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Cycles triggered with GnRH agonist: exploring low-dose HCG for luteal support
Reproductive BioMedicine Online (2010) 20, 175– 181
www.sciencedirect.com www.rbmonline.com
ARTICLE
Cycles triggered with GnRH agonist: exploring low-dose HCG for luteal support ˜, F Bonilla-Musoles JC Castillo *, M Dolz, E Bienvenido, L Abad, EM Casan Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Hospital Clı´nico Universitario de Valencia, ˜ez Avenue 17, Valencia 46010, Spain Blasco Iban * Corresponding author. E-mail address: [email protected] (JC Castillo). Juan Carlos Castillo obtained his MD degree in 1998 and his speciality degree in Obstetrics and Gynaecology in 2005 at the Grau Emergency Hospital, Lima, Peru ´. In 2006, he received his Master’s degree in Human Reproduction from the Universidad de Valencia, Spain. Since then he has been working as a consultant specialist in reproductive medicine at Hospital Clı´nico Universitario de Valencia, Spain. His PhD thesis at Universidad de Valencia is focused on the prevention of ovarian hyperstimulation syndrome and will be defended in late 2009.
Abstract The aim of this study in patients at risk of ovarian hyperstimulation syndrome (OHSS) was to determine the efficacy and
safety of luteal support using human chorionic gonadotrophin (HCG) after triggering ovulation with gonadotrophin-releasing hormone (GnRH) agonist in IVF/intracytoplasmic sperm injection antagonist cycles. A total of 192 OHSS-risk patients, following a GnRH antagonist protocol (0.25 mg/day cetrorelix) during recombinant FSH stimulation, were triggered with 1.5 mg s.c. leuproreline for ovulation. A total of three boluses of HCG were used for luteal support, 1000 IU (group A, n = 44), 500 IU (group B, n = 115) or 250 IU (group C, n = 33) every third day, starting the day after oocyte retrieval. For the reproductive outcome, main variables were biochemical and clinical pregnancy rates, and for OHSS, the variables were the numbers of moderate and severe OHSS cases. Overall pregnancy rate was 51.8% and clinical pregnancy rate was 43.4%. This study observed eight cases of moderate (4.2%) and seven of severe OHSS (3.6%). Six out of the seven (85.7%) severe cases were late-onset OHSS, related to pregnancy. In conclusion, GnRH agonist single dose for triggering ovulation and low doses of HCG used as luteal-phase support seem to secure a normal pregnancy outcome without increasing the OHSS risk. RBMOnline ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: GnRH agonist, GnRH antagonist, HCG, OHSS
Introduction Third generation gonadotrophin-releasing hormone (GnRH) antagonists were re-introduced into the market for use in ovarian stimulation protocols during the 1990s (Albano et al., 1997; Diedrich et al., 1994; Macklon et al., 2006). A particular property of the GnRH antagonist is its reversible
effect, rapid action and short duration, which allows the pituitary to remain reactive to the action of a single bolus of GnRH agonist for triggering ovulation as an alternative to human chorionic gonadotrophin (HCG) (Nakano et al., 1973). Many studies show that GnRH agonists are as effective as HCG to induce an adequate final follicular maturation (Gonen et al., 1990; Humaidan et al., 2005;
1472-6483/$ - see front matter ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2009.11.018
176 Imoedemhe et al., 1991). Moreover, agonists induce the release of FSH in addition to LH (the flare-up effect), comparable to the natural cycle. However, there are some differences; in the natural cycle the surge of gonadotrophins lasts approximately 48 h, as compared with 24–36 h when GnRH agonists are used to induce ovulation (Hoff et al., 1983; Itskovitz et al., 1991). Previous randomized controlled trials (Humaidan et al., 2005; Kolibianakis et al., 2005) showed that the use of GnRH agonist for triggering ovulation was associated to negative clinical results: low implantation rate, low clinical pregnancy rate and a high rate of early pregnancy loss; all of them presumably related to luteal-phase insufficiency despite standard supplementation with progesterone and oestradiol. Other claimed explanations for these results like oocyte or embryo toxic effects of GnRH agonists seem unlikely because published studies show that implantation rates remain normal in oocyte recipients and frozen-thawed cycles post agonist trigger (Acevedo et al., 2006; Bodri et al., 2008; Griesinger et al., 2007). In this context, luteal-phase support with HCG arises as an alternative; its potent luteotrophic action is able to restore corpus luteum function and normalize luteal phase after GnRH agonist trigger. This effect has been demonstrated in intrauterine insemination cycles (Emperaire et al., 2004) and more recently a pilot study showed that a small bolus of HCG seems to secure the reproductive outcome in IVF cycles (Humaidan et al., 2006). It is well known that standard doses of HCG in the luteal phase are associated with the risk of ovarian hyperstimulation syndrome (OHSS); however, its use in high responders after a single dose of GnRH agonist for triggering ovulation, may still be an efficient and safe alternative if lower doses of HCG are given for luteal support. During cycles with GnRH antagonists, the use of a single dose of GnRH agonist for triggering ovulation has been related to low frequency and severity of OHSS (Kol, 2003; Orvieto, 2005; Revel and Casper, 2001). OHSS, is classified in three clinical entities: mild, moderate and severe forms (Golan et al., 1989). Mild forms can be present in most patients undergoing ovarian stimulation but its importance is not clinically relevant. However, moderate and severe forms, especially the latter, can be life threatening. Moreover, OHSS is subdivided in an early (closely related to exogenous HCG for triggering ovulation) and a late form (related to endogenous HCG (trophoblastic) production). In IVF cycles, the general incidence of OHSS is estimated to be 0.6–14% (Hugues, 2002; Rizk, 2006; Rizk and Smitz, 1992). Nonetheless, the incidence of severe forms of OHSS in high responders (the risk population for this event) is not well defined, although data extracted from published trials suggest that may be as high as 30%. In contrast, recent studies in IVF cycles as well as in donor cycles show a preventive effect against OHSS when GnRH agonist is used as trigger agent. In these studies, patients at risk for OHSS (with polycystic ovaries or being oocyte donors with high response) were triggered with GnRH agonist showing a low incidence of OHSS, in contrast to the high rate (ranging from 16% to 30%) in the HCG arm (Acevedo et al., 2006; Babayof et al., 2006; Dhont et al., 1998; Engmann et al., 2008). The objectives of the present study were to investigate the efficacy of a single dose of GnRH agonist, followed by
JC Castillo et al. repeated boluses of HCG, on the reproductive outcome and the possible prevention of OHSS (moderate and severe forms) in patients at risk for OHSS.
Materials and methods Study design A retrospective, cohort-based, observational, sequential, non-controlled study was performed between 2002 and 2006 at the Unit of Assisted Reproduction, Hospital Clı´nico Universitario of Valencia, including a total of 192 patients.
