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Written by Mst. Noorjahan Begum, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing, 1 , 2 , 3   Rumana Mahtarin , Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing, 1 , 4   Md. Tarikul Islam , Formal analysis, Investigation, 1   Sinthyia Ahmed , Formal analysis, Investigation, Methodology, 5   Tasnia Kawsar Konika , Investigation, 6   Kaiissar Mannoor , Conceptualization, Supervision, Writing – review & editing, 1   Sharif Akhteruzzaman , Conceptualization, Supervision, Writing – review & editing, 2  and Firdausi Qadri , Conceptualization, Supervision 1 , 7 ,* Dhanusha Yesudhas, Editor PLoS One. 2023; 18(8): e0282553. Published online 2023 Aug 10.   doi:  10.1371/journal.pone.0282553 Abstract The disorder of thyroid gland development or thyroid dysgenesis accounts for 80–85% of congenital hypothyroidism (CH) cases. Mutations in the TSHR gene are mostly associated with thyroid dysgenesis, and prevent or disrupt normal development of the gland. There is limited data available on the genetic spectrum of congenital hypothyroid children in Bangladesh. Thus, an understanding of the molecular aetiology of thyroid dysgenesis is a prerequisite. The aim of the study was to investigate the effect of mutations in the TSHR  gene on the small molecule thyrogenic drug-binding site of the protein. We identified two nonsynonymous mutations (p.Ser508Leu, p.Glu727Asp) in the exon 10 of the TSHR gene in 21 patients with dysgenesis by sequencing-based analysis. Later, the TSHR368-764 protein was modeled by the I-TASSER server for wild-type and mutant structures. The model proteins were targeted by thyrogenic drugs, MS437 and MS438 to perceive the effect of mutations. The damaging effect in drug-protein complexes of mutants was explored by molecular docking and molecular dynamics simulations. The binding affinity of wild-type protein was much higher than the mutant cases for both of the drug ligands (MS437 and MS438). Molecular dynamics simulates the dynamic behavior of wild-type and mutant complexes. MS437-TSHR368-764MT2 and MS438-TSHR368-764MT1 showed stable conformations in biological environments. Finally, Principle Component Analysis revealed structural and energy profile discrepancies. TSHR368-764MT1 exhibited much more variations than TSHR368-764WT and TSHR368-764MT2, emphasizing a more damaging pattern in TSHR368-764MT1. This genetic study might be helpful to explore the mutational impact on drug binding sites of TSHR protein which is important for future drug design and selection for the treatment of congenital hypothyroid children with dysgenesis. 1. Introduction Congenital hypothyroidism is associated with various factors including genetics. Genetic causes account for about 15 to 20 percent of congenital hypothyroidism (CH). Although CH is a genetically heterogeneous disorder, the candidate genes divide the disorder into two main groups namely thyroid dysgenesis and thyroid dyshormonogenesis. Different studies including online databases such as Genetics Home Reference and Online Mendelian Inheritance in Men (OMIM) suggested that about 10–20 percent of total cases with CH were associated with thyroid dyshormonogenesis that would result from mutations in one of several genes involved in the biosynthesis of thyroid hormones [ 1 ]. The above-mentioned databases also described that about 80–85% of CH cases are associated with disorders of thyroid gland development (Dysgenesis) which is categorized as ectopic (located in a distant region, 40%), agenesis (absent of thyroid gland, 40%), and the other cases are accompanying with hypoplasia (small size). Although the actual cause of thyroid dysgenesis is still under investigation, some studies have suggested that 4 major genes that play roles in the proper growth and development of the thyroid gland, such as TSHR  (Thyroid 3 stimulating hormone receptor) and three transcription factors- TTF-1 , TTF-2 , and PAX8  (paired box-8, transcription factor) [ 2 ]. Mutations in these genes prevent or disrupt the normal development of the gland. The TSHR  gene is predominantly related to thyroid dysgenesis, as most of the mutations occurs in the gene in CH patients [ 2 ]. TSHR is a G protein-coupled transmembrane receptor which is present on the surface of thyroid follicular cells. TSH, secreted by the anterior pituitary, mediates its effect through TSHR which is crucial for thyroid gland development and function. The TSHR  gene is located on chromosome 14q31 and contains 11 exons code for a receptor protein of 764 amino acid residues [ 3 , 4 ]. TSHR has high affinity binding sites for TSH. Mutations in the TSHR gene result in mutant TSHR protein which lacks its binding affinity to TSH or loses its ability to activate adenylate cyclase. Thus, mutant TSHR protein disrupts thyroid gland development and proper functioning. TSHR  mutation may also be present in a normally placed thyroid gland. TSHR gene mutation is reported to be inherited as an autosomal recessive manner and exon 10 is known to carry the majority of the mutations [ 5 ]. In a high-throughput screening system, two small molecule agonists (MS437 and MS438) exhibited pharmacotherapeutic potential with the highest potency (EC50 of 13x10-8 M, and EC50 of 5.3x10-8 M respectively) [ 6 ]. Very limited data are available on genetic study of Bangladeshi hypothyroid patients. Therefore, the present study tried to explore the effect of two non-synonymous mutations in the 3D structure of TSHR368-764 targeted by thyrogenic drugs, MS437 and MS438 which will help to update any future treatment strategy including suitable drug design for Congenital Hypothyroid children. 2. Methods and materials 2.1. Study design, clinical settings, and ethical clearance The study was designed and carried out on 21 confirmed cases of Congenital Hypothyroid children with dysgenesis who were kept under treatment of Levothyroxine (LT4) drug in the Department of Endocrinology and National Institute of Nuclear Medicine and Allied Sciences (NINMAS) of Bangabandhu Shaikh Mujib Medical University (BSMMU). Ethical permission was obtained from the Ethical Review Committee of University of Dhaka (CP-4029) and the study was collaborated with NINMAS and Dept. of Endocrinology, BSMMU for specimen collection. Prior to enrollment of study participants, a written informed consent along with the clinical information was collected from the parent(s) or legal guardian(s) of each patient. 2.2. Collection and processing of blood specimens Blood Specimens were collected from the participants to conduct the molecular, biochemical and metabolic profiling tests. A total of 3ml blood was collected from each participant. All the samples were transported to the laboratory immediately. After the genomic DNA isolation, EDTA containing blood was stored at -70°C freezer. 2.3. Molecular analysis of TSHR  gene Now-a-days, gene-based study plays the key role to explore the actual cause of a particular disease. The present study was designed to perform the molecular analysis in various steps. 2.3.1. Genomic DNA isolation to perform PCR Genomic DNA was isolated from the EDTA blood by using Qiagen DNAeasy mini kit according to the manufacturer’s instruction. 500 μl of FG1 buffer was taken in a 1.5 ml microcentrifuge tube. 200 μl of whole blood was added to the FG1 buffer and mixed by inverting the tube 5–10 times. The mixture was then centrifuged at 10,000×g for 5 minutes in fixed angle rotor. The supernatant was carefully removed so that the pellet remained in the tube. 1μl of QIAGEN protease was added to 100 μl of FG2 buffer and mixed by vortex in a fresh Eppendorf tube. Then 100 μL of FG2/QIAGEN protease was added to the pellet and vortexed immediately until the pellet was completely dissolved and the color was changed into olive green so that all the protein components were degraded. The mixture was then incubated in a water bath or heat block at 65°C for 5 minutes. After incubation, 100 μl of isopropanol (100%) was added and mixed by inversion until DNA was precipitated as visible threads. The tube was centrifuged for 5 minutes at 10000×g. The supernatant was discarded and the pellet was dried by keeping the tube inverted state on a clean tissue paper for one minute. 100 μl of 70% ethanol was added and vortexed for 5 seconds. The tube was centrifuged for 5 minutes at 10000×g. The supernatant was carefully aspirated using a micropipette and keeping the micro-centrifuge tube in the inverted state on the tissue paper to allow the pellet to air dry for at least 5 minutes. Over-drying was avoided as the process can make it difficult to dissolve the DNA. Depending on the pellet size, 25–50 μl of nuclease free water was added and the tube was vortexed for 5 seconds and the mixture was incubated at 65°C for one hour in water bath for dissolving DNA or left overnight at room temperature. Finally, the concentration and the purity of the DNA was measured using a Nano drop machine and adjusted the concentration for PCR. 2.3.2. Polymerase Chain Reaction (PCR) amplification of TSHR  gene The isolated DNA was then amplified by PCR using TSHR gene-specific primers. At first, we performed PCR using primers set that could flank the sequence between exon 1 to exon 10, since global data showed that most of the common mutations in the TSHR  gene of the patients with Congenital Hypothyroidism were confined in this region. Next, we conducted PCR for other regions of the TSHR  gene. The primer sequences are listed in the Table 1  as follows. To amplify the desired target sequence of TSHR gene, PCR amplification was conducted on a thermal cycler (Bio-Rad, USA). The final reaction volume was 10 μl for each of the reactions which contained 1 μL 10X PCR buffer, 0.3 μL 25mM MgCl2, 2 μL 5X Q-solution, 1.6 μL 2.5 mM dNTPs mixture, 0.2 μL 10mM Forward primer and 0.2 μL Reverse primer, 0.05 μL Taq DNA Polymerase, 50 ng of genomic DNA and total reaction volume was made up to 10μL by addition of nuclease free water. The thermal cycling condition included (a) initial denaturation at 95°C for 5 minutes, (b) cyclic denaturation at 95°C for 40 seconds and annealing at 58°C for 35 seconds and extension at 72°C for 40 seconds; and (c) final extension at 72°C for 5 minutes for 35 cycles. 2.3.3. Sanger Sequencing of PCR products Before sequencing, the PCR products were purified using a Qiagen PCR purification kit (Qiagen) following manufacturer’s instruction. The cycle sequencing PCR was then performed by BigDye Chain Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, USA) applying manufacturer’s instructions. The thermal cycling profile comprised (i) initial denaturation at 94°C for 1 minute, (ii) 25 cycles of denaturation at 94°C for 10 seconds, annealing at 58°C for 5 seconds and extension at 60°C for 4 minutes, and (iii) final extension at 60°C for 10 minutes. After cycle sequencing PCR, the products were purified using BigDye XTerminator® Purification Kit (Applied Biosystems). Then, sequencing of the purified cycle sequencing products was executed on the ABI PRISM 310 automated sequencer (Applied Biosystems, USA) [ 7 ]. 2.3.4. Sequencing data analysis The Sequencing data were obtained from ABI PRISM 310 data collection software version 3.1.0. FASTA format of sequencing data were utilized to identify mutations in the TSHR  gene by alignment with the reference sequence (Accession number; NG_009206.1 retrieved from the NCBI database) through the basic local alignment search tool (BLAST). The nucleotides sequence was converted into corresponding amino acids by ExPASy translate tool [ 7 ]. 2.4. Prediction of 3D structure of TSHR protein and ligand selection After performing the Sanger Sequencing, we detected two mutations, one was in the transmembrane (TM)-domain and another was in cytoplasmic (CT)-domain of TSHR protein. TSHR protein is composed of a total of 764 amino acids where the TM-domain and CT-domain belong to 368 to 764 amino acids of the full length TSHR protein. I-TASSER server was used to predict the 3D structures of wildtype and mutant TSHR protein (TM and CT domains) due to lack of the full-length experimental structure. I-TASSER provided the five models for TSHR368-764 based on the detected template rhodopsin x-ray crystal structure (PDB:1F88) [ 6 ] by LOMETS (local meta-threading-server) from the PDB library [ 8 ]. Target-template alignment was also provided for each model structure. On the basis of Confidence score (C-score), template modelling (TM) score, root mean square deviation (RMSD) score, and target-template aligned structures, we obtained best model for each structure. The I-TASSER predicted structure was compared with AlphaFold predicted structure by TM-align algorithm which detects the best structural alignment ( https://zhanggroup.org/TM-align/ ) [ 9 ]. Two promising small molecule ligands (MS437 and MS 438) were selected which act as agonist to TSHR protein. Since among the small molecules MS437 interacts with threonine 501 (T501), and MS438 interacts with residues serine 505 (S505) and glutamic acid 506 (E506) bind to the intrahelical region of TM3 of TSHR protein [ 6 ], we targeted the region to see the effect of mutations on that particular site. 2.5 Molecular docking, protein-ligand interactions, and molecular dynamics (MD) simulation Finally, the molecular docking was performed for I-TASSER predicted and AlphaFold predicted wild-type and mutant proteins using PyRx software [ 10 , 13 , 14 ]. Grid box center was x = 72.5922; y = 72.4245; z = 72.6927 and Grid box size was 25 in every axis during docking encompassing the active site residues Thr134 (501) for MS437, and Ser138 (505) and Glu139 (506) for MS438 in the intrahelical region of TM3 of TSHR protein. The binding affinity for both I-TASSER predicted and AlphaFold predicted structures was analyzed using PyRx software and PRODIGY [ 11 , 12 ]. Non-covalent interactions were also observed both for of MS437 and MS438 molecules using BIOVIA Discovery Studio version 4.5. The MD simulation was implemented through YASARA suits [ 13 ] employing AMBER14 force field [ 14 ] for calculations. The membrane was built during simulation. YASARA scanned the plausible transmembrane region comprising hydrophobic residues among the secondary structure elements of proteins. The protein was projected to the membrane, YASARA presented the recommended membrane insertion with required size (69.2 Å × 7.3 Å) containing phosphatidyl-ethanolamine, -choline, and -serine lipid constituents. The whole simulation system was equilibrated for 250 ps. During the equilibration phase, membrane was artificially stabilized. The entire environment was equilibrated at 310K temperature with 0.9% NaCl and water solvent. The temperature was controlled by Berendsen thermostat process during simulation. The particle Mesh Ewald algorithm maintained long-range electrostatic interactions. The periodic boundary condition was applied for the whole simulation. The time step was 1.25 fs during 50 ns MD simulation. The snapshots were collected at every 100 ps [ 12 ]. Diverse data containing root mean square deviation (RMSD), root mean square fluctuation (RMSF), total number of hydrogen bonds, radius of gyration, solvent-accessible surface area (SASA), and molecular surface area (MolSA) were retrieved from MD simulations, following our earlier MD data analysis [ 13 , 14 ]. 2.6. Principal Component Analysis (PCA) MD simulation data were applied to explore the structural and energy variabilities via principal component analysis (PCA) among TSHR-small molecule ligand (MS437 and MS438) complexes. The different multivariate energy factors were employed to explore the existing variability in the MD trajectory applying the low-dimensional space [ 13 ]. The variables from MD data were bond distances, bond angles, dihedral angles, planarity, van der Waals energies, and electrostatic energies considered for the structural and energy factors [ 14 ]. The data pre-processing were implemented by centering and scaling. In the analysis, final 45 ns MD trajectories were applied for exploration of the variations. The PCA model is reproduced by the following equation: 𝑋= 𝑇𝑘𝑃𝑇𝑘 + 𝐸 Where, multivariate factors are presented into the resultant of two new matrices by X  matrix i.e. Tk  and Pk ; Tk  matrix of scores correlates the samples; Pk  matrix of loadings associates the variables, k  is the number of factors available in the model and E demonstrates the matrix of residuals. The trajectories were analysed through R, RStudio and essential codes. The R package ggplot2 was utilized for PCA plots generation. 3. Result 3.1. Investigation of mutation in TSHR  gene All the 21 patients with dysgenesis had mutation in exon 10 among a total of 11 exons in TSHR gene. The mutations we found namely, c.1523C>T (p.Ser508Leu) and c.2181G>C (p.Glu727Asp). Among the 21 patients, only one patient had mutation c.1523C>T (p.Ser508Leu) and 20 patients had the other variant c.2181G>C (p.Glu727Asp). The variants were then analyzed by bioinformatics tools to explore the pathogenic effect. Firstly, the mutations were tested by Polyphen 2, Mutation taster, and PROVEAN bioinformatics tools to see whether they possessed damaging effect or not. We found that the mutation c.1523C>T probably had damaging effect and c.2181G>C variant showed benign effect. Fig 1  represents a chromatogram showing the mutation (c.1523C>T) for the specific participant and Table 2  shows the mutations found in TSHR  gene. 3.2. Effect of mutations on predicted 3D structure of TSHR protein The I-TASSER predicted best structures were designated as wild-type TSHR368-764WT, p.Ser508Leu variant as TSHR368-764 MT1 and p.Glu727Asp variant as TSHR368-764 MT2. Table 3 shows the C-score (Confidence score) for wild-type, MT1 and MT2 was -0.71, -0.61 and -0.63, respectively. The TM score (Template Modelling score) and RMSD were 0.62±0.14, 8.4±4.5Å for TSHR368-764 WT; 0.64±0.13 and 8.2±4.5Å for TSHR368-764 MT1; 0.63±0.13 and 8.2±4.5Å for TSHR368-764 MT2. C-score indicated confidence score to assess the global accuracy of predicted models which is calculated based on the significance of threading template alignments and the convergence parameters of the structure assembly simulations. C-score of higher value [–5,2] suggests a model with a high confidence [ 8 ]. The global quality of the protein model prediction had been assessed by the TM-score. The TM-score represents the similarity between two protein structures and the accuracy of structure modeling. The TM-score (TM-score > 0.5) of the predicted proteins indicated structures were in correct global topology [ 15 ]. From the analysis of TM-score, the target and template (rhodopsin x-ray crystal structure, PDB:1F88) alignments for TSHR368-764 WT, TSHR368-764 MT1, and TSHR368-764 MT2 were 62%, 64%, and 63% respectively. The target-template superimposed structures were displayed in Fig 2A–2C . Later, I-TASSER predicted structure was compared with AlphaFold predicted structure based on analysis of their TM score (0.68) by TM-align algorithm. TM score (0.68) indicated AlphaFold and I-TASSER predicted structures were in same fold with correct global topologies (TM score>0.5). AlphaFold and I-TASSER both provide highly accurate structures [ 8 , 16 , 17 ] while I-TASSER ( https://zhanggroup.org/I-TASSER/ ) got recognition as the No 1 server for protein structure prediction in community-wide CASP experiments. Hence, I-TASSER predicted models were utilized for further analysis. Moreover, the full length TSHR protein predicted by AlphaFold was shown ( Fig 2D ) and the structural alignment of TSHR368-764 for AlphaFold, and I-TASSER was shown in superimposed pose ( Fig 2E ). Fig 3  depicts the structures of the TSHR protein and the small molecules MS437 and MS438. 