E2 Assay Research Articles

PD04-01: Predictors of Recovery of Ovarian Function during Aromatase Inhibitor (AI) Therapy.

Abstract Background: AIs may cause a paradoxical rise in estrogen levels due to re-activation of ovarian function in women with chemotherapy-induced ovarian failure (CIOF). Therefore, identification of residual ovarian estradiol production is critical if such women are treated with adjuvant AI therapy rather than tamoxifen. We performed a prospective registry trial to identify predictors of recovery of ovarian function during AI therapy. Methods: Women with hormone receptor (HR) positive breast cancer who were pre- or peri-menopausal at diagnosis and who remained amenorrheic for ≥8 weeks after cyclophosphamide-containing adjuvant chemotherapy were enrolled in a multi-institutional, open-label clinical trial of anastrozole (1 mg/day). Following confirmation that serum estradiol (E2) levels were <20 pg/ml using an ultrasensitive E2 assay (Quest Diagnostics), subjects initiated anastrozole. Serum E2 was assessed biweekly for 12 wks, then less frequently, for 72 wks. Multivariable logistic regression was used to evaluate clinical predictors (age at AI initiation OR chemotherapy, menopausal status at chemotherapy, body mass index (BMI), baseline E2) of recovery of ovarian function defined as elevated serum E2 levels or return of menses. Results: Sixty-nine women were enrolled; current status is given in Table 1. Median age at initiation of chemotherapy was 47.2 yrs (range 37–55), median time since chemotherapy was 0.8 yrs (range 0.3–6.4), and median age at enrollment was 49.8 yrs (range 40–58). Thirty-six had received tamoxifen. We observed elevated E2 concentrations or return of menses during AI therapy in 21 subjects after a median 2.0 mo (range 0.6-17); for that cohort, median age at chemotherapy was 43.8 yrs (range 37–51) and median age at AI initiation was 45.8 yrs (range 40–56). In contrast, for the 15 subjects who had postmenopausal E2 levels for at least 48 wks, median age at chemotherapy was 49.2 yrs (range 44–52) and median age at AI initiation was 50.7 yrs (range 44–55). Age at chemotherapy (p=0.0006) and age at AI initiation (p=0.001) were statistically significant different between the 2 cohorts. On multivariable analysis, age at chemotherapy and age at AI initiation remained significant when each was adjusted for menopausal status, BMI, and baseline E2 (odds ratio (OR) 1.64, p=0.0102 and OR 1.47, p=0.015, respectively). Conclusions: A significant proportion of women who develop CIOF recover ovarian function during AI therapy. Although recovery is usually rapid, it can occur at least one year following initiation of AI therapy. Younger age was the strongest predictor of recovery of ovarian function, although 2 of 21 women who developed elevated E2 levels or return of menses were older than 50 yrs at the time of chemotherapy. Tamoxifen remains the standard of care for women with CIOF; if use of an AI is necessary, patients should be monitored frequently with high-quality E2 assays for recurrent ovarian function. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD04-01.

Estimation of Estradiol in Mouse Serum Samples: Evaluation of Commercial Estradiol Immunoassays

The University of Virginia Center for Research in Reproduction Ligand Core performed an evaluation of nine commercial estradiol (E2) immunoassays for use with mouse serum. The evaluation had two components. 1) Recovery Studies: a mouse pool was spiked with E2 concentrations across the assay range, and percent recovery and parallelism to the assay standard curve were determined. 2) Correlation Studies: serum pools were collected from intact females, ovariectomized (OVX) and OVX-E2 treated mice and E2 assayed, then measured by gas chromatography/tandem mass spectrometry (GC/MSMS) for comparison to a gold standard method. Recovery results showed that E2 recovery from spiked mouse pools varied greatly (from <18% to >640%) among kits tested. However, three kits (DiaSorin Radioimmunoassay, Siemens Double Antibody RIA, and CalBiotech Enzyme Immunoassay) showed reasonable recoveries and parallelism. Data collected from the Correlation Study showed that values from intact, OVX and OVX-E2-treated mouse pools varied by several fold vs. GC/MSMS for most of the kits tested. The DiaSorin RIA and CalBiotech Enzyme Immunoassay Kits showed the best correlation to GC/MSMS. Unfortunately, while this evaluation was ongoing, the DiaSorin Kit was discontinued. In summary, the CalBiotech Kit was the only available assay tested that demonstrated good E2 parallelism to the assay standard curve and accuracy vs. a gold standard method (i.e. GC/MSMS). Also of note, the CalBiotech assay is sensitive and requires minimal sample volume. Therefore, based on these findings the CalBiotech E2 assay has been implemented for use in mouse serum samples within the Ligand Core.

