Comparison of day 3 FSH serum values as determined by six different immunoassays

Joëlle Taieb1,3, François Olivennes2, Anne Sophie Birr1, Clarisse Benattar1, Claudia Righini2, René Frydman2 and Albert Lindenbaum1

1 Department of Biochemistry and Hormonology and 2 Department of Obstetrics and Gynecology and Reproductive Endocrinology, Hôpital Antoine Béclère, Clamart, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The accurate assessment of FSH concentration is important for evaluating ovarian function prior to IVF. However, a number of different assay techniques are currently in use, leading to inconsistencies in the hormone data being reported. To address this problem, we measured FSH concentration using a number of commercially available systems. METHODS: Day 3 serum FSH levels were measured in 215 healthy fertile women using six different immunoassays: Coatria 125I (Bio-Mérieux), ACS-180 (Bayer Diagnostics), Advia-Centaur (Bayer Diagnostics), Vitros ECi (Ortho-Clinical Diagnostics), Architect i2000 (Abbott) and Elecsys 2010 (Roche Diagnostics). RESULTS: According to the immunoassay, means ± SD of FSH concentrations were: 6.5 ± 2.2 mIU/ml for Coatria 125I, 6.8 ± 2.7 mIU/ml for Advia-Centaur, 6.7 ± 3.0 mIU/ml for Vitros ECi, 7.6 ± 3.0 mIU/ml for ACS-180, 8.2 ± 3.3 mIU/ml for Architect i2000 and 8.8 ± 3.0 mIU/ml for Elecsys 2010. CONCLUSION: Day 3 FSH values determined by six different immunoassays were significantly different (P < 0.01, paired t-test). Physicians must take care when interpreting results from different clinical laboratories.

Key words: FSH/immunoassay/menstrual cycle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The assessment of ovarian reserve before ovarian stimulation is a major factor in the outcome of IVF. Currently, serum estradiol (E2), FSH and inhibin B levels are examined and combined at the beginning of the menstrual cycle (day 3 or 4) to evaluate the functional status of the ovaries, providing information for appropriate ovarian stimulation treatment and prognosis for IVF outcome (Scott et al., 1989Go, 1990Go; Licciardi et al., 1995Go; Sharara et al. 1998Go).

Several studies agree about the usefulness of the specific two-site enzyme-linked immunosorbent assay (ELISA) developed by Groome and O'Brien for inhibin B determination (Groome and O'Brien, 1993Go). The cut-off value generally used for day 3 serum inhibin B is 45 pg/ml (Seifer et al., 1997Go).

Non-isotopic or isotopic immunoassays for E2 and FSH measurements are currently used in clinical laboratories and inconsistent hormone data are frequently reported, particularly for FSH, making the results difficult to interpret. These discrepancies are due to differences in methods (resulting from FSH polymorphism, antibody specificities and preparation of the standards) and result in decision limit values being different for different immunoassays.

The aim of this study was to compare FSH values obtained on day 3 of the menstrual cycle using six different immunoassays.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study was a retrospective analytical investigation. We analysed serum from women donating oocytes in our IVF centre. These women underwent ovarian stimulation for oocyte retrieval. All were <35 years old, had regular menstrual cycles and were selected for the oocyte donation programme according to their day 3 hormonal status (E2, FSH and inhibin B concentrations). Blood samples were collected in Vacutainer® (Becton-Dickinson, Plymouth, UK) glass systems without anticoagulant (serum) and were separated by centrifugation. All samples were placed in 300 µl aliquots in screw-capped Eppendorf® tubes, frozen and stored at –70°C. A serum bank of 215 sera was thus constituted. As the volume of the samples obtained was limiting, the number of samples tested by each assay was between 77 and 215. The immunoassays tested included one isotopic method (FSH Coatria 125I; Bio-Mérieux, Marcy l'Etoile, France) and five non-isotopic immunometric methods performed on automated multi-analysers: ACS-180 (Bayer Diagnostics, Tarrytown, NY, USA), Advia-Centaur (Bayer Diagnostics), Vitros ECi (Ortho-Clinical Diagnostics Inc., Rochester, NY, USA), Architect i2000 (Abbott Laboratories, Abbott Park, IL, USA) and Elecsys 2010 (Roche Diagnostics, IND, USA). All are two-site immunoassays based on a one step method (except Architecht i2000, which is a two step method). The five non-isotopic assays are visualized by direct (Architect i2000, Advia-Centaur, ACS-180) or indirect (Vitros ECi) chemiluminescence or electrochemiluminescence (Elecsys 2010).

