Phenol and Phthalate Effects on Thyroid Hormone Levels during Pregnancy: Relying on In Vitro Assays and Adverse Outcome Pathways to Inform an Epidemiological Analysis.

BACKGROUND: Studies characterizing associations between phenols, phthalates and thyroid hormones during pregnancy produce inconsistent results. This divergence may be partly attributable to false positives due to multiple comparison testing of large numbers of chemicals, and measurement error as studies rely on small numbers of biospecimens despite high intra-individual variability in urinary chemical metabolite concentrations. OBJECTIVES: This study employs a priori chemical filtering and expanded urinary biomonitoring to evaluate associations between phenol/phthalate exposures and serum thyroid hormones assessed during pregnancy. METHODS: A two-tiered approach was implemented: a) In vitro high-throughput screening results from the ToxCast/Tox21 database, as informed by a thyroid Adverse Outcome Pathway network, were evaluated to select phenols/phthalates with activity on known and putative molecular initiating events in the thyroid pathway; and b) Adjusted linear regressions were used to study associations between filtered compounds and serum thyroid hormones measured in 437 pregnant women recruited in Grenoble area (France) between 2014 and 2017. Phenol/phthalate metabolites were measured in repeated spot urine sample pools (median: 21 samples/women). RESULTS: The ToxCast/Tox21 screening reduced the chemical set from 16 to 13 and the associated number of statistical comparisons by 19%. Parabens were negatively associated with free triiodothyronine (T3) and the T3/T4 (total thyroxine) ratio. Monobenzyl phthalate was positively associated with total T4 and negatively with the T3/T4 ratio. Effect modification by iodine status was detected for several compounds (among them ΣDEHP and mono-n-butyl phthalate) that were associated with some hormones among women with normal iodine levels. CONCLUSION: For these chemicals, screening for compounds with an increased likelihood for thyroid-related effects and relying on repeated urine samples to assess exposures improved the overall performance of multichemical analyses of thyroid disruption. This approach may improve future evaluations of human data for the thyroid pathway with implication for fetal health and may serve as a model for evaluating other toxicity outcomes. https://doi.org/10.1289/EHP10239.

Analytical validation of eight methods of thyroglobulin measurement in fine-needle aspiration washouts.

BACKGROUND: Thyroglobulin (Tg) assay in washout fluids of fine needles, after cervical lymph nodes aspiration, is used for detecting metastases from differentiated thyroid carcinomas. Assay methods are the same as for Tg in serum. However, with non-serum samples, methods require extensive validation to notably check for the absence of matrix effect. This study fits this context. Our objectives were to assess analytic performances, in washout fluid, of eight different Tg assay methods and to compare them to validated data in serum. METHODS: Eleven medical laboratories participated in this study. The matrix tested was phosphate-buffer saline containing 1% bovine serum albumin (PBS-1% BSA). Samples used were dilutions, in this buffer, of Certified Reference Material (CRM 457). We verified, for all methods, the limit of detection, precision, linearity, trueness and accuracy. RESULTS: In PBS-1% BSA, the functional sensitivities (FS) were comparable to those expected for serum. All the methods were linear. The relative biases of trueness were between -24.5 and 10.2% around 1 µg/L. Total analytical error was ≤40% near the functional sensitivity values. CONCLUSION: No quantitatively important matrix effect was observed. All the methods showed their ability to measure Tg in PBS-1% BSA, over the concentration range of interest, with acceptable total analytical error. We validated the functional sensitivity value as a decision threshold in thyroidectomized patients after treatment and with low concentrations of serum Tg.

Improved human islets' viability and functionality with mesenchymal stem cells and arg-gly-asp tripeptides supplementation of alginate micro-encapsulated islets in vitro.

INTRODUCTION: The extension of islet transplantation to a wider number of type 1 diabetes patients is compromised by severe adverse events related to the immunosuppressant therapy required for allogenic islet transplantation. In this context, microencapsulation offers the prospects of immunosuppressive-free therapy by physically isolating islets from the immune system. However, current biomaterials need to be optimized to: improve biocompatibility, guaranty the maintenance of graft viability and functionality, and prevent fibrosis overgrowth around the capsule in vivo. Accumulating evidence suggest that mesenchymal stem cells (MSCs) and anchor points consisting of tripeptides arg-gly-asp (RGD) have cytoprotective effects on pancreatic islets. Here, we investigated the effect of supplementing reference M-rich alginate microcapsules with MSCs and RGD-G rich alginate on bioprocessing as well as on human pancreatic islets viability and functionality. METHODS: We characterized the microcapsules components, and then for the new microcapsule composite product: we analyzed the empty capsules biocompatibility and then investigated the benefits of MSCs and RGD-G rich alginate on viability and functionality on the encapsulated human pancreatic islets in vitro. We performed viability tests by confocal microscopy and glucose stimulated insulin secretion (GSIS) test in vitro to assess the functionality of naked and encapsulated islets. RESULTS: Encapsulation in reference M-rich alginate capsules induced a reduction in viability and functionality compared to naked islets. This side-effect of encapsulation was in part counteracted by the presence of MSCs but the restoration was complete with the combination of both MSCs and the RGD-G rich alginate. CONCLUSIONS: The present findings show that bioprocessing a favorable composite environment inside the M-rich alginate capsule with both MSCs and RGD-G rich alginate improves human islets survival and functionality in vitro.