3.3. Molecular docking and protein-ligand interactions of MS437 and MS438 with TSHR proteins (wild-type and mutant) The structures of the small molecules MS437 and MS438 were optimized. After molecular docking best docking poses for the protein-ligand complexes were selected evalutating their binding affinity and interaction. The binding affinities for the small molecules were obtained from both PyRx software and PRODIGY. The Table 4  showed that the binding affinities of the wild -type TSHR protein (-6 kcal/mol, -5.45 kcal/mol for TSHR368-764WT) were higher compared to the mutant cases (-4.8 kcal/mol, -5.28 kcal/mol for TSHR368-764 MT1 and -5.7 kcal/mol, -5.27 kcal/mol for TSHR368-764 MT2) in both PyRx software and PRODIGY. Also, the binding affinities for the small molecules were obtained from both PyRx software and PRODIGY for AlphaFold predicted structures and the values were included in Table 4 . Total non-covalent interactions were 11, 19 and 12 for wild-type, MT1 and MT2, respectively ( Table 5 ). MS437 bound to Threonine 501 and MS438 bound to Serine 505 and Glutamic acid 506 of transmembrane helix3 (TMH3) in full length TSHR protein [ 6 ] with corresponding amino acid position Thr134, Ser138 and Glu139, respectively in TM-region ( Fig 3 ). We tried to investigate whether these crucial amino acids could interact with small molecule thyrogenic drugs. We found that in case of MS437 none of these three amino acids could interact with both wild-type and mutant cases. On the other hand, MS438 interacted with all the crucial amino acids including Thr134, Ser138 and Glu139 for wild-type case and for the mutant cases (TSHR368-764 MT1 and TSHR368-764 MT2), it could interact only with Ser138. The binding affinities were -7.1 kcal/mol; -5.59 kcal/mol, -5.4 kcal/mol; -5.77 kcal/mol, and -2.6 kcal/mol; -5.50 kcal/mol for TSHR368-764WT, TSHR368-764 MT1, and TSHR368-764 MT2, in both PyRx software and PRODIGY respectively. In Table 6 the binding affinity of wild-type protein was much higher for MS438 than mutants. Also, the binding affinities for the small molecules were obtained from both PyRx software and PRODIGY for AlphaFold predicted structures and the value were included in Table 6 . All the non-covalent interactions ( Table 7 ) were depicted in Figs ​Figs44  and ​and55  for MS437 and MS438 respectively. 3.4. Molecular dynamics simulation MD simulation was performed for each complex of TSHR368-764WT, TSHR368-764 MT1, and TSHR368-764MT2 with two designated drugs (MS437 and MS438) for 50 ns time range. In case of MS437 ( Fig 6 ), the RMSDs for TSHR368-764MT2 (0.973–4.965 Å) displayed less fluctuations for α-carbon atoms than TSHR368-764WT (0.906–5.91 Å), and TSHR368-764 MT1 (0.939–5.504 Å) in Fig 6A . Thus, suggesting that, comparatively MS437-TSHR368-764 MT2 was stable in physiological conditions, while more fluctuations were visible in TSHR368-764WT at 43.8 ns (RMSD 5.91 Å) and TSHR368-764 MT1 till 26.5 ns (RMSD ⁓5.2 Å). The Rg manifested quite similar pattern in TSHR368-764 MT1 and TSHR368-764 MT2. However, TSHR368-764WT exhibited more fluctuations initially up to 5.5 ns and later from 46 ns. The average value remained ⁓25.40 Å for the three protein complexes. However, low compactness in ligand-mutant complexes was observed during simulation ( Fig 6B ). In case of SASA, more deviations were found in TSHR368-764WT (18587.716–24360.945 Å2) compared to TSHR368-764MT1 (18106.599–22314.102 Å2) and TSHR368-764 MT2 (19148.648–22851.2 Å2) complexes. However, TSHR368-764 MT1 and TSHR368-764 MT2 manifested some deviations at 7–19 ns, and 24–43 ns during simulation. Overall, mutant structures TSHR368-764 MT1 and TSHR368-764 MT2 were more stable as MS437 bound complexes than TSHR368-764WT ( Fig 6C ). For MolSA in Fig 6D , TSHR368-764WT showed (21208.043–25437.671 Å2) much fluctuations in the whole run than TSHR368-764MT1 (21135.321–24580.781 Å2) and TSHR368-764 MT2 (21739.906–24397.155Å2). TSHR368-764WT was unstable, but TSHR368-764 MT1 and TSHR368-764 MT2 were more stable as the ligand bound complexes in physiological condition. The RMSF value deviated most for TSHR368-764WT in between 1(368)-30(397) and 326(693)-397(764) residues, where, TSHR368-764 MT1 exhibited more fluctuations in 1(368)-47(415) residues than TSHR368-764 MT2. Overall, TSHR368-764 MT2 was more stable as a complex than others due to least deviation in the whole run ( Fig 6E ). The total number of hydrogen bonds indicated structural rigidity of protein. In case of TSHR368-764WT high frequency of hydrogen bonds (average ⁓633) was observed during interaction while TSHR368-764 MT1 exhibited average 593, and TSHR368-764 MT2 manifested average 600 hydrogen bonds. Among the mutant structures, TSHR368-764 MT2 displayed more structural stability in the simulation ( Fig 6F ). During simulation, for MS438 ( Fig 7 ), the RMSD values of α-carbon atoms remained ⁓5.251 Å in TSHR368-764-WT, ⁓5.53 Å in TSHR368-764 MT1, and ⁓5.39 Å in TSHR368-764 MT2. The fluctuations had been observed in TSHR368-764 MT1 and TSHR368-764 MT2 during 30–50 ns while least was found in TSHR368-764-WT. However, the average RMSD values of all the complexes were almost close ( Fig 7A ). The Rg manifested quite high deviations among three complexes. However, TSHR368-764 MT1 exhibited maximum 27.068 Å, which indicated higher stability than other complexes ( Fig 7B ). The SASA values remained close among three complexes. However, TSHR368-764 WT manifested least deviations during 10–20 ns and 30–50 ns ( Fig 7C ). In case of MolSA, the graphical patterns for three complexes were almost same through the whole MD run ( Fig 7D ). The RMSF value more diverged in TSHR368-764 MT2 till first 15 residues while least deviation was observed for TSHR368-764 WT through whole run. However, three complexes showed quite similar pattern between 130(498)-240(608) residues ( Fig 7E ). The highest number of hydrogen bonds (about 678) was observed for TSHR368-764 MT1 while TSHR368-764 WT exhibited about 669 hydrogen bonds and TSHR368-764 MT2 showed almost 665 to maintain stable conformation. Thus, TSHR368-764MT1 showed highest structural stability among the complexes ( Fig 7F ). Moreover, we had visualized the binding pattern of MS437 and MS438 ligands with the wild-type and mutants TSHR368-764 through the snapshots from MD simulation ( Fig 8 ). In simulation, MS437 exhibited persistent interaction with the residues LEU302(669), ALA306(673), LEU310(677) of TSHR368-764MT2. The MS438 ligand mostly showed stable interactions with the residues LEU100(467), VAL135(502), SER138(505), LEU203(570), PRO204(571), LYS293(660). Both ligands remained within the binding site in stable mutant proteins. 3.5. Principal Component Analysis (PCA) Two PCA models were generated for structural and energy profiles of the protein-ligand complexes to assess and realize the dissimilarities among wild-type and mutant proteins during MD simulation. The scores plot for MS437-protein ( Fig 9A ) and MS438-protein ( Fig 9C ) complexes had exhibited the different clusters for the wild-type and mutants of TSHR368-764. It was observed that in both protein-ligand complexes, TSHR368-764WT and TSHR368-764MT1 were remotely situated. Consequently, pathogenic TSHR368-764 MT1 was liable for the differences. However, TSHR368-764WT and TSHR368-764 MT2 were overlapped. The loading plots ( Fig 9B and 9D ) demonstrated that bond, bond angles, van der Waals energies, and dihedral angles were closely distributed and displayed quite similar graphical pattern. The distribution mainly contributed for PC1 variance while coulomb energy difference contributed to PC2 variance. In MS437-protein complexes, the total 92.7% of the variance had been unveiled by PC1 and PC2, where PC1 expressed 76.1% and PC2 expressed 16.6% of the variance. Moreover, in MS438-protein complexes, the total 88.3% of the variance had been disclosed by PC1 and PC2, where PC1 expressed 72.1% and PC2 expressed 16.3% of the variance. 4. Discussion The newborn screening for endocrine disorders is not frequently practiced in Bangladesh. In this study, we focused on the etiology of dysgenesis types of Congenital Hypothyroid patients having small glands or ectopic gland or agenesis (absent of thyroid gland). Different studies suggested that TSHR  was the major gene responsible for growth and development of thyroid gland [ 2 , 18 ]. TSH binds to the receptor and creates the signaling pathway through G-protein coupled-receptor and Cyclic AMP-mediated adenylate cyclase. The full-length protein structure of TSHR is still under investigation through crystallography. The available structures do not include the all-cytoplasmic residues. Mutations in the TSHR  gene results from loss or gain of function of the protein that causes different phenotypic variations and lead to hyperthyrotopinemia to severe Congenital Hypothyroidism [ 18 , 19 ]. Analysis of TSHR gene showed that two mutations were found, namely c.1523C>T and c.2181G>C in the patients and we analyzed the effect of mutations by using different bioinformatics tools. Almost all the tools such as Polyphen 2, Mutation Taster and PROVEAN were very much popular to analyze the mutational effect. The mutation c.1523C>T was found to be damaging, disease causing or deleterious and c.2181G>C was found to be benign or neutral. Since MS437 and MS438 are thyrogenic potent molecules, we selected these two molecules as ligands for molecular docking with the wild-type and mutant structures of TSHR protein [ 6 ]. The molecular docking analysis showed that the binding affinity for both of the ligands with mutant cases was decreased compared to the wild-type TSHR protein. MD simulation indicated that, the RMSDs for MS437-TSHR368-764 MT2 (average 4.37Å) showed less deviations for α-carbon atoms. Thus, proposing the complex as most stable in biological environments. However, MS438-protein complexes manifested quite close average RMSD values during simulation. The Rg of MS437-TSHR368-764WT exhibited more instabilities at start and end of the simulation. Conversely, TSHR368-764 MT1 and TSHR368-764 MT2 displayed quite similar pattern of lesser compactness as well as more stability for interaction with MS437. In case of MS438, TSHR368-764 MT1 exhibited highest Rg value, which identified higher stability than other complexes. The analysis of SASA and MolSA values revealed that TSHR368-764 MT1 and TSHR368-764 MT2 mutant structures were more stable in their complex form with MS437 than TSHR368-764WT. However, the SASA presented close pattern and MolSA exhibited almost same graphical pattern among the three complexes for the interaction with MS438. In case of hydrogen bonds, MS437-TSHR368-764MT2 manifested average 600 hydrogen bonds which was close to TSHR368-764WT (average ⁓633). The complex was more stable than the other mutant. On the other hand, MS438-TSHR368-764 MT1 displayed maximum structural stability compared to other complexes. Considering RMSF values, MS437 rendered more stability to TSHR368-764MT2 than others. The RMSF value more diverged in TSHR368-764MT2 while minimum deviance was detected for TSHR368-764-WT and TSHR368-764 MT1 mostly remained between both complexes. However, three complexes displayed almost similar stability between 130(498)-240(608) residues while interacting with MS438. Moreover, PCA analysis for MS437-protein and MS438-protein complexes had revealed the existing differences among structural and energy profiles of the structures. It was observable that TSHR368-764 MT1 exhibited much variations than TSHR368-764WT and TSHR368-764 MT2, emphasizing more damaging pattern in TSHR368-764 MT1. In the study, we had utilized allosteric ligands MS437 and MS438 as agonists against the identified mutants for TSHR368-764. These two ligands had ‘drug-likeness’ as well as previously confirmed their efficacy by conducting in vivo  animal studies [ 6 ]. The agonists (MS437 and MS438) displayed different binding sites in the TSHR protein [ 6 ]. After analyzing all data, it can be proposed that low-affinity binding infers, a comparatively high concentration of the ligands can maximally occupy the binding sites to achieve maximum physiological response. Moreover, modifying chemical properties or ligands with novel scaffolds targeting signal-sensitive amino acids surrounding the allosteric binding sites might lead to design agonists with even higher efficiency to activate TSHR [ 19 ]. 5. 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Written by Hai-Yang Zhang, M.D.,#1,*  Feng-Yao Wu , M.D.,#1,*  Xue-Song Li , M.D.,2  Ping-Hui Tu , M.D.,1  Cao-Xu Zhang , M.D.,1  Rui-Meng Yang , M.D., Ph.D.,1  Ren-Jie Cui , Ph.D.,1  Chen-Yang Wu , M.D.,1  Ya Fang , M.D., Ph.D.,1  Liu Yang , M.S.,1  Huai-Dong Song , M.D., Ph.D.,1 and Shuang-Xia Zhao , M.D., Ph.D.1 Ann Lab Med. 2024 Jul 1; 44(4): 343–353. Published online 2024 Mar 4.   doi:  10.3343/alm.2023.0337 Abstract Background Genetic defects in the human thyroid-stimulating hormone (TSH) receptor ( TSHR ) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype–phenotype relationships for most TSHR  variants associated with CH remain unexplored. We aimed to identify TSHR  variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR  genotypes and clinical phenotypes. Methods In total, 367 patients with CH were recruited for TSHR  variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results Among the 367 patients with CH, 17 TSHR  variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR  variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR  biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2  biallelic variants. Conclusions We found a high frequency of TSHR  variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR  biallelic variants is relatively mild. Our study expands the TSHR  variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH. Keywords: Congenital hypothyroidism, Recessive inheritance, Thyroid-stimulating hormone receptor, Variant, Whole-exome sequencing INTRODUCTION Congenital hypothyroidism (CH) is a disease characterized by impairments in neurodevelopment and physical growth and development owing to dysfunction of the hypothalamic-pituitary-thyroid axis present at birth [ 1 ]. CH is the most common congenital endocrine metabolic disease, with a global prevalence of 1/2,000–1/3,000 [ 1 ]. With the recent developments in gene sequencing technologies, an increasing number of pathogenic genes related to CH, including genes related to thyroid dysgenesis and dyshormonogenesis, have been reported. Among these, the thyroid-stimulating hormone (TSH) receptor ( TSHR ) gene is one of the widely investigated candidate genes [ 2 , 3 ]. The human TSHR gene is located on chromosome 14q31 and encodes a G-protein-coupled receptor that consists of a seven-transmembrane domain (TMD) and a large extracellular domain (ECD) responsible for high-affinity hormone binding. The TSHR is activated upon binding to TSH and induces two signal transduction pathways: the Gs/cyclic AMP (cAMP) pathway and the Gq/11 phospholipase C pathway, which contribute to thyroglobulin iodination and cell proliferation, whereas the Gs pathway is also responsible for iodine uptake regulation in thyrocytes [ 4 , 5 ]. Both pathways are important for thyroid hormone synthesis and thyroid development [ 4 , 6 ]. Loss-of-function (LOF) variants in the TSHR can cause TSH resistance, which leads to congenital nongoitrous hypothyroidism 1 (OMIM: 275200), which presents a broad spectrum of phenotypes, ranging from severe congenital hypothyroidism to mild euthyroid hyperthyrotropinemia [ 2 , 7 - 10 ]. These LOF variants may result in a thyroid gland of normal position and size or in thyroid dysgenesis [ 11 ]. LOF variants in TSHR  were first described in patients with TSH resistance in 1995 [ 12 ]. Up to April 2021, 202 TSHR variants have been reported and documented in the Human Gene Mutation Database (HGMD). However, the biological functions of most TSHR  variants remain unknown, and genotype–phenotype relationships have not yet been clearly established. We previously identified 15 TSHR  variants in 13 out of 220 Chinese patients with CH [ 13 ]. In the present study, we enrolled an additional 367 patients with CH, expanding the sample for screening TSHR  variants and characterizing the phenotypes of patients with CH carrying TSHR  variants. The biological functions of the variants were investigated through a series of in vitro  experiments. We expected this study to deepen our understanding of the genetic landscape and functional consequences of TSHR  variants and to provide valuable insights into the clinical management of patients harboring TSHR  variants. MATERIALS AND METHODS Patients In total, 367 patients were enrolled from the Chinese Han populations in Jiangsu province, Fujian province, Anhui province, and Shanghai. Among them, 362 patients (98.7%) received neonatal CH screening, which was performed using filter-paper blood spots (obtained through a heel prick) within 3–5 days after birth. Patients with TSH levels ≥10 μIU/mL at initial screening were recalled for re-examination using an immune-chemiluminescence assay (UniCelDxI 800; Beckman, Indianapolis, IN, USA) to determine the levels of TSH, free triiodothyronine (FT3), and free thyroxine (FT4). The details of the diagnostic standards for CH have been described in our previous study [ 14 ]. In addition, five patients who were on l-thyroxine replacement therapy were recruited from outpatient clinics. Although these patients were not neonatally screened, they had a clear history of CH. Thyroid morphology was determined by experienced radiologists through thyroid ultrasound or technetium-99m scanning. Written informed consent to participate was provided by the participants’ legal guardians, and the study was approved by the Ethics Committee of Shanghai Ninth People’s Hospital affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China (approval number: 2016-76-T33). Whole-exome sequencing (WES) WES was performed as previously described [ 15 ]. Genomic DNA was extracted from peripheral blood, fragmented to 200–300 bp, and ligated to adapters using the KAPA HyperPrep Kit (Roche, Basel, Switzerland). Exonic hybrid capture was performed according to the instructions in the Roche SeqCap EZ Library SR User’s Guide. Library quality and levels were determined using the MAN CLS140145 DNA 1 K Chip (PerkinElmer, Waltham, MA, USA) and the PE LabChip GXII Touch (PerkinElmer, Waltham, MA, USA). The Illumina HiSeq 3000 system (Illumina, San Diego, CA, USA) was then used to sequence the paired-end libraries with 150-bp paired-end reads, averaging approximately 100× depth. Statistical analysis IBM SPSS Statistics version 25.