The diseases of thyroid gland are common around the globe, and India has contributed in this case significnatly. There are significant number of cases with thyroid disorder.¹ Around half of the population with thyroid disorders have microscopic nodules, overt “hypothyroidism” along with “hyperthyroidism” is reported in 5% of the population, goiters which are palpable are reported in 15% of the population, primary occult carcinoma is reported amongst the 3.5% of the population, and nearly 10% have abnormal TSH levels. There are 42 million thyroid gland disorders reported in India.² Amongst the thyroid gland disorder, “hypothyroidism” is the most common. A study conducted in Cochin involved 971 adults and 3.9% of them were found to have “hypothyroidism”. 11.4% of the “hypothyroidism” patients were women and 6.2% were male. The peculiar characteristic of the “hypothyroidism” is high level of TSH and low levels of “thyroid hormones”.³ “thyroid dysfunction” and irregularities in menstrual cycle is a significant clinical topic. “thyroid hormone” has variety of roles including the metabolism, maintenance of homeostasis, and it mainly effects reproductive function of female.⁷ “thyroid hormones” affect the fertility of females, thus the dysfunction of thyroid gland hampers the reproductive function during the reproductive age which marks its onset from menarche until menopause.⁸ “thyroid dysfunction”, encompassing both “hypothyroidism” and “hyperthyroidism”, has been linked to several menstrual disorders, including oligomenorrhea, polymenorrhea, menorrhagia, and amenorrhea.⁹ These associations highlight the significance of understanding interrelation of thyroid with reproductive system and its implications for women's health. Majority of the women found with thyroid gland disorders are in their reproductive age.¹⁰ The anemia has many types. The most reported type of anemia is IDA which occurs because of deficiency in iron. “iron deficiency” has many adverse effects in all age groups. In older children and adults especially in young females, it decreases work capacity and output and disturbs the immune system. It is also associated with reduced reproduction capacity. This can result in symptoms like fatigue, weakness, and shortness of breath. Anemia is a worldwide health problem affecting 33% of non-pregnant women and 38% of pregnant women.⁸ “Iron deficiency” (ID) is a common nutritional problem among reproductive age group females worldwide. IDA can impair psychomotor and cognitive development in children. It can reduce immunity, leading to infections. IDA during pregnancy can lead to increased bleeding, sepsis, low birth weight, and increased maternal and perinatal mortality.⁹ Plasma ferritin is a measure of iron stores and the best single test to confirm “iron deficiency”. Low hemoglobin concentration is the most readily available sign of anemia, but a significant fall in circulating hemoglobin cannot be detected until the final stage of “iron deficiency”.¹⁰ In tissues iron is stored inside the cell with the help of protein known as ferritin. This ferritin also carries iron into the serum. Moreover, it prevents the body from the symptoms of deficiency and overload of iron, acting as a buffer. Ferritin has been used to treat “iron deficiency” and for diagnosis of “iron deficiency”.¹¹ Various kind minerals are required for the normal functioning of the thyroid gland which includes selenium, iron, zinc, and iodine. For the synthesis of “thyroid hormone”, iodine is the most important component considering it is required in the basic structure of the hormone, selenium is the component required for conversion of T3 into T4. Selenium plays a role in the protection of the “thyroid gland” from excessive exposure of iodine. As for the zinc also has a role in the conversion of “thyroid hormone”. Low ferritin’s association with lower thyroid function has been reported.¹³ T3 hormone has a crucial role in controlling the “basal metabolic rate” and maintaining homeostasis. Method of working of T3 is by intracellular receptors, like steroids it modulates the transcription by binding to a specific gene. Even after transcription T3 modulates gene expression.¹⁴ A growing body of evidence suggests that IDA may play a significant role in the pathogenesis of “thyroid dysfunction”. The production of “thyroid hormones” is negatively affected by ID, and their deficiency reduces the proliferation of erythrocyte precursors, both directly and through reduced secretion of erythropoietin by the kidneys.¹⁵ Additionally, ID can affect the hypothalamic–pituitary–thyroid axis, leading to altered “thyroid hormone” levels and a decreased response to thyroid-stimulating hormone. Iron is also essential for the activity of thyroid peroxidase, an enzyme required to iodinate tyrosine in thyroglobulin a precursor protein for “thyroid hormone” synthesis.¹⁶ Despite growing interest in the relationship between “iron deficiency” and “thyroid dysfunction”, scientific evidence is still inconclusive. The association between iron status and thyroid function has been evaluated many times, but the results are conflicting. Whilst a few researches propose that IDA is associated with an increased risk of “thyroid dysfunction”, others have found no significant association.¹⁷ The relationship between “iron deficiency” and “thyroid hormone” has been researched a lot among pregnant or other healthy populations, but there are scarce studies on reproductive age group females. So we are conducting a study to evaluate the Iron profile including serum levels of iron, ferritin, and TIBC in clinically diagnosed cases of hypothyroid patients of reproductive age group females. This study also aimed to find the relationship between various parameters in the iron profile to that of the thyroid profile in hypothyroid and euthyroid pregnant females. AIM To establish the Biochemical relationship between the iron metabolism and thyroid profile in hypothyroid patients. OBJECTIVES 1. To estimate the levels of T3,T4 and TSH in clinically diagnosed hypothyroid patients of reproductive age group women. 2. To estimate the level of hemoglobin ,”serum iron” ,TIBC, ferritin and Transferrin saturation ratio in hypothyroid patients. 3. To find out statistical significant correlation between iron metabolism and “hypothyroidism”.

