RECURRENT MISCARRIAGE CURRENT CONCEPTS

1. RECURRENT MISCARRIAGECURRENT CONCEPTS SUSHANTA BHADRA FEBRUARY 2004 WEXHAM PARK Distressing for the patient and frustrating for the physician Cause is usually not apparent and often requires intensive and expensive clinical and laboratory investigations, despite which there is only limited understanding of the subjectDistressing for the patient and frustrating for the physician Cause is usually not apparent and often requires intensive and expensive clinical and laboratory investigations, despite which there is only limited understanding of the subject

2. DEFINITION Loss of three or more clinically recognized pregnancy losses before 20 wks gestation. Clinical investigation should however be initiated after 2 consecutive losses specially when fetal heart activity has been identified before any pregnancy losses, when the woman is >35 yr or when the couple has difficulty conceiving.

3. epidemiology Affects 0.5-3% of all women Risk of subsequent loss - 24% after 2 30% after 3 40% after 4 Definitions not adhered to at all times Some have included only first trimester losses, some only 2nd trim Specify the type of loss- preclinical- demise before 6wks , embryonic loss 6-10wks fetal loss 10-20 wks Important to understand different definitions before comparing dataDefinitions not adhered to at all times Some have included only first trimester losses, some only 2nd trim Specify the type of loss- preclinical- demise before 6wks , embryonic loss 6-10wks fetal loss 10-20 wks Important to understand different definitions before comparing data

4. PROPOSED ETIOLOGIES Genetic - 5 % Anatomic- 15% Endocrine- 20% Infections- 5% Immunologic/ Thrombotic -30% Other factors � 10% Unknown

5. Genetic mechanisms Chromosomal abnormalities numerical � aneuploidies structural - translocations Single gene ( Mendelian) Polygenic ( single anatomic defect)

6. Chromosomal abnormalities Spontaneous abortions Normal chromosomes � 40-50% Abnormal chromosomes- 50-60%

7. aneuploidies Trisomies (extra chromosome) and monosomies (missing chromosome) Segregation errors during cell division Sporadic Nonrecurrent Trisomies associated with maternal age

8. Abnormal chromosomes Autosomal trisomy �50% Monosomy X � 25% Polyploidy �20% Sex chromosome polysomy - rare Translocations - < 5%

9. Autosomal trisomies Chromosomes 1�0% 13� 5.8% 2�5% 14, 18 �5% 3,5,6 ,11,12,17 - <1% 15�7.2% 4 , 20 �2.5% 16�31% 7�4.5% 21 � 8.4% 8,9�3.5% 22 ---11%

10. Parental origin - trisomy Maternal �90�95% -- age related recurrent Paternal � 5�10%

11. Aneuploid screening There is an increased rate of numerical chromosomal abnormalities in human periimplantation embryos in women with RSA There is also an increased incidence of chromosomal abnormalities in the sperm from RSA couples Role of preimplantation genetic diagnosis (day3 - blastomeres ) using FISH

12. Structural chromosomal abnormalities Defect in structure of 1 or more chromosomes Inversions, translocations 7% couples affected Risk of spontaneous abortions vary from 25-50% May be passed from parent to child Karyotype indicated When a parent carries a balanced chromosome rearrangement the chance of having a live birth with unbalanced chromosomal complement is usually about 1 -15%. The exact risk depends on the specific chromosomes involved , size of the segments involved , sex of the transmitting parent, family history and mode of ascertainment.When a parent carries a balanced chromosome rearrangement the chance of having a live birth with unbalanced chromosomal complement is usually about 1 -15%. The exact risk depends on the specific chromosomes involved , size of the segments involved , sex of the transmitting parent, family history and mode of ascertainment.