Stimulative effects of Polygonum amplexicaule var. sinense on osteoblastic MC3T3-E1 cells

Context: Polygonum amplexicaule D. Don var. sinense Forb. (Polygonaceae) (PAF) is a well known traditional herb used to treat some diseases, such as fractures, rheumatoid arthritis, muscle injury, and pain. However, its pharmacological mechanism of promoting the healing of fractures is still unknown.Objective: The present study was designed to investigate the effects of PAF ethanol extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cell in vitro, thereby to illuminate the pharmacological mechanism to promote the healing of fractures.Materials and methods: The effects of PAF ethanol extracts on MC3T3-E1 cell proliferation and differentiation were detected by using CCK-8, cell cycle, alkaline phosphatase (ALP), and prostaglandin E2 (PGE2) assays in vitro.Results: The results showed that PAF ethanol extracts significantly stimulated cell proliferation at 0.1–100 μg/mL and the proportion of cells in S-phase increased from 16.33 to 27.29% in osteoblastic MC3T3-E1 cells. Moreover, PAF ethanol extracts increased ALP expression in MC3T3-E1 cells at the concentration from 0.1 to 100 μg/mL and inhibited PGE2 production induced by TNF-α in osteoblasts at the concentrations ranging from 10 to 100 μg/mL in MC3T3-E1 osteoblasts.Discussion and conclusion: These results indicated that PAF directly stimulates cell proliferation and differentiation of osteoblasts; therefore, this study preliminarily explored the pharmacological mechanism of PAF to promote the healing of bone rheumatism and various fractures.

Incidence and predictors of ovarian function recovery (OFR) in exemestane patients with breast cancer (BC) with chemotherapy-induced amenorrhea (CIA) using two estradiol assays.

521 Background: Aromatase inhibitors (AIs) may promote OFR in patients with CIA. True incidence of OFR is unknown partly because estradiol (E2) assays used in the clinical setting lacking sensitivity. We studied incidence and predictors of OFR measuring E2 by a clinical assay (direct) or by a highly sensitive (indirect) immunoassay, together with other markers of ovarian reserve. Methods: Prospective exploratory study in ER positive BC patients, who had CIA lasting at least two years and postmenopausal E2 levels, while on tamoxifen. Patients received exemestane and underwent assessments of E2 (indirect and direct assay), FSH, antimullerian hormone (AMH), and antral follicule count (AFC) at baseline and at month 1, 3 and 6. We considered OFR if: resumption of menses, or developed (i) both E2 levels inconsistent with postmenopausal women on AI (indirect: >8 pmol/L; direct >165 pmol/L) and FSH (<26 IU/L) levels), or (ii) frankly non-postmenopausal E2 values (indirect >55 pmol/L; direct >220 pmol/L). Results: Fifty-three consecutive patients were enrolled; median age: 48y (41-55). By indirect assay, 15 patients (28.3%; CI95%:16.1-40.4) had OFR, (11 menses, 4 OFR without menses). By direct assay 14 had OFR (11 menses, 3 OFR without menses). Mean time to OFR by indirect assay was 5.7 months (CI95%: 3.1-8.3) and 6.4 months (CI95%: 3.7-9.1) by direct assay. There was no difference in baseline E2 levels among those who recover or not OFR. Mean age was the only baseline marker different between patients who had OFR or not (45.7 vs 49.1 years, t-test p<0.0001). In patients ≤48 years (n=26) rate of OFR was 46%. AMH at baseline was below detection limit in all patients and it did not change during exemestane. Baseline and serial values for AFC were not different among patients who had OFR or not. Conclusions: There is a high incidence (28%) of OFR, in patients with CIA treated with AIs, especially in women ≤48y. The two E2 assays showed similar incidence of OFR and time to OFR, despite having markedly different performance characteristics. AMH was not helpful in defining ovarian reserve and likelihood of OFR.

