ASSISTED

REPRODUCTION:
STIMULATION
PROTOCOLS

Yasser Orief MD, PhD.

Assistant Professor of Obstetrics & Gynecology, Alexandria
University, Egypt
Fellow, Lubeck University, Germany
DOGE, Auvergne university, France.
First successful human IVF
pregnancy resulted from
a spontaneous cycle.
Now, COH is routinely
employed to effect multiple
follicular recruitment.
 Dominance

Ovulation
Recruitment

Selection
Corpus Luteum

50

40 LH
P
FSH, LH E2
30

E2, P
20
FSH

10

0
1 2 3 4 5 6 7 8 9 101112131415161718192021222324262728

Days
 COH Protocols
The Cornet Model
10

9
FSH Level

6
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
 Dominance
Recruitment

Ovulation
Selection

Corpus Luteum
D5

50

40 LH
P
FSH, LH

E2, P
30 E2
FSH

20

10

0
1 2 3 4 5 6 7 8 9 101112131415161718192021222324262728

Days
 CC
Conventional Low dose step
Gonadotropin protocol up
Non GnRH
analouges only
Chronic low doe Low dose step
protocol down
CC / hMG
Natural cycle sequential

Induction
CC
protocols
Single dose
Antagonist protocol
FSH & / HMG Fixed
Multiple dose
protocol
CC + FSH
&/HMG Flexible
GnRH analouges
Ultralong
Follicular
Long
Agonist Luteal
Short

Ultrashort
onadotropins Trade Name
Human Menopausal GnRH antagonists
Gonadotropins Antagon,
Pergonal Ganirelix,
Menogon Cetrotide
Merional Orgalutran
Purified hMG Cetrorelix
Menopur Recombinant FSH
Repronex Gonal-f
Purified FSH Gonal-f Pen
Metrodin Puregon
Bravelle Puregon Pen
Fertinex Recombinant LH
GnRH agonist Luveris
Lupron Urinary Human chorionic
Luprolide gonadotropin (HCG) Novarel,
Decapeptyl Pregnyl,
Nafarelin Profasi,
Buserelin (suprefact) Chorex,
Zoladex Choriomon
Synarel Recombinant HCG
Enantone Ovidrel
Procin
NON GNRH
ANALOUGE
PROTOCOLS
The human FSH
molecule.
It consists of an
alpha and a
beta chain with
four charbohydrate
sidechains (red).
No GnRH Analogue Protocols:
hMG, FSH, or both

17
95 96
E2
75 75 77 78
70
65 66 65
Premature surge
50 50

33
27 27 28 29 29 29 30 31
23 FSH Premature luteinization
18 15 15 15 15 15
8 9LH10 10 10 10 11 12 12 12 13 13

hMG injections

menstruation

h
C
G
Problems of protocols without
GnRH Analogues:

1. CC: oocyte quality and

endometrial receptivity.
2. Asynchroneity
3. Spont. LH surge (15-35%)
4. Premature luteinization
5. Short or inadequate luteal phase
 GONADOTROPIN
THERAPY
Preparations
hMG
Purified hMG
Purified FSH
Recombinant FSH

There is little or no difference between the

different preparations as regards the
clinical outcome.

(Sagle et al., 1991; Aboulghar et al., 1996; Yarali et al., 1999;

Hugues et al., 2001; Nugent et al., 2003; and Bayram et al.,
2003)
 GONADOTROPIN
THERAPY
Indications:

Clomiphene resistant & failure cases.

Obvious antiestrogenic side effects with
CC.

Problems with gonadotrophin O/I:

High incidence of OHSS.

