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Aromatase inhibitors are proving to be more effective than tamoxifen for postmenopausal patients with breast cancer. Estrogen concentrations in the breast are similar in both premenopausal and postmenopausal women, and several fold higher than circulating levels in postmenopausal women. In order to investigate the importance of intratumoral aromatase in stimulating the proliferation of the tumor, we used immunocytochemistry to determine the extent of aromatase expression in relationship to the response of the patient to aromatase inhibitor treatment. The relationship between positive staining for aromatase in the primary tumor and response to treatment with an aromatase inhibitor was investigated in a retrospective study of 102 patients with advanced breast cancer. Immunohistochemical staining using a monoclonal antibody against aromatase was performed on paraffin embedded tumor tissue. Response was evaluated using UICC criteria. Nine out of 13 patients with objective response to treatment stained positive and 49 of 89 patients with stable or progressive disease stained positive. No significant relationship between positive staining and objective response to treatment could be found. When patients with ‘clinical benefit’ (i.e. objective response plus prolonged stable disease of at least 6 months) were considered, also no relationship could be found. Further analysis of subgroups with positive hormone receptors, treatment with newer generation aromatase inhibitors, single metastatic site, non-visceral metastases and previous treatment only with tamoxifen did not show any relationship. Tumor aromatase expression did not correlate with response of patients with advanced breast cancer to aromatase inhibitor treatment. Most patients had relapsed from other treatments before receiving an aromatase inhibitor. It seems likely that many of these patients had tumors that may have progressed to hormone independence at this stage of the disease. Research in patients who have received treatment with aromatase inhibitors in earlier stages of disease (first line and adjuvant treatment) may provide further information on the relationship between tumor aromatase, steroid receptors and response to inhibitor treatment. [Copyright &y& Elsevier]

Abstract: Background: Dehydroepiandrosterone sulphate (DHEAS) is an important marker of the adrenal gland. Its measurement is required in several adrenal diseases, such as adrenal tumours, adrenal insufficiency and congenital adrenal hyperplasia. Most clinical laboratories measure DHEAS using commercially available immunoassays. The aim of the present study was to investigate the accuracy of currently available DHEAS methods. Methods: Seven commercially available DHEAS assays were compared to Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS) by measuring 75 serum samples (concentration range 0.06–20.6μmol/L measured by ID-LC-MS/MS) with each method. Moreover, recovery and linearity experiments were performed. Data from our present study were compared to DHEAS data of the Dutch, German and British External Quality Assessment Schemes (EQAS's). Results: Three methods agreed well with ID-LC-MS/MS (R between 0.93 and 0.99 and slopes ranging from 0.92 to 1.07) and showed good recoveries. Four methods showed standardization problems (slopes were 0.84, 1.14, 1.20 and 1.28). Linearity was good in all methods. Intra-assay coefficient of variation was 4.1% using ID-LC-MS/MS and below 5.5% in immunometric methods; one assay had an unacceptably high intra-assay coefficient of variation of 18%. Our data are in agreement with data obtained in three EQAS's. Conclusion: Some of the commercially available DHEAS methods show standardization problems and/or a high imprecision. These problems may potentially have clinically adverse consequences. We advise the manufacturers to improve their assays and laboratory specialists to scrutinize the DHEAS method they employ. [Copyright &y& Elsevier]

Abstract: Background: Decreased amyloid β (1-42) (Aβ42) and increased (phosphorylated) tau in cerebrospinal fluid (CSF) are considered to be a reflection of plaques, tangles, and neuronal degeneration in Alzheimer''s disease (AD). Atrophy of the medial temporal lobe (MTA) on magnetic resonance imaging (MRI) reflects neuronal loss in this area. Objective: To compare diagnostic accuracy of CSF biomarkers and MTA in AD versus controls. Methods: Aβ42, tau and tau phosphorylated at threonine 181 (Ptau-181) were measured in CSF from 61 AD patients and 32 controls by sandwich enzyme-linked immunosorbent assay. A CSF biomarker profile for AD was constructed. MTA was rated visually on MRI. Results: When AD patients and controls were evaluated separately, no correlations were present between the CSF markers and MTA score. Both MTA and CSF biomarker profile were independently associated with the diagnosis AD (MTA: OR (95% CI)=28 (3–239); CSF biomarker profile: OR (95% CI)=57 (13–262)). Among individuals younger than 65 years old and without MTA 60% suffered AD, and the finding of an abnormal CSF biomarker profile was limited to AD patients only. Conclusions: MTA and CSF biomarkers seem to be of incremental value for the diagnosis AD. CSF analysis is most sensitive in the absence of MTA, and especially among early-onset AD patients. [Copyright &y& Elsevier]

