Unconjugated E2 Research Articles

Levels of angiogenic markers in second-trimester maternal serum from in vitro fertilization pregnancies with oocyte donation

To determine whether differences exist in angiogenic placental growth factor (PlGF) and antiangiogenic soluble vascular endothelial growth factor receptor 1 (sVEGFR-1; both being early markers of placental ischemic disease) in oocyte-donation (OD) pregnancies, compared with autologous invitro fertilization (aIVF) and spontaneous pregnancies. Case-control study of residual second-trimester serum samples from women undergoing prenatal screening. Academic medical center. Fifty-seven OD pregnancies were identified. Each OD pregnancy was matched to two spontaneous pregnancies (n = 114) and one aIVF pregnancy (n = 57). None. Second-trimester serum PlGF and sVEGFR-1 levels. sVEGFR-1, PlGF, and unconjugated E2 levels were similar among the three study groups. The ratio of sVEGFR-1 to PlGF was significantly higher in the OD group. Consistently with previous studies, alpha-fetoprotein (AFP) in the OD group was significantly elevated compared with spontaneous pregnancy. Both aIVF and OD groups had greater levels of inhibin A than the spontaneous pregnancy group, and the OD group had significantly higher levels of inhibin A than the aIVF group. hCG levels were significantly elevated in aIVF compared with spontaneous pregnancy; however, levels were not different between aIVF and OD. Second-trimester serum sVEGFR-1 and PlGF levels were not significantly altered in OD pregnancies. Our data support previous findings that OD pregnancies have uniquely increased second-trimester AFP, hCG, and inhibin A levels compared with aIVF. However, the biologic basis of these marker elevations in OD may not be related to placental angiogenesis.

Seasonal transfer and quantification of urinary estradiol in the big brown bat (Eptesicus fuscus)

Growing evidence shows that sex steroids not only act within the individual whose glands produce them; they can also act on proximate conspecifics. Previous studies show that exogenous 17β-estradiol (E2) can be absorbed both nasally and percutaneously, arriving in blood, neural, reproductive, and peripheral tissues. When male bats were injected with radiolabeled E2 (3H-E2) and housed with females during the mating season, radioactivity was reliably measured in the females’ tissues. The present study was designed to compare E2 transfer from male to female bats at three time points in the annual reproductive cycle: spring (ovulation and fertilization), summer (maternal season), and autumn (mating season). Pairs of mature female bats were housed with a mature 3H-E2-treated male (50 μCi). Following 48 h of communal housing, radioactivity was measured in the tissues of female bats. Higher levels of radioactivity were present in the uterus and other tissues during the spring and autumn seasons compared to the summer season. We also measured natural levels of bioactive, unconjugated E2 in the urine of male bats using enzyme immunoassays, and found that it was present in all three seasons but at lower levels during the summer. Male-excreted E2 could transfer to females within the close confines of a roost, potentially influencing their reproductive physiology and behavior. These results suggest increased E2 transfer coincides with female reproduction, with urine as a likely vector. We suggest that sex steroid transfer among interacting individuals may explain several mammalian phenomena historically viewed as “pheromonal”.

Abstract P4-10-14: Impact of route of administration of estradiol (oral vs. transdermal) on genotoxic estrogens concentrations in girls with ovarian failure due to Turner syndrome: Potential implications for breast cancer prevention

