Sandro C. Esteves, MD, PhD Director, ANDROFERT
Andrology & Human Reproduction Clinic Campinas, Brazil
Ovarian Biomarkers in Ovulation Induction
XVIII Annual Ob-Gyn Conference, Kuwait 2013
Individualization of Controlled Ovarian
Stimulation (iCOS)
Optimal Endometrial Receptivity
Maximize beneficial effects of
treatment
Minimize complications
and risks
Central Paradigm
High-quality Gametes and
Embryos
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Know the best biomarkers
Understand how they work
How to use biomarkers in Ovulation Induction
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http://www.androfert.com.br/review
Ovarian Biomarkers in Ovulation Induction
Esteves SC – Kuwait’s XVIII Annual Ob-Gyn Conference, 2013
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Why Predict Ovarian Response in OI?
Avoid over-aggressive stimulation in ‘true’ high responders Avoid over-conservative stimulation in ‘false’ high responders Ex
cess
ive
Ovar
ian
Resp
onse
Avoid over-conservative stimulation in ‘true’ DOR Avoid over-aggressive stimulation in ‘false’ DOR
Dim
inis
hed
Ovar
ian
Rese
rve
(DOR
)
For P
atie
nts
• Poor or Negligible Response • Cycle cancellation • Egg donation or adoption
• Chances of Pregnancy and Live Birth
Realistic Prognosis
Prediction of Ovarian Response in OI
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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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A Valid Biomarker Should be Highly Sensitive and Highly Specific
Esteves, 8
+
-
+ -
Bio
mar
ker T
est R
esul
t
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)
Who is Who Before OI Evidence Level 1a
Esteves, 9
Population Cut-off Sensitivity Specificity Accuracy
AMH*ng/mL
High-responder1 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
Biomarkers in OI
In a group of 131 women undergoing conventional COS after pituitary down-regulation for IVF:
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013
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Esteves, 11
Evidence Level 1a
AMH and AFC are not accurate for pregnancy prediction
Broer et al. Fertil Steril 2009 ; Broer et al. Hum Reprod Update, 17:46; 2011
How AMH and AFC Work
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Esteves, 13
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
.
Reflect No. pre-antral and small antral follicles
(≤4-8mm)
AMH
AF
C 2D-TVUS early follicular phase 2-10 mm (mean diameter)
No. AF at a given time that can be stimulated by medication
Esteves, 14
AMH
Low Inter-cycle Fluctuations (Fanchin et al, Hum Reprod 2005;20:923)
Low Intra-cycle Fluctuations (Hehenkamp et al. JCEM 2006;91:4057)
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%
Can be assessed at any cycle day with a single measurement
Esteves, 15
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
AMH
Esteves, 16
AMH
*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 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
Accurate to Predict Ovarian Response
Esteves, 17
AM
H
Fleming et al. RBM online 2013;26:130; Nelson SM. Fertil Steril. 2013 Jan 8; Nelson & La Marca. RBM online 2011;23:411;
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, 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
High Inter- and Intra-observer Reproducibility Scheffer et al. Ultrasound Obstet Gynecol 2002;20:270
Esteves, 19
AFC
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
1>20 oocytes retrieved in conventional COS; 2≤4 oocytes retrieved
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
Accurate to Predict Ovarian Response
Esteves, 20
AFC
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 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 • Costly
How to Use AMH and AFC in OI
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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, 22
Biomarkers for iCOS in High Responders
Evidence Level 2b
Evidence Level 1a
Esteves, 23
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 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
Pundir J et al. RBM Online 2012; 24:6-22.
~40% reduction in moderate/severe OHSS by using antagonists rather than agonists
4 RCT; 2377 pts. OR [95% CI]
Clinical PR, Miscarriage, LBR Not different
Risk of OHSS 1.29 (0.78; 2.26)
Cancellation 5.67 (1.07; 30.13)*
*p=0.04; risk of OHSS
GnRH Antagonist Protocol with Long-acting recFSH vs recFSH
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
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
Up to 45% of Infertility Patients in ART
Biomarkers for iCOS in Poor Responders
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Impaired Oocyte Quality
Reduced Fertilization Rate
Reduced Embryo Quality
Increased Miscarriage Rates
Westergaard et al., 2000; Esposito et al., 2001; Humaidan et al., 2002
Ovarian Aging
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• Normal androgen and estrogen biosynthesis • Normal follicular growth and development • Normal oocyte maturation N
orm
al
LH “Window” Concept
Reduced ovarian
paracrine activity
Hurwitz & Santoro 2004
Androgen secretory capacity reduced
• Piltonen et al., 2003
Decreased numbers of functional
LH receptors
• Vihko et al. 1996
Reduced LH bioactivity
• Mitchell et al. 1995; Marama et al 1984
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LH Supplementation in DOR
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, 28
Level 1a
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.