Objective: To investigate the effect of GnRH treatment on estrogen levels and sulfatase activity in leiomyoma and myometrium tissue.Design: Retrospective analyses of tissue obtained in a prospective randomized clinical study.Setting: Gynecology departments of eight hospitals in the Netherlands.Patient(s): Thirty-two patients scheduled for leiomyoma surgery.Intervention(s): Patients were randomized to receive either GnRHa (3.75 mg/4 weeks of triptorelin or 3.6 mg/4 weeks of goserelin) or no GnRHa for 4 months. At subsequent surgery, leiomyoma and myometrium samples were collected.Main Outcome Measure(s): Estrone, estradiol, and sulfatase activity levels in leiomyoma and myometrium.Result(s): In myometrium, levels of estrone, estradiol, and sulfatase activity were significantly lower in the treated group (to median values of 46%, 21%, and 61%, respectively). In leiomyomas of treated patients, the reduction in median estrone level (to 65% of untreated value) was comparable to that in myometrium. The reduction in estradiol level in leiomyoma, however, was significantly less than in myometrium (median to 58% vs. 21%), and no significantly lower sulfatase activity was found.Conclusion(s): Estradiol and sulfatase results show that the effect of GnRHa treatment on leiomyoma differs from the effect on myometrium, suggesting a continuing estrogenic stimulus in leiomyoma tissue despite treatment. [ABSTRACT FROM AUTHOR]

Puberty is highly important for the accumulation of bone mass. Bone turnover and bone mineral density (BMD) can be affected in transgender adolescents when puberty is suppressed by gonadotropin-releasing hormone analogues (GnRHa), followed by treatment with cross-sex hormone therapy (CSHT). We aimed to investigate the effect of GnRHa and CSHT on bone turnover markers (BTMs) and bone mineral apparent density (BMAD) in transgender adolescents. Gender dysphoria was diagnosed based on diagnostic criteria according to the DSM-IV (TR). Thirty four female-to-male persons (transmen) and 22 male-to-female persons (transwomen)were included. Patients were allocated to a young (bone age of < 15 years in transwomen or < 14 in transmen) or old group (bone age of ≥ 15 years in transwomen or ≥ 14 years in transmen). All were treated with GnRHa triptorelin and CSHT was added in incremental doses from the age of 16 years. Transmen received testosterone esters (Sustanon, MSD) and transwomen received 17-β estradiol. P1NP, osteocalcin, ICTP and BMD of lumbar spine (LS) and femoral neck (FN) were measured at three time points. In addition, BMAD and Z-scores were calculated. We found a decrease of P1NP and 1CTP during GnRHa treatment, indicating decreased bone turnover (young transmen 95% CI − 74 to − 50%, p = 0.02, young transwomen 95% CI − 73 to − 43, p = 0.008). The decrease in bone turnover upon GnRHa treatment was accompanied by an unchanged BMAD of FN and LS, whereas BMAD Z-scores of predominantly the LS decreased especially in the young transwomen. Twenty-four months after CSHT the BTMs P1NP and ICTP were even more decreased in all groups except for the old transmen. During CSHT BMAD increased and Z-scores returned towards normal, especially of the LS (young transwomen CI 95% 0.1 to 0.6, p = 0.01, old transwomen 95% CI 0.3 to 0.8, p = 0.04). To conclude, suppressing puberty by GnRHa leads to a decrease of BTMs in both transwomen and transmen transgender adolescents. The increase of BMAD and BMAD Z-scores predominantly in the LS as a result of treatment with CSHT is accompanied by decreasing BTM concentrations after 24 months of CSHT. Therefore, the added value of evaluating BTMs seems to be limited and DXA-scans remain important in follow-up of bone health of transgender adolescents. [ABSTRACT FROM AUTHOR]

Background To assess a patient’s vitamin D status the precursor metabolite 25-hydroxyvitamin D can be determined. However, measurement of 1,25-dihydroxyvitamin D is required when disorders of 1a-hydroxylation, extrarenal 1a-hydroxylation, or vitamin D receptor defects are suspected. Methods The aim of this study was to determine reference values for 1,25-dihydroxyvitamin D 3 and D 2 using a 2D ID-UPLC–MS/MS method. Results The LC–MS/MS method, able to measure picomolar concentrations of both 1,25-dihydroxyvitamin D 3 and D 2 in human serum, was extensively validated. Intra-assay variations were <5% and 8.5% and <7.5% and 11%, for 1,25-dihydroxyvitamin D 3 and D 2 , respectively, over the whole dynamic range (3.1–376 and 3.1–652 pmol/L). Limit of quantitation was 3.4 pmol/L for both compounds. Our method correlated well with a published LC–MS/MS method ( r = 0.87) and with the average 1,25-dihydroxyvitamin D 3 results of the vitamin D External Quality Assessment Scheme (DEQAS) determined with LC–MS/MS ( r = 0.93). Reference ranges, determined in 96 plasma samples of healthy volunteers were 59–159 pmol/L and <17 pmol/L for respectively 1,25-dihydroxyvitamin D 3 and D 2 . The female part of the reference group showed a statistically significant decrease of 1,25-dihydroxyvitamin D 3 concentrations with age. The presence of significantly higher average 1,25-dihydroxyvitamin D 3 levels in premenopausal women taking oral contraceptive pills compared to postmenopausal women suggests that this effect is estrogen-related, as estrogens lead to a higher vitamin D binding protein. Conclusions The major finding of the present study is a reference interval of 59–159 pmol/L for 1,25-dihydroxyvitamin D 3 determined with a highly sensitive and precise LC–MS/MS method. [ABSTRACT FROM AUTHOR]

