LH in Human Reproduction (Updated)

Sandro Esteves

Medical Director, ANDROFERTCampinas, Brazil

LH in Human Reproduction

Mexico and Panama Lecture Tour 2014

http://www.androfert.com.br/review

LH in Human Reproduction

Mexico 2014

ANDROFERTandrofert.com.br

ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION

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2014 June

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Learning objectives

At the completion of this presentation, participants should be able to:

1. Understand the role of LH in reproductive cycles

2. Identify patient subgroups to whom LH supplementation is beneficial

3. Understand the differences in LH supplementation available gonadotropin preparations



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Is LH important in reproductive

cycles?a. Absolutely true

b. Maybe true

c. False



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0

9Endometrium (mm)

0

5

10

15

0 5 10 15 20Days of Stimulation

50100

Fo

llicl

e si

ze(m

m)

and

FS

H (

IU/L

)



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025

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

Rec-hLH administration (IU):



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0

25

75225

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

Rec-hLH (IU):



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Early follicular phaseSteroidogenesis (TC)

Late follicular phaseSteroidogenesis (TC)

Up-regulates FSHr expression (GC)

Sustains follicular growth and final follicular

maturation (GC)

Role of LH in reproductive cyclesPhysiology



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Balasch & Fábreques 2002

•Adequate androgen and estrogen biosynthesis, normal follicular development and oocyte maturationN

orm

al

•Follicular atresia•Premature luteinization•Oocyte development compromisedH

igh

•Low (and estrogen) synthesis• Impaired follicular maturation• Inadequate endometrial proliferationLo

wLH Window



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What is the minimum needed

LH level?S

eru

mLH

UI/

L 1.5

1.0

0.5 0.5 Westergaard 20010.7 Fleming 1998

1.2 O’Dea 20001.35 Mahmoud 2001

Injected

rec-hLH

LH Cmax

75 UI 0.5 – 1.35 UI/L



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Is LH important in

reproductive cycles?

a. Absolutely true



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Who need LH supplementation

during ovarian stimulation?

a. All patients

b. Poor responders

c. Hypo-responders

d. Older women (>35)

e. GnRH antagonist protocol



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Natural cycle5.4

3.1

1.68

0.75

0

1

2

3

4

5

6

Seru

m L

H I

U/l

Sd1 Sd8 hCG OPU

0.15

GnRH agonist

Hypo-hypo

GnRH antagonist

LH levels in natural and stimulated cycles

1.6

4.8



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Bioactive LH Levels

30-45% have less sensitive ovariesOlder patients (≥35 years)3

Poor responders4

Slow/Hypo-responders5

Deeply suppressed endogenous LH levels(hypo-hypo; endometriosis treated with GnRH-a)6

Lo

w

1Tarlatzis et al. Hum Reprod 2006; 2Esteves et al. Reprod Biol Endocrinol 2009; 3Marrs et al. Reprod

Biomed Online 2004;4Mochtar MH, Cochrane Database, 2007; 5Alviggi, et al. RBMOnline 2009;6De Placido et al. Clin Endocrinol (Oxf) 2004

No

rma

l

~55-70% normogonadotropic women

undergoing COS1,2



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Among patients treated with FSH and GnRH analogues for in vitro fertilization, is the addition of recombinant LH

associated with the probability of live birth?

0.01 0.1 10 100

Study FSH + LH FSH OR (fixed) Weight OR (fixed)

n/N n/N 95% CI % 95% CI

Agonist

Sills 1999 3/13 10/17 10.00 0.21 [0.04, 1.05]

Balasch 2001 0/16 1/14 2.32 0.27 [0.01, 7.25]

Humaidan 2004 39/116 31/115 31.00 1.37 [0.78, 2.41]

Fabregues 2006 24/60 25/60 22.50 0.93 [0.45, 1.93]

Tarlatzis 2006 6/55 10/59 12.90 0.60 [0.20, 1.78]

Subtotal (95% CI) 72/260 77/265 78.72 0.94 [0.64,1.39]

Antagonist

Sauer 2004 9/25 10/24 9.80 0.79 [0.25, 2.49]

Griesinger 2005 8/62 9/65 11.48 0.92 [0.33, 2.56]

Subtotal (95% CI) 17/87 19/89 21.28 0.86 [0.40,1.85]

Total (95% CI) 89/347 96/354 100.00

]

advantage r-hFSH Advantage r-hFSH + r-hLH

No patient preselection

Kolibianakis, et al. Hum Reprod Update 2007;13:445-452

Is LH needed in unselected women treated with FSH and GnRH

antagonists in IVF?

