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This article describes the development of a short pre-treatment method that allows the simultaneous analysis of free estrogens (estrone, 17beta-estradiol, estriol and 17 alpha-ethynylestradiol) and their sulphate and glucuronide conjugated forms. For a range of matrices, from sewage effluent to river water, the developed methodology based on solid-phase extraction and fractionation technique with ultra-performance liquid chromatography system showed effective separation of the targeted estrogens. The detection limits of this method ranged from 0.2 to 0.8 ng L(-1) for river water. The recoveries for river water and sewage effluent varied from 63% to 127%. The problems of matrix effects and ion suppression or enhancement were allowed quantitatively for in the analysis using standard addition. The developed method was used successfully to detect estrogens and their conjugates in both raw and treated wastewater, and river water at a location in Japan. High concentrations of the free estrogens estrone, 17beta-estradiol and estriol were found in the influent (22.6, 77.2, 64.6 ng L(-1), respectively) but only E1 was still present at a high concentration in the effluent which was reflected in the downstream river concentration. Estrone-3-sulphate was detected up to 18.0 ng L(-1) in influent water sample and 1.1 ng L(-1) in downstream water. For the sulphate conjugates, removal efficiencies varied from 35 to 88%. Glucuronide conjugates were detected only once in the sewage influent.

The highly complex matrix of activated sludge in sewage treatment plants (STPs) makes it difficult to detect endocrine-disrupting chemicals (EDCs) which are usually present at low concentration levels. To date, no literature has reported the concentrations of steroid estrogens in activated sludge in China and very limited data are available worldwide. In this work, a highly selective and sensitive analytical method was developed for simultaneous determination of two classes of EDCs, including estrone (E1), 17beta-estradiol (E2), estriol (E3), 17 alpha-ethynylestradiol (EE2), 4-nonylphenol (NP) and bisphenol A (BPA), in the liquid and solid phases of activated sludge. The procedures for sample preparation, extracts derivatization, and gas chromatography-mass spectrometry (GC-MS) quantification were all optimized to effectively determine target EDCs while minimizing matrix interference. The developed method showed good calibration linearity, recovery, precision, and a low limit of quantification (LOQ) for all selected EDCs in both liquid and solid phases of activated sludge. It was successfully applied to determine the concentrations of EDCs in activated sludge samples from two STPs located in Beijing and Shanghai of China, respectively.

Saliva progesterone and oestriol concentrations were determined weekly from 24 weeks of gestation in women at increased risk of preterm delivery. Samples were analysed from 28 women with spontaneous onset of labour and delivery before 37 weeks of gestation, and 64 who delivered at term. Saliva progesterone was lower in the 12 women delivering before 34 weeks than in those delivering later, between 34 and 37 weeks (P = 0.007) or at term (P = 0.009). Measurement of saliva progesterone may be of value in the prediction of early preterm labour and in determining which women might benefit from progesterone supplementation.

For the quantitative evaluation of low levels of an estriol metabolite of estriol (estriol-16-glucuronide (E3-16G)) in liquid media, we developed a simple and highly sensitive immunoassay using a surface plasmon resonance (SPR) biosensor which did not require any time-consuming sample pretreatment steps. E3-16G was conjugated to ovalbumin (OVA) through an oligoethylene glycol (OEG) linker to form protein conjugates (E3-16G-OEG-OVA), which were then immobilized on a carboxymethyl dextran-coated sensor chip via amine coupling to develop inhibition immunoassays. A limit of detection (LOD) of 76 pg/mL was achieved using a rabbit anti-sheep primary antibody as a binding agent. The detection limit was further improved by using synthesized gold colloids (15 nm) as high mass labels conjugated to the primary antibody. In this Au nanoparticle-enhanced assay, the concentration of E3-16G in aqueous samples could be determined in 7.5 min at a level as low as 14 pg/mL. In addition, the high stability of the E3-16G-OEG-OVA surface gave no obvious drop in antibody-binding capability after more than 1000 binding/regeneration cycles which significantly lowered the research cost.

Progesterone and estrogens are hormones produced in the human body that are essential for regulating many vital functions. The three major estrogens produced by women are estriol, estradiol, and estrone. Progesterone is a naturally occurring hormone in both men and women. Pharmaceuticals containing estrogens alone or estrogens in combination with progesterone are commonly used in therapy. Patients requiring unique combinations of the drugs rely on pharmacies to compound the ingredients. In order to assess the potency of drugs containing combinations of estrogens and progesterone, a method was developed to determine all four ingredients simultaneously. The liquid chromatographic method utilized a microBondapak C18 column with an isocratic mobile phase of acetonitrile-water (50 + 50, v/v) at a flow rate of 1.0 mL/min and temperature of 30 degrees C. Under these conditions, the order of elution was estriol, estradiol, and estrone, followed by progesterone. UV detection was at 205 nm to monitor elution of the estrogens, then switched to 270 nm to monitor progesterone. The method was applied to the analysis of pharmacy-compounded drugs containing combinations of the hormones. Validation studies demonstrated that the method is accurate and precise.

Ecological risk quotients and estradiol equivalent concentrations of 32 endocrine disrupting chemicals (EDCs) in 4 categories were calculated and analyzed using reported concentrations in secondary effluents from municipal wastewater treatment plants in China, considering both extreme scenario for maximum concentration and moderate scenario for median concentration. These EDCs include industrial chemicals, pesticides, natural estrogens, and pharmaceuticals. In extreme scenario, 12 chemicals of ecological risk and 9 chemicals of estrogenicity were identified. While in moderate scenario, 6 chemicals of ecological risk and 5 chemicals of estrogenicity were identified. After further scoring and ranking, 4 priority EDCs have been suggested for control in municipal wastewater treatment plants in China, including 17a-ethynylestradiol, estrone, nonylphenol, and bisphenol A.

