Principles and Practices of LH Use in ART

Principles and Practices of LH administration in ART

Sandro C. Esteves, MD., PhD. Medical Director, ANDROFERT

Andrology & Human Reproduction Clinic Campinas, BRAZIL

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. Appraise the differences in LH

supplementation using the available gonadotropin preparations

ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 2 2015

ANDROFERT

Principles and Practices of LH administration in ART

Kingdom of Saudi Arabia 2014


ANDROFERT

This presentation is available at http://www.slideshare.net/

sandroesteves

Is LH important in reproductive

cycles? a.  True b.  Maybe true c.  False


ANDROFERT

0

9 Endometrium (mm)

0

5

10

15

0 5 10 15 20 Days of Stimulation

50 100

Follic

le siz

e (m

m)

and

FSH

(IU/L

)


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WHO type I treated with r-hFSH (150 IU) + r-hLH (75 IU) in a 2:1 ratio combination

17 patients; 27 cycles IU FSH ± SEM 1922 ± 266 IU LH ± SEM 961 ± 133 Days stimulation ± SEM 13.8 ± 1.8 % PR cycle 55.5% % PR patient 88.3% N follicles >17mm ± SEM 4.3 ± 2.4 E2 hCG day (pmol/l) 541 ± 299 Mid-luteal P4 (nmol/l) 40 ± 14 Endometrium sd10 (mm) 11 ± 3 E2/follicle (pmol/l) 152 ± 64

Carone et al. J Endocrinol Invest 2012


ANDROFERT

Early follicular phase Steroidogenesis (TC)

Late follicular phase Steroidogenesis (TC)

Up-regulates FSHr expression (GC) Sustains follicular growth and final

follicular maturation (GC)

Physiology of LH in reproductive cycles


ANDROFERT

Balasch & Fábreques 2002

• Adequate androgen and estrogen biosynthesis

• Normal follicular development and oocyte maturation N

orm

al

• Follicular atresia • Premature luteinization • Oocyte development compromised H

igh

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

w

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 2001 0.7 Fleming 1998

1.2 O’Dea 2000 1.35 Mahmoud 2001

Injected rec-hLH

LH Cmax

75 IU 0.5 – 1.35 IU/L


ANDROFERT

Is LH important in reproductive cycles?


ANDROFERT

a.  True b.  Maybe true c.  False

Who need LH supplementation

during ovarian stimulation?

a.  All patients b.  Poor responders c.  Hypo-responders d.  Older women (>35) e.  GnRH antagonist protocol


ANDROFERT

u Natural cycle 5.4

3.1

1.68 0.75

0

1

2

3

4

5

6

Seru

m L

H IU

/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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Among patients treated with FSH and GnRH analogues for IVF, is the addition of rec-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

Unselected Patient Population

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


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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

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


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

mal

~55-70% normogonadotropic women undergoing COS1,2


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LH supplementation improves clinical pregnancy in women >35 yo.


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Fertil Steril 2011

Impl

anta

tion

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

r-FSH + r-hLH*


ANDROFERT

*75 IU r-hLH form Sd1

Poor responders

Pregnancy rates

increased by 30% in poor responders

treated with rLH+rFSH

Lehert et al Reprod Biol Endocrinol 2014, 12:17


ANDROFERT

Lehert et al 2012

Significant increase of

+0.75 oocytes in poor

responders treated with

r-hFSH + r-hLH

Lehert et al Reprod Biol Endocrinol 2014, 12:17


ANDROFERT

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

Live

birt

h ra

te (%

)

Oocyte number

Observed live birth rate Predicted live birth rate

Sunkara et al. Hum. Reprod., 2011

400,135 IVF cycles

Number of Oocytes and LBR


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On average, one additional embryo for transfer or cryopreservation

Air Quality Control and GMP 2,315 patients; 14,660 embryos


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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

•  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)


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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

Hypo Responders

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-LHβ Carrier frequency 0-52% in various ethnic groups

ü 13 % in Sweden ü 12-13 % 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. Reproduc0ve 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 11 10 14 18 22 32

40

FSH step-up (+150 UI) LH supplementation (Sd8)

Normal Responders

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


ANDROFERT

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


ANDROFERT

What product to use for LH

supplementation? a.  hMG/HP-hMG b.  rec-hLH c.  Either of the above; they

are similar


ANDROFERT

Gonadotropins containing LH activity

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

Product LH activity (IU/vial)

LH content* Purity

hMG 75 hCG ~5% HP-hMG 75 hCG ~70% Lutroprin alfa (rec-hLH) 75 LH >99% Follitropin alfa + lutroprin alfa in a 2:1 ratio combination

75 LH >99%

*hCG concentrated or added during purification process (8IU hCG ~ 75IU LH)


ANDROFERT

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


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Extracellular fluid

Cytoplasm

Plasma membrane

LH hCG

LH/hCG receptor

Sharing the same α subunit and 81% of AA residues of β subunit, LH and hCG bind to the same receptor

