“Understanding Infertility”: A CME Course in Reproductive Medicine August 12-14th, 2011 New Delhi, India

1. Improving Success in Ovulation
Induction by Treatment
Individualization

2. Learning Objectives

3. UN Census Estimates, 2008

7. Ovulation Induction
for ART
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. Ovulation Induction
One size fits all?
Patient is the main
variable of OI response
 Demographics and
anthropometrics (Age,
BMI, Race)
 Genetics profile
 Cause of Infertility
 Years of Infertility
 Health status
 Nutritional status

9. What we really want to know in OI is...
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
Esteves, 9

10. 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, 10

11. Learning Objectives
Gonadotropins:
better now

12. Markers of Ovarian Response
Can we predict ovarian response?
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
Esteves, 12

13. Who has the highest chance of a live
birth following IVF?
Hana Maria
Age 26 Age 37
Basal FSH 9 Basal FSH 5
Esteves, 13

14. Age and FSH
chronological vs biological in IVF
20
FSH IU/L
Hana Maria
<3
15
Live births (%)
3–5.9
6–8.9
10
9–11.9
5
≥12
(n = 1019)
0
20–24 25–29 30–34 35–39 40–44 45–49
Age (years)
Esteves, 14 1. Akande et al. Hum Reprod 2002;17:2003–2008

15. Why do ovaries age at different rates?
Multifactorial, but genetics important
FSH-R: Ser680 genotype
Single nucleotide polymorphisms Human FSH Receptor Mutations
(SNPs) linked to: - NH2
● Ovarian response to gonadotrophins Ile160Thr Ala189Val (Asn191Ile)
● Premature menopause
Asp224Val
*
Pro346Arg
Thr307Ala
Val341Ala Pro519Thr
*
Both activating and inactivating
Leu 601Val
mutations identified in the LH and Arg573Cys
FSH receptor genes1
Ala419Thr
Asp567Gly??
*
- COOH
* Ser680Asn
Esteves, 15 1. Themmen and Huhtaniemi. Endocr Rev 2000;21:551–583

16. Markers of Ovarian Response
Biomarkers and follicular development
AMH levels are
correlated with
the number of
follicles at
gonadotropin
independent
stage
La Marca, et al. Hum Reprod 2009.
Esteves, 16

17. Markers of Ovarian Response
anti-Mullerian hormone (AMH)
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
AMH: a cut-off 1.26 ng/ml was able to predict
poor response (<4 oocytes) with 97% sensitivity
Esteves, 17 Gnoth, et al. Hum Reprod 2008.

18. Markers of Ovarian Response
Prediction of response by AMH
AMH category 0.14 to <0.7 0.7 to <2.1 >2.1
(ng/mL) (N=74) (N=128) (N=148)
Agonist protocol + 375 225 150
rFSH
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, 18 Verhagenet al. 2008; Broer et al., 2010

19. Markers of Ovarian Response
Antral Follicle Count (AFC)
No. of antral
Mean number of oocytes retreived
25 <3 4-10 > 10
follicles
20 No. of cycles 16 76 57
Mean age
15 36.8 36.3 32.8
(years)
r=0.64
10 p<0.001
Day 3 FSH
12.7 7.1 5.6
(IU/l)
5 Cx rate 68.8% 5.3% 0%
0 Peak E2
432 1,001 1,912
0 5 10 15 20 25 (pg/ml)
Mean No. of
Number of antral follicles 2.0 ± 0.9 6.3 ± 4.4 14.1 ± 8.5
eggs
Hansen KR, et al. Fertil Steril Pregnancy
0% 13.2% 26.3%
2003;80:577–83 rate
Esteves, 19
Chang, et al. Fertil Steril. 1998;69:505.

20. Markers of Ovarian Response
Prediction of response
AMH = AFC >Inhibin B >FSH >Age
Esteves, 20 Broer et al. , 2010

21. Markers of Ovarian Response
Summary
The patient individual
factors play a crucial
role in predicting ovarian
response.
AFC and AMH are helpful
to predict ovarian
response to stimulation.
Esteves, 21

22. Learning Objectives

23. Gonadotropins:
 Recombinant
FSH/LH/hCG
 Urinary
FSH/LH/hCG
GnRH
Analogues:
 Agonist
Other:  Antagonist
 Progesterone
 Estradiol
 Aromatase inibitor
 Contraceptive pill
 Antioxidants/vitamins
Esteves, 23

24. Gonadotropins: an overview
What is available?
Product Technology Brand name Manufacturer
hMG Urine-derived Menogon®; Repronex® Ferring
Merional® 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® Ferring
Choriomon® IBSA
r-hFSH (follitropin Recombinant Puregon®; Follistim® MSD
beta)
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

25. Gonadotropins: an overview
Urinary-derived products

26. Gonadotropins: an overview
Recombinants
Bioreactor Harvest
Culture media
Production Purification
Cell attachment and Concentration of
proliferation supernatant
r-hFSH production and Chromatographic
secretion purification
steps
Collection of cell
culture supernatant Ultrasterile filtration
medium containing
Characterization
r-hFSH
and full QC of
In-process QC bulk r-hFSH
Esteves, 26

