The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA 23507-1627, USA
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Abstract |
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Key words: gonadotrophins/oestradiol/ovarian response/ovarian stimulation/pituitary desensitization
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Introduction |
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Besides the fact that these few reports are far from leading to firm conclusions regarding the predictive value of down-regulated gonadotrophin concentrations, they all employed human menopausal gonadotrophins (HMG), either alone or in combination with FSH. While FSH is established as the main force driving the ovary to folliculogenesis, studies utilizing highly purified or recombinant FSH in hypogonadotrophic women would also imply a facilitating, if not essential, role for LH in ovarian function (Couzinet et al., 1988; Shoham et al., 1993
). Even though the minimal absolute concentration of LH necessary for adequate ovarian function remains yet to be defined, low mean follicular phase LH concentrations (<1.0 IU/l) following pituitary suppression have been shown in the clinical in-vitro fertilization (IVF) setting to be associated with compromised follicular growth, as displayed by a lower oestradiol output from day 5 of stimulation onwards and a longer follicular phase, when only highly-purified FSH (FSH-HP) was used for stimulation (Fleming et al., 1996
). This study was undertaken to elucidate the respective roles of serum FSH and LH, as well as that of oestradiol concentrations following down-regulation by pituitary suppression, in predicting ovarian response to stimulation with FSH-HP alone.
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Materials and methods |
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All women underwent pituitary down-regulation with a long protocol, using leuprolide acetate (Lupron, TAP Pharmaceuticals, Abbott Park, IL, USA), starting in the midluteal phase of the preceding cycle with a subcutaneous daily dose of 1.0 mg in oocyte donors and 0.5 mg in IVF patients. Lupron was discontinued on the first day of menstruation in 33 IVF patients pre-judged to be `poor' responders (IVF-`stop Lupron' protocol; IVF-SL), based on elevated basal FSH concentrations on day 3 of a previous unstimulated cycle and/or a history of low response to prior stimulations (peak oestradiol 600 pg/ml and yield of
4 mature oocytes; Faber et al., 1998). In the remaining 38 IVF patients (IVF-`luteal Lupron' protocol; IVF-LL), as well as in all oocyte donors, the Lupron dose was reduced to 0.25 mg/day at onset of menses and maintained until human chorionic gonadotrophin (HCG) administration.
Apart from the fact that all women had menstruated while on Lupron, and that all had oestradiol < 100 pg/ml at start of stimulation, no absolute value of oestradiol after pituitary down-regulation was considered mandatory so as to designate adequate pituitary ovarian suppression. Thus, ovarian stimulation with FSH-HP (Fertinex, Serono Laboratories, Randolph, MA, USA) was started in all patients on cycle day 3, regardless of the oestradiol value on the same day, given that there was no cystic structure in the ovaries on ultrasound. All patients with cysts on day 3 were excluded from the study. There were no other exclusion criteria. The starting dose of Fertinex, 450600 IU/day in the IVF-SL group, and 150300 IU/day in all other patients, was kept constant for the first 4 days of stimulation in each patient, after which it was individualized according to ovarian response based on follicular growth on ultrasound and oestradiol concentrations. Transvaginal oocyte retrieval was performed 3536 h after i.m. administration of 10 000 IU of HCG (Profasi, Serono Laboratories), given when at least three follicles were 16 mm. Evaluation of oocyte maturity, gamete processing, culture techniques and transfer methods were in accordance with previous publications (Veeck et al., 1983
; Muasher et al., 1985
).
Oestradiol concentrations were measured on day 3 and at intervals deemed clinically necessary for monitoring of ovarian response to stimulation, utilizing a microparticle enzyme immunoassay (IMx Estradiol, Abbott Laboratories, Abbott Park, IL, USA), which was sensitive at 25 pg/ml. Sera from day 3 bloods were frozen-stored and assayed for FSH and LH at the end of the study period, using microparticle enzyme immunoassays (IMx FSH and IMx LH, Abbott Laboratories), with sensitivities of 0.2 IU/l and 0.5 IU/l respectively.
All hormonal data were log transformed prior to analysis to correct for skewness. Differences between the three study groups in patient characteristics, down-regulated hormone concentrations and outcome measures of ovarian response to stimulation, including serum oestradiol on day 5 of stimulation and day of HCG administration, as well as duration of stimulation, numbers of ampoules used and oocytes retrieved, were examined by analysis of variance (ANOVA). Within each patient group, multiple regression was used as a first step to evaluate relationships of down-regulated hormone concentrations with the outcome measures. Age, weight and body mass index (BMI) were included in the regression models as potential confounding variables. To facilitate presentation, furthermore, patients in each study group were divided into subsets, using cut-offs for FSH, LH and oestradiol concentrations after down-regulation set arbitrarily at the 33rd, 50th and 66th percentiles due to the skewness of the distribution of each hormone. Differences in outcome measures between subsets of patients thus generated were examined by ANOVA, with Tukey's honest significant difference test for post hoc comparisons. Effects of potential confounding variables were controlled for using the general linear model. Any significant cut-off identified by ANOVA was used to partition patients into two discrete groups, for which results were presented. P < 0.05 was considered statistically significant. Results of regression analyses were presented as coefficients of determination (R2) and regression coefficients (B), and descriptive data as means with standard deviations (SD), means with standard errors of the means (SEM) or means with 95% confidence intervals (CI), whichever was appropriate.