Aciculatin inhibits lipopolysaccharide-mediated inducible nitric oxide synthase and cyclooxygenase-2 expression via suppressing NF-κB and JNK/p38 MAPK activation pathways

ObjectivesNatural products have played a significant role in drug discovery and development. Inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been suggested to connect with various inflammatory diseases. In this study, we explored the anti-inflammatory potential of aciculatin (8-((2R,4S,5S,6R)-tetrahydro-4,5-dihydroxy-6-methyl-2H-pyran-2-yl)-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one), one of main components of Chrysopogon aciculatis, by examining its effects on the expression and activity of iNOS and COX-2 in lipopolysaccharide (LPS)-activated macrophages.MethodsWe used nitrate and prostaglandin E2 (PGE2) assays to examine inhibitory effect of aciculatin on nitric oxide (NO) and PGE2 levels in LPS-activated mouse RAW264.7 macrophages and further investigated the mechanisms of aciculatin suppressed LPS-mediated iNOS/COX-2 expression by western blot, RT-PCR, reporter gene assay and confocal microscope analysis.ResultsAciculatin remarkably decreased the LPS (1 μg/mL)-induced mRNA and protein expression of iNOS and COX-2 as well as their downstream products, NO and PGE2 respectively, in a concentration-dependent manner (1-10 μM). Such inhibition was found, via immunoblot analyses, reporter gene assays, and confocal microscope observations that aciculatin not only acts through significant suppression of LPS-induced NF-κB activation, an effect highly correlated with its inhibitory effect on LPS-induced IκB kinase (IKK) activation, IκB degradation, NF-κB phosphorylation, nuclear translocation and binding of NF-κB to the κB motif of the iNOS and COX-2 promoters, but also suppressed phosphorylation of JNK/p38 mitogen-activated protein kinases (MAPKs).ConclusionOur results demonstrated that aciculatin exerts potent anti-inflammatory activity through its dual inhibitory effects on iNOS and COX-2 by regulating NF-κB and JNK/p38 MAPK pathways.

Down-regulation of PROS1 Gene Expression by 17β-Estradiol via Estrogen Receptor α (ERα)-Sp1 Interaction Recruiting Receptor-interacting Protein 140 and the Corepressor-HDAC3 Complex

Pregnant women show a low level of protein S (PS) in plasma, which is known to be a risk for deep venous thrombosis. 17Beta-estradiol (E(2)), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E(2) in HepG2-ERalpha cells, which stably express ERalpha, and also the genomic ER signaling pathway, which modulates the ligand-dependent repression of the PSalpha gene (PROS1). We observed that E(2) repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E(2) down-regulated PROS1 promoter activity and that this E(2)-dependent repression disappeared upon the deletion or mutation of two adjacent GC-rich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ERalpha. In a chromatin immunoprecipitation assay, we found ERalpha-Sp protein-promoter interaction involved in the E(2)-dependent repression of PROS1 transcription. Furthermore, we demonstrated that E(2) treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E(2) might repress PROS1 transcription depending upon ERalpha-Sp1 recruiting transcriptional repressors in HepG2-ERalpha cells and, consequently, that high levels of E(2) leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women.

