Biomarkers of OvarianResponseSandro Esteves, M.D., Ph.D.Director, ANDROFERTAndrology & Human Reproduction ClinicReferral Center for Male ReproductionCampinas, BRAZILMerck Serono Symposium – La Red Annual Meeting 2013May 3, 2013, Panama City"360 degrees in ART"
Esteves, 3Know which biomarkers are best for clinicaluseUnderstand how they work including theiradvantages and shortcomingsLearn how to use their results to individualizeovarian stimulationWhat is in it for me?
Biomarkers of Ovarian ResponseLecture StructureEsteves, 4Level Type of evidence1a Obtained from meta-analysis of randomised trials1b Obtained from at least one randomised trial2a Obtained from one well-designed controlled study withoutrandomisation2b Obtained from at least one other type of well-designed quasi-experimental study3 Obtained from well-designed non-experimental studies(comparative and correlation studies, case series)4 Obtained from expert committee reports or opinions or clinicalexperience of respected authoritiesModified from Sackett et al. Oxford Centre for EBM Levels of Evidence (2009)Level ofEvidence Review of Biomarkers applied to the general IVF population Studies with Best Levels of Evidence
Why Do We Need Biomarkers toPredict Ovarian Response to COS?Esteves, 5ForDoctors• Avoid over-aggressive stimulation in‘true’ high responders• Avoid over-conservative stimulation in‘false’ high respondersExcessiveOvarianResponse• Avoid over-conservative stimulation in‘true’ DOR• Avoid over-aggressive stimulation in‘false’ DORDiminishedOvarianReserve(DOR)
Esteves, 6Realistic Prognostic Information• Poor or Negligible Response• Cycle cancellation• Egg donation or adoption• Chances of Pregnancy and Live BirthForPatients Why Do We Need Biomarkers toPredict Ovarian Response in COS?
Esteves, 7Which are the Biomarkers?Biomarkers●Hormonal Biomarkers: FSH, Clomiphene citratechallenge test, Inhibin-B, Anti-MullerianHormone (AMH)●Functional Biomarkers: Antral Follicle Count(AFC)●Genetic Biomarkers: Single NucleotidePolymorphisms for FSH-R; LH/LH-R; E2-R;AMH-R
A Valid Biomarker Should beHighly Sensitive and Highly SpecificEsteves, 8+-+ -BiomarkerTestResultDiminished or ExcessiveOvarian ResponseAdapted from: ASRM Practice Committee, Fertil Steril 2012;98:147FalsePositive(B)FalseNegative(C)TrueNegative(D)TruePositive(A)Sensitivity (A/A+C)Specificity (D/B+D)Predictive Value(PPV=A/A+B; NPV=D/C+D)Accuracy(A+D/A+B+C+D)
Esteves, 9Understand How TheyWork And What Are TheirAdvantages andShortcomingsBiomarkers of Ovarian Response
Esteves, 10Understanding BiomarkersBasic ConceptsFSHWhat is known?*Standardized assays by WHO IRP 78/549; Esposito et al.Hum Reprod 2002;17:118; ASRM Practice Committee, Fertil Steril 2012;98:147.• Basal (days 2-3) serum FSH levels increase withaging• Indirect biomarker of ovarian reserveInfluenced by Inhibin B and Estradiol• FSH assays are well standardized* but... High Inter- and Intra-cycle Variability (Low Reliability)