Patients and treatment groups The main inclusion criterion was a high response to ovarian stimulation in IVF cycles: presence of 15 or more follicles 14 mm on the triggering day (Papanikolaou et al., 2006). This study was initiated in 2002 and patients were grouped as follows: group A, from 2002 to 2003 (44 patients); group B, from 2003 to 2005 (115 patients); and group C, from 2005 to 2006 (33 patients). All patients received a fixed dose of 150–200 IU recombinant FSH (Gonal-F; Serono Laboratories, Madrid, Spain) for ovarian stimulation according to age and antral follicle count (AFC). After 6 days of stimulation, FSH was adjusted according to ovarian response. Premature LH surge was prevented with 0.25 mg of a GnRH antagonist (Cetrotide; Serono International, Geneva, Switzerland) starting when the leading follicle was 14 mm of maximum diameter until and including the day of triggering ovulation. Triggering of ovulation was performed with a single dose of 1.5 mg s.c. of leuprolide acetate (Procrin; Abbott, Madrid, Spain) when two or more follicles reached a size of 18–20 mm. Luteal-phase support started the day after oocyte pick up (OPU) and was performed with a fixed low intramuscular dose of HCG (HCG-Lepori, Farma-Lepori, Barcelona, Spain) every third day (OPU+1, OPU+4 and OPU+7): three doses in total. In addition, micronized progesterone 600 mg vaginally (Progeffik; EFFIK Laboratories, Madrid, Spain) was administered. Three different doses of HCG in the luteal phase were evaluated in groups A–C during consecutive years: group A (n = 44): 1000 IU; group B (n = 115): 500 IU and group C (n = 33): 250 IU (Figure 1). At the beginning of the trial, the dose of HCG in the luteal phase was set by taking into account the regular dose used for IVF cycles: 2500 IU (Pen ˜arrubia et al., 1998). However, since these were high-responder patients, a fractionated administration of HCG (three doses of 1000 IU) was established. After 1 year of using this protocol, an internal analysis was made and the decision to reduce the dose to 500 IU was taken; the same process was followed 2 years later, to finally reach 250 IU of HCG for luteal support. Oocyte retrieval under conscious sedation was scheduled 35 h after the administration of the agonist. A 5 MHz vaginal probe (Aloka ultrasound 1400) and 18G aspiration needles (Cook Medical, USA) with aspiration pressures within 160– 180 mmHg were used. Oocytes recovered were inseminated using IVF or ICSI, depending on sperm quality. Embryo selection was made according to morphological criteria previously described (Veeck, 1990). All embryos
HCG as luteal support following agonist trigger
177
Figure 1 Treatment protocol and luteal-phase supplementation scheme. Recombinant FSH (rFSH; 150–200 IU) was used for stimulation. Gonadotrophin-releasing hormone antagonist (0.25 mg/day) started when follicles reached 14 mm. Ovulation was triggered with leuprolide acetate (1.5 mg). Luteal phase was supported with three doses of human chorionic gonadotrophin (HCG; 250–1000 IU).
were transferred using a soft catheter (Labotect; LaborTechnik, Go ¨ttingen) under ultrasound guidance (Aloka 1400 and 3.5 MHz abdominal probe). Biochemical pregnancy was defined as a positive test in urine (HCG in urine with onboard controls (Inverness Medical; Unipath, Bedford, UK) 14 days after embryo transfer. Clinical pregnancy was defined as the verification of an intrauterine gestational sac with positive fetal heart beat. OHSS was classified according to symptoms, clinical evaluation, ultrasound and laboratory findings following previously described classification criteria (Golan et al., 1989). As mild forms of OHSS are clinically irrelevant, only moderate and severe forms of the syndrome were included for statistical analysis.
concentrations at triggering day. Oestradiol concentrations were 2371 ± 1205, 2506 ± 1460 and 2834 ± 1332 pg/ml, in groups A, B and C, respectively (Table 1).
Clinical results
Hormonal assay
Oocytes and embryos A similar number of oocytes were retrieved in groups A and C (15.9 ± 6.0 and 17.8 ± 8.1, respectively). Significantly fewer oocytes were retrieved in group B (13.4 ± 7.1; P = 0.001). Significantly more embryos were obtained in group C (8.3 ± 5.6 versus group A 6.2 ± 6.2 and group B 5.4 ± 3.6; P = 0.001). Mean number of embryos transferred were 2.6 ± 0.5 in group A, 2.1 ± 0.4 in group B and 1.9 ± 0.5 in group C, showing differences in all three groups (P = 0.0001). However, this finding is related to actual trends of transferring fewer embryos.
Oestradiol concentrations were analyzed on the day of GnRH agonist triggering (enzyme immunoassay kit, Vidas, Biomerieux, France). Intra and interassay variability for oestradiol were 2.3% and 3.9%, respectively. Units were expressed in pg/ml.
Reproductive outcome The biochemical pregnancy rates were 54.5%, 51.3% and 50% in groups A, B and C, respectively, and the clinical pregnancy rates were 47.7%, 42.6% and 39.4% (Table 2).
Statistical analysis Data was processed using Statistical Package for Social Sciences version 13.0 (SPSS, USA). Values were expressed as mean ± SD and percentage where applicable. Taking into consideration that group samples are not equivalent neither in number nor in time of evaluation, inter-group analysis was made using the non-parametrical Kruskall–Wallis test and chi-squared test for proportions. A P value <0.05 was considered as statistically significant.
Results
OHSS A total of 15/192 patients (7.8%) developed either moderate or severe OHSS. Four patients (9.1%) in group A, three patients (2.6%) in group B and one patient (3.0%) in group C developed moderate OHSS, with a trend for more cases in group A. Regarding severe OHSS, two patients (4.5%) in group A, four patients (3.5%) in group B and one patient (3.0%) in group C developed severe symptoms; showing again no differences between groups (Table 3). The vast majority (6/7, 85.7%) of severe cases were late-onset OHSS and, of these, four (4/6, 66.7%) occurred in twin pregnancies (data not shown).
General data
Discussion
The number of patients included was 44, 115 and 33 in groups A, B and C respectively. All three groups were comparable regarding age, number of stimulation days and oestradiol
As far as is known, this is the largest observational study in OHSS-risk patients published to date. It assessed the efficacy and safety of low-dose HCG supplementation during
178
JC Castillo et al. Table 1
Patient age and ovarian stimulation characteristics.
No. of patients Age (years) Stimulation (days) Oestradiol on triggering day (pg/ml)
Group A (1000 IU HCG)
Group B (500 IU HCG)
Group C (250 IU HCG)
Total
44 33.5 ± 3.4 8.5 ± 1.3 2371 ± 1205
115 32.6 ± 3.5 9.3 ± 2.1 2506 ± 1460
33 32.1 ± 3.4 9.4 ± 2.7 2834 ± 1332
192 32.7 ± 3.5 9.1 ± 2.1 2531 ± 1366
Values are presented as mean ± SD. No statistically significant differences were found using the Kruskal–Wallis test. HCG, human chorionic gonadotrophin.
Table 2
Treatment cycle outcomes.
