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
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Abstract |
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Key words:
free subunit/GnRH agonists/IVF/ovarian stimulation
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Introduction |
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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., 1989), does not totally prevent the risk of endogenous LH surge during the late follicular phase (Acharya et al., 1992
). 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., 1993
). 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, 1995) and interruption of GnRH agonist administration is usually followed by an abrupt fall in
subunit concentrations (Oppenheimer et al., 1992
). Although
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., 1997
). 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
and LH ß subunits.
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Materials and methods |
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Protocol
The short-term protocol routinely used in our unit included the following: (i) a preliminary phase of 1220 days of treatment with a progestogen [norethisterone (Norluten®) 10 mg/day; Smith KlineBeecham, 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., 1992). 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 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
subunit and HCG were achieved as previously described (Ozturk et al., 1987
). We recently reported the development of an assay for the specific detection of the free ß subunit of LH (Chanson et al., 1997
). 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-
monoclonal antibody as the tracer. These immunoassays did not show cross-reactivity with common free
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 2 test, as appropriate. They were expressed as mean ± SEM. A probability < 0.05 was considered statistically significant.
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Results |
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Discussion |
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In normo-ovulatory women, it is well documented that, while free 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
subunits remain high (Meldrum et al., 1984
; Evans et al., 1984
). Similar effects of a chronic GnRH agonist administration on
subunit secretion have been observed in several situations: in patients with glycoprotein hormone secreting pituitary adenomas (Roman et al., 1984
; Klibanski et al., 1989
), in a number of hormone-dependent disorders (Lahlou et al., 1987
; Kwekkeboom et al., 1990
) and in menopausal women (Hagen and McNeilly, 1975
). Furthermore, during this period of hypophyseal desensitization, GnRH receptors still seem to be responsive to native GnRH as suggested by the sustained release of
subunit to a GnRH stimulation test (Lahlou et al., 1987
). 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
subunit (Fluker et al., 1994
), attesting to a role for GnRH as a trophic hormone for
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., 1996
) as well as in plasma free
subunit concentrations (Oppenheimer et al., 1992
). 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, 1995) 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, 1996), 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., 1998
), 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., 1984; Lemay and Lourdusamy, 1991
). 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., 1988
). Changes in glycosylation of LH may also lead to decreasing bioactivity (Bhasin et al., 1984
). 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., 1988
). 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
subunit plasma concentrations during daily GnRH agonist injection and after its cessation may suggest that
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., 1988; Matikainen et al., 1992
). 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., 1995). 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.
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Notes |
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References |
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Submitted on August 9, 1999; accepted on January 24, 2000.