Gonadotropin-Releasing Hormone-Antagonist in Human In Vitro Fertilization doc

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Gonadotropin-Releasing
Hormone-Antagonist in Human
In Vitro Fertilization
F. Olivennes
Department of Obstetrics and Gynecology, Hospital Cochin, Paris, France

INTRODUCTION
Gonadotropin-releasing hormone antagonists (GnRH-nt) available for clinical use are GnRH molecules with amino acid modifications in positions 1, 2,
3, 6, and 10. They are not associated with the histaminic-release effects of
previous compounds (1). These compounds immediately block GnRH receptor in a competitive fashion (2). They decrease the luteinizing hormone (LH)
and follicle-stimulating hormone (FSH) secretion within a period of eight
hours. Inhibition of LH secretion is more important than FSH. This is probably due to the different forms of gonadotropin regulation, the prolonged
FSH half-life, or the immunoactive and bioactive forms of FSH (3,4).
Administered during the follicular phase, GnRH-nt can prevent or
interrupt LH surges (5). In addition, their use has been proposed in in vitro
fertilization (IVF)–embryo transfer (ET) cycles to obtain results similar to
those obtained with GnRH-a, however with the simplest protocol and fewer
side effects (6).
Two different compounds are available: the Cetrorelix (Cetrotide1,
formerly ASTA Medica, now Serono) and the Ganirelix (Antagon1 or
Orgalutran1, Organon). Two different protocols of administration (Fig. 1)
have been proposed in the literature for using GnRH-nt in controlled
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Figure 1 Gonadotropin-releasing hormone antagonist multiple- and single-dose

protocols. Fixed day regimens. Abbreviations: FSH, follicle-stimulating hormone;
hMG, human menopausal gonadotropin; hCG, human chorionic gonadotropin.

ovarian stimulation (COH). In the multiple-dose protocol, small doses
(0.25 mg) of the GnRH-nt are injected in the middle of the follicular phase
(7–9). In the single-dose protocol, a higher dose (3 mg) is injected during the
late follicular phase, when the LH surge is most feared (10,11).

PHASE II DOSE-FINDING STUDIES
Single-Dose Protocol
In the first investigation with Cetrorelix, we simply reproduced the previously published Nal–Glu protocol consisting of two 5 mg injection 48 hr
apart in the late follicular phase (12,13). We therefore proposed two
administrations of 5 mg Cetrorelix 48 hour apart, the first injection being
administered on stimulation day 7. We observed that the second injection
was often unnecessary as hCG was given on the same day. We concluded
also that the 5 mg dose induced a deep suppression of LH and that a lower
dose should be tried (10). A single-dose protocol was designed where a single
injection of 3 mg of the GnRH-nt is performed on stimulation day 7 (11).
To determine the minimal effective dose, we conducted a dose-finding
study. We compared the use of 2 and 3 mg to investigate the ‘‘protection
period,’’ the time during which an LH surge is prevented after the antagonist
administration. The IVF-ET results were strictly comparable between the


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two doses and the 2 mg dose prevented LH surges for three days in all the
patients. However, we observed that the suppression of LH tended to

be reduced three days after the injection in the 2 mg dose and an LH surge
was observed four days after the 2 mg Cetrorelix administration. The 3 mg
dose was therefore selected as a safer choice, as a ‘‘protection period’’ of
at least four days can be obtained (14). No LH surge was observed in all
the patients treated with the 3 mg dose. In some patients, an LH rise
(LH > 10 IU/L) was observed on the day of the antagonist administration.
The Cetrorelix was able to prevent any further rise in LH, lowering immediately the LH levels and no surge was observed in these patients. Moreover,
the interruption of LH rises does not seem to have a deleterious effect on
IVF-ET results (15). The consumption of hMG was clearly reduced
(24–30 hMG ampoules) as compared to the use of GnRH-a in the long
protocol using a depot preparation (11).
The tolerance of the GnRH-nt Cetrorelix was excellent with only
transient erythema at the injection site in 15% of the patients.
Multiple-dose
The two GnRH-nts (Cetrorelix or Ganirelix) were studied in order to
achieve the best dose that blocks the premature LH rise and does not
oversuppress the pituitary. Sommer et al. (16) were the first in describing
the suppression of gonadotropin and estradiol secretion after 3 mg of
Cetrorelix daily in normal cycling women. After that, the dose-finding studies generally initiated the gonadotropins (recombinant or urinary) on day 2
of menstrual cycle and the antagonist daily administration was initiated on
stimulation day 6 (7–9,17). The risk for premature LH surge is higher after
the sixth day of ovarian stimulation, and this day was chosen to initiate the
antagonist injections (7). The authors of the different studies compared
different doses of Cetrorelix or Ganirelix to achieve the best dose with most
appropriate assisted reproductive technology (ART) results.
Comparing the Cetrorelix administration of three and 1 mg or 0.5 and
0.25 mg after day 6 of stimulation protocol, the authors showed that all
patients had a decline on LH serum levels. The group of patients receiving
0.5 and 0.25 mg/day showed the best ART results in terms of pregnancy and
implantation rates without the risk of a pituitary oversuppression which

