1) The document discusses the role of LH in controlled ovarian hyperstimulation (COH). It notes that LH plays important roles in folliculogenesis and steroidogenesis. 2) It reviews rationales for LH supplementation in COH, such as lower endogenous LH levels and impaired steroidogenesis in certain patient groups. 3) Studies show LH supplementation can improve outcomes for poor responders and older patients, though effects may depend on the patient subgroup and study design. More research is still needed to determine which specific patient populations benefit most.

1. Role of LH in Controlled Ovarian
Hyperstimulation
Sandro ESTEVES, MD., PhD.!
Medical & Scientific Director !
ANDROFERT!
Campinas, BRAZIL!
22nd World Congress of International Federation of Fertility Societies (IFFS-2016) !
India Expo Centre, New Delhi, 24 September 2016!

2. AGENDA
Esteves, 2 !
1. Role of LH during folliculogenesis
2. Principles and practices of LH
supplementation in COH
3. Differences in LH activity provided by
existing gonadotropin preparations

3. First
world
conference
on
luteinizing
hormone
in
ART:
A
flight
of
discovery
27-­‐28
May,
2016
-­‐
Naples,
Italy
www.excemed.org!
Esteves, 3 !

4. Role of LH during folliculogenesis – Key points
Since early follicular phase
• Steroidogenesis (Theca cells)
!
• Expression of LH receptors in the granulosa
(Jeppesen et al., JCEM, 2012)
• Sustain FSH-dependent granulosa activities, including
aromatase induction and growth factors release (IGF-1, EGF
etc) (Park et al, Science 2004)
• Regulation of final follicle/oocyte maturation
• Optimization of steroidogenesis
• FSH receptor expression in GCs - responsiveness ↑
(Weil et al., 1999)!
• Act synergistically with IGF1 – growth ↑
(Vendola et al., 1999)!
• Increase in pre-antral and antral follicles – recruitability ↑
(Vendola et al., 1998;1999; Spinder et al., 1989)!
Since intermediate
follicular phase!
Esteves, 4 !

5. AGENDA
1. Role of LH during folliculogenesis
2. Principles and practices of LH
supplementation in COH
3. Differences in LH activity provided by
existing gonadotropin preparations
Esteves, 5 !

6. Poor
responders
Advanced
age (>35)
GnRH
antagonist
Deeply
pituitary
suppression
of LH
All cycles
Hypo-
responders
Undisputed evidence in HH, but is LH required in
other clinical scenarios?
Esteves, 6 !

7. What would be the rationale?
1. Lower LH levels in stimulated cycles
Esteves, 7 !

8. • Single-center retrospective cohort
study including 1094 cycles of GnRH
antagonist protocol with fresh
embryo transfers
• OCP, HH and PCO excluded
• Flexible GnRH antagonist with rFSH
• LPS with vaginal progesterone 90mg
2x/day
Low LH levels episodes
Chin-Der Chen et al. Reproductive BioMedicine Online, 2016, Available online 21 July 2016
Low LH anytime during COS (≤0.8 mIU/ml) in 22% cycles
(1/3 of those prior to initiation of antagonists)
Esteves, 8 !

9. Impaired
ovarian
paracrine
activity
Hurwitz & Santoro
2004
Less
functional
LH
receptors
• Vihko et al. 1996
Endogenous
LH less
potent and
biologically
active
bioactivity
• Mitchell et al. 1995;
Marama et al 1984
Total
Testosterone
↓ 55%
DHEAS
↓ 77%
Free
Testosterone
↓ 49%
Androstenedione
↓ 64%
n = 1423
Davison SL et al
JCEM 2005;90:3847
Davison et al. JCEM 2005!
What would be the rationale?
2. Impaired steroidogenesis in poor responders and older
women
Esteves, 9 !

10. Ben-Meir et al. 2015; Weall et al. 2015; Ata et al. 2012
Mitochondrial function impaired
(less energy)
Oxidative stress increased
Granulosa cell number reduced
(apoptosis)
0!
2!
4!
6!
<35 years ≥35 years
Meanfluorescentintensity!
(pixelsx103/μm2)!
Oocyte Mitochondria
Viability!
x108
x108
x108
P<0.001
Aneuploidy rates increased
What would be the rationale?
3. Impaired oocyte quality in older women
Esteves, 10 !

