This document discusses optimizing treatment outcomes in assisted reproductive technology (ART). It begins with an outline of predictors of pregnancy in IVF and individualizing controlled ovarian stimulation (COS). The author then discusses evidence that the optimal number of oocytes retrieved is around 15 to maximize live birth rates. Strategies are presented for tailoring COS to individual phenotypes, including using biomarkers like AMH to predict response and adjusting gonadotropin preparations and protocols. Evidence is provided for approaches to optimize COS in both high and poor responders, such as using GnRH antagonists and LH supplementation respectively.

1. Doha, Qatar
Amman, Jordan
Tehran, Iran
2013
Optimizing Treatment
Outcome in ART
Sandro C. Esteves, MD, PhD
Director, ANDROFERT
Andrology & Human Reproduction Clinic
Campinas, Brazil

2. Maximize
Beneficial Effects
Minimize Complications
and Risks
Cycle
Multiple
Cancellation Pregnancy
Risk of OHSS
Poor Response
OHSS
Singleton
live birth at
term
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.
Esteves, 2
ANDROFERT, Referral Center for Male Reproduction

3. High-quality
Gametes and
Embryos
Individualization of
Controlled Ovarian
Stimulation
(iCOS)
Optimal
Endometrial Receptivity
Esteves, 3
ANDROFERT, Referral Center for Male Reproduction

4. Outline
Predictors of pregnancy in IVF
Individualization of COS
Cumulative live birth rates
Esteves, 4
ANDROFERT, Referral Center for Male Reproduction

5. Evidence
Level
1a
Predictive Factors for Pregnancy
in ART
Female Age
Duration of infertility
Basal FSH
Type of infertility
Indication
Fertilization method
Number of oocytes retrieved
Number of embryos transferred
Embryo quality
Negative
Predictors
Positive
Predictor
van Loendersloot et al. Hum Reprod Update 2010
Esteves, 5
ANDROFERT, Referral Center for Male Reproduction

6. Number of Oocytes Retrieved and Live
Birth Rates
Live birth rate (%)
Observed live birth rate
45%
40%
35%
30%
25%
20%
15%
10%
5%
0%
Predicted live birth rate
number of oocytes that best
optimized LBR was 15
450,135 Cycles
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 20 25 30 35 40
Oocyte number
Sunkara et al. Hum. Reprod., 2011
Esteves, 6
ANDROFERT, Referral Center for Male Reproduction

7. ...in all Age Groups
Esteves, 7
ANDROFERT, Referral Center for Male Reproduction

8. Key Points (1)
There is a strong association between the
number of oocytes retrieved and live birth
rates in IVF
The optimum number of oocytes needed to
maximize IVF outcomes seems to be about 15
COS should be tailored to the individual
phenotype, maximizing the number of oocyte
yield for poor responders and fine-tuning for
hyper-responders
Esteves, 8
ANDROFERT, Referral Center for Male Reproduction

9. How can we tailor COS?
Esteves, 9
ANDROFERT, Referral Center for Male Reproduction

10. Define Who is Who
Esteves, 10
ANDROFERT, Referral Center for Male Reproduction

11. Who is Who in ART
Young and older patients
Polycystic ovaries/PCOS
High basal FSH/small ovaries
Easily
Recognized
Previous OHSS/poor response
High/Decreased Ovary Sensitivity
BIOMARKERS of
Ovarian Response
Fiedler & Ezcurra Reprod Biol and Endocrinol 2012;
Humaidan et al. Fertil Steril. 2010.
Esteves, 11
ANDROFERT, Referral Center for Male Reproduction

12. AFC AMH
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; Fleming et al. Fertil Steril 2012;
Broekmans et al. Fertil Steril 2010; Scheffer et al. Hum Reprod 2003.
..
Esteves, 12
ANDROFERT, Referral Center for Male Reproduction

13. Non-growing
follicles (NGF)
recruited per
month
Kelsey et al. Mol Hum Reprod 2012
Esteves, 13
ANDROFERT, Referral Center for Male Reproduction

14. Evidence
Level
1a
Esteves, 14
ANDROFERT, Referral Center for Male Reproduction

15. Low Inter-cycle Fluctuations (Fanchin et al. Hum Reprod 2005)
AMH
ICC: 0.89; 95% IC: 0.83–0.94
Esteves, 15
ICC: 0.55; 95% IC: 0.39–0.71
Can be assessed at any cycle day
with a single measurement
Low Intra-cycle Fluctuations (Hehenkamp et al. JCEM 2006)
Max. Variation: 17.4%
Max. Variation: 108%
ANDROFERT, Referral Center for Male Reproduction

16. Biomarkers in COS
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, 16
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013