Interoceptive Abilities in Inflammatory Bowel Diseases and Irritable Bowel Syndrome.

Alexithymia is usually described by three main dimensions difficulty identifying feelings (DIF), difficulty describing feelings (DDF), and externally oriented thinking (EOT). The most commonly used questionnaire investigating alexithymia, the Toronto Alexithymia Scale (TAS-20), supports this three-factor structure. One important assumption is that alexithymia severity is associated to vulnerability to somatic diseases, among them gastrointestinal disorders. However, the association between alexithymia and gastrointestinal disorders is not systematic, thus questioning the role of alexithymia as a vulnerability factor for those illnesses. A recent factor analysis suggested another four-factor structure for the TAS-20: difficulties in awareness of feelings (DAF), difficulties in interoceptive abilities (DIA), externally oriented thinking (EOT), and poor affective sharing (PAS). We assume that DIA and DAF might be more relevant to investigate the association between alexithymia and gastrointestinal disorders. The rationale is that DIA and DAF reflect impairments in emotion regulation that could contribute to an inappropriate autonomic and HPA axis homeostasis in irritable bowel syndrome (IBS), ulcerative colitis (UC), or Crohn's disease (CD). The aim of this study was to investigate whether DIA and DAF are associated with the presence of IBS, UC or CD, while checking for anxiety, depression, parasympathetic (vagus nerve) activity and cortisol levels. We recruited control participants (n=26), and patients in remission who were diagnosed with IBS (n=24), UC (n=18), or CD (n=21). Participants completed questionnaires to assess anxiety, depression, and alexithymia. A blood sample and an electrocardiogram were used to measure the level of cortisol and parasympathetic activity, respectively. Logistic regressions with the four-factor structure of the TAS-20 revealed that DIA was a significant predictor of IBS (W(1)=6.27, p=.01). Conversely, DIA and DAF were not significant predictors in CD and UC patients. However, low cortisol level was a significant predictor of UC (W(1)=4.67, p=.035). Additional logistic regressions based on the original 3-factor structure of TAS-20 (DIF, DDF, and EOT) showed that only DDF was a significant predictor of CD [W(1)=6.16, p < .001]. The present study suggests that DIA is an important dimension for assessing potential risk for gastrointestinal diseases, in particular for IBS.

Subclinical Hypothyroidism: is it Really Subclinical with Aging?

No recent study has focused on clinical features of subclinical hypothyroidism (SCH), especially in older patients. TSH measurement has remarkably evolved these last 20 years and thus reconsideration is needed. In our prospective multicenter study (2012-2014) including 807 subjects aged <60 years (<60y) and 531 subjects ≥60 years (≥60y), we have monitored 11 hypothyroidism-related clinical signs (hCS) together with TSH, FT4, FT3 and anti-thyroperoxidase antibodies values. hCS expression has been compared in patients with SCH vs euthyroidism in each age group. The number of hCS above 60y of age were found to be more elevated in the euthyroid population (1.9 vs 1.6, p<0.01) than in the SCH population (2.3 vs 2.6, p=0.41) while increase in hCS is limited to SCH subjects in the <60y group (p<0.01). The percentage of subjects with at least 3 signs increased with SCH in the <60y group (42.6% vs 25.0%, p<0.01) but not ≥60y (34.4% vs 33.9%, p=0.96). In older individuals, only three hCS could be related to both SCH and a decreased T3/T4-ratio (0.26 vs 0.27, p<0.01), suggesting either a reduced activity of TSH, or an adaptive response with aging. While hCS are clearly associated with SCH in patients <60y, they are not so informative in older subjects. TSH measurements carried out on the basis of hCS need to be interpreted with caution in aged patients. A reassessment of the TSH reference range in older patients is clearly needed and should be associated to more appropriate monitoring of thyroid dysfunction.

