optimizing treatment outcome in art

Sandro C. Esteves, MD, PhD Director, ANDROFERT

Andrology & Human Reproduction Clinic Campinas, Brazil

Optimizing Treatment Outcome in ART

Doha, Qatar Amman, Jordan

Tehran, Iran 2013

Singleton live birth at

term

Maximize Beneficial Effects

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.

Multiple Pregnancy

Cycle

Cancellation Risk of OHSS

Poor Response

OHSS

Minimize Complications and Risks

Esteves, 2 ANDROFERT, Referral Center for Male Reproduction

Individualization of Controlled Ovarian

Stimulation (iCOS)

Optimal Endometrial Receptivity

High-quality Gametes and

Embryos


Outline


Predictors of pregnancy in IVF Individualization of COS

Cumulative live birth rates

Positive Predictor

van Loendersloot et al. Hum Reprod Update 2010

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

Predictive Factors for Pregnancy in ART


Evidence Level 1a

Number of oocytes retrieved

0% 5%

10% 15% 20% 25% 30% 35% 40% 45%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 20 25 30 35 40

Live

birt

h ra

te (%

)

Oocyte number

Observed live birth rate Predicted live birth rate

Sunkara et al. Hum. Reprod., 2011

450,135 Cycles

Number of Oocytes Retrieved and Live Birth Rates

number of oocytes that best optimized LBR was 15



...in all Age Groups

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


How can we tailor COS?


Define Who is Who


Young and older patients

Polycystic ovaries/PCOS

High basal FSH/small ovaries

Previous OHSS/poor response

Easily Recognized

Fiedler & Ezcurra Reprod Biol and Endocrinol 2012; Humaidan et al. Fertil Steril. 2010.

BIOMARKERS of Ovarian Response

High/Decreased Ovary Sensitivity

Who is Who in ART


No. pre-antral and small antral follicles (≤4-8mm)

AMH

AF

C


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.

..

2D-TVUS early follicular phase 2-10 mm (mean diameter)

No. AF at a given time that can be stimulated by medication

Non-growing follicles (NGF) recruited per month

Kelsey et al. Mol Hum Reprod 2012



Evidence Level 1a

AMH

Low Inter-cycle Fluctuations (Fanchin et al. Hum Reprod 2005)

Low Intra-cycle Fluctuations (Hehenkamp et al. JCEM 2006)

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, 16

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 COS

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

Optimize the number of oocytes retrieved


Gonadotropin Preparations and Oocyte Yield


Rec-hFSH has greater potency compared to both uFSH and HP-HMG

↑ 1.5 oocytes (GnRH antagonist cycles) Devroey et al., 2012

↑ 3.1 oocytes (GnRH antagonist cycles) Bosch et al., 2008

↑ 1.8 oocytes (GnRH agonist cycles) MERIT Study, 2006

↑ 2.8 oocytes (GnRH agonist cycles) Hompes et al., 2008

Evidence Level 1b

↑ 2.1 oocytes (16 RCT; different protocols) Lehert et al., 2010


*Follitropin alfa; Bassett et al. Reprod Biomed Online 2005;10:169–177.

Protein content

Specific activity

(IU/mg protein)

Injected protein per 75 IU (mcg)

hMG < 5% ~100 ~750 hMG-HP < 70% 2,000–2,500 ~33

rec-hFSH* > 99% 13,645 6.1

Size Exclusion High Performance Liquid

Chromatography (SE- HPLC)

Protein content in solution by mass (FbM)


Accurate dose delivery Adjustments by small increments Self-administration


75%

25%

Folitropin alfa prefilled ready-to-use pen

Needle-free reconstitution, conventional syringe

Easy of use 58%

Dosing mechanism 43%

Less chance of error 26%

Weiss N. RBMonline 2007

Individualize COS protocol


High Responders (N=70)

AMH >2.1

iCOS: rec-hFSH FbM 112.5 to 150 IU daily + GnRH antagonist


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 *p<0.05

* *

iCOS using AMH vs cCOS

Excessive response >20 oocytes retrieved; Mild/severe OHSS reported; Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013.

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


Risk for OHSS markedly reduced: 3% ! 0%-2.6%

GnRH-agonist vs hCG: 11 RCT – 1,055 women

Fresh autologous cycles (8 RCT)

Live birth 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)

Youssef et al. Cochrane Database Syst Rev. 2011

Chance of Pregnancy also reduced: 30% ! 12%-22%


Humaidan et al. Fertil Steril 2012; Engmann & Benadiva Fertil Steril 2012

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


LH Trigger with GnRH-agonist

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

Intervention Outcome Evidence

Ovarian biomarkers Identify who are at risk 1a

Recombinant rather than urinary gonadotropins Oocyte yield 1a

Biomarkers + low starting doses of rec-hFSH FbM

Fine-tune COS + oocyte yield 1b

GnRH Antagonists OHSS 1a

GnRH Agonist for LH Triggering OHSS 1a Two-step IVF OHSS 1b

Evidence-based Strategies to Optimize COS in High Responders


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


Intervention Meta-analyses Effect on Pregnancy

Growth Hormone Kyrou et al,20091 Kolibianakis et al, 20092 Duffy et al, 20103

Higher LBR1,2,3 Higher PR2

Higher CPR3

Testosterone Bosdou et al , 2012 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;


