryo differentiates into a blastocyst .
Blastocyst:
inner cell mass that will form the embryo proper
outer trophectoderm will become the placenta and chorion.
Attachment differentiation of the trophectoderm into two layers:
an inner cytotrophoblast layer
an outer syncytium of syncytiotrophoblast
It is the syncytiotrophoblast that invades into the decidua, and the blastocyst quickly becomes submerged in the maternal tissue.

7. This primitive network will eventually become the intervillous space
Erosion of maternal capillaries by the invading trophoblast
Filling of spaces within the syncytium with maternal blood.
Fusion of these lacunae and the formation of a sponge-like network
Maternal blood circulates bathing the blastocyst in nutrients and allowing gaseous exchange.
This primitive network will eventually become the intervillous space

8. Villous Formation
2 weeks after attachment, the underlying cytotrophoblast proliferates into buds, which will protrude through the syncytium.
Cytotrophoblast will have two cell fates known as
Villous trophoblast
Extravillous trophoblast
Villous trophoblast covers the chorionic villi, providing the barrier through which metabolic exchange between mother and fetus occurs, and it interacts with maternal blood in the intervillous space.
Maternal Blood

9. Extravillous cytotrophoblast
The primary role of extravillous cytotrophoblast is regulation maternal blood flow into the intervillous space by their invasion and transformation of decidual arteries.
A population of extravillous trophoblast migrate into the decidua and surround the maternal spiral arteries.
Where this occurs, the muscular walls of the arteries are destroyed and the endothelial cells swell.

11. Maternal blood with syncytiotrophoblast
Maternal decidua with extravillous trophoblast
Endovascular trophoblast and maternal blood

12. Mechanisms of fetomaternal tolerance
Role of MHC
Cytokines (LIF,TGF-B,IL10,…)
T cell apoptosis
Immunoregulation
Indoleamine-2,3-dioxygenase
B7 family of costimulatory molecules
Role of complement and complement regulators

13. MHC class Ia (HLA-A, HLA-B &HLA-C)
On all nucleated cells
presenting antigens to Tc
are involved in the inhibition and activation of NK cells via inhibitory NK-cell receptors (KIR) and activating NK-cell receptors (KAR).

14. MHC class Ib proteins
Play a role in the immunological acceptance of the fetus
HLA-G and HLA-E are expressed by some trophoblast populations

16. HLA-G HLA-G Induction of immunosuppressive APCs
Induction of immunosuppressive T cells
Induction of Th2 cytokines
Induction of apoptosis in CD8+ cytotoxic cells

17. Uterine natural killer cells (uNK)
90% are CD56 bright CD16-
Present in the decidua during the 1st and 2nd trimesters
Modify the uterine arteries
Critical role in acceptance/rejection of the fetus
Regulate invasion of extra villous trophoblasts
Low cytotoxic
More cytokine producing

18. HLA-E Major effect is suppression of NK cell activity
Expression of the HLA-E receptor, CD94/NKG2A, on uNK cells is abundant so the role of HLA-E may be of great importance in repression of NK cytotoxicity in the uterus
HLA-G in humans is thought to facilitate expression of HLA-E

19. Cytokines

20. Pregnancy ?
Hormones

21. Other cytokines
The gp130 cytokines
LIF
IL-6
IL-11
TGFβ

22. T cell apoptosis
Clonal deletion of immune cells that recognize paternal Ags
Fas-L is expressed on
Fetal trophoblast
Maternal decidual cells

23. TReg cells Estrogen and trophoblast- derived chemokines
Expanded during first two trimesters (systemic and decidual)
Maternal antigen-presenting cells in the decidua could present fetal alloantigens to TReg cells
Production of TGF-β,IL-10
Induce IDO production in DCs

24. Treg cell expansion

25. Treg cells in pregnancy

26. Indoleamine-2,3-dioxygenase
IDO is an enzyme that degrades the essential amino acid tryptophan and can generate downstream tryptophan metabolites.
It is expressed both extravillous trophoblasts and villous trophoblasts in humans.
IFN-gamma induce IDO.

27. Mechanisms of IDO
Inhibit maternal T cell activation by depriving T cells of tryptophan
IDO affects nature of APCs
suppressive ligands (PDL1 or CD95 ligands)
triggering the secretion of immunoregulatory cytokines (IL-10 or TGF-β)
Expression of HLA-G on DCs can be induced by IDO

28. B7 family of costimulatory molecules
At least five of the seven known B7 family proteins are expressed in the human placenta, including on trophoblast cells.