INCLUSION CRITERIA Women of “hypothyroidism” aged between 20 to 45 years of age. EXCLUSION CRITERIA Persons suffering from any other endocrine disorders. Persons having acute or chronic illness. Patients taking drugs which includes lithium or steroid which can interfere with thyroid function tests. Pregnant and lactating women will also be excluded from the study. METHODS TYPE OF STUDY: A cross-sectional study. STUDY PEROID: 18 months. SAMPLE SIZE: 60 cases of hypothyroid women from Guntur Government General Hospital during study period are included in the study period are included in the study. 40 age and sex matched controls included. SAMPLE COLLECTION: under aseptic precautions, 5ml of venous blood is collected from peripheral vein and allowed to clot and serum is separated. ESTIMATION METHODS USED FOR THYROID PROFILE AND IRON PROFILE Parameter Method TSH and Ferritin Chemiluminescence T3 and T4 Chemiluminescence Iron and TIBC Spectrophotometry The principles of Chemiluminescence Immunoassay (CLIA) are based on the specific binding between antibodies and antigens, combined with the generation of light through a chemical reaction. Immunometric methods based on chemiluminescence are capable of measuring TSH even at low concentration. SPECTROPHOTOMETRY The excess iron is added to the serum to bind all the ferritin in the serum, and the excess iron is adsorbed by adding the iron adsorbent. The iron bound with the ferritin is separated from the protein by the action of acid solution and reductant. Fe +3in serum is reduced to Fe+2, and Fe+2 binds with bipyridine to form the pink complex. In a certain range, the amount of TIBC is positively correlated with the depth of color. Sample Type- Serum, Plasma Detection Method- Colorimetric method Machine used- AU480 machine. TRANSFERRIN SATURATION ESTIMATION It is done using the formula: “serum iron” level/Total iron binding capacity x100.

Numerical variables were presented as mean ± standard deviation (mean ± S.D). Correlation analysis was done by using pearson correlation analysis. The data was entered in excel and analyzed in SPSS version 22. These were divided in to 2 groups: Group1: 60 test group with “hypothyroidism” Group 2: 40 controls P value<0.05 was statistically significant. P value >0.05 was statistically nonsignificant. P value< 0.001 was considered statistically highly significant. Table 1. Age and thyroid profile of study participants Parameter Case (n=60) Control (n=40) P value Age, Mean ± SD 31.83 ± 7.60 28.83 ± 4.21 0.025 T3 (ng/mL), Mean ± SD 0.68 ± 0.22 1.46 ± 0.33 <0.001 T4 (µg/dL), Mean ± SD 5.44 ± 1.62 9.41 ± 1.91 <0.001 TSH (mIU/L), Mean ± SD 15.50 ± 14.34 3.29 ± 1.15 <0.001 In our study, statistically significant (p value <0.05) more mean age was seen in case group compared to control group. Statistically significant (p value <0.001) lower mean T3 (ng/mL) level was seen in case group compared to control group. Statistically significant (p value <0.001) lower mean T4 (µg/dL)level was seen in case group compared to control group. Statistically significant (p value <0.001) more mean TSH (mIU/L) level was seen in case group compared to control group. Table 2. Distribution according to thyroid profile Parameter Category Case n (%) Control n (%) P value T3 High 0 (0.0%) 8 (20.0%) <0.001 T3 Low 48 (80.0%) 0 (0.0%) T3 Normal 12 (20.0%) 32 (80.0%) T4 Low 25 (41.7%) 0 (0.0%) <0.001 T4 Normal 35 (58.3%) 40 (100.0%) TSH High 59 (98.3%) 0 (0.0%) <0.001 TSH