Dr. Milton Leong Director IVFCENTRE Hong Kong Sanatorium & Hospital

The Gonadotrophin Releasing Hormone Antagonists

Schally AV, Arimura A, Bowers CY et al. 1968 Synthesis of GnRH pGlu1-His2-Trp3-Ser4-Tyr5-Gly6-Leu7-Arg8- Pro9-Gly10NH2 by Schally in 1968 GnRH could restore ovulatory functions in hypogonadotrophic amenorrheas. Schally AV, Arimura A, Bowers CY et al. 1968

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)

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

Results of first application of GnRH-agonists in the long protocol 11 patients eligible for IVF GnRH agonist s.c. (buserelin) started at day of menstruation or 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

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

Action of GnRH agonists downregulation 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 pituitary suppression flare up effect GnRH - agonist

GnRH agonist Over-suppression: 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

Structure of GnRH antagonists

Characteristics of GnRH 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.

Antagonists in controlled ovarian stimulation - the first steps Administration of the GnRH antagonist leads to suppression of estradiol, stop of follicular growth and avoidance of a LH or FSH surge. Ditkoff EC, Cassidenti DL, Paulson RJ, et al. The gonadotropin-releasing hormone antagonist (Nal-Glu) acutely blocks the luteinizing hormone surge but allows for resumption of folliculogenesis in normal women. Am J Obstet Gynecol 1991; 165: 1811-1817. control Days relative to ovulation Nal-Glu cycles Ditkoff et al., 1991

Cetrotide® Cetrorelix

Dose finding studies to identify the minimal effective dose in the multiple dose and single dose protocol

The development of the multiple dose antagonist protocol 20 patients in IVF cycles gonadotropins were started on cycle day 2 Cetrorelix in a daily dosage of either 3 mg or 1 mg started on day 7 of stimulation no spontaneous LH surge was observed This study was done using HMG Diedrich K, Diedrich C, Santos E, et al. Suppression of the endogenous luteinizing hormone surge by the gonadotrophin-releasing hormone antagonist Cetrorelix during ovarian stimulation. Hum Reprod 1994; 9: 788-791 Diedrich et al., 1994

The development of the multiple dose antagonist protocol dose finding study using Cetrorelix in a daily dosage of 3 mg, 1 mg, and 0.5 mg This study was done using HMG. Felberbaum R, Reissmann T, Küpker W, et al. Hormone profiles under ovarian stimulation with human menopausal gonadotropin (hMG) and concomitant administration of the gonadotropin releasing hormone (GnRH)- Antagonist Cetrorelix at different dosages. J Assist Reprod Genet 1996; 13: 216-222 no premature LH surge Felberbaum et al., 1996

The development of the multiple dose antagonist protocol dose finding study using Cetrorelix in a daily dosage of 0.5 mg, 0.25 mg, and 0.1 mg premature LH surge occured in the 0.1 mg group This study was done using HMG. Albano C, Smitz J, Camus M, Riethmuller-Winzen H, Van Steirteghem A, Devroey P. Comparison of different doses of gonadotropin-releasing hormone antagonist Cetrorelix during controlled ovarian hyperstimulation. Fertil Steril 1997; 67: 917-922 Albano et al., 1997

The development of the multiple dose antagonist protocol Cetrotide® 0.25 mg is the minimal effective dose in the multiple dose antagonist protocol Albano C, Smitz J, Camus M, Riethmuller-Winzen H, Van Steirteghem A, Devroey P. Comparison of different doses of gonadotropin-releasing hormone antagonist Cetrorelix during controlled ovarian hyperstimulation. Fertil Steril 1997; 67: 917-922

The development of the single dose antagonist protocol 11 patients for IVF first injection of 3 mg Cetrorelix always on day 8 of the cycle second injection, if no hCG injection latest 72 hours later possible 3 patients received a second injection of Cetrorelix these 3 patients had low estradiol levels on day of first Cetrorelix administration Olivennes, F., Fanchin, R., Bouchard, P., Taieb, J., Selva, J., Frydman, R. (1995). Scheduled administration of a gonadotrophin-releasing hormone antagonist (Cetrorelix) on day 8 of in-vitro fertilization cycles: a pilot study. Hum Reprod, 10, 1382-1386 Olivennes et al., 1995

The development of the single dose antagonist protocol Cetrotide® 3 mg is the minimal effective dose in the single dose antagonist protocol . Olivennes F., Alvarez S., Bouchard P., Fanchin R., Salat-Baroux J., Frydman R. (1998) The use of a GnRH antagonist (Cetrorelix) in a single dose protocol in IVF-embryo transfer: a dose finding study of 3 versus 2 mg. Hum Reprod 13; 2411-2414 .

