A randomized controlled trial of the GnRH antagonist ganirelix in Chinese normal responders: high efficacy and pregnancy rates

Gonadotropin-releasing hormone (GnRH) antagonists for controlled ovarian stimulation (COS) were only recently introduced into China. The efficacy and safety of the GnRH antagonist ganirelix was assessed in a multicenter, controlled, open-label study, in which Chinese women were randomized to either ganirelix (n = 113) or a long GnRH agonist protocol of triptorelin (n = 120). The primary end point was the amount of recombinant follicle-stimulating hormone (rFSH) required to meet the human chorionic gonadotropin criterion (three follicles ≥17 mm). The amount of rFSH needed was significantly lower for ganirelix (1272 IU) vs. triptorelin (1416 IU; P< 0.001). Ongoing pregnancy rates per started cycle were 39.8% (ganirelix) and 39.2% (triptorelin). Although both treatments were well tolerated, cancellation due to risk of ovarian hyperstimulation syndrome (OHSS) was less frequent with ganirelix (1.8%) than triptorelin (7.5%) (P = 0.06). Less rFSH was needed in the ganirelix protocol than the long GnRH agonist protocol, with fewer reported cases of OHSS and similar pregnancy rates.     

冻融胚胎移植中三种不同内膜准备方法的妊娠结局比较

目的：比较三种不同内膜准备方法中复苏胚胎移植的妊娠结局。方法：对因女方输卵管因素不孕的248个冻融囊胚移植周期的临床资料进行回顾性分析。将内膜准备方法分为自然周期组、全激素替代周期组和半激素替代周期组三种,分别对各组的着床率、临床妊娠率、生化妊娠率和早期流产率进行分析比较。结果：自然周期组的子宫内膜厚度显著高于全激素周期组和半激素替代周期组（P〈0．05）,差异有统计学意义。而这三组病人在年龄、不孕年限和移植胚胎数目上则均无明显差异（P〉0．05）。另外,三种内膜准备方法的着床率、临床妊娠率、早期流产率及生化妊娠流产率亦无显著性差异（P〉O．05）。结论：冻融胚胎移植中,对于排卵正常患者我们建议采用自然周期法更为经济、方便。而对于月经周期不正常,排卵障碍的病人应依实际情况采用全激素周期或半激素周期方案。     

Comparative response of rams and bulls to long-term treatment with gonadotropin-releasing hormone analogs

The objective was to compare the relative response between rams and bulls in characteristics of LH, FSH and testosterone (T) secretion, during and after long-term treatment with GnRH analogs. Animals were treated with GnRH agonist, GnRH antagonist, or vehicle (Control) for 28 days. Serial blood samples were collected on day 21 of treatment, and at several intervals after treatment. Injections of natural sequence GnRH were used to evaluate the capacity of the pituitary to release gonadotropins during and after treatment. Treatment with GnRH agonist increased basal LH and T concentrations in both rams and bulls, with a greater relative increase in bulls. Endogenous LH pulses and LH release after administration of GnRH were suppressed during treatment with GnRH agonist. Treatment with GnRH antagonist decreased mean hormone concentrations, LH and T pulse frequency, and the release of LH and T after exogenous GnRH, with greater relative effects in bulls. Rams previously treated with antagonist had a greater release of LH after administration of GnRH compared with control rams, while rams previously treated with agonist showed a reduced LH response. Bulls previously treated with agonist had reduced FSH concentrations and LH pulse amplitudes compared with control bulls while bulls previously treated with antagonist had greater T concentrations and pulse frequency. The present study was the first direct comparison between domestic species of the response in males to treatment with GnRH analogs. The findings demonstrated that differences do occur between rams and bulls in LH, FSH and testosterone secretion during and after treatment. Also, the consequences of treatment with either GnRH analog can persist for a considerable time after discontinuation of treatment.     