Abstract Objective: The established link between estrogen and breast cancer occurs via both estrogen receptor (ER)-mediated and non ER-mediated mechanisms. The term genotoxic estrogens describes mutagenic metabolites, including estrogen catechols and quinones, which have been linked to breast carcinogenesis in post-menopausal women. Recent data showed that childhood obesity is associated with significantly higher levels of genotoxic estrogens in the blood compared with lean children, raising the possibility of a potential pathogenic roles of these metabolites in breast cancer starting even prior to the onset of puberty (Mauras et al: J Clin Endocrinol Metab. 100:2322, 2015). A finding of higher levels of genotoxic estrogens associated with oral estradiol may have breast cancer prevention implications.We hence aimed to assess if the route of administration of 17β estradiol (E2) affects the accumulation of genotoxic estrogen metabolites in a model of ovarian failure in young girls with Turner Syndrome. Methods: Stored plasma were used from 40 adolescents with Turner's who participated in a previous 12 months randomized controlled trial of the metabolic impact of E2 orally (2mg/d) vs. transdermally (100μg/d). The doses of oral and transdermal E2 were determined to result in similar plasma levels of unconjugated E2. Previously we had reported that despite the similar plasma levels of unconjugated E2, the oral E2 administration route was associated with higher levels of biologically active estrogen activity than the transdermal route (Torres-Santiago L et al:J Clin Endocrinol Metab. 98:2716, 2013). In this study, we measured 12 estrogen metabolites (conjugated and unconjugated) using a highly sensitive LCMSMS assay. Results from 48 normally menstruating adolescents were used for comparison. Results: After treatment, least square mean (SE) total (conjugated plus unconjugated) E2 and estrone (E1) concentrations were higher in the oral vs. transdermal group (p<0·0001), as were catechol-estrogens 4-OH-E2 (149 vs. 28 (49) pmol/L), 2-OH-E2 (300 vs 76 (52)), 4-OH-E1 (450 vs 105 (113)), 2-OH-E1 (304 vs 740 (684)) and 16α-OH-E1 (3007 vs 157 (534)) (<0·001 between groups). Levels were much closer to controls in the transdermal group. Conclusions: Common feminizing doses of oral estradiol for 12 months result in greater accumulation of unphysiologic, genotoxic estrogens than transdermal estradiol, expanding concerns about oral estrogens' first hepatic passage contributing to the accumulation of these metabolites. These metabolites have the potential for inducing breast cancer in post-menopausal women. These results suggest the potential benefit of preferential use of transdermal versus oral estrogens as replacement and possibly contraceptive options, in the prevention of breast cancer. Further studies to assess long-term risks of these metabolites in women taking different forms of estrogen replacement are needed. Citation Format: Colon-Otero G, Torres-Santiago L, Santen R, Mericq V, Ross J, Damaso L, Hossain J, Wang Q, Mesaros C, Blair IA, Mauras N. Impact of route of administration of estradiol (oral vs. transdermal) on genotoxic estrogens concentrations in girls with ovarian failure due to Turner syndrome: Potential implications for breast cancer prevention [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-10-14.

Exposure of pregnant sows to low doses of estradiol-17β impacts on the transcriptome of the endometrium and the female preimplantation embryos†.

Maternal exposure to estrogens can induce long-term adverse effects in the offspring. The epigenetic programming may start as early as the period of preimplantation development. We analyzed the effects of gestational estradiol-17β (E2) exposure with two distinct low doses, corresponding to the acceptable daily intake "ADI" and close to the no-observed-effect level "NOEL", and a high dose (0.05, 10, and 1000 μg E2/kg body weight daily, respectively). The E2 doses were orally applied to sows from insemination until sampling at day 10 of pregnancy and compared to carrier-treated controls leading to a significant increase in E2 in plasma, bile and selected somatic tissues including the endometrium in the high-dose group. Conjugated and unconjugated E2 metabolites were as well elevated in the NOEL group. Although RNA-sequencing revealed a dose-dependent effect of 14, 17, and 27 differentially expressed genes (DEG) in the endometrium, single embryos were much more affected with 982 DEG in female blastocysts of the high-dose group, while none were present in the corresponding male embryos. Moreover, the NOEL treatment caused 62 and 3 DEG in female and male embryos, respectively. Thus, we detected a perturbed sex-specific gene expression profile leading to a leveling of the transcriptome profiles of female and male embryos. The preimplantation period therefore demonstrates a vulnerable time window for estrogen exposure, potentially constituting the cause for lasting consequences. The molecular fingerprint of low-dose estrogen exposure on developing embryos warrants a careful revisit of effect level thresholds.

Triclosan causes spontaneous abortion accompanied by decline of estrogen sulfotransferase activity in humans and mice.

