Embryo implantation and GnRH antagonists

Ovarian actions of GnRH antagonists

O. Ortmann,1, J.M. Weiss and K. Diedrich

Department of Obstetrics and Gynecology, Medical University Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany


    Abstract
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 Abstract
 Introduction
 GnRH receptors in human...
 Actions of GnRH agonists...
 Actions of GnRH antagonists...
 References
 
The gonadotrophin-releasing hormone (GnRH) antagonists, cetrorelix and ganirelix, have both been approved for ovarian stimulation to prevent a premature LH surge. Since GnRH receptors and their gene expression have been detected in human ovary, concern has risen over whether GnRH antagonists might affect ovarian function. Three large trials which compared GnRH agonists (used in the standard protocol worldwide), with the new antagonist treatment found no significant differences concerning the most important goals, e.g. pregnancy rate, fertilization and quality of oocytes. However, the concentration of oestradiol, and the pregnancy and implantation rates were lower in GnRH antagonist-treated patients. These findings again fuelled the debate about the possible extrapituitary effects of GnRH antagonists. Here, we review the conflicting data in the literature on the ovarian effects of GnRH antagonists and discuss our own results. In our view, it is unlikely that GnRH antagonists have a relevant impact on ovarian steroidogenesis and function; however, GnRH antagonists may exert other effects on the ovary.

Key words: embryo implantation/GnRH antagonists/ovarian effects/ovarian stimulation protocols/steroidogenesis


    Introduction
 Top
 Abstract
 Introduction
 GnRH receptors in human...
 Actions of GnRH agonists...
 Actions of GnRH antagonists...
 References
 
Gonadotrophin-releasing hormone (GnRH) plays a pivotal role in the control of female reproduction and is secreted by hypothalamic neurons in a pulsatile way. It binds to specific receptors on pituitary gonadotrophs, which is followed by the secretion of the gonadotrophins, LH and FSH, which regulate steroidogenesis and gametogenesis in the ovary (Knobil, 1980Go).

GnRH analogues are able to suppress gonadotrophin release and, subsequently, gonadal function. This is the basis for their clinical application. Several agonistic or antagonistic GnRH analogues have been developed for this purpose.

Over the last two decades, GnRH agonists have been used in ovarian stimulation protocols in assisted reproductive techniques (ART) in combination with gonadotrophins to prevent a premature LH surge. GnRH agonists induce an initial rise of gonadotrophins (flare-up) before they achieve suppression through desensitization. Recently, GnRH antagonists have been introduced into ovarian stimulation protocols.

The concept of suppressing gonadotrophins by competitive receptor blockage rather than through desensitization with its inevitable flare-up is compelling. The first antagonist was synthesized >20 years ago (Karten and Rivier, 1986Go). Its clinical application was hampered by a high histaminergic potential. Recently, new substances were developed which lacked the histaminergic side-effects (Schally et al., 1989Go; Reissmann et al., 1995Go). These antagonists lead to a rapid inhibition of LH release due to competitive binding to pituitary GnRH receptors. Two of these compounds are cetrorelix and ganirelix. Cetrorelix has recently been introduced into ovarian stimulation protocols to prevent premature LH surges. Ganirelix and cetrorelix proved their safety and effectiveness in clinical trials, showing comparable results with the agonists (Diedrich et al., 1994Go; Felberbaum et al., 1996Go; Ganirelix Dose Finding Study Group, 1998Go; Weiss et al., 1999Go; Albano et al., 2000Go; Borm and Mannaerts, 2000Go; Olivennes et al., 2000Go). No significant differences in fertilization rates, embryo quality and oocytes were found between agonist and antagonist treatment. One advantage of the antagonists seems to be a lower incidence of ovarian hyperstimulation syndrome (OHSS), a serious side-effect of ovarian stimulation treatment (De Jong et al., 1998Go). There is no evident mechanism that explains this finding, but it is consistent in the studies performed so far. In a novel study (Albano et al., 2000Go), the effects of cetrorelix and buserelin in patients treated in ovarian stimulation protocols were compared. Significantly lower oestradiol serum concentrations on the day of human chorionic gonadotrophin (HCG) administration were found in patients treated with GnRH antagonists, which could be due to the lower number of small follicles observed on the day of HCG administration. These observations and the shorter human menopausal gonadotrophin (HMG) treatment period could contribute to the rationale of the lower incidence of OHSS in GnRH antagonist-treated patients. Additional findings indicated that the number of HMG ampoules administered and the duration of ovarian stimulation were lower in GnRH antagonist-treated patients (Olivennes et al., 2000Go). In a recent large European multicentre study, the impact of ganirelix on oocyte numbers, mean duration of stimulation, number of good quality embryos and embryos transferred compared with a long protocol using buserelin was less striking, although again the implantation (15.7 versus 21.8%) and pregnancy rates (20.3 versus 25.7%) in the ganirelix group were lower (Borm and Mannaerts, 2000Go).

