Consequences on gonadotrophin secretion of an early discontinuation of gonadotrophin-releasing hormone agonist administration in short-term protocol for in-vitro fertilization

Isabelle Cedrin-Durnerin1,5, Jean-Michel Bidart4, Philippe Robert4, Jean-Philippe Wolf2, Michele Uzan3 and Jean-Noël Hugues1

1 Reproductive Medicine Unit, 2 Laboratory of Reproductive Biology, 3 Department of Obstetrics and Gynecology, Jean Verdier Hospital, Av. du 14 Juillet, Bondy 93143, University Paris XIII and 4 Department of Clinical Biology, Gustave-Roussy Institute, University Paris XI, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Administration of gonadotrophin-releasing hormone (GnRHa) agonists, used in IVF short-term protocols to initiate follicular recruitment, may be restricted to the early follicular phase without any further risk of LH surge. However, consequences of an early discontinuation upon residual endogenous gonadotrophin secretion are still unknown. Here, the effects of early cessation of GnRH agonist upon gonadotrophin secretion and ovarian parameters of IVF cycles were investigated. A total of 230 normo-ovulatory women were prospectively allocated to one of the two regimens: decapeptyl-GnRH (100 µg) was daily injected either from day 1 to the triggering of ovulation (group 1) or for the first 7 days (group 2). Exogenous gonadotrophins (150 IU) were administered on day 4 and 5 with a subsequent adjustment. Detections of free {alpha} subunit and dimeric LH were performed by highly specific `two site' monoclonal immunoradiometric assays. The results show that early discontinuation of GnRH agonist administration was associated with a sharp decrease in both plasma free {alpha} subunit and dimeric LH concentrations while plasma oestradiol response to exogenous gonadotrophins was reduced. Other ovarian parameters and pregnancy rate were unchanged. These data indicate that endogenous LH secretion is maintained by a daily administration of GnRH agonist and may contribute to the final follicular maturation.

Key words: free {alpha} subunit/GnRH agonists/IVF/ovarian stimulation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Among several protocols currently used for IVF programmes, the short-term gonadotrophin-releasing hormone (GnRH) agonist regimen has been recognized as effective and convenient (Frydman et al., 1988Go; Hughes et al., 1992Go). In contrast with the long-term protocol, it favours the dual effect of the agonist administration with an initial release of endogenous gonadotrophins which participate in follicular recruitment and a subsequent decline in gonadotrophin secretion related to hypophyseal desensitization. This latter effect starts shortly after the flare-up effect of the GnRH agonist, lasts as long as its administration is continued and prevents any further endogenous LH surge (Broekmans et al., 1996Go). During this period, the pattern of LH secretion is characterized by a decrease in both dimeric LH and free LH ß subunit while free {alpha} subunit release is maintained (Meldrum et al., 1984Go; Lahlou et al., 1987Go; Lemay and Lourdusamy, 1991Go). Following cessation of GnRH agonist daily injection, a recovery period of hypophyseal synthesis is usually observed for 5–8 days (Barriere et al., 1991Go; Winslow et al., 1992Go) and thereafter gonadotrophin secretion is completely restored.

Although this biphasic effect of GnRH agonist accounts for the effectiveness of the so-called short-term protocol in assisted reproductive technologies, several aspects of this regimen remain unclear. Among them, the duration of GnRH agonist administration was questioned: a daily injection for the first 3 days of the cycle (ultra-short protocol), as proposed (Macnamee et al., 1989Go), does not totally prevent the risk of endogenous LH surge during the late follicular phase (Acharya et al., 1992Go). In contrast, a 7 day period of GnRH agonist administration seems sufficient to preclude any premature endogenous LH surge and is associated with good fertilization and pregnancy rates (Hazout et al., 1993Go). This regimen is cost-effective and may avoid follicular exposure to the potential deleterious effects of GnRH agonist in the late follicular phase.