Triclosan (TCS), an antibacterial agent, is identified in serum and urine of humans. Here, we show that the level of urinary TCS in 28.3% patients who had spontaneous abortion in mid-gestation were increased by 11.3-fold (high-TCS) compared with normal pregnancies. Oral administration of TCS (10 mg/kg/day) in mice (TCS mice) caused an equivalent urinary TCS level as those in the high-TCS abortion patients. The TCS-exposure from gestation day (GD) 5.5 caused dose-dependently fetal death during GD12.5–16.5 with decline of live fetal weight. GD15.5 TCS mice appeared placental thrombus and tissue necrosis with enhancement of platelet aggregation. The levels of placenta and plasma estrogen sulfotransferase (EST) mRNA and protein in TCS mice or high-TCS abortion patients were not altered, but their EST activities were significantly reduced compared to controls. Although the levels of serum estrogen (E2) in TCS mice and high-TCS abortion patients had no difference from controls, their ratio of sulfo-conjugated E2 and unconjugated E2 was reduced. The estrogen receptor antagonist ICI-182,780 prevented the enhanced platelet aggregation and placental thrombosis and attenuated the fetal death in TCS mice. The findings indicate that TCS-exposure might cause spontaneous abortion probably through inhibition of EST activity to produce placental thrombosis.

Association of estrogen measurements in serum and urine of premenopausal women.

We evaluated conjugated and unconjugated urinary estrogen metabolites as surrogate biomarkers for serum levels of unconjugated E1 and E2 in premenopausal women. Repeated blood and urine samples were analyzed for estrogens and their metabolites using radioimmunoassays and liquid chromatography/mass spectrometry. The strongest correlation (r = 0.39) was observed between serum E1 and urinary E1 and E2. The correlations of urinary E2 (r = 0.35), E1 (r = 0.29), all E2 metabolites (r = 0.30), all E1 metabolites (r = 0.23) and total estrogens (r = 0.26) with serum E2 were only moderate although statistically significant. All correlations were substantially stronger for Whites than Asians. Urinary E2 emerged as the best predictor for serum E1 and E2, but the large intra-subject variability in urinary estrogen levels limits its use as a biomarker.

Ultrasensitive quantification of serum estrogens in postmenopausal women and older men by liquid chromatography–tandem mass spectrometry

An ultrasensitive stable isotope dilution liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for multiplexed quantitative analysis of six unconjugated and conjugated estrogens in human serum. The quantification utilized a new derivatization procedure, which formed analytes as pre-ionized N-methyl pyridinium-3-sulfonyl (NMPS) derivatives. This method required only 0.1mL of human serum, yet was capable of simultaneously quantifying six estrogens within 20min. The lower limit of quantitation (LLOQ) for estradiol (E2), 16α-hydroxy (OH)-E2, 4-methoxy (MeO)-E2 and 2-MeO-E2 was 1fg on column, and was 10fg on column for 4-OH-E2 and 2-OH-E2. All analytes demonstrated a linear response from 0.5 to 200pg/mL (5–2000pg/mL for 4-OH-E2 and 2-OH-E2). Using this validated method, the estrogen levels in human serum samples from 20 female patients and 20 male patients were analyzed and compared. The levels found for unconjugated serum E2 from postmenopausal women (mean 2.7pg/mL) were very similar to those obtained by highly sensitive gas chromatography–mass spectrometry (GC–MS) methodology. However, the level obtained in serum from older men (mean 9.5pg/mL) was lower than has been reported previously by both GC–MS and LC–MS procedures. The total (unconjugated+conjugated) 4-MeO-E2 levels were significantly higher in female samples compared with males (p<0.05). The enhanced sensitivity offered by the present method will allow for a more specific analysis of estrogens and their metabolites. Our observations might suggest that the level of total 4-MeO-E2 could be a potential biomarker for breast cancer cases.

Abstract 2237: Synthesis and characterization of novel, membrane permeable estradiol fluorophores that maintain estrogenic activity.