High incidence of multiple gestations.
 FACTORS AFFECTING
OUTCOME OF
GONADOTROPHIN THERAPY
Females age. (McClure et al., 1993; Mulders et al., 2003)
Elevated LH levels (~40%)
Hyperandrogenic state. (Mulders et al., 2003)
Obesity (BMI>28-30 kg/m2) and insulin resistance
may result in hyperandrogenism and cause poor
response;
insulin resistance and the compensatory
hyperinsulinemia may increase the risk for OHSS.
(Vicino et al., 2000; Mulders et al., 2003(Dale et al., 1998)
Preceding response to CC. (Imani et al., 2002)
Polycystic ovarian morphology (Jonard et al., 2003)
 GONADOTROPIN
THERAPY
Treatment strategy:
The aim is to give the
minimum
amount of FSH which will
induce the development of
a single ovulatory follicle.
 CC
Conventional Low dose step
Gonadotropin protocol up
Non GnRH
analouges only
Chronic low doe Low dose step
protocol down
CC / hMG
Natural cycle sequential

Induction
CC
protocols
Single dose
Antagonist protocol
FSH & / HMG Fixed
Multiple dose
protocol
CC + FSH
&/HMG Flexible
GnRH analouges
Ultralong
Follicular
Long
Agonist Luteal
Short

Ultrashort
GONADOTROPIN
THERAPY
Gonadotro
pin only

Chronic
Convention
low doe
al protocol
protocol

Low dose
step up

Low dose
step down

sequential
 GONADOTROPIN
THERAPY
In all regimens hCG (5000-10000 IU)
is given to induce ovulation when
there is at least one follicle 18
mm.
HCG may be withheld if :

More than 3 follicles 15 mm in

diameter are present
Or
E2 is >1500 pg/mL.
 GONADOTROPIN
THERAPY
CONVENTIONAL
This protocol involves initial daily doses of 75
PROTOCOL
150 IU FSH treatment with increases of 75 IU
FSH every 57 days, when needed.
Supraphysiological doses of FSH in this protocol
provoke initial development of a large cohort.
While ovulation rates of 70% were achieved,
multiple pregnancy rates were observed to
occur in 36% of pregnancies, and potentially
life-threatening OHSS in 14% (Dor et al., 1980)
Reported cumulative pregnancy rates ranged
from 21 to 75%
(Hamilton-Fairley and Franks, 1990; Farhi et al., 1993; Fauser and Van
Heusden, 1997).
 GONADOTROPIN
THERAPY
CONVENTIONAL
PROTOCOL
The conventional protocol, which is
associated with unacceptably high
multiple follicle development, multiple
pregnancy and a significant incidence of
OHSS, should NOT be employed to induce
ovulation in patients with PCOS.
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
Essential toPROTOCOLS
minimizes the rate of multi-
follicular development.
Low dose
step up

Chronic
low doe Low dose
protocol step down

sequential
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
The most commonly employed chronic
PROTOCOLS
low-dose regiment.
A. LOW
The aim DOSE
is to give the STEP-UP
minimum dose of
PROTOCOL
gonadotrophin necessary to induce
normal follicular development.
Injection of a small amount of FSH is
often sufficient to supplement
endogenous FSH, so that the
concentration rises above the threshold.
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
Once so, growing follicle(s) secrete sufficient E2
PROTOCOLS
& inhibin to suppress the endogenous FSH so
that the A. LOW
overall levelDOSE STEP-UP
of FSH drops below the
threshold required to recruit
PROTOCOL additional
follicles.

Start of treatment:

Treatment starts any time, provided low ovarian

activity is present and is monitored by U/S.
Usual start is after spontaneous or P induced
withdrawal bleeding.
 GONADOTROPIN
THERAPY
CHRONIC
Starting dose:
LOW DOSE
PROTOCOLS
Standard starting FSH dose is 75 IU/d. It is maintained for
up to 14 days unless follicle maturity is reached (Polson et
A. LOW DOSE STEP-UP
al., 1987).

Starting dose of 50PROTOCOL

IU/d (White et al., 1996) is as effective as
75 IU/d as regards ovulation, pregnancy, and miscarriage.

Monofollicular ovulation is slightly increased (84% vs 72%).

Starting dose of 37.5 IU/d of recFSH (Balasch et al., 2000),
can be equally effective.

Starting dose must be adjusted according to BMI and

previous response (Franks and White, 2002).
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
PROTOCOLS
Monitoring of ovarian response:
A. LOW DOSE STEP-UP
Start of ovarianPROTOCOL
response is diagnosed
when a follicle 12 mm is seen in the
ovaries.