17. Optimize the number of
oocytes retrieved
Esteves, 17
ANDROFERT, Referral Center for Male Reproduction

18. Gonadotropin Preparations
and Oocyte Yield
Esteves, 18
ANDROFERT, Referral Center for Male Reproduction

19. Rec-hFSH has greater potency compared
to both uFSH and HP-HMG
Evidence
Level
1b
↑ 1.5 oocytes (GnRH antagonist cycles)
Devroey et al., 2012
↑ 2.1 oocytes (16 RCT; different protocols)
Lehert et al., 2010
↑ 3.1 oocytes (GnRH antagonist cycles)
Bosch et al., 2008
↑ 2.8 oocytes (GnRH agonist cycles)
Hompes et al., 2008
↑ 1.8 oocytes (GnRH agonist cycles)
MERIT Study, 2006
Esteves, 19
ANDROFERT, Referral Center for Male Reproduction

20. Protein content in solution
by mass (FbM)
Protein
content
hMG
hMG-HP
Specific
activity
(IU/mg protein)
Injected
protein per
75 IU (mcg)
< 5%
~100
~750
< 70%
2,000–2,500
~33
13,645
6.1
rec-hFSH* > 99%
Size Exclusion High
Performance Liquid
Chromatography
(SE- HPLC)
*Follitropin alfa; Bassett et al. Reprod Biomed Online 2005;10:169–177.
Esteves, 20
ANDROFERT, Referral Center for Male Reproduction

21. Accurate dose delivery
Adjustments by small increments
Self-administration
75%
Easy of use
58%
Dosing mechanism
43%
Less chance of error
26%
25%
Folitropin alfa prefilled ready-touse pen
Needle-free reconstitution,
conventional syringe
Weiss N. RBMonline 2007
Esteves, 21
ANDROFERT, Referral Center for Male Reproduction

22. Individualize COS
protocol
Esteves, 22
ANDROFERT, Referral Center for Male Reproduction

23. iCOS using AMH vs cCOS
High Responders
(N=70)
AMH >2.1
60
50
40
30
20
10
0
57.0
39.3
cCOS
*p<0.05
*
14.3
18.5
*
14.7
56.0
iCOS
14.0
4.8
Observed
Excessive
Response (%)
Esteves, 23
iCOS: rec-hFSH FbM 112.5 to 150 IU
daily + GnRH antagonist
Oocytes retrieved
(N)
OHSS (%)
Pregnancy (%)
Excessive response >20 oocytes retrieved; Mild/severe OHSS reported;
Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013.
ANDROFERT, Referral Center for Male Reproduction

24. Evidence
Level
1a
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 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 (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
Esteves, 24
ANDROFERT, Referral Center for Male Reproduction

25. GnRH-agonist vs hCG: 11 RCT – 1,055 women
Live birth
Fresh autologous
cycles (8 RCT)
Pregnancy
Moderate/
severe OHSS
OR 0.44
(0.29 - 0.68)
OR 0.45
(0.31 - 0.65)
OR 0.10,
(0.01 to 0.82)
Risk for OHSS markedly reduced:
3% ! 0%-2.6%
Chance of Pregnancy also reduced:
30% ! 12%-22%
Youssef et al. Cochrane Database Syst Rev. 2011
Esteves, 25
ANDROFERT, Referral Center for Male Reproduction

26. LH Trigger with GnRH-agonist
Modified Luteal Support
hCG bolus OPU day (1,500 UI) or 3x 500 UI
boluses; recLH; intense progesterone +
estradiol; combined
Risk Difference for Pregnancy
(hCG vs. GnRHa)
18% (Before) vs 6% (After) Modified LPS
Humaidan et al. Fertil Steril 2012;
Engmann & Benadiva Fertil Steril 2012
Embryo
cryopreservation
Meta-analysis of 5 RCT
Vitrification vs. Slow-freezing
OPR = 35% x 27%;
OR: 1.82; 95% CI: 1.04-3.20
IR = 29% x 24%;
OR: 1.49, 95% CI: 1.03-2.15
AbdelFahez et al . RBM Online 2010
Esteves, 26
ANDROFERT, Referral Center for Male Reproduction

27. Evidence-based Strategies to
Optimize COS in High Responders
Intervention
Outcome
Evidence
Identify who are at risk
1a
Oocyte yield
1a
Fine-tune COS +
oocyte yield
1b
GnRH Antagonists
OHSS
1a
GnRH Agonist for LH Triggering
OHSS
1a
Two-step IVF
OHSS
1b
Ovarian biomarkers
Recombinant rather than
urinary gonadotropins
Biomarkers + low starting doses
of rec-hFSH FbM
Esteves, 27
ANDROFERT, Referral Center for Male Reproduction

28. 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, 28
ANDROFERT, Referral Center for Male Reproduction

