1. Middle East Lecture Tour, 2012
Use of LH in IVF and IUI
Differences between rec-hLH and LH
Activity in HMG Preparations
Sandro Esteves, MD, PhD
Director, ANDROFERT
Center for Male Reproduction
Campinas, BRAZIL

2. What is in it for me?
Role of LH in Reproductive Cycles
 LH window concept
To Whom to Give LH Supplementation
Recent Advances in Injectable
Gonadotropin Preparations
 Rec-LH Products
 Differences between rec-hLH and LH
Activity in HMG Preparations
Esteves, 2

3. Level of
evidence
Individualization of Patient Treatment
Lecture Structure
 Points I Consider Highly Relevant in Clinical Practice;
 Arguments Supported by Studies with High Level of Evidence.
Level Type of evidence
1a Obtained from meta-analysis of randomised trials
1b Obtained from at least one randomised trial
2a Obtained from one well-designed controlled study without
randomisation
2b Obtained from at least one other type of well-designed quasi-
experimental study
3 Obtained from well-designed non-experimental studies
(comparative and correlation studies, case series)
4 Obtained from expert committee reports or opinions or clinical
experience of respected authorities
Esteves, 3 Modified from Sackett et al. Oxford Centre for EBM Levels of Evidence (2009)

4. Use of LH in IVF and IUI
Differences between rec-hLH and LH
Activity in HMG Preparations
Review this Lecture at:
http://www.androfert.com.br/review
Esteves, 4

5. What is in it for me?
Role of LH in Reproductive Cycles
2 To Whom to Give LH Supplementation
3
Recent Advances in Injectable
Gonadotropin Preparations
 Rec-LH Products
 Differences between rec-hLH and LH
Activity in HMG Preparations
Esteves, 5

6. • 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, 6
Esteves, 6 Alviggi et al.Hum Reprod Update 2009; 15: 5–12.
Reprod Biomed Online 2006;12:221.

7. Hypogonadotropic Hypogonadism
Treated with FSH Alone
15 Follicles
Follicle Size
(mm) 10 Estradiol
and FSH Serum FSH (pg/ml)
(IU/L)
5
Estradiol levels 100
50
0
0 5 10 15 20
Days of Stimulation
Endometrial 9
Thickness
(mm) 0
r-hFSH
Esteves, 7

8. Evidence for LH threshold (1)
Rec-hLH suppementation (UI): 0 25 75 225
3000
Serum Estradiol Levels
2500 225
2000
(pmol/L)
1500 75
1000
500
25
0 0
Day 1 Day 5 Day 10 hCG
Day of Stimulation
Esteves, 8 The European Recombinant Human LH Study Group, JCEM 1998; 83:1507

9. Evidence for LH threshold (2)
0 25 75 225 rLH
Endometrial Thickeness (mm) 75
8 Injected rLH LH Cmax
225
dose (UI)
6 75 UI 0.5 – 1.35 UI/L
4
25
2 0
0
Day 1 Day 5 Day 10 hCG
Day of Stimulation
Esteves, 9 The European Recombinant Human LH Study Group, JCEM 1998; 83:1507

10. • 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, 10
Esteves, Balasch J, Fábreques F. Curr Opin Obstet Gynecol 2002, 14:265.
Hum Reprod Update 2009; 15: 5–12.

11. Both FSH and LH are essential for normal
estradiol biosynthesis.
75 UI recLH is sufficient to promote optimal
follicular and endometrial growth as well as
androgen production in most HH patients.
Evidence suggests that in reproductive cycles
optimal follicular development occurs within an
‘LH window’, above a certain ‘LH threshold’ and
below an ‘LH ceiling’ (1.2 to ? UI/L).
Esteves, 11

12. What is in it for me?
Role of LH in Reproductive Cycles
To Whom to Give LH Supplementation
Recent Advances in Injectable
Gonadotropin Preparations
 Rec-LH Products
 Differences between rec-hLH and LH
Activity in HMG Preparations
Esteves, 12

13. Central
Paradigm
Maximize Minimize
beneficial effects complications
of treatment and risks
High-quality Cycle cancellation,
oocyte yield OHSS, multiple
pregnancy
Fauser BC et al: Predictors of ovarian response: progress towards individualized treatment in ovulation
Esteves, 13 induction and ovarian stimulation. Hum Reprod Update 2008;14:1-14.