Alternatively response can be initially

monitored by serum E2 every 2-3 days.
When E2 is >100 pg/ml, monitoring is
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
PROTOCOLS
Increments:
A. LOW DOSE STEP-UP
If no ovarian response is seen after 14 days,
PROTOCOL
the dose is increased by 37.5 IU/day at
weekly intervals up to a maximum dose of
225 IU/day

In women who develop multiple follicles

using the above protocol, the treatment is
modified to a smaller starting dose (37.5 IU)
 GONADOTROPIN
THERAPY
CHRONIC
Maintenance:
LOW DOSE
If a dominant PROTOCOLS
follicle emerges, the dose of FSH
(threshold dose) is maintained until the follicle
A. LOW DOSE STEP-UP
reaches a mean diameter of 17 mm.
HCG: PROTOCOL
HCG in a dose of 10,000 IU is injected when the
leading follicle is 18 mm in diameter.
Cancellation: (9%)
If there are >3 follicles of 15 mm, to avoid the
risk of multiple pregnancy and/or OHSS.
No response after 35 days of treatment.
If E2 is >1500pg/mL.
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
Follow up:
PROTOCOLS
SerumA. LOW
P 68 daysDOSE
after hCGSTEP-UP
administration to
PROTOCOL
confirmed ovulation (P >5 ng/ml)
Serum pregnancy test is done 14 days after
administration of HCG.
If, despite ovulation, pregnancy has not occurred
during the first course of treatment, FSH is
reintroduced at a subthreshold dose (37.5 IU less
than the preceding threshold dose), as the
threshold dose within-subject varies from cycle to
cycle.
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
Results:
PROTOCOLS
Strict adherence to a 14-day starting period using a
persistent dose is essential to minimize the risk of
A. LOW DOSE STEP-UP
multifollicular development.
PROTOCOL
Monofollicular response is found in 5488% of cases
(Homburg and Insler, 2002).
Very low incidence of OHSS (1.4%)
Low incidence of multiple pregnancy rate of only 5.7%
(Homburg and Howles, 1999; Franks and White, 2002).

No response in about 6% of cycles.

Low responders have characteristics of more severe
PCOS, such as obesity, hyperandrogenism and polycystic
ovaries as compared with good responders (Mulders et al.,
2003).
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
Results:
PROTOCOLS
There is A. LOW
a need DOSE STEP-UP
for optimization of the
PROTOCOL
starting doses. Such optimization will prevent

1. hyperstimulation due to a starting dose far

above the FSH threshold as well as
2. minimize the time-consuming low-dose
increments by starting with a higher
dose in women with augmented FSH
threshold.
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
Results:
PROTOCOLS
A. LOW
The pregnancy ratesDOSE STEP-UP
are 17, 26, 34 and 26%
when 1, 2, 3 andPROTOCOL
>3 follicles exceeding 15
mm in diameter are present on hCG day
(Ares-Serono, 1995).

However, multiple pregnancy rates increase

dramatically; the respective figures are 5, 12,
20 and 50%.
Obese patients have a poor ongoing
pregnancy rate of <10%.
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
Results:
PROTOCOLS
WhiteA. LOW
et al. DOSE
(1996) STEP-UP
reported the cumulative
pregnancy ratePROTOCOL
as 57% after 6 cycles; 75% of
pregnancies occurred in the first 3 cycles of
treatment and only one pregnancy occurred
after the 6th cycle.

IVF may be offered to those who fail to

conceive after a maximum of 6 cycles of
gonadotrophin treatment.
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
PROTOCOLS
In an attempt to mimic physiology more
B. in
closely LOW
womenDOSE STEP-
with PCOS, DOWN
a step-down
PROTOCOL
regimen involves reducing the dose of
gonadotrophins administered (van Santbrink and
Fauser, 1997).

Monitoring of a step-down cycle may need more

experience compared with a low- dose step-up
regimen.
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
150 IU/d FSHPROTOCOLS
is started and continued until a
dominant follicle (10 mm) is seen on TVS
B. LOW DOSE STEP- DOWN
(Macklon and Fauser, 2002).

