Genomics, transcriptomics, proteomics- used for investigating possible genes or their products associated with pathogenesis of RPL which may occur due to aberrant expression of several different genes, proteins
Critical process during placental development and differentiation and for maintaining tissue homeostasis
Antithrombin III , fibrinogen levels are downregulated in follicular fluid in RPL patients
Immunology and recurrent pregnancy loss
Dr. Indira Devi Ponugoti MD,DGO,FICOG,FIAMS,FCGP.•• MD.DGO , Osmania University, Hyderabad - 1965,1969,1970• FICOG 2009• FAMS 2010•Professor of Obstetrics and Gyneaecology - Osmania Medical College 1991--1999•Kamineni institute of Medical Sciences HOD 2001 - 06•Under and post graduate Examiner 1970 - 2010•Chair person womens wing IMA - Hyderabad 2007 - 08•Vice President OGSH 2007•President OGSH 2008 - 2009•Coordinator APCOG 2008 - 09•Vice President IMA –HYD 2010 - 11•Presented papers at national conferences•Chaired the sessions at national and international conferences•Contributed to FOGSI focus on Maternal Nutrition•Life member IMA ISOPARB OGA AIAORAO•Presently•State council member IMA Hyderabad•Organizing Co- Chair person & Chair Person Scientific Committee - AICOG 2011•Dean Of Faculty CGP-IMA 2011
DefinitionDefined as 3 or more clinical pregnancies lost before the 20th week of gestation from the last menstrual period Incidence 1 – 2% (3 or more losses) 5% (2 or more losses) Only 30% pregnancies result in live birth Albemann, SalatBarour 1988
SignificanceNo. of previous pregnancy Frequency losses 2 24%--30% 3 37%----40% 40% 4
Etiology Cause Frequency Anatomical 10-15% Chromosomal 2-5% Endocrinal 17-20% Autoimmune 20% Infections 0.5-5% Unexplained 40-50%
Reproductive ImmunologyPregnancy is Pregnancy – associated an adaptive The with B cells form of principle suppression with immunity target forof humeral & immuno involves T cells this human cell recognition recognize response globulin of specific antigen mediated receptors are theimmunologic antigen & as MHC recognize confers peptide function to antigenic moleculesaccommodat specificity & bound to expressed position memory MHC e the semi- of intact on donor allogenic effect by T& cells(allo- molecules B graft- the MHC) fetus* lymphocyts. Many causes of RPL- maternal transplant rejection *Thellin& Henwm
Normal ImplantationDepends on Diminishedcontrolled This inturn Decidua is immunologictrophoblastic depends on supposed to al response ofinvasion of uterine large be an the pregnantmaternal granular immunologic woman mayendometrium- cells-LGLs & ally be a cause fordecidua & expression of privileged survival ofspiral 3 of HLA tissue site semi alloarterioles* class genes graft * Meffeh –King- 2002
Protective mechanisms in pregnancy The primary cellular 3 response that develops against transplanted The proteins from HLA 1 tissue is directed against major histo genes are not compatability expressed co complex(MHC),proteins dominantly on donor tissue.* on trophoblast cell membrane 2 In humans MHC unlike other proteins are human cell types. leukocyte antigens (HLA)Tilburg.T,Scherjonsa,Reprod immol,2010;85:58
Protective mechanisms inpregnancy Strict regulation of the expression of HLA class 1 molecules in sub population of trophoblast is supposed to protect the semi allograft against immune cells which 4 are programmed to attack cells expressing paternal HLA class 1 antigens.* Trophoblasts contain indoleamine 2,3 5 disoxynase(IDO , inhibits tryptophan metabolism) there by inactivates T cells *362514Lebo tiller P.Mallet V-HLA G& preg-Reprod 1997;2:7 Role of HLA G in Human preg Reprd Bio Endo 2006;4 Supp,1:510
Expressions and Reactions At Fetomaternal interphase by placenta & fetal membranes Endocrine system & Under the immurne system interact influence of sex closely during steroids ,dramatic implantation and increase occurs in maintenance of unique population pregnancy. of lymphocytes Recruitment of Role –not clear uterine natural probably, promote killer cells, ( which growth of placenta, and are derived from trophoblast ,providing peripheral NK immune modulation cells * *Dysregulation of above expressions occurs in RPL
Immune Response T cell Activated T induce CD recognition cells undergo cell + T cell is the clonal mediated primary expansion & cytotoxicity, event of influence provide help antigen interleukin 2- for B cell growth antibody factor, production, Signal 1 is provided by the interaction of T cell Signal 2 –by a receptor(TCR) receptor provide help for with antigen legend macrophages to present as a interaction on induce delayed peptide by T cell/APC cell hypersensitivity antigen presenting surface cell(APC).