The Ganirelix dose-finding Study Group, Hum Reprod 1998;13:3023-31 Dose Finding Studies With Ganirelix : 2 mg, 1 mg, 0.5 mg, 0.25 mg, 0.125 mg and 0.0625 mg were used 0.25 mg daily was the preferred dosage With Ganirelix, increasing dosage related with drop in pregnancy rate and increase in abortion rate. 2 mg daily dosage slowed follicular growth as well as almost stopping ay increase in estradiol secretion. The Ganirelix dose-finding Study Group, Hum Reprod 1998;13:3023-31

Comparison of the long protocol and the antagonist protocols more physiologic no cyst formation no hormonal withdrawal antagonist administration gonadotropin administration multiple dose protocol less gona- dotropins early pregnancy? Advantages of the antagonist protocol: - no cyst formation (since there is no flare up effect) - no hormonal withdrawal symptoms (since pituitary suppression is achieved after ovarian stimulation has been started and not before) - the antagonist cannot be given in early pregnancy, since this can be excluded by a pregnancy test - less gonadotropins necessary - shorter treatment duration - more physiologic, since it can be integrated in the spontaneous menstrual cycle flare up effect pituitary suppression agonist administration gonadotropin administration long protocol longer treatment pre-treatment cycle treatment cycle

Comparison of antagonist protocols and the long luteal protocol Prospective, randomized trials

The multiple dose antagonist protocol compared to the long luteal protocol Prospective, randomized phase III study 7 European centres 273 patients for IVF or IVF/ICSI Stimulation procedures long luteal protocol: buserelin nasal spray (4 x 150µg) multiple dose antagonist protocol: Cetrotide® 0.25 mg start with 150 IU FSH per day in the antagonist and agonist group This study was done using HMG. Albano, C., Felberbaum, R.E., Smitz, J., Riethmüller-Winzen, H., Engel, J., Diedrich, K., and Devroey, P. (2000) Controlled ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the GnRH-antagonist Cetrorelix (Cetrotide) and the GnRH-agonist Buserelin. Hum Reprod, 15:526-531 Albano et al., 2000

The multiple dose antagonist protocol compared to the long luteal protocol inclusion criteria age: 18 - 39 years normal menstrual cycle (range: 24 - 35 days) with an intraindividual variation of max. ± 3 days no more than 3 IVF procedures normal uterus and at least one functioning ovary exclusion criteria severe endometriosis (AFS III/IV) PCO syndrome Albano, C., Felberbaum, R.E., Smitz, J., Riethmüller-Winzen, H., Engel, J., Diedrich, K., and Devroey, P. (2000) Controlled ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the GnRH-antagonist Cetrorelix (Cetrotide) and the GnRH-agonist Buserelin. Hum Reprod, 15:526-531 Albano et al., 2000

The multiple dose antagonist protocol compared to the long luteal protocol Albano, C., Felberbaum, R.E., Smitz, J., Riethmüller-Winzen, H., Engel, J., Diedrich, K., and Devroey, P. (2000) Controlled ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the GnRH-antagonist Cetrorelix (Cetrotide) and the GnRH-agonist Buserelin. Hum Reprod, 15:526-531 n.s. not significant Albano et al., 2000

The multiple dose antagonist protocol compared to the long luteal protocol Albano, C., Felberbaum, R.E., Smitz, J., Riethmüller-Winzen, H., Engel, J., Diedrich, K., and Devroey, P. (2000) Controlled ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the GnRH-antagonist Cetrorelix (Cetrotide) and the GnRH-agonist Buserelin. Hum Reprod, 15:526-531 n.s. not significant Albano et al., 2000

