1 Department of Endocrine Gynaecology and Reproductive Medicine, Hôpital Jeanne de Flandre, C.H.R.U., 59037 Lille, France and Faculty of Medicine, 59000 Lille, 2 Laboratory of Endocrinology, Clinique Marc Linquette, C.H.R.U., 59037 Lille, 3 Department of Radiology, Hôpital Jeanne de Flandre, C.H.R.U., 59037 Lille, France
4 To whom correspondence should be addressed. Email: ddewailly{at}chru-lille.fr
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Abstract |
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Key words: anti-Müllerian hormone/FSH/functional hypothalamic amenorrhoea/inhibin B/ovarian follicle
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Introduction |
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The primum movens of FHA is a disordered frequency pattern of hypothalamic GnRH secretion (Santoro et al., 1986; Perkins et al., 2001
). Consequently, the LH pulse frequency and amplitude are significantly reduced compared to controls leading to a low serum level of this hormone, while its response to the GnRH stimulation test is variable (Leyendecker and Wildt, 1983
). Although the LH pattern has been extensively evaluated in FHA, less is known about the FSH secretion. It is commonly thought that FSH deficiency acts in concert with the disruption of LH pulsatility to impair ovarian function in FHA. However, most of the authors reported normal serum FSH levels in FHA (Alvero et al., 1998
; Couzinet et al., 1999
; Tschugguel and Berga, 2003
). In one study about recreational women runners, a situation close to FHA but with fewer menstrual disturbances, FSH insufficiency was restricted to a blunted elevation of the serum level during the luteal-follicular transition (De Souza et al., 1998
). So, the question arises as to whether there is truly a quantitative FSH deficiency in FHA.
Knowing the physiological effects of FSH on antral follicle growth, if there were such a deficiency, one would expect the ovaries of women with FHA to be depleted from visible non-dominant follicles (i.e. 29 mm in diameter) at ovarian ultrasound (ultrasound). Although the data available in the literature are scarce, the ovaries actually appeared to be multifollicular in some studies (Adams et al., 1985; Futterweit et al., 1988
), making them difficult to distinguish from polycystic ovaries (Futterweit et al., 1988
). It is only in severe states of FHA, such as anorexia nervosa, that the ovaries appear small and amorphous at ultrasound (Treasure et al., 1988
). In such patients, weight gain leads to the appearance of multifollicular ovaries (Treasure et al., 1988
). Therefore, we hypothesize that the FSH deficiency in FHA is most often subtle and does not impair the antral follicle growth, until follicles reach the stage of being selectable at a diameter of
5 mm (Gougeon, 1996
). Ultimately, however, it would impair the shift from non-selectable to selectable follicles, which occurs in normal women during the lutealfollicular transition (so-called FSH window) (Macklon and Fauser, 2001
). This phenomenon is highly FSH dependent and it precedes the selection process which is under the influence of LH (Gougeon, 1996
). The multifollicular appearance of the ovaries in women with FHA could thus be explained by the accumulation of non-selectable follicles, due to the lack of inter-cycle FSH rise.
The validity of ultrasound to analyse the above-mentioned shift had been previously reported by Pache et al. (1990) in normal women. They showed that the 25 mm (non-selectable) follicles are observed at each stage of the cycle while the 69 mm (selectable) follicles appear only in the early follicular phase (EFP). Accordingly, in our study, we considered that a follicle size of 5 mm at ultrasound represented the threshold between selectable and non-selectable follicles, as also suggested by our previous studies documenting the follicular arrest in polycystic ovary syndrome (PCOS) (Pigny et al., 2003
; Jonard et al., 2003
). Therefore, we used again this stratification of ultrasound data to document the putative FSH insufficiency in FHA.
Besides ultrasound, some biological ovarian markers may also be helpful in evaluating the gonadotrophin defect of FHA. In normal women in EFP, inhibin B is secreted by granulosa cells in response to the inter-cycle FSH rise, just before the induction of estradiol (E2) secretion (Hayes et al., 1998). Only two studies have previously addressed the inhibin B status in FHA (Petraglia et al., 1998
; Casper et al., 2000
). The basal level was considered as normal in the study by Petraglia et al. (1998)
, but their norms were significantly lower than those published later in a larger series (Pigny et al., 2000
). In the study by Casper et al. (2000)
, the increase of inhibin B was not significant after GnRH stimulation while FSH levels doubled and LH levels increased 12-fold. However, the studied population did not exclusively contain patients with FHA. Lastly, there are no data concerning the anti-Müllerian hormone (AMH) in FHA. Recent studies indicated that it is a good marker of the small (i.e. 25 mm in diameter) antral follicule number (FN), both in normal women in EFP (Van Rooij et al., 2002
; Fanchin et al., 2003
) and in patients with PCOS (Pigny et al., 2003
). In these studies, the serum AMH level also appeared to be negatively linked to the FSH level, which is in agreement with experimental data showing a negative effect of FSH on AMH secretion (Baarends et al., 1995
).
