- Email: [email protected]
High-dose recombinant LH add-back strategy using high-dose GnRH antagonist is an innovative protocol compared with standard GnRH antagonist
RBMOnline - Vol 15. No 3. 2007 280-287 Reproductive BioMedicine Online; www.rbmonline.com/Article/2554 on web 16 July 2007
!RTICLE (IGH DOSE RECOMBINANT ,( ADD BACK STRATEGY USING HIGH DOSE 'N2( ANTAGONIST IS AN INNOVATIVE PROTOCOL COMPARED WITH STANDARD 'N2( ANTAGONIST *UAN 'ARCIA 6ELASCO DID HIS TRAINING IN /BSTETRICS AND 'YNECOLOGY IN -ADRID !UTONOMA 5NIVERSITY AND THEN HIS 2EPRODUCTIVE %NDOCRINOLOGY AND )NFERTILITY &ELLOWSHIP AT 9ALE 5NIVERSITY 53! n (E IS NOW $IRECTOR OF )6) -ADRID AND !SSISTANT 0ROFESSOR AT 2EY *UAN #ARLOS 5NIVERSITY (E HAS PUBLISHED OVER PEER REVIEWED ARTICLES (E IS AN AD HOC REVIEWER FOR THE MAIN JOURNALS IN THE lELD AND SERVES IN THE EDITORIAL BOARD OF 2EPRODUCTIVE "IO-EDICINE /NLINE AND (UMAN 2EPRODUCTION (IS MAIN RESEARCH INTERESTS ARE ENDOMETRIOSIS /(33 LOW RESPONDERS AND HUMAN IMPLANTATION
$R *UAN 'ARCIA 6ELASCO *! 'ARCIA 6ELASCO (*4 #OELINGH "ENNINK 2 %PIFANIO % %SCUDERO ! 0ELLICER # 3IMØN )NSTITUTO 6ALENCIANO DE )NFERTILIDAD -ADRID )6) -ADRID -ADRID 3PAIN )NSTITUTO 6ALENCIANO DE )NFERTILIDAD &OUNDATION &)6) 5NIVERSITY OF 6ALENCIA 6ALENCIA 3PAIN )NSTITUTO 6ALENCIANO DE )NFERTILIDAD )6) 6ALENCIA 3PAIN 0ANTARHEI "IOSCIENCE "6 )NSTITUTE FOR #LINICAL #ONCEPT 2ESEARCH 0/ "OX AL :EIST 4HE .ETHERLANDS #ORRESPONDENCE 4EL &AX E MAIL CSIMON IVIES
!BSTRACT High daily doses of gonadotrophin-releasing hormone (GnRH) antagonists during the follicular phase of ovarian stimulation were associated with low implantation rates. To test if this occurred because of profound pituitary suppression, the pituitary response was suppressed with a high-dose GnRH antagonist and recombinant LH (rLH) was added back to correct the implantation rate. An open-label, randomized, controlled, prospective clinical study in 60 patients undergoing IVF was performed. GnRH antagonist was initiated on day 6 of stimulation (2 mg/day) together with 375 IU rLH, and maintained until the day of HCG administration. Controls received 0.25 mg/day GnRH antagonist. Fluctuating LH concentrations were present on days 3 and 6 in both groups. This strong uctuation continued on day 8 and on the day of HCG administration in the control (low-dose) group, where 30% of patients had LH concentrations <1 IU/l on the HCG day. The study (high-dose) group showed stable LH concentrations on day 8 and on the HCG day, with no LH surges. No clinical differences were found between groups. The LH add-back strategy (375 IU/day) rescued the adverse effects that high doses of GnRH imposed on implantation. These results suggest that rLH should be considered during ovarian stimulation with GnRH antagonist. Keywords: GnRH antagonist, IVF, recombinant LH
)NTRODUCTION
Gonadotrophin-releasing hormone (GnRH) antagonists have been proved to be effective in the prevention of the premature LH surge in ovarian stimulation. Enough evidence exists in the literature to demonstrate their benecial effects when compared with GnRH agonists beyond effectively suppressing endogenous LH, such as a decrease in the amount of gonadotrophins required and a shorter period of stimulation (Albano et al., 2000; Borm et al., 2000; Olivennes et al., 2000; European and Middle East Orgalutran Study Group, 2001; Fluker et al., 2001). This newer GnRH antagonist
strategy was expected to yield similar clinical outcomes, but this expectation was not met in the initial ve randomized studies that indicated a trend towards lower implantation and pregnancy rates for the GnRH-antagonist treatment group compared with those in the GnRH-agonist group (Al Inany and Aboulghar, 2002). These results were probably due to differences in stimulation protocols, different requirements of LH in different patient populations, hypothetical effects on the endometrium, as well as the physicians’ learning curve. These difculties motivated several changes in
© 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
S52
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
ovarian stimulation and patient management, to obtain comparable implantation rates in recent times (Kolibinanakis, 2005). The dose-nding study of Devroey et al. (1998) investigated the effects of six different dosages of ganirelix, and showed a dosedependent decrease in implantation, with a complete absence of pregnancies at doses above 1 mg/day, whereas no impact on the number of oocytes and good quality embryos was found. Followup of the frozen embryos obtained in that dose-nding study revealed that ongoing pregnancies were subsequently achieved in 11 patients, of which six were treated with a high ganirelix dose of 1.0 or 2.0 mg (Kol et al., 1999). A recent study by Huirne et al. (2005) addressing a similar question reached similar conclusions, with a lower implantation and pregnancy rate when a GnRH antagonist was administered at high doses. It was postulated that this unwanted effect was due to either direct action of GnRH antagonist on the endometrium and/ or to a profound suppression of the pituitary that may lead to extremely low LH concentrations, which may be detrimental to the implantation process. In an attempt to address the rst possibility, the authors investigated the effect of both low- (0.25 mg) and high-dose (2.0 mg) GnRH antagonist as well as a GnRH-agonist treatment on the endometrial development in women undergoing ovarian stimulation for oocyte donation (Simón et al., 2005). No relevant alteration was observed in the endometrial development in the early- and mid-luteal phases in women undergoing ovarian stimulation for oocyte donation following daily treatment with a standard- or high-dose GnRH antagonist. In addition, the endometrial development after GnRH antagonist mimics the natural endometrium more closely than after GnRH agonist. The current study was conducted to test the second hypothesis by suppressing the pituitary response with a high-dose GnRH antagonist (2 mg/day) and recombinant LH add-back strategy (375 IU/day) to correct the LH bioactivity for adequate follicle development and preparation of the endometrium.
