Hypothyroidism is one of the most common endocrine disorders worldwide and is characterized by insufficient production of thyroid hormones, resulting in a generalized reduction in metabolic activity.¹ The condition affects approximately 3–10% of the adult population, with a higher prevalence among women and older individuals.² The most frequent cause of primary hypothyroidism in iodine-sufficient regions is autoimmune thyroiditis (Hashimoto's thyroiditis), whereas iodine deficiency remains an important cause in many developing countries.³

Thyroid hormones play a fundamental role in regulating

carbohydrate metabolism, protein synthesis, lipid metabolism, erythropoiesis, and immune function. Consequently, thyroid hormone deficiency leads to widespread metabolic and hematological disturbances that complicate the interpretation of several routinely used laboratory biomarkers.⁴

The relationship between thyroid dysfunction and glucose metabolism has attracted increasing attention because thyroid hormones directly influence insulin secretion, hepatic glucose production, intestinal glucose absorption, and peripheral glucose utilization.⁵ In hypothyroidism, decreased hepatic gluconeogenesis and reduced glucose disposal may alter glucose homeostasis.⁶ Although fasting blood glucose (FBG) and random blood glucose (RBG) frequently remain within normal limits in patients without diabetes mellitus, glycated hemoglobin (HbA1c) may be unexpectedly elevated. This discrepancy raises concerns regarding the reliability of HbA1c as an indicator of glycemic status in individuals with thyroid dysfunction.

HbA1c is considered the gold standard biomarker for long-term glycemic assessment because it reflects the average blood glucose concentration over the preceding 8–12 weeks.⁷ The formation of HbA1c depends on the non-enzymatic glycation of hemoglobin throughout the lifespan of circulating erythrocytes. However, HbA1c is influenced not only by blood glucose concentration but also by conditions that affect erythrocyte survival. Any disorder that prolongs red blood cell lifespan may falsely increase HbA1c values, whereas conditions associated with shortened erythrocyte survival may lead to falsely reduced HbA1c concentrations. Therefore, diseases affecting erythropoiesis or red blood cell turnover can significantly compromise the diagnostic and prognostic utility of HbA1c.

Hypothyroidism is frequently associated with hematological abnormalities that may influence erythrocyte lifespan and consequently alter HbA1c measurements. Thyroid hormones stimulate erythropoietin production and bone marrow activity; therefore, thyroid hormone deficiency often results in decreased erythropoiesis and various forms of anemia.⁸ Normocytic normochromic anemia is the most common hematological manifestation, although microcytic anemia secondary to iron deficiency and macrocytic anemia due to vitamin B12 or folate deficiency may also occur, particularly in autoimmune thyroid disease.⁹ Reduced erythrocyte turnover increases the exposure time of hemoglobin to circulating glucose, potentially leading to falsely elevated HbA1c despite normal plasma glucose concentrations. These alterations may contribute to misclassification of glycemic status and inappropriate diagnosis or management of diabetes mellitus in patients with hypothyroidism.

Routine plasma glucose measurements such as fasting blood glucose and random blood glucose directly reflect circulating glucose concentrations and are not influenced by erythrocyte lifespan. However, these measurements represent glucose levels at a single time point and may not adequately reflect intermediate-term glycemic exposure. Alternative glycemic biomarkers, including fructosamine and glycated albumin, have therefore emerged as valuable adjuncts in situations where HbA1c may be unreliable. Fructosamine represents glycated serum proteins, predominantly albumin, whereas glycated albumin specifically reflects glycation of serum albumin.¹⁰ Because albumin has a biological half-life of approximately 2–3 weeks, both biomarkers provide an assessment of short-term glycemic control and remain unaffected by disorders that alter erythrocyte survival. These characteristics make fructosamine and glycated albumin particularly useful in patients with hematological disorders, hemoglobinopathies, chronic kidney disease, pregnancy, and endocrine disorders such as hypothyroidism.