Mechanisms Associated with AlloGraft Rejection Hypothesis Non immunologic Once activated CD4+ T injury responses (,induce cells initiate non specific inflammation) macrophage mediated delayed hypersensivity Increased antigen response & provide help to B production to T cells cells ,for allo antibody production. (by up regulating the expression of adhesion molecules ,Class 11 MHC, Chemokines and cytokines) CD 8 cells induce apoptosis By shedding of intact Jabs WJ etall J .infet 2004;190:1604, Wyburn KR,Jose et ,soluble HIA,( which may all,J.tranplantation,2005;80:164 prime the indirect allo recognition path way.)
Alloimmunity and RPL Mechanisms - Postulated Immune Maternal anti mediated and fetal blocking suppressor antibody cell Sharing of mechanism HLA deficiencyExistence of immunologicaldifferences among theindividual of the samespecies*
OMICS - Studies RPL *Molecular, genomics, These studies possibly reveal the transcripto genes associated with mics and pathogenesis witch might occur proteomics due to aberrant expression of studies are genes and proteins. required to under stand etiology of Also revealed immune response Recurrent thrombosis, steroid bio pregnancy synthesis,apoptosis,and angiogenesis loss. related genes*Laird Smetal 2003.Hum Reprd.update 9,163-174Molecular medicine.vol;13:7
Transcriptomic analysis Chromosomally normal chorionic villi from RPL women showed Expression levels of five groups of immune suppression related genes Embryo Other etiology related attachment genes related Angiogenesis Apoptosis related relatedChoi HK et al 2003 Mol Reprd Dev 66,24-31,Back HH2002.Reprd Fertil dev,14,235-240 ,Lee,J 2005 Fertil Sterl 83,1047-49
Proteomic analysis RPL Revealed Follicular aberrant fluid is Cc3 is expression of identified regulated by C3& C4thrombophilic with membrane levels are factors as differentially co factor found to fibrinogen – expressed protein be high in y& anti proteins, (MCP) and RPL with 3 thrombin including decay losess. which are compliment accelerating associated component factor (DAF) with RPL C3c.
Role of cytokines in RPL When HLA-G expression is down regulated, Th 1 cells are activated and release cytokines-IFNȣ, TNF-α, IL-2 Cytokines inhibit human placental trophoblast cell growth and metabolic activity. IFN-ȣ inhibit secretion of (GM-CSF) which promotes growth, differentiation of trophoblast during normal pregnancy. Ratio of Th1/Th2 activity is critical for normal pregnancy. Dysregulation of NK cytotoxicity and cytokine production might be involved in RPL.Expression of natural cytotoxicity receptors (NCRs)- NKp46, NKp44, NKp30 and A2V-ATPase on CD56 NK cells were up- regulated in RPL patients.
Role of HLA -G in RPL Non classic MHC molecules Expressed in extravillous cytotrophoblast Invasion of extravillous cytotrophoblast into the uterus is a vital stage in the establishment of pregnancy HLA-G polymorphism → pregnancy complications- RPL
Possible Immunological Mechanisms Involved In RPL HLA G molecules of trophoblast cells inter act with killer activator receptor (KAR)of uterine natural killer cells (NK) Cytokines released from NK cells attack trophoblast cells. Inter action of HLA G with killer inhibitory cell ( KIR) has opposite effect. Th1 cytokines induce cytotoxic activity in uterine NK cells & cytotoxic T cells. B cells release auto antibodies –APA,ANA,ATA
Possible Immunological Mechanism Involved In RPLHLA G molecules of trophoblast Cytokines releasedcells inter act with from NK cells killer activator attack trophoblast receptor (KAR)of cells. uterine natural killer cells (NK) Inter action of Th1 cytokinesHLA G with killer induce cytotoxic inhibitory cell ( activity in uterineKIR) has opposite NK cells & effect. cytotoxic T cells. B cells release auto antibodies – APA,ANA,ATA
Role of Angiogenesis Expression levels of Insufficient or abnormal angiogenesis related gestational angiogenesis genes MMP- resulting from aberrant 2,PAI, Integrin, TGF- expression of angiogenesisβ, VEGF, FGF were lower related genes lead toin intact chorionic villi abnormal growth of fetus derived from RPL or pregnancy loss. patients.