occurred with one and 3 mg (7,17). Another study compared the starting
doses of 0.5, 0.25, and 0.1 mg/day (8). The authors demonstrated that
patients receiving 0.5 or 0.25 mg/day during the follicular phase did not
showed premature LH surge, evidenced by the lower LH serum levels.
However, one out of seven patients with 0.1 mg/day protocol showed a
premature LH rise with progesterone elevation and the 0.1 mg dose was
therefore abandoned. The results were similar between the patients receiving
0.25 and 0.5 mg/day in terms of clinical pregnancy and implantation rates.


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The group of investigators concluded that the minimal necessary and effective dose to prevent premature LH surge was 0.25 mg/day with Cetrorelix.
The Ganirelix Study Group (9) also investigated the minimal safe/
effective dose to achieve good IVF results. This study also showed that,
during the multiple dose protocol with Ganirelix, the minimal effective dose
was 0.25 mg/day, inhibiting the premature LH secretion without compromising IVF results in stimulated cycles with recombinant FSH. This group
of patients, receiving Ganirelix 0.25 mg/day had the highest vital pregnancy
rate per transfer (40.3%) as the main clinical outcome if compared with the
others doses (0.0625–2 mg).
Observing very low implantation rates in the groups of daily 1 or 2 mg,
Kol et al. (18) analyzed the database from the Ganirelix dose-finding study
(9) concerning the effect of GnRH-nt in freeze–thaw cycles. The authors
concluded that high doses (1.0 and 2.0 mg/day) of Ganirelix did not affect
the biologic potential of embryos to develop clinical pregnancy.

PHASE III RANDOMIZED CONTROLLED TRIALS
AND OPEN STUDIES

Single-Dose Protocol
We have compared our single-dose protocol to the GnRH-a long protocol
using a depot formula of Triptorelin in a prospective randomized study (6).
A 3:1 randomization was selected including 115 patients in the Cetrorelix
group and 36 in the agonist long protocol group. No difference was
observed between the GnRH agonist and antagonist groups for demographic and baseline data.
One hundred and four patients (90.4%) out of 115 patients received
only one 3 mg dose of Cetrorelix. If the criteria for triggering of ovulation
were not reached within four days (the protection period), we administered
an additional dose of Cetrorelix (0.25 mg). Only nine (7.9%) of the patients
received one additional dose on the morning of the hCG and two patients (1.7%) received two additional doses of 0.25 mg.
Moreover, a total of 18 patients of the Cetrorelix group (15.7%)
presented an LH rise (LH > 10 IU/L) on the day of Cetrorelix injection.
The administration of the Cetrorelix inhibited LH secretion. Four of them
became pregnant (22.2%). These interrupted LH rises seem to have no
measurable deleterious effect in this study. Only one patient in the Triptorelin group (2.8%) experienced an LH surge. None of the 115 patients of the
Cetrorelix group experienced an LH surge after the Cetrorelix administration. No LH surge has been reported so far within the four days following
the single administration of 3 mg Cetrorelix.
The mean length of stimulation was significantly lower in the Cetrorelix group. The mean number of ampoules was significantly higher in the


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Triptorelin group. The E2 levels on the day of hCG were significantly lower
in the Cetrorelix than in the Triptorelin group. The total number of follicles
!15 and 17 mm was higher in the Triptorelin group (5.0 Æ 3.9 vs.
3.4 Æ 2.6; CI 0.5–2.8). The long GnRH agonist protocol resulted in more
oocytes and more embryos as already demonstrated in the literature when

