THE USE OF HUMAN GONADOTROPINS IN ART CYCLES: IMPORTANCE OF FSH ISOFORMS AND HMG WITH PLACENTAL HCG

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1 THE USE OF HUMAN GONADOTROPINS IN ART CYCLES: IMPORTANCE OF FSH ISOFORMS AND HMG WITH PLACENTAL HCG Sandro Gerli Associate Professor Dept. Obstetrics and Gynecology University of Perugia, Italy

What do doctors and patients expect from ART adequate number of mature oocytes sufficient number of good quality embryos high live birth rate minimal complications fewer multiple

2 What do doctors and patients expect from ART adequate number of mature oocytes sufficient number of good quality embryos high live birth rate minimal complications fewer multiple pregnancies highest possible pregnancy rates lowest possible risks shortest possible time lowest possible cost (in case medication costs are not reimbursed by public health care) Ovarian Stimulation

3 The start of gonadotropin history 1947 Piero Donini, an IFS* chemist, successfully extracted and purified the first gonadotropin preparation from the urine of postmenopausal women * Istituto Farmacologico Serono (IFS), Rome (Italy)

Serono was registered in Italy on May 22 Lunenfeld, B.

4 The first HMG 1950 REGISTRATION OF PERGONAL SERONO The first human menopausal gonadotropin (hmg) preparation for clinical use Pergonal Serono was registered in Italy on May 22 Lunenfeld, B. Historical perspectives in gonadotrophin therapy. Hum. Reprod. Update (6):

Evolution of human gonadotropins by technology

FSH & LH Large amount of impurities High content

Generation FSH Large amount of impurities

Generation FSH or FSH & LH High purification

5 Evolution of human gonadotropins by technology purification The First Generation FSH & LH Large amount of impurities High content of LH LH-activity of pituitary hcg The Second Generation FSH Large amount of impurities Minimal LHactivity of pituitary hcg The Third Generation FSH or FSH & LH High purification process Reduced amount of impurities LH-activity of pituitary hcg

7 Evolution of human gonadotropins by technology purification 1990 The last Generation 1950 The First Generation FSH & LH Large amount of impurities High content of LH LH-activity of pituitary hcg 1970 The Second Generation FSH Large amount of impurities Minimal LHactivity of pituitary hcg The Third Generation FSH or FSH & LH High purification process Reduced amount of impurities LH-activity of pituitary hcg FSH or FSH & LH Extremely high purification process Human FSH isoforms LH-activity of placental hcg

8 What is the origin of human FSH and LH contained in pharmaceuticals preparations? human body? OR laboratory?

9 FSH A complex glycoprotein Beta = Asn 7 Asn 24 FSH is a complex glycoprotein made of a proteic part and a glycidic part which is about 30 % of the molecular mass Two non-identical proteic subunits: alpha (common to FSH, LH, CG) beta (biological specificity = specific biological properties of gonadotropin) Alpha = Asn 52 Asn 78 4 Asn-glycosylation sites for the glycidic chains

10 The oligosaccharide chains structure 4 branches of sialic acid residues Asn Mannose N-Acetyl-Glucosamine Galactose Sialic Acid Negative charge of the carboxillic terminal -

11 sialic acid = glycosylation = acidity sialic acid = glycosylation = acidity

FSH isoforms: pattern of release in FSH dependent phase Least acidic isoforms PREOVULATORY Acidic isoforms DOMINANT SELECTED Atresia Atresia

12 FSH isoforms: pattern of release in FSH dependent phase Least acidic isoforms PREOVULATORY Acidic isoforms DOMINANT SELECTED Atresia Atresia Recruitment Selection Early dominance Late dominance LATE LUTEAL EARLY FOLLICULAR MID FOLLICULAR LATE FOLLICULAR day day 1-5 day 6-10 day 11-14

13 Estrogen driven shift

Ovary) cells CHO cells are cultured into a big tank called

14 Recombinant FSH The gene for human FSH (synthesis of the proteic backbone) was inserted into the genome of a CHO (Chinese Hamster Ovary) cells CHO cells are cultured into a big tank called bioreactor CHO cells produce, together with their proteins, also FSH

15 Recombinant FSH Low sugar content at the origin Further loss of sugars during purification Low glycosylated Less acidic ( Gonal-F, Puregon )

[FSH] FSH glycosylation in human-derived FSH 10 20 30 40 45 50 60 Very acidic Low acidic Total FSH Year Due to the high blood concentration, the urine

