1. LH Forum I, Kingdom of Saudi Arabia, Jun 2013

3. Esteves, 3


4. Review this Lecture at:
http://www.androfert.com.br/review
Esteves, 4
Esteves SC – June 2013

5. Esteves, 5 Modified from Sackett et al. Oxford Centre for EBM Levels of Evidence (2009)
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
Grade A
recommendation
• Good and
consistent
scientific evidence
 Information concerning IVF population
 Evidence-based medicine

6. Esteves, 6
Role of LH in Reproductive
Cycles
2
3
Who Need LH?

7. LH
FSH
LH
Esteves, 7

8. Esteves, 8
• 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.Esteves, 8 Alviggi et al. Reprod Biomed Online 2006;12:221.
Early Follicular Phase:
Promotion of steroidogenesis
(theca cells)
• Adequate estrogen production
• Uterine/endometrial changes
Late Follicular Phase:
Steroidogenesis and stimulation
of final follicular maturation
(granulosa cells)

9. Balasch J, Fábreques F. Curr Opin Obstet Gynecol 2002, 14:265.Esteves, 9
• Normal androgen and estrogen biosynthesis
• Normal follicular growth and development
• Normal oocyte maturation
Normal
• Suppression of GC proliferation
• Follicular atresia (non-dominant follicles)
• Premature luteinization
• Oocyte development compromised
High
• Insufficient androgen (and estrogen) synthesis
• Follicular maturation impaired
• Inadequate endometrial proliferation
Low

10. Esteves, 10
0
5
10
15
0 5 10 15 20
Days of Stimulation
50
100
Follicle Size
(mm)
and FSH
(IU/L)
0
9
Endometrial
Thickness
(mm)

11. Esteves, 11
0
25
75
225
0
500
1000
1500
2000
2500
3000
Day 1 Day 5 Day 10 hCG
0 25 75 225
The European Recombinant Human LH Study Group, JCEM 1998; 83:1507
Addition of LH (UI):

12. Esteves, 12
0
25
75
225
0
2
4
6
8
Day 1 Day 5 Day 10 hCG
0 25 75 225 rLH
The European Recombinant Human LH Study Group, JCEM 1998; 83:1507
Evidence for LH threshold (2)
Injected rec-hLH LH Cmax
75 UI 0.5 – 1.35 UI/L

13. Esteves, 13
LH is essential for normal ovarian steroidogenesis.
75 UI rec-hLH is sufficient to promote optimal
follicular and endometrial growth, as well as
androgen production, in HH women (WHO I).
In reproductive cycles optimal follicular
development occurs within an ‘LH window’,
above a certain ‘LH threshold’ and below an ‘LH
ceiling’ (1.1 to 5.1 UI/L).

14. Esteves, 14

15. Esteves, 15
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;
• Most normogonadotropic women
undergoing Ovarian Stimulation1-3
Normal
• ~20% of NG women have less sensitive
ovaries
• Older patients (≥35 years)4
• Poor responders5
• Slow/Hypo-responders6
• Deeply suppressed endogenous LH levels
(hypo-hypo; endometriosis treated with GnRH-a)7
Low

16. Esteves, 16
• Older patients (≥35 years)
• Poor responders
• Slow/Hypo-responders
• Deeply suppressed LH
LessSensitiveOvaries
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; Alviggi, et al. RBMOnline 2012
Poor Responders
At least 2 of the following:
Maternal age ≥40 years
Previous DOR (≤3 oocytes with a
conventional stimulation)
Abnormal ovarian reserve
biomarker (AFC<5; AMH <1.1)
Or:
2 episodes of DOR after maximal
stimulation
Hypo/Slow Responders
Normal markers of ovarian reserve
Hypo-responders:
D1-D7: normal follicular recruitment
using fixed dose of FSH;
D7-D10: follicular growth plateau
despite stimulation with FSH.
Slow responders:
Require high doses of FSH (>3,000UI)
to achieve follicular growth;
May indicate genetic polymorphisms of
LH and/or FSH receptors.
Prevalence of
infertility patients
aged 35 or above
is growing

17. Impaired Oocyte Quality
Reduced Fertilization Rate
Reduced Embryo Quality
Increased Miscarriage Rates
Reduced
ovarian
paracrine
activity
Hurwitz &
Santoro 2004
LH
receptor
poly-
morphisms
Alviggi et al.,
2006
Androgen
secretory
capacity
reduced
• Piltonen et al.,
2003
Decreased
numbers of
functional
LH
receptors
• Vihko et al. 1996
Reduced
LH
bioactivity
while
immuno-
reactivity
unchanged
• Mitchell et al.
1995; Marama et
al 1984Esteves, 17
LessSensitiveOvaries
Westergaard et al., 2000; Esposito et al.,
2001; Humaidan et al., 2002

