Lilly Chang

Progesterone and Estrogen Regulate Alzheimer-Like Neuropathology in Female 3xTg-AD Mice

Estrogen depletion in postmenopausal women is a significant risk factor for the development of Alzheimers disease (AD), and estrogen-based hormone therapy may reduce this risk. However, the effects of progesterone both alone and in combination with estrogen on AD neuropathology remain unknown. In this study, we used the triple transgenic mouse model of AD (3xTg-AD) to investigate the individual and combined effects of estrogen and progesterone on β-amyloid (Aβ) accumulation, tau hyperphosphorylation, and hippocampal-dependent behavioral impairments. In gonadally intact female 3xTg-AD mice, AD-like neuropathology was apparent by 3 months of age and progressively increased through age 12 months, a time course that was paralleled by behavioral impairment. Ovariectomy-induced depletion of sex steroid hormones in adult female 3xTg-AD mice significantly increased Aβ accumulation and worsened memory performance. Treatment of ovariectomized 3xTg-AD mice with estrogen, but not progesterone, prevented these effects. When estrogen and progesterone were administered in combination, progesterone blocked the beneficial effect of estrogen on Aβ accumulation but not on behavioral performance. Interestingly, progesterone significantly reduced tau hyperphosphorylation when administered both alone and in combination with estrogen. These results demonstrate that estrogen and progesterone independently and interactively regulate AD-like neuropathology and suggest that an optimized hormone therapy may be useful in reducing the risk of AD in postmenopausal women.

Pregnancy, the postpartum, and steroid hormones: effects on cognition and mood

The effects of pregnancy on cognition and mood were examined using a repeated-measures design. Nineteen women, average age 33, were tested with a comprehensive neuropsychological battery during their last 2 months of pregnancy and again within 2 months of delivery. Blood samples were obtained from all subjects and assayed for a variety of steroid hormones implicated in cognitive and mood functioning. Most participants also completed several self-report measures of mood. In comparison with performance after delivery, women showed significantly more impairment in aspects of verbal memory during pregnancy and also tended to report more negative mood states. Memory deficits were not explained by mood disturbances. No hormone assayed consistently related to cognitive performance during pregnancy. During pregnancy, higher levels of progesterone (P) were associated with greater mood disturbances and higher levels of dehydroepiandrosterone (DHEA) with better mood. After delivery, testosterone (T) was strongly and consistently associated with greater reported mood disturbances. Our results confirm a peripartal memory deficit, which cannot be explained by the dramatic rise in circulating steroid hormones, or by mood status during pregnancy. Steroidal hormones, namely P, DHEA and T, appear to play a role in mood disturbances during, and after, pregnancy. Studies beginning earlier in pregnancy and continuing for an extended period of time after delivery are needed to confirm and expand these observations.

Reproducibility of Serum Sex Steroid Assays in Men by RIA and Mass Spectrometry

There is an increasing trend to apply gas chromatography combined with mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS) assay methods to large-scale epidemiologic studies for the measurement of serum sex steroids. These methods are generally considered the gold standard for sex steroid measurements because of their accuracy, sensitivity, turnaround time, and ability to assess a more complete panel of steroid metabolites in the same run. In this report, we evaluated the precision, including within-batch (intra) and between-batch (inter) reproducibility, of steroid hormone measurements determined by GC-MS and LC-MS/MS assays and RIA and compared measurements among these methods. Specifically, 282 overnight fasting serum samples from 20 male volunteers were analyzed for 12 steroid metabolites by GC-MS or LC-MS/MS in one lab over a 4-month period. Six of the analytes were also measured by RIA in another lab. Unconjugated hormones, including testosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione, androst-5-ene-3β,17β-diol, estrone, and estradiol, were measured by GC-MS, whereas conjugated hormones, including DHEA sulfate, androsterone glucuronide, 5α-androstane-3α,17β-diol 3-glucuronide, 5α-androstane-3α,17β-diol 17-glucuronide, and estrone sulfate, were measured by LC-MS/MS. A subset of these hormones, including testosterone, dihydrotestosterone, androstenedione, 5α-androstane-3α,17β-diol 17-glucuronide, estrone, and estradiol, were also measured by RIA following extraction and chromatography. We used the coefficient of variation (CV) and the intraclass correlation coefficient (ICC) to assess within- and between-batch assay variations. For the 12 analytes measured by GC-MS or LC-MS/MS, CVs and ICCs for within- and between-batch measurements were similar, with CVs ranging from 6.1% to 21.4% and ICCs ranging from 87.6% to 99.2%. The six analytes measured by RIA had good CVs and ICCs, with CVs <10% and ICCs >70% (range, 71.7-99.7%). For the six metabolites that were measured by both methods, the CVs were similar, whereas the ICCs were generally higher with the GC-MS method. The absolute values for each analyte measured by RIA and GC-MS differed, with RIAs usually yielding markedly higher levels than GC-MS, although the Pearson and Spearman correlation coefficients for these six analytes were near one and all were significant (P < 0.001). Our results show that RIA, GC-MS, and LC-MS/MS assays for androgens and estrogens in the two labs included in the study have good reproducibility, as measured by small CVs (<15%) and high ICCs (>80%), with the exception of estradiol (71.7%) when measured by RIA. Despite substantial differences in absolute measurements of sex steroid hormones by RIA and MS methods, correlations between the two assays for the six sex steroids measured in the two labs were high (>0.9). However, it is important for future large epidemiologic studies to incorporate MS with high reproducibility and specificity to measure a more complete profile of androgen and estrogen metabolites to clarify the role of sex steroids in prostate cancer. (Cancer Epidemiol Biomarkers Prev 2007;16(5):1004–8)