Abstract The ability to label biomolecules with fluorescent tags has become essential in basic cancer research and offers great promise for improvements in clinical diagnostics. The structure and physicochemical properties of the fluorescent tag have the potential of altering binding and activity characteristics of the conjugates, therefore it is desirable to use small, neutral, membrane permeable and biocompatible fluorescent tags with favorable photophysical properties, and versatile coupling chemistries. . Estradiol (E2) is a well-characterized ovarian steroid hormone with known measureable biological endpoints at the cellular, transcriptional, and signal transduction levels. We synthesized a new class of fluorescent estrogen derivatives from 17-alpha-ethynylestradiol conjugated to triazaborolopyridinium (HPY) dyes. The estrogenic activity of the covalently linked E2-HPY fluorophores were characterized using the MCF-7 breast cancer cell line, an E2-dependent cell line that is often used as a model system to study E2-induced cell responses. The MCF-7 cells were treated with a series of E2-HPY derivatives (10nM), E2 (10nM) or vehicle control. The time points for treatment was as short as 15 minutes for effect on signal transduction to 5 days for cell proliferation endpoints. After treatment for specified time points, E2-induced proliferation was determined by growth curve analysis (1-5 days), E2-mediated progesterone receptor (PR) transcription was determined by real-time PCR (24 hours), and E2-dependent accumulation of pERK was measured by immunoblot analysis (15min). Our results indicate that the bioactivity of E2-HPY was not significantly different from unconjugated E2 for the three endpoints tested. The accumulation of E2-HPY in cells was demonstrated by flow cytometry and fluorescence microscopy. These data indicate that E2-HPY is taken up by cells and is easily detectable. Additionally, transcriptome analysis was performed to identify potential differences in general transcriptional activation between E2 and E2-HPY. These data suggest the exciting possibility that HPY fluorophores can be covalently linked to cancer-related biomolecules to enhance basic research and for potential use as a cancer diagnostic tool in the clinical. Citation Format: Jason E. Stailey, Sudath Hapuarachchige, Chinnasamy Ramesh, Sanjay Shrestha, Angelique M. Wimbley, Charles B. Shuster, Jeffrey B. Arterburn, Kevin D. Houston. Synthesis and characterization of novel, membrane permeable estradiol fluorophores that maintain estrogenic activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2237. doi:10.1158/1538-7445.AM2013-2237

Selective Recruitment of an E2∼Ubiquitin Complex by an E3 Ubiquitin Ligase

RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40-108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12-39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.

Membrane estrogen receptors mediate calcium signaling and MAP kinase activation in individual hippocampal neurons

Previously we demonstrated that 17β-Estradiol (E2) induced rapid Ca 2+ influx via L-type calcium channel activation, which was required for activation of Src/ERK/CREB/Bcl2 signaling cascade and subsequent induction of neuroprotective and neurotrophic responses in rat hippocampal and cortical neurons (Wu et al., 2005; Zhao et al., 2005). The current study determined the presence and specificity of membrane E2 binding sites and the functional consequence of E2 binding to membrane receptors in individual neurons. Using E2-BSA-FITC (fluorescein isothiocyanate) macromolecular complex, membrane E2 binding sites were observed in hippocampal neurons. Punctate FITC signal was observed on plasma membrane of soma and neuronal processes in E2-BSA-FITC binding neurons. No membrane binding was observed with BSA-FITC. Specificity of binding was demonstrated by competition with excess un-conjugated E2. An ERa specific agonist, PPT, and an ERb agonist, DPN, partially competed for E2-BSA-FITC binding. Imaging of intracellular Ca 2+ ([Ca 2+]i) in live neurons, revealed rapid Ca 2+ responses in E2-BSA-FITC binding neurons within minutes that culminated in a greater [Ca 2+]i rise and [Ca 2+]i spikes at > 20 min. The same neurons in which E2-BSA-FITC induced a [Ca 2+]i rise also exhibited activated pERK (extracellular signal-regulated kinase) that was translocated to the nucleus. Immunofluorescent analyses demonstrated that both excitatory and inhibitory neuronal markers labeled subpopulations of E2-BSA-FITC binding neurons. All E2-BSA-FITC binding neurons expressed L-type calcium channels. These results demonstrate, at a single cell level, that E2 membrane receptors mediate the rapid signaling cascades required for E2 neuroprotective and neurotrophic effects in hippocampal neurons. These results are discussed with respect to therapeutic targets of estrogen therapy in brain.

