Effects of HCG and LH on ovarian stimulationPituitary hCG in the postmenopausal woman 12 Snyder JA et al. Clin Chem. 2005;51(10):1830-1835.Nonpregnant cohort n hCG range, IU/L 97.5

Effects of HCG and LH on ovarian stimulation

Are they bioequivalent ?

Know the type of gonadotrophin required to have enough oocytes of good quality to achieve a healthy child

FSH

IVF

Rob Gilchrist courtesy, adapted

MAXIMIZE EFFICIENCY

MINIMIZE RISK

Pleiotropic effects of gonadotrophins

• rescue follicle(s) from atresia : “recruitment”

• Influence steroid balance : A-E-P

• Effect on ovary

• Effect on implantation

• Diverse effect in total body :

• Risks : OHSS

3

ART treatment pursues several goals at once

• Focus on ovary : many mature follicles

• Endometrium development

• Implantation window

• Minimum of side effects

Can all these goals be reconciled ?

CLEAR TREND TO SEGMENTATION

4

Objectives of the lecture

• LH and hCG: functions, mechanisms of action,

available formulations ( hMG & HP-hMG)

• LH and hCG: understanding the differences

• Defining optimal LH levels

• Results from the daily practice

5

• Molecular structures : gonadotrophin dimers

• The LH effect on the ovarian follicle

6

LH and hCG:

The family of pituitary glycoproteins

7

Strauss J et al, eds. Yen and Jaffe’s Reproductive Endocrinology. 5th ed. 2004.

hLH

&

hCG

hFSH

ɑ-chain

hFSH

hLH

hCG

Asn 52

92 AA

Asn 78

Asn 7

hFSHAsn 24 111 AA

hLH Asn 30115 AA

Ser 121Ser 132

Ser 127Ser 138

Asn 30Asn 13hCG

β-chain

145 AA

2-cell, 2-gonadotropin model

of hormone production

8

Blood GRANULOSA CELLTHECA CELL

ANDROSTENEDIONE

DHEA

cAMP

Cholesterol

Pregnenolone

17-OH Pregnenolone

Protein

Kinase A

17βHSD1CYP17

CYP17

3βHSD

Protein

Kinase AP450arom

Basement Membrane

ESTRADIOL-17β

ESTRONE

ANDROSTENEDIONE

cAMP

CYP11A1

FSHRRLH R

Adapted from Wilson JD et al, eds.

Carr BR. In: Wilson JD et al, eds. Williams Textbook of Endocrinology. 9th ed. Philadelphia, PA: WB Saunders; 1998:751-817.

(Has2,'Tnfaip6,'Ptgs2)

Ccnb1,'Mos,'Mad2,'Bub1b,'Sogl2,'Tex19.1,'Tpx2,'Dazl,'others'

'

Mural&Cells&

LH bioactivity relation to the oocyte

Cumulus-oocyte ‘capacitation’ for development

• hMG–human menopausal gonadotropin— 75 IUs of FSH activity and 75 IUs of LH activity

— HP-hMG–highly purified urinary hMG

• Urinary purified hCG

• Recombinant hCG

• Recombinant LH

Products that provide LH bioactivity

10

Origin of hCG in HP-hMG• Pituitary gonadotrophs produce hCG

– Up to 1/3 of follicular-stage LH activity may derive

from hCG in a natural cycle1

– Lack of E2 feedback during menopause/ovarian

failure increases pituitary hCG production2,3

• Pituitary hCG and placental/urinary hCG are largely

similar, according to4:

– Amino acid analysis

– Mass spectrometric studies

11

1. Cole LA. Reprod Biol Endocrinol. 2010;8:102-116.2. Stenman U-H et al. J Clin Endocrinol Metab. 1987;64(4):730-736.3. Cole LA et al. J Reprod Med. 2004;49(6):423-432. 4. Birken S et al. Endocrinology. 1996;137(4):1402-1411.