29. PD1,PD-L1,PD-L2
PD1 interaction with PDL1 and PDL2 plays an important role in peripheral tolerance
PDL1 (B7-H1) is present on all trophoblast populations
PDL2 (B7-DC) is expressed by the syncytiotrophoblast
Blockade as well as deficiency of PDL1 resulted in decreased fetal survival and a shift to Th1 cytokines

30. Role of complement and complement regulators
Regulatory proteins expressed on trophoblast, which prevent damage inflicted by complement activation.
Decay-accelerating factor (CD55)
Membrane cofactor protein (CD46)

32. Pregnancy associated Immune Diseases

33. Recurrent Spontaneous abortion
three or more consecutive miscarriages before 20th week of gestation
about 1% of all pregnancies
~50% remains unknown

34. RSA etiology Antiphospholipid syndrome
Parental chromosomal abnormalities
Uterine anatomical abnormalities
Endocrine imbalance
Thrombophilias
Immunological factors

35. Antiphospholipid syndrome (APS)
A disorder characterized clinically by:
Recurrent venous and/or arterial thromboembolic events
Pregnancy morbidity
Collection of autoantibodies that target specific phospholipid-binding proteins

36. Anti phospholipid Abs (aPL)
a heterogeneous group of autoantibodies
may be present in healthy persons
The antigenic targets of aPL are uncertain
Phospholipids such as cardiolipin
Phospholipid-binding proteins, or protein cofactors, bound to phospholipids (β2GPI)

38. Clinical criteria Vascular thrombosis Pregnancy morbidity
≥1 unexplained death of morphologic normal fetus at or after 10 weeks
≥1 premature birth of a morphologic normal neonate before 34 week because of eclampsia, severe preeclampsia, placental insufficiency
≥3 unexplained consecutive spontaneous abortions before 10 week (anatomic, endocrine and chromosomal causes should be excluded)

39. Laboratory criteria
Lupus anticoagulant present in plasma on 2 or more occasions (at least 12 week apart)
Anticardiolipin antibody (IgM or IgG) in serum or plasma in medium or high titer (>40 GPL or MPL or 99th percentile) on 2 or more occasions (at least 12 week apart)
Anti β2 glycoprotein 1 Ab (IgG or IgM)(> 99th percentile) on 2 or more occasions (at least 12 week apart)

40. Fetal loss mechanisms related to aPLs
Placental thrombosis
aPL might induce a procoagulant state at the placental level
ability of the aPL antibodies (specifically, anti‑β2GPI antibodies) to disrupt the anticoagulant annexin A5 shield on trophoblast and endothelial cell monolayers.
Histopathological findings suggestive of thrombosis cannot be detected in most samples from miscarried fetuses and placentas from women with APS
Inflammatory responses
Defective placentation

41. Pathogenesis aPL have a variety of effects on trophoblasts
Inhibition of villous cytotrophoblast differentiation
Inhibition of extravillous cytotrophoblast invasion into the decidua
Induction of syncytiotrophoblast apoptosis
Initiation of maternal inflammatory pathways on the syncytiotrophoblast surface

42. ↑Adhesion of leukocytes
Thrombosis in APS
Cytokine secretion
PGE2 synthesis
↑Adhesion of leukocytes
aPL actions favor clot formation through several routes. (1) aPL interact with endothelial cells, primarily through binding of β2GPI on the cell surface, and induce a procoagulant and proinflammatory endothelial phenotype. (2) aPL upregulate tissue factor expression on endothelial cells and blood monocytes, and promote endothelial leukocyte adhesion, cytokine secretion and PGE2 synthesis. (3) aPL recognize phospholipid-binding proteins expressed on platelets—aPL binding potentiates platelet aggregation induced by another agonist. (4) aPL interfere with plasma components of the coagulation cascade, by inhibiting anticoagulant activity, by affecting fibrinolysis, and by displacing the binding of the natural anticoagulant annexin A5 to anionic structures. These mechanisms all contribute to a procoagulant state that is necessary but not sufficient for clotting. Clot formation seems to require two steps: the presence of aPL provides the 'first hit', which produces clotting when accompanied by another procoagulant condition, a 'second hit'. Complement activation seems to be necessary for clot formation in vivo. 

43. Main effects of aPL on placenta

44. Endothelial cell activation by anti‑β2GPI autoantibodies
aPL react with β2GPI expressed on the endothelial cell membrane and induce cell signaling. β2GPI adheres to endothelial cell membranes: (i) through the electrostatic interaction between the cationic phospholipid-binding site (located in the fifth domain of the molecule) and anionic structures, such as heparan sulfate, on the cell membrane; or (ii) as a ligand for annexin A2. Binding of anti-β2GPI antibody induces clustering of β2GPI with its potential receptors, and induces cell signaling, resulting in activation of NFκB or p38 MAPK or both. The interaction of the β2GPI clusters with TLR2/TLR4 might be responsible for MyD88 and TRAF6-dependent signaling

45. aPL effects on trophoblasts

46. Endometriosis
Presence of endometrial glands and stroma in ectopic locations
Pelvic peritoneum (primarily)
Ovaries
Rectovaginal septum
6%–10% of women of reproductive age

47. Endometriosis 1. Recurrent pelvic pain 2. Infertility Dysmenorrhea
Dysparunia
Chronic pelvic pain
dysuria
1. Recurrent pelvic pain
Cramping or stabbing pain, pain does not correlate with dis extention
2. Infertility