-ATERIALS AND METHODS 3TUDY DESIGN AND PATIENTS The study was designed as an open-label, randomized, controlled, prospective clinical study in patients undergoing IVF/ intracytoplasmic sperm injection (ICSI). The primary objective of the study was to compare the clinical outcome in the study group, using high-dose GnRH antagonist (2 mg/day) and adding-back 375 IU/day recombinant LH (rLH), with the control group, using similar rFSH doses and low-dose GnRH antagonist (0.25 mg/ day). The secondary objective was to investigate serum oestradiol, progesterone and LH concentrations during the treatment cycle on stimulation days 3, 6 and 8, the day of HCG, the day of oocyte retrieval and HCG + 12 days, in the control versus the study group. The study was conducted in accordance with the last revision of the Declaration of Helsinki and the International Conference on Harmonisation (ICH) Guideline for Good Clinical Practice (1996). Written informed consent was obtained from all subjects and the study was approved by the internal institutional review board. A total of 60 patients were assessed for eligibility, with the following inclusion criteria: 18–35 years of age, rst IVF cycle
at our institution and basal oestradiol < 60 pg/ml, FSH < 10 IU/ l and prolactin < 20 ng/ml. Exclusion criteria were history of pelvic endometriosis, polycystic ovarian syndrome, body mass index (BMI) < 35 kg/m2, thyroid dysfunction, or gross uterine or ovarian alterations found by transvaginal ultrasound. Sample size was estimated by proof-of-concept, and randomization was performed on-site using a computer generated list, not blocked and not concealed. Of those patients allocated to intervention, nine were lost to followup: three patients discontinued intervention due to risk of ovarian hyperstimulation syndrome (OHSS) (one in the study group, and two in the control group), three were cancelled due to insufcient ovarian response (study group n = 2, control group n = 1), one patient from the study group was excluded due to poor quality oocytes, another patient in the control group due to premature luteinization, and one more patient abandoned the study for personal reasons before receiving the GnRH antagonist. Thus, a total of 51 patients were analysed, 26 in the study group and 25 in the control group Both groups of patients were initiated with recombinant FSH (rFSH; 150 IU) on cycle day 2 after ovarian quiescence was ascertained (absence of ovarian cyst > 10 mm diameter on transvaginal ultrasound and/or serum oestradiol < 60 pg/ml) and adjusted after 2 days, according to oestradiol concentration. On treatment day 3 the patients underwent blood analysis for LH and oestradiol concentration. rFSH was increased to 200 IU/day if oestradiol <100 pg/ml, decreased to 100 IU/day if oestradiol > 200 pg/ml, or maintained at the same dose if oestradiol ranged from 100 to 200 pg/ ml. On day 6 and onwards, rFSH was again adjusted according to oestradiol concentration and ultrasonographic ndings every other day. In the study group (n = 26) high-dose GnRH antagonist cetrorelix (Cetrotide, Serono, Geneve, Switzerland) 2 mg/day plus rLH (Luveris, Serono, Geneve, Switzerland) 375 IU/day were added s.c. at day 6 of stimulation and maintained until the HCG day. The dose of rLH was chosen according to the previous experience of other groups (Hillier, 2000; Balasch and Fabregues, 2003). In the control group (n = 25) a regular daily dose of antagonist (0.25 mg/ day) was started s.c. at day 6 and maintained until the HCG day. rHCG (Ovitrelle; Serono, Geneva, Switzerland) 250 g was given by a single s.c. injection if at least two follicles reached 18 mm mean diameter, and ovum retrieval was then scheduled for 36–38 h later. Premature LH surge was dened as LH > 10 IU/l on the day of HCG administration. The standard IVF procedure has been described elsewhere (GarciaVelasco et al., 2000). Embryo transfer (ET) was performed on day 3 of embryo development under ultrasound guidance, with a halffull bladder. Luteal phase was supported with natural micronized progesterone (Utrogestan, Seid, Barcelona, Spain) 200 mg twice daily, starting the evening after oocyte retrieval. Serum -HCG was evaluated 2 weeks after embryo transfer and in those who tested positive, a transvaginal ultrasound was scheduled 4 weeks after ET to detect clinical intrauterine pregnancy, and a followup ultrasound was scheduled 10 weeks after ET to document an ongoing pregnancy.
(ORMONAL ANALYSIS Serum oestradiol, progesterone and LH were evaluated during the treatment cycle on stimulation days 0, 3 and 6, every other day thereafter until day of rHCG administration, the day after
RBMOnline®
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
S53
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
HCG and the day of oocyte retrieval. Blood was taken between approximately 08.00 and 10.00 hours in all patients. Hormones were analysed using a commercially available microparticle enzyme immunoassay kit (Abbot Laboratories, Abbot Park, IL, USA). Inter- and intra-assay coefcients of variation for oestradiol at a concentration less than 40 pg/ml were 2.8 and 4.3%, respectively. The serum progesterone kit had a sensitivity of 0.2 ng/ml, with inter- and intraobserver coefcients of variation of 3.9% and 9.6%, respectively. Inter- and intra-assay coefcients of variation for LH were 1.9 and 4.6%, respectively, with a sensitivity of 0.5 IU/l.
3TATISTICAL ANALYSIS Data were expressed as mean ± SD. Student’s t test, chi-squared or Fisher’s exact test, and Wilcoxon/Mann–Whitney U-test, were used as appropriate. P < 0.05 was considered statistically signicant. The statistical analysis was performed with Statistics Package for Social Sciences.
2ESULTS #LINICAL OUTCOME A total of 318 eggs was retrieved in the study group and 467 in the control group. Patients in both groups were comparable in terms of age (31.3 versus 30.8), BMI (23 versus 23.5), and basal hormone determinations. No signicant differences were found between groups in terms of days of stimulation, days of GnRH antagonist treatment (4.6 versus 4.7), number of oocytes inseminated for IVF (8.7 versus 9.4) or microinjected for ICSI (7.0 versus 8.9), IVF fertilization rate (51% versus 44%), ICSI fertilization rate (71.3% versus 70.9%), overall fertilization rate (63.9% versus 58.9%) or mean number of embryos transferred (2.1 versus 2.2) (Table 1). The only signicant nding was a
lower number of oocytes retrieved in the study group compared with the control group (11.4 versus 16.1, P = 0.04). Again, no signicant differences were observed in the implantation rate (32.1% versus 23.2%) and clinical pregnancy rate per transfer (44.4 versus 38.5%).
(ORMONAL PROlLE Similar patterns of uctuating LH concentrations were observed on days 3 and 6 in both groups before the introduction of the GnRH antagonists and rLH add-back therapy (Figure 1a). This rather strong day-to-day variation continued on day 8 in the low-dose cetrorelix group (median 1, range < 0.5–6.7 IU/l, under detection limit of 0.5 IU/l in 20% of patients). However, in the high-dose cetrorelix/LH add-back group there were remarkably stable LH concentrations on day 8 (median 2, range 0.6–4.0 IU/l), which were signicantly higher when compared with control group LH concentrations (P < 0.001). This trend was similar at the day of HCG administration, with serum LH concentrations signicantly higher in the study group versus the control group (2.6 versus 1.4, P < 0.01). (Figure 1b) LH concentrations from day to day were extremely well controlled in the high-dose cetrorelix/LH add-back group (median 2.6, range 1.0–4.8 IU/l) as opposed to the control group (median 1.45, range 0.5–14 IU/l), and 17.2% of patients had LH concentrations under the detection limit of 0.5 IU/l (Figure 2). Once medication was stopped, it was observed that the majority of patients (89.2%) from the study group displayed serum LH concentrations under the detection limit as opposed to 31.0% of control patients. This study group showed signican tly lower LH serum concentrations when compared with controls (<0.5 versus 1.1, P < 0.0001). Similarly, high-dose cetrorelix/LH add-backtreated patients had lower serum LH concentrations on HCG + 12 (1.2 versus 2.1, P < 0.05) (Figure 1c).
Table 1. Clinical outcome in patients undergoing high-dose (2 mg/day) GnRH antagonist plus add-back LH therapy compared with low-dose (0.25 mg/day) GnRH antagonist.
Age (years) Oocytes retrieved IVF: oocytes incubated ICSI: metaphase II IVF fertilization rate (%) ICSI fertilization rate (%) GQE Embryos replaced Implantation rate (%) CPR/transfer (%) MPR (%) Miscarriage
Study group (n = 26)
Control group (n = 25)
31.3 ± 2.4 11.4 ± 8.7 8.7 ± 4.2 7.0 ± 3 51 71.3 4.8 ± 3 2.1 ± 0.6 18/56 (32.1) 12/26 (46.1) 6 (50) 0
30.8 ± 3.6 16.1 ± 9.9a 9.4 ± 5.5 8.9 ± 6.3 44 70.9 6.3 ± 4.6 2.2 ± 0.5 13/56 (23.2) 10/25 (40.0) 3 (30) 0
Values are mean ± SD unless otherwise stated. CPR = clinical pregnancy rate; GQE = good quality embryos; ICSI = intracytoplasmic sperm injection; MPR = multiple pregnancy rate. a The number of oocytes retrieved was signicantly higher in the control group (P = 0.04).
RBMOnline®
S54
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
1a2
"OX PLOT
"OX PLOT
$,(
$,(
1al
'
'ROUPS
'
'
1b2
"OX PLOT
'
'
'ROUPS
'
1c2
"OX PLOT
'
'ROUPS
'
"OX PLOT
(#'?,(
,(
'ROUPS
"OX PLOT
(#',(
$,(
1bl
1cl
'
'ROUPS
'
'
'ROUPS
'
Figure 1. Serum LH concentrations on stimulation days (a) 3 and 6, (b) day 8 and day of human chorionic gonadotrophin (HCG) administration, and (c) day of oocyte retrieval and HCG + 12 days. After the introduction of the gonadotrophin-releasing hormone (GnRH) antagonist, serum LH concentrations were well controlled in the study group (G1) (high-dose GnRH antagonist + rLH) when compared with the control group (G2) (no add-back LH + low-dose GnRH antagonist), being signicantly lower (P < 0.001) in controls, except on the day of oocyte retrieval and HCG + 12 days. The black dots show outlying values not included in the ranges of the box plots.
