Individualization of Cycle Control Dr. Milton Leong MDCM DSc (McGill) Director, IVF Center, HKSH Specialist in Reproductive Medicine Adjunct Professor, OBS-GYN, McGill University

The first IVF Baby Drs. Steptoe and Edwards decided to abandon the use of fertility medications and try aspirating a single egg in a natural menstrual cycle. On their second attempt, Louise Brown was conceived

Preparation for Ovum Collection Natural Cycles Minimal stimulation (clomiphene/FSH) IVM FSH stimulation with agonists FSH stimulation with antagonists

Ovulation Stimulation WHAT GOES AROUND COMES AROUND *American idiom

Stimulated ovary

Technology and product development timeline: gonadotrophins Horse PMSG Pig FSH Pituitary FSH u-hMG u-FSH u-FSH r-hFSH (HP) r-hFSH FbM 1930 1940 1950 1960 1980 1990 1995 2003 Consistency Quality Local reactions Potential side-effects Antibodies Creutzfeldt–Jacob disease Local, systemic reactions Adapted from: Lunenfeld. Reprod Biomed Online 2002;4:11

Premature LH surge Poor quality No fertilization or very poor pregnancy rate Cancel egg retrieval 5-20% 5-20% All cycles treated in early 1980’s

GnRHa Long Protocol vs No Suppression meta-analysis IVF cases Odds ratios for IVF clinical pregnancy after GnRH-a versus clomiphene/FSH/hMG ovulation induction protocols

GnRHa Long Protocol vs No Suppression meta-analysis GIFT cases Odds ratios for GIFT clinical pregnancy after GnRH-a versus clomiphene/FSH/hMG ovulation induction protocols

Results of first application of GnRH-agonists in the long protocol 11 patients eligible for IVF GnRH agonists s.c. (busereline) started at day of menstruation of one day before Ovarian stimulation started with HMG or purified FSH when all ovarian follicles and the endometrial lining has disappeared on ultrasound (average 15 days) One ongoing pregnancy achieved Porter RN, Smith W., Craft IL., Abdulwahid NA., JAcobs HS (1984) Induction of ovulation for in-vitro fertilization using buserelin and gonadotropins. Lancet 2; 1284-1285 Porter et al., 1984

OVARIAN STIMULATION FSH with agonist down regulation FSH with antagonists Low dose clomid/FSH stimulation Delayed stimulation IVM Natural cycles

Structure of GnRH agonists Modifications of natural GnRH to have GnRH agonistic properties 1 2 4 3 6 5 9 8 10 7 pyro (Glu) – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2 activation of the GnRH receptor regulation of GnRH receptor affinity regulation of biologic activity Modification of position 6 and 10 is typical for all GnRH agonists. This leads to a change in - GnRH receptor affinity (which is increased) - regulation of biologic activity (which is increased due to a longer half life)

Action of GnRH agonists Down regulation Action of GnRH agonists 1. Binding of GnRH leads to a post-receptor-cascade and this consecuitively to the release of LH and FSH 2. Adding of GnRH agonists will lead - because they have a higher affinitiy to the receptors and have a higher biologic potency - to binding of the agonists to the receptors instead of the natural GnRH. 3. Initially, this will lead to an increase in the receptor action, number and post-receptor-cascade with a consecutive increase in the release of LH and FSH (flare up effect). 4. After that, however, receptors are internalized, lysed, the number of receptors decreases, the post-receptor-cascade is downregulated and the stimulus to release LH and FSH will also be suppressed. 5. Downregulation and pituitary suppression will result. GnRH LH + FSH GnRH - receptor post-receptor-cascade GnRH - agonist Flare up effect Pituitary suppression

Schematic representation of different protocols using GnRH agonists in combination with gonadotrophins for ovarian stimulation in IVF

The long luteal protocol ovulation induction oocyte pick up embryo transfer gonadotropin administration in an individualized dosage start of GnRH agonist In the long luteal protocol a GnRH agonist depot preparation is administered during the mid-luteal phase of the preceeding cycle, or a GnRH agonist is started with a daily administration at that time. Two weeks later, in between menstruation will start, pituitary suppression is achieved. At that time point a transvaginal ultrasound should be done to exclude cyst formation, since the flare up effect of the agonist may lead to ovarian cyst formation. If pituitary suppression has been achieved and the ovaries do not show cysts, gonadotropin stimulation can be started at that day. It will go on until hCG can be administered for ovulation induction. Luteal phase support is necessay for these protocols. 22nd day of previous cycle 1st day of gonado- tropins luteal phase support 14 days