Why We Should Still Treat by Neurosurgery Patients With Cushing Disease and a Normal or Inconclusive Pituitary MRI.

CONTEXT: In patients with Cushing disease (CD) and a typical image of adenoma at MRI, transsphenoidal surgery is consensual. However, when MRI is inconclusive or normal, some authors now advocate medical treatment instead. The implicit assumption is that modern MRI should miss only very small microadenomas that are too difficult to visualize at surgery. OBJECTIVE: To analyze the evolution with time of the performances of MRI and the outcomes of surgery in patients with CD with a typical image of adenoma vs an inconclusive or normal MRI. DESIGN AND PATIENTS: Retrospective single center study of 195 patients with CD treated by transsphenoidal surgery between 1992 and 2018, using first a translabial microscopic and then a transnasal endoscopic approach. Patients with inconclusive or normal MRI were explored by bilateral inferior petrosal sinus sampling. Four MRI groups were defined: microadenomas (n = 89), macroadenomas (n = 18), or MRI either inconclusive (n = 44) or normal (n = 44). RESULTS: The proportion of inconclusive/normal MRI decreased with time, from 60% (21/35) in 1992 to 1996 to 27% (14/51) in 2012 to 2018 (P = 0.037). In the four MRI groups, the per-operatory adenoma visualization rate was only slightly lower when MRI was normal (95%, 100%, 86%, 79%; P = 0.012) and postoperative remission rates were not different (85%, 94%, 73%, 75%; P = 0.11). CONCLUSION: The diagnostic performances of MRI have improved but remain inferior to the eye of an expert neurosurgeon, best assisted by endoscopy. We propose that patients with CD and an inconclusive/normal MRI be addressed by an expert neurosurgeon for transsphenoidal surgery rather than being treated medically.

Human mesenchymal stem cells improve rat islet functionality under cytokine stress with combined upregulation of heme oxygenase-1 and ferritin.

BACKGROUND: Islets of Langerhans transplantation is a promising therapy for type 1 diabetes mellitus, but this technique is compromised by transplantation stresses including inflammation. In other tissues, co-transplantation with mesenchymal stem cells has been shown to reduce damage by improving anti-inflammatory and anti-oxidant defences. Therefore, we probed the protection afforded by bone marrow mesenchymal stem cells to islets under pro-inflammatory cytokine stress. METHODS: In order to evaluate the cytoprotective potential of mesenchymal stem cells on rat islets, co-cultures were exposed to the interleukin-1, tumour necrosis factor α and interferon γ cocktail for 24 h. Islet viability and functionality tests were performed. Reactive oxygen species and malondialdehyde were measured. Expression of stress-inducible genes acting as anti-oxidants and detoxifiers, such as superoxide dismutases 1 and 2, NAD(P)H quinone oxidoreductase 1, heme oxygenase-1 and ferritin H, was compared to non-stressed cells, and the corresponding proteins were measured. Data were analysed by a two-way ANOVA followed by a Holm-Sidak post hoc analysis. RESULTS: Exposure of rat islets to cytokines induces a reduction in islet viability and functionality concomitant with an oxidative status shift with an increase of cytosolic ROS production. Mesenchymal stem cells did not significantly increase rat islet viability under exposure to cytokines but protected islets from the loss of insulin secretion. A drastic reduction of the antioxidant factors heme oxygenase-1 and ferritin H protein levels was observed in islets exposed to the cytokine cocktail with a prevention of this effect by the presence of mesenchymal stem cells. CONCLUSIONS: Our data evidenced that MSCs are able to preserve islet insulin secretion through a modulation of the oxidative imbalance mediated by heme and iron via heme oxygenase-1 and ferritin in a context of cytokine exposure.

Prognostic Biomarkers Used for Localised Prostate Cancer Management: A Systematic Review.