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Quantitative variables are presented as mean±SE. The normality of the data was assessed using the Shapiro–Wilk test and intergroup comparisons were performed using Student’s unpaired t -test (for normally distributed data) or the Mann–Whitney U test (for non-normally distributed data), as appropriate. Categorical variables are presented as percentages and were compared using the chi-square test or Fisher exact test, as appropriate. P <0.05 was considered statistically significant. Additional methods are available in the Supplemental Materials . RESULTS Clinical characteristics of 367 patients with CH In total, 367 patients with CH, including 196 boys and 171 girls, were recruited in this study. The mean serum FT3, FT4, and TSH levels at diagnosis were 4.87 pmol/L, 10.55 pmol/L, and 77.57 μIU/mL, respectively (reference ranges: FT3, 3.85–6.01 pmol/L; FT4, 7.46–21.11 pmol/L; TSH, 0.34–5.60 μIU/mL). Based on the serum FT4 level at diagnosis, CH was classified as severe (FT4<5 pmol/L), moderate (5 pmol/L≤FT4<10 pmol/L), or mild (FT4≥10 pmol/L) [ 16 ]. In the present cohort, 49.1% of patients had moderate or severe CH, and 50.9% of patients presented with mild CH. There were no significant differences in hormone levels and other clinical characteristics between boys and girls ( Table 1 ). Screening for TSHR  variants in Chinese patients with CH and pedigree analysis Among the 367 patients with CH, 45 patients carried 17 non-synonymous TSHR  variants, including 16 missense variants and one nonsense variant. The TSHR variant frequency was 12.3% (45/367). Out of 17 variants, three (p.S237G, p.W546C, and p.M728T) were first reported in this study, and 10 were recurrent variants (p.G132R, p.G245S, p.S305R, p.N432S, p.R450H, p.F525S, p.R609X, p.Y613C, p.V689G, and p.E758K). p.R450H, which is a hotspot variant in the Chinese population, had the highest frequency (2.7%) ( Table 2 ). Four of the 17 variants were located in the leucine-rich repeat (LRR) domain of the TSHR protein ( Fig. 1A ). Conservation analysis of the three novel variants showed that p.W546C was highly conserved across species, whereas p.M728T was less conserved ( Fig. 1B ). Out of the 45 patients with TSHR  variants, 18 patients carried biallelic variants. We conducted a long-term follow-up of two patients with TSHR biallelic variants (CHT558 and CHT573) and collected blood samples from their parents for pedigree analysis. Sanger sequencing showed that the biallelic variants carried by the patients were inherited from their father and mother separately, which is in line with an autosomal recessive inheritance pattern ( Fig. 2 ). Pathogenicity prediction of detected TSHR  variants The potential effects of the 17 variants identified were assessed using in silico programs (SIFT, Polyphen-2, Mutation Taster, and M-CAP). All four programs predicted that the variants p.I216T, p.G245S, p.A275T, p.N432S, p.R450H, p.A526T, p.R531W, p.W546C, p.Y613C, and p.V689G were detrimental to TSHR protein function and that the novel p.M728T variant was harmless. The prediction results of the other variants were inconsistent among the four programs ( Supplemental Data Table S1 ). Subsequently, we predicted the three-dimensional structure of the wild-type (WT) and three novel mutant proteins using in silico tools. In the novel variant p.S237G, a polar neutral serine was replaced with a non-polar aliphatic glycine, disrupting the hydrogen bond between serine 237 and lysine 211 ( Supplemental Data Fig. S1A ). For the p.W546C variant, the aromatic tryptophan residue at 546 was mutated to a neutral cysteine, disrupting the hydrogen bond between tryptophan 546 and asparagine 455, destabilizing the helix ( Supplemental Data Fig. S1B ). As for p.M728T, the model confidence at amino acid 738 was very low; therefore, it was not analyzed. The American College of Medical Genetics (ACMG) issued new guidelines for the interpretation of sequence variants in 2015, describing a process for classifying variants into five categories based on criteria related to typical variant evidence types (such as population data, computational data, functional data, and segregation data). Variants are classified as pathogenic (P), likely pathogenic (LP), variants of uncertain significance (VUS), likely benign (LB), or benign (B) [ 17 ]. Based on the available evidence, the pathogenicity of the 17 variants identified was classified according to the ACMG guidelines and standards. Five variants (p.G132R, p.N432S, p.R450H, p.F525S, and p.R609X) were classified as P or LP, and p.M728T was classified as LB. The remaining 11 variants were classified as VUS ( Supplemental Data Table S1 ). Clinical characteristics of CH patients with TSHR  variants The clinical phenotypes of the 45 CH patients with TSHR  variants were compared with those of the 322 CH patients without TSHR variants. There were no significant differences between the two groups in terms of hormone levels, age at diagnosis, and initial levothyroxine dose ( Supplemental Data Table S2 ). Thyroid functional information at diagnosis was collected for 25 out of 45 patients who harbored TSHR  variants, including four patients with severe CH, seven with moderate CH, and 14 with mild CH. One patient (CHT241) harboring TSHR  variants had thyroid dysgenesis ( Supplemental Data Table S3 ). Among the seven patients with TSHR biallelic variants, only one patient showed moderate CH, and the remaining six patients presented with mild CH. However, patients with the TSHR  monoallelic variant can present with mild to severe CH. Surprisingly, we found that the patients with the TSHR  monoallelic variant had more severe hypothyroidism, with lower FT4 levels (9.58±1.50 vs. 15.87±1.18, P =0.020) at diagnosis, than patients with TSHR  biallelic variants ( Fig. 3A–3C ). Dual oxidase 2 ( DUOX2 ) is a key protein for thyroid hormone synthesis, and DUOX2  is the most frequently mutated gene in Chinese patients with CH [ 14 , 18 ]. We compared the clinical characteristics of patients with TSHR  or DUOX2  biallelic variants in the present cohort. Compared with CH patients with DUOX2  biallelic variants, patients with TSHR  biallelic variants had lower serum TSH levels and higher FT3 and FT4 levels at diagnosis (TSH: 52.96±17.84 vs. 105.77±5.48, P =0.012; FT3: 5.71±0.43 vs. 4.47±0.15, P =0.025; FT4: 15.87±1.18 vs. 7.20±0.45, P <0.001) ( Fig. 3D–3F ). In patients with DUOX2  variants, hypothyroidism may vary with age, whereas in patients with TSHR variants, it tends to remain stable over time. Therefore, we compared thyroid function at 6 months and 3 yrs of age between patients carrying TSHR  biallelic variants and those carrying DUOX2  biallelic variants. Interestingly, patients harboring TSHR  biallelic variants exhibited higher FT4 levels at both 6 months and 3 yrs of age than patients carrying DUOX2  biallelic variants (6 months: 20.60±1.27 vs. 16.77±0.65, P =0.041; 3 yrs: 22.72±1.73 vs. 16.83±0.96, P =0.019) ( Supplemental Data Fig. S2 ). Functional assessment of the TSHR  variants in vitro The eight variants (p.G132R, p.I216T, p.G245S, p.N432S, p.R450H, p.F525S, p.A526T, and p.V689G) detected in 18 patients with TSHR biallelic variants and the three novel variants (p.S237G, p.W546C, and p.M728T) were selected for molecular function assessment. The variants were transiently transfected into 293T cells, and Gs/cAMP and Gq/11 signal transduction were investigated by measuring cAMP levels and luciferase activity, respectively. Compared with 293T cells transfected with the WT plasmid, cAMP production in response to bovine TSH (bTSH) was significantly reduced in cells transfected with the p.G132R, p.I216T, p.S237G, p.G245S, p.N432S, p.R450H, p.F525S, p.A526T, and p.W546C mutant plasmids. However, the p.V689G and p.M728T variants did not affect cAMP production ( Fig. 4A ). The p.G132R, p.I216T, p.S237G, p.G245S, p.F525S, p.A526T, p.W546C, and p.V689G variants showed partial Gq/11 signaling activity (14%–57%), whereas activity was almost abrogated for the p.N432S and p.R450H variants (<10%) after stimulation with 100 U/L bTSH. The p.M728T variant had no effect on Gq/11 signaling ( Fig. 4B ). We next investigated the protein expression and subcellular localization of the three novel variants (p.S237G, p.W546C, and p.M728T) in 293T cells. Western blot analysis showed no significant differences in protein expression between the WT and the three mutants ( Supplemental Data Fig. S3A and 3B ). Subcellular localization analysis showed that the WT and three mutant TSHR proteins all localized to the cell membrane in an intact manner ( Supplemental Data Fig. S3C ). DISCUSSION Through comprehensive screening, we identified 17 distinct TSHR  variants in 367 CH patients in China. We found a high frequency of TSHR  variants in Chinese patients with CH (45/367, 12.3%), with 4.9% of patients carrying biallelic TSHR variants. We identified three novel variants (p.S237G, p.W546C, and p.M728T), two of which (p.S237G and p.W546C) impaired TSHR biological functions in the Gs/cAMP and Gq/11 pathways. Seventeen non-synonymous TSHR variants were identified in 45 CH patients, with a detection rate of 12.3%, which is higher than the rates reported in most domestic studies [ 13 , 19 - 21 ] but lower than those in two cohort studies in Italy and Korea [ 22 , 23 ]. Most TSHR  LOF variants reported to date are located in exons 1, 4, 6, and 10 [ 11 ]. In this study, 12 of the 17 identified variants were located in exon 10, whereas none were located in exons 1, 4, and 6. These findings suggest that there may be regional and ethnic differences in the spectrum of TSHR  variants. In addition, we found a hotspot variant, p.R450H, which is one of the most common TSHR  LOF variants and has been demonstrated to have a founder effect in Japan [ 24 ]. Notably, among the 45 patients carrying TSHR  variants, 18 carried biallelic variants. The total residual Gs/cAMP and Gq/11 pathway signaling activities in CH patients with TSHR  biallelic variants were calculated as the sum of pathway signaling activities from both TSHR variant alleles divided by two. Two patients (CHT385 and CHT573) harboring the p.R450H homozygous variant, who had 35% Gs/cAMP signaling pathway activity and 6% Gq/11 signaling pathway activity, had clinically similar phenotypes and presented with mild hypothyroidism. Patient CHT506, who carried the p.G132R homozygous variant, had a 62% reduction in Gs/cAMP signaling pathway activity and 70% Gq/11 signaling pathway activity and was diagnosed as having moderate CH, with serum TSH and T4 levels of 150.00 μIU/mL and 9.27 pmol/L, respectively. Patient CHT436, who harbored the p.N432S/p.R450H biallelic variants with residual Gs/cAMP and Gq/11 signaling pathway activities of 32% and 8%, respectively, presented with mild CH, with serum TSH and FT4 levels of 27.26 μIU/mL and 18.66 pmol/L. Patients CHT445 and CHT516 carried the p.G132R/p.R450H biallelic variants, with residual Gs/cAMP and Gq/11 signaling pathway activities of 37% and 18%, respectively. They both exhibited mild CH. Patient CHT553 harboring the p.R450H/p.F525S biallelic variants had 30% and 32% residual Gs/cAMP and Gq/11 signaling pathway activities, respectively, and was diagnosed as having mild CH, with serum TSH and FT4 levels of 25.56 μIU/mL and 15.96 pmol/L, respectively ( Supplemental Data Table S4 ). These functional experimental results in patients with TSHR  biallelic variants support the hypothesis that TSHR  variants can cause the onset of CH. Pedigree analysis of two patients showed that CH caused by TSHR  variants is inherited in an autosomal recessive manner, which is consistent with previous findings [ 12 , 25 , 26 ]. LOF TSHR variants result in variable TSH resistance manifested as euthyroid hyperthyrotropinemia with a normal thyroid gland (fully compensated TSH resistance), mild hypothyroidism with a normal thyroid gland (partially compensated TSH resistance), or severe hypothyroidism with thyroid dysgenesis (uncompensated TSH resistance) [ 2 ]. In the present study, out of seven patients with TSHR biallelic variants, six patients presented with mild hypothyroidism, and only one patient presented with moderate hypothyroidism. Moreover, patients with TSHR  biallelic variants had milder hypothyroidism than those with DUOX2  biallelic variants. These findings indicate that the phenotypes of CH caused by TSHR  defects are milder and associated with completely or partially compensated TSH resistance. The TSHR is a G-protein-coupled receptor with a TMD domain and a large ECD, which comprises an LRR domain involved in hormone binding specificity and a hinge region, linking the LRR domain to the TMD [ 11 , 27 ]. TSHR activation results in intracellular signaling via the Gs protein, which leads to cAMP cascade activation, and via the Gq protein, which leads to phospholipase C cascade activation. In the present study, four variants (p.G132R, p.I216T, p.S237G, and p.G245S) were located in the LRR domain, and they caused varying degrees of impairment to the Gs/cAMP and Gq/11 signaling pathways. The novel p.S237G variant had no effect on the expression and membrane localization of the TSHR protein but partially hindered Gs/cAMP and Gq/11 signaling. This may be attributed to the replacement of amino acids altering the TSHR protein structure, thereby decreasing its ability to bind to TSH. The novel p.W546C variant, located in the fourth TMD of TSHR, is highly conserved among species. In silico  tools predicted that this missense variant is detrimental to protein stability and function. In vitro experiments demonstrated that the p.W546C variant damages receptor function by affecting the Gs/cAMP and Gq/11 signaling pathways. p.M728T, another novel variant identified in the present study, is located in the C-terminal intracellular region of TSHR. Chazenbalk, et al . [ 28 ] confirmed that the removal of the C-terminal 2/3 residues (Q709–L764) of TSHR did not impair receptor function. Concurrently, functional experiments in the present study showed that the p.M728T variant did not interfere with the Gs/cAMP or Gq/11 pathway. Numerous studies have confirmed that the hotspot variant p.R450H not only results in reduced cAMP activity and severely impaired Gq/11 pathway activity but also reduces the TSH binding ability of TSHR [ 5 , 13 , 24 , 29 ], which is consistent with our findings. The phenotypes of the TSHR  monoallelic variant are reportedly always mild, whereas biallelic variants are often associated with a more severe phenotype [ 7 ]. However, we found that patients with the TSHR monoallelic variant presented with mild to severe CH. Therefore, we compared thyroid function at diagnosis in patients with monoallelic and biallelic TSHR  variants. Surprisingly, patients with the TSHR monoallelic variant had lower FT4 levels, which may be because of the following reasons. First, in our cohort, 12 of 27 patients with the TSHR  monoallelic variant harbored biallelic variants in 21 other CH pathogenic genes ( NKX2-1 , NKX2-5 , FOXE1 , PAX8 , HHEX , TPO , SLC5A5 , TG , DUOX2 , DUOXA2 , TSHR , SLC26A4 , IYD , DIO1 , DIO2 , THRA , THRB , DUOX1 , DUOXA1 , GNAS , and SLC16A2 ) as described in our previous study [ 14 ] ( Supplemental Data Table S3 ). Compared with patients with TSHR  biallelic variants, patients with oligogenic variants, including in TSHR , had lower FT4 levels and higher TSH levels at diagnosis, whereas patients with only a TSHR  monoallelic variant showed no difference in thyroid function at diagnosis ( Supplemental Data Fig. S4 ), which partially explains why patients with the TSHR  monoallelic variant presented more severe hypothyroidism than those with TSHR  biallelic variants. Second, the genetic etiology of CH is largely unknown, and patients with the TSHR monoallelic variant may also carry novel CH-causative genes, leading to a more severe phenotype. Finally, environmental modifiers, such as iodine intake and ethnicity, should be considered in addition to genetic factors to explain this phenotypic variation. For example, Vigone, et al . [ 30 ] reported phenotypic differences between two brothers harboring the same genetic variants attributed to different neonatal iodine supplies, which suggested that the different neonatal iodine supplies acted as disease modifiers. This study had some limitations. First, among the 18 patients carrying biallelic TSHR  variants, pedigree analysis was conducted for only two families. Second, we did not clarify whether patients carrying TSHR variants require lifelong thyroxine therapy. Third, the pathogenic mechanism related to the presence of a heterozygous sequence variant in TSHR in patients with CH was not fully identified. In future work, we will mine and analyze unknown pathogenic genes in CH to gain insight into the molecular mechanisms of CH pathogenesis. In conclusion, we reported 17 TSHR variants in 367 Chinese patients with CH and investigated the biological function of 11 variants (eight biallelic and three novel variants). Two novel variants (p.S237G and p.W546C) impair TSHR protein biological function by interfering with Gs/cAMP and Gq/11 signaling. Characterization of the phenotypes of patients with TSHR  variants revealed that TSHR  biallelic variants cause mild CH. The present study expanded the TSHR  variant spectrum and provided further evidence for the elucidation of the genetic etiology of CH. References 1. van Trotsenburg P, Stoupa A, Léger J, Rohrer T, Peters C, Fugazzola L, et al. 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Written by Hopkins Medicine https://www.hopkinsmedicine.org/health/conditions-and-diseases/hashimotos-thyroiditis Original language: English Thyroiditis is when your thyroid gland becomes irritated. Hashimoto's thyroiditis is the most common type of this health problem. It is an autoimmune disease. It occurs when your body makes antibodies that attack the cells in your thyroid. The thyroid then can't make enough of the thyroid hormone. Many people with this problem have an underactive thyroid gland. That's also known as hypothyroidism. They have to take medicine to keep their thyroid hormone levels normal. What is the cause of Hashimoto’s thyroiditis? Hashimoto's thyroiditis is an autoimmune disorder. Normally, your autoimmune system protects your body by attacking bacteria and viruses. But with this disease, your immune system attacks your thyroid gland by mistake. Your thyroid then can't make enough thyroid hormone, so your body can't work as well. Who is at risk for Hashimoto’s thyroiditis? Things that may make it more likely to you for to get Hashimoto’s thyroiditis are: Being a woman. Women are about 7 times more likely to have the disease. Hashimoto's thyroiditis sometimes begins during pregnancy. Middle age. Most cases happen between 40 to 60 years of age. But it has been seen in younger people. Heredity. The disease tends to run in families. But no gene has been found that carries it. Autoimmune diseases. These health problems raise a person’s risk. Some examples are rheumatoid arthritis and type 1 diabetes. Having this type of thyroiditis puts you at higher risk for other autoimmune illnesses. What are the symptoms of Hashimoto's thyroiditis? Each person's symptoms may vary. Symptoms may include: Goiter This is an enlargement of your thyroid gland. It causes a bulge on your neck. It is not cancer. But it can cause problems like pain or trouble with swallowing, breathing, or speaking. Underactive thyroid When your thyroid doesn’t make enough thyroid hormone, it can cause these symptoms: Tiredness Muscle weakness Weight gain Being cold bothers you Depression Hair and skin changes Overactive thyroid When the thyroid is attacked by antibodies, it may at first make more thyroid hormone. This is called Hashitoxicosis. It does not happen to everyone. But it can cause these symptoms: Being hot bothers you Rapid heart rate Sweating Weight loss Tremors Anxiety These symptoms may look like other health problems. Always see your healthcare provider for a diagnosis. How is Hashimoto thyroiditis diagnosed? Your healthcare provider will ask about your medical history and give you a physical exam. You will also have blood tests. These can measure your thyroid hormone levels and check for some antibodies to proteins in the thyroid. How is Hashimoto's thyroiditis treated? Your healthcare provider will figure out the best treatment for you based on: Your age, overall health, and medical history How sick you are How well you handle certain medicines, treatments, or therapies If your condition is expected to get worse Your opinion or preference You will not need treatment if your thyroid hormone levels are normal. But Hashimoto's thyroiditis often looks like an underactive thyroid gland. If so, it can be treated with medicine. The medicine replaces lost thyroid hormone. That should stop your symptoms. It can also ease a goiter if you have one. A goiter can cause problems like pain or trouble swallowing, breathing, or speaking. If these symptoms don't get better, you may need surgery to remove the goiter. When should I call my healthcare provider? Tell your healthcare provider if your symptoms get worse or you have new symptoms. Key points about Hashimoto’s thyroiditis Hashimoto's thyroiditis can cause your thyroid to not make enough thyroid hormone. It is an autoimmune disease. It occurs when your body makes antibodies that attack the cells in your thyroid. Symptoms may include an enlarged thyroid gland (goiter), tiredness, weight gain, and muscle weakness. You don’t need treatment if your thyroid hormone levels are normal. If you have an underactive thyroid, medicine can help. Next steps Tips to help you get the most from a visit to your healthcare provider: Know the reason for your visit and what you want to happen. Before your visit, write down questions you want answered. Bring someone with you to help you ask questions and remember what your provider tells you. At the visit, write down the name of a new diagnosis, and any new medicines, treatments, or tests. Also write down any new instructions your provider gives you. Know why a new medicine or treatment is prescribed, and how it will help you. Also know what the side effects are. Ask if your condition can be treated in other ways. Know why a test or procedure is recommended and what the results could mean. Know what to expect if you do not take the medicine or have the test or procedure. If you have a follow-up appointment, write down the date, time, and purpose for that visit. Know how you can contact your provider if you have questions.

