Effects of HCG and LH on ovarian stimulation. Are they bioequivalent?

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 MAXIMIZE EFFICIENCY MINIMIZE RISK IVF Rob Gilchrist courtesy, adapted

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 ɑ-chain Asn 52 hfsh hlh hcg 92 AA Asn 78 β-chain Asn 7 hfsh hfsh Asn 24 111 AA hlh & hcg hlh Asn 30 Ser 127 115 AA Ser 138 hcg Asn 13 Asn 30 Ser 121 145 AA Ser 132 Strauss J et al, eds. Yen and Jaffe s Reproductive Endocrinology. 5th ed. 2004. 7

2-cell, 2-gonadotropin model of hormone production THECA CELL Blood GRANULOSA CELL Cholesterol LH R camp CYP11A1 Pregnenolone ESTRADIOL-17β R FSH Protein Kinase A CYP17 17-OH Pregnenolone CYP17 17βHSD1 ESTRONE camp 3βHSD DHEA P450arom Protein Kinase A ANDROSTENEDIONE ANDROSTENEDIONE Adapted from Wilson JD et al, eds. Basement Membrane Carr BR. In: Wilson JD et al, eds. Williams Textbook of Endocrinology. 9th ed. Philadelphia, PA: WB Saunders; 1998:751-817. 8

LH bioactivity relation to the oocyte Mural&Cells& Ccnb1,'Mos,'Mad2,'Bub1b,'Sogl2,' Tex19.1,'Tpx2,'Dazl,'others' ' (Has2,' Tnfaip6,'Ptgs2) Cumulus-oocyte capacitation for development

Products that provide LH bioactivity 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 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 cycle 1 Lack of E 2 feedback during menopause/ovarian failure increases pituitary hcg production 2,3 Pituitary hcg and placental/urinary hcg are largely similar, according to 4 : Amino acid analysis Mass spectrometric studies 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. 11

hcg (IU/L) Pituitary hcg in the postmenopausal woman 14 12 10 8 6 4 2 0 20 40 60 80 100 Age (years) Table 1. hcg concentration ranges and the 97.5 percentile values for the nonpregnant cohorts in the study. Nonpregnant cohort Premenopausal, 18-40 years Perimenopausal, 41-55 years Postmenopausal, >55 years n hcg range, IU/L 97.5 percentile, IU/L 240 <2.0 to 4.6 2.5 240 <2.0 to 7.7 4.8 0.07 * 240 <2.0 to 13.1 7.7 <0.000 * Compared with the nonpregnant premenopausal cohort. Compared with the nonpregnant premenopausal and nonpregnant perimenopausal cohorts. P Snyder JA et al. Clin Chem. 2005;51(10):1830-1835. 12

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 Final chromatography step Fraction 1 FSH, little LH Fraction 3 hcg Fraction 2 FSH, LH, and hcg 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 HP-hMG is human-derived FSH with approximately 10 IUs of hcg driving the majority of the 75 IUs of LH activity. 13

HP-hMG is NOT hmg Source of various gonadotropins since 1985 1985 1 1994 1 1999 1 2004 1 2004 1 hmg 2 hmg 2 hmg 2 rfsh 2 HP-hMG 2 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 hcg 3 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. 14

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

LH and hcg: understanding the differences in effects 16

Differences LH and hcg LH hcg AA number beta subunit 121 145 Receptor binding affinity Low High* No. Glycosylation sites 1 3 6 Initial half-life (h) 0.6-1.3 3.9-5.5 Serum halflife (h) 2 1.5 34 Bioequivalency 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 2014 Choi and Smitz. Mol Cell Endocrinol 2014 2 Mann K. Oncodev Biol Med 1980

EVALUATE EFFECTS LH - hcg IN VITRO: on Proximal or Distal output parameters Proximal : camp intracellular proteins ( CREB, ERK, ) Distal : gene expression ( QRT-PCR ) proteins : Amphiregulin (AREG), Epiregulin (EREG) steroids : progesterone, estradiol

hlhcg receptor

Exposure to GC in vivo? 10 IU/L= 10.7pM 10 IU/L is dose when 3Amps Menopur injected Casarini et al., Mol Cell Endocrinol 2016

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

Casarini et al., Mol Cell Endocrinol 2016

Casarini et al., Mol Cell Endocrinol 2016

Casarini et al., Mol Cell Endocrinol 2016

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

Therapy injections: pharmacodynamic profiles HCG and LH *P<0.001 Adapted from Thuesen LL et al. Hum Reprod. 2012;27(10):3074-3084. 29