However, consequences of an early discontinuation of the agonist administration upon gonadotrophin secretion are still unclear. A profound LH suppression after stopping leuprolide acetate injection early in the follicular phase has been reported (Sungurtekin and Jansen, 1995Go) and interruption of GnRH agonist administration is usually followed by an abrupt fall in {alpha} subunit concentrations (Oppenheimer et al., 1992Go). Although {alpha} subunit is commonly considered as biologically inactive, such a decrease in LH secretion after cessation of GnRH agonist daily injection may be clinically relevant, especially for stimulation regimens using recombinant FSH preparations free of LH activity. Indeed, it has been reported that an early discontinuation of GnRH agonist during a long-term protocol may be detrimental to follicular development and steroid synthesis and that a higher amount of exogenous gonadotrophins may be required (Fuji et al., 1997Go). Collectively these data thus suggest that the duration of GnRH agonist administration may play a pivotal role for the effectiveness of short-term protocol in relation to the residual endogenous gonadotrophin secretion. Therefore a comparative prospective and randomized study was performed to evaluate the effects of an early discontinuation of administration of a short-acting GnRH agonist on gonadotrophin secretion and IVF outcome. For this purpose, highly specific immunoassays were used to detect dimeric LH, FSH and free {alpha} and LH ß subunits.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A total of 230 infertile women undergoing new or repeated IVF cycles in our infertility clinic were asked to participate in this study after being thoroughly informed. This study was performed according to local ethical committee rules. Women who were 43 years of age or older and who suffered from chronic anovulation were excluded from the study.

Protocol
The short-term protocol routinely used in our unit included the following: (i) a preliminary phase of 12–20 days of treatment with a progestogen [norethisterone (Norluten®) 10 mg/day; Smith Kline–Beecham, Nanterre, France] from the 15th day of the cycle preceding IVF. The first day of the IVF cycle was arbitrarily designated as the third day after the cessation of progestogen administration and it was actually the first day of menstrual flow in about 90% of patients. This procedure allowed us to programme oocyte retrieval on a week day; (ii) a daily s.c. injection of Dtrp6-GnRH (Decapeptyl®, 100 µg/day; Ipsen-Biotech, Paris, France) in the evening from the first day of the IVF cycle; (iii) ovarian stimulation with exogenous gonadotrophins (Neopergonal® or Metrodine® H.P.75 IU; Serono SA, Boulogne, France), 150 IU i.m. in the evening of day 4 and 5 of the cycle, the subsequent doses being adjusted according to hormonal measurements and ultrasound data with strictly defined criteria as follows. Although doctors carrying out the IVF cycle were not blinded to patient group, the decision to raise the FSH dose was based on both the percentage increase in oestradiol concentration and the day of stimulation, applied similarly in the two groups. It is therefore unlikely that knowledge of patient randomization could have led to bias. A pelvic ultrasound was performed every 2 days from day 7 until the time of human chorionic gonadotrophin (HCG) administration; and (iv) HCG (10 000 IU, gonadotrophine chorionique endo; Organon, St Denis, France), injected when at least three follicles reached a diameter of 17 mm and oestradiol values were about 200 pg/ml per follicle >15 mm.

Oocytes were retrieved 36 h after triggering of ovulation by transvaginal aspiration and cultured as previously reported (Hugues et al., 1992Go). Luteal phase was supported similarly in both groups. When plasma oestradiol was <2500 pg/ml at the time of ovulation triggering, HCG was administered i.m. on the day of oocyte retrieval (5000 IU) and thereafter on day 1 and day 4 following embryo transfer (2500 IU). In every situation, the luteal phase was supported by daily vaginal administration of 400 mg of micronized progesterone (Utrogestan®; Besins-Iscovesco Pharmaceutics, Paris, France).

Participating women were randomly assigned by a random number table to two treatment groups. Group 1 (n = 115) received a standard short-term protocol with GnRH agonist being daily injected from day 1 of the IVF cycle to the time of HCG administration. Group 2 (n = 115) received a 7 day GnRH agonist protocol, administration of the agonist being stopped on the 7th day of the IVF cycle.

Hormonal measurements
In both treatment groups, the first blood sampling occurred on cycle day 6 after 2 days of exogenous gonadotrophin therapy. Thereafter, blood samples were obtained every morning until HCG administration. Plasma was assayed for LH, oestradiol and progesterone in order to adjust therapy while a fraction was frozen and stored for subsequent determination of FSH, HCG and LH subunits. All samples were run in duplicate in different assays.

