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Biomarkers of Ovarian Response
1. Biomarkers of Ovarian
Response
Sandro Esteves, M.D., Ph.D.
Director, ANDROFERT
Andrology & Human Reproduction Clinic
Referral Center for Male Reproduction
Campinas, BRAZIL
Merck Serono Symposium – La Red Annual Meeting 2013
May 3, 2013, Panama City
"360 degrees in ART"
3. Esteves, 3
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
What is in it for me?
4. Biomarkers of Ovarian Response
Lecture Structure
Esteves, 4
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
5. Why Do We Need Biomarkers to
Predict Ovarian Response to COS?
Esteves, 5
ForDoctors
• Avoid over-aggressive stimulation in
‘true’ high responders
• Avoid over-conservative stimulation in
‘false’ high responders
Excessive
Ovarian
Response
• Avoid over-conservative stimulation in
‘true’ DOR
• Avoid over-aggressive stimulation in
‘false’ DOR
Diminished
Ovarian
Reserve
(DOR)
6. Esteves, 6
Realistic Prognostic Information
• Poor or Negligible Response
• Cycle cancellation
• Egg donation or adoption
• Chances of Pregnancy and Live Birth
ForPatients Why Do We Need Biomarkers to
Predict Ovarian Response in COS?
7. Esteves, 7
Which are the Biomarkers?
Biomarkers
●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
8. A Valid Biomarker Should be
Highly Sensitive and Highly Specific
Esteves, 8
+
-
+ -
BiomarkerTestResult
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)
9. Esteves, 9
Understand How They
Work And What Are Their
Advantages and
Shortcomings
Biomarkers of Ovarian Response
10. Esteves, 10
Understanding Biomarkers
Basic Concepts
FSH
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 reserve
Influenced by Inhibin B and Estradiol
• FSH assays are well standardized* but...
High Inter- and Intra-cycle Variability (Low Reliability)
11. Esteves, 11
Evidence
Level
2b
Understanding Biomarkers
FSH is not very accurate to assess ovarian
response or pregnancy risk in ART
FSH
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.
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 oocytes
Low sensitivity (10%-30%) to predict DOR and failure to
achieve pregnancy in ART
• Cannot Identify High Responders
Low number
of false
positives
12. Esteves, 12
Understanding Biomarkers
AMH
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
13. Esteves, 13
Understanding Biomarkers
Basic ConceptsAMH
AMH 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
15. Esteves, 15
Evidence
Level
2a
Understanding Biomarkers
AMH is accurate to assess ovarian response
AMH
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
16. Esteves, 16
Understanding BiomarkersAMH
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
17. Esteves, 17
Understanding Biomarkers
AFC
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
18. Esteves, 18
AFC
Moderate 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 Biomarkers
Evidence
Level
2a
High Inter- and Intra-observer Reproducibility:
Scheffer et al. Ultrasound Obstet Gynecol 2002;20:270
19. Esteves, 19
Evidence
Level
1b
Understanding Biomarkers
AFC is accurate to assess ovarian response
AFC
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
20. Esteves, 20
Understanding BiomarkersAFC
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
21. Esteves, 21
Biomarkers
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.)
+++ +++ +++
24. Esteves, 24
AFC 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
26. 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
Esteves, 26
Ovarian Stimulation in High
Responders According to AMH
Level
2b
27. Esteves, 27
Gonadotropin Releasing Hormone
(GnRH) Antagonists in High Responders
9 RCT; 966 PCOS women
GnRH 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 OHSS
Mild
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
Level
1a
28. 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
Esteves, 28
LessSensitiveOvaries 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
29. LH Supplementation to COS in
Poor Responders
Regimen Outcome
Effect on
Pregnancy
Mochtar et al, 2007
3 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, 2012
7 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, 2012
7 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
Level
1a
30. Esteves, 30
Population 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
31. Esteves, 31
Individualization of COS Using
AMH Results
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
Level
2b
32. Esteves, 32
1Excessive 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
OHSS
CPR/ongoing
39.3%
14.3%
57.1%
14.3%
4.8%*
55.6%
0.04
0.38
0.93
Poor2
Cancellation
CPR/ongoing
64.2%
22.5%
35.0%
34.0%
10.0%
36.3%
0.02
0.21
0.92
Individualization of COS Using
AMH Results
Level
2b
33. Esteves, 33
Biomarkers of Ovarian Response
KeyPoints
AMH and AFC are currently the best biomarkers to
predict ovarian response to COS.
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.
After 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.
Merck Serono Symposium "360 degrees in ART"
Editor's Notes
I will dedicate the last few minutes of this lecture to explore how we can improve patient care by adapting ovarian stimulation regimens according to biomarkers’ results.
More robust evidence comes from this meta-analysis, in which GnRH antagonists were used in PCOS patients undergoing IVF. It confirms the benefit of this approach not only in reducing duration of stimulation and gonadotropincomsumption, but also in reducing complications, and at the same time maintaining sustainable pregnancy results. A total of 966 women were included in nine randomized controlled trials. There was inconsistency in definition, classification of severity and reporting of the OHSS rate. There was no difference in the incidence of severe OHSS in the antagonist group compared with the long agonist group (relative risk 0.61; 95% CI 0.23 to 1.64). However, when all moderate and severe OHSS cases were pooled, the antagonist protocol was associated with significantly lower risk of OHSS (relative risk 0.60; 95% CI 0.48-0.76; P<0.0001). A possible reduction in the incidence of severe OHSS with the GnRH antagonist protocol should be viewed with caution since the data is inconclusive.
Before I conclude, I want to summarize this lecture in 3 key points: first…