No. of patients Oocytes recovered Embryos per oocyte (%) Embryos transferred Pregnancy rate (%) Clinical pregnancy (%)
Group A (1000 IU HCG)
Group B (500 IU HCG)
Group C (250 IU HCG)
P-valuea
Total
44 15.9 ± 6.0b 39c 2.6 ± 0.5c 24/44 (54.5) 21/44 (47.7)
115 13.4 ± 7.1c 40c 2.1 ± 0.4b 59/115 (51.3) 49/115 (42.6)
33 17.8 ± 8.1b 46b 1.9 ± 0.5d 16/33 (48.5) 13/33 (39.4)
– 0.001 0.0001 0.0001 NS NS
192 14.8 ± 7.2 41.6 2.1 ± 0.5 99/192 (51.6) 83/192 (43.2)
Values are mean ± SD or number/total (%) unless otherwise stated. HCG, human chorionic gonadotrophin; NS, not statistically significant. a Values shown as mean ± SD were analyzed with Kruskal–Wallis test. Values shown as a percentage were analyzed with chisquared test. b,c,d Within rows, values with different superscript letters are significantly different.
Table 3
Incidence of ovarian hyperstimulation syndrome.
No. of patients Moderate OHSS Severe OHSS
Group A (1000 IU HCG)
Group B (500 IU HCG)
Group C (250 IU HCG)
Total
44 4 (9.1) 2 (4.5)
115 3 (2.6) 4 (3.5)
33 1 (3.0) 1 (3.0)
192 8 (4.2) 7 (3.6)
Values are number (%) unless otherwise stated. Percentages were evaluated with chi-squared test and no statistically significant differences were found. HCG, human chorionic gonadotrophin; OHSS, ovarian hyperstimulation syndrome.
the luteal phase after triggering of final oocyte maturation with a GnRH agonist. This study showed that low-dose HCG in the luteal phase after GnRH-agonist triggering is effective in normalizing the reproductive outcome. Regarding OHSS, overall frequencies of 4.2% and 3.6% of moderate and severe cases of OHSS, respectively, were detected in this group of OHSS-risk patients; these results show a clear trend towards fewer cases of OHSS of clinical relevance linked to the use of smaller doses of HCG as luteal support, and more importantly, it shows a clear relationship between severe OHSS and multiple pregnancy within this protocol. Previous publications showed a low pregnancy rate with GnRH agonist as the trigger agent (Babayof et al., 2006; Humaidan et al., 2005; Itskovitz-Eldor et al., 2000; Kol and Muchtar, 2005; Kolibianakis et al., 2005). This agent is responsible for a suboptimal luteal phase associated with low concentrations of steroidal hormones and shortening
of the luteal phase (Balasch et al., 1995; Segal and Casper, 1992); the decrease in steroid hormone concentrations may be abrupt (especially in high responders) causing endometrial instability and compromising receptivity and implantation (Sharara and McClamrock, 1999). However, this deleterious process may be overcome by the potent luteotrophic action of HCG, restoring the luteal phase and stabilizing steroid hormone concentrations. The clinical consequence is the normalization in pregnancy rates showed in this trial with an overall biochemical pregnancy rate of 51.8% and clinical pregnancy rate of 43.4%. These results constitute the rates expected for this population and support recent conclusions published by others (Humaidan et al., 2006). Recently, with the introduction of antagonists in IVF cycles some reviews showed a significant reduction in severe OHSS (Al-Inany et al., 2007). Moreover, by combining
HCG as luteal support following agonist trigger antagonist protocols with GnRH triggering, the incidence of OHSS might be reduced even further, as was demonstrated in oocyte donor cycles (Acevedo et al., 2006; Bodri et al., 2008) and IVF cycles (Humaidan et al., 2005; Kolibianakis et al., 2005). Severe OHSS, as calculated by the World Health Organization, is present in 0.2–1% of all stimulation cycles in assisted reproduction (Binder et al., 2007), but in at-risk patients (high responders) the exact frequency of this lifethreatening complication remains unknown. Moderate– severe cases in high responders triggered with standard HCG could be as high as 30% according to the scarce publications regarding this issue. In the present study, a total of 15/192 patients (7.8%) developed either moderate or severe OHSS (Table 3); these results apparently seem to defeat the purpose of using GnRH agonist for trigger, which is to reduce OHSS; however, the fact that these are high responders (mean 14.8 oocytes retrieved) must be taken into consideration; this means that the frequency of OHSS might be reduced by at least by half if the results are compared with the frequency of OHSS reported for high responders triggered with standard HCG (Babayof et al., 2006; Dhont et al., 1998; Engmann et al., 2008). In fact, in group C, severe OHSS was only 3.0% (1/33 patients) and this finding is especially relevant for this risk group. Moreover, a subgroup analysis of all severe cases in this trial showed that the vast majority (6/7, 85.7%) were late forms of the syndrome with symptoms appearing 11.8 days (mean) after ovulation triggering (data not shown) and twin pregnancy was present in 66.7% (4/6) of these cases. These findings suggest that severe forms of OHSS shown in this trial are clearly related to multiple pregnancy and not to the protocol used. It is well known that multiple pregnancy raises endogenous concentrations of circulating HCG that will act over the multiple corpora lutea produced by high responders; the clinical consequence may be occurrence of late-onset OHSS or the complication of a pre-existent early form. This knowledge raises the need to minimize multiple pregnancies especially in high responders and, because generally speaking this group is of good prognosis, elective single embryo transfer is therefore highly recommended when using HCG for luteal support after GnRH agonist for triggering. Many GnRH agonists are described as efficient inducers of final follicular maturation (buserelin, triptorelin, leuprorelin, etc.), all of them with similar characteristics. The most important in clinical practice are triptorelin and leuprorelin and no study has shown a clear advantage of one specific agonist over another. There is also a lack of studies focusing on the optimal dose of agonists as the ovulation trigger. A recent meta-analysis showed that 0.2 mg triptorelin and 1–2 mg of leuprorelin are the most common dosage employed (Griesinger et al., 2006). This study’s group employed 1.5 mg of leuprorelin due to its efficacy (Bracero et al., 2001; Parneix et al., 2001; Romeu et al., 1997) and clinical experience. The present study shows that leuprorelin is not only an efficient triggering agent but, more importantly, its use is related to lower rates of OHSS of clinical relevance. A lower incidence of OHSS is one of the main reasons to trigger final oocyte maturation with GnRH agonists (Kol and Muchtar, 2005; Revel and Casper, 2001). However, the exact mechanism of OHSS prevention after GnRH agonist trig-