Autoimmune thyroid disease constitutes the predominant cause of primary hypothyroidism in many populations. Hashimoto's thyroiditis is characterized by chronic lymphocytic infiltration of the thyroid gland accompanied by the production of thyroid-specific autoantibodies, particularly anti-thyroid peroxidase (anti-TPO, also known as anti-microsomal antibodies) and anti-thyroglobulin antibodies (TgAb). Anti-TPO antibodies are considered the most sensitive marker of autoimmune thyroiditis and are detected in the majority of affected individuals, while TgAb further supports the diagnosis and reflects ongoing autoimmune destruction of thyroid tissue. Persistent autoimmune inflammation contributes to progressive thyroid dysfunction and has been associated with metabolic abnormalities extending beyond thyroid hormone deficiency.¹¹

Autoimmune thyroid disease frequently coexists with other autoimmune disorders, reflecting shared genetic susceptibility and immune dysregulation. Antinuclear antibodies (ANA), commonly regarded as markers of systemic autoimmunity, may be detected in a subset of patients with autoimmune hypothyroidism even in the absence of clinically overt connective tissue disease. The presence of ANA may indicate generalized immune activation and identify patients who warrant further evaluation for concomitant autoimmune conditions such as systemic lupus erythematosus, Sjögren syndrome, rheumatoid arthritis, or autoimmune hepatitis. Simultaneous assessment of thyroid-specific autoantibodies and ANA may therefore provide a broader understanding of the autoimmune profile associated with hypothyroidism.¹²

Complete blood count (CBC) parameters offer additional clinical information that may explain alterations in HbA1c observed in hypothyroid patients. Evaluation of hemoglobin concentration, hematocrit, red blood cell count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), leukocyte count, differential leukocyte count, and platelet count provides insight into underlying hematological abnormalities that influence erythrocyte kinetics. Identification of anemia or abnormal erythrocyte indices may assist clinicians in distinguishing falsely elevated HbA1c values from genuine deterioration in glycemic control.

Although several previous studies have demonstrated elevated HbA1c levels in hypothyroid patients despite normal plasma glucose concentrations, most investigations have focused only on HbA1c and fasting glucose or evaluated limited thyroid parameters.¹³ Few studies have simultaneously compared HbA1c with fasting blood glucose, random blood glucose, fructosamine, glycated albumin, thyroid function tests, thyroid-specific autoantibodies, antinuclear antibodies, and detailed hematological indices within the same study population. Consequently, the mechanisms underlying HbA1c elevation in hypothyroidism remain incompletely understood, and evidence regarding the most reliable glycemic marker in these patients remains limited.

The present comparative cross-sectional study is designed to comprehensively evaluate glycemic biomarkers in hypothyroid patients by simultaneously assessing HbA1c, fasting blood glucose, random blood glucose, fructosamine, and glycated albumin together with thyroid function tests, anti-thyroid peroxidase antibodies, anti-thyroglobulin antibodies, antinuclear antibodies, and complete blood count parameters. By examining the relationships among thyroid dysfunction, autoimmune status, hematological alterations, and glycemic markers, this study aims to determine whether HbA1c accurately reflects glycemic status in hypothyroidism and whether alternative biomarkers provide a more reliable assessment. The findings are expected to improve diagnostic accuracy, prevent misinterpretation of HbA1c results, and support evidence-based selection of glycemic markers in patients with hypothyroidism, particularly those with autoimmune thyroid disease and associated hematological abnormalities.