These findings have generated the hypothesis that GnRH antagonists might have direct ovarian actions. Although there are conflicting data, GnRH receptors have been demonstrated in the human ovary (see below). Even if the concentration of native GnRH is too low in the peripheral circulation to interfere with extrapituitary GnRH receptors, it is possible that the application of 0.25 mg GnRH antagonist daily is sufficient to induce effects on ovarian GnRH receptors. In addition, the actions of GnRH and GnRH-like peptides produced directly by the ovary might be influenced by GnRH antagonists (Aten et al., 1986Go; Rivier et al., 1996Go). It is still unclear whether ovarian GnRH receptors are functional. Conflicting data exist on the actions of GnRH antagonist on steroidogenesis in human granulosa lutein cells.


    GnRH receptors in human ovary
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 Abstract
 Introduction
 GnRH receptors in human...
 Actions of GnRH agonists...
 Actions of GnRH antagonists...
 References
 
The prerequisite for GnRH action in the human ovary is the presence of functional GnRH receptors. GnRH receptors and their gene expression have been described in a variety of benign and malignant extrapituitary tissues. The GnRH receptor contains seven membrane-spanning domains which is characteristic of G protein-coupled receptors. They can be regulated by GnRH itself or other hormones. GnRH binding to its pituitary receptor activates the Gq/G11 subfamily of G-proteins. This leads to an increase in phospholipase C (PLC) activity which results in phosphoinositide breakdown with the generation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 releases Ca2+ from intracellular stores. DAG activates protein kinase C (PKC). These events lead to synthesis and secretion of gonadotrophins (Stojilkovic and Catt, 1995Go).

The rodent ovary contains GnRH receptors. Most of the studies were performed in rat ovaries where high affinity binding sites for GnRH have been demonstrated (Clayton et al., 1979Go; Jones et al., 1980Go; Pieper et al., 1981Go). In rats, GnRH has inhibitory effects on granulosa and luteal cell functions. The peptide reduces LH-induced steroidogenesis and agonist-induced cAMP-production (Hsueh and Jones, 1981Go).

However, conflicting data exist on the presence of GnRH receptors in the human ovary, their gene expression and function. Binding studies with human luteal tissue or cells revealed no, low or high affinity binding sites for GnRH. GnRH receptors have been found in oocytes (Dekel et al., 1988Go). High affinity GnRH receptors have been shown by quantitative autoradiography in human granulosa cells (Latouche et al., 1989Go). Binding sites with low affinity have been found in the corpus luteum (Bramley et al., 1985Go), whereas other authors (Clayton and Huhtaniemi, 1982Go) detected no GnRH binding in human corpus luteum.

