Prospective analysis of the relationships between the ovarian follicle cohort and basal FSH concentration, the inhibin response to exogenous FSH and ovarian follicle number at different stages of the normal menstrual cycle and after pituitary down-regulation

Peter Y.K. Yong1, David T. Baird2, K.Joo Thong1,2, Alan S. McNeilly3 and Richard A. Anderson3,4

1 Assisted Conception Unit, Royal Infirmary of Edinburgh, 2 Department of Reproductive and Developmental Sciences and 3 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, UK 4 To whom correspondence should be addressed. e-mail: r.a.anderson{at}hrsu.mrc.ac.uk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Analyses of the follicular reserve and activity of the ovary are central to our understanding of the regulation of follicular development. We have carried out a prospective analysis of endocrine and biophysical assessments under three differing basal conditions: the early follicular and mid-luteal phases, and following GnRH analogue down-regulation. METHODS: Hormonal analyses were carried out before and after a single dose of FSH on spontaneously ovulating women (n = 58). Ovarian volume and antral follicle count (AFC) were also determined. RESULTS: Inhibin B and estradiol concentrations were increased by FSH under all three conditions, and inhibin A in the follicular phase and after down-regulation. Basal hormone concentrations, except inhibin A and B after down-regulation, did not generally correlate with AFC. A close relationship between inhibin B and AFC was evident at all stages after FSH administration (r = 0.70–0.77). AFC and inhibin B after FSH stimulation were well correlated with the number of oocytes recovered after superovulation. Multivariate analysis demonstrated that inhibin B after FSH administration in the down-regulated state showed the closest correlation with oocyte number. In the more clinically useful early follicular and luteal phases, basal FSH was the most significant contributor to the number of oocytes, with a significant contribution from luteal phase AFC. CONCLUSIONS: These data extend our understanding of the relationships between follicular number, follicular functional activity, and the recruitable follicular population. Down-regulation and subsequent FSH stimulation was required to clearly demonstrate the close relationship between inhibin B and the ovarian reserve. Without such complex manipulation, early follicular phase FSH (supplemented by AFC in the relatively hypogonadotrophic luteal phase) remains of greater value in predicting the ovarian reserve than the currently known direct products of the ovary.

Key words: FSH/follicular development/inhibin B/ovarian reserve/pituitary down-regulation


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovarian follicular development is a continuous process throughout the reproductive lifespan. It is estimated that in a young woman ~10–20 follicles leave the pool of resting primordial follicles each day and start to develop as growing follicles (Gougeon, 1996Go). Since only one follicle is destined to complete maturation and undergo ovulation during each menstrual cycle, the vast majority of oocytes will become atretic. The number of growing follicles at any time-point is believed to be related to the total number of follicles present in the ovary, although the proportion growing increases as the pool of primordial follicles falls approaching the menopause (Faddy et al., 1992Go; Gougeon et al., 1994Go; Faddy and Gosden, 1996Go). The pre-menopausal ovary therefore always contains cohorts of growing follicles that may potentially be stimulated to mature. Treatment with supraphysiological doses of FSH stimulates the development of many large antral follicles by preventing atresia of those secondary follicles that would normally not ovulate (Erickson, 1986Go). The number of follicles developing under such circumstances reflects the total growing pool and thus the total number of follicles present in the ovary. Therefore the major factor determining the response to exogenous gonadotrophins is the number of follicles in the ovary (Baird and Pearson, 1998Go).

There is currently much interest in determining the size of the growing follicular pool. This is of particular value in the context of controlled ovarian stimulation (COS) and IVF to identify those women from whom few oocytes will be recovered or conversely are at increased risk of ovarian hyperstimulation syndrome (OHSS) (Gulekli et al., 1999Go; Bukman and Heineman, 2001Go). The serum concentration of FSH during the cycle is regulated by the negative feedback effect of ovarian hormones, i.e. estradiol and the inhibins (Hayes et al., 1998Go; Baird, 1999Go). FSH concentrations in the early follicular phase show an inverse relationship to the size of the follicular cohort (Chang et al., 1998Go). The rise in FSH concentration after clomiphene citrate administration (clomiphene challenge test) has been shown to be of value in unmasking poor responders to COS who would not have been detected by basal FSH alone (Navot et al., 1987Go; Tanbo et al., 1989Go; Loumaye et al., 1990Go; Gulekli et al., 1999Go). It might be anticipated that direct hormonal products of the ovary would be better markers of ovarian response than indirect markers such as age and FSH. The rise in estradiol concentrations after FSH (Fanchin et al., 1994Go; Phelps et al., 1998Go; Syrop et al., 1999Go; Fabregues et al., 2000Go) or GnRH analogue (Padilla et al., 1990Go; Winslow et al., 1991Go; Ranieri et al., 1998Go; Ravhon et al., 2000Go) administration has been shown to be predictive of IVF success, although high basal estradiol concentrations were associated with increased IVF cycle cancellation and lower pregnancy rates (Licciardi et al., 1995Go; Smotrich et al., 1995Go).