14. Factors Determining Response
to Ovarian Stimulation
 Demographics and
anthropometrics (Age,
BMI, Race)
 Genetic profile
 Cause of Infertility
 Years of Infertility
 Health status
 Nutritional status
Esteves, 14

15. Level
1a
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
Esteves, 15 van Loendersloot et al. Hum Reprod Update 2010; 16: 577–589.

16. Normal
• ~80% normogonadotropic women undergoing
Ovarian Stimulation1-3
• 15-20% of NG women have less sensitive
ovaries
• Older patients (≥35 years)4
Low
• Poor responders5
• Slow/Hypo-responders6
• Deeply suppressed endogenous LH levels
(endometriosis)7
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.
Esteves, 16 RBMOnline 2009; 7. De Placido et al. Clin Endocrinol (Oxf) 2004;60:637;

17. Up to 45%
Infertility
Patients
• Older patients (≥35 years) aged 35 or
Less Sensitive Ovaries
• Poor responders above
• Slow/Hypo-responders
• Deeply suppressed endogenous LH (endometriosis)
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-
Abnormal ovarian reserve test (AFC<5; d10: plateau on follicullar growth
AMH <1.1) despite continuing same FSH dosage
Or: Slow responders:
2 episodes of POR after maximal High doses of FSH (>3,000UI) to promote
stimulation follicular growth;
May indicate genetic polymorphisms
of LH and/or FSH receptor
Marrs et al. Reprod Biomed Online 2004;8:175
De Placido et al. Clin Endocrinol (Oxf) 2004;60:637; Ferraretti et al. Fertil Steril. 2004; 82:1521-6;
Esteves, 17 Mochtar MH, Cochrane Database, 2007; Alviggi, et al. RBMOnline 2012

18. Theca cells
Increase in LH
LH drive
LH
Granulosa
Increase in cells
FSH drive FSH
Increasing the Number % Cycle Pregnancy
Level Stimulation Dose of oocytes cancellation rates
1b FSH… retrieved
Manzi et al, 1994 …is not associated with better
Klinkert et al, 2004 IVF outcome
Berkkanoglu & Ozgur,
2010
Esteves, 18

19. Reduced oocyte quality
Less Sensitive Ovaries
Reduced Fertilization Rate
Reduced Embryo Quality
Increase Miscarriage Rates
Westergaard et al., 2000; Esposito et
al., 2001; Humaidan et al., 2002
Reduced Androgen Decreased Reduced
ovarian LH receptor LH
secretory numbers of
paracrine poly- bioactivity
capacity functional
activity morphisms while
reduced LH
receptors imnuno-
• Piltonen et al.,
reactivity
Hurwitz & Alviggi et al., unchanged
Santoro 2004 2006 2003
• Vihko et al. 1996
• Mitchell et al.
1995; Marama et
al 1984
Esteves, 19

20. Level LH Supplementation in Poor
1a Responders…
Effect on
Regimen Outcome
Pregnancy
Mochtar et al, 2007
r-hFSH+rLH vs. OR 1.85
3 RCT (N=310) OPR
r-hFSH alone* (95% CI: 1.10; 3.11)
Poor responders
CPR RD: +6%,
Bosdou et al, 2012 r-hFSH+rLH vs. (95% CI: -0.3; +13.0)
7 RCT (N= 603) r-hFSH alone*
Poor responders LBR RD: +19%
(only 1 RCT) (95% CI: +1.0; +36.0%)
Hill et al, 2012
r-hFSH+rLH vs.
7 RCT (N=902) OR 1.37
r-hFSH alone CPR
Women advanced (95% CI: 1.03; 1.83)
age ≥35 yrs.
*long GnRH-a protocol; OR=odds-ratio; RD=risk difference
Mochtar MH et al. Cochrane Database Syst Rev. 2007;2:CD005070; Bosdou JK et al,
Esteves, 20 Hum Reprod Update 2012; 8(2):127-45. Hill MJ et al. Fertil Steril 2012; 97:1108-4.

