The Role of LH in COS
Hamburg 23.9. 2016
Robert Fischer
MVZ Fertility Center Hamburg GmbH
ISO 9001:2015 ISO 17025
Outline:
LH mode of action
Who will need LH
What are the sources of LH
LH and hCG are different
Conclusions
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 20 25 30 35 40
Liv
e b
irth
rat
e (%
)
Oocyte number
Observed live birth rate Predicted live birth rate
Sunkara et al. Hum Reprod 2011
450,135 IVF cycles
Number of oocytes retrieved and
live birth rates
number of oocytes that best optimized LBR was 15
RCT and meta-analyses comparing oocyte yield with different gonadotropins
↑ 1.5 oocytes (GnRH antagonist cycles)
Devroey et al., 2012
↑ 3.1 oocytes (GnRH antagonist cycles)
Bosch et al., 2008
↑ 1.8 oocytes (GnRH agonist cycles)
MERIT Study, 2006
↑ 2.8 oocytes (GnRH agonist cycles)
Hompes et al., 2008
Evidence Level
1b
↑ 2.1 oocytes (16 RCT; different protocols)
Lehert et al., 2010 No. Oocytes
retrieved higher with Rec-FSH vs.
hMG, HP-hMG, and uFSH
Follicular phase Midcycle Luteal phase
Maintenance of
corpus luteum and
P4 secretion
Endometrial
receptivity and
implantation
Promotes
androgen secretion
theca.
Synergic action with
FSH in producing
estrogens
Support dominance
once FSH levels
decrease
Resumption of oocyte
meiosis
Maturation of COC
Follicular rupture
Granullosa luteinization
Role of LH in menstrual cycle
Recruit a new pool of follicles (FSH)
Primordial
and primary
Preantral Antral
FSH-LH
independent
Preovulatory
LH
dependent
FSH dependent
Expression of LH Receptors on both theca and granulosa Zeleznik et al 1974
10mm
Expression of Receptors : FSH: granulosa cells LH : theca cells Adashi 1996
LH is able to exert its action on both : granulosa and theca cells Hillier 1994
2mm
Two cell two gonadotropin hypothesis Ken Ryan 1979 LH receptor is needed for the progression from antral follicle to preovulatory
follicle
1,2-10 IU/ml
LH ceiling
LH threshold
LH therapeutic window in follicular development
Adapted from Hiller SG,1994
Under-exposure to LH
Diminished steroidProduction
Follicular maturation impaired
Endometrial proliferation
inadequate
Over-exposure to LH
High LH
Follicular atresia
Premature luteinizaion
Oocyte development compromised ↑ P4 synthesis ↓ aromatase ↓ cellular prolif
Balasch and Fabreques 2002
75IU rLH is sufficient
for promoting optimal follicular development The European Recombinant Human LH Study
Group,
JCEM 1998; 83:1507
Do patients need LH supplementation during COS? Who might benefit from LH treatment?
• LH supplementation is mandatory in the hypogonadotrophic
hypogonadal patient
(FSH and LH < 1.2 IU/l)
• For most women « FSH-only » stimulation is sufficient as their
endogenous LH level , even after down-regulation, will support
follicular development and steroidogenic activity.
BUT…
There are Patients with ‘LH deficiency’ during COS
LH supplementation in ART
– No benefit in unselected population (Kobilianakis et al. 2007)
– Benefit in profound LH suppression in GnRH agonist long protocol – Potential benefit in initial poor response – Potential benefit and better outcome in patients > 35 years old
• Controverted results • Confusing evidences • Lack of consensus
• Studies use different doses, starting day, duration, type of LH activity, LH assay
Mochtar et al, 2007 Cochrane Database Syst Rev.18: 2
< 35 years old ≥ 35 years old
GnRH
agonist
Marrs et al, 2004
Humaidan et al, 2004
NyboeAndersen et al,
2008
Fábregues et al, 2006
Matorras et al, 2009
FSH = FSH + LH(n=310)
FSH = FSH + LH(n=192)
FSH = FSH + LH(n=426)
FSH + LH > FSH (n=88)
FSH + LH > FSH (n=38)
FSH + LH = FSH(n=100)
FSH + LH = FSH(n=120)
FSH + LH > FSH(n=131)
GnRH
antag .