Plasma estrogen concentrations after oral and vaginal estrogen administration in women with atrophic vaginitis

In this open-label, randomized, multiple-dose, two-treatment crossover study, 24 postmenopausal women with moderate to severe atrophic vaginitis received 0.3 mg conjugated estrogens daily for 14 days: 7 days orally (0.3 mg tablet) and 7 days vaginally (0.5 g cream). Steady-state plasma concentrations of E2 and estrone were one-third lower after vaginal versus oral administration of conjugated estrogens.

Reproductive Cycle, Ovarian Development, and Vertebrate-Type Steroids Profile in the Freshwater Prawn Macrobrachium Rosenbergii

Abstract This study is intended to characterize the ovarian development and hemolymph vertebrate-type steroids concentration during the reproductive cycle of the freshwater prawn, Macrobrachium rosenbergii. A five-stage classification based on the external observation of the ovary's size and color as seen through the tegument was used. The results showed the existence of a direct correspondence between the ovarian stages and the gonadosomatic index, the oocyte diameter, and the characteristics of ovarian histology. In each stage total bleeding of the prawns was conducted and the hemolymph concentrations of 17β-estradiol (E2), testosterone (T) and 17α-hydroxyprogesterone (17-OHP; conjugated and unconjugated) were determined by solid-phase radioimmunoassay (RIA). High levels of unconjugated 17-OHP, relatively constant concentrations of unconjugated T, and null concentration of unconjugated E2 were found throughout the five stages considered. Nonetheless, low levels of E2 were determined in all stages, in co...

Binding of estrogen receptor with estrogen conjugated to bovine serum albumin (BSA)

BackgroundThe classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E2-BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E2-BSA does not compete for E2 binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E2 and E2-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E2-BSA preparations with unconjugated E2 (usually between 3–5%), the latter was carefully removed by ultrafiltration.ResultsAs previously reported, a 10-to 1000-fold molar excess of E2-BSA was unable to compete with 3H-E2 binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E2-BSA, the binding of 3H-E2 was significantly reduced. E2-BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E2-BSA (E2-BSA-FITC). Staining was restricted to the cell membrane when E2-BSA-FITC was incubated with stable transfectants of the murine ERα within ER-negative HeLa cells and with MC7 cells that endogenously produce ERα. This staining appeared highly specific since it was competed by pre-incubation with E2 in a dose dependent manner and with the competitor ICI-182,780.ConclusionsThese results demonstrate that E2-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E2-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E2. More importantly, these findings demonstrate that in intact cells that express ER, E2-BSA binding is localized to the cell membrane, strongly suggesting a membrane bound form of the ER.

Specific radioimmunoassay of estrone sulfate: Application to measurement in male plasma

We described a new, specific and easy to use radioimmunoassay (RIA) of estrone sulfate (E1S) in males. After synthesis of an E1S-6-( O-carboxymethyl) oxime hapten then coupling to BSA, we obtained a specific anti-E1S antiserum. Although the cross-reactivity of DHEAS with our anti-E1S antiserum was low (CR=0.002%), we confirmed the absolute necessity of separating plasma DHEAS from plasma E1S, before E1S RIA, because in plasma, DHEAS is present at levels 3–6000-fold higher than E1S, which generally is ignored. Thus, we elicited an easy separation of DHEAS from E1S, by a fast chromatography on in-house minicolumns. This new RIA, was applied to the determination of E1S plasma normal values in males. In 27 young men (<35 years), mean±S.D. were 1.97 nmol/l±1.07 nmol/l and in 63 untreated healthy aged men (>55 years), 1.80 nmol/l±1.21 nmol/l . No significant difference was seen between young and older subjects. The ranges of E1S plasma levels in these subjects were rather large and the ratios between the highest and the lowest E1S plasma levels were seven in the young group and 23.4 in the older group. No decrease of E1S plasma levels was observed with ages. Contrary to large interindividual E1S plasma level variations, the intraindividual variations have been found to be no significant. Correlations between E1S and unconjugated estrogens, E2 and E1 were 0.22 ( P=0.016) and 0.51 (0.001), respectively.