The study population of 129 patients does not include nine patients who were cancelled prior to oocyte retrieval. Cancellation was caused by non-response to either ovarian stimulation (n = 8) or hyperstimulation (n = 1), and involved one patient in the IVF-LL group, five in the IVF-SL group, and three donors.
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Results |
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Discussion |
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It is generally accepted that FSH and LH differ with regard to mechanisms regulating their synthesis and release, as evidenced by relatively little effect of anti-GnRHa serum on plasma FSH, although its administration acutely abolishes pulsatile LH secretion (Lincoln and Fraser, 1979; Culler and Negro-Vilar, 1986
). Likewise, administration of GnRHa results in a dose-dependent and progressive fall in LH concentrations, which lasts for at least 2 weeks after discontinuation of treatment, while a relative resistance of FSH to suppression has been demonstrated by various investigators (Calogero et al., 1987
; Smitz et al., 1988
; Broekmans et al., 1996
; Yamashita et al., 1996
). This relative independence of FSH from GnRHa could explain the findings of Cahill et al. (Cahill et al., 1994
), showing FSH concentrations following pituitary suppression to reflect basal concentrations and to similarly predict the number of oocytes recovered. The data presented in this study are in accordance with the above, in that high FSH after pituitary down-regulation was predictive of diminished yield of oocytes in the IVF-LL and donor groups, even though it did not provide additional information in the pre-selected group of `poor' responders treated with the SL protocol.
We believe the finding of higher oestradiol on day 5 of stimulation, a shorter duration of therapy, and need for fewer ampoules in patients with higher FSH after down-regulation in the IVF-LL and IVF-SL groups could imply early follicular recruitment, and deserves further consideration regarding the possibility of incomplete pituitary suppression in women destined to produce fewer oocytes. In fact, a premature increase in FSH, with resultant early follicular recruitment, has been suggested as an explanation for the higher basal oestradiol concentrations commonly observed in women with diminished ovarian reserve (Licciardi et al., 1995; Smotrich et al., 1995
). Whereas a positive correlation in the IVF-LL group between FSH after down-regulation and follicular size on ultrasound on day 5 of stimulation (R2 = 0.274; P = 0.007) seems to favour the above implication, it remains to be seen if oocyte yield can be improved by overcoming any untoward effect of possible early follicular recruitment, which might hamper the recruitment by FSH-HP of the desired cohort of follicles.
The finding of lower LH concentrations after pituitary down-regulation in the donor group treated with a higher Lupron dose, compared with the IVF-LL group, was predictable in the light of the above literature. It was tempting, on the other hand, to view the unexpected finding of lower mean LH in the IVF-SL group as inherent to this patient population and hence predictive of poor ovarian response. Yet LH after down-regulation did not prognosticate, within any group, outcome of stimulation in terms of peak oestradiol concentrations or number of oocytes retrieved. Furthermore, the vast differences in almost all outcome measures between the IVF-SL and IVF-LL groups, both down-regulated with the same dose of Lupron, could not be explained by a difference in LH concentrations alone, whereas there was no difference in any parameter, except number of ampoules used, between the IVF-LL and donor groups with significantly differing LH concentrations. Thus, it appears likely that the lower mean LH following down-regulation in the IVF-SL group was primarily due to the discrete pituitary suppression protocol used. A similar fall in LH concentrations following withdrawal of the GnRHa has also been reported by others (Sungurtekin and Jansen, 1995; Broekmans et al., 1996
), who have ascribed this phenomenon to some residual agonistic action of GnRHa, withdrawal of which would leave the pituitary devoid of any stimulation until it regained sensitivity to endogenous GnRH.