Brain levels of sex steroid hormones in men and women during normal aging and in Alzheimer’s disease

We examined the relationships between normal aging, Alzheimers disease (AD), and brain levels of sex steroid hormones in men and women. In postmortem brain tissue from neuropathologically normal, postmenopausal women, we found no age-related changes in brain levels of either androgens or estrogens. In comparing women with and without AD at different ages, brain levels of estrogens and androgens were lower in AD cases aged 80 years and older but not significantly different in the 60-79 year age range. In male brains, we observed that normal aging was associated with significant decreases in androgens but not estrogens. Further, in men aged 60-79 years, brain levels of testosterone but not estrogens were lower in cases with mild neuropathological changes as well as those with advanced AD neuropathology. In male cases over age 80, brain levels hormones did not significantly vary by neuropathological status. To begin investigating the relationships between hormone levels and indices of AD neuropathology, we measured brain levels of soluble β-amyloid (Aβ). In male cases with mild neuropathological changes, we found an inverse relationship between brain levels of testosterone and soluble Aβ. Collectively, these findings demonstrate sex-specific relationships between normal, age-related depletion of androgens and estrogens in men and women, which may be relevant to development of AD.

Selective Loss of AKR1C1 and AKR1C2 in Breast Cancer and Their Potential Effect on Progesterone Signaling

Progesterone plays an essential role in breast development and cancer formation. The local metabolism of progesterone may limit its interactions with the progesterone receptor (PR) and thereby act as a prereceptor regulator. Selective loss of AKR1C1, which encodes a 20α-hydroxysteroid dehydrogenase [20α-HSD (EC 1.1.1.149)], and AKR1C2, which encodes a 3α-hydroxysteroid dehydrogenase [3α-HSD (EC 1.1.1.52)], was found in 24 paired breast cancer samples as compared with paired normal tissues from the same individuals. In contrast, AKR1C3, which shares 84% sequence identity, and 5α-reductase type I (SRD5A1) were minimally affected. Breast cancer cell lines T-47D and MCF-7 also expressed reduced AKR1C1, whereas the breast epithelial cell line MCF-10A expressed AKR1C1 at levels comparable with those of normal breast tissues. Immunohistochemical staining confirmed loss of AKR1C1 expression in breast tumors. AKR1C3 and AKR1C1 were localized on the same myoepithelial and luminal epithelial cell layers. Suppression of ARK1C1 and AKR1C2 by selective small interfering RNAs inhibited production of 20α-dihydroprogesterone and was associated with increased progesterone in MCF-10A cells. Suppression of AKR1C1 alone or with AKR1C2 in T-47D cells led to decreased growth in the presence of progesterone. Overexpression of AKR1C1 and, to a lesser extent, AKR1C2 (but not AKR1C3) decreased progesterone-dependent PR activation of a mouse mammary tumor virus promoter in both prostate (PC-3) and breast (T-47D) cancer cell lines. We speculate that loss of AKR1C1 and AKR1C2 in breast cancer results in decreased progesterone catabolism, which, in combination with increased PR expression, may augment progesterone signaling by its nuclear receptors.

Is 11β-hydroxyandrostenedione a better marker of adrenal androgen excess than dehydroepiandrosterone sulfate?