The human GnRH receptor was cloned in 1992 (Kakar et al., 1992Go). Since then molecular approaches have become possible. GnRH receptor mRNA was detected in the human ovary using reverse transcriptase–polymerase chain reaction (RT–PCR) (Kakar et al., 1992Go; Peng et al., 1994Go; Minaretzis et al., 1995bGo). The level of GnRH receptor mRNA in the ovary is ~200-fold lower than in the pituitary. Another author (Fraser et al., 1996Go) found very low levels of GnRH receptor mRNA in the corpus luteum by in-situ hybridization. Studies on the regulation of the GnRH receptor mRNA found that GnRH up-regulates and HCG down-regulates GnRH receptor and GnRH receptor gene expression (Peng et al., 1994Go). A more recent study (Brus et al., 1997Go) found GnRH receptors predominantly after the LH surge. They could not detect GnRH receptors in preovulatory follicles. Other authors (Kang et al., 2000Go) showed that GnRH and GnRH receptor mRNA are expressed in human ovarian surface epithelium. In human granulosa–luteal cells it was shown (Nathwani et al., 2000Go) that oestradiol (24 h) inhibits GnRH mRNA and regulated its receptor gene expression in a biphasic way such as short-term oestradiol treatment (6 h) increases and long-term (48 h) decreases GnRH mRNA levels. Their finding indicates an autocrine role for GnRH in the human ovary and that gonadal steroids might be important regulators of the ovarian GnRH/GnRH-receptor system.


    Actions of GnRH agonists in the human ovary
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 Abstract
 Introduction
 GnRH receptors in human...
 Actions of GnRH agonists...
 Actions of GnRH antagonists...
 References
 
The expression of GnRH receptors in the human ovary does not necessarily indicate that they are functional. In the past, a number of studies investigated whether GnRH agonists influence ovarian function. Most of the experiments were performed in granulosa lutein cells obtained from patients undergoing follicle puncture during IVF cycles. They compared steroidogenesis and gained contradictory results. GnRH agonist exerted different effects of steroidogenesis in granulosa lutein cells in vitro. To shed light on that controversy we summarize the main findings of most studies: Tureck et al. (1982) found an inhibition of progesterone secretion by GnRH agonists. Bussenot et al. (1993) stated that some GnRH agonists enhance oestradiol secretion and another (triptorelin) did not. Gaetje (1994) regarded a decrease in steroid production as an indirect clue to an inhibitory effect of the GnRH agonists decapeptyl on ovarian steroidogenesis. Miro et al. (1992) demonstrated alterations of progesterone accumulation by GnRH agonists. Pellicer and Miro (1990) found a stimulation of oestradiol production, whereas progesterone release was lower in cells from patients treated with a GnRH agonist compared with clomiphene and gonadotrophin stimulation only. In a study by Uemura et al. (1994) GnRH agonists seemed to inhibit steroidogenesis directly. Parinaud et al. (1988) suggested that GnRH agonists could modulate steroidogenesis by a direct ovarian action. The agonist, buserelin, increased basal and decreased LH-induced progesterone secretion in vitro. Guerrero et al. (1993) found an increase in progesterone and decrease in oestradiol production, which seemed to be related to a decrease of LH receptor numbers and aromatase activity in GnRH agonist-treated cells. In the latest study by Dor et al. (2000) FSH-induced oestradiol production by granulosa lutein cells was significantly decreased in agonist-treated patients compared with patients treated exclusively with HMG for ART. However, they could detect no effect in cells that were not stimulated by FSH. Others studies have shown that GnRH agonists have no direct effects on steroidogenesis (Casper et al., 1982Go; Lanzone et al., 1989Go). Metha and Kumar (2000) suggest that GnRH agonists directly act on the ovary and contribute to cyst formation there.