Inhibins are also products of the ovary. Tissue localization studies (Rabinovici et al., 1992Go; Roberts et al., 1993Go; Yamoto et al., 1993Go; Jaatinen et al., 1994Go) and changes in serum levels of inhibin A and B during the menstrual cycle and in response to gonadotrophin manipulation, suggest that inhibin B is secreted predominantly by smaller antral follicles (Groome et al., 1996Go; Welt et al., 1997Go; Anderson et al., 1998Go; Lahlou et al., 1999Go) and inhibin A mainly by the dominant follicle (Groome et al., 1994Go; Lambert-Messerlian et al., 1994Go). Dynamic inhibin B testing with FSH (Lockwood et al., 1996Go; Dzik et al., 2000Go) or GnRH analogue (Ravhon et al., 2000Go) appears to correlate with ovarian response to COS, but the value of basal inhibin B measured in the early follicular phase remains uncertain (Seifer et al., 1997Go; Tinkanen et al., 1999Go; Creus et al., 2000Go; Dumesic et al., 2001Go). Female age was shown to be superior to basal inhibin B at predicting pregnancy following IVF and embryo transfer (Hall et al., 1999Go; Creus et al., 2000Go). Inhibin B measured 4–6 days after starting COS was highly correlated with the number of oocytes retrieved (Eldar-Geva et al., 2000Go; Fawzy et al., 2002Go). However, it would be of greater clinical value to be able to predict follicular cohort size prior to starting GnRH analogue/FSH treatment, and it has been suggested that the inhibin B and estradiol responses to FSH administration can predict later oocyte collection (Dzik et al., 2000Go; Elting et al., 2001Go). Measurement of the number of antral follicles or ovarian volume by high resolution ultrasound scanning have also been shown in some but not all studies to predict ovarian response (Lass et al., 1997Go; Tomas et al., 1997Go; Chang et al., 1998Go; Syrop et al., 1999Go; Dumesic et al., 2001Go). Generally, however, these endocrine and biophysical measures have not been studied prospectively or under basal conditions of differing endogenous FSH concentrations.

The primary objectives of our study were therefore (i) to investigate the relationships between the concentration of FSH in the early follicular phase, the endocrine responses of the ovary (inhibins, estradiol) to exogenous FSH stimulation and the number of antral follicles and ovarian volume, under different hormonal milieu, and (ii) to correlate these endocrine and biophysical measures with the number of oocytes retrieved following COS. The hormonal milieu under which these measures were investigated were the early follicular phase, i.e. during rising/high endogenous FSH concentrations, the luteal phase, i.e. during endogenous FSH suppression by ovarian hormones, and following GnRH analogue down-regulation, i.e. more marked hypogonadotrophism.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
A total of 58 spontaneously ovulating women about to undergo IVF treatment was recruited. Exclusion criteria were: endometriosis (other than minimal); the presence of only one ovary or previous ovarian surgery; polycystic ovary syndrome or other endocrine disorders; exposure to study drugs within 3 months of participation in the study. Serum prolactin and thyroid function test were determined to be normal before inclusion in the study. Ethical committee approval for the study was obtained, and all subjects gave written informed consent. Eight women completed only the pre-IVF component of the study (first four visits) as they had either cancelled or deferred their IVF treatment. A further four women started the study but did not complete the first four visits for the same reasons. Complete data sets were therefore available on 46 women. Subject characteristics are given in Table I.


