1. XVIII Annual Ob-Gyn Conference, Kuwait 2013
Ovarian Biomarkers in
Ovulation Induction
Sandro C. Esteves, MD, PhD
Director, ANDROFERT
Andrology & Human Reproduction Clinic
Campinas, Brazil

2. Maximize
beneficial effects of
treatment
Central
Paradigm
Individualization of
Controlled Ovarian
Stimulation
(iCOS)
Minimize
complications
and risks
High-quality
Gametes and
Embryos
Optimal
Endometrial Receptivity
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3. Know the best
biomarkers
Understand how they
work
How to use biomarkers
in Ovulation Induction
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4. Ovarian Biomarkers in Ovulation
Induction
Esteves SC – Kuwait’s XVIII Annual Ob-Gyn Conference, 2013
http://www.androfert.com.br/review
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5. Excessive
Ovarian
Response
Avoid over-aggressive stimulation in ‘true’ high
responders
Diminished
Ovarian
Reserve (DOR)
Why Predict Ovarian
Response in OI?
Avoid over-conservative stimulation in ‘true’
DOR
Avoid over-conservative stimulation in ‘false’
high responders
Avoid over-aggressive stimulation in ‘false’
DOR
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6. For Patients
Prediction of Ovarian
Response in OI
Realistic Prognosis
• Poor or Negligible Response
• Cycle cancellation
• Egg donation or adoption
• Chances of Pregnancy and Live
Birth
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7. Know the 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, LH,
E2 and AMH receptor genes
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8. A Valid Biomarker Should be
Highly Sensitive and Highly Specific
Diminished or Excessive
Ovarian Response
Specificity (D/B+D)
Predictive Value
(PPV=A/A+B; NPV=D/C+D)
Accuracy
(A+D/A+B+C+D)
Esteves, 8
Biomarker Test Result
Sensitivity (A/A+C)
+
-
+
True
Positive
(A)
False
Positive
(B)
-
False
Negative
(C)
True
Negative
(D)
Adapted from: ASRM Practice Committee, Fertil Steril 2012;98:147

9. Evidence
Level
1a
Esteves, 9
Who is Who Before OI

10. Biomarkers in OI
In a group of 131 women
undergoing conventional
COS after pituitary downregulation for IVF:
Population
AMH*
ng/mL
Cut-off
Sensitivity
Specificity
Accuracy
Highresponder1
2.1
85%
79%
0.82
Poor
responder2
0.82
76%
86%
0.88
*Beckman-Couter generation II assay; 1>20 oocytes retrieved; 2≤4 oocytes retrieved
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013
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11. AMH and AFC are not
accurate for pregnancy
prediction
Evidence
Level
1a
Broer et al. Fertil Steril 2009 ; Broer et al. Hum Reprod Update, 17:46; 2011
Esteves, 11

12. How AMH and
AFC Work
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13. AMH
AFC
Esteves, 13
Reflect No. pre-antral and
small antral follicles
(≤4-8mm)
2D-TVUS early follicular phase
2-10 mm (mean diameter)
No. AF at a given time that can
be stimulated by medication
La Marca et al, Hum Reprod 2009;24:2264; Fleming et al, Fertil Steril 2012;98:1097;
Broekmans et al. Fertil Steril, 2010; 94:1044-51; Scheffer et al. Hum Reprod 2003;18:700
.

14. Low Inter-cycle Fluctuations (Fanchin et al, Hum Reprod 2005;20:923)
AMH
ICC: 0.89; 95% IC: 0.83–0.94
Can be assessed at any cycle day
with a single measurement
Low Intra-cycle Fluctuations (Hehenkamp et al. JCEM 2006;91:4057)
Max. Variation: 17.4%
Esteves, 14
ICC: 0.55; 95% IC: 0.39–0.71
Max. Variation: 108%

15. Serum Levels:
AMH
Peak at age 25 and decrease with aging
Early marker of diminished ovarian reserve
Non-growing
follicles (NGF)
recruited per
month
Esteves, 15
Kelsey et al. Mol Hum Reprod 2012;18:79