RBMOnline®
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
S55
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
,( CONCENTRATIONS ON DAY OF (#' ,( 'ROUP N
)5L
3UBJECT NUMBER
,( CONCENTRATIONS ON DAY OF (#' CONTROL GROUP N
)5L
MISSING VALUE BELOW DETECTION LIMIT )5L
3UBJECT NUMBER
Figure 2. Serum LH concentrations on the day of HCG administration. LH variation from day to day was very well controlled in the high-dose cetrorelix/LH add-back group (median 2.6, range 1.0–4.8 IU/l) as opposed to the control group (median 1.45, range 0.5–14 IU/l).
RBMOnline®
S56
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
One patient from the control group had a premature LH surge and progesterone rise; no premature LH rise was observed in the highdose cetrorelix/LH add-back treated patients.
$ISCUSSION In the present study, the obvious proper control group was to use a daily dose of 2 mg cetrorelix without rLH add-back. However, it would be unethical to include such a control group in the light of the available data on high-dose ganirelix treatment, showing an almost complete inhibition of implantation in the human (Devroey et al., 1998) as well as in animal experiments. There is no reason to expect that cetrorelix will behave differently from ganirelix, because the mechanisms of action of both GnRH antagonists are exactly the same. This study clearly shows that supplementation with rLH in patients treated with high doses of GnRH antagonist (2 mg/day) for IVF/ICSI is able to rescue the adverse effects imposed on implantation reported in the literature, as both the implantation and pregnancy rate were similar in both groups. Such an outcome opens new questions for debate, as this deleterious effect of high doses of GnRH antagonist has been linked to negatively impact on oocyte/embryo quality, systemic hormonal milieu – oestradiol, progesterone and LH concentrations – and/ or endometrium (Hernandez, 2000). Recent evidence shows that LH supplementation during ovarian stimulation may be helpful in selected patients. Well-calibrated LH administration improves the ovarian response in advanced age women (>35 years old) and in young, normogonadotrophic patients with steady response to rFSH (Alviggi et al., 2006; De Placido et al., 2006). As immunoreactive LH is not related to the concentration of bioactive LH, it is quite difcult to estimate prior to ovarian stimulation which patients may benet from LH supplementation. However, this issue is still controversial, especially in relation to those who may benet from LH cotreatment, in what type of protocol, at what time point, and in what doses of LH (Griesinger et al., 2006). Clinical results from different studies – including the dosending reports – have shown comparable numbers of metaphase II (MII) oocytes collected regardless of the dose of GnRH antagonist administered. This being – nuclear maturity status – a limited aspect of the complex oocyte cell, it is a good estimate of the effect of the GnRH antagonist on the ovarian response. Considering that the oocyte expresses GnRH receptors (Dekel et al., 1988), an unlikely effect in the maturation process of the female gamete cannot be ruled out. In any case, both oocyte quality and maturity, as well as fertilization rates were similar to those found when using GnRH agonists, in all studies. An interesting hypothesis would be that high doses of GnRH antagonist may be affecting embryo development, and that LH add-back therapy could reverse this effect. Considering that the GnRH antagonist half-life is around 30 h (Hermann et al., 1996), as the embryo is transferred back into the uterus 4–7 days after the last dose is administered, a direct effect on the embryo is very unlikely. More robust data to discard this hypothesis comes from the dose-nding studies. The morphological quality of the embryos obtained from patients exposed to high-dose antagonist was comparable to that of
the other groups, but obviously morphological assessment of embryo quality does not reect the true potential of a human embryo to implant. Interestingly, frozen–thawed embryos from these patients implanted as well as embryos from patients who received a much lower dose of GnRH antagonist (Kol et al., 1999), suggesting that there is no negative effect of a high antagonist dose on embryo developmental potential. A second hypothesis of the detrimental effect of high-dose GnRH antagonist administration that could be reverted by LH add back therapy could be a systemic effect inducing an altered hormonal milieu during the late follicular phase with profound suppression in both LH and oestradiol production, which may affect the implantation rate. Aberrant hormonal concentrations may induce abnormal oocyte development and suboptimal embryo implantation (Simón et al., 1995; Pellicer et al., 1996). Although still a matter of discussion timing and doses, it seems clear that a subgroup of IVF patients require additional LH activity in order to adequately respond to FSH ovarian stimulation (De Placido et al., 2005), as not all patients benet from this supplementation (Griesinger et al., 2005). A specic group of patients may be more sensitive to LH concentration uctuations, and thus interfere with the correct sequence of maturational changes and correct synchronization between nuclear and cytoplasmic maturation (Mattioli and Barboni, 2000; Huirne et al., 2005). Our data are in accordance with this hypothesis, as LH add-back therapy could normalize the aberrant hormonal milieu and, thus, induce correct maturational changes. High doses of GnRH antagonist may profoundly suppress LH pituitary secretion, but this is a difcult parameter to evaluate as concentrations of circulating LH during ovarian stimulation do not correlate with ovarian outcome parameters (Balasch et al., 2001). In fact, bioactive LH may not be the hormone evaluated in the laboratory as immunoreactive LH, which complicates this issue even further. Taking into account these considerations that make clinicians use an empirical approach towards LH supplementation, it is important to note that no direct relationship between serum LH or oestradiol concentrations and clinical outcome was observed in the dose-nding studies (Kolibianakis et al., 2006). As a third explanation for the effect of high-dose GnRH antagonists on implantation, it has been postulated that they exert a direct effect on the endometrium. Our group has investigated the effect of both low- (0.25 mg) and high-dose (2.0 mg) GnRH antagonist, as well as a GnRH-agonist treatment, on the endometrial development in women undergoing ovarian stimulation for oocyte donation (Simón et al., 2005). The endometrial development in a natural cycle was studied as a reference. Parameters used in this study as markers of endometrial receptivity were evaluation of endometrial thickness and pattern by ultrasound, and endometrial biopsy assessments in terms of endometrial dating, oestrogen and progesterone receptor expression and surface structure (pinopodes). In addition, gene expression proles were investigated. The endometrial development in the early and mid-luteal phase revealed no relevant differences in the parameters investigated. Interestingly, both ganirelix regimens were associated with endometrial development that more closely resembled that in natural (unstimulated) cycles compared with the buserelin regimen (Simón et al., 2005). A possible explanation for this
RBMOnline®
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
S57
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
nding is that although GnRH receptors have been described to be expressed in human endometrial tissue (Dong et al., 1998; Raga et al., 1998), it may be that GnRH antagonists lack intrinsic effects at the GnRH receptor concentration, and they can only counteract the actions of native GnRH analogues (Mannaerts and Gordon, 2000). It is believed that the deleterious effect induced by a higher GnRH antagonist dose is caused by the extreme LH suppression induced. Comparable uctuating LH concentrations on days 3 and 6 can be observed in both study and control groups. This rather strong uctuation continues on day 8 and the day of HCG in the low-dose cetrorelix group, with very low LH concentrations on the day of HCG that were below 1 IU/l in six out of 16 subjects, and one clear LH surge. The high-dose cetrorelix/rLH group, in contrast, showed remarkably stable LH concentrations both on day 8 and on the day of HCG, with values between 2 and 4 IU/l. There were no LH surges in this group and there was just one subject out of 16 with an LH concentration below 1 IU/l. The LH concentrations in the rLH group conrm that the 375 IU dose of rLH chosen, which was based on other authors’ previous experiences (Hillier, 2000; Balasch and Fabregues, 2003), was indeed the right dose. A hypothetical concern in clinical practice could be the high cost of this new regimen. Extrapolating data from this study, it is estimated that this new regimen might pose an unfavourable ‘cost/benet’ ratio to the patient due to the large rLH doses needed. Rather than investigating a daily-use protocol as a substitute for classical ovarian stimulation protocols, the aim of this study was to delineate how rLH add-back therapy might rescue the profound pituitary suppression achieved by the highdose GnRH antagonist, to offer new insights into new ways of stimulation or improving the actual protocols. One of the advantages of the antagonist protocols is the possibility of triggering oocyte maturation and ovulation with the GnRH agonist, thereby avoiding the use of HCG and, thus, beneting patients at high risk of OHSS. However, recent results conrm that both implantation and pregnancy rates may be affected (Orvieto et al., 2006). To conclude, these results strongly suggest that rLH is an important player to be considered during ovarian stimulation with GnRH antagonists. Both non-pituitary as well as profound pituitary effects of high-dose GnRH antagonist effects may be reverted by LH add-back therapy. At clinically used concentrations, this work conrms that the high-dose cetrorelix/ rLH regimen is a very reliable regimen for ovarian stimulation, providing the lowest risk of cycle cancellation and the most stable LH concentrations. The present randomized study opens the possibility to perform a much bigger study to document the advantages of this new regimen, such as less premature LH surges, less intermediary size follicles, better implantation rate, and a high robustness of this regimen, which makes it very suitable for the management of large IVF units. Nevertheless, it can be argued that there are higher costs for this regimen, and this needs to be addressed.