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Table I. Demographic characteristics of study population
 
Methods
Subjects were studied in the early follicular (days 3–5) and mid-luteal phases (days 20–24) of a spontaneous ovulatory cycle shortly before they started a cycle of IVF as set out in Figure 1. They attended for venesection in the early follicular phase before (visit 1) and 24 h after (visit 2) the s.c. administration of 225 IU recombinant human FSH (rFSH, Gonal-F; Serono, Welwyn Garden City, UK). Venesection was repeated in the mid-luteal phase, again before (visit 3) and 24 h after (visit 4) administration (s.c.) of 225 IU rFSH. Confirmation of an ovulatory cycle was determined by serum progesterone measurement at visit 3 without detailed monitoring of the precise date of ovulation in that cycle; data from two women were excluded because of low progesterone concentrations. Subjects subsequently commenced down-regulation with nafarelin (200 mg intranasally twice a day; Synarel; Searle, Milton Keynes, UK) in the mid-luteal phase within a further 2 cycles. When adequate down-regulation had been achieved following 2 weeks of nafarelin (endometrial thickness <4 mm or serum estradiol <150 pmol/l), venesection was performed before (visit 5) and 24 h after (visit 6) further s.c. administration of 225 IU rFSH, the latter comprising the first day of COS. A final blood sample was taken on the day of administration of hCG. Serum samples were stored at –20°C for subsequent assays of hormone concentrations.



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Figure 1. Schematic representation of the study protocol. Cycle A, first intervention cycle: cycle B, one or two subsequent cycles without intervention: cycle C, pre-IVF cycle, during which down-regulation started in the mid-luteal phase. The timings of study visits in the early follicular phase and luteal phase of cycle A and at the initiation of controlled ovarian stimulation (COS) are indicated. The number of subjects completing each stage of the study is also indicated.

 
Biophysical (ovarian volume and antral follicle count) measurements
Transvaginal ultrasound scans were performed by a single investigator (P.Y.) at visits 1, 3 and 5 to determine ovarian volume and antral follicle count (AFC), defined as number of follicles <10 mm in all diameters, in both ovaries. All ultrasound scans were performed using the same equipment (7 mHz probe; Toshiba Eccocee, Stirling, UK). The limit of sensitivity was 2 mm, and the intra-observer coefficient of variation was 7%. Ovarian volume was calculated using the formula for the volume of an ellipsoid ({pi}/6xlengthxwidthxheight), and the mean volume was determined. Ovaries with cystic enlargements >=15 mm were excluded from the analysis of ovarian volume.

Controlled ovarian stimulation/IVF
Recombinant FSH was continued at a dose of 225 or 150 IU daily, depending on the subject’s previous response to COS and the risk of OHSS. When three or more follicles with diameter >=17 mm were observed, hCG (Profasi; Serono) s.c. 5000 or 10 000 IU was administered and transvaginal oocyte recovery performed ~36 h later. Following IVF, not more than two embryos were transferred 48 h after oocyte recovery. Luteal support was provided with progesterone pessaries (Cyclogest; Shire, Basingstoke, UK) given for 2 weeks following embryo transfer. All subjects completed IVF treatment within 4 months of the first study visit. The women returned 2 weeks after embryo transfer for a pregnancy test. A positive result was followed up 2 weeks later by an ultrasound scan to confirm fetal viability.

Hormonal assays
Inhibin A and B and inhibin forms containing pro- and {alpha}C immunoreactivity (pro-{alpha}C) were measured using two-site enzyme-linked immunosorbent assays using plates coated with specific monoclonal antibodies to the ßA and ßB subunits for inhibin A and B and the {alpha} subunit respectively as previously described (Groome et al., 1994Go, 1995, 1996). Assay sensitivity for inhibin A was 2 pg/ml, for inhibin B was 7 pg/ml and for pro-{alpha}C was 3 pg/ml. Inter- and intra-plate coefficients of variation were <8% and <5% respectively.

Other hormones (FSH, estradiol and progesterone) were measured by radioimmunoassay as previously described (Backstrom et al., 1982Go).