CONTEXT: Prostate cancer stratification is based on tumour size, pretreatment PSA level, and Gleason score, but it remains imperfect. Current research focuses on the discovery and validation of novel prognostic biomarkers to improve the identification of patients at risk of aggressive cancer or of tumour relapse. OBJECTIVE: This systematic review by the Intergroupe Coopérateur Francophone de Recherche en Onco-urologie (ICFuro) analysed new evidence on the analytical validity and clinical validity and utility of six prognostic biomarkers (PHI, 4Kscore, MiPS, GPS, Prolaris, Decipher). EVIDENCE ACQUISITION: All available data for the six biomarkers published between January 2002 and April 2015 were systematically searched and reviewed. The main endpoints were aggressive prostate cancer prediction, additional value compared to classical prognostic parameters, and clinical benefit for patients with localised prostate cancer. EVIDENCE SYNTHESIS: The preanalytical and analytical validations were heterogeneous for all tests and often not adequate for the molecular signatures. Each biomarker was studied for specific indications (candidates for a first or second biopsy, and potential candidates for active surveillance, radical prostatectomy, or adjuvant treatment) for which the level of evidence (LOE) was variable. PHI and 4Kscore were the biomarkers with the highest LOE for discriminating aggressive and indolent tumours in different indications. CONCLUSIONS: Blood biomarkers (PHI and 4Kscore) have the highest LOE for the prediction of more aggressive prostate cancer and could help clinicians to manage patients with localised prostate cancer. The other biomarkers show a potential prognostic value; however, they should be evaluated in additional studies to confirm their clinical validity. PATIENT SUMMARY: We reviewed studies assessing the value of six prognostic biomarkers for prostate cancer. On the basis of the available evidence, some biomarkers could help in discriminating between aggressive and non-aggressive tumours with an additional value compared to the prognostic parameters currently used by clinicians.

Variability among TSH Measurements Can Be Reduced by Combining a Glycoengineered Calibrator to Epitope-Defined Immunoassays.

OBJECTIVES: Measuring protein markers with variable glycosylation, such as thyroid-stimulating hormone (TSH), with high accuracy is not an easy task. Despite highly sensitive third-generation tests, discrepancies among TSH assays still remain unsolved and are the focus of important standardization efforts. Earlier work from our group showed that a lack of similarity in epitope expression between standards and samples may account for discordant hormone measurements. In this study, we aimed at producing a glycoengineered TSH with serum-type glycosylation and compared its immunological behavior to that of the international standards. STUDY DESIGN: Recombinant glycoengineered TSH (rgTSH) was produced in glycoengineered Chinese hamster ovary cells to express a highly sialylated TSH and tested in newly designed assays. Two groups of assays targeting defined epitopes were constructed and TSH levels were estimated in a panel of 84 clinical samples (2.1-22.4 mIU/l) based on the use of the current 3rd IS 81/565, the 1st IRP 94/674 and rgTSH calibrations. RESULTS: Calibration based on rgTSH was found to significantly reduce the percentage difference means of assays compared to the pituitary standard. We also found that a switch from a mIU/l (3rd IS 81/565) to ng/l (rgTSH) basis can be established within the normal as well as in the mid to upper normal range of TSH levels. Of interest, TSH assays targeting the main immunogenic region displayed variable TSH values, indicating that, in this region, epitopes should be defined for assays to deliver similar values. CONCLUSIONS: A glycoengineered TSH with serum-type glycosylation proved to be a new calibrator efficient in harmonizing TSH values.

Thyroglobulin assay in fluids from lymph node fine needle-aspiration washout: influence of pre-analytical conditions.

The aim of this study was to evaluate the pre-analytical factors contributing to uncertainty in thyroglobulin measurement in fluids from fine-needle aspiration (FNA) washout of cervical lymph nodes. We studied pre-analytical stability, in different conditions, of 41 samples prepared with concentrated solutions of thyroglobulin (FNA washout or certified standard) diluted in physiological saline solution or buffer containing 6% albumin. In this buffer, over time, no changes in thyroglobulin concentrations were observed in all storage conditions tested. In albumin free saline solution, thyroglobulin recovery rates depended on initial sample concentrations and on modalities of their conservation (in conventional storage tubes, recovery mean was 56% after 3 hours-storage at room temperature and 19% after 24 hours-storage for concentrations ranged from 2 to 183 µg/L; recovery was 95%, after 3 hours or 24 hours-storage at room temperature, for a concentration of 5,656 µg/L). We show here that these results are due to non-specific adsorption of thyroglobulin in storage tubes, which depends on sample protein concentrations. We also show that possible contamination of fluids from FNA washout by plasma proteins do not always adequately prevent this adsorption. In conclusion, non-specific adsorption in storage tubes strongly contributes to uncertainty in thyroglobulin measurement in physiological saline solution. It is therefore recommended, for FNA washout, to use a buffer containing proteins provided by the laboratory.

Middle Iron-Enriched Fructose Diet on Gestational Diabetes Risk and on Oxidative Stress in Offspring Rats.

