Human ovarian steroid secretion in vivo: effects of GnRH agonist versus antagonist (cetrorelix)

Juan A. Garcia-Velasco1,4, Verónica Isaza1, Carmen Vidal2, Adriana Landazábal1, José Remohí2,3, Carlos Simón2,3 and Antonio Pellicer2,3

1 IVI-Madrid, Madrid, 2 Instituto Valenciano de Infertilidad (IVI) and 3 Department of Paediatrics, Obstetrics and Gynaecology, Valencia University School of Medicine, Valencia, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: In order to investigate whether gonadotrophin-releasing hormone (GnRH) antagonists exert a significant effect on steroid secretion in vivo compared with GnRH agonists, concentrations of sex steroid hormones (oestradiol, progesterone and testosterone) were studied in follicular fluid from women undergoing ovarian stimulation and treated with either GnRH agonist or antagonist. In addition, the correlation between follicular fluid steroid hormone concentrations and variables of follicular and oocyte development was evaluated. METHODS: Microparticle enzyme immunoassay and radioimmunoassays were used. RESULTS: The mean (SEM) follicular fluid oestradiol concentration was significantly lower in patients treated with GnRH antagonist than in those treated with GnRH agonist (542.0 ± 76.9 versus 873.0 ± 105.1 pg/ml, P = 0.02), which correlates with the mean serum oestradiol concentrations found in these two groups. No significant differences were found between groups in follicular fluid progesterone concentrations. Women undergoing GnRH antagonist treatment showed similar concentrations of follicular fluid testosterone compared with GnRH agonist-treated women (14.8 ± 1.1 versus 13.3 ± 2.7 ng/ml). The oestradiol:testosterone ratio was markedly reduced in women treated with GnRH antagonist (49.1 ± 2.3 versus 60.1 ± 4.4, P = 0.04). In contrast, no differences were found either in the progesterone:testosterone ratio, or in the oestradiol:progesterone ratio. CONCLUSIONS: GnRH antagonist therapy in women undergoing ovarian stimulation had a significant effect on ovarian follicular steroidogenesis.

Key words: follicular fluid/GnRH antagonist/IVF/steroid hormones


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Gonadotrophin-releasing hormone (GnRH) agonists were introduced into IVF protocols as a means of avoiding premature luteinization, the incidence of which fell from 20 to 2% (Fauser et al., 1999Go). Chronic administration of GnRH agonists induces a hypogonadotrophic state as a consequence of the down-regulation and desensitization of pituitary gonadotrophin receptors, the rationale for clinical use of GnRH agonists. Conversely, GnRH antagonists achieve a rapid decline in gonadotrophin secretion by competitive blockade of the GnRH receptors (Diedrich et al., 1994Go). The recently introduced GnRH antagonist cetrorelix has been shown to be effective in preventing the LH rise during ovarian stimulation in IVF (Diedrich et al., 1994Go; Albano et al., 1996Go; Felberbaum et al., 2000Go; Olivennes et al., 2000Go), and its clinical efficacy has been confirmed by a large multicentre phase III clinical trial (Felberbaum et al., 2000Go). However, a lower serum oestradiol concentration was observed in GnRH antagonist-stimulated cycles.

It has been shown that GnRH analogues may have a direct effect on the ovary. Specific GnRH binding sites have been identified in rat and human granulosa (Latouche et al., 1989Go; Minaretzis et al., 1995bGo). Activation of these receptors causes stimulatory effects on oocyte meiosis and ovulation (Hillensjo and LeMaire, 1980Go) and a direct inhibition of ovarian steroidogenesis (Hsueh and Erickson, 1979Go). However, conflicting reports have appeared regarding the effects of GnRH agonists on human granulosa cells (Casper et al., 1982Go; Tureck et al., 1982Go; Pellicer and Miró, 1990Go; Bussenot et al., 1993Go; Valbuena et al., 1997Go). Whether the GnRH antagonists have any direct effect on the ovarian cells is not known.