16. AMH
Accurate to Predict Ovarian Response
Cut-off point 3.5 ng/mL* (Nardo et al, Fertil Steril 2009;92:1586)
Ø High sensitivity (88%), specificity (70%)
and accuracy (0.81) to predict excessive response1
Cut-off point 1.4 ng/mL* (Kwee et al, Fertil Steril 2008;90:737)
Ø High sensitivity (76%) and specificity (86%) for DOR2
Caution to apply AMH cut-off points!
Make sure the assay you rely on is the
same used in the reference population
Esteves, 16
*DSL assay; 1>20 oocytes retrieved; 2≤5 oocytes retrieved;
Conversion: ng/mL to pmol/L = value in ng/mL X7.14

17. AMH
ELISA assays with different
performances:
DSL and Immunotech
Beckman-Couter gen II (AB DSL + Curves Im.)
Fully automated ELISA (to be released)
Lack international standardization
and EQC
Sample instability; 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, 17
Fleming et al. RBM online 2013;26:130; Nelson SM. Fertil Steril. 2013 Jan 8;
Nelson & La Marca. RBM online 2011;23:411;

18. Moderate to Low Inter-cycle
Fluctuations
AFC
van Disseldorp et al, Hum Reprod 2010;25:221
Esteves, 18
ICC: 0.71 (95% CI: 0.63–0.77);
29% individual cycle
variation
High Inter- and Intra-observer Reproducibility
Scheffer et al. Ultrasound Obstet Gynecol 2002;20:270

19. Accurate to Predict Ovarian Response
Cut-off point of 14
Kwee et al, Fertil Steril 2008;90:737
AFC
High sensitivity (81%) and specificity (89%) to predict
excessive response1
Cut-off point of 4 Bancsi et al, Fertil Steril 2002;77:328
Moderate sensitivity (61%) and High specificity
(88%) and to predict DOR2
Caution to Apply Cut-off Points to Predict No.
of Oocytes to be Retrieved
For any given AFC there is a potential oocyte yield, but it
can be altered by the stimulation strategy
Esteves, 19
1>20
oocytes retrieved in conventional COS; 2≤4 oocytes retrieved

20. Lack of standardization1
• Inclusion criteria for antral follicles
AFC
Ø 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
1Nelson SM. Fertil Steril. 2013 Jan 8;
• Costly
2
Esteves, 20
Broekmans et al., Fertil Steril, 2010; 94(3):1044-51;
3Raine-Fenning et al., Fertil Steril 2009;91:1469.

21. How to Use AMH
and AFC in OI
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22. Evidence
Level
2b
Biomarkers for iCOS in High
Responders
AMH (ng/mL) >2.1¶
GnRH Agonist
Low-starting FSH dose (150 UI)
(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%)*
4 (2.7%)
1 (2.9%)
40.1%
63.6%*
Cancellation
CPR per transfer
*P ≤ 0.01
Esteves, 22
¶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.

23. Evidence
Level
GnRH Antagonists in High
Responders
1a
9 RCT; 966 PCOS women
GnRH Antagonist X Agonist
Weight Mean Difference (WMD)1;
Relative Risk (RR)2
Duration of OS
-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 (Moderate & Severe)
20% vs 32%
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
~40% reduction in moderate/severe OHSS by using
antagonists rather than agonists
Esteves, 23
Pundir J et al. RBM Online 2012; 24:6-22.

24. GnRH Antagonist Protocol with
Long-acting recFSH vs recFSH
4 RCT; 2377 pts.
Clinical PR, Miscarriage, LBR
Risk of OHSS
Cancellation
OR [95% CI]
Not different
1.29 (0.78; 2.26)
5.67 (1.07; 30.13)*
*p=0.04; risk of OHSS
Mahmoud Youssef et al. van Fertil Steril 2012; 97(4): 876-85; Pouwer AW et al.
Cochrane Database Syst Rev 2012; 6: CD009577.
Esteves, 24

