Dynamic changes in the gut microbiota during three consecutive trimesters of pregnancy and their correlation with abnormal glucose and lipid metabolism.

INTRODUCTION: During normal pregnancy, changes in the gut microbiota (GM) in response to physiological alterations in hormonal secretion, immune functions and homeostasis have received extensive attention. However, the dynamic changes in the GM during three consecutive trimesters of pregnancy and their relationship with glucose and lipid metabolism have not been reported. In this study, we aimed to investigate the dynamic changes in the diversity and species of the GM during three consecutive trimesters in women who naturally conceived, and their relationships with abnormal fasting blood glucose (FBG) and serum lipid levels. METHODS: A total of 30 pregnant women without any known chronic or autoimmune inflammatory disease history before pregnancy were enrolled during the first trimester. Serum and stool samples were collected during the first trimester, the second trimester, and the third trimester. Serum samples were tested for FBG and blood lipid levels, and stool specimens were analyzed by 16S rDNA sequencing. RESULTS: The abundance ratio of bacteroidetes/firmicutes showed an increasing tendency in most of the subjects (19/30, 63.3%) from the first to the third trimester. LEfSe analysis showed that the abundance of Bilophila was significantly increased from the first to the third trimester. In addition, at the genus level, the increased relative abundance of Mitsuokella, Clostridium sensu stricto and Weissella were potentially involved in the development of high FBG during pregnancy. The raised relative abundance of Corynebacterium, Rothia and Granulicatella potentially contributed to the occurrence of dyslipidemia during pregnancy. CONCLUSIONS: There are dynamic changes in the GM during the three trimesters, and the alterations in some bacterium abundance may contribute to the development of high FBG and dyslipidemia during pregnancy. Monitoring enterotypes and correcting dysbiosis in the first trimester may become new strategies for predicting and preventing glucolipid metabolism disorders during pregnancy.

Changing Iodine Status and the Incidence of Thyroid Disease in Mainland China: A Prospective 20-Year Follow-Up Study.

Background: We aimed to assess the long-term effects of the transition in iodine status on the incidence of thyroid disorders over 20 years of follow-up. Methods: The original prospective cohort study, started in 1999 (n = 3761), classified three regions in north China based on iodine status (insufficient iodine, more than adequate iodine, and excessive iodine, respectively) for 5 years. Subsequently, participants were followed for up to another 15 years to assess the long-term effects of shifts to adequate iodine on the incidence of thyroid disorders. Panshan transitioned from insufficient to adequate iodine, and Huanghua transitioned from excessive to more than adequate iodine. Both regions were compared with Zhangwu, in which iodine status changed from more than adequate to adequate iodine (from 214 to 167.2 µg/L). A cluster sampling method was used to select participants in the three regions. Participants completed questionnaires and underwent thyroid ultrasonography. Urinary iodine concentrations (UICs), serum thyroid hormone concentration, and thyroid antibodies were measured. Results: When the iodine status changed from insufficient to adequate (with the median UIC increasing from 88 to 141.9 µg/L), the incidence density of subclinical hyperthyroidism, positive thyroperoxidase antibody, positive thyroglobulin antibody (TgAb), and goiter decreased significantly (p < 0.05 for all). Additionally, the cumulative incidence of subclinical hypothyroidism was significantly lower compared with the region where the iodine status changed from being more than adequate to adequate (1.9% vs. 6.0%, p < 0.001). When the iodine status changed from excessive to more than adequate (median UIC from 634 to 266.7 µg/L), a significant decrease in the incidence density of subclinical hyperthyroidism, positive thyroid antibodies, positive TgAb, and goiter (p < 0.05 for all) were also found. However, an increase in thyroid nodule incidence density (17.26 vs. 28.25 per 1000 person-years, p < 0.001) was seen. Conclusions: The incidence of thyroid disorders (except for thyroid nodules) stabilized or decreased among adults in the three communities from year 5 to year 15 of follow-up. Appropriate iodine fortification is safe and effective over the long term. Restoring urinary iodine to appropriate levels reduces population risk for thyroid disorders.

Determination of the Predictive Roles and Potentially Pathogenic Antigen Epitopes of α-Enolase Related to the Development of Miscarriage in Females with Autoimmune Thyroiditis.