Numerous reports have dealt with intrafollicular concentrations of ovarian steroids in patients undergoing IVF, attempting to identify a relationship between the steroid concentrations and oocyte quality/cycle outcome (Franchimont et al., 1989Go; Hartshorne, 1989Go; Andersen, 1993Go; Enien et al., 1995Go; Minaretzis et al., 1995aGo; Mendoza et al., 1999Go; Teissier et al., 1999Go; Dor et al., 2000Go). However, to the best of our knowledge, no data have been published regarding intrafollicular steroid concentrations in patients undergoing IVF cycles with GnRH antagonists.

The specific aims of this study were to quantify follicular fluid concentrations of oestradiol, progesterone and testosterone in IVF cycles on the day of oocyte retrieval in patients stimulated with GnRH agonist or antagonist, and to assess the relationship between them.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
Thirty-six women with primary infertility undergoing IVF consented to participate in the study, which was approved by the Institutional Review Board. All patients had regular menstrual cycles every 26 to 32 days. The mean (± SEM) patient age was 32.6 ± 0.9 years (range 23–40), and the mean duration of infertility was 3.1 ± 0.6 years (range 1–6). All women had normal body mass index (BMI) (22.3 ± 1.5 kg/m2) and were not receiving any medication. The aetiologies of infertility were similar in both groups studied (41% male factor, 12% tubal disease, 15% unexplained, and a combination of male and female factors in 32%). No patient had any uterine anomaly.

Subjects selected for this study were a subset of women enrolled in a phase III clinical study (D-20761-3119; Asta Médica AG, Frankfurt, Germany). In order to control for potential selection factors that might influence the choice of GnRH agonist/antagonist regimen, a matching process was established on key factors that might influence outcome, including primary diagnosis, age, duration of infertility and BMI. The matching process for patients treated with GnRH agonist consisted of including the next IVF cycle performed in a patient who fulfilled the inclusion criteria and matching requirements right after a GnRH antagonist-treated patient. After completing the match, 16 pairs of women remained in which one woman had been treated with a GnRH antagonist and the other had been treated with a GnRH agonist. In addition, four more patients were included who fulfilled the inclusion criteria and who received the GnRH agonist, two at the beginning of the study and two at the end.

Stimulation protocol
Two different protocols were established: (i) long mid-luteal suppression with GnRH agonist and recombinant gonadotrophins; and (ii) GnRH antagonist with recombinant FSH.

The protocol for ovarian stimulation was initiated with pituitary desensitization by the administration of leuprolide acetate, 1 mg per day, s.c. (Procrin; Abbot S.A., Madrid, Spain), starting in the luteal phase of the previous cycle, on cycle day 21, and this was adjusted according to the length of patient's cycle in order to start the analogue 7 days prior to menstruation. Serum oestradiol concentrations <60 pg/ml (220 pmol/l) and negative findings (absence of ovarian cysts >10 mm diameter) on vaginal ultrasound scans were used to define ovarian quiescence. If a cyst >10 mm diameter was observed, then a serum oestradiol concentration <60 pg/ml was sufficient to confirm ovarian quiescence. If serum oestradiol concentrations were beyond the cut-off point, the patient was excluded from the study.

Briefly, after pituitary desensitization, on days 1 and 2 of ovarian stimulation, 300 IU of recombinant FSH (rFSH) (Gonal-F 75; Serono Laboratories S.A., Madrid, Spain) was administered. On days 3, 4 and 5, 150 IU of rFSH were given to each patient. Beginning on day 6, rFSH was administered on an individual basis according to serum oestradiol concentrations and transvaginal ovarian ultrasound scans.