compared to other stimulation regimens. However, the percentage of mature
oocytes, fertilization rate, clinical and ongoing pregnancy rates, and miscarriage rates were not statistically different between the two groups. The
incidence of ovarian hyperstimulation syndrome (OHSS) was lower in
the GnRH-nt group. This difference did not reach statistical significance
but some patients of the GnRH-a group were cancelled for being at risk of
OHSS. Adding these patients brought the difference to significance.
In conclusion, this study has confirmed the efficacy of a single dose of
3 mg of Cetrorelix, administered in the late follicular phase, in preventing
premature ovulation as indicated by LH surges. The single-dose protocol
is easy to use and assures patient compliance. The 3 mg dose of Cetrorelix
was tolerated well, with only mild and transitory reactions at the injection
site. This protocol provides a shorter duration of treatment, uses less gonadotrophins, and has a lower incidence of OHSS. In some of the patients
treated with rec-FSH, a decrease in the E2 level is observed after the injection of the Cetrorelix. This was also observed in our first study using a
higher dose of Cetrorelix (5 mg) with human menopausal gonadotrophin
(hMG) (11). An increase of the hMG dose, on the day of the antagonist
administration, suppresses most of these E2 decreases, probably related to
the LH suppression but not exclusively (19). However, no difference is
observed in our experience in the IVF-ET results in the patients with or
without an E2 decrease following the Cetrorelix administration (unpublished results). One study done in an oocyte donor model (20) found a lower
implantation rate of embryos coming from oocytes collected in patients
with an E2 drop as compared to patients with continuous rise of E2.
Multiple-Dose Protocol
In all the studies presented, the multiple-dose protocol uses 0.25 mg/day of
Cetrorelix or Ganirelix. To compare the antagonist multiple dose protocol
(0.25 mg/day) to the GnRH-a in IVF cycles, the European Cetrorelix Study
Group (21) published the results of an open randomized trial. They studied
188 patients treated with Cetrorelix and 85 patients treated with the long
(Buserilin) agonist protocol; both groups received hMG. The authors transferred embryos in 83.5% of Cetrorelix group versus 79% of Buserelin group.
The clinical pregnancy rate was 22.3% and 25.9% per started cycle in the
Cetrorelix and Buserilin groups, respectively; these differences were not

statistically significant. The duration of treatment with gonadotropins and
the estradiol serum levels on the day of hCG were lower in the antagonist


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group. The incidence of ovarian hyperstimulation syndrome (OHSS II and
III) was higher in patients using agonist treatment.
The European Ganirelix Study Group (22) also performed a
controlled, multicentric, randomized trial to compare two treatment regimens for ovarian stimulation (multiple-dose antagonist vs. long-agonist)
in women receiving recombinant FSH. A total of 672 patients were investigated and randomized. The total dose of FSH administered was higher in
the Buserilin group (1500 and 1800 IU). In addition, patients receiving antagonist had a shorter stimulation duration than the agonist group. The estradiol
serum levels on the day of hCG administration were higher in patients using
Buserelin than Ganirelix and the incidence of OHSS was higher (5.9 vs. 2.4%)
in the Buserelin group. Otherwise, the number of good quality embryos, fertilization rate (62.1% in both groups), and replaced embryos were similar
between the two treatments schemes. The implantation rate was lower in
the Ganirelix group (15.7%) than in Buserelin group (21.8%), however the
clinical pregnancy rates per attempt were not statistically significant.
The North American Ganirelix Study Group (23) was organized to
evaluate the efficacy and safety of Ganirelix (multiple-dose protocol) versus
leuprolide (long-protocol) in IVF patients. This multicenter (United States
and Canada) trial demonstrated that the mean number of retrieved oocytes
was similar between the groups (11.6 in antagonist group and 14.1 in agonist
group). Moreover, the fertilization rates (62.4% and 61.9%) and implantation rates (21.6% and 26.1%) were also similar in both groups. The
ongoing pregnancy rates per attempt were 30.8% in ganirelix group and
36.4% in leuprolide group; however, the antagonist group showed fewer
local site reactions after injection administration (12.5%) than the leuprolide
group (25.5%). The authors proved the effectiveness and safety of multiple

antagonist drug protocol with a shorter stimulation period and fewer side
effects when compared with the long agonist (leuprolide) protocol.
Another multicentric European (The European and Middle East)
Orgalutran Study Group (24) trial, comparing two treatment schemes
(Ganirelix and Triptorelin) in 337 women, demonstrated that the median
dose of FSH recombinant was lower in the antagonist protocol. The authors
showed also that the estradiol serum levels were lower in Ganirelix group on
the day of hCG. The fertilization rates (64% Ganirelix and 64.9% Triptorelin), the mean number of good quality embryos (2.7 and 2.9, respectively),
the implantation rates (22.9% both treatments), and finally the ongoing
pregnancy rate per attempt were similar between the two treatments (31%
and 33.9%, Ganirelix and Triptorelin, respectively).
The multiple-dose protocol, compared with the long-agonist regimen,
offers a simple, safe, and efficient option, with comparable IVF results. The
OHSS risk is decreased (25), the total dose of gonadotrophin needed to
stimulate the ovulation is lower, and the stimulation period is also shorter
than in the long protocol. Patients receiving antagonist treatment had lower