In this study we present a pressurized liquid extraction/liquid chromatography-tandem mass spectrometry (PLE/LC-MS-MS) method to determine a group of estrogens and conjugated estrogens in sewage sludge. Parameters that affect the extraction step such as extraction solvent, temperature, pressure, static extraction time, number of cycles, purge time and flush volume have been optimized. In the LC-MS-MS system, electrospray ionization and a triple quadrupole analyzer have been used, and the multiple reaction monitoring mode has enabled low levels of target analytes to be detected. All recoveries were higher than 81% except for estrone 3-glucoronide and estradiol 17-glucoronide which were not extracted and consequently, they were not considered in the present study. The repeatability and reproducibility between days expressed as %RSD (n=3), were lower than 6% and 9%, respectively. The method developed allowed the target analytes to be quantified at low levels of microg/kg. The limits of detection were lower than 26 microg/kg of dry weight (d.w.) of sewage sludge, except for 17 alpha-estradiol, 17beta-estradiol, 17 alpha-ethinylestradiol and estradiol 17-acetate whose values were between 150 and 175 microg/kg (d.w.). The method was applied to determine these compounds in sewage sludge from two domestic sewage treatment plants. Estrone 3-sulfate, estradiol 3-sulfate, diethylstilbestrol, estrone and estriol were determined in some samples and estriol showed the highest value (406 microg/kg d.w.).

Objective: The objectives of the study was to determine whether salivary progesterone (P) or estriol (E3) concentration at 16-20 weeks' gestation predicts preterm birth or the response to 17alpha-hydroxyprogesterone caproate (17OHPC) and whether 17OHPC treatment affected the trajectory of salivary P and E3 as pregnancy progressed., Study Design: This was a secondary analysis of a clinical trial of 17OHPC to prevent preterm birth. Baseline saliva was assayed for P and E3. Weekly salivary samples were obtained from 40 women who received 17OHPC and 40 who received placebo in a multicenter randomized trial of 17OHPC to prevent recurrent preterm delivery., Results: Both low and high baseline saliva P and E3 were associated with a slightly increased risk of preterm birth. However, 17OHPC prevented preterm birth comparably, regardless of baseline salivary hormone concentrations. 17OHPC did not alter the trajectory of salivary P over pregnancy, but it significantly blunted the rise in salivary E3 as well as the rise in the E3/P ratio., Conclusion: 17OHPC flattened the trajectory of E3 in the second half of pregnancy, suggesting that the drug influences the fetoplacental unit.

Background: Prenatal antidepressant use has been associated with shorter pregnancy duration and an increased risk for preterm birth. This study measured saliva levels of estriol, a hormone that increases exponentially in the few weeks before spontaneous labor, in pregnant women with and without antidepressant treatment., Methods: Saliva estriol levels were obtained across the day at three time points during pregnancy in 77 subjects with a history of DSM-IV major depressive disorder (MDD) who were treated with antidepressants in pregnancy (Group 1), a history of DSM-IV MDD who were not treated or had limited exposure to antidepressants during pregnancy (Group 2), and a normal control group (Group 3)., Results: Mean estriol levels in the second half of pregnancy were significantly higher for Group 1 (history of MDD, on meds) than Group 2 (history of MDD, off meds) or Group 3 (control)., Conclusions: Prenatal antidepressant use was associated with significantly higher saliva estriol levels in the second half of pregnancy. Whether estriol reflects a causal mechanism by which women on antidepressants have shorter pregnancy duration remains to be further studied.

The decomposition of endocrine-disrupting chemicals (EDCs) including estrone (E1), 17beta-estradiol (E2), estriol (E3), nonylphenol (NP), and bisphenol A (BPA) during ozonation of municipal sewage grabbed from the outlets of primary sedimentation tanks was studied through laboratory-scale experiments. A newly developed in vitro bioassay called nuclear receptor-ligand assay and GC-MS were both utilized to respectively determine the estrogenicity and individual EDCs in the wastewater samples. The original estrogenicity, expressed as the E2 equivalent concentration (EEQC), in the primary effluents was 315-1018 ng/L. Results indicate that the EEQC can be reduced rapidly to below 10 ng/L after ozonation. The appearance of 0.1 mg/L dissolved ozone (DO3), which corresponds to a consumed ozone amount of 0.4 mg per initial TOC (total organic carbon) of wastewater samples, was an appropriate operational parameter to simultaneously achieve efficient EDC removal and control of BrO3- and total organic bromine (TOBr). The presence of suspended solids in the range of 38-67 mg/L exhibited no obvious impact on the removal of nonsorbed estrogenicity. A complete decomposition of E2, E3 and BPA was achieved once 0.1 mg/L DO3 appeared in the primary effluent. The oxidative decomposition of NP was relatively less efficient with a residual concentration of 100 ng/L. This work investigates the feasibility of EDC removal and brominated byproduct control during ozonation of original municipal sewage prior to biological treatment.

Biological degradation of natural estrogens was investigated by separately spiking 17 beta-estradiol (E2) and estrone (E1) into activated sludge reactors operated in a semi-continuous flow mode under aerobic conditions. By conducting runs for simultaneous addition of glucose over the designated initial concentrations of 0, 10, 30, 50 and 100 mg l(-1), respectively, the likely effect of the easily biodegradable organic constituent on the degradation rate of the natural estrogens was also studied. The spiked E2 disappeared in a manner much faster than E1; and its disappearance occurred through elimination of its metabolic byproduct of E1. The apparent first-order disappearance rate of E2 and E1 fell in the ranges of 0.84-4.31h(-1) and 0.15-0.84 h(-1), respectively when spiked separately into respective reactors. A general trend of decreases in the disappearance rate with increases of the initial glucose concentration was revealed. The presence of glucose was also found capable of lowering the conversion ratio from E2 to E1.

A set of simple HPLC methods employing UV detection were developed for detection of counterfeit drugs by the qualitative and quantitative analysis of nine steroidal drugs, ethinylestradiol, diethylstilbestrol, norethisterone, norgestrel, methyltestosterone, medroxyprogesterone acetate, progesterone, testosterone propionate and nilestriol. The methods were based on studies of the relationships between the retention factors (k) of the nine compounds and the percentages of water to methanol in the mobile phases on a reverse phase Alltima C(18) column giving reliable separation of the compounds under three sets of chromatographic conditions. The methods were validated using statistical tests and were used on nine commercial samples for detection of possible counterfeit drugs.

