1. Identifying the signs for Implantation Failure and Miscarriage By Roy Farquharson Liverpool Women's Hospital UK Contact: rgfarquharson@yahoo.com 1

2. Declaration of Interests Executive Committee member, European Society of Human Reproduction and Embryology (2011 - 2015) NICE Guideline Development Group for ectopic pregnancy and miscarriage (CG 154; 2010-2013) Chair, Association of Early Pregnancy Units (2006-2011) Co-ordinator, ESHRE Special Interest Group for Early Pregnancy (2007-2010) Associate Editor, Human Reproduction Update (20102014) 2 3. Educational Objectives Learning Objectives At the conclusion of this presentation, participants should be able to: Describe the assessment for the diagnosis of implantation failure, ectopic pregnancy and miscarriage Acknowledge the limitations of available diagnostic methods Develop a practical approach to using relevant tests and management protocols. 3 4. 4 5. Predictive Modelling for Early Pregnancy Best Area of Interest Diagnostic UtilityParameter(s)OvulationBiomarkerD21 ProgesteronePregnancy of Unknown Location (PUL)Transvaginal (TVU) Scan and BiomarkerTVU Scan + HCG doubling time/ratio +/- Progesterone Pregnancy of Scan Fetal heart action Uncertain Viability Scan plus Crown-rump (PUV) ExclusivelyScan length5 6. Practical Advice - PULs 3. Predicting outcome Hormones Human chorionic gonadotrophin (hCG) Progesterone Other: Creatine kinase CA 125 Activin A Inhibin A Mathematical Models6 7. HCG changes in normal pregnancyMean (SE) serum concentrations of human chorionic gonadotrophin (adapted from Braunstein et al 1976)7 8. Haemodynamically stable Pain free Expectant managementPUL Haemodynamically stableHaemodynamically unstablePainPain? Serum hCG Serum hCG levels at 0 and 48 hrs +/progesterone ? Intra-uterine PregnancyConsider laparoscopy/laparotomyConsider laparoscopy? Ectopic Pregnancy? Failing PUL8 9. Practical Advice - PULs 3. Predicting outcome Hormones Human chorionic gonadotrophin (hCG) Progesterone Other: Creatine kinase CA 125 Activin A Inhibin A Mathematical Models9 10. Serum hCG Levels Single LevelsSerial LevelsDiscriminatory Zone10 11. Serum hCG Levels Single LevelsSerial LevelsDiscriminatory Zone Developed with respect to transabdominal USS Lower levels of hCG used with TVS Using a single value of hCG in a PUL population is of limited value: Many ectopic pregnancies have a low hCG Clinicians may be falsely reassured 11 12. Serum hCG Levels Single LevelsSerial Levels Change over 48hrs (hCG ratio)Intrauterine Pregnancies (IUPs) Kadar et al. (1981) first to describe the minimal rate of rise for an IUP to be 66% over 48hrs More recently minimal rise reported to be 53% (Barnhart et al. 2004) In clinical practice a more conservative cut-off of 35% has been suggested 12 13. Serum hCG Levels Single LevelsSerial Levels Change over 48hrs (hCG ratio)Failing PULs A decline of 21-35% at 48 hours depending on initial hCG level ( levels at presentation rate of decrease) (Barnhart et al. 2004) An hCG decrease of >13% (hCG ratio < 0.87) has been shown to have a sensitivity of 92.7% and a specificity of 96.7% for the prediction of a failing PUL (Condous et al., 2006)13 14. Serum hCG Levels Single LevelsSerial Levels Change over 48hrs (hCG ratio)Ectopic Pregnancies (EPs) No single way to characterize the pattern of serum hCG behaviour (Silva et al., 2006) hCG profile mimicked IUP in 21% and a spontaneous miscarriage in 8% (Silva et al., 2006) Sensitivity of 83% for EP when IUP excluded by hCG rise < 35% and failing PUL excluded by hCG decrease > 14 21-35% (Seeber et al., 2006) 15. Evidence based management of PULsPredicting outcome Hormones Human chorionic gonadotrophin (hCG) Progesterone Other: Creatine kinase CA 125 Activin A Inhibin A Mathematical Models15 16. Serum Progesterone Levels Serum Progesterone < 20 nmol/L PPV > 95% to predict pregnancy failure (Banerjee et al., 2001)Viable IUPs reported with levels < 16nmol/L> 