To determine whether the adrenal androgen 11 beta-hydroxyandrostenedione is a more sensitive and specific marker than dehydroepiandrosterone sulfate, we compared these serum androgens in 81 women with anovulatory hyperandrogenism before treatment, after corticotropin and corticotropin-releasing-factor stimulation, and after short- and long-term dexamethasone suppression. Of all subjects, 65% and 57% had elevated levels of 11 beta-hydroxyandrostenedione (greater than 2.0 ng/ml) and dehydroepiandrosterone sulfate (greater than 2.8 micrograms/ml), respectively. However, 11 beta-hydroxyandrostenedione and dehydroepiandrosterone sulfate levels did not correlate in either the women with hyperandrogenism (r = 0.12) or the 26 normal women (r = 0.29). After 0.25 mg corticotropin was administered intravenously (n = 16), 11 beta-hydroxyandrostenedione increased by 157% +/- 53% (mean +/- SEM), whereas dehydroepiandrosterone sulfate, androstenedione, dehydroepiandrosterone, and cortisol increased by 6% +/- 2%, 46% +/- 10%, 416% +/- 80%, and 2326% +/- 371%, respectively. After intravenous administration of 100 micrograms corticotropin-releasing factor to eight patients, the percent change from baseline level to peak was 148% +/- 26%, 24% +/- 5%, 61% +/- 15%, 117% +/- 15%, and 116% +/- 18% for 11 beta-hydroxyandrostenedione, dehydroepiandrosterone sulfate, androstenedione, dehydroepiandrosterone, and cortisol, respectively. After 2 mg dexamethasone for 3 days (n = 10), 11 beta-hydroxyandrostenedione, dehydroepiandrosterone sulfate, androstenedione, and testosterone were suppressed by 95% +/- 2%, 74% +/- 3%, 51% +/- 9%, and 32% +/- 9%, respectively. Suppression with 0.5 mg dexamethasone for 3 months lowered 11 beta-hydroxyandrostenedione and dehydroepiandrosterone sulfate levels equally by 50% +/- 14% and 62% +/- 12%, respectively. 11 beta-Hydroxyandrostenedione is a useful marker of adrenal androgen secretion with a calculated sensitivity and specificity greater than that of dehydroepiandrosterone sulfate. The greater sensitivity of 11 beta-hydroxyandrostenedione over dehydroepiandrosterone sulfate to adrenal stimulation and suppression suggests its unique diagnostic use.

Impaired Dihydrotestosterone Catabolism in Human Prostate Cancer: Critical Role of AKR1C2 as a Pre-Receptor Regulator of Androgen Receptor Signaling

We previously reported the selective loss of AKR1C2 and AKR1C1 in prostate cancers compared with their expression in paired benign tissues. We now report that dihydrotestosterone (DHT) levels are significantly greater in prostate cancer tumors compared with their paired benign tissues. Decreased catabolism seems to account for the increased DHT levels as expression of AKR1C2 and SRD5A2 was reduced in these tumors compared with their paired benign tissues. After 4 h of incubation with benign tissue samples, (3)H-DHT was predominantly catabolized to the 5alpha-androstane-3alpha,17beta-diol metabolite. Reduced capacity to metabolize DHT was observed in tumor samples from four of five freshly isolated pairs of tissue samples, which paralleled loss of AKR1C2 and AKR1C1 expression. LAPC-4 cells transiently transfected with AKR1C1 and AKR1C2, but not AKR1C3, were able to significantly inhibit a dose-dependent, DHT-stimulated proliferation, which was associated with a significant reduction in the concentration of DHT remaining in the media. R1881-stimulated proliferation was equivalent in all transfected cells, showing that metabolism of DHT was responsible for the inhibition of proliferation. PC-3 cells overexpressing AKR1C2 and, to a lesser extent, AKR1C1 were able to significantly inhibit DHT-dependent androgen receptor reporter activity, which was abrogated by increasing DHT levels. We speculate that selective loss of AKR1C2 in prostate cancer promotes clonal expansion of tumor cells by enhancement of androgen-dependent cellular proliferation by reducing DHT metabolism.