Mochtar et al.

3 RCT (N=216)

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 oocytesWMD 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 0.89

(95% CI: 0.57; 1.39)

Mochtar et al. Cochrane Database Syst Rev. 2007;2:CD005070;

Baruffi et al, Reprod Biomed Online. 2007;14:14-25.

WMD weight mean difference



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Total Dose per Live Birth

(IU)*

0

3,000

7,000

10,000

21.6%

Rec-FSHHP-hMG

6,324

7,739

hMG

9,69052.2%

*Mean total dose per cycle/Live

birth rate (≤35 years)

Esteves SC et al. Reprod Biol Endocrinol 2009

N=865; GnRH agonist cycles

30.1 32.4

24.4

rec-FSH HP-HMG HMG

LBR (%)p=NS



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Who need LH supplementation during ovarian stimulation?

Key points (1)

Mandatory in the hypogonadotrophic hypogonadal (HH) patients

(FSH and LH<1.2 IU/l)

For most women in IVF, endogenous LH levels, irrespective of the GnRH analogue, is sufficient to support follicular development and steroidogenic activity, so «FSH-only« stimulation is enough



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Impaired oocyte quality

Decreased fertilization rate

Reduced embryo quality

Increased miscarriage rates

Reduced ovarian

paracrine activity

Hurwitz &

Santoro 2004

Androgen secretory capacity reduced

Piltonen et al., 2003

Decreased number of

functional LH receptors

Vihko et al.

1996

Reduced LH bioactivity

Mitchell et al. 1995; Marama et al 1984

3-5 in every 10 treated women have aged ovaries



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3-5 in every 10 treated women have aged ovaries



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LH supplementation improves clinical pregnancy in women >35

years old



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Fertil Steril 2011

Imp

lan

tati

on

rate

(%)

p=0.03

OR: 1.56 (1.04-2.33)

p=0.84

OR: 1.03 (0.73-1.47)

27.8

18.9

28.6

26.7

<=35

36-39

FSH+LH FSH



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Bologna Criteria for Poor Responders Ferraretti et al. ESHRE Consensus, Hum Reprod 2011

At least 2 of the following:

1. Advanced maternal age ≥40 years or risk factor for POR

2. Previous POR ≤3 oocytes with conventional stimulation

3. Abnormal ovarian reserve biomarker AFC<5-7; AMH <0.5-1.1ng/mL

Or Two episodes of POR after maximal stimulation



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Lehert et al Reprod Biol

Endocrinol 2014, 12:17



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Pregnancy rates

increased by 30% in poor responders treated with

rLH+rFSH

Lehert et al 2012ANDROFERT

androfert.com.br


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Significant

increase of

0.75 oocytes

per 1,000 UI

gonadotropin

administered

Lehert et al Reprod Biol

Endocrinol 2014, 12:17

rec-hLH improves oocyte yield in Poor Responders

Why is LH beneficial in aged women and poor responders?

Total

Testosterone

55%

DHEAS

77%

Free

Testosterone

49%

Androstenedione

64%

n = 1423

Davison SL et al JCEM 2005;90:3847

It seems to be a matter of androgensand the anti-apoptoticeffect of LH

• Action of LH at the follicular level in a dose dependent manner increases androgen production

• Androgens are then aromatized to estrogensand help restore the follicular milieu

Rationale of LH supplementation (1)



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Rationale of LH supplementation (2)

Anti-apoptotic effect on

granulosacells

Up-regulate growth factors

Increase FSH receptor

responsiveness

Act synergistically

with IGF-1

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

Evidence of a beneficial effect in older women (≥35 yrs.) and poor responders

Benefit related to increased androgen production and direct efect on the ovary

better follicular recruitment higher number of oocytesbetter implantation rate


Key points (2)



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Definition of hypo-responders (initial poor responders) Alviggi et al. RBM online 2006;

2009

• Normal ovarian reserve

• May present follicular growth plateau on D7-D10

• Achieve ‘adequate’ number of oocytesretrieved and estradiol production

• But at the expense of an increased cumulative rFSH dose (i.e. >3000 IU) and duration of stimulation



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Why is there a suboptimal response to exogenous FSH in

hypo-responders?