Autoimmune thyroiditis (AIT) is a common endocrine disease which causes a significantly increased risk of miscarriage. Our recent study has shown that the increased ENO1 autoantibody (ENO1Ab) expression in an experimental AIT mouse model was induced by thyroglobulin (Tg) immunization only. In this study, we explored the potential roles of ENO1Ab in miscarriage occurrence among AIT women, and the specific epitopes of ENO1 targeted by ENO1Ab. A total of 432 euthyroid pregnant participants were selected from the project of Subclinical Hypothyroid during Early Pregnancy, including 48 women with AIT and miscarriage, 96 with miscarriage but no AIT, 96 with AIT but no miscarriage, and 192 without either AIT or miscarriage. The enzyme-linked immunosorbent assay was used to determine the serum levels of total IgG against ENO1 and 18 predicted antigen epitopes of ENO1. The results showed that women with AIT and miscarriage had the highest serum levels of ENO1Ab compared to the other groups. Logistic regression analysis showed that the serum ENO1Ab was an independent risk factor for miscarriage, especially among AIT females. The serum level of total IgG against the predicted epitope peptide 6 (i.e., P6 and aa168-183) of ENO1 was significantly increased in women with AIT and miscarriage when compared with those of both the AIT non-miscarriage group and non-AIT miscarriage group. This pilot study suggests that serum ENO1Ab may have a fair predictive value for AIT-related miscarriage, and the autoantibody specific to P6 epitope may especially be more specifically related to this disorder.

Molecular Optimization on Polymer Acceptor Enables Efficient All-Polymer Solar Cell with High Open-Circuit Voltage of 1.10 V.

Currently, rational design of polymer acceptors is desirable but there is still a challenge to develop high-performance all-polymer solar cells (all-PSCs). In this work, brominated thienyl-fused malononitrile-based monomer is employed to copolymerize with indacenodithiophene (IDT) and benzodithiophene (BDT)-based linking units to develop two polymerized small molecule acceptors (PSMAs) PIDT and PBDT, respectively, for all-PSCs. The two PSMAs show similar absorption edges, while PBDT shows a slightly higher lowest unoccupied molecular orbital (LUMO) energy level than PIDT. Benefitted from the relatively high LUMO levels of the two polymer acceptors, notable open-circuit voltage (Voc ) values over 1.0 V are achieved when using them as acceptor to blend with PTQ10 as polymer donor. Particularly, the all-PSC based on PTQ10:PIDT demonstrates a power conversion efficiency of 10.19%, with an outstanding Voc of 1.10 V benefitted from the higher LUMO energy level of PIDT acceptor. The results demonstrate a feasible strategy to design PSMAs by selecting appropriate linking units for increasing the Voc and improving the efficiency of all-PSCs.

In Vivo Inhibition of MicroRNA-326 in a NOD.H-2h4 Mouse Model of Autoimmune Thyroiditis.

Background: Previous studies reported that various miRNAs participate in autoimmune diseases, but the potential regulatory mechanism of miRNAs in autoimmune thyroiditis (AIT) needs further exploration. Objective: This study aimed to further verify that miR-326 contributes to AIT by regulating Th17/Treg balance through Ets-1 using lentiviral gene delivery through tail vein and thyroid injection in NOD.H-2h4 mice. Materials and Methods: Five-week-old NOD.H-2h4 mice were divided randomly into tail vein and thyroid injection groups, and each received either mmu-miR-326 sponge (LV-sponge) or lentiviral vector control. Mice were divided for tail vein injection: the therapeutic LV-ctrl, therapeutic LV-sponge, prophylactic LV-ctrl, and prophylactic LV-sponge groups. The control group was fed high-iodine water without vein injection. The thyroid infiltration of lymphocytes and serum TgAb value were investigated by thyroid hematoxylin and eosin (HE) staining and ELISA, respectively. Ets-1 and lymphocyte counts were measured by RT-PCR, western blotting, and flow cytometry. The thyroid CD4+IL-17a+ cells and CD4+Ets-1+ cells were detected by immunofluorescence, and the serum cytokines were tested by ELISA. Results: In the tail vein injection groups, the thyroid inflammatory score and serum TgAb titer were significantly lower in the LV-sponge groups than in the control and LV-ctrl groups while Ets-1 protein expression in mouse spleens was increased in the LV-sponge groups. Moreover, Th17/Treg ratio declined in the LV-sponge group and decreased significantly in the prophylactic LV-sponge group (P = 0.036) tested by flow cytometry. Immunofluorescence showed that, in LV-sponge groups, CD4+IL-17a+ cells were decreased significantly (P = 0.001), while CD4+Ets-1+ cells were increased significantly in the LV-sponge group (P = 0.029). The serum IL-17/IL-10 was decreased significantly in the LV-sponge group (P < 0.05). In the thyroid injection groups, the thyroid inflammatory score and serum TgAb titer in the LV-sponge group decreased significantly compared with those in the LV-ctrl group (P < 0.05). In addition, in LV-sponge groups, CD4+IL-17a+ cells were decreased, while CD4+Ets-1+ cells were increased significantly in the inhibition group evaluated by immunofluorescence. Moreover, tail vein injection of LV-sponge resulted in much lower TgAb levels in thyroiditis compared with thyroid injection. Conclusion: MiR-326 targeted therapy may be a promising approach for AIT. In addition, tail vein injection may achieve a better intervention effect than thyroid injection.