13. TRANSLOCATIONS Reciprocal --- any chromosome Robertsonian �(centric fusion) only acrocentric chromosomes 13,14,15,21,22 Cryptic translocations - balanced translocations involving only the telomeric regions of the chromosomes � not detectable by conventional cytogenetics In reciprocal translocation pieces from 2 non-homologous chromosomes have switched places with each other, in Robertsonian 2 acrocentric chromosomes are fused togetherIn reciprocal translocation pieces from 2 non-homologous chromosomes have switched places with each other, in Robertsonian 2 acrocentric chromosomes are fused together

14. Other chromosomal rearrangements Inversions Balanced complex translocations Interchromosomal insertions Jumping chromosomes

15. X Chromosome inactivation Occurs in female mammals Random inactivation of a X chromosome to compensate for the difference in x linked gene dosage Preferential inactivation of x chromosome is directly correlated with RM Underlying causes include cryptic x chromosome aberrations, gene microdeletions, gene mutations and genetic imprinting

16. Chromosomal causesConclusions Aneuploidies are responsible in 55-85% of EPL Trisomies are usually maternal meiotic in origin and age related. Polyploidy(67%) and Monosomy X(80%) are usually paternal in origin Trisomies can be recurrent Parental translocations found more often in female, not highly correlated with number of losses and show 2-5% unbalanced offspring

17. Single gene defects Maternal � endometrial, immunologic, vascular Embryonic � developmental Genes conferring pharmacologic susceptibility to toxins or infections Genes causing aneuploidy

18. Polygenic 2 or more genes cumulatively affect presence or absence of a given trait Unequivocal relationship to 2nd and 3rd trimester losses Associated with anatomic defects involving single organ system Associated with subsequent live born ntd and prior polygenic defects Fetuses with anatomic defects (embryoscopy) usually show cytogenetic abnormalities Recurrence risk 1-5% limited to first degree relatives

19. Maternal gene perturbations Mutant maternal gene likely to be associated with consecutive losses not interspersed as in genes acting through embryos Endometrial receptivity (PR) Luteal Function (CYP 17) Alloimmune (HLA G promoter polymorphism)

20. Lethal genes affecting fetus Early lethal � Surf 1 , ETA2 , OCT 4( mice models � human analogies present � neurodevelopmental problems ) Placental � trophoblast differentiation , fetoplacental vascular development , trophoblast transcription factors Homebox and other developmental : HOX PAX Lethal recessive genes usually produce non consecutive losses If lethal dominant factors are resonsible for deleterios embryonic effect would cause repeted losses if parental gonadal mosaicism existsLethal recessive genes usually produce non consecutive losses If lethal dominant factors are resonsible for deleterios embryonic effect would cause repeted losses if parental gonadal mosaicism exists

21. Hla genotypesThe REMIS Trial analysis of 12 HLA g alleles in prospectively followed cohorts of couples with recurrent miscarriages using PCR sequence specific oligonucleotides for 12 alleles 113 couples studied- 63 with successful pregnancy, 50 with rm

22. Remis trial HLA g gene genotype 0104 and 0105n is predictive of low successful pregnancy rates Presence of HLA G isoform 1 and 725C/G polymorphism in promoter regions are associated with an increased risk of recurrent miscarriages if both partners carried the allele

23. The Paternal contribution Balanced structural chromosomal abnormalities Sperm abnormalities Sub chromosomal abnormalities subtle chromosome rearrangements gene dosage imbalances Mutations Early studies using conventional semen analysisi showed that semen from male partners of women with rm were not significantly abnormal Recent studies have shown higher aneuploidy rates Paternal genome plays a role in early embryogenesis sperm chromatic structure assay(SCSA) meeasures chromatin susceptibility to acid denaturation. High scsa values are associated with rm Increaased rates of miscarriages has been observed in icsi cyles in patients known to have meiotic disorders.Early studies using conventional semen analysisi showed that semen from male partners of women with rm were not significantly abnormal Recent studies have shown higher aneuploidy rates Paternal genome plays a role in early embryogenesis sperm chromatic structure assay(SCSA) meeasures chromatin susceptibility to acid denaturation. High scsa values are associated with rm Increaased rates of miscarriages has been observed in icsi cyles in patients known to have meiotic disorders.

