Biomarkers of Ovarian Response

Sandro Esteves, M.D., Ph.D.Director, ANDROFERT

Andrology & Human Reproduction ClinicReferral Center for Male Reproduction

Campinas, BRAZIL

Merck Serono Symposium – La Red Annual Meeting 2013 May 3, 2013, Panama City 

"360 degrees in ART" 

Goals of ART in 2013

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Individualization of ovarian stimulation

Optimalendometrial receptivity

Maximize beneficial effects

of treatment

Minimize complications

and risks

Central Paradigm Shift

High-quality gametes and

embryos

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Know which biomarkers are best for clinical use

Understand how they work including their advantages and shortcomings

Learn how to use their results to individualize ovarian stimulation

3What is in it for me?

Biomarkers of Ovarian ResponseLecture Structure

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Level Type of evidence

1a Obtained from meta-analysis of randomised trials

1b Obtained from at least one randomised trial

2a Obtained from one well-designed controlled study without randomisation

2b Obtained from at least one other type of well-designed quasi-experimental study

3 Obtained from well-designed non-experimental studies (comparative and correlation studies, case series)

4 Obtained from expert committee reports or opinions or clinical experience of respected authorities

Modified from Sackett et al. Oxford Centre for EBM Levels of Evidence (2009)

Level of Evidence

Review of Biomarkers applied to the general IVF population Studies with Best Levels of Evidence

Why Do We Need Biomarkers to Predict Ovarian Response to COS?

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Fo

r D

oct

ors • Avoid over-aggressive stimulation in

‘true’ high responders• Avoid over-conservative stimulation in

‘false’ high respondersExc

essi

ve

Ova

rian

R

esp

on

se

• Avoid over-conservative stimulation in ‘true’ DOR

• Avoid over-aggressive stimulation in ‘false’ DOR

Dim

inis

hed

O

vari

an

Res

erve

(D

OR

)

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Realistic Prognostic Information

• Poor or Negligible Response• Cycle cancellation• Egg donation or adoption

• Chances of Pregnancy and Live BirthFo

r P

atie

nts

Why Do We Need Biomarkers to Predict Ovarian Response in COS?

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Which are the Biomarkers?

1Biomarkers

●Hormonal Biomarkers: FSH, Clomiphene citrate challenge test, Inhibin-B, Anti-Mullerian Hormone (AMH)

●Functional Biomarkers: Antral Follicle Count (AFC)

●Genetic Biomarkers: Single Nucleotide Polymorphisms for FSH-R; LH/LH-R; E2-R; AMH-R

A Valid Biomarker Should be Highly Sensitive and Highly Specific

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+

-

+ -

Bio

mar

ker

Test

Res

ult

Diminished or Excessive Ovarian Response

Adapted from: ASRM Practice Committee, Fertil Steril 2012;98:147

False Positive

(B)

False Negative

(C)

True Negative

(D)

True Positive

(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)

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Understand How They Work And What Are Their

Advantages and Shortcomings

2

Biomarkers of Ovarian Response

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Understanding BiomarkersBasic Concepts

FS

H

What 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 with aging

• Indirect biomarker of ovarian reserveInfluenced by Inhibin B and Estradiol

• FSH assays are well standardized* but... High Inter- and Intra-cycle Variability (Low Reliability)

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Evidence Level2b

Understanding BiomarkersFSH is not very accurate to assess ovarian response or pregnancy risk in ART

FS

H

What 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 number of false

negatives

‘Normal’ FSH values

DOR or failure to

pregnancy

• 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 to

achieve pregnancy in ART

• Cannot Identify High Responders

Low number of false

positives

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Understanding BiomarkersA

MH

La Marca et, Hum Reprod 2009;24:2264; Fleming et al, Fertil Steril 2012;98:1097.

What is known?

Direct Biomarker of Ovarian Reserve: Dimeric glycoprotein; ~140KDa

Product of GCs of early follicles Pre-antral and small antral (≤ 4-8mm)

Reflect both the number of small growing follicles and the primordial pool at gonadotropin-independent folliculogenesis

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Understanding BiomarkersBasic Concepts

AM

HAMH Serum Levels:

Peak at age 25 and decrease with aging Early marker of diminished ovarian reserve

Non-growing follicles (NGF)recruited per month

Kelsey et al. Mol Hum Reprod 2012;18:79

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AM

HLow Inter-cycle Fluctuations (Fanchin et al, Hum Reprod 2005;20:923)

Understanding Biomarkers

Low Intra-cycle Fluctuations (Hehenkamp et al. JCEM 2006;91:4057)

Evidence Level2a

ICC: 0.89; 95% IC: 0.83–0.94 ICC: 0.55; 95% IC: 0.39–0.71

Max. Variation: 17.4% Max. Variation: 108%

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Evidence Level2a

Understanding BiomarkersAMH is accurate to assess ovarian response

AM

H

What 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 DOR2

Caution to apply AMH cut-off points!