21. Action of LH at the follicular level that increases
androgen production for its later aromatization to
estrogens in a dose dependent manner may
restore the follicular milieu in these patients to
recover oocyte quality and, therefore, embryo
quality and implantation rates.
Jamnongjit M et al. PNAS 2005;102:16257-16262

22. Level LH Supplementation in
1b
Hypo/Slow Responders (1)
• RCT 260 pts with “steady” response on COS D8
(E2 <180pg/mL; >6 follicles <10mm)
• 3 groups:
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, 22 De Placido et al. Hum Reprod. 2004; 20: 390-6.

23. Level
1b LH Supplementation in
Hypo/Slow Responders (2)
• RCT
• 126 pts. follicular stagnation during d7-d10 COS
• 4 groups:
Mean No. oocytes retrieved LBR (%)
41 37
22 18
8 11 11 10
increase in r- increase in r- increase in r- controls
hFSH dose hFSH dose + r- hFSH dose + LH
(max. 450UI) hLH (75-150UI) supplementation
supplementation with HMG
Esteves, 23 Ferraretti et al. Fertil Steril. 2004; 82: 1521-6.

24. Level LH Supplementation in
1b OI and IUI
LH levels 1.2 UI/L (WHO group I)
Higher follicular development pts. receiving LH (67% vs 20%;
p=0.02): Shoham et al., 2008.
Similar follicular development HMG vs FSH+rLH; higher
cumulative PR after 3 cycles in FSH+LH (56% vs 23%; p=0.01):
Carone et al., 2012.
WHO group II
Clomiphene-resistant: fewer intermediate-sized follicles and OHSS in
LH-supl. vs FSH group; similar ovulation rate (Plateau, 2006);
Previous over-response: higher monofollicular development in LH group
(32% vs 13%; p=0.04): Hughes et al., 2005;
IUI: higher monofollicular development in LH group without
intermediate-size (42% vs 11%; p=0.03); lower cycle cancellation due
to risk OHSS (-7% difference): Segnella et al., 2011.
Esteves, 24

25. What is the optimal LH
supplementation protocol?
 Existing studies give us some clues but the
optimal LH protocol has yet to be established
 How much LH should be used?
 Should the dose be fixed or flexible?
 At what stage of the cycle should LH be
administered?
 Is LH needed in a GnRH antagonist Protocol?
FSH
LH
2:1? 1:1? Fixed? Mimic of
natural LH levels?
Esteves, 25

26. Level
Is LH needed in a GnRH
1a
antagonist Protocol?
Unselected women undergoing COS;
r-hFSH+r-hLH vs. r-hFSH alone in antagonist cycles
Mochtar et al. Kolibianakis et al. Baruffi et al.
3 RCT (N=216) 2 RCT (N=176) 5 RCT (N= 434)
Estradiol on WMD 571 - WMD 514
hCG day (pg/ml) (95% CI 259; 882) (95% CI 368; 660)
No. retrieved WMD 0.50 WMD 0.41
-
oocytes (95% CI -0.68; 1.68) (95% CI -0.44; 1.3)
†OR 0.79 *OR 0.86 †OR 0.89
CPR†/LBR*
(95% CI: 0.26; 2.43) (95% CI: 0.04; 1.85) (95% CI: 0.57; 1.39)
WMD weight mean difference
Mochtar MH et al. Cochrane Database Syst Rev. 2007;2:CD005070; Kolibianakis et al, Hum Reprod
Update. 2007;13:445-52; Baruffi RL et al, Reprod Biomed Online. 2007;14:14-25.
Esteves, 26

