1. Dr. Milton Leong Director
IVFCENTRE
Hong Kong Sanatorium & Hospital

2. The Gonadotrophin Releasing Hormone Antagonists

3. 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

4. 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)

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

6. 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;
Porter et al., 1984

7. 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

8. 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

9. 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.

10. 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

11. 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

12. Structure of GnRH antagonists

13. 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.

14. 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:
control
Days relative to ovulation
Nal-Glu cycles
Ditkoff et al., 1991

15. Cetrotide®
Cetrorelix

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

17. 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:
Diedrich et al., 1994

18. 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:
no premature LH surge
Felberbaum et al., 1996

19. 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:
Albano et al., 1997

20. 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:

21. 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,
Olivennes et al., 1995

22. 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; .

23. 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, mg and 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:

24. 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

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

26. 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:
Albano et al., 2000

27. The multiple dose antagonist protocol compared to the long luteal protocol
inclusion criteria
age: years
normal menstrual cycle (range: 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:
Albano et al., 2000

28. 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:
n.s. not significant
Albano et al., 2000

29. 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:
n.s. not significant
Albano et al., 2000

30. 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:
n.s. not significant
Albano et al., 2000

31. 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: )
Ludwig et al., 2000

32. The multiple dose antagonist protocol compared to the long luteal protocol
Significantly less OHSS °II and °III (RR 6.2, 95% CI: , p = 0.03)
Less patients with threatened OHSS (no hCG - administration when  12 follicles  15 mm and/or estradiol  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

33. 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

34. 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

35. 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

36. 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
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

37. 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: , p = 0.03
Single dose protocol
rate of OHSS: 11.1% vs. 3.5% (agonist vs. antagonist protocol) 95% CI: to 3.2
patients requiring hospitalisation: 5.6% vs. 1.8% (agonist vs. antagonist protocol) 95% CI: to 4.1
With both Cetrotide® protocols a clear reduction of OHSS was be achieved

38. 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

39. 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

40. 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 mg daily
Switching to a half dosage of ml per day gives:
Normal LH levels
Expected follicular growth
Better ovum quality
No premature LH surge or progesterone rise