Statistical analysis
All data are presented as mean ± SD values. Comparisons of paired hormone variables before and after administration of rFSH were performed with the Wilcoxon signed-ranks test, and the Mann–Whitney U-test was used for unpaired values. Respective differences in ovarian volume and AFC at visits 1, 3 and 5 were evaluated using the Friedman test. Linear regression analysis was employed to determine correlations hormone variables, AFC, subject’s age and the number of oocytes recovered. Multiple regression analysis performed in stepwise fashion was used to evaluate the relative contribution of each variable to the number of oocytes recovered. Data were not transformed prior to analysis. Bonferroni correction was applied to P-values for correlations with multiple variables, hence P < 0.005 was taken as being statistically significant. P < 0.05 was considered significant for all other analyses.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Changes in inhibin responses to rFSH
Compared to the other stages investigated, the concentration of inhibin A was highest during the luteal phase and of inhibin B during the early follicular phase (Figure 2). The concentration of all three inhibins was lowest in the sample collected after down-regulation (visit 5). Administration of 225 IU rFSH resulted in significant increases in inhibin B and estradiol at all phases and in the concentration of inhibin A in the early follicular and down-regulated phases. In contrast, the concentration of inhibin A was unchanged following rFSH injection in the luteal phase. There was relatively little change in the concentration of pro-{alpha}C after rFSH administration at any stage although there was a statistically significant rise in the early follicular phase. The mean percentage increase in the concentrations of inhibin A and B and estradiol were similar in the early follicular phase (82, 94 and 73% respectively), but at the other endocrine phases the mean rise in inhibin B was much greater than that in the other hormones. The mean rise in inhibin B in the luteal phase was 245% and after down-regulation was 303%, compared to a rise in inhibin A of 105% after down-regulation. All hormones were markedly elevated at the time of hCG administration at the conclusion of COS: estradiol, 5387 ± 3167pmol/l; inhibin A, 1343 ± 627 pg/ml; inhibin B, 1123 ± 853 pg/ml; pro-{alpha}C, 3237 ± 2087 pg/ml.



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Figure 2. Serum concentrations of inhibin A, inhibin B, pro-{alpha}C and estradiol in the early follicular phase, mid-luteal phase and after GnRH analogue down-regulation. Open columns, basal concentrations; hatched columns, 24 h after administration of 225 IU rFSH. Values are mean ± SD. *P < 0.005, **P < 0.0005 versus pre-FSH concentrations.

 
Inhibin A and B were significantly correlated with each other in the early follicular phase (r = 0.54, P < 0.0005), but not in the mid-luteal phase (r = –0.12, P = 0.43). The best correlation was found in the down-regulated phase (r = 0.73, P < 0.0005). Recombinant FSH administration did not alter these correlations appreciably (early follicular r = 0.46, mid-luteal r = 0.15, after down-regulation r = 0.77). The strong correlation following down-regulation may reflect the relative synchrony of follicular development following gonadotrophin withdrawal, whereas follicular development is more heterogeneous under the physiological conditions of the early follicular phase.

Changes in biophysical measures
Significant differences in ovarian volume were detected with highest volume found in the luteal phase, and lowest volume after down-regulation (Figure 3). AFC, however, showed little variation between the endocrine phases (Figure 3).



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Figure 3. Ovarian volume (a) and antral follicle count (AFC) (b) in the early follicular phase, mid-luteal phase and after GnRH analogue down-regulation. Mean ± SD. There was a significant variation in ovarian volume (P < 0.0005) but not AFC between the three endocrine states.

 
COS and IVF outcome measures
The duration of rFSH stimulation was 12.1 ± 2.2 days (range 9–17). The number of oocytes collected was 7.7 ± 0.7 (0–24), fertilization rate was 66.7 ± 3.6% (0–100) and the number of embryos formed was 4.6 ± 0.4 (0–11). Two subjects had cycle cancellation prior to oocyte retrieval due to poor ovarian response. Embryo transfer was not performed in two other women, the reasons being failed fertilization and OHSS respectively. There were 10 resulting clinical pregnancies.

Correlations between hormonal responses and antral follicle count
Early follicular FSH concentration was weakly negatively correlated with AFC at all three endocrine phases investigated (Table II). The ovarian hormones showed variable correlations with AFC depending on the endocrine environment, and correlations with inhibin B in particular were increased following rFSH administration at all three time-points assesed. Inhibin A, pro-{alpha}C and estradiol concentrations were poorly correlated with AFC other than between inhibin A and AFC following down-regulation and this was not increased by FSH. Thus rFSH stimulation of the small antral follicles in the early follicular phase uncovered the functionality–morphology relationship for inhibin B, which did not exist with basal inhibin B or with the other hormones. The poor correlation between AFC and inhibin A and estradiol in the luteal phase is in keeping with the corpus luteum being the major source of these hormones at this stage of the menstrual cycle.


