High Estradiol Levels During Postmenopause - Pitfalls in Laboratory Analysis.
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ABSTRACT:
SUBMITTER: Mebes I
PROVIDER: S-EPMC4596695 | biostudies-other | 2015 Sep
REPOSITORIES: biostudies-other
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Project description:Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom-up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enabling RP HPLC separation. When histone dynamics are studied in a quantitative manner, specificity, and efficiency of this chemical derivatization are crucial. Therefore we examined eight different protocols, including two different propionylation reagents. This revealed amidation (up to 70%) and methylation (up to 9%) of carboxyl groups as a side reaction. Moreover, incomplete (up to 85%) as well as a specific propionylation (up to 63%) can occur, depending on the protocol. These results highlight the possible pitfalls and implications for data analysis when doing bottom-up MS on histones.
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Project description:BACKGROUND:Adenosine-to-inosine (A-to-I) editing is a site-selective post-transcriptional alteration of double-stranded RNA by ADAR deaminases that is crucial for homeostasis and development. Recently the Mouse Genomes Project generated genome sequences for 17 laboratory mouse strains and rich catalogues of variants. We also generated RNA-seq data from whole brain RNA from 15 of the sequenced strains. RESULTS:Here we present a computational approach that takes an initial set of transcriptome/genome mismatch sites and filters these calls taking into account systematic biases in alignment, single nucleotide variant calling, and sequencing depth to identify RNA editing sites with high accuracy. We applied this approach to our panel of mouse strain transcriptomes identifying 7,389 editing sites with an estimated false-discovery rate of between 2.9 and 10.5%. The overwhelming majority of these edits were of the A-to-I type, with less than 2.4% not of this class, and only three of these edits could not be explained as alignment artifacts. We validated 24 novel RNA editing sites in coding sequence, including two non-synonymous edits in the Cacna1d gene that fell into the IQ domain portion of the Cav1.2 voltage-gated calcium channel, indicating a potential role for editing in the generation of transcript diversity. CONCLUSIONS:We show that despite over two million years of evolutionary divergence, the sites edited and the level of editing at each site is remarkably consistent across the 15 strains. In the Cds2 gene we find evidence for RNA editing acting to preserve the ancestral transcript sequence despite genomic sequence divergence.
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Project description:ObjectivesAmong postmenopausal women taking hormone therapy (HT), the estradiol (E2) dose and E2 levels were differentially associated with change in metabolic measures. We evaluated determinants of attained E2 levels in response to HT.MethodsPostmenopausal women from the REPLENISH trial tested four formulations of oral combined E2 and progesterone compared with placebo. Mixed-effects linear models assessed characteristics associated with E2 levels among women with ≥80% HT compliance, adjusted for E2 dose and baseline E2 level.ResultsAmong 1173 postmenopausal women with mean (standard deviation) age 55 (4.3) years and 5.2 (4.8) years since menopause, higher treated E2 levels were significantly related to younger age, more recent menopause, and current alcohol use, while lower E2 levels were related to current smoking. Both age and time since menopause were significantly inversely associated with E2 levels; time since menopause had a stronger association with E2 levels. In the final multivariable model, E2 levels were positively associated with current alcohol use, and inversely associated with time since menopause and current smoking.ConclusionAdjusting for E2 dose and baseline E2 level, on-trial E2 levels were significantly associated with time since menopause, current smoking, and current alcohol use. Practitioners should consider these factors in individual women to achieve a desirable E2 level during HT.
