Elisabet Gentzschein

Sex differences in β-amyloid accumulation in 3xTg-AD mice: Role of neonatal sex steroid hormone exposure

The risk of Alzheimers disease (AD) is higher in women than in men, a sex difference that likely results from the effects of sex steroid hormones. To investigate this relationship, we first compared progression of β-amyloid (Aβ) pathology in male and female triple transgenic (3xTg-AD) mice. We found that female 3xTg-AD mice exhibit significantly greater Aβ burden and larger behavioral deficits than age-matched males. Next, we evaluated how the organizational effects of sex steroid hormones during postnatal development may affect adult vulnerability to Aβ pathology. We observed that male 3xTg-AD mice demasculinized during early development exhibit significantly increased Aβ accumulation in adulthood. In contrast, female mice defeminized during early development exhibit a more male-like pattern of Aβ pathology in adulthood. Taken together, these results demonstrate significant sex differences in pathology in 3xTg-AD mice and suggest that these differences may be mediated by organizational actions of sex steroid hormones during development.

Endothelin levels decrease after oral and nonoral estrogen in postmenopausal women with increased cardiovascular risk factors

OBJECTIVE To establish levels of plasma endothelin-1 in postmenopausal women with increased CV risk as compared with healthy premenopausal women and to measure the effects of different forms of estrogen replacement on plasma endothelin-1. DESIGN Prospective randomized study. SETTING University of Southern California Medical Center. PATIENT(S) We studied 18 postmenopausal women (mean age 53.4 +/- 4.9 years) with total cholesterol levels > 240 mg/dL divided into those with and without hypertension as well as in 10 healthy premenopausal women. INTERVENTION(S) The postmenopausal women were randomized to receive oral estrone sulfate, transdermal E2, or placebo for 30 days. MAIN OUTCOME MEASURE(S) We measured the endothelin-1 levels and total cholesterol at baseline and after 30 days of estrogen treatment. RESULT(S) In the postmenopausal women, endothelin-1 was higher (4.58 +/- 0.46 pg/mL) compared with premenopausal levels (2.80 +/- 0.46 pg/mL). In hypertensive postmenopausal women, endothelin-1 was 5.56 +/- 0.44 pg/mL. After estrogen, plasma endothelin-1 values decreased from 5.38 +/- 0.66 to 4.82 +/- 0.9 pg/mL with oral estrone sulfate, 4.84 +/- 0.25 to 4.54 +/- 0.49 pg/mL with transdermal E2, and did not change after placebo 4.76 +/- 0.71 to 4.81 +/- 0.46 pg/mL. In evaluating hypertensive women alone with estrogen therapy, plasma endothelin-1 showed the greatest decrement from 5.39 +/- 0.49 to 4.4 +/- 0.59 pg/mL (18.4%). The decrease in endothelin-1 with estrogen, which was statistically significant for the entire group, did appear to be influenced by the route of administration. Baseline plasma endothelin-1 levels were correlated positively to plasma cholesterol levels with a correlation coefficient of 0.632. CONCLUSION(S) These data provide another potential mechanism explaining the cardioprotective effects of hormone replacement therapy.

Locus-Wide Chromatin Remodeling and Enhanced Androgen Receptor-Mediated Transcription in Recurrent Prostate Tumor Cells

ABSTRACT Prostate cancers (PCas) become resistant to hormone withdrawal through increased androgen receptor (AR) signaling. Here we show increased AR-mediated transcription efficiency in PCa cells that have acquired the ability to grow in low concentrations of androgen. Compared to androgen-dependent PCa cells, these cells showed increased activity of transiently transfected reporters and increased mRNA synthesis relative to levels of AR occupancy of the prostate-specific antigen (PSA) gene. The locus also displayed up to 10-fold-higher levels of histone H3-K9/K14 acetylation and H3-K4 methylation across the entire body of the gene. Although similar increased mRNA expression and locus-wide histone acetylation were also observed at another kallikrein locus (KLK2), at a third AR target locus (TMPRSS2) increased gene expression and locus-wide histone acetylation were not seen in the absence of ligand. Androgen-independent PCa cells have thus evolved three distinctive alterations in AR-mediated transcription. First, increased RNA polymerase initiation and processivity contributed to increased gene expression. Second, AR signaling was more sensitive to ligand. Third, locus-wide chromatin remodeling conducive to the increased gene expression in the absence of ligand was apparent and depended on sustained AR activity. Therefore, increased AR ligand sensitivity as well as locus-specific chromatin alterations contribute to basal gene expression of a subpopulation of specific AR target genes in androgen-independent PCa cells. These features contribute to the androgen-independent phenotype of these cells.

