1 Department of Obstetrics and Gynaecology, University of Alberta, Edmonton and 2 Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, Canada
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Abstract |
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Key words: androgens/ovarian stimulation/PCOS/recombinant FSH/recombinant LH
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Introduction |
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Clinically, persistent elevation of LH during the follicular phase has been associated with decreased pregnancy rates and increased spontaneous pregnancy loss rates in PCOS, (Homburg et al., 1988; Regan et al., 1990
) although other investigators have not found any significant differences in pregnancy rates (Larsen et al., 1990
; Sagle et al., 1991
). Nevertheless, these associations underpin the rationale for using highly purified urinary FSH (containing negligible amounts of LH) and more recently, recombinant FSH (devoid of LH), for ovulation induction in women with PCOS instead of hMG which contain FSH:LH in a 1:1 ratio. On the other hand, profound suppression of LH has been negatively associated with poorer oocyte retrieval and fertilization indices in women undergoing IVF treatment (Fleming et al., 1998
; Anderiesz et al., 2000
; Westergaard et al., 2000
). Currently, the optimal LH range during gonadotrophin treatment for the various clinical categories, including PCOS, is unknown; nor is the impact of altered LH levels on steroidogenesis, particularly ovarian thecal androgen production that may impact on normal follicle development. The recent availability of recombinant LH (rLH) and FSH (rFSH) preparations has provided clinical in-vivo probes to examine both the individual and combined effects of these two gonadotrophins on ovarian androgen secretion.
The main purpose of this study, therefore, was to compare androgen secretion in PCOS women, during ovarian stimulation with either rFSH alone or combined with rLH in a 1:1 ratio, similar to that found in hMG. Previous studies examining the additional effects of LH by comparing highly purified urinary FSH with hMG have been hampered by residual LH activity and/or measurable hCG in these two gonadotrophin preparations (Larsen et al., 1990; Sagle et al., 1991
; Filicori et al., 2001
). Because small changes in circulating LH levels following rLH administration and corresponding thecal androgen secretion could be masked by endogenous LH release and adrenal androgen production respectively, this study included subgroups of women with PCOS pre-treated with GnRH agonist (to suppress pituitary LH release) and GnRH agonist combined with dexamethasone [to suppress adrenocorticotrophic hormone (ACTH) and adrenal androgens] to specifically examine these important hormonal interactions.
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Materials and methods |
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Study design and treatment protocol
This was a prospective, controlled study conducted at a university-based fertility centre. The study was approved by the Research Ethics Committee of the University of Alberta and informed consent was obtained from all subjects. Forty-eight subjects were recruited for this study. The study design was a 2x3 (between-group) balanced factorial design (Winer, 1971) with time as a repeated-measures component (Figure 1
). The first between-group factor of the factorial design was the two gonadotrophin regimens, rFSH alone (n = 24) versus rFSH + rLH in a 1:1 ratio (n = 24) as in conventional hMG preparations. These two gonadotrophin regimens were then further divided into three subgroups (the second between-group factor) as follows: (i) gonadotrophin stimulation alone (None); (ii) nafarelin suppression (Naf); and (iii) nafarelin suppression with dexamethasone (Naf + Dex).
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All subjects had a transvaginal ultrasound evaluation in addition to a serum progesterone test to confirm anovulation before initiating any hormone treatment. Subjects in the None subgroup began gonadotrophin stimulation on the second or third day of withdrawal bleeding following a 10 day course of medroxyprogesterone acetate 10 mg per os daily (Provera®; Upjohn). In the Naf and Naf + Dex subgroups, intranasal nafarelin was started at the higher dose of 400µg twice a day for 6 weeks, to ensure maximal pituitaryovarian suppression before gonadotrophin administration. On the first day of gonadotrophin therapy, nafarelin was reduced to the standard dose of 200 µg twice daily and continued up to the day of hCG. Dexamethasone 0.5 mg per os four times daily was initiated 4 days before gonadotrophin stimulation; on the second day of stimulation the dosage was reduced to 0.5 mg nightly and continued until the onset of menstrual flow or confirmation of a pregnancy. There were two phases of gonadotrophin induction. During the initial 5 days of stimulation, subjects received a fixed dose of 150 IU rFSH (2 ampoules) daily with or without the equivalent dose of rLH. In the next phase, the gonadotrophin dosage was adjusted according to serial plasma estradiol (E2) and ultrasound monitoring. Doses were adjusted by 0.51 ampoules (37.575 IU) when the exponential rise in E2 was anticipated, based on the trend of E2 and ultrasound results from preceding days. Twenty-four hours after the last gonadotrophin dose, the patient received hCG 10 000 IU when there was one leading follicle with a minimal mean diameter of 15 mm.