24. Sperm abnormalities 24 couples with rm � semen analysis&Fish Characteristic rec misc fertile donors Motile 46% 49% Tapered 38% 16% Amorphous 9% 5% Viable 56% 71% (Carrell 2003)

25. Sperm abnormalities Disomy rec misc sperm donors Xy 0.77% 0.31% 13 1.02% 0.39% 18 0.51% 0.25% 21 0.47% 0.28%

26. Sperm aneuploidy Mechanisms Quality marker ? Carrier of a defect that influences post zygotic aneuploidy , implantation, embryonic growth

27. The role of the trophoblast Placental development Continuous turnover CT proliferation � differentiation � fusion � aging � shedding as syncitial knots into maternal circulation over 3- 4 weeks CT / ST ratio � reduced in apl pregnancies and rsas Tenney Parker changes Dvillous trophoblast displays a continuous turnover from proliferation of cytotrophoblast cells to late apoptosis inside th syncytiotrophobblast leading to extrusion of syncitial knots in the maternal circulation In 1st trimester ratio of number of ct and the no of nuclei absorbed in st could be predictive of placental survival and growth � ct st ratio During late pregnancy extrusion of syncitial material is achieved by a variety of methods tenney parker changes are syncitial knotting described as histological term to describe the histological appearance of increased numbers of seemingly multinucleated trophoblastic outgrowths at the villous surface. - widely accepted as diagnostic marers of placental ischemia and pet Patterns of shedding -- arrested shedding � IUGR Necrotic shedding in PETDvillous trophoblast displays a continuous turnover from proliferation of cytotrophoblast cells to late apoptosis inside th syncytiotrophobblast leading to extrusion of syncitial knots in the maternal circulation In 1st trimester ratio of number of ct and the no of nuclei absorbed in st could be predictive of placental survival and growth � ct st ratio During late pregnancy extrusion of syncitial material is achieved by a variety of methods tenney parker changes are syncitial knotting described as histological term to describe the histological appearance of increased numbers of seemingly multinucleated trophoblastic outgrowths at the villous surface. - widely accepted as diagnostic marers of placental ischemia and pet Patterns of shedding -- arrested shedding � IUGR Necrotic shedding in PET

28. Placental oxidative stress Human fetus develops in a low oxygen environment Intraplacental oxygen conc increases from < 20mm Hg at 10 wks to > 50 at 12 wks Trophoblastic cells are extremely sensitive to oxidative stress Mounting evidence that in most miscarriages the onset of intervillous circulation is premature and widespread due to incomplete transformation of uteroplacental arteries leading to high oxygen concentrations in early pregnancy

29. Placental oxidative stress

30. Endometrial receptivity INFERTILITY RM 50-75% of pregnancies lost represent a failure of implantation Failure of implantation may result from a non receptive endometrium Involves a complex synchronous interaction between embryo , endometrium and ovary IM PLANTATION REQUIRES INTERACTION BETWEEN EMBRYO AND ENDOMETRIUM. DESCRIBED IN 50S BASED ON ANIMAL STUDIES IM PLANTATION REQUIRES INTERACTION BETWEEN EMBRYO AND ENDOMETRIUM. DESCRIBED IN 50S BASED ON ANIMAL STUDIES

31. Endometrial receptivity Growth factors �LIF,HB EGF Cytokines Adhesion molecules- integrins A5, B3 Steriod hormones and receptors Immunologic factors-- NK Cells, T cells Prostaglandins LIF � leukemia inhibiting factor hBEGF - heparin binding epidermal growth factor LIF PRESENT IN ENDOMETRIUM AT TIME OF IMPLANTATION ( d20-24), DECREASED LIF ASSOCIATED WITH RM Decreased lif is associated with rm antiprogesterones decrease lif expression. Hbgef increased by est and prog, stimulates embryo development and trophoblast growth steroid hormones regulates proliferation and diff of endometrial stromal celss , regulates expression of hox genes, stimulates embryo adhesionLIF � leukemia inhibiting factor hBEGF - heparin binding epidermal growth factor LIF PRESENT IN ENDOMETRIUM AT TIME OF IMPLANTATION ( d20-24), DECREASED LIF ASSOCIATED WITH RM Decreased lif is associated with rm antiprogesterones decrease lif expression. Hbgef increased by est and prog, stimulates embryo development and trophoblast growth steroid hormones regulates proliferation and diff of endometrial stromal celss , regulates expression of hox genes, stimulates embryo adhesion