A radioimmunoassay for norethindrone (NET): Measurement of serum net concentrations following ingestion of net-containing oral contraceptive steroids

A sensitive and reliable radioimmunoassay (RIA) for the measurement of norethindrone (NET) in serum has been established employing anti-11 alpha-hydroxynorethindrone 11-hemisuccinyl-bovine serum albumin serum in conjunction with norethindrone-3-(0-carboxymethyl) oximino-[125I]-iodohistamine. Of a number of ring A reduced NET metabolites, only 17 beta-hydroxy-17 alpha-ethinyl-5 beta-estran-3-one (43%) and 17 alpha-ethinyl-5 alpha-estrane-3 beta, 17 beta-diol (15.7%) cross-reacted appreciably in this RIA. Ethinyl estradiol (EE2) and mestranol (MEE2) exhibited cross-reactions of only 1.1 and 0.4%, respectively. Serum NET levels were measured in four groups of 3 women, each ingesting either 1 mg NET plus 0.05 mg MEE2 (Norinyl 1 + 50 or Ortho Novum 1/50), 0.5 mg NET plus 0.035 mg EE2 (Brevicon) or only 0.35 mg NET (Micronor) daily for 5 consecutive days. Peak serum NET levels were observed within 1/2 to 4 hours after oral intake and fell precipitously thereafter. After reaching a maximum, serum NET concentrations declined in a manner consistent with at least two disposition phases. The average half-life for the first disposition phase was 2.3, 3.4, 3.9 and 4.4 hours in subjects ingesting Norinyl 1 + 50, Ortho Novum 1/50, Brevicon and Micronor, respectively. Peak and 3-hour post-ingestion serum NET concentrations were dose-related but showed considerable subject-to-subject variations. Following discontinuation of tablet intake, serum NET levels remained detectable (greater than 0.05 ng/ml) for at least 5 days in all 3 women who had taken Ortho Novum 1/50, but in none of the other 9 volunteers. These results suggest that different preparations of identical doses and combinations of oral contraceptive steroids may yield different serum NET profiles. However, due to considerable subject-to-subject variations, larger numbers of subjects are required for a conclusive investigation.

SERUM NORETHINDRONE (NET) CONCENTRATIONS FOLLOWING INTRAMUSCULAR NET ENANTHATE INJECTION. EFFECT UPON SERUM LH, FSH, ESTRADIOL AND PROGESTERONE

Serum norethindrone (NET), estradiol-17 beta (E), progesterone (P), and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were radioimmunoassayed in 15 women after injection of 300 mg norethindrone enanthate (NET-EN) in 2 ml of oil (5 women), 200 mg NET-EN in 2 ml of oil (5 women), and 200 mg NET-EN in 1 ml of oil (5 women). These values were measured twice a week for 4-6 months to determine 1) the effect of various serum NET concentrations upon hypothalamic/pituitary-ovarian function; 2) intersubject variability of this effect; 3) between-subject variability of serum NET values; 4) the effect of an increase in NET-EN dosage or in the volume of the vehicle; and 5) the correlation between uterine bleeding and serum NET, E and P patterns. Peak serum NET levels were reached 4-15 days (median 7 days) postinjection. These high levels lasted about 20 days postinjection and then decreased, quickly at first and then gradually. Serum NET averaged 1.0 and .38 ng/ml 60 and 120 days postinjection and continued to be measurable in some of the subjects for up to a year. Calculations of day levels of serum NET revealed 63% in the 1st 20 days, 26% in the 2nd, and 11% in the 3rd. Follicle development occurred between 33-116 days postinjection (median 43 days) and was followed by ovulation in only 1 subject (45 days postinjection) with the 2nd earliest ovulation at 80 days postinjection. Uterine bleeding patterns were independent of dosage of NET-EN. The higher dose of NET-EN provides no contraceptive advantage, and the larger vehicle has no effect. A variability of positive feedback inhibition among subjects is thought to be responsible for differing patterns of return to ovulation, suggesting that for contraceptive purposes a regimen of 150 mg or less NET-EN every 6 weeks may be appropriate.