Study Design and Setting: A hospital-based comparative cross-sectional study will be conducted over 12 months at the Department of Biochemistry in collaboration with the Departments of Endocrinology and Pathology in Abbottabad international medical hospital after obtaining approval from the Institutional Review Board (IRB). Written informed consent will be obtained from all participants prior to enrollment. Study Population: The study will include adult patients (18–65 years) with biochemically confirmed primary hypothyroidism and age- and sex-matched healthy euthyroid controls. Participants with diabetes mellitus, chronic kidney or liver disease, pregnancy, hemoglobinopathies, recent blood transfusion, malignancy, acute infection, or other conditions affecting HbA1c or erythrocyte lifespan will be excluded. Sample Size: A total of 150 participants (75 hypothyroid patients and 75 euthyroid controls) will be recruited using consecutive sampling. The sample size will be calculated using OpenEpi software with a 95% confidence level, 80% study power, and an expected difference in HbA1c between the study groups. Data and Sample Collection: Demographic and clinical information, including age, sex, body mass index (BMI), duration of hypothyroidism, medication history, and family history, will be recorded using a structured questionnaire. Following an overnight fast (8–12 hours), venous blood samples will be collected for biochemical, immunological, and hematological investigations. Serum samples will be separated by centrifugation and stored at −80°C until analysis. Laboratory Investigations: Thyroid function will be assessed by measuring serum thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) using chemiluminescent immunoassays. Glycemic status will be evaluated by fasting blood glucose (FBG), random blood glucose (RBG), glycated hemoglobin (HbA1c), fructosamine, and glycated albumin using standardized enzymatic and immunochemical methods. Autoimmune status will be determined by measuring anti-thyroid peroxidase (anti-TPO), anti-thyroglobulin (TgAb), and antinuclear antibodies (ANA). Complete blood count (CBC), including hemoglobin, hematocrit, red blood cell indices, white blood cell count, and platelet count, will be analyzed using an automated hematology analyzer. Statistical Analysis: Data will be analyzed using IBM SPSS Statistics version 26. Continuous variables will be expressed as mean ± standard deviation and categorical variables as frequencies and percentages. Group comparisons will be performed using the independent t-test, Mann–Whitney U test, or Chi-square test as appropriate. Correlations between glycemic biomarkers, thyroid function, autoimmune markers, and CBC parameters will be assessed using Pearson's or Spearman's correlation analysis, while multiple linear regression will identify independent predictors of HbA1c. A p-value <0.05 will be considered statistically significant.

Below are hypothetical (expected) results consistent with your proposed study. These data are realistic for a comparative cross-sectional study and can be used as a model for manuscript preparation. Replace them with your actual findings after data collection.

Table 1. Baseline Demographic and Clinical Characteristics of the Study Population

Table 2. Thyroid Function Tests

Table 3. Comparison of Glycemic Biomarkers

Graph 1. Comparison of glycemic biomarkers between patients with hypothyroidism and healthy controls.

Table 4. Thyroid Autoantibodies and Antinuclear Antibodies

Table 5. Complete Blood Count Parameters

Graph 2. Relative Distribution of Mean Glycemic Biomarker Values in Patients with Hypothyroidism

Table 6. Correlation of HbA1c with Thyroid Function, Autoimmune Markers, and CBC Parameters in Patients with Hypothyroidism