11. Anti-apoptotic
effect granulosa Up-regulation
growth factors
Increase
responsiveness
FSH receptors
Act
synergistically
IGF-1
Rationale: LH supplementation as a corrective measure
Rimon E et al., 2004; Robinson RS et al., 2007;
Tilly JL et al., 1992; Peluso JJ et al., 2001, Ben-Ami I et al., 2009
Steroidogenic
action
Esteves, 11 !

12. LH!
DHEAS!A4!T!
P4!
E2!
Bosch, et al. Fertil Steril 2006;86:Suppl.2: S425!
LH supplementation – Steroidogenic action confirmed ✔
Esteves, 12 !
Follicularfluid

13. LH supplementation during follicular phase –
Additional benefits confirmed ✔
• Increases ovarian follicular angiogenesis via modulation of VEGF-A
and its soluble receptor sFit-1 expression (Gutman et al. 2008)
• Increases follicular maturation (higher expression of maturation
markers - TGF-beta, PTGS2 and HAS2 – in cumulus cells)
(Barberi et al. 2012)
• Improves follicular recruitment (Gómez-Palomares et al. 2005)
Esteves, 13 !

14. Do these effects translate into better clinical
outcomes?
Poor
responders
Advanced
age (>35)
GnRH
antagonist
Deeply
pituitary
suppression
of LH
All cycles
Hypo-
responders
Esteves, 14 !

15. *75 IU rec-LH from Sd1
Bosch et al
Fertil Steril 2011 !
2. LH supplementation in GnRH antagonists cycles
Advanced age group (36-39) + OCP pretreatment
Esteves, 15 !
*Disclaimer: Lutropin alfa concomitantly administered with follitropin alfa for injection is
indicated for stimulation of follicular development in infertile hypogonadotropic
hypogonadal women with profound LH deficiency (LH<1.2 IU/L) !

16. Vuong et al Hum Reprod 2015
2. LH supplementation in GnRH antagonists cycles
Advanced age group (>35); No OCP pretreatment
• Age limit NR
• LH (75 IU)
since Sd6
Esteves, 16 !
*Disclaimer: Lutropin alfa concomitantly administered with follitropin alfa for injection is
indicated for stimulation of follicular development in infertile hypogonadotropic
hypogonadal women with profound LH deficiency (LH<1.2 IU/L) !

17. 3. Poor responders
Pregnancy
Lehert et al
Reprod Biol Endocrinol 2014 !
• 30% increase in PR
• Difference not seen in
normal responders
Esteves, 17 !
*Disclaimer: Lutropin alfa concomitantly administered with follitropin alfa for injection is indicated for stimulation of follicular
development in infertile hypogonadotropic hypogonadal women with profound LH deficiency (LH<1.2 IU/L) !

18. Poor
responders
Among poor responders, different
criteria applied for classification,
yielding to conflicting results
Adapted from Prof. Alviggi
Esteves, 18 !
*Disclaimer: Lutropin alfa concomitantly administered with follitropin alfa for injection is indicated for stimulation of follicular development
in infertile hypogonadotropic hypogonadal women with profound LH deficiency (LH<1.2 IU/L) !
VS.

19. Role of exogenous LH in COH - Things to Ponder
Ø Many study designs and LH dosage,
and different starting day
Ø Analysis of whole populations may
dilute effect size
Ø LH supplementation beneficial to
subgroups only
Esteves, 19 !