2EFERENCES Albano C, Felberbaum R, Smitz J et al. 2000 Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. European Cetrorelix Study Group. Human Reproduction 15, 526–531. Al-Inany H, Aboulghar M 2002 GnRH antagonist in assisted reproduction: a Cochrane review. Human Reproduction 17, 874–885. Alviggi C, Clarizia R, Mollo A et al. 2006 Who needs LH in ovarian stimulation? Reproductive BioMedicine Online 12, 599–607. Balasch J, Fabregues F 2003 Pregnancy after administration of high dose recombinant human LH alone to support nal stages of follicular maturation in a woman with long-standing hypogonadotrophic hypogonadism. Reproductive BioMedicine Online 6, 427–431. Balasch J, Vidal E, Peñarrubia J et al. 2001 Suppression of LH during ovarian stimulation: analysing threshold values and effects on ovarian response and the outcome of assisted reproduction in down-regulated women stimulated with recombinant FSH. Human Reproduction 16, 1636–1643. Borm G, Mannaerts B 2000 Treatment with the gonadotrophinreleasing hormone antagonist ganirelix in women undergoing ovarian stimulation with recombinant follicle stimulating hormone is effective, safe and convenient: results of a controlled, randomized, multicentre trial. The European Orgalutran Study Group. Human Reproduction 15, 1490–1498. De Placido G, Mollo A, Clarizia R et al. 2006 Gonadotropin-releasing hormone (GnRH) antagonist plus recombinant luteinizing hormone vs. a standard GnRH agonist short protocol in patients at risk for poor ovarian response. Fertility and Sterility 85, 247–250. De Placido G, Alviggi C, Perino A et al. on behalf of the Italian Collaborative Group on Recombinant Human Luteinizing Hormone 2005 Recombinant human LH supplementation versus recombinant human FSH (rFSH) step-up protocol during controlled ovarian stimulation in normogonadotrophic women with initial inadequate ovarian response to rFSH. A multicentre, prospective, randomized controlled trial. Human Reproduction 20, 390–396. Dekel N, Lewysohn O, Ayalon D et al. 1988 Receptors for gonadotropin releasing hormone are present in rat oocytes. Endocrinology 123, 1205–1207. Devroey P, Abyholm T, Diedrich K et al. 1998 A double-blind, randomized, dose-nding study to assess the efcacy of the gonadotrophin-releasing hormone antagonist ganirelix (Org 37462) to prevent premature luteinizing hormone surges in women undergoing ovarian stimulation with recombinant follicle stimulating hormone (Puregon™). Human Reproduction 13, 3023–3031. Dong K, Marcelin K, Hsu M et al. 1998 Expression of gonadotropinreleasing hormone (GnRH) gene in human uterine endometrial tissue. Molecular Human Reproduction 4, 893–898. European and Middle East Orgalutran Study Group 2001 Comparable clinical outcome using the GnRH antagonist ganirelix or a long protocol of the GnRH agonist triptorelin for the prevention of premature LH surges in women undergoing ovarian stimulation. Human Reproduction 16, 644–651. Fluker M, Grifo J, Leader A et al. North American Ganirelix Study Group 2001 Efcacy and safety of ganirelix acetate versus leuprolide acetate in women undergoing controlled ovarian hyperstimulation. Fertility and Sterility 75, 38–45. Garcia-Velasco JA, Isaza V, Requena A et al. 2000 High doses of gonadotrophins combined with stop versus non-stop protocol of GnRH analogue administration in low responder IVF patients: a prospective, randomized, controlled trial. Human Reproduction 15, 2292–2296. Griesinger G, Diedrich K 2006 Role of LH in ovarian stimulation: considerations. Reproductive BioMedicine Online 12, 404–406.
RBMOnline®
S58
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
Griesinger G, Schultze-Mosgau A, Dafopoulos K et al. 2005 Recombinant luteinizing hormone supplementation to recombinant follicle-stimulating hormone induced ovarian hyperstimulation in the GnRH-antagonist multiple-dose protocol. Human Reproduction 20, 1200–1206. Hermann R, Scheider E, Romeis P et al. 1996 Pharmacokinetics and pharmacodynamics of the LH-RH-antagonist Cetrorelix in healthy male and female subjects. Gynecologic Endocrinology 10 (Suppl. 1), 9–15. Hernandez ER 2000 Embryo implantation: the Rubicon for GnRH antagonists. Human Reproduction 15, 1211–1216. Hillier SG 2000 Controlled ovarian stimulation in women. Journal of Reproduction and Fertility 120, 201–210. Huirne J, van Loenen A, Schats R et al. 2005 Dose-nding study of daily GnRH antagonist for the prevention of premature LH surges in IVF/ICSI patients: optimal changes in LH and progesterone for clinical pregnancy. Human Reproduction 20, 359–367. ICH 1996 International Conference on Harmonisation of technical requirements for registration of pharmaceuticals for human use: ICH Harmonised tripartite guideline. Guideline for good clinical practice E6(R1). See http://www.ich.org/LOB/media/MEDIA482. pdf [accessed 12 July 2007]. Kol S, Lightman A, Hillensjo T et al. 1999 High doses of gonadotrophin-releasing hormone antagonist in in-vitro fertilization cycles do not adversely affect the outcome of subsequent freeze–thaw cycles. Human Reproduction 14, 2242– 2244. Kolibianakis E 2005 GnRh antagonists in IVF. Reproductive BioMedicine Online 10, 705–712. Kolibianakis EM, Collins J, Tarlatzis B et al. 2006 Are endogenous LH levels during ovarian stimulation for IVF using GnRH analogues associated with the probability of ongoing pregnancy? A systematic review. Human Reproduction Update 12, 3–12.
Mannaerts B, Gordon K 2000 GnRH antagonists do not activate the GnRH receptor. Human Reproduction 15, 1882–1883. Mattioli M, Barboni B 2000 Signal transduction mechanism for LH in the cumulus–oocyte complex. Molecular Cellular Endocrinology 161, 19–23. Olivennes F, Belaisch-Allart J, Emperaire JC et al. 2000 Prospective, randomized, controlled study of in vitro fertilization–embryo transfer with a single dose of a luteinizing hormone-releasing hormone (LH-RH) antagonist (cetrorelix) or a depot formula of an LH-RH agonist (triptorelin). Fertility and Sterility 73, 314–320. Orvieto R, Rabinson J, Meltzer S et al. 2006 Substituting HCG with GnRH agonist to trigger nal follicular maturation – a retrospective comparison of three different protocols. Reproductive BioMedicine Online 13, 198–201. Pellicer A, Valbuena D, Cano F et al. 1996 Lower implantation rates in high responders: evidence for an altered endocrine milieu during the preimplantation period. Fertility and Sterility 65, 1190–1195. Raga F, Casañ E, Kruessel J et al. 1998 Quantitative GnRH gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biology of Reproduction 59, 661–669. Simón C, Cano F, Valbuena D et al. 1995 Clinical evidence for a detrimental effect on uterine receptivity of high serum oestradiol concentrations in high and normal responder patients. Human Reproduction 10, 2432–2437. Simón C, Oberyé J, Bellver J et al. 2005 Similar endometrial development in oocyte donors treated with either high- or standard-dose of Gn-RH antagonist compared to treatment with a GnRH agonist or in natural cycles. Human Reproduction 20, 3318–3327. Received 10 August 2006; refereed 20 September 2006; accepted 20 June 2007.