PROTOCOL
The dose is then decreased to 112.5 IU/d
followed by a further decrease to 75 IU/d 3
days later, which is continued until HCG is
administered to induce ovulation (van Santbrink et
al., 1995; van Santbrink and Fauser, 1997).
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
PROTOCOLS
Pregnancy outcomes
step-up regimens.
are comparable to low-dose

B. LOW DOSE
Substantially STEP- DOWN
shorter stimulation period (9 days
PROTOCOL
versus 18 days) is required.
More multifollicular cycles.
More hyperstimulation.
(Christin-Maitre and Hugues, 2003).
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
PROTOCOLS
In order to avoid the tendency to hyper-
response in step-down protocols, it is suggested
B. LOW
offering DOSE STEP-
first a dose-finding DOWN
low-dose step-
PROTOCOL
up induction cycle during which the FSH
threshold dose is determined (Van Santbrink and
Fauser, 2003).

After the first cycle, step-down cycles are

undertaken with a starting dose 37.5 IU above
the threshold dose in the initial low-dose
step-up cycle.
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
PROTOCOLS
The
C. FSH dependence of
SEQUENTIAL the leading
STEP-UP ANDfollicle
decreases as the follicle
STEP-DOWN grows.
PROTOCOL
This decrease in FSH threshold
contributes to the escape of the
leading follicle from atresia when FSH
concentrations start to decrease due to
negative feedback of rising oestrogen
concentrations during the follicular phase.
 GONADOTROPIN
THERAPY
CHRONIC LOW DOSE
PROTOCOLS
In the sequential protocol, using this
C. SEQUENTIAL STEP-UP AND
physiological principle, a step-up approach is
STEP-DOWN
utilized, and thereafter thePROTOCOL
FSH dose is
reduced by 37.5 IU when the leading follicle
reaches a diameter of 14 mm (Hugues et al.,
1996).

Decreasing the FSH dose following step-up

follicular selection might be an
alternative method to avoid multifollicular
development
 CC
Conventional Low dose step
Gonadotropin protocol up
Non GnRH
analouges only
Chronic low doe Low dose step
protocol down
CC / hMG
Natural cycle sequential

Induction
CC
protocols
Single dose
Antagonist protocol
FSH & / HMG Fixed
Multiple dose
protocol
CC + FSH
&/HMG Flexible
GnRH analouges
Ultralong
Follicular
Long
Agonist Luteal
Short

Ultrashort
 Non GnRH
analouges
Induction
protocols Antagonist
GnRH
analouges
Agonist
 Gonadotropin releasing
hormone

Pyro
Glu His Trp Ser Tyr Gly Leu Arg Pro Gly

1 2 3 4 5 6 7 8 9 10
GnRH Agonists

Pyro
Glu His Trp Ser Tyr Leu Arg Pro

GnRH Antagonists

Ac-D- D-Phe D-Ala-

Nal(2) D/PAL Ser Tyr Leu Pro
(4CL) NH2
GnRH Receptor: G protein-coupled receptor
 Induction
protocols
Non
GnRH
GnRH
analouge
analouge
s
s
Antagoni
Agonist
st

Ultrashor
Ultralong Long Short
2-7 Start D2 or D3
Start D2 or D3 t D2,3 or
Agonist
hMG with or 1D 4 only
4 only
months after hMG D2 or 3

Follicular Luteal
Starts D1 Starts D21 23
hMG after hMG after
suppression suppression
 Endopeptidase

Gonadotropin releasing
hormone
Pyro
Glu His Trp Ser Tyr Gly Leu Arg Pro Gly CONH2

Carboxyamide
peptidase
D-Leu D-Trp
GnRH agonists
Pyro
Glu
His Trp Ser Tyr Leu Arg Pro NH-Ethylamide

D-His D-Ser (Buserelin)

Suprefact
 AGONIST PROTOCOL

Selective amino acid substitution at

positions
6 & 10 of GnRH result in:

High binding affinity to the GnRH

receptors
Reduced susceptibility to degradation by
pituitary endopeptidases
Proloned plasma half-life
Increased biologic activity
 AGONIST PROTOCOL