Blood samples were drawn at 0800 h following 20 min of rest at the Clinical Investigation Unit of the University of Alberta. Just before receiving the first gonadotrophin dose, three baseline samples were collected 20 min apart between 0700 and 0800 hours and were pooled for subsequent hormone assays. All samples were taken after an overnight fast and before the morning dose of nafarelin and gonadotrophins. An aliquot of each subjects serum sample was immediately used for E2 measurement in accordance with the clinical protocol outlined above; the remaining serum was stored at 20°C until gonadotrophin and androgen assays at study completion.
Hormone assays
LH and FSH levels were measured by microparticle enzyme immunoassay based on chromofocusing techniques (ImxTM; Abbott Diagnostics, IL, USA) (Cheung and Chang, 1995) which incorporates two high-affinity antibodies for higher sensitivity and specificity compared to the traditional radioimmunoassay. The assay sensitivity for LH was 0.5 IU/l and the intra- and inter-assay coefficients of variation (CV) were 36 and 6% respectively. Corresponding values for FSH were 0.2 IU/l, 2 and 46%. Steroids were measured using commercial radioimmunoassay kits: 17-OHP, testosterone and E2 from Diagnostic Products Corporation (Los Angeles, CA, USA) and
4 from Diagnostic Systems Laboratories, Inc. (Webster, TX, USA) which had assay sensitivities of 0.21 nmol/l, 0.13 nmol/l, 29 pmol/l and 0.10 nmol/l respectively. The corresponding intra- and inter-assay CV were 3.56.7 and 511% for 17-OHP; 3.811 and 6.411% for testosterone; 4.07.0 and 4.28.1% for E2; and 2.46.3 and 8.212.5% for
4.
Statistical analysis
The results were reported as means ± SEM. Between-gonadotrophin regimens and between-subgroup differences and their interactions were compared by analysis of variance (ANOVA). Time-related differences in hormone levels were evaluated by repeated measures ANOVA. Significant overall effects were followed by pairwise comparison using the Bonferroni t-procedure. Specific cycle parameters were also compared by ANOVA: the duration of gonadotrophin induction (days); the total number of ampoules of rFSH required; and on the day of hCG injection, the number of mature (15 mm) follicles, medium-sized (1014 mm) follicles and endometrial thickness on ultrasound assessment and the E2 levels. Statistical analysis was performed using Statistica® 6.0 (Statsoft Inc., 2001; Tulsa, OK, USA) and the level of significance was defined at P < 0.05 (two-tailed).
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Results |
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Despite this measurable LH increase with rFSH + rLH stimulation in the Naf subgroup, 17-OHP, 4 and testosterone levels were not significantly different between the two gonadotrophin regimens in the early, fixed-dose phase of the study (day 1 and day 5, Figure 3B, E, H
). In contrast, when the adrenal contribution of these steroids was suppressed by concomitant dexamethasone treatment, there was an early rise of 17-OHP,
4 and testosterone levels on day 1 with rFSH + rLH which was absent with rFSH stimulation (Naf + Dex; Figure 3C, F, I
). There was also a trend toward higher steroid levels in the pre-ovulatory, dose-adjusted phase (day hCG 1, day hCG) with rFSH + rLH which did not reach statistical significance, except for testosterone levels in the Naf subgroup (Figure 3H
) and 17-OHP levels in the Naf + Dex subgroup (Figure 3C
). In the latter subgroup, 17-OHP levels fell markedly on day hCG in those receiving only rFSH (Figure 3C
).