Sauer et al , 2004
Griesinger et al, 2005
Levi-Setti et al, 2006
Bosch et al, 2011
König et al, 2014
FSH = FSH + LH (n=49)
FSH = FSH + LH(n=126)
FSH = FSH + LH(n=40)
FSH =FSH + LH (n=333)
FSH+LH > FSH (n=292)
FSH+LH=FSH (n=253)
Is there a benefit in advanced age ?
Increased IR in women > 35 years of age
Long vs
Short protocol
0
10
20
30
40
<=35 36-39
Implantation rate
p=0.84
OR: 1.03 (0.73-1.47)
p=0.03
OR: 1.56 (1.04-2.33)
27.8 28.6 18.9 26.7
0
10
20
30
40
<=35 36-39
FSH alone FSH + LH
OR: 1.49 (0.93-2.38); p=0.09 OR: 1.0 (0.65-1.57); p=1.0
37.3 37.3 25.3 33.5
Ongoing Pregnancy rate per
Randomized patient (ITT analysis)
Bosch et al (2011) Fertil Steril 95; 1031-6;
Why is LH Supplementation benefitial in advanced age(>35)?
Ovarian ageing :
It is the roll of LH in Androgen and anti-apoptotic effect She looks like 45 Feels like 35 But is 55
Ovarian Aging
• ↓ Androgens secretion Piltonen 2003
• ↓ Functional LH receptors Vihko 1996
• ↓ LH immunorreactivity Mitchell 1995, Marama 1984
• ↓Ovarian paracrine activity Hurwitz & Santoro 2004
• LH supplementation reduces the apoptosis of cumulus and granulosa cells • LH supplementation enhances the expression of anti-apoptotic proteins
Rimon et al., 2004; Ruvolo et al., 2007
Tilly JL et al., 1992; Peluso JJ et al., 2001,Grondahl et al., 2008; Ben Ami et al., 2009
Expected Role of LH
• LH supplementation increases FSH receptor
responsiveness (Weil et al., 1999)
• LH supplementation up-regulates growth factors FGF2,AR,ER and act synergistically with IGF1
Increase in pre-antral and antral follicles – recruitability ↑ (Vendola et al., 1998; 1999; Spinder et al., 1989)
Which improvement could be expected in poor responders?
Ovarian effect
better follicular recruitment
higher number of oocytes - higher quality embryos?
Implantation
improved implantation rate
LH - an androgen modulating agent
Androgens enhance FSH responsiveness of immature follicles and promote follicle selection and maturation
Androgens augment FSH receptors in the granulosa cells
Assumption:
LH as an androgen modulating agent improves stimulation outcome in poor responders
ESPART-Study Efficacy and Safety of Pergoveris® in
Assisted Reproductive Technology –
ESPART: rationale and design of a
randomised controlled trial in poor
ovarian responders undergoing
IVF/ICSI treatment
Running title: RCT of r-hFSH with/without r-hLH in POR patients
P. Humaidan, J. Schertz, R. Fischer
A phase III, randomized, controlled, single-blind, multicentre, parallel arm trial to
assess the efficacy and safety of PERGOVERIS™ (follitropin alfa and lutropin
alfa) and GONAL-f® (follitropin alfa) for multifollicular development as part of an
assisted reproductive technology treatment cycle in poor ovarian responders,
as defined by the European Society of Human Reproduction and Embryology
criteria
Multicentre multination.