Written by: Hari Krishnan Krishnamurthy , Swarnkumar Reddy, Vasanth Jayaraman, Karthik Krishna, Qi Song, Karenah E. Rajasekaran, Tianhao Wang, Kang Bei and John J. Rajasekaran Vibrant Sciences LLC., San Carlos, CA, USA/Vibrant America LLC., San Carlos, CA, USA Abstract Micronutrients are involved in various vital cellular metabolic processes including thyroid hormone metabolism. This study aimed to investigate the correlation between serum levels of micronutrients and their effects on thyroid parameters. The correlation of serum levels of micronutrients and thyroid markers was studied in a group of 387 healthy individuals tested for thyroid markers (T4, T3, FT4, FT3, TSH, anti-TPO, RT3, and anti-Tg) and their micronutrient profile at Vibrant America Clinical Laboratory. The subjects were rationalized into three groups (deficient, normal, or excess levels of micronutrients), and the levels of their thyroid markers were compared. According to our results, deficiency of vitamin B2, B12, B9 and Vit-D25[OH] (p < 0.05) significantly affected thyroid functioning. Other elemental micronutrients such as calcium, copper, choline, iron, and zinc (p < 0.05) have a significant correlation with serum levels of free T3. Amino acids asparagine ( r  = 0.1765, p < 0.001) and serine ( r  = 0.1186, p < 0.05) were found to have a strong positive correlation with TSH. Valine, leucine, and arginine (p < 0.05) also exhibited a significant positive correlation with serum levels of T4 and FT4. No other significant correlations were observed with other micronutrients. Our study suggests strong evidence for the association of the levels of micronutrients with thyroid markers with a special note on the effect of serum levels of certain amino acids. 1. Introduction The deficiency of micronutrients such as vitamins and minerals is of great concern in public health. The World Health Organization (WHO) reported more than 2 billion people are affected by micronutrient deficiency and its related health consequences [ 1 ]. Iodine, iron, vitamin A, and zinc are the primary micronutrients that have been the focus of development efforts since they have major health implications. Micronutrient deficiency is regarded as a preventable cause of various nonspecific physiological impartments such as suppressed immune responses, metabolic disorders, and delayed or impaired physical and psychomotor development [ 2 ]. Elimination of micronutrient deficiencies through nutrition supplementation programs is widely seen as the most promising and cost-effective way to eradicate nutrition deficiency. The optimal metabolic functioning of an individual requires a proper supply of micronutrients such as vitamins, coenzymes, and intracellular elements. Micronutrients play a crucial role in catalyzing various enzymatic reactions, regulating the permeability of cell membranes, and various other physiological activities [ 3 ]. Nutritional alterations result in various endocrine dysfunctions with a prime effect on thyroid functioning. Thyroid disorders are the most common endocrine disorders and are known to affect 5% to 6% of the US population. Thyroid hormone is a sensitive hormone and synthesized by an autoregulated feedback loop mechanism regulated by the hypothalamus-pituitary-thyroid (HPT) axis. Thyroid hormones are involved in various developmental and physiological functioning. Regular functioning of a thyroid gland is characterized by the synthesis of the appropriate amount of triiodothyronine (T3) and thyroxine (T4) in response to thyroid-stimulating hormone (TSH) synthesized by the pituitary gland. Any physiological or biochemical alterations in the feedback loop result in thyroid dysfunctions and result in catastrophic health consequences. These alterations may arise from several pathologies; autoimmune disorders are the most common cause of thyroid disorders which results in excess (hyperthyroidism) or diminished (hypothyroidism) levels of thyroid hormones. Other reasons may include various environmental factors and demographic and intrinsic factors [ 4 ]. Autoimmune responses, thyroid surgery, radiation therapy, congenital hypothyroidism, etc. are the most commonly studied factors of thyroid dysfunctions. The pathogenesis of thyroid disorders has also been shown to be highly influenced by dietary factors, i.e., the availability of micronutrients such as iodine, vitamin D, iron, selenium, copper, zinc, vitamin B12, etc. Micronutrients are involved in physiological functioning like hormone synthesis, hormone transportation, and its binding to a target receptor. Micronutrients also play a pivotal role in regulating autoimmune thyroid disorders (AITD). Hypothyroidism is an autoimmune thyroid disorder resulting from iodine deficiency. The synthesis of both thyroid hormones triiodothyronine (T3) and thyroxine (T4) is inhibited by iodine deficiency which in turn induces the autoantibodies against the thyroid gland and results in goiter [ 3 ]. Hashimoto’s thyroiditis (HT) is an autoimmune disorder characterized by hypothyroid functioning resulting from vitamin D deficiency. In addition to these nutrients, several other micronutrients such as amino acids, cofactors, and metal ions are essential for thyroid functioning. But only a few studies have reported the marginal possibilities of these micronutrients in altering thyroid functions. The present study is designed to evaluate the significant correlation between the serum levels of vital micronutrients and thyroid function. 2. Materials and Methods 2.1. Subjects and Study Design The study population comprised 387 individuals aged between 13 and 85 subjects who were tested for various thyroid markers (thyroxine (T4), triiodothyronine (T3), free T4 hormone (FT4), thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), antithyroid peroxidase (anti-TPO), reverse T3 (RT3), and antithyroglobulin (anti-Tg)) and micronutrient panel at Vibrant America Clinical Laboratory. The female to male ratio was 2 : 1 (69% female, 31% male), and the mean age (±SD) of the subjects was 48 ± 16 years. The study was exempted from formal ethical reviews by Western IRB (Washington, USA) since the study comprises the retrospective analysis of deidentified clinical data and test results. The subjects were categorized on the serum levels of thyroid markers listed in Table 1 . 2.2. Reference Range of Thyroid Markers and Micronutrients The reference ranges of thyroid markers and micronutrients tested depend on the lab where the test is performed. The present study followed the reference ranges widely followed by commercial diagnostic labs and hospital labs. The reference range of thyroid hormones and autoantibodies is shown in Table 1 . The optimum serum levels of essential micronutrients are provided in Table S1 . 2.3. Serum Analysis Serum levels of TSH, FT4, anti-TPO, and anti-Tg tests were measured using a commercial Roche e601 analyzer (Roche Diagnostics, Indianapolis, IN, USA) following the manufacturer’s instructions. All reagents were procured from Roche Diagnostics (Indianapolis, IN, USA). Monoclonal antibodies specifically directed against human TSH were employed for the Elecsys TSH assay. The presence of chimeric construct from human- and mouse-specific components in antibodies labeled with ruthenium complex results in the elimination of interfering effects of HAMA (human anti-mouse antibodies). For the Elecsys T4 and FT4 test, a specific anti-T4 antibody labeled with a ruthenium complex was used for the determination of free thyroxine. The use of a small quantity of the antibodies (equivalent to approx. 1-2% of the total T4 content of a normal serum sample) enables the equilibrium between bound and free T4 virtually unaffected. Serum levels of free triiodothyronine and bound triiodothyronine were determined using Elecsys FT3 assay, a specific anti-T3 antibody with a ruthenium complex. Human antigen and monoclonal human anti-Tg antibodies were employed for the Elecsys anti-Tg assay whereas Elecsys anti-TPO assay used recombinant antigens and polyclonal anti-TPO antibodies for the determination of serum levels of anti-TPO. Serum levels of RT3 were determined by a sensitive and reliable LC-MS/MS technique. Analytical standards of thyroid hormones were procured from Cerilliant Corporation (Round Rock, Texas), and the serum samples were analyzed using Waters TQ-S Tandem Mass Spectrometer. Serum levels of micronutrients were analyzed using Waters TQ-XS Tandem mass spectrometer (LC-MS MS), Waters GC-MS, and Perkin Elmer NexION ICP-MS using standard protocols. 2.4. Statistical Analysis The processing of clinical data from deidentified subjects was performed via Java for windows version 1.8.161, and statistical analysis was performed using GraphPad Prism version 7.00 (Windows). Descriptive statistics were used to define continuous variables (mean ± SD, and median, minimum and maximum) with statistical significance set at p < 0.05. Mann–Whitney U test was used to compare two independent groups without normal distribution, and this method offers the advantage of possible comparison of small samples of subjects. Univariate relationships between variables were analyzed using Pearson’s correlation analysis with significance set at p < 0.05. 3. Results The present study aimed to evaluate the significance of 37 micronutrients on selected thyroid parameters. The study was conducted on the general population without any clinical prevalence of thyroid disorders. The mean age of the candidates involved in the study was 44 ± 14.5, and the study includes 70% females (Table 1 ). The subjects were categorized into three groups based on the serum concentrations of micronutrients being less than the reference range, within the reference range, and higher than the reference. The analysis showed a significant relationship of selected micronutrients on the expression of thyroid hormones; decreased levels of amino acids such as asparagine, glutamine, serine, valine, citrulline, and arginine had a significant effect on thyroid parameters. Deficiency in these amino acids significantly alters the thyroid functioning, specifically, the deficiency of citrulline increased the serum levels of T4 (p < 0.001), and low levels of arginine decreased the serum levels of T3 (<0.0001). Thyroid functions were significantly affected by the various vitamin deficiencies; the present study observed that Vit B2, Vit B12, Vit B9(Folate), and Vit-D25[OH] are the most significant for normal thyroid functioning. Serum levels of T4 were significantly lower in subjects with low Vit B2 (p < 0.01). Vitamin B9 (folate) deficiency is the most significant factor affecting thyroid functioning as it increases the serum TSH level (p < 0.01), increases anti-TPO levels (p < 0.05), and also elevates the serum levels of anti-Tg (p < 0.001). Apart from amino acids and vitamins, other micronutrients such as calcium, copper, chromium, selenium, inositol, and carnitine have significance on thyroid functioning. Decreased serum levels of carnitine were characterized by a significant increase in the levels of anti-TPO levels (p < 0.001) and anti-Tg (p < 0.01). While low levels of micronutrients are a concern, high levels can also result in adverse effects. Elevated levels of inositol and copper beyond the reference range have a significant increase in the serum levels of T4 and T3 (p < 0.001). Increased levels of selenium also show considerable significance in decreasing the levels of T3 and Free T3 (p < 0.01) (Table 2 ).

Collected from Merck's websites https://www.merckgroup.com/en/company/history/the-living-memory-of-merck/organ-therapy.html In 1894, Emanuel August Merck introduced one of the first thyroid preparations in the world. Since then, E. Merck has played a substantial role in thyroid research and development: 1894 : Thyroidinum siccatum Introduction of desiccated sheep thyroid gland (powder, later tablets) in Germany. Available as Thyroidin Merck until 1983. 1896 : Thyroidin Merck (USA) Offered in the price list published in Merck's 1896 Index (New York). 1901 : Antithyroidin Merck Produced from the serum of thyroidectomized rams (Moebius). 1911 : Glandula Thyroideae siccata "Merck" Tabletten Dessiccated thyroid gland available as tablets. 1912 : Research on a standardized thyroid extract product (Annual report Scientific Laboratory 1912). 1921 : Thyreoidinum depurat. Notkin Tabletten Thyroid protein extract. 1925 : Evaluation of Kendall's Thyroxine "Obviously thyroxine is not the only active thyroid product". 1925 : Thyroidea Opton Introduction of protein-free degradation product of the thyroid gland. 1928 : Novothyral Introduction of water soluble standardized thyroid extract (Axolotl). 1941 : Thyroidin Merck Standardized also with Axolotl method. 1948 : Methicil Brand of Methylthiouracil available from Merck in Germany. 1965 : Kalium jodatum Compretten (100 mg KI) Indicated for prevention of radioactive iodine incorporporation in nuclear accidents. 1968 : Novothyral Newly introduced as a T4/T3 combination in Germany. 1973 : Euthyrox Mono T4 tablets introduced in Germany, later in many other countries. 1975 : Jodid Merck Iodide 100 µg tablets (later 200 and 500 µg) introduced in Germany. 1985 : Jodthyrox T4/Iodide combination introduced in Germany for iodine deficiency goiter. 1990 : Thyrozol Thiamazole (Methimazole) introduced in Germany. 1993 : Beginning globalization of Merck KGaA's thyroid business. Help for the thyroid gland in the 19th century In the last quarter of the 19th century, a Swiss surgeon swears by radical removal of the thyroid in severe cases of goiter. Physical and mental limitations were potential consequences of treatment. Preserving residual glandular tissue in the patient is an initial attempt at a solution. In the early 1890s, research scientists all over Europe advocate internal administration of animal thyroid. Suggested delivery forms include extracts and "material delivered raw with some seasoning or cooked in various ways". The age of modern organ therapy has dawned. The notion of curing human illnesses with animal organs has been around since antiquity, but the question regarding the mechanism of action is now being reframed in an entirely new way. "E. Merck’s Annual Pharmacy Reviews" dating from 1896 points out the revolutionary nature of current developments: Although the customary use of organs may have been successful in many cases, it was "not rational". Moreover, the "compound-bound" mechanism of action has already been noted: "Not the organs as such should be used, but their secretions (…). Regrettably, our knowledge (…) is very limited to date." The first Merck thyroid product comes onto the market in 1894. Freshly slaughtered sheep thyroid is dried, powdered and also made into tablets. With "Thyreoidinum siccatum", the company hopes to overcome patients’ reluctance to use other dosage forms. Merck applies the latest scientific insights to develop a product. The highest standards of quality, both with respect to the raw material and its processing, are essential to the product’s efficacy. A dedicated manufacturing facility is built and "every conceivable precaution is observed" to stop the ever so precarious starting material from becoming infected. Research in the following decades is driven by two phenomena: a growing understanding of thyroid gland function, and standardization and checking active ingredient quantity. The research scientists are struck by the variety of symptoms that respond to thyroid medicinal products but do not know the precise mechanism of action. This makes it impossible to isolate specifically effective substances in a chemically pure state and determine their therapeutic value "with clinical accuracy". Standardization is by the biological route. Only in the late 1960s and early 1970s do medicines based on purified thyroid hormone preparations usher in a new era in thyroid disease management. The first Merck thyroid products enter the market in 1894. The packaging is from a later date. "E. Merck's Annual Pharmacy Reviews", 1894, states that the whole thyroid is used for the purpose, since it was not known for sure "to which component (...) the specific effect is attributable." Production records are available since 1895. The importance of the organ products business grows. A new building is erected in 1935 in the southern part of the company premises on Frankfurter Strasse. The employee newpaper comments: "The size and features of this production unit indicate that the products manufactured here must be of significant importance." New products are also launched on the basis of collaboration with external scientists. A clear theoretical underpinning of the clear therapeutic success is still lacking, however. The active ingredient is standardized in the 1930's, one contributor being the axolotl. Organ products are a success, both nationally and internationally.