Limitations of Direct Immunoassays for Measuring Circulating Estradiol Levels in Postmenopausal Women and Men in Epidemiologic Studies

Serum estradiol (E(2)) serves as an important diagnostic marker in a variety of clinical conditions. In epidemiologic studies, E(2) is commonly used to define the etiologic role of estrogen in hormone-related cancers and chronic conditions. Having an accurate and reliable E(2) assay is of critical importance in these studies, especially when measuring the very low E(2) levels (<30 pg/mL) common in postmenopausal women and men, and for discerning the relatively small (usually <20%) case-control differences in E(2) levels. Because E(2) is metabolized to >100 metabolites in the body, some of which cross-react with E(2) antibodies, direct RIAs without purification steps lack specificity for E(2) and can substantially overestimate E(2) levels. Although direct E(2) RIAs using commercial kits are simpler, less time consuming, and less expensive and require less sample volume than conventional RIAs with preceding purification steps, their lack of sensitivity and specificity makes them invalid for measuring circulating E(2) levels in epidemiologic studies of postmenopausal women or men. Instead, we recommend the use of a well-validated RIA with purification steps to improve sensitivity and specificity and to help achieve the necessary accuracy and reliability needed for epidemiologic studies.

Terbium(III) Chelate as an Efficient Donor for Multiple-Wavelength Fluorescent Acceptors

The emission spectra of the lanthanide chelates enable them to act as a donor for several acceptors emitting at different wavelengths. Fluorescence resonance energy transfer between terbium(III) chelate labeled antibody Fab fragment (donor) and a 17beta-estradiol conjugated to Alexa Fluor 488, 555, 594 or 680 (acceptor) was employed to study the functionality of the terbium(III) chelate as an efficient donor for several acceptors emitting from green to far-red. During measurement, the sensitized emission of the acceptor was measured at acceptor specific wavelength. All the tested dyes proved to be efficient acceptors, and they were successfully used in the competitive homogeneous E2 assay. The highest signal to background ratio and the best assay performance was obtained with Alexa Fluor 680, due to the very low donor emission background at the far-red area. In addition, the sensitized emission of both Alexa Fluor 488 and 680 could be measured simultaneously without significant cross talk.

Serum inhibin B during controlled ovarian hyperstimulation: an additional criterion for deciding whether to proceed with egg retrieval

To determine the predictive value of the inhibin B increment during controlled ovarian hyperstimulation (COH) for differentiating between poor and normal responders and for deciding whether to continue or stop an IVF attempt. Prospective study. Assisted reproduction unit at a university hospital. A total of 110 women undergoing IVF for idiopathic, tubal, and/or male infertility. Blood samples were collected on days 6 and 8 of COH. Inhibin B and E(2) assays were performed. The degree of inhibin B increment was defined as Delta IB = day-8 value minus day-6 value. We analyzed the correlation of day-6 and day-8 inhibin B values and Delta IB with the number of oocytes retrieved. The predictive value of each parameter was determined by using the receiving operator characteristics curve analysis. The Delta IB correlated best with the number of oocytes retrieved (r = 0.5) and with the number of embryos obtained (r = 0.26), independently of age. From the receiving operator characteristics curve analysis, a Delta IB cutoff value of 300 pg/mL discriminated poor (few than four oocytes retrieved, n = 16) from normal (more than four oocytes retrieved, n = 94) responders, with a sensitivity of 70% and a specificity of 94%. The degree of inhibin B increment during COH provides additional information for predicting ovarian response to COH. An increment >300 pg/mL is required to rule out the eventuality of a poor ovarian response.