New protocol for commencing the GnRH antagonist in assisted conception treatment cycles: elimination of the premature LH surge with similar pregnancy rates

GnRH antagonists have been used with increasing frequency in assisted reproduction treatments over the past few years and have been associated with quicker and more profound LH suppression and shorter treatment cycles than conventional GnRH agonists. Usually, these are commenced on day 6 of FSH stimulation without allowing for patient variation in response to treatment. The study was aimed at individualising this protocol to the patients' ovarian response. The control group included 215 treatment cycles where the GnRH antagonist was commenced on day 6 of FSH stimulation. A new individualised protocol was formulated, applied to practice and 172 treatment cycles following that were analysed. The study group had no premature LH surges (LH > 10 iu x mL(-1)) compared to the control group who had a rate of 4.1%. There was also a higher fertilisation and clinical pregnancy rate in the study group (P = 0.06). It is concluded that the new individualised GnRH antagonist protocol eliminates premature LH surges in assisted conception treatment cycles and may improve clinical pregnancy rates compared to the conventional protocol of "day 6 commencement".     

Effects of chronic treatment with a GnRH agonist on oestrous behaviour and on the secretion of LH and progesterone in the ewe

A study was carried out to investigate a novel approach to oestrus synchronization in the ewe by treatment with a gonadotrophin releasing hormone (GnRH) agonist. Groups of ewes were initially treated on Day 2, 10 or 14 of the oestrous cycle with 10 mug GnRH analogue (D-Ser(Bu(t)) 6 des Gly GnRH ethylamide) per ewe per day for 14 days. Behavioural oestrus was inhibited during GnRH agonist treatment and recurred from 8 to 38 days after the treatment in an unsynchronized manner. Luteal activity during treatment was not impaired but reduced progesterone concentrations occurred in cycles after the treatment. The rhythm of ovarian function, generally characterized by prolonged follicular development, was impaired. During the treatment and subsequent recovery period, integrity of pituitary function was examined by measuring luteinizing hormone (LH) after GnRH agonist was injected, and after stimulation test doses of 150 ng natural GnRH were administered. During treatment there was, with time, a decline in pituitary response to the agonist which suggested that pituitary release of LH was exhausted. After the 14-day treatment the stimulation test with GnRH revealed a gradual return to normal responsiveness although this was not complete three weeks after the treatment when compared to control ewes. This lowered pituitary activity could cause the impaired ovarian function.     

Agonist-induced regulation of pituitary receptors for gonadotropin-releasing hormone. Dissociation of receptor recruitment from hormone release in cultured gonadotrophs

The regulation of receptors for gonadotropin-releasing hormone (GnRH) by the homologous decapeptide ligand was analyzed in cultured rat anterior pituitary cells. Assay of GnRH receptors in both intact and disrupted cells showed that GnRH binding to gonadotrophs was rapidly followed by dose-dependent loss of sites that was maximal within 1 h. This early loss of GnRH receptors was not dependent on protein synthesis, and was attributable to ligand-induced processing of the peptide binding sites. No loss of GnRH sites was observed after receptor occupancy by a GnRH antagonist, or after target cell activation by exposure to a depolarizing concentration of KCl to stimulate luteinizing hormone release. After their initial down-regulation, GnRH receptors returned to normal and subsequently increased in concentration after 6 h of incubation. The delayed phase of receptor up-regulation was prevented by treatment with cycloheximide or actinomycin D and was calcium-dependent, being induced by 50 mM KCl and by low concentrations of the calcium ionophore, A23187. Conversely, calcium antagonists such as verapamil and MgCl2 impaired the agonist-induced increase of GnRH receptor sites. These findings have demonstrated that pituitary GnRH receptors undergo two distinct phases of regulation after interaction with the homologous ligand. The initial phase of agonist-dependent receptor loss is followed by a postsecretory phase of receptor recruitment that is dependent on protein synthesis. The expression of GnRH receptors can be completely dissociated from gonadotropin secretion, indicating that fusion of luteinizing hormone secretory granules with the plasma membrane is not a major pathway for transport of GnRH receptors to the cell surface in cultured gonadotrophs. Such changes in cell surface GnRH receptors during activation by the peptide agonist are relevant to the alterations in gonadotroph sensitivity that occur in vivo during physiological regulation of the pituitary gland by GnRH.     