Normal 1 (1.7%) 40 (100.0%) In our study, statistically significant (p value <0.001) more proportion of low T3 level was seen in case group compared to control group. Statistically significant (p value <0.001) more proportion of low T4 level was seen in case group compared to control group. Statistically significant (p value <0.001) moe proportion of high TSH level was seen in case group compared to control group. Table 3. Iron profile and haemoglobin Parameter Case (n=60) Control (n=40) P value Ferritin (ng/mL), Mean ± SD 35.01 ± 19.19 107.25 ± 35.00 <0.001 Iron (µg/dL), Mean ± SD 45.93 ± 21.182 115.20 ± 38.900 <0.001 TIBC (µg/dL), Mean ± SD 461.85 ± 107.71 313.68 ± 94.14 <0.001 Haemoglobin (g/dL), Mean ± SD 8.38 ± 1.71 11.79 ± 0.73 <0.001 In our study, statistically significant (p value <0.001) lower mean Ferritin (ng/mL) level was seen in case group compared to control group. In our study, statistically significant (p value <0.001) lower mean Iron (µg/dL)level was seen in case group compared to control group. In our study, statistically significant (p value <0.001) more mean TIBC (µg/dL) level was seen in case group compared to control group. In our study, statistically significant (p value <0.001) lower mean Haemoglobin (g/dl) level was seen in case group compared to control group. Table 4. Categorical distribution of iron profile and haemoglobin Parameter Category Case n (%) Control n (%) P value Ferritin Low 7 (11.7%) 0 (0.0%) 0.066 Ferritin Normal 53 (88.3%) 40 (100.0%) Iron High 0 (0.0%) 5 (12.5%) <0.001 Iron Low 56 (93.3%) 3 (7.5%) Iron Normal 4 (6.7%) 32 (80.0%) TIBC High 38 (63.3%) 6 (15.0%) <0.001 TIBC Low 1 (1.7%) 9 (22.5%) TIBC Normal 21 (35.0%) 25 (62.5%) Haemoglobin <11 g/dL 58 (96.7%) 2 (5.0%) <0.001 Haemoglobin ≥11 g/dL 2 (3.3%) 38 (95.0%) In our study, statistically insignificant (p value >0.05) more proportion of low ferritin level was seen in case group compared to control group. Statistically significant (p value <0.001) more proportion of low iron level was seen in case group compared to control group. Statistically significant (p value <0.001) more proportion of high TIBC level was seen in case group compared to control group. Statistically significant (p value <0.001) more proportion of Haemoglobin level of <11 g/dlwas seen in case group compared to controlgroup. Table 5. Correlation between thyroid profile and iron profile parameters Thyroid parameter Iron profile parameter Pearson correlation (r) P value TSH Ferritin −0.338 0.001 TSH TIBC 0.169 0.093 TSH Iron −0.294 0.003 TSH Haemoglobin −0.226 0.024 TSH Transferrin saturation ratio −0.266 0.008 T3 Ferritin 0.701 <0.001 T3 TIBC −0.440 <0.001 T3 Iron 0.620 <0.001 T3 Haemoglobin 0.646 <0.001 T3 Transferrin saturation ratio 0.552 <0.001 T4 Ferritin 0.655 <0.001 T4 TIBC −0.506 <0.001 T4 Iron 0.611 <0.001 T4 Haemoglobin 0.612 <0.001 T4 Transferrin saturation ratio 0.594 <0.001 In our study a negative and statistically significant (p value <0.05) correlation between Thyroid Stimulating Hormone (TSH) and all ironprofile parameters(ferritin, iron, haemoglobin transferrin saturationratio) except TIBC was found. In our study a positive and statistically significant (p value <0.001) correlation between T3, T4 and alliron profile parameters except TIBC wasfound. A negative and statistically significant (p value <0.001) correlation with TIBC was foundAlsoapositivecorrelationbetweenFerritinandT4incase group was found which was statistically significant.

