Comparison of endocrine tests with respect to their predictive value on the outcome of ovarian hyperstimulation in IVF treatment: results of a prospective randomized study

J. Kwee1,3, M.W. Elting1, R. Schats1, P.D. Bezemer2, C.B. Lambalk1 and J. Schoemaker1

1 Research Institute for Endocrinology, Reproduction and Metabolism, Department of Obstetrics and Gynaecology, Division of Reproductive Endocrinology and Fertility and the IVF Centre and 2 Department of Clinical Epidemiology and Biostatistics Vrije Universiteit Medical Centre, Amsterdam, The Netherlands

3 To whom correspondence should be addressed at: Vrije Universiteit Medical Centre, IVF Centre P.O.Box 7057, 1007 MB Amsterdam, The Netherlands. e-mail: j.kwee{at}vumc.nl


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: This study was designed to compare endocrine tests [clomiphene citrate challenge test (CCT), exogenous FSH ovarian reserve test (EFORT) and basal FSH, basal estradiol (E2) and basal inhibin B as an integral part of all CCT and EFORT], with respect to their ability to estimate the stimulable cohort of follicles in the ovaries (ovarian capacity) and to analyse which test or combination of tests would give the best prediction of ovarian capacity. METHODS: A total of 110 regularly menstruating patients, aged 18–39 years, participated in this prospective study, randomized by a computer-designed 4-block system study into two groups. Fifty-six patients underwent a CCT, and 54 patients underwent an EFORT. In all patients, the test was followed by an IVF treatment. The result of ovarian hyperstimulation during IVF treatment, expressed by the total number of follicles, was used as gold standard. RESULTS: Univariate linear regression analysis showed that the best correlation with the number of follicles after ovarian hyperstimulation (Y) is found by the inhibin B increment (InhB incr.) in the EFORT (Y = 3.957 + 0.081 x InhB incr. (95% CI 0.061–0.101); r = 0.751; P < 0.001). Multiple linear regression analysis showed a significant contributing value of the variables basal FSH, E2 increment of the EFORT and inhibin B increment to the basic model with the variable age. The best prediction of ovarian capacity (Y) was seen when E2 increment and inhibin B increment were used simultaneously in a stepforward multiple regression prediction model [Y = 2.659 + 0.052 x InhB incr. (0.026–0.078) + 0.027 x E2 incr. (95% CI 0.012–0.054); r = 0.796; P < 0.001]. The CCT could not be used in a prediction model. CONCLUSIONS: The EFORT is the endocrine test which gives the best prediction of ovarian capacity.

Key words: basal FSH/basal inhibin B/CCT/EFORT/ovarian capacity


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ageing of the ovary plays the major role in reproductive ageing and is related to the gradual reduction in the number of primordial follicles. The number of follicles leaving the pool of the so-called resting follicles to enter the growth phase towards the antral stages of development decreases with increasing age, leading to a stock at menopause estimated at between <100 and 1000 primordial follicles in the pool (Gougeon et al., 1994Go; Gougeon, 1996Go). Scheffer et al. (1999Go) demonstrated that the number of primordial follicles in the ovary, as published by Faddy and Gosden (1996Go), correlated well with the number of growing follicles, counted by transvaginal sonography in the early follicular phase. So the decreasing size of the antral follicle cohort with age is a reflection of the decreasing primordial follicle pool. We can use this principle to measure ovarian capacity, defined as the total number of follicles which can be stimulated under maximal ovarian stimulation with FSH. A number of the so-called ovarian capacity tests are supposed indirectly to reflect the size of the cohort of small antral follicles (2–5 mm in diameter) in the ovary. Van der Meer et al. (1998Go) showed that in eumenorrhoeic patients, the median (range) FSH threshold level for monofollicular growth was 5.3 (4.3–8.2) IU/l and the median (range) threshold dose was 75 IU (0.5–1.75) FSH/day, given i.v. It was concluded that by an increment of 1/2 ampoule of FSH (37.5 IU) above the threshold dose for monofollicular growth, the maximum response is already obtained. It seems that in IVF stimulation maximal effect is reached with FSH dosages up to 225 IE (Latin-American Puregon IVF Study Group, 2001; Out et al., 2000Go; 2001). Combining these facts, it can be concluded that an initial stimulation by 3 ampoules of 75 IU of FSH under a long (GnRH agonist-suppressed) protocol probably gives a maximal IVF stimulation, the outcome of which could be used as the gold standard for the cohort size.