179 ger remains to be fully elucidated. Prevention seems to be related to luteolysis that induces a decline in vasoactive agents responsible for inducing OHSS (e.g. vascular endothelial growth factor) (Kol, 2004). However, it remains to be fully explored if luteolysis is caused by a direct action of the agonist over the corpus luteum or an indirect action mediated by pituitary function. A direct effect is plausible; extra pituitary GnRH agonist receptors have been described (Casan ˜ et al., 2000) including granulosa cells (Latouche et al., 1989; Minaretzis et al., 1995); moreover, in-vitro studies showed that GnRH agonists are able to initiate the apoptosis cascade in granulosa cells (Takekida et al., 2000). On the other hand, an indirect effect may be related to the shorter half-life of the endogenous LH surge and subsequent pituitary suppression leading to early luteolysis (Hoff et al., 1983; Itskovitz et al., 1991). Finally, the effect caused by the agonist must be added to other causes presumably related to luteal-phase insufficiency described in IVF cycles (Macklon et al., 2006). When using GnRH agonist for triggering ovulation, Pen ˜arrubia et al. (1998) described HCG for luteal support with good results in intrauterine insemination cycles. However, for high responders in IVF cycles and due to the long life of HCG, a fractionated application was established, starting 24 h after oocyte retrieval, this time interval was set empirically due to the fact that ovarian puncture for retrieval constitutes a deleterious procedure for granulosa cells; hence HCG may be more efficient after this event. In line with this argument, recent studies by Humaidan et al. showed that HCG administered 35 h after GnRH agonist, effectively restores an optimal luteal phase and pregnancy rates in IVF cycles (Humaidan et al., 2006); this pregnancy rate normalization is also reported after using a dual trigger: a GnRH agonist plus low-dose HCG for triggering (Shapiro et al., 2008). Two other studies (Engmann et al., 2006, 2008) also confirm the low incidence of OHSS and pregnancy rate normalization using GnRH agonist for triggering, in these studies the luteal phase was aggressively supported with oestradiol and i.m. progesterone. Although the implantation rate was restored to normal in both trials, there are some potential biases comparing long GnRH-agonist protocol versus GnRH antagonist/GnRH agonist and supporting the luteal phase with individualized doses of oestradiol patches and progesterone only in the study group. Another issue to consider is that daily supplementation using i.m. progesterone until early pregnancy could be more inconvenient for many patients. The target population in the present trial were patients at risk for OHSS. It may be argued that, according to oestradiol concentrations shown in this study (2371–2834 pg/ml), this was not the case. However, a recent study showed that oestradiol concentrations at triggering day is not superior to follicular count in identifying patients at risk for developing OHSS (Papanikolaou et al., 2006). The number of oocytes collected from the population in this trial was sufficient to put them at risk for OHSS (14.8 ± 7.2) and this finding is more reliable in terms of prediction than oestradiol concentrations alone. In order to optimize this protocol, the optimal dose of HCG for luteal support must be found. In the present study, biochemical pregnancy and clinical pregnancy rates were similar between groups. Nonetheless, there was a clear
180 trend to have fewer cases of OHSS related to the use of minor doses of HCG. Although not statistically significant, this finding suggests that three doses of 1000 IU of HCG in luteal phase may be unadvisable. Three doses of 250 or 500 IU allowed the transfer of embryos in fresh cycles with similar pregnancy rates and its use deserves more research. To further diminish OHSS, a strict policy of single embryo transfer within this protocol is highly recommended. In order to draw firm conclusions, an on-going randomized trial in the study hospital is comparing these two doses in the triggering agonist protocol including an analysis of the impact of elective single embryo transfer. In conclusion, the present study has shown that the use of low doses of HCG in the luteal phase post agonist trigger is associated with normalization of pregnancy rates and also supports the concept of its effectiveness for high responders in reducing OHSS risk. Elective single embryo transfer may be an important complement to this protocol and is therefore highly recommended. More trials are needed to validate these observations and for fine tuning HCG for optimal luteal-phase supplementation in the GnRH agonist trigger protocol. Low doses of HCG as luteal support warrants further investigation. In summary, low-dose HCG in the luteal phase after GnRH agonist triggering (days OPU +1, +4 and +7) is effective in normalizing the reproductive outcome and three doses of 250 or 500 IU allow the transfer of embryos in fresh cycles with similar pregnancy rates and seem to reduce the frequency of clinically relevant OHSS in risk patients. To further diminish OHSS, a strict policy of single embryo transfer within this protocol is highly recommended.
Acknowledgements The authors thank Dr Peter Humaidan from The Fertility Clinic, Viborg Hospital (Skive), DK 7800 Skive, Denmark, for his collaboration and insightful comments.
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HCG as luteal support following agonist trigger triggered with a GnRH agonist: a pilot study. Reprod. Biomed. Online 13, 173–178. Imoedemhe, D.A., Sigue, A.B., Pacpaco, E.L., et al., 1991. Stimulation of endogenous surge of luteinizing hormone with gonadotropin-releasing hormone analog after ovarian stimulation for in-vitro fertilization. Fertil. Steril. 55, 328–332. Itskovitz, J., Boldes, R., Levron, J., et al., 1991. Induction of preovulatory luteinizing hormone surge and prevention of ovarian hyperstimulation syndrome by gonadotropin-releasing hormone agonist. Fertil. Steril. 56, 213–220. Itskovitz-Eldor, J., Kol, S., Mannaerts, B., 2000. Use of a single bolus of GnRH agonist triptorelin to trigger ovulation after GnRH antagonist ganirelix treatment in women undergoing ovarian stimulation for assisted reproduction, with special reference to the prevention of ovarian hyperstimulation syndrome: preliminary report: short communication. Hum. Reprod. 15, 1965–1968. Kol, S., 2003. Prediction of ovarian hyperstimulation syndrome: why predict if we can prevent! Hum. Reprod. 18, 1557–1558. Kol, S., 2004. Luteolysis induced by a gonadotropin-releasing hormone agonist is the key to prevention of ovarian hyperstimulation syndrome. Fertil. Steril. 81, 1–5. Kol, S., Muchtar, M., 2005. Recombinant gonadotrophin-based, ovarian hyperstimulation syndrome-free stimulation of the high responder: suggested protocol for further research. Reprod. Biomed. Online 10, 575–577. Kolibianakis, E.M., Schultze-Mosgau, A., Schroer, A., et al., 2005. A lower ongoing pregnancy rate can be expected when GnRH agonist is used for triggering final oocyte maturation instead of HCG in patients undergoing IVF with GnRH antagonists. Hum. Reprod. 20, 2887–2892. Latouche, J., Crumeyrolle-Arias, M., Jordan, D., et al., 1989. GnRH receptors in human granulosa cells: anatomical localization and characterization by autoradiographic study. Endocrinology 125, 1739–1741. Macklon, N.S., Stouffer, R.L., Giudice, L.C., et al., 2006. The science behind 25 years of ovarian stimulation for in-vitro fertilization. Endocr. Rev. 27, 170–207. Minaretzis, D., Jakubowski, M., Mortola, J.F., et al., 1995. Gonadotrophin releasing hormone receptor gene expression in human ovary and granulosa lutein cells. J. Clin. Endocrinol. Metab. 80, 430–434. Nakano, R., Mizuno, T., Kotsuji, F., et al., 1973. ‘Triggering’ of ovulation after infusion of synthetic luteinizing hormone releasing factor (LRF). Acta Obstet. Gynecol. Scand. 52, 269–272. Orvieto, R., 2005. Can we eliminate severe ovarian hyperstimulation syndrome? Hum. Reprod. 20, 320–322. Papanikolaou, E.G., Pozzobon, C., Kolibianakis, E.M., et al., 2006. Incidence and prediction of ovarian hyperstimulation syndrome in women undergoing gonadotropin-releasing hormone antagonist in-vitro fertilization cycles. Fertil. Steril. 85, 112–120.