Dynamic Changes in Antithyroperoxidase and Antithyroglobulin Antibodies Suggest an Increased Risk for Abnormal Thyrotropin Levels.

Background: Antithyroperoxidase (TPOAb) and antithyroglobulin (TgAb) antibodies are associated with abnormal thyrotropin (TSH) levels. However, the effect of dynamic changes in TPOAb and TgAb on incident abnormal TSH is unknown. Methods: A total of 2,387 euthyroid participants aged 18 years or older from three rural areas in northern China were enrolled in this cohort study. Questionnaire interviews and laboratory measurements were performed at baseline in 1999 and at follow-up in 2004. Multinomial logistic regression was used to examine the relationship between changes in thyroid antibodies and incident abnormal TSH levels. Results: In this 5 year follow-up study, TPOAb tier gain was significantly associated with an increased risk of subnormal TSH levels (adjusted RR, 1.535; 95% CI: 1.357-1.736) and supranormal TSH levels (adjusted RR, 1.378; 95% CI: 1.196-1.587), and TgAb tier gain was significantly associated with an increased risk of supranormal (adjusted RR, 1.090; 95% CI: 1.007-1.179) TSH levels. Both thyroid antibody-positive seroconversion and persistent positivity were significantly associated with an increased risk of incident abnormal TSH levels. Thyroid antibody positive seroconversion was associated with a higher risk of incident subnormal TSH than incident supranormal TSH, whereas persistent positive thyroid antibody was associated with a higher risk of incident supranormal TSH than incident subnormal TSH. Conclusions: Dynamic thyroid antibody changes may be related to incident abnormal TSH levels. Those with persistent positive thyroid antibody were more likely to have supranormal TSH than subnormal TSH, and those with positive seroconversion were more likely to have subnormal TSH than supranormal TSH. Further studies are needed to confirm this conclusion and to explore this association mediated by TSH receptor antibodies.

Roles of Endogenous IL-10 and IL-10-Competent and CD5+ B Cells in Autoimmune Thyroiditis in NOD.H-2h4 Mice.

Interleukin (IL)-10 is a highly important anti-inflammatory cytokine in the immune system. CD1dhi and CD5+ B cells are both traditionally defined IL-10-secreting B cells. In recent years, a B cell group with combined markers of CD1dhi and CD5+ has been widely studied as it has been reported to suppress autoimmunity in mouse models of autoimmune diseases through IL-10 mechanisms. From the perspective of origination, CD1dhi and CD5+ B cells are developed from different B cell lineages. Whether the regulatory capacity of these 2 B cell groups is consistent with their ability to secrete IL-10 has not been determined. In this study, we generated IL-10 knockout NOD.H-2h4 mice to investigate the function of endogenous IL-10 in autoimmune thyroiditis and conducted adoptive transfer experiments to explore the respective roles of CD5+ and CD1dhi B cells. In our results, the IL-10-/- NOD.H-2h4 mice developed thyroiditis, similar to wild-type NOD.H-2h4 mice. The CD5+ B cells were more capable of secreting IL-10 than CD1dhi B cells in flow cytometric analysis, but the CD1dhi B cells showed more suppressive effects on thyroiditis development and autoantibody production, as well as Th17 cell response. In conclusion, endogenous IL-10 does not play an important role in autoimmune thyroiditis. CD1dhi B cells may play regulatory roles through mechanisms other than secreting IL-10.

Circulating Exosomes Activate Dendritic Cells and Induce Unbalanced CD4+ T Cell Differentiation in Hashimoto Thyroiditis.

OBJECTIVE: This study explored whether circulating exosomes effectively participate in the inflammatory response in Hashimoto thyroiditis (HT). DESIGN: Exosomes were extracted from the serum of 30 patients with HT and 30 healthy control (HC) subjects. The expression of thyroperoxidase (TPO), thyroglobulin, high mobility group box 1 (HMGB1), heat shock protein 60 (HSP60), major histocompatibility complex class II (MHC-II), and intercellular adhesion molecule 1 (ICAM1) in exosomes was determined by Western blotting. Flow cytometry and immunofluorescence were performed to confirm that exosomes were taken up by healthy peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs). Then, either DCs or PBMCs were stimulated with HT exosomes (serum exosomes from patients with HT) or HC exosomes (serum exosomes from HC subjects) in the presence or absence of Toll-like receptor (TLR)2/3 inhibitors. RESULTS: TPO, HSP60, and MHC-II expression was higher in HT exosomes than in HC exosomes. Exosomes were mainly taken up by CD14+ monocytes and CD11c+ DCs. After DCs were stimulated by HT exosomes, significant elevations were observed in MyD88, TRIF, and p-P65 expression; median fluorescence intensity of CD40 and CD83; and IL-6 production. After stimulating PBMCs with HT exosomes, CD11c+TLR2+/TLR3+ and CD4+IFN-γ+Th1/IL-17A+Th17A cell percentages were significantly elevated, and CD4+CD25+Foxp3+ Treg cell percentage was significantly decreased. HT exosomes induced increased IL-17A and IFN-γ production, whereas IL-10 production was suppressed. However, addition of TLR2 or TLR3 inhibitor reversed most of the abovementioned results. CONCLUSIONS: Our study demonstrates that HT exosomes can present antigens to DCs and bind TLR2/3, causing DC activation via the nuclear factor κB signaling pathway, leading to an imbalance in CD4+ T lymphocyte differentiation, and potentially contributing to HT onset.