Apoptosis Fas ligand (FasL)-Fas interaction between decidualcells expressing FasL-Fas bearing leukocytes leads toapoptosis of activated leukocytes → down regulation of production of cytokines TGF-β and IL-10 (↓extravillous trophoblast invasion) High expression levels of apoptosis related genes caspase 3,6,7,8,9,10,12,BAD, BAX, BID, FasL, Fas in women with RPL. Apoptosis genes directly regulate embryonic development during normal pregnancy.
Regulatory T Cells (Tregs)*Women with RPL showedlow no. and functionallydeficient T regs at both thefollicular,and luteal phases Immunity related genes, those encoding PP14, HCG, mucin 1 were aberrantly expressed in intact chorionic villi from RPL patients
Type 2 T Helper cells IL-4 a growth factor for B Th1 to cell T cell Th 2 Type 17 Produc antibody e IL- production 3 cytokin T cells subse e may4,5,10,1 ,also t express contribu 3 and inhibits T ion is te to help B cell TH17 associa allograft cell maturation secret ted with rejectiofunction into Th1 . path es IL- allograft n is not 17 toleranc clear. way(Clears e parasitic infections in mammals)
HLA Class 11 Contains genes encoding HLA molecule region – HLA-DR,DQ &DP These are expressed on B cells ,dendrite & monocytes can be induced during inflammation. Also contain alpha & B chains on MHC.
Genetic predisposition RPL Predisposition to venous thrombosis - specific single nucleotide polymorphism s(SNPs)in the Elevation of homocystein venous genes coding levels thrombosis & for –factor v attributed to elevation in Leiden& mutation in homocystein prothrombin- MTHFR in levels G20210A particular to C677T SNP *Nelen 1998,Lissak 1999, Finan 2002 Wang 2004,
Antiphospholipid Antibodies(2%Auto immune ) Incidence of APA 2% LA, ACl in RPL 15-20% Prevent trophoblast proliferation, cause early fetal loss by complement activation Cause thrombosis by interrupting fibrinolysis, inhibits the anticoagulant pathway, causes LA& aCLAthrombin generation elevated require plasma protein co factors Interact with placental interphase resulting in decidual vasculopathy ,deposition of immune complexes leading to lowered
Thrombophilias Pregnancy –a hypercoaguable state Factor VII, VIII & X shifts the thromboxane & prostacyclin ratio , vasospasm& platelet aggregation leading to micro thrombi and placental necrosis. Hypercoaguability is aggravated by thrombophilia –RPL Deficiency of Protein C ,S & anti thrombin III results in Platelet aggregation, generation of thromboxane, lowered platelet reactivity to anti aggregating response of prostacyclin
Schematic representation of the coagulation pathway.•The circles depict theprothrombin–convertasecomplex (FVa + FXa) that drivescoagulation by convertingprothrombin into thrombin.• Thrombin converts fibrinogento fibrinand this is stabilized by thecrosslinking of fibrin polymers byFXIII.•Thrombin is inactivated byantithrombin III (ATIII). Blackarrows indicate modification,white arrows indicate catalyticor modifying effects and the
Thyroid Disease & RPL *Pain less thyroiditis Positive TPO one year after antibody is pregnancy loss can associated with cause immunological high incidence of changes and RPL. hypothyroidism 20% of TPO + will Auto antibodies develop subclinical might causehypothyroidism by term growth inhibition of if untreated. trophoblast and thrombosis. *Investigation RPL Fertil,Steril,2005;83:821.
Summary Programming the uterus for semi allo genic pregnancy my occur with introduction of semen* Possible causes for RPL Secondary immune response due to dys regulation of HlA Expressions , cytokines and exposed paternal antigens .are the. Lack of immunological protection to the embryo Lack of appropriate expression of compliment regulatory proteins , Apoptosis-inducing TNF super family members, HLA G or HLA E .
Summary Extraordinary variation in upstream regulatory regions of HLA G explains the fetal loss rates and polymorphism. Polymorphism -725 C/G allele in both partners has increased the rates of fetal loss. Increased pro inflammatory cytokines and up regulated thrombophilic tendency –a
Conclusion With introduction of” OMIC “ studies RPL etiology has become more complex than thought earlier. Functional analysis of genes and or their products will help to recognize the pregnancy with RPL A definitive approach to etiologic cal factor and immune modulators may help. Understanding the agony ,counseling ,assurance on future with sympathy, are necessary for better out come.
The gift of life is a marvel of divinity, and no words can expressthe awesome moment of its creation.