To ensure that interpretation of the above findings was not confounded by differences in the degree of ovarian stimulation between rFSH and rFSH + rLH treatment, FSH and E2 levels were compared as a reference. As expected, significant, time-related increments in FSH and E2 levels were seen during gonadotrophin stimulation (P < 0.0001 for both) but the levels achieved were not significantly different between the two gonadotrophin regimens at any time-point; nor did nafarelin (with or without dexamethasone) influence the absolute levels of either FSH (Figure 4A, B, C) and E2 (Figure 4D, E, F
). However, there was a trend to lower E2 levels with rFSH stimulation in subjects receiving nafarelin but this trend was less obvious in those also receiving dexamethasone. There was also a more rapid rate of E2 increase on day 5 in the Naf + Dex subgroups.
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Discussion |
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The differential effects of rFSH alone versus rFSH + rLH treatment have directly confirmed the role of LH in stimulating 17-OHP, 4 and testosterone secretion in vivo. Previous studies have only indirectly shown this effect using the simultaneous release of supraphysiological levels of both FSH and LH with a potent GnRH agonist (Barnes et al., 1989
) in which LH (if using an identical immunometric assay as the current study) can increase to >100 and >40 IU/l at 4 and 24 h respectively. In contrast, LH levels following rLH administration in our study are well within the range observed in the normal menstrual cycle, and, in those subgroups pre-treated with nafarelin, are actually below the nadir observed in the luteal/early follicular phase of spontaneous cycles. LH levels in the rFSH group treated with nafarelin remained close to the assay sensitivity of 0.5 IU/l of a newer monoclonal immunometric assay based on chromofocusing techniques. When rLH was added, mean LH levels increased to between 1.37 and 1.73 IU/l (Figure 2
). It is likely that peak LH levels were higher given the short half-life of circulating LH (Catt and Dufau, 1991
; le Cotonnec et al., 1998
) and that blood samples were collected 24 h after rLH injection. Combined with observations in a previous study that showed LH levels could increase by 34%, peaking at 4 h after a single i.m. injection of hMG (150 IU) (Anderson et al., 1989
), the estimated peak levels following rLH administration in our study would still fall to <23 IU/l. Filicori et al. observed a similar rise in LH levels which were measured at 16 h after hMG injection in ovulatory women undergoing ovarian stimulation and intrauterine insemination (Filicori et al., 2001
). In addition to using different study populations, their findings must be distinguished from our data by the measurable hCG in the hMG preparation used which would confound any assessment of a pure LH effect, by the inability to exclude residual LH activity in highly purified urinary FSH preparations, and finally by the lesser degree of LH suppression where baseline LH levels were >1 IU/l.
With LH and concomitant adrenal androgen suppression, we observed a difference in temporal patterns in steroid secretion. The early day 1 rise in 17-OHP, 4 and testosterone was absent when only rFSH was given (Figure 3C, F, I
), but by day 5 these steroid levels were comparable to those observed with rFSH + rLH. In addition, despite profoundly suppressed LH levels in Naf and Naf + Dex subgroups stimulated with rFSH alone, a small rise in these three steroids was still observed, albeit delayed. This rise continued to day hCG 1/day hCG (Figure 3C, F, I
). One possibility for this delay might be a greater reliance on evolving paracrine mechanisms (involving factors such as inhibin, insulin-like growth factor and androgens) as follicles mature under profound LH suppression (Hillier, 1994
, 2001
). In monkeys, androgen receptor mRNA and FSH receptor mRNA have been co-localized and positively correlated in granulosa cells of growing follicles (Weil et al., 1999
). Further, FSH can induce acquisition of androgen receptors whereas androgen stimulates FSH receptors in granulosa cells of small follicles (Weil et al., 1999
). It is therefore tempting to speculate that similar interactions between FSH and androgen might also be in place in humans and compensate for suppressed LH effects during follicular development. Such interactions would potentially have a greater effect in PCOS subjects, known to have exaggerated intrinsic basal and LH-stimulated thecal androgen output (Gilling-Smith et al., 1994
).