Different Thyroidal Responses to Human Chorionic Gonadotropin Under Different Thyroid Peroxidase Antibody and/or Thyroglobulin Antibody Positivity Conditions During the First Half of Pregnancy.

BACKGROUND: Thyroid peroxidase antibody (TPOAb) positivity can attenuate gestational thyroid responses to human chorionic gonadotropin (hCG) during pregnancy, whereas the effects of thyroglobulin antibodies (TgAb) remain unknown. The aim of our study was to explore the thyroid response to hCG in women with thyrotropin (TSH) levels within the method-specific reference range under different conditions of thyroid autoimmunity. METHODS: The study screened 822 women at 7-20 weeks of gestation using the pregnancy-specific reference range for TSH. Serum TSH, free thyroxine (fT4), TPOAb, TgAb, and ß-hCG levels were measured using electrochemiluminescence immunoassays. RESULTS: The enrolled pregnant women were subdivided into four subgroups based on TPOAb/TgAb positivity: co-positive for TPOAb and TgAb (group 1), isolated TPOAb positive (group 2), isolated TgAb positive (group 3), and co-negative for TPOAb and TgAb (group 4). TSH was negatively associated with hCG in all four groups (p < 0.05). fT4 was positively associated with hCG in groups 3 and 4 (p < 0.01) but not in groups 1 (p = 0.096) and 2 (p = 0.758). Group 2 was further stratified into tertiles according to TPOAb concentrations. No negative TSH/hCG association was observed in the middle- and upper-tertile groups when TPOAb were ≥53 IU/mL (p > 0.05). There was no positive fT4/hCG association in any of the three subgroups (p > 0.05). Similarly, group 3 was further stratified into tertiles according to TgAb levels. TSH was negatively associated with hCG in the lower and middle tertiles (p < 0.01), but the association was not found in the upper tertile when TgAb was ≥356 IU/mL (p = 0.191). fT4 was positively associated with hCG in the lower tertile (p = 0.027) but not in subgroups when TgAb was ≥219 IU/mL (p > 0.05). CONCLUSIONS: When TSH was within the pregnancy-specific reference range, high concentrations of TPOAb and TgAb attenuated the fT4 stimulation and suppression of TSH by hCG. The results imply that TgAb, in addition to TPOAb, could also interfere with thyroidal responses to hCG during the first half of pregnancy.

Smoking Is Positively Associated with Antithyroperoxidase Antibodies and Antithyroglobulin Antibodies in Populations with Mildly Deficient Iodine Intake.

To evaluate the relationship between smoking and both antithyroperoxidase antibody (TPOAb) and antithyroglobulin antibody (TgAb) positivity in subjects from Panshan, Zhangwu, and Huanghua with mildly deficient, more than adequate, and excessive iodine intake, respectively. Smoking-related data were collected by questionnaire, and laboratory measurements of TPOAb, TgAb, and thyrotropin (TSH) were determined at baseline and follow-up. (1) A 1.48-fold increased risk of TPOAb positivity was found in smokers than in non-smokers after adjusting for confounders (age, sex, and areas) (OR[95% CI] = 1.48[1.12-1.95], p = 0.01). (2) Among female subjects, the prevalence of thyroid autoantibodies in smokers was increased than that in non-smokers in Panshan, Zhangwu, and Huanghua (TPOAb): 16.79 vs. 8.89%, 14.14 vs. 11.09%, 19.53 vs. 9.57%; TgAb 15.32 vs. 9.29%, 12.79 vs. 11.94%, 17.19 vs. 10.55%, respectively). The difference was significant in Panshan after adjusting for age. (3) Female long-term smokers (> 20 years) had an increased frequency of thyroid autoantibody positivity than non-smokers after adjusting for confounders (TPOAb OR[95% CI] = 1.60[1.10-2.34]; TgAb OR[95% CI] = 1.31[0.88-1.94]). (4) There was no difference in the incidence of thyroid autoantibodies among non-smokers, new smokers, and long-term smokers at follow-up. (5) TSH was greater in TPOAb-positive subjects than in seronegative smokers (1.56 vs. 1.20 mU/L, p < 0.001) and non-smokers (1.97 vs. 1.58 mU/L, p < 0.001). However, TSH was also greater in non-smokers than in smokers, regardless of whether subjects were positive (1.97 vs. 1.56 mU/L, p = 0.04) or negative (1.58 vs. 1.20 mU/L, p < 0.001) for TPOAb. Long-term smoking could increase the prevalence of thyroid autoantibodies in a population with mildly deficient iodine intake. TSH levels were lesser in smokers than in non-smokers and greater in subjects with thyroid autoantibody positivity than in seronegative subjects. The influence of smoking on TSH levels was independent of thyroid autoantibody levels.

