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Context Epidemiologic studies have demonstrated that overweight/obese girls (OW/OB) undergo thelarche and menarche earlier than normal weight girls (NW). There have been no longitudinal studies to specifically investigate how body weight/fat affects both clinical and biochemical pubertal markers in girls. Objective To investigate the effect of total body fat on reproductive hormones and on the maturation of estrogen-sensitive tissues during puberty in girls. Methods Ninety girls (36 OW/OB, 54 NW), aged 8.2 to 14.7 years, completed 2.8 ± 1.7 study visits over 4 years. Visits included dual-energy x-ray absorptiometry to calculate total body fat (TBF), Tanner staging, breast ultrasound for morphological staging (BMORPH; A-E), pelvic ultrasound, hormone tests, and assessment of menarchal status. The effect of TBF on pubertal markers was determined using a mixed, multistate, or Cox proportional hazards model, controlling for baseline BMORPH. Results NW were older than OW/OB (11.3 vs 10.2 years, P Conclusion In late puberty, girls with higher TBF demonstrate differences in standard hormonal and clinical markers of puberty. Investigation of the underlying causes and clinical consequences of these differences in girls with higher TBF deserves further study.

Background Girls who are overweight/obese (OB) develop breast tissue but do not undergo menarche (the first menstrual period) significantly earlier than girls of normal weight (NW). It has been proposed that estrogen synthesized by adipose tissue may be contributory, yet OB do not have higher serum estrogen levels than NW matched on breast stage. We hypothesized that estrogen synthesized locally, in mammary fat, may contribute to breast development. This hypothesis would predict that breast development would be more advanced than other estrogen-sensitive tissues as a function of obesity and body fat. Methods 80 pre-menarchal girls (26 OB, 54 NW), aged 8.2–14.7 yrs, underwent dual-energy x-ray absorptiometry to calculate percent body fat (%BF), Tanner staging of the breast, breast ultrasound for morphological staging, trans-abdominal pelvic ultrasound, hand x-ray (bone age), a blood test for reproductive hormones, and urine collection to determine the vaginal maturation index (VMI), an index of estrogen exposure in urogenital epithelial cells. Results When controlling for breast morphological stage determined by ultrasound, %BF was not associated with serum estrogen or gonadotropin (LH and FSH) levels or on indices of systemic estrogen action (uterine volume, endometrial thickness, bone age advancement, and VMI). Tanner breast stage did not correlate with breast morphological stage and led to misclassification of chest fatty tissue as breast tissue in some OB. Conclusions These studies do not support the hypothesis that estrogen derived from total body fat or local (mammary) fat contributes to breast development in overweight/obese girls.

Background: Studies comparing the timing and pace of puberty in overweight/obese girls (OW/OB) vs normal weight girls (NW) have produced conflicting results; some suggest earlier activation of the central components of the reproductive axis in OB while others are more consistent with a peripheral source of estrogen (e.g. adipose tissue) driving puberty in OB. Importantly, there have been no longitudinal assessments of both clinical and biochemical pubertal markers in OB vs. NW. Methods: 90 healthy pre-menarchal girls (26 OW/OB, 54 NW) from the community, aged 8.2-14.7 years, completed 2.8 ± 1.7 (mean, SD) study visits over the course of 4 years. Visits included dual-energy x-ray absorptiometry to calculate percent total body fat (TBF), Tanner staging, breast ultrasound for morphological staging (BMORPH; stages A-E), pelvic ultrasound, hand x-ray (bone age, BA), blood tests for reproductive hormones, and urine collection to determine a vaginal maturation index (VMI), an index of estrogen exposure in urogenital epithelial cells. Menarchal status was determined at each visit and via follow-up questionnaires. The effect of TBF on hormones and markers of estrogen action, the pace of breast maturation, and age at menarche were determined using a mixed, multi-state, or Cox proportional hazards model, respectively. Mixed and Cox models controlled for BMORPH at visit 1 (V1) and race. Results: NW girls were older than OW/OB (11.3 vs. 10.2 yrs, p

Overweight/obese girls (OB) undergo thelarche but not menarche significantly earlier than normal weight girls (NW). It has been proposed that estrogen synthesized by adipose tissue may be contributory, yet OB do not have higher serum estrogen levels than NW matched on breast stage. We hypothesized that in OB, adipose tissue overlying the breast bud may be a ready source of estrogen that is sufficient to initiate thelarche because the primitive breast bud is intimately associated with the mammary fat pad during development (Anbazhagan Am J Anat ’91, Neville J Mammary Gland Biol Neoplasia ’98). Estrogen derived from mammary fat could act locally to induce thelarche but would not be detected in the circulation and therefore would not act systemically to induce uterine maturation/menses. To address this hypothesis, we measured reproductive hormones and imaged the breast, uterus, and ovaries in 80 pubertal, pre-menarchal girls (26 OB, 54 NW) aged 8.2-14.7 yrs. We used total percent body fat (%BF), determined by DXA, as a proxy for mammary fat, taking advantage of the positive correlation between these two measures (Novotny Am J Hum Biol ’11, Schautz Eur J Clin Nutr ’11, Zhu Eur Radiol ’16). We predicted that if estrogen derived from mammary fat contributes to breast maturation, then girls with higher %BF (and thus more mammary fat) would demonstrate immaturity of other estrogen-sensitive tissues (e.g. uterus, endometrial lining, vaginal epithelium) compared with girls of the same breast morphological stage (stages A-E based on Bruni et al. Adolesc Pediatr Gynecol ‘90) with lower %BF. OB were younger than NW (10.1 ± 1.1 vs. 11.3 ± 1.3 yrs, p