Antagonistic and agonistic GnRH analogue treatment of precocious puberty: tracking gonadotropin concentrations in urine

BACKGROUND: The pharmacodynamics of gonadotropin-releasing hormone (GnRH) agonists includes an initial 'flare-up' of the pituitary-gonadal axis, followed by reduced luteinizing hormone (LH) secretion. The question is if combining a short-acting antagonist with a long-acting agonist can diminish gonadotropin flare-up. METHODS: To achieve quick downregulation in patients with recently diagnosed central precocious puberty (CPP, 7 patients) or short stature with short predicted final height (3 patients), we combined the GnRH antagonist cetrorelix (3 subcutaneous injections every 72 h) at the beginning of GnRH agonist treatment (leuprorelin or triptorelin) in 6 patients and compared the effect to 4 patients treated solely with GnRH agonist. To monitor effects, we measured LH and FSH concentrations in urine collected from initial morning urination during the first month of treatment. RESULTS: In both treatment groups, gonadotropin flare-up could be detected in urine levels increased due to the flare-up phenomenon which was of short duration (<5 days) in the majority (5 of 6) of combined-treated patients and in the minority (1 of 4) of patients treated by agonist alone. During the first 10 days of treatment, mean LH concentration measured in urine was significantly lower in 4 CPP patients treated by the combined therapy compared to 3 CPP patients treated by the agonist only (mean LH combined therapy: 10.4 +/- 2.8 vs. 20.1 +/- 11.0 mU/ml in the agonist-only group, mean +/- SEM, p < 0.05). Significant correlations between stimulated serum LH in GnRH test prior to treatment and maximum urine LH after initiating GnRH analogue treatment (r = 0.547, p = 0.043), as well as basal serum LH and basal urine LH (r = 0.685, p = 0.014) were found. CONCLUSION: Combined GnRH agonist and antagonist treatment led to rapid gonadotropin suppression. Also, urine measurements of LH and FSH seemed suitable for monitoring gonadotropin-inhibiting or -stimulating properties of GnRH analogues in individual patients. However, a controlled trial of a larger patient cohort is required to decide which treatment is the most effective.     

Characterization of Leydig cell gonadotropin-releasing hormone binding sites utilizing radiolabeled agonist and antagonist

Gonadotropin-releasing hormone (GnRH) binding sites in intact Leydig cells and in membrane preparations were investigated using 125I-labeled GnRH agonist and antagonist. Binding was saturable and involved a single class of high affinity sites. Intact Leydig cells and a membrane preparation had a higher affinity for GnRH agonist (Kd 3.0 +/- 1.7 X 10(-10) M) than for GnRH antagonist (Kd 10.0 +/- 1.8 X 10(-10) M). With anterior pituitary membranes the Kd was 2.8 +/- 0.7 X 10(-10) M for the agonist and 2.4 +/- 1.4 X 10(-10) M for the antagonist. The Kd for GnRH was similar for Leydig cells and the anterior pituitary. Chymotrypsin and trypsin digestion decreased receptor binding, but neuraminidase increased Leydig cell binding in contrast to the decrease in binding observed with pituitary receptors. The results suggest that the Leydig cell GnRH binding sites may differ from the pituitary receptor which may be related to structural differences in GnRH-like peptides recently described in extracts of rat testis.     

Receptor-binding properties of gonadotropin-releasing hormone derivatives. Prolonged receptor occupancy and cell-surface localization of a potent antagonist analog

The receptor-binding properties and in vitro biological effects of a highly active gonadotropin-releasing hormone (GnRH) antagonist, [N-acetyl-D-p-chloro-Phe1,2D-Trp3,D-Lys6,D-Ala10]GnRH, were compared with those of the GnRH superagonist analog, [D-Ala6] des-Gly10-GnRH-N-ethylamide. In rat pituitary particles and isolated pituitary cells, the 125I-labeled GnRH antagonist showed saturable high-affinity binding (Ka v 8.4 +/- 1.4 X 10(9) M-1) to the same receptor sites which bound the GnRH agonist. The rate of dissociation of the receptor-bound antagonist from pituitary particles and cells was extremely slow in comparison with that of the agonist ligand. Also, dissociation of the antagonist analog was incomplete, with a residual fraction of tightly bound ligand that was proportional to the duration of preincubation. The [D-Lys6]GnRH antagonist prevented GnRH-induced luteinizing hormone release during static incubation and superfusion of cultured pituitary cells, but in contrast to the agonist did not cause desensitization of the gonadotroph. Although the antagonist caused a prolonged reduction in available GnRH receptor sites, this was attributable to persistent occupancy by the slowly dissociating ligand rather than to receptor loss. Autoradiographic analysis of [D-Lys6]GnRH-antagonist uptake by cultured pituitary cells revealed that the peptide remained bound at the cell membrane for up to 2 h, in contrast with the rapid endocytosis of GnRH agonists. The slow dissociation of receptor-bound antagonist was consistent with its ability to cause sustained blockade of GnRH actions, and its prolonged cell-surface location suggests that receptor activation is necessary to initiate the rapid internalization of hormone-receptor complexes that is a feature of the agonist-stimulated gonadotroph.     