939 Patients recruited. EMR200061-005
Demographics, baseline, and efficacy assessments (MITT) – 939 randomized patients
Characteristics PERGOVERIS
(N=462) GONAL-f
(N=477)
Age (years) (mean, SD)* • 40 to <41 years (n, %)
38.3 (2.9) • 229 (49.6)
38.3 (3.0) • 236 (49.5)
AMH (ng/mL) (mean, SD)* 0.580 (0.4976) 0.603 (0.4847)
Antral follicle count (mean, SD) 4.9 (2.30) 4.8 (2.19)
Prior POR with ≤3 oocytes (n, %)* 379 (82.0) 402 (84.3)
Outcome
Number of oocytes retrieved (mean, SD) 3.3 (2.71) 3.6 (2.82)
Biochemical pregnancy (n, %) 80 (17.3) 114 (23.9)
Ongoing pregnancy (n, %) 51 (11.0) 59 (12.4)
*Subjects had to meet 2 of 3 POR criteria
R-hLH versus in r-hFSH dose in POOR RESPONDERS
Statistically significant increase in ongoing PR
Favours r-hFSH Favours r-hFSH + r-hLH
Mochtar MH, Cochrane Database, 2007, Issue 2
Ongoing PR per woman randomized
No difference in LH endogenous levels during stimulation
ALL STUDIES PERFORMED
IN HYPO-RESPONDERS!!!
R-hLH versus in r-hFSH dose in POOR RESPONDERS
Statistically significant increase in ongoing PR
Lehert et al., 2014
Relative increase
30% in poor
responders
Poor respond
ers
R-h (LH+FSH) vs
r-hFSH:
Meta-analysis 6443
patients
Normal respond
ers
MANY STUDIES PERFORMED
IN HYPO-RESPONDERS!!!
~30%
The meaning of hypo-response: Conclusions
Day stimulation 5 - 8
What to do?
Increasing FSH?
Adding LH?
• In 15% of normogonadotrophic – good prognosis women (normal AMH and AFC) an initial inadequate (poor) response to standard FSH doses is observed
• This phenomenon reflects an hypo-sensitivity of granulosa cells to standard FSH doses. For this reason it has been defined ”hypo-response”
• Hypo-response is associated to higher FSH consumption, low FORT, unexpected poor response (i.e. <3 eggs retrieved) and lower PRs
• If hypo-response is identified early (i.e., day 5-8 of COS), r-hLH is effective in rescuing follicle/oocyte number (FORT) and embryo competence
LH
Greb, et al. JCEM, 2005; Gromoll & Simoni TEM 2005.
Asn/Asn Asn/Ser Ser/Ser0
10
20
30
40
50
n=46 n=72 n=43
FS
H a
mp
ou
les
(n
)
*
*
p < 0.05
*
Asn/Asn Asn/Ser Ser/Ser0.0
2.5
5.0
7.5
10.0
n=46 n=72 n=43
*
*
p < 0.05
FS
H (
IU/l)
FSH receptor Ser680 genotype and ovarian response to FSH
Perez Mayorga, et al. 2000; Sudo, et al. 2002; Choi, et al. 2004; Falconer, et al. 2005.
• 11.6 %] v-LH carriers found • 10.2 % heterozygotes • 1.4% homozygotes
V-betaLH genotype and ovarian response to FSH
Alviggi et al., RBMOnline
2009;
Alviggi and Humaidan RB and
E, 2012.
FSH consumption is higher in carriers of FSH-R Ser680 and v-betaLH variants
Two amino acid changes in b-chain Additional sulphated sugar at asn-13
Hypo-response is related to genetic characteristics: less bioactive LH variant?