RBMOnline®
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
S59
!RTICLE (IGH DOSE RECOMBINANT ,( ADD BACK STRATEGY USING HIGH DOSE 'N2( ANTAGONIST IS AN INNOVATIVE PROTOCOL COMPARED WITH STANDARD 'N2( ANTAGONIST *UAN 'ARCIA 6ELASCO DID HIS TRAINING IN /BSTETRICS AND 'YNECOLOGY IN -ADRID !UTONOMA 5NIVERSITY AND THEN HIS 2EPRODUCTIVE %NDOCRINOLOGY AND )NFERTILITY &ELLOWSHIP AT 9ALE 5NIVERSITY 53! n (E IS NOW $IRECTOR OF )6) -ADRID AND !SSISTANT 0ROFESSOR AT 2EY *UAN #ARLOS 5NIVERSITY (E HAS PUBLISHED OVER PEER REVIEWED ARTICLES (E IS AN AD HOC REVIEWER FOR THE MAIN JOURNALS IN THE lELD AND SERVES IN THE EDITORIAL BOARD OF 2EPRODUCTIVE "IO-EDICINE /NLINE AND (UMAN 2EPRODUCTION (IS MAIN RESEARCH INTERESTS ARE ENDOMETRIOSIS /(33 LOW RESPONDERS AND HUMAN IMPLANTATION
$R *UAN 'ARCIA 6ELASCO *! 'ARCIA 6ELASCO (*4 #OELINGH "ENNINK 2 %PIFANIO % %SCUDERO ! 0ELLICER # 3IMØN )NSTITUTO 6ALENCIANO DE )NFERTILIDAD -ADRID )6) -ADRID -ADRID 3PAIN )NSTITUTO 6ALENCIANO DE )NFERTILIDAD &OUNDATION &)6) 5NIVERSITY OF 6ALENCIA 6ALENCIA 3PAIN )NSTITUTO 6ALENCIANO DE )NFERTILIDAD )6) 6ALENCIA 3PAIN 0ANTARHEI "IOSCIENCE "6 )NSTITUTE FOR #LINICAL #ONCEPT 2ESEARCH 0/ "OX AL :EIST 4HE .ETHERLANDS #ORRESPONDENCE 4EL &AX E MAIL CSIMON IVIES
!BSTRACT High daily doses of gonadotrophin-releasing hormone (GnRH) antagonists during the follicular phase of ovarian stimulation were associated with low implantation rates. To test if this occurred because of profound pituitary suppression, the pituitary response was suppressed with a high-dose GnRH antagonist and recombinant LH (rLH) was added back to correct the implantation rate. An open-label, randomized, controlled, prospective clinical study in 60 patients undergoing IVF was performed. GnRH antagonist was initiated on day 6 of stimulation (2 mg/day) together with 375 IU rLH, and maintained until the day of HCG administration. Controls received 0.25 mg/day GnRH antagonist. Fluctuating LH concentrations were present on days 3 and 6 in both groups. This strong uctuation continued on day 8 and on the day of HCG administration in the control (low-dose) group, where 30% of patients had LH concentrations <1 IU/l on the HCG day. The study (high-dose) group showed stable LH concentrations on day 8 and on the HCG day, with no LH surges. No clinical differences were found between groups. The LH add-back strategy (375 IU/day) rescued the adverse effects that high doses of GnRH imposed on implantation. These results suggest that rLH should be considered during ovarian stimulation with GnRH antagonist. Keywords: GnRH antagonist, IVF, recombinant LH
)NTRODUCTION
Gonadotrophin-releasing hormone (GnRH) antagonists have been proved to be effective in the prevention of the premature LH surge in ovarian stimulation. Enough evidence exists in the literature to demonstrate their benecial effects when compared with GnRH agonists beyond effectively suppressing endogenous LH, such as a decrease in the amount of gonadotrophins required and a shorter period of stimulation (Albano et al., 2000; Borm et al., 2000; Olivennes et al., 2000; European and Middle East Orgalutran Study Group, 2001; Fluker et al., 2001). This newer GnRH antagonist
strategy was expected to yield similar clinical outcomes, but this expectation was not met in the initial ve randomized studies that indicated a trend towards lower implantation and pregnancy rates for the GnRH-antagonist treatment group compared with those in the GnRH-agonist group (Al Inany and Aboulghar, 2002). These results were probably due to differences in stimulation protocols, different requirements of LH in different patient populations, hypothetical effects on the endometrium, as well as the physicians’ learning curve. These difculties motivated several changes in
© 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
S52
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
ovarian stimulation and patient management, to obtain comparable implantation rates in recent times (Kolibinanakis, 2005). The dose-nding study of Devroey et al. (1998) investigated the effects of six different dosages of ganirelix, and showed a dosedependent decrease in implantation, with a complete absence of pregnancies at doses above 1 mg/day, whereas no impact on the number of oocytes and good quality embryos was found. Followup of the frozen embryos obtained in that dose-nding study revealed that ongoing pregnancies were subsequently achieved in 11 patients, of which six were treated with a high ganirelix dose of 1.0 or 2.0 mg (Kol et al., 1999). A recent study by Huirne et al. (2005) addressing a similar question reached similar conclusions, with a lower implantation and pregnancy rate when a GnRH antagonist was administered at high doses. It was postulated that this unwanted effect was due to either direct action of GnRH antagonist on the endometrium and/ or to a profound suppression of the pituitary that may lead to extremely low LH concentrations, which may be detrimental to the implantation process. In an attempt to address the rst possibility, the authors investigated the effect of both low- (0.25 mg) and high-dose (2.0 mg) GnRH antagonist as well as a GnRH-agonist treatment on the endometrial development in women undergoing ovarian stimulation for oocyte donation (Simón et al., 2005). No relevant alteration was observed in the endometrial development in the early- and mid-luteal phases in women undergoing ovarian stimulation for oocyte donation following daily treatment with a standard- or high-dose GnRH antagonist. In addition, the endometrial development after GnRH antagonist mimics the natural endometrium more closely than after GnRH agonist. The current study was conducted to test the second hypothesis by suppressing the pituitary response with a high-dose GnRH antagonist (2 mg/day) and recombinant LH add-back strategy (375 IU/day) to correct the LH bioactivity for adequate follicle development and preparation of the endometrium.