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estradiol serum levels at the time of hCG administration, probably because
of the lower number of follicles. The impact of this finding in implantation
rates is disputed and unknown.
In the multiple-dose protocol, there is a small incidence of LH surge
(between 1% and 2.5%). These surges were often associated with a lack of
compliance by the patients, forgetting one antagonist administration. This
point is important to stress to the patients. More recently, some centers have
observed a higher incidence of LH surge in poor responders using the

multiple-dose protocol (unpublished data). These reports should be confirmed and documented. The dose of 0.25 mg might not be always sufficient.
This dose might also have to be adapted to the weight of the patients.
The follicular development was also studied in a controlled randomized multicentric study, in patients using Ganirelix with different doses
(0.0625–2.0 mg/day). Patients received recombinant FSH after day 2 of
menstrual cycle and Ganirelix were administrated daily after day 6 of ovarian stimulation protocol (26). Overall, 311 patients were studied and
compared in terms of number of follicles, total follicular surface area, serum
gonadotropin, and steroid hormones levels. Increasing GnRH-nt doses
demonstrated an additional suppressive action on estradiol and androstenedione serum levels, probably by an important inhibition of LH secretion,
which may have exerted a harmful effect. The follicular growing pattern
was not affected by the dose of GnRH-nt. The decreased secretion of
androstenedione and estradiol was not totally explained by the LH
inhibition. Other mechanisms could be involved in GnRH-nt action and
influence the cycles stimulated with this regimen protocol.
Wikland et al. (27), in a prospective randomized trial, evaluate two
starting doses (150 vs. 225 IU) of recombinant FSH with the multiple-dose
Cetrorelix protocol. The purpose was to increase the number of follicles,
oocytes, and embryos to increase the pregnancy rates. Despite a higher
number of recovered oocytes in the group of patients receiving 225 IU of
recombinant FSH, pregnancy and implantation rates were similar.
The effect of GnRH-nt on oocyte and embryo quality could also be measured by studying the implantation and pregnancy rates after cryopreservation of
pronuclear oocytes or embryos. The first study to evaluate this aspect was conducted with 62 patients divided into two groups (28). One group received the
multiple GnRH-nt dose protocol (group I) and the other group the conventional
GnRH long protocol (group II). The implantation and pregnancy rates, after
frozen-thawed procedure in pronucleous oocyte stage, were similar between
the groups (3.26% and 8.33% for group I; 3.73% and 10.25% for the group II).
GnRH-nt IN MILD STIMULATION
Mild stimulation protocol is aimed at reducing the intensity of stimulation
to obtain a lower number of oocytes. The advantages of those regimens

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are a reduction in the intensity of side effects, a reduction in the incidence of
OHSS, and a reduction in costs and complication. Other advantages lie in
the potential adverse effects on endometrium of heavy stimulation regimens
and the possible benefit of a natural selection of the best oocytes.
Those protocols include mainly the association between citrate of clomiphene and gonadotrophins, the modified natural cycle, and the reduced
amount of gonadotrophin dose. In both these protocols, the GnRH-nts
were used to prevent LH surges.
Very few data are available on the use of CC-hMG/rec-FSH and
antagonists in IVF (29,30). Pregnancy rates appear satisfactory but a high
incidence of LH surges was observed (31). It was demonstrated in an animal
model that CC increases the sensitivity of the pituitary to GnRH and that
the dose of antagonists might need to be increased when CC is used. A
prospective study found a lower pregnancy rate in CC-hMG cycles and
did not support the interest in this protocol (32).
The revival of the natural cycle was proposed as the use of GnRH-nts
could prevent premature LH surges. The objective of this regimen is to combine the possible prevention of an LH surge by the administration of the
GnRH-nt and the simplicity of the natural cycle with minimal stimulation
(Fig. 2). We investigated the administration of the Cetrorelix in the late
follicular phase of minimally stimulated cycles in women of good prognosis.
These patients had an age between 26 and 36 years old (mean 34.1 Ỉ 1.4),
normal menstrual cycles, day 3 FSH < 8 UI/l, day 3 E 2 < 50 pg/ml, less
than three previous IVF procedures, male factor infertility requiring IVF,
and ICSI (33). A single subcutaneous injection of 1 or 0.5 mg Cetrorelix
was administered when plasma estradiol levels reached 100–150 pg/ml,
and a lead follicle was between 12 and 14 mm to assess the minimal effective
dose. Since studies with Nal–Glu (34) and Cetrorelix (35) demonstrated that

estradiol secretion can be reduced after the GnRH-nt administration, daily
administrations of 150 IU of hMG were performed at the time of the first
injection of Cetrorelix and repeated thereafter until hCG administration.

Figure 2 Modified natural cycle using Gonadotropin-releasing hormone antagonists. Abbreviations: hCG, human chorionic gonadotropin; FSH, follicle-stimulating
hormone; hMG, human menopausal gonadotropin; OPU, oocyte pick-up.