V-beta-LH and native molecule Worldwide occurrence
The common Trp8Arg/Ile15Thr LH β1 121
Y 30
Trp8Arg Ile15Thr
K. Pettersson and I. Huhtaniemi, 1998 Jiang et al., 1999; Ropelato et al., 1999
Percent V/V + V/WT
0
0 10 20 30 40 50 60
13.6%
Australia/Aboriginals Finland (Lapp)
Finland
Faroe Islands Iceland
Greenland
Estonia Poland
Sweden (Stockholm) South Africa (black) United Kingdom
United States (black)
The Netherlands China Sweden (Göteborg)
Italy Thailand Jordan Jordan United States (Hispanic)
Spain (Vasco) Mexico (Mayan) Western India (Kota)
Increased in vitro bioactivity Decreased circulatory half-life Increased promoter activity Associated with reduced bioactivity of LH Association with ovulatory disorders and infertility
POSEIDON Working Group Proposes a new definition of POR based on
the concept of “Low Prognosis” to guide
patient management
(Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number)
Carlo Alviggi,
Claus Y. Andersen,
Klaus Buhler, Alessandro Conforti,
Giuseppe De Placido, Sandro Esteves,
Robert Fischer, Daniela Galliano,
Nikolaos Polyzos, Sesh Sunkara,
Filippo Ubaldi, Peter Humaidan
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 3 • Young patients (<35 years) with
poor ovarian reserve pre-stimulation
parameters (AFC<5; AMH<1.2 ng/ml)
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 4 Older patients (≥35 years) with poor
ovarian reserve pre-stimulation
parameters (AFC<5; AMH<1.2 ng/ml)
Poseidon Group, F&S 2016
Conclusion: This study, though
relatively small, demonstrated
significantly higher number of eggs and
higher live birth rates in women given
LH supplementation in antagonist
cycles in which their intra-cycle LH
levels were very low. Monitoring
indicates this occurs in over half of
antagonist cycles.
What are the potential sources of LH?
For Real LH molecules
• Recombinant human LH (r-hLH) 75 IU,99% pure
• (r-hFSH+r-hLH) 150:75 IU, 99% pure
For LH Activity (LH and/or hCG molecules)
• Human Chorionic Gonadotropin
– Recombinant – 250 mcg = 6750 IU of hCG, 99% pure
– Urinary 10,000 IU - >50% impuritites
• Human Menop. Gonadotropin
– 75 IU FSH: 75 IU of LH activity, 95% hCG derived and >30% impurities
hpHMG is spiked with u-hCG for LH activity
The presence of hCG in hMG is due to the artificial addition to compensate for the lost LH
Publications
1. van de Weijer et al. RBM Online 2003;7:547–557;
2. Wolfenson C. et al., RBM Online2005:4:442-454
3. Giudice et al. J Clin Res 2001;4:27–33
4. Basset et al.. RBM Online 2009
36
hCG - α 92 aa - β 145 aa
8 Glycosylation sites - 37 kDa
Trophoblastic embryonic cells
hCG LH
Cotonnec, et al. FS 1998;69:201-9 - Trinchard, et al. RBMonline 2002;4:106-15
LH - α 92 aa - β 121 aa
3 Glycosylation sites – 28kDa
Anterior Pituitary Gland
Molecule and Dose rLH
150 IU
rhCG
250mcg
Initial or Distribution Half-life (hrs) 1.0 ± 0.2 4.7 ± 0.8
Terminal Half-life (hrs) IV route 11 ± 8 28 ± 3
Terminal Half-life (hrs) SC route 21-24 72-96
They attach to the same receptor (LHCG-R)
Research Institute, Inc
Sharing the same α subunit and 81% of the aminoacid residues of the β subunit, LH and hCG bind to the same receptor: LH/hCG receptor (Kessler et al., 1979)
Constitutively expressed on theca cells Expressed on granulosa cells at a follicle size of 8-12 mm
LHCGR
Cell membrane
PKA P-AKT P-ERK1/2
Adenylyl
cyclase
cAMP
PIP3
LH/hCG
G-proteins
Sterodogenesis, cell proliferation/apoptosis, DNA
transcription (AREG, EREG, NRG1, CYP19A1)…..)