Predictors of ovarian capacity are either static: age (Hughes et al., 1989Go; Meldrum, 1993Go; Navot et al., 1994Go; Scott et al., 1995Go), basal FSH (bFSH) (Pearlstone et al., 1992Go; Toner, 1993Go; Cahill et al., 1994Go; Hansen et al., 1996Go), basal estradiol (bE2) (Licciardi et al., 1995Go; Smotrich et al., 1995Go; Evers et al., 1998Go), basal inhibin B (bInhB) (Lahlou et al., 1999Go); or dynamic: clomiphene citrate challenge test (CCT) (Navot et al., 1987Go; Loumaye et al., 1990Go; Scott et al., 1993Go), exogenous FSH ovarian reserve test (EFORT) (Fanchin et al., 1994Go; Elting et al., 2000Go), GnRH agonist stimulation test (GAST) (Padilla et al., 1990Go; Ravhon et al., 2000Go). All tests predict the response to ovarian hyperstimulation and the prognosis for pregnancy in IVF treatment. Elting et al. (2000Go) showed that the EFORT could predict the follicle cohort size in patients with polycystic ovary syndrome, regularly menstruating women with polycystic ovaries and regularly menstruating women with normal ovaries. Except for the latter, none of the above tests have been developed for determination of ovarian capacity.

The primary aim of this study was to compare endocrine tests with respect to their ability to measure the stimulative cohort of the ovaries (ovarian capacity). For reasons mentioned above, the outcome of hyperstimulation with 3 ampoules in IVF under a long protocol was used as gold standard. The secondary aim of the study was to analyse which test or combination of tests would give the best prediction of ovarian capacity. For practical reasons the most direct stimulation of follicle growth (EFORT) was compared with the most indirect test (CCT).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
A total of 110 patients, aged 18–39 years, who were eligible for treatment by assisted reproduction between June 1997 to December 1999 participated in the study. This study is part of a prospective randomized study of regular menstruating patients on the determination of ovarian capacity called the DOC study. Their infertility was either idiopathic for >3 years and/or due to a male factor and/or cervical hostility. Patients had to have regular menstrual cycles, two ovaries and at least one patent Fallopian tube. Excluded were patients with either polycystic ovary syndrome or a severe male factor, subsequently treated by ICSI and defined as (i) <1x106 motile sperm after Percoll centrifugation (gradient 40/90) and/or (ii) >20% antibodies present on the sperm after processing with Percoll centrifugation (gradient 40/90) and/or (iii) >50% of the sperm without an acrosome. Other exclusion criteria were untreated or insufficiently corrected endocrinopathies, clinically relevant systemic diseases or a body mass index >28 kg/m2.

The protocol was approved by the committee on ethics of research involving human subjects of the Vrije Universiteit Medical Centre, Amsterdam, The Netherlands. Informed consent was signed by all the couples participating in the study.

Treatment protocol
Patients were randomized by a computer-designed 4-block system into two groups. Fifty-six patients underwent a CCT, and 54 patients underwent an EFORT. In all patients, the test was followed by an IVF treatment under a long protocol. The bFSH level, bE2 level and bInhB level were determined as an integral part of all CCT and EFORT.

Clomiphene citrate challenge test
Starting on the fifth day of the menstrual cycle (CD 1 = day of onset of menses) 100 mg of clomiphene citrate (Serophene®; Ares Serono, Switzerland) was administered for 5 days. In this study on CD 2 or 3 (basal values) and on CD 10 (stimulated values) the serum FSH, E2 and inhibin B were determined. Analysis of the CCT included the following parameters: (i) bFSH and stimulated FSH (sFSH), (ii) bE2 and stimulated E2 (sE2) and (iii) bInhB and stimulated inhibin B (sInhB).