-ATERIALS AND METHODS 3TUDY DESIGN AND PATIENTS The study was designed as an open-label, randomized, controlled, prospective clinical study in patients undergoing IVF/ intracytoplasmic sperm injection (ICSI). The primary objective of the study was to compare the clinical outcome in the study group, using high-dose GnRH antagonist (2 mg/day) and adding-back 375 IU/day recombinant LH (rLH), with the control group, using similar rFSH doses and low-dose GnRH antagonist (0.25 mg/ day). The secondary objective was to investigate serum oestradiol, progesterone and LH concentrations during the treatment cycle on stimulation days 3, 6 and 8, the day of HCG, the day of oocyte retrieval and HCG + 12 days, in the control versus the study group. The study was conducted in accordance with the last revision of the Declaration of Helsinki and the International Conference on Harmonisation (ICH) Guideline for Good Clinical Practice (1996). Written informed consent was obtained from all subjects and the study was approved by the internal institutional review board. A total of 60 patients were assessed for eligibility, with the following inclusion criteria: 18–35 years of age, rst IVF cycle
at our institution and basal oestradiol < 60 pg/ml, FSH < 10 IU/ l and prolactin < 20 ng/ml. Exclusion criteria were history of pelvic endometriosis, polycystic ovarian syndrome, body mass index (BMI) < 35 kg/m2, thyroid dysfunction, or gross uterine or ovarian alterations found by transvaginal ultrasound. Sample size was estimated by proof-of-concept, and randomization was performed on-site using a computer generated list, not blocked and not concealed. Of those patients allocated to intervention, nine were lost to followup: three patients discontinued intervention due to risk of ovarian hyperstimulation syndrome (OHSS) (one in the study group, and two in the control group), three were cancelled due to insufcient ovarian response (study group n = 2, control group n = 1), one patient from the study group was excluded due to poor quality oocytes, another patient in the control group due to premature luteinization, and one more patient abandoned the study for personal reasons before receiving the GnRH antagonist. Thus, a total of 51 patients were analysed, 26 in the study group and 25 in the control group Both groups of patients were initiated with recombinant FSH (rFSH; 150 IU) on cycle day 2 after ovarian quiescence was ascertained (absence of ovarian cyst > 10 mm diameter on transvaginal ultrasound and/or serum oestradiol < 60 pg/ml) and adjusted after 2 days, according to oestradiol concentration. On treatment day 3 the patients underwent blood analysis for LH and oestradiol concentration. rFSH was increased to 200 IU/day if oestradiol <100 pg/ml, decreased to 100 IU/day if oestradiol > 200 pg/ml, or maintained at the same dose if oestradiol ranged from 100 to 200 pg/ ml. On day 6 and onwards, rFSH was again adjusted according to oestradiol concentration and ultrasonographic ndings every other day. In the study group (n = 26) high-dose GnRH antagonist cetrorelix (Cetrotide, Serono, Geneve, Switzerland) 2 mg/day plus rLH (Luveris, Serono, Geneve, Switzerland) 375 IU/day were added s.c. at day 6 of stimulation and maintained until the HCG day. The dose of rLH was chosen according to the previous experience of other groups (Hillier, 2000; Balasch and Fabregues, 2003). In the control group (n = 25) a regular daily dose of antagonist (0.25 mg/ day) was started s.c. at day 6 and maintained until the HCG day. rHCG (Ovitrelle; Serono, Geneva, Switzerland) 250 g was given by a single s.c. injection if at least two follicles reached 18 mm mean diameter, and ovum retrieval was then scheduled for 36–38 h later. Premature LH surge was dened as LH > 10 IU/l on the day of HCG administration. The standard IVF procedure has been described elsewhere (GarciaVelasco et al., 2000). Embryo transfer (ET) was performed on day 3 of embryo development under ultrasound guidance, with a halffull bladder. Luteal phase was supported with natural micronized progesterone (Utrogestan, Seid, Barcelona, Spain) 200 mg twice daily, starting the evening after oocyte retrieval. Serum -HCG was evaluated 2 weeks after embryo transfer and in those who tested positive, a transvaginal ultrasound was scheduled 4 weeks after ET to detect clinical intrauterine pregnancy, and a followup ultrasound was scheduled 10 weeks after ET to document an ongoing pregnancy.
(ORMONAL ANALYSIS Serum oestradiol, progesterone and LH were evaluated during the treatment cycle on stimulation days 0, 3 and 6, every other day thereafter until day of rHCG administration, the day after
RBMOnline®
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
S53
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
HCG and the day of oocyte retrieval. Blood was taken between approximately 08.00 and 10.00 hours in all patients. Hormones were analysed using a commercially available microparticle enzyme immunoassay kit (Abbot Laboratories, Abbot Park, IL, USA). Inter- and intra-assay coefcients of variation for oestradiol at a concentration less than 40 pg/ml were 2.8 and 4.3%, respectively. The serum progesterone kit had a sensitivity of 0.2 ng/ml, with inter- and intraobserver coefcients of variation of 3.9% and 9.6%, respectively. Inter- and intra-assay coefcients of variation for LH were 1.9 and 4.6%, respectively, with a sensitivity of 0.5 IU/l.
3TATISTICAL ANALYSIS Data were expressed as mean ± SD. Student’s t test, chi-squared or Fisher’s exact test, and Wilcoxon/Mann–Whitney U-test, were used as appropriate. P < 0.05 was considered statistically signicant. The statistical analysis was performed with Statistics Package for Social Sciences.
2ESULTS #LINICAL OUTCOME A total of 318 eggs was retrieved in the study group and 467 in the control group. Patients in both groups were comparable in terms of age (31.3 versus 30.8), BMI (23 versus 23.5), and basal hormone determinations. No signicant differences were found between groups in terms of days of stimulation, days of GnRH antagonist treatment (4.6 versus 4.7), number of oocytes inseminated for IVF (8.7 versus 9.4) or microinjected for ICSI (7.0 versus 8.9), IVF fertilization rate (51% versus 44%), ICSI fertilization rate (71.3% versus 70.9%), overall fertilization rate (63.9% versus 58.9%) or mean number of embryos transferred (2.1 versus 2.2) (Table 1). The only signicant nding was a
lower number of oocytes retrieved in the study group compared with the control group (11.4 versus 16.1, P = 0.04). Again, no signicant differences were observed in the implantation rate (32.1% versus 23.2%) and clinical pregnancy rate per transfer (44.4 versus 38.5%).
(ORMONAL PROlLE Similar patterns of uctuating LH concentrations were observed on days 3 and 6 in both groups before the introduction of the GnRH antagonists and rLH add-back therapy (Figure 1a). This rather strong day-to-day variation continued on day 8 in the low-dose cetrorelix group (median 1, range < 0.5–6.7 IU/l, under detection limit of 0.5 IU/l in 20% of patients). However, in the high-dose cetrorelix/LH add-back group there were remarkably stable LH concentrations on day 8 (median 2, range 0.6–4.0 IU/l), which were signicantly higher when compared with control group LH concentrations (P < 0.001). This trend was similar at the day of HCG administration, with serum LH concentrations signicantly higher in the study group versus the control group (2.6 versus 1.4, P < 0.01). (Figure 1b) LH concentrations from day to day were extremely well controlled in the high-dose cetrorelix/LH add-back group (median 2.6, range 1.0–4.8 IU/l) as opposed to the control group (median 1.45, range 0.5–14 IU/l), and 17.2% of patients had LH concentrations under the detection limit of 0.5 IU/l (Figure 2). Once medication was stopped, it was observed that the majority of patients (89.2%) from the study group displayed serum LH concentrations under the detection limit as opposed to 31.0% of control patients. This study group showed signican tly lower LH serum concentrations when compared with controls (<0.5 versus 1.1, P < 0.0001). Similarly, high-dose cetrorelix/LH add-backtreated patients had lower serum LH concentrations on HCG + 12 (1.2 versus 2.1, P < 0.05) (Figure 1c).
Table 1. Clinical outcome in patients undergoing high-dose (2 mg/day) GnRH antagonist plus add-back LH therapy compared with low-dose (0.25 mg/day) GnRH antagonist.
Age (years) Oocytes retrieved IVF: oocytes incubated ICSI: metaphase II IVF fertilization rate (%) ICSI fertilization rate (%) GQE Embryos replaced Implantation rate (%) CPR/transfer (%) MPR (%) Miscarriage
Study group (n = 26)
Control group (n = 25)
31.3 ± 2.4 11.4 ± 8.7 8.7 ± 4.2 7.0 ± 3 51 71.3 4.8 ± 3 2.1 ± 0.6 18/56 (32.1) 12/26 (46.1) 6 (50) 0
30.8 ± 3.6 16.1 ± 9.9a 9.4 ± 5.5 8.9 ± 6.3 44 70.9 6.3 ± 4.6 2.2 ± 0.5 13/56 (23.2) 10/25 (40.0) 3 (30) 0
Values are mean ± SD unless otherwise stated. CPR = clinical pregnancy rate; GQE = good quality embryos; ICSI = intracytoplasmic sperm injection; MPR = multiple pregnancy rate. a The number of oocytes retrieved was signicantly higher in the control group (P = 0.04).
RBMOnline®
S54
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
1a2
"OX PLOT
"OX PLOT
$,(
$,(
1al
'
'ROUPS
'
'
1b2
"OX PLOT
'
'
'ROUPS
'
1c2
"OX PLOT
'
'ROUPS
'
"OX PLOT
(#'?,(
,(
'ROUPS
"OX PLOT
(#',(
$,(
1bl
1cl
'
'ROUPS
'
'
'ROUPS
'
Figure 1. Serum LH concentrations on stimulation days (a) 3 and 6, (b) day 8 and day of human chorionic gonadotrophin (HCG) administration, and (c) day of oocyte retrieval and HCG + 12 days. After the introduction of the gonadotrophin-releasing hormone (GnRH) antagonist, serum LH concentrations were well controlled in the study group (G1) (high-dose GnRH antagonist + rLH) when compared with the control group (G2) (no add-back LH + low-dose GnRH antagonist), being signicantly lower (P < 0.001) in controls, except on the day of oocyte retrieval and HCG + 12 days. The black dots show outlying values not included in the ranges of the box plots.