In this study, we combined biological follicle markers to ultrasound FN within two different size ranges to test the hypothesis that the putative FSH deficiency in FHA impairs the terminal growth and/or functions of antral follicles.
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Materials and methods |
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Controls
The control population consisted of 30 healthy women, whose mean age and body mass index (BMI) are indicated in Table I. They were referred for IVF because of tubal and/or male infertility. Exclusion criteria included a history of menstrual disturbances (i.e. cycle length either <25 days or >35 days), hirsutism, abnormal serum level of prolactin or,rogens (i.e. serum testosterone and/or and rostenedione >0.7 or 2.2 ng/ml respectively), polycystic ovaries (PCO) at ultrasound according to our criteria (Jonard et al., 2003), BMI >25 kg/m2 and hormonal treatment during the 3 months prior to the study.
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Exclusion criteria were hirsutism and/or elevated serum levels of androgens (i.e. serum testosterone and/or androstenedione >0.7 or 2.2 ng/ml respectively) and/or PCOS and/or PCO features at ultrasound (Jonard et al., 2003), hyperprolactinaemia, pituitary insufficiency, idiopathic hypogonadotrophic hypogonadism, Kallmann's syndrome, premature ovarian failure and a history of drug abuse or hormonal treatment during the 3 months prior to the study. Any patient had ultrasonic exclusion criteria (see below).
Blood sampling was performed in the EFP (i.e. between days 2 and 7 after beginning of bleeding) in control women, as previously described (Pigny et al., 2000). All FHA patients received didrogesterone (10 mg/day for 7 days). In the case of uterine bleeding (39% of patients), blood sampling was performed between days 2 and 7 after the beginning of the bleeding. In the absence of bleeding, blood sampling was performed 7 days after didrogesterone withdrawal.
Hormonal immunoassays
E2, inhibin B, testosterone, and rostenedione, LH and FSH were measured prospectively by immunoassays as described previously (Pigny et al., 2000).
Serum AMH levels were measured retrospectively after thawing stored serum samples. They were performed in duplicate using an ultra-sensitive enzyme-linked immunosorbent assay (AMH-EIA; Beckman Coulter, France) according to the supplier's instructions. Results are expressed in pmol/l using human recombinant AMH as a standard. The detection limit of this assay using the ultra-sensitive protocol is 0.7 pmol/l (Pigny et al., 2003). This assay could not be performed in 10 patients with FHA, due to the absence or insufficient quantity of stored serum sample.
Pelvic ultrasound examination
ultrasound examination was performed on the same day as blood sampling with a 7 MHz transvaginal transducer (Logic 400; General Electric, USA). Ultrasound measurements were taken in real time, according to a standardized protocol, as previously reported (Jonard et al., 2003). All follicles of <9 mm but >2 mm were counted according to our previously reported method (Jonard et al., 2003
).
Patients in whom transvaginal ultrasonography was inappropriate (virgin or refusing patients) were excluded, as well as those in whom no follicle was seen in either the right or the left ovary and/or in whom the ovarian area was below the lower normal limit, i.e. 2.5 cm2. Patients with at least one follicle >9 mm in diameter at ultrasound, or a serum E2 level >80 pg/ml, were also excluded from the study so as not to confound the data with the presence of a dominant follicle.
Statistical methods
Within each follicle size range (i.e. 25 and 69 mm), the data used for statistical analysis was the mean of observed FN for the left and right ovaries. Statistical significance between mean values was attributed to two-tailed P<0.05. Comparisons of two independent groups were made using Student's t-test or the 2-test, as appropriate. Significant relationships between the various parameters were evaluated by the non-parametric Spearman correlation coefficient. Influence of the day of sampling was assessed by analysis of variance (ANOVA). All statistical procedures were run on Statview 4.5 (Abacus Concepts Inc., USA).
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Results |
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The separate counts of the 25 and the 69 mm follicles indicated that the mean value of the former was similar between the two groups, while the mean value of the latter was significantly less in FHA than in the control group (Figure 1A and Table I). Univariate analysis using the Spearman correlation test indicated significant negative and positive relationships between FSH and the 25 and 69 mm FN respectively in the FHA group exclusively (Table II and Figure 2A and B respectively).
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Nine (29%) and 22 (71%) patients with FHA had a basal serum FSH level below and above 4.5 IU/l (5th percentile of control values) respectively. The mean value of the 69 mm FN was significantly lower in the low FSH group than in the normal FSH group (0.3±0.7 versus 1.5±1.5 IU/l respectively, P=0.02), the latter being similar to controls (Figure 4A). On the other hand, no significant difference was found for the mean AMH and inhibin B serum levels between the two subgroups, every mean value remaining significantly higher and lower than in controls, in both subgroups, respectively (Figure 4B). In patients with normal FSH (>4.5 IU/l), the ratio AMH:25 mm FN was significantly higher than in controls (8.4±3.8 versus 5.4±2.9 respectively, P=0.007), whereas it was similar to controls in the low FSH group (5.3±1.6, not significant).
