The Role of LH in COS

Hamburg 23.9. 2016

Robert Fischer

MVZ Fertility Center Hamburg GmbH

rfischer@fertility-center-hh.de

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

To LH or not to LH ?

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

Jeppepesen et al.J Clin Endocrinol Metab. 2012 Aug. 97(8) 1524-1531

Courtesy of C.Alviggi

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

LH supplementation for women >35 years is beneficial

Hill et al Fertil Steril 2012 ”

< 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;

König et al. Hum Reprod 2014

Different findings

when LH is started

on day 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

Gatta et al.,F&S 2013

Courtesy J.N.Hugues

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

Courtesy J.N.Hugues

Courtesy J.N.Hugues

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

Thank you for your time