16 [FSH] FSH glycosylation in human-derived FSH Very acidic Low acidic Total FSH Year Due to the high blood concentration, the urine of menopausal women contain big amounts of highly glycosylated FSH The urine undergo a complex purification process that may cause detachment of the sugars

17 Recombinant FSH Human-derived FSH Low sugar content at the origin High sugar content at the origin Further loss of sugars during purification Big loss of sugars during purification Hypoglycosylated Less acidic ( Gonal-F, Puregon ) Mid glycosylated Mid acidic (Metrodin, Bravelle, Menopur)

during purification Mid glycosylated Mid acidic (Metrodin, Bravelle, Menopur) Different purification process

18 Recombinant FSH Human-derived FSH Low sugar content at the origin High sugar content at the origin Further loss of sugars during purification Hypoglycosylated Less acidic ( Gonal-F, Puregon ) Big loss of sugars during purification Mid glycosylated Mid acidic (Metrodin, Bravelle, Menopur) Different purification process Minor loss of sugars during purification (IBSA process) Hyperglycosylated More acidic ( Alterpur, IBSA Menotropin)

19 Glycoforms according to isoelectric focusing Fostimon Gonal-F Lispi et al., 2006 More acidic Hyperglycosylated Less acidic Hypoglycosylated

20 Oligosaccharide composition and estimated charge number (Z) of Fostimon, Gonal-F and Puregon The Z number : quantitative index for sialic acid content Lombardi, Electrophoresis, 2013

21 Recombinant FSH Low glycosylation Less acidic Human FSH Mid glycosylation, mid acidic (Bravelle) High glycosylation, more acidic (Alterpur)

22 Human FSH preparations: Conclusions All human gonadotropins derive from urine of menopausal women FSH is only natural FSH activity and no generics are present in these pharmaceutical preparations The difference is represented by a specific purification process which characterizes some biochemical properties (e.g. glycosilation) and therefore the biological activity of each one

23 What is the biological effect of different FSH isoforms on oocyte and embryo quality?

24 MORE ACIDIC LESS ACIDIC Murine intact follicles culture Nayudu, RBMO, 2002 Antral FSH threshold /ml Dose tolerance Antral FSH threshold (20 times higher) /ml Antral threshold Higher antral FSH threshold dose Larger dose tolerance, therefore a more difficult degeneration of OCC if higher levels of FSH are present Dose tolerance (10 times larger)

25 Embryonic development after follicle culture is reduced with higher concentrations of hypoglycosilated FSH isoforms Vitt et al., 2001

26 GONADOTROPINS IN ART evidences of clinical studies

27 day 2 day 6 monitoring 75 IU (Fostimon) HP-hFSH hcg IUI 50 IU (Puregon) r-fsh Gerli S, Recombinant versus urinary folliclestimulating hormone in intrauterine insemination cycles: a prospective, randomized analysis of cost effectiveness; Fertil Steril, 2004

28 No statistical differences were found in clinical parameters analysed ufsh rfsh 12 h-fsh r-fsh Follicles >17 mm Days of stimulation No. of ampules per cycle 2 0 clinical pregnancy rate miscarriage rate cancellation rate Gerli S, Recombinant versus urinary folliclestimulating hormone in intrauterine insemination cycles: a prospective, randomized analysis of cost effectiveness; Fertil Steril, 2004

29 Gonadotropins in ovarian stimulation for IUI: pregnancy rate per couple Cantineau, 2008

32 rfsh vs HP-hFSH Live birth rfsh vs. Metrodin HP Van Wely, 2011 rfsh vs. Fostimon

33 Are all human-derived FSH products the same? Fostimon vs. Metrodin-HP: live birth rate Adjusted indirect comparison of Fostimon vs. Metrodin-HP using recfsh as a common comparator demonstrated a significantly higher live-birth rate with Fostimon (OR:1.72; ). Al Inany, 2011

34 rfsh (Gonal F) vs. HP-hFSH (Fostimon) Summary of RCT in IVF/ICSI cycles Reference # patients Objectives of the study Conclusions Selman, Oocyte and embryo quality Equally effective Mohamed, Efficacy in older patients HP-uFSH performed better Pacchiarotti, Efficacy of a sequential protocol Improvement in oocyte/embryo quality and clinical outcome with the sequential protocol Baker, Efficacy in biological and clinical outcomes No differences Abate, Oocyte and embryo quality More oocytes with shorter stimulation and less IU used with HP-uFSH Selman, Biological and clinical efficacy with a sequential protocol Aboulghar, Biological and clinical outcomes in PCOS patients Selman, Biological and clinical outcomes in a combined protocol Gurgan, Biological/clinical outcomes and CC gene expression in rfsh, HP-uFSH and sequential protocol Better oocyte quality, embryo cleavage, clinical outcomes with the sequential protocol Higher fertilization rate, better embryos with HP-uFSH Better oocyte quality, embryo cleavage, clinical outcomes with HPuFSH No significant differences