The measurement of testosterone in saliva is an attractive alternative to serum analysis due to the simple and non-invasive sample collection. In children and adolescents salivary testosterone is mainly measured to investigate whether puberty has started or not. This study aimed to establish reference values for salivary testosterone during puberty in boys and girls. We measured salivary testosterone using ID-LC–MS/MS in a cohort of 131 girls and 123 boys of whom each had salivary testosterone measured at two time points during puberty. Salivary testosterone concentrations start to increase with the start of puberty around eight years and continuously increase up to adult concentrations in the following ten years. Reference values were calculated using the Lambda–Mu–Sigma (LMS)-curve fitting method and provided per year from 8 to 26 years of age in boys and girls. These reference ranges may help clinicians and researchers to interpret salivary testosterone results in both individual patients and study subjects. [ABSTRACT FROM AUTHOR]

Background Liquid-chromatography tandem mass spectrometry (LC–MS/MS) has become the method of choice in steroid hormone measurement. However, little information on the mutual agreement of LC–MS/MS methods is available. We compared eight routine unpublished LC–MS/MS methods for the simultaneous measurement of testosterone and androstenedione. Methods Sixty random serum samples from male and female volunteers were analysed in duplicate by eight routine LC–MS/MS methods. We performed Passing–Bablok regression analyses and calculated Pearson's correlation coefficients to assess the agreement of the methods investigated with one published method known to be accurate. Intra-assay CV of each method was calculated from duplicate results, recoveries for each method were calculated from six spiked samples. Furthermore, a CV between the investigated methods was calculated. Results The concentrations ranged from 0.05–1.26 nmol/L, 6.15–24.44 nmol/L and 0.15–4.78 nmol/L for testosterone in females, testosterone in males and androstenedione, respectively. The intra-assay CVs were between 3.7–16.0%, 0.9–5.2% and 1.2–9.5% for testosterone in females, testosterone in males and androstenedione, respectively. The slopes of the regression lines ranged between 0.90–1.25, 0.87–1.24 and 0.94–1.31 for testosterone concentrations in females, all testosterone values and androstenedione, respectively. Inter-method CVs were 24%, 14% and 29% for testosterone for concentrations in females and males and androstenedione, respectively. These compare unfavourably to the variation found earlier in published methods. Conclusion Although most routine LC–MS/MS methods investigated here showed a reasonable agreement, some of the assays showed a high variation. The observed differences in standardization should be taken into account when applying reference values, or should, preferably, be solved. [ABSTRACT FROM AUTHOR]

Abstract: BRI2 protein binds amyloid precursor protein to halt amyloid-β production and inhibits amyloid-β aggregation via its BRICHOS-domain suggesting a link between BRI2 and Alzheimer's disease (AD). Here, we investigate the possible involvement of BRI2 in human AD pathogenesis. BRI2 containing BRICHOS-domain was increased up to 3-fold in AD hippocampus (p = 0.003, n = 14/group). Immunohistochemistry showed BRI2 deposits associated with amyloid-β plaques in early pathologic stages (Braak-III; Thal-2/3). We observed a decrease in the protein levels of ADAM10 (p = 0.02) and furin (p = 0.066), as well as an increase in SPPL2b (p < 0.0001) in AD hippocampus. Because these enzymes are involved in BRI2 processing, their changes may lead to aberrant processing of BRI2 promoting its deposition and likely affecting BRI2 function. Loss of BRI2 function in AD was supported by the decreased presence of BRI2-amyloid precursor protein complexes in the hippocampus of AD patients compared with control subjects. In conclusion, our data obtained from human samples indicate that in early stages of AD there is an increased deposition of BRI2, which likely leads to impaired BRI2 function thereby influencing AD pathophysiology. [Copyright &y& Elsevier]

Abstract: Total testosterone is considered to be decreased during the use of combined oral contraceptives. There is, however, considerable concern about the quality of testosterone assays, especially at low levels. We aimed to confirm testosterone levels measured by direct radioimmunoassay in a recent clinical trial with a state-of-the-art LC–MSMS method. Surplus specimens with known testosterone levels collected during the study (Clinical Trial Registration number ISRCTN06414473) were reanalyzed with an LC–MSMS method. This method was compared to another LC–MSMS method that had shown to concur excellently to a reference method. Follow-up experiments were designed to explain the results. In contrast to our expectation, LC–MSMS measurements did not corroborate the data obtained by radioimmunoassay. Subsequent experiments showed that this could be attributed to a strong dependency of the radioimmunoassay on SHBG. Testosterone results (n=198) obtained by direct radioimmunoassay showed a negative correlation to SHBG levels (r=−0.676; p<0.001). By contrast, testosterone results obtained by LC–MSMS were not related to SHBG (r=0.100; NS). In conclusion, our results indicate that total testosterone measurements during oral contraceptive use are unreliable when performed with assays sensitive to the SHBG concentration. The discrepancy with the literature can most likely be explained by the sensitivity of the immunoassay used to SHBG. Given the sharp increase in SHBG during the use of many oral contraceptives, total testosterone may not decrease, whereas its bioavailability, estimated by free testosterone levels, will be diminished. Studies aiming at restoration of testosterone homeostasis during oral contraception need to take this into account. [Copyright &y& Elsevier]