29. Evidence
Level
1a
Adjuvant Therapy in Poor
Responders
Intervention
Growth Hormone
Testosterone
Effect on
Pregnancy
Meta-analyses
Kyrou et al,20091
Kolibianakis et al, 20092
Duffy et al, 20103
Bosdou et al , 2012
Higher LBR1,2,3
Higher PR2
Higher CPR3
Higher LBR
Higher CPR
Kolibianakis et al, Hum Reprod Update 2009,15:613-22; Kyrou et al, Fertil Steril 2009;91: 749–66; Duffy et al,
Cochrane Database Syst Rev 2010;1:CD000099; Bosdou JK et al, Hum Reprod Update 2012;8:127-45;
Esteves, 29
ANDROFERT, Referral Center for Male Reproduction

30. Evidence
Level
1a
GnRH Antagonists in Poor
Responders
14 RCT (1,127 patients)
Duration of
stimulation
-1.9 days
(-3.6; -0.12)
Number Oocytes Cycle cancellation
retrieved
-0.17
(-0.69; 0.34)
1.01
(0.71; 1.42)
Clinical
Pregnancy
1.23
(0.92, 1.66)
ü Limited Clinical Benefit
Pu D et al. Hum Reprod. 2011
Esteves, 30
ANDROFERT, Referral Center for Male Reproduction

31. Evidence
Level
1b
Increasing the Stimulation Dose
of Gonadotropin in Poor
Responders…
RCT
Manzi et al, 1994
Klinkert et al, 2004
Berkkanoglu & Ozgur,
2010
Number
oocytes
retrieved
Cycle
Pregnancy
cancellation
rates
…is not associated with
better IVF outcome
Manzi DL et al. Fertil Steril. 1994; Klinkert ER et al. Hum Reprod. 2005;
Berkkanoglu & Ozgur Fertil Steril. 2010.
Esteves, 31
ANDROFERT, Referral Center for Male Reproduction

32. Evidence
Level
1a
LH Supplementation in OS
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*
(95% CI: 1.10; 3.11)
RD: +6%,
CPR
(95% CI: -0.3; +13.0)
LBR
(only 1 RCT)
Hill et al, 2012
7 RCT (N=902)
Age ≥35 yo.
r-hFSH+rLH
vs.
r-hFSH alone
CPR
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
Mochtar et al. Cochrane Database 2007; Bosdou et al, Hum Reprod Update 2012; Hill et al. Fertil Steril 2012.
Esteves, 32
ANDROFERT, Referral Center for Male Reproduction

33. Normal
LH “Window” Concept
• Normal androgen and estrogen biosynthesis
• Normal follicular growth and development
• Normal oocyte maturation
Reduced
ovarian
paracrine
activity
Decreased
numbers of
functional
LH receptors
Reduced LH
bioactivity
Hurwitz & Santoro
2004
Esteves, 33
Androgen
secretory
capacity
reduced
• Piltonen et al.,
2003
• Vihko et al. 1996
• Mitchell et al. 1995;
Marama et al 1984
ANDROFERT, Referral Center for Male Reproduction

34. LH Supplementation in Poor Responders
Late follicular phase
Early follicular phase
TC: Androgen production
TC: Androgen
GC: Enhance FSH action (estrogen
production
production) and Progesterone synthesis
Alviggi et al. Reprod Biomed Online 2006
Esteves, 34
ANDROFERT, Referral Center for Male Reproduction

35. LH How to Use LH in S Who and How
supplementation:
Patients (≥35 years)
Diminished Ovarian Reserve (AMH ≤0.82 ng/mL)
Rec-LH; GnRH antagonist flexible protocol
DOR: Recombinant FSH/LH (2:1 or 3:1 ratio) from stimulation D1
Normal reserve: 75 IU recLH added to rec-hFSH since D6
3
1
2
4
5
6
7
8
9
10
11
3
4
5
6
7
8
9
10
11
12
Menses
Esteves, 35
ANDROFERT, Referral Center for Male Reproduction

36. Individualized vs. Conventional COS
Poor Responders
(N=118)
rec-hFSH FbM + 75 IU rec-hLH
+ GnRH antagonist
• Total daily dose: 262.5 to 375 IU
AMH ≤ 0.82
80
60
40
20
72.0
*
46.6
45.0
*p<0.05
3.5
*
cCOS
*
23.3
20.0
iCOS
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. Fertil Steril 2013; 100 (Suppl.): S16.
Esteves, 36
ANDROFERT, Referral Center for Male Reproduction

37. 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
Esteves, 37
ANDROFERT, Referral Center for Male Reproduction

38. Evidence
Level
2a
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)
HMG
rec-hFSH + HMG
CPR per
transfer (%)
Buhler KF, Fisher R. Gynecol Endocrinol 2011
Esteves, 38
ANDROFERT, Referral Center for Male Reproduction

