Aula ministrada pelo Dr. Sandro Esteves no "Gulf Lecture Tour" em Kuwait and Bahrain, dias 6 e 7 de novembro.

1. Improving Success by Tailoring
Ovarian Stimulation
- We are all individuals -

2. Lecture Outline

3. UN Census Estimates, 2008

7. Ovarian Stimulation
Pharmaceutical industry
One size fits all protocol for OS
► suppress LH surge: GnRHa
► ovarian stimulation with HMG/FSH
● high doses of gonadotropin
● high number oocytes
● high number of embryos
Results not the same for all
● poor response and OHSS
● side effects
● patient satisfaction neglected

8. Psychological burden 49%-26%
Prognosis 40%-23%
Cost of treatment 23%-0%
Relationship/divorce 15%-9%
Physical burden 7-6%
Up to 65% of couples dropout
from IVF without achieving
pregnancy before they complete
3 cycles1-5
Oocyte retrieval 52%
Embryo transfer 29%
Injections 29%
Physical pain 20%
Blood tests 14%
1. Olivius K t al, Fertil Steril 2004;81:258; 2. Land JA et al, Fertil Steril 1997; 68:278; 3. Schroder AK, et al, RBM Online 2004; 5:600; 4.
Osmanangaoglu K et al, Hum Reprod 2002; 17:2655; 5. Rajkhowa M et al, Hum Reprod 2006; 21:358; 6. Brandes M et al, Hum Reprod 2009;
24:3127; 7. Hammarberg K et al, Hum Reprod 2001; 16:374.
Reasons1,5,6
IVF events women find stressful7
Pregnancy loss 94%
Unsuccessful cycle 87%
Waiting after ET 81%
Waiting to find out how many
eggs fertilized
68%
Result of pregnancy scan 47%
Patient Satisfaction
Why should I care?

9. Ovarian Stimulation
One size fits all?
Patient is the main
variable of OS response
z Demographics and
anthropometrics (Age,
BMI, Race)
z Genetics profile
z Cause of Infertility
z Years of Infertility
z Health status
z Nutritional status

10. How to define the right individual
treatment for the right patient to:
●Prevent poor response and
OHSS (reduce cancellation)
●Reduce side effects
●Increase pregnancy rates
●Reduce physical, psychological
and financial burden
Understanding the Problem
What we really want to know is...
Esteves, 10

11. Understanding the Problem
From cookery to science
Individualizing ovarian
stimulation according to
patients is important
But how ?
There are several predictors
of ovarian response
Can we make prediction
more scientific but simple ?
Esteves, 11

12. Lecture Outline
Gonadotropins:
better now

13. Age
Biomarkers
● Hormonal Biomarkers, FSH, Inhibin-B, AMH
● Functional Biomarkers: Antral Follicle Count (AFC)
● Genetic Biomarkers: Single Nucleotide Polymorphisms for
FSH-R/LH/LH-R/E2-R/AMH-R
Markers of Ovarian Response
Can we predict ovarian response?
Esteves, 13

14. 1. CDC Report December 2006 (2004 results)
10
20
30
40
50
0
<21 22 24 26 28 30 32 34 36
Live birth rate
Maternal age (years)
38 40 42 44 46 48
35 years
ART pregnancy and live birth rates
decline with increasing age
Esteves, 14

15. Who has the highest chance of a live
birth following IVF?
Hana
Age 26
Basal FSH 9
Maria
Age 37
Basal FSH 5
Esteves, 15

16. 1. Akande et al. Hum Reprod 2002;17:2003–2008
(n = 1019)
20–24 25–29 30–34 35–39 40–44 45–49
5
0
10
15
20
Age (years)
6–8.9
3–5.9
<3
FSH IU/L
≥12
9–11.9
Age and FSH
chronological vs biological in IVF
MariaHana
Esteves, 16

17. Why do ovaries age at different rates?
Multifactorial, but genetics important
Single nucleotide polymorphisms
(SNPs) linked to:
●Ovarian response to gonadotrophins
●Premature menopause
Both activating and inactivating
mutations identified in the LH and
FSH receptor genes1
1. Themmen and Huhtaniemi. Endocr Rev 2000;21:551–583
Human FSH Receptor Mutations
FSH-R: Ser680 genotype
- NH2
- COOH
Ala189Val
Asp567Gly??
(Asn191Ile)Ile160Thr
Asp224Val
Arg573Cys
Leu 601Val
Ala419Thr
Pro346Arg
Val341Ala
*
Pro519Thr Thr307Ala
Ser680Asn
*
*
*
Esteves, 17

