1. The Evolution of Ovarian Stimulation for ART
“Meet the Expert” - May 2012
Sandro Esteves, MD, PhD
Director, ANDROFERT
Center for Male Reproduction
Campinas, BRAZIL

2. 1.
Historical perspective of gonadotropins development.
2.
Primary factors affecting IVF success and ovarian response to stimulation.
3.
Taking advantage of new products and clinical strategies to individualize COS.
Esteves, 2

3. www.slideshare.net/sandroesteves

4. UN Census Estimates, 2008

7. High-quality oocyte yield
Cycle cancellation, OHSS, multiple pregnancy
Central Paradigm
Minimize complications and risks
Maximize beneficial effects of treatment
Fauser et al., 2008
Esteves, 7

8. Bassett et al. Reprod Biomed Online 2005;10:169–177; Lunenfeld. Hum Reprod Update 2004;10:453–467. Bosch. Expert Opin. Biol. Ther. 2010;10:1001-1009.
Milestones in the development of gonadotrophins
1949
First hMG extracted from urine pools
1940
First hCG extracted from human urine
1962
Purified u-hMG (Pergonal®) and u- hCG (Profasi®) become available
1980s
First FSH-only product launched (Metrodin®)
1993
First highly purified FSH-only product launched (Metrodin HP®)
2001
First r-hCG launched (Ovidrel®/Ovitrelle)
2001
Full recombinant gonadotropin portfolio available
Milestones in the development of r-hFSH
1980
α-subunit
sequenced
1983
β-subunit
sequenced
1985
β-FSH gene cloned and expressed in fibroblasts
1988
Human FSH expressed in Chinese hamster ovary (CHO) cells
1992
First pregnancy with r-hFSH
2000
First r-hLH launched (Luveris®)
1995
First r-hFSH launched
(GONAL-f®)
2002
First filled-by-mass product launched (GONAL-f® FbM)
2008
First
r-hLH+r-FSH combined (Pergoveris®)
Esteves, 8

9. 1. Demand increased

10. From urinary to recombinant
2. Safety - Impurities in Urinary-derived Drugs
FSH
Protein impurities
hMG HP
30% of impurities per vial with
(different proteins identified) varying from batch to batch
van de Weijer et al. Reprod Biomed Online 2003;7:547–557
Kuwabara Y et al, J Reprod Med 2009; 54:459–466
Impossibility to trace donor source
Quality cannot be checked during
transportation
Decontamination may denature proteins
Cross‐contamination cannot be avoided
Many of the protein contaminants are active and have unknown effects
Suboptimal testing for consistency and purity

11. Cell attachment and proliferation
r-hFSH production and secretion
Collection of cell culture supernatant medium containing r-hFSH
In-process QC
Esteves, 11
Concentration of supernatant
Chromatographic purification steps
Ultrasterile filtration
Characterization and full QC of bulk r-hFSH
Culture media
Harvest
Bioreactor

12. u-hMG HP (5 batches)
r-hFSH (follitropin alfa)
Merck Serono data on file
Molecular weight markers
Esteves, 12
2. Safety - Impurities in Urinary-derived Drugs
Impurities cannot be associated with a better or worse outcome but certainly are not needed for COH

13. Typical Cycle (long protocol):
Daily SC GnRH-a: x21
HMG/FSH: x10-15
hCG: x1
Progesterone: x14
Blood tests: x4-7
Number of sticks: 36-57

14. 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, 14

15. 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
Rat ovary weight gain
FbM: Novel analitycal method
Protein content by mass
Minimal batch-to- batch variability (1.6%)1,2
Urinary gonadotropins
Follitropin beta
Follitropin alfa
Esteves, 15

16. Esteves, 16
1930s
1950
1980
1995
2003
Horse
PMSG
Pituitary FSH
u-hMG
u-FSH
u-FSH HP
r-hFSH
r-hFSH FbM
Intramuscular administration
sc
Injector pens
sc, subcutaneous; FbM, filled by Mass; HP, highly-purified
Safety, Quality, Consistency and Patient Convenience

18. For clinicians:
Manufactured to the highest standards of quality and consistency;
Delivers a guaranteed dose.
For patients:
Best convenience;
Improve satisfaction & treatment compliance.
Advantages of Novel Products
Esteves, 18

19. 2.
Primary factors affecting IVF success and ovarian response to stimulation.
Esteves, 19

20. Esteves, 20
Negative Predictors
Positive Predictor
van Loendersloot et al. Hum Reprod Update 2010; 16: 577–589.
Female Age
Duration of infertility
Basal FSH
Type of infertility
Indication
Fertilization method
Number of oocytes retrieved
Number of embryos transferred Embryo quality
All reflecting ovarian reserve

21. Ovarian Response to Gonadotropin Stimulation
Demographics and anthropometrics (Age, BMI, Race)
Genetics profile
Cause of Infertility
Years of Infertility
Health status
Nutritional status

22. 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
Live births (%)
Age (years)
6–8.9
3–5.9
<3
FSH IU/L
≥12
9–11.9
Chronological vs Biological Ageing
Esteves, 22

23. La Marca et al. Hum Reprod 2009.
= remaining population of primordial and resting follicles
Esteves, 23
Anti-Mullerian Hormone levels are correlated with the number of follicles at gonadotropin independent stage.