Esteves, 11EvidenceLevel2bUnderstanding BiomarkersFSH is not very accurate to assess ovarianresponse or pregnancy risk in ARTFSHWhat is known?*Standardized assays by WHO IRP 78/549Esposito et al. Hum Reprod 2002;17:118; ASRM Practice Committee, Fertil Steril 2012;98:147.High numberof falsenegatives‘Normal’FSH valuesDOR orfailure topregnancy• Cut-off point of 11.4 IU/L*:High specificity (83%-100%) to predict ≤4 retrieved oocytesLow sensitivity (10%-30%) to predict DOR and failure toachieve pregnancy in ART• Cannot Identify High RespondersLow numberof falsepositives
Esteves, 12Understanding BiomarkersAMHLa Marca et, Hum Reprod 2009;24:2264; Fleming et al, Fertil Steril 2012;98:1097.What is known?Direct Biomarker of OvarianReserve: Dimeric glycoprotein; ~140KDaProduct of GCs of early folliclesPre-antral and small antral (≤ 4-8mm) Reflect both the number ofsmall growing follicles and theprimordial pool at gonadotropin-independent folliculogenesis
Esteves, 13Understanding BiomarkersBasic ConceptsAMHAMH Serum Levels:Peak at age 25 and decrease with aging Early marker of diminished ovarian reserveNon-growingfollicles (NGF)recruited permonthKelsey et al. Mol Hum Reprod 2012;18:79
Esteves, 15EvidenceLevel2aUnderstanding BiomarkersAMH is accurate to assess ovarian responseAMHWhat is known?*DSL assay; 1>20 oocytes retrieved; 2≤5 oocytes retrieved;Conversion: ng/mL to pmol/L = value in ng/mL X7.14• Cut-off point of 3.5 ng/mL* (Nardo et al, Fertil Steril 2009;92:1586) High sensitivity (88%), specificity (70%) and accuracy (0.81)to predict excessive response1• Cut-off point of 1.4 ng/mL* (Kwee et al, Fertil Steril 2008;90:737) High sensitivity (76%) and specificity (86%) to predict DOR2Caution to apply AMH cut-off points!Make sure the assay you rely on is the sameused in the reference population
Esteves, 16Understanding BiomarkersAMHShortcomings and PitfallsFleming et al. RBM online 2013;26:130; Nelson SM. Fertil Steril. 2013 Jan 8;Nelson & La Marca. RBM online 2011;23:411;Several ELISA assays with different performances• DSL and Immunotech• Beckman-Couter generation II (hybrid of DSL and Immunotech)• Fully automated ELISA assay (to be released)No international standardization and EQCSample instability and measured levels altered by handling• Collection in EDTA• Storage at room temperature (up to 40% increase)• No separation of serum from blood before postage
Esteves, 17Understanding BiomarkersAFCBroekmans et al. Fertil Steril, 2010; 94(3):1044-51; Scheffer et al. Hum Reprod 2003;18:700What is known?Direct Biomarker of FunctionalOvarian Reserve: Sum of antral follicles in both ovaries onTVUS at early follicular phase (D2-D4): 2-10 mm (mean diameter) Greatest 2D-plane Decrease in the number of detectable(TVUS) antral follicles with aging Reflect the number of antral follicles inthe ovaries at a given time that can bestimulated by exogenous gonadotropins
Esteves, 18AFCModerate to Low Inter-cycleFluctuations:van Disseldorp et al, Hum Reprod 2010;25:221ICC: 0.71 (95% CI: 0.63–0.77);29% individual cycle variationUnderstanding BiomarkersEvidenceLevel2aHigh Inter- and Intra-observer Reproducibility:Scheffer et al. Ultrasound Obstet Gynecol 2002;20:270