Decreased Thyroid Peroxidase Antibody Titer in Response to Selenium Supplementation in Autoimmune Thyroiditis and the Influence of a Selenoprotein P Gene Polymorphism: A Prospective, Multicenter Study in China.

Background: Recent intervention studies have suggested that selenium (Se) is an effective treatment for autoimmune thyroiditis (AIT). However, the exact effect of Se on AIT is unclear as well as the mechanism of action. The aim of the present study was to explore the effect of Se on thyroid peroxidase antibody (TPOAb) titers in patients with AIT and to analyze the potential impact of the genetic background on the effect of Se supplementation. Methods: This was a randomized, placebo-controlled, double-blind trial. Three hundred and sixty-four patients with elevated TPOAb (>300 IU/mL) were recruited and randomized to receive Se yeast 200 µg/day supplementation or placebo. Urinary iodine concentration, serum thyrotropin, free thyroxine, TPOAb, Se, malondialdehyde, and serum glutathione peroxidase activity were measured at baseline and follow-up. Ninety-six patients were genotyped for single nucleotide polymorphism r25191G/A in the selenoprotein P (SEPP1/SELENOP) gene. Results: The median urinary iodine concentration was 182 µg/L. Serum Se increased significantly (p < 0.001) after Se treatment. TPOAb titer decreased by 10.0% at 3 months and by 10.7% at 6 months after Se supplementation, while there was a moderate increase in TPOAb titers over the follow-up period in patients receiving placebo. Glutathione peroxidase activity significantly increased (p < 0.001), and malondialdehyde significantly decreased (p < 0.001) after 6 months of Se supplementation. TPOAb titers decreased to variable extents in patients with different genotypes of single nucleotide polymorphism r25191G/A after Se supplementation. Serum TPOAb titers in patients with the AA genotype showed a more significant decrease (by 46.2%) than those with the GA and GG genotypes (by 14.5 and 9.8% respectively) at 3 months of Se supplementation (p = 0.070). Conclusions: Se supplementation significantly reduced TPOAb titers in patients with AIT, and there may be an important genetic component influencing interindividual differences in the decrease in TPOAb titers.

An Age-Specific Serum Thyrotropin Reference Range for the Diagnosis of Thyroid Diseases in Older Adults: A Cross-Sectional Survey in China.

Background: The fact that serum thyrotropin (TSH) levels increase with age may influence the diagnosis of thyroid diseases in older adults. This study aimed to establish an age-specific serum TSH reference range, examine the prevalence of thyroid diseases in older adults ≥65 years, and analyze the risk factors. Methods: A cross-sectional study of adult populations in 10 cities in China was conducted from 2010 to 2011. A total of 15,008 subjects were randomly selected and completed the present study. Urinary iodine concentration, serum TSH, thyroid peroxidase antibody (TPOAb), and thyroglobulin antibody (TgAb) titers were measured. Thyroid ultrasonography and questionnaires were completed by all the subjects. When the TSH level was abnormal, free thyroxine and/or free triiodothyronine levels were measured. Results: When the reference range of the general population was used, the prevalence rates of overt hypothyroidism (Ohypo) and subclinical hypothyroidism (Shypo) in older adults ≥65 years were significantly higher than those in younger adults <65 years (2.09% vs. 0.80% and 19.87% vs. 16.23%, respectively; p < 0.001). Positive TPOAb and positive TgAb were associated with the prevalence of Shypo in older adults. An age-specific serum TSH reference range was formulated according to guidelines set forth by the National Academy of Clinical Biochemistry. Both the median and upper limit values of serum TSH in older adults were higher than those in younger adults (2.58 [0.75-8.86] mIU/L vs. 2.38 [0.76-6.57] mIU/L; p < 0.001). Using the age-specific serum TSH reference range, the prevalence of Shypo in older adults was 3.3%, which was significantly lower than the prevalence based on the reference range of the general population (3.3% vs. 19.87%). The prevalence rates of Ohypo, overt hyperthyroidism (Ohyper), and subclinical hyperthyroidism (Shyper) did not change much (Ohypo: 1.6% vs. 2.09%; Ohyper: 0.7% vs. 0.52%; and Shyper: 3.8% vs. 0.73%). Positive TPOAb, but not positive TgAb, was also associated with the prevalence of Shypo as diagnosed with the age-specific serum TSH reference range. Conclusion: The serum TSH level increases with age, which may represent a normal compensatory phenomenon in older adults ≥65 years. To prevent misdiagnosis and mistreatment, the use of an age-specific serum TSH reference range is recommended in older adults for the diagnosis of thyroid diseases.