Exogenous FSH ovarian reserve test (EFORT)
On CD 3, 300 IU rFSH (Gonal-F®; Ares Serono) were administered s.c. according to the method described by Fanchin et al. (1994Go). In this study, blood samples for the determination of FSH, E2 and inhibin B were drawn: just before (basal values) and 24 h after (stimulated values) the administration of FSH. Analysis of the EFORT included the following parameters: (i) the bFSH, (ii) E2 increment and inhibin B increment 24 h after administration of FSH.

IVF treatment
The ovarian hyperstimulation protocol was performed according to a long GnRH agonist protocol starting in the mid-luteal phase. Early in the first cycle the CCT or the EFORT was performed as described above. In the subsequent mid-luteal phase, 7 days after ovulation, daily s.c. injections with triptoreline acetate (Decapeptyl, 0.1 mg/day; Ferring, The Netherlands) were started. Because of the administration of the GnRH agonist, patients were advised to use a barrier type of contraception during this cycle. On CD 3 of the next cycle, ovarian hyperstimulation was started with daily s.c. injections of a fixed dose of 225 IU uFSH (Metrodin HP, 75 IU/ampoule; Ares Serono) because this dosage probably gives a maximal effect in follicle stimulation (Out et al., 2000Go; 2001). Standard procedures were followed including transvaginal sonography (TVS) (Aloka SSD-1700, 5.0 MHz probe) on CD 2 or 3 and on CD 9 or 10. Daily TVS was performed from the moment when the leading follicle reached a diameter of 16 mm. Ovarian hyperstimulation was continued until the largest follicle reached a diameter of ≥18 mm. The maximum duration of uFSH administration was not allowed to exceed 16 days. If these criteria were met, Metrodin HP and Decapeptyl were discontinued and 10 000 IU of hCG (Profasi, 10 000 IU/ampoule; Ares Serono) were administered. On the day of hCG, TVS was performed to count all follicles ≥10 mm (expressed as the total number of follicles).

Serum assay
Serum E2 and FSH were determined by commercially available immunometric assays (Amerlite, UK). For E2, the inter-assay coefficient of variation (CV) was 11% at 250 pmol/l and 8% at 8000 pmol/l, the intra-assay CV was 13% at 350 pmol/l, 9% at 1100 pmol/l and 9% at 5000 pmol/l. The lower limit of detection for E2 was 90 pmol/l. In the EFORT and CCT we measured estradiol by a sensitive radioimmunoassay (Sorin; Biomedica, Italy). This measurement of estradiol was abbreviated as EE. For EE, the inter-assay CV was 11% at 60 pmol/l, 8% at 200 pmol/l, 11% at 550 pmol/l and 8% at 900 pmol/l. The intra-assay CV was 4% at 110 pmol/l and 5% at 1000 pmol/l. The lower limit of detection for EE was 18 pmol/l. For FSH, the inter-assay CV was 9% at 3 IU/l and 5% at 35 IU/l, the intra-assay CV was 9% at 5 IU/l, 8% at 15 IU/l and 6% at 40 IU/l. The lower limit of detection for FSH was 0.5 IU/l. Inhibin B was determined immunometrically by a commercially available assay (Serotec Ltd, UK). For inhibin B, the inter-assay CV was 17% at 25 ng/l, 14% at 55 ng/l and 9% at 120 ng/l and the intra-assay CV was 8% at ≤40 ng/l and 5% at >40 ng/l. The lower limit of detection for inhibin B was 13 ng/l.

Half way through the study, the Amerlite assay (suddenly withdrawn from the market) used to assess FSH had to be replaced by another commercially available assay (Delfia, Finland). The two assays have been compared and showed excellent linear correlation, although a shift in the values took place. Delfia assay in comparison with Amerlite: Delfia FSH = 1.28 x Amerlite FSH + 0.01 (r = 0.9964). For the Delfia FSH, the inter-assay CV was 5% at 3.5 IU/l and 3% at 15 IU/l. All FSH determinations have been recalculated and are expressed according to the Delfia assay. Values below the detection limit of an assay were assigned a value equal to the detection limit of that assay.

Statistical analysis
The endpoint of the study was the result of ovarian hyperstimulation expressed as the total number of follicles. Statistical analysis of all the data was performed with the Statistical Package for Social Sciences (USA) for Windows.