32. RX to improve endometrial receptivity Progesterone�at best controversial , at worst ineffective Immunomodulation paternal cell immunization intravenous immunoglobulin PROGESTERONE AND IMMUNOSUPPRESSION STIMULATES GROWTH FACTORS, HOXA GENE EXPRESSION AND ADHESION MOLECULES INHIBITS LYMPHOCYTE CYTOTOXICITY, PROINFLAMMATORY CYOTKINES , NK CELL DEGRANULATION AND CYTOKINE PRODUCTIONPROGESTERONE AND IMMUNOSUPPRESSION STIMULATES GROWTH FACTORS, HOXA GENE EXPRESSION AND ADHESION MOLECULES INHIBITS LYMPHOCYTE CYTOTOXICITY, PROINFLAMMATORY CYOTKINES , NK CELL DEGRANULATION AND CYTOKINE PRODUCTION

33. Novel Therapies Intrauterine Prostaglandins Intrauterine steroids Intrauterine Peripheral blood mononuclear cells L arginine Glue� Fibrin!

34. Infections 1 in 20 women are exposed to pathogens Majority are harmless Early infection � congenital problems Delayed infection -

35. Infections - spectrum MISCARRIAGES CONGENITAL INFECTIONS STILL BIRTH NEONATAL DEATHS ASYMPTOMATIC INFECTIONS NORMAL FINDINGS

36. INFECTIONS Rubella CMV HBV VZ HSV HIV GBS Syph

37. Infections � What do they do ? Direct effect on ova Endometrial infection � implantation defects Embryopathy Placental infections Amniotic fluid infection

38. UTERINE PATHOLOGY Septate uterus- Asherman�s Syndrome- Uterine Fibroids- esp. sub mucous Primary endometrial defects Des exposure Septate uterus is most common and there is impairment of implantation due to avascularity- also reduced sensitivity to steroids Ashermans- reduced rewponsiveness to steroids- extensive dense fibrosis carries a poor prognosis Fibroids � observational data from six ivf series � modestly compromised with intramural,possibly with sebserous Removal of submucous fibroids reduces miscarriage rates- possibly also with intramural Hsgs in rm- normal- 43 septum � 22 ashermans- 23 polyps -1 Conflicting results on relationships between ER and PR receptors and RM- links to renewed interest after discovery of Era and PRB receptore no reltionship to AR receptorsSeptate uterus is most common and there is impairment of implantation due to avascularity- also reduced sensitivity to steroids Ashermans- reduced rewponsiveness to steroids- extensive dense fibrosis carries a poor prognosis Fibroids � observational data from six ivf series � modestly compromised with intramural,possibly with sebserous Removal of submucous fibroids reduces miscarriage rates- possibly also with intramural Hsgs in rm- normal- 43 septum � 22 ashermans- 23 polyps -1 Conflicting results on relationships between ER and PR receptors and RM- links to renewed interest after discovery of Era and PRB receptore no reltionship to AR receptors

39. Cervical Cerclage Shirodhkar McDonalds Lash Benson Durfee

40. Indication for abd cerclage Congenital short cx Amputated cx Torn cx Severe scarring Chronic cervicitis Cervicovaginal fistula Failed shirodhkar Rec pproms Cervical dysfunction

41. Cervical cerclage Steer Modifications Nuchal first USS guidance � before , during and after No bladder dissection Straight blunt needle

42. PROTHROMBOTIC STATES Antiphospholipid syndromes Heritable Thrombophilia-antithrombin def protein C & S def Factor V Leiden Prothrombin20210 A Thrombocythemia

43. ANTIPHOSPHOLIPID SYNDROME 7-42 % OF WOMEN Wide variation Poor laboratory standardization

44. APL � diagnostic criteria 3 or more unexplained consecutive spontaneous abortions before 10 wks with exclusion of maternal anatomic or hormonal abnormalities and maternal and paternal chromosomal abnormalities OR One or more unexplained deaths of a morphologically normal fetus at or beyond 10 wks with normal fetal morphology documented by USS or direct examination of the fetus OR One or more PTBs of a morphologically normal neonate at or before 34wks gest because of severe PET or Placental Insufficiency