Evidence Level 1a Adjuvant Therapy in Poor

Responders

14 RCT (1,127 patients) Duration of stimulation

Number Oocytes retrieved

Cycle cancellation Clinical Pregnancy

-1.9 days (-3.6; -0.12)

-0.17 (-0.69; 0.34)

1.01 (0.71; 1.42)

1.23 (0.92, 1.66)

Pu D et al. Hum Reprod. 2011

GnRH Antagonists in Poor Responders

ü  Limited Clinical Benefit


Evidence Level 1a

Pregnancy rates

Cycle cancellation

Number oocytes

retrieved

RCT

…is not associated with

better IVF outcome

Manzi et al, 1994 Klinkert et al, 2004 Berkkanoglu & Ozgur, 2010

Manzi DL et al. Fertil Steril. 1994; Klinkert ER et al. Hum Reprod. 2005; Berkkanoglu & Ozgur Fertil Steril. 2010.

Increasing the Stimulation Dose of Gonadotropin in Poor

Responders…


Evidence Level 1b

LH Supplementation in OS 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) Age ≥35 yo.

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 et al. Cochrane Database 2007; Bosdou et al, Hum Reprod Update 2012; Hill et al. Fertil Steril 2012.

Evidence Level 1a


• Normal androgen and estrogen biosynthesis • Normal follicular growth and development • Normal oocyte maturation

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

Nor

mal


Early follicular phase TC: Androgen

production

Late follicular phase TC: Androgen production

GC: Enhance FSH action (estrogen production) and Progesterone synthesis

Alviggi et al. Reprod Biomed Online 2006


LH Supplementation in Poor Responders

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

2 3 4 5 7 6 8 9 10 11 1

Menses

12

How to Use LH in S LH supplementation: Who and How

7 6 8 9 10 11 3 4 5

Normal reserve: 75 IU recLH added to rec-hFSH since D6


Individualized vs. Conventional COS Poor Responders

(N=118)

AMH ≤ 0.82

rec-hFSH FbM + 75 IU rec-hLH + GnRH antagonist

• Total daily dose: 262.5 to 375 IU 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

Poor response <5 oocytes retrieved;

Leão RBF, Nakano FY, Esteves SC. Fertil Steril 2013; 100 (Suppl.): S16.


*p<0.05 *

* *

*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

Rec-hLH

hMG-HP*

Adapted from ASRM Practice Committee. Fertil Steril. 2008


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

Matched case-control study; N=4,719 IVF pts.

P=0.02


Evidence Level 2a

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


Esteves, 40

r-FS

H

hMG

Intervention Outcome Evidence

Ovarian biomarkers Identify who are at risk 1a

Recombinant rather than urinary gonadotropin Oocyte yield 1a

Adjuvant therapy Pregnancy rate 1a

GnRH antagonist protocol Duration of stimulation 1a

LH supplementation Pregnancy rate 1a

Biomarkers + rec-LH Oocyte yield/cancellation/PR 2a LH supplementation with rec-LH rather than hMG Pregnancy rate 2a

Evidence-based Strategies to Optimize COS in Poor Responders


Cumulative live birth as a key

strategy to optimize outcome in IVF


Cumulative live birth rates

ANDROFERT ANDROFERT ANDROFERT

ANDROFERT ANDROFERT

Are they critical for success?

• Hyaruronic acid binding • Polarization microscopy • MSOME • Electroforetic sperm isolation • Magnetic-activated cell sorting • Microfluids • Microarray technology • Proteomics

• Polscope • Oxygen consumption • Cumulus cells gene expression

(mRNA transcripts) • Molecular mining of follicular fluid

(metabolomics, proteomics)

Oocyte Selection

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 micro-

array, Next-generation Sequencing (single gene), Quantitative Real-time PCR (qPCR)

Embryo Selection Sperm Selection



Cumulative pregnancy to optimize treatment outcome in ART Pillar #1 – IVF facilities

Esteves & Bento. RBM Online 2013

Identification of embryos with optimal development potential

Papanikolaou E et al. Hum Reprod 2008

Meta-analysis of eight RCT with 1,654 patients


Cumulative pregnancy to optimize treatment outcome in ART

Pillar #2 – Blastocyst Culture

LBR with Blastocyst vs. Cleavage-stage ET 35% x 28%; OR: 1.39; 95% CI: 1.10-1.76

Time-lapse Technology Videomicrography + Computer Vision Software

(Eeva; Auxogyn)

Wong et al, 2010


Identification of Embryos with Optimal Development Potential

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

Cumulative pregnancy to optimize treatment outcome in ART

Pillar #3 – Vitrification

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

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

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)

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optimizing treatment outcome in art

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