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Use of the architect-i2000 estradiol immunoassay during in vitro fertilization.

In women undergoing in vitro fertilization and embryo transfer (IVF-ET), serial measurements of 17[beta]-estradiol ([E.sub.2]) can be used to monitor follicular growth, and serum

[E.sub.2] has been shown to be correlated with the number and diameter of preovulatory follicles observed on transvaginal ultrasound scan (1-6). The serum [E.sub.2] 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 [E.sub.2] 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 [E.sub.2] 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 [E.sub.2] (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 [E.sub.2] 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 [E.sub.2] immunoassays. The two methods are linear up to 3670 pmol/L and use monoclonal antibody derived by coupling the [E.sub.2] 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 (12-15). 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 17[beta]-[E.sub.2] or [E.sub.2] valerate, respectively. [E.sub.2] 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 [E.sub.2] 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 [E.sub.2] 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 [E.sub.2] 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 [E.sub.2] assay gave higher estimates of [E.sub.2] concentration than did ACS-180 over this concentration range (slopes >1 and intercept at +62 pmol/L for high and low [E.sub.2] 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.

The values obtained for serum [E.sub.2] depend on the method used (17-20). Various factors may account for the differences between the two methods (21). These factors include differences in calibration curves, antibody specificity, the matrix effect (22-24), and the mathematical relationship permitting the conversion of the signal obtained into [E.sub.2] concentration in the system used.

The results obtained with the two [E.sub.2] immunoassays for specimens from patients receiving micronized 17[beta]-[E.sub.2] or [E.sub.2] 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 [E.sub.2] valerate were ~10% higher than those obtained with ACS-180, which corresponded to the cross-reaction of 6% obtained in vitro by the manufacturer.

[FIGURE 1 OMITTED]

Before beginning ovarian stimulation, it is necessary to check that down-regulation has been successful to prevent the stimulation of apoptotic follicles. The [E.sub.2] 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, [E.sub.2] concentrations obtained with the ACS-180 method were in all cases [less than or equal to]184 pmol/L. The mean [E.sub.2] 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 [E.sub.2] 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 [E.sub.2] concentration, mean final [E.sub.2] concentration before hCG administration, mean number of mature follicles (with a diameter >15 mm, as measured by transvaginal ultrasound), and the relationship between [E.sub.2] concentration and the number of mature follicles are presented in Table 1. The two [E.sub.2] immunoassays gave similar results. The mean (SD) increase in [E.sub.2] 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 [E.sub.2] 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 [E.sub.2] 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 [E.sub.2] 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 [E.sub.2]/mature follicle for the ACS-180 and 1132 pmol [E.sub.2]/mature follicle for the Architect-i2000. The results obtained with [E.sub.2] Architect-i2000 fell within the expected range of values for the amount of [E.sub.2] per mature follicle.

For 80 sera obtained on day 3 of the menstrual cycle from women donating oocytes at our IVF center, the mean [E.sub.2] concentration obtained with the ACS-180 was 142 pmol/L (range, <110-337 pmol/L). Eight patients had [E.sub.2] concentrations >220 pmol/L, corresponding to our cutoff point for the selection of patients for inclusion in the oocyte donation program. The mean [E.sub.2] 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 [kappa] coefficient; a good agreement between the results of the two tests was observed ([kappa] = 0.6875) (27).

In conclusion, [E.sub.2] measurements with the automated Architect-i2000 system could be used to monitor ovulation in combination with transvaginal ultrasound. Although the mean [E.sub.2] 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 [E.sub.2] 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 downregulation 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.

Abbott Laboratories provided assay reagent and the assay system without charge.

References

(1.) Howles CM, Macnamee MC. Endocrine monitoring for assisted human conception. Br Med Bull 1990;46:616-27.

(2.) Balos O, Lundkvist O, Wide L, Bergh T. Ultrasonographical and hormonal description of the normal ovulatory menstrual cycle. Acta Obstet Gynecol Scand 1994;73:790-6.

(3.) Muse MD, Wilson EA. Monitoring ovulation: use of biochemical and biophysical parameters. Semin Reprod Endocrinol 1986;4:301-9.

(4.) Fossum G, Vermesh M, Kletzky OA. Biochemical and biophysical indices of follicular development in spontaneous and stimulated ovulatory cycles. Obstet Gynecol 1990;75:407-11.

(5.) Sushanek E, Huderer K, Dobec D, Hlavati V, Simunic V, Grizelj V. Number of follicles, oocytes and embryos in human in vitro fertilization is relative to serum estradiol and progesterone patterns during different types of ovarian hyperstimulation. Eur J Obstet Gynecol Reprod Biol 1994;56:121-7.