Radioimmunoassays were used to measure plasma oestradiol and progesterone concentrations (Coatria®; Biomerieux, Lyon, France). The lower limit of sensitivity was 7 pg/ml for the oestradiol assay and 0.05 ng/ml for the progesterone assay. The intra-assay coefficients of variation (CVmax) were 12 and 8.5% respectively. Interassay variabilities were 16 and 11% respectively. Plasma FSH and LH concentrations were determined by a specific (125I) radioimmunoassay without extraction (Bio-Merieux, Marcy l'étoile, France). The FSH assay had intra- and interassay maximum coefficients of variation (CVmax) of 6% with minimum detectable value of 0.37 IU/l. The LH assay had intra- and interassay CVmax of 8% with minimum detectable value of 0.5 IU/l.

In order to investigate further the nature of the gonadotrophins secreted during the follicular phase, detection of common free {alpha} subunit, free LH ß subunit, dimeric LH, FSH and HCG was performed in 29 patients by highly specific `two-site' monoclonal immunoradiometric assays. Briefly, measurements of free {alpha} subunit and HCG were achieved as previously described (Ozturk et al., 1987Go). We recently reported the development of an assay for the specific detection of the free ß subunit of LH (Chanson et al., 1997Go). Serum dimeric LH and FSH were detected by in-house immunoassays based on specific anti-ß subunit antibodies, as capture antibodies, and a radiolabelled anti-{alpha} monoclonal antibody as the tracer. These immunoassays did not show cross-reactivity with common free {alpha} and respective ß subunits and displayed a sensitivity of 0.7 IU/l and 0.4 IU/l for LH and FSH respectively. Intra-assay and interassay CVmax were 8 and 9% respectively for dimeric LH and 9 and 11% for dimeric FSH.

Statistical analysis
Results were analysed by unpaired t-test and {chi}2 test, as appropriate. They were expressed as mean ± SEM. A probability < 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical characteristics of the 115 women allocated to each group are presented in Table IGo. Infertility resulted either from a tubal factor (56%), male factor (47%) or was unexplained (11%). Infertility was primary and secondary in 35% and 65% respectively. There was no difference between the two treatment groups (Table IGo). During the ovarian stimulation period, 30 cycles were cancelled for poor ovarian response (eight in each group), or error in treatment (one in each group) or cyst formation (two in group 1 and three in group 2) or a drop in oestradiol concentrations (two in group 1 and five in group 2).


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Table I. Main characteristics of patients in both treatment groupsa
 
Overall results of the 200 women (n = 102 in group 1; n = 98 in group 2) who achieved oocyte retrieval are presented in Table IIGo. As compared with group 1, patients from group 2 were characterized by a longer period of ovarian stimulation (P < 0.05) and by a greater total amount of ampoules required to get final follicular maturation (P < 0.004). In contrast, there were no differences between the two groups as regards the numbers of retrieved or mature oocytes, total or transferred embryos and pregnancy or miscarriage rates.


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Table II. Comparison of IVF outcome in the two treatment groupsa
 
In group 2 women treated with a 7 day GnRH agonist administration, the main endocrine feature was a sharp and highly significant decrease in plasma LH concentrations from day 8 to the time of HCG administration (Figure 1Go). Concomitantly, plasma oestradiol concentrations tended to increase with a lower slope from day 10 and difference between plasma oestradiol values of the two groups was highly significant at the time of HCG (1837 ± 82 versus 1338 ± 66 pg/ml for groups 1 and 2 respectively; P < 0.001). Accordingly, the ratio between the maximal plasma oestradiol concentration and the number of exogenous gonadotrophin ampoules (75 IU) (calculated for each patient, then averaged over group) was significantly lower in group 2 as compared with group 1 (46.9 ± 4.6 versus 74.1 ± 4.8 respectively; P < 0.0001). Moreover, the oestradiol/oocyte ratio (calculated for each patient, then averaged over group) was significantly lower in group 2 than in group 1 (196 ± 14 versus 317 ± 32 respectively; P < 0.0009). Finally, there was no difference between the two groups regarding plasma progesterone values during the stimulation period and at the time of HCG administration (data not shown).



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Figure 1. Comparison of plasma concentrations of luteinizing hormone (LH) (upper panel) and oestradiol (lower panel) between patients from group 1, • [Decapeptyl® until human chorionic gonadotrophin (HCG) administration] and group 2, {circ} (Decapeptyl® for 7 days) during treatment. Results are expressed as mean ± SEM; °P < 0.01; *P < 0.001.