The cetrorelix single-dose protocol, in which the GnRH antagonist is administered in the late follicular phase, was used as described. Ovarian stimulation was started on day 3 of the menstrual cycle with three ampoules of rFSH (Gonal-F 75) for the first 4 days of treatment. From day 7 of the menstrual cycle onwards the dose was adjusted according to the observed follicle growth. Monitoring of the cycle was carried out every second day, starting from day 7 of the menstrual cycle until the lead follicle reached a diameter of 15 mm; from this day onwards daily ultrasound was performed until the day of human chorionic gonadotrophin (HCG) administration, when plasma concentrations of oestradiol were assessed. A single 3 mg dose of the GnRH antagonist cetrorelix (Cetrotide®; Serono International S.A., Geneva, Switzerland) was administered on day 9 of the menstrual cycle (day 7 of rFSH stimulation). If triggering of ovulation was not performed within 96 h of administration of the 3 mg dose of cetrorelix, a daily injection of 0.25 mg was given until the time of HCG administration. HCG was administered when there were three or more follicles with a maximum diameter >18 mm, as determined by transvaginal ultrasound.

IVF/intracytoplasmic sperm injection (ICSI)
The standard IVF/ICSI procedure was used and has been described previously (Pellicer et al., 1989Go; Gil-Salom et al., 1995Go). Briefly, cumulus–oocyte complexes (COC) were evaluated under the dissecting microscope and classified. COC were incubated at 37°C under 5% CO2 in atmospheric air. Embryos were scored on the day of transfer (day 2) according to their morphology under the dissecting microscope. Four types of embryos were established, ranging from types I to IV. Type I embryos were the best, and defined as round and well-shaped blastomeres without fragments. The study policy for embryo transfer was to select three type I and type II day 2 embryos wherever possible: the remaining embryos were cryopreserved with 1,2-propanediol and sucrose. Only patients with freshly transferred embryos at day 2 were included in the study.

Sample collection and processing
The content of all mature follicles (>14 mm diameter) containing the COC was collected into sterile plastic tubes by electronic suction. Only follicular fluid from follicles in which an oocyte was clearly identified was used for the study. After isolation of the oocyte, the clear follicular fluid from each patient was pooled and immediately centrifuged (1500 g) for 10 min to separate out cellular contents and debris. Follicular fluid supernatant was then transferred to sterile polypropylene tubes and frozen at –20°C until taken for analysis. As a wide interfollicular variation in intrafollicular steroid concentrations has been reported (this reflects interfollicular asynchrony during ovarian stimulation for IVF (Barak et al., 1992; Mendoza et al., 1999Go), the decision was made to use pooled aspirated follicular fluid from each patient in an attempt to assess whole ovarian production, as previously recommended.

Hormone measurements
The samples were stored at –20°C in aliquots for subsequent analysis. Serum oestradiol and LH were analysed using a commercially available microparticle enzyme immunoassay kit (Abbot Laboratories, Abbot Park, IL, USA). Inter- and intra-assay variability for oestradiol at a concentration of <40 pg/ml was 2.8 and 4.3% respectively. Inter- and intra-assay variability for LH at a concentration of <10 IU/ml was 2.49 and 4.28% respectively. Follicular fluid oestradiol, testosterone and progesterone, as well as serum progesterone, were evaluated by radioimmunoassay (BYKSangtec, Dietzenbach, Germany for oestradiol; and Diagnostic Systems Laboratories Inc., Webster, TX, USA for progesterone and testosterone). Inter- and intra-assay variability for oestradiol, progesterone and testosterone was 2.9 and 8.3%, 8.1 and 9.1%, and 4.8 and 9.2% respectively.

Statistical analysis
Data were expressed as mean ± SEM. Student's t-test, {chi}2 test and Pearson's correlation coefficient were used as appropriate. A P-value < 0.05 was defined as being statistically significant. The statistical analysis was performed with use of Sigmastat for Windows, version 2.0 (Jandel Scientific Corporation, San Rafael, CA, USA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The epidemiological characteristics of patients allocated to the two treatment protocols were similar (Table IGo).