From a clinical standpoint, very low LH concentrations after down-regulation could cause concern regarding the adequacy of ovarian response to stimulation, since the stimulation agent used contained negligible LH activity (Howles et al., 1994). Treating patients similarly with FSH-HP, it has been shown (Fleming et al., 1996
) that follicular growth in women with mean follicular phase LH < 1.0 IU/l can be compromised, with a slower rise in oestradiol and a longer follicular phase. These investigators, however, were unable to show a concurrent decline in oestradiol concentrations on day of HCG administration or in number of oocytes retrieved in this subset of patients. Likewise, Loumaye et al. (Loumaye et al., 1997
) could not attribute any significance to LH after down-regulation in predicting number of oocytes or oestradiol per oocyte retrieved on day of HCG administration, and concluded that even very low LH concentrations (<0.5 IU/l) do not prognosticate a less favourable outcome. The results of this study agree with the above, in that patients with lower LH after down-regulation in both the IVF-LL and IVF-SL groups had lower oestradiol during the early phase of the cycle. The difference in oestradiol was attenuated, however, by the end of the follicular phase, and patients with lower LH achieved the criteria for HCG administration at the same time as their counterparts, albeit at the expense of, on average, 57 additional ampoules of FSH-HP. These findings are compatible with previous observations that FSH-driven signalling mechanisms from the granulosa can act on the theca in a paracrine fashion to enhance androgen and hence oestrogen synthesis (Smyth et al., 1993
). It should be emphasized, furthermore, that in the donor group, even women with LH < 1.0 IU/l after down-regulation responded adequately to stimulation, without any compromise in oestradiol secretion or need for supplementary medication. Whether the minimal amount of LH necessary for satisfactory ovarian function is even less in younger women with unrestricted ovarian reserve, possibly due to offsetting of any unfavourable effect of inadequate LH concentrations by ample paracrine signalling from an abundant number of follicles, is a tempting postulation that awaits further investigation in view of the higher inhibin concentrations in younger women (Hughes et al., 1990
), and a proven role for inhibin as a modulator of the above-mentioned signalling mechanisms (Hillier et al., 1991
).
Low response to ovarian stimulation early on in the cycle has long been associated with poor IVF results (Pellicer et al., 1987), and dynamic tests of ovarian reserve have been devised based on the observation that a higher oestradiol output in response to stimulation by endogenous (GAST) or exogenous gonadotrophins (EFORT) is predictive of a more favourable outcome (Winslow et al., 1991
; Fanchin et al., 1994
). When stimulating patients with GnRHa on cycle days 24, it has been observed (Winslow et al., 1991
) that patterns of oestradiol secretion on days 3 and 4 differ distinctly among groups of patients with similar concentrations of FSH and LH, and it can be concluded that ovarian rather than pituitary factors determined the oestradiol pattern. Because the protocol used did not mandate achievement of any strict cut-off of oestradiol for confirmation of pituitary suppression, it was possible to estimate the value of oestradiol after down-regulation as well, which we postulated could serve as a marker of baseline ovarian activity, and hence as a predictor of ovarian reserve. In fact, higher oestradiol concentrations after down-regulation were found to prognosticate higher oestradiol on day 5 of stimulation and on day of HCG administration, as well as a shorter duration of treatment and need for fewer ampoules of FSH-HP. Oestradiol concentration after down-regulation could also prove useful in predicting the number of oocytes retrieved, with which it was weakly correlated in the IVF-LL group. In this group of non-selected patients, oestradiol substantially improved the value of FSH after down-regulation in predicting number of follicles as assessed by ultrasound, favouring the above proposition (ß = 0.390; P = 0.04 and ß = 0.813; P < 0.0001 for FSH and oestradiol respectively; R2 = 0.553; P = 0.005).
From a physiological standpoint, on the other hand, it can be argued that the `ovarian factor' cannot be viewed separately from gonadotrophin influence, as evidenced by failure of previous studies in attributing independent significance to basal oestradiol concentrations, distinct from FSH, as a prognosticator of ovarian response (Scott et al., 1989; Toner et al., 1991
). In fact, oestradiol concentrations were found to be significantly correlated with FSH concentrations after down-regulation in the IVF-SL (r = 0.437; P = 0.03) and insignificantly so in the IVF-LL groups (r = 0.300; P = 0.090). On the other hand, no such correlation could be shown in the donor group, which can be explained by a more consistent suppression of FSH secretion, possibly due to the more active negative feedback mechanisms operative in these younger women. These findings do not imply, however, that oestradiol concentrations in the former two groups were driven to any considerable extent by FSH, as after down-regulation differences in FSH concentrations could account for only a small percentage of the variance in oestradiol concentrations among patients in both the IVF-SL (R2 = 0.191) and IVF-LL groups (R2 = 0.090). We believe that it is the larger part of the variance in oestradiol concentrations after down-regulation, which is not accounted for by the residual variance following pituitary down-regulation in either gonadotrophin, that reflects differences in the `ovarian factor' between individuals and proves valuable as yet another, but less complicated and costly, means to evaluate the potential of the ovary to respond to stimulation.
It can be concluded that higher oestradiol concentrations following pituitary desensitization predict a more favourable pattern of ovarian response to ovarian stimulation for IVF, whereas high down-regulated FSH concentrations predict a diminished oocyte yield, and may also imply early follicular recruitment. While the data in this study are in line with the notion of a facilitating role for LH in ovarian steroidogenesis, down-regulated LH concentrations are not further predictive of outcome of stimulation.
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Notes |
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1 To whom correspondence should be addressed
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References |
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Submitted on June 2, 1998; accepted on January 21, 1999.