(6.) Dor J, Seidman DS, Ben-Shlomo I, Levran D, Karasik A, Mashiach S. The prognostic importance of the number of oocytes retrieved and estradiol levels in poor and normal responders in in vitro fertilization (IVF) treatment. J Assist Reprod Genet 1992;9:228-32.

(7.) Levran D, Lopata A, Nayudu PL, Martin MJ, McBain JC, Bayly CM, et al. Analysis of the outcome of in-vitro fertilization in relation to the timing of human chorionic gonadotropin administration by the duration of estradiol rise in stimulated cycles. Fertil Steril 1985;44:335-41.

(8.) Navot D, Bergh PA, Laufer N. Ovarian hyperstimulation syndrome in novel reproductive technologies: prevention and treatment. Fertil Steril 1992;58: 249-61.

(9.) Morris RS, Paulson RJ, Sauer MV, Lobo RA. Predictive value of serum estradiol concentrations and oocyte number in severe ovarian hyperstimulation syndrome. Hum Reprod 1995;10:811-4.

(10.) Hughes EG, Fedorkow DM, Daya S, Sagle MA, Van de Koppel P, Collins JA. The routine use of gonadotropin-releasing hormone agonists prior to in vitro fertilization and gamete intra-Fallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril 1992;58:888-96.

(11.) Licciardi FL, Liu HC, Rosenwaks Z. Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil Steril 1995;64:991-4.

(12.) Spencer CA, Takeuchi M, Kararosyan M. Current status and performance goals for serum thyrotropin (TSH) assays. Clin Chem 1997;42:140-5.

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(14.) Taieb J, Sarnel C, Benattar C, Lindenbaum A. A new technique for measuring 17[beta]-estradiol using Kryptor (Cis Bio International): utilization to monitor ovulation stimulation. Ann Biol Clin 2000;58:71-9.

(15.) Taieb J, Benattar C, Chalas J, Messaoudi C, Lindenbaum A. Comparaison de cinq techniques de dosage direct du 17[beta] estradiol. Immunoanal Biol Spec 1997;12:267-74.

(16.) Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307-10.

(17.) Schroder V, Thode J. Six direct radioimmunoassays of estradiol evaluated. Clin Chem 1988;34:949-52.

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(19.) Mikkelsen AL, Borggaard B, Lebech PE. Results of serial measurement of estradiol in serum with six different methods during ovarian stimulation. Gynecol Obstet Invest 1996;41:34-40.

(20.) Tummon I, Stemp J, Rose C, Vandenberghe H, Bany B, Tekpetey F, et al. Precision and method bias of two assays for oestradiol: consequences for decisions in assisted reproduction. Hum Reprod 1999;14:1175-7.

(21.) Buttner J. Philosophy of measurement by means of immunoassays. Scand J Clin Lab Invest 1991;51(Suppl 205):11-20.

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Joelle Taieb, * Clarisse Benattar, Rokhaya Diop, Anne Sophie Birr, and Albert Lindenbaum

(Hopital Antoine Beclere, Clamart, France, Department of Biochemistry and Hormonology, 157 rue de la porte de Trivaux, 92141 Clamart cedex, France; * author for correspondence: fax 33-1-45374745, e-mail joelle.taieb@abc.aphop-paris.fr)
Table 1. Mean increase in [E.sub.2] concentration during 25 cycles of
ovarian induction, mean final [E.sub.2] concentration, mean number of
mature follicles, and relationship between E2 concentration and the
number of mature follicles for the [E.sub.2] Architect-i2000 and
[E.sub.2] ACS-180 immunoassays.

 Architect-
 ACS-180 i2000

Mean (SD) increase in [E.sub.2]
concentration, pmol/L (a)
 From day 1 to day 6 1601 (828) 1680 (833)
 From day 6 to day 8 2026 (928) 2345 (1119)
 From day 8 to day 10 4030 (2257) 4734 (3170)
 From day 10 to day of hCG 3997 (2015) 4819 (2932)
 administration
Mean (SD) final [E.sub.2] 10 335 (2917) 12 111 (3853)
 concentration, pmol/L
Mean number of mature follicles 10.7 (range, 5-18)
 (diameter >15 mm)
Mean amount of [E.sub.2] (pmol) 965 1132
 per mature follicle

(a) 1 pmol/L = 3.67 pg/mL.
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Title Annotation:Technical Briefs
Author:Taieb, Joelle; Benattar, Clarisse; Diop, Rokhaya; Birr, Anne Sophie; Lindenbaum, Albert
Publication:Clinical Chemistry
Date:Jan 1, 2003
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