18. Mochtar et al,
2007
3 RCT (N=310)
r-hFSH+rLH vs.
r-hFSH alone*
OPR
OR 1.85
(95% CI: 1.10; 3.11)
Bosdou et al,
2012
7 RCT (N= 603)
r-hFSH+rLH vs.
r-hFSH alone*
CPR
LBR
(only 1 RCT)
RD: +6%,
(95% CI: -0.3; +13.0)
RD: +19%
(95% CI: +1.0; +36.0%)
Hill et al, 2012
7 RCT (N=902)
r-hFSH+rLH vs.
r-hFSH alone CPR
OR 1.37
(95% CI: 1.03; 1.83)
*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,
Hum Reprod Update 2012; 8(2):127-45. Hill MJ et al. Fertil Steril 2012; 97:1108-4.Esteves, 18

19. Esteves, 19
6 9 1110
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. with “steady” response on
stimulation D8 (E2 <180pg/mL; >6 follicles <10mm)
3 groups:

20. Esteves, 20
8
11 11 10
22
41
18
37
r-hFSH dose
increased (max.
450UI)
r-hFSH dose
increased; r-hLH
(75-150UI) added
r-hFSH dose
increased; HMG
added
controls
Mean No. oocytes retrieved LBR (%)
Ferraretti et al. Fertil Steril. 2004; 82:1521-6.
RCT of 126 pts. showing follicular stagnation at
stimulation d7-d10;
4 groups:

21. FSH
Theca cells
Granulosa
cells
Esteves, 21
Pregnancy
rates
% Cycle
cancellation
Number
oocytes
retrieved
Increasing the
Stimulation Dose
of FSH…
…is not associated with
better IVF outcome
Manzi et al, 1994
Klinkert et al, 2004
Berkkanoglu & Ozgur, 2010

22. Jamnongjit M et al. PNAS 2005;102:16257-16262
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 selected patients
to recover oocyte quality and, therefore, embryo
quality and implantation rates.

23. Esteves, 23
Androgen secretory capacity decreases with ovarian aging.
Mechanisms include decreased number of functional LH
receptors and ovarian paracrine activity. LH-r
polymorphisms possibly involved in hypo-responders.
LH supplementation in COS is an evidence-based strategy
to maximize pregnancy results.
4 subgroups benefit of LH supplementation in COS:
Women with less sensitive ovaries (ovarian
aging) have poor IVF outcomes.

24. Esteves, 24
Role of LH in Reproductive Cycles
Who Need LH

25. 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?
What kind of LH – recombinant LH or
HMG?

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

27. Yes, for women aged >35 yo
61%
25%
19%
68%
33%
27%
%2PN Ongoing PR Implantation
300 rec-hFSH 225 IU rec-hFSH + 75 IU rec-hLH
Esteves, 27
RCT; 292 NG women aged 36-39; GnRH antagonist D6 (fixed)
LH started on day 1
Bosch et al. Fertil Steril. 2011; 95:1031-6.

28. Esteves, 28
Matched case-control study;
N=4,719 pts.; long GnRH-a protocol
3 groups:
19
14 14
31
26 25
0
5
10
15
20
25
30
35
Fixed 2:1 r-hFSH
(150IU)/r-hLH
(75IU)
HMG rec-hFSH + HMG
Duration of
Stimulation
(days)
Mean No.
oocytes
retrieved
IR (%)
CPR per
transfer (%)
P=0.02
Buhler KF, Fisher R. Gynecol Endocrinol 2011;1-6.

29. *Steelman-Pohley Rat Bioassay, 1953; Bassett et al. Reprod Biomed Online 2005;10:169–
177; Driebergen et al. Curr Med Res Opin 2003;19:41–46.
Conventional
Bioassay*
High
variability
Rat ovary
weight
gain
Urinary
Esteves, 29
FbM: Novel
analitycal method
Protein content in
solution by mass
Minimal batch-to-
batch variability
(1.6%)
Recombinant
Size Exclusion
High Performance
Liquid
Chromatography

30. Beta unit Carboxyl terminal segment
Longer in hCG; higher
receptor affinity
Absent in LH and present in
hCG (Longer Half-life)
Purity
(LH
content)
hCG
content
(IU/vial)
LH
activity
(IU/vial)
Specific
activity
(LH/mg
protein)
Rec-hLH >99% 0 75 22,000 IU
hMG-HP 3% ~70 75* ≥ 60 IU
Adapted from ASRM Practice Committee. Fertil Steril. 2008; 90:S13-20.Esteves, 30
*derives primarily from hCG, which is concentrated during
purification or added to achieve the desired LH-like
biological activity.

