1. Advances in OBGYN Conference, Oman 2013
Tailoring Ovarian
Stimulation
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)
High-quality
Gametes and
Embryos
Optimal
Endometrial Receptivity
Esteves, 2
Minimize
complications
and risks

3. Maximize
Beneficial Effects
Singleton
live birth at
term
Esteves, 3
Minimize Complications
and Risks
Cycle
Multiple
Cancellation Pregnancy
Risk of OHSS
Poor Response
OHSS
Delvigne & Rozenberg Hum Reprod Update. 2003;9:77-96; Cantineau et al.,
Cochrane Database Syst Rev. 2007; 18:CD005356;
Aboulghar. Fertil Steril. 2012;97:523-6.

4. Who is Who in ART
Up to
68%
Infertile Patients (WHO II) with
PCO in Clinical Practice
Up to 45% Patients
Aged ≥35 have Poor
Response to
Stimulation
Esteves, 4
Reproductive Hormones Report - GCC Countries (Feb 2011)
Bologna criteria: Ferraretti et al. Hum Reprod 2011.

5. How to Tailor Ovarian
Stimulation for IVF Using
Ovarian Biomarkers
Know the best biomarkers
Understand how they work
How to use them in COS
Esteves, 5

6. Tailoring Ovarian Stimulation
Esteves SC – Oman Conference Nov 2013
http://www.androfert.com.br/review
Esteves, 6

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
Esteves, 7

8. Evidence
Level
1a
Esteves, 8

9. How AMH and
AFC Work
Esteves, 9

10. AMH
AFC
Esteves, 10
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
.

11. 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, 11
ICC: 0.55; 95% IC: 0.39–0.71
Max. Variation: 108%

12. 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, 12
Kelsey et al. Mol Hum Reprod 2012;18:79

13. 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, 13
Fleming et al. RBM online 2013;26:130; Nelson SM. Fertil Steril. 2013 Jan 8;
Nelson & La Marca. RBM online 2011;23:411;

14. Moderate to Low Inter-cycle
Fluctuations
AFC
van Disseldorp et al, Hum Reprod 2010;25:221
Esteves, 14
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

15. 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
Broekmans et al., Fertil Steril, 2010; 94(3):1044-51;
3Raine-Fenning et al., Fertil Steril 2009;91:1469.
Esteves, 15

16. 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, 16

17. How to Use AMH and
AFC to Tailor OS
Esteves, 17

18. Biomarkers in OI
In a group of 131 women
undergoing conventional
COS after pituitary downregulation for IVF:
Population
High-
AMH* responder1
ng/mL
Poor
responder2
Cut-off
Sensitivity
Specificity
Accuracy
2.1
85%
79%
0.82
0.82
76%
86%
0.88
*Beckman-Couter generation II assay; 1>20 oocytes retrieved; 2≤4 oocytes retrieved
Esteves, 18
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013

19. iCOS Using Biomarkers
High
Responders
AMH >2.1
Poor
Responders
AMH ≤ 0.82
Esteves, 19
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

20. iCOS Using AMH vs. cCOS
High Responders (N=70)
60
50
40
57.0 56.0
p=0.03
39.3
p=0.04
30
20
p=0.92
18.5
14.3
14.7
p=0.38
14.0
4.8
10
0
Observed
Excessive
Response (%)
Oocytes
retrieved (N)
cCOS
OHSS (%)
Pregnancy (%)
iCOS
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013
1Excessive
Esteves, 20
response: >20 oocytes retrieved;
*Pts. received GnRH-a trigger + embryo vitrification; Mild/severe OHSS reported

21. 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, 21
¶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.

22. Accurate to Predict Ovarian Response
AFC
Cut-off point of 14
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
1>20
Esteves, 22
Kwee et al, Fertil Steril 2008;90:737
oocytes retrieved in conventional COS; 2≤4 oocytes retrieved

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. 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
Esteves, 24

25. 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
Balasch J, Fábreques F. Curr Opin Obstet Gynecol 2002, 14:265.
Esteves, 25

26. 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, 26
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.

27. 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.
Esteves, 27

28. Sources of LH Activity
Matched case-control study; N=4,719 IVF pts.
35
30
P=0.02
25
Duration of
Stimulation
(days)
31
26
20
15
25
19
14
10
14
Mean No.
oocytes
retrieved
IR (%)
5
0
Fixed 2:1 r-hFSH
(150IU)/r-hLH
(75IU)
Esteves, 28
HMG
rec-hFSH + HMG
CPR per
transfer (%)
Buhler KF, Fisher R. Gynecol Endocrinol 2011;1-6.

29. Sources of LH Activity
Sources of LH Activity
Beta unit
hCG
Longer in hCG;
(Higher
receptor
affinity)
LH
Carboxyl
terminal
segment
Absent in LH and
present in hCG
(Longer Half-life)

30. 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
Esteves, 30
Grondal ML et al. Fertil Steril 2009; 91: 1820-1830.
Menon KM et al. Biol Reprod 2004; 70:861-866

31. How to Use LH in supplementation
Our Method for LHCOS
Patients (≥35 years)
Diminished Ovarian Reserve (AMH ≤0.82 ng/mL)
GnRH antagonist flexible protocol
DOR: Recombinant FSH/LH (2:1 or 3:1 ratio) from stimulation D1;
Normal ovarian reserve: 75 IU recLH added to rec-hFSH from D6 on
3
1
2
Menses
Esteves, 31
4
5
6
7
8
9
10
11
3
4
5
6
7
8
9
10
11
12

32. iCOS Using AMH vs cCOS
Poor Responders (N=49)
80
60
72.0
p=0.02
46.6
45.0
40
20
p=0.06
23.3
p=0.03
3.5
p=0.51
20.0
26.8
4.8
0
Expected Poor Oocytes retrieved Cancellation (%) Pregnancy/cycle
Response (%)
(N)
(%)
cCOS
iCOS
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013
Esteves, 32
Poor response: <5 oocytes retrieved;

33. 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,
are useful strategies to optimize outcomes in ART
cycles.
Esteves, 33