An hCG antagonist reduces ovulation and diminishes OHSS in a rat model through down-regulation of VEGF

OHSS is a complication of IVF, attributable to changes following hCG treatment to induce final oocyte maturation. These changes increase vascular endothelial growth factor (VEGF) resulting in increased vascular permeability (VP). We have shown that a modified hCG (hCG-Ant) binds competitively to the LH receptor, blocking LH activity in vitro. In a rat OHSS model, hCG-Ant reduces ovulation and significantly diminishes OHSS by reducing VP. We quantified the effect of hCG-Ant on ovulation and OHSS by measuring hormone levels and determining VEGF and VEGF receptor 2 (VEGFR-2) protein localization and mRNA expression in tissue, as well as changes in ovarian weight between treatment groups. In vivo studies used standard rat models for ovulation and OHSS. For ovulation studies, immature female Wistar rats were randomized to 2 groups. 11 rats received 10 IU pregnant mare serum gonadotropin (PMSG) ip for 1d, followed by 10 IU hCG or hCG-Ant 48h later. Rats were sacrificed 24h post-hCG and serum was obtained for E2, P4, and inhibin assays. For OHSS studies, 12 rats were randomized to receive 10 IU PMSG for 4d, followed by 0.1 mL saline (control, n = 4), 30 IU hCG to induce OHSS (n = 4), or 30 IU hCG followed by 30 IU hCG-Ant 24 h later (treatment group, n = 4). After 48 h, tissue was prepared for Western blotting and RT-PCR using standard methods. Ovarian weight was higher in the hCG group vs. hCG-Ant (0.0361 ± 0.014 g vs. 0.019 ± 0.007 g, P<.05). E2 and P4 levels were higher in the hCG vs. hCG-Ant group (91.45 ± 30.84 pg/mL and 6.48 ± 4.13 ng/mL vs. 53.12 ± 18.55 pg/mL and 3.43 ± 2.03 ng/mL, P<.05). Inhibin A increased linearly as luteal E2 and P4 rose. Average inhibin A was higher in the hCG vs. hCG-Ant group (553.63 ± 107.18 pg/mL vs. 247.2 ± 74.19 pg/mL, P<.05). Western blot showed VEGF was present in ovaries treated with hCG and saline and absent in ovaries treated with hCG-Ant following hCG. VEGFR-2 was present in the ovary by Western blot for all groups. RT-PCR confirmed increased ovarian mRNA expression of VEGF in the hCG and saline groups vs. hCG-Ant following hCG. HCG-Ant linearly decreases luteal E2, P4, and inhibin A, reflecting a reduction in ovulation, suggesting a dose-dependent effect. Controlling ovulation may diminish OHSS risk. Ovarian VEGF expression is abolished by giving hCG-Ant after hCG in an OHSS model. Clinically, during an IVF cycle, hCG-Ant may be given following hCG treatment and oocyte retrieval to reduce OHSS while preserving IVF cycle outcome.

Standardization of Steroid Hormone Assays: Why, How, and When?

Lack of standardization of high-quality steroid hormone assays is a major deficiency in epidemiologic studies. In postmenopausal women, reported levels of serum 17beta-estradiol (E(2)) are highly variable and median normal values differ by approximately a 6-fold factor. A particular problem is the use of E(2) assays for prediction of breast cancer risk and osteoporotic fractures, where assay sensitivity may be the most important factor. Identification of women in the lowest categories of E(2) levels will likely provide prognostic information that would not be available in a large group of women in whom E(2) levels are undetectable by less sensitive assays. Detailed and costly methods involving extraction and chromatography in conjunction with RIA provide generally acceptable E(2) results in postmenopausal serum, whereas less tedious, direct immunoassays suffer from inadequate specificity and sensitivity. Studies comparing the two types of methods generally report higher E(2) values with the direct methods as a result of cross-reactivity with other steroids and reduced correlation with biological variables such as body mass index. Similar problems exist with measurements of E(2) and estrone in men, and estrone and testosterone in women. Interest in mass spectrometry-based assays is increasing as potential gold standard methods with enhanced sensitivity and specificity; however, these assays require costly instrumentation and highly trained personnel. Taking all of these issues into consideration, we propose establishment of standard pools of premenopausal, postmenopausal, and male serum, and utilization of these for cross-comparison of various methods on an international basis. An oversight group could then establish standards based on these comparisons and set agreed upon confidence limits of various hormones in the pools. These criteria would allow validation of sensitivity, specificity, precision, and accuracy of current steroid hormone assay methodology and provide surrogates until a true gold standard can be developed.