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

Defining optimal LH-bioactivity levels Treshold & Ceiling 31

Therapeutic LH window 1-3 Threshold 1,2 Optimal window 1-3 Ceiling 1,3 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. 32

LH Concentration IU/L What is the THRESHOLD dose of LH? Lessons from hypogonadotropic hypogonadism patients 3 IU/L Esposito 2001 1 LH Threshold 1.2 IU/L Lahoud 2006 2, O Dea 2008 3 0.7 IU/L Fleming 2000 4 0.5 IU/L Fleming 1998 5 ; Westergaard 2000 6, 2001 7 ; Pezzuto 2010 8 Unequivocal LH cut-off values that identify patients who require supplemental LH have yet to be determined 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. 33

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 ) 34

hcg ceiling? Thuesen LL et al. 2012 60 patients, 25-37 years of age, undergoing IVF stimulated with 150 IU/day of rfsh (N=62) 0 IU hcg (n=16) 50 IU hcg (n=15) 100 IU hcg (n=16) *Two patients were withdrawn after randomization due to 10-fold hcg dosing errors. The results are based on the per protocol (PP) analysis. 150 IU hcg (n=13)* Primary endpoint: number of top-quality embryos at day 3 Thuesen LL et al. Hum Reprod. 2012;27(10):3074-3084. 35

HCG injections: pharmacodynamic profiles *P<0.001 Thuesen LL et al. Hum Reprod. 2012;27(10):3074-3084. 36

Effects on ovary of the increasing hcg doses Dose 0 Dose 50 Dose 100 Dose 150 Thuesen LL et al. Hum Reprod. 2012;27(10):3074-3084. 37

Follicles 11-14 mm Effects of increasing hcg doses on follicle growth and clinical outcome Small follicles (11-14 mm): day of hcg P=0.58 Cumulative no. live births/started fresh cycle Dose 0 (n=16) n (%) Dose 50 (n=15) n (%) Dose 100 (n=16) n (%) Dose 150 (n=13) n (%) P value 5 (31) 5 (33) 7 (44) 5 (39) 0.89 Thuesen LL et al. Hum Reprod. 2012;27(10):3074-3084. 38

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

How do these data translate in practice? 40

Andersen A.N. et al, 2006 (MERiT) 731 premenopausal patients, 21-37 years of age, with regular menstrual cycles undergoing IVF HP-hMG 225-450 IU/day (n=363) rfsh 225-450 IU/day (n=368) 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 Andersen AN et al. Hum Reprod. 2006;21(12):3217-3227. 41

Results: primary endpoint Ongoing pregnancy rate P=NS (n=363) (n=368) Andersen AN et al. Hum Reprod. 2006;21(12):3217-3227. 42

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 Smitz J et al. Hum Reprod. 2007;22(3):676-687. 43

Results Serum hormone levels at day 6 FSH (IU/I) LH (IU/I) hcg (IU/I) E 2 (pg/ml) P 4 (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 Serum hormone levels on last stimulation day HP-hMG (n=363) rfsh (n=368) 18.3 ± 6.0 1.8 ± 0.9 2.94 ±1.18 1960.4 ± 1170.8 0.81 ± 0.41 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 Smitz J et al. Hum Reprod. 2007;22(3):676-687. 44

Results: day 6 serum hcg concentrations and outcomes 100 80 60 P=0.008 P=0.003 Serum hcg on day 6 <25% 25%-50% 40 50%-75% >75% 20 0 Smitz J et al. Hum Reprod. 2007;22(3):676-687. 45

Endocrine data on Day 6 1 hcg on Day 6 <25% 25 50% 50 75% >75% (N=87) (N=88) (N=90) (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 MERIT DATASET ( Data on File ) 1. Data on file MERiT

Results: embryo quality P=0.044 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 Ziebe S et al. Hum Reprod. 2007;22(9):2404-2413. 47

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 E 2 and lower P 4 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 R 1: 1 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 34 38 hours 2 3 days r-hcg Oocyte Embryo retrieval transfer Luteal Phase support within 48 hours after oocyte retrieval for 7 weeks Vaginal progesterone gel r-hfsh Start dose 300 IU Max. dose 450 IU r-hcg Oocyte Embryo retrieval transfer Vaginal progesterone gel 51

Results of the ESPART trial Secondary and other efficacy endpoints 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 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