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Table II. Univariate linear regression analysis of hormone variables and antral follicle count (AFC) in the three endocrine states investigated
 
Relationship with number of oocytes recovered
Simple regression analysis
The hormone variables in each of the three endocrine phases were compared with the number of oocytes recovered following COS using linear regression analysis (Table III). The concentration of FSH in the early follicular phase showed a highly significant negative correlation with the number of oocytes recovered, but there were no significant correlations with the other basal hormones measured in this phase. Following rFSH administration in the early follicular phase, correlations with inhibin A, inhibin B and estradiol were increased, but only the correlation with inhibin B achieved statistical significance. Absolute inhibin concentrations following rFSH showed better correlations with number of oocytes than did the rises between visits 1 and 2. In keeping with these findings, both inhibin A and B following rFSH administration in the early follicular phase showed significant correlations with the number of large (>10 mm) follicles present on the day of hCG administration (r = 0.45, P < 0.005 and r = 0.53, P < 0.0005 respectively).


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Table III. Univariate linear regression analysis of hormone variables and number of oocytes recovered
 
In the mid-luteal phase, basal hormone concentrations did not correlate with the number of oocytes recovered. Recombinant FSH administration markedly enhanced the correlation of inhibin B, but not the other hormones.

After down-regulation, both inhibin A and B showed stronger correlations with oocyte number. These correlations were further increased after rFSH administration. The strongest correlation was with inhibin B. Estradiol and pro-{alpha}C showed poor correlations with oocyte number, and there was no significant change after FSH injection.

Of the biophysical variables, AFC showed highly significant correlations with the number of oocytes recovered (Table IV). The best correlation was observed in the mid-luteal phase and was roughly similar to that of inhibin A after rFSH administration in the down-regulated phase (visit 6). Ovarian volume showed weaker correlations, close to the limit of statistical significance at all three states although highest after down-regulation.


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Table IV. Univariate linear regression analysis of biophysical measures and number of oocytes retrieved
 
Dichotomization of the data by number of oocytes (cut-off of three oocytes) confirmed the predictive value of early follicular FSH and stimulated inhibin B concentrations in the early follicular phase (Table V). In contrast, the difference in inhibin A concentration after rFSH administration in this phase was not appreciably different. Inhibin A and B concentrations in the down-regulated state did not differ significantly between the two groups, either before or after rFSH administration as there was a wide variation within each group. AFC differed significantly between the two groups in the early follicular and luteal phases, but not after down-regulation.


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Table V. Comparison between groups following dichotomization according to number of oocytes recovered after controlled ovarian stimulation
 
Multiple regression analysis
The endocrine and biophysical variables at all three endocrine phases were evaluated using multiple regression analysis to ascertain the relative contribution of each variable to the number of oocytes retrieved. In this analysis, inhibin B at visit 6 (post-FSH in the down-regulated phase) was by far the most important determinant, while early follicular FSH contributed to a lesser but still statistically significant degree (Table VI). All other variables did not add to the prediction of the number of oocytes retrieved, which is described by the equation: 9.4 + 0.04(inhibin B visit 6) – 0.6 (FSH).


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Table VI. Multiple regression analysis showing relationship between hormone and biophysical variables, and number of oocytes retrieveda
 
When endocrine and biophysical variables at the clinically more useful pre-COS/IVF time-points of the early follicular and mid-luteal phases were evaluated, basal FSH emerged as the most significant contributor to the number of oocytes retrieved (Table VI). AFC in the mid-luteal phase (visit 3) also contributed significantly (P = 0.009), but none of the other variables significantly improved the prediction of the number of oocytes retrieved. The number of oocytes is described by the regression equation: 10.6 – 0.8(FSH) + 0.2(AFC visit 3).