https://www.britannica.com/biography/George-Redmayne-Murray George Redmayne Murray , (born June 20, 1865, Newcastle-upon-Tyne, Northumberland , Eng.—died Sept. 21, 1939, Mobberley, Cheshire), English physician who pioneered in the treatment of endocrine disorders. He was one of the first to use extractions of animal thyroid to relieve myxedema (severe hypothyroidism) in humans. George, the son of a prominent physician, William Murray, received clinical training at University College Hospital, London. He was awarded both his M.B. (1889) and M.D. (1896) by the University of Cambridge . Determined to pursue a career in experimental medicine , Murray in 1891 became pathologist to the Hospital for Sick Children in Newcastle. He also lectured in bacteriology and comparative anatomy at Durham University. From 1893 to 1908 he was Heath professor of comparative pathology at Durham. Appointed to the chair of medicine at Manchester University , he remained there to the end of his career. In 1891 Murray published his most important research, a report in the British Medical Journal on the effectiveness of sheep thyroid extract in treating myxedema in humans. Thyroid deficiency had been recognized as the cause of myxedema in the 1880s, and several researchers had established that an animal could survive the usually fatal effects of thyroidectomy if part of the excised thyroid gland was transplanted to another body location. Sir Victor Horsley, a colleague of Murray’s, later suggested that part of a sheep’s thyroid could be transplanted into human patients to relieve myxedema. Murray surmised, however, that a hypodermic injection of thyroid extract could more effectively be used to correct myxedema in humans, and he was completely successful in his first such attempt at treatment. Subsequent tests substantiated his approach. George Murray was born at Newcastle, the son of William Murray, F.R.C.P. He was educated at Eton and Trinity College, Cambridge, being placed in the first class of the natural sciences tripos of 1886. He qualified in medicine at University College London, in 1888, receiving the Fellowes gold medal in the same year, and completed his training with visits to Berlin and Paris. He acted as house physician in University College Hospital before starting practice in his native city, where he was appointed in 1891 pathologist to the Hospital for Sick Children and lecturer on bacteriology at Durham University. In 1891, too, he made his reputation by being the first to treat myxoedema with thyroid extract given by injection. In 1893 he was made Heath professor of comparative pathology in the University and in 1898 physician to the Royal Victoria Hospital, Newcastle. He relinquished both appointments in 1908 when he was chosen to fill the chair of medicine at Manchester University, which carried with it the office of physician to the Manchester Royal Infirmary. In the 1914—1918 War he served with the 2nd Western General and 57th General Hospitals, and from 1918 to 1919 as consulting physician, with the rank of colonel, to the British forces in Italy. He was a member of the Medical Research Council from 1916 to 1918. He was a contributor to Quain’s Dictionary and Allbutt’s System of Medicine , and at the Royal College of Physicians delivered the Goulstonian Lectures in 1899 and the Bradshaw Lecture in 1905. He resigned his offices at Manchester in 1925, and in retirement lived at Mobberley in Cheshire. Murray was a man of high ability and unassuming friendliness — qualities which effectively dispelled the opposition to his appointment at Manchester in 1908. He married in 1892 Annie, daughter of Edward Robert Bickersteth of Liverpool, and had three sons and one daughter. He died at Mobberley. G H Brown

Written by Lisa Victoria Larsen The endocrine system is in charge of creating and releasing the hormones that is needed to maintain countless bodily functions. The tissues of the endocrine system includes your hypothalamus, pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands, pancreas and reproductive organs. Hormones are chemicals. They carry instructions through your blood to your organs, skin, muscles and other tissues. There are more than 50 different hormones and they affect almost every aspect of your body. They tell your organs what to do and when to do it. Glands are tissues that create and release substances. They send hormones directly into your bloodstream. The endocrine system controls the metabolism, internal balance (homeostasis), growth, development, reproduction, sleep, mood, energy, digestion, blood sugar, sexual drive, blood pressure, and the nervous system. Metabolism provides energy for essential body functions like breathing, digestion, circulating blood, regulating body temperature and growing and repairing cells. It refers to the chemical processes that happens in your body when you eat, drink, rest, and breathe. The process is complex, regulating conversion of the things you digest to combine calories and oxygen to create and release energy. And it never stops. We have this thing called Basal Metabolic Rate (BMR), which refers to the absolute minimum amount of energy you need to exist. This amount is individual. There is a difference between a fast metabolism and a slow metabolism. People with fast metabolisms burns many calories even while resting. The ones with a slow metabolism needs fewer calories to keep everything going. A fast BMR does not necessarily lead to a thinner body. They just need more energy to maintain their essential body functions. Many factors can weigh in on how well your metabolism works. For instance: - Muscle mass ; People with more muscle mass tend to have faster metabolisms, and it takes more energy to build and upkeep muscle mass than fat. - Age ; When getting older, muscle mass tends to get smaller. This slows down our metabolism. - Gender ; Men usually have faster metabolisms than women because they have more muscle mass, bigger bones and less body fat. Women generally needs more body fat to reproduce. - Genes ; What you inherit from your parents can be a factor when it comes to your ability to build and store muscle mass. - Activity ; Exercise in all forms cause your body to burn more energy than it does resting. This includes walking, sports, raising small kids etc. - Smoking ; One reason people who quit smoking may gain weight is that nicotine speeds up your metabolism. Although it may be very negative for other health issues like cancer, high blood pressure, coronary artery disease. Homeostasis is, in simple terms, a self-regulating process. It involves three mechanisms; a receptor, a control center and an effector. These three work together to keep your body in balance, noticing changes in your body and then starting processes to regulate your system. It refers to any automatic process that anyone needs to stay balanced on the inside, and the body does this to make sure that everything works the right way and we stay alive. It really is quite complex. In a state of homeostasis, your levels are rising and falling as responses to changing enviroments, for instance blood sugar, blood pressure, energy level, acid levels, oxygen, proteins, temperature, hormones and electrolytes. When the system is disturbed, your body will react to create balance again. This can be made possible by the nervous system, the hormonal system or electrical currents. Allostasis is another term that is used to describe our body's ability to foresee, adapt and deal with future events of balance. It really is preparing for needs and managing resources so that your body can adjust before the problems arise. - The receptor of homeostasis; these are cells, tissues and organs that track your system and spots any changes. When the change comes, they notify the control center. - The control centers are often found in the brain, and are the determinators of what is "your normal" and what to do to achieve this. The control center will notify the effector. - The effector is your cells, tissues and organs that then will react to the signals and start the correcting process to achieve the wanted balance. Growth ; three different ways that we grow are through cell count increase, cell mass increase and a rise of non-cellular particles that circles the cell. We grow from birth until death in one way or another. It is a biological process that happens as a result of formation of new cells and packing proteins or other materials into cells that already are there. The growth is not constant in every part of the body, as it is different rates of maturing in different tissues and regions of the body. Human growth hormones (HGH) are produced in your brain's pituitary gland, which controls your height, bone lenght and muscle growth. These hormones increase during childhood and peaks at puberty. Throughout life, the hormones regulates fat, muscle mass, tissue and bones, in addition to insulin and blood sugar levels. These hormones tells your liver to produce a substance called insulin-like growth factor (IGF-1). Acromegaly is a condition that is caused by excess levels of growth hormones, typically as a result of a pituitary tumour. Development ; human organs and organ systems develop in a process called organogenesis. The pituitary gland sends out two hormones concerning development, follicle stimulating hormone (FSH) and luteinizing hormone (LH). These make sure that we develop male or female reproduction abilities. The thyroid is another important gland when it comes to development. Without thyroid hormones, your body's development will not be normal. The thyroid is the body's first endocrine gland to develop in the gestation process. In adults, thyroid hormones can influence mood and behavior. Thyroid dysfunction can affect neurotransmitter systems and lead to psychiatric disorders. Adrenocorticotropic hormone (ACTH) controls the coordinated development of the vasculature and endocrine tissue mass. ACTH is a tropic hormone that indirectly affects target cells by first stimulating other endocrine glands. ACTH stimulates the adrenal glands to produce cortisol, the stress hormone, which again plays a role in glucose metabolism and immune function. Reproduction ; the main hormones of reproduction are estrogen, testosterone and progesterone. They are responsible for puberty, menstruation, menopause, sex drive, sperm production and fetal egg production. They are produced in the female ovaries and male testes. Other hormones that play a part in this are human chorionic gonadotropin (HcG), prolactin, luteinizing hormone (LH) and follicle stimulating hormone (FSH), which are produced, stored and stimulated by the pituitary gland. Estrogen causes eggs to mature in the ovaries when women hits puberty. These are then released during the menstrual cycle. In males, testosterone stimulates sperm production in the testes. In males, the endocrine regulation is as follows: - The hypothalamus releases gonadotropin-releasing hormone (GnRH), which stimulates the pituitary gland to release follicle-stimulating hormone (FSH) and luteinizing hormone (LH). - FSH and LH travel through your blood and bind to receptors in the testes. - FSH stimulates the production of sperm cells (spermatogenesis). LH stimulates production of testosterone in the testes. - Together, FSH and LH controls the function of the testes. - Your adrenal glands also produce a hormone (DHEA), which your body transforms into testosterone. - Testosterone also signals your body to make new red blood cells, ensures that you bones and muscles stay strong, and enhances libido (the sex drive). In females, the endocrine regulation is as follows: - The hypothalamus releases gonadotropin-releasing hormone (GnRH), which stimulates the pituitary gland to release follicle-stimulating hormone (FSH) and luteinizing hormone (LH). - FSH and LH travel through your blood and bind to receptors in the ovaries. - FSH and LH promote ovulation and stimulate secretion of estradiol (an estrogen) and progesterone from the ovaries. - Estrogen and progesterone circulate in the bloodstream, almost entirely bound to plasma proteins. Only unbound estrogen and progesterone appears to be biologically active. - They stimulate the uterus, vagina and breasts to prepare for pregnancy. Sleep interacts with the endocrine system over a wide range of hormones. But what is really sleep? It is a state in which the consciousness is lost and motoric function is reduced. It also comprises different stages of brain waves of different patterns, with the most well known being rapid eye movement (REM) and non-rapid eye movement (NREM) sleep. NREM is characterized by orderly synchronized brain activity, with the brain moving from phases of light to deep sleep. Deeper sleep means slower brain waves. While your muscles are relaxed, they are not paralyzed. On the other hand is REM, which occurs after slow-wave sleep is completed. In the REM stage, the brain shows very disorderly brain waves, similar to the awake brain. Skeletal muscles are paralyzed. Sleep is regulated by the circadian system and the sleep pressure system. Sleep pressure is the need to sleep of any organism at a given moment. Both these systems work in parallel and are responsive to the hours in a day and the organism's homeostatic processes. Circadian rhytms are shifted by external cues like light and dark, exercise, food intake, temperature and various chemicals/medicines. The master circadian clock is the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN enables the body to sleep in long periods related to external light and dark, which are translated into biological day and night, and also regulates the daily rhythm of hormonal secretion and other biological processes. The sleep pressure system determines how much sleep each person needs to return to the normal state. When it comes to keeping track of the day-night cycle, the SCN helps with the secretion of high cortisol early in the morning, to be ready and alert for the activity of the wake cycle. Several hormones are involved in sleep and our circadian rhythm. Growth hormone levels are increased during deep sleep and are vital for cell growth and repair. Growth hormones in turn, affects the regulation and metabolism of glucose, lipids and proteins. Melatonin levels are high during the biological night versus day. It plays an important role in regulating human sleep patterns. Basically it tells your body when to sleep. Melatonin also controls more than 500 genes in the human body, including the genes involved in the immune system. Thyroid stimulating hormone (TSH) concentrations reach their maximum and minimum in the middle of the biological night and afternoon. T3 and T4 concentrations are not associated with circadian rhytmicity. Mood is affected by almost all of your hormones in one way or another. The hormones that perhaps impacts your mental health more than others are serotonin, dopamine, thyroid hormones and sex hormones. - Serotonin is a chemical that carries messages between nerve cells in the central nervous system in the brain and throughout your body. It's techically a monoamine neurotransmitter known as hydroxytryptamine (5-HT), which also acts as a hormone. Most of the serotonine found in your body is in your gut. Actually, about 90% of it is found in the cells lining your gastrointestinal tract. It's made from the essential amino acid tryptophan. An essential amino acid means it can't be made by your body. It has to be obtained by the food you eat. Serotonin is often called one of the "feelgood" chemicals. When it's at a normal level, you feel more focused, emotionally stable, happier and calmer. Low levels are associated with depression. - Dopamine is also a monoamine neurotransmitter. It plays a role in the "fight-or-flight" response. It also causes blood vessels to relax or constrict. Dopamine increases salt and urine removal from your body, as well as reduces insulin production in the pancreas. Dopamine is another "feelgood" chemical, being a part of your reward system. This system is designed from an evolutionary standpoint, to reward you when doing the things you need to do to survive, like eat, drink, reproduce and overcome obstacles. As humans, we are hard-wired to seek out situations that releases dopamine in our system. It makes us feel good and we seek more of that feeling. This is why junk food and sugar is so addictive. They trigger the release of a large amount of dopamine into your brain and gives a feeling that you want to repeat. Dopamine makes you experience happiness, motivation, alertness and focus. - Thyroid hormones commonly affect your mood, and the more severe the thyroid disease, the more severe the mood changes. It can cause anxiety, nervousness, irritability, depression, unusual tiredness, loss of apetite, lack of concentration, short temper and anger. - Sex hormones have both organizational and activational effects in the central nervous system. Energy levels are heavily dependent on our hormones. And with so many hormones involved, it can be difficult to isolate exactly which ones are responsible for changes in the energy level. Here are a few to consider when detangling your decreased energy health; - Thyroid hormones ; they are responsible for maintaining balance in the body, affecting temperature, metabolism, heart rate, mood, and energy levels. When you experience hypothyroidism, which is abnormally low thyroid hormones, many body systems slow down, leading to fatigue and low energy. Less energy consumed can lead to weight gain, slower digestion leads to constipation, and the decreasing serotonin levels may result in depression. - Adrenal hormones ; cortisol responds to the body's energy needs by regulating metabolism and hunger. Chronic stress can disrupt this system and damage your body's ability to function optimally. - Estrogen and progesterone ; when these levels fluctate or drop sharply, many women experience uncomfortable symptoms like hot flashes, mood swings, vaginal dryness, and bone thinning/increased fracture risk. Both low and high estrogen levels may also effect your energy levels significantly. For women, hormone levels can shift dramatically during perimenopause and menopause, whereas men typically experience a slow and gradual decline beginning around age 30. So both men and women can develop symptoms and see their energy levels decline as they approach middle age. Unfortunately, imbalances in a variety of hormones often have similar symptoms. Additionally, an imbalance of one hormone may cascade into more complex conditions since many hormones directly impact each other. Digestion hormones regulate the system that produces them, functioning largely independent from the rest of the endocrine system. When food enters the stomach, the wall where the stomach joins the small intestine, gastrin is released, which promotes the flow of acid from the gastric glands in the stomach. These glands also release pepsinogen which is the inactive form of the protein digesting enzyme pepsin, but this process is primarily under nervous control. The entry of the acidified stomach contents into the first part of the small intestine releases secretin (a digestive hormone) and cholecystokinin. Secretin promotes the discharge of fluid and bicarbonate ions from the pancreas and promotes the secretion of bile from the liver, which aids in the digestion of fats. Blood sugar is influenced by several hormones. These are: - Insulin ; Enhances entry of glucose into cells and storage of glucose as glycogen, or conversion to fatty acids. It also enhances synthesis of fatty acids and proteins and suppresses breakdown of protein into amino acids and triglycerids into free fatty acids. - Amylin ; Suppresses glucagon secretion after eating and slows gastric emptying. It also reduces food intake. - GIP ; Induces insulin secretion, inhibits apoptosis of the pancreatic beta cells and promotes their proliferation. It also stimulates glucagon secretion and fat accumulation. - Glucagon ; Enhances release of glucose from glycogen and synthesis of glucose from amino acids or fats. - Asprosin ; Enhances release of liver glucose during fasting. - Somatostatin ; Suppresses release of insulin, pituitary tropic hormones, gastrin and secretin. It also decreases stomach acid production by preventing the release of other hormones (gastrin and histamine), thus slowing down the digestive process. - Epinephrine ; Enhances release of glucose from glycogen and fatty acids from adipose tissue. - Cortisol ; Enhances gluconeogenesis and antagonizes insulin. - ACTH ; Enhances release of cortisol and fatty acids from adipose tissue. - Growth hormone ; Antagonizes insulin. - Thyroxine ; Enhances release of glucose from glycogen and absorption of sugars from intestine. Sexual drive : Libido naturally varies from person to person. It can also change throughout life. However, the sex hormones testosterone and estrogen, together with the neurotransmitters dopamine and oxytocin regulates libido. Blood pressure is regulated by the hormone aldosterone (ALD) by managing the levels of salt (sodium) and potassium in your blood and impacting blood volume. Your blood pressure can also be impacted by several other hormones: - Adrenal glands ; if the adrenal glands make too much aldosterone, cortisol or adrenaline-like hormones, it can cause high blood pressure. - Thyroid gland ; high blood pressure can be caused by an underactive or overactive thyroid gland. - Pituitary gland ; sometimes problems with the adrenal glands and thyroid gland are due to problems with the pituitary gland. If the pituitary gland sends too much signal to the adrenal galnds or thyroid gland, it can result in high blood pressure. - Parathyroid glands ; if the parathyroid glands make too much parathyroid hormone, it can cause high blood pressure. - Pancreas ; high blood pressure in adults with obesity may be partially due to elevated insulin levels and insulin resistance. The nervous system is designed to protect us from danger through its interpretation of, and reactions to, stimuli. But a primary function of the sympathetic and parasympathetic nervous systems is to interact with the endocrine system to elicit chemicals that provide another system for influencing our feelings and behaviours. When the hormones released by one gland arrive at receptor tissues or other glands, these recieving receptors may trigger the release of other hormones, resulting in a series of complex chemical chain reactions. The endocrine system works together with the nervous system to influence many aspects of human behaviour, including growth, reproduction and metabolism. And the endocrine system also plays a vital role in emotions.

Found at https://pubmed.ncbi.nlm.nih.gov/15253676/ By Alan R Gaby Abstract Evidence is presented that many people have hypothyroidism undetected by conventional laboratory thyroid-function tests, and cases are reported to support the empirical use of Armour thyroid. Clinical evaluation can identify individuals with sub-laboratory hypothyroidism who are likely to benefit from thyroid-replacement therapy. In a significant proportion of cases, treatment with thyroid hormone has resulted in marked improvement in chronic symptoms that had failed to respond to a wide array of conventional and alternative treatments. In some cases, treatment with desiccated thyroid has produced better clinical results than levothyroxine. Research supporting the existence of sub-laboratory hypothyroidism is reviewed, and the author's clinical approach to the diagnosis and treatment of this condition is described. "Objections to the Use of Armour Thyroid The main objections voiced in textbooks and editorials 1,73 regarding the use of desiccated thyroid are: (1) its potency varies from batch to batch, and (2) the use of T3-containing preparations causes the serum T3 concentration to rise to supraphysiological levels. Regarding between-batch variability, there may have been some problems with quality control a half-century or more ago, and in a 1980 study a number of generic versions of desiccated thyroid were still found to be unreliable in their potency. The amounts of T4 and T3 in Armour thyroid, on the other hand, were found to be constant.74 Moreover, two-year old tablets of Armour thyroid contained similar amounts of T4 and T3 as did fresh tablets. Three studies are typically cited to support the contention that T3 containing preparations should not be used. Smith et al reported a levothyroxine-plus-T3 product caused adverse side effects in 46 percent of patients; whereas, side effects occurred in only 10 percent of those receiving levothyroxine alone.75 In that study, however, the combination product and the levothyroxine product differed substantially in potency. For the combination treatment, each 100 mcg of levothyroxine was replaced by 80 mcg of levothyroxine plus 20 mcg of T3. Considering 20mcg of T3 is equivalent to 80 mcg of levothyroxine, the total hormone dose in the combination product was 60-percent greater than that in the levothyroxine preparation. Therefore, the high incidence of adverse side effects may not have been due to the T3, but to the higher total dose of thyroid hormones. In the second study, by Surks et al, the administration of T3-containing preparations to hypothyroid patients caused the plasma T3 concentration to become markedly elevated for several hours after ingestion of the medication.76 In most cases, however, the amount of T3 administered (50-75 mcg) was considerably greater than that contained in a typical dose of desiccated thyroid (9 mcg T3 per 60 mg),77 and/or the total dose of thyroid hormones given was excessive (180 mcg of levothyroxine plus 45 mcg of T3). By contrast, in a patient given 60 mg of desiccated thyroid, the plasma T3 concentration increased from a hypothyroid level to a euthyroid level. Of two hypothyroid patients treated with 120 mg per day of desiccated thyroid, one showed a relatively constant plasma concentration of T3. In the other patient, the T3 level increased by a maximum of 80 percent, to the bottom of the range seen in hyperthyroid patients, and returned to the baseline value within 24 hours. In that patient, the pre-dose plasma T3 concentration was near the top of the normal range, suggesting that this patient may have been receiving too high a dose of desiccated thyroid. Finally, Jackson and Cobb reported that the serum T3 concentration (measured 2-5 hours after a dose) was above normal in most patients receiving desiccated thyroid.2 They concluded there is little use for desiccated thyroid in clinical medicine. Most of the patients (87.5%) in that study, however, were taking a relatively large dose of desiccated thyroid (120-180 mg daily). Moreover, 57.5 percent of the patients were not being treated for hypothyroidism, but rather to suppress the thyroid gland.  Nearly half of the patients continued to have an elevated serum T3 concentration after they were switched to levothyroxine, even though the equivalent dose was reduced in 62.5 percent of patients. Thus, the elevated serum T3 concentrations found in this study can be explained in large part by the high doses used and by the selection of patients, the majority of whom were not hypothyroid.  What this study does suggest is that desiccated thyroid should not be used for thyroid-suppression therapy. Although the oral administration of T3 causes a transient increase in serum T3 concentrations, that fact does not appear to be of significance for hypothyroid patients receiving usual replacement doses of Armour thyroid. In this author's experience, reports of post-dose symptoms of hyperthyroidism are extremely rare, even among patients taking larger doses of desiccated thyroid. An occasional patient reports feeling better when he or she takes Armour thyroid in two divided doses daily. The nature of that improvement, however, is usually an increase in effectiveness, rather than a reduction in side effects. For patients taking relatively large amounts of desiccated thyroid (such as 120 mg daily or more), splitting the daily dose would obviate any potential concern about transient elevations of T3 levels. In practice, however, splitting the daily dose is rarely necessary."