Administration of GnRH agonists results in:

Initial stimulation orflare up effect with

surge of gonadotropins.
Followed by desensitization and
downregulation of the pituitary due to
prolonged receptor occupancy, with a
resultant reversible hypogonadism.
 AGONIST PROTOCOL

Advantages

Suppression of endogenous LH
Better FSH/LH ratio
More oocytes
Better synchroneity
Better oocyte and embryo quality
Higher pregnancy rate & lower
abortions
SHORT AGONIST PROTOCOL

18
95 96
E2
75 75 77 78
70
65 LH 65 66 65
50 50 50
40 40 44 FSH
36 33 35 34 36
33
30 31 29
30 29
27 27 28 29 31
28 31
30 30 27 26 26

hMG injections

GnRHa
menstruation

h
C
G
ULTRA-SHORT AGONIST
PROTOCOL

18
95 96
E2
75 75 77 78
70
65 LH 65 66 65
50 50 50
40 40 44 FSH
36 33 35 34 36
33
30 31 29
30 29
27 27 28 29 31
28 31
30 30 27 26 26

hMG injections
GnRHa

menstruation

h
C
G
LONG AGONIST PROTOCOL

18
95 96

75 75 77 78
70
65 62 E2 65 66 65
55
50 50
43 45
40 43 46 FSH 40 44
31 35 33 39
31
37 36 35 35 33 34 35 36 36
31 34 27 29 31 29
27 27 26 30
26 30
26 29
26 29
26 29
26 29
26 29
26 29
26 29
26 29 26 30
26 29 LH
26 26 26

hMG injections

GnRHa
menses

h
C
G
 LONG LUTEAL
AGONIST PROTOCOL

A- The suppression phase:

Start on the D1 (long follicular)
or D21 to 23 (long luteal)
Give the appropriate type of
agonist; ovarian suppression is
more with Zoladex, less with
Suprefact, and least with
Decapeptyl.
 LONG LUTEAL
AGONIST PROTOCOL

A) The suppression phase:

Different agonists are available:

Decapeptyl 0.1 mg (triptorelin) S.C. daily.

Decapeptyl CR 3.75 mg single I.M. depot
injection.
Suprefact vials (buserelin acetate) 3/4 mg
S.C. daily.
Suprefact nasal spray (3x300g/d).
Zoladex (goserelin; 3.6 mg) single S.C.
implant.
 LONG LUTEAL
AGONIST PROTOCOL

A)The suppression phase:

) When menses comes, continue

administering the agonist if it is a
short acting type that is given daily.
) U/S on 1st or 2nd menstrual day:
endometrium < 5 mm & no follicles
>10 mm in the ovaries.
) E2: if <35 pg/mL = suppressed.
 Long protocol:
Suppression, ovaries
Long protocol:
Suppression,
endometrium
 LONG LUTEAL
AGONIST PROTOCOL

B) The stimulation phase:

The dose of daily given agonist can be
reduced (e.g. to mL Suprefact, 0.05 mg
Decapeptyl).

Decide the starting dose of hMG:

Usual dose is 4 amps of hMG/d I.M. (FSH
&/or recFSH &/or hMG)
Lower doses (2-3 amps/d) are needed
for high responders e.g. young &/or PCO
patients.
 LONG LUTEAL
AGONIST PROTOCOL
B) The stimulation phase (cont.):
U/S after 3 to 5 days to monitor response.
(potential modifications)
HCG criteria:
at least 3 follicles >18 mm
endometrium >8 mm & tripple line.
stimulation days > 8
if in doubt: E2>150-200
pg/mL/follicle>16 mm
 Long protocol
Stimulation day 5
 Long protocol:
Stimulation day 8
 Long protocol:
Stimulation day 5
 Long protocol:
Stimulation day 6
tripple line endometrium
7 mm
 Long protocol:
Stimulation: day hCG
tripple line endometrium
9 mm
 Long protocol:
Stimulation: day hCG
early secretory
endometrium
 LONG LUTEAL
AGONIST PROTOCOL
B) The stimulation phase (cont.):
hCG (Pregnyl, Choragon, Profasi) is
given in the dose of 10,000 IU I.M.
(2x5000 IU amp) usually 9:30 in the
evening.