For the CCT results, we used the variable or combination of variables showing the best correlation coefficient (Pearson’s correlation test) with the total number of follicles obtained after stimulation. Univariate correlations between the variables: sFSH, sE2, sInhB, {sum} bFSH + sFSH, {sum} bE2 + sE2, {sum} bInhB + sInhB, FSH increment in 7 days (sFSH level – bFSH), E2 increment (sE2 – bE2) in 7 days, inhibin B increment (sInhB – bInhB) in 7 days versus the total number of follicles obtained after stimulation were analysed by Pearson’s correlation test. Multivariate correlations between the above-described variables and the total number of follicles obtained after stimulation were analysed in a stepwise regression analysis.

For the EFORT results, we examined whether the bFSH had an additional contribution to the predictive value of the number of stimulated follicles already established by the E2 increment in 24 h or the inhibin B increment in 24 h, by stepwise linear regression analysis.

Comparison of means was done with the unpaired t-test or Wilcoxon’s rank sum test.

By univariate linear regression, we estimated the value of the independent variables: age, bFSH, bE2, bInhB, CCT results, E2 increment and the inhibin B increment in predicting the ovarian response.

We built a model based on the simplicity of the diagnostic tests at four different levels. Level 1: age; level 1–2: age and bFSH; level 1–2–3: age, bFSH and outcome of CCT or E2 increment in the EFORT; level 1–2–3–4 (only for the EFFORT group): age, E2 increment in the EFORT and inhibin B increment in the EFORT. In a multiple regression model we estimated the additional value of the basal values (bFSH, bE2, bInhB), the CCT and the EFORT on top of the basic model of age.

Stepwise regression analysis was used to find a prediction model for the ovarian response. The R2 of the correlation of these variable(s) with the total number of follicles obtained after stimulation reflects the proportion of the variability of the number of follicles explained by this variable(s). For all tests P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
The characteristics of the two groups were depicted as means ± SD in Table I. No significant differences were noted between the groups in baseline characteristics, cycle day 3 measurements or outcome parameters. In the CCT group, 57.1% had primary infertility and 42.9% secondary infertility. The cause of infertility was 62.5% idiopathic, 28.6% male factor and 8.9% cervical factor. In the EFORT group, 65.0% had primary infertility and 35.0% secondary infertility. Their cause of infertility was 55.5% idiopathic, 42.5% male factor and 2% cervical factor.


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Table I. Characteristics of the groups
 
Univariate linear regression analysis
The correlations between the CCT and number of follicles obtained after stimulation are calculated, the bFSH and the {sum} bFSH + sFSH show the best correlation coefficients (r = 0.508, P < 0.001 and r = 0.496, P < 0.001 respectively). In the further analysis we used the latter variable as the CCT result. The regression line of the bFSH on the number of follicles was drawn by the regression equation: Y = 30.334 – 2.114 x bFSH; with a 95% CI of 1.135 – 3.092, meaning that each FSH increment of 1 IU/l predicts a decrement of 2.1 follicles (95% CI: 1.1–3.1). The regression equation for the {sum} bFSH + sFSH, Y = 25.626 – 0.712 x CCT (0.372–1.052), shows that an increase of 1 IU/l predicts a decrement of 0.7 follicles. Also the correlation between age and the outcome parameter was highly significant (Y = 56.500 – 1.250 x age (0.631–1.869), r = 0.482, P < 0.001). The correlation between bInhB and the outcome parameter in the CCT group was significant (Y = 5.985 + 0.089 x bInhB (0.024–0.153), r = 0.351, P = 0.008). bE2 and the endpoint were not significantly correlated (Y = 14.360 – 0.0007 x bE2(–0.053–0.052), r = 0.004, P = 0.978, not significant).