Table 7. Multiple Linear Regression Analysis for Independent Predictors of HbA1c

The present study demonstrated that patients with hypothyroidism had significantly higher HbA1c levels compared with euthyroid controls despite having comparable fasting blood glucose (FBG), random blood glucose (RBG), fructosamine, and glycated albumin levels. This finding suggests that elevated HbA1c in hypothyroidism may not always indicate impaired glycemic control but may partly reflect thyroid-related alterations in erythrocyte physiology. Previous studies have similarly reported increased HbA1c concentrations in non-diabetic hypothyroid individuals despite normal glucose levels, supporting the concept that thyroid dysfunction can influence HbA1c independently of glycemia.14 In the current study, HbA1c was significantly increased in hypothyroid patients (5.92 ± 0.49%) compared with controls (5.41 ± 0.36%), whereas FBG and RBG showed no significant differences. These findings are consistent with the observations of Babu et al., who reported elevated HbA1c in hypothyroid individuals without diabetes and suggested that anemia and reduced erythrocyte turnover may act as connecting mechanisms. Their study demonstrated higher HbA1c levels in hypothyroid patients with anemia compared with matched controls, emphasizing that HbA1c interpretation should consider hematological status.15 The mechanism underlying this discrepancy is primarily related to thyroid hormone-mediated regulation of erythropoiesis. Thyroid hormones stimulate erythropoietin production and bone marrow activity; therefore, reduced thyroid hormone availability decreases erythropoiesis and prolongs erythrocyte survival. Longer exposure of erythrocytes to circulating glucose results in increased hemoglobin glycation and falsely elevated HbA1c values. Ambalavanan et al. demonstrated that altered thyroid status affects HbA1c values through changes in red blood cell turnover, supporting the biological explanation observed in the present study. 16 Unlike HbA1c, fructosamine and glycated albumin did not differ significantly between hypothyroid patients and controls in our study. This finding indicates that these biomarkers may more accurately represent actual glycemic exposure because they reflect glycation of serum proteins rather than erythrocyte-dependent hemoglobin glycation. Previous investigations have suggested that glycated albumin and fructosamine are useful alternatives when HbA1c reliability is compromised by conditions affecting red blood cell lifespan, including anemia and endocrine disorders.14 The thyroid profile findings confirmed significant endocrine dysfunction, with markedly elevated TSH and reduced FT3 and FT4 levels among hypothyroid patients. Similar studies have shown that thyroid hormone deficiency is associated with metabolic disturbances, including altered glucose utilization and insulin sensitivity. However, the present findings indicate that thyroid dysfunction affects HbA1c interpretation more strongly through hematological mechanisms rather than direct changes in plasma glucose concentrations.17 Autoimmune evaluation revealed significantly increased anti-TPO and anti-thyroglobulin antibody positivity among hypothyroid patients compared with controls, confirming autoimmune thyroiditis as the predominant underlying mechanism. Additionally, ANA positivity was higher among hypothyroid individuals, suggesting enhanced autoimmune activation. Autoimmune thyroid diseases frequently coexist with systemic autoimmune abnormalities due to shared immunological pathways and genetic susceptibility. The coexistence of thyroid-specific antibodies and ANA positivity highlights the importance of comprehensive autoimmune assessment in patients with hypothyroidism. Hematological analysis revealed significantly lower hemoglobin, hematocrit, and RBC count with increased RDW among hypothyroid patients. These findings are comparable with previous reports demonstrating that hypothyroidism is commonly associated with anemia due to impaired erythropoiesis, iron deficiency, or autoimmune mechanisms.18 Increased RDW reflects heterogeneous erythrocyte populations and altered red cell maturation, which may further influence HbA1c accuracy. The strong negative correlation between HbA1c and hemoglobin and the positive correlation between HbA1c and RDW observed in our study further support this relationship. Correlation analysis demonstrated that HbA1c was positively associated with TSH, anti-TPO antibodies, and RDW, while negatively associated with FT3, FT4, hemoglobin, and RBC count. These findings indicate that worsening thyroid dysfunction and autoimmune activity are accompanied by increased HbA1c values, independent of plasma glucose. Similar observations have been reported in studies showing reduction of HbA1c after restoration of euthyroid status, suggesting that thyroid correction can normalize HbA1c values without major changes in glucose levels.19 Multiple regression analysis in the present study identified TSH, hemoglobin, RDW, and anti-TPO antibodies as independent predictors of HbA1c. This finding provides additional evidence that HbA1c variation in hypothyroidism is influenced by a combination of thyroid dysfunction, autoimmune activity, and erythrocyte characteristics. Therefore, clinicians should avoid interpreting HbA1c values in isolation in hypothyroid patients, particularly those with anemia or abnormal red cell indices. The clinical significance of these findings is substantial. Overreliance on HbA1c may result in overdiagnosis of prediabetes or diabetes in hypothyroid patients, leading to unnecessary investigations and treatment. A combined approach incorporating plasma glucose measurements, fructosamine, glycated albumin, thyroid function tests, autoimmune markers, and CBC parameters may provide a more accurate assessment of metabolic status. Overall, the present study extends previous findings by simultaneously evaluating conventional glycemic markers, alternative glycemic biomarkers, thyroid autoantibodies, ANA, and detailed hematological parameters within the same population. The results support the concept that HbA1c should be interpreted cautiously in hypothyroidism and that fructosamine and glycated albumin may serve as valuable complementary markers for assessing true glycemic status.

Patients with hypothyroidism exhibited significantly higher HbA1c levels despite having normal fasting blood glucose, random blood glucose, fructosamine, and glycated albumin, suggesting that HbA1c may overestimate glycemic status because of altered erythrocyte turnover and associated hematological abnormalities. Elevated thyroid autoantibodies and ANA further reflected the autoimmune nature of the disease. These findings indicate that HbA1c should be interpreted cautiously in hypothyroid patients, and alternative glycemic markers such as fructosamine and glycated albumin may provide a more accurate assessment of glycemic control, particularly in individuals with anemia or autoimmune thyroid disease.

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