39. Sources of LH Activity
Sources of LH Activity
Beta unit
hCG
Carboxyl
terminal
segment
Longer in hCG Absent in LH and
present in hCG
(Higher receptor
affinity)
(Longer Half-life)
LH
Esteves, 39
ANDROFERT, Referral Center for Male Reproduction

40. 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, 40
Grondal ML et al. Fertil Steril 2009; 91: 1820-1830.
Menon KM et al. Biol Reprod 2004; 70:861-866

41. Evidence-based Strategies to
Optimize COS in Poor Responders
Intervention
Outcome
Evidence
Identify who are at risk
1a
Oocyte yield
1a
Pregnancy rate
1a
Duration of stimulation
1a
LH supplementation
Pregnancy rate
1a
Biomarkers + rec-LH
Oocyte yield/cancellation/PR
2a
Pregnancy rate
2a
Ovarian biomarkers
Recombinant rather than
urinary gonadotropin
Adjuvant therapy
GnRH antagonist protocol
LH supplementation with
rec-LH rather than hMG
Esteves, 41
ANDROFERT, Referral Center for Male Reproduction

42. Cumulative live
birth as a key
strategy to optimize
outcome in IVF
Esteves, 42
ANDROFERT, Referral Center for Male Reproduction

43. Cumulative live birth rates

44. Are they critical for success?
Sperm Selection
• Hyaruronic acid binding
• Polarization microscopy
• MSOME
• Electroforetic sperm isolation
• Magnetic-activated cell sorting
• Microfluids
• Microarray technology
• Proteomics
Esteves, 44
Oocyte Selection
Embryo Selection
• Polscope
• Oxygen consumption
• Cumulus cells gene expression
(mRNA transcripts)
• Molecular mining of follicular fluid
(metabolomics, proteomics)
Metabolic Profile:
• Glucose and pyruvate uptake
• Amino acid turnover
• Oxygen consumption
Proteomics
• Mass Spectroscopy
• Raman Spectroscopy
• Nuclear Magnetic Resonance
Embryo biopsy
• FISH, PCR, CGH, SNS microarray, Next-generation
Sequencing (single gene),
Quantitative Real-time PCR
(qPCR)
ANDROFERT, Referral Center for Male Reproduction

45. Cumulative pregnancy to optimize
treatment outcome in ART
Pillar #1 – IVF facilities
Esteves, 45
ANDROFERT, Referral Center for Male Reproduction

46. Esteves & Bento. RBM Online 2013

47. Esteves & Bento. RBM Online 2013

48. Cumulative pregnancy to optimize
treatment outcome in ART
Pillar #2 – Blastocyst Culture
Identification of embryos with
optimal development potential
Meta-analysis of eight RCT with
1,654 patients
LBR with Blastocyst vs. Cleavage-stage ET
35% x 28%; OR: 1.39; 95% CI: 1.10-1.76
Papanikolaou E et al. Hum Reprod 2008
Esteves, 48
ANDROFERT, Referral Center for Male Reproduction

49. Identification of Embryos with Optimal
Development Potential
Time-lapse Technology
Videomicrography + Computer Vision Software
(Eeva; Auxogyn)
Wong et al, 2010
Esteves, 49
ANDROFERT, Referral Center for Male Reproduction

50. Cumulative pregnancy to optimize
treatment outcome in ART
Pillar #3 – Vitrification
Embryo and Oocyte
Vitrification vs. Slow-freezing
Ongoing PR: 35% x 27%;
OR: 1.82; 95% CI: 1.04-3.20
Meta-analysis of five RCT with 765 cycles
AbdelFahez et al . RBM Online 2010
Vitrification is simpler and faster than
Slow Freezing
Esteves, 50
ANDROFERT, Referral Center for Male Reproduction

53. Conclusions (1)
The number of oocytes obtained is a key factor
for optimizing Live Birth rates
COS should be tailored to the individual phenotype,
maximizing the number of oocyte yield for poor
responders and fine-tuning for hyper-responders
COS with recombinant FSH results in an
increased oocyte yield compared with hMG/uFSH
Higher FSH bioactivity, which is related to the way the
drug is made, filled and delivered
Esteves, 53
ANDROFERT, Referral Center for Male Reproduction

54. Conclusions (2)
AMH and AFC are currently the best tools to
predict ovarian response to COS
Similar accuracy to determine who is at risk of excessive
and poor response
Evidence-based strategies to optimize COS
combine biomarkers and stimulation protocol
Low recFSH doses and GnRH antagonists
recLH supplementation
Esteves, 54
ANDROFERT, Referral Center for Male Reproduction

55. Conclusions (3)
Cumulative pregnancy is a key strategy to
optimize success in ART
Stimulation Protocol and Oocyte Yield
Laboratory Facilities
Identification Embryos with Development Potential
Cryopreservation Program (Vitrification)
Esteves, 55
ANDROFERT, Referral Center for Male Reproduction

56. obrigado
Thank You