Abstract: Objective: To study the effect of different government prenatal screening (PNS) policies on the uptake of PNS and prenatal diagnostic testing (PND) over the periods 2001–2003 (PNS on request), 2004–2006 (permission to offer the first-trimester combined test (FCT) to women of advanced maternal age (AMA), with women aged <36 years informed on explicit request) and 2007–2010 (introduction of population screening) and to evaluate whether trends in uptake are related to maternal age. The indication AMA for PND is still warranted, and the costs for FCT are only reimbursed for AMA women. Study design: Analysis of data on the first- and second-trimester screening program (n =41,600) for Down syndrome (DS) and on PND (n =10,795) performed from 2001 to 2010 in the region North-Holland of the Netherlands. To evaluate the actual participation in PNS and PND in different maternal age groups, estimation of the age distribution of women who underwent a fetal anomaly scan in 2009 (n =14,481) was used as a reference population (participation of 85.2%). Results: The overall uptake of FCT was 35.2% in 2010. Over the years the number of FCT in all age groups increased significantly (P <0.001). Overall the number of PND decreased significantly; the number of PND for AMA decreased and the number of PND for increased risk at FCT (in women <36 and ≥36 years) increased (P <0.05). Since 2004 significantly more DS cases were detected with FCT in AMA women and fewer with PND for AMA, and since 2007 more DS cases were detected with FCT in women <36 years (P <0.001). Conclusion: The effect of the national screening program is limited. Significantly more women opt for PNS but the overall uptake remains low, especially in younger women. A significant number of AMA women still opt for PND for AMA. The choice for FCT and PND for AMA seems dependent on background risk. To accomplish a more effective screening policy, reimbursement of the cost of the test should apply to all women and the indication for PND for AMA should be abolished. [Copyright &y& Elsevier]

Background: Neurofilament (Nf) proteins have been shown to be promising biomarkers for monitoring and predicting disease progression for various neurological diseases. The aim of this study was to evaluate the effects of pre-analytical variables on the concentration of neurofilament heavy (NfH) and neurofilament light (NfL) proteins. Methods: For NfH an in-house newly-developed and validated SinglePlex Luminex assay was used; ELISA was used to analyze NfL. Results: For the NfL ELISA assay, the intra- and inter-assay variation was respectively, 1.5% and 16.7%. Analytical performance of the NfH SinglePlex Luminex assay in terms of sensitivity (6.6pg/mL), recovery in cerebrospinal fluid (CSF) (between 90 and 104%), linearity (from 6.6–1250pg/mL), and inter- and intra-assay variation (<8%) were good. Concentrations of both NfL and NfH appeared not negatively affected by blood contamination, repeated freeze–thaw cycles (up to 4), delayed processing (up to 24hours) and during long-term storage at −20°C, 4°C, and room temperature. A decrease in concentration was observed during storage of both neurofilament proteins up to 21days at 37°C, which was significant by day 5. Conclusions: The newly developed NfH SinglePlex Luminex assay has a good sensitivity and is robust. Moreover, both NfH and NfL are stable under the most prevalent pre-analytical variations. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was to quantitatively assess the relationship between apolipoprotein (APOE) genotype and electroencephalographic oscillatory brain dynamics in Alzheimer's disease (AD) patients and control subjects and its regional distribution. We obtained resting-state electroencephalographs of 320 AD patients and 246 control subjects, categorized into APOE ε4 carriers and noncarriers. Peak frequency and relative power in 4 different frequency bands were calculated. We tested the associations between APOE genotype and relative power in 4 brain regions. Peak frequency was comparable in APOE ε4 carrying and noncarrying control subjects, but lower in APOE ε4 noncarrying AD patients. In control subjects, APOE ε4 carriers had a different regional distribution of alpha power than noncarriers. We found no APOE effect in beta, delta, and theta bands. In AD, APOE ε4 noncarriers had lower alpha and higher delta power than carriers. This difference was most pronounced in the parieto-occipital region. In the theta band, APOE ε4 noncarriers had a different regional distribution of power compared with carriers. In conclusion, the most pronounced effect of genotype was seen in AD patients, and APOE ε4 noncarriers showed slower activity, especially in parieto-occipital regions. [Copyright &y& Elsevier]