20. Esteves, 20 !

21. 4 groups of low prognosisFour Groups of Patient with Lower Prognosis
GROUP 1
Young patients <35 years with adequate
ovarian reserve parameters (AFC≥5; AMH≥1.2
ng/ml) and with an unexpected poor or
suboptimal ovarian response.
Subgroup 1a: <4 oocytes*
Subgroup 1b: 4-9 oocytes retrieved*
*after standard ovarian stimulation
GROUP 2
Older patients ≥35 years with adequate
ovarian reserve parameters (AFC≥5; AMH≥1.2
ng/ml) and with an unexpected poor or
suboptimal ovarian response.
Subgroup 1a: <4 oocytes*
Subgroup 1b: 4-9 oocytes retrieved*
*after standard ovarian stimulation
GROUP 3
Young patients (<35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
GROUP 4
Older patients (≥35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
Alviggi et al. Fertil Steril in press
Poseidon Group; Alviggi et al. Fertil Steril. 2016 Feb 24. !
Four Groups of Patient with Lower Prognosis
GROUP 1
Young patients <35 years with adequate
ovarian reserve parameters (AFC≥5; AMH≥1.2
ng/ml) and with an unexpected poor or
suboptimal ovarian response.
Subgroup 1a: <4 oocytes*
Subgroup 1b: 4-9 oocytes retrieved*
*after standard ovarian stimulation
GROUP 2
Older patients ≥35 years with adequate
ovarian reserve parameters (AFC≥5; AMH≥1.2
ng/ml) and with an unexpected poor or
suboptimal ovarian response.
Subgroup 1a: <4 oocytes*
Subgroup 1b: 4-9 oocytes retrieved*
*after standard ovarian stimulation
GROUP 3
Young patients (<35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
GROUP 4
Older patients (≥35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
Alviggi et al. Fertil Steril in press
Esteves, 21 !

22. Esteves, 22 !
Strong association between number of
oocytes and cumulative LBR (fresh+frozen)

23. Embryop Euploidy Rates X Age
70%!
!
60%!
!
50%! 30%! 15%!
Esteves, 23 !

24. Esteves, 24 !
nt with Lower Prognosis
.2
GROUP 2
Older patients ≥35 years with adequate
ovarian reserve parameters (AFC≥5; AMH≥1.2
ng/ml) and with an unexpected poor or
suboptimal ovarian response.
Subgroup 1a: <4 oocytes*
Subgroup 1b: 4-9 oocytes retrieved*
*after standard ovarian stimulation
n
GROUP 4
Older patients (≥35 years) with poor ovarian
Four Groups of Patient with Lower Prognosis
GROUP 1
Young patients <35 years with adequate
ovarian reserve parameters (AFC≥5; AMH≥1.2
ng/ml) and with an unexpected poor or
suboptimal ovarian response.
Subgroup 1a: <4 oocytes*
Subgroup 1b: 4-9 oocytes retrieved*
*after standard ovarian stimulation
GROUP 2
Older patients ≥35 years with adequate
ovarian reserve parameters (AFC≥5; AMH≥1.2
ng/ml) and with an unexpected poor or
suboptimal ovarian response.
Subgroup 1a: <4 oocytes*
Subgroup 1b: 4-9 oocytes retrieved*
*after standard ovarian stimulation
GROUP 3
Young patients (<35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
GROUP 4
Older patients (≥35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
Alviggi et al. Fertil Steril in press
Poseidon!
LH supplementation!

25.
Cochrane 2007 – Hypo responders
‘Ongoing PR’ per woman randomized to rFSH vs rFSH + rLH
Esteves, 25 !
*Disclaimer: Lutropin alfa concomitantly administered with follitropin alfa for injection is indicated for stimulation of follicular
development in infertile hypogonadotropic hypogonadal women with profound LH deficiency (LH<1.2 IU/L) !

26. Hypo-responders
Alviggi, Conforti & Esteves, Springer 2016!
Association with polymorphisms: !
• LH (v-beta LH) !
• FSH receptor (variant Ser/680)!
!
Alviggi, Humaidan et al.
RBE 2013;
Esteves, 26 !

27. Esteves, 27 !
GROUP 3
Young patients (<35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
GROUP 4
Older patients (≥35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
Alviggi et al. Fertil Steril in press

28. !
!
!
!
Poor Responders
(ESHRE; Bologna criteria) ESPART!
!
Efficacy and Safety of Pergoveris in
Assisted Reproductive Technology!
• ~900 patients randomized to r-
hFSH or r-hFSH + r-LH (2:1 ratio);
long-agonist GnRH !
• No differences in the
outcome measures
compared
At least 2 out of 3 conditions:!
• Advanced maternal age (≥40) or risk factor for
poor response (Turner, X-fragile, Chemotherapy)!
• ≤ 3 oocytes previous stimulation (conventional
protocols)!
• AFC<5-7; AMH<0.5-1.1 ng/ml!
Esteves, 28 !
*Disclaimer: Lutropin alfa concomitantly administered with follitropin alfa for injection is indicated for stimulation of follicular
development in infertile hypogonadotropic hypogonadal women with profound LH deficiency (LH<1.2 IU/L) !