Relationships with age
The correlations between age and the endocrine measures were generally poor (e.g. early follicular FSH; r = 0.14, P = 0.33). In contrast, there was a significant relationship between age and AFC in the luteal and down-regulated phases (r = –0.47, P < 0.0005 and r = –0.50, P < 0.0005 respectively). The correlation with the other biophysical measures (early follicular AFC, ovarian volume at all three phases) were weak (r = –0.11 to –0.28). There was only a poor relationship between age and the number of oocytes recovered (r = –0.18, P = 0.22).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study explores the relationships between the hormonal activity of the ovary and ultrasound measures of size and activity under differing conditions of gonadotrophin stimulation, varying from low concentrations, physiologically in the luteal phase and following pharmacological down-regulation, to relatively high concentrations in the early follicular phase. AFC in particular was investigated as a biophysical measure of ovarian activity. Both the down-regulated state and the luteal phase were included to allow comparison of these two relatively hypogonadotrophic conditions, and only women with regular ovulatory cycles were included to assess physiological relationships without complicating pathophysiological influences. It is, however, possible that recruitment from an infertile population (31% with unexplained infertility) may have had a subtle influence on the results. Basal activity was compared with the response to exogenous rFSH, with hormone concentrations determined 24 h after rFSH administration as this time-point has been shown to give maximal rises in inhibin concentrations (Burger et al., 1998Go) as well as being of practical convenience. Subsequent analysis compared these data with the number of oocytes collected after COS–IVF treatment, both for its importance as an index of the follicular pool and therefore the number of follicles within the ovary, and in clinical practice. It is important to emphasize that these data do not address the question of oocyte quality, which is of prime importance in determining true reproductive potential rather than the more limited concept of assessment of the ovarian reserve.

Serum inhibin concentrations showed the expected patterns, inhibin B being highest in the early follicular phase, inhibin A being highest in the luteal phase, and both being low after down-regulation (Groome et al., 1994Go, 1996; Lockwood et al., 1996Go). Administration of rFSH resulted in increases in inhibin B concentration at all time-points. The proportional rise in inhibin B was greatest following down-regulation, with a similar rise during the luteal phase. Inhibin B is produced by the granulosa cells of antral follicles. When concentrations of FSH are low, such as occurs during the luteal phase of the cycle and after down-regulation with GnRH analogue, the secretion of inhibin B is minimal because even the healthy follicles are deprived of FSH. Following injection of rFSH, there was a prompt increase in the secretion of inhibin B, the concentration of which showed a close correlation with AFC reflecting the number of healthy follicles as they express their potential. As AFC differed little between the three time-points examined, these data indicate that the granulosa cells of small follicles are most sensitive to a rise in FSH when endogenous concentrations are low.

The changes in inhibin A were relatively unremarkable, although the relationships with inhibin B particularly after rFSH administration confirm the common source of both hormones, i.e. from the antral follicle population. The measurement of pro-{alpha}C was of no predictive value either under basal conditions or after rFSH stimulation. The physiological role of this precursor peptide (if any) in regulation of ovarian or pituitary function is unknown. The correlation between estradiol and AFC was much weaker than that of inhibin B. The secretion of estradiol by the follicle is dependent on an adequate supply of androgen precursor from the theca layer which is stimulated by LH (Hillier, 1994Go). Thus when the levels of gonadotrophins are low, such as during the luteal phase and after down-regulation, injection of LH as well as FSH is required to realise the full potential for estradiol secretion (Illingworth et al., 1996Go). In contrast, LH has little effect on the production of inhibin B by the follicle although inhibin A secretion by the corpus luteum is sensitive to LH and hCG (but not FSH).

Interpretation of these data is based on the premise that the inhibins and estradiol are products of the ovary, and may therefore be anticipated to reflect ovarian activity. FSH is under feedback control by these hormones. FSH secretion thus reflects a summation of several positive and negative influences, yet is the prime determinant of follicular recruitment and growth. The present data show a relatively weak relationship between FSH concentration in the early follicular phase and AFC in all three endocrine states investigated. AFC did not vary significantly between the three endocrine states. Close correlations between inhibin B and AFC were observed at all three endocrine states after rFSH administration, despite mean inhibin B concentrations varying by >100% between them. Directly comparable data have not been previously obtained. In contrast, ovarian volume was demonstrated to differ between the endocrine states examined, being biggest during the luteal phase, reflecting the contribution of the corpus luteum, and lowest after down-regulation.