25. Biomarkers for iCOS in Poor
Responders
Up to 45% of Infertility Patients in ART
Older patients (≥35 years)
Poor responders
Slow/Hypo-responders
Deeply suppressed endogenous LH
Marrs et al. Reprod Biomed Online 2004;8:175;Mochtar MH, Cochrane Database, 2007; Alviggi,
et al. RBMOnline 2009; De Placido et al. Clin Endocrinol (Oxf) 2004;60:637
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26. Ovarian Aging
Impaired Oocyte Quality
Reduced Fertilization Rate
Reduced Embryo Quality
Increased Miscarriage Rates
Westergaard et al., 2000; Esposito et al., 2001; Humaidan et al., 2002
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27. Normal
LH “Window” Concept
• Normal androgen and estrogen biosynthesis
• Normal follicular growth and development
• Normal oocyte maturation
Reduced
ovarian
paracrine
activity
Androgen
secretory
capacity
reduced
Decreased
numbers of
functional
LH receptors
Reduced LH
bioactivity
Hurwitz & Santoro
2004
• Piltonen et al.,
2003
• Vihko et al. 1996
• Mitchell et al. 1995;
Marama et al 1984
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28. Level
1a
LH Supplementation in DOR
Regimen
Mochtar et al, 2007
3 RCT (N=310)
Poor responders
Bosdou et al, 2012
7 RCT (N= 603)
Poor responders
Outcome
Effect on Pregnancy
r-hFSH+rLH
vs.
r-hFSH alone*
OPR
OR: 1.85
r-hFSH+rLH
vs.
r-hFSH alone*
CPR
LBR
(only 1 RCT)
Hill et al, 2012
7 RCT (N=902)
Women advanced age ≥35
yrs.
r-hFSH+rLH
vs.
r-hFSH alone
CPR
(95% CI: 1.10; 3.11)
RD: +6%,
(95% CI: -0.3; +13.0)
RD: +19%
(95% CI: +1.0; +36.0%)
OR: 1.37
(95% CI: 1.03; 1.83)
*long GnRH-a protocol; OR=odds-ratio; RD=risk difference
Esteves, 28
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.

29. Rationale of LH supplementation
Action of LH at the follicular level in a dose dependent
manner increases androgen production;
Androgens are later aromatized to estrogens and may help
restore the follicular milieu;
LH has also a direct positive effect on final follicular
maturation;
Altogether, positive effect in oocyte quality and, therefore,
embryo quality and implantation.
Esteves, 29

30. Sources of LH Activity
Purity
(LH content)
hCG
content
(IU/vial)
LH activity
(IU/vial)
Specific activity
(LH/mg protein)
Rec-hLH
>99%
0
75
22,000 IU
hMG-HP*
3%
~70
75*
≥ 60 IU
*derives from hCG
Adapted from ASRM Practice Committee. Fertil Steril. 2008; 90:S13-20.
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31. Sources of LH Activity
Sources of LH Activity
Beta unit
hCG
Longer in hCG;
(Higher
receptor
affinity)
Carboxyl
terminal
segment
Absent in LH and
present in hCG
(Longer Half-life)
LH
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32. hMG
Grondal et al. 2009:
r-FSH
Sources of LH Activity
GCs gene expression in pts. treated with
hMG and rec-hFSH
q Lower expression of LH/hCG receptor
gene and other genes involved in
steroids biosynthesis in hMG group
Down-regulation of receptors owed to
constant ligand exposure to hCG
(Menon et al. 2004)
CYP11A activity decreased by 2.4 fold
Lower steroids synthesis and P levels
q Higher potency of rec-hFSH inducing
more LH/hCG receptors
Grondal ML et al. Fertil Steril 2009; 91: 1820-1830.
Menon KM et al. Biol Reprod 2004; 70:861-866
Esteves, 32

33. Sources of LH Activity
Matched case-control study; N=4,719 IVF pts.
35
30
25
P=0.02
31
26
20
15
25
19
14
10
14
5
Duration of
Stimulation
(days)
Mean No.
oocytes
retrieved
IR (%)
CPR per
transfer (%)
0
Fixed 2:1 r-hFSH
(150IU)/r-hLH
(75IU)
HMG
rec-hFSH + HMG
Buhler KF, Fisher R. Gynecol Endocrinol 2011;1-6.
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34. Individualization of OI with AMH
AMH cut-off points used to individualize COS in 118
women undergoing IVF;
Outcome compared with a group of 131 women who
received conventional stimulation
High
Responders
AMH >2.1
Poor
Responders
AMH ≤ 0.82
Esteves, 34
rec-hFSH FbM 112.5 to 150 IU daily +
GnRH antagonist
rec-hFSH FbM + 75 IU rec-hLH
+ GnRH antagonist
• Total daily dose: 262.5 to 375 IU
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013