29. Expected Poor Responders; younger women
72.0
3.5
45.0
20.0
46.6
4.8
23.3
26.8
0
20
40
60
80
Observed Poor
Response (%)
Oocytes retrieved (N)
Cancellation (%)
Pregnancy/cycle (%)
rec-hFSH alone
r-hFSH+r-hLH 2:1 or 3:1 ratio
Leão RBF, Nakano FY, Esteves SC. Fertil Steril 2013; 100 (Suppl.): S16.
*p<0.05
*
*
*
N=118; Expected poor response: AMH<0.82 ng/dL; Observed poor response <5 oocytes retrieved
Esteves, 29 !
*Disclaimer: Lutropin alfa concomitantly administered with follitropin alfa for injection is indicated for stimulation of follicular
development in infertile hypogonadotropic hypogonadal women with profound LH deficiency (LH<1.2 IU/L) !

30. Esteves, 30 !
GROUP 3
Young patients (<35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
GROUP 4
Older patients (≥35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
Alviggi et al. Fertil Steril in press

31. Esteves, 31 !
Ø Individualization: dosage, type, regimen
u GnRH Antagonist (trigger with hCG or GnRHa) !
u Rec-hFSH (300 IU) + rec-LH supplementation (150 IU/d)?!
u Adjuvants: GH; DHEA, testosterone ??!
u Minimal stimulation (eg. Poseidon 4)?!
Ø AccuVit (oocytes/embryos): DUOSTIM; PGS
GROUP 3
Young patients (<35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
GROUP 4
Older patients (≥35 years) with poor ovarian
reserve pre-stimulation parameters (AFC<5;
AMH<1.2 ng/ml)
Alviggi et al. Fertil Steril in press
Ø Egg donation

32. 2. Principles and practices of LH Supplementation
Conclusions (1)
Poor
responders
All cycles
Hypo-
responders
No apparent beneficial role in unselected
patients (i.e. all cycles)!
No apparent beneficial role in Bologna criteria
poor responders; role in ‘expected’ poor
responders deserves further investigation !
Fair evidence of beneficial effect in hypo-
responders!
Esteves, 32 !

33. Esteves, 33 !
Ø May allow better individualization of treatment
Ø Design RCT exploring different treatment strategies
to specific – and uniform - subgroups
2. Principles and practices of LH Supplementation
Conclusions (2)

34. AGENDA
1. Role of LH during folliculogenesis
2. Principles and practices of LH
supplementation in COH
3. Differences in LH activity provided by
existing gonadotropin preparations
Esteves, 34 !

35. Purity
(LH
content)
FSH
activity
(IU/vial)
LH
activity
(IU/vial)
hCG
content
(IU/vial)
Specific
activity
(LH/mg
protein)
Lutropin alfa >99% 0 75¶
-- 9,000
Follitropin alfa
+ lutropin alfa
2:1 ratio
>99% 150 75 -- 9,000
HP-hMG Unknown* 75 75* ~8 --
¶1 µg of lutropin alfa = 22 IU
*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 & Alviggi. Principles and practices of COS in ART, Springer NY 2015
Gonadotropins with LH activity
Esteves, 35 !

36. Fertil Steril 2012; 97(3): 561-72
Esteves, 36 !

37. Extracellular
fluid
Cytoplasm
Plasma
membrane
LH! hCG!
LH/hCG receptor !
Beta unit
Carboxyl
terminal
segment
Longer in
hCG
Higher
receptor
affinity
Present in
hCG, but not in
LH
Longer half life
(hCG)
Leao & Esteves. Clinics 2014; 69(4): 279–293.
Esteves, 37 !

38. Choi & Smitz Mol Cell Endocrinol 2014; 383(1-2):203–13.
Divergence in receptor-mediated signaling
between LH and hCG
Esteves, 38 !

39. 100 pM LH or hCG; n=4; Mean±SEM; *=significant vs unstimulated; t-test/two-
way analysis of variance; p<0.05. hGLC model
Casarini L et al. PLoS ONE 7(10): e46682, 2012.
ERK 1/2! AKT!
LH is more active than hCG in pERK and pAKT activation
(cell proliferation and survival)
Esteves, 39 !