Individualizing protocols Our contribution to 1. low dose short term agonist down regulation using decapeptyl 2. flexible low dose antagonist Aims: - to simplify treatment - to minimize drug usage

Agonist Studies 2000 - 2001 Deca Long Luc Long Bus <40 Number of OPU 69 76 61 Number of Eggs Retrieved 881 885 726 Number of MTII 647, 73% 642, 73% 552, 76% Number of MTI 136, 15% 44, 5% 101, 14% Fertilization Rate 74% 76% 71% Mean # of Embryos Transferred per ET 3.1 3.2 2.8 Pregnancy Rate per ET 51% 49% 44% Implantation Rate 20% 22% 18% Average Age 34.4 33.2 34.9

Decapeptyl Down Regulation 2000-2002 Total < 40 ≥ 40 # of patients 90 76 (32.9) 14(40.8) # of pregnancy 42 40 2 Pregnancy % 46.7 52.6 14 # of twins+ 10 # of babies 43 1 Miscarriage rate 16% 50%

Decapeptyl Down Regulation 2000-2003 Laboratory Data # of eggs 831 MTII 539 (67%) MTI 139 (16.7%) # of eggs ICSI 551 # of fertilized 427 Fert. % 76.4 # of E.T. 244 Mean transferred 2.7 # of preg. (F.H.) 46 Implantation rate 21%

Down Regulation

GnRH agonists Undesirable effects: Over-suppression: Also it is: LH becomes so low that it affects the production of estrogen, and possibly progesterone in the luteal phase Leads to poor response, poor pregnancy outcome due to early abortion. Also it is: Too long and too much drug use, cost, cancelled cycles and it is unnatural.

Structure of GnRH antagonists to achieve antagonistic properties of natural GnRH more modifications than only in position 6 and 10 are necessary 1 2 4 3 6 5 9 8 10 7 pyro (Glu) – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2 activation of the GnRH receptor regulation of GnRH receptor affinity regulation of biologic activity In contrast to GnRH agonists, there are more changes necessary in the structure of the natural GnRH molecule, to achieve antagonistic properties. These antagonistic properties mean: - no intrinsic effect - competitive action

Action of GnRH antagonists 1. Binding of GnRH leads to a post-receptor-cascade and this consecutively to the release of LH and FSH 2. Adding of GnRH antagonists will lead to a competitive action of GnRH and GnRH antagonists - which do not have any intrinsic activity. 3. A sudden downregulation of the post-receptor-cascade is the result with a consecutive decrease in the stimulus to release LH and FSH. 4. Pituitary suppression is achieved within a few hours without any initial flare up effect. GnRH LH + FSH GnRH - receptor post-receptor-cascade pituitary suppression GnRH - antagonist

Characteristics of GnRH antag Ganirelix Fully effective within 4 hours, with a half-life of about 13 hours Cetrorelix Fully effective within 8 hours, with a half-life of about 36 hours R.E. Felberbaum and K. Diedrich, 1999.

The Cetrotide® 0.25 mg multiple dose protocol ovulation induction oocyte pick up embryo transfer gonadotropin administration in an individualized dosage 1st day of menstruation In the multiple dose antagonist protocol ovarian stimulation is started with the second or third day of the menstrual cycle. Cetrotide® 0.25 mg is started on the 6th day of ovarian stimulation in the morning or on the 5th day in the evening. And is administered up to and including the day of hCG, if given in the morning. 1st day of gonado- tropins luteal phase support Cetrotide® 0.25 mg administration daily s.c. starting on day 6 of stimulation

Possibilities to individualize the multiple dose protocol To avoid a premature LH rise the administration of cetrotide® 0.25 mg on day 6 of stimulation should be the standard procedure Using the standard procedure, a mean of 6.3 injections are necessary This is in accordance with the package size of 7 ampoules cetrotide® 0.25 mg per patient

Possibilities to individualize the multiple dose protocol Individualized administration of Cetrotide® 0.25 mg can be done According to follicle size: only if leading follicle is  14 mm Thereby, the multiple dose protocol can also be adapted to patients with a lower response

Cetrorelix 0.125mg Flexible Dose Trial Selection Criteria: 1. Previous over-suppression with agonist 2. Previous poor response 3. Previous LH surge if no agonist

