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Technical Briefs |
1 Hôpital Antoine Béclère, Clamart, France, Department of Biochemistry and Hormonology, 157 rue de la porte de Trivaux, 92141 Clamart cedex, France
aauthor for correspondence: fax 33-1-45374745, e-mail joelle.taieb{at}abc.ap-hop-paris.fr
In women undergoing in vitro fertilization and embryo transfer (IVF-ET), serial measurements of 17ß-estradiol (E2) can be used to monitor follicular growth, and serum E2 has been shown to be correlated with the number and diameter of preovulatory follicles observed on transvaginal ultrasound scan (1)(2)(3)(4)(5)(6). The serum E2 concentration is therefore an essential variable for evaluating the progression of stimulation, adjusting daily gonadotropin therapy, predicting the optimal day for induction of ovulation [administration of human chorionic gonadotropin (hCG)] (7), preventing ovarian hyperstimulation syndrome (8)(9), and ensuring that pituitary function is adequately suppressed if a long-acting gonadoliberin-releasing hormone agonist (GnRHa) is used before stimulation (10). Another major use of E2 measurements is to evaluate ovarian function at day 3 of the menstrual cycle to determine the prognosis of IVF-ET (11).
Because results must be available within a few hours, rapid and automated assays are required. In this study, we measured E2 with a new automated assay that could be used in random, continuous access mode and assessed its usefulness for monitoring ovulation stimulation for IVF-ET.
The Architect-i2000 E2 (Abbott Laboratories) assay is a competitive two-step immunoassay based on chemiluminescent microparticle immunoassay technology and can be performed in 29 min. The ACS-180 E2 assay (Bayer Diagnostics) is a competitive one-step immunoassay based on solid-phase antigen-linked technology and chemiluminescence detection and can be completed in 15 min. The luminescence reaction and the calibration procedure are identical for the two E2 immunoassays. The two methods are linear up to 3670 pmol/L and use monoclonal antibody derived by coupling the E2 molecule at the specificity-enhancing sixth position.
The within- and between-run imprecision, detection limits, and functional sensitivities of these assays have been reported previously (12)(13)(14)(15). The two methods were compared for 190 serum samples, and their agreement was assessed by the method of Bland and Altman (16). The specificity of each assay was evaluated by assaying 14 and 10 sera from patients receiving 2 mg of micronized 17ß-E2 or E2 valerate, respectively. E2 concentrations were determined for 166 serum samples from 25 patients undergoing ovarian stimulation. All patients were treated with a single injection of GnRHa to abolish the activity of gonadotropin hormones during the 2 weeks preceding exogenous gonadotropin administration. In all cases, ovulation was stimulated with recombinant follicle-stimulating hormone (FSHr). All patients received an initial dose of 225 IU FSHr/day during the first 5 days. Subsequent doses of FSHr and the timing of hCG administration were determined according to the usual criteria for follicular maturation (serum E2 concentrations and transvaginal ultrasound). All sera were assayed simultaneously by the two methods, with single determinations as recommended by the manufacturers. Investigating physicians were blind to the results of Architect-i2000 for monitoring stimulation of ovulation. We also retrospectively analyzed and compared the results obtained with the two methods for 80 sera obtained on day 3 of the menstrual cycle from women selected for the oocyte donation program at our IVF center (11).
The Architect-i2000 E2 assay was linear within the calibration range. The regression equation was (SD given in parentheses): y (observed) = 0.988(0.019)x (expected) - 11.3(22.6) pmol/L; r = 0.999.
Linear regression analysis for 190 serum samples with E2 concentrations of 0–16 500 pmol/L yielded: Architect-i2000 = 1.16(0.013) ACS-180 - 29(92) pmol/L; r = 0.988. For concentrations of 0–367 pmol/L (n = 58) and 367–16 500 pmol/L (n = 132), we obtained the following equations, respectively: Architect-i2000 = 0.94(0.072) ACS-180 + 62 (3) pmol/L (r = 0.870); and Architect-i2000 = 1.17 (0.018) ACS-180 - 79 (97) pmol/L (r = 0.985). The Architect-i2000 E2 assay gave higher estimates of E2 concentration than did ACS-180 over this concentration range (slopes >1 and intercept at +62 pmol/L for high and low E2 concentrations, respectively). The differences between the results obtained with the two methods were statistically significant (P <0.001) in the nonparametric Wilcoxon matched-pairs signed-rank test. Because the ACS-180 method is not a reference method, we analyzed the results by the method of Bland and Altman (16), taking into account the imprecision of the two methods. The results of this analysis are shown in Fig. 1
. A marked positive difference was observed overall for this range of concentrations. This difference was of consequence only in the low concentration range.
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The values obtained for serum E2 depend on the method used (17)(18)(19)(20). Various factors may account for the differences between the two methods (21). These factors include differences in calibration curves, antibody specificity, the matrix effect (22)(23)(24), and the mathematical relationship permitting the conversion of the signal obtained into E2 concentration in the system used.