The multiple dose antagonist protocol compared to the long luteal protocol Albano, C., Felberbaum, R.E., Smitz, J., Riethmüller-Winzen, H., Engel, J., Diedrich, K., and Devroey, P. (2000) Controlled ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the GnRH-antagonist Cetrorelix (Cetrotide) and the GnRH-agonist Buserelin. Hum Reprod, 15:526-531 n.s. not significant Albano et al., 2000

The multiple dose antagonist protocol compared to the long luteal protocol: significant reduction of OHSS Ludwig M., Felberbaum R.E., Devroey P., Albano C., Riethmüller-Winzen, H., Schüler A., Engel W., Diedrich K. (2000) Significant reduction of the incidence of ovarian hyperstimulation syndrome (OHSS) by using the LHRH antagonist Cetrorelix (Cetrotide®) in controlled ovarian stimulation for assisted reproduction. Arch Gynecol Obstet 264; 29-32 * p = 0.03, Fishers exact test, relative risk: 6.2 (95% CI: 1.4 - 27.1) Ludwig et al., 2000

The multiple dose antagonist protocol compared to the long luteal protocol Significantly less OHSS °II and °III (RR 6.2, 95% CI: 1.4 - 27.1, p = 0.03) Less patients with threatened OHSS (no hCG - administration when  12 follicles  15 mm and/or estradiol  4.000 pg/ml) Cetrotide® 0.25: 3 patients (1.6%) buserelin: 5 patients (5.9%) One more patient in the buserelin group did not have an embryo transfer because of a threatened OHSS Ludwig M., Felberbaum R.E., Devroey P., Albano C., Riethmüller-Winzen, H., Schüler A., Engel W., Diedrich K. (2000) Significant reduction of the incidence of ovarian hyperstimulation syndrome (OHSS) by using the LHRH antagonist Cetrorelix (Cetrotide®) in controlled ovarian stimulation for assisted reproduction. Arch Gynecol Obstet 264; 29-32 Ludwig et al., 2000

The multiple dose antagonist protocol compared to the long luteal protocol Ganirelix vs. Buserelin No. of patients 463 237 No. of patients who reached the day of hCG 448 224 No. of patients with oocyte pick-up 440 221 No. of patients with embryo transfer 399 208 Cancellation rate (%) 13.8 12.6

The multiple dose antagonist protocol compared to the long luteal protocol Ganirelix vs. Buserelin LH rises during treatment (%) 2.8 1.3 Days of analogue treatment 5 26 Fertilization rate (%) 62.1 Clinical pregnancy rate per embryo transfer (%) 25.1 31.7 Overall incidence of OHSS (%) 2.4 5.9

The multiple dose antagonist protocol compared to the long luteal protocol Cetrorelix vs. Triptorelin depot Cetrorelix Triptorelin depot No. of patients 115 39 hCG administered (%) 98.3 92.3 Patients with OPU (%) Patients with embryo transfer (%) 86.1 84.6 Incidence of LH surges 2.6 Days of stimulation 9.4 10.7

The multiple dose antagonist protocol compared to the long luteal protocol Cetrorelix vs. Triptorelin depot Cetrorelix Triptorelin depot No. of hMG ampules 24.3 35.6 E2 (pg/ml) on the day of hCG 1786 + 808 2549 + 1194 COC per patient 9.2 10.7 Fertilization rate (%) 50.5 54.7 Clinical pregnancy rateper embryo transfer (%) 21.2 27.3 Babies born per embryos replaced (%) 10.6 13.3 OHSS grades II-III (%) 1.8 5.6

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 be achieved

Personal experience with multiple dose of Cetrorelix 0.25 mg Patient group: Over suppression with agonist long protocol (LH < 1mlU) Patient over 40 Poor response to agonists suppression

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

No premature LH surge or progesterone rise Results Check the stimulation day 7th LH level LH > 1.5 mIU/ml, 0.25 mg daily was given LH < 1.5 mIU/ml, reduce to 0.125 mg daily Switching to a half dosage of 0.125 ml per day gives: Normal LH levels Expected follicular growth Better ovum quality No premature LH surge or progesterone rise