Measurement of Estradiol—Challenges Ahead

SPECIAL FEATURE

Commentary

Frank Z. Stanczyk and Nigel J. Clarke

Obstetrics and Gynecology, and Preventive Medicine (F.Z.S.), University of Southern California Keck School of Medicine, Los Angeles, California 90033; and Steroids Department (N.J.C.), Quest Diagnostics Nichols Institute, San Juan Capistrano, California 92675 Downloaded from https://academic.oup.com/jcem/article/99/1/56/2836184 by guest on 28 September 2021 n their recent Endocrine Society position statement on quired to be accurate and precise across several orders of I the measurement of estradiol, Rosner et al (1) provide concentration. a good overview of the current status of estradiol assays in the context of their clinical and research applications. The position statement includes both published data and the Estradiol Measurements in Relevant expert opinions of the authors, and it is long overdue be- Populations cause estradiol assay methods that lack accuracy and precision are being used in many clinical and research settings It is obvious from the position statement that standardiz- (2, 3). Problems of inaccuracy are particularly found when ing estradiol assays to encompass such a wide range of measuring the low estradiol levels commonly seen in pre- estradiol levels will be a formidable task and could take pubertal children, postmenopausal women, and men, as years to accomplish, although the difficulties already en- well as in patients treated with aromatase inhibitors. The countered and overcome in standardizing the T assay position statement outlines the many challenges associ- should provide useful information. Our recommended ap- ated with standardizing estradiol measurements using proach is to focus on standardizing assays that measure mass spectrometry assay methodology. The purpose of the estradiol levels in the range of l–1000 pg/mL. This range Ͻ present commentary is to point out: 1) the relevant pop- would include the very low estradiol levels ( 20 pg/mL) ulations in which estradiol measurements are critical; and found in prepubertal children, postmenopausal women, 2) the problematic ranges of these measurements. and patients treated with aromatase inhibitors. Also in- cluded in that range would be the estradiol levels found in adolescent boys and in men (Ͻ40 pg/mL); in premeno- Range of Estradiol Measurements pausal women during the follicular, periovulatory, and luteal phases of the menstrual cycle (30–800 pg/mL); and A very wide range of estradiol levels are seen in clinical and in postmenopausal women treated with estrogenic prep- research settings (Table 1). Physiological estradiol levels in arations, for example, conjugated equine estrogens or mi- prepubertal children, adolescents, and adult women (pre- cronized estradiol (20–100 pg/mL). menopausal, pregnant, and postmenopausal) and men en- At the present time, it is not necessary to standardize compass a range of approximately 1–20 000 pg/mL. This assays that measure estradiol levels Ͼ1000 pg/mL, which range also includes estradiol levels obtained in breast can- are found in pregnancy and in premenopausal women un- cer patients treated with aromatase inhibitors, in post- dergoing fertility treatments involving controlled ovarian menopausal women treated with estrogen, and in patients stimulation. Although circulating levels of estradiol in- undergoing ovarian stimulation treatment. The range can crease dramatically during the second and third trimesters be extended at the lower end to Ͻ1 pg/mL in patients of pregnancy and reach levels as high as 20 ng/mL (4), treated with aromatase inhibitors. Thus, estradiol assays estradiol is not a good marker of fetal well-being because employed by clinical and research laboratories are re- its androgenic precursor dehydroepiandrosterone sulfate

56 jcem.endojournals.org J Clin Endocrinol Metab, January 2014, 99(1):56–58 doi: 10.1210/jc.2013-2905 doi: 10.1210/jc.2013-2905 jcem.endojournals.org 57

