Short Communication The Optimal Timing of Blood Collection during the Menstrual Cycle for the Assessment of Endogenous Sex Hormones: Can Interindividual Differences in Levels over the Whole Cycle Be Assessed on a Single Day? 1 Naseer Ahmad, Tessa M. Pollard, and Nigel Unwin 2 Departments of Diabetes [N. U.] and Epidemiology and Public Health [N. A., N. U.], University of Newcastle, Newcastle NE2 4HH, United Kingdom, and Department of Anthropology, University of Durham, Durham, United Kingdom [T. M. P.] Abstract The objective of this study was to identify the optimal timing of sampling during the menstrual cycle for assessment of interindividual variation in exposure to endogenous sex hormones, including estradiol, progesterone, and the free androgen index. Twenty-four healthy premenopausal women with regular periods were recruited, and alternate day venous blood samples were taken in the morning throughout one menstrual cycle. Spearman rank correlation coefficients were calculated for the estimates of average hormone levels (based on area under the curve) over one menstrual cycle against values on single days within that cycle. Days within the menstrual cycle were identified that provided the best assessment of interindividual differences. The most consistent correlation for estradiol was seen between days 9 and 11 (e.g., r  0.53 and P  0.01, day 10), the most consistent correlation for progesterone was seen between days 17 and 21 (e.g., r  0.80 and P < 0.001, day 20), and the most consistent correlation for free androgen index was seen between days 12 and 15 (e.g., r  0.90 and P < 0.001, day 15). Post hoc analysis of estradiol and progesterone levels on days counted back from the start of the next menstrual cycle identified marginally stronger associations. On repeat hormone measurements (not done for progesterone) on days 10 and 15, two to five menstrual cycles later, correlation coefficients with the original hormone levels remained reasonable (>0.55) for most. In conclusion, a reasonable characterization of interindividual differences in premenopausal estradiol, androgen, and progesterone levels may be achieved with single blood samples taken on specific days. This provides a useful approach for future epidemiological studies. Introduction Endogenous sex hormones have been related to the risk of several diseases in women, including breast and ovarian cancer (1, 2), osteoporosis (3), and cardiovascular disease (4, 5). Most evidence for the effects of these hormones comes from com- paring risk in women before and after the menopause [e.g., Rannevik et al. (6)], from our increasing understanding of the effects of postmenopausal hormone replacement therapy [e.g., Anonymous (7)], or from comparing risk according to endog- enous postmenopausal hormone levels [e.g., Hankinson et al. (8)]. However, there is good evidence that variation between populations in premenopausal endogenous sex hormone levels correlates with variation in reproductive cancers in women (9) and also that within modern industrial populations, overall exposure to estrogen over the reproductive years is an important risk factor for the same cancers (8) and affects the degree of protection against cardiovascular disease (10). It is important, therefore, that we understand the variation in premenopausal sex hormone levels between and within popula- tions and its causes. Unfortunately, such comparisons are plagued by methodological difficulties associated with changes in sex hormones levels during the menstrual cycle (2). Thus, some re- searchers have chosen the labor-intensive course of collection of multiple samples of either serum or saliva through at least one menstrual cycle (11–13). Gann et al. (14) suggest that small and intensive studies may find repeated salivary samples, which are expensive, most useful, but that timed one-off serum samples are potentially more useful for larger studies. However, some studies have failed to address the timing of samples (15), whereas others have sampled at one stage of the menstrual cycle, with [e.g., Bernstein et al. (16)] or without [Kamath et al. (17)] attempts to justify their sampling strategy. Michaud et al. (18) recognized the importance of evaluating whether a single plasma hormone measurement can be used to estimate stable differences between individuals, given that most epidemiological studies can only collect one sample per partici- pant. They compared the reproducibility of plasma estrogen and progesterone levels over 1 year, with estrogens assessed either during the follicular phase (on the 3 rd ,4 th , or 5 th day of the menstrual cycle) or in the luteal phase (7–9 days before the anticipated start of the next cycle), and progesterone assessed only in the luteal phase. They found that reproducibility was good, except for estradiol in the luteal phase, although after the exclusion of women who were anovulatory (identified by low progesterone levels) and those whose samples were collected 4 days or 10 days before their next menses, reproducibility in luteal phase samples was also moderately good. However, to our knowledge, no study has investigated in greater depth than this the extent to which interindividual differences can be reliably characterized using measurements on any one day during the menstrual cycle. Received 2/23/01; revised 10/12/01; accepted 11/5/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by The Northern Regional Health Authority. 2 To whom requests for reprints should be addressed, at Department of Diabetes, University of Newcastle, Medical School, Newcastle NE2 4HH, United Kingdom. Phone: 44-191-222-5407; Fax: 44-191-261-1763; E-mail: n.c.unwin@ncl.ac.uk. 147 Vol. 11, 147–151, January 2002 Cancer Epidemiology, Biomarkers & Prevention on January 21, 2022. © 2002 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from on January 21, 2022. © 2002 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from on January 21, 2022. © 2002 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from