Pituitary hCG in the postmenopausal woman

12

Snyder JA et al. Clin Chem. 2005;51(10):1830-1835.

Nonpregnant cohort n

hCG range,

IU/L

97.5

percentile,

IU/L P

Premenopausal,

18-40 years240 <2.0 to 4.6 2.5

Perimenopausal,

41-55 years240 <2.0 to 7.7 4.8 0.07*

Postmenopausal,

>55 years240 <2.0 to 13.1 7.7 <0.000†

*Compared with the nonpregnant premenopausal cohort.

†Compared with the nonpregnant premenopausal and nonpregnant

perimenopausal cohorts.

Table 1. hCG concentration ranges and the 97.5 percentile

values for the nonpregnant cohorts in the study.

0

2

4

6

8

10

12

14

20 40 60 80 100Age (years)

hC

G (

IU/L

)

The purification process and why hCG is

in HP-hMG

• Postmenopausal pituitary cells secrete gonadotropin into the body’s circulation,

where it is filtered and excreted by the kidneys

• During purification, LH is sequentially moved away from hCG, resulting in

3 fractions

• Fraction 1 is then combined with the other 2 fractions to reach a 1:1 ratio of FSH

and LH activity, as measured by bioassay, producing HP-hMG

13

Fraction 1FSH, little LH

Fraction 3hCG

Finalchromatography

step

Fraction 2FSH, LH, and hCG

HP-hMG is human-derived FSH with approximately 10 IUs of hCGdriving the majority of the 75 IUs of LH activity.

HP-hMG is NOT hMG

14

Source of various gonadotropins since 1985

hMG2 hMG2 hMG2 rFSH2 HP-hMG2

LH (IU/vial) 13.5 5.8 7.4 - 0.4

hCG (IU/vial) 3.4 6.9 7.2 - 9.9

Approximately 95% of the LH bioactivity in HP-hMG is

provided by hCG3

1. FDA Orange Book. www.accessdata.fda.gov. 2. Wolfenson C et al. Reprod Biomed Online. 2005;10(4):442-454. 3. van de Weijer BHM et al. Reprod Biomed Online. 2003;7(5):547-557.

19851 19941 19991 20041 20041

http://www.accessdata.fda.gov

Conclusions

• The gonadotropin content of HP-hMG

is distinct from hMG

• HP-hMG is a highly purified product derived from the

urine of postmenopausal women

– suitable for SC injection

– the majority of the LH activity is "hCG-driven"

15

16

LH and hCG:

understanding the differences in effects

Differences LH and hCG

LH hCG

AA number beta subunit 121 145Receptor binding affinity Low High*No. Glycosylation sites1 3 6Initial half-life (h) 0.6-1.3 3.9-5.5Serum halflife (h) 2 1.5 34Bioequivalency 6-8 IU 1 IU

1 N-linked and O-linked glycosylation sites in beta subunit* 2-3x higher than LH

Leao and Esteves. Clinics 2014Choi and Smitz. Mol Cell Endocrinol 20142Mann K. Oncodev Biol Med 1980

• Proximal :• cAMP• intracellular proteins ( CREB , ERK ,…)

• Distal :• gene expression ( QRT-PCR )• proteins : Amphiregulin (AREG) , Epiregulin (EREG)• steroids : progesterone , estradiol

EVALUATE EFFECTS LH - hCG IN VITRO: on Proximal or Distal output parameters

hLHCG receptor

Casarini et al., Mol Cell Endocrinol 2016

Exposure to GC in vivo ?10 IU/L= 10.7pM10 IU/L is dose when3Amps Menopur injected

Therefore, on cAMP:

• Higher in vitro potency of hCG vs LH (about 5-fold, extractive or recombinant, any cell model)

• Faster maximal response after LH vs hCG(10 min vs. 60 min, COS7-LHCGR and hGLC)

