LH in Human Reproduction

Sandro C. EstevesDirector, ANDROFERT

Campinas, Brazil

LH in Human Reproduction

Sesiones Científicas - Sociedad Peruana de FertilidadJunio 2014 - Lima PERU

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

LH in Human Reproduction

ANDROFERTandrofert.com.br

ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTIONS ESTEVES, 22014 June

ANDROFERT

Sesiones Científicas - Sociedad Peruana de FertilidadJunio 2014 - Lima PERU

Learning objectivesAt the completion of this presentation, participants should be able to: 1. Understand the role of LH in

reproductive cycles2. Identify patient subgroups to whom

LH supplementation is beneficial3. Understand the differences in LH

supplementation according to gonadotropin preparations



ANDROFERT

Is LH important in reproductive

cycles?

1a. Absolutely trueb. Maybe truec. False



ANDROFERT

Hypogonadotropic hypogonadism treated with FSH

alone

FSH dose0

9Endometrium (mm)

0

5

10

15

0 5 10 15 20Days of Stimulation

Serum FSH

50100

Follicles

Estradiol(pg/mL)

Folli

cle

size

(mm

)an

d FS

H (IU

/L)

Estradiol levels



ANDROFERT

Day 1 Day 5 Day 10 hCG0

50010001500200025003000

025

75

225

Day of Stimulation

Seru

m E

stra

diol

Lev

els

(pm

ol/L

)

The European Recombinant Human LH Study Group, JCEM 1998; 83:1507

Rec-hLH administration (IU):

Evidence of a LH threshold (1)



ANDROFERT

Day 1 Day 5 Day 10 hCG0

2

4

6

8

025

75225 0 25 75 225 rLH

Day of StimulationThe European Recombinant Human LH Study Group, JCEM 1998; 83:1507

Endo

met

rial T

hick

enes

s (m

m)

Rec-hLH (IU):

Evidence of a LH threshold (2)



ANDROFERT

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 cycles

Physiology



ANDROFERT

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 proliferationLow

LH Window



ANDROFERT

What is the minimum needed LH level?

Seru

m L

H U

I/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



ANDROFERT

Is LH important in reproductive

cycles?1

a. Absolutely true



ANDROFERT

Who need LH supplementation

during ovarian stimulation?

2a. All patientsb. Poor respondersc. Hypo-respondersd. Older women (>35)e. GnRH antagonist protocol



ANDROFERT

Natural cycle5.4

3.1

1.68 0.7

50

1

2

3

4

5

6

Seru

m L

H IU

/l

Sd1 Sd8 hCG OPU0.15

GnRH agonistHypo-hypoGnRH antagonist

LH levels in natural and stimulated cycles

1.6

4.8



ANDROFERT

threshold

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

No, for unselected

pts.

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 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 0.89

(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



ANDROFERT

No, it is not.

Total Dose per Live Birth (IU)*

0

3,000

7,000

10,000

21.6%

Rec-FSHHP-hMG

6,3247,739

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

rec-F

SH

HP-HMG

HMG

30.1 32.4 24.4LBR (%)

p=NS



ANDROFERT

Who need LH supplementation during

ovarian stimulation?Key points (1)

2Mandatory 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



ANDROFERT

Bioactive LH Levels

30-45% have less sensitive ovaries

Older patients (≥35 years)3

Poor responders4

Slow/Hypo-responders5

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

Low

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

Nor

ma

l~55-70% normogonadotropic women undergoing COS1,2



ANDROFERT

Impaired oocyte qualityDecreased fertilization

rateReduced embryo qualityIncreased miscarriage

rates Reduced ovarian

paracrine activity

Hurwitz & Santoro 2004

Androgen

secretory

capacity reducedPiltonen 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



ANDROFERT

LH supplementation improves outcome in women

>35 yo



ANDROFERT

Fertil Steril 2011Im

plan

tati

on r

ate

(%)

p=0.03OR: 1.56 (1.04-2.33)

p=0.84OR: 1.03 (0.73-1.47)