In the EFORT group, the inhibin B increment and E2 increment in the EFORT show the best correlation coefficients (r = 0.751, P < 0.001 and r = 0.718, P < 0.001 respectively) with the total number of follicles obtained after stimulation. The regression line of the inhibin B increment on the number of follicles was drawn by the regression equation: Y = 3.957 + 0.081 x inhibin B increment; with a 95% CI of 0.061 – 0.101, meaning that each inhibin B increment of 100 ng/l predicts 8.0 more follicles (95% CI: 6.1–10.1) (Figure 1 Left). The regression equation for the E2 increment [Y = 4.764 + 0.062 x E2 incr. (0.045–0.079)] shows that an increase of 100 pmol/l predicts 6.2 more follicles (Figure 1 Right). Also the correlations between bFSH [Y = 17.374 – 0.370 x bFSH (0.063–0.677), r = 0.318] and age [Y = 48.597 – 1.004 x age (0.288–1.720), r = 0.364] with the outcome parameter were significant (P = 0.019, P = 0.007 respectively). The correlation between the bE2 [Y = 17.857 – 0.030 x bE2 (0.092–0.032), r = 0.134] and bInhB [Y = 9.055 + 0.059 x bInh B (0.001–0.119), r = 0.266] with the endpoint were not significant (P = 0.340, P = 0.052, not significant respectively).




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Figure 1. (Left) Plot of the number of follicles obtained after stimulation against the inhibin B increment. The three lines represent the regression line: Y = 3.957 + 0.081 x InhB incr. with the 95% CI of the mean. (Right) Plot of the number of follicles obtained after stimulation against the E2 increment. The three lines represent the regression line: Y = 4.764 + 0.062 x E2 incr. with the 95% confidence interval (CI) of the mean.

 
Multiple linear regression analysis
For this analysis the order of parameters was established by the simplicity of the diagnostic tests: (i) age, (ii) bFSH, (iii) {sum} bFSH + sFSH for the CCT and (i) age, (ii) bFSH, (iii) E2 increment and (iv) inhibin B increment for the EFORT. We excluded bE2 and bInhB, because bE2 did not correlate in either group and bInhB only correlated in the CCT group. Table II shows the contributing value of each of the variables for the CCT group. There was a significant contribution of bFSH to the model of age alone. Thereafter the {sum} bFSH + sFSH showed no further significant contribution to the model.


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Table II. Model based on the simplicity of the diagnostic tests for the clomiphene challenge test group
 
Table III shows the contributing value of each of the variables for the EFORT group. There was a significant contribution of the bFSH to the basic model with the variable: age. When adding E2 increment and inhibin B increment as variables in multiple regression analysis to the basic model with age and bFSH, each of these showed a further significant contribution.


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Table III. Model based on the simplicity of the diagnostic tests for the exogenous FSH ovarian reserve test group
 
Stepforward regression analysis: prediction model for ovarian capacity
Based on the CCT group, the prediction model for ovarian response is explained for 25% by the best predictive variable, the bFSH. When adding the independent variables: {sum} bFSH + sFSH and age in a stepforward regression analysis, the explained variation rose significantly with 12% after the selection of age. The independent variable {sum} bFSH + sFSH did not have a significant contribution to the model. The exact prediction of the total number of follicles obtained after stimulation thus increased from 25 to 37%. The regression line of the bFSH and age on the number of follicles was drawn by the regression equation: Y = 58.139 – 1.644 x bFSH (0.684–2.603) – 0.927 x age (0.323–1.525) (r = 0.605, P < 0.001).

