Percent difference in testosterone (T), androstenedione (AD) and 17-hydroxyprogesterone.

<p>(17OHP) levels measured in a RIA and LC-MS/MS measured in women with PCOS (black circles) and controls (white circles) as a function of mean levels measured in the two assays (Bland-Altman Plot). The solid lines indicate 2 standard deviations above and below the mean difference.</p

Simultaneous Measurement of Thirteen Steroid Hormones in Women with Polycystic Ovary Syndrome and Control Women Using Liquid Chromatography-Tandem Mass Spectrometry

<div><p>Background</p><p>The measurement of adrenal and ovarian androgens in women with PCOS has been difficult based on poor specificity and sensitivity of assays in the female range.</p><p>Methods</p><p>Women with PCOS (NIH criteria; n = 52) and control subjects with 25–35 day menstrual cycles, no evidence of hyperandrogenism and matched for BMI (n = 42) underwent morning blood sampling. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to simultaneously measure 13 steroids from a single blood sample to measure adrenal and ovarian steroids. Androgen and progesterone results were compared in the same samples using RIA.</p><p>Results</p><p>Testosterone, androstenedione, progesterone and 17OH progesterone levels were higher when measured using RIA compared to LC-MS/MS, although the testosterone RIA demonstrated the best agreement with the LC-MS/MS using a Bland-Altman analysis. Results using LC-MS/MS demonstrated that the concentration of androgens and their precursors were higher in women with PCOS than controls [median (2.5, 97.5th %ile); 1607 (638, 3085) vs. 1143 (511, 4784) ng/dL; p = 0.03]. Women with PCOS had higher testosterone [49 (16, 125) vs. 24 (10, 59) ng/dL], androstenedione [203 (98, 476) vs. 106 (69, 223) ng/dL] and 17OH progesterone levels [80 (17, 176) vs. 44 (17, 142) ng/dL] compared to controls (all P<0.02), but no differences in serum concentrations of the adrenal steroids DHEAS, cortisol, corticosterone and their 11 deoxy precursors. Women with PCOS also had an increase in the product:precursor ratio for 3β-hydroxysteroid dehydrogenase [22% (6, 92) vs. 20% (4, 43); p = 0.009].</p><p>Conclusion</p><p>LC-MS/MS was superior to RIA in measuring androstenedione, progesterone and 17OH progesterone levels, while testosterone measurements were better matched in the two assays. Androgen levels were higher in women with PCOS in the absence of a difference in adrenal-predominant steroids. These data support previous findings that the ovary is an important source for the androgen excess in women with PCOS.</p></div

Age, BMI and steroid concentrations in women with PCOS and controls in the follicular phase.

1<p>p value corrected controlled for age and BMI, as appropriate.</p>2<p>Values reported as median (2.5, 97.5th %ile).</p>3<p>p value not corrected for BMI.</p

Percent product-to-precursor concentrations for adrenal and ovarian enzymes in women with PCOS and controls. Data are expressed as % (2.5, 97.5% ile).

1<p>Results are expressed as % product to precursor.</p

Hormone Levels Assayed Using LC-MS/MS and RIA.

<p>Testosterone (T), androstenedione (AD) and 17OH progesterone (17OHP) levels in individual subjects with PCOS (black circles) and controls (white circles) as measured in an RIA and LC-MS/MS. Linear regression is plotted for each hormone. Testosterone, androstenedione and 17-hydroxyprogesterone levels are consistently higher using the RIA.</p

Hormones Across the Cycle in Controls and on a Single Day in Women with PCOS.

<p>A) LH, B) 17OH progesterone, C) testosterone and D) androstenedione levels in controls across the menstrual cycle (white circles) and women with PCOS on a day at least 10 days after the last menstrual cycle (black circles). Line plots are smoothly changed ranges of smooth model fittings using raw cycle day as the independent variable and transformed steroid as dependent variable. Solid black lines are back-transformed means and dashed lines are back-transformed mean±2SD. SDs are estimated using model residuals. The levels in women with PCOS are higher at all cycle phases indicated by the dotted lines and asterisks.</p

Seasonal Variation of 25-Hydroxyvitamin D₃ (bottom panel) and Intact PTH (iPTH) (top panel) Weekly Mean Values.

<p>The maximum seasonal variation of 25(OH)D₃ (peak to trough) was 6.8 ng/mL, reaching its trough in the 8^th week (early March) of each year and its peak in the 34^th week (early September). Peak iPTH values occurred at week 12 (early April) and trough values at week 37 (late September), a pattern that is roughly reciprocal to that of 25(OH)D₃, but lags by 3.5 weeks. Individual points represent the mean of the normalized distribution for each week. The solid lines represent the fit. Dark vertical dashed lines represent 25-hydroxyvitamin D₃ peaks and troughs, and light vertical dashed lines represent the iPTH peaks and troughs. To convert 25-hydroxyvitamin D₃ from ng/mL to nmol/L, multiply by 2.496 (rounded as 2.5).</p

Seasonal Variation in Weekly Mean Values of 25-Hydroxyvitamin D₃ (panels A and B) and Intact PTH (iPTH; panels C and D) by Gender and Region.

<p>A. Women show higher average values for 25(OH)D₃ than men. B. North and Central regions show similar average values for 25(OH)D₃ but the South region shows higher average values in winter, even though all three regions show similar values in summer. C. Men show higher average values than women for iPTH. D. The Central region shows the highest average values for iPTH, followed by the Northern region and then the South region.</p

Percentages of patients with 25(OH)D₃ deficiency (<20 ng/mL), insufficiency (20–29 ng/mL), and sufficiency (≥30 ng/mL), by month.

<p>The percentiles are categorized by month. The upper portion (blue) of each month shows the percentage of patients with sufficient 25(OH)D₃; the central portion (yellow), insufficient but not deficient; and the lower portion (red), deficient. The percentage of patients considered deficient or insufficient depends on the season, lower in summer (approximately 50%) and higher in winter (approximately 70%). The sun symbol indicates the summer solstice and the crescent moon symbol, the winter solstice. The central panel shows the seasonal similarity of total 25(OH)D with that of 25(OH)D2. The lower panel shows the rhythmic pattern across regions and gender. The Central and Northern regions were combined because they were similar. Their trough to peak difference is greater than that for the Southern region. Women and men percentages show similar patterns, except that the trough to peak difference is greater for men than women. The sun symbol indicates the summer solstice and the crescent moon, the winter solstice.</p

Categorical percentage of patients for total 25(OH)D for all patients who had detectable 25(OH)D₂.

<p>The percentiles are categorized by month. The upper portion (blue) of each month shows the percentage of patients with sufficient 25(OH)D₃; the central portion (yellow), insufficient but not deficient; and the lower portion (red), deficient.</p