18. La Marca, et al. Hum Reprod 2009.
AMH levels are
correlated with
the number of
follicles at
gonadotropin
independent
stage
Markers of Ovarian Response
Biomarkers and follicular development
Esteves, 18

19. AMH: a cut-off 1.26 ng/ml was able to predict
poor response (<4 oocytes) with 97% sensitivity
Gnoth, et al. Hum Reprod 2008.
Retrospective analysis, 316
patients (1st IVF cycle) in
GnRH-a long protocol
Variables: age, basal FSH, AMH,
Inhibin-B
Endpoint: number of oocytes
Cut-off of poor response: 4 oocytes
Markers of Ovarian Response
anti-Mullerian hormone (AMH)
Esteves, 19

20. Verhagenet al. 2008; Broer et al., 2010
Markers of Ovarian Response
Prediction of response by AMH
AMH category (ng/mL) 0.14 to <0.7 (N=74) 0.7 to <2.1 (N=128) >2.1 (N=148)
Agonist protocol + rFSH 375 225 150
Oocytes (n) 5 (3-7) 10 (7-15) 14 (10-19)
Severe OHSS 0 (0%) 3 (2%) 20 (13.9%)
Cancellation 19 (25.7%) 3 (2.3%) 4 (2.7%)
CPR per transfer 11.1% 34.6% 40.1%
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.
Esteves, 20

21. Markers of Ovarian Response
Antral Follicle Count (AFC)
No. of antral
follicles
< 3 4-10 > 10
No. of cycles 16 76 57
Mean age (years) 36.8 ± 2.9 36.3 ± 4.0 32.8 ± 3.8
Day 3 FSH (IU/l) 12.7 ± 8.5 7.1 ± 4.1 5.6 ± 1.7
Cx rate 68.8% 5.3% 0%
Peak E2 (pg/ml) 432 ± 157 1.001 ± 627
1.912 ±
1.355
Mean No. of eggs 2.0 ± 0.9 6.3 ± 4.4 14.1 ± 8.5
OG pregnancy
rate
0% 13.2% 26.3%
Chang, et al. Fertil Steril. 1998;69:505.
Hansen KR, et al. Fertil Steril
2003;80:577–83
Number of antral follicles
r=0.64
p<0.001
0 5 10 15 20 25
25
20
15
10
5
0
Esteves, 21

22. Markers of Ovarian Response
AFC: limitations
Number of ovaries
evaluated
Min/max follicular
diameter (<6? Or<11)
Type of scansCut off points
Ultrasound Axes
Number of ovaries evaluated
Min/max follic. diameter (<6? or<11)
Type of scans; cut off points

23. Markers of Ovarian Response
Antral Follicle Count (AFC)
Broekmans et al., Fertil Steril, 2009
Clinical considerations
● Cycle day 2-4
● Count all AF 2-10mm
Technical considerations
● Real-time 2 dimension
image adequate
● Transvaginal probe 7Mhz
minimum
Esteves, 23

24. Broer et al. , 2010
AMH = AFC >Inhibin B >FSH >Age
Markers of Ovarian Response
Prediction of response
Esteves, 24

25. The patient individual
factors play a crucial
role in predicting ovarian
response.
AFC and AMH are helpful
to predict ovarian
response to stimulation.
Markers of Ovarian Response
Summary
Esteves, 25

26. Lecture Outline

27. Other:
z Progesterone
z Estradiol
z Aromatase inibitor
z Contraceptive pill
z Antioxidants/vitamins
Gonadotropins:
z Recombinant
FSH/LH/hCG
z Urinary
FSH/LH/hCG
GnRH
Analogues:
z Agonist
z Antagonist
Esteves, 27

28. Product Technology Brand name Manufacturer
hMG Urine-derived Menogon®; Repronex®
Merional®
Ferring
IBSA
hMG HP Urine-derived Menopur® Ferring
u-FSH Urine-derived Fostimon® IBSA
u-FSH HP Urine-derived Bravelle® Ferring
u-hCG Urine-derived Choragon®
Choriomon®
Ferring
IBSA
r-hFSH (follitropin
beta)
Recombinant Puregon®; Follistim® MSD
r-hFSH (follitropin alfa) Recombinant GONAL-f® MerckSerono
r-hLH Recombinant Luveris® MerckSerono
r-FSH + r-hLH Recombinant Pergoveris® MerckSerono
r-hCG Recombinant Ovidrel®; Ovitrelle® MerckSerono
Gonadotropins: an overview
What is available?