45. AND Persistent abnormality of the following tests when measured twice at least 6 wks apart Lupus anticoagulant Antiphospholipid antibodies � IgG or IgM

46. Pathophysiology of APS Thrombotic Lack of Trophoblastic invasion in 1st trimester decidua

47. APL � Maternal Risks Thrombosis �Heparin RX Access to prenatal care and pt education Hypertension � antenatal care and pt education Thrombocytopenia Secondary conditions rheumatologist involvement Treatment Complications� hge, osteopenia, thrombocytopenia Catastrophic APS

48. FETAL RISKS Miscarriage Uteroplacental insufficiency IUD IUGR Fetal Distress Preterm birth SLE and Thrombosis

49. Heritable thrombophilias 5 recognized defects � antithrombin def, protein c def, protein s def, v leiden, prothrombin 20210A variant EPCOT � European study analysed pregnancy outcome in women with known thrombophilia � v leiden not associated with rm, better association with activated protein c resistance Essential thrombocythemia

50. ENDOCRINOLOGICAL FACTORS Hypersecretion of LH(>10IU/L)In the follicular phase is a marker for RM Androgen levels in the follicular phase have been shown to be high in pts with RM- This correlates negatively with the conc. of Placental Protein 14 a biochemical marker for endometrial function Hyperprolactinemia � no firm evidence Difficult to evalauate role of LH for different assay methods,sifferent samples, different timings pulsatile release of LH- hypersecretion mainly in urine samples- may be an overestimate as suppression of LH does not have an observable beneficial effect � occurs in only 8% of women PCO not predictive of pregnancy loss- Rai 2000 prevalance of PCO in rm is 40% - live birth rate similar in pco(60%) to normal women (59%)Difficult to evalauate role of LH for different assay methods,sifferent samples, different timings pulsatile release of LH- hypersecretion mainly in urine samples- may be an overestimate as suppression of LH does not have an observable beneficial effect � occurs in only 8% of women PCO not predictive of pregnancy loss- Rai 2000 prevalance of PCO in rm is 40% - live birth rate similar in pco(60%) to normal women (59%)

51. IMMUNOLOGICAL FACTORS Autoantibodies � 18-43% of pts with RM APL --14% ANA � 7% Antisperm AB Thyroid Peroxidase 31 % of patients of rm have positive test results with one or both thyroid ab- peroxidase and thyroglobulin-may be aresult direct effect of autoab on fetal tissue or representing a more generalized defect in auto immunity Peroxidase ab has prognostic value- titre declines with positive outcome of pregnancy31 % of patients of rm have positive test results with one or both thyroid ab- peroxidase and thyroglobulin-may be aresult direct effect of autoab on fetal tissue or representing a more generalized defect in auto immunity Peroxidase ab has prognostic value- titre declines with positive outcome of pregnancy

52. Immunology � Alterations in Cellular Immune Function NK cells- LGL cells � CD56+ increased in endometrium of RM pts Around time of implantation 20% cells of endometrial stroma are leucocytes � majority being of large granular types with surface markers for cd56,cd16 and cd3 like nk celss- staining for cd56,4,14,16, and mhc class2 are increased in rm( data from endometrial biopsies0Around time of implantation 20% cells of endometrial stroma are leucocytes � majority being of large granular types with surface markers for cd56,cd16 and cd3 like nk celss- staining for cd56,4,14,16, and mhc class2 are increased in rm( data from endometrial biopsies0

53. OTHER FACTORS COFFEE SMOKING AND ALSCOHOL HYPERHOMOCYSTENEMIA- Interferes with embryonic development SELENIUM DEFICIENCY CELIAC DS STRESS PCP EXPOSURE MATERNAL DS Hyperhomocysteinemia associated with arterial and venous thrombosis- levels fall in pregnancy- high levels are associated with pregnancy complications including ntds , placental infardcts, iugr, placental abruption Meta analysis of dat\a shows significvant correlation between hyperhomocystenemia and rm Folate deficiency is the commonest acquired causes � genetic causes \mthfr gene defect( polymorphism at position 677 of gene ) causes a thermolabile variant- take fasting blood process within 1 hr Stress may be linked to immunological imbalancesHyperhomocysteinemia associated with arterial and venous thrombosis- levels fall in pregnancy- high levels are associated with pregnancy complications including ntds , placental infardcts, iugr, placental abruption Meta analysis of dat\a shows significvant correlation between hyperhomocystenemia and rm Folate deficiency is the commonest acquired causes � genetic causes \mthfr gene defect( polymorphism at position 677 of gene ) causes a thermolabile variant- take fasting blood process within 1 hr Stress may be linked to immunological imbalances