24. Antral Follicle Count (AFC)
Devroey et al. Hum Reprod Update 2009; Broekmans et al. Fertil Steril 2009.
Hansen KR, et al. Fertil Steril 2003;80:577–83
Number of antral follicles
Mean number of oocytes retreived
r=0.64
p<0.001
0
5
10
15
20
25
25
20
15
10
5
0
Esteves, 24
Number of antral follicles present in the ovaries at a given time that can be stimulated into dominant follicle growth by exogenous gonadotropins.

25. AMH = AFC >Inhibin B >FSH >Age
Esteves, 25
Excessive Response Predictor
Poor Response Predictor
Broer et al. Fertil Steril, 2009; Broer et al. Hum Reprod Update 2011.

26. *Bologna criteria: Ferraretti et al. Hum Reprod 2011; Broer et al. Hum Reprod Update 2011; Nelson et al. Hum Reprod. 2009;
Broer et al. Fertil Steril. 2009; Hendricks et al. Fertil Steril 2007.
AMH and AFC – Operational Purposes
Esteves, 26
Response to Ovarian Stimulation
Anti- Mullerian Hormone
(ng/mL)
Antral Follicle Count
False Positive Rate
Risk of Excessive Response (≥15 oocytes or OHSS)
≥ 3.5
> 15
~15%
Risk of Poor Response
(≤ 4 oocytes)*
< 1.1
< 5

27. Esteves, 27

Tailoring gonadotropin dose using recombinant FSH fbM pre-filled ready-to- use pen devices.

Exploring the flexibility of GnRH antagonist protocols.

Improving success in IVF by identifying the subgroups of patients who benefit from LH supplementation.

28. Unselected group of NG down-regulated women (n=865)
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, 28
Reproductive Biology and Endocrinology 2009; 7:111.

29. 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
Esteves et al, Reprod Biol Endocrinol. 2009; 7:111

30. To achieve a live birth, 21-52% more HP-hMG and hMG was required compared with r-hFSH
Total Dose per Live Birth (IU)*
0
3,000
7,000
10,000
21.6%
r-hFSH
HP-hMG
6,324*
7,739
hMG
9,690
52.2%
* Mean total dose per cycle/Live birth rate (≤35 years)

31. 18.7
20.3
53.4*
% Cycles with “Step-down” during ovarian stimulation
HMG
HP-HMG
rec-hFSH (fbm)
*P<0.01

32. Evidence-based truth:
Rec-hFSH is more potent
↑ 3.1 oocytes
(Bosch, 2008)
↑ 1.8 oocytes
(MERIT, 2006)
↑ 2.8 oocytes
(Hompes, 2007)
Scientific truth:
Rec-hFSH is purer
Non urine- extracted product
Recombinant technology
Esteves, 32

33. 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)
Batch variability
± 2%
Risk of OHSS
Poor response

34. 62%
•Incidence of Infertility (WHO II)
67%
•Infertile Patients with PCOS (WHO II)
41%
•Prevalence of Patients with PCOS in Clinical Practice
Treatment Management of Infertility GCC Countries (IPSOS May 2008) Yeko et al. Fertil Steril 2004; Keck et al. RBM Online 2005.

35. 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;18:95–204.
Individualized dosing in increments of 37.5 IU of Folitropin alfa 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)
Esteves, 35
CONSORT = CONsistency in r-hFSH Starting dOses for Individualized tReatmenT: ART results

36. Esteves, 36
1.
Rec-hFSH fbM is purer, safer and more potent than urinary gonadotropins.
2.
Lower starting doses and step- up/step-down (by 37.5 UI) COS is advisable.