RBMOnline®
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
S55
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
,( CONCENTRATIONS ON DAY OF (#' ,( 'ROUP N
)5L
3UBJECT NUMBER
,( CONCENTRATIONS ON DAY OF (#' CONTROL GROUP N
)5L
MISSING VALUE BELOW DETECTION LIMIT )5L
3UBJECT NUMBER
Figure 2. Serum LH concentrations on the day of HCG administration. LH variation from day to day was very well controlled in the high-dose cetrorelix/LH add-back group (median 2.6, range 1.0–4.8 IU/l) as opposed to the control group (median 1.45, range 0.5–14 IU/l).
RBMOnline®
S56
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
One patient from the control group had a premature LH surge and progesterone rise; no premature LH rise was observed in the highdose cetrorelix/LH add-back treated patients.
$ISCUSSION In the present study, the obvious proper control group was to use a daily dose of 2 mg cetrorelix without rLH add-back. However, it would be unethical to include such a control group in the light of the available data on high-dose ganirelix treatment, showing an almost complete inhibition of implantation in the human (Devroey et al., 1998) as well as in animal experiments. There is no reason to expect that cetrorelix will behave differently from ganirelix, because the mechanisms of action of both GnRH antagonists are exactly the same. This study clearly shows that supplementation with rLH in patients treated with high doses of GnRH antagonist (2 mg/day) for IVF/ICSI is able to rescue the adverse effects imposed on implantation reported in the literature, as both the implantation and pregnancy rate were similar in both groups. Such an outcome opens new questions for debate, as this deleterious effect of high doses of GnRH antagonist has been linked to negatively impact on oocyte/embryo quality, systemic hormonal milieu – oestradiol, progesterone and LH concentrations – and/ or endometrium (Hernandez, 2000). Recent evidence shows that LH supplementation during ovarian stimulation may be helpful in selected patients. Well-calibrated LH administration improves the ovarian response in advanced age women (>35 years old) and in young, normogonadotrophic patients with steady response to rFSH (Alviggi et al., 2006; De Placido et al., 2006). As immunoreactive LH is not related to the concentration of bioactive LH, it is quite difcult to estimate prior to ovarian stimulation which patients may benet from LH supplementation. However, this issue is still controversial, especially in relation to those who may benet from LH cotreatment, in what type of protocol, at what time point, and in what doses of LH (Griesinger et al., 2006). Clinical results from different studies – including the dosending reports – have shown comparable numbers of metaphase II (MII) oocytes collected regardless of the dose of GnRH antagonist administered. This being – nuclear maturity status – a limited aspect of the complex oocyte cell, it is a good estimate of the effect of the GnRH antagonist on the ovarian response. Considering that the oocyte expresses GnRH receptors (Dekel et al., 1988), an unlikely effect in the maturation process of the female gamete cannot be ruled out. In any case, both oocyte quality and maturity, as well as fertilization rates were similar to those found when using GnRH agonists, in all studies. An interesting hypothesis would be that high doses of GnRH antagonist may be affecting embryo development, and that LH add-back therapy could reverse this effect. Considering that the GnRH antagonist half-life is around 30 h (Hermann et al., 1996), as the embryo is transferred back into the uterus 4–7 days after the last dose is administered, a direct effect on the embryo is very unlikely. More robust data to discard this hypothesis comes from the dose-nding studies. The morphological quality of the embryos obtained from patients exposed to high-dose antagonist was comparable to that of
the other groups, but obviously morphological assessment of embryo quality does not reect the true potential of a human embryo to implant. Interestingly, frozen–thawed embryos from these patients implanted as well as embryos from patients who received a much lower dose of GnRH antagonist (Kol et al., 1999), suggesting that there is no negative effect of a high antagonist dose on embryo developmental potential. A second hypothesis of the detrimental effect of high-dose GnRH antagonist administration that could be reverted by LH add back therapy could be a systemic effect inducing an altered hormonal milieu during the late follicular phase with profound suppression in both LH and oestradiol production, which may affect the implantation rate. Aberrant hormonal concentrations may induce abnormal oocyte development and suboptimal embryo implantation (Simón et al., 1995; Pellicer et al., 1996). Although still a matter of discussion timing and doses, it seems clear that a subgroup of IVF patients require additional LH activity in order to adequately respond to FSH ovarian stimulation (De Placido et al., 2005), as not all patients benet from this supplementation (Griesinger et al., 2005). A specic group of patients may be more sensitive to LH concentration uctuations, and thus interfere with the correct sequence of maturational changes and correct synchronization between nuclear and cytoplasmic maturation (Mattioli and Barboni, 2000; Huirne et al., 2005). Our data are in accordance with this hypothesis, as LH add-back therapy could normalize the aberrant hormonal milieu and, thus, induce correct maturational changes. High doses of GnRH antagonist may profoundly suppress LH pituitary secretion, but this is a difcult parameter to evaluate as concentrations of circulating LH during ovarian stimulation do not correlate with ovarian outcome parameters (Balasch et al., 2001). In fact, bioactive LH may not be the hormone evaluated in the laboratory as immunoreactive LH, which complicates this issue even further. Taking into account these considerations that make clinicians use an empirical approach towards LH supplementation, it is important to note that no direct relationship between serum LH or oestradiol concentrations and clinical outcome was observed in the dose-nding studies (Kolibianakis et al., 2006). As a third explanation for the effect of high-dose GnRH antagonists on implantation, it has been postulated that they exert a direct effect on the endometrium. Our group has investigated the effect of both low- (0.25 mg) and high-dose (2.0 mg) GnRH antagonist, as well as a GnRH-agonist treatment, on the endometrial development in women undergoing ovarian stimulation for oocyte donation (Simón et al., 2005). The endometrial development in a natural cycle was studied as a reference. Parameters used in this study as markers of endometrial receptivity were evaluation of endometrial thickness and pattern by ultrasound, and endometrial biopsy assessments in terms of endometrial dating, oestrogen and progesterone receptor expression and surface structure (pinopodes). In addition, gene expression proles were investigated. The endometrial development in the early and mid-luteal phase revealed no relevant differences in the parameters investigated. Interestingly, both ganirelix regimens were associated with endometrial development that more closely resembled that in natural (unstimulated) cycles compared with the buserelin regimen (Simón et al., 2005). A possible explanation for this
RBMOnline®
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
S57
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
nding is that although GnRH receptors have been described to be expressed in human endometrial tissue (Dong et al., 1998; Raga et al., 1998), it may be that GnRH antagonists lack intrinsic effects at the GnRH receptor concentration, and they can only counteract the actions of native GnRH analogues (Mannaerts and Gordon, 2000). It is believed that the deleterious effect induced by a higher GnRH antagonist dose is caused by the extreme LH suppression induced. Comparable uctuating LH concentrations on days 3 and 6 can be observed in both study and control groups. This rather strong uctuation continues on day 8 and the day of HCG in the low-dose cetrorelix group, with very low LH concentrations on the day of HCG that were below 1 IU/l in six out of 16 subjects, and one clear LH surge. The high-dose cetrorelix/rLH group, in contrast, showed remarkably stable LH concentrations both on day 8 and on the day of HCG, with values between 2 and 4 IU/l. There were no LH surges in this group and there was just one subject out of 16 with an LH concentration below 1 IU/l. The LH concentrations in the rLH group conrm that the 375 IU dose of rLH chosen, which was based on other authors’ previous experiences (Hillier, 2000; Balasch and Fabregues, 2003), was indeed the right dose. A hypothetical concern in clinical practice could be the high cost of this new regimen. Extrapolating data from this study, it is estimated that this new regimen might pose an unfavourable ‘cost/benet’ ratio to the patient due to the large rLH doses needed. Rather than investigating a daily-use protocol as a substitute for classical ovarian stimulation protocols, the aim of this study was to delineate how rLH add-back therapy might rescue the profound pituitary suppression achieved by the highdose GnRH antagonist, to offer new insights into new ways of stimulation or improving the actual protocols. One of the advantages of the antagonist protocols is the possibility of triggering oocyte maturation and ovulation with the GnRH agonist, thereby avoiding the use of HCG and, thus, beneting patients at high risk of OHSS. However, recent results conrm that both implantation and pregnancy rates may be affected (Orvieto et al., 2006). To conclude, these results strongly suggest that rLH is an important player to be considered during ovarian stimulation with GnRH antagonists. Both non-pituitary as well as profound pituitary effects of high-dose GnRH antagonist effects may be reverted by LH add-back therapy. At clinically used concentrations, this work conrms that the high-dose cetrorelix/ rLH regimen is a very reliable regimen for ovarian stimulation, providing the lowest risk of cycle cancellation and the most stable LH concentrations. The present randomized study opens the possibility to perform a much bigger study to document the advantages of this new regimen, such as less premature LH surges, less intermediary size follicles, better implantation rate, and a high robustness of this regimen, which makes it very suitable for the management of large IVF units. Nevertheless, it can be argued that there are higher costs for this regimen, and this needs to be addressed.