27. Level Is LH needed in a GnRH
1b antagonist Protocol?
RCT; 292 NG women aged 36-39; Fixed (D6) antagonist COH protocol
rFSH rFSH + rLH
P= 0.027
68%
61% OR=1.49
OR=1.56
95% CI 0.93-2.38
95% CI 1.04-2.33
33%
25% 27%
19%
%2PN Ongoing PR Implantation
Yes, for women aged >35 yo
Esteves, 27 Bosch et al. Fertil Steril. 2011; 95:1031-6.

28. Women with less sensitive ovaries (ovarian aging) have poorer
IVF outcomes.
Androgen secretory capacity decreases with ovarian ageing.
Mechanisms include decreased number of functional LH
receptors and ovarian paracrine activity resulting in reduced
LH bioactivity. LH-r polymorphisms possibly involved in hypo-
responders.
LH supplementation to COS is an evidence-based strategy to
maximize pregnancy results.
4 subgroups benefit of LH supplementation in COS:
 Poor responders
 Slow/hypo-responders
 Age >35 years
 Deeply suppressed endogenous LH levels
Esteves, 28

29. 3 subgroups clearly benefit of LH supplementation in
OI and IUI:
 WHO group I anovulation
 WHO group II clomiphene resistant
 WHO group II with previous over-response to OS
Other potential indications include:
Poor responders
Slow/hypo-responders
Age >35 years
Deeply suppressed endogenous LH levels
Esteves, 29

30. What is in it for me?
Role of LH in Reproductive Cycles
To Whom to Give LH Supplementation
Recent Advances in Injectable
Gonadotropin Preparations
 Rec-LH Products
 Differences between rec-hLH and LH
Activity in HMG Preparations
Esteves, 30

31. r-hFSH Long-
FbM r-hFSH acting
u-FSH HP +r-hLH r-hFSH;
Pituitary r-hFSH FbM
u-FSH
FSH r-hLH
u-hMG
Puriity
Horse and
Safety, Quality,
PMSG Specific
Consistency and Patient
Activity
Convenience
1930s 1950 1962 1980 1993 1995 2000 2003 2007 2010
Intramuscular administration sc Injector
pens
sc, subcutaneous; FbM, filled by Mass; HP, highly-purified
Esteves, 31
Adapted from Lunenfeld. Hum Reprod Update 2004;10:453–67

32. *
Launched in Germany, 2007
*Pergoveris™: rec-hFSH (150 UI) + rec-hLH (75 UI);
Bosch et al. Expert Opin Biol Ther 2010;10:1001-9.
Esteves, 32

33. • Same injection device
design for all
gonadotropins;
• Color-coded for
differentiation;
• Pre-filled, ready-to-
use family of pens for
fertility treatment.
Esteves, 33

34. Conventional FbM: Novel
Bioassay analitycal method
High
Protein content by
Rat ovary mass
weight variability
gain Minimal batch-to-
batch variability
(1.6%)
Urinary gonadotropins
Follitropin beta Follitropin alfa and rec-hLH
Bassett et al. Reprod Biomed Online 2005;10:169–177;
Esteves, 34 Driebergen et al. Curr Med Res Opin 2003;19:41–46.

35. Alfa Unit Beta unit Carboxyl terminal
(biological action segment
and receptor (determines half-life)
affinity)
LH 92 AA; 121 AA Absent; half life of 20’
hCG Identical to LH 144 AA Present; half-life of
Higher receptor affinity 24h
Purity FSH LH activity
(LH content) activity (IU/vial) (IU/vial)
Rec-hLH >99% 0 75
Rec-hLH + rec-hFSH >99% 150 75
hMG-HP Unknown* 75 75*
*derives primarily from the hCG component, which preferentially is
concentrated during the purification process and sometimes was added
to achieve the desired amount of LH-like biological activity.
Esteves, 35 ASRM Practice Committee. Fertil Steril. 2008; 90:S13-20.