Sex steroid hormone levels in breast adipose tissue and serum in postmenopausal women

Elevated levels of circulating estrogens and androgens are linked to higher breast cancer risk among postmenopausal women; however, little is known about hormone levels within the breast. Hormone concentrations within the breast may not be reflected in the blood and are likely important contributors to breast carcinogenesis. We used a previously validated method to measure levels of estrone, estradiol, androstenedione, and testosterone in adipose tissue removed as part of breast excisions performed for cancer in 100 postmenopausal women (69 ER/PR +/+ and 31 ER/PR −/−) participating in a breast cancer case–control study. We also measured the same steroid hormones, as well as estrone sulfate, and sex hormone-binding globulin (SHBG) in serum from these patients and 100 controls matched on ages at blood collection and on menopause. Overall, concentrations of serum hormones did not vary significantly between controls and cases. However, women with ER−/PR− breast cancers had lower circulating levels of all measured sex steroid hormones and higher SHBG levels than women with ER+/PR+ breast cancers and controls. Similarly, hormone concentrations in breast adipose tissue were higher among women with ER+/PR+ compared to ER−/PR− breast cancer, although differences were only significant for testosterone. These data demonstrate that high sex steroid concentrations in both serum and adipose tissues are more strongly related to ER+/PR+ than ER−/PR− breast cancers. Measurement of sex hormones in serum and in the microenvironment may help in understanding the hormonal etiology of breast cancer, suggest methods for prevention, and have value in gauging treatment response and prognosis.

Estrogen Levels in Nipple Aspirate Fluid and Serum during a Randomized Soy Trial

Background: On the basis of hypothesized protective effect, we examined the effect of soy foods on estrogens in nipple aspirate fluid (NAF) and serum, possible indicators of breast cancer risk. Methods: In a crossover design, we randomized 96 women who produced 10 μL or more NAF to a high- or low-soy diet for 6 months. During the high-soy diet, participants consumed 2 soy servings of soy milk, tofu, or soy nuts (∼50 mg of isoflavones per day); during the low-soy diet, they maintained their usual diet. Six NAF samples were obtained using a FirstCyte aspirator. Estradiol (E2) and estrone sulfate (E1S) were assessed in NAF and estrone (E1) in serum only, using highly sensitive radioimmunoassays. Mixed-effects regression models accounting for repeated measures and left-censoring limits were applied. Results: Mean E2 and E1S were lower during the high-soy than the low-soy diet (113 vs. 313 pg/mL and 46 vs. 68 ng/mL, respectively) without reaching significance (P = 0.07); the interaction between group and diet was not significant. There was no effect of the soy treatment on serum levels of E2 (P = 0.76), E1 (P = 0.86), or E1S (P = 0.56). Within individuals, NAF and serum levels of E2 (rs = 0.37; P < 0.001) but not of E1S (rs = 0.004; P = 0.97) were correlated. E2 and E1S in NAF and serum were strongly associated (rs = 0.78 and rs = 0.48; P < 0.001). Conclusion: Soy foods in amounts consumed by Asians did not significantly modify estrogen levels in NAF and serum. Impact: The trend toward lower estrogen levels in NAF during the high-soy diet counters concerns about adverse effects of soy foods on breast cancer risk. Cancer Epidemiol Biomarkers Prev; 20(9); 1815–21. ©2011 AACR.

Measurement of Sex Steroid Hormones in Breast Adipocytes: Methods and Implications

Background: The lack of validated methods for measuring sex steroid hormones in breast tissue has limited our knowledge of their role in the development of breast cancer. We explored the feasibility of measuring hormones in breast adipocytes for epidemiologic and clinical studies by refining an existing assay procedure and assessing the reliability of hormone measurements using the modified assay. This report presents the reproducibility of measurements of androstenedione (A), testosterone (T), estrone (E1), and estradiol (E2), using breast adipose tissue samples obtained from women undergoing surgical resection for a variety of pathologic conditions. Methods: Breast adipose tissues were obtained from 20 women. Measurements of the steroid hormones were carried out by harvesting oil from adipocytes following enzymatic digestion of the adipose tissue, extracting and chromatographing the steroids, and quantifying them by RIA. The study was conducted in three phases: first, samples from five women were used to assess the assay procedure; following this, tissues from an additional five women were assayed repeatedly to determine reproducibility of the hormone measurements. Finally, using samples from 10 women undergoing surgical resection of a breast tumor, we evaluated hormone concentrations in samples distal and proximal to the tumor. The assay coefficient of variation and intraclass correlation coefficient were used to assess hormone reproducibility. Results: The within-batch coefficients of variation ranged from 5% to 17%, and between-batch estimates were between 2% and 10%, suggesting that E1, E2, A, and T can be reliably measured in breast adipocytes. Among samples obtained from women undergoing surgical resection of a breast tumor, hormone levels did not differ between adipose tissue fragments that were distal or proximal to the tumor, with the possible exception of E1 in which levels were 10% higher in distal fragments. Conclusion: These data support the feasibility of measuring steroid hormone concentrations in breast adipocytes in epidemiologic studies. Future investigations that include the measurement of hormones in the breast microenvironment may have value in understanding breast carcinogenesis, developing prevention strategies, and assessing hormonal treatments. (Cancer Epidemiol Biomarkers Prev 2008;17(8):1891–5)