LH and hCG act with different potency and kinetics

Molecular effects are not SIMILAR

Signal transduction / gene expression




Conclusions of in vitro experiments :LH and hCG are NOT equivalent in vitro

• LH and hCG are biochemically different

• 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

• LH and hCG both potentiate FSH action

Effects of LH and hCG when adminstered in-vivo

Hypothalamo-pituitary production is pulsatile to allow sustained secretion of Testo , Prog

*P<0.001

Adapted from Thuesen LL et al. Hum Reprod. 2012;27(10):3074-3084. 29

†

Therapy injections: pharmacodynamic profiles

HCG and LH

What is RELEVANCE “in-vivo” : for ART

LH and hCG are …. not equivalent for ART ?Can we understand why ?

Heterogeneity of COS protocols

• FSH alone, vs. FSH + HMG, vs. FSH + LH, vs. FSH + HCG

• Agonist vs. Antagonist

Litterature is available

31

Defining optimal

LH-bioactivity levels

Treshold

&

Ceiling

Therapeutic LH window1-3

32

LH <1.2

Impaired follicular

development

Inadequate thecal

androgen synthesis

and hence, reduced

granulosa

aromatization to

estrogen

No full oocyte

maturation

LH 1.2-10.0

Optimal follicular

growth and

development

Full oocyte

maturation

LH >10.0

LH receptor

downregulation

Suppression of

granulosa cell

proliferation

Follicular atresia

(nondominant

follicles) premature

luteinization

(preovulatory follicle)

1. Balasch J et al. Curr Opin Obstet Gynecol. 2002;14(3):265-274. 2. O’Dea L et al. Curr Med Res Opin. 2008;24(10):2785-2793. 3. Regan L et al. Lancet. 1990;336(8724):1141-1144.

Threshold1,2 Optimal window1-3 Ceiling1,3

1. Esposito MA et al. Fertil Steril. 2001;75(3):519-524.

2. Lahoud R et al. Hum Reprod. 2006;21(10):2645-2649.

3. O’Dea L et al. Curr Med Res Opin. 2008;24(10):2785-2793.

4. Fleming R et al. Hum Reprod. 1998;13(7):1788-1792.

5. Fleming R et al. Hum Reprod. 2000;15(7):1440-1445.

6. Westergaard LG et al. Hum Reprod. 2000;15(5):1003-1008.

7. Westergaard LG et al. Fertil Steril. 2001;76(3): 543-549.

8. Pezzuto A et al. Gynecol Endocrinol. 2010;26(2):118-124.

Unequivocal LH cut-off values that identify patients who require supplemental LH have yet to be determined

What is the THRESHOLD dose of LH?

33

3 IU/L Esposito 20011

1.2 IU/L Lahoud 20062, O’Dea 20083

0.7 IU/L Fleming 20004

0.5 IU/L Fleming 19985; Westergaard 20006, 20017; Pezzuto20108

LH Threshold

LH C

on

cen

trat

ion

IU

/LLessons from hypogonadotropic hypogonadism patients

34

LH Treshold influenced by ovarian ageing

Insensitivity of theca cells to LH resulting in low androgen precursor production

Deficient 17 OH-Progesterone production when challenged with HCG ( sensitive marker )

( French and Finnish Studies )

60 patients, 25-37 years of age, undergoing IVFstimulated with 150 IU/day of rFSH (N=62)

0 IU hCG

(n=16)

50 IU hCG

(n=15)

100 IU hCG

(n=16)

150 IU hCG

(n=13)*

*Two patients were withdrawn after randomization due to 10-fold hCG dosing errors.

The results are based on the per protocol (PP) analysis.

hCG ceiling?

• Primary endpoint: number of top-quality

embryos at day 3

35

Thuesen LL et al. 2012

Thuesen LL et al. Hum Reprod. 2012;27(10):3074-3084.