36. Level
2a
• Matched case-control study;
• N=4,719 pts.; long GnRH-a protocol
• 3 groups
35
30 P=0.02 Duration of
31 Stimulation (days)
25
26 25 Mean No. oocytes
20 retrieved
15
IR (%)
10
5 CPR per transfer
(%)
0
2:1 r-hFSH+r- HMG rec-hFSH +
hLH HMG
Esteves, 36 Buhler KF, Fisher R. Gynecol Endocrinol 2011; 1-6.

37. Level
1a
Lower expression of LH/hCG receptor gene as well
as genes involved in in biosynthesis of cholesterol
and steroids in granulosa cells in pts. treated with
HMG preparations
May reflect down-regulation of LH receptors, as shown in animals:
Caused by a constant ligand exposure during the follicular
phase due to longer half life and higher binding affinity of
hCG to LHr
May explain the observed lower progesterone levels:
Caused by lower LH-induced cholesterol uptake, a decrease in
the novo cholesterol synthesis and a decrease in steroid
synthesis.
Trinchard-Lugan I et al. Reprod Biomed Online 2002; 4:106-115; Menon KM et al. Biol Reprod
Esteves, 37
2004; 70:861-866; Grondal ML et al. Fertil Steril 2009; 91: 1820-1830.

38. Recombinants vs Urinary products
Recombinant LH preparations have 3 major
differences compared to urinary products:
 Higher purity and specific activity (SC delivery in
very small volumes))
 Higher dose precision (FbM)
 LH activity in u-HMG is hCG dependent:
hCG is concentrated during purification or added to
achieve the desired amount of LH-like biological activity;
hCG has higher half-life and biological activity compared to
rec-hLH.
Esteves, 38

39. Differences between rec-hLH and LH
Activity in HMG Preparations
 Lower expression of LH receptor gene
in pts. treated with HMG (LH-r down-
regulation).
 Preparations used for COS are
important for granulosa cell function and
may influence the developmental
competence of the oocyte and the
function of corpus luteum.
Esteves, 39

41. Use of LH in IVF and IUI
Progesterone Issues
Supplementary Material
Esteves, 41

42. Steroidogenesis During Normal
Follicular Phase and Following COS
The expression of LH-R (GCs) is linked to the synthesis of
progesterone during COS.
Levels of LH-R and progesterone synthesis vary depending on
the hormones used during the stimulation protocol.
Esteves, 42 Steroidogenesis Consensus Meeting III, Copenhagen, Denmark, May 2011.

43. Steroidogenesis in COS
Endogenous LH then results in higher levels of progesterone
synthesis following treatment with FSH than hMG:
higher levels of LHR expressed on granulosa cells and
increased number of granulosa cells.
Esteves, 43 Steroidogenesis Consensus Meeting III, Copenhagen, Denmark, May 2011.

44. Level
1a Progesterone on the Day of hCG
and Probability of Pregnancy in IVF
Progesterone Elevation x No Progesterone Elevation
Venetis et al, 2007 Kolibianakis et al, 2012
GnRH Agonists Antagonists Antagonists
analogue n = 2,624 n = 109 n = 109
OR: 0.86 OR: 0.57 WMD: -9%
CPR (95% CI: 0.59; 1.25) (95% CI: 0.09; 3.56) (95% CI: -17; -2)
E2 levels on
WMD: 413.06 WMD: 956
the day of (95% CI: 240.14; 585.99) (95% CI 248; 1664)
hCG (pg/mL)
Number of
WMD: +2.96 WMD: 0.00 WMD: +2.9
retrieved (95% CI: +1.74; +4.18) (95% CI: -2.98; +2.99) (95% CI: +1.5; +4.4)
oocytes
heterogeneity of the studies included;
arbitrary serum progesterone threshold values
Venetis et al, Hum Reprod Update. 2007;13:343-55;
Esteves, 44 Kolibianakis et al, Curr Pharm Biotechnol. 2012;13:464-70.