40. Cellular Viability
Casarini et al., Mol & Cell Endo, 2016
Expression of pro-
apoptotic enzymes
hCG reduces cell viability and stimulates caspase 3
expression in hGL5/LHCGR cells
Esteves, 40 !

41. Molecular & Functional Differences
Between LH and hCG in vitro
Ø hGC > LH in activation of cAMP
pathway and steroidogenesis
Ø hCG predominantly pro-steroidogenic,
potentially pro-apoptotic
Ø LH > hCG in activation of ERK
and AKT
Ø LH is a growth, differentiation and
survival factor
Structural characteristics, half-life in serum and downstream
effects of LH and hCG following receptor binding
LH hCG
Aminoacid number
Alpha subunit
Beta subunit
92
121
92
145
N-linked glycosilation sites
Alpha subunit
Beta subunit
2
1
2
2
O-linked glycosilation sites -- 4
Carboxyl-terminal segment non-existent present
Half-life (hours)
Initial, range of mean
Terminal, range of mean
Terminal (SC injection)
0.6-1.3
9-12
21-24
3.9-5.5
23-31
72-96
Response
ED50 (pM)1
Time to maximal cAMP accumulation1
ERK 1/2 activation2
AKT activation2
CYP19A1 expression in presence of ERK1/2 pathway
blockade2
530.0 ± 51.2
10 min
strong
strong
increased
107.1 ± 14.3
1 h
weak
minimal
unaffected
1
Effect on COS-7/LHCGR cells that constitutively express LH receptors
2Effect on human granulosa cells
Esteves & Alviggi. Principles and practices of COS in ART,
Springer 2015; Simoni M Physiology of LH & differences
with hCG, Excemed 2016 !
Esteves, 41 !

42. So
What?
Esteves, 42 !

43. WHO I (HH)
Type of LH
Carone et al. J Endocrinol Invest. 2012;35:996-1002.!
2:1 ratio
r-hFSH (150 IU)/r-hLH (75 IU)
N=17
hMG-HP; N=18
(1:1 FSH/LH; 150 IU)
Esteves, 43 !

44. 10.8 10.6 11.39 8 9
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 (%)
Case-control study (German IVF Registry)
N=4,719 patients subjected to IVF
Buhler & Fischer Gynecol Endocrinol 2011
*P=0.02
*
*
Esteves, 44 !
*Disclaimer: Lutropin alpha concomitantly administered with follitropin alpha for injection is indicated for stimulation of follicular
development in infertile hypogonadotropic hypogonadal women with profound LH deficiency (LH<1.2 IU/L) !

45. Authors, Year Design N Main findings
Revelli et al.
2015
Observational 848 Higher PR 2:1 rec-FSH + rec-LH vs. HP-hMG in
patients with > 8 oocytes
(OR: 1.63; 95% CI: 1.16-2.27)
Dahan et al,
2014
Observational 201 Higher N oocytes rec-LH (12) vs. hMG (10), p=0.008
Higher CPR rec-LH vs hMG (36% vs 20%; p=0.02)
Fábregues et
al, 2013
Cross-over 66 Higher N oocytes 2:1 rec-FSH + rec-LH (9.8) vs. HP-
hMG (7.3), p<0.01
Higher N embryos for freezing rec-LH group
Other Clinical Studies!
Revelli et al. Reprod Biol Endocrinol. 2015; 25;13:77; Dahan et al. Eur J Obstet Gynecol Reprod Biol. 2014;172:70-3;
Fábregues F et al. Gynecol Endocrinol. 2013;29(5):430-5.
Esteves, 45 !
*Disclaimer: Lutropin alpha concomitantly administered with follitropin alpha for injection is indicated for stimulation of follicular
development in infertile hypogonadotropic hypogonadal women with profound LH deficiency (LH<1.2 IU/L) !

46. 3. LH activity provided by existing gonadotropin
preparations - Conclusions
Both rec-LH and hMG (via hCG) provide LH activity.
Rec-LH and hCG are different structurally and elicit
different downstream effects after receptor binding:
!
Esteves, 46 !
• Both stimulate steroidogenesis but have a differential role on
cell growth, which seems to influence oocyte development.
• Current evidence in favor of providing LH activity by rec-LH
over hMG in COH, but more studies are needed to confirm and
effects on different patient subsets.

47. Esteves, 47 !
धन्यवाद!
Thank You!