Objective: Congenital lipoid adrenal hyperplasia is an autosomal recessive condition due to mutations in Steroidogenic Acute Regulatory Protein (StAR) associated with the inability to synthesize all adrenal and gonadal steroids, resulting in adrenal insufficiency and failure to develop male genitalia in affected 46,XY individuals. We used two independent methods of prenatal diagnosis for CLAH., Method: CLAH was diagnosed prenatally by measuring amniotic fluid estriol, adrenal, and gonadal hormone levels, and by mutation analysis for the c.201-202 delCT mutation in StAR., Results: Prenatal testing diagnosed one affected and three unaffected fetuses in two families at risk for CLAH., Conclusion: Prenatal testing for CLAH is effective, and targeted molecular genetic analysis should be considered in Palestinian families with a fetus with discordant 46,XY karyotype and female genitalia on ultrasound, and low maternal serum estriol.

This paper discusses the requirement for, and presents an analytical procedure for, the determination of four steroid hormones and a conjugated steroid (estrone-3-sulfate) in wastewaters. The method utilizes LC/MS/MS following solid phase extraction and a two stage clean-up procedure, achieving limits of detection of 0.2 ng l(-1) for estriol, 17beta-estradiol and 17 alpha-ethinylestradiol, and 0.1 ng l(-1) for estrone and the conjugate. The approach demonstrates that using appropriate clean-up and deuterated internal standards, the impact of matrix effects on ionization can be overcome to reliably determine estrogens at environmentally relevant concentrations. The robustness of the method was demonstrated by achieving recoveries of >83% for all steroids in settled sewage and final effluent samples with relative standard deviations of 0.5-12%.

Objectives: To summarize the experience and evaluate the performance of the Hat Yai maternal serum screening (MSS) program., Setting: The Hat Yai MSS program between 16 February 2003 and 11 March 2004., Material and Method: The uptake of screening was 999 in 1,040 women (96.0%), between 14 to 20 weeks of gestation with the triple markers: Alpha-fetoprotein (AFP), human Chorionic Gonadotropin (hCG), and unconjugated Estriol (uE3) by Immulite chemiluminescent immunoassay system, Diagnostic Product Corporation (DPC). The risk cut-off for Down 's syndrome is one in 250 or greater, based on software for prenatal Down's syndrome risk calculation, by Prisca 3.5 DPC., Results: There were 119 in 999 cases (11.9%) of the triple test positive. Amniocentesis had been performed on voluntary basis, and the uptake rate of amniocentesis following a positive Down's syndrome screening was 104 in 119 cases (87.3%). Based on clinical diagnosis of Down's syndrome in the newborns of non-amniocentesis mothers, assuming that normal looking babies were not Down's syndrome, the sensitivity (SENS), specificity (SPEC), positive predictive value (PPV), and negative predictive value (NPV) of all chromosomal abnormalities were 85.7%, 88.6%, 5.0%, and 99.8% respectively. The false positive rate was 113 in 992 cases (11.4%). Whereas, the SENS, SPEC, PPV and NPV of Down's syndrome were 100%, 88.4%, 3.4%, and 100% respectively. The false positive rate was 115 in 995 cases (11.6%). The mean level, median level, and multiple of median (MoM) of triple markers were demonstrated., Conclusion: The Down's syndrome screening is a systematic application of a test to identify subjects at increased risk of a specific disorder; of course it is not diagnostic, but to benefit making decision regarding further amniocentesis. The sensitivity of Prisca 3.5 software was satisfactory but false positive rate was remarkably high. It needs further standardization with adjusted MoM values.

An analytical method based on ultra performance liquid chromatography has been developed for the simultaneous determination of 15 hormones (triamcinolone, hydrocortisone, prednisone, cortisone, 6a-methylprednisolone, betamethasone, dexamethasone, prednisolone acetate, hydrocortisone acetate, estriol, beta-estradiol, estrone, diethylstilbestrol, testosterone and progesterone) in cosmetics. Various cosmetic samples were extracted with methanol under ultrasonication, and then cleaned up by a Oasis HLB solid phase extraction cartridge. The 15 hormones were separated and detected within 6 min on a Waters ACQUITY UPLC BEH C18 column (1.7 microm, 2.1 mm x 50 mm) by gradient elution with acetonitrile and water system. In the range of 1-25 ng, the calibration curves of 15 hormones showed good linearity with the correlation coefficients larger than 0.9995. The mean recoveries at the three spiked levels (2, 10, 20 mg/kg) were 88.2%-102.4% with the relative standard deviations of 1.6%-7.4%.

A rapid microwave-accelerated derivatization process for the GC-MS analysis of steroid estrogens, estrone (E1), 17beta-estradiol (E2), estriol (E3), 17alpha-ethynylestradiol (EE2) and mestranol (MeEE2), was developed. Under microwave irradiation, the five estrogenic hormones studied were simultaneously derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+trimethylchlorosilane (TMCS) in pyridine solution. Effects of irradiation time (15-120 s) and power level (240-800 W) on the yield of the derivatization were investigated. The derivatization under the irradiation of 800 W microwave for 60s produced comparable results when compared with the conventional heating process in a sand bath for 30 min at 80 degrees C in terms of derivatization yield, linearity and precision for all steroid hormones tested. The calibration curves are linear between 3.00 and 3.00 x 10(2) microg mL(-1). The square of the regression coefficients (R(2)) range from 0.979 to 1.000. The applicability of the method was evaluated on spiked river and distilled water samples at two concentrations, 25.0 and 2.00 x 10(2) ng mL(-1). The recoveries obtained by using microwave heating (60s, 800 W) were similar to those by conventional heating. When combined solid-phase extraction (SPE) with the application of the microwave-accelerated derivatization proposed here, the detection limits of 0.02-0.1 ng L(-1) for the steroid hormones have been achieved. The results demonstrated that microwave-accelerated derivatization is an efficient and suitable sample preparation method for the GC-MS analysis of estrogenic steroids.