*P<0.001


36

†

HCG injections: pharmacodynamic profiles


37

Dose 0 Dose 50 Dose 100 Dose 150

Effects on ovary of the increasing hCG doses

P=0.58

Small follicles (11-14 mm): day of hCG

Dose 0 (n=16)

n (%)

Dose 50 (n=15)

n (%)

Dose 100 (n=16)

n (%)

Dose 150 (n=13)

n (%)P value

Cumulative no.

live births/started

“fresh cycle”5 (31) 5 (33) 7 (44) 5 (39) 0.89


38

Folli

cle

s 1

1-1

4 m

m

Effects of increasing hCG doses on follicle

growth and clinical outcome

Conclusion:

does an LH ceiling exist during ovarian stimulation?• No negative effects on pregnancy outcomes seen up to

exogenous 150 IU hCG per day

• No negative effects on pregnancy outcomes seen with >1300 IU added rLH (Hugues et al. 2005;20(3):629-635)

• Exogenous sources of LH activity do not appear to have the

same negative impact on ovarian stimulation as does

pathologically elevated endogenous LH

39

40

How do these data

translate in practice?

Andersen A.N. et al, 2006 (MERiT)

• Randomized, open-label, assessor-blind, parallel-group, multicenter,

multinational, phase 3 study

• Patients received long-protocol downregulation with a GnRH agonist

• Primary objective was ongoing pregnancy rate per started cycle

41Andersen AN et al. Hum Reprod. 2006;21(12):3217-3227.

HP-hMG 225-450 IU/day

(n=363)

rFSH 225-450 IU/day

(n=368)

731 premenopausal patients, 21-37 years of age, with regular

menstrual cycles undergoing IVF

Results: primary endpoint

42

(n=368)

P=NS

(n=363)

Ongoing pregnancy rate

Andersen AN et al. Hum Reprod. 2006;21(12):3217-3227.

MERiT endocrinology

• A published report of the MERiT study

investigated the endocrine status of study

subjects during IVF with HP-hMG vs rFSH

• Blood samples were obtained on days 1 and 6 of

stimulation, last stimulation day, and at oocyte

retrieval

• Follicular fluid was collected at retrieval from at

least 1 follicle of ≥17 mm from which an oocyte

had been retrieved

43

Smitz J et al. Hum Reprod. 2007;22(3):676-687.

Results

Serum hormone levels at day 6

Serum hormone levels on last stimulation day

44


FSH

(IU/I)

LH

(IU/I)

hCG

(IU/I)

E2

(pg/mL)

P4

(ng/mL)

HP-hMG

(n=363)15.8 ± 3.5 1.4 ± 0.8

2.45 ±0.80

272.3 ± 245 0.44 ± 0.19

rFSH

(n=368)15.2 ± 3.5 1.4 ± 0.8 -- 299.5 ± 272.3 0.47 ± 0.22

P value 0.009 NS N/A 0.004 NS

HP-hMG

(n=363)18.3 ± 6.0 1.8 ± 0.9 2.94 ±1.18 1960.4 ± 1170.8 0.81 ± 0.41

rFSH

(n=368)16.3 ± 4.7 1.7 ± 0.9 -- 1797.0 ± 1089.1 1.1 ± 0.53

P value <0.001 NS N/A 0.031 <0.001

100

80

60

40

20

0

Results: day 6 serum hCG concentrations

and outcomes

45

Serum hCG on day 6P=0.008

P=0.003


<25%

25%-50%

50%-75%

>75%

MERIT DATASET ( Data on File )

Endocrine data on Day 61

hCG on Day 6

<25%

(N=87)

25–50%

(N=88)

50–75%

(N=90)

>75%

(N=90)