35. iCOS Using AMH
High Responders
60
50
40
57.0 56.0
p=0.03
cCOS
39.3
p=0.04
30
20
p=0.92
18.5
14.3
14.7
10
iCOS
p=0.38
14.0
4.8
0
Observed
Excessive
Response (%)
Oocytes
retrieved (N)
OHSS (%)
Pregnancy (%)
1Excessive
response: >20 oocytes retrieved;
*Pts. received GnRH-a trigger + embryo vitrification; Mild/severe OHSS reported
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013
Esteves, 35

36. iCOS Using AMH
Poor Responders
80
60
72.0
cCOS
p=0.02
46.6
45.0
40
20
iCOS
p=0.03
3.5
p=0.06
23.3
p=0.51
20.0
26.8
4.8
0
Expected Poor Oocytes retrieved Cancellation (%) Pregnancy/cycle
Response (%)
(N)
(%)
Poor response <5 oocytes retrieved
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013
Esteves, 36

37. Take Home Messages
AMH and AFC are currently the best biomarkers
to predict ovarian response to COS.
AMH and AFC are direct biomarkers of ovarian
reserve. Both markers have similar accuracy to
predict who is at risk of excessive and poor
response in COS.
After identifying ‘Who is Who’, mild stimulation and
GnRH antagonists in pts. at risk of excessive
response, and rec-hLH supplementation in DOR,
maximize treatment benefits and minimize risks.
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38. Thank you

39. iCOS Using AMH
Excessive1
Oocytes retrieved
OHSS
Pregnancy
Poor2
Oocytes retrieved
Cancellation
Pregnancy/ET
1Excessive
Esteves, 39
iCOS
(n=118)
P
value
39.3%
Response to COS
Conventional COS
(n=131)
14.3%
18.5 ± 6.7
14.3%
57.1%
14.7± 6.2
4.8%*
55.6%
0.03
0.04
0.38
0.92
72.0%
3.5 ± 3.1
45.0%
20.0%
46.6%
4.8 ± 3.5
23.3%
26.8%
0.02
0.03
0.06
0.51
response: >20 oocytes retrieved; 2Poor response: <5 oocytes retrieved;
*Pts. received GnRH-a trigger + embryo vitrification; No severe OHSS reported

40. Progesterone Rise
What we have learned…
Number of oocytes
Estradiol levels on hCG day
FSH dose
Rec-hFSH vs. hMG
positively
associated
with P levels
P levels not associated with oocyte and embryo
quality, nor with fertilization and cleavage rates
Bosch et al. 2008, 2010; Xu et al, 2012;
Kolibianakis et al 2012; Venetis et al. 2012; Griesinger et al 2013
Esteves, 40
ANDROFERT, Referral Center for Male Reproduction

41. No CYP17
LH
Esteves, 41
FSH
LH

42. Progesterone thresholds
affecting PR controversial
Bosch et al. 2010 (N=4,032)
Irrespective of GnRH analogue;
CUT-OFF = 1.5 ng/mL
Xu et al, 2012 (N=11,055)
GnRH agonist
Ovarian
response
Number of
oocytes
Serum P
threshold
(ng/mL)
Poor
≤4
1.5
Intermediate
5-19
1.75
High
≥20
■ Fresh
■ FET
2.25
Esteves, 42
ANDROFERT, Referral Center for Male Reproduction

43. Effect of progesterone levels on day of
hCG administration on pregnancy
Griesinger et al, 2013 (6 RCT, N=1866; Antagonist cycles)
P4 cut-off: 1.5 ng/mL
P4 rise related to
ovarian response:
Low-responder: 4.5%
High-responder: 19%
Overall: 8.4%
OPR not impaired in
high responders with P
elevation
Ongoing PR: OR = 0.55 (0.37–0.81)
Griesinger et al. Fertil Steril 2013
Esteves, 43
ANDROFERT, Referral Center for Male Reproduction