    Actions of GnRH antagonists on steroidogenesis in the human ovary
 Top
 Abstract
 Introduction
 GnRH receptors in human...
 Actions of GnRH agonists...
 Actions of GnRH antagonists...
 References
 
Limited studies were performed to test the effects of GnRH antagonists on the putative GnRH receptors in human ovaries, although it is still controversial, whether they are functional (Hsueh and Jones, 1981Go; Stojilkovic and Catt, 1995Go). Granulosa lutein cells obtained from patients undergoing ovarian stimulation for ART in different stimulation protocols with or without GnRH antagonists were cultured and the steroidogenesis was determined. Minaretzis et al. (1995a) reported reduced aromatase activity in granulosa lutein cells from patients treated with GnRH antagonist. However, the basal and gonadotrophin-stimulated progesterone responses were similar in both antagonist and agonist treatments, suggesting that the reduced aromatase activity might explain the lower oestradiol concentrations observed in patients treated with antagonists. The high concentrations (5 mg) of the GnRH antagonist, Nal-Glu, used in this study, and the small number of patients (n = 15) in this group could limit these results. Lin et al. (1999) conducted a study to evaluate the effect of the agonist, buserelin, and the antagonist, cetrorelix, on the function of granulosa cells. They could not detect any differences in the amount of steroidogenesis between patients treated either with agonist (n = 12) or antagonist (n = 13). They only showed that granulosa cells from women treated with antagonists responded earlier (culture days 2–4 compared with 4–6) in terms of progesterone and better in terms of oestradiol accumulation to the in-vitro HCG and cAMP stimulation than women treated with the agonist. The main findings of the only two studies published so far are contradictory. This may be due to different antagonists used, divergent methodological setting and aims of the studies. In our own experimental studies, we compared ganirelix and cetrorelix treatments in vivo and in vitro with the GnRH agonist triptorelin and controls. We measured basal and HCG-induced oestradiol and progesterone accumulation from granulosa lutein cells obtained from a total of 40 patients that were treated with GnRH analogues. The in-vivo treatments with triptorelin, ganirelix, cetrorelix and no GnRH analogue did not significantly affect basal or HCG-stimulated steroidogenesis. Cells from patients that received triptorelin secreted slightly lower amounts of progesterone. GnRH antagonists did not change the steroid production, even if measured during the first hours of culture period. To exclude the possibility that the in-vivo treatment with GnRH analogues did not alter steroidogenesis because of their absence during the culture period, we added the GnRH analogues directly to the medium. We found no differences in steroid release after different treatments with ganirelix or cetrorelix (Ortmann et al., 1998Go). Concerning the GnRH agonist, triptorelin, our study is in agreement with many other studies (see above). Our finding that GnRH antagonist did not affect steroidogenesis is also in accord with previous studies (Minaretzis et al., 1995aGo; Lin et al., 1999Go), which found insignificant effects on steroidogenesis in cells from a smaller number of patients treated with GnRH antagonists.

The finding that antagonists do not alter the steroidogenesis of granulosa lutein cells, does not exclude the existence of other effects on ovarian functions. It has been established that cAMP-mediated signalling is of importance in granulosa lutein cells. Early studies (Ranta et al., 1983Go) suggested that GnRH reduces ovarian adenylate cyclase, while a later study (Furger et al., 1996Go) indicated that the GnRH agonist, triptorelin, interacts with FSH-induced cAMP-dependent signal transduction. Therefore, we investigated whether GnRH antagonists exert any effects on cAMP accumulation. We found that ganirelix did not affect basal or HCG-stimulated cAMP accumulation in human granulosa and cumulus cells (Demirel et al., 2000Go).

In conclusion, there is little evidence that antagonists exert direct effects on ovarian steroidogenesis. This assumption is mainly derived from extensive in-vitro work with granulosa lutein cells. The action of gonadotrophins is not altered. Both stimulated steroid secretion and cAMP accumulation were not significantly influenced by antagonists. Our observations do not support the hypothesis that lower concentrations of serum oestradiol during ovarian stimulation in combination with a GnRH antagonist are due to direct ovarian action of the peptide analogue. However, other ovarian functions may be altered by direct GnRH analogue effects.


    Notes
 
1 To whom correspondence should be addressed. E-mail: ortmann{at}medinf.mu-luebeck.de Back

This debate was previously published on Webtrack (www.oup.co.uk/humrep/comment) on December 7, 2000


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 Actions of GnRH agonists...
 Actions of GnRH antagonists...
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