The relationship between these endocrine and biophysical measures and the number of oocytes recovered after COS was also investigated. This is of both scientific and clinical value. Non-invasive determination of the follicular pool may allow an improvement in prediction of women who may under- or over-respond to COS protocols. This has been the subject of many investigations (Gulekli et al., 1999Go; Bukman and Heineman, 2001Go) but no clinically useful predictive test (i.e. sufficiently accurate and distinct in time from COS treatment) has emerged. The realization that inhibin B concentrations reflect the number of antral follicles present has allowed the demonstration that inhibin B concentrations during COS show a close relationship with the number of oocytes recovered (Eldar-Geva et al., 2000Go; Fawzy et al., 2002Go), and such a relationship, although much weaker, can also be demonstrated using basal early follicular phase inhibin B concentration in some (Seifer et al., 1997Go; Hall et al., 1999Go; Tinkanen et al., 1999Go) but not all studies (Dumesic et al., 2001Go). The present data confirm the lack of predictive value of basal ovarian hormone concentrations, with neither inhibin A, inhibin B nor estradiol showing a significant relationship to subsequent oocyte recovery although early follicular FSH concentration showed a strong correlation. However, following rFSH administration in the early follicular phase, inhibin B concentration showed a highly significant relationship. In the luteal phase, despite the larger increase in inhibin B concentration after rFSH administration, only a weak relationship with oocyte recovery was detected. The closest correlations were observed after down-regulation, both before and after rFSH administration. This is to be expected because it is these particular small follicles which in the next few days will be stimulated to pre-ovulatory size. This observation indicates the intrinsic variability over the period of a few weeks of the size of the recruitable cohort of follicles. In the clinical setting, the greater predictive power of endocrine measurements following down-regulation would need to be weighed against the value of prediction at an earlier time-point prior to initiation of COS treatment. Our findings extend previous demonstrations of relationships between inhibin B concentrations during the later stages of COS and the number of oocytes recovered (Hall et al., 1999Go; Eldar-Geva et al., 2000Go; Dumesic et al., 2001Go) to both before the start of COS and after the first administration of rFSH. This indicates that assessing the hormonal sensitivity of follicular granulosa cells both prior to and after initial FSH administration has similar accuracy as a predictor of the number of oocytes recovered, as assessment delayed until follicular growth is well established. AFC showed a significant and similar correlation with oocyte number at all three states, as previously demonstrated in the early follicular phase (Chang et al., 1998Go). Ovarian volume, however, showed relatively weak correlations with the number of oocytes recovered. Ovarian volume in the early follicular phase and after down-regulation was found to be a poor predictor of the number of oocytes recovered after COS (Tomas et al., 1997Go; Tinkanen et al., 1999Go; Dumesic et al., 2001Go) although luteal phase ovarian volume did show predictive value in one study (Syrop et al., 1999Go). Of the two biophysical measures assessed, AFC therefore appears to reflect more closely the ovarian reserve than does ovarian volume.

The investigation of both hormonal and biophysical variables allowed assessment of the relative value of these measures as predictors of oocyte number, both in the early follicular phase and after down-regulation. AFC and ovarian volume showed significant correlations with oocyte number, with coefficients of regression for AFC being similar to those of inhibin B after rFSH administration, consistent with the initial analysis showing close relationships between these markers of follicular activity. However, multiple regression analysis demonstrated that early follicular phase FSH and mid-luteal AFC were the only factors independently associated with oocyte number. Stimulated inhibin B concentrations reflected the potential follicular development of the ovary, but were not significantly associated with oocyte number when the biophysical data were included in the model.

In conclusion, these data extend our understanding of the relationships between follicular number, follicular functional activity and sensitivity to FSH, and the recruitable follicular population. FSH-stimulated inhibin B concentration may add to, but will not supplant, measurement of FSH concentration in the early follicular phase, and AFC appears to be of greater value in assessing the ovarian reserve than direct ovarian hormones. That the summative analysis of ovarian activity determined by the hypothalamus and gonadotroph as reflected in FSH secretion is superior to measurement of ovarian products should not be surprising, but illustrates present limitations in assessment of ovarian function.


    Acknowledgements
 
We are grateful to Joan Creiger and the staff of the Assisted Conception Unit for their expert care of the women in this study, to Professor Nigel Groome for the supply of reagents for the inhibin immunoassays, and to Ian Swanston for carrying out the immunoassays.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anderson, R.A., Groome, N.P. and Baird, D.T. (1998) Inhibin A and inhibin B in women with polycystic ovarian syndrome during treatment with FSH to induce mono-ovulation. Clin. Endocrinol., 48, 577–584.[CrossRef][ISI][Medline]

Backstrom, C.T., McNeilly, A.S., Leask, R. and Baird, D.T. (1982) Pulsatile secretion of LH, FSH, prolactin, oestradiol and progesterone during the human menstrual cycle. Clin. Endocrinol., 7, 29–42.

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Submitted on July 2002; accepted on September 6, 2002