Reassessment of adrenal androgen secretion in women with polycystic ovary syndrome

Objective To reevaluate the clinical significance of elevations of adrenal androgens in polycystic ovary syndrome (PCOS). Methods Thirty women with PCOS and ten ovulatory controls were evaluated. Serum dehydroepiandrosterone (DHEA) sulfate and 11β-hydroxyandrostenedione were measured before and after 3 and 6 months of GnRH agonist (GnRH-A) therapy. All controls and 15 women with PCOS received intravenous ACTH before and after GnRH-A therapy Results Twenty-one (70%) of the women with PCOS had elevations of DHEA sulfate, and 16 (53%) had elevations in 11β-hydroxyandrostenedione. Only two women with PCOS had normal values of both adrenal androgens. After GnRH-A therapy, only 11 subjects (37%) had elevated values of DHEA sulfate. Four of 16 women had reductions in 11β-hydroxyandrostenedione. Only those with elevated baseline DHEA sulfate levels had reductions after GnRH-A therapy. The reduction of DHEA sulfate with GnRH-A correlated with the reduction in androstenedione. Of the subjects who had reductions in DHEA sulfate with GnRH-A therapy, there was a blunted response of DHEA to ACTH after treatment. Conclusion Our findings suggest that the ovary may influence the prevalence and magnitude of adrenal androgen excess in PCOS.

The ratio of androstenedione: 11β-hydroxyandrostenedione is an important marker of adrenal androgen excess in women*

Objective To determine if the ratio of serum androstenedione (A):11 β -hydroxyandrostenedione (OHA) would be helpful in differentiating adrenal from ovarian hyperandrogenism. Design/Setting Prospective study of outpatients being evaluated for hyperandrogenism. Patients/Participants Normal women (n=27), those with hyperandrogenie chronic anovulation (n=25), and 7 with adult onset of congenital adrenal hyperplasia (CAH) because of 21-hydroxylase deficiency. Interventions Fasting serum between 8:00 A.M. and 9:00 A.M. Patients with hyperandrogenic chronic anovulation and CAH received dexamethasone (DEX) 2mg for 7 days. Main Outcome Measures Serum testosterone (T), unbound T, dehydroepiandrosterone sulfate (DHEAS), A, and 11 β -OHA by radioimmunoassay. Results Serum 11 β -OHA and DHEAS were elevated in 52% and 40% of patients with hyperandrogenic chronic anovulation and in 7 of 7 and 1 of 7 patients with CAH. The ratio of A:11 β -OHA was significantly higher ( P P β -OHA correlated with T ( r =0.58, P β -OHA were similar and significantly lower in CAH and hyperandrogenic chronic anovulation patients who were DEX sensitive compared with those who were not DEX sensitive. The ratio correlated with the percentage suppression of T, unbound T, and A after DEX ( P β -OHA. Using the mean ratio of controls (1.3) as a cutoff value, the sensitivity of the A:11 β -OHA in detecting adrenal hyperandrogenism, as assessed by DEX sensitivity, was 100%, the specificity was 84%, and the predictive value was 67%. Conclusions The ratio of A:11 β -OHA appears to be an excellent marker for identifying patients with adrenal hyperandrogenism and CAH.

Evidence for heterogeneous etiologies of adrenal dysfunction in polycystic ovary syndrome

OBJECTIVE To examine the hypothesis that, in polycystic ovary syndrome (PCOS), ovarian steroids induce adrenal enzyme dysfunction or adrenal androgen hyperresponsiveness to ACTH. DESIGN Prospective controlled clinical study. SETTING Reproductive endocrinology unit of an academic medical center. PATIENTS Twelve women with PCOS who had adrenal androgen excess were compared with five weight-matched ovulatory women. In half of the women with PCOS, prestudy screening was suggestive of mild 3 beta-hydroxysteroid dehydrogenase (HSD) deficiency. INTERVENTIONS Basal and adrenal dynamic blood sampling before and after GnRH agonist (GnRH-a) administration for 6 months. MAIN OUTCOME MEASURES Basal E2 and androgen levels as well as dexamethasone-suppressed, ACTH-stimulated 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, and androgen levels before and after ovarian suppression. RESULTS Although none of the subjects with PCOS proved to have mild 3 beta-HSD deficiency, the majority of them (58%) met the criteria for 17,20 lyase hyperactivity before and after GnRH-a therapy. As a group, the remaining subjects with PCOS exhibited an elevated DHEAS response to ACTH before GnRH-a treatment, which may have normalized after GnRH-a treatment. CONCLUSION Adrenal androgen excess in PCOS may be heterogeneous in etiology, whereas 17,20 lyase hyperactivity appears to be an intrinsic adrenal disorder, adrenal androgen hyperresponsiveness to ACTH may be ovarian induced. Reliance on historical controls may lead to overdiagnosis of mild 3 beta-HSD deficiency.