45. Level
2b Progesterone on the Day of hCG
and Probability of Pregnancy in IVF
Bosch et al. 2010 (N=4,032)
OPR: inversely associated with serum P levels on the day of hCG irrespective
of the GnRH analogue: CUT-OFF: 1.5 ng/mL
Serum P levels ≤1.5 ng/ml: ↑OPR
31% versus 19.1%; P = 0.00006;
OR: 0.53 (95% CI, 0.38 – 0.72)
positively
FSH dose
associated
No. oocytes with P levels
Estradiol (day of hCG) (P < 0.0001 for
all).
Serum P levels:
agonists: 0.84 ± 0.67 vs antagonists: 0.75 ± 0.66 (P = 0.0003)
Esteves, 45 Bosch et al. Hum Reprod. 2010; 25(8):2092-100.

46. Level
Progesterone on the Day of hCG
2b
and Probability of Pregnancy in IVF
Xu et al, 2012 (N=11,055 long agonist protocol)
For fresh cycles, OPR inversely associated with serum P levels on hCG day
FSH dose
Positively
No. oocytes associated
with P levels ■ Fresh
Estradiol (day of hCG)
■ FET
Serum P
Ovarian Number of
threshold
response oocytes
(ng/mL)
Poor ≤4 1.5
Intermediate 5-19 1.75
High ≥20 2.25
Xu et al. Fertil Steril 2012;97:1321–7.
Esteves, 46

47. Progesterone on the Day of hCG
and Probability of Pregnancy in IVF
The rise in progesterone levels seen during COS for
IVF/ICSI cycles cannot be explained by luteinization of
granulosa cells
 Conversion
 FSH activity Granulosa of cholesterol to
cell progesterone
 LH  Conversion
Teca
bioactivity of progesterone
cell
to androgens
 FSH dose,  number of follicles and rec-hFSH (x HMG):
correlation to P increase on hCG day
Bosch et al. Hum Reprod. 2010;25:2092-100;
Esteves, 47 Xu et al. Fertil Steril 2012;97:1321–7; Smitz J et al. Hum Reprod 2007;22:676–87.

48. LevelsProgesterone on the Day of hCG
2b, 3 and Probability of Pregnancy in IVF
Hofmann et al, Fertil Steril. 1993;60:675-9; Xu et al. Fertil Steril 2012;97:1321–7; Huang et al.
Fertil Steril 2012; 98:664–70; Melo et al. Hum Reprod 2006; 21:1503–1507.
Esteves, 48

49. Serum Progesterone and IVF Outcome
Most circulating P4 (95%) is produced in the intrafollicular
compartment by the granulosa cells;
Intrafollicular P4 and Hydroxi-progesterone are terminal
products and cannot be converted to estradiol by GCs
under the effect of LH/hCG activity contained in hMG, due to
lack of expression of an hydrogenase and P450-17α needed
for this pathway;
Higher serum Progesterone increments are related with
more follicles developed and more oocytes retrieved and it’s
effect in pregnancy still controversial. Increments up to
>7nmol/L seems not to affect clinical pregnancy rates.

50. Serum Progesterone and IVF Outcome
 Treatment with FSH results in higher levels of
progesterone than treatment with hMG.
 A large number of developing follicles leads to
increased levels of progesterone.
 The higher the level of LH present, the higher the
level of progesterone.
 The effect of high progesterone levels at the time
of hCG administration on pregnancy outcome is
still controversial. Further detailed analyses are
required to understand why, when and how much
progesterone is detrimental for implantation rates.
Esteves, 50