181 Parneix, I., Emperaire, J.C., Ruffie, A., et al., 2001. Comparison of different protocols of ovulation induction, by GnRH agonists and chorionic gonadotropin. Gyne ´col. Obste ´t. Fertil. 29, 100– 105. Pen ˜arrubia, J., Balasch, J., Fa ´bregues, F., et al., 1998. Human chorionic gonadotrophin luteal support overcomes luteal phase inadequacy after gonadotrophin-releasing hormone agonistinduced ovulation in gonadotrophin-stimulated cycles. Hum. Reprod. 13, 3315–3318. Revel, A., Casper, R.F., 2001. The use of LHRH agonists to induce ovulation. Infertil. Reprod. Med. Clin. North Am. 12, 105–118. Rizk, B., 2006. Epidemiology of ovarian hyperstimulation syndrome. In: Rizk, B. (Ed.), Ovarian Hyperstimulation Syndrome. Cambridge University Press, pp. 10–42 (Chapter 2). Rizk, B., Smitz, J., 1992. Ovarian hyperstimulation syndrome after superovulation for IVF and related procedures. Hum. Reprod. 7, 320–327. Romeu, A., Monzo ´, A., Peiro ´, T., et al., 1997. Endogenous LH surge versus hCG as ovulation trigger after low-dose highly purified FSH in IUI: a comparison of 761 cycles. J. Assist. Reprod. Genet. 14, 518–524. Segal, S., Casper, R.F., 1992. Gonadotropin-releasing hormone agonist versus human chorionic gonadotropin for triggering follicular maturation in in-vitro fertilization. Fertil. Steril. 57, 1254–1258. Shapiro, B.S., Daneshmand, S.T., Garner, F.C., et al., 2008. Gonadotropin-releasing hormone agonis combined with a reduced dose of human chorionic gonadotropin for final oocyte maturation in fresh autologous cycles of in-vitro fertilization. Fertil. Steril. 90, 231–233. Sharara, F.I., McClamrock, H.D., 1999. Ratio of oestradiol concentration on the day of human chorionic gonadotrophin administration to mid-luteal oestradiol concentration is predictive of invitro fertilization outcome. Hum. Reprod. 14, 2777–2782. Takekida, S., Deguchi, J., Samoto, T., et al., 2000. Comparative analysis of the effects of gonadotropin-releasing hormone agonist on the proliferative activity, apoptosis, and steroidogenesis in cultured porcine granulosa cells at varying stages of follicular growth. Endocr. 12, 61–67. Veeck, L.L., 1990. The Morphological Assessment of Human Oocytes and Early Concepts. Handbook of the Laboratory Diagnosis and Treatment of Infertility. CRC Press, Boca Raton, FL, pp. 353– 369. Declaration: The authors report no financial or commercial conflicts of interest. Received 28 January 2009; refereed 31 March 2009; accepted 11 November 2009.
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ARTICLE
Cycles triggered with GnRH agonist: exploring low-dose HCG for luteal support ˜, F Bonilla-Musoles JC Castillo *, M Dolz, E Bienvenido, L Abad, EM Casan Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Hospital Clı´nico Universitario de Valencia, ˜ez Avenue 17, Valencia 46010, Spain Blasco Iban * Corresponding author. E-mail address: [email protected] (JC Castillo). Juan Carlos Castillo obtained his MD degree in 1998 and his speciality degree in Obstetrics and Gynaecology in 2005 at the Grau Emergency Hospital, Lima, Peru ´. In 2006, he received his Master’s degree in Human Reproduction from the Universidad de Valencia, Spain. Since then he has been working as a consultant specialist in reproductive medicine at Hospital Clı´nico Universitario de Valencia, Spain. His PhD thesis at Universidad de Valencia is focused on the prevention of ovarian hyperstimulation syndrome and will be defended in late 2009.
Abstract The aim of this study in patients at risk of ovarian hyperstimulation syndrome (OHSS) was to determine the efficacy and
safety of luteal support using human chorionic gonadotrophin (HCG) after triggering ovulation with gonadotrophin-releasing hormone (GnRH) agonist in IVF/intracytoplasmic sperm injection antagonist cycles. A total of 192 OHSS-risk patients, following a GnRH antagonist protocol (0.25 mg/day cetrorelix) during recombinant FSH stimulation, were triggered with 1.5 mg s.c. leuproreline for ovulation. A total of three boluses of HCG were used for luteal support, 1000 IU (group A, n = 44), 500 IU (group B, n = 115) or 250 IU (group C, n = 33) every third day, starting the day after oocyte retrieval. For the reproductive outcome, main variables were biochemical and clinical pregnancy rates, and for OHSS, the variables were the numbers of moderate and severe OHSS cases. Overall pregnancy rate was 51.8% and clinical pregnancy rate was 43.4%. This study observed eight cases of moderate (4.2%) and seven of severe OHSS (3.6%). Six out of the seven (85.7%) severe cases were late-onset OHSS, related to pregnancy. In conclusion, GnRH agonist single dose for triggering ovulation and low doses of HCG used as luteal-phase support seem to secure a normal pregnancy outcome without increasing the OHSS risk. RBMOnline ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: GnRH agonist, GnRH antagonist, HCG, OHSS
Introduction Third generation gonadotrophin-releasing hormone (GnRH) antagonists were re-introduced into the market for use in ovarian stimulation protocols during the 1990s (Albano et al., 1997; Diedrich et al., 1994; Macklon et al., 2006). A particular property of the GnRH antagonist is its reversible
effect, rapid action and short duration, which allows the pituitary to remain reactive to the action of a single bolus of GnRH agonist for triggering ovulation as an alternative to human chorionic gonadotrophin (HCG) (Nakano et al., 1973). Many studies show that GnRH agonists are as effective as HCG to induce an adequate final follicular maturation (Gonen et al., 1990; Humaidan et al., 2005;
1472-6483/$ - see front matter ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2009.11.018
176 Imoedemhe et al., 1991). Moreover, agonists induce the release of FSH in addition to LH (the flare-up effect), comparable to the natural cycle. However, there are some differences; in the natural cycle the surge of gonadotrophins lasts approximately 48 h, as compared with 24–36 h when GnRH agonists are used to induce ovulation (Hoff et al., 1983; Itskovitz et al., 1991). Previous randomized controlled trials (Humaidan et al., 2005; Kolibianakis et al., 2005) showed that the use of GnRH agonist for triggering ovulation was associated to negative clinical results: low implantation rate, low clinical pregnancy rate and a high rate of early pregnancy loss; all of them presumably related to luteal-phase insufficiency despite standard supplementation with progesterone and oestradiol. Other claimed explanations for these results like oocyte or embryo toxic effects of GnRH agonists seem unlikely because published studies show that implantation rates remain normal in oocyte recipients and frozen-thawed cycles post agonist trigger (Acevedo et al., 2006; Bodri et al., 2008; Griesinger et al., 2007). In this context, luteal-phase support with HCG arises as an alternative; its potent luteotrophic action is able to restore corpus luteum function and normalize luteal phase after GnRH agonist trigger. This effect has been demonstrated in intrauterine insemination cycles (Emperaire et