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This treatment scheme is not a complete natural cycle since a low
gonadotrophin support is associated (minimal stimulation). Triggering of
ovulation (5000 IU of hCG) was decided when the lead follicle reached
16–20 mm and estradiol values were above 200 pg/ml. Oocyte pick-up was
performed 36 to 40 hours later without anesthesia (36).
A total of 33 patients (44 cycles) were included. The mean number of
hMG ampoules was 4.7 Ỉ 1.4 and the mean time between the Cetrorelix and
hCG administration was 2.0 Ỉ 0.7 days. We canceled four cycles (9.0%).
Follicular growth and E2 secretion were not altered by the Cetrorelix
administration. A total of 40 oocytes retrievals leading to 22 transfers
(55%) were performed. In 10 cycles, no oocyte was obtained. Fertilization
failure occurred in six cycles, and in two patients the transfer was not
performed because of a developmental arrest of the embryo at the two pronuclear stages. The fertilization rate was 80% (24 embryos/30 oocytes). A total
of five clinical pregnancies were obtained (32.0%/transfer, 17.5%/retrieval) of
which four are ongoing.
The number of patients in whom the cycle was canceled for premature
LH surge was very low (9.0%) as compared to previous reports on natural
cycles, confirming the efficacy of the antagonist administration. In addition,

the pregnancy rate (17.5% clinical pregnancy rate per retrieval; 32.0% per
transfer) seems interesting, even though it has to be confirmed in larger series. The same protocol was used in a recent study with rec-FSH as follicular
growth support (37).
Another form of mild stimulation was proposed recently by delaying the
start of gonadotrophin stimulation. The mild ovarian stimulation protocol
resulted in pregnancy rates per started IVF cycle similar to those observed
after profound stimulation with GnRH agonist cotreatment, despite shorter
stimulation and a 27% reduction in exogenous FSH. A higher cancellation rate
before oocyte retrieval was compensated by improved embryo quality concomitant with a higher chance of undergoing ET (38).
The high burden and drawbacks of ‘‘heavy’’ (stimulated) COH
protocol (side effects, multiple pregnancies, and potential serious health
complications) make a clear demand for softer protocols (39,40), a ‘‘friendly
IVF’’ (41).
If these preliminary results with spontaneous cycle and hMG support are confirmed on large numbers, the repetition of two or three of
these cycles could lead to acceptable cumulative pregnancy rates without
the potential adverse effects of COH (42–44) and be more costeffective (45).
Another use of natural cycle with GnRH-nt was proposed recently.
The protocol was proposed in poor responders (46). The number of studies
evaluating this approach is very low so far and results are controversial (47).
Further studies are needed to analyze the possible interest of the modified
natural cycle in poor responders.


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REMAINING QUESTIONS ON GnRH-nt
Pregnancy Rates
There is a trend in most of the controlled studies using GnRH-nt (with both

compounds and protocols) to find slightly lower pregnancy rates as compared
to the GnRH-a long protocol. This led to the questioning of IVF-ET results
of GnRH-nts. A meta-analysis concluded there were significantly lower pregnancy rates in GnRH-nt cycles as compared to GnRH agonists (48). However,
the difference was very close to being nonsignificant (OR 0.79; 95% CI 0.63–
0.99). Adding one study, Ludwig et al. (49) did not find a significant difference
in pregnancy rates for Cetrorelix. A recent meta-analysis presented by Daya did
not find a difference in ongoing pregnancy rate (unpublished data). Care should
be taken in drawing conclusions on these observations. Some population factors
were not equivalent in the groups despite randomization. In addition, the learning curve, inherent to the use of new treatment schemes, could have influenced
some of the studied outcome. The trend towards higher pregnancy rate (PR) in
the GnRH-a group may be associated with the relatively higher number of
obtained embryos due to the higher number of oocytes. This hypothesis was
not confirmed by the study presented by Wikland et al. (27). The difference
could be related to the absence of desensitization of the previous luteal phase.
In fact, a difference of the same magnitude in the pregnancy rates is found
between the short and long protocols (50). The clear reasons for this difference
are still not clear. The potential deleterious effect of the GnRH-nt on the endometrium or even on the fertilization process has been presented (51). However,
there is no clinical data to confirm these hypotheses in the human species (52,53).
Therefore, a careful analysis is needed before drawing conclusions on the PR. In
fact, a comparative study designed to assess a 5% difference for PR situated in
the region of 20% will require over 1200 patients in each treatment group.
The important issue about GnRH-nt pregnancy rates is that there are
still major questions to be answered on the best way to use them.
Single Versus Multiple-Dose Protocols
The two protocols have not been prospectively compared in large studies.
The only large available study presents the results of two large multicentric
studies using the single- and multiple-dose Cetrorelix protocols (54). No
differences are observed between the two regimens but as this is not a prospective randomized study; firm conclusions cannot be drawn from those
data. A propspective randomized study compared the Ganirelix multiple
dose protocol to the Cetroreliw single dose regimen and did not find a statistical difference in the PR between the two protocols (55,56).