Before the clinical evaluation presented in this paper, for each method we analysed the precision (within and between runs) as described by the French National Committee for clinical laboratories (Vassault et al., 1999Go). It was studied using pools of serum ranging over the calibration curves. Between-run precision was analysed per 30 days using two sets of reagents.

All sera (n = 215) were evaluated by ACS-180, a multi-analyser system routinely used in our Hormonology department. For the other methods tested, the number of samples analysed was between 77 for Elecsys 2010 (these samples were also evaluated by Coatria 125I) and 133 for Vitros ECi (these samples included the 129 and 99 samples evaluated by Architect i200 and Advia-Centaur respectively). For each assay, mean and SD at day 3 were calculated. The paired Student's t-test, under Statview SE Software, was used for statistical analysis. A P-value < 0.05 was considered statistically significant.

Ethics
This study did not need approval by the French ethical committee. FSH measurements are required to evaluate the functional status of the ovaries before inclusion in an oocyte donation programme.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All methods show coefficients of variation of <3% (intra-assay) and <5% (inter-assay) for all analytic ranges (<5 to >50 mIU/ml; data not shown).

For day 3 FSH measurements, concentration ranges tested, mean results and SDs for each method are presented in Table IGo. For each assay, the concentration ranges analysed were similar: 3.2–17.4 mIU/ml for Elecsys 2010, 3.1–15.8 mIU/ml for Coatria 125I, 3.4–18.4 mIU/ml for Advia-Centaur, 3.6–21.5 mIU/ml for Architect i2000, 2.8–19.8 mIU/ml for Vitros ECi and 3.4–20.8 mIU/ml for ACS-180. The means ranged between 6.5 (FSH Coatria 125I) and 8.8 mIU/ml (Elecsys 2010) and the SD between 2.2 (FSH Coatria 125I) and 3.3 mIU/ml (Architect i2000).


View this table:
[in this window]
[in a new window]
 
Table I. Comparison of means and SDs for day 3 serum FSH concentrations as determined by six immunoassays
 
For ACS-180, two populations of samples (n = 82 and 133) were tested. The difference between the two sets of sera was not statistically significant (P = 0.11, non-paired Student's t-test) and they were consequently pooled for the study. The differences between all methods compared (on one hand, ACS-180, Coatria 125I and Elecsys 2010 and on other hand, ACS-180, Advia-Centaur, Vitros ECi and Architect i2000) were statistically significant (P < 0.01, paired Student's t-test).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Differences observed between the different methods tested may be due to the overall structure of FSH, a glycoprotein assembled from two distinct {alpha} and ß subunits and which presents heterogeneities in carbohydrate and peptide structures [different isoforms of FSH circulate (Stanton et al., 1992Go)] affecting immunoreactivity. The specificity of the monoclonal and/or polyclonal antibodies used in the assay may be such that some FSH isoforms are quantified by some, but not all, immunoassays. Although, some manufacturers had included in their immunoassay procedures two monoclonal (anti-ßFSH and anti-{alpha}FSH) antibodies (Architect i2000 and Coatria 125I) or one monoclonal (anti-ßFSH) and one polyclonal antibody (anti-{alpha}FSH; ACS-180, Advia-Centaur, Vitros ECi and Elecsys 2010), this approach was not sufficient to improve the immunoassay specificity.

The presence in the serum of various glycoproteins (LH, thyroid-stimulating hormone, HCG) structurally closely related to FSH (the same {alpha} subunit and homologies of the ß subunits) could contribute to the observed discrepancies. However, it should be noted that all manufacturers have tested cross-reactivities against these molecules and found no significant cross-reactivity. A central part of a measuring system is the standardization of assay. Although all manufacturers have tested their standard curve calibration (`secondary standard') against the same International Standard (NIBSC, 1998Go), the matrix of the standard is required to be identical to the matrix of the specimen and this condition is met only rarely [note that a FSH human recombinant standard is in preparation (Rose and Gaines-Das, 1998Go), which could diminish the influence of imperfect test standardization].