al., 2004) and more recently a pilot study showed that a small bolus of HCG seems to secure the reproductive outcome in IVF cycles (Humaidan et al., 2006). It is well known that standard doses of HCG in the luteal phase are associated with the risk of ovarian hyperstimulation syndrome (OHSS); however, its use in high responders after a single dose of GnRH agonist for triggering ovulation, may still be an efficient and safe alternative if lower doses of HCG are given for luteal support. During cycles with GnRH antagonists, the use of a single dose of GnRH agonist for triggering ovulation has been related to low frequency and severity of OHSS (Kol, 2003; Orvieto, 2005; Revel and Casper, 2001). OHSS, is classified in three clinical entities: mild, moderate and severe forms (Golan et al., 1989). Mild forms can be present in most patients undergoing ovarian stimulation but its importance is not clinically relevant. However, moderate and severe forms, especially the latter, can be life threatening. Moreover, OHSS is subdivided in an early (closely related to exogenous HCG for triggering ovulation) and a late form (related to endogenous HCG (trophoblastic) production). In IVF cycles, the general incidence of OHSS is estimated to be 0.6–14% (Hugues, 2002; Rizk, 2006; Rizk and Smitz, 1992). Nonetheless, the incidence of severe forms of OHSS in high responders (the risk population for this event) is not well defined, although data extracted from published trials suggest that may be as high as 30%. In contrast, recent studies in IVF cycles as well as in donor cycles show a preventive effect against OHSS when GnRH agonist is used as trigger agent. In these studies, patients at risk for OHSS (with polycystic ovaries or being oocyte donors with high response) were triggered with GnRH agonist showing a low incidence of OHSS, in contrast to the high rate (ranging from 16% to 30%) in the HCG arm (Acevedo et al., 2006; Babayof et al., 2006; Dhont et al., 1998; Engmann et al., 2008). The objectives of the present study were to investigate the efficacy of a single dose of GnRH agonist, followed by
JC Castillo et al. repeated boluses of HCG, on the reproductive outcome and the possible prevention of OHSS (moderate and severe forms) in patients at risk for OHSS.
Materials and methods Study design A retrospective, cohort-based, observational, sequential, non-controlled study was performed between 2002 and 2006 at the Unit of Assisted Reproduction, Hospital Clı´nico Universitario of Valencia, including a total of 192 patients.
Patients and treatment groups The main inclusion criterion was a high response to ovarian stimulation in IVF cycles: presence of 15 or more follicles 14 mm on the triggering day (Papanikolaou et al., 2006). This study was initiated in 2002 and patients were grouped as follows: group A, from 2002 to 2003 (44 patients); group B, from 2003 to 2005 (115 patients); and group C, from 2005 to 2006 (33 patients). All patients received a fixed dose of 150–200 IU recombinant FSH (Gonal-F; Serono Laboratories, Madrid, Spain) for ovarian stimulation according to age and antral follicle count (AFC). After 6 days of stimulation, FSH was adjusted according to ovarian response. Premature LH surge was prevented with 0.25 mg of a GnRH antagonist (Cetrotide; Serono International, Geneva, Switzerland) starting when the leading follicle was 14 mm of maximum diameter until and including the day of triggering ovulation. Triggering of ovulation was performed with a single dose of 1.5 mg s.c. of leuprolide acetate (Procrin; Abbott, Madrid, Spain) when two or more follicles reached a size of 18–20 mm. Luteal-phase support started the day after oocyte pick up (OPU) and was performed with a fixed low intramuscular dose of HCG (HCG-Lepori, Farma-Lepori, Barcelona, Spain) every third day (OPU+1, OPU+4 and OPU+7): three doses in total. In addition, micronized progesterone 600 mg vaginally (Progeffik; EFFIK Laboratories, Madrid, Spain) was administered. Three different doses of HCG in the luteal phase were evaluated in groups A–C during consecutive years: group A (n = 44): 1000 IU; group B (n = 115): 500 IU and group C (n = 33): 250 IU (Figure 1). At the beginning of the trial, the dose of HCG in the luteal phase was set by taking into account the regular dose used for IVF cycles: 2500 IU (Pen ˜arrubia et al., 1998). However, since these were high-responder patients, a fractionated administration of HCG (three doses of 1000 IU) was established. After 1 year of using this protocol, an internal analysis was made and the decision to reduce the dose to 500 IU was taken; the same process was followed 2 years later, to finally reach 250 IU of HCG for luteal support. Oocyte retrieval under conscious sedation was scheduled 35 h after the administration of the agonist. A 5 MHz vaginal probe (Aloka ultrasound 1400) and 18G aspiration needles (Cook Medical, USA) with aspiration pressures within 160– 180 mmHg were used. Oocytes recovered were inseminated using IVF or ICSI, depending on sperm quality. Embryo selection was made according to morphological criteria previously described (Veeck, 1990). All embryos
HCG as luteal support following agonist trigger
177
Figure 1 Treatment protocol and luteal-phase supplementation scheme. Recombinant FSH (rFSH; 150–200 IU) was used for stimulation. Gonadotrophin-releasing hormone antagonist (0.25 mg/day) started when follicles reached 14 mm. Ovulation was triggered with leuprolide acetate (1.5 mg). Luteal phase was supported with three doses of human chorionic gonadotrophin (HCG; 250–1000 IU).
were transferred using a soft catheter (Labotect; LaborTechnik, Go ¨ttingen) under ultrasound guidance (Aloka 1400 and 3.5 MHz abdominal probe). Biochemical pregnancy was defined as a positive test in urine (HCG in urine with onboard controls (Inverness Medical; Unipath, Bedford, UK) 14 days after embryo transfer. Clinical pregnancy was defined as the verification of an intrauterine gestational sac with positive fetal heart beat. OHSS was classified according to symptoms, clinical evaluation, ultrasound and laboratory findings following previously described classification criteria (Golan et al., 1989). As mild forms of OHSS are clinically irrelevant, only moderate and severe forms of the syndrome were included for statistical analysis.
concentrations at triggering day. Oestradiol concentrations were 2371 ± 1205, 2506 ± 1460 and 2834 ± 1332 pg/ml, in groups A, B and C, respectively (Table 1).
Clinical results
Hormonal assay
Oocytes and embryos A similar number of oocytes were retrieved in groups A and C (15.9 ± 6.0 and 17.8 ± 8.1, respectively). Significantly fewer oocytes were retrieved in group B (13.4 ± 7.1; P = 0.001). Significantly more embryos were obtained in group C (8.3 ± 5.6 versus group A 6.2 ± 6.2 and group B 5.4 ± 3.6; P = 0.001). Mean number of embryos transferred were 2.6 ± 0.5 in group A, 2.1 ± 0.4 in group B and 1.9 ± 0.5 in group C, showing differences in all three groups (P = 0.0001). However, this finding is related to actual trends of transferring fewer embryos.