 
Comparisons between plasma LH concentrations are presented in Figure 2Go. Discontinuation of GnRH agonist administration was associated with a parallel and sharp decrease in plasma concentrations of both dimeric LH and free {alpha} subunits. By contrast, when GnRH agonist administration was maintained, {alpha} subunit concentrations remained unchanged and dimeric LH values only slightly decreased. Differences between groups were highly significant (P < 0.001) at day 10 and day 13. In both groups, plasma free LH ß subunits were undetectable and plasma HCG and FSH concentrations remained unchanged (Table IIIGo).



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Figure 2. Comparison of plasma concentrations of dimeric LH and free {alpha} subunits between patients from group 1, • (Decapeptyl® until HCG administration) and group 2, {circ} (Decapeptyl® for 7 days) during treatment. Results are expressed as mean ± SEM; *P < 0.001.

 

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Table III. Plasma concentrations of dimeric LH, free {alpha} subunits, free LH ß subunits, HCG and FSH in the two treatment groupsa
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The main finding of this study was that an early discontinuation of GnRH agonist administration during a short-term protocol for IVF induces a significant decline in both dimeric LH and free {alpha} subunit secretion with a concomitant decrease in ovarian steroid secretion and a need for a higher amount of exogenous gonadotrophins.

In normo-ovulatory women, it is well documented that, while free {alpha} subunit and dimeric LH are co-secreted in response to an acute injection of native GnRH, the pattern of LH secretion differs greatly during a chronic GnRH agonist administration which induces a hypophyseal desensitization. Indeed, bioactive dimeric LH secretion is markedly reduced but plasma concentrations of free {alpha} subunits remain high (Meldrum et al., 1984Go; Evans et al., 1984Go). Similar effects of a chronic GnRH agonist administration on {alpha} subunit secretion have been observed in several situations: in patients with glycoprotein hormone secreting pituitary adenomas (Roman et al., 1984Go; Klibanski et al., 1989Go), in a number of hormone-dependent disorders (Lahlou et al., 1987Go; Kwekkeboom et al., 1990Go) and in menopausal women (Hagen and McNeilly, 1975Go). Furthermore, during this period of hypophyseal desensitization, GnRH receptors still seem to be responsive to native GnRH as suggested by the sustained release of {alpha} subunit to a GnRH stimulation test (Lahlou et al., 1987Go). Thus, the effects of GnRH agonists on LH secretion differ greatly from those of GnRH antagonists which induce a parallel decrease in both dimeric LH and free {alpha} subunit (Fluker et al., 1994Go), attesting to a role for GnRH as a trophic hormone for {alpha} subunit secretion. This partial agonistic effect of short-acting GnRH agonist on LH secretion has also been indirectly demonstrated by observations that discontinuation of GnRH agonist injection induces a rapid decline in plasma LH concentrations (Broekmans et al., 1996Go) as well as in plasma free {alpha} subunit concentrations (Oppenheimer et al., 1992Go). Thus, there is some evidence that a daily administration of a short-acting GnRH agonist is able to sustain some degree of LH release from the pituitary and that withdrawal of the agonist leaves the pituitary in a refractory state of LH secretion.

The results of this study are in line with a previous report (Sungurtekin and Jansen, 1995Go) which compares the effects of a daily leuprolide acetate administration for 5 days or during the whole follicular phase. It was also observed that gonadotrophin responsiveness to daily GnRH agonist administration was preserved and that discontinuation of the agonist injection induced a marked decrease in plasma LH concentrations. While the authors suggested that the low amount of gonadotrophin release after stopping GnRH agonist may result from a low endogenous GnRH activity, it is more likely that it results from the loss of the agonist effects of the GnRH agonist in combination with a submaximal pituitary desensitization. Collectively these studies thus confirm that stopping administration of GnRH agonist may adversely affect the residual endogenous LH secretion maintained by a daily administration of a short-acting agonist.