Esteves, 19EvidenceLevel1bUnderstanding BiomarkersAFC is accurate to assess ovarian responseAFCWhat is known?Cut-off point of 4:(Bancsi et al, Fertil Steril 2002;77:328)Moderate sensitivity (61%)and High specificity (88%)and to predict DOR2Cut-off point of 14:(Kwee et al, Fertil Steril 2008;90:737)High sensitivity (81%) andspecificity (89%) to predictexcessive response1Eldar-Geva et al.Hum Reprod 20051>20 oocytes retrieved in conventional COS; 2≤4 oocytes retrievedCaution to Apply AFC Cut-offPoints to Predict Number ofOocytes Retrieved!For any given AFC there is apotential oocyte yield, but it can bealtered by the stimulation strategy
Esteves, 20Understanding BiomarkersAFCShortcomings and Pitfalls1Nelson SM. Fertil Steril. 2013 Jan 8;2Broekmans et al., Fertil Steril, 2010; 94(3):1044-51;3Raine-Fenning et al., Fertil Steril 2009;91:1469.Lack of standardization1• Inclusion criteria for antral follicles e.g., 2–5 mm or 2–10 mm• Method for counting and measuring follicles• Variable scanning techniques• Image optimizationImproved standardization proposed2Three-dimensional automatedfollicular tracking3• Reduce intra- and inter-observer variability• Requires offline analysis• Costly
Esteves, 21BiomarkersFSH AMH AFCClinical Utility in Ovarian StimulationExcessiveOvarianResponseAvoid over-aggressivestimulation in ‘true’ highresponders (↑Sensit.)- +++ +++Avoid over-conservativestimulation in ‘false’ highresponders (↑Specif.)- +++ +++DiminishedOvarianReserve(DOR)Avoid over-conservativestimulation in ‘true’ DOR(↑Sensit.)+ +++ +++Avoid over-aggressivestimulation in ‘false’ DOR(↑Specif.)+++ +++ +++
Esteves, 22Determining Who is WhoBefore COSEvidenceLevel1aBiomarkers
Esteves, 23EvidenceLevel1aBiomarkers AMH and AFC are notaccurate for pregnancypredictionBroer et al. Fertil Steril 2009 ; Broer et al. Hum Reprod Update, 17:46; 2011
Esteves, 24AFC and AMH are accurate biomarkers to predictovarian response to ovarian stimulation.Given their limitations clinicians should exercisecaution to interpret cut-off points when using AMHand AFC as sole predictors of ovarian response.AMH and AFC are not accurate predictors of failure toconceive after COS and IVF.There is insufficient evidence to recommend the use ofa combination of biomarkers to improve predictionpower.Understand How Biomakers Work,their Advantages and Shortcomings
Esteves, 25Learn How to UseBiomarkers toIndividualize OvarianStimulation
Low-starting FSH dose(150 UI)AMH (ng/mL) >2.1¶GnRHAgonist(n=148)GnRHAntagonist(n=34)Days of Stimulation 13 (12-14) 9 (8-11)*No. Oocytes retrieved (n) 14 (10-19) 10 (8.5-13.5)*OHSS requiringhospitalization20 (13.9%) 0 (0%)*Cancellation 4 (2.7%) 1 (2.9%)CPR per transfer 40.1% 63.6%*¶DSL assay; Adapted from Nelson SM et al . Anti-Müllerian hormone-based approach tocontrolled ovarian stimulation for assisted conception. Hum Reprod. 2009; 24(4):867-75.*P ≤ 0.01Esteves, 26Ovarian Stimulation in HighResponders According to AMHLevel2b
Esteves, 27Gonadotropin Releasing Hormone(GnRH) Antagonists in High Responders9 RCT; 966 PCOS womenGnRH Antagonist X AgonistWeight Mean Difference (WMD)1;Relative Risk (RR)2Duration of ovarian stimulation -0.74 (95% CI: -1.12; -0.36)1Gonadotropin dose -0.28 (95% CI: -0.43; -0.13)1Oocytes retrieved 0.01 (95% CI: -0.24; 0.26)1Risk of OHSSMildModerate and Severe20% vs 32%1.23 (95% CI: 0.67-2.26)20.59 (95% CI: 0.45-0.76)2Clinical PR 1.01 (95% CI: 0.88; 1.15)2Miscarriage rate 0.79 (95% CI: 0.49; 1.28)2Pundir J et al. RBM Online 2012; 24:6-22.~40% reduction in moderate/severe OHSS byusing antagonists rather than agonistsEvidenceLevel1a