pCREB
Courtesy M.Simoni
On cAMP:
Higher in vitro potency of hCG vs LH
(about 5-fold, extractive or recombinant,
any cell model)
Faster maximal response to LH vs hCG
(10 min vs. 60 min, COS7-LHCGR)
By chronic stimulation :
Higher intracellular cAMP production by hCG vs LH but
same progesterone accumulation (up to 36 hrs, )
Casarini L et al. PLoS ONE 7(10): e46682, 2012
Courtesy M.Simoni
LH and hCG act on the same receptor with
different potency and kinetics
hCG reduces cell viability and stimulates
caspase 3 expression in hGL5/LHCGR cells
Casarini et al., 2016 Courtesy M.Simoni
The effect of LH and hCG and changes in the presence of FSH
In the presence of FSH:
-LH stimulates expression of oocyte maturing factors
-hCG stimulates steroidogenesis
Casarini et al., 2016 Courtesy M.Simoni
Conclusions LH and hCG in vitro
• HCG is more active than LH in activation of the cAMP pathway and steroidogenesis
• LH is more active than HCG in pERK and pAKT activation
• HCG is a predominantly pro-steroidogenic factor, potentially pro-apoptotic
• LH is a growth, differentiation and survival factor
LH and hCG are NOT equivalent in vitro
Does it play a role whether HMG (hCG) or rLH? Gene Expression in Granulosa cells
• 30 IVF/ICSI patients randomized to rLH or HMG treatment
• At aspiration granulosa cells collected for gene expression analysis
Results:
• 85 genes statistically significantly different in expression
• Expression levels of LH/hCG receptor gene and genes involved in biosynthesis of cholesterol and steroids were expressed at a lower level in HMG-treated granulosa cells
Conclusion:
• Preparation used for COS significantly influences the developmental competence of the oocyte and the function of the corpus luteum
Grønlund ML et al., Fert Ster 2008
Serum hCG levels increase during hMG stimulation (16 IU/L hCG=116 IU/L LH) hCG stronger affinity to receptor than LH and 3x time longer half life time: down regulation LH-R on granulosa cells
0.1
1
10
O P U 8 1
HMG/ GnRHa intranasal HMG/ GnRHa sc
FSH/GnRHa intranasal FSH/ GnRHa sc
Sere
um
HC
G(I
U/L
)
Westergaard et al, 2001
Stimulation day
LH versus hCG activity
Significant differences exist between LH and hCG at the: Molecular and functional level
This will influence clinical outcomes
matched case-controlled study 4.719 women included
1.573 per group
matched by: age, BMI, indication, previous ART cycles
( German multicenter observational study & FCH) 3 groups:
r-[FSH-LH] (2:1) u-hMG
u-hMG & r-FSH
Bühler, Fischer 2011, GynecolEndocrinol
Bühler & Fischer (Gynecol Endocrinol 2011)
10 10,5
11 11,5
r-hFSH + r-hLH
hMG r-hFSH + hMG
10,8 10,65
11,28
Mean duration of treatment (days)
0 20 40 60
r-hFSH + r-hLH
hMG r-hFSH + hMG
34,35 36,38 46,31
Mean number of 75 IU ampoules
Bühler & Fischer (Gynecol Endocrinol 2011)
7
7,5
8
8,5
9
r-hFSH + r-hLH
hMG r-hFSH + hMG
9
7,8
8,8
Number of oocytes retrieved
0%
5%
10%
15%
20%
r-hFSH + r-hLH
hMG r-hFSH + hMG
19%
13,90%
13,80%
Implantation rate (%)
* *
*
LBR per started cycle sig. higher with rFSH+rLH 2:1
Effect seen in both age groups <35 & >35 y.o.
rh-FSH + rh-LH (2:1) vs uFSH + hCG (hMG) ( HP-hMG )in ART Fabregues 2013 (n=33 per group)
Conclusions: • rFSH+rLH(2:1) produced more oocytes/embryos • 2/3 of the patients in rFSH+rLH group have frozen embryos to transfer if fresh transfer failed • 1/3 of the patients in hMG have frozen embryos
Conclusions
• LH is different than hCG at molecular, functional and clinical levels
• LH is the natural molecule to support the follicular phase • “Consensus” on benefit in patient sub-populations:
– Hypogonadotropic – hypogonadism – Profound LH suppression in GnRH agonist long protocol – Poor responders – Older than 35 years – Suboptimal response to FSH (LH gene polymorphism) One should give r-LH during stimulation for these patients
• Start LH Supplementation on First day of COS