BMI Distribution Mean = 21.8 (range 19-30) Mean = 21.8 (range 19-30)

# Days Cetrorelix Used Mean = 2.2 days (range 1-3)

LH and Cetrorelix 0.125mg/day Range mIU/ml Pre 1.2 - 7.8 Day 1 post 0.9 - 4.9 Day HCG 1.8 - 6

Cetrotide 0.125 mg vs 0.25 mg, 2004 – Sep 2006 0.125 mg/day Cycles 121 331 Average age 37.1±4.0 37.5±4.2 NS Days of stimulation 9.3±1.7 9.4±1.8 Total dose of FSH used (amp) 31.4±14.4 36.0±14.5 0.004 E2 on HCG day (pg/ml) 1943±941.8 2028.0±1376.0 LH on HCG day (IU/L) 3.5±3.9 2.1±1.9 0.001 Oocytes collected 1160 (9.6) 3198 (9.7) MTII 902 (77.75%) 2503 (78.26) Fertilized oocytes (fertilization rate) 770 (85.4%) 2085 (83.3%) Embryos transferred 2.8±0.8 2.9±0.8 Pregnancy rate/ET 50/121 (41.3%) 106/331 (32.0%) NS (P=0.066) Implantation rate 17.3% 13.4% NS (P=0.081)

Cetrotide 0.125 mg vs 0.25 mg, 2004 – Sep 2006 (age <40) 0.125 mg/day 0.25 mg/day P Cycles 86 215 Average age 35.1±3.1 35.2±2.9 NS Days of stimulation 9.4±1.7 9.3±1.8 Total dose of FSH used (amp) 29.6±11.9 33.2±11.6 0.016 E2 on HCG day (pg/ml) 2081.5±977.6 2040.6±1300.2 LH on HCG day (IU/L) 3.7±4.4 2.1±1.8 0.002 Oocytes collected 941 (10.9) 2240 (10.4) MTII 732 (77.78%) 1742 (77.76) Fertilized oocytes (fertilization rate) 623 (85.1%) 1448 (83.1%) Embryos transferred 2.8±0.6 2.8±0.7 Pregnancy rate/ET 43/86 (50.0%) 84/215 (39.1%) NS (P=0.083) Implantation rate 21.8% 17.4% NS (P=0.144)

Cetrotide 0.125 mg vs 0.25 mg, 2004 – Sep 2006 (age ≥40) 0.125 mg/day 0.25 mg/day P Cycles 35 116 Average age 41.6±1.7 42.0±2.3 NS Days of stimulation 9.1±1.8 9.4±1.9 Total dose of FSH used (amp) 36.0±18.6 41.1±17.7 E2 on HCG day (pg/ml) 1602.2±756.1 2003.9±1517.8 LH on HCG day (IU/L) 3.0±2.4 2.2±2.1 Oocytes collected 219 (6.26) 958 (8.25) MTII 170 (77.6%) 761 (79.4%) Fertilized oocytes (fertilization rate) 147 (86.5%) 637 (83.7%) Embryos transferred 2.9±1.1 3.0±1.0 Pregnancy rate/ET 7/35 (20.0%) 22/116 (19.0%) Implantation rate 6.9% 6.6%

Antagonist vs Agonists Cet Agonist <40 ≥40 Number of OPU 371 184 171 23 Number of Eggs Retrieved 3994 1388 2126 199 Number of MTII 2984(75%) 1055(76%) 1575(74%) 152(76%) Number of MTI 526 (13%) 160 (12%) 205 (10%) 25 (13%) Number of ICSI’d 3269 1131 1729 173 Number of 2PN 2472 870 1303 126 Fertilization Rate 76% 77% 75% 73% Total # of Embryos Transferred 1039 521 532 62 Mean # of Embryos Transferred per ET 2.8 3.1 2.7 Number of Pregnancy 145 25 82 5 Pregnancy Rate per ET 39% 14% 48% 22% Implantation Rate 17% 5% 20% 10% Average Age 35.1 41.8 33.7 41.5

Comparison: Mode of Actions Antagonists Agonists Immediate onset of actions (shortens treatment durations) Prevents hormonal withdrawal symptoms No recovery time of the pituitary long pre-treatment Hormonal (estrogen) withdrawal symptoms through desensitization of pituitary Recovery of the pituitary gonadotrophin secretion, after stopping the treatment takes about 2 weeks.