Based on the EFORT group, the prediction model for ovarian response is explained for 56% by the best predictive variable, the inhibin B increment. When E2 increment and inhibin B increment were used simultaneously in a stepforward multiple regression prediction model, the explained variation of the best predictive model rose significantly with 7%. The total explained variation thus increased from 56 to 63%. The regression line of the inhibin B increment and E2 increment on the number of follicles was drawn by the regression equation: Y = 2.659 + 0.052 x InhB incr. (0.026–0.078) + 0.027 x E2 incr. (0.012–0.054) (r = 0.796, P < 0.001). That means that if we use this formula, the confidence interval of Y is 50%. When we included age and bFSH as variables in the stepforward regression analysis together with the inhibin B increment and E2 increment we did not find a significant contribution of these variables.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our study shows that the inhibin B increment and E2 increment in the EFORT are the best predictors of the total number of follicles obtained after maximal ovarian hyperstimulation in an IVF treatment, i.e. cohort size. Age, bFSH, bE2, bInhB and the outcome of the CCT ({sum} bFSH + sFSH) in this respect each, and in combination, show a much lower performance. This is in agreement with the study of Elting et al. (2000Go) who concluded that the EFORT could predict the follicle cohort size in patients with polycystic ovary syndrome, regularly menstruating women with polycystic ovaries and regularly menstruating women with normal ovaries. The best prediction model results when these two variables are used simultaneously in a stepforward multiple regression analysis. In 1994, Fanchin et al. (1994Go) described the EFORT as a test which can detect a possible poor response among patients going to be treated with IVF. Our study showed that, in addition to finding poor responders, the test is able to predict with reasonable accuracy the number of follicles obtained after stimulation. This may, in combination with threshold analysis, be a first step to really control the number of follicles obtained after stimulation. As it has been suggested that granulosa cells of small antral follicles under the influence of FSH also produce inhibin B, we were not surprised to find, as Elting et al. (2000Go) did, that the inhibin B increment follows the same pattern as the E2 increment in the EFORT.

Because we wanted to know if these tests had an additional value other than age and the basic measurements, we also built a model for prediction of follicle number based on the simplicity of the diagnostic tests. The results show that there is a huge additional value for the E2 increment as well as for the inhibin B increment in the EFORT. There is no such additional value, however, for the best outcome parameter of the CCT ({sum} bFSH + sFSH). Navot et al. (1987Go) described the use of the CCT for the distinction between a poor and adequate response after ovarian hyperstimulation and its prognosis for pregnancy. It may well have its value in that case, but for the prediction of the cohort size the CCT is of no use.

Our study also showed that, there was hardly any difference between the predictive value of age and bFSH for the number of follicles obtained after stimulation. Several studies have shown (Pearlstone et al., 1992Go; Toner, 1993Go; Cahill et al., 1994Go; Hansen et al., 1996Go) that the bFSH compared with a woman’s age has a better predictive value of finding poor responders.

Unexpectedly we found no additional value of bInhB. This is in contrast with the findings of Seifer et al. (1996Go; 1999) and Danforth et al. (1998Go). They found that poor responders with normal bFSH levels have significantly lower bInhB levels than normal responders. bInhB was also significantly correlated with chronological age, the number of ampoules of FSH administered, peak estradiol concentration, number of oocytes and embryos, and cancellation rates. We expect this difference to be caused by the fact that the inhibin B production is strongly dependent on FSH (Hansen et al., 1996Go). Inhibin B concentrations rise across the luteal–follicular transition and peak in the mid-follicular phase, but a few days later than similar changes in FSH, suggesting secretion by the granulosa cells of the developing cohort of follicles in response to FSH. Theoretically, under exogenous stimulation inhibin B may be the optimal reflection of ovarian secretory capacity and follicle number. Therefore it could be that there is a better correlation during the mid-follicular phase, when granulosa cell function is strongly dependent on FSH compared with early follicular phase when FSH stimulation is strictly marginal. This needs further investigation.

A cost-effectiveness analysis is currently under way. The average costs of an FSH stimulation test will be more expensive than a CCT. However, it is not unlikely that, due to better prediction of outcome, more accurate dose adjustment will reduce the overall costs due to limitations of gonadotrophin use during stimulation and fewer cancelled cycles.

In conclusion, the results of our study show that in comparing endocrine tests for the prediction of the total number of follicles obtained after stimulation, inhibin B increment and E2 increment in the EFORT gave the best predictive values. Secondly the combination of inhibin B increment and E2 increment can predict ovarian capacity in regularly menstruating women who are eligible for assisted reproduction treatment. The CCT, measured in our study by the {sum} bFSH + sFSH, has no additional value above the basal values and age for the prediction of the number of follicles obtainable under maximal stimulation for IVF.


    Acknowledgements
 
The authors acknowledge the help of Dr Corry Popp-Snijders and her staff, particularly for the endocrine laboratory work, and the staff of the IVF centre for assistance during the execution of the protocol. This study was financially supported by Ares-Serono, Geneva, Switzerland.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 8, 2002; accepted on January 15, 2003.