5 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 67 68 Olivennes Figure 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 mg injection 48 hr apart in the late follicular phase (12,13) We therefore proposed two administrations of mg Cetrorelix 48 hour apart, the first injection being administered on stimulation day We observed that the second injection was often unnecessary as hCG was given on the same day We concluded also that the 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 mg of the GnRH-nt is performed on stimulation day (11) To determine the minimal effective dose, we conducted a dose-finding study We compared the use of and 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 GnRH-Antagonist in Human In Vitro Fertilization 69 two doses and the 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 mg dose and an LH surge was observed four days after the mg Cetrorelix administration The 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 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 mg of Cetrorelix daily in normal cycling women After that, the dose-finding studies generally initiated the gonadotropins (recombinant or urinary) on day of menstrual cycle and the antagonist daily administration was initiated on stimulation day (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 mg or 0.5 and 0.25 mg after day 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 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 70 Olivennes 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 or 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 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 mg Cetrorelix The mean length of stimulation was significantly lower in the Cetrorelix group The mean number of ampoules was significantly higher in the GnRH-Antagonist in Human In Vitro Fertilization 71 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 Ỉ 3.9 vs 3.4 Ỉ 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 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 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 72 Olivennes 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 GnRH-Antagonist in Human In Vitro Fertilization 73 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 of menstrual cycle and Ganirelix were administrated daily after day 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 74 Olivennes 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 FSH < UI/l, day E < 50 pg/ml, less than three previous IVF procedures, male factor infertility requiring IVF, and ICSI (33) A single subcutaneous injection of 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 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 GnRH-Antagonist in Human In Vitro Fertilization 75 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 76 Olivennes 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 GnRH-Antagonist in Human In Vitro Fertilization 77 Figure 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 In the single dose, the mg administration of Cetrorelix was proposed on day 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 78 Olivennes 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 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 GnRH-Antagonist in Human In Vitro Fertilization 79 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 80 Olivennes 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 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 GnRH-Antagonist in Human In Vitro Fertilization 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 82 Olivennes 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 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 GnRH-Antagonist in Human In Vitro Fertilization 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 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