Reexamination of testosterone, dihydrotestosterone, estradiol and estrone levels across the menstrual cycle and in postmenopausal women measured by liquid chromatography-tandem mass spectrometry

Abstract

Measuring serum androgen levels in women has been challenging due to limitations in method accuracy, precision sensitivity and specificity at low hormone levels. The clinical significance of changes in sex steroids across the menstrual cycle and lifespan has remained controversial, in part due to these limitations. We used validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays to determine testosterone (T) and dihydrotestosterone (DHT) along with estradiol (E2) and estrone (E1) levels across the menstrual cycle of 31 healthy premenopausal females and in 19 postmenopausal females. Samples were obtained in ovulatory women in the early follicular phase (EFP), midcycle and mid luteal phase (MLP). Overall, the levels of T, DHT, E2 and E1 in premenopausal women measured by LC-MS/MS were lower overall than previously reported with immunoassays. In premenopausal women, serum T, free T, E2, E1 and SHBG levels peaked at midcycle and remained higher in the MLP, whereas DHT did not change. In postmenopausal women, T, free T, SHBG and DHT were significantly lower than in premenopausal women, concomitant with declines in E2 and E1. These data support the hypothesis that the changes in T and DHT that occur across the cycle may reflect changes in SHBG and estrogen, whereas in menopause, androgen levels decrease. LC-MS/MS may provide more accurate and precise measurement of sex steroid hormones than prior immunoassay methods and can be useful to assess the clinical significance of changes in T, DHT, E2 and E1 levels in females.

Figure 1

Panel A LH and SHBG levels during the EFP, midcycle and MLP) measured by fluoroimmunometric assay. (*p<0.01) Panel B E2 and E1 levels during the EFP, midcycle and MLP measured by LCMS/MS. (*p<0.01) Panel C T, free T and DHT levels during the EFP, midcycle and MLP. T and DHT were measured by LCMS/MS (**p<0.001 and p=0.59). Free T was calculated as described in methods. (*p<0.01).

Figure 2

Panel A E2, E1, SHBG and FSH levels during EFP in premenopausal women compared to postmenopausal women. E2 and E1 were measured by LCMS/MS (**p<0.0001). SHBG and FSH levels were measured by fluroimmunometric assay (*p<0.05 and **p<0.0001). Panel B T and DHT levels during EFP in premenopausal women compared to postmenopausal women. T and DHT were measured by LCMS/MS (**p<0.001). Panel C Free T during EFP in premenopausal women compared to postmenopausal women. Free T was calculated as described in methods. (*p<0.05).