Fixed Versus Flexible Administration of the Antagonist
In the first study using GnRH-nt, the protocol included a fixed day of
GnRH-nt administration. In the multiple-dose protocol, the antagonist


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Figure 3 Gonadotropin-releasing hormone antagonist multiple- and single-dose
protocols. Fixed day regimens. Abbreviations: FSH, follicle-stimulating hormone;
hCG, human chorionic gonadotropin; hMG, human menopausal gonadotropin.

was started on day 6. In the single dose, the 3 mg administration of Cetrorelix was proposed on day 7. We proposed a more flexible approach by
adapting the moment of antagonist administration to ovarian response
(Fig. 3) (11). The antagonist could be administered when the leading follicle
reached 14 mm and/or E2 reached 600 pg/ml. The same approach was later
proposed by Ludwig et al. (57) in the multiple-dose protocol (Fig. 3). Some
authors have predicted a lower pregnancy rate when the flexible approach
was proposed (58). Data remain controversial on this question. A recent
meta-analysis did not find differences in the pregnancy rates between the
flexible and fixed approaches (59).
A very early start of the antagonist, injected at the beginning of the
stimulation, was proposed to reduce the LH levels at the beginning of
the follicular phase. A prospective randomized study failed to demonstrate
an advantage to this proposal (60). Moreover, a long period of injection of
antagonist will really alter the main benefit of GnRH-nts.
INDICATIONS
PCOS Patients
The use of GnRH-nts in large series of polycystic ovarian syndrome (PCOS)

patients has not been published so far. One of the most important hormonal


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aspects of PCOS patients is the increased LH tone secretion. This group of
patients is characterized by anovulation, and ovarian ovulation induction is
usually performed using clomiphene citrate, FSH associated or not with
GnRH agonists. The rationale for the use of GnRH-nt in PCOS patients
is the fact that the LH/FSH ratio will be decreased since LH secretion is
more affected by the antagonist administration than FSH secretion (2). In
IVF, another clear advantage is the reduced incidence of OHSS with the utilization of GnRH-nt. The use of GnRH-nt protocol allows also to induce
the oocyte final maturation with GnRH agonist, to elicit an endogenous LH
surge and, subsequently, decreasing the risk of OHSS (61). However, a large
prospective trial is necessary to confirm these physiological hypotheses (see
infra). Lubin et al. (62) described two case reports of PCOS patients treated
with GnRH-nt before the treatment with GnRH agonist to induce ovulation. The patients showed a normalization of LH and testosterone serum
levels, however, the authors failed to induce an appropriate ovarian response.
Two recent prospective randomized studies compared GnRH-nt to
agonists in PCO patients (63,64). Those studies found similar pregnancy rate
but failed to find a significant advantage to GnRH-nts.
Larger studies are needed to further evaluate the potential benefits of
the association of GnRH-nt in PCOS patients.
POOR RESPONDERS
The definition of poor responders and the heterogeneity of this group of
patients cause an important bias in published series. The rational for using
ovarian stimulation protocols with GnRH-nt in poor responders is that
GnRH-nts do not require desensitization and are not causing an important

depression on gonadotropin secretion during the stimulation.
Forty-two patients who are poor responders were divided into two
groups for ICSI treatment (long GnRH agonist or Cetrorelix multiple-dose
protocols) (65). The stimulation protocol also included, in some patients,
clomiphene citrate associated with gonadotropins. Age, number of oocytes
retrieved, number of fertilized oocytes, transferred embryos, score of
embryo quality, and clinical pregnancy were not significantly different
between the groups. A trend was observed in the pregnancy rates (14.28%
for Cetrorelix vs. 9.52% for GnRH agonist treatment) but the difference
was not significant. The authors discussed the sample size utilized. Probably,
with an adequate power calculation this difference in terms of pregnancy
rate can become statistically important.
With the same objective of the above-mentioned paper, Akman et al.
(66) presented a randomized trial comparing the microdose flare-up GnRH-a
protocol versus the antagonist multiple dose protocol. Forty-eight patients
were divided into the two regimen protocols. The implantation rates (15.07%
for flare-up protocol and 11.36% for Cetrorelix) and the ongoing pregnancy


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rates per transfer (21.05% and 16.6% for flare-up and Cetrorelix protocol,
respectively) were similar between the two groups.
Other small studies have been published recently (67–69). Those
studies did not find an improvement in pregnancy rates, but similar results
were achieved.
The Need for LH Activity Adjunction
The need to counteract the dramatic decrease in LH observed in the