2EFERENCES Albano C, Felberbaum R, Smitz J et al. 2000 Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. European Cetrorelix Study Group. Human Reproduction 15, 526–531. Al-Inany H, Aboulghar M 2002 GnRH antagonist in assisted reproduction: a Cochrane review. Human Reproduction 17, 874–885. Alviggi C, Clarizia R, Mollo A et al. 2006 Who needs LH in ovarian stimulation? Reproductive BioMedicine Online 12, 599–607. Balasch J, Fabregues F 2003 Pregnancy after administration of high dose recombinant human LH alone to support nal stages of follicular maturation in a woman with long-standing hypogonadotrophic hypogonadism. Reproductive BioMedicine Online 6, 427–431. Balasch J, Vidal E, Peñarrubia J et al. 2001 Suppression of LH during ovarian stimulation: analysing threshold values and effects on ovarian response and the outcome of assisted reproduction in down-regulated women stimulated with recombinant FSH. Human Reproduction 16, 1636–1643. Borm G, Mannaerts B 2000 Treatment with the gonadotrophinreleasing hormone antagonist ganirelix in women undergoing ovarian stimulation with recombinant follicle stimulating hormone is effective, safe and convenient: results of a controlled, randomized, multicentre trial. The European Orgalutran Study Group. Human Reproduction 15, 1490–1498. De Placido G, Mollo A, Clarizia R et al. 2006 Gonadotropin-releasing hormone (GnRH) antagonist plus recombinant luteinizing hormone vs. a standard GnRH agonist short protocol in patients at risk for poor ovarian response. Fertility and Sterility 85, 247–250. De Placido G, Alviggi C, Perino A et al. on behalf of the Italian Collaborative Group on Recombinant Human Luteinizing Hormone 2005 Recombinant human LH supplementation versus recombinant human FSH (rFSH) step-up protocol during controlled ovarian stimulation in normogonadotrophic women with initial inadequate ovarian response to rFSH. A multicentre, prospective, randomized controlled trial. Human Reproduction 20, 390–396. Dekel N, Lewysohn O, Ayalon D et al. 1988 Receptors for gonadotropin releasing hormone are present in rat oocytes. Endocrinology 123, 1205–1207. Devroey P, Abyholm T, Diedrich K et al. 1998 A double-blind, randomized, dose-nding study to assess the efcacy of the gonadotrophin-releasing hormone antagonist ganirelix (Org 37462) to prevent premature luteinizing hormone surges in women undergoing ovarian stimulation with recombinant follicle stimulating hormone (Puregon™). Human Reproduction 13, 3023–3031. Dong K, Marcelin K, Hsu M et al. 1998 Expression of gonadotropinreleasing hormone (GnRH) gene in human uterine endometrial tissue. Molecular Human Reproduction 4, 893–898. European and Middle East Orgalutran Study Group 2001 Comparable clinical outcome using the GnRH antagonist ganirelix or a long protocol of the GnRH agonist triptorelin for the prevention of premature LH surges in women undergoing ovarian stimulation. Human Reproduction 16, 644–651. Fluker M, Grifo J, Leader A et al. North American Ganirelix Study Group 2001 Efcacy and safety of ganirelix acetate versus leuprolide acetate in women undergoing controlled ovarian hyperstimulation. Fertility and Sterility 75, 38–45. Garcia-Velasco JA, Isaza V, Requena A et al. 2000 High doses of gonadotrophins combined with stop versus non-stop protocol of GnRH analogue administration in low responder IVF patients: a prospective, randomized, controlled trial. Human Reproduction 15, 2292–2296. Griesinger G, Diedrich K 2006 Role of LH in ovarian stimulation: considerations. Reproductive BioMedicine Online 12, 404–406.
RBMOnline®
S58
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
Article - rLH add-back strategy in high-dose GnRH antagonist cycles - JA Garcia-Velasco et al.
Griesinger G, Schultze-Mosgau A, Dafopoulos K et al. 2005 Recombinant luteinizing hormone supplementation to recombinant follicle-stimulating hormone induced ovarian hyperstimulation in the GnRH-antagonist multiple-dose protocol. Human Reproduction 20, 1200–1206. Hermann R, Scheider E, Romeis P et al. 1996 Pharmacokinetics and pharmacodynamics of the LH-RH-antagonist Cetrorelix in healthy male and female subjects. Gynecologic Endocrinology 10 (Suppl. 1), 9–15. Hernandez ER 2000 Embryo implantation: the Rubicon for GnRH antagonists. Human Reproduction 15, 1211–1216. Hillier SG 2000 Controlled ovarian stimulation in women. Journal of Reproduction and Fertility 120, 201–210. Huirne J, van Loenen A, Schats R et al. 2005 Dose-nding study of daily GnRH antagonist for the prevention of premature LH surges in IVF/ICSI patients: optimal changes in LH and progesterone for clinical pregnancy. Human Reproduction 20, 359–367. ICH 1996 International Conference on Harmonisation of technical requirements for registration of pharmaceuticals for human use: ICH Harmonised tripartite guideline. Guideline for good clinical practice E6(R1). See http://www.ich.org/LOB/media/MEDIA482. pdf [accessed 12 July 2007]. Kol S, Lightman A, Hillensjo T et al. 1999 High doses of gonadotrophin-releasing hormone antagonist in in-vitro fertilization cycles do not adversely affect the outcome of subsequent freeze–thaw cycles. Human Reproduction 14, 2242– 2244. Kolibianakis E 2005 GnRh antagonists in IVF. Reproductive BioMedicine Online 10, 705–712. Kolibianakis EM, Collins J, Tarlatzis B et al. 2006 Are endogenous LH levels during ovarian stimulation for IVF using GnRH analogues associated with the probability of ongoing pregnancy? A systematic review. Human Reproduction Update 12, 3–12.
Mannaerts B, Gordon K 2000 GnRH antagonists do not activate the GnRH receptor. Human Reproduction 15, 1882–1883. Mattioli M, Barboni B 2000 Signal transduction mechanism for LH in the cumulus–oocyte complex. Molecular Cellular Endocrinology 161, 19–23. Olivennes F, Belaisch-Allart J, Emperaire JC et al. 2000 Prospective, randomized, controlled study of in vitro fertilization–embryo transfer with a single dose of a luteinizing hormone-releasing hormone (LH-RH) antagonist (cetrorelix) or a depot formula of an LH-RH agonist (triptorelin). Fertility and Sterility 73, 314–320. Orvieto R, Rabinson J, Meltzer S et al. 2006 Substituting HCG with GnRH agonist to trigger nal follicular maturation – a retrospective comparison of three different protocols. Reproductive BioMedicine Online 13, 198–201. Pellicer A, Valbuena D, Cano F et al. 1996 Lower implantation rates in high responders: evidence for an altered endocrine milieu during the preimplantation period. Fertility and Sterility 65, 1190–1195. Raga F, Casañ E, Kruessel J et al. 1998 Quantitative GnRH gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biology of Reproduction 59, 661–669. Simón C, Cano F, Valbuena D et al. 1995 Clinical evidence for a detrimental effect on uterine receptivity of high serum oestradiol concentrations in high and normal responder patients. Human Reproduction 10, 2432–2437. Simón C, Oberyé J, Bellver J et al. 2005 Similar endometrial development in oocyte donors treated with either high- or standard-dose of Gn-RH antagonist compared to treatment with a GnRH agonist or in natural cycles. Human Reproduction 20, 3318–3327. Received 10 August 2006; refereed 20 September 2006; accepted 20 June 2007.
RBMOnline®
Reprinted from Vol. 15, No. 3 (2007) pp. 280 287.
S59