ANDROFERT

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 POR2. Previous POR

≤3 oocytes with conventional stimulation

3. Abnormal ovarian reserve biomarker

AFC<5-7; AMH <0.5-1.1ng/mLOr Two episodes of POR after

maximal stimulation



ANDROFERT

Pregnancy rates

increase by 30% in

poor responders

treated with rec-

hLH

Lehert et al 2012



ANDROFERT

rec-hLH improves oocyte yield in Poor Responders

Significant increase of 0.75 oocytes per 1,000 UI gonadotropin administered

Lehert et al 2012

Lehert et al 2012



ANDROFERT

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 in part a matter of androgens

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

• Androgens are then aromatized to estrogens and help restore the follicular milieu

Rationale of LH supplementation (1)



ANDROFERT

Rationale of LH supplementation (2)

Anti-apoptotic effect on

granulosa cells

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

2Evidence 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





ANDROFERT

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

oocytes retrieved and estradiol production

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



ANDROFERT

Why is there a suboptimal response to exogenous FSH

in hypo-responders? LH gene polymorphism: V-LHbCarrier frequency 0-52% in various ethnic groups

13 % in Sweden12-13 % in Denmark and Italy

Associated with reduced bioactivity of LH

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



ANDROFERT

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

polymorphism

Alviggi et al. Reproductive Biology and Endocrinology, 2013



ANDROFERT

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)



ANDROFERT

6 9 1110 14 1822 32 40

Mean No. oocytes retrievedIR (%)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)

P<0.05

LH supplementation in Hypo-responders



ANDROFERT

2Evidence 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





ANDROFERT

Who need LH supplementation

during ovarian stimulation?

2a. All patientsb. Poor respondersc. Hypo-respondersd. Older women (>35 yrs.)e. GnRH antagonist protocol



ANDROFERT

What product to use for LH

supplementation?

3a. hMG/HP-hMGb. rec-hLHc. Either of the above; they

are similar



ANDROFERT

Products containing LH Activity

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



ANDROFERT

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



ANDROFERT



ANDROFERT

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

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

hCG

Although they attach to the same receptor (LHCG-

R)…

Courtesy of Xuliang Jiang, EMD Serono 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



ANDROFERT

…LH and hCG downstreamcascade pathways are

differentLH hC

GLHR 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 steroidogenesis (TRA : RXRB, TTR, ALDH8A1)Embryo development & survival (AKT3)

Aromatase inhibition (PPARS)

Apoptosis enhanceme

nt (DNAsi)

LH hCG

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

Fixed 2:1 r-hFSH (150IU)/r-

hLH (75IU)

HMG rec-hFSH + HMG

0

5

10

15

20

25

30

35

19

14 14

3126 25

Duration of Stimulation (days)

Mean No. oocytes re-trieved

IR (%)

CPR per trans-fer (%)

Buhler KF, Fisher R. Gynecol Endocrinol 2011

Matched case-control study; N=4,719 IVF pts.P=0.0

2

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



ANDROFERT

• 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?



ANDROFERT

3Significant differences exist

between LH and hCG at boh the molecular and functional level

Limited evidence indicates that the choice of products containing LH activity impact IVF clinical outcome

What product to use for LH supplementation?

Key points



ANDROFERT

What product to use for LH

supplementation?

3a. hMG/HP-hMGb. rec-hLHc. Either of the above; they

are similar



ANDROFERT



ANDROFERT

How we use LH supplementati

on at Androfert

4

Ovarian stimulation protocol

• Clinical features• Age• Ovarian volume• AMH/AFC

Identify who is who

• Patient friendly• Efficacy• Effectiveness• Efficiency • Safety

Protocol



ANDROFERT

Population Cut-off Sensitivity

Specificity

Accuracy

AMH*ng/mL

High-responder1

2.1 85% 79% 0.82Poor 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



ANDROFERT

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

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

Rec-hCG 250mcg

12



ANDROFERT

Individualized vs. Conventional COSin Expected Poor

Responders (N=118)

020406080 72.0

3.5

45.020.0

46.6

4.823.3 26.8

cCOS (Long GnRH with recFSH)

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

*

**



ANDROFERT

GnRH antagonist flexible protocolRec-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

Rec-hCG 250mcg

12



ANDROFERT

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



ANDROFERT

Patients that could 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



ANDROFERT

Than

k Yo

u

grac

ias

LH in Human Reproduction

Health & Medicine

Transcript of LH in Human Reproduction