The last component which explains differences in assay measurements could result from the mathematical relationship permitting, on the system used, the conversion of the signal into the concentration of hormone. In our study, we did not find a clear difference in serum FSH between assays using monoclonal antibodies or monoclonal and polyclonal antibodies. We also did not find differences according to the signal measured (isotope, direct or indirect chemiluminescence). It is likely that a combination of all these factors, involved in the immunoassay procedure, contributes to the divergence of the results obtained (Büttner, 1991Go). This lack of agreement between FSH immunoassays is also illustrated by the French National Quality Control which clearly shows two major and distinct populations of results (République Française, 1999Go).

The present study, revealing statistically significant differences for day 3 FSH values according to the immunoassay used, raises issues for the interpretation of results from different clinical laboratories. FSH is widely used by physicians (in association with E2 and inhibin B) as a criterion for inclusion in assisted reproductive technology programmes. The discrepancies observed indicate that it is advisable to refer patients to selected laboratories using analytical methods for which they have defined reference values and decision limits for this clinical situation. We hope that, as soon as possible, manufacturers will harmonize their routine immunoassay systems. These efforts toward standardization would be beneficial for both physicians and clinical laboratories and could avoid travelling and expense for the patients.


    Notes
 
3 To whom correspondence should be addressed at: Department of Biochemistry and Hormonology, Hôpital Antoine Béclère, 157 rue de la porte de Trivaux, 92141 Clamart, France. E-mail: joelle.taieb{at}abc.ap-hop-paris.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Büttner, J. (1991) Philosophy of measurement by means of immunoassays. Scand. J. Clin. Lab. Invest., 51 (Suppl. 205), 11–20.[Medline]

Groome, N.P. and O'Brien, M. (1993) Immunoassays for inhibin and its subunits. Further applications of the synthetic peptide approach. J. Immunol. Methods, 165, 167–176.[ISI][Medline]

Licciardi, F.L., Liu, H.C. and Rosenwaks, Z. (1995) Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil. Steril., 64, 991–994.[ISI][Medline]

NIBSC (1998) National Institute for Biological Standards and Controls, WHO International Laboratory for Biological Standards. Follicle stimulating hormone pituitary for bioassay. 2nd International Reference Preparation. South Mimms, Potters Bar, Herts, UK.

République Française (1999) Ministère de l'Emploi et de la Solidarité, Secrétariat d'Etat à la Santé, Agence Française de Sécurité Sanitaire des Produits de Santé. Annales du contrôle national de qualité, 16, 78–79.

Rose, M.P. and Gaines-Das, R.E. (1998) Characterisation, calibration and comparison by international collaborative study of international standards for the calibration of FSH. J. Endocrinol., 158, 94–114.

Scott, R.T., Toner, J.P., Muasher, S.J., Oehninger, S., Robinson, S. and Rosenwaks, Z. (1989) Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil. Steril., 51, 651–654.[ISI][Medline]

Scott, R.T., Hofmann, G.E., Oehninger, S. and Muasher S.J. (1990) Intercycle variability of day 3 follicle-stimulating hormone levels and its effect on stimulation quality in in vitro fertilization. Fertil. Steril., 54, 297–302.[ISI][Medline]

Seifer, D.B., Lambert-Masserlian, G., Hogan, J.W., Gardiner, A.C., Blazar, A.S. and Berk, C.A. (1997) Day 3 serum inhibin B is predictive of assisted reproductive technologies outcome. Fertil. Steril., 67, 110–114.[ISI][Medline]

Sharara, F.I., Scott, R.T. Jr and Seifer, D.B. (1998) The detection of diminished ovarian reserve in infertile women. Am. J. Obstet. Gynecol., 179, 804–812.[ISI][Medline]

Stanton, P.G., Robertson, D.M. and Burgon, P.G. (1992) Isolation and physicochemical characterization of human follicle-stimulating hormone isoforms. Endocrinology, 130, 2820–2832.[Abstract]

Vassault, A., Grafmeyer, D., de Graeve, J., Cohen, R., Beaudonnet, A. and Bienvenu, J. (1999) Analyses de biologie médicale: spécifications et normes d'acceptibitilé à l'usage de la validation de techniques. Ann. Biol. Clin., 57, 685–695.[ISI]

Submitted on July 2, 2001; accepted on November 12, 2001.





This Article
Abstract
FREE Full Text (PDF )
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Request Permissions
Google Scholar
Articles by Taieb, J.
Articles by Lindenbaum, A.
PubMed
PubMed Citation
Articles by Taieb, J.
Articles by Lindenbaum, A.