Purpose: It was hypothesized that the risk for testicular germ cell tumors (TGCTs) is associated with maternal hormone levels. To examine the hypothesis, some studies used perinatal factors as surrogates for hormone levels. To determine the validity of this assumption, hormone-perinatal factor relationships were examined in the Collaborative Perinatal Project., Methods: Maternal estradiol, estriol, and testosterone levels in first- and third-trimester serum samples were correlated with perinatal factors in 300 mothers representative of populations at high (white Americans) or low (black Americans) risk for TGCT., Results: For white participants, testosterone levels were associated negatively with maternal height (p < 0.01) and age (p = 0.02) and positively with maternal weight (p = 0.02) and body mass index (BMI; p < 0.01), whereas estradiol levels were associated negatively with height (p = 0.03) and positively with son's birth weight (p = 0.04). For black participants, estriol levels were associated negatively with maternal weight (p = 0.01), BMI (p = 0.02), and gestational age p < 0.01) and positively with son's birth weight (p < 0.01), length (p = 0.04), and head circumference (p = 0.03)., Conclusions: These findings indicate that use of perinatal characteristics as surrogates for hormone levels should be limited to a specific ethnic group. For white men, previously reported associations of TGCT with maternal weight and age may be caused by lower maternal testosterone levels.

The fate and behavior of estrogenic substances in various biological wastewater treatment processes and several advanced sewage treatment processes were examined. The removal of 17beta-estradiol (E2), estrone (E1) and estriol (E3) was investigated by using a pilot-scale activated sludge plant supplied with domestic sewage. Several sewage treatment plants adopting the activated sludge process were evaluated for the removal of estrogenic substances using an in vitro recombinant yeast assay and chemical analysis. The results indicated that E2 significantly contributed to estrogen-like activity particularly in secondary treated effluents. The removal rate of E2 was found to be sufficiently high throughout a one-year study on estrogens in domestic sewage, whereas E1 often remained in the effluent. The optimization of operational conditions based on E1 removal is important for reducing estrogenic activity in treated water.

Steroid hormones are naturally synthesized by both humans and animals and are released into the environment. Significant levels of steroid hormones have been detected in sewage effluent around the world. The potential problem is that these hormones may interfere with the normal function of the endocrine systems, thus affecting reproduction and development in wildlife. Due to the major shortage of water in Western Cape, South Africa there is a great need to recycle water by either direct or indirect methods. The treated sewage effluent-natural surface water mixture found in the Kuils and Eerste Rivers is used directly for irrigation of agricultural areas. Sewage effluents were collected from four sites (Jonkershoek, Belville, Zandvliet, and Macassar) and subjected to C(18) solid phase extraction. Commercially available rapid ELISA kits were validated for the quantification of estrogens in these sewage effluent samples. Analysis of estrone, estradiol, and estriol levels showed a significant difference between the control site (Jonkershoek) and sewage effluent from the three sewage treatment works. Steroid hormone concentrations detected in these sewage effluents were similar to reports from Britain, Italy, Germany, Canada, and Netherlands.

The occurrence of the steroid hormones estrone (E1), 17alpha-estradiol (alphaE2), 17beta-estradiol (betaE2), and estriol (E3) in processed bovine milk with different fat contents and in raw milk from (non)gestated cows was investigated. Following liquid extraction, optional enzymatical deconjugation, C18 solid-phase extraction, and derivatization, estrogens were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Free and deconjugated E1 (6.2-1266 ng/L) was the major estrogen followed by alphaE2 (7.2-322 ng/L) and betaE2 (5.6-51 ng/L), whereas E3 was detected regularly at the detection limit of 10 ng/L. The lowest and highest concentrations were determined in raw milk from nonpregnant and from cows in the third trimester of gestation, respectively. The estrogen concentration in processed milk coincides with that of raw milk between first and second trimesters, reflecting the contribution of lactating pregnant cows to the final consumable product. The daily intake of total investigated estrogens through milk is 372 ng, which is dramatically more than currently recognized.

The primary objective of our research was to establish the technical feasibility of using the horseradish peroxidase (HRP) enzyme for natural and synthetic estrogens-estrone (E1), 17beta-estradiol (E2), estriol (E3), and 17alpha-ethinylestradiol (EE2)-removal. The effects of temperature and pH on enzymatic treatment kinetics were investigated. Residual estrogen concentrations were quantified by liquid chromatography, coupled with mass spectrometry analysis. In a synthetic solution at pH 7 and 25+/-1 degrees C, the HRP enzyme-catalyzed process was capable of achieving 92-100% removal of E1, E2, E3, and EE2 within 1h of treatment with an HRP activity of 0.017 U/ml. The influence of the pH (5-9) and temperature (5-35 degrees C) on estrogen removal was observed to be significant, with the optimum pH near neutral conditions. The results also showed that wastewater constituents significantly impact the HRP-catalyzed estrogen removal. The experimental research proved that the HRP-catalyzed system is technically feasible for the removal of the main estrogens present in the environment at low concentrations.

In this work four different commercially available enzyme-linked immunosorbent assays (ELISA) (from Japan EnviroChemicals, Ltd., Tokyo, Japan) were evaluated in terms of performance for the rapid screening of estrogens in different water matrices, including natural and spiked samples from urban wastewater, river water and ground water. All four test kits are based on monoclonal antibodies. The compounds detected by these immunoassays are (1) 17-beta-estradiol, (2) estrone, (3) 17-alpha-ethynyl estradiol and (4) estrogens in general, with high recognition properties for 17-beta-estradiol, estrone and estriol. Standards were prepared in water containing 10% (v/v) methanol. The IC50 (corresponding to the 50% of the effective concentration) values, the dynamic ranges, and the limits of detection of the ELISA kits were 0.060-0.304 microg/L, 0.05-5 microg/L and 0.05 microg/L, respectively. All samples were extracted by solid-phase extraction (SPE) beforehand, and the evaluation was carried out by comparing the results obtained by ELISA with those obtained by HPLC-MS/MS using a triple quadrupole (QqQ) instrument. In addition, two different solid-phase extraction procedures were carried out and compared. Except for moderate overestimation in the results observed with the ELISA kits in the analysis of complex wastewater samples, the results obtained using all of the tested techniques were generally in very good agreement.