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Table I. Epidemiological data of the patients and hormonal concentrations on the day of human chorionic gonadotrophin administration
 
Results of IVF treatment
Patients treated with GnRH antagonist, when compared with those who received rFSH together with GnRH agonist, required a shorter stimulation time (8.4 and 10.1 days respectively, P = 0.004) with a comparable total FSH dose (2012.5 versus 2558.3 IU, not significant). The mean peak serum oestradiol concentration was significantly reduced (846 versus 2481 pg/ml, P = 0.001), and fewer oocytes were retrieved (9.3 versus 16.6, P = 0.004) (Tables I and IIGoGo). The number of days under the influence of the GnRH-analogue was reduced in the antagonist-treated women (3.4 versus 12.2, P < 0.001), as well as the total dose of GnRH-analogue (3.1 versus 18.6 mg, P < 0.001). No differences were found in serum LH concentrations and serum progesterone concentrations on the day of oocyte retrieval. Patients treated with GnRH antagonist showed a higher fertilization rate (91.4 versus 75.3%, P < 0.05). Although the number of oocytes retrieved was lower in the antagonist-treated group (P = 0.004), there was a trend towards a higher number of correctly fertilized 2PN oocytes (91.0 versus 82.5%, NS), whilst the rate of triploids was reduced in this group, although the differences did not reach statistical significance (Table IIGo). The clinical pregnancy rate did not vary significantly between the two groups.


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Table II. 2IVF outcome
 
Follicular fluid steroid concentrations in vivo
Mean concentrations of some follicular fluid steroid hormones were markedly affected by GnRH antagonist treatment (Table IIIGo), with follicular fluid oestradiol significantly lower in these patients when compared with the GnRH agonist-treated group (542.0 ± 76.9 versus 873.0 ± 105.1 pg/ml; P = 0.02); these data correlate with the mean serum oestradiol concentrations determined in these two groups. No significant differences were found between the two groups in follicular fluid concentrations of progesterone or testosterone. These differences were exacerbated when the steroid ratios were calculated. The oestradiol:testosterone ratio was reduced in women treated with GnRH antagonist (P = 0.04). A trend was also observed towards a higher progesterone:testosterone ratio in this group of women, although no significant differences were found. The oestradiol:progesterone ratio was the same in both groups of patients.


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Table III. Follicular fluid steroid hormone concentrations and ratios
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Follicular fluid steroids are secreted by granulosa and theca cells under the control of gonadotrophins, and the hormonal microenvironment provided may have a relevance in the subsequent pre-ovulatory development of the follicle, oocyte quality and IVF outcome (Pellicer et al., 1987Go). The present study provides some of the first evidence suggesting that GnRH antagonists exert significant actions on ovarian steroidogenesis.

Follicular growth is mandatory for intrafollicular oestradiol production, as oestradiol has a role in granulosa cell proliferation, enhancement of aromatase activity, increasing expression of FSH receptors, and induction of LH receptors in granulosa cells (Macklon and Fauser, 1999Go). This important role for oestradiol in follicular maturation has been supported by classical studies demonstrating a correlation between follicular oestradiol production, follicular fluid content, and the health of the follicle (Tonetta and diZerega, 1989). However, recent data from in-vivo studies on humans have questioned the role of oestradiol, suggesting that it may act as a marker of oocyte maturation rather than as a factor controlling maturation (Schoot et al., 1992Go). In the present study, significantly reduced follicular fluid oestradiol concentrations were found in antagonist-treated compared with agonist-treated women, most likely due to a direct action on the ovarian cells, inducing a lower granulosa cells aromatase activity in the antagonist-treated patients (Minaretzis et al., 1995aGo). A shorter stimulation period in the antagonist-treated group does not seem to explain these findings, as both groups of patients required identical HCG administration criteria. A direct action on ovarian physiological mechanisms, influencing enzymes or pathways that control the aromatase activity of granulosa cells is a challenging hypothesis. The in-vivo administration of GnRH antagonist may differentially regulate aromatase activity pathways in granulosa-lutein cells. The fact that granulosa-lutein cells express GnRH receptor mRNA (Minaretzis et al., 1995bGo) as well as the different in-vitro effects on steroidogenesis of granulosa-lutein cells previously exposed to GnRH analogues (Lin et al., 1999Go; Dor et al., 2000Go) support this hypothesis. Considering that the oestradiol:testosterone ratio serves as an approximate measure of aromatase activity (Dor et al., 2000Go), the findings of the present study are consistent with a diminished aromatase activity in antagonist-treated women. However, no effect was observed on c-AMP (a mediator of postreceptor signalling) after ganirelix treatment of in-vitro-cultured human granulosa-lutein cells (Demirel et al., 2000Go).