60 nmol/L Strongly associated with viable pregnancies Discriminative capacity insufficient to diagnose ectopic pregnancy with certainty (Mol et al., 1998)Good at predicting viability but not location16 17. Pregnancies of Unknown Location (PULs) The majority of PULs fail and resolve spontaneously (44% 69%) RCOG green top guideline on Tubal Pregnancy 2004 sourcing five observational studies Of the remainder, ectopic pregnancy was subsequently diagnosed in 14 to 28% Intervention (medical or surgical) was required in approx 25% cases17 18. HCG in practice (NICE 2012) Clinical symptoms more important than HCG results HCG levels do not locate the pregnancy nor assess viability 2 levels 48 hours apart are useful for risk stratification and act as best evidence for subsequent management Limitations of prediction should be shared and acknowledged to patients (eg ectopic pregnancy HCG levels mimic viable IUP in 21% and EPL in 8%) Ectopic pregnancy and miscarriage: diagnosis and initial management in early pregnancy of ectopic pregnancy and miscarriage. (NICE Clinical guideline 154; 2012; www.nice.org.uk) 18 19. Sites of ectopic pregnanciesIllustration: John Yanson.Seeber. Suspected Ectopic Pregnancy. Obstet Gynecol 2006. From: Seeber: Obstet Gynecol, Volume 107(2, Part 1).February 2006.399-41319 20. Ectopic Pregnancy Variable mode of presentation Mask of invisibility High index of suspicion and vigilance eg against diagnosis of complete early pregnancy loss All areas of emergency care provision will receive cases of undiagnosed ectopic pregnancy 20 21. Ectopic Pregnancy presentation ACUTE (typical) Collapse with lower abdominal pain, tachycardia and hypotension Pain, amenorrhoea and sign of pelvic tenderness EPU presentation with positive pregnancy test, scan showing empty uterus and adenexal inhomogeneous mass CHRONIC (atypical) Symptoms mimicking gastroenteritis Light irregular bleeding >1/3rd of all patients have no risk factors21 22. HCG studies Review question What is the diagnostic accuracy of two or more hCG measurements for determining an ectopic pregnancy in women with pain and bleeding and pregnancy of unknown location? Description of included studies Nine studies were included in this review (Barnhart et al., 2010; Condous et al., 2004; Condous et al., 2007; Dart et al., 1999; Daus et al., 1989; Hahlin et al., 1991; Mol et al., 1998; Stewart et al., 1995; Thorburn et al., 1992). Five prospective cohort studies (Condous et al., 2004; Condous et al., 2007; Hahlin et al., 1991; Mol et al., 1998; Thorburn et al., 1992) Four retrospective cohort studies (Barnhart et al., 2010; Dart et al., 1999; Daus et al., 1989; Stewart et al., 1995). Conducted in the UK (Condous et al., 2004; Condous et al., 2007), the USA (Dart et al., 1999; Daus et al., 1989; Stewart et al., 1995), the Netherlands (Mol et al., 1998) and Sweden (Hahlin et al., 1991; Thorburn et al., 1992). One study (Barnhart et al., 2010) was conducted in both the UK and USA.22 23. GRADE system GRADE (Grading of Recommendations Assessment,Development and Evaluation) assesses evidence on an outcome-by-outcome basis Quality can vary within a study and is based on 5 factors: Study designLimitations Inconsistency Indirectness Imprecision 23 2323 24. Summary of findingsQuality assessmentNo. of studiesDesignLimitati onsIncon siste ncyIndirect nessImprec isionOther consider ationsNu m be r of w o m enMeasure of diagnostic accuracy Sensiti vity % (95% CI)Specifi city % (95% CI)Positi ve predic tive value % (95% CI)Negative predictiv e value % (95% CI)Positiv e likelih ood ratio % (95% CI)Negati ve likelih ood ratio % (95% CI)Qualit y% (95% CI)GRADE findings for the diagnosis of ectopic pregnancy using two or more hCG measurements Model M4 1 study Condous et al., 2007prospec tive study1 study Barnhart et al., 2010retrospe ctive study (2 included cohorts: UK and adjuste d USA)serious1, 6serious4, 6serious4, 6no seriou s incons