Analysis of 17β-Estadiol from Sewage in Coastal Marine Environment by Surface Plasmon Resonance Technique

In this study, surface plasmon resonance (SPR) for monitoring 17β-eatradiol (E2) was developed. The small molecule E2 was immobilized on a CM5 sensor chip for an indirect competitive immunoassay to detect E2. The SPR response based on the antigen-antibody reaction was measured by injecting the sample solution into the flow system. The limitation of detection was 0. 445 μg/L. The developed SPE-SPR system was applied to analyze the seawater samples. Recovery of E2 was 91. 6%–93. 1%. Relative standard deviations (RSD) for the E2 assay were between 10. 9%–15. 1% (n = 3). The range of determination of E2 samples from the sewage in the coastal marine environment was between ND (lower than detection limit) and ca. 11. 78 ng/L.

Features of follicle-stimulating hormone–stimulated follicles in a sheep model: keys to elucidate embryo failure in assisted reproductive technique cycles

To evaluate the individual functionality of gonadotropin-stimulated preovulatory follicles, for understanding embryo failure in assisted reproductive technique cycles, in a sheep model. Observational, model study. Public research unit. Fifteen adult Manchega ewes. Synchronization of the estrous cycle with intravaginal progestagens and ovarian stimulation with FSH; evaluation of reproductive activity, plasma sampling, ovarian ultrasonography, and ovariectomies. Determination of estrus behavior, plasma and intrafollicular concentrations of E(2) and inhibin A, number and size of ovarian follicles, and developmental competence of oocytes. These results support the usefulness of serial measurements of plasma inhibin A for assessment of follicular growth during the FSH treatment, rather than of E(2) assays commonly used. Functionality of FSH-stimulated preovulatory follicles is clearly disturbed, as confirmed by a negative correlation between follicular size and intrafollicular concentrations of inhibin A and E(2) in preovulatory follicles after individual dissection; moreover, the ability of their oocytes to resume meiosis was diminished. Functionality of follicles in controlled ovarian stimulation (COS), and developmental competence of their oocytes, is disturbed by the high doses of gonadotropin supplied and finally determined by follicular sizes at starting FSH treatment.

Substance P augments PGE2 and IL‐6 production in titanium particles‐stimulated fibroblasts from hip periprosthetic membrane

Aseptic loosening remains the primary cause of failure in total joint arthroplasty. Implant-derived particles are thought to be a main cause of osteolysis that leads to failure of total joint arthroplasty. The nervous system has been implicated in the etiology and pathogenesis of joint diseases. Substance P (SP) immunoreactive nerve fibers have been detected in the pseudomembrane and pseudocapsular tissues of aseptic loose hip prostheses, suggesting that SP might be involved in the process of aseptic loosening. Fibroblasts are abundant in periprosthetic membrane. Neuropeptides are able to modulate cytokine production by fibroblasts. In this study, we isolated fibroblasts from periprosthetic membrane at the time of revision hip arthroplasty performed because of aseptic loosening. Fibroblasts were stimulated with titanium (Ti) particles or SP. Prostaglandin (PG) E2 and interleukin-6 (IL-6) assays were performed using enzyme-linked immunosorbent assay kit. PGE2 and IL-6 secretion by fibroblasts have been significantly increased in the presence of Ti particles or SP. Moreover SP caused significant increase in PGE2 and IL-6 production by Ti particles-stimulated fibroblasts. Thus, SP and Ti particles acted synergistically to increase PGE2 and IL-6 secretion in fibroblasts from periprosthetic membrane.