Abstract: Background: Testosterone concentrations in normally cycling women are assumed to be elevated around the time of ovulation. The clinical relevance of changing testosterone concentrations during the menstrual cycle, however, is unclear. Poor performance of current direct immunoassays for testosterone at low concentrations confounds this issue. Therefore, our objective was to assess daily testosterone fluctuation during the menstrual cycle by a thoroughly validated isotope dilution-liquid chromatography–tandem mass spectrometry (ID-LC–MS/MS) method and to evaluate whether an ARCHITECT® 2nd Generation Testosterone fully automated immunoassay is equally suited for this purpose. Methods: Testosterone was measured in serum obtained daily during the menstrual cycle of 25 healthy women, characterized by biochemical and physical examination. Results: Performance of the ID-LC–MS/MS method was concordant with a published reference method (y =1.007x −0.056nmol/L; r =0.9998). Comparison of the immunoassay to ID-LC–MS/MS yielded y =1.095x +0.104nmol/L (r =0.9031). Overall, testosterone concentrations were higher mid-cycle, but a peak was not discernible in each individual. Apart from a persistent positive bias, the immunoassay measured the same testosterone profiles as the ID-LC–MS/MS method. The reference interval in women was 0.30–1.69nmol/L (8.7–48.7ng/dL) for ID-LC–MS/MS and 0.50–2.00nmol/L (14.4–57.7ng/dL) for the immunoassay. Conclusion: The elevation of mid-cycle testosterone concentrations is statistically significant, although not clinically relevant since day-to-day variation is higher and independent of the menstrual cycle. In this light, a single testosterone measurement might not be reflective of the overall testosterone status in an individual. Measurements obtained using the 2nd generation immunoassay gave comparable results across the menstrual cycle. [Copyright &y& Elsevier]

Abstract: Introduction: In our hospital, female-to-male (FtM) transgender adolescents from the age of 16 are treated with two- or four-weekly intra-muscular injections of testosterone-esters. Some patients treated with four-weekly injections have complaints of fatigue and experience mood swings towards the end of the inter-injection period, which calls for an evaluation of the time-course of testosterone levels between injections. Evaluation of salivary testosterone is a practical approach for sequential measurements. Since only ∼2% of total serum testosterone is present in saliva, a sensitive assay is necessary. The objective was to develop an isotope dilution-liquid chromatography–tandem mass spectrometry method (ID-LC–MS/MS) for salivary testosterone measurements and to evaluate the testosterone profiles after testosterone-ester mixture injections in FtM-adolescents. Experimental: FtM treated with 125mg/2weeks or with 250mg/4weeks depots of testosterone-ester mixture collected saliva at different time intervals. Salivary testosterone was measured by a thoroughly validated ID-LC–MS/MS assay. Results: An ID-LC–MS/MS method for measuring salivary testosterone was developed with adequate accuracy and specificity. The reference range was established at 135–400pmol/L. Testosterone levels peaked supra-physiologically immediately post-injection, and decreased to levels within the male reference range after nine days in all patients. 250mg/4weeks depots resulted in values below the reference range at the end of the 4weeks. Discussion: The development of an adequate ID-LC–MS/MS method for measuring salivary testosterone allowed us to investigate the testosterone profile in FtM-adolescents after testosterone-esters mixture injections. These injections lead to extreme concentrations which may affect the wellbeing of the patients. [Copyright &y& Elsevier]

Abstract: In this longitudinal study we investigated change over time in cerebrospinal fluid (CSF) levels of amyloid-beta 40 and 42 (Aβ40 and Aβ42), total tau (tau), tau phosphorylated at threonine 181 (ptau-181), isoprostane, neurofilaments heavy (NfH) and light (NfL). Twenty-four nondemented subjects, 62 mild cognitive impairment (MCI) and 68 Alzheimer''s disease (AD) patients underwent 2 lumbar punctures, with minimum interval of 6, and a mean ± SD of 24 ± 13 months. Linear mixed models were used to assess change over time. Amyloid-beta 42, tau, and tau phosphorylated at threonine 181, differentiated between diagnosis groups (p < 0.05), whereas isoprostane, neurofilaments heavy, and NfL did not. In contrast, effects of follow-up time were only found for nonspecific CSF biomarkers: levels of NfL decreased, and levels of isoprostane, amyloid-beta 40, and tau increased over time (p < 0.05). Isoprostane showed the largest increase. In addition, increase in isoprostane was associated with progression of mild cognitive impairment to AD, and with cognitive decline as reflected by change in Mini Mental State Examination (MMSE). Contrary to AD-specific markers, nonspecific CSF biomarkers, most notably isoprostane, showed change over time. These markers could potentially be used to monitor disease progression in AD. [Copyright &y& Elsevier]

Abstract: Objective: To study CSF biomarkers amyloid-beta 1–42 (Aβ42) and total tau (tau) in relation to APOE genotype in their ability to predict progression from mild cognitive impairment (MCI) to Alzheimer''s disease (AD). Methods: In 100 MCI patients CSF Aβ42, tau and APOE genotype were determined. At follow-up of 18 (13–24) months 58 patients remained non-progressive and 42 progressed to AD. Results: Cox proportional hazards models showed an interaction between Aβ42 and APOE genotype (p <0.05). Stratification for APOE revealed HR (95% CI) for abnormal Aβ42 of 8.2 (2.1–31.9) for ε4 non-carriers, 3.9 (0.8–18.5) for heterozygotes and 0.3 (0.0–1.7) for homozygotes. Inversely, stratification for Aβ42 revealed that in patients with normal levels of Aβ42, ε4 homozygotes had a strongly increased risk of progression to AD with HR (95% CI) 20.8 (2.4–182.8). Tau and APOE independently predicted progression to AD. Conclusions: Aβ42 was a stronger predictor of progression to AD in APOE ε4 non-carriers than in carriers. Furthermore, the risk of progression for ε4 homozygotes was very high, also in patients with normal levels of Aβ42. [Copyright &y& Elsevier]