From a clinical point of view, consequences of this decreased LH secretion after discontinuation of GnRH agonist on ovarian function remain unclear. In the current study, steroidogenic response to gonadotrophins was reduced in women who stopped GnRH agonist injections early. Indeed, in spite of a higher supply of exogenous gonadotrophins, plasma oestradiol values at the time of HCG administration were significantly lower, indicating a decreased ovarian sensitivity. In contrast, follicular development which is primarily FSH-dependent was similar in both groups. As both FSH and LH are required to get final production of oestradiol by granulosa cells (Hillier, 1996Go), this dissociation between steroid production and follicular growth strongly suggests that the residual endogenous LH secretion observed during GnRH agonist administration is also involved in the final follicular maturation. Furthermore, oestradiol/oocyte ratio, a better index of exposure to LH during the follicular phase than punctual assessments for serum LH concentrations (Loumaye et al., 1998Go), was significantly lower in patients who discontinued GnRH agonist treatment. Altogether these data would thus suggest that the residual endogenous LH secretion during a short-term GnRH agonist protocol is still biologically active and may contribute to the final follicular maturation.

These findings are not in line with previous reports showing that concentrations of bioactive LH measured by testosterone production from dispersed mouse Leydig cells in vitro are reduced during chronic GnRH agonist therapy (Meldrum et al., 1984Go; Lemay and Lourdusamy, 1991Go). Inhibition of bioactive LH biosynthesis during long-term desensitization is usually considered to be a consequence of a marked reduction of LH ß gene expression (Lalloz et al., 1988Go). Changes in glycosylation of LH may also lead to decreasing bioactivity (Bhasin et al., 1984Go). As the pituitary LH content falls in concert with LH ß mRNA, it is usually considered that ß subunit synthesis is the rate-limiting step of dimeric LH production. In contrast, the short-term effects of GnRH agonist administration on hypophyseal secretion have been investigated less. Some experiments in castrated rats treated for 7 days by GnRH infusion indicated that LH ß mRNA was unchanged while pituitary LH content was reduced (Lalloz et al., 1988Go). This may indicate that inhibition of LH ß gene expression is not the only mechanism involved in the reduction in pituitary LH content observed during the first days of GnRH agonist administration. In the current study, measurements of free ß subunit plasma concentrations did not give additional information because plasma concentrations were undetectable after 7 days of GnRH agonist administration and did not reflect the degree of suppression in LH ß synthesis. However, the strongly correlated variations in dimeric LH and free {alpha} subunit plasma concentrations during daily GnRH agonist injection and after its cessation may suggest that {alpha} subunit synthesis is also involved in the regulation of dimeric LH synthesis during this period.

Another alternative to explain the discrepancy between steroid production between the two groups could be that the residual endogenous FSH secretion participates in the final ovarian maturation. While plasma FSH concentrations were similar, it cannot be excluded that FSH bioactivity was different in both groups. Indeed, some studies provide evidence for a certain degree of bioactive FSH secretion during GnRH agonist therapy with a sustained release following daily administration (Huhtaniemi et al., 1988Go; Matikainen et al., 1992Go). This latter point needs further confirmation in other studies.

Finally, this study shows that, in spite of a decreased steroid ovarian responsiveness to exogenous gonadotrophins in patients who stopped GnRH agonist administration early, other ovarian parameters and IVF outcome were similar. Indeed the groups did not differ as regards the total number of oocytes, the oocyte fertilization rate, the number of embryos transferred or the pregnancy rate. Thus, the relatively lower LH exposure during the late follicular phase was not detrimental to IVF outcome, indicating that a minimal value for the oestradiol/oocyte ratio is required for succeeding in implantation as suggested by Loumaye et al. (1998). The authors found that the pregnancy rate appeared to be decreased only in patients with an oestradiol/oocyte ratio lower than 70. Therefore, as the ratio was above this threshold in both groups, this may account for the lack of significant difference in pregnancy rates. Nevertheless, as the pregnancy rate tended to be higher in group 2 patients, it may be speculated that exposure of endometrium to lower amount of oestradiol may be a contributory factor to implantation (Simon et al., 1995Go). Finally the clinical implication of this study is that none of these patients would benefit from exogenous LH administration to contribute to the oestradiol synthesis during the late follicular phase. This conclusion is clinically relevant in the new perspective of a worldwide use of recombinant FSH molecules.


    Notes
 
5 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on August 9, 1999; accepted on January 24, 2000.