GnRH-nts cycle has been proposed. No large prospective study is yet published comparing patients treated with GnRH-nt in which LH activity was
added. LH activity could be added by the adjunction of rec-LH or by a
small dose of hCG (70). It could also be obtained by using hMG. Profound
suppression of LH was not found to be associated with lower pregnancy
rates (71). No significant advantage was found by adding rec-LH on a systematic basis in GnRH-nts single and multiple-dose protocols (72,73). This
remains to be confirmed in large published studies.
Programmation of GnRH-nt Cycles
The programmation of GnRH-nt cycles is important to organize the work
load of large IVF centers. Programmation could be obtained with progesterone administration in the late luteal phase or oral contraceptive pill (74).
A more original way of programming the cycle was proposed by Fanchin
et al. (75). The authors proposed luteal administration of estradiol in the late
luteal phase as proposed by de Ziegler (76). The administration of estradiol
prevents the FSH rise induced by the luteolysis and could synchronize the
follicular cohort (75). Some authors (Kolibianakis et al., unpublished data)
found lower pregnancy rates when an estro-progestative pill is used. The
results of this study are not yet confirmed by other authors. In poor responders, the use of contraceptive pills to program GnRH-nt cycle was found to
be associated with increased cancellation rate (77).
Luteal Phase
The LH secretion is fundamental for the development of a normal luteal
phase and for progesterone secretion. The luteal-phase defect induced by
agonist administration is well known and studied, caused mainly by the profound pituitary suppression (78). The antagonist exerts a transitory LH
inhibition and, hypothetically, the luteal phase is less disturbed. Some
authors (78,79), comparing LH serum levels in the early and mid-luteal
phase of hMG treated cycles with or without antagonist (Cetrorelix multiple
dose), concluded that there is a decrease in LH serum levels in Cetrorelix
groups. However, the implications of this phenomenon were not studied.
In a small group of patients treated with Cetrorelix multiple doses without


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luteal support, no pregnancy was obtained (80). A recent study comparing
triggering of ovulation with hCG, rec-LH, and Triptoreline in IVF patients
without any luteal support and treated with GnRH-nts showed an abnormal
luteal phase in those three regimens (81). However, it must be emphasized
that the authors used Antide as an antagonist and those results might
not be extrapolated to Cetrorelix or Ganirelix. Others (82) demonstrated
that, comparing antagonist and agonist treatment, the granulosa cells
cultured in vitro from IVF patients were less impaired, in terms of progesterone secretion, in the antagonist group. Ragni et al. (83) showed, on intrauterine insemination (IUI) cycles, that the utilization of GnRH-nt is safe
and do not affect the luteal-phase duration or progesterone secretion. The
fact that the intensity of the stimulation is much lower in IIU cycles could
explain those contradictory results, as luteal phase is affected by strong
stimulation regimens.
Until full scientific data and controlled studies are available, it seems
preferable to maintain luteal support of GnRH-nt treated cycles.
Triggering Ovulation with GnRH-a
in GnRH-nts Cycles
We described the use of GnRH agonist to induce endogenous LH surge
during an ovarian stimulation cycle with GnRH-nt (61). All patients showed
an appropriate LH and progesterone rise after the GnRH agonist administration, confirming that this approach can be used to induce LH secretion
during the final stage of COH.
Others (84–87) previously proposed this strategy to decrease the risk of
OHSS, as the endogenous LH has a lower half-life than hCG. However, this
approach is not suitable in patients previously down-regulated with
GnRH agonist.
A recent study, comparing hCG, Lupron (0.2 mg), and Triptorelin
(0.1 mg) to trigger ovulation in IVF patients treated with Ganirelix, found
similar IVF-ET results between the three groups of patients (88). Other

studies found lower pregnancy rates in patients treated with a combination
of GnRH-nts and agonists (89). A small group of high responders were
treated with a combination of GnRH-nt and agonists and no OHSS was
observed in this preliminary report (90). As mentioned earlier, a study using
Antide as GnRH-nt showed an altered luteal phase in cycles combining
GnRH-nt and agonist (81). Some authors advocate that the luteal phase
needs to be supported by both progesterone and estrogens (Itskowitz, personal communication). In a prospective randomized study, reduced pregnancy rate was found in GnRH-nt cycle in which GnRH-a was used to
trigger ovulation, despite using progesterone and estradiol as luteal supplementation (91).
More studies are needed before recommending this approach.


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81

IUI
Few papers on the use and potential benefits of GnRH-nt protocol in IUI
cycles are yet available. Some found higher pregnancy rates (92,93). Others
found only a trend in higher success rate (94).
Some of the advantages shown in IVF cycles can be applied for IUI.
In case of premature LH surge when criteria of optimal follicular maturation are not obtained, GnRH-nts could be proposed to prevent and
postpone ovulation. The luteal phase of stimulated cycle in IUI cycles was
studied by Ragni et al. (83).
Programmation of the timing of IUI could also be obtained with
GnRH-nt. Of course, this is not a medical indication and IUI can be
advanced if an LH surge is detected, however this is not always possible.
It remains to be demonstrated that postponing the triggering of ovulation
with GnRH-nt when adequate follicular size and E2 levels are reached does
not adversely affect the results.
Perinatal Outcome of Pregnancy After GnRH-nt for