35 total oocytes mature oocytes Effect size meta-analysis plot [random effects] Effect size meta-analysis plot [random effects] Selman HA et al., 2002 Selman HA et al., 2002 ohamed MA et al., 2006 Antoine JM et al., 2007 Antoine JM et al., 2007 Abate A et al Baker VL et al., 2009 Baker VL et al., 2009 Selman H et al., 2010 Selman H et al., 2010 Aboulghar M et al., 2010 Aboulghar M, DL pooled weighted mean difference = (95% CI = to ) DL pooled weighted mean difference = (95% CI = to ) Gerli et al., Gynecol Endocrinol, 2013

36 Meta-analysis: rfsh (Gonal-F) vs. HP-hFSH (Fostimon) in IVF Live birth rate Study rfsh HP-hFSH OR (random) Weight (random) Odds ratio meta-analysis plot [random effects] x/n x/n 95%CI % Selman HA et al., /134 52/ ( ) 31.6 Mohamed M et al. 18/129 20/ ( ) 17.0 Baker VL et al., 2009 Aboulghar M et al., 2010 Selman H et al., /76 29/ ( ) /42 19/ ( ) /65 20/ ( ) 13.9 Murber A et al., /35 11/ ( ) 7.6 Combined (random) 136/ / ( ) odds ratio (95% confidence interval) Favours HP-hFSH Favours rfsh Gerli et al., Gynecol Endocrinol, 2013

37 Secretion of LH and pituitary HCG Choi J and Smitz J, Gynecol Endocrinol, 2014

38 Presence of LH/hCG activity in human menotropins Choi, Molecular and Cellular Endocrinology, 2014 Casarini, PLoS ONE, 2012

39 The human chorionic gonadotropin may have a different origin It is synthesized by the pituitary gland and the placenta

40 Comparative content of pituitary and placental hcg subunits: hcg isoforms In vitro studies demonstrated that placental hcg displayed both a higher biological activity and receptor binding potency in comparison to pituitary hcg Birken S, Mol Cell Endocrinol. 1996

41 Reduced duration of gonadotropin administration in case of LH presence

Reduced occurrence of small follicles in case of LH presence preovulatory follicles <10mm 10 8 6 4 2 0 1000 2000 3000 4000 5000 FSH activity administered

42 Reduced occurrence of small follicles in case of LH presence preovulatory follicles <10mm FSH activity administered (IU) r = P < 0.05 preovulatory follicles <10mm r= P< LH activity administered (IU) Filicori, 2002

43 Better IVF outcome with HP-hMG vs. rfsh Platteau, RBMOnline, 2008

44 Two highly purified hmg in Russia HP-HMG origin INSTITUTO MASSONE S.A. Arias 4431 C1430CSE - Buenos Aires Argentina PCT/EP97/06058 Exclusive purification protocol Endproduct LHactivity

45 HP-hMG IBSA vs. HP-hMG Ferring IBSA HP-hMG FERRING HP-hMG

46 MENOTROPHINS BIOCHEMICAL COMPARISON GLYCOSYLATION ISOELECTRIC FOCUSING/WB IBSA s HP-HMG Comparator HP-HMG IBSA s HP-HMG Comparator HP-HMG IEF Western Blotting Antibodies Anti-FSH IEF - Coomassie Blue Staining

47 IBSA HP-hMG shows a more balanced and preserved glycosylation profile HP-hMG IBSA Wolfenson C, et al. Reprod Biomed Online 2005;10(4):442-54

48 SDS-PAGE analysis HP-hMG IBSA HP-hMG IBSA HP-hMG IBSA HP-hMG IBSA

49 hmg preparations: Conclusions All human gonadotropins derive from urine of women hmg is only natural LH/hCG activity and no generics are present in these pharmaceutical preparations The difference is represented by the provenience (menopausal or pregnant women) and by a specific purification process which characterizes some biochemical properties (e.g. glycosilation) and therefore the biological activity of each one