Estrogens in Intrahepatic Cholestasis of Pregnancy

In Brief Objective To determine whether estrogen production and excretion are impaired in gravidas with intrahepatic cholestasis. Methods Plasma and urine samples were collected from 13 women from the United States and Chile at 35–38 weeks' gestation with mild (n = 9) or severe (n = 4) intrahepatic cholestasis of pregnancy. Urinary and plasma steroid levels from women with cholestasis were compared with levels from 27 normal pregnant women within the same gestational age range. Urinary concentrations of dehydroepiandrosterone (DHEA), estrone (E1), estradiol (E2), estriol (E3), estetrol, progesterone, and 16-hydroxy-pregnenolone were measured by gas chromatography mass spectrometry, and plasma concentrations of DHEA sulfate, progesterone, unconjugated E1, unconjugated E2, unconjugated E3, sulfated E3 derivatives, glucuronidated E3 derivatives, and total E3 were measured by radioimmunoassay. Results Compared with normal pregnant women, women with cholestasis had significantly lower plasma levels of estrogens and DHEA sulfate, the precursor to placental estrogen production synthesized by the fetal adrenal gland (Hotelling-Lawley trace = 0.81; F4,19= 3.9; P = .02). The mean plasma DHEA sulfate, unconjugated E2, unconjugated E3, and total E3 concentrations were 0.271, 10.21, 9.80, and 99.53 ng/mL, respectively, in women with cholestasis compared with 0.802, 18.98, 16.28, and 145.07 ng/mL for controls. Conclusion Fetal adrenal production of DHEA sulfate, and in response, downstream placental production of estrogens, was compromised by intrahepatic cholestasis of pregnancy. Intrahepatic cholestasis of pregnancy is associated with decreased maternal plasma levels of estrogens and the estrogen precursor dehydroepiandrosterone sulfate.

Estrogens in intrahepatic cholestasis of pregnancy

Objective: To determine whether estrogen production and excretion are impaired in gravidas with intrahepatic cholestasis. Methods: Plasma and urine samples were collected from 13 women from the United States and Chile at 35–38 weeks’ gestation with mild ( n = 9) or severe ( n = 4) intrahepatic cholestasis of pregnancy. Urinary and plasma steroid levels from women with cholestasis were compared with levels from 27 normal pregnant women within the same gestational age range. Urinary concentrations of dehydroepiandrosterone (DHEA), estrone (E1), estradiol (E2), estriol (E3), estetrol, progesterone, and 16-hydroxy-pregnenolone were measured by gas chromatography mass spectrometry, and plasma concentrations of DHEA sulfate, progesterone, unconjugated E1, unconjugated E2, unconjugated E3, sulfated E3 derivatives, glucuronidated E3 derivatives, and total E3 were measured by radioimmunoassay. Results: Compared with normal pregnant women, women with cholestasis had significantly lower plasma levels of estrogens and DHEA sulfate, the precursor to placental estrogen production synthesized by the fetal adrenal gland (Hotelling-Lawley trace = 0.81; F 4,19 = 3.9; P = .02). The mean plasma DHEA sulfate, unconjugated E2, unconjugated E3, and total E3 concentrations were 0.271, 10.21, 9.80, and 99.53 ng/mL, respectively, in women with cholestasis compared with 0.802, 18.98, 16.28, and 145.07 ng/mL for controls. Conclusion: Fetal adrenal production of DHEA sulfate, and in response, downstream placental production of estrogens, was compromised by intrahepatic cholestasis of pregnancy.

Differential effects of estradiol and estradiol-BSA conjugates.