Make sure the assay you rely on is the same used in the reference population

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Understanding BiomarkersA

MH

Shortcomings and Pitfalls

Fleming 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 EQC

Sample 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

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Understanding BiomarkersA

FC

Broekmans et al. Fertil Steril, 2010; 94(3):1044-51; Scheffer et al. Hum Reprod 2003;18:700

What is known?

Direct Biomarker of Functional Ovarian Reserve: Sum of antral follicles in both ovaries on

TVUS 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 in the ovaries at a given time that can be stimulated by exogenous gonadotropins

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AF

CModerate to Low Inter-cycle

Fluctuations: van Disseldorp et al, Hum Reprod 2010;25:221

ICC: 0.71 (95% CI: 0.63–0.77); 29% individual cycle variation

Understanding BiomarkersEvidence

Level2a

High Inter- and Intra-observer Reproducibility: Scheffer et al. Ultrasound Obstet Gynecol 2002;20:270

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Evidence Level1b

Understanding BiomarkersAFC is accurate to assess ovarian response

AF

C

What 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 DOR2

Cut-off point of 14:(Kwee et al, Fertil

Steril 2008;90:737)

High sensitivity (81%) and specificity (89%) to predict excessive response1

Eldar-Geva et al. Hum Reprod 2005

1>20 oocytes retrieved in conventional COS; 2≤4 oocytes retrieved

Caution to Apply AFC Cut-off Points to Predict Number of

Oocytes Retrieved! For any given AFC there is a

potential oocyte yield, but it can be altered by the stimulation strategy

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Understanding BiomarkersA

FC

Shortcomings and Pitfalls

1Nelson 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 optimization

Improved standardization proposed2

Three-dimensional automated follicular tracking3

• Reduce intra- and inter-observer variability• Requires offline analysis• Costly

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Bio

mar

kers FSH AMH AFC

Clinical Utility in Ovarian Stimulation

Excessive Ovarian Response

Avoid over-aggressive stimulation in ‘true’ high responders (↑Sensit.) - +++ +++

Avoid over-conservative stimulation in ‘false’ high responders (↑Specif.) - +++ +++

Diminished Ovarian Reserve (DOR)

Avoid over-conservative stimulation in ‘true’ DOR (↑Sensit.) + +++ +++

Avoid over-aggressive stimulation in ‘false’ DOR(↑Specif.) +++ +++ +++

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Determining Who is Who Before COS

Evidence Level1a

Bio

mar

kers

AFC

FSH

AMH

AMH

AFC

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Evidence Level1a

Bio

mar

kers

AMH and AFC are not accurate for pregnancy prediction

Broer et al. Fertil Steril 2009 ; Broer et al. Hum Reprod Update, 17:46; 2011

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2AFC and AMH are accurate biomarkers to predict

ovarian response to ovarian stimulation.

Given their limitations clinicians should exercise caution to interpret cut-off points when using AMH and AFC as sole predictors of ovarian response.

AMH and AFC are not accurate predictors of failure to conceive after COS and IVF.

There is insufficient evidence to recommend the use of a combination of biomarkers to improve prediction power.

Understand How Biomakers Work, their Advantages and Shortcomings

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3Learn How to Use

Biomarkers to

Individualize Ovarian

Stimulation

Low-starting FSH dose (150 UI)

AMH (ng/mL) >2.1¶

GnRH Agonist(n=148)

GnRH Antagonist

(n=34) Days of Stimulation 13 (12-14) 9 (8-11)*

No. Oocytes retrieved (n) 14 (10-19) 10 (8.5-13.5)*

OHSS requiring hospitalization 20 (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 to controlled ovarian stimulation for assisted conception. Hum Reprod. 2009; 24(4):867-75.