Table 1. Approximate Range of Serum Estradiol Levels Over time they will undoubtedly morph into smaller, sim- in Prepubertal Children, Adolescents, Women, and Men pler, and less expensive instruments destined for the and During Certain Treatments smaller practice laboratories and physicians’ offices. Some of the challenges that the vendors will face will depend on Estradiol Levels, Estradiol Source pg/mL the type of instrument involved, but it will be important for all of these instruments to be user-friendly, fully inte- Prepubertal children Ͻ20 Adolescent girls 20–300 grated, and comparable to the currently used immunoas- Menstrual cycle 30–800 say platforms in terms of expense and skill set. It will also Pregnancy Up to 20 000 be important for the instrument vendors to do as much as Postmenopausal women Ͻ20 Adolescent boys Ͻ40 possible in supplying fully validated clinical mass spec- Ͻ Men 40 trometry assays. This is essential for labs that don’t have Downloaded from https://academic.oup.com/jcem/article/99/1/56/2836184 by guest on 28 September 2021 Ͻ Patients on aromatase inhibitors 5 access to research and development personnel. Postmenopausal estrogen therapy 20–100 Ovarian stimulation treatment Up to 10 000

is derived equally from the maternal and fetal compart- Problematic Ranges of Estradiol ments (5). Instead, estriol has been used as the marker Measurements because its androgenic precursor (16␣-hydroxy dehydro- An important and difficult challenge identified in the po- epiandrosterone sulfate) is derived predominantly from sition statement is the standardization of estradiol mea- the fetus (5). By contrast, measurement of estradiol plays surements at the very low concentrations (Ͻ30 pg/mL) an important role in monitoring response during ovarian found in prepubertal girls and boys and in postmeno- stimulation in women with infertility. pausal women, as well as the extremely low concentra- Owing to the very high estradiol levels (up to 10 ng/mL) tions that can be achieved in patients undergoing treat- achieved during ovarian stimulation treatment, estradiol ment with aromatase inhibitors. Standardizing estradiol is measured predominantly by direct immunoassays on measurements is especially important for patients with automated platforms, which are now frequently located in breast cancer receiving aromatase inhibitors because their doctors’ offices. Although estradiol levels are overesti- serum samples are often sent to diagnostic testing labora- mated by these direct assays owing to cross-reaction with tories that use direct estradiol immunoassays—assays in high levels of estradiol metabolites, for example, estrone which estradiol is not purified before its quantitation (6). sulfate, clinicians are aided in their interpretation of es- Antibodies in direct immunoassays often cross-react with tradiol levels by concurrent ultrasound monitoring of ovarian follicular development. Moreover, fertility clinics aromatase inhibitors and their metabolites and affect the require rapid, cost-effective, same-day results to facilitate test results, which is especially true of exemestane (7). The clinical management of their patients; therefore, sending falsely high results from such assays could affect the treat- the samples away for mass spectrometry assessment ment outcome and safety of patients. would be inconvenient and too costly. Furthermore, in The validity of very low estradiol measurements is addition to measuring estradiol, immunoassay-based in- questionable, even in the research setting when these mea- struments are also capable of measuring reproductive pro- surements are obtained by conventional RIA methods in teins, eg, FSH and human chorionic gonadotropin, which which estradiol first undergoes one or two purification are essential for monitoring patients undergoing ovarian steps. These assays are often used in studies that determine stimulation treatment. the suppression of estradiol levels by aromatase inhibitors. The increasing permeation of mass spectrometry assays Notable deficiencies in some of these assay methods in- throughout the large diagnostic clinical laboratories clude the following: extrapolation of estradiol concentra- means that in the near future mass spectrometry-based tions below the lowest point on the standard curve; use of measurements will probably be available on the same day an insufficient number of estradiol concentrations at the to clinicians. The advent of affordable, accessible, and low end to generate an accurate standard curve; lack of a accurate mass spectrometry-based assays for use in phy- chromatographic step to separate potential unconjugated sicians’ offices and smaller laboratories will no doubt pres- cross-reacting compounds (the aromatase inhibitor and its ent challenges at first. However, it is becoming clear that metabolites, and estradiol metabolites); and addition of this is the inevitable direction in which vendors of mass too much mass of estradiol to each sample when 3H-es- spectrometry instruments are moving. The first fully in- tradiol is used as the internal standard to follow proce- tegrated diagnostic instruments will most likely be tar- dural losses. Also, the claimed estradiol assay sensitivity in geted at reference labs or larger hospital laboratories. some studies is Ͻ1 pg/mL, yet no information is given 58 Stanczyk and Clarke Estradiol Measurements J Clin Endocrinol Metab, January 2014, 99(1):56–58 regarding how the sensitivity was determined. Such dis- curacy and precision, which is in the best interest of the crepancies have become increasingly clear with the adop- patient. tion of analysis using mass spectrometry.