istenc yserious2,no seriou s incons istenc yserious2,no seriou s incons istenc yserious11212no serious impreci sionnoneno serious impreci sionnone17343180.0 (59.8, 100)80.8 (65.6, 95.9)88.6 (83.7, 93.6)88.9 (85.8, 92.0)40.0 (22.5, 57.5)31.8 (20.6, 43.1)97.9 (95.6, 100)98.6 (97.4, 99.8)7.02 (4.25, 11.61)7.27 (5.21, 10.14)0.23 (0.08 , 0.62)0.22 (0.10 , 0.48)LOWV. LOW3no serious impreci sionnone54454.8 (45.2, 64.4)87.7 (84.7, 90.8)51.4 (42.1, 60.7)89.2 (86.2, 92.1)4.47 (3.29, 6.06)0.52 (0.46 , 0.64)V. LOW24 25. Treatment Options for EcP and PUL Laparoscopic surgery ESEP RCT 2013 (NL) Salpingectomy versus SalpingostomySystemic Methotrexate (MTX) - DEMETER RCT 2013 (Fr)Expectant management - METEX RCT 2013 (NL)25 26. When can expectant management be employed? Clinically stable Minimal symptoms Discriminatory HCG zone: 1000-2000iu/l Weekly USS Twice weekly HCG until 7mm; NICE GDG & RCOG 2012) Acknowledgement of inherent, wide biological variation of embryo growth velocities Specificity of viability assessment is 99.9% 29 30. Comparison of the CRL curve (solid line) with the Robinson curve (dashdotted) and the Hadlock curve (dotted) 90 80 70CRL (in mm)60 50 40 30 20 10 0 40506070 GA (in days)809010030 31. Updated Gestational Age Measurement in early pregnancy Total number of pregnancies: 6666 (2002-2008) No. Excluded = 2956 (uncertain dates, redated, infertility treatment, miscarriage, stillbirth, genetic or congenital abnormalities) No. Included = 3710 normal singleton pregnancies dated according to known and recorded last menstrual period (LMP) with confirmed viability at the time of the nuchal scan Predominantly transvaginal ultrasound below 10 weeks by contrast with Robinson transabdominal derived CRL curve (BMJ, 1972) The gestational age (GA) ranged between 35 and 98 days Linear mixed-effects model in order to account for possible codependency of multiple CRL measurements in the same patientReference: Bottomley C ,Bourne T. Dating and growth in the first trimester. Best Practice and research Clin Obstet Gynaecol 2009 ; ESHRE precongress course, Roma, 201031 32. TV Ultrasound Fetal loss with CRL =7mm32 33. Embryoscopy the close-up H=head/heart prominence, Y=yolk sac, B=bubble33 34. TVU small embryonic structure in disproportionately large sac34 35. Embryoscopy short body stalk with 6mm CRL - cytogenetics = 47XY+735 36. Fetal loss at 7 weeks CRL = 19mm36 37. Cytogenetics = 47XY+15 Small head compared to CRL, dysplastic face, partial encephalocele37 38. Is Treatment Failure in RM a valid concept? - Cytogenetic Analysis of Pregnancy Loss in RM38 39. Opportunityisnowhere39 40. microarrays technique high resolution WHOLE genome scancytogeneticsFISHarrays40 41. Microarray Advantages - SINGLE test vs Karyotype + 5 FISH tests - DNA extraction directly vs cell culture - detect low level fetal cells vs maternal cell contamination - higher resolution vs lower resolutionDisadvantages - CANNOT detect balanced rearrangements- confirmatory follow up studies41 42. Trisomy 10 - TR Karyotype = Normal Female Array = Abnormal MALE result +10 FISH = confirmed +10 (70% MCC)42 43. 14q deletion - JS Karyotype = Normal Female Array = Abnormal Female deletion 14qFISH = confirm deletion in 11% of cells (89% MCC)43 44. RM Evaluation of Array CGH v Conventional Cytogenetics (McNamee et al, British Journal of Hospital Medicine, 2013, 74, 36-40 )Array CGH and conventional cytogeneticsN=50Triploidy on FISH N=4Normal result N=23(46%)Abnormal result N=27 (54%)Diagnosed with conventional cytogeneticsMissed withN=14N=9NUMERICAL+16 x3 +15 x2 +21 x2 +13 x2 +22+10 +14 -X x2conventional cytogeneticsNUMERICAL +22 +10 +15 +8 +16 STRUCTURAL >dup(22)(q11.2q11.2) , >del(14q)(q31.1) ,t(1:q1 6)mat >del(13q)12.3-q34 44 45. Pregnancy Success Prediction MatrixFollowing idiopathic RM, the predicted probability (%) of successful pregnancy is determined by age and previous miscarriage history ( 95% confidence interval