Objective: Triple test with measured maternal serum alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol combination as a routine procedure for fetal Down's syndrome, trisomy 18 and neural tube defect screening has some intrinsic problems, such as precision. The aim of this study was to evaluate the effect of analytical variation of triple test on prenatal risk estimation., Method: Five different serum pools were prepared and triple test was performed seven times for within run and five times for between run precision determination., Result: Within run and between run, precision values of risk estimations by measuring the same sample for Triple test were calculated to be 7.9-21.4% and 14.1-31.0% for trisomy 21, 13.2-23.7% and 14.2-15.1% for trisomy 18, 47.2 and 42.0 % for neural tube defect, respectively., Conclusion: These results demonstrated that analytical variations have great impact on second trimester risk estimation procedures; therefore, triple test analyses should be carried out in laboratories using strict internal and external quality control programs. Moreover, triple test results should always be interpreted by considering analytical and biological variations.

This paper describes a way of differentiating between the three isomers of estriol glucuronide by the use of chemical derivatization and liquid chromatography/electrospray tandem mass spectrometry (MS/MS). In their native form, these isomers gave rise to almost identical product ion spectra, involving the neutral loss of 176 Da (i.e. monodehydrated glucuronic acid), which made it impossible to determine the position of conjugation by MS/MS alone. In order to change the fragmentation pathways, positive charges were introduced into the analytes by chemical derivatization. The following reagents were tested: 2-chloro-1-methylpyridinium iodide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2-picolylamine. Interestingly, derivatization using a combination of all three reagents gave a selective fragmentation pattern that could differentiate between the isomers estriol-16-glucuronide and estriol-17-glucuronide. Estriol-3-glucuronide, which lacks a free phenolic group, could be differentiated through a different type of reaction product when exposed to 2-chloro-1-methylpyridinium iodide. Furthermore, in order to assist structural assignment of the fragments, their accurate masses were determined using a hybrid quadrupole time-of-flight mass spectrometer and fragmentation pathways were elucidated by the use of MS3 on an ion trap mass spectrometer., (Copyright 2006 John Wiley & Sons, Ltd.)

Objective: To describe a method for deciding whether an individual's first-trimester Down syndrome screening test result justifies further testing in the second trimester., Methods: Statistical modelling was used to estimate the distribution of second-trimester marker profiles for a given first-trimester profile and hence the probability of a final positive result, using a 1 in 250 term cut-off. A multi-variate log Gaussian model was used with published parameters. Markers were maternal serum pregnancy-associated plasma protein-A and free beta-human chorionic gonadotrophin (hCG) at 10 weeks, nuchal translucency at 11 weeks, and second-trimester maternal serum alpha-fetoprotein, total hCG, unconjugated estriol and inhibin-A. To illustrate the method, the model was applied to a published series of 24 Down syndrome and 367 unaffected pregnancies., Results: Modelling predicts that for 63% Down syndrome and 0.4% unaffected pregnancies having first-trimester tests, there is a 50% or more probability of a final positive result. A step-wise sequential screening policy based on immediate prenatal diagnosis for those with high probability and second-trimester testing for the remainder would have a 90% detection rate and 1.7% false-positive rate. Modelling also predicts 8.0% Down syndrome and 89% unaffected pregnancies with probabilities below 3%. A contingent screening policy restricting second-trimester testing to those with 3-49% probabilities would have an 88% detection rate and 1.4% false-positive rate., Conclusion: Predicting the probability of a positive final result from the first-trimester marker profile has potential utility, either as a decision aide for individual women or as a formal part of screening policy in selecting a subset of women for second-trimester testing., (Copyright 2005 John Wiley & Sons, Ltd)

Steroid hormones play a key role in the beginning, development and termination of gestation. This reveiw is devoted for physiological effects of estrogens, progesterone, cortisole, ACTH, CRH in various pregnancy events: implantation, fetus development, maternal adaptation and birth initiation. Priority is fixed for estrogens--steroids that vastly increase maternal circulating blood value, induce progesterone action on uterus, regulate fetal "hypothalamic-pituitary-adrenocortical" axis, control free cortisole level in feminine blood. Diagnostic criterions of steroid hormone determination durijng pregnancy are presented. To day unconjugated estriol is the only steroid hormone that implicated in total pregnancy screening programs. Its concentration reduction has been noted in pregnancies with Dawn syndrome, some child enzyme defetcs, intrauterine growth retardation and fetal death incidents.

An efficient method for analyzing seven sexual hormones in cosmetics, namely, estriol, estradiol-17beta, estrone, testosterone, methyl-testosterone, progesterone and diethylstilbestrol by reversed-phase high performance liquid chromatography was developed. Samples were saponified with 20 g/L sodium hydroxide. The sexual hormones were then extracted with dichloromethane-acetic acetate (40:1, v/v) under acidic conditions (pH 3, adjusted with 1 mol/L HCl). An XTerra RP18 column was employed and a mixture of water-methanol-acetonitrile (50:32:18, v/v) was used as mobile phase. The seven sexual hormones were detected at 230 nm. The average spiked recoveries for the seven sexual hormones ranged from 75.6% to 97.8% with relative standard deviations of 1.9% to 7.2%. The linear ranges of determination were from 5 to 50 mg/L with correlation coefficients of 0.9999, and the limits of detection were from 3.7 to 12 ng. The method is suitable to simultaneously assay the seven sexual hormones in cosmetics with simplicity and accuracy in a single injection.

Estriol (EO) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method using HPLC for EO was investigated. After placing 1.0 ml of EO solution at 50 microg/ml and 0.5 g of the lotion into a 10-ml volumetric flask, the methanol was added to make until that volume and this solution was used as the testing solution. Milky lotion was procedured as follows: After placing 1.0 ml of EO solution at 50 microg/ml and 0.5 g of the milky lotion into a 10-ml volumetric flask, the methanol was added to make until that volume. The suspending mixture was moved to a centrifuging tube with a cap. After centrifuging at 3000 rpm for 5 minutes, the supernatant was used as the testing solution. The testing solution of 20 microl was determined by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250 mm), the mixture of water and acetonitrile (31:9) and the detection wavelength of 285 nm. The working curve from 1.0 to 6.0 microg/ml showed a linear line between the concentrations of EO and the peak area. There was no interference of peak of EO from the lotion and milky lotion.