29. Gonadotropins: an overview
Urinary-derived products

30. Culture media
HarvestBioreactor
Production
Cell attachment and
proliferation
r-hFSH production and
secretion
Collection of cell
culture supernatant
medium containing
r-hFSH
In-process QC
Purification
Concentration of
supernatant
Chromatographic
purification
steps
Ultrasterile filtration
Characterization
and full QC of
bulk r-hFSH
Esteves, 30
Gonadotropins: an overview
Recombinants

31. Gonadotropins: an overview
Differences
Bassett et al. Reprod Biomed Online 2005;10:169–177
Purity
(FSH
content)
Mean specific
FSH activity
(IU/mg protein)
Injected
protein
per 75 IU
(mcg)
hMG < 5% ~100 ~750*
hMG-HP < 70% 2000–2500 ~33*
r-hFSH
Follitropin beta – 7000–10,000 8.1*
Follitropin alfa > 99% 13,645 6.1
Esteves, 31

32. 1. Bassett et al. Reprod Biomed Online 2005;10:169–177
2. Driebergen et al. Curr Med Res Opin 2003;19:41–46
Conventional
Bioassay
High
variability
(~20%)
in vivo (rat)
Novel analitycal
method
Physiochemical
technique
Minimal batch-to-
batch variability
(1.6%)1,2
Gonadotropins: an overview
Product Quality: Filled by Mass (FbM)
Esteves, 32

33. Concept of Dose Precision
Clinical implications
Batch variability
+20%, -25%
225
270
170
IU
Bioassay
Urinary and Follitropin beta
16.5 mcg
(225 IU)
Filled by Mass
Folitropin alfa (Gonal-f FbM)
Batch variability
rrrr 2%
Risk of OHSS
Poor response

34. Portable, ready-to-use device
Precise dose delivered
Gonal-f FbM

35. Group A (hMG; N=299)
Group B (HP-hMG; N=330)
Group C (r-hFSH; N=236)
Gonadotropin rFSH/hMG
112.5-450 UI
Individualized dose
Agonist (nasal spray): Nafarelin acetate (400 mcg/day; fixed)
Day 1 Day 6
Day
of hCG
Cycle
day 21
Day 2-5 of menses
menses
Vaginal
progesterone
Esteves, 35

36. Outcome Measure HMG
n=299
HP-hMG
N=330
r-hFSH
n=236
P-
value
Total gonadotropin dose (IU) 2,685 2,903 2,268 <0.01
Retrieved oocytes (N) 10.9 10.7 10.8 NS
MII oocytes (N) 8.9 8.9 8.7 NS
2PN fertilization rate (%) 72 72 71 NS
Implantation rate (%) 24 27 23 NS
Live birth rate per cycle (%) 24.4 32.4 30.1 NS
Moderate/severe OHSS(%) 2.3 1.8 1.3 NS
r-hFSH vs hMG/HP-hMG in ART
Esteves et al. (observational study 2009)
Esteves et al, Reprod Biol Endocrinol. 2009; 7:111

37. 18.7 20.3
53.4*
% Cycles with “Step-down”
during ovarian stimulation
HMG HP-HMG rec-hFSH (fbm)
*P<0.01
r-hFSH vs hMG and HP-hMG in ART
Esteves et al. (observational study 2009)
Esteves et al, Reprod Biol Endocrinol. 2009; 7:111

38. To achieve a
live birth,
21-52% more
HP-hMG and
hMG was
required
compared
with r-hFSH
0
3.000
7.000
10.000
21.6%
r-hFSH HP-hMG
6,324*
7,739
hMG
9,69052.2%
* Mean total dose per cycle/Live birth rate (≤35 years)
r-hFSH vs hMG and HP-hMG in ART
Esteves et al. (observational study 2009)
Esteves et al, Reprod Biol Endocrinol. 2009; 7:111