Human albumin does not prevent ovarian hyperstimulation syndrome in assisted reproductive technology program: a prospective randomized placebo-controlled double blind study

We aimed to clarify the efficiency of IV human albumin in the prevention of ovarian hyperstimulation syndrome (OHSS). We found that human albumin at the described strength does not seem to either prevent or reduce the incidence of severe OHSS in high risk patients undergoing intracytoplasmic sperm injection (ICSI).

The Long-Term Hormone Levels and Sexual Function of Monorchid Patients

The purpose of this study was to evaluate monorchid patients’ long-term hormone levels and sexual function. Materials and Methods Between September and December 2005, 20 patients were reassessed, having undergone orchiectomy for testicular tumors at the Policlinico and at the National Tumor Institute in Milan between 1986 and 1996. Four patients had seminoma, three of them underwent radiotherapy; 15 patients with embryonal carcinoma subsequently underwent retroperitoneal lymphadenectomy, and one of them underwent chemotherapy; one patient had a Leydig cell tumour. The patients’ case histories were analysed; they underwent urological examination, endocrine test, scrotal ultrasound, and blood samples were taken for testosterone, FSH, LH, prolactin, E2, total cholesterol, triglyceride, beta-HCG, alpha-FP and LDH assays. Body mass index was calculated. Patients were also asked to fill in the IIEF questionnaire. Results Patients’ mean age was 30.7 years at the time of orchiectomy, and 44.7 at the time of endocrine/sexology follow up. Case histories showed that 2 patients suffered from arterial hypertension, for which they were receiving treatment, 2 patients were obese, none of the patients suffered from endocrine disorders. Upon ultrasound evaluation, the remaining testicle mean volume was 22.04 mL. All patients presented testosterone levels at the lower limits of the normal range (mean value 3.3 ng/ml). In 5 patients values were below the lower limit, indicating marked hypogonadism. Only one patient suffered from moderate erectile dysfunction. Conclusions Twenty-five per cent of patients showed low testosterone levels. In view of the long life expectancy of patients suffering from testicular tumors, it is fundamental to consider the long-term problems that a mutilation such as orchiectomy can involve. Patients undergoing orchidectomy in general should be considered to be at higher risk of andropause.

New fluorometric enzyme immunoassay for 17β-estradiol by homogeneous reaction using biotinylated estradiol

A new fluorometric enzyme immunoassay for 17β-estradiol (E2) using biotinylated estradiol (BE) as a probe ligand, is described. In this method, E2 is detected indirectly by a solid-phase avidin–biotin binding assay, in which the biotin is immobilized on a microtiter plate (biotin-plate). After the competitive reaction between E2 and BE for the anti-E2 antibody in solution, the free E2 and BE are separated from the bound forms by means of ultrafiltration. The concentration of BE in the solution is determined from the reaction between the biotin immobilized on the plate and the free BE for the limited biotin binding sites of avidin conjugated with horseradish peroxidase (avidin–HRP), which is added to the solution. The enzymatic reaction of HRP was measured by a fluorometric analysis with the QuantaBlu™ Fluorogenic Peroxidase Substrate (QFPS) in order to detect of the avidin–biotin binding with a high degree of sensitivity. The detection limit and linear range for the determination of E2 were 0.12 nM and from 0.12 to 25 nM, respectively. The relative standard deviations (R.S.D.) for the E2 assay were between 2.2 and 9.1% ( n = 3). The cross-reactivity for several other estrogens was also evaluated.