31. In pts. treated with HMG (hCG-LH activity),
expression of LH/hCG receptor and other genes
involved in steroids biosynthesis in GCs is
lowered:
Reflect LH receptors down-regulation:
May explain the observed lower progesterone levels:
Trinchard-Lugan I et al. Reprod Biomed Online 2002; 4:106-115; Menon KM et al. Biol
Reprod 2004; 70:861-866; Grondal ML et al. Fertil Steril 2009; 91: 1820-1830.Esteves, 31

32. Esteves, 32
Population Biomarker Cut-off Sensitivity Specificity Accuracy
DOR1
AMH 0.82 76% 88% 0.88
AFC 5 92% 58% 0.75
*Beckman-Couter generation II assay; 1DOR: diminished ovarian response
(≤4 oocytes retrieved)
Leão RBF, Nakano FY, Esteves SC. ASRM 2013, submitted
Define “Who is
Who” Before
COS Using
Biomarkers
• Group of 131 women
undergoing
conventional COS
after pituitary down-
regulation for IVF
Individualization
of COS strategy

33. Esteves, 33
La Marca et al, Hum Reprod 2009;24:2264; Fleming et al, Fertil Steril 2012;98:1097;
Broekmans et al. Fertil Steril, 2010; 94:1044-51; Scheffer et al. Hum Reprod 2003;18:700
Reflect No. Pre-antral and Small Antral
Follicles
(≤4-8mm); gonadotropin-independent
Low inter and intra-cycle variation
assessment at any cycle day in a
single measurement
AMHAFC
TVUS at early follicular phase (D2-D4)
2-10 mm (mean diameter); 2D-plane
Reflect No. AF at a given time that can be
stimulated by gonadotropins
Low inter-cycle variation

34. Esteves, 34
Define “Who is
Who” Before COS
Using Biomarkers
AMH ≤ 0.82
“Poor
Responders”
0.25 mg/day GnRH
Antagonist (flexible)
Rec-hLH (75-150 IU)
FSH dose decreased
by the same amount
LH added
OCP
Vaginal
Progesterone
gel 8%
2 3 4 5 76 8 9 10 11 12 131
Menses
Start day
14 15
rec-FSH (Fbm)
262.5 - 375 IU
16 17
D2 or D3 ET
or FET (based
on P4 levels
hCG day)
Individualization
of COS strategy

35. Esteves, 35
Poor
Responders
AMH ≤ 0.82
rec-hFSH FbM + rec-hLH 75 IU (D6)
+ GnRH antagonist
• Total daily dose: 262.5 to 375 IU
1Poor response: ≤4 oocytes retrieved
Leão RBF, Nakano FY, Esteves SC. ASRM 2013, submitted
Prospective study involving 118 women
undergoing IVF/ICSI
Response to
COS
Conventional
down-regulation
COS (n=131)
Individualized
COS
(n=118)
P
value
Poor1 64.2% 34.0% 0.02
CPR per ET
Cancellation
35.0%
22.5%
36.3%
10.0%
0.92
0.21

36. Esteves, 36
A fixed dose of 75-150 IU rec-hLH seems adequate
to restore androgen secretory capacity in most
patients with ovarian aging.
LH supplementation can start either from
stimulation day 1 or day 6; maximum beneficial
effect yet to be determined;
LH supplementation is beneficial to women aged
>35 in a GnRH antagonist protocol.

37. Esteves, 37
Recombinant LH has 3 major differences compared to
urinary LH (HMG):
1. Higher purity and specific activity in rec-hLH
SC delivery in very small volumes
2. Higher dose precision in rec-hLH
Protein content in solution by mass (FbM)
3. LH activity is hCG dependent in u-HMG
hCG concentrated/added to achieve LH-like biological activity;
hCG has higher half-life and biological activity than rec-hLH;
Lower expression of LH receptor gene (down-regulation) after
hCG exposure; may influence GC function.

38. AMH seems to be the best biomarker to identify
patients at risk of poor response in COS.
AMH results can be used to individualize COS.
Our experience with poor responders shows that
iCOS using rec-hLH supplementation in
association with GnRH antagonists is a valid
strategy to maximize the beneficial effects of
treatment while minimizing the risk of cycle
cancellation.