39. Other products for ART
What is available?
Product Brand name Manufacturer
GnRH-analogue
Nafareline Synarel® Pfizer
Leuprolide Lupron® Abbott
Triptoreline Decapeptyl® Ferring
Gosereline Zoladex® Astra-Zeneca
Busereline Suprefact®, Suprecur® Sanofi-Aventis
GnRH antagonist
Cetrorelix Cetrotide® Merck Serono
Ganirelix Orgalutran® MSD
Progesterone
8% gel Crinone® Merck Serono
100 capsules Utrogestan® Ferring
Oil solution 50mg Several Several

40. LH surge prevention
GnRH agonists
pyro (Glu) – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2
Activation of the
GnRH receptor
Regulation of
receptor affinity
Regulation of receptor
biological activity

41. LH surge prevention
GnRH antagonists
pyro (Glu) – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2
Activation of the
GnRH receptor
Regulation of
receptor affinity
Regulation of receptor
biological activity
Antagonistic
effect
1 32

42. LH surge prevention
GnRH antagonists
Follicular
Luteal
0
10
20
30
2-4 weeks
Synchronized follicles
Agonist
Antagonist
• Half-life ~20h (Cetrorelix)
• Suppress LH by 80% of
baseline levels

43. A comparison of Nafarelin and Cetrorelix for
LH suppression in COH-ICSI cycles with
Follitropin alfa
• Retrospective (2002-2008)
• Unselected group of NG women – COS with r-hFSH
• Group 1 (Nafarelin; N=1,362); Group 2 (Cetrorelix; N=414)
Individualized dose
Agonist: Nafarelin acetate (400 mcg/day; fixed)
Gonadotropin dose
112.5-450 UI
Day 1
of rFSH
Day 6
of rFSH
Cycle
day 21
Day 2-5 of menses
menses
Vaginal
progesterone
Day
of hCG
Follitropin alfa dose
112.5-450 UI
Individualized
rFSH dose
0.25 mg/day of
Cetrorelix (flexible)
Follicle
13 mm
Day
of hCG
Day 2 or 3
of menses
Day 1
of rFSH
menses
Vaginal
progesterone
Esteves et al., JBRA Assist Reprod (Suppl 1), 2010Esteves, 43

44. A comparison of Nafarelin and Cetrorelix for
LH suppression in COH-ICSI cycles with
Follitropin alfa
1st ICSI cycles Cetrorelix
N=163
Nafarelin
N=948
P-value
Age (yrs) 34.5 33.4 0.002
Total r-hFSH dose (IU) 2,313 2,453 0.001
Days of -hrFSH 9.9 10.3 0.01
E2 hCG day (pmol/L) 1,585 2,371 <0.001
Oocytes retrieved (n) 9.5 11.3 <0.001
2PN Fertilization (%) 63.3 62.5 NS
Transfer (n) 2.4 2.5 NS
Live birth (%) 35.5 36.3 NS
Embryo cryopreserved (%) 47.1 48.4 NS
85
64
54 50
15
36
46 50
cycle no.1
(n=1111)
cycle no.2
(n=378)
cycle no.3
(n=194)
cycle no.
≥4 (n=93)
Nafarelin Cetrorelix
Esteves et al., JBRA Assist Reprod (Suppl 1), 2010
Distribution by ICSI cycle rank (%)

45. Kolibianakis et al (2006)2
N studies 22
Included non peer-reviewed data No
Included IUI cycles No
N patients 3176
Odds ratio 0.86 (0.72-1.02; p=.08)*
Duration of stimulation -1.54 days (OR: -2.42; -0.66; p=.0006)
Oocytes retrieved -1.19 (OR: -1.82; -0.56)
Risk of severe OHSS OR=0.61 (0.42; 0.89; p=.01)*
GnRH antagonists vs agonists
Meta-analysis
*For every 59 women treated with a GnRH agonist vs GnRH
antagonist, one additional case of severe OHSS will occur.
Esteves, 45