The day of hCG is the LAST day in

which agonist and hMG are given.

Oocyte retrieval is scheduled 36 hours

after hCG (in the morning).
 LONG LUTEAL
AGONIST PROTOCOL

Disadvantages

Higher cost
Possible over-suppression with diminished
ovarian reserve
Higher OHSS in high responders
Ovarian cysts
More luteal phase defects
 Non GnRH
analouges
Induction
protocols Antagonist
GnRH
analouges
Agonist
 Induction
protocols

Non GnRH GnRH

analouges analouges

Antagonist Agonist

Natural FSH & / CC + FSH

cycle CC HMG &/HMG

Multiple
Single dose
dose
protocol
protocol

Fixed

Flexible
 ANTAGONIST
PROTOCOL
Native GnRH

Pyro
Glu His Trp Ser Tyr Gly Leu Arg Pro Gly

1 2 3 4 5 6 7 8 9 10
Cetrorelix

Ac-D- D-Phe D-Ala-

Nal(2) D/PAL Ser Tyr D-CIT Leu Arg Pro
(4CL) NH2

Ganirelix

Ac-D- D-Phe D-hArg L-hArg D-Ala-

Nal(2) D/PAL Ser Tyr Leu Pro
(4CL) (Et2) (Et2) NH2
 ANTAGONIST
PROTOCOL
Mechanism of action of antagonists:

Competitive blockade of GnRH-R

leading to IMMEDIATE SUPPRESSION OF
LH & FSH SECRETION.
Antagonists are free of any agonistic
activity, so there is NO FLARE UP
EFFECT.
Recent data indicate that prolonged
treatment leads to down regulation of
GnRH-R.
ANTAGONIST PROTOCOL
I- THE MULTIPLE DOSE
PROTOCOL.
hMG is started on D2 (2 amp/d)
On D5 hMG dose may be adjusted
according to response.
On D7 GnRHant is administered daily
(0.25 mg/d of Cetrorelix S.C.) till the hCG
criteria are met.
hCG 10,000 IU when leading follicles
are 18-20 mm & appropriate E2 level.
ANTAGONIST PROTOCOL
I- THE MULTIPLE DOSE
PROTOCOL.
20

1500
1450
1200
LH 1300
1000 1000

Cetrorelix 700 650 700

600 600
0.25 mg/d 300380300320250
E2
200
50120 10050100
From D7
till hCG day Cetrorelix
hMG injections
menstruation

h
C
G
ANTAGONIST PROTOCOL
I- THE SINGLE DOSE
PROTOCOL.
hMG is started on D2 (2 amp/d).

On D5 hMG dose may be adjusted

according to response.

GnRHant is administered in a single dose

of 3 mg S.C. When E2 is 150-200 pg/ml
& follicles >14 mm (usually about D8 or
D9).
ANTAGONIST PROTOCOL
I- THE SINGLE DOSE
PROTOCOL.

GnRHant dose is repeated after 96 h

if hCG criteria were not yet
achieved.

hCG 10,000 IU is given I.M. when

leading follicles reach 18-20 mm &
E2 reaches appropriate levels.
ANTAGONIST PROTOCOL
I- THE SINGLE DOSE
PROTOCOL.
20

1500
1450
LH
1200
1300
1000 1000
700 700
600650600
300380300320250
E2
50 120200
100 50 100

hMG injections
menstruation

Cetrorelix 3 mg S.C. A h
D9 (repeat in 96 h if n C
t G
hCG criteria not met)
ANTAGONIST PROTOCOL
Advantages of Antagonist
Protocols:
Shorter stimulation period.
Less hMG ampoules.
Lower incidence of severe OHSS.
Probably less luteal phase defect.
Comparable fertilization & cleavage rates,
embryo quality, & pregnancy rates.
Preserved pituitary responsiveness.
Natural cycle & simpler protocols may be used for
ART.
THANK YOU FOR
YOUR ATTENTION