Objective: To determine the precise degree of variability that is represented by the reproducibility and reliability of semen analysis. The general assumption is that semen analyses need to be repeated because of a high degree of within-individual variability. However, the precise degree of variability is not well established in male partners of subfertile couples. Design: Retrospective cohort study. Setting: Two university hospitals in the Netherlands, which routinely perform two semen analyses in the male partner of subfertile couples. Patient(s): Male partners of subfertile couples. Interventions: None. Main Outcome Measure(s): We assessed the test-retest reproducibility, by calculating the coefficient of variation (CVw) for five semen parameters. The CVw expresses, on a relative scale, the degree of closeness of repeated measurements taken in the same subject. We also estimated the reliability of these semen parameters, in terms of the intraclass correlation coefficient, which expresses the ratio of the between-subject variability over the total variability. Result(s): We analyzed the data of 5,240 men and found that the CVw of all semen parameters ranged from 28% to 34%. The intraclass correlation coefficients of these semen parameters were moderate to high: volume: 0.70; concentration: 0.89; motility: 0.58; morphology: 0.60; total motile count: 0.73. Conclusion(s): This study affirmed the presumed large within-subject variability and the limited reproducibility of semen analyses in subfertile men. Whether this degree of variability within men justifies one or more repetitions of the semen analysis in view of consequences for clinical management should be the topic of future studies. Until then it seems reasonable to perform two semen analyses. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: To assess associations between cerebrospinal fluid (CSF) biomarker levels and MRI-based whole-brain atrophy rate in mild cognitive impairment (MCI) and Alzheimer''s disease (AD). Methods: We included 99 patients (47 AD, 29 MCI, 23 controls) who underwent lumbar puncture at baseline and repeat MRI. A subgroup of 48 patients underwent a second lumbar puncture. CSF levels of beta-amyloid1–42 (Aβ1–42), tau and tau phosphorylated at threonine-181 (P-tau181), and whole-brain atrophy rate were measured. Results: Across groups, baseline Aβ1–42 and tau were modestly associated with whole-brain atrophy rate. Adjusted for age, sex and diagnosis, we found no association between Aβ1–42 or tau, and whole-brain atrophy rate. By contrast, high CSF levels of P-tau181 showed a mild association with a lower whole-brain atrophy rate in AD but not in controls or MCI patients. Finally, whole-brain atrophy rate was associated with change in MMSE, but change in CSF biomarker levels was not. Conclusions: Whole-brain atrophy rate and CSF levels of Aβ1–42, tau or P-tau181 provide complementary information in patients with MCI and AD. [Copyright &y& Elsevier]

Abstract: Background: Neurofilament proteins (Nf) are highly specific biomarkers for neuronal death and axonal degeneration. As these markers become more widely used, an inter-laboratory validation study is required to identify assay criteria for high quality performance. Methods: The UmanDiagnostics NF-light ®enzyme-linked immunoabsorbent assays (ELISA) for the neurofilament light chain (NfL, 68kDa) was used to test the intra-assay and inter-laboratory coefficient of variation (CV) between 35 laboratories worldwide on 15 cerebrospinal fluid (CSF) samples. Critical factors, such as sample transport and storage, analytical delays, reaction temperature and time, the laboratories'' accuracy and preparation of standards were documented and used for the statistical analyses. Results: The intra-laboratory CV averaged 3.3% and the inter-laboratory CV 59%. The results from the test laboratories correlated with those from the reference laboratory (R =0.60, p <0.0001). Correcting for critical factors improved the strength of the correlation. Differences in the accuracy of standard preparation were identified as the most critical factor. Correcting for the error introduced by variation in the protein standards improved the correlation to R =0.98, p <0.0001 with an averaged inter-laboratory CV of 14%. The corrected overall inter-rater agreement was subtantial (0.6) according to Fleiss'' multi-rater kappa and Gwet''s AC1 statistics. Conclusion: This multi-center validation study identified the lack of preparation of accurate and consistent protein standards as the main reason for a poor inter-laboratory CV. This issue is also relevant to other protein biomarkers based on this type of assay and will need to be solved in order to achieve an acceptable level of analytical accuracy. The raw data of this study is available online. [Copyright &y& Elsevier]