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Discussion |
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Our ultrasound data document for the first time that the ovarian marker best related to low FSH serum levels (i.e. <4.5 IU/l) in FHA patients is the paucity in 69 mm follicles. Presumably, this finding reflects the impaired shift from the 25 mm to the 69 mm follicles, which is known to be highly FSH dependent and occurs during the FSH inter-cycle peak in normal women (Macklon and Fauser, 2001). That the 25 mm FN was normal in our patients with FHA having a low basal FSH level could seem paradoxical since the growth of these small antral follicles is also FSH dependent (McGee and Hsueh, 2000
). To explain this, we hypothesize that the aforementioned impaired shift was responsible for the stagnation of these follicles. The negative relationship that we found between the FSH serum level and the 25 mm FN in our patients with FHA argues further for the responsibility of the relative FSH insufficiency in this impaired shift. This phenomenon may explain why ovaries in FHA frequently appear as multifollicular (Adams et al., 1985
; Futterweit et al., 1988
). Also, that no relationship was found between serum FSH level and the 25 mm or the 69 mm FN in controls could be explained by the fact that most of them were investigated during the second part of their FSH window; a time when the role of FSH on follicle maturation declines (Macklon and Fauser, 2001
). This emphasizes the need for an early work-up in the cycle (no later than day 3) if one wishes to analyse the relationships between FSH and follicle maturation or number during the FSH window in normal women.
If one relied on their normal basal FSH level, the majority (71%) of our patients with FHA could be considered as having no FSH insufficiency. However, at the follicle level, they displayed features of FSH insufficiency, as reflected by the lower mean inhibin B serum level and the lack of significant relationship between this parameter and the 69 mm FN, whereas in controls this relationship tended to be significant. Indeed, the positive association between inhibin B and the 69 mm FN in our controls indicates that these follicles are the main source of this hormone, which is in agreement with physiological studies (Hayes et al., 1998). The fact that patients with a normal serum FSH level have a low serum inhibin B level despite the presence of a normal number of 69 mm follicles suggests that the FSH-dependent secretion and growth functions are dissociated in these follicles. It has been theorized that the FSH threshold is lower for inducing follicle growth than for inducing GC differentiation (Gougeon, 1996
). According to this theory, such a finding could indicate a partial loss of FSH activity at the follicle level which is not properly reflected by the routine assay of its serum level.
AMH was positively related to the 25 mm FN in our whole group of patients with FHA, as previously reported in normal women (Seifer et al., 2002; Van Rooij et al., 2002
; Fanchin et al., 2003
) and in patients with PCOS (Cook et al., 2002
; Pigny et al., 2003
). However, the absolute mean AMH level was significantly higher than in controls despite similar 25 mm FN. One could relate this finding to FSH insufficiency since experimental data have shown a suppressive effect of FSH on the AMH secretion by GC from 25 mm follicles (Baarends et al., 1995
). Nevertheless, the increased AMH serum levels were independent from the serum FSH levels, which is in contrast to controls. In addition, the mean AMH level was still excessive in the subgroup of patients with normal serum FSH and the mean ratio AMH:25 mm FN was higher than in controls. Although we cannot exclude the possibility that higher AMH levels in FHA are due to an excess of follicles that are below the range of detection by ultrasound, our data rather suggest a higher AMH secretion by each follicle and therefore an impaired FSH activity at the follicle level. Whether both inhibin B and AMH abnormalities are secondary to alteration in the pulsatile secretion of FSH and/or in its bioactivity and/or in follicle sensitivity was not addressed in our study and would deserve further investigation. Nevertheless, the fact that the ratio AMH:25 mm FN was normal in patients with a low FSH serum level argues against an intrinsic FSH resistance in the follicles of women with FHA. These findings in FHA differ from those that we previously reported in PCOS (Pigny et al., 2003
). Indeed, in the latter situation, the increase in AMH serum level could be explained by the excess in the 25 mm FN per se and it occurred despite the maintenance of the negative effect of FSH on AMH (Pigny et al., 2003
). From both data in FHA and PCOS, we hypothesize therefore that the serum AMH level in women not only reflects the number of small antral follicles (25 mm), but also the maturation stage of these follicles, and more especially their degree of exposure and/or sensitivity to bioactive FSH.
In conclusion, our ultrasound data document for the first time that the paucity in the 69 mm FN directly reflects the low FSH level in FHA, which is infrequent, however. Despite a normal serum FSH level and 69 mm FN in the majority of patients with FHA, the functional follicle markers of the FSH sensitivity during the EFP (inhibin B, AMH) are abnormal, suggesting that FSH is not fully active.
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Acknowledgements |
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References |
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Submitted on June 2, 2004; resubmitted on August 10, 2004; accepted on September 17, 2004.