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

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

1Effect 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


ANDROFERT

Divergence in receptor-mediated signaling between LH and hCG

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


ANDROFERT

•  ERK/PKA & AKT cell survivor regulators and apoptosis blockers

•  P produc0on in preovulatory GCs mainly modulated by ERK/PKA

•  In vitro ac0va0on of cAMP pathway associated with apopto0c events

ERK/PKA & AKT pathway (LH) cAMP (hCG)

ERK/PKA & AKT pathways

Casarini et al., 2012; Grzesik et al., 2014


ANDROFERT

•  LH significanly more potent to induce EREG gene expression

•  Epiregulin plays a key role in oocyte matura:on

Epiregulin (EREG) pathway

Chin & Abayasekara, 2004; Sekiguchi et al., 2004


ANDROFERT

0"20"40"60"80"100"120"

2PN$ Preg.$ IR$ DNA$fragmenta2on$

r4$FSH$hMG$r4FSH$+$r$LH$

*P<0.01

*

* *

Lower apoptosis rate (marker of oocyte quality) in human cumulus cells aQer administra0on of

rec-‐LH to women undergoing COS for IVF

Ruvolo et al. Fertil Steril 2007; 87:542-6


ANDROFERT

• Cross-over study (n=66) comparing rec-hFSH + rec-hLH (2:1) vs. HP-hMG

•  All 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;29(5):430-5.

Type of LH supplementation and number of oocytes retrieved

7.3 9.8

No. oocytes retrieved

HP-HMG rec-FSH + rec-LH

p<0.01


ANDROFERT

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 (%)

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?


ANDROFERT


ANDROFERT

ü Significant differences exist between LH and hCG at boh the molecular and functional level

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

What product to use for LH supplementation?

Key points


ANDROFERT

How we use LH supplementation

at Androfert


ANDROFERT

Ovarian stimulation protocol


ANDROFERT

Population Cut-off Sensitivity Specificity Accuracy

AMH*ng/mL

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 response AMH


ANDROFERT

Rec-hFSH + rec-hLH (2:1 ratio) from Sd1 Gonadotropin dose per day 450 IU: Ø  rec-hFSH 300 IU + rec-hLH 150 IU)

GnRH antagonist (flexible): mean 13mm LH trigger with rec-hCG (mean 17-18 mm

Our preferred regimen in expected poor responders

(AMH≤0.82 and/or history of POR)

2 3 4 5 7 6 8 9 10 11 1

Menses

13


ANDROFERT

12

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 Poor Response (%)

Oocytes retrieved (N)

Cancellation (%) Pregnancy/cycle (%)

cCOS (Long GnRH with r-hFSH) iCOS (GnRH Antag. with r-hFSH+r-hLH)

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 protocol Rec-hFSH + rec-hLH (2:1 ratio) from Sd1

Gonadotropin dose/day 225 IU: Ø  rec-hFSH 150 IU + rec-hLH 75 IU

How tse LH in Coin S Our preferred regimen in women ≥35yr. and normal ovarian reserve

(AMH>0.82)

2 3 4 5 7 6 8 9 10 1

Menses

13


ANDROFERT

11 12

GnRH antagonist flexible protocol; i.  r-hFSH + r-hLH (2:1 ratio) from Sd6-7


ANDROFERT

Our preference in hypo-responders (Age <35yr.; AMH >0.82; follicular stagnation

(<10mm) Sd5-7)

Gonadotropin dose per day: 225 IU

2 3 4 5 7 6 8 9 10 11 1 Menses

14

ii.  r-hFSH + r-hLH (2:1 ratio) from Sd1 2 3 4 5 7 6 8 9 10 1 13 11 12

12 13

Expected poor responders

§  AMH ≤ 0.82 ng/ml §  History of previous IVF a_empt with poor response at a conven0onal s0mula0on

Hypo responders §  < 35 yr. §  AMH >0.82 ng/ml

§  Follicular stagna0on aQer 6-‐7 days of s0mula0on with r-‐hFSH

2

Start from Sd6-‐7 (1st cycle) Start Sd1 (subsequent

cycles) (1 vial/day)

Start from s0mula0on day 1

(2 vials/day)

Our strategy for LH supplementa0on using 2:1 combina0on of r-‐hFSH + r-‐hLH

§  Expected normo-‐responder (AMH >0.82 ng/ml and no history POR)

Age ≥ 35

Start from s0mula0on day 1

(1 vial/day)

3 1


ANDROFERT

40.4% 48.0%

ET #3 (FET) 49

ET #2 (FET) 239

ET #1 (fresh) 822

50.5% +18.8%

+25.0% Female Age ≤39 ANDROFERT

332/822 63/239 17/49

Cumulative LBR – IVF/ICSI

Year 2012


ANDROFERT

Conclusions 1. Adequate LH levels critical for

steroidogenesis, follicular development and oocyte maturation

2. 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

3. Patients most likely to benefit from 2:1

fixed FSH/LH combination during COS: Poor/hypo responders

Age >35 years; hypo-hypo 4. rec-hLH and hMG sources of LH-

acitivity LH and hCG differ at molecular and functional levels

5. iCOS with 2:1 FSH/LH combination has been one of our strategies to maximize success in IVF


ANDROFERT

Conclusions

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Principles and Practices of LH Use in ART

Health & Medicine

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