Quantitative Analysis of 17β-Estradiol in River Water by Fluorometric Enzyme Immunoassay Using Biotinylated Estradiol

A sensitive and simple immunoassay to determine 17beta-estradiol (E2) in fresh water was developed. The method is based on a solid-phase avidin-biotin binding assay and solid phase extraction. The binding event of E2 to the antibody is detected indirectly by the competitive reaction between E2 and biotinylated estradiol (BE) as a tracer for the limited binding sites of antibodies immobilized onto the wall of a microtiter plate. Namely, E2 concentrations are determined from the strong interaction between BE and avidin conjugated with horseradish peroxidase (avidin-HRP). In order to achieve a sensitive measurement for the binding of BE to the antibody immobilized on the microtiter plate substrate, QuantaBlu fluorogenic peroxidase substrate (QFPS) was employed. The detection limit and the linear range of E2 determination were 27 pM and 27 - 7480 pM, respectively. The relative standard deviations (RSD) for the E2 assay were between 0.3 and 12.0% (n = 3). The cross-reactivities of several other estrogens in this assay system were also investigated. No serious influences from any cross-reaction caused by other estrogens tested in this experiment were observed. The determination of E2 in water samples from eight rivers and a marsh in Hokkaido was performed by the immunoassay combined with solid phase extraction. It was found that the concentration of E2 was in the range between 0.06 and 67 pM.

Estradiol levels in prepubertal boys and girls--analytical challenges.

Increasing evidence points at an important function of low concentrations of estradiol (E2) in prepubertal boys and girls. E2 serum levels in prepubertal children are, however, often immeasurable in conventional E2 assays. This strongly hampers further investigation of the physiological relevance of E2 in children. In addition, there is an increasing concern of the potential effect of exposure to endocrine disrupters with estrogenic or antiandrogenic activity on pubertal development. A requirement of assessing the instance for this concern, adds further to the demands for applicable methodologies for the evaluation of the sensitivity of the organism to low E2 concentrations. Traditionally, E2 is measured by use of the radioimmunoassay (RIA). As an ultrasensitive alternative to the RIA, a recombinant cell bioassay has been developed. In this review, methodological aspects for these methods of analysis are examined and their applicability for evaluation of low E2 serum concentrations in children is estimated. Furthermore, available data on E2 levels in prepubertal boys and girls are evaluated and discussed, taking into consideration the limitations of the methods of analysis. In conclusion, there is a pronounced demand for new and improved methods of analysis for accurate and sensitive evaluation of low concentrations of E2.

Use of the Architect-i2000 estradiol immunoassay during in vitro fertilization.

In women undergoing in vitro fertilization and embryo transfer (IVF-ET), serial measurements of 17β-estradiol (E2) can be used to monitor follicular growth, and serum E2 has been shown to be correlated with the number and diameter of preovulatory follicles observed on transvaginal ultrasound scan (1)(2)(3)(4)(5)(6). The serum E2 concentration is therefore an essential variable for evaluating the progression of stimulation, adjusting daily gonadotropin therapy, predicting the optimal day for induction of ovulation \[administration of human chorionic gonadotropin (hCG)\] (7), preventing ovarian hyperstimulation syndrome (8)(9), and ensuring that pituitary function is adequately suppressed if a long-acting gonadoliberin-releasing hormone agonist (GnRHa) is used before stimulation (10). Another major use of E2 measurements is to evaluate ovarian function at day 3 of the menstrual cycle to determine the prognosis of IVF-ET (11). Because results must be available within a few hours, rapid and automated assays are required. In this study, we measured E2 with a new automated assay that could be used in random, continuous access mode and assessed its usefulness for monitoring ovulation stimulation for IVF-ET. The Architect-i2000 E2 (Abbott Laboratories) assay is a competitive two-step immunoassay based on chemiluminescent microparticle immunoassay technology and can be performed in 29 min. The ACS-180 E2 assay (Bayer Diagnostics) is a competitive one-step immunoassay based on solid-phase antigen-linked technology and chemiluminescence detection and can be completed in 15 min. The luminescence reaction and the calibration procedure are identical for the two E2 immunoassays. The two methods are linear up to 3670 pmol/L and use monoclonal antibody derived by coupling the E2 molecule at the specificity-enhancing sixth position. The within- and between-run imprecision, detection limits, and functional sensitivities of these assays have been reported …