In order to investigate the potential for microbial degradation of estrogens, and the products formed, activated sludge collected from Korea (ASK) and night soil-composting microorganisms (NSCM) were used to degrade estrogens. Results showed that both ASK and NSCM degraded almost 100% of the natural estrogens estrone (E1), 17beta-estradiol (E2), and estriol (E3) from initial concentrations of 20-25 mg/L, while synthetic estrogen, ethynylestradiol (EE2), was not degraded. Analysis of degradation products of E2 by using HPLC-ECD and a consecutive first-order reaction calculation confirmed that E2 was sequentially degraded to E1, which was further degraded to other unknown compounds by ASK and NSCM. We then used the yeast two-hybrid assay to show that the unknown degradation products did not appear to possess estrogenic activity when E1, E2 or E3 were degraded to below the detection limit after 14 days of incubation, indicating that ASK and NSCM not only degrade natural estrogens, but also remove their estrogenic activities.

Preterm birth is a major complication of pregnancy and remains a leading cause of neonatal morbidity and mortality worldwide. Improvements in the authors' understanding of the pathophysiology of preterm labor have led to the development of novel diagnostic tools of use to identify women at greatest risk for preterm birth. Currently two FDA-approved biochemical tests are available in the United States: (1) fetal fibronectin and (2) salivary estriol. The presence of a positive fetal fibronectin test in the midtrimester of pregnancy is strongly associated with early spontaneous preterm birth. In contrast, a positive salivary estriol test is associated with late preterm birth, thus limiting its clinical use. Both tests have low test sensitivity and are currently used clinically for their negative predictive values. That is, women who screen negative are at very low risk for preterm birth and, thus, no interventions are indicated to prevent preterm birth. Women with a positive test are at increased risk and would be candidates for intervention. One of the main limitations of fetal fibronectin and salivary estriol, and an array of other proposed markers, is the fact that while these markers may aid in identification of women at increased risk for preterm birth, the authors currently have no clearly effective obstetric interventions for preterm-birth prevention in these high-risk women. Use of tocolytics, antimicrobials, or progesterone therapy currently has limited or unproven benefit in the management of women deemed at increased risk using these markers. Thus, until effective targeted obstetric interventions are available, the use of biochemical markers to identify women at increase risk for preterm birth remains largely research tools.

Over the past decade, many studies have argued the relative merits of different chromosome abnormality biochemical screening protocols in different labs. Results and interpretations have varied markedly (e.g., double vs. triple screening). In this study we sought to compare coefficients of variation (CV) among 12 laboratories in one system, using identical and different methodologies for the three parameters. Ten identical specimens were processed as part of the 1999 College of American Pathologists (CAP) (FP-A, FP-B) proficiency tests. Results were compared among 12 laboratories using the Abbott ELISA for alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG), and two methods for estriol [Diagnostic Services Laboratory (DSL) and an "in house" assay]. The range on the 10 specimens of means for AFP varied from 12.56 to 117.87; hCG 14.05-68.08; and estriol 0.61-2.73. CV for AFP specimens averaged 10%, hCG 8%, and estriol 35% (F = 22.4). However, when only DSL was used for estriol, the CV was reduced to 8.7%. Standardization of AFP and hCG across 12 labs has reduced CV to <10%, which is similar to accepted between run results. Wide variation of uE3 among different methods may explain the widely divergent results in the literature. With national standardization of all parameters, the widely divergent results seen in the literature should narrow, and regional medians will no longer be necessary.

Objective: Method comparison studies were performed in order to move a semi-automated prostate specific antigen (PSA) immunoassay and a manual unconjugated estriol (uE3) immunoassay to an automated chemistry immunoassay analyzer. The results of the two method comparison studies are compared., Design: Serum samples collected on patients with physician orders for PSA or uE3 were assayed by both methods. PSA samples were assayed on a Hybritech Tandem Photon ERA and on two Beckman Coulter Access instruments. UE3 samples were assayed by RIA and on two Beckman Coulter Access instruments. Linear regression analysis was performed on both sets of data and within-run precision and dilution studies were performed on the PSA Access method., Setting: Clinical chemistry laboratory, West Virginia University Hospitals Inc, Morgantown WV., Results: PSA linear regression analysis for the two methods (ERA and Access 1) were y = 1.0008x + 0.0393, r = 0.9976, SE = 0.1319, n = 37 and (ERA and Access 2), y = 1.0019x + 0.0486, r = 0.9964, SE = 0.1632, n = 37. Within-run precision studies for both Access instruments produced acceptable coefficient variations and dilution study results were in PSA reportable range. uE3 linear regression analysis for the two methods (RIA and Access 1) were y = 1.4105x - 0.3741, r = 0.8696, SE = 0.8330, n = 33 and (RIA and Access 2) were y = 1.315x - 0.2292, r = 0.8643, SE = 0.7964, n = 33., Conclusion: The results of the method comparison studies for PSA were acceptable and the automated PSA immunoassay method was adopted. The results of the uE3 comparison studies did not show good correlation; the automated method was not adopted.

Here, we describe the coordinated use of two antibodies with different affinities in a single immunoassay to extend the dynamic range and to enable detection of multiple analytes. The combination of dual antibodies was permitted with a flow-based assay at the antibody concentration below the dissociation constant, enabling affinity to govern the antibody-antigen binding. Both high and low affinity antibodies to estriol were used in combination to extend the range. The binding of each antibody was mutually independent and individually occurred over concentration ranges of 10 pM(-1) nM and 100 pM(-1) microM. The wide dynamic range of 10 pM(-1) microM was thus achieved as summation of the proportional signals to the total binding. When a combination of antibodies toward different antigens was used, it effectively detected multiple analytes within a mixture. In simultaneous analysis of a mixture of estradiol and estriol, the total signal was the sum of the binding signals from anti-estradiol and anti-estriol antibodies. In a further refinement, the individual antibodies were flowed through the flow cell sequentially, allowing the quantification of each binding signal within the combination. With this sequential format, measurement of the individual hormones in the range of 1.6 pM(-1) nM was shown. Furthermore, the same flow format was successfully applied to assay estriol and estradiol hormones in mixtures of six related compounds.