54. BASELINE INVESTIGATIONS ENDOCRINE�LH,FSH,TSH,PRL,PRG BIOCHEMICAL� BLOOD SUGAR , HOMOCYSTEINE UTERINE� USS, HSG IMMUNOLOGICAL � LUPUS,APL,C3,4 THROMBOPHILIA SCREEN GENETIC

55. PROGNOSTIC FACTORS Fetal Heart Beats No of prev misc Age Underlying etiology History of live birth Underlying infertility BMI Menstrual cycles In a low risk population risk of miscarriage is 2-6% once fhr is seen. In high risk populations this ranges from10-30% esp in older women , so do not put undue stress on this Live birth rate falls from 65% in women with 2 misc to 43% in women with 6 miscarriages Higher fetal loss in older women Bmi not related to misc Regular cycles positively correted with live birthrateIn a low risk population risk of miscarriage is 2-6% once fhr is seen. In high risk populations this ranges from10-30% esp in older women , so do not put undue stress on this Live birth rate falls from 65% in women with 2 misc to 43% in women with 6 miscarriages Higher fetal loss in older women Bmi not related to misc Regular cycles positively correted with live birthrate

56. Neonatal Outcome Increased Risk of SFD PTL PNM LSCS Reginald -97 women in 87- small for gest age in 30%,preterm del in 28%.mortlqaity in 2% -no controls and underlying cause and treatment not documented . Hughes (91) compared to a control gr and found small for gest age in 3.4%, preterm del in 12% and perinatal mortality in 0- no different from control gr.- again incomplete data re diagnosisand treatment Recently Jivraj(�))!)- in cohort of women with rm compared to controls preterm del 14%,small for dates 13%,pperinatal mortality 2.5% , lscs 40%- higher than in controlReginald -97 women in 87- small for gest age in 30%,preterm del in 28%.mortlqaity in 2% -no controls and underlying cause and treatment not documented . Hughes (91) compared to a control gr and found small for gest age in 3.4%, preterm del in 12% and perinatal mortality in 0- no different from control gr.- again incomplete data re diagnosisand treatment Recently Jivraj(�))!)- in cohort of women with rm compared to controls preterm del 14%,small for dates 13%,pperinatal mortality 2.5% , lscs 40%- higher than in control

57. MANAGEMENT CAUSE SPECIFIC Uterine anomalies � metroplasty, hysteroscopic surgery Endometrial defect- prime endometrium in follicular phase with estrogen, GnRH Prothrombotic states- aspirin, heparin, steroids PCOS � laparoscopic drilling associated with reduced miscarriage rate Aspirin and heparin together results in a successful outcome in 70% cases � start aspirin at positive pregnancy test and heparin at detection of fhr stop at 34 wks- monitor platelets weekly for 4 wks and 3 wks thereafter- postnatal prophylaxis not reqiured for rmAspirin and heparin together results in a successful outcome in 70% cases � start aspirin at positive pregnancy test and heparin at detection of fhr stop at 34 wks- monitor platelets weekly for 4 wks and 3 wks thereafter- postnatal prophylaxis not reqiured for rm

58. MANAGEMENT TLC including serial uss Progestogens- no clear benefit Hcg- no evidence of benefit Immunotherapy- Unproven Aspirin � empirical use not justified Thyroid hormones Folic acid Immunotherapy �active and passive paternal leucocyte immunizaation, trophoblast membrane infusion, iv immunoglobulinsImmunotherapy �active and passive paternal leucocyte immunizaation, trophoblast membrane infusion, iv immunoglobulins

59. THANK YOU