Meta-analyses consistently show that postmenopausal women with elevated levels of estradiol have an increased risk for certain cancers, such as breast cancer. At the same time, estradiol therapy is used to treat menopausal symptoms. Data on blood levels of estradiol are highly variable, especially in postmenopausal women, which prevents the formulation of generally recognized normal ranges and the consistent treatment of postmenopausal women. Similarly, no generally recognized reference ranges for estradiol exist for men and children. One reason for this variability in estradiol blood levels is the lack of appropriate analytical methods. To overcome these challenges, new, highly sensitive, specific, and standardized methods for measuring estradiol are needed. This study describes a new analytical method for measuring estradiol in serum from post and premenopausal women, men and children, which is standardized to the CDC Hormone Standardization Program (HoSt). Data produced by this analytical method can be compared with data produced by other methods standardized to CDC HoSt. The analytical method optimizes the extraction of estradiol from serum using two liquid-liquid extractions with pH and polarity adjustments prior to LC-MS/MS analysis. All sample handling and extraction procedures are automated using 96-well plates. Chromatographic separation is carried out using a phenyl-hexyl HPLC column and a gradient of methanol and methanol:water. E2 and its C13 internal standard were analyzed by selected reaction monitoring (SRM) in the negative ion mode with transitions of m/z 271 to 145 and 274 to 148, respectively. The mean biases of this method to certified reference materials ranged between −1.3% and −1.7% and were statistically not significant. No significant difference to established metrological reference methods was determined. The limit of detection for estradiol using 200 µL of serum is 11.0 pM (2.99 pg/mL). The method is highly precise with a within-run, among-day (determined over 68 days), and total within-laboratory imprecision ranging between 2.9-5.0%, 1.5-1.8%, and 3.3-5.3% CV, respectively [1]. The sensitivity of the method was found to be suitable for determining estradiol levels in postmenopausal women, men, and children, and is being applied to measure estradiol in postmenopausal women in NHANES 2013-2014. Data generated by this method are comparable to those of other analytical methods that are standardized to the CDC HoSt program. This method allows for the development of generally recognized normal ranges of estradiol in postmenopausal women and other population subgroups. References: 1. Zhou, Hui et al. “Simultaneous measurement of total estradiol and testosterone in human serum by isotope dilution liquid chromatography tandem mass spectrometry” Analytical and bioanalytical chemistry vol. 409,25 (2017): 5943-5954. Citation Format: Lumi Duke, Paul H. Kim, Julianne Cook Botelho, Hui Zhou, Candice Z. Ulmer, Hubert W. Vesper. Highly sensitive and accurate method for measuring serum estradiol in postmenopausal women and other population subgroups [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 587.

Endogenous steroid hormones play a critical role in the etiology of breast cancer. However, the interrelationships among estrogens, androgens, and progesterone in determining risk are not well understood. Relative strengths of associations, independent and joint effects, differential effects for tumor subtypes, and effect modification by lifestyle and genetic profile remain unclear. A major reason is the limited accuracy, specificity, and sensitivity of many steroid hormone assays, especially at the low circulating hormone concentrations characteristic of postmenopausal women. CDC developed a new sensitive, accurate isotope dilution LC-MS/MS method capable of measuring eight important steroid hormones in 200 µL of female serum. The method uses sequential liquid-liquid extractions for isolation of estradiol (E2), estrone (E1), estrone sulfate (E1S), testosterone (TT), androstenedione (A), dehydroepiandrosterone sulfate (DHEAS), progesterone (P), and 17-hydroxyprogesterone (17-OHP) without derivatization or hydrolysis. Chromatographic separation of the steroids is carried out using a phenyl-hexyl HPLC column and a gradient of methanol and methanol:water. Scheduled selected reaction monitoring by electrospray ionization in the positive and negative ion modes is applied to quantify the analytes. Two mass transitions, each specific to the fragmentation of the analyte, are monitored for each analyte: quantitation ion (QI) and confirmation ion (CI). The QI to CI ratios are used as criteria to identify possible interferences. The method is able to measure each steroid in nearly all postmenopausal women as well as premenopausal women. Limits of detection in serum-based materials are: E2 1.72 pg/mL, E1 0.13 ng/dL, E1S 2.04 pg/mL, TT 0.57 ng/dL, A 0.82 ng/dL, DHEAS 0.22 µg/dL, P 0.86 ng/dL, and 17-OHP 0.41 ng/dL. Precision, expressed as coefficients of variation and evaluated at three levels for each analyte, ranged from 1.2 to 9.9%. Average bias, a measure of accuracy, ranged from -0.3 to 8.8%. The method is certified through the CDC Hormone Standardization Program for estradiol and testosterone and meets the requirements of the College of American Pathologists Proficiency Testing Program for androgens and progesterone. This new, highly specific and sensitive method demonstrates high accuracy and precision while requiring small amounts of specimens. It can assay up to 140 samples at once. Thus, the method is suitable for large epidemiologic studies investigating the hormonal etiology of breast, endometrial, and ovarian cancer. Citation Format: Lumi Duke, Paul H. Kim, Julianne Cook Botelho, Regina G. Ziegler, Hubert Vesper. A new sensitive, accurate liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to measure circulating estrogens, androgens, and progesterone in postmenopausal and premenopausal women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2206.