48. Retrograde menstruation
Coelomic Metaplasia
MSCs theory
Endometriosis
Mullerianosis
coelomic metaplasia: involves the transformation of normal peritoneal tissue to ectopic endometrial tissue under influence of hormonal and immunologic factors
Mullerianosis: cells residual from embryologic Mullerian duct migration maintain the capacity to develop into endometriotic lesions under the influence of estrogen beginning at puberty
extrauterine stem/progenitor cells originating from bone marrow may differentiate into endometriotic tissue

49. Endometriosis 1. Genetic predisposition 2. Estrogen dependence
3. Progesterone resistance
4. Inflammation
It is the propensity for implantation that best accounts for the discrepancy between the 90% prevalence of retrograde menstruation and the nearly 10% prevalence of the disease

50. balance between MMPs and TIMPs
NK cell function ↓
Macrophage function ↓
TGF-β
Mesothelial injury
Refluxed endometrial tissue is cleared from the peritoneum by the immune system, and the dysregulation of this clearance mechanism has been implicated in the predisposition to implantation and growth of endometrial cells.
Larger tissue fragments demonstrate an increased capacity to implant (resistant to clearance)
eutopic endometrium from women with endometriosis was found to be more resistant to lysis by natural killer (NK) cells
Sheding of ICAM-1 resistence to clearance by NK cells
balance between MMPs and TIMPs
MMP expression increase ↑

51. NEUROANGIOGENESIS
A rich vascular supply is necessary for the development and sustenance of endometriotic lesions
Neoangiogenesis and capillary recruitment are visibly associated with endometriotic lesions
Nerves frequently accompany angiogenesis
particularly in the peritoneal microenvironment, which is relatively avascular compared with the eutopic endometrium

52. Growth factors Vascular endothelial growth factor (VEGF)
Glandular endometrial cells
Activated peritoneal macrophage
platelet-derived endothelial growth factor
Epidermal GF
Insulin-like growth factors (IGF): antiapoptotic
Vascular endothelial growth factor (VEGF) has been consistently detected in high concentrations in peritoneal fluid.
VEGF exhibits a cycle phase dependence (Estrogen dependent)

53. Cytokines
Expression of tumor necrosis factor-α (TNF-α), IL-8 increased in menstrual endometrium.
TNF-α and IL-8 promote:
proliferation and adhesion of endometrial cells
angiogenesis

54. Cytokines Macrophage migration inhibitory factor TNF-α IL-1β IL-6 IL-8
RANTES
MCP-1
Chemoattractant

55. IL-6
eutopic endometrium from women with endometriosis showed an increased basal production of IL-6
IL-6 plays a prominent role in many chronic inflammatory conditions and is secreted by macrophages as well as epithelial endometrial cells.

56. Inflammation
The peritoneal microenvironment in the setting of endometriosis is notably rich in prostaglandins.
Peritoneal macrophages from women with endometriosis express higher levels of cyclo-oxygenase-2 (COX-2).
Inflammation is not only present in the peritoneal microenvironment but also in the eutopic endometrium of women with endometriosis

57. Preeclampsia (PE)
Preeclampsia (PE), a pregnancy-related potentially life threatening condition, which affects 3–5% of pregnancies
New-onset hypertension
Oedema
Proteinuria
Develop after 20 weeks of gestation
Events leading to its onset seem to occur early in pregnancy

59. Preeclampsia is a 2 or 3 stages diseases.
Pre-clinical (symptomless)
Clinical stages

60. Role of maternal-fetal recognition
Maternal–fetal immune recognition at the site of placentation is highly individualized by two polymorphic gene systems:
HLA‑C molecules of trophoblasts
Their cognate receptors, killer cell immunoglobulin-like receptors (KIRs) of natural killer cells

61. uNK and HLA-C
Uterine natural killer cells release chemokines, angiogenic factors and cytokines that promote trophoblast invasion.
These secretions are increased upon binding of HLA‑C antigens to stimulatory KIRs (haplotype B).
KIR BB mothers carrying HLA‑C1 fetuses best placentation

63. ER stress
Narrow spiral arteries create conditions for ischaemia–reperfusion injury in the intervillous space.
Hypoxia endoplasmic Reticulum stress suspension of protein folding cessation of cell proliferation and severe apoptosis of trophoblasts release particles intravascular inflammatory response

64. Oxidative stress
The cause of oxidative stress in the placentas of women with pre-eclampsia is thought to be intermittent hypoxia and reoxygenation.
Deficient conversion of the myometrial segment of the spiral arteries, which contains a contractile portion of the artery.

65. Antibodies to type‑1 angiotensin II receptor
A subset of women with pre-eclampsia have detectable serum autoantibodies against type‑1 angiotensin II receptor (AT1)
autoantibodies can activate AT1 in endothelial cells, vascular smooth muscle cells and mesangial cell.

66. Intravascular inflammation
Increased release of microparticles and nanoparticles from the syncytiotrophoblast into the maternal circulation
Proinflammatory cytokines and chemokines released upon activation of NF‑κB in the context of ER and oxidative stress