Acknowledgments Reference Intervals Address all correspondence and requests for reprints to: Dr Another major challenge discussed in the position state- Frank Z. Stanczyk, Reproductive Endocrine Research Labo- ment is the requirement for estradiol reference intervals ratory, Livingston Research Building, 1321 North Mission Road, Room 201, Los Angeles, CA 90033. E-mail: fstanczyk@ derived from well-characterized, adequately sized popu- att.net. lations using standardized procedures, such as those for- Disclosure Summary: F.Z.S. has nothing to disclose. N.J.C. is Downloaded from https://academic.oup.com/jcem/article/99/1/56/2836184 by guest on 28 September 2021 mulated by the Clinical and Laboratory Standards Insti- an employee of Quest Diagnostics and owns stock in Quest. tute. A particular challenge will be the establishment of reference ranges in a sufficient number of postmenopausal women by age groups, stage of postmenopause, and type References of menopause (natural vs surgical). Because adipose tissue is the predominant source of estradiol in postmenopausal 1. Rosner W, Hankinson SE, Sluss PM, Vesper HW, Wierman ME. women, the estradiol reference ranges will have to be ad- Challenges to the measurement of estradiol: an Endocrine Society position statement. J Clin Endocrinol Metab. 2013;98:1376–1387. justed for level of obesity. Serum estradiol levels in post- 2. Stanczyk FZ, Lee JS, Santen RJ. Standardization of steroid hormone menopausal women are positively associated with body assays: why, how, and when? Cancer Epidemiol Biomarkers Prev. mass index (8). Of note, the interaction of age and body 2007;16:1713–1719. 3. Stanczyk FZ, Clarke NJ. Advantages and challenges of mass spec- mass index can also affect estradiol concentrations in es- trometry assays for steroid hormones. J Steroid Biochem Mol Biol. trogen-treated postmenopausal women (8). Taking these 2010;121:491–495. factors into account, accurate estradiol reference ranges 4. Levitz M, Young BK. Estrogens in pregnancy. Vitam Horm. 1977; 35:109–147. for postmenopausal women can help guide individualized 5. Goebelsmann U. Protein and steroid hormones in pregnancy. J Re- hormone replacement therapy to ensure that levels are prod Med. 1979;23:166–177. kept within a physiological range. 6. Jaque J, Macdonald H, Brueggmann D, et al. Deficiencies in immunoassay methods used to monitor estradiol levels during aromatase inhibitor treatment in postmenopausal breast cancer patients. SpringerPlus. 2013;2:5. Conclusion 7. Geisler J, King N, Anker G, et al. In vivo inhibition of aromatization by exemestane, a novel irreversible aromatase inhibitor, in postmenopausal breast cancer patients. Clin Cancer Res. 1998;4:2089– Ultimately, meeting the challenges specified in The Endo- 2093. crine Society position statement will have a major impact 8. Karim R, Mack WJ, Hodis HN, Roy S, Stanczyk FZ. Influence of age on the diagnosis and treatment of endocrine-related dis- and obesity on serum estradiol, estrone, and sex hormone binding globulin concentrations following oral estrogen administration in orders and disease. The position statement should moti- postmenopausal women. J Clin Endocrinol Metab. 2009;94:4136– vate those involved with estradiol assays to strive for ac- 4143.