27. Gonadotropins: an overview
Differences
Purity Mean specific Injected
(FSH FSH activity protein
content) (IU/mg 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, 27 Bassett et al. Reprod Biomed Online 2005;10:169–177

28. Gonadotropins: an overview
Product Quality: Filled by Mass (FbM)
Novel analitycal
Conventional method
Bioassay
Physiochemical
technique
High
in vivo (rat) variability Minimal batch-to-
(~20%) batch variability
(1.6%)1,2
1. Bassett et al. Reprod Biomed Online 2005;10:169–177
2. Driebergen et al. Curr Med Res Opin 2003;19:41–46
Esteves, 28

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

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

31. Group A (hMG; N=299)
Group B (HP-hMG; N=330)
Group C (r-hFSH; N=236)
Day
Day 1 Day 6 of hCG
Cycle
day 21 Gonadotropin rFSH/hMG
Individualized dose
112.5-450 UI Vaginal
progesterone
Agonist (nasal spray): Nafarelin acetate (400 mcg/day; fixed)
menses
Esteves, 31 Day 2-5 of menses

32. r-hFSH vs hMG/HP-hMG in ART
Esteves et al. (observational study 2009)
Outcome Measure HMG HP-hMG r-hFSH P-
n=299 N=330 n=236 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
Esteves et al, Reprod Biol Endocrinol. 2009; 7:111

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

34. r-hFSH vs hMG and HP-hMG in ART
Esteves et al. (observational study 2009)
Total Dose per Live Birth (IU)*
To achieve a
10.000 live birth,
52.2% 9,690
21-52% more
7.000 21.6% 7,739
HP-hMG and
6,324*
3.000 hMG was
required
0 compared
r-hFSH HP-hMG hMG
with r-hFSH
* Mean total dose per cycle/Live birth rate (≤35 years)
Esteves et al, Reprod Biol Endocrinol. 2009; 7:111

35. 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

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

37. LH surge prevention
GnRH antagonists
1 2 3
pyro (Glu) – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2
Activation of the
Antagonistic Regulation of Regulation of receptor
GnRH receptor
effect receptor affinity biological activity

38. LH surge prevention
GnRH antagonists
Agonist
Follicular
LH, FSH
30
E2 , P 4
Luteal
20
2-4 weeks
10 Synchronized follicles
0
6
• Half-life ~20h (Cetrorelix)
Antagonist 5 • Suppress LH by 80% of
4 baseline levels
LH (IU/L)
3 Antagonist
2
1
0
-6 0 6 12 18 24 30 36 42 48
Hours

39. 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)
Day 1 Follicle Day
of rFSH 13 mm of hCG
Follitropin alfa dose Individualized
112.5-450 UI rFSH dose Vaginal
menses 0.25 mg/day of progesterone
Day 2 or 3 Cetrorelix (flexible)
of menses
Day 6 Day
Day 1 of hCG
of rFSH of rFSH
Cycle Gonadotropin dose
day 21 Individualized dose
112.5-450 UI Vaginal
progesterone
Agonist: Nafarelin acetate (400 mcg/day; fixed)
menses
Day 2-5 of menses
Esteves, 39 Esteves et al., JBRA Assist Reprod (Suppl 1), 2010

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

41. GnRH antagonists vs agonists
Meta-analysis
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)*
*For every 59 women treated with a GnRH agonist vs GnRH
antagonist, one additional case of severe OHSS will occur.
Esteves, 41

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

43. Learning Objectives

44. Individualized Treatment with AMH
AMH + antagonists in hyper-responders
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%*
*P<0.01
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, 44

45. CONSORT = CONsistency in r-hFSH
Starting dOses for Individualized
tReatmenT
Individualized dosing in Clinical pregnancy rates/cycle
increments of 37.5 IU of started
60%
Gonal-f possible by FbM
technology 50%
50.0%
40%
Use of algorithm of
30% 35.3%
patients characteristics 31.3% 31.1%
● basal FSH 20%
● body mass index (BMI) 20.0%
● age
10%
● antral follicle count 0%
75 IU 112.5 IU 150 IU 187.5 IU 225 IU
Age (28-32)
Oocytes retrieved (8-12)
Olivennes F, et al. The CONSORT study. Reprod Biomed Online. 2009;18:195–204.
Esteves, 45

46. LH supplementation in ART
What do we know today
 The majority of patients do not need LH
supplementation as endogenous LH levels are
sufficient1–3
 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
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.
Esteves, 46

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

48. LH supplementation in ART
Biologic older (less sensitive) ovaries
LH • Theca cells
Consider
increasing LH
drive
LH • Granulosa
cells
Increasing FSH
drive of limited FSH
value
There is a potential role for r-hLH in this
population
Esteves, 48

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

50. From cookery to science – Practical Points
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
GnRHa LH triggering
LH supplementation
Esteves, 50

51. Thank you...