Differential Expression of MicroRNAs and miR-206-Mediated Downregulation of BDNF Expression in the Rat Fetal Brain Following Maternal Hypothyroidism.

To investigate the mechanism responsible for the neurological alterations, miRNA expression profile and brain-derived neurotrophic factor (BDNF) were evaluated in brain tissues of fetal or neonatal rats and from maternal rats with hypothyroidism. Ninety female Wistar rats were divided into a control and a hypothyroid group, which were mated. Brain samples of the offspring were obtained at maternal embryonic day (E) E13 and E17 as well as postnatal day (P) P0 and P7, and the hippocampus and cortex were separated at P7. BDNF mRNA at E13 was tested by real-time PCR and protein expression by Western blot. Luciferase assays were used to confirm that miR-206 targets the 3'-untranslated region (3'-UTR) of BDNF. In the brain tissues of fetal and neonatal rats from maternal rats with hypothyroidism, differentiation miRNAs profile were found at E13, E17, P0, and P7. Compared with the control group, miR-206 levels in the hypothyroidism group were increased by 3.1-fold by micro-array, and were higher as measured by SYBR green real-time qRT-PCR (p<0.01). There was no significant difference in the BDNF mRNA levels at E13 between the hypothyroidism group and the control group (1.767±0.477 vs. 1.798±0.462, respectively; p>0.05), but pro-BDNF and mature BDNF protein levels in the hypothyroid group at E13 were significantly lower than those in the control group (p<0.05). miR-206 targeted 3'-UTR of BDNF. Our data highlight the role of miR-206 as a post-transcriptional inhibitor of BDNF at E13 in pregnant hypothyroid rats.

Iron Deficiency May Predict Greater Risk for Hypothyroxinemia: A Retrospective Cohort Study of Pregnant Women in China.

BACKGROUND: Pregnant women are highly vulnerable to iron deficiency (ID) due to the increased iron needs during pregnancy. ID decreases circulating thyroid hormone concentrations likely through impairment of iron-dependent thyroid peroxidase. The present study aimed to explore the association between ID and hypothyroxinemia in a retrospective cohort of pregnant women in China. METHODS: To investigate the relationship between ID and hypothyroxinemia, 723 pregnant women were retrospectively analyzed, including 675 and 309 women in the second and third trimesters, respectively. Trimester-specific hypothyroxinemia was defined as free thyroxine (fT4) levels below the 2.5th percentile of the reference range with normal serum thyrotropin (TSH) or TSH higher than the 97.5th percentile of the reference range in each trimester of pregnancy. Serum TSH, fT4, thyroid peroxidase antibodies, thyroglobulin antibodies, serum ferritin, soluble transferrin receptor, and urinary iodine concentrations were measured. Serum ferritin, soluble transferrin receptor, and total body iron were used to indicate the nutritional iron status. RESULTS: Cross-sectional multiple linear regression analysis showed that iron status was positively associated with serum fT4 levels in the first and second trimesters of pregnancy, but not in the third trimester. Logistic regression analysis showed that ID was an independent risk factor for hypothyroxinemia (odds ratio = 14.86 [confidence interval 2.31-95.81], p = 0.005 in the first trimester and odds ratio = 3.36 [confidence interval 1.01-11.21], p = 0.048 in the second trimester). The prospective analysis showed that pregnant women with ID during the first trimester of pregnancy had lower serum fT4 levels and a higher rate of hypothyroxinemia in the second or third trimester than those without ID. CONCLUSIONS: ID appears to be a risk factor to predict hypothyroxinemia in the first and second trimesters of pregnancy, but not in the third trimester. Pregnant women with ID in the first and second trimesters should be regarded as a high-risk group for maternal hypothyroxinemia.

Targeting glutaminase-mediated glutamine dependence in papillary thyroid cancer.