A sensitive method for detecting 17 beta-estradiol (E2) and estriol (E3) in river water has been developed, based on the time-resolved fluoroimmunoassay by using a fluorescent europium chelate label, 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedion-6"-yl)- chlorosulfo-o-terphenyl (BHHCT)-Eu3+. In the E2 assay, microtiter plates were coated with the E2-bovine serum albumin (BSA) conjugate. The anti-17 beta-estradiol antibody, the biotinylated goat anti-rabbit IgG antibody and the BHHCT-Eu3+ labeled streptavidin (SA)-BSA conjugate were used. In the E3 assay, the goat anti-rabbit IgG antibody was coated on a microtiter plate. The anti-estriol antibody and the BHHCT-Eu3+ labeled E3-BSA conjugate were used. The detection limits for E2 and E3 were 2.3 pg/ml and 4.3 pg/ml, respectively, and the analytical recoveries were 95-120%. Quantitative measurement of estrogens in river water was carried out for Kanda River (Tokyo, Japan) by using the method. The E2 and E3 levels were 32 pg/ml and 5.5 pg/ml, respectively. The detection limits of the present method are in the same orders of magnitude as those of ELISA for E2, and are 1-2 orders of magnitude better for E3.

A precise and reliable method, using liquid chromatography combined with ion trap based mass spectrometry, for the determination of three endogenous estrogens, namely, estrone, estradiol, and estriol, and two synthetic estrogens, ethinyl estradiol and diethylstilbestrol, in environmental water samples was developed. Optimization of the parameter settings of the ion source and mass analyzer as well as evaluation of solvent composition were carried out by continuous introduction of standards through a syringe pump. In negative ion mode the electrospray ionization source gave acceptable results. The optimum solvent used consisted of water/acetonitrile, with no volatile bases or buffers added. A simple, off-line, manual solid-phase extraction method was developed for sample preparation of environmental water samples. Recoveries were over 86% for all compounds. The method was validated and found to be linear, selective, and robust. For analysis of a 50-mL sample, the limit of detection (LOD) ranged from 3.2 to 10.6 ng/L for all compounds, and the limit of quantitation (LOQ) from 10.6 to 35.0 ng/L. Within-day (n = 5) and total (n = 5) reproducibility were investigated at three different concentration levels and ranged from 6.2 to 9.5% and 9.4 to 12.1%, respectively. Finally, the method was applied to real-world samples., (Copyright 2002 John Wiley & Sons, Ltd.)

Preterm birth is the second leading cause of neonatal mortality in the United States (1) (second only to birth defects), and preterm labor is the cause of most preterm births (2). Neonatal intensive care has improved the survival rate for babies at the cusp of viability, but it also has increased the proportion of survivors with disabilities. The incidence of multiple births also has increased along with the associated risk of preterm delivery (4). Interventions to delay preterm delivery in these settings have not shown conclusive effectiveness. Because the morbidity of babies born after 34-35 weeks of gestation has diminished, most efforts to identify preterm deliveries have focused on deliveries before this age. This document describes the various methods proposed for predicting preterm birth and the evidence for their roles in clinical practice.

Preterm birth is a leading cause of peripheral morbidity and mortality. The national rate of prematurity approaches 11%. In spite of widespread tocolytic use, the preterm birth rate has actually increased over the past 30 years in the United States. Preterm birth appears to have a multifactorial etiology. Leading theories include infectious, inflammatory or ischemic insult to the uteroplacental barrier, activation of the fetal hypothalamic-pituitary pathway, decreased cervical competence, and pathologic uterine distention. Multiple biochemical and biophysical markers have been studied for their potential to correctly identify women at risk of preterm delivery. Of these, fetal fibronectin and endovaginal ultrasound examination of the cervix have proven effective in predicting which symptomatic women are actually at low risk of preterm birth. Salivary estriol is being studied as a marker for preterm labor and delivery and it too will likely be found to be a reliable risk identifier in a high risk population. However, home uterine activity monitoring has not been shown to decrease the frequency of preterm birth or its neonatal complications.

A flow-based immunoassay that uses microspheres as the solid phase accomplished the theoretical limit of detectability achievable with the antibody. An equilibrated mixture of anti-estriol monoclonal antibody and estriol was briefly exposed to a bead pack containing immobilized estriol in a flow cell. A small portion of free antibody was separated rapidly from the mixture by binding it to immobilized hormone, but the antibody-hormone complex was kinetically excluded from binding. This rapid separation prevented shift in the equilibrium of the liquid phase binding. Signals were generated by labeling the separated antibodies on the beads with a Cy5-conjugated antispecies secondary antibody. By labeling after the separation step, perturbing the liquid-phase or solid-phase binding was prevented. This assay allowed the reduction of the concentration of primary antibody by continuously accumulating free antibody onto the beads prior to quantification and, thus, offered ideal conditions to achieve theoretical limits of detectability. The optimum achievable dynamic range of this immunoassay was 4-300 pM. Because the proportion of free anti-estriol antibody in the mixture was controlled by the Kd of the antibody-estriol interaction, when the concentration of the antibody was below the Kd, the smallest detectable estriol concentration approached the theoretical limit of detectability achievable with this antibody.

Aim: To constitute a method to determine the estradiol metabolites in human liver microsome in low concentration of estradiol., Methods: Use high performance liquid chromatography after solvent extraction, evaporation, and reconstitution to separate the metabolites and use a electrochemistry detector to detect the metabolites., Results: With a mobile phase of acetic acid buffer-acetonitrile (50:50, v/v, pH 4.5) at flow rate of 1.0 mL/min and a potential of +0.7 V vs Ag/AgCl, all six composition were well separated and satisfactorily detected. There are E3, 16alpha-OHE1, 2-OHE2, E1, and two unidentified composition. The minimum detectable amount is about 100 p g on column. This method is sensitive enough to detect E1 in a substrate concentration of 1 micromol/L., Conclusion: The method can be used to study the metabolism mechanism of estradiol in liver microsome.