The normal functioning of the thyroid gland depends on many trace elements such as iron, selenium, zinc and iodine. Deficiencies in these elements, particularly iron, impairs thyroid functions. There is abundant literature evidence that “iron deficiency” decreases the circulating levels of both thyroxine and triiodothyronine and can also reduce peripheral conversion of T4 to T3.¹⁷ The normal range of T3 is 0.98-1.78 ng/ml. In this study, statistically significant (p value <0.001) lower mean T3 levels were seen in the case group compare to control group. Statistically significant (p value <0.001) more proportion of low T3 level was seen in case group compare to control group. Similar results were reported by Santhosh Kumar et al.,¹⁴ p<0.001, Sachdeva et al¹² p<0.001 Suman Chatterjee et al¹³ (p=0.000) in their studies associating thyroid profiles with iron profiles in hypothyroid patients. Rad et al¹⁶ in 2016 evaluated the effect of “hypothyroidism” on hematological parameters and also body iron store in an Iranian population in 45 hypothyroid patients with 45 matched controls. This study showed statistically significant (p value <0.05) more mean TSH (µIU/L) levels (p=0.024) in cases group as compared to controls. Similar findings have also been reported by Aminorroaya et al¹⁷ in 2017, Santhosh Kumar et al.,¹⁴ Sachdeva et al¹² and Suman Chatterjee et al¹³ in their studies associating thyroid profiles with iron profiles in hypothyroid patients. Serum iron levels (normal range 80-180 µg/dl) in this study were found to be statistically significantly low (p value <0.001) in cases as compared to controls. This finding was similarly reported in a study by Santosh Kumar et al¹⁴ which was done to explore any relationship between iron and thyroid profiles in hypothyroid patients as compared to healthy subjects. In the current study, statistically significant (p value<0.001) more mean TIBC (µg/dL) level was seen in case group compare to control group. This is in concurrence with a cross-sectional study¹⁸ which correlated “iron deficiency” Status with Thyroid Profile among Subclinical and Overt “hypothyroidism” Patients where TIBC was more, in both the subclinical and overt hypothyroid groups compared to healthy subjects. In another study by Manger et al¹⁹ found correlations between iron and copper levels as well as total iron binding capacity (TIBC) and triiodothyronine (T3), T4, and thyroid-stimulating hormone (TSH) levels in healthy and hypothyroid subjects. In the current study, statistically significant (p value <0.001) lower mean Haemoglobin (g/dl) level of 8.44g/dL was seen in case group compare to control group. Also, statistically significant (p value <0.001) more proportion of low Haemoglobin level 91.7% was seen in case group compare to 5% in the control group. Our findings reiterate the findings of Ahmed et al 2020²⁰, who did a case-controlled study on effect of thyroid function on blood parameters. The level of hemoglobin was significantly associated with “thyroid dysfunction” (P value 0.000) and anemia was present in 31.3% of the patients in this study. In our study, statistically significant (p value <0.001) lower mean Transferrin saturation ratio level was seen in case group (11.48% versus 43.07%) compare to control group. In a similar study by Khatiwada et al in 2016²¹, which studied the association between iron status and thyroid function in Nepalese children, the transferrin saturation was also statistically significant (P=0.002). In our study a negative and statistically significant (p value <0.05) correlation between Thyroid Stimulating Hormone (TSH) and all iron profile parameters (ferritin, iron, haemoglobin transferrin saturation ratio) except TIBC was found. In contrast, in a study by Dahiya et al, (2016)²² TIBC was found to be positively correlated with TSH (r=0.063, p=0.623) though, it was not significant statistically. The correlation of TSH was found to be weakly positive but insignificant with TIBC in the hypothyroid cases. In our study a positive and statistically significant (p value <0.001) correlation between T3, T4 and all iron profile parameters except TIBC was found. A negative and statistically significant (p value <0.001) correlation with TIBC was found. Also a positive correlation between Ferritin and T4 in case group was found which was statistically significant. Similar results were also reported by Dedic et al in 2023²³, in female subjects of the hypothyroid group, a weak positive correlation was found between “serum ferritin” concentration and fT3 (Rho=0.488; p<0.05). In the euthyroid group, a correlation of the same strength and direction was found for fT4 (Rho = 0.366; p < 0.05). Eftekari et al in 2006²⁴ using stepwise regression analysis, concluded that only ferritin contributed significantly to the rT3 concentration (r=-0.35, P<0.01). Limitations of this study include a small sample size. This could contribute to the disparity in results that are observed when compared to other studies with larger sample sizes. Also, assessment of anti-Thyroid Peroxidase Antibody (anti-TPO) would have helped to rule out autoimmune thyroid disorders such as Hashimoto’s thyroiditis which can also impact the iron profile and cause iron deficiency anaemia.

Iron profile of hypothyroid women in the reproductive age was evidenced to be pointing towards “iron deficiency”anaemia. Future studies with a larger sample size, and anti-ThyroidPeroxidaseAntibody (anti-TPO) to ruleoutautoimmunethyroid disorders such as Hashimoto’s thyroiditis can confirm that “hypothyroidism” can be a risk factor for development of iron-deficiency anemia in women of reproductive age. This information will be valuable in the management of “hypothyroidism” in these individuals.

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