37. 
Exploring the flexibility of GnRH antagonist protocols.
Esteves, 37

38. 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
3
2

39. Agonist administration
Gonadotropin administration
Long GnRH agonist protocol
Antagonist administration
Gonadotropin administration
GnRH antagonist protocol
Longer
treatment
Can exclude early
pregnancy
Can be integrated in spontaneous
and OI cycles
Pre-treatment cycle
Treatment cycle
Flare up
effect
Pituitary
suppression
No hormonal
withdrawal
No flare
effect with possible cyst formation
Shorter duration of stimulation
Prevent OHSS by GnRH-a

40. Esteves, 40
N studies
45
22
Included IUI cycles
Yes
No
N patients
7511
3176
Primary outcome
OPR or LBR
LBR
Odds-ratio
0.86
(95% CI: 0.69-1.08)
0.86
(95% CI: 0.72-1.02)
1. Al-Inany et al. Cochrane Database Syst Rev. 2011; 5:CD001750.
2. Kolibianakis et al. Hum Reprod Update. 2006;12:651.
Probability of Live Birth

41. Esteves, 41
Duration of OS
-1.13 days
(-1.83; -0.44)
-1.54 days
(-2.42; -0.66; p=.0006)
Oocytes retrieved
--
-1.19 (-1.82; -0.56)
Risk of severe OHSS
0.43*
(95% CI 0.33-0.57)
0.61
(0.42; 0.89; p=.01)
1. Al-Inany et al. Cochrane Database Syst Rev. 2011; 5:CD001750.
2. Kolibianakis et al. Hum Reprod Update. 2006;12:651.
Duration of OS and Risk of OHSS

42. Esteves, 42
POOR RESPONDERS
14 RCT (1127 patients); Pu et al. 2011
Duration of stimulation
Oocytes retrieved
Cycle cancellation
CPR
-1.9 days
(-3.6; -0.12)
-0.17
(-2.42; -0.66)
1.01
(0.71; 1.42)
1.23
(0.92, 1.66)
Lainas et al. Hum Reprod. 2010;25:683; Pu D et al. Hum Reprod. 2011; 26: 2742.
PCOS
RCT; 220 patients; Lainas et al. 2010
Days of stimulation
Oocytes retrieved; N
Grades II + III OHSS (%)
CPR (%)
10 vs 12 (P<.001)
27 vs 28
(P=0.22)
44 vs 65
(P=0.006)
50.9 vs 47.3
(P=0.68)

43. 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, 43

44. 
GnRH-a triggering (0.2-1.5 mg): antagonist protocol;

Reduced if not eliminated risk for OHSS;

In specific high risk patients for OHSS and egg donation programs should become the choice

Challenge is to rescue luteal phase insufficiency;

Modified luteal support improved delivery rate: hCG bolus OPU day (1,500 UI) or 3x 500 UI boluses; recLH; intense progesterone + estradiol; combined Delivery rates: 18% risk difference favoring hCG (before) X 6% risk (after modified luteal support)
Esteves, 44
Humaidan et al. Hum Reprod Update 2011.

45. Esteves, 45
1.
GnRH antagonists improve COS flexibility.
2.
Duration of stimulation is decreased by 1-2 days (gonadotropin total dose by 10-20%).
3.
Use of GnRH antagonists in COS reduces (or eliminate) the risk of severe OHSS.

46. Esteves, 46
1st Level: Antagonist rather than Agonists.
2nd Level: In patients on antagonist protocol at risk of OHSS, replace hCG with GnRH-a for oocyte maturation trigger.
3rd Level: In patients with early OHSS onset, use of GnRH-ant luteal phase.
OHSS: Three Levels of Protection

47. Esteves, 47
1999
2009
15%
60%
Cycles with GnRH Antagonists
9%
39%
52%
r-hFSH
r-hFSH+hMG
hMG
Data supplied by REDLARA and ICMART
2009

48. Esteves, 48

49. Esteves, 49
•
Mild Stimulation (low dose rec-hFSH + GnRH ant.):
•
5 oocytes retrieved;
•
IR = 31%
•
Conventional Stimulation :
•
10 oocytes retrieved;
•
IR = 29%
Verberg et al. Hum Reprod Update 2009; 15: 5–12.
Promotion of Steroidogenesis (TCs) early FP
•Adequate estrogen production
•Uterine/endometrial changes
Stimulation of final Follicular Maturation (GCs) late FP
Alviggi et al. Reprod Biomed Online 2006;12:221.