LH gene polymorphism: V-LHb

Carrier frequency 0-52% in various ethnic groups

13 % in Sweden

12-13 % in Denmark and Italy

Associated with reduced bioactivity of LH

Huhtaniemi et al., 1999; Jiang et al., 1999; Ropelato et al., 1999



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The cumulative FSH consumption is higher in carriers of v-beta LH

polymorphism

Alviggi et al. Reproductive Biology and Endocrinology, 2013



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Hypo-responders benefit from LH Cochrane review 2007

Mochtar MH, Cochrane Database, 2007 issue 2

Favours r-hFSH Favours r-hFSH + r-hLH

Ongoing PR per woman randomized

(COS in a GnRH-agonist dow-regulated IVF/ICSI cycle)



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6 9 111014

1822

3240

FSH step-up (+150UI)

LHsupplementation

(+150 UI)

NormalResponders

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)

LH supplementation in Hypo-responders



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Evidence of a beneficial effect of LH supplementation in hypo-responders (initial poor responders)

Dose-related increased LH bioactivity with a positive effect on androgen production and ovarian function


Key points (3)



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Who need LH supplementation

during ovarian stimulation?a. All patients

b. Poor responders

c. Hypo-responders

d. Older women (>35 yrs.)

e. GnRH antagonist protocol



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ANDROFERT

What product to use for LH

supplementation?

a. hMG/HP-hMG

b. rec-hLH

c. Either of the above; they

are similar



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ANDROFERT

Products containing LH Activity

Leao & Esteves. Clinics 2014; 69(4): 279–293.



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Fertil Steril 2012; 97(3): 561-72



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Beta unit Carboxyl

terminal

segment

Longer in

hCG

Higher

receptor

affinity in hCG

Absent in LH and present

in hCG

Longer half-life in

hCG

Sources of LH ActivitySources of LH

hCG

LH

Leao & Esteves. Clinics 2014; 69(4): 279–293.

Courtesy of Xuliang Jiang

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 receptors:• Constitutively expressed

on theca cells

• Expressed on granulosacells at a follicle size of 8-12 mm



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Divergence in receptor-mediated signaling between LH and hCG

Choi & Smitz Mol Cell Endocrinol 2014; 383(1-2):203–13.



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Casarini et al PloS One 2012;7:e46682

Half-life in serum and differential downstream effects of r-hLH and r-hCG binding to LHCGR.

Cell type r-hLH r-hCG

Half-life

t1/2Î±<comma> range of mean<comma> hrs 0.6â€“1.3a<comma>b 3.9â€“5.5c

t1/2Î²<comma> range of mean<comma> hrs 9â€“12a<comma>b 23â€“31c

Response

ED50 (pM) COS-7/LHCGRe 530.0 Â± 51.2 107.1 Â± 14.3

Time to maximal cAMP accumulationd COS-7/LHCGRe 10 min 1 h

ERK1/2 activationf hGLC Strong Weak

AKT activationf hGLC Strong Minimal

Neuregulin 1 inhibition in presence of ERK1/2 and AKT pathway blockade hGLC Abrogated Unaffected

CYP19A1 expression in presence of ERK1/2 pathway blockade hGLC Increased Unaffected

…LH and hCG downstreamcascade pathways are different

LH hCG

LHR and FSHR expression (Trafficking of retinoic acid : RXRB, TTR, ALDH8A1)

Meiosis and follicular maturation (TRA : RXRB, TTR, ALDH8A1; IL11; AKT3)

Follicular development (IL11; AKT3)

Cellular growth (RXRB, TTR, ALDH8A1;

IL11;AKT3)

Ovarian stereodogenesis(TRA : RXRB, TTR, ALDH8A1)

Embryo development & survival (AKT3)

Aromataseinhibition(PPARS)

Apoptosis enhancement

(DNAsi)

LH hCG

Grondal ML et al. Fertil Steril 2009; Menon KM et al. Biol Reprod 2004;; Ruvolo et al. Fertil Steril 2007

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 ofStimulation(days)

Mean No.oocytesretrieved

IR (%)

CPR pertransfer (%)

Buhler KF, Fisher R. Gynecol Endocrinol 2011

Matched case-control study; N=4,719 IVF pts.

P=0.02

Does it matter whether hMG hCG(hMG) or rec-hLH?