*P ≤ 0.01

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Ovarian Stimulation in High Responders According to AMH

Level2b

3

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Gonadotropin Releasing Hormone (GnRH) Antagonists in High Responders

9 RCT; 966 PCOS womenGnRH Antagonist X Agonist

Weight Mean Difference (WMD)1; Relative Risk (RR)2

Duration of ovarian stimulation -0.74 (95% CI: -1.12; -0.36)1

Gonadotropin dose -0.28 (95% CI: -0.43; -0.13)1

Oocytes retrieved 0.01 (95% CI: -0.24; 0.26)1

Risk of OHSSMild

Moderate and Severe

20% vs 32% 1.23 (95% CI: 0.67-2.26)2

0.59 (95% CI: 0.45-0.76)2

Clinical PR 1.01 (95% CI: 0.88; 1.15)2

Miscarriage rate 0.79 (95% CI: 0.49; 1.28)2

Pundir J et al. RBM Online 2012; 24:6-22.

~40% reduction in moderate/severe OHSS by using antagonists rather than agonists

Evidence Level1a

3

Reduced oocyte quality

Reduced Fertilization Rate

Reduced Embryo Quality

Increase Miscarriage Rates

Reduced ovarian

paracrine activity

Hurwitz & Santoro, 2004

LH receptor poly-

morphisms

Alviggi et al., 2006

Androgen secretory capacity reduced

• Piltonen et al., 2003

Decreased numbers of functional

LH receptors

• Vihko et al., 1996

Reduced LH

bioactivity while

imnuno-reactivity

unchanged

• Mitchell et al.,1995; Marama et al., 1984

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Les

s S

ensi

tive

Ova

ries

Poor Responders

Westergaard et al., 2000; Esposito et al., 2001; Humaidan et al., 2002

• Older patients (≥35 years)• Poor responders• Slow/Hypo-responders• Deeply suppressed

endogenous LH

Up to 45% infertility patients are aged 35 or

above

LH Supplementation to COS in Poor Responders

Regimen OutcomeEffect on

Pregnancy

Mochtar et al, 20073 RCT (N=310)Poor responders

r-hFSH+rLH vs. r-hFSH alone* OPR OR 1.85

(95% CI: 1.10; 3.11)

Bosdou et al, 20127 RCT (N= 603)Poor responders

r-hFSH+rLH vs. r-hFSH alone*

CPR

LBR(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 advanced age ≥35 yrs.

r-hFSH+rLH vs. r-hFSH alone CPR OR 1.37

(95% CI: 1.03; 1.83)

*long GnRH-a protocol; OR=odds-ratio; RD=risk difference

Mochtar 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, 29

Level1a

3

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3Population Cut-off Sensitivity Specificity Accuracy

AMH*

High-responder1 2.1 85% 86% 0.90

Poor responder2 0.82 76% 88% 0.88

*Beckman-Couter generation II assay; 1>20 oocytes retrieved; 2≤4 oocytes retrieved

AMH to Determine Who is Who Before COS

In a group of 131 women undergoing conventional COS after pituitary down-regulation for IVF:

Leão RBF, Nakano FY, Esteves SC. ASRM 2013, submitted

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Individualization of COS Using AMH Results

3 High Responders

AMH >2.1

Poor Responders

AMH ≤ 0.82

rec-hFSH FbM 112.5 to 150 IU daily+ GnRH antagonist

rec-hFSH FbM + rec-hLH

+ GnRH antagonist • Total daily dose: 262.5 to 375 IU

AMH cut-off points were used to individualize treatment strategy in a group of 118 women undergoing IVF:

Leão RBF, Nakano FY, Esteves SC. ASRM 2013, submitted

Level2b

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31Excessive response: >20 oocytes retrieved; 2Poor response: <5 oocytes retrieved;*Pts. received GnRH-a trigger + embryo vitrification; No severe OHSS reported

Leão RBF, Nakano FY, Esteves SC. ASRM 2013, submitted

Response to COS

Conventional COS (n=131)

Individualized COS (n=118)

P value

Excessive1

OHSSCPR/ongoing

39.3%14.3%57.1%

14.3%4.8%*55.6%

0.040.380.93

Poor2

CancellationCPR/ongoing

64.2%22.5%35.0%

34.0%10.0%36.3%

0.020.210.92

Individualization of COS Using AMH Results

Level2b

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Biomarkers of Ovarian ResponseK

ey P

oin

ts

AMH and AFC are currently the best biomarkers to predict ovarian response to COS. 1

Individualization of COS guided by such biomarkers is sound, and it is aimed to maximize the beneficial effects of treatment while minimizing complications and risks.

2After identifying ‘Who is Who’ before COS, there is

fair evidence to support the use of mild stimulation and GnRH antagonists in high-responders, and rec-hLH supplementation in poor responders.

3

Merck Serono Symposium "360 degrees in ART"