Evaluation of the possible direct effects of gonadotrophin-releasing hormone analogues on the monkey (Macaca mulatta) testis

In Exp. 1, the effect of treatment with a GnRH agonist on basal concentrations of serum testosterone and peak values of serum testosterone after administration of hCG was determined. One group of adult male monkeys was treated with a low dose (5-10 micrograms/day) and a second group with a high dose (25 micrograms/day) of a GnRH agonist for 44 weeks. Basal and peak testosterone concentrations were both significantly reduced by GnRH agonist treatment in all groups compared to untreated control animals, but the % rise in serum testosterone above basal values in response to hCG administration was unchanged by agonist treatment. In Exp. 2, the GnRH agonist (100 or 400 ng) or a GnRH antagonist (4 micrograms) was infused into the testicular arteries of adult monkeys. The agonist did not alter testosterone concentrations in the testicular vein or testosterone and LH values in the femoral vein. In Exp. 3, testicular interstitial cells from monkeys were incubated with three concentrations (10(-9), 10(-7) and 10(-5)M) of the GnRH agonist or a GnRH antagonist with and without hCG. After 24 h, neither basal nor hCG-stimulated testosterone production was affected by the presence of the GnRH agonist or antagonist. The results from all 3 experiments clearly suggest that GnRH agonist treatment does not directly alter steroid production by the monkey testis.     

Comparisons of GnRH Antagonist versus GnRH Agonist Protocol in Supposed Normal Ovarian Responders Undergoing IVF: A Systematic Review and Meta-Analysis

Objective

To evaluate the effectiveness and safety of GnRH antagonist and GnRH agonist in supposed normal ovarian responders undergoing IVF.

Methods

Data from 6 databases were retrieved for this study. The RCTs of GnRH agonist and GnRH antagonist use during IVF-EF therapy for patients with supposed normal ovarian response were included. A meta-analysis was performed with Revman 5.1software.

Results

Twenty-three RCTs met the inclusion criteria. The number of stimulation days (mean difference (MD): −0.66, 95% confidence interval (CI): −1.04∼−0.27), Gn amount (MD: −2.92, 95% CI: −5.0∼−0.85), E2 values on the day of HCG (MD: −330.39, 95% CI: −510.51∼−150.26), Number of oocytes retrieved (MD: −1.33, 95% CI: −2.02∼−0.64), clinical pregnancy rate (odds ratio (OR): 0.87, 95% CI: 0.75−1.0), and ovarian hyperstimulation syndrome (OHSS) incidence (OR: 0.59, 95% CI: 0.42∼0.82) were significantly lower in GnRH antagonist protocol than GnRH agonist protocol. However, the endometrial thickness on the day of HCG (MD: −0.04, 95% CI: −0.23∼0.14), the ongoing pregnancy rate (OR: 0.87, 95% CI: 0.74∼1.03), live birth rate (OR: 0.89, 95% CI: 0.64∼1.24), miscarriage rate (OR: 1.17, 95% CI: 0.85∼1.61), and cycle cancellation rate (OR: 1.11, 95% CI: 0.90∼1.37) did not significantly differ between the 2 groups.

Conclusions

During IVF treatment for patients with supposed normal responses, the incidence of OHSS were significantly lower, whereas the ongoing pregnancy and live birth rates were similar in the GnRH antagonist compared with the standard long GnRH agonist protocols.     