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• RCT comparing rec-hFSH + rec-hLH

(2:1) vs. HP-hMG

• Higher No. oocytes retrieved in the rFSH

+ rLH (2:1) group (9.8 vs 7.3; p<0.01)

• 2/3 of the patients in rFSH+rLH group

(vs. 1/3 hMG group) had frozen embryos

to transfer if fresh transfer failed

Fábregues F et al. Gynecol Endocrinol. 2013 May;29(5):430-5.

Does it matter whether hMG hCG(hMG) or rec-hLH?



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Significant differences exist between

LH and hCG at boh the molecular and

functional level

Preliminary evidence indicates that the

choice of products containing LH

activity impact IVF clinical outcome

What product to use for LH supplementation?

Key points



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What product to use for LH

supplementation?

a. hMG/HP-hMG

b. rec-hLH

c. Either of the above; they

are similar



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ANDROFERT



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How we use LH

supplementation

at Androfert

Ovarian stimulation protocol



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iCOS at Androfert

Dosage based on clinical profile and AMH/AFC

Morning injections

rec-hFSH 112.5-150 IU high responders

rec-hFSH 187.5-225 IU normal responders

rec-hFSH+rec-hLH (2:1 ratio) poor/hypo-responders; women ≥35 yrs.

• Antagonist regimen Daily evening injections Flexible protocol (when follicle reaches 12-14mm) Last injection day of trigger (morning)

Individualization of COS Androfert



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• Monitoring: TVUS + E2 days 1; 6; 9-10

• Ovulation trigger not before stimulation D9Trigger when 2-3 follicles =>17 mm

Rec-hCG or GnRH-agonist (triptorelin 0.2mg SC)

No (FSH/LH) stimulation on triggering day

Freeze all in GnRH-a cycles

• Oocyte pick-up: 35h post-trigger

• LPS early pregnancy (9-12 weeks)Transdermal estradiol + vaginal progesterone gel (90mg)

Individualization of COS Androfert



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Population Cut-off Sensitivity Specificity Accuracy

AMH*

ng/m

L

High-

responder1 2.1 85% 79% 0.82

Poor

responder2 0.82 76% 86% 0.88

*Beckman-Couter generation II assay; 1>20 oocytes retrieved; 2≤4 oocytes retrieved

Leão RBF, Nakano FY, Esteves SC. Fertil Steril 2013; 100 (Suppl.): S16

Biomarkers of ovarian responseAMH



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Rec-hFSH + rec-hLH (2:1 ratio) from stimulation D1

Total dose: 300 IU FSH + 150 IU LHGnRH antagonist (flexible): mean diameter 13mm

LH trigger with rec-hCG (mean diameter 17-18 mm)

Our Preferred Stimulation Regimen in Expected Poor Responders

2 3 4 5 76 8 9 10 111

Menses

12



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Individualized vs. Conventional COS

in Expected Poor Responders (N=118)

72.0

3.5

45.0

20.0

46.6

4.8

23.3 26.8

0

20

40

60

80

Observed PoorResponse (%)

Oocytesretrieved (N)

Cancellation (%) Pregnancy/cycle(%)

cCOS (Long GnRH with recFSH)iCOS (GnRH Antag. with rFSH+rLH)

Expected poor response: AMH<0.82 ng/dL; Observed poor response <5 oocytes retrieved;

Leão RBF, Nakano FY, Esteves SC. Fertil Steril 2013; 100 (Suppl.): S16.

*p<0.05

*

*

*



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GnRH antagonist flexible protocol

Rec-hFSH + rec-hLH (2:1 or 3:1 ratio) from D1

Total dose: 150-225 IU FSH + 75 IU LH

How tse LH in Coin SLH supplementation in women ≥35 years and hypo-responders

(normal ovarian biomarkers)

2 3 4 5 76 8 9 10 111

Menses

12



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LH in Human Reproduction Conclusions

Adequate LH levels critical for steroidogenesis, follicular development and oocyte maturation

Androgen secretory capacity decreases with ovarian aging

Mechanisms include decreased number of functional LH receptors and ovarian paracrine activity. LHr polymorphisms involved in hypo-responders



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Many patients will benefit from LH supplementation during COS:

Poor/hypo respondersAge >35 years; hypo-hypo

Sources are rec-hLH and hMGLH and hCG differ at molecular, functional and clinical levels

iCOS with rec-hLH is one of our strategies to maximize pregnancy in IVF

LH in Human Reproduction Conclusions



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Thank Y

ou

gra

cia

s

LH in Human Reproduction (Updated)

Health & Medicine

Transcript of LH in Human Reproduction (Updated)