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Project description:Ovarian hyperstimulation syndrome (OHSS) is one of the most life-threatening and potentially fatal complications associated with controlled ovarian hyperstimulation (COH) during in vitro fertilization (IVF) treatment. Although the pathogenesis of OHSS remains unclear, elevated serum estradiol (E2) levels before human chorionic gonadotropin (hCG) administration are associated with the risk of OHSS. The pineal hormone melatonin and its receptors are expressed in human granulosa cells and have been shown to stimulate E2 production. However, the effect of melatonin on the expression of aromatase, an enzyme responsible for a key step in the biosynthesis of E2, in human granulosa cells remains to be determined. Here, we demonstrate that melatonin upregulates aromatase expression in primary cultured human granulosa-lutein (hGL) cells through the melatonin receptor-mediated PKA-CREB pathway. Using a mouse model of OHSS, we demonstrate that administration of the melatonin receptor inhibitor luzindole inhibits the development of OHSS. In addition, the expression of ovarian aromatase and serum E2 levels are upregulated in OHSS mice compared to control mice, but this upregulation is attenuated by inhibition of the function of melatonin. Moreover, clinical results reveal that aromatase expression levels are upregulated in hGL cells from OHSS patients. Melatonin and E2 levels in the follicular fluid are significantly higher in OHSS patients than in non-OHSS patients. Furthermore, melatonin levels are positively correlated with E2 levels in follicular fluid. This study helps to elucidate the mechanisms mediating the expression of aromatase in hGL cells and provides a potential mechanism explaining the high E2 levels in patients with OHSS.
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Project description:We have previously reported that hexamethylene bis-acetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound estrogen receptor α (ERα) and the androgen receptor (AR) by disrupting the interaction between these receptors and positive transcriptional elongation factor b (P-TEFb) and attenuating RNA polymerase II (RNAPII) phosphorylation at serine 2. Functional consequences of the inhibition of transcriptional activity of ERα and AR by HEXIM1 include the inhibition of ERα- and AR-dependent gene expression, respectively, and the resulting attenuation of breast cancer (BCa) and prostate cancer (PCa) cell proliferation and growth. In our present study, we determined that HEXIM1 inhibited AKR1C3 expression in BCa and PCa cells. AKR1C3, also known as 17β-hydroxysteroid dehydrogenase (17β-HSD) type 5, is a key enzyme involved in the synthesis of 17β-estradiol (E2) and 5-dihydrotestosterone (DHT). Downregulation of AKR1C3 by HEXIM1 influenced E2 and DHT production, estrogen- and androgen-dependent gene expression, and cell proliferation. Our studies indicate that HEXIM1 has the unique ability to inhibit both the transcriptional activity of the ER and AR and the synthesis of the endogenous ligands of these receptors.
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Project description:ObjectiveTo determine differences in plasma sex hormone levels in male and female coronavirus disease 2019 (COVID-19) patients and healthy volunteers (HVs) because cell entry of severe acute respiratory syndrome coronavirus 2 occurs via the angiotensin-converting enzyme 2 receptor which is downregulated by 17β-estradiol.Patients and methodsCitrated plasma samples were collected from 101 patients with COVID-19 upon presentation to the emergency department and from 40 HVs between November 1, 2020, and May 30, 2021. Plasma 17β-estradiol and 5α-dihydrotestosterone (DHT) levels were measured using enzyme-linked immunosorbent assay (pg/mL). Data are presented as median and quartiles (IQR). Wilcoxon rank sum test with a P value less than .05 was considered significant.ResultsPatients with COVID-19 (median age, 49 years) included 51 males and 50 females (25 postmenopausal). Hospital admission was required for 58.8% of male patients (n = 30) and 48.0% of female patients (n = 24) (66.7% postmenopausal, n = 16) Healthy volunteers (median age, 41 years) included 20 males and 20 females (9 postmenopausal). Female patients with COVID-19 were found to have decreased 17β-estradiol levels (18.5 [IQR, 10.5-32.3] pg/mL; 41.4 [IQR, 15.5-111.0] pg/mL, P=.025), and lower 17β-estradiol to DHT ratios (0.073 [IQR, 0.052-0.159] pg/mL; 0.207 [IQR, 0.104-0.538] pg/mL, P=.015) than female HVs. Male patients with COVID-19 were found to have decreased DHT levels (302.8 [IQR, 249.9-470.8] pg/mL; 457.2 [IQR, 368.7-844.3] pg/mL, P=.005), compared with male HVs. Levels of DHT did not differ between female patients with COVID-19 and female HVs, whereas 17β-estradiol levels did not differ between male patients with COVID-19 and male HVs.ConclusionSex hormone levels differ between patients with COVID-19 and HVs, with sex-specific patterns of hypogonadism in males and females. These alterations may be associated with disease development and severity.
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