The presentation discusses the evolution of ovarian stimulation for assisted reproductive technology (ART). It begins with a historical perspective of gonadotropin development from urinary sources to recombinant products. It then examines primary factors affecting IVF success like age, ovarian reserve markers, and cause of infertility. The presentation concludes by exploring more individualized clinical strategies like tailoring gonadotropin doses, flexible GnRH antagonist protocols, and LH supplementation to improve IVF outcomes.

1. “Meet the Expert” - May 2012
The Evolution of Ovarian
Stimulation for ART
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. Central
Paradigm
Maximize Minimize
beneficial effects complications
of treatment and risks
High-quality Cycle cancellation,
oocyte yield OHSS, multiple
pregnancy
Esteves, 7 Fauser et al., 2008

8. Milestones in the development of gonadotrophins
2001 2008
1940 1962 Full recombinant First
First hCG 1993 2000
Purified u-hMG gonadotropin r-hLH+r-FSH
extracted from First highly purified First r-hLH
(Pergonal®) and u- portfolio available combined
human urine FSH-only product launched
hCG (Profasi®) (Pergoveris®)
launched (Luveris®)
become available
(Metrodin HP®)
1949 1980s 1995 2001 2002
First hMG extracted First FSH-only First r-hFSH First r-hCG First filled-by-mass
from urine pools product launched launched launched product launched
(Metrodin®) (GONAL-f®) (Ovidrel®/Ovitrelle) (GONAL-f® FbM)
Milestones in the development of r-hFSH
1980 1983 1985 1988 1992
α-subunit β-subunit β-FSH gene cloned and Human FSH expressed First pregnancy
sequenced sequenced expressed in fibroblasts in Chinese hamster ovary with r-hFSH
(CHO) cells
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.
Esteves, 8

9. 1. Demand increased

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

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

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 Molecular u-hMG HP
(5 batches)
r-hFSH
weight (follitropin
markers alfa)
Esteves, 12 Merck Serono data on file

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. 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, 14 Bassett et al. Reprod Biomed Online 2005;10:169–177.

15. Conventional FbM: Novel
Bioassay analitycal method
High
Protein content by
Rat ovary mass
weight variability
gain Minimal batch-to-
batch variability
(1.6%)1,2
Urinary gonadotropins
Follitropin beta Follitropin alfa
1. Bassett et al. Reprod Biomed Online 2005;10:169–177; 2. Driebergen et al.
Esteves, 15 Curr Med Res Opin 2003;19:41–46.

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

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

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

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

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. Chronological vs Biological Ageing
20
FSH IU/L
<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, 22 Akande et al. Hum Reprod 2002;17:2003–2008.

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

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

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

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

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.
Esteves, 27

28. Reproductive Biology and Endocrinology 2009; 7:111.
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)
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, 28 Day 2-5 of menses

29. 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

30. 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* hMG was
3,000
required
0 compared with
r-hFSH HP-hMG hMG
r-hFSH
* Mean total dose per cycle/Live birth rate (≤35 years)

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

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

33. 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)

34. • Incidence of
62% Infertility (WHO II)
• Infertile Patients with PCOS
67% (WHO II)
• Prevalence of Patients with PCOS
41% 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. CONSORT = CONsistency in r-hFSH
Starting dOses for Individualized
tReatmenT: ART results
Individualized dosing in Clinical pregnancy rates/cycle
increments of 37.5 IU of started
60%
Folitropin alfa 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)
Esteves, 35 Olivennes F, et al. The CONSORT study. Reprod Biomed Online. 2009;18:95–204.

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.
Esteves, 36

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

38. 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

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

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

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

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

43. 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, 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. 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.
Esteves, 45

46. OHSS: Three Levels of Protection
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.
Esteves, 46

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

48. Esteves, 48

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

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

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

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

53. 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 women with
less sensitive ovaries
Esteves, 53

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

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

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

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

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.
Esteves, 58

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. Up to 65% of couples dropout from
IVF without achieving pregnancy
before they complete 3 cycles
Reasons
Psychological burden 49%-26%
Oocyte retrieval 52%
Prognosis 40%-23%
Embryo transfer 29%
Cost of treatment 23%-0% Injections 29%
Relationship/divorce 15%-9% Physical pain 20%
Physical burden 7-6% 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.

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