Oestradiol concentrations were analyzed on the day of GnRH agonist triggering (enzyme immunoassay kit, Vidas, Biomerieux, France). Intra and interassay variability for oestradiol were 2.3% and 3.9%, respectively. Units were expressed in pg/ml.
Reproductive outcome The biochemical pregnancy rates were 54.5%, 51.3% and 50% in groups A, B and C, respectively, and the clinical pregnancy rates were 47.7%, 42.6% and 39.4% (Table 2).
Statistical analysis Data was processed using Statistical Package for Social Sciences version 13.0 (SPSS, USA). Values were expressed as mean ± SD and percentage where applicable. Taking into consideration that group samples are not equivalent neither in number nor in time of evaluation, inter-group analysis was made using the non-parametrical Kruskall–Wallis test and chi-squared test for proportions. A P value <0.05 was considered as statistically significant.
Results
OHSS A total of 15/192 patients (7.8%) developed either moderate or severe OHSS. Four patients (9.1%) in group A, three patients (2.6%) in group B and one patient (3.0%) in group C developed moderate OHSS, with a trend for more cases in group A. Regarding severe OHSS, two patients (4.5%) in group A, four patients (3.5%) in group B and one patient (3.0%) in group C developed severe symptoms; showing again no differences between groups (Table 3). The vast majority (6/7, 85.7%) of severe cases were late-onset OHSS and, of these, four (4/6, 66.7%) occurred in twin pregnancies (data not shown).
General data
Discussion
The number of patients included was 44, 115 and 33 in groups A, B and C respectively. All three groups were comparable regarding age, number of stimulation days and oestradiol
As far as is known, this is the largest observational study in OHSS-risk patients published to date. It assessed the efficacy and safety of low-dose HCG supplementation during
178
JC Castillo et al. Table 1
Patient age and ovarian stimulation characteristics.
No. of patients Age (years) Stimulation (days) Oestradiol on triggering day (pg/ml)
Group A (1000 IU HCG)
Group B (500 IU HCG)
Group C (250 IU HCG)
Total
44 33.5 ± 3.4 8.5 ± 1.3 2371 ± 1205
115 32.6 ± 3.5 9.3 ± 2.1 2506 ± 1460
33 32.1 ± 3.4 9.4 ± 2.7 2834 ± 1332
192 32.7 ± 3.5 9.1 ± 2.1 2531 ± 1366
Values are presented as mean ± SD. No statistically significant differences were found using the Kruskal–Wallis test. HCG, human chorionic gonadotrophin.
Table 2
Treatment cycle outcomes.
No. of patients Oocytes recovered Embryos per oocyte (%) Embryos transferred Pregnancy rate (%) Clinical pregnancy (%)
Group A (1000 IU HCG)
Group B (500 IU HCG)
Group C (250 IU HCG)
P-valuea
Total
44 15.9 ± 6.0b 39c 2.6 ± 0.5c 24/44 (54.5) 21/44 (47.7)
115 13.4 ± 7.1c 40c 2.1 ± 0.4b 59/115 (51.3) 49/115 (42.6)
33 17.8 ± 8.1b 46b 1.9 ± 0.5d 16/33 (48.5) 13/33 (39.4)
– 0.001 0.0001 0.0001 NS NS
192 14.8 ± 7.2 41.6 2.1 ± 0.5 99/192 (51.6) 83/192 (43.2)
Values are mean ± SD or number/total (%) unless otherwise stated. HCG, human chorionic gonadotrophin; NS, not statistically significant. a Values shown as mean ± SD were analyzed with Kruskal–Wallis test. Values shown as a percentage were analyzed with chisquared test. b,c,d Within rows, values with different superscript letters are significantly different.
Table 3
Incidence of ovarian hyperstimulation syndrome.
No. of patients Moderate OHSS Severe OHSS
Group A (1000 IU HCG)
Group B (500 IU HCG)
Group C (250 IU HCG)
Total
44 4 (9.1) 2 (4.5)
115 3 (2.6) 4 (3.5)
33 1 (3.0) 1 (3.0)
192 8 (4.2) 7 (3.6)
Values are number (%) unless otherwise stated. Percentages were evaluated with chi-squared test and no statistically significant differences were found. HCG, human chorionic gonadotrophin; OHSS, ovarian hyperstimulation syndrome.
the luteal phase after triggering of final oocyte maturation with a GnRH agonist. This study showed that low-dose HCG in the luteal phase after GnRH-agonist triggering is effective in normalizing the reproductive outcome. Regarding OHSS, overall frequencies of 4.2% and 3.6% of moderate and severe cases of OHSS, respectively, were detected in this group of OHSS-risk patients; these results show a clear trend towards fewer cases of OHSS of clinical relevance linked to the use of smaller doses of HCG as luteal support, and more importantly, it shows a clear relationship between severe OHSS and multiple pregnancy within this protocol. Previous publications showed a low pregnancy rate with GnRH agonist as the trigger agent (Babayof et al., 2006; Humaidan et al., 2005; Itskovitz-Eldor et al., 2000; Kol and Muchtar, 2005; Kolibianakis et al., 2005). This agent is responsible for a suboptimal luteal phase associated with low concentrations of steroidal hormones and shortening
of the luteal phase (Balasch et al., 1995; Segal and Casper, 1992); the decrease in steroid hormone concentrations may be abrupt (especially in high responders) causing endometrial instability and compromising receptivity and implantation (Sharara and McClamrock, 1999). However, this deleterious process may be overcome by the potent luteotrophic action of HCG, restoring the luteal phase and stabilizing steroid hormone concentrations. The clinical consequence is the normalization in pregnancy rates showed in this trial with an overall biochemical pregnancy rate of 51.8% and clinical pregnancy rate of 43.4%. These results constitute the rates expected for this population and support recent conclusions published by others (Humaidan et al., 2006). Recently, with the introduction of antagonists in IVF cycles some reviews showed a significant reduction in severe OHSS (Al-Inany et al., 2007). Moreover, by combining
HCG as luteal support following agonist trigger antagonist protocols with GnRH triggering, the incidence of OHSS might be reduced even further, as was demonstrated in oocyte donor cycles (Acevedo et al., 2006; Bodri et al., 2008) and IVF cycles (Humaidan et al., 2005; Kolibianakis et al., 2005). Severe OHSS, as calculated by the World Health Organization, is present in 0.2–1% of all stimulation cycles in assisted reproduction (Binder et al., 2007), but in at-risk patients (high responders) the exact frequency of this lifethreatening complication remains unknown. Moderate– severe cases in high responders triggered with standard HCG could be as high as 30% according to the scarce publications regarding this issue. In the present study, a total of 15/192 patients (7.8%) developed either moderate or severe OHSS (Table 3); these results apparently seem to defeat the purpose of using GnRH agonist for trigger, which is to reduce OHSS; however, the fact that these are high responders (mean 14.8 oocytes retrieved) must be taken into consideration; this means that the frequency of OHSS might be reduced by at least by half if the results are compared with the frequency of OHSS reported for high responders triggered with standard HCG (Babayof et al., 2006; Dhont et al., 1998; Engmann et al., 2008). In fact, in group C, severe OHSS was only 3.0% (1/33 patients) and this finding is especially relevant for this risk group. Moreover, a subgroup analysis of all severe cases in this trial showed that the vast majority (6/7, 85.7%) were late forms of the syndrome with symptoms appearing 11.8 days (mean) after ovulation triggering (data not shown) and twin pregnancy was present in 66.7% (4/6) of these