The results obtained with the two E2 immunoassays for specimens from patients receiving micronized 17ß-E2 or E2 valerate (25) did not differ significantly (P = 0.55 and 0.06, respectively, in the nonparametric Wilcoxon test). However, all of the values obtained with Architect-i2000 for patients treated with E2 valerate were 
10% higher than those obtained with ACS-180, which corresponded to the cross-reaction of 6% obtained in vitro by the manufacturer.
Before beginning ovarian stimulation, it is necessary to check that down-regulation has been successful to prevent the stimulation of apoptotic follicles. The E2 concentration must be <150 pmol/L by RIA (26) and <184 pmol/L with the ACS-180 system (unpublished data). In 25 samples from women treated with GnRHa for 2 weeks, E2 concentrations obtained with the ACS-180 method were in all cases 
184 pmol/L. The mean E2 concentration obtained with the Architect-i2000 assay was 132 pmol/L (range, <169–224 pmol/L). Nineteen samples gave values <169 pmol/L, corresponding to the cutoff for functional sensitivity. Six samples gave E2 concentrations slightly higher than 184 pmol/L (187–224 pmol/L). Thus, the cutoff point for ovarian down-regulation with the Architect-i2000 assay should be set at 225 pmol/L.
For 128 sera from 25 patients undergoing ovarian stimulation (mean of 5.1 measurements per patient), the mean increase in E2 concentration, mean final E2 concentration before hCG administration, mean number of mature follicles (with a diameter >15 mm, as measured by transvaginal ultrasound), and the relationship between E2 concentration and the number of mature follicles are presented in Table 1
. The two E2 immunoassays gave similar results. The mean (SD) increase in E2 was 1601 (828) pmol/L for the ACS-180 and 1680 (833) pmol/L for the Architect-i2000 during the first 5 days of stimulation. Between day 6 of stimulation and the day on which hCG was administered, the mean increase in E2 concentration in the ACS-180 assay was 2026 (928) pmol/L from day 6 to day 8, 4030 (2257) pmol/L from day 8 to day 10, and 3997 (2015) pmol/L from day 10 to the day of hCG administration. The mean increase in E2 concentration in the Architect-i2000 assay was 2345 (1119) pmol/L from day 6 to day 8, 4734 (3170) pmol/L from day 8 to day 10, and 4819 (2932) pmol/L from day 10 to the day of hCG administration. The mean final E2 concentrations were 10 335 (2917) pmol/L for the ACS-180 and 12 111 (3853) pmol/L for the Architect-i2000. The mean number of follicles per patient was 17.2 (range, 10–30), with a mean of 10.7 (range, 5–18) mature follicles. We obtained a value of 965 pmol E2/mature follicle for the ACS-180 and 1132 pmol E2/mature follicle for the Architect-i2000. The results obtained with E2 Architect-i2000 fell within the expected range of values for the amount of E2 per mature follicle.
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For 80 sera obtained on day 3 of the menstrual cycle from women donating oocytes at our IVF center, the mean E2 concentration obtained with the ACS-180 was 142 pmol/L (range, <110–337 pmol/L). Eight patients had E2 concentrations >220 pmol/L, corresponding to our cutoff point for the selection of patients for inclusion in the oocyte donation program. The mean E2 concentration obtained with the Architect-i2000 was 334 pmol/L (range, <169–572 pmol/L). The relationship between the results obtained with the two methods may be expressed as: Architect-i2000 = 1.025 ACS-180 + 187 pmol/L. Because of the positive difference, the cutoff point for the day 3 concentration limit (<220 pmol/L) for ACS-180 should be increased to 400 pmol/L for the Architect-i2000. We checked for agreement between the results obtained with the two assays after implementing this adjustment by calculating the 
coefficient; a good agreement between the results of the two tests was observed ( = 0.6875) (27).
In conclusion, E2 measurements with the automated Architect-i2000 system could be used to monitor ovulation in combination with transvaginal ultrasound. Although the mean E2 values obtained per mature follicle were slightly higher than with the ACS-180, the results obtained with the Architect-i2000 system fell into the range generally expected. As shown previously (28), the functional sensitivity of this method is insufficient for the evaluation of E2 in sera from children, men, or menopausal women. This assay has been optimized for clinical applications in which high concentrations are expected. It could be used for determinations in sera from women at the beginning of the menstrual cycle, to evaluate the functional status of the ovaries, and to evaluate down-regulation before ovarian stimulation. However, such applications require the upward modification of clinical cutoff points. Until assays are better standardized, clinical decision criteria (reference ranges, cutoff points) must be evaluated and, if necessary, modified for each new assay. Collaboration between laboratories and physicians is essential in the setting up of new immunoassays.
Acknowledgments
Abbott Laboratories provided assay reagent and the assay system without charge.
References
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