Abstract: Objective: To compare CSF levels of beta-amyloid 1–42 (Aβ1–42), total tau (tau) and tau phosphorylated at threonine 181 (ptau-181) between AD patients and controls according to age. Methods: 248 AD patients (48% men) and 127 controls (51% men, 22 volunteers and 105 subjective complainers) underwent lumbar puncture. Both patients and controls were divided into a young (<65 years) and old (≥65 years) group. Results: All three biomarkers showed main effects of diagnosis (p <0.001). There was an interaction between diagnosis and age for all three biomarkers (p <0.05), as old controls had lower Aβ1–42 and higher (p)tau than young controls (Aβ1–42 699±250 versus 866±191pg/ml, tau 408±245 versus 243±102pg/ml, ptau-181 60±28 versus 42±15pg/ml), but there was no difference according to age among AD patients (Aβ1–42 451±178 versus 425±146pg/ml, tau 741±460 versus 798±467pg/ml, ptau-181 91±42 versus 91±41pg/ml). Conclusion: We found that the older control group had lower Aβ1–42 and higher (p)tau compared to the younger control group. This suggests that older individuals may have AD pathology, even in the absence of objective cognitive impairment. [Copyright &y& Elsevier]

Abstract: Objective: The aim of this study was to demonstrate the involvement of the inflammatory proteins IL-6, ACT and CRP early in the pathology process of AD in patients with mild cognitive impairment (MCI) and AD. Methods: IL-6, ACT, CRP, Aβ42, phospho-tau (p-tau) and total tau concentrations in serum and CSF of 145 patients with probable AD and 67 patients with MCI were measured by sandwich ELISA. MCI patients were characterized as high- respectively low-risk MCI according to their Aβ42/tau risk profile. Results: CSF and serum CRP levels were significantly higher in MCI compared to AD patients after adjustment for age, ApoE ɛ4 genotype and cardiovascular diseases (p <0.01). This difference remained present in patients with a low-risk biomarker profile for AD after adjustment for abovementioned covariates. CSF IL-6 levels were also significantly higher in MCI patients with a low-risk CSF profile. Conclusions: These findings suggest that inflammatory processes might be involved in early stages of AD, even before Aβ and tau changes are present in CSF of MCI patients. [Copyright &y& Elsevier]

Abstract: Background: Truncated forms and full-length forms of the amyloid-beta 40 (Aβ40) are key molecules in the pathogenesis of dementia, and are detectable in CSF. Reliable methods to detect these biomarkers in CSF are of great importance for understanding the disease mechanisms and for diagnostic purposes. Methods: VU-α-Aβ40, a monoclonal antibody (mAb) specifically detecting Aβ40, was generated and characterized by solid and fluid phase ELISA, surface plasmon resonance spectroscopy (SPRS), immunoprecipitation (IP), immunohistochemical and Western blot (WB) analysis. In addition, an ELISA with VU-α-Aβ40 as catching and 6E10 as detecting mAbs was set up and validated. This ELISA was used to measure Aβ40 in CSF of controls (N =27), patients with Alzheimer''s disease (AD; N =20), frontotemporal lobe dementia (FTLD; N =14), noninflammatory (N =15) and inflammatory (N =15) neurological conditions. Results: VU-α-Aβ40 specifically recognizes Aβ40 with high affinity (K A =1.3×109 M−1) and detects Aβ40 in AD brain specimens. The developed sandwich ELISA has a detection limit of 0.21 ng/mL, a mean recovery of 90%, and an intra- and inter-assay CV of 1.4% and 7.3%. FTLD patients had a lower mean level of Aβ40 (8.8 (1.9) ng/mL) than controls (12.0 (1.7) ng/mL); p <0.01). Conclusions: VU-α-Aβ40 was successfully implemented in an ELISA which enables us to measure Aβ40 accurately in human CSF. Clinical validation revealed lower levels of Aβ40 in FTLD patients. This finding opens new possibilities for early and differential diagnosis of dementia. [Copyright &y& Elsevier]

Abstract: Objective: We examined the relation between Apolipoprotein E ε4 allele (ApoE ε4) genotype and functional connectivity measured by Electroencephalography (EEG) in patients with Alzheimer’s disease (AD) and patients with subjective complaints (SC). Methods: We included 43 patients with AD (age (SD)=74.2 (4.0), m/f=22/21; 30 of ApoE ε4 carriers) and 21 patients with SC (age (SD)=73.2 (5.2), m/f=13/8; 7 ApoE ε4 carriers) for this study. Resting state EEGs were recorded in all subjects. Synchronisation likelihood (SL) between local cortical areas was compared in the alpha and beta band according to ApoE ε4 status and diagnosis. Results: ApoE ε4 carriers had higher SL values in lower and upper alpha band, in both diagnostic groups. In upper alpha band and beta band AD patients had lower SL than patients with SC, was irrespective of ApoE status. Conclusion: The effects of AD and ApoE ε4 on functional connectivity are opposite and independent. Significance: The observed increase in SL in both AD and patients with SC carrying ApoE ε4 suggests a strong genetic impact of ApoE ε4 on brain function. [Copyright &y& Elsevier]