cases. These findings suggest that severe forms of OHSS shown in this trial are clearly related to multiple pregnancy and not to the protocol used. It is well known that multiple pregnancy raises endogenous concentrations of circulating HCG that will act over the multiple corpora lutea produced by high responders; the clinical consequence may be occurrence of late-onset OHSS or the complication of a pre-existent early form. This knowledge raises the need to minimize multiple pregnancies especially in high responders and, because generally speaking this group is of good prognosis, elective single embryo transfer is therefore highly recommended when using HCG for luteal support after GnRH agonist for triggering. Many GnRH agonists are described as efficient inducers of final follicular maturation (buserelin, triptorelin, leuprorelin, etc.), all of them with similar characteristics. The most important in clinical practice are triptorelin and leuprorelin and no study has shown a clear advantage of one specific agonist over another. There is also a lack of studies focusing on the optimal dose of agonists as the ovulation trigger. A recent meta-analysis showed that 0.2 mg triptorelin and 1–2 mg of leuprorelin are the most common dosage employed (Griesinger et al., 2006). This study’s group employed 1.5 mg of leuprorelin due to its efficacy (Bracero et al., 2001; Parneix et al., 2001; Romeu et al., 1997) and clinical experience. The present study shows that leuprorelin is not only an efficient triggering agent but, more importantly, its use is related to lower rates of OHSS of clinical relevance. A lower incidence of OHSS is one of the main reasons to trigger final oocyte maturation with GnRH agonists (Kol and Muchtar, 2005; Revel and Casper, 2001). However, the exact mechanism of OHSS prevention after GnRH agonist trig-
179 ger remains to be fully elucidated. Prevention seems to be related to luteolysis that induces a decline in vasoactive agents responsible for inducing OHSS (e.g. vascular endothelial growth factor) (Kol, 2004). However, it remains to be fully explored if luteolysis is caused by a direct action of the agonist over the corpus luteum or an indirect action mediated by pituitary function. A direct effect is plausible; extra pituitary GnRH agonist receptors have been described (Casan ˜ et al., 2000) including granulosa cells (Latouche et al., 1989; Minaretzis et al., 1995); moreover, in-vitro studies showed that GnRH agonists are able to initiate the apoptosis cascade in granulosa cells (Takekida et al., 2000). On the other hand, an indirect effect may be related to the shorter half-life of the endogenous LH surge and subsequent pituitary suppression leading to early luteolysis (Hoff et al., 1983; Itskovitz et al., 1991). Finally, the effect caused by the agonist must be added to other causes presumably related to luteal-phase insufficiency described in IVF cycles (Macklon et al., 2006). When using GnRH agonist for triggering ovulation, Pen ˜arrubia et al. (1998) described HCG for luteal support with good results in intrauterine insemination cycles. However, for high responders in IVF cycles and due to the long life of HCG, a fractionated application was established, starting 24 h after oocyte retrieval, this time interval was set empirically due to the fact that ovarian puncture for retrieval constitutes a deleterious procedure for granulosa cells; hence HCG may be more efficient after this event. In line with this argument, recent studies by Humaidan et al. showed that HCG administered 35 h after GnRH agonist, effectively restores an optimal luteal phase and pregnancy rates in IVF cycles (Humaidan et al., 2006); this pregnancy rate normalization is also reported after using a dual trigger: a GnRH agonist plus low-dose HCG for triggering (Shapiro et al., 2008). Two other studies (Engmann et al., 2006, 2008) also confirm the low incidence of OHSS and pregnancy rate normalization using GnRH agonist for triggering, in these studies the luteal phase was aggressively supported with oestradiol and i.m. progesterone. Although the implantation rate was restored to normal in both trials, there are some potential biases comparing long GnRH-agonist protocol versus GnRH antagonist/GnRH agonist and supporting the luteal phase with individualized doses of oestradiol patches and progesterone only in the study group. Another issue to consider is that daily supplementation using i.m. progesterone until early pregnancy could be more inconvenient for many patients. The target population in the present trial were patients at risk for OHSS. It may be argued that, according to oestradiol concentrations shown in this study (2371–2834 pg/ml), this was not the case. However, a recent study showed that oestradiol concentrations at triggering day is not superior to follicular count in identifying patients at risk for developing OHSS (Papanikolaou et al., 2006). The number of oocytes collected from the population in this trial was sufficient to put them at risk for OHSS (14.8 ± 7.2) and this finding is more reliable in terms of prediction than oestradiol concentrations alone. In order to optimize this protocol, the optimal dose of HCG for luteal support must be found. In the present study, biochemical pregnancy and clinical pregnancy rates were similar between groups. Nonetheless, there was a clear
180 trend to have fewer cases of OHSS related to the use of minor doses of HCG. Although not statistically significant, this finding suggests that three doses of 1000 IU of HCG in luteal phase may be unadvisable. Three doses of 250 or 500 IU allowed the transfer of embryos in fresh cycles with similar pregnancy rates and its use deserves more research. To further diminish OHSS, a strict policy of single embryo transfer within this protocol is highly recommended. In order to draw firm conclusions, an on-going randomized trial in the study hospital is comparing these two doses in the triggering agonist protocol including an analysis of the impact of elective single embryo transfer. In conclusion, the present study has shown that the use of low doses of HCG in the luteal phase post agonist trigger is associated with normalization of pregnancy rates and also supports the concept of its effectiveness for high responders in reducing OHSS risk. Elective single embryo transfer may be an important complement to this protocol and is therefore highly recommended. More trials are needed to validate these observations and for fine tuning HCG for optimal luteal-phase supplementation in the GnRH agonist trigger protocol. Low doses of HCG as luteal support warrants further investigation. In summary, low-dose HCG in the luteal phase after GnRH agonist triggering (days OPU +1, +4 and +7) is effective in normalizing the reproductive outcome and three doses of 250 or 500 IU allow the transfer of embryos in fresh cycles with similar pregnancy rates and seem to reduce the frequency of clinically relevant OHSS in risk patients. To further diminish OHSS, a strict policy of single embryo transfer within this protocol is highly recommended.
Acknowledgements The authors thank Dr Peter Humaidan from The Fertility Clinic, Viborg Hospital (Skive), DK 7800 Skive, Denmark, for his collaboration and insightful comments.
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