BMC Endocr Disord. 2019; 19: 37. Published online 2019 Apr 18. doi:  10.1186/s12902-019-0365-4 Written by John E. M. Midgley,  Anthony D. Toft ,  Rolf Larisch ,  Johannes W. Dietrich  and Rudolf Hoermann Background In the treatment for hypothyroidism, a historically symptom-orientated approach has given way to reliance on a single biochemical parameter, thyroid stimulating hormone (TSH). Main body The historical developments and motivation leading to that decision and its potential implications are explored from pathophysiological, clinical and statistical viewpoints. An increasing frequency of hypothyroid-like complaints is noted in patients in the wake of this directional shift, together with relaxation of treatment targets. Recent prospective and retrospective studies suggested a changing pattern in patient complaints associated with recent guideline-led low-dose policies. A resulting dramatic rise has ensued in patients, expressing in various ways dissatisfaction with the standard treatment. Contributing factors may include raised problem awareness, overlap of thyroid-related complaints with numerous non-specific symptoms, and apparent deficiencies in the diagnostic process itself. Assuming that maintaining TSH anywhere within its broad reference limits may achieve a satisfactory outcome is challenged. The interrelationship between TSH, free thyroxine (FT4) and free triiodothyronine (FT3) is patient specific and highly individual. Population-based statistical analysis is therefore subject to amalgamation problems (Simpson’s paradox, collider stratification bias). This invalidates group-averaged and range-bound approaches, rather demanding a subject-related statistical approach. Randomised clinical trial (RCT) outcomes may be equally distorted by intra-class clustering. Analytical distinction between an averaged versus typical outcome becomes clinically relevant, because doctors and patients are more interested in the latter. It follows that population-based diagnostic cut-offs for TSH may not be an appropriate treatment target. Studies relating TSH and thyroid hormone concentrations to adverse effects such as osteoporosis and atrial fibrillation invite similar caveats, as measuring TSH within the euthyroid range cannot substitute for FT4 and FT3 concentrations in the risk assessment. Direct markers of thyroid tissue effects and thyroid-specific quality of life instruments are required, but need methodological improvement. Conclusion It appears that we are witnessing a consequential historic shift in the treatment of thyroid disease, driven by over-reliance on a single laboratory parameter TSH. The focus on biochemistry rather than patient symptom relief should be re-assessed. A joint consideration together with a more personalized approach may be required to address the recent surge in patient complaint rates. Background The clinical state of hypothyroidism (then known as myxoedema) was described around 1870, and 10 years later it was recognised as being due to loss of function of the thyroid gland [ 1 – 4 ]. While the Chinese may have been treating goitre in cretins with sheep’s thyroid in the sixth century BCE [ 5 ], initial attempts at treating hypothyroidism were made by transplantation of animal thyroid tissue, followed by injectable and oral formulations [ 5 – 7 ]. In 1914, Kendall [ 8 ] was the first to purify the hormone thyroxine at Mayo Laboratories, which was synthesised as levothyroxine (LT4) in 1926 [ 9 ]. Despite this early chemical breakthrough in drug manufacturing, desiccated animal thyroid extract remained widely used, and even at this time some patients still regard it as the most satisfactory treatment of hypothyroidism for them [ 10 , 11 ]. A policy was adopted by endocrinologists in the 1960s to replace thyroid extract with synthetic levothyroxine as the latter was then more consistent in its content [ 12 – 15 ]. More recently, thyroid extracts have been standardized by modern high pressure liquid chromatography (HPLC) techniques to maintain their content of thyroid hormones in different batches within USP specifications. Few clinical trials have been performed to compare the efficacy of the two products [ 15 ], and an exploratory RCT was conducted only in 2013 [ 16 ]. From its very beginnings replacement therapy was individualised and guided by the measurement of basal metabolic rate, a peripheral marker of the adequacy of thyroid hormone action [ 17 ]. Such a test was cumbersome and operator-dependent and was supplanted by biochemical tests such as protein-bound iodine measurements initially in the 1950s, followed in the early 1970s by radioimmunoassay methods for measuring serum concentrations of T3, T4 and TSH [ 18 – 21 ]. Despite a historically late start in its recognition as a disease entity, hypothyroidism has remarkably become one of the most frequently diagnosed diseases in the Western world, and levothyroxine one of the most frequently used drugs worldwide [ 22 – 24 ]. How could a condition that had been overlooked throughout the centuries of human culture rise to such prominence in such a short time period? Clearly, this was related to the convenient and sensitive measurement of serum TSH [ 25 ], which has achieved a pre-eminent position in defining primary hypothyroidism [ 26 ]. Consequently, a new disease class of subclinical hypothyroidism was introduced, which is solely based on the presence of an elevated TSH while the thyroid hormones FT3 and FT4 remain within their respective reference ranges [ 26 ]. This strategy has not remained unchallenged and the deficiencies of this diagnostic approach have been reviewed elsewhere [ 27 ]. In an attempt to scale back on the avalanche of purported thyroid diseases created by this strategy, the TSH threshold for treatment was raised in recent guidelines [ 26 ]. In doing so, another problem was created by dissociating the TSH-based diagnosis of the disease from the requirement of therapeutic intervention. However, doctors and patients find it incomprehensible that a thyroid condition labelled as a disease would not therefore require suitable intervention. This questions the practical value of designation and appropriateness of the current diagnostic entity of subclinical hypothyroidism. Main text In this article, we take a closer look how these technical changes may have impacted on patient care. A transition occurred from the era of low metabolic rate regarded as synonymous with hypothyroidism to a purely biochemically based definition [ 28 ]. Hence, TSH measurement became the new determinant of hypothyroidism [ 26 ]. Consequently, treatment habits changed over the last decade and were more related to laboratory records than subjective patient experience. In particular, LT4 replacement doses tended to decrease, as a suppressed TSH was viewed as evidence of overtreatment [ 24 , 26 ]. However, for two reasons this is an area of considerable uncertainty. Firstly, thyroid-related patient complaints overlap with a plethora of non-specific symptoms caused by other conditions and diseases [ 29 – 36 ]. Thyroid tests are also more likely to be obtained in patients with unspecific symptoms [ 37 – 39 ]. In these conditions, LT4 treatment may not be superior to placebo in symptom alleviation [ 40 – 42 ]. Secondly, TSH is increasingly recognised to be less reliable as a definitive diagnostic tool than previously assumed [ 27 ]. Not only is its reference interval not universally agreed on or adjusted for various influences, such as ethnicity, iodine supply, age, but the univariate statistical derivation of a TSH reference range is inherently ill-defined owing to its nature as a controlling element [ 43 ]. Physiologically, stimulation by TSH raises thyroid hormones to a level appropriate to the optimal well-being of a person. Because TSH, FT4 and FT3 are interrelated through the operation of hypothalamic-pituitary-thyroid feedback regulation, integrated pairs of TSH and FT4 values define the so-called individual set points [ 43 , 44 ]. Unlike a population-based univariate reference interval, set points are subject to multivariate normality and narrow homeostatic ranges [ 43 ]. When plotting TSH against FT4 concentrations the resulting distribution in a healthy population does not describe the familiar rectangle, but a kite-shaped area [ 43 ]. Accordingly, a TSH value can be indicative of true euthyroidism in an individual despite it slightly exceeding the upper reference limit, while a TSH measurement within that reference interval may represent a truly hypothyroid subject [ 43 ]. Isolated TSH interpretation thereby becomes ambiguous, resulting in unacceptable diagnostic and therapeutic uncertainty surrounding a given TSH measurement when it approaches the TSH euthyroid range [ 43 , 44 ]. As a consequence, this strategy divorces diagnostic disease definitions from treatment targets. Rationally therefore, the triple roles of TSH as a screening test, diagnostic tool and therapeutic target require separate assessment. Diagnostic reliability for patients may be improved by reconstructing personal TSH-FT4 set points, depending on whether this novel approach can be confirmed in clinical trials [ 45 ]. Both the non-specific nature of complaints and inherent deficiencies in the diagnostic process raise an unsettling dilemma for patients and thyroid specialists alike. The issues are exemplified and particularly pertinent to an etiological disease entity whose consequences are paralleled in similar outcomes: primary hypothyroidism due to total thyroidectomy in patients with differentiated thyroid cancer. Treatment requirements and dosing of the drug LT4 changed when guidelines relaxed the need for TSH-suppressive treatment targets for these patients [ 46 , 47 ]. The reason for this shift was not primarily motivated by any improvement in the replacement strategy but by a revision of the long-held tenet that TSH may act as a thyroid growth stimulating hormone. Even when only present at a low level in the circulation it was believed that it could potentially stimulate the growth of remaining tumour cells and thereby promote the relapse of the thyroid cancer in the long-term [ 48 ]. This view has recently been revised, and TSH suppression is now deemed unnecessary for many thyroid cancer patients [ 49 ]. However, this remarkable strategy change has presented a unique opportunity to study the implications for patient complaints of such a far-reaching decision to abandon TSH-suppressive LT4 treatment in low-risk thyroid cancer [ 47 ]. Although this could not be done in a prospective study, careful retrospective analysis revealed some interesting trends [ 50 ]. Over the years when replacement therapy aimed at complete TSH suppression a relatively low rate of persistent hypothyroid complaints was reported by patients followed at a single institution, much lower than in the subsequent years when the relaxed TSH policy came into effect (Fig. ​(Fig.1).1 ). The reverse was true for hyperthyroid complaints reported by patients, which were relatively higher in the first and much lower in the second time period (Fig. ​(Fig.1).1 ). The symptom reporting by these patients indicates a historical shift in the trend from a lack of hypothyroid symptoms on LT4 towards an increased awareness of the persisting symptomatology. While the nature, reliability and accuracy of freely communicated symptoms may be questionable it appears that the opposing trends in these rates in the same patients are well documented in this cohort, and they occurred in association with an important change in the treatment policy during follow-up [ 46 – 50 ]. We are not aware of any prospective studies that followed this historic shift in the pattern of patient complaints during the last decade. The changing pattern in patient complaints observed in this cohort [ 50 ] and associated with the low-dose policy promoted by recent guidelines is mirrored in a recent prospective study [ 51 ] and by a dramatic increase in patients worldwide expressing their concerns and dissatisfaction with the standard treatment in various ways including over the internet and through patient advocacies [ 52 ]. This sentiment was confirmed by a large online survey of 12,146 hypothyroid patients conducted by the American Thyroid Association [ 11 ]. The expression of dissatisfaction may be partly explained by raised awareness of the problem, based on unspecific subjective criteria, and the possible contribution of a lack in certainty of the diagnostic process discussed above [ 11 , 29 – 45 ]. Patient expectations introduce a confounding influence on perceived outcomes [ 11 , 53 , 54 ]. This is difficult to address, particularly since expectation bias extends to RCTs, regarded as the highest class of evidence in Evidenced-Based Medicine [ 53 ]. A conflict arises between Evidenced-Based Medicine and FDA regulations, the latter mandating that drug evaluation is strictly done under conditions of actual use [ 53 , 55 ]. A statistical remedy (R2R) has been proposed to adjust for expectation bias, but we are not aware of any thyroid-related analysis following such a rigorous protocol [ 53 ]. A question may be asked as to why such a renunciation of a previous protocol has not been accompanied by the initiation of appropriate trials to monitor the consequences of the new recommendations and the transition period in a suitable way. We strongly believe that this should become a priority from a public health perspective and an important joint task of the stakeholders advocating for change in the best interest of patients. This would make any discussion surrounding this important topic better grounded in evidence. As most of our patients were otherwise healthy and free of comorbidity it does not seem to be plausible or fair to blame a host of other possible influences for their complaints [ 50 ]. Similarly, in patients with autoimmune thyroiditis, LT4 treatment did not restore quality of life assessed with a validated state-of-the-art and thyroid-specific instrument to that of the healthy population in a large Danish open label study [ 51 ]. It remains however questionable whether these patients received optimum treatment, since some patients did not have their TSH “normalised” and the pituitary hormone may also be an unreliable marker in this particular setting [ 51 ]. Using the observed historical narrower therapeutic range for an individual patient we note that the treatment targets may overlap for patients in a group. If that is true the general assumption that maintaining TSH anywhere within its broad reference limits to routinely achieve a satisfactory outcome for each and every patient may be ill advised. We have refuted the applicability of treatment targets based on the consideration of the reference ranges in the healthy population, by demonstrating dissociations between FT3 and FT4, and FT3 and TSH in LT4-treated athyreotic patients, and documenting altered equilibria between the hormones on LT4, compared to the healthy state [ 27 , 56 ]. Others have arrived at similar conclusions [ 57 ]. In laboratory diagnostics, the high individuality of TSH and thyroid hormones has long been recognised since the pioneering work of Andersen and colleagues [ 58 ]. However, this applies equally to the statistical analysis of associations involving thyroid parameters. Data clustering, be it in groups with similar properties or in subjects where multiple measurements are obtained over time, potentially masks the true relationship, abolishing the strong associations at the group level when the data are combined for analysis. This phenomenon, known as Simpson’s paradox, is readily demonstrated with a fictitious random sample of two groups with a slightly shifted centre showing the same strong inverse correlation. Unlike the correct analysis by individual groups, a combined analysis of the total cohort artificially weakens the correlation (Fig. ​(Fig.2).2 ). The analytical distinction between the averaged versus the typical outcome is clinically relevant for all thyroid drug trials, independently of evidence class and study design, because doctors are naturally more interested in the latter. In a large retrospective longitudinal study, relying on a multilevel model and accounting for both within-subject and between-subject variation, symptomatic outcomes were associated with serum FT3 concentrations, and differed according to the placement of biochemical parameters within the reference range or noticeably beyond its limits in the case of TSH and FT4 [ 50 ]. Treatment-related displacement of the equilibria between thyroid parameters, wide variations in the biochemical treatment response, and individually adjusted dose requirement pose particular challenges for thyroid trials [ 27 , 45 , 59 , 60 ]. Demonstration of averaged equivalency cannot therefore be a satisfactory analytical goal [ 61 ]. Accordingly, the value of statistical evidence derived from historical meta-analyses [ 62 – 66 ] and RCTs on the acceptability of T3/T4 combination therapy is severely weakened and requires careful reconsideration [ 60 ]. Many RCTs were conducted with inferior quality of life instruments available at the time and relied on statistical techniques both less suited for highly individual parameters and in addition susceptible to Simpson’s paradox. Using the overall preference expressed by patients at the end of double-blind studies as a proxy, patients mostly favoured T3/T4 combination therapy [ 52 ]. A thyroid-specific QoL has only recently been developed and validated [ 51 ]. Simpson’s paradox (also known as amalgamation bias or collider stratification bias) may explain, at least in part, why otherwise well-performed studies failed to provide a convincing relationship between symptoms and thyroid function tests [ 27 , 54 , 59 – 61 ]. The paradox is a relevant factor for the relationship of patient complaints, biochemical markers and treatment response to LT4 [ 60 ]. This bias - unless properly accounted for statistically - dissociates the personal treatment responses from the statistical group effect, thereby masking individual treatment success or failure in an unchanged grouped outcome. A lack of group to individual generalizability has been increasingly recognized in other fields, requiring explicit testing for equivalence of processes both at the individual and group level [ 61 ]. Trials purporting to relate TSH and thyroid hormone levels to the incidence of osteoporosis and atrial fibrillation fall under the same fundamental caveats [ 24 , 27 ]. In particular, the Rotterdam study has shown that within the euthyroid range the prognostic implications of thyroid hormones and TSH differ, and, that TSH measurements therefore cannot substitute for FT4 concentrations in predicting the risk of atrial fibrillation [ 67 ]. The cause of atrial fibrillation poses a complex problem, as its occurrence has been physiologically and statistically associated with both high and low FT3 concentrations [ 68 ]. Thyrotoxicosis due to exogenous thyroid hormone intake and endogenous hyperthyroidism have different physiological roots. This traditional distinction should be noted because the interrelationships between TSH and thyroid hormones differ on LT4 treatment from those in thyroid health [ 24 , 56 , 57 , 59 , 69 ]. This may explain why a prospective study measuring surrogate markers of thyroid tissue effects in athyreotic patients found a slightly suppressed TSH to be optimum for these patients rather than constituting overtreatment [ 57 ]. This problem is paralleled in FT4 measurements, which also overlap significantly at the hypothyroid-euthyroid borderline, both in untreated states and even more so in LT4-treated patients [ 24 , 26 , 59 , 67 ]. However, this neither implies that TSH suppression is universally desirable, nor that a suppressed TSH is without risk [ 24 ]. Rather TSH by itself, unaccompanied by measurements of FT4 and FT3, is an unsuitable risk measure in LT4-treated patients, displaying considerable inherent uncertainty in an individual about the risk - benefit ratio for TSH values close to the lower reference limit [ 27 , 69 ]. Taken together, a combination of nonspecific complaints, statistical group-to-individual bias and limited diagnostic performance of TSH testing obfuscates the transition between diseased and healthy state and fosters disagreement of interpretation depending on the respective focal points. Serious correction of scientific evidence is not unprecedented in medicine. Notably, some cholesterol trials have undergone re-interpretation, reversing previous conclusions, following re-analysis of recovered crude data with improved statistical methods [ 70 ]. Market retraction of the antidiabetic drug rosiglitazone is just one noteworthy example of an initially overlooked effect reversion due to Simpson’s paradox [ 71 ]. New studies could be performed in the light of changes in the treatment habits, consequential shifts in symptom reporting and the complaint spectrum as well as recent developments in statistical analysis which favour greater stratification of disease aetiology and individual outcome before commencing suitable analytic procedures. Emphasis should be more strongly concentrated on personalised treatment strategies, reflected by appropriate protocols and statistical instruments favouring multilevel analysis or latent class hierarchical models. Range-based use of biochemical thyroid parameters, though having an essential role in diagnosis, should not automatically dominate patient presentation and surrogate markers for tissue T3 effects [ 26 , 57 , 60 , 72 , 73 ]. When rejecting patient preference as an objective criterion, standard LT4 and combination therapy performed equally on average on QoL measures in several metanalyses [ 62 – 66 ]. However, heterogeneity of the observed treatment response and collider stratification bias require targeting homogenous subgroups and performing statistical latent class analysis [ 60 , 61 ]. This may identify patients that preferentially benefit from the two modalities [ 60 ]. TSH and FT3 dissociate under LT4 treatment, particularly in athyreotic patients where equilibria are formed between TSH and FT4/FT3 different from the healthy state [ 56 , 57 , 59 ]. Poor T3 converters with persisting symptoms may thus be the most suitable candidates for trials of T3/T4 combinations. T3 addition may also avoid LT4 dose escalation resulting in T4 excess, as T4 has been implicated in non-genomic actions, not mediated via T3, such as actin-related cell migration [ 74 ]. Following the timeless wise words of Paracelsus “Dosis solum facit venenum” (“Only the dose makes the poison”). and in keeping with the historic practice to adjust LT4 dose based on a metabolic marker, individual dosing regimens and personalised treatment targets have to be reconsidered [ 27 ]. This is another area where current TSH based LT4 dosing guidelines fall short, as carefully conducted experiments in rodents, which cannot be performed in humans, have shown [ 72 , 73 ]. LT4 monotherapy was unable to restore euthyroidism at the level of various tissues in the animals despite bringing TSH within its reference range [ 72 , 73 ]. Conclusions Until the situation is clarified all currently available treatment options should remain on the table and the focus should remain on facilitating the free choice of prescriptible treatment options rather than imposing new restrictions. The biochemically based reason for the rise in patient complaints has to be addressed, not a shift on to them of blame and burden of proof. This invites a resume of the current state of affairs. It appears that what we are witnessing constitutes an unprecedented historic change in the diagnosis and treatment of thyroid disease, driven by over-reliance on a single laboratory parameter TSH and supported by persuasive guidelines. This has resulted in a mass experiment in disease definition and a massive swing of the pendulum from a fear of drug-induced thyrotoxicosis to the new actuality of unresolved designation of hypothyroidism. All of this has occurred in a relatively short period of time without any epidemiological monitoring of the situation. Evidence has become ephemeral and many recommendations lag behind the changing demographic patterns addressing issues that are no longer of high priority as the pendulum has already moved in the opposite direction. 