Lower LH circulating concentrations result in lower thecal cell androgen production and thus, decreased substrate for conversion to oestradiol by granulosa-lutein cells. LH-depleted gonadotrophin preparations were used in both the GnRH agonist and antagonist groups, and significant differences were found in oestradiol concentrations. Variations in exogenous gonadotrophin administration may partially account for such a difference. A trend was observed towards lower FSH doses in antagonist-treated women, in accordance with previously published reports (Felberbaum et al., 2000Go), but differences in total gonadotrophin doses did not reach statistical significance. A striking feature was the low serum oestradiol concentration after GnRH antagonist administration; however, this did not disrupt follicular growth, as observed by the high rate of metaphase II (MII) oocytes retrieved and the high fertilization rate achieved. Serum oestradiol concentrations were significantly lower than those observed with the agonist, and this may have positive implications reducing the risk of ovarian hyperstimulation syndrome (OHSS) and improving endometrial receptivity (Simón et al., 1998Go). These lower serum oestradiol concentrations correlate with the lower follicular fluid oestradiol concentration, thereby suggesting a possible direct role of GnRH antagonist on oestradiol production in the ovary, without affecting follicular development.

The ovaries are the major sites of androgen production and metabolism. Androgens produced by LH-stimulated theca cells influence granulosa cell function, including inhibin formation. Granulosa cell androgen receptor mRNA and protein are down-regulated during FSH-induced pre-ovulatory follicular development, suggesting a paracrine signalling (granulosa on theca) (Hillier, 1999Go). In the present study, no significant differences in testosterone concentrations were found in the follicular fluid of women who received GnRH antagonist versus GnRH agonist. Previously published studies reported reduced testosterone concentrations in stimulated cycles compared with natural cycles (Enien et al., 1995Go). Down-regulation allows the strict control of premature luteinization due to spontaneous LH surges, and theca cells produce less androgens in the presence of minimum amounts of LH. This pattern has also been reported in patients receiving rFSH (Chappel and Howles, 1991Go). When GnRH antagonist is administered, LH circulating concentrations are successfully suppressed, which results in a blockage of thecal stimulus for androgen production, thereby reducing intrafollicular androgen concentrations. Similar serum LH concentrations were found in both groups of patients, which partly explains why follicular fluid testosterone concentrations were also similar.

A correlation between oestrogen:androgen ratio and follicular health and maturity has been reported (Andersen, 1993Go), with pregnancy-associated follicles being characterized by a high oestrogen:androgen ratio. The ratio of oestrogen to androgen in follicular fluid may reflect the effectiveness of the granulosa cell to convert androgens into oestrogen, so that the healthiest follicle would show a high ratio of androgen conversion to oestradiol. Moreover, androgens enhance ovarian granulosa cell apoptosis, while oestradiol exerts an inhibitory effect (Billig et al., 1993Go). Among the two groups of the present study, the oestradiol:testosterone ratio was lower in antagonist-treated patients, this ratio being more likely due to the low oestradiol concentration consistently found in antagonist-treated patients, than to high testosterone concentrations in follicular fluid that would suggest early atretic changes. The high MII and fertilization rates observed in oocytes retrieved from these antagonist-treated women support this contention, as suggested also by others (Minaretzis et al., 1995aGo). This previous observation, when combined with the present data, expand the evidence that classical indices of follicular health such as oestradiol concentrations may not necessarily be valid. An additional system, such as the GnRH-GnRH receptor, may influence oocyte maturation and oestradiol concentrations. The presence of high-affinity GnRH receptors in human luteinized granulosa cells has been described (Brus et al., 1997Go), and these authors proposed a possible pharmacological modulation of the follicular GnRH receptor in the ovary. This may have relevant implications in selecting the correct protocol for inducing ovulation in IVF.