ReducedovarianparacrineactivityHurwitz &Santoro, 2004LH receptorpoly-morphismsAlviggi et al.,2006Androgensecretorycapacityreduced• Piltonen et al.,2003Decreasednumbers offunctionalLHreceptors• Vihko et al., 1996ReducedLHbioactivitywhileimnuno-reactivityunchanged• Mitchell etal.,1995; Maramaet al., 1984Esteves, 28LessSensitiveOvaries Poor RespondersWestergaard et al., 2000;Esposito et al., 2001; Humaidan et al., 2002• Older patients (≥35 years)• Poor responders• Slow/Hypo-responders• Deeply suppressedendogenous LHUp to 45%infertility patientsare aged 35 orabove
LH Supplementation to COS inPoor RespondersRegimen OutcomeEffect onPregnancyMochtar et al, 20073 RCT (N=310)Poor respondersr-hFSH+rLH vs.r-hFSH alone*OPROR 1.85(95% CI: 1.10; 3.11)Bosdou et al, 20127 RCT (N= 603)Poor respondersr-hFSH+rLH vs.r-hFSH alone*CPRLBR(only 1 RCT)RD: +6%,(95% CI: -0.3; +13.0)RD: +19%(95% CI: +1.0; +36.0%)Hill et al, 20127 RCT (N=902)Women advancedage ≥35 yrs.r-hFSH+rLH vs.r-hFSH alone CPROR 1.37(95% CI: 1.03; 1.83)*long GnRH-a protocol; OR=odds-ratio; RD=risk differenceMochtar MH et al. Cochrane Database Syst Rev. 2007;2:CD005070; Bosdou JK et al,Hum Reprod Update 2012; 8(2):127-45. Hill MJ et al. Fertil Steril 2012; 97:1108-4.Esteves, 29Level1a
Esteves, 30Population Cut-off Sensitivity Specificity AccuracyAMH*High-responder1 2.1 85% 86% 0.90Poorresponder2 0.82 76% 88% 0.88*Beckman-Couter generation II assay; 1>20 oocytes retrieved; 2≤4 oocytes retrievedAMH to DetermineWho is WhoBefore COSIn a group of 131 womenundergoing conventionalCOS after pituitary down-regulation for IVF:Leão RBF, Nakano FY, Esteves SC. ASRM 2013, submitted
Esteves, 31Individualization of COS UsingAMH ResultsHigh RespondersAMH >2.1PoorRespondersAMH ≤ 0.82rec-hFSH FbM 112.5 to 150 IU daily+ GnRH antagonistrec-hFSH FbM + rec-hLH+ GnRH antagonist• Total daily dose: 262.5 to 375 IUAMH cut-off points were used to individualizetreatment strategy in a group of 118 womenundergoing IVF:Leão RBF, Nakano FY, Esteves SC. ASRM 2013, submittedLevel2b
Esteves, 321Excessive response: >20 oocytes retrieved; 2Poor response: <5 oocytes retrieved;*Pts. received GnRH-a trigger + embryo vitrification; No severe OHSS reportedLeão RBF, Nakano FY, Esteves SC. ASRM 2013, submittedResponse toCOSConventionalCOS (n=131)IndividualizedCOS (n=118)PvalueExcessive1OHSSCPR/ongoing39.3%14.3%57.1%14.3%4.8%*55.6%0.040.380.93Poor2CancellationCPR/ongoing64.2%22.5%35.0%34.0%10.0%36.3%0.020.210.92Individualization of COS UsingAMH ResultsLevel2b
Esteves, 33Biomarkers of Ovarian ResponseKeyPointsAMH and AFC are currently the best biomarkers topredict ovarian response to COS.Individualization of COS guided by such biomarkersis sound, and it is aimed to maximize the beneficialeffects of treatment while minimizing complicationsand risks.After identifying ‘Who is Who’ before COS, there isfair evidence to support the use of mild stimulationand GnRH antagonists in high-responders, and rec-hLH supplementation in poor responders.Merck Serono Symposium "360 degrees in ART"