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Written by Dr. Westin Childs August 17, 2023 Will taking Armour thyroid help you lose weight, feel better, and have more energy? That is the question that many patients have and one of the main reasons they want to at least give Armour thyroid a try.  Armour thyroid is a special thyroid medication that is formulated to include ALL of the thyroid hormones  – both biologically active and biologically inactive forms.  These special properties may be one of the reasons that patients who switch to Armour thyroid experience significant improvement in their hypothyroid symptoms.  But will it work for you? This guide will teach you everything you need to know about using Armour thyroid ranging from who should use it, how to use it safely, how to use it for weight loss, and common pitfalls patients experience. Let’s jump in:   Everything you Need to Know About Armour Thyroid What is Armour thyroid? Armour thyroid is a prescription medication that is used to treat patients with the diagnosis of hypothyroidism .  As you probably know hypothyroidism is a condition in the body that results in a decreased production or utilization of thyroid hormone in the body.  As a result patients with a “sluggish” thyroid often experience symptoms such as weight gain, fatigue, cold extremities, constipation, and so on. These patients (those with hypothyroidism) also notoriously struggle with the ability to lose weight and keep it off.  So where does Armour thyroid fit in here? Armour thyroid is unique among thyroid medications in that it contains both active AND inactive thyroid hormones  (1). This is actually quite important because the active thyroid hormone T3 is what makes thyroid hormone do its job .  Armour thyroid is, therefore, a significantly more potent thyroid medication when compared to thyroid medications such as levothyroxine  and Synthroid .  Patients can take advantage of this increased potency and may actually feel much better when switching from other medications to Armour thyroid.  The question is why? Armour Thyroid vs Levothyroxine & Synthroid I mentioned that Armour thyroid is unique among thyroid medications so let me explain a little bit further here.  Armour thyroid is ranked among a class of thyroid medications known as Natural Desiccated thyroid medications.  These medications are created by desiccating (or crushing) up the thyroid glands of certain animals – in this case, pigs.  Pharmaceutical companies then encapsulate the desiccated pig thyroid gland and standardize the hormones in each capsule and humans then take it.  I know it sounds weird but it works incredibly well for several reasons: #1. It contains the ACTIVE and INACTIVE thyroid hormones – Both T3 and T4 When your thyroid is functioning at 100% it naturally produces 2 thyroid hormones each and every day.  Normally you wouldn’t think twice about this, but it matters when you take thyroid medication.  Why? Because the conventional treatment for patients with hypothyroidism is to supplement with only the inactive thyroid hormone T4.  Physicians, such as endocrinologists, give patients T4 only thyroid medications because they assume that each patient  (2) will be able to perfectly convert the inactive thyroid hormone T4 into the active thyroid hormone T3.  This may work for many patients but it absolutely does not work for everyone! In fact, it has been estimated that at least up to 15% of patients may have trouble tolerating T4-only medications  (3) and they manifest this “trouble” with low serum T3 levels despite a “normal” TSH.  Let’s recap this information because it can be confusing: The healthy thyroid gland naturally produces about 80% T4 thyroid hormone and about 20% T3 thyroid hormone  (4) each and every day. Your body lives in harmony and functions optimally when it can create the amount of T4 and T3 that it needs on a daily basis.  Your body has created a special mechanism to “control” the amount of thyroid hormone that it needs on a daily basis through a system known as thyroid hormone conversion.  In this system, your body takes the inactive thyroid hormone T4 and it can activate it by turning it into T3.  It can also “inactive” it if it doesn’t need any extra thyroid hormone by turning it into reverse T3.  This is just thyroid physiology 101  (5). Armour thyroid is special when compared to levothyroxine because it contains at least two types of thyroid hormone: T4 and T3.  Levothyroxine, on the other hand, only contains T4.  This means Armour thyroid may more closely approximate what your body produces in a natural and healthy state.  #2. Armour also contains less biologically active thyroid hormones such as T1 and T2 Another very important beneficial aspect of taking Armour thyroid is that you are getting more than just the combination of T3 and T4 thyroid hormones.  In physiology, we give all of the credit to T3 because it is the most biological thyroid compound floating around in your blood.  But did you know that there are other thyroid hormones?  These other thyroid hormones, known as T1 and T2, also exist in your body but they don’t get much credit because they are considered to be “less biologically active” than T4 and T3.  But emerging research  (6) is showing that these compounds are still important nonetheless, even if we don’t completely understand what they do or how they work.  It would be silly of us to assume that simply because we don’t know what they do or how they work that they are unimportant, but that is kind of the mentality that we have when it comes to the human body.  No matter how you look at it though there may be some individuals who benefit from the “extra” thyroid hormones T1 and T2 that come with the whole Armour thyroid package.  These hormones (along with probably other enzymes and hormone precursors) are present in the thyroid gland of animals and therefore make it into the pharmaceutical medication as it is prepared.  These extra hormones may not be of benefit to each and every person but it is certainly possible that patients who don’t have a functioning thyroid (such as those who have had it surgically removed) may benefit from the extra T1 and T2 that comes in this medication.  Patients who still have a thyroid gland that functions may be able to produce T1 and T2 in sufficient amounts to not notice a difference, but either way it’s worth discussing.  #3. Armour thyroid requires less “activation” and is, therefore, more active when compared to Levothyroxine and Synthroid I mentioned previously that Armour thyroid is more “potent” and more “active” than T4 medications such as Levothyroxine and Synthroid and part of this is because it contains the active T3 hormone but another important part is that it doesn’t require as much activation.  Activation or conversion are both words for the same process: The turning of inactive T4 into active T3.  This process is controlled by various factors including your genetics so it may come as no surprise that some people are “better” at converting thyroid hormone than others.  New research has shown  (7) that there are some existing genes known as SNPs  that may alter the efficiency of this conversion process.  This means that some of you out there are just not very good at converting T4 into T3  (8) while some of you out there are incredible at doing it.  So how does this relate to Armour thyroid? Because Armour thyroid contains T3 it can directly bypass this conversion process  which allows the body to use some of the thyroid hormones immediately after ingestion.  If we could reliably test for these genetic differences we would be able to more easily determine what kind of thyroid medication that each person needs .  Since this testing is not mainstream (yet!) it may be beneficial for certain individuals to use Armour thyroid over traditional T4 medications like Synthroid.  Is Armour Better than Levothyroxine/Synthroid?  When you lay out the benefits of Armour thyroid and compare it to T4-only medications it’s easy to come to the conclusion that Armour thyroid may be superior, but is this really true? I wouldn’t jump to that conclusion right away, but there are certainly some reasons that you should consider using Armour thyroid.  Reasons to try or consider using Armour thyroid: You’ve tried Synthroid and Levothyroxine and you still experience hypothyroid symptoms even with a “normal” TSH.  You have low reverse T3 levels or low serum T3 levels with a “normal” TSH.  You are gaining weight or remaining weight neutral on T4-only medications even with normal labs and after trying dietary changes and adding in exercise.  You are suffering from infertility or menstrual problems despite taking thyroid medication. This is not a complete list but instead focuses on our current understanding of thyroid physiology, genetics , and thyroid conversion and attempts to come up with logical reasons why some patients might feel better on Armour thyroid compared to other medications.  While many patients report almost immediate improvement in their hypothyroid symptoms when switching to Armour thyroid there are plenty of patients who also report negative symptoms (more on that below).  And these patients may simply do better on traditional T4 medications.  Reasons to use or stick with Levothyroxine/Synthroid: T4 medications may be better for people who experience heart palpitations, anxiety, or other symptoms when taking Armour thyroid or NDT.  T4 medications have many different tablets and variations which means that you can adjust dosing easily.  Patients may benefit from taking the combination of T4 and T3 in individual doses and medications to titrate specifically to the needs of the patient.  Some patients have no issues with peripheral thyroid conversion and they readily and actively convert T4 to T3 when it is supplied. Instead of focusing on which is the “best” thyroid medication, it’s more helpful to determine (through testing and symptom management) which is the best thyroid medication for YOUR body.  This approach will help you achieve long-lasting results and help improve your symptoms more than any other.  Armour Thyroid and Weight Loss – Will it Help you? One of the biggest (and perhaps more important) questions that most patients have when considering Armour thyroid is this:  Will Armour thyroid actually help me lose weight? Most people assume when they switch to Armour thyroid that their extra weight will magically start shedding off… And about 90% of people who make the switch don’t see those results. Why does this happen? While Armour Thyroid is a great medication (that will probably help reduce your symptoms even if it doesn’t help you lose weight)  it usually isn’t enough for weight loss by itself . After all – If weight loss were that simple, then Doctors would be prescribing low doses of Armour to every patient! Why this happens is a long story but the short version is that your thyroid is probably only contributing to about 10-20 pounds of extra weight you may be carrying right now. Another hormone imbalance… Poor diet… Too much stress… Lack of sleep… Menopause… Environmental exposure… You get the idea. While your thyroid certainly contributes (at least initially) to weight gain, it almost always isn’t the ONLY contributing factor.  I’ve discussed the thyroid hormone obesity myth in detail and you can read more about this unique approach to weight loss in thyroid patients here .  So let’s talk about the 5 main reasons that patients who make the switch to Armour Thyroid don’t lose weight and what you can do to FINALLY lose those extra pounds:  #1. Insufficient Dosing One of the biggest issues is that many patients may be undertreated or underdosed.  If a patient is hypothyroid, meaning their thyroid gland is not producing enough thyroid hormone naturally, then they must undergo thyroid hormone replacement therapy.  In order for this therapy to be effective, you must be getting ENOUGH thyroid hormone .  For various reasons, physicians may be relying on tests that may not accurately identify the status of thyroid hormone in the body.  You can read more about this idea in detail here .  Relevant to this article it’s important to understand that dosing based on the TSH alone may not be the most accurate way to manage Armour thyroid dosing.  Another reason that physicians may not be adequately dosing certain patients is that they are unfamiliar with dosing thyroid medications that contain T3.  As we’ve discussed previously, Armour thyroid contains a combination of T4 (the inactive thyroid hormone) and T3 (the active thyroid hormone).  The portion of T3 thyroid hormone in each “grain” (which is a unit of Armour thyroid dosing) is actually still quite small:  1 grain  = 38mcg of T4 and 9mcg of T3 Doctors know that T3 it is approximately 3.3 times more effective at dropping the TSH than T4 medication alone  (9) (Synthroid and levothyroxine).  This means that when transitioning from T4-only medication to Armour thyroid many patients are usually under-treated, especially if physicians rely solely on the TSH as an indicator of thyroid status in the body.  Other factors such as your sex hormone binding globulin  may be a better indicator of tissue levels of thyroid hormone.  It’s also important to note that many providers aren’t as comfortable dosing Armour Thyroid as they are levothyroxine and so they will intentionally underdose patients because they are concerned about side effects.  Transitioning from T4 only Medication to Armour Thyroid​ ​If you transitioned from Levothyroxine (T4-only medication) then you should know how to properly convert your medication. Most conversion charts will show that 100mcg of T4 = 1 grain of Armour thyroid. But remember that each grain of Armour thyroid only has 38mcg of T4 and 9 mcg of T3.  Even if you assume that T3 is 3x more potent (which isn’t the best indicator of biological activity in the body) then you still come up short with this conversion – let’s break down the math: 38 mcg of T4 = 38mcg of T4 9 mcg of T3 x 3 = 27 mcg of T4  “ equivalents ” in each grain of Armour thyroid 38mcg + 27mcg = 65 mcg of T4  “ equivalents ” in each grain of Armour thyroid total So suggesting that 1 grain of T4 (which equals about 65mcg) is a substitute for 100mcg of T4 is not necessarily a fair assumption .   Using this conversion you can see how many people may end up under-treated. It may be no wonder why some patients who “transition” to Armour thyroid often feel symptomatically worse during the transition.  This is sometimes used by the physician as “proof” that Armour thyroid doesn’t work. I’ve even had patients who report that when they switch to Armour thyroid their TSH increases and the physician then states that Armour thyroid is unreliable.  In reality, it may be more accurate that the patient was underdosed which resulted in a hypothyroid state and a subsequent increase in the TSH.  This change is not necessarily indicative of the failure of Armour thyroid but more of a failure with the conversion and dosing process. A more accurate conversion process may look something like this: 50 mcg of T4 = About 1 grain of Armour 100mcg of T4 = About 2 grains of Armour 150mcg of T4 = About 3 grains of Armour   A couple of important points when considering this information: First: If you are transitioning from a T4-only medication do NOT start yourself off at the maximum dose of Armour. If you dose your Armour too quickly you are likely to end up with side effects from the T3: Heart palpitations, Anxiety, Heat intolerance, etc. Instead, start off on a lower dose and titrate up SLOWLY every 10-14 days . Second:   Thyroid dosing is highly individualized and depends on factors such as your current metabolic rate, your sensitivity to T3, and your peripheral thyroid conversion status. This means that you can never use a “ dosing calculator ” to simply determine what your dose should be, instead you may need to use a combination of trial and error along with frequent laboratory testing.  #2. The Ratio of T3 to T4 in Thyroid Hormone Replacement Therapy While Armour does contain some T3 it is possible that your body may actually need more. Along this same vein, it’s also possible that your dose of T4 may be either too high or too low relative to your dose of T3.  If this sounds confusing, don’t let it be:   Armour thyroid (and other forms of Natural Desiccated thyroid) contain fixed ratios of T4 to T3. Meaning the amount of T3 in each grain of Armour is always the same (this is a good thing from a pharmaceutical and consistency standpoint, but may be a problem due to the inability to titrate individual T3 and T4 doses).  Unfortunately, some people may require higher amounts of T3 due to reasons such  tissue level hypothyroidism ,  euthyroid sick syndrome/low T3 syndrome   (10), or peripheral thyroid conversion disorders.   These conditions are characterized by physiologic changes in which the patient may require higher doses of T3 to saturate cellular receptors and allow thyroid hormone to get into the cells. How do you know if you are someone who needs more T3? Patients ​who need more T3 usually fall into one or more of the following categories: They have high levels of Reverse T3 They have Leptin Resistance They have Diabetes, Prediabetes, or Insulin Resistance They have extremely low body temperatures They have a personal history of bipolar disorder or a strong family history of mental health disorders They have a personal history of Fibromyalgia or Chronic fatigue syndrome If you fall into any of the categories above AND you aren’t losing weight on Armour thyroid then you may want to consider adjusting the individual dosing of T3 and T4 in your medication.  In some cases, this may be as easy as adding a small dose of T3, in others, it may mean switching to individual T4 and T3 prescriptions.  Adding T3 to Armour Thyroid One problem with Armour thyroid (and NDT in general) is the static dosing in each individual grain of hormone.  Each grain gives you 38mcg of T4 and 9mcg of T3 . This is ok for some patients but doesn’t allow for altering individual dosing of the T4 and T3 if necessary. Patients with high stress , high levels of inflammation, leptin resistance , insulin resistance , etc. may require more T3 relative to T4.  Some studies even suggest  (11) that temporary supraphysiologic dosing of T3 may help to overcome these hormone imbalances, provided it is used safely and correctly.  In my experience, (and if used safely) lowering T4 dosing and increasing T3 dosing provides improved weight loss and improved lipid metabolism in a subset of patients.  This is also confirmed by studies (12) which have shown that the transition from T4 to T3 dosing (based on pituitary and TSH testing) improves all of these markers. In some patients dropping Armour thyroid total dosing and adding T3 can be sufficient to improve symptoms and provide improved weight loss without causing negative symptoms.  T3 can be added to Armour thyroid with the following medications: liothyronine , Cytomel , or SR T3 .  #3. The presence of Hormone Imbalances such as Leptin Resistance ​If you aren’t familiar with   Leptin resistance or the hormone leptin please read this article .  ​As a quick primer: Leptin  is a hormone that is pumped out by your fat cells (yes they pump out hormones!). ​As fat cells grow, leptin levels increase. As they increase they are supposed to tell your brain to increase metabolism and start burning off that extra fat you just gained.  When you have leptin resistance (13) the exact opposite happens: your body thinks you’re starving so it decreases metabolism and increases appetite (even though you have plenty of fat).  The presence of leptin resistance creates hormonal havoc which results in weight loss resistance despite changes to your diet and to the amount that you exercise.  You simply won’t lose weight until it is treated. And, to make matters worse – high levels of leptin actually decrease T4 to T3 conversion  (14) in the body. How do you know if you have Leptin resistance?  Leptin levels can be tested easily in the serum and fasting leptin levels higher than 10-12 indicate the presence of leptin resistance.  Patients with leptin resistance generally require higher levels of T3-only medications to help reduce the T4 to reverse T3 conversion that occurs with this condition.  It’s important to note that leptin resistance is generally a late finding and usually indicates that heavy metabolic damage has already occurred.  Leptin resistance is often accompanied by insulin resistance and thyroid resistance (all three conditions are caused by the same thing). ​ Patients with leptin resistance generally require aggressive treatment to reverse leptin​ levels to help with weight loss.  To see a case study example treatment plan please refer to this post .  You can find supplements to treat leptin resistance  here. ​ ​I’ve outlined how to treat leptin resistance in this article .  #4. You are Reacting to Inactive Ingredients in Armour Thyroid Not all forms of Natural Desiccated thyroid are created equal. Beyond Armour thyroid, there are other types of natural desiccated thyroid such as Nature-throid  and WP Thyroid .  While it’s true that 1 grain of Armour thyroid contains the same amount of active thyroid hormone (T3 and T4) as a grain of WP thyroid or Naturethroid – these medications differ in the inactive ingredients that they contain. And it is these inactive ingredients that may cause certain people to respond well (or not) to these medications due to how they react to these inactive ingredients.  