50. Esteves, 50
•
Mild Stimulation (low dose rec-hFSH + GnRH ant.):
•
5 oocytes retrieved;
•
IR = 31%
•
Conventional Stimulation :
•
10 oocytes retrieved;
•
IR = 29%
Verberg et al. Hum Reprod Update 2009; 15: 5–12.
Balasch J, Fábreques F. Curr Opin Obstet Gynecol 2002, 14:265.
THRESHOLD
CEILING
•Suppression of GC proliferation
•Follicular atresia (non-dominant follicles)
•Premature luteinization
•Oocyte development compromised
High
•Normal androgen and estrogen biosynthesis
•Normal follicular growth and development
•Normal oocyte maturation
Normal
•Insufficient androgen (and estrogen) synthesis
•Follicular growth and maturation impaired
•Inadequate endometrial proliferation
Low

51. Esteves, 51
•
Mild Stimulation (low dose rec-hFSH + GnRH ant.):
•
5 oocytes retrieved;
•
IR = 31%
•
Conventional Stimulation :
•
10 oocytes retrieved;
•
IR = 29%
Verberg et al.
Hum Reprod Update 2009; 15: 5–12.
•~80% normogonadotropic women undergoing ART1-3
Normal
•15-20% of NG women have less sensitive ovaries
•Older patients (≥35 years)4
•Poor responders5
•Slow/Hypo-responders6
•Deeply suppressed endogenous LH (endometriosis)7
Low
1. Alviggi et al. Reprod Biomed Online 2006;12:221; 2. Tarlatzis et al. Hum Reprod 2006;21:90;
3. Esteves et al. Reprod Biol Endocrinol 2009;7:111; 4. Marrs et al. Reprod Biomed Online 2004;8:175
5. Mochtar MH, Cochrane Database, 2007; 6. Alviggi, et al. RBMOnline 2009.
7. De Placido et al. Clin Endocrinol (Oxf) 2004;60:637;
7

52. Esteves, 52
Women with Less Sensitive Ovaries
Poor Responders* At least 2 of the following: Advanced maternal age (≥40 years) Previous POR (≤3 oocytes with a conventional stimulation protocol) Abnormal ovarian reserve test (AFC<5; AMH <1.1) Or: 2 episodes of POR after maximal stimulation
Hypo/Slow Responders Normal markers of ovarian reserve; Hypo-responders: d1-d7: normal initial follicullar recruitment using fixed starting dose of FSH; d7- d10: plateau on follicullar growth despite continuing same FSH dosage Slow responders: High doses of FSH (>3,000UI) to promote follicular growth; May indicate genetic polymorphisms of LH and/or FSH receptor
*Bologna criteria: Ferraretti et al. Hum Reprod 2011; Alviggi, et al. RBM Online 2009; De Placido et al. Hum Reprod. 2004; 20: 390-6; Ferraretti et al. Fertil Steril. 2004; 82: 1521-6.

53. 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 women with less sensitive ovaries
Esteves, 53

54. Hill MJ et al. Fertil Steril 2012; 97: 1108-4.
Comparison of Clinical Pregnancy Rates
Rec-hLH for older women (≥35 years)
Esteves, 54

55. Rec-hLH for Poor Responders
Cochrane review 2007:
Poor-responders using r-hFSH vs r-hLH + r-hFSH (Ongoing PR)
Esteves, 55

56. Esteves, 56
Deeply Suppressed Endogenous LH
6
9
11
10
14
18
22
32
40
FSH step-up (+150 UI)
LH supplementation
(+150 UI)
Normal Responders
Mean No. oocytes retrieved
IR (%)
OPR (%)
De Placido et al. Hum Reprod. 2004; 20: 390-6.
RCT 260 pts; “Steady” response on D8 (E2 <180pg/mL; >6 follicles <10mm)

57. Esteves, 57
8
11
11
14
37
35
22
41
37
rec-hFSH step-up
rec-hLH
supplementation
Control
Mean No. oocytes retrieved
IR (%)
LBR (%)
Ferraretti et al. Fertil Steril. 2004; 82: 1521-6.
RCT 180 pts; follicular stagnation d7-d10
LH for Slow/Hypo Responders

58. Esteves, 58
1.
LH supplementation to COS increase IVF success in patients subgroups:
i.
Advanced female age (≥35 years)
ii.
Poor responders
iii.
Slow/Hypo-responders
iv.
Profound LH suppression after down- regulation (endometriosis pts.)
2.
75-150 UI/day is sufficient.
3.
Take advantage of rec-hLH fbM.

59. The Evolution of Ovarian Stimulation for ART
1. Using markers of ovarian reserve (AMH; AFC)
2. Using better drugs (rec-gonadotropins FbM)
3. Mild stimulation (PCOS)
4. Flexibility of antagonist protocols
5. LH supplementation
6. Integrate strategies to maximize beneficial effects of treatment and minimize risks and complications.
Esteves, 59

60. 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 cycles
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.
Reasons

61. Color-coded for differentiation
The New Family of PensTM:
•
same injection device design for all gonadotropins;
•
first & only pre-filled, ready-to-use family of pens for fertility treatment.
Esteves, 61

62. Consider a change...
Esteves, 62