Papillary thyroid cancer is a prevalent endocrine malignancy. Although alterations in glutamine metabolism have been reported in several types of hematological and solid tumors, little is known about the functions of glutamine and glutaminolysis-associated proteins in papillary thyroid cancer. Here, we demonstrated the glutamine dependence of papillary thyroid cancer cells, and with the use of RT2-PCR arrays, we screened for the aberrant overexpression of glutaminase in human papillary thyroid cancer tissues and cells. These results were later confirmed via real-time PCR, Western blots, and immunohistochemical staining. We found that the levels of glutaminase were significantly correlated with extrathyroidal extension. Inhibition of GLS suppressed glutaminolysis and reduced mitochondrial respiration. The proliferative, viable, migratory, and invasive abilities of papillary thyroid cancer cells were impaired by both the pharmacological inhibition and the genetic knockdown of glutaminase. Additionally, the inhibition of glutaminase deactivated the mechanistic target of the rapamycin complex 1 (mTORC1) signaling pathway, promoting autophagy and apoptosis. Collectively, these findings show that glutaminase-mediated glutamine dependence may be a potential therapeutic target for papillary thyroid cancer. KEY MESSAGES: PTC cells are glutamine-dependent, and GLS is aberrantly overexpressed in PTC. Inhibition of GLS suppressed glutaminolysis and reduced mitochondrial respiration. Inhibition of GLS impairs the viability of PTC cells. GLS blockade causes deactivation of mTORC1 and induction of autophagy and apoptosis. GLS may be a potential therapeutic target for PTC.

Cytokine Secretion and Pyroptosis of Thyroid Follicular Cells Mediated by Enhanced NLRP3, NLRP1, NLRC4, and AIM2 Inflammasomes Are Associated With Autoimmune Thyroiditis.

Background: Inflammasomes, which mediate maturation of interleukin-1ß (IL-ß) and interleukin-18 (IL-18) and lead to pyroptosis, have been linked to various autoimmune disorders. This study investigated whether they are involved in the pathogenesis of autoimmune thyroiditis (AIT). Methods: We collected thyroid tissues from 50 patients with AIT and 50 sex- and age-matched controls. Serum levels of free T3, free T4, thyrotropin, thyroid peroxidase antibody (TPOAb), and thyroglobulin antibody (TgAb) were measured by electrochemiluminescent immunoassays. Expression of several inflammasome components, the NOD-like receptor (NLR) family pyrin domain containing 1 (NLRP1), NLRP3, CARD-domain containing 4 (NLRC4), absent in melanoma 2 (AIM2), the apoptosis-associated speck-like protein that contains a caspase recruitment domain (ASC), caspase-1, IL-1ß, and IL-18 was determined by real-time PCR and western blot. Immunohistochemistry was used to localize the expression of NLRP1, NLRP3, NLRC4, and AIM2. The Nthy-ori 3-1 thyroid cell line was stimulated with tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-17A, interleukin-6, and poly(dA:dT). The levels of IL-18 and IL-1ß in the cell supernatant were measured by enzyme-linked immunosorbent assay, and lactate dehydrogenase was quantified by absorptiometry. ASC specks were examined by confocal immunofluorescence microscopic analysis. Cell death was examined by flow cytometry, and the N-terminal domain of gasdermin D was detected by western blot analysis. Results: Expression of NLRP1, NLRP3, NLRC4, AIM2, ASC, caspase-1, pro IL-1ß, pro IL-18, mRNA, and protein was significantly increased in thyroid tissues from patients with AIT, and enhanced posttranslational maturation of caspase-1, IL-18 and IL-1ß was also observed. Expression of NLRP1, NLRP3, NLRC4, and AIM2 was localized mainly in thyroid follicular cells adjacent to areas of lymphatic infiltration. The thyroid mRNA level of NLRP1 and ASC was correlated to the serum TPOAb and TgAb levels in the AIT group. TNF-α and IFN-γ had a priming effect on the expression of multiple inflammasome components in thyroid cells. IFN-γ was found to strengthen poly(dA:dT)-induced cell pyroptosis and bioactive IL-18 release. Conclusion: Our work has demonstrated for the first time that multiple inflammasomes are associated with AIT pathogenesis. The identified NLRP3, NLRP1, NLRC4, AIM2 inflammasomes and their downstream cytokines may represent potential therapeutic targets and biomarkers of AIT.

Estrogen receptor ß activation stimulates the development of experimental autoimmune thyroiditis through up-regulation of Th17-type responses.

Estrogens play important roles in autoimmune thyroiditis, but it remains unknown which estrogen receptor (ER) subtype mediates the stimulatory effects. Herein we treated ovariectomized mice with ERα or ERß selective agonist followed by thyroglobulin-immunization to induce experimental autoimmune thyroiditis (EAT), and observed the aggravation of EAT after diarylpropionitrile (DPN, ERß selective agonist) administration. The mRNA levels of interleukin(IL)-17A, IL-21 and RORγt and percentages of T helper (Th) 17 cells were up-regulated in the splenocytes of DPN-treated mice. Activated ERß was found directly binding to IL-17A and IL-21 gene promoters, and also indirectly promoting IL-21 and RORγt gene transcription through interaction with NF-κB. The expressions of co-stimulatory molecules were increased on antigen-presenting cells (APCs) after DPN administration. It suggests that ERß is the predominant ER subtype responsible for EAT development, and its activation may enhance Th17-type responses through genomic pathways and alteration of APCs' activities.

Physiological low-dose oestrogen promotes the development of experimental autoimmune thyroiditis through the up-regulation of Th1/Th17 responses.

