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
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Abstract |
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Key words: basal FSH/basal inhibin B/CCT/EFORT/ovarian capacity
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Introduction |
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Predictors of ovarian capacity are either static: age (Hughes et al., 1989; Meldrum, 1993
; Navot et al., 1994
; Scott et al., 1995
), basal FSH (bFSH) (Pearlstone et al., 1992
; Toner, 1993
; Cahill et al., 1994
; Hansen et al., 1996
), basal estradiol (bE2) (Licciardi et al., 1995
; Smotrich et al., 1995
; Evers et al., 1998
), basal inhibin B (bInhB) (Lahlou et al., 1999
); or dynamic: clomiphene citrate challenge test (CCT) (Navot et al., 1987
; Loumaye et al., 1990
; Scott et al., 1993
), exogenous FSH ovarian reserve test (EFORT) (Fanchin et al., 1994
; Elting et al., 2000
), GnRH agonist stimulation test (GAST) (Padilla et al., 1990
; Ravhon et al., 2000
). All tests predict the response to ovarian hyperstimulation and the prognosis for pregnancy in IVF treatment. Elting et al. (2000
) 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).
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Materials and methods |
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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. (1994). 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., 2000; 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 (Pearsons correlation test) with the total number of follicles obtained after stimulation. Univariate correlations between the variables: sFSH, sE2, sInhB, bFSH + sFSH,
bE2 + sE2,
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 Pearsons 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 Wilcoxons 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 12: age and bFSH; level 123: age, bFSH and outcome of CCT or E2 increment in the EFORT; level 1234 (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.
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Results |
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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: 6.110.1) (Figure 1 Left). The regression equation for the E2 increment [Y = 4.764 + 0.062 x E2 incr. (0.0450.079)] 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 (0.0630.677), r = 0.318] and age [Y = 48.597 1.004 x age (0.2881.720), 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 (0.0920.032), r = 0.134] and bInhB [Y = 9.055 + 0.059 x bInh B (0.0010.119), r = 0.266] with the endpoint were not significant (P = 0.340, P = 0.052, not significant respectively).
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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. (0.0260.078) + 0.027 x E2 incr. (0.0120.054) (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.
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Discussion |
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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 ( bFSH + sFSH). Navot et al. (1987
) 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., 1992; Toner, 1993
; Cahill et al., 1994
; Hansen et al., 1996
) that the bFSH compared with a womans 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. (1996; 1999) and Danforth et al. (1998
). 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., 1996
). Inhibin B concentrations rise across the lutealfollicular 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 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.
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Acknowledgements |
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References |
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Submitted on July 8, 2002; accepted on January 15, 2003.