principles and practices of lh administration in controlled ovarian stimulation

Principles and Practices of LH administration in

COS 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. Review the principles of LH

supplementation in reproductive cycles 2. Understand the molecular and functional

differences in LH supplementation using the available gonadotropin preparations

3. Appraise the clinical outcome of using LH activity driven by these gonadotropin preparations

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

ANDROFERT

How do we practice? We use LH supplementation during COS in… i.  All patients ii.  Poor responders iii. Hypo-responders iv. Older women (>35) v. Hypo-hypo vi. GnRH antagonist protocol

What are the principles of LH supplementation in

COS?


ANDROFERT

Early follicular phase Steroidogenesis (TC)

Late follicular phase Steroidogenesis (TC)

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

follicular maturation (GC)

LH physiology in reproductive cycles


ANDROFERT

A minimum level of LH is needed to ensure adequate follicular growth and

androgen production Se

rum

LH

UI/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 or hMG LH Cmax 75 IU 0.5 – 1.35 IU/l


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

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

Endogenous LH levels in natural and stimulated cycles

1.6

4.8


ANDROFERT

Among unselected patients treated with FSH and GnRH analogues for IVF, LH supplementation is NOT

associated with a higher 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

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


ANDROFERT

There seems to be NO need of LH supplementation to unselected women treated with FSH and GnRH antagonists

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


ANDROFERT

Androgen levels markedly lower in aged women

Total Testosterone

↓ 55%

DHEAS ↓ 77%

Free Testosterone

↓ 49%

Androstenedione ↓ 64%

n = 1423

Davison SL et al JCEM 2005;90:3847

LH supplementation improves clinical pregnancy in women >35 yo.


ANDROFERT

Pregnancy rates

increased by 30% in poor responders

treated with r-hLH + r-

hFSH

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

rec-hLH improves oocyte yield in poor Responders


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


ANDROFERT

Definition of hypo-responders (initial poor responders) Alviggi et al. RBM online 2006; 2009

•  Normal ovarian reserve •  Follicular growth stagnation D7-D10

with FSH-only stimulation •  Achieve ‘adequate’ response at the

expense of high FSH consumption •  Likely to harbor genetic polymorphism

of LH gene (V-LHβ)


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

•  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


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

How do we practice? We prescribe recombinant LH or hMG, which are the gonadotropin containing LH activity

What are the principles of LH supplementation

using rec-hLH or hMG?


ANDROFERT

Gonadotropins containing LH activity

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

Product LH activity (IU/vial)

LH content Purity

hMG (FSH:LH ratio 1:1) 75 hCG* ~5% HP-hMG (FSH:LH ratio1:1) 75 hCG* ~70% Lutroprin alfa (rec-hLH) 75 LH >99% Follitropin alfa + lutroprin alfa 2:1 ratio (150IU recFSH + 75IU recLH)

75 LH >99%

*95% LH bioactivity in hMG from hCG (concentrated or added during purification process; 8 IU hCG ~ 75 IU LH)


ANDROFERT

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


ANDROFERT

Human Chorionic Gonadotropin •  Glycoprotein produced by

syncytiotrophoblast cells of early embryo and by the pituitary in menopause women

•  In early pregnancy, hCG rescues the corpus luteum and maintains progesterone production until placental steroidogenesis is established


ANDROFERT

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.

How do we practice? In general, it is assumed that LH activity driven by either recLH or hCG (hMG) is similar, and many of us opt to prescribe the less costly medication

What are the principles of LH activity driven by

either rec-hLH or hMG?


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 Activity Sources of LH

hCG

LH 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 produc;on in preovulatory GCs mainly modulated by ERK/PKA

•  In vitro ac;va;on of cAMP pathway associated with apopto;c 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

LH and hCG elicit different gene expression

LH hCG

LHR and FSHR expression Meiosis and follicular matura;on Follicular development Cellular growth Ovarian steroidogenesis Embryo development & survival

Aromatase inhibition Apoptosis

enhancement

LH hCG

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


ANDROFERT

What are the clinical outcomes of using LH

activity driven by either rec-hLH or hMG?


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

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 aOer administra;on of

rec-‐LH to women undergoing COS for IVF

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


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 patients

P=0.02

Type of LH supplementation and pregnancy outcome


ANDROFERT

How we use LH supplementation in

COS for IVF


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 conven;onal s;mula;on

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

§  Follicular stagna;on aOer 6-‐7 days of s;mula;on with r-‐hFSH

2

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

cycles) (1 vial/day)

Start from s;mula;on day 1

(2 vials/day)

Our strategy for LH supplementa;on using 2:1 combina;on of r-‐hFSH + r-‐hLH

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

Age ≥ 35

Start from s;mula;on 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


ANDROFERT

ü LH ac;vity has a crucial role in steroidogenesis, follicular growth and matura;on

ü LH supplementa;on to COS in IVF especially benefi;al to aged women (>35), and poor & hypo-‐responders

ü Significant differences exist between LH and hCG at boh the molecular and func;onal level

ü Preliminary evidence indicates that the choice of products containing LH ac;vity impact IVF clinical outcome


ANDROFERT

Conclusions

Thank you

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

sandroesteves

principles and practices of lh administration in controlled ovarian stimulation

Health & Medicine

lh activity

male reproductions esteves

practices of lh administration

study fsh lh fsh

hmg lh cmax75 iu

androferta minimum level

adequate follicular

serum lh iulsd1 sd8