46. Agonist administrationAgonist administration
Gonadotropin administrationGonadotropin administration
Long GnRH
agonist protocol
Antagonist
administration
Antagonist
administration
Gonadotropin administrationGonadotropin administration
Single or multiple
dose GnRH
antagonist protocol
Flare up
effect
Pituitary
suppression
Longer
treatment
Can exclude
early
pregnancy
Can be integrated
in spontaneous
and OI cycles
Pre-treatment cycle Treatment cycle
No hormonal
withdrawal
No flare
effect with
possible cyst
formation
Less gona-
dotropins
Prevent OHSS
by GnRH-a
LH surge prevention
GnRH antagonists vs agonists

47. Lecture Outline

48. AMH category (ng/mL) >2.1
GnRH analogue + r-hFSH 150UI Agonist Antagonist
Oocytes (n) 14 (10-19) 10 (8.5-13.5)
Severe OHSS 20 (13.9%) 0 (0%)*
Cancellation 4 (2.7%) 1 (2.9%)
CPR per transfer 40.1% 63.6%*
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
Individualized Treatment with AMH
AMH + antagonists in hyper-responders
Esteves, 48

49. 31.3% 31.1%
35.3%
50.0%
20.0%
0%
10%
20%
30%
40%
50%
60%
75 IU 112.5 IU 150 IU 187.5 IU 225 IU
Clinical pregnancy rates/cycle
started
Olivennes F, et al. The CONSORT study. Reprod Biomed Online. 2009;18:195–204.
Individualized dosing in
increments of 37.5 IU of
Gonal-f possible by FbM
technology
Use of algorithm of
patients characteristics
● basal FSH
● body mass index (BMI)
● age
● antral follicle count
Age (28-32)
Oocytes retrieved (8-12)
CONSORT = CONsistency in r-hFSH
Starting dOses for Individualized
tReatmenT
Esteves, 49

50. 1. Alviggi et al. Reprod Biomed Online 2006;12:221–233; 2. Tarlatzis et al. Hum Reprod 2006;21:90–94
3. Esteves et al. Reprod Biol Endocrinol 2009;7:111; 4. Marrs et al. Reprod Biomed Online 2004;8:175–182
5. Mochtar MH, Cochrane Database, 2007; 6. De Placido et al. Clin Endocrinol (Oxf) 2004;60:637–643
7. Alviggi, et al. RBMOnline 2009.
LH supplementation in ART
What do we know today
z The majority of patients do not need LH
supplementation as endogenous LH levels are
sufficient1–3
z 15-20% of women have less sensitive ovaries
Older patients (> 35 years)4
Low responders5
Deeply suppressed endogenous LH6
Hypo-responders7
FSH and AFC considered adequate
Genetic characteristics
Single nucleotide polymorphisms of FSH-R and LH-R
Esteves, 50

51. Mochtar MH, Cochrane Database, 2007
No difference in basal LH levels.
Less bioactive LH/LH receptor polymorphism ?
LH supplementation in ART
Cochrane review 2007: hypo-responders
r-hFSH vs r-hLH + r-hFSH (Ongoing PR)

52. Increasing FSH
drive of limited
value
LH
LH
FSH
• Theca cells
• Granulosa
cells
Consider
increasing LH
drive
There is a potential role for r-hLH in this
population
Esteves, 52
LH supplementation in ART
Biologic older (less sensitive) ovaries

53. Tailoring Ovarian Stimulation
Treatment individualization strategies
• Antagonist + r-FSH FbM 112.5-150 UI
• Normal oocyte yield
• Very low cancellation/OHSS
• Adequate LBR
High
Responders
AFC >10
AMH >2.1
• Antagonist or Agonist + r-hFSH 187.5-262.5 UI
• Low cancellation & OHSS
• Adequate LBR
Normal
Responders
AFC 4-10
AMH 0.7-2.1
• Antagonist + r-hFSH (+r-hLH) 300-375 UI
• Short stimulation
Moderate cancellation
Low LBR
Poor
Responders
AFC <4
AMH <0.7

54. Understanding the Problem
From cookery to science - Summary
We can we make prediction
more scientific but simple
AMH and AFC
We can tailor OS according to
patients characteristics
Using markers
Using better drugs (FbM)
Dose reduction (PCOS)
Antagonist protocol
LH supplementation
Esteves, 54

55. Thank you...