Gestational diabetes mellitus (GDM) is associated with increased insulin resistance and a heightened level of oxidative stress (OS). Additionally, high iron consumption could also increase insulin resistance and OS, which could aggravate GDM risk. The aim of this study is to evaluate a high fructose diet (F) as an alternative experimental model of GDM on rats. We also have evaluated the worst effect of a fructose iron-enriched diet (FI) on glucose tolerance and OS status during pregnancy. Anthropometric parameters, plasma glucose levels, insulin, and lipid profile were assessed after delivery in rats fed an F diet. The effects observed in mothers (hyperglycemia, and hyperlipidemia) and on pups (macrosomia and hypoglycemia) are similar to those observed in women with GDM. Therefore, the fructose diet could be proposed as an experimental model of GDM. In this way, we can compare the effect of an iron-enriched diet on the metabolic and redox status of mother rats and their pups. The mothers' glycemic was similar in the F and FI groups, whereas the glycemic was significantly different in the newborn. In rat pups born to mothers fed on an FI diet, the activities of the antioxidant enzyme glutathione peroxidase (GPx) and glutathione-S-transferase in livers and GPx in brains were altered and the gender analysis showed significant differences. Thus, alterations in the glycemic and redox status in newborns suggest that fetuses are more sensitive than their mothers to the effect of an iron-enriched diet in the case of GDM pregnancy. This study proposed a novel experimental model for GDM and provided insights on the effect of a moderate iron intake in adding to the risk of glucose disorder and oxidative damage on newborns.

Association between iron level, glucose impairment and increased DNA damage during pregnancy.

Elevated circulating ferritin has been reported to increase the risk of gestational diabetes mellitus (GDM). When high ferritin translates into high iron stores, iron excess is also a condition leading to free radical damage. We aimed to evaluate the relationship between oxidative stress (OS) induced by iron status and GDM risk in non iron-supplemented pregnant women. METHODS: This was a pilot observational study conducted on 93 non-anemic pregnant women. Iron status was assessed at the first trimester of gestation. Blood sampling was done at 24-28 weeks' gestation for oral glucose tolerance test (OGTT), insulin and biological markers of oxidative damage tests. RESULTS: A significant increase in DNA damage was found in patients who developed GDM. Women with elevated DNA damage had a six-fold increased risk of developing GDM (Exp (B)=6.851, P=0.038; 95% CI [1.108-42.375]). The serum ferritin levels at first trimester were significantly correlated to lipid peroxidation (rho=0.24, p=0.012). The stratified analysis suggests that ferritin is a modifying factor for the correlation of oxidative stress (OS) and glucose intolerance. CONCLUSION: Moderate ferritin levels due to iron intake without iron-supplement, at early pregnancy is a modifying factor for the correlation of oxidative damage and glucose intolerance in pregnant women. Larger studies to evaluate the risk of food iron intake induced increased oxidative damage in offspring are warranted to propose nutrition advice regarding iron intake in women with a high risk of GDM.

Impact of sleep behavior on glycemic control in type 1 diabetes: the role of social jetlag.

BACKGROUND: Sleep behavior is changing toward shorter sleep duration and a later chronotype. It results in a sleep debt that is acquitted on work-free days, inducing a small but recurrent sleep misalignment each week, referred to as "social jetlag". These sleep habits could affect health through misalignment with circadian rhythms. OBJECTIVES: The primary objective is to address the impact of sleep behavior on glycemic control, assessed by HbA1c, in patients with type 1 diabetes, independently of other lifestyle or sleep-related factors. The secondary objective is to address whether circadian phase affects glycemic control. DESIGN: In total, 80 adult patients with type 1 diabetes (46% female) were included in a clinical cohort study. METHODS: Sleep behavior was addressed objectively by a 7-day actimetry, lifestyle by questionnaires, sleep breathing disorders by nocturnal oximetry and circadian phase by dim light melatonin onset (DLMO). RESULTS: Univariate analyses showed that chronotype (r = 0.23, P = 0.042) and social jetlag (r = 0.30, P = 0.008) were significantly associated with HbA1c. In multivariable analysis, social jetlag was the only sleep habit independently associated with HbA1c (ß = 0.012 (0.006; 0.017), P < 0.001). HbA1c was lower in patients with a social jetlag below versus above the median (7.7% (7.1-8.7) and 8.7% (7.6-9.8), P = 0.011). DLMO was not associated with HbA1c. However, the later the DLMO, the worse the sleep efficiency (r = -0.41, P < 0.001) and fragmentation index (r = 0.35, P = 0.005). CONCLUSIONS: Social jetlag, a small but recurrent circadian misalignment, is associated with worse glycemic control in type 1 diabetes, whereas circadian phase is not. Further intervention studies should address the potential improvement of glycemic control by correcting social jetlag.