Extra-pituitary action of gonadotropin releasing hormone: possible application

Chronic administration of a potent gonadotropin releasing hormone inhibits ovulation in women. The suppression of gonadal function during long term treatment with the GnRH analogues is ascribable to inhibition of gonadotropin secretion caused by the down regulatory action of the decapeptide at the pituitary level. Reduced progesterone production with premature onset of menstruation has been observed in women injected with the agonist during the midluteal phase. The decapeptide however, has no effect onin vitro human ovarian steroidogenesis. Specific receptors for GnRH have been located on rodent ovarian cells, but corpora lutea of rhesus monkey and human ovaries seem to lack these receptors. The luteolytic effect in women thus appears to be central in origin and not a direct effect on the corpus luteum. Recently, a superactive agonist of GnRH given around the peri-implantation period has been shown to terminate pregnancy in baboons. Monoclonal antibodies against GnRH administered during the same period in a fertile cycle also abrogated pregnancy in these animals. Using immuno-enzymatic techniques GnRH has been localized on the placenta. GnRH also exerts a stimulatory effect on hCG production by the placental villi maintained in culture. Addition of anti-luteinizing hormone releasing hormone antibodies blocks this effect completely. It seems that placenta is the only other tissue besides the pituitary where GnRH has probably a regulatory role in the human female.     

Direct ovarian effects of a GnRH agonist in hypophysectomized immature rats: inhibition of theca-interstitial luteinizing hormone receptor mRNA expression and induction of interstitial cell differentiation

The effect of a gonadotropin-releasing hormone (GnRH) agonist on luteinizing hormone (LH) receptor mRNA expression was examined histologically in the ovaries of immature hypophysectomized (HPX) rats by in situ hybridization. In the ovaries of HPX rats treated with diethylstilbestrol (DES) and pregnant mare serum gonadotropin (PMSG), LH receptor mRNA was expressed in the granulosa cells of mature follicles as well as the theca-interstitial cells. In DES-primed ovaries of rats treated with both GnRH agonist plus PMSG, many follicles were luteinized without ovulation, and the signal of LH receptor mRNA disappeared completely in the theca-interstitial cells as well as the luteinized cells, but remained in the granulosa cells of unaffected mature follicles. The complete suppression of the theca-interstitial LH receptor expression by GnRH agonist was also observed in HPX rats that received no other treatment. On the other hand, the coadministration of a GnRH antagonist with PMSG resulted in the hyperstimulation of follicular growth, accompanied by very strong expression of LH receptor mRNA in the granulosa cells as well as the thecainterstitial cells. In addition, morphological changes in the ovarian interstitial cells were also induced by the administration of GnRH agonist in HPX rats: loose connective tissue decreased and the interstitial cell mass markedly increased. The increase of the interstitial cells became more prominent when rats were treated with GnRH agonist and testosterone simultaneously. These results suggest that GnRH may be an important factor for modulating the interstitial cell function and differentiation in the rat ovary.     

Dissecting autocrine effects on pulsatile release of gonadotropin-releasing hormone in cultured rat hypothalamic tissue

The control of reproductive function is manifested centrally through the control of hypothalamic release of gonadotropin-releasing hormone (GnRH) in episodic events or pulses. For GnRH release to occur in pulses, GnRH neurons must coordinate release events periodically to elicit a bolus of GnRH. We used a perifusion culture system to examine the release of GnRH from both intact hypothalami and enzymatically dispersed hypothalamic cells after challenge with GnRH analogs to evaluate the role of anatomical neuronal connections on autocrine/paracrine signals by GnRH on GnRH neurons. The potent GnRH agonist des-Gly(10)-D-Ala(6)-GnRH N-ethylamide, potent GnRH antagonists D-Phe(2)-D-Ala(6)-GnRH and D-Phe(2,6)-Pro(3)-GnRH or vehicle were infused, whereas GnRH release from hypothalamic tissue and cells were measured. PULSAR analysis of GnRH release profiles was conducted to evaluate parameters of pulsatile GnRH release. Infusion of the GnRH agonist resulted in a decrease in mean GnRH (P < 0.001), pulse nadir (P < 0.01), and pulse frequency (P < 0.05) but no effect on pulse amplitude. Infusion of GnRH antagonists resulted in an increase in mean GnRH (P < 0.001), pulse nadir (P < 0.05), and pulse frequency (P < 0.05) and in GnRH pulse amplitude only in dispersed cells (P < 0.05). These results are consistent with the hypothesis that GnRH inhibits endogenous GnRH release by an ultrashort-loop feedback mechanism and that treatment of hypothalamic tissue or cells with GnRH agonist inhibits ultrashort-loop feedback, whereas treatment with antagonists disrupts normal feedback to GnRH neurons and elicits an increased GnRH signal.     