As reported previously (Minaretzis et al., 1995aGo), follicular fluid progesterone concentrations were similar in all groups, suggesting that administration in vivo of these GnRH analogues might have similar effects on progesterone production pathways. Progesterone production is mainly affected by LH and HCG (Rabinovici, 1993Go). In the present study, no differences were found in concentrations of either serum LH or progesterone in both groups of patients. Interestingly, antagonist-treated patients showed a lower follicular fluid progesterone concentration, though these differences were not significant. This is in accordance with recent data that suggested a better developmental competence of oocytes retrieved from follicles with a high progesterone concentration (Mendoza et al., 1999Go). However, the present data showed a similar luteinization process in both groups of GnRH-analogue-treated patients.

An interesting observation was the fact that with similar FSH doses, and with a significantly reduced period of stimulation, fewer oocytes were retrieved in antagonist-treated women. It might be expected that follicle recruitment would be greater if a higher dose of FSH were to be administered, mainly as the result of administering the same total FSH dose over fewer days. However, similar numbers of mature oocytes were obtained in both groups, which may indicate a better synchrony in follicular growth. Although this finding should be checked in a larger study, it may be the result of a more physiological approach to ovarian stimulation, as no previous down-regulation is required, thus avoiding the (hypothetical) direct effects that GnRH analogues may exert on the ovary (Hsueh and Erickson, 1979Go; Hillensjo and LeMaire, 1980Go). Thus, it may be postulated that a GnRH antagonist may induce a different pattern of follicle growth and oocyte maturation. In fact, this has been previously reported (Albano et al., 2000Go; Ludwig et al, 2000Go; The European Orgalutran Study Group et al, 2000), suggesting that GnRH antagonists induce different follicular growth, with fewer small follicles when compared with GnRH agonists, and indeed this is in agreement with the lower oestradiol concentrations and lower incidence of OHSS. Nonetheless, it should be highlighted that FSH dosage was not a primary end-point of the present study, and sample size limitations may therefore hinder the drawing of valid conclusions in this respect. Indeed, an inter-group difference of 500 IU in the total FSH dose administered was reported, though this was not statistically significant.

Several limitations of the present study should be mentioned. First, this was an observational study and not a randomized controlled clinical trial; hence, patients were selected who were undergoing different GnRH analogue treatments during the same timeframe in order to avoid confounding variables that may influence the results. Second, no studies were performed in vitro, such as granulosa or theca cell steroidogenesis under agonist or antagonist treatment, as the present study evaluated only follicular fluid specimens obtained from women who had received either drug. This may narrow the conclusions of the study, although further in-vitro experiments are being performed to investigate further the role of GnRH antagonists on ovarian granulosa/theca cells. Third, two different regimens of FSH were used in both groups: step-down and fixed dose with gradual increments as needed. Although, hypothetically, this may influence the results, recent data have confirmed that serum and follicular fluid hormone concentrations do not vary when different protocols of FSH are used, but do so when different GnRH agonist regimens are used (Bo-Abbas et al, 2001).

In conclusion, these data suggest that GnRH antagonist therapy in women undergoing ovarian stimulation had a significant effect on ovarian follicular steroidogenesis. The lower oestradiol concentration may improve the support of the oocyte, as these patients showed excellent oocyte maturation and fertilization rate. The correct combination of gonadotrophins with the GnRH antagonist cetrorelix will enable healthy follicles to be grown and for the oocyte to be exposed to the best conditions in order to increase the chances of fertilization.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank Dr M.Carmen Díez and Dr Ignacio Tagarro from ASTA Médica and Dr José Antonio Peinado from Serono Laboratories for their critical review of the manuscript.


    Notes
 
4 To whom correspondence should be addressed at: IVI-Madrid, C/Santiago de Compostela 88, 28035 Madrid, Spain. E-mail: jgvelasco{at}ivi.es Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on May 2, 2001; accepted on September 3, 2001.