LH (IU/L) 1.4 ± 0.9 1.4 ± 0.8 1.5 ± 0.9 1.5 ± 0.7

Estradiol (nmol/L) 0.6 ± 0.7 0.8 ± 0.7 1.0 ± 1.0 1.3 ± 1.0

Androstenedione (nmol/L) 5.6 ± 2.1 5.8 ± 2.2 6.1 ± 2.8 6.4 ± 2.4

Total testosterone (nmol/L) 0.9 ± 0.4 0.9 ± 0.4 0.9 ± 0.5 0.9 ± 0.4

SHBG (nmol/L) 45 ± 19 52 ± 21 58 ± 24 63 ± 22

FAI 2.5 ± 1.8 1.9 ± 1.1 1.8 ± 1.7 1.6 ± 1.0

Progesterone (nmol/L) 1.3 ± 0.5 1.5 ± 0.6 1.4 ± 0.6 1.5 ± 0.5

1. Data on file

MERiT®

Results: embryo quality

• In the HP-hMG group, 11.3% of embryos assessed were top-quality, versus 9% with rFSH (P=0.044), as assessed by local embryologists

47

P=0.044

Ziebe S et al. Hum Reprod. 2007;22(9):2404-2413.

Conclusions

• Evidence suggests that exogenous LH activity does not have the same negative impact on outcomes as does pathologically elevated endogenous LH

• In the MERiT study, HP-hMG treatment resulted in:— Comparable pregnancy outcomes vs rFSH

— Endocrine profile differed at the end of stimulation – higher E2 and lower P4 with hMG

— Fewer oocytes retrieved but a greater proportion of top-quality embryos vs rFSH

— Day 6 serum hCG levels but NOT LH levels were correlated with pregnancy outcomes

48

Quid with rLH ?

• Evidence suggests that exogenous rLH activity does not have

the same impact on outcomes as does the hCG in HP-hMG

• No improved outcomes when systematically added to rFSH

• Only ‘tendency‘ of improved outcome in :

– Older patients

– Poor responders

• Effect in poor responders (?)

49

50

ESPART: dosage regimen design

Pituitary

downregulation

up to 21 days

Triptorelin

0.1 mg daily

Ovarian stimulation for up to

21 days, until follicle(s) ≥17

mm

r-hFSH + r-hLH (2:1)

Start dose 300/150 IU

Max. dose 450/225 IU

+ daily triptorelin

r-hFSH

Start dose 300 IU

Max. dose 450 IU

R

1:

1

r-hCG Oocyte Embryo

retrieval transfer

r-hCG Oocyte Embryo

retrieval transfer

Luteal Phase support within

48 hours after oocyte

retrieval for

≥7 weeks

Vaginal progesterone gel

Vaginal progesterone gel

34–38

hours

2–3 days

51

Results of the ESPART trial

Outcome

r-hFSH + r-

hLH

(n=462)

r-hFSH

(n=477)

p

value

Biochemical pregnancy, n (%) 80 (17.3) 114 (23.9) 0.020

Embryo implantation rate, n/N* (%)

79/538 (14.7) 93/597 (15.6) 0.675

Clinical pregnancy, n (%) 65 (14.1) 80 (16.8) 0.320

Ongoing pregnancy, n (%) 51 (11.0) 59 (12.4) 0.599

Live birth, n (%) 49 (10.6) 56 (11.7) 0.663

Cancelled cycles,† n (%) 35 (7.6) 32 (6.7) 0.648

*n is the number of fetal sacs identified by transvaginal ultrasound and N is the total number of embryos transferred†All cycle cancellations were due to lack of ovarian response

Secondary and other efficacy endpoints

52

General Conclusions

• exogenous LH bioactivity supplementation does not have the same negative impact on outcomes as elevated endogenous LH

• hCG and LH are different molecules, with a different in-vitro and in-vivo effect— Half-life in circulation

— Binding to receptor

— Post-receptor triggering

• hCG low dose therapy in combination with FSH (HP-hMG)

– influences the ovarian hormone profiles,

– reduces the amount of small follicles (with incompetent oocytes / embryos )

– Is correlated with a favourable safety profile in ART

53

Documents

Effects of HCG and LH on ovarian stimulationPituitary hCG in the postmenopausal woman 12 Snyder JA et al. Clin Chem. 2005;51(10):1830-1835.Nonpregnant cohort n hCG range, IU/L 97.5