Defining optimal FSH and LH relationships and establishing optimal LH ranges for ovarian stimulation of various clinical categories is a therapeutic consideration receiving wider attention (Anonymous, 1998). Hillier et al. first described the concept of an optimal LH ceiling for the follicular phase, which, if exceeded, would result in suboptimal ovulatory function and fertility rates (Hillier, 1994
). Westergaard et al. proposed a minimal LH requirement or threshold using a level of 0.5 IU/l to demarcate profound and normal LH suppression in ovulatory women undergoing IVF (Westergaard et al., 2000
). In our study, baseline LH levels were suppressed to
0.5 IU/l (below assay sensitivity), corresponding to Westergaards profound category.
The European Recombinant Human LH Study Group has recently demonstrated the beneficial effects of adding LH to the ovarian stimulation regimen for IVF patients which underscores the clinical importance of determining optimal LH thresholds during ovarian stimulation (Anonymous, 1998; Anderiesz et al., 2000
). Correspondingly, our data provide some relevant information specifically for women with PCOS. The fact that E2 response, follicle development and successful conceptions were comparable with both rFSH and rFSH + rLH regimens after pituitary suppression indicates indirectly that sufficient theca androgen secretion was present to provide substrate for adequate estrogen production and follicle growth, a finding also noted in IVF patients (Balasch et al., 2001
). Sullivan et al. observed that once the follicle achieved a diameter of 14 mm, FSH or LH was equally effective in sustaining granulosa E2 output (Sullivan et al., 1999
). Our findings therefore suggest that provision of continued rFSH stimulation in the face of profoundly suppressed endogenous LH levels (
0.5 IU/l) is still adequate for follicular function and pregnancy in women with PCOS, even when ovarian and adrenal androgen secretion are concomitantly suppressed.
The lack of significantly different E2 levels and mature follicle numbers between the gonadotrophin regimens might also reflect our stimulation protocol of inducing ovulation with hCG once a follicle of 15 mm had been identified, inter-individual variations in response to FSH stimulation relative to the subject sample size. Although not statistically significant, the increased number of medium-sized follicles in the Naf + Dex subgroup receiving only rFSH (Table III
) raises the possibility that dexamethasone may exert an anti-apoptotic effect on granulosa cells as reported in cell culture experiments (Sasson et al., 2001
).
The abrupt increase in 17-OHP and 4 24 h after rLH was added further confirms that the corresponding enzyme system, CPY17, is indeed LH responsive as suggested by previous in-vitro observations (Bogovich and Richards, 1982
). Furthermore, we have demonstrated that this enzyme complex is responsive in the low LH milieu of our study and in the early follicular phase of ovulation induction. Whether this enzyme system in subjects with PCOS is more sensitive to LH is being investigated in a parallel study involving normal ovulatory women. It should be noted that this study only measured steroid intermediates of the
4 pathway and that specific contributions from the
5 pathway via 3ß-hydroxysteroid dehydrogenase/isomerase for the relevant androgens in the follicular phase are unknown.
In conclusion, by contrasting the effects of rFSH and rFSH + rLH with and without nafarelin, and nafarelin with and without dexamethasone, we have successfully demonstrated a significant rise in LH when rLH is added during ovulation induction. This LH increase, although small, is sufficient to stimulate 17-OHP, 4 and testosterone secretion early in folliculogenesis, an effect only revealed by first suppressing endogenous LH and ACTH. Despite profound LH and 17-OHP,
4 and testosterone suppression, comparable E2 response, follicle development and successful pregnancies in PCOS subjects receiving rFSH alone to those receiving rFSH + rLH, would argue that circulating LH at levels as low as 0.5IU/l are sufficient to sustain adequate follicular development and function when FSH is present in abundance. Definition of optimal LH ranges for various clinical conditions remains to be elucidated, but is clearly an evolving concept that could change our clinical approach to gonadotrophin therapy.
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Acknowledgements |
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Notes |
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References |
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Submitted on February 15, 2002; resubmitted on June 20, 2002; accepted on July 7, 2002.