The steroid 17beta-estradiol (E2) acts to modulate transcription through classical nuclear estrogen receptors (ER-alpha and ER-beta). However, E2 also induces a number of rapid responses (<10 min) within cells, including cells devoid of classical ERs, consistent with the presence of a membrane receptor for E2. Membrane impermeable steroids, typically bovine serum albumin (BSA) conjugates, are commonly used to characterize these non-genomic actions of E2 to exclude the involvement of nuclear ERs. Here we report that E2-BSA conjugate preparations, but not unconjugated E2, activate extracellular signal-regulated protein kinases (ERK1 and ERK2) in the SK-N-SH neuroblastoma cell line, raising concerns regarding the use of these reagents as E2 mimics. Freshly prepared solutions of E2-BSA were found to contain free immunoassayable E2 (iE2), which could be removed by filtration. E2-BSA solutions devoid of free iE2 failed to compete for binding of 125I16alpha-iodo-E2 to ER-alpha or ER-beta. Furthermore, in contrast to E2, E2-BSA conjugates did not bind to ER-alpha or ER-beta as assessed by electrophoretic mobility shift analyses. Protein analysis demonstrated that certain E2-BSA preparations were of very high molecular weight, suggesting extreme protein cross-linking. These findings suggest that E2-BSA does not mimic E2 and is not an appropriate ligand for investigating estrogen receptors. This underscores the need to design stable, cell impermeable analogs of estrogen for the characterization of membrane estrogen receptors.

ESTERASE, ESTRADIOL AND SEX HORMONE-BINDING GLOBULIN IN BREAST CYST FLUID

Women who have palpable breast cysts lined by apocrine epithelium (intracystic Na/K < 3) may have a higher risk of developing breast cancer than those with breast cysts lined by flattened epithelium (intracystic Na/K > 3). Esterase converts fatty acid esters of E2 to unconjugated E2 and could conceivably modulate intracystic E2 concentrations. The amount of SHBG in BCF may regulate the amount of unbound, biologically active E2 present. In this study the relationships amongst E2, esterase, SHBG and Na/K in BCF were assessed. Negative correlations were found between E2 and esterase (P<0.05), E2 and SHBG (P<0.001), and E2 and Na/K (P<0.001). A positive correlation was found between SHBG and Na/K (P<0.001). No correlation was found between esterase and Na/K. The findings of this study suggest that esterase is unlikely to be important in modulating intracystic E2 concentrations.

Measurement of serum unconjugated estriol and estradiol by high‐performance liquid chromatography

Measurement of Estriol (E3) and estradiol (E2) within 22 min by high-performance liquid chromatography (HPLC) was achieved in this study, and the values were compared with those of a radioimmunoassay (RIA). A totally computerized HPLC method was developed for measuring unconjugated E3 (u-E3) and E2 (u-E2) in the sera of pregnant women. The serum samples were injected directly into the apparatus and transferred to a pretreatment column where the estrogens were absorbed while hydrophilic components such as proteins and carbohydrates were excluded. The estrogens were then passed through another separation column containing a new type of polymer gel. The mobile phase consisted of an acetonitrile-water mixture, and separation was achieved by means of a reversed-phase procedure. The eluate was monitored for fluorescence. All procedures were monitored and controlled with a built-in microcomputer. Serum samples from 97 normal pregnant women at 20-41 wk gestation were simultaneously assayed by HPLC and RIA. The correlations obtained by HPLC and RIA were as follows: u-E3, y = 0.905x - 0.385, with a coefficient of correlation of r = 0.912; for u-E2, y = 0.964x + 5.024, with a coefficient of correlation of r = 0.841 (y, RIA value; x, HPLC value). The quick measurement of u-E3 and u-E2 by HPLC can be a useful method for evaluating fetoplacental function.

Unconjugated estradiol, estriol and total estriol in maternal peripheral vein, cord vein, and cord artery serum at delivery in pregnancies with intrauterine growth retardation.

The levels of unconjugated estradiol (E2), estriol (E3) and total (conjugated plus unconjugated) E3 in maternal vein serum during labor, cord vein serum, and cord artery serum were measured in normal singleton and twin pregnancies with appropriate for dates babies (AFD) and with light for dates babies (LFD). The mean level of total E3 in the maternal vein serum in singleton pregnancy was significantly lower in the LFD group than in the AFD group, but no differences were seen in the mean levels of unconjugated E2 or E3 between the groups. The concentration of unconjugated E2 in the maternal vein serum was significantly higher in the twin group with a large placenta than in the singleton group with a smaller placenta, while the concentration of total E3 in the case of twin pregnancy with LFD was lower than that in singleton pregnancy with AFD but not significantly. No difference in the concentration of total E3 was observed between the cord vein serum and cord artery serum. The present data suggest that the total E3 level in maternal vein serum may be used in evaluating fetal states such as intrauterine growth retardation.