Alterations in androgen conjugate levels in women and men with alopecia

OBJECTIVE To assess levels of androgen metabolites thought to reflect, at least in part, peripheral androgen activity in women with androgenic alopecia and men with premature balding in an effort to determine if a common abnormality exists. DESIGN Prospective study in various groups of women and men. SETTING Reproductive Endocrine Clinic at our university medical center. PATIENTS Ten normal ovulatory female controls and 50 hyperandrogenic women divided on the basis of hirsutism and alopecia as follows: [1] 8 hirsute women with androgenic alopecia; [2] 12 nonhirsute women with androgenic alopecia; [3] 18 hirsute women without androgenic alopecia; and [4] 12 nonhirsute women without androgenic alopecia. Ten normal men and 10 young premature balding men matched for age and weight also were compared. INTERVENTION Blood was obtained from all subjects. MAIN OUTCOME MEASURE Comparison of blood hormone levels in the various groups. RESULTS Serum T, androstenedione, and DHEAS were similarly elevated in hyperandrogenic women with and without alopecia, compared with controls. The female groups were then divided on the basis of hirsutism. Hirsute groups with and without alopecia had similarly elevated levels of unconjugated 3 alpha-androstanediol, 3 alpha-androstanediol glucuronide, 3 alpha-androstanediol sulfate, androsterone glucuronide, and androsterone sulfate compared with controls. In the nonhirsute groups, androgenic alopecia patients were compared with hyperandrogenic females and cycling controls. The androgenic alopecia patients had elevated levels of 3 alpha androstanediol (0.75 +/- 0.12 versus 0.46 +/- 0.1 and 0.41 +/- 0.1 nmol/L), 3 alpha-androstanediol sulfate (200 +/- 31 versus 79.6 +/- 6 and 67.0 +/- 4.0 nmol/L), elevated ratios of 3 alpha-androstanediol sulfate:3 alpha-androstanediol (267 +/- 49 versus 170 +/- 20 and 164 +/- 49 nmol/L), elevated ratios of 3 alpha-androstanediol sulfate:3 alpha-androstanediol glucuronide (32.2 +/- 6 versus 10.8 +/- 1 and 10.0 +/- 1) and lower ratios of 3 alpha-androstanediol glucuronide:3 alpha-androstanediol glucuronide (8.3 +/- 1.8 versus 17 +/- 1.7 and 15.2 +/- 1.6 nmol/L). In men the premature balding group had lower levels of 3 alpha-androstanediol glucuronide compared with the male controls (29.8 +/- 4.4 versus 15.2 +/- 1.6 nmol/L). Also, the ratio of 3 alpha-androstanediol glucuronide:3 alpha-androstanediol was significantly decreased, whereas the ratio of 3 alpha-androstanediol sulfate:3 alpha-androstanediol glucuronide was elevated. CONCLUSIONS These data provided evidence confirming that enhanced 5 alpha-reductase activity occurs in androgenic alopecia but also suggests that a disorder of androgen conjugation, favoring sulfurylation over glucuronidation, may be a characteristic feature of scalp hair loss.

Temporal changes in cervical mucus after insertion of the levonorgestrel-releasing intrauterine system.

BACKGROUND The major contraceptive action of the levonorgestrel-releasing intrauterine system (LNG-IUS) is cervical mucus (CM) thickening, which prevents sperm penetration. No study to date has examined the temporal relationship between the insertion of the LNG-IUS and changes in CM quality and sperm penetration. STUDY DESIGN Participants were enrolled in a clinically descriptive study to compare the quality of CM and three parameters of sperm penetration prior to insertion of the LNG-IUS and on Days 1, 3 and 5 after insertion. Measurements of estradiol, progesterone and levonorgestrel (LNG) in serum and LNG in CM were also carried out at these times. CM was analyzed using the World Health Organization CM grading criteria. Sperm penetration was determined using an in vitro sperm-CM penetration test. RESULTS All 10 participants underwent LNG-IUS insertion during midcycle when CM quality was good and sperm penetration was excellent. On Day 1 after LNG-IUS insertion, the majority of participants demonstrated poor CM quality and poor sperm penetration. On Day 3, all participants had poor CM quality, and all but one subject had poor sperm penetration. By Day 5, all participants had poor CM quality and poor sperm penetration. LNG levels in CM peaked on the day after LNG-IUS insertion. CONCLUSION Significant changes in quality of CM and sperm penetration were observed shortly after LNG-IUS insertion; however, CM can remain penetrable for up to 5 days when the LNG-IUS is inserted midcycle.