In addition, these inactive ingredients may alter breakdown and digestion which then alters the absorption of thyroid hormone in the body.  Some individuals who take NDT (and Armour thyroid) may experience negative side effects while taking this medication which they may wrongly attribute to the active thyroid hormone.  Instead, it may be possible that they would do much better by switching to a different formulation rather than switching back to T4 medication.  Using the table below you can see that the NDT form of thyroid hormone with the least amount of ingredients is WP thyroid – though this doesn’t necessarily mean that it is the “best”.  These changes in inactive ingredients help to explain why some patients react poorly to Armour thyroid with symptoms like headaches, worsening fatigue, or rashes – only to find that these symptoms completely resolve upon switching to Naturethroid.  Again it’s not the difference in the concentration of thyroid hormone in each individual medication but most likely a reflection of digestion and absorption.  As an example Armour thyroid is known to contain both methylcellulose and dextrose  and these inactive ingredients are notoriously difficult for some patients to digest and break down.  You can think about methylcellulose as a glue that holds on tight to the thyroid hormone component of the medication and in order for your body to absorb (and properly utilize) your intestinal tract must separate the methylcellulose from the active thyroid hormone.  Unfortunately, many patients with hypothyroidism also have gastrointestinal issues including low stomach acid  (15). This may result in decreased absorption of thyroid hormone or cause a “delayed” release of the medication into the bloodstream. This exact process is what happens when patients take Sustained release T3. ​The “slow release” comes from the fact that it is harder to digest and absorb in the body, but this also reduces the overall amount of hormone available in each dosage.  In general patients with GI-related issues may do better on WP thyroid  or Tirosint  which both have fewer inactive ingredients and absorption tends to be better.  Bottom line : If Armour thyroid isn’t helping you (either with weight loss or you still remain symptomatic) you might consider switching to a different form of NDT like Naturethroid or WP thyroid.  #5. Your Reverse T3 is too High  If you aren’t familiar with Reverse T3 please read this article .  To recap: T4 = INACTIVE storage form of thyroid.  T3 = ACTIVE thyroid hormone.  Reverse T3 = INACTIVE thyroid metabolite that competes with T3 for cellular binding.  It should come as no surprise then to hear that you don’t want high levels of Reverse T3 because it competes for binding with T3 which creates tissue-level hypothyroidism  or thyroid resistance .  In general, the higher your reverse T3 the more hypothyroid you will feel and the less active thyroid hormone is in your body (unless the Reverse T3 is compensatory due to supraphysiologic levels of free T3). High levels of reverse T3 may lead you to experience weight gain, a slower-than-normal metabolism, and other hypothyroid symptoms .   Because T4 has the option to turn into T3 or Reverse T3 we need to concern ourselves with what causes the body to preferentially create more reverse T3.  It turns out that many conditions may be sabotaging your thyroid conversion and the presence of these conditions may be causing your body to turn T4 into reverse T3.  These conditions include:  Taking thyroid medications containing T4 (Synthroid, levothyroxine but also NDT like Armour thyroid) – This acts as a substrate, and in the presence of inflammation or other hormone imbalances your body may produce Reverse T3 over T3 Inflammation – From conditions like SIBO, Hashimoto’s, Food allergies, nutrient deficiencies, medications, etc. Leptin resistance Insulin resistance Obesity LPS  – Usually caused by increased intestinal permeability (SIBO, SIFO, SIBO, leaky gut, etc.)  Certain Medications: Anti-depressants, diabetic medications, antiseizure medications, blood pressure medications, Narcotics, etc. (Please don’t stop taking these medications if you are on them, but consider discussing options with your Doctor) Two things should pop out to you after reading that list:  1) Thyroid medications containing T4 may contribute to elevated reverse T3 levels And… 2) If you fall into one or more of the categories listed above AND you are taking T4 medication (including Armour thyroid, but especially T4-only medications like Synthroid or Levothyroxine) then your medication may be contributing to your inability to lose weight.  What happens when Reverse T3 levels are too high? As Reverse T3 levels climb your cells will have more difficulty taking up active thyroid hormone. This leads to a situation where you will have the symptoms of hypothyroidism  but your lab tests may be “normal”. Your temperature will drop, your metabolism will drop and your appetite will increase in an attempt to compensate.  All of these changes ultimately lead to weight gain. ​ The real question is what do you do about it?  ​How do you fix high Reverse T3 levels?  Because high levels of reverse T3 can be caused by different factors, the best way to treat it is to identify the cause and focus on that issue.  For instance: If you have high levels of insulin – your focus should be on reversing this condition. If your leptin is too high – then you should undergo therapies designed to reduce leptin resistance. If your ​T4-containing medication is too high you may need to decrease your T4 dose and/or increase your T3 dose. If you suffer from chronic inflammation then you may need to address whatever issue is causing inflammation in your body.   And so on. If you can identify and treat these conditions then your body will naturally be able to convert T4 into T3  which is exactly what you want.  Dosing Armour Thyroid If you decide that Armour thyroid is worth trying, then how do you dose it? Dosing Armour thyroid is not much different than using traditional or conventional thyroid medications such as levothyroxine or Synthroid.  The primary difficulties come when transitioning from an existing thyroid medication over to Armour thyroid.  If you fall into this category then you need to make sure that you are “converting” your dose correctly as we outlined at the beginning of this post.  Many physicians will tend to underdose you on the transition because they aren’t familiar with using medications that contain T3.  This underdosing may have the unintended consequence of exacerbating (at least temporarily) hypothyroid symptoms.  If this happens to you don’t let it scare you from using the medication further! Instead, make sure you re-evaluate your lab tests and determine if you are on a sufficient dose or if you need a higher dose.  Each person will require a unique amount of thyroid hormone but the average dose of Armour thyroid is somewhere between 2-3 grains or 180mg.  Armour thyroid can be dosed in milligrams or in grains and it’s important to understand the difference. 1 grain of Armour thyroid is equal to 60 mg.  6 0mg of Armour thyroid contains about 38mcg of T4 and 9 mcg of T3.  So 1 grain = 60mg = 38mcg of T4 and 9 mcg of T3 .  If you are taking 2 grains of Armour thyroid then you are taking 120mg.  2 grains of Armour thyroid = 120 mg of Armour thyroid = 76 mcg of T4 and 18mcg of T3.   If you use the calculation above then 2 grains is equal to 120mg of Armour thyroid or 130mcg of T4 equivalents (if you convert the 18mcg of T3 into T4).  This is helpful because it can help you determine how much Armour thyroid to start with and what kind of dose you should be looking to hit.  As a general rule of thumb (though not always accurate) certain patients tend to need higher doses of thyroid hormone.  Patients in this category include those with higher metabolic demands  (those with a higher metabolism), patients who are overweight or who have a higher than average BMI,  and patients who may be taking medications  that block or limit thyroid hormone action.  If you fall into any of these categories make sure you monitor your dose very closely.  If you are transitioning from an existing T4 medication then starting your dose off at about 25% of the maximum and then increasing slowly every few weeks is a safe and effective method.  If you are naive to thyroid medication (meaning that you have never tried thyroid medication before) then starting off at 15-30mg and titrating slowly up every few weeks may also be appropriate.  While none of these are hard and fast rules they can help you get started.  In addition to these general rules there are also other factors that you should consider when using armour:  Tips for Taking Armour Thyroid #1. Take Armour thyroid on an empty stomach. No doubt you’ve heard this one before! Taking thyroid medication on an empty stomach is usually recommended by physicians and pharmacists because this action may help your body increase absorption.  Thyroid hormone can bind to certain substances which may reduce the dose of thyroid medication that is delivered to your body.  For instance: If you are taking 1 grain of Armour thyroid but you take it with a meal, it’s possible you may only be absorbing 70% of that 1 grain.  Taking your medication on an empty stomach helps fight this.  #2. Take away from supplements that contain Calcium and Iron but pretty much avoid all supplements if possible. Along the same vein as taking your medication on an empty stomach is to avoid taking your medication with other supplements – especially those that contain calcium or iron.  Calcium and iron are notorious for binding to and preventing the absorption of thyroid hormone, but this can also occur with other supplements.  To play it safe I generally recommend that you wait 4 hours after taking any supplement before you also take your thyroid medication.  #3. Splitting your dose. Another strategy worth considering is to split your dose throughout the day.  Splitting your dose may help reduce the massive swings in thyroid hormone that occur when you take an entire day’s worth of thyroid hormone at once.  So if you are taking 2 grains total each day you might consider taking 1 grain in the morning and then another in the afternoon.  So your total dose stays the same but you are “splitting” that dose throughout the day.  This can help maintain constant serum levels of thyroid hormone and may be especially helpful for patients who are sensitive to T3.  #4. Consider taking it at night. Another strategy that I often recommend is to simply take your thyroid medication at night.  Taking your medication at night offers several advantages: First is that you don’t have to worry about taking it on an empty stomach because most people generally don’t eat right before bed.  And second is that taking your thyroid hormone in the evening may actually improve absorption because the GI tract tends to be slower in the evening  (16). Side Effects and Symptoms Are there any downsides to using Armour thyroid? Well, yes.  It should be stated that Armour thyroid is not a perfect thyroid medication nor will it necessarily work for every person.  It’s important to remember that Armour thyroid comes from an animal and animal tissue is considered “foreign” to your body.  The actual thyroid hormones from the porcine-derived thyroid gland are EXACTLY the same as the ones your body produces but, as we stated previously, Armour also contains other inactive ingredients that may cause issues when ingested by humans.  It’s theoretically possible that certain patients taking Armour thyroid may experience an “antigenic” reaction.  What is this? Basically, it’s possible that your immune system may recognize Armour thyroid as “foreign” tissue and react or attempt to destroy it.  Some people believe that taking Armour thyroid may cause a “flare up” of existing Hashimoto’s disease.  While the research is limited in this area I have seen several patients who develop a rapid and excessive increase in thyroid antibodies immediately after starting Armour thyroid.  These patients also often experience a worsening in hypothyroid symptoms such as an acute increase in fatigue an increase in weight gain, etc.  In my experience, this kind of reaction is quite rare but it’s worth pointing out for this discussion.   Symptoms of excessive Armour thyroid dosing (Your dose is too high) Anxiety or Jittery sensation Heart palpitations Excessive sweating Intolerance to heat Rapid heart rate Diarrhea Trembling hands or extremities Increased blood pressure Irritability Insomnia Weight loss If you experience any of these symptoms you should seek help from your physician immediately.  This sort of reaction tends to be rare,  provided you slowly increase your dose over time and monitor your serum thyroid levels as you go.  More common than excessive dosing is insufficient dosing characterized by hypothyroid symptoms:  Symptoms of insufficient Armour thyroid dosing (Your dose is too low) Persistent fatigue Weight gain (even while taking thyroid medication which is never a normal symptom) Continued infertility or persistent menstrual disturbances Continued brain fog or reduced mental focus/clarity Constipation Swelling of the neck Swelling of the extremities and around the eyes (especially in the morning) Dry skin Hair loss Cold intolerance Depression/anxiety The presence of these symptoms may indicate that your dose is insufficient or not high enough for your body.  They also might be a sign that you are having difficulty absorbing or utilizing thyroid hormone after it is absorbed.  If you’ve been experiencing any of these symptoms then you should have your blood work retested and evaluated.   While these symptoms tend to be easy to diagnose there is actually a third reason you might not tolerate Armour thyroid and that has to do with sensitivities to inactive fillers and dyes.  Symptoms of reacting to the fillers or dyes in the medication itself (You might want to switch medications) Rashes Acid reflux Abdominal pain Bloating or excessive gas Headaches These symptoms tend to be unique in the sense that they are NOT caused by the active thyroid hormone but instead caused by the other “stuff” that the medication is formulated with.  This includes inactive ingredients such as methylcellulose and dextrose (and many others).  The good news is that these symptoms often disappear when you switch to “cleaner” medications such as WP thyroid or Tirosint.  Wrapping it up ​Armour Thyroid is a great medication and it CAN help you lose weight but it is not a magic weight loss pill.  At most, it will help you lose 10-20 pounds. If you have hypothyroidism and are more than 20 pounds overweight there is a high chance your weight gain is due to some other hormone imbalance . ​If you switched to Armour recently and haven’t lost ANY weight then make sure you have addressed these 5 areas: your dose, how much T3 you are taking in addition to Armour, your leptin levels, your allergies and sensitivities to inactive ingredients in medications and finally your reverse T3 level.  Remember: You won’t be able to lose weight unless all 5 of those areas have been addressed and reversed (if necessary). Scientific References #1. https://www.ncbi.nlm.nih.gov/pubmed/909397 #2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267409/ #3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980994/ #4. https://www.ncbi.nlm.nih.gov/pubmed/12915350 #5. https://www.ncbi.nlm.nih.gov/books/NBK285547/ #6. https://www.ncbi.nlm.nih.gov/pubmed/28192176 #7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509195/ #8. https://www.ncbi.nlm.nih.gov/pubmed/6479377 #9. http://www.ncbi.nlm.nih.gov/pubmed/402379 #10. http://emedicine.medscape.com/article/118651-overview #11. https://www.ncbi.nlm.nih.gov/pubmed/16883675 #12. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3205882/ #13. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069066/ #14. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608008/ #15. https://www.ncbi.nlm.nih.gov/pubmed/7435122 #16. https://www.ncbi.nlm.nih.gov/pubmed/21149757

Frontiers, Press Release, 20th March 2018. Discovery of a crucial link between chronic fatigue syndrome and lower levels of key thyroid hormones raises hopes for treating this common yet debilitating disease Frontiers in Endocrinology New research demonstrates a link between chronic fatigue syndrome (CFS) symptoms and lower thyroid hormone levels. Published in  Frontiers in Endocrinology , the study indicates that CFS, a condition with unknown causes, can be explained by lower thyroid hormones — but may be distinct from thyroidal disease. This finding can be seen as a first step to finding treatment for a debilitating illness for which there is no recognized treatment. Chronic fatigue syndrome is a common disease marked by lengthy spells of weakness, fatigue and depression. Its diagnosis is predominantly based on symptoms and on ruling out any underlying medical condition, rather than on laboratory tests and physical examination. Interestingly, several symptoms resemble those of hypothyroidism — a condition where the thyroid gland does not produce enough thyroid hormone. In hypothyroidism, the body tries to encourage thyroid hormone activity by releasing more thyroid-stimulating hormone — however, this does not happen in patients with chronic fatigue syndrome. This contrast in thyroid-stimulating activity led the study's authors to hypothesize that chronic fatigue syndrome is caused by low activity of thyroid hormones in the absence of thyroidal disease. Led by  Dr. Begoña Ruiz-Núñez  at the University Medical Center Groningen, The Netherlands, the researchers compared thyroid function and markers of inflammation between 98 CFS patients and 99 healthy controls. Remarkably, the CFS patients had lower serum levels of certain key thyroid hormones such as triiodothyronine (T3) and thyroxine (T4), but normal levels of thyroid-stimulating hormone. Additional analyses indicated that CFS patients had a lower urinary iodine status and low-grade inflammation, which possibly mirrored the symptoms of patients with hypothyroidism. These CFS patients, however, had relatively higher levels of another thyroid hormone called “reverse T3” or rT3. This appeared to be due to a shift in hormone production, where the body preferred to convert T4 to rT3 instead of producing T3. The low T3 levels found in CFS patients coupled with this switchover to rT3 could mean that T3 levels are severely reduced in tissue. “One of the key elements of our study is that our observations persisted in the face of two sensitivity analyses to check the strength of the association between CFS and thyroid parameters and low-grade inflammation,” says Dr. Ruiz-Núñez. “This strengthens our test results considerably.” The researchers believe inclusion of patient information, such as duration of illness, would enable a correlation with their biochemical profiles. Further, even though the study demonstrates a link between chronic fatigue syndrome symptoms and low levels of key thyroid hormones, a definitive cause for CFS remains unknown. If the study findings are confirmed by additional research, it may pave the way for a treatment for chronic fatigue syndrome. For more details, and the full journal report, contact Frontiers: Emma Duncanpress@frontiersin.org Science Communications ManagerT  +41 21 510 17 04 ME Association Comment: Dr Charles Shepherd, Hon. Medical Adviser, ME Association. This new research into thyroid gland hormones in ME/CFS represents an important advance in our understanding of hormonal abnormalities in this illness. We already know that there are abnormalities involving the hormone cortisol in ME/CFS. However, routine blood tests for thyroid function have always indicated that thyroid gland hormones are not affected. Consequently, thyroid hormone treatment (i.e. thyroxine) is not recommended for ME/CFS – as this can cause serious side effects in people who have normal thyroid function. This new research demonstrates a defect in thyroid hormone activity (involving the conversion of one thyroid hormone to another – T4 to T3) rather than actual thyroid gland disease. If these findings can be replicated by other independent research groups, it suggests that the cautious use of thyroid hormone treatment needs to be assessed in a clinical trial – as it could be an effective form of treatment for at least a subgroup of people with ME/CFS. The ME Association Ramsay Research Fund funds research into the cause and management of ME/CFS and would welcome research grant applications that aim to follow up these findings. The current ME Association position on thyroid gland function and the use of thyroxine in ME/CFS  can be read in a new leaflet that is available to download from our website. We also featured information relation to thyroid function and hypothyroidism , on our website last year. The research: Higher prevalence of ‘low T3 syndrome’ in patients with chronic fatigue syndrome: A case-control study Authors:Begoña Ruiz-Núñez1, 2*, Rabab Tarasse1, Emar Vogelaar3, Janneke Dijck-Brouwer1, FritsMuskiet1 Laboratory Medicine, University Medical Center Groningen, Netherlands, Healthy Institute, Spain, European Laboratory of Nutriënts (ELN), Netherlands. Abstract: Chronic fatigue syndrome (CFS) is a heterogeneous disease with unknown cause(s). CFS symptoms resemble a hypothyroid state, possibly secondary to chronic (low-grade) (metabolic) inflammation. We studied 98 CFS patients (21-69 years, 21 males) and 99 age- and sex-matched controls (19-65 years, 23 males). We measured parameters of thyroid function, (metabolic) inflammation, gut wall integrity and nutrients influencing thyroid function and/or inflammation. Most remarkably, CFS patients exhibited similar TSH, but lower FT3 (difference of medians 0.1%), TT4 (11.9%), TT3 (12.5%), %TT3 (4.7%), SPINA-GD (14.4%), SPINA-GT (14.9%), 24-hour urinary iodine (27.6%) and higher %rT3 (13.3%). FT3 below the reference range, consistent with the ‘low T3 syndrome', was found in 16/98 CFS patients vs. 7/99 controls (OR 2.56; 95% CI=1.00 – 6.54). Most observations persisted in two sensitivity analyses with more stringent cut-off values for BMI, hsCRP and WBC. We found possible evidence of (chronic) low-grade metabolic inflammation (ferritin and HDL-C). FT3, TT3, TT4 and rT3 correlated positively with hsCRP in CFS patients and all subjects. TT3 and TT4 were positively related to hsCRP in controls. Low circulating T3 and the apparent shift from T3 to rT3 may reflect more severely depressed tissue T3 levels. The present findings might be in line with recent metabolomic studies pointing at a hypometabolic state. They resemble a mild form of ‘non thyroidal illness syndrome' and ‘low T3 syndrome' experienced by a subgroup of hypothyroid patients receiving T4 monotherapy. Our study needs confirmation and extension by others. If confirmed, trials with e.g. T3 and iodide supplements might be indicated.