Reduction of OHSS using Cetrotide® Multiple dose protocol rate of OHSS: 6.5% vs. 1.1% (agonist vs. antagonist protocol) RR 6.2, 95% CI: 1.4 - 27.1, p = 0.03 Single dose protocol rate of OHSS: 11.1% vs. 3.5% (agonist vs. antagonist protocol) 95% CI: - 18.4 to 3.2 patients requiring hospitalisation: 5.6% vs. 1.8% (agonist vs. antagonist protocol) 95% CI: - 11.7 to 4.1 With both Cetrotide® protocols a clear reduction of OHSS was achieved

The GnRH Antagonists Conclusions: Why treat 100% of patients when we are trying to prevent 5-10% LH surge Avoid over-suppression and poor response Effective in preventing LH surge Reduction of hyper-stimulation Lower costs

Ovum Preparation for IVF FSH/GnRH Down Regulation FSH/GnRH Antagonists Clomid, Clomid/FSH Minimal Stimulation IVM Natural Cycles

Problems with Ovarian Stimulation Drug Costs Side effects: immediate and delayed Future long term risks Not “User Friendly”

Problems with Ovarian Stimulation Waste of Human Resources Excess eggs ? how to deal with Excess embryos - even worse Multiple pregnancies and their associated complications

Individualized stimulation

Individualized Stimulation

Individualizing Stimulation

Individualized Stimulation

Over responders Risk of OHSS Treatment options Cancel cycle Coasting No embryo transfer Convert to IVM

Individualizing protocols For over responders For low responders

Over responders Prolonged Coasting Aim: To prevent hyperstimulation Practice: Coast till E2 ≤ 3000 pg/mL Sher, 1995 Start when 30% follices > 15 mm Nilsson, 1999 When 3 follicles > 17mm

IVM stimulation

Poor responders Age (average age of ML patient 38.7 yrs) Decrease ovarian reserve (↑D2 FSH) Decrease preantral follicles Previous ovarian surgery (Laparoscopic ovarian cystectomy)

Poor responders High dose Microdose flare Low dose clomid/FSH stimulation Delayed stimulation IVM

Microdose Flare Regimen (1) Oral Contraceptive 20 mcg leuprolide cs bd x 2 d uFSH for ovarian stimulation Results: ↑oocytes Less ampoules FSH Source: Scott et al, 1994

Microdose Flare Regimen (2) Oral Contraceptive 40 mcg leuprolide sc bd 4 IU/d growth hormone IM Followed by uFSH 2 days later Results: ↓Cancellation rate ↑E2 levels, number of oocytes Source: Schoolcraft et al, 1997

Microdose Flare Regimen (3) Oral Contraceptive 40 mcg leuprolide sc bd uFSH starting 2 days later Results ↓Cancellation rate ↑E2 levels, number of oocytes Source: Surrey et al, 1998

Poor responders High dose Microdose flare Low dose clomid/FSH stimulation Delayed stimulation IVM

Minimal stimulation

Poor responders High dose Microdose flare Low dose clomid/FSH stimulation Delayed stimulation IVM

Delayed Stimulation

Poor responders High dose Microdose flare Low dose clomid/FSH stimulation Delayed stimulation IVM

IVM stimulation

IVM results 2004 Aug to 2007 Jun <38 ≥38 Patients (n) 33 16 Average age 32.6 40.0 Total eggs 420 (12.7 ) 160 (10.0) MTII stage 314 (74.8%) 123( 76.9%) Fertilization rate 254 (80.9%) 107 (87.0%) Pregnancy rate 33.3% 37.5% Embryos transferred 84 34 Implantation rate 14.3% 17.6%

Modern Trend in ART Minimize multiple pregnancies Minimize number of embryos transfer Minimize patients’ load and stress Physiological Psychological Financial

Question Is it time to revisit the aim and clinical practice of so called Controlled Ovarian Hyperstimulation. Should we be heading towards a modified direction

Answer We should look at the clinical aim of “Preparing Eggs for the treatment of IVF” rather than Ovarian Stimulation

Preparation for Ovum Collection Natural Cycles Minimal stimulation (clomiphene/FSH) IVM FSH stimulation with agonists FSH stimulation with antagonists

Conclusions: It is possible to choose stimulation procotol according to: age Ovarian status Previous history We should aim for minimal stress (in all senses) for the patients provided similar result can be obtained. Individualization of stimulation should be considered for every case.

Stimulated ovary

Stimulated ovary