Purpose: To determine whether the results of triple-test analysis differ between spontaneous and IVF pregnancies., Methods: The study population consisted of 140 women with singleton pregnancies, 70 by IVF, and 70 by spontaneous conception. The groups were matched for maternal age, gestational week, and laboratory batch. The levels of all triple-test markers--alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG), and unconjugated estriol (u-E3)--were compared between the groups., Results: Significantly higher HCG levels were detected in the patients with IVF pregnancies than in the control group (1.31 +/- 0.8 vs. 0.95 +/- 0.5 multiple of the medians, respectively, (p < 0.006), but there were no significant differences in AFP or u-E3 levels. Overall, 18.5% of the IVF group were found to be screen-positive as compared with 11.4% of the control group (difference not statistically significant). Only 8 IVF pregnancies (11.4%) reached the lowest calculated risk possible (1:9999) compared with 17 (24.2%) in the control group (p < 0.05)., Conclusions: Our findings support previous data demonstrating elevated maternal serum HCG in IVF patients in comparison with spontaneous ones.

Objective: The aim of this study was to characterize the increases of salivary estriol concentrations before the onset of labor at term., Study Design: Salivary estriol concentrations were measured in weekly patient-collected samples by means of a sensitive (mean +/- SD threshold, 0.025 +/- 0.001 ng/mL; coefficient of variation, 3.8%) direct enzyme immunoassay in a microtiter plate format. The salivary estriol concentrations in 16 healthy pregnant women were characterized from 30 weeks' gestation until the time of parturition and delivery. Samples were stored frozen at collection and analyzed in batches after delivery., Results: The median salivary estriol concentration profile revealed a nonlinear rise beginning from 30 weeks' gestation (0.89 ng/mL) until term (2.70 ng/mL, an increase of 201%). At 35 weeks' gestation the salivary estriol concentration median value increased sharply (positive inflection point, 50%-93% increase) at a demarcation between a slower increase during early pregnancy and a more rapid increase during late pregnancy. This positive inflection point associated with a late pregnancy increase characterized subgroups of pregnancies according to the lengths of gestation as follows: early term (delivered at <38 weeks 1 day's gestation), middle term (delivered at 38 weeks 1 day-40 weeks' gestation), and late term (delivered at >40 weeks' gestation). Five weeks before delivery the mean (+/-SEM) rate of rise in salivary estriol concentration was 0.50 +/- 0.13 ng/mL per week to 0.84 +/- 0.26 ng/mL per week in the early term group. The increase in rate for the middle term group was 0.32 +/- 0.06 ng/mL per week to 0.37 +/- 0.26 ng/mL per week, whereas in the late term group the rate of salivary estriol concentration rise was 0.37 +/- 0.03 ng/mL per week to -0.03 +/- 0.25 ng/mL per week., Conclusion: These data demonstrate in normal pregnancies (1) that a direct, nonradiometric measure of salivary estriol concentration can be used to monitor the late pregnancy increase in estriol production, (2) that 35 weeks' gestation marks a positive inflection point of the onset of increased estriol production, and (3) that the late pregnancy rise in salivary estriol concentration shows distinct patterns that tend to be characteristic of the length of pregnancy. These data support the concept that the rate of increase of estriol production is related to the timing of the onset of labor.

Background: New biochemical markers for the risk of spontaneous preterm birth (SPB) give a more precise and earlier diagnosis than the usual ones. We reviewed the data about the principal markers., Results: Using studies with good methodology and a large number of subjects, we observe that the predictive value of these new markers is somewhat higher than those of the usual markers. Fetal fibro-nectin (FNf) and cervical ultrasound undeniably improve the identification of patients at risk of preterm birth, both in the general population and in these threatened preterm delivery. However no management has yet been demonstrated efficacious, especially in a general population so any recommendations for their systematic utilisation is premature. Other biochemical markers (salivary estriol, serum CRH, etc.) are still under assessment and should not be used outside research protocols., Implication for Practice: It is appropriate to integrate either FNf or cervical ultrasound into daily clinical practice for patients with signs of preterm labor. These new indicators are of special use when the diagnosis is uncertain with the standard markers (uterine contractions, digital examination). Among these patients, they should reduce the number of hospitalizations and of useless treatments, because of their good negative predictive value. At the same time, for patients poorly 'labeled' by the clinical examination, they should allow the application of intensive management (intravenous tocolysis, corticoids, in utero transfers)., Conclusion: Future studies should evaluate these tests in everyday practice. The objective is not to predict preterm birth but to prevent either it or its negative consequences. This goal will be met when we have an effective treatment, without associated adverse effects, to offer patients after a positive test result.

The clinician is faced with many dilemmas in the diagnosis of preterm labor. The tools at hand (no pun intended) yield subjective information and it is difficult to distinguish true labor from false labor. Because preterm labor is multifactorial in cause and can occur in primiparas, estimating risk for the individual patient is difficult. The cause of the preterm labor in the majority of patients is idiopathic; in this group, the use of salivary estriol as a biochemical marker for preterm labor can increase the accuracy of the diagnosis of true labor. It may also lend confidence to the diagnosis of false labor and may allay anxiety and prevent unnecessary interventions. On the horizon are two noninvasive methods: the EMG, to evaluate uterine contractility, and the collascope, to evaluate the cervix. Both show promise and may provide a more objective assessment of risk for preterm delivery among women with symptoms of preterm labor.

Objective: To evaluate serial measurements of salivary estriol (E3) to detect increased risk of spontaneous preterm labor and preterm birth., Methods: A masked, prospective, multicenter trial of 956 women with singleton pregnancies was completed at eight United States medical centers. Saliva was collected weekly, beginning at the 22nd week of gestation until birth, and tested for unconjugated E3 by enzyme-linked immunosorbent assay. Women were separated into high-risk and low-risk groups using the Creasy scoring system., Results: A single, positive (at or above 2.1 ng/mL) salivary E3 test predicted an increased risk of spontaneous preterm labor and delivery in the total population (relative risk [RR] 4.0, P <.005), in the low-risk population (RR 4.0, P < or =.05), and in the high-risk population (RR 3.4, P =.05). Two consecutive positive tests significantly increased the RR in all study groups, with a dramatic improvement in test specificity and positive predictive value but only a modest decrease in sensitivity. In women who presented with symptomatic preterm labor, salivary E3 identified 61% of those who delivered within 2 weeks, using a threshold of 1.4 ng/mL., Conclusion: Elevated salivary E3 is associated with increased risk of preterm birth in asymptomatic women and symptomatic women who present for evaluation of preterm labor.