The αE-catenin is a well-known invasion suppressor. A recently described novel α-catenin, i.e. αT-catenin (CTNNA3), shows related functions being necessary for the formation of cell–cell adhesion complexes. We recently demonstrated that the 10q21.3 region containing the CTNNA3 gene shows a parent-of-origin effect and that transcription of the CTNNA3 gene is downregulated in placental tissues of complete androgenetic origin. As this suggests that the CTNNA3 gene is subject to imprinting, we performed allele-specific RT-PCR on early placenta tissues using informative heterozygous samples. This was supplemented by immunostaining for αT-catenin, p57KIP2 and low molecular weight cytokeratin in tissues of a partial hydatidiform mole. As shown here we demonstrate that the CTNNA3 gene is subject to imprinting with preferential expression of the maternal allele in first trimester placental tissues. Imprinting, however, is trophoblast cell type-dependent: expression in extravillus trophoblast is biallelic; expression in villus cytotrophoblast is from the maternal allele only. Expression of αT-catenin is lost in villus syncytiotrophoblast as well as in extravillus trophoblast following epithelial–mesenchymal transition. The trophoblast cell type-dependent imprinting of CTNNA3 is identical to p57KIP2 imprinting with respect to trophoblast cell type (villus) and parental origin of the expressed allele (maternal). This suggests that gene dosage compensation of CTNNA3 and p57KIP2 in the placenta shares a conserved regulatory mechanism that correlates with an early step in trophoblast determination, i.e. differentiation into villus or extravillus trophoblast. [Copyright &y& Elsevier]

Objective: To study the association between hepatocyte growth factor (HGF) levels and pregnancy outcome. Study design: Hepatocyte growth factor levels were measured in 42 plasma samples between weeks 14 and 21 of gestation using an enzyme-linked immunosorbent assay (ELISA). Results were correlated to pregnancy outcome and Mann–Whitney U-test applied to study the differences. Results: Hepatocyte growth factor values in pregnancies that develop preeclampsia (n=12) were not significantly different from unaffected pregnancies (n=21, multiples of the median (MoM)=1.38, P=0.47). However, hepatocyte growth factor values were significantly elevated in pregnancies of small-for-gestational age (SGA) fetuses (n=9) compared to uncomplicated pregnancies (MoM=2.66, P<0.001). Conclusion: Measurement of hepatocyte growth factor in peripheral blood between 14 and 21 weeks gestation may offer new possibilities in the early diagnosis and prediction of fetal birth weight but not of preeclampsia. [Copyright &y& Elsevier]

The adrenal and gonadal androgens, testosterone, androstenedione and dehydroepiandrosterone (DHEA) play an important role in sexual development as well as in other processes. We developed a method for simultaneous quantitative analysis of serum and plasma testosterone, androstenedione and DHEA levels using Isotope-Dilution Liquid-Chromatography Tandem Mass Spectrometry (ID-LC–MS/MS). Samples underwent liquid–liquid extraction and were analyzed on an Acquity 2D-UPLC-System and a Xevo TQ-S tandem mass spectrometer (Waters). The intra-assay and inter-assay coefficients of variation were < 4.0%, < 6.3% and < 7.0% and < 6.0%, < 8.1% and < 7.7% for testosterone, androstenedione and DHEA, respectively. Inter-assay CVs at the lower limit were 10.6%, 16.9% and 9.0% for testosterone (0.10 nmol/L), androstenedione (0.10 nmol/L) and DHEA (1.0 nmol/L), respectively. Recoveries of spiked analytes were 93–107%. The present testosterone method compared well (y = 1.00x − 0.04; r = 0.998) to a published ID-LC–MS/MS method for testosterone in our lab. The latter method being concordant with a published reference method (Bui et al., 2013). The present method compared well to a published ID-LC–MS/MS method (Kushnir et al., 2010) (y = 1.06x − 0.06; r = 0.996 for testosterone; y = 1.04x − 0.04; r = 0.995 for androstenedione and y = 1.03x + 0.01; r = 0.991 for DHEA). In conclusion, we developed a sensitive and accurate ID-LC–MS/MS method to simultaneously measure serum testosterone, androstenedione and DHEA in serum and plasma. [ABSTRACT FROM AUTHOR]

Abstract: Cerebral microbleeds (MBs) may relate to amyloid in dementia. We selected 26 probable Alzheimer''s disease (AD) patients with MBs, 26 age- and sex-matched AD patients without MBs, 11 vascular dementia (VaD) patients, and 22 patients with subjective complaints. We measured amyloid beta 1–42 (Aβ42) and 1–40 (Aβ40) in cerebrospinal fluid (CSF) and plasma, and blood-brain barrier (BBB) function using albumin ratios. CSF Aβ42 was lowest in AD with MBs, whereas Aβ40 was selectively decreased in VaD. In plasma, amyloid-beta was nonsignificantly elevated in VaD compared with controls. Higher albumin ratios in VaD suggested blood-brain barrier dysfunction. A MB pattern suggestive of cerebral amyloid angiopathy (CAA) related to lower CSF Aβ42, while a non-cerebral amyloid angiopathy specific MB distribution related to higher plasma Aβ40. Amyloid-beta is differentially implicated in AD with MBs and VaD. MB distribution related to different amyloid profiles, supporting distinct etiologies. Our results suggest that Aβ42 is retained in cerebrovasculature of AD patients with MBs, while in contrast, VaD patients may possibly drain amyloid. [Copyright &y& Elsevier]