Rationale of LH supplementation
Esteves, 29
*derives from hCG
Purity (LH content)
hCG content (IU/vial)
LH activity (IU/vial)
Specific activity (LH/mg protein)
>99% 0 75 22,000 IU
3% ~70 75* ≥ 60 IU
Sources of LH Activity
Rec-hLH
hMG-HP*
Adapted from ASRM Practice Committee. Fertil Steril. 2008; 90:S13-20.
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Beta unit
Carboxyl terminal segment
Longer in hCG;
(Higher receptor affinity)
Absent in LH and present in hCG
(Longer Half-life)
Sources of LH Activity Sources of LH Activity
hCG
LH
Grondal et al. 2009: 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
Sources of LH Activity
Esteves, 32
r-FS
H
hMG
Sources of LH Activity
19 14 14
31 26 25
0 5
10 15 20 25 30 35
Fixed 2:1 r-hFSH (150IU)/r-hLH
(75IU)
HMG rec-hFSH + HMG
Duration of Stimulation (days) Mean No. oocytes retrieved IR (%)
CPR per transfer (%)
Buhler KF, Fisher R. Gynecol Endocrinol 2011;1-6.
Matched case-control study; N=4,719 IVF pts.
P=0.02
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Esteves, 34
Individualization of OI with AMH
High Responders
AMH >2.1
Poor Responders
AMH ≤ 0.82
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
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
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013
39.3
18.5 14.0
57.0
14.3 14.7
4.8
56.0
0
10
20
30
40
50
60
Observed Excessive
Response (%)
Oocytes retrieved (N)
OHSS (%) Pregnancy (%)
cCOS iCOS
1Excessive response: >20 oocytes retrieved; *Pts. received GnRH-a trigger + embryo vitrification; Mild/severe OHSS reported
p=0.03
p=0.04 p=0.38
p=0.92 iCOS Using AMH
High Responders
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013 Esteves, 35
72.0
3.5
45.0
20.0
46.6
4.8
23.3 26.8
0
20
40
60
80
Expected Poor Response (%)
Oocytes retrieved (N)
Cancellation (%) Pregnancy/cycle (%)
cCOS iCOS p=0.02
p=0.03
p=0.06 p=0.51
iCOS Using AMH Poor Responders
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013
Poor response <5 oocytes retrieved
Esteves, 36
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.
Take Home Messages
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Thank you
Esteves, 39
1Excessive response: >20 oocytes retrieved; 2Poor response: <5 oocytes retrieved; *Pts. received GnRH-a trigger + embryo vitrification; No severe OHSS reported
Response to COS Conventional COS
(n=131) iCOS
(n=118) P
value
Excessive1
Oocytes retrieved OHSS
Pregnancy
39.3% 18.5 ± 6.7
14.3% 57.1%
14.3% 14.7± 6.2
4.8%* 55.6%
0.03 0.04 0.38 0.92
Poor2
Oocytes retrieved Cancellation
Pregnancy/ET
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
iCOS Using AMH
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
Bosch et al. 2008, 2010; Xu et al, 2012; Kolibianakis et al 2012; Venetis et al. 2012; Griesinger et al 2013
P levels not associated with oocyte and embryo quality, nor with fertilization and cleavage rates
Esteves, 40 ANDROFERT, Referral Center for Male Reproduction
LH
FSH
LH
Esteves, 41
No CYP17
Esteves, 42 ANDROFERT, Referral Center for Male Reproduction
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
Progesterone thresholds affecting PR controversial
Ovarian response
Number of oocytes
Serum P threshold (ng/mL)
Poor ≤4 1.5
Intermediate 5-19 1.75
High ≥20 2.25
■ Fresh ■ FET
Esteves, 43 ANDROFERT, Referral Center for Male Reproduction
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
Effect of progesterone levels on day of hCG administration on pregnancy
Griesinger et al. Fertil Steril 2013
Ongoing PR: OR = 0.55 (0.37–0.81)
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