50 GONADOTROPINS IN ART evidences of clinical studies

51 HP-hMG IBSA vs. HP-hMG Ferring Two randomised, controlled, multicentre clinical trials Indication Study design Sample size 1. Italian study Alviggi et al. Gynecol Endocrinol, 2013 COH Prospective, randomised, controlled vs Menopur (Ferring), multicentre, single blind. 151 patients 3 study sites in Italy 2. European study Lockwood et. al RBMOnline, 2017 COH Prospective, randomised, controlled vs Menopur (Ferring), multicentre, single blind. 270 patients 6 study sites in different countries

non-inferiority study 3 IVF Centers (Universities of Naples, Bologna, and Siena) patients characteristics

52 HP-hMG IBSA vs. HP-hMG Ferring The Italian Study type of study prospective, randomized, investigator-blind, controlled non-inferiority study 3 IVF Centers (Universities of Naples, Bologna, and Siena) patients characteristics years old FSH < 10 IU/L normal uterus, no previous poor response, no PCOS, no hydrosalpinx, no stage III-IV endometriosis, normal PRL 157 patients randomized 78 HP hmg I (test group) 79 HP hmg F (control group)

53 Study outcome: main parameters HP-hMG I HP-hMG F

54 Results hmg-i and hmg-f administration resulted in comparable : numbers of retrieved oocytes (primary endpoint) oocyte fertilization rates embryo quality embryo implantation rates biochemical and clinical pregnancy rates administration of hmg-i vs. hmg-f was associated with: shorter COS duration lower gonadotropin dose administration (daily & per cycle) improved retrieval of mature oocytes

55 HP-hMG IBSA vs. HP-hMG Ferring The European Study 6 study sites Lockwood, RBMO, in Denmark 1 in Hungary 1 in UK 1 in Switzerland 1 in France

56 Population 270 patients were included with the following characteristics: Age: 18 and <40 years old BMI: 18 and 30 kg/m2 Less than 3 previously completed IVF cycles Basal FSH <10 IU/L and E2 <80 pg/ml Normal uterine cavity Normal haematology and blood chemistry values

57 Efficacy Results Variables, mean (SD) HP-hMG I HP-hMG F p value* Total hmg units (980.0) (906.4) 0.25 Daily hmg units (77.3) (69.7) 0.51 COH duration (days) 10.2 (1.3) 10.6 (1.5) 0.02 Follicles>16 mm at hcg day 5.0 (3.8) 4.5 (3.2) 0.23

58 Efficacy Results Variabile HP-hMG I HP-hMG F P value Number of oocytes retrieved 11.6 (6.6) 9.7 (5.9) Mean nr of metaphase II oocytes (SD) 10.3 (6.0) 8.2 (5.0) Rate Metaphase II/Total retrieved (%) Mean nr of Immature oocytes (SD) 1.3 (1.9) 1.6 (2.2) 0.35 Rate Immature/Total retrieved (%) <0.001

59 Efficacy Results Variabile HP-hMG I HP-hMG F P Value Implantation rate* Positive Pregnancy test (n) /OPU (%) /ET (%) Clinical Pregnancy (n) /OPU (%) /ET (%) * total nr of gestational sacs/total nr of embryos transferred P = statistically not significat for all the parameters

60 Cumulative Pregnancy Rates ( including FETs) HP-hMG I HP-hMG F

61 Conclusions HP-hMG IBSA is a viable alternative for controlled ovarian stimulation in ART cycles where a urinary derived hmg is chosen European multicentre study demonstrated: same quantity of drug administered higher number of oocytes and mature oocytes retrieved shorter period of stimulation similar safety profile

62 Conclusions Hyperglycosylated human FSH represents a natural alternative at the beginning of the stimulation The biological effects on oocyte and embryo development show the importance of hyperglycosylated FSH isoforms in ovarian stimulation The COH with IBSA HP-hMG containing placental hcg seems to perform better than HP-hMG Ferring

63 Спасибо за внимание!

- Meta. : (rfsh); (ufsh); (IVF); : R711.6 : A : X(2015) : hmg( FSH LH) [ufsh, (ufsh-p) (ufsh-hp)] (rfsh) [1] 80, rfsh, 90, :

- Meta. : (rfsh); (ufsh); (IVF); : R711.6 : A : X(2015) : hmg( FSH LH) [ufsh, (ufsh-p) (ufsh-hp)] (rfsh) [1] 80, rfsh, 90, : 35 2 Vol.35 No.2 2015 2 Feb. 2015 Reproduction & Contraception doi: 10.7669/j.issn.0253-357X.2015.02.0099 E-mail: randc_journal@163.com (FSH) - Meta FSH ( 400010) : (IVF) (ICSI) (rfsh) (ufsh) (COS) : PubMed