Ovarian Induction
Recently, two papers have been published on the perinatal outcome of IVF
pregnancies obtained with GnRH-nt.
One report followed 67 pregnant patients after ovarian induction with
Ganirelix multiple-dose protocol (95). The miscarriage rate was 9%, and full
data on perinatal outcome was obtained in 61 patients. The mean gestational age was 39.4 week for singleton pregnancies and 36.6 week for
multiple pregnancies. A birth weight lower than 2500 g was present in
8.7%, one baby had a major congenital malformation, and seven minor
malformations were reported in five infants. These results were not different
from data available on IVF pregnancies.
Another study addresses the same objective with the use of Cetrorelix
(multiple- and single-dose protocols). Pregnancies that resulted from phase
II and III trials were followed to investigate the safety of GnRH-nt (96). A
total of 227 children born were evaluated in terms of outcome of pregnancy,
delivery, birth weight, and after one and two years of age to search for some
developmental disorder. The incidence of major congenital malformation
was 3.1% and minor malformations occurred in 2.6% of the cases. The
clinical abortion rate was 16.8% and the ectopic pregnancy rate 3.4%.
The follow-up data on physical development did not show any significantly
abnormality.
The authors of both studies concluded that the use of GnRH-nt in
ovarian stimulation protocols did not cause a harm or detrimental effect
on the pregnancy course or perinatal outcome of those patients. These
two studies concern a too small number of cases to discuss the malformations rates.


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Recommended Regimens
As we saw in this paper we still have questions on the best protocol to be used
with GnRH-nts. From available literature, we can suggest that GnRH-nt
can be used in a single- or multiple-dose regimen. The flexible or fixed
approach appears also to give similar results. Programming the cycle can
be obtained with OC pill or luteal estrogen. The latter lacks a prospective
study to confirm its interest as opposite to the OC pill which is already used
in many IVF cycles. The supplementation with LH activity is not supported
by available scientific data. The supplementation of the luteal phase is
recommended. If GnRH-nt appears to give comparable results in good
responders, it should be studied in poor responders, patients with PCOS
or IUI deserve further study.

CONCLUSION
In COH, the different studies presented have confirmed the efficacy of a
single dose of 3 mg of Cetrorelix to prevent premature LH surges when
administered in the late follicular phase. The single-dose protocol is easy
to use and assures patient compliance. When compared with the long protocol using a depot formula of Triptorelin, the IVF-ET results showed a
shorter duration of treatment, less amount of hMG used, and a lower occurrence of OHSS in the group of patients treated with Cetrorelix. Clinical
trials showed that the multiple-dose protocol using Cetrorelix or Ganirelix
is effective and safe. A shorter duration of treatment, less amount of gonadotropins, and a lower occurrence of OHSS was observed in the group of
patients treated with Cetrorelix or Ganirelix. Single- and multiple-dose
protocols have not yet been compared prospectively. Fixed day or flexible
day schemes have not been compared either. The single dose is simple but
requires monitoring of the cycle. The multiple doses, in a fixed regimen,
could reduce the need for hormone assessments but compliance is mandatory. Patients report a better quality of life with GnRH-nt protocol as
compared to the long GnRH-a regimen, but this aspect was not
scientifically evaluated.
The pregnancy rates are not statistically different within the GnRH
agonist treatment. A trend in lower pregnancy rates observed in most of

the antagonists group of the controlled studies suggests further data are
needed on this point. Meta-analyses found the difference significant in favor
of the GnRH agonist.
However, there are still major questions to be answered to define the
best way to use GnRH-nts (97). Therefore, it should be mentioned that there
is room for optimization of the antagonist protocol. The antagonist administration can be proposed on a fixed day of the stimulation or based on
monitoring with a flexible approach. This could reduce the amount of


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83

antagonists. However, the optimal timing of the flexible approach of
GnRH-nt administration is difficult since prediction of LH surge is difficult.
The programmation of the treatment through manipulation of the luteal
phase with progestative and/or estrogen has to be evaluated. The lutealphase supplementation remains mandatory so far.
The use of the GnRH-nt in mild stimulation regimen (CC/gonadotropins or natural cycle with hMG support) allows reduction of the rate of
premature LH surges and therefore the cancellation rate. Stimulation can
be minimal and pregnancy rates, in some preliminary report, are satisfactory.
If larger studies confirm these results, mild stimulation protocols associated
with GnRH-nt single or multiple dose administration could represent an
interesting first-choice IVF treatment regimen in selected indications. These
protocols could reduce the complications and risks of the actual COH protocols. The reduction of the costs associated with the possibility of offering
the retrieval on an outpatient procedure is also of clear interest. Successive
cycles with an acceptable success rate could result in interesting cumulative
pregnancy rates. More studies are needed to clarify some important clinical
questions regarding the utilization of antagonist in PCO patients and in poor
responders. In IUI cycles, small studies showed an increase in pregnancy rate
when GnRH-nt multiple-dose regimens were use in the stimulation regimen.

These results need to be confirmed on larger samples.
New GnRH-nts are already available for clinical use in most countries. It
will certainly change our protocols of ovarian stimulation. If similar pregnancy
rates are achieved by a better understanding of the effect of GnRH-nts, the
main advantage of these compounds is the reduction of the side effects and
complications of our actual stimulation protocols, a clear benefit for our
patients. They could also allow us to design softer stimulation schemes.
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