Early-cleavage is a reliable predictor for embryo implantation in the GnRH agonist protocols but not in the GnRH antagonist protocols

Background  

To test if early-cleavage was a strong predictor of pregnancy in patients receiving either a GnRH agonist long protocol or a GnRH antagonist protocol for in-vitro fertilization treatment (IVF) and intracytoplasmic sperm injection (ICSI).     

GnRH analogues--agonists and antagonists

GnRH analogues have achieved widespread clinical use for the control of reproduction in animals. Over 2000 analogues of GnRH have been developed and tested over the last 30 years. Paradoxical anti-fertility effects are seen when the more potent agonists are delivered continuously to animals. The evaluation of agonist potency depends largely on the model used and wide varying potencies are reported for the same agonist. The design of analogues has centered on improving the receptor-binding and subsequent activation for agonists. Antagonists have been produced with strong receptor binding but without activation. Deslorelin is classified as a superagonist, with a potency perhaps 100 times that of GnRH. The interactions between agonist potency, dose and duration of treatment largely determine whether pro- or anti-fertility effects are induced. Due to the peptide nature of the synthetic analogues oral administration and potential gastrointestinal enzymatic degradation poor bioavailability results necessitating a parenteral delivery system. Some GnRH antagonists have been associated with significant histamine release, inhibiting their widespread use. More recently, antagonists have been developed that avoid this side effect without compromising potency. However the GnRH antagonist development has lagged behind that of the agonists, in part related to their high cost of production. In conclusion, GnRH agonists have achieved widespread clinical use in animals for controlling reproduction in either pro- or anti-fertility roles, yet antagonist development has been slower.     

Two mutations in extracellular loop 2 of the human GnRH receptor convert an antagonist to an agonist

GnRH regulates the reproductive system through cognate G protein-coupled receptors in vertebrates. Certain GnRH analogs that are antagonists at mammalian receptors behave as agonists at Xenopus laevis and chicken receptors. This phenomenon provides the opportunity to elucidate interactions and the mechanism underlying receptor activation. A D-Lys(iPr) in position 6 of the mammalian GnRH receptor antagonist is required for this agonist activity (inositol phosphate production) in the chicken and X. laevis GnRH receptors. Chimeric receptors, in which extracellular loop domains of the human GnRH receptor were substituted with the equivalent domains of the X. laevis GnRH receptor, identified extracellular loop 2 as the determinant for agonist activity of one of the mammalian antagonists: antagonist 135-18. Site-directed mutagenesis of nine nonconserved residues in the C-terminal domain of extracellular loop 2 of the human GnRH receptor showed that a minimum of two mutations (Val(5.24(197))Ala and Trp(5.32(205))His) is needed in this region for agonist activity of antagonist 135-18. Agonist activity of antagonist 135-18 was markedly decreased by low pH (<7.0) compared with GnRH agonists. These findings indicate that D-Lys(iPr)(6) forms a charge-supported hydrogen bond with His(5.32(205)) to stabilize the receptor in the active conformation. This discovery highlights the importance of EL-2 in ligand binding and receptor activation in G protein-coupled receptors.     

Preparation and characterization of photoreactive derivatives of GnRH. Persistent activation of function by photoaffinity labeling

A photoreactive derivative of the highly potent gonadotropin releasing hormone (GnRH) agonist, D-Lys6-GnRH(1-9)-ethylamide, was prepared by selective modification of the epsilon-amino group with 2-nitro-4-azidophenyl sulfenyl chloride (2,4-NAPS C1). The modified peptide [D-Lys(NAPS)]6-GnRH-(1-9)-ethylamide was found to be a full agonist of LH release from rat pituitary cells with a relative potency 23 compared to GnRH. Covalent attachment of the photoreactive analog to rat pituitary cells resulted in prolonged activation of LH secretion which could not be inhibited by a potent GnRH antagonist. Persistent stimulation of pituitary gonadotrophs caused by covalently bound hormone led to desensitization of the LH releasing mechanism.