Previous studies have reported a preponderance of autoimmune thyroiditis (AIT) in females, but the detailed mechanisms have not been elucidated. In this study, we explored the effects of oestrogen on experimental AIT (EAT) and its potential mechanisms in an ovariectomised mouse model through the supplementation of high (equivalent to the level during pregnancy) or low (equivalent to the level at the oestrus stage) doses of oestradiol (E2). We found that EAT incidence, the intrathyroidal inflammatory score, serum anti-thyroglobulin IgG2b levels, splenic mRNA expression of Th1- and Th17-specific transcription factors and typical cytokines and the proportion of IL-12-producing dendritic cells were significantly increased in EAT mice treated with low-dose E2 compared with those in the control group. However, they were not changed when administered with high-dose E2. These findings indicate that low physiological levels of E2 can stimulate the occurrence and development of EAT through the up-regulation of Th1/Th17 responses.

Effect of Iodine Nutrition on Pregnancy Outcomes in an Iodine-Sufficient Area in China.

Many studies focused on the association between thyroid disease and pregnancy outcomes. The present study explored the effect of iodine nutrition during the first trimester on pregnancy outcomes. One thousand five hundred sixty-nine pregnant, euthyroid women at ≤12 weeks of gestation in an iodine-sufficient area in China were recruited. According to the World Health Organization (WHO) criteria for iodine nutrition during pregnancy, participants were divided into four groups: adequate iodine (median urinary iodine concentration (UIC), 150-249 µg/L), mild deficiency (UIC, 100-150 µg/L), moderate and severe deficiency (UIC, <100 µg/L), and more than adequate and excessive (UIC, ≥250 µg/L) groups. Pregnancy outcomes, including abortion, gestational hypertension, pre-eclampsia, gestational diabetes mellitus (GDM), placenta previa, placental abruption, preterm labor, low birth weight infants, macrosomia, breech presentation, and cord entanglement, were obtained during follow-up. The results showed that there was no significant difference in general characteristics, including age, body mass index, abdominal circumference, systolic blood pressure, diastolic blood pressure, heart rate, smoking rate, and drinking rate, among the four groups. In the more than adequate and excessive group, thyroid-stimulating hormone (TSH) was greater and free thyroxine (FT4) was lower than any other groups but still within normal range. The thyroglobulin (Tg) level was greater in the moderate and severe deficiency group. The incidence of GDM was significantly greater in women with mild iodine deficiency than in women with adequate iodine nutriture (18.38 vs. 13.70%, p < 0.05). Compared with the adequate group, incidence of macrosomia was significantly greater in the more than adequate and excessive group (12.42 vs. 9.79%, p < 0.05). Mild iodine deficiency was an independent risk factor for GDM (odds ratio = 1.566, 95% confidence interval = 1.060-2.313, p = 0.024); more than adequate and excessive iodine was an independent risk factor for macrosomia (OR = 1.917, CI = 1.128-3.256, p = 0.016). In summary, during 1st trimester, both mild iodine deficiency and excessive iodine intake had adverse impacts on pregnancy outcomes in an iodine-sufficient area.

MicroRNA-326 contributes to autoimmune thyroiditis by targeting the Ets-1 protein.

PURPOSE: MicroRNA-326 (miR-326), as a member of the microRNA (miRNA) family, which includes endogenous single-stranded, conserved, noncoding small RNAs, has been reported to play important roles in autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus. However, few studies of the role of miR-326 in autoimmune thyroiditis (AIT) have been published. Here, we explored the roles of miR-326 and the involved pathway in iodine-induced AIT. METHODS: NOD.H-2h4 mice, which are a model of human AIT, were randomly divided into a normal water control group and a high-iodine group. Mice in the high-iodine group were administered 0.05% NaI (~1000 times the normal daily iodine intake), and mice in the control group received sterile water. Furthermore, we evaluated small interfering RNA (siRNA) interference in spleen mononuclear cell experiments in vitro. RESULTS: In this study, we found that Th17 cells were significantly increased with a high expression of miR-326 in an iodine-induced thyroiditis NOD.H-2h4 mouse model. In addition, the expression of Ets-1 protein, a negative regulator of Th17 differentiation, was significantly decreased. Intriguingly, our analysis showed that Ets-1 protein expression was negatively correlated with miR-326 levels in AIT mice (r = -0.814, p < 0.01). Our study indicated that miR-326 inhibited Ets-1 protein expression and promoted the differentiation of Th17 cells during the onset and development of AIT. The addition of a miR-326 inhibitor reversed Th17 cell production and Ets-1 protein expression, supporting this hypothesis. CONCLUSIONS: The results of our study suggest that miR-326 may target the Ets-1 protein to contribute to iodide-induced thyroiditis, providing a new theoretical basis for the use of miRNA targeting therapy for the treatment of autoimmune diseases.