Iodine deficiency is still a significant global public health problem and affects both developed and developing countries. It is estimated that iodine intake is insufficient in about 2 billion people worldwide [1]. Iodine is an essential micronutrient required for thyroid hormone synthesis and its role in fetal and early childhood brain development is emphasized by studies showing iodine deficiency in pregnancy is associated with stunted growth and neuromotor, intellectual, behavioural, and cognitive impairment, as well as, cretinism in severe cases [2,3]. Iodine nutrition in community is accessed by goiter rate, median urinary iodine level, serum TSH and thyroglobulin, although key indicator is median urinary iodine levels among school children [4]. Since majority of dietary iodine (>90%) is excreted in the urine, urinary iodine excretion (UIE) is largely a passive process dependent on glomerular filtration rate (GFR) [5]. The UIE in a non-pregnant individual on a stable diet represents a dynamic equilibrium between dietary intake, thyroidal iodine extraction, the total body thyroid hormone pool, and GFR. The adequacy of iodine nutrition is defined by the following criteria: A median MUI of at least 100 μg/L (with <20% of the population having MUI <50 μg/L) represents adequate population iodine nutrition; a median MUI between 50 μg/L and 99 μg/L represents mild iodine deficiency; and medians of 20-49 μg/L and <20 μg/L represent moderate and severe iodine deficiency, respectively [4].

During normal pregnancy, GFR increases within the first month following conception, peaking by the end of the first trimester by approximately 40-50% above prepregnant levels [6]. Hence, pregnancy can be expected to result in increased renal iodine loss. Taking these physiological changes into consideration, the WHO and the American Thyroid Association have recommended higher pregnancyspecific urinary iodine ranges as marker of adequacy of iodine nutrition i.e. MUI of <150 μg/L represent insufficient iodine nutrition; MUI of 150-249 μg/L represent adequate iodine nutrition and ≥ 250 represent above requirement [1]. Studies on iodine nutrition in pregnancy, based on median urinary iodine excretion, have been far and few with inter trimester variation, with no study being from Haryana (India). So we conducted this cross sectional study to know the iodine status of pregnant women in Haryana, which is supposed to be an iodine sufficient belt where all households are consuming iodized salt.

This was a cross-sectional study, conducted at Department of Endocrinology & Medicine Unit VI of PGIMS Rohtak for a period of 1 year after approval from institutional ethical review board. 381 consecutive pregnant women attending antenatal clinic in the obstetrics and gynecology department were included for study. All women who were carrying a healthy singleton uncomplicated intrauterine pregnancy and consuming iodized salt were recruited. On enrolment of participants, informed consent was obtained from each patient after explaining the purpose of the study. Then detailed history was enquired and participants were subjected to relevant general physical examination which included presence or absence of goiter and general and systemic examinations. Participants having any history of chronic illness, goiter on physical examination, thyroid illness in the past or present, consuming thyroid medications (current and past), consuming iodine containing vitamins and minerals, having family history of thyroid illness, poor obstetrics history included 3 or more abortions were excluded from the study. Blood sample and spot urine samples were collected.

Estimation for FT3, FT4 TSH, and TPO Ab was done using the electrochemiluminescence (ECL) technique using commercially available kits by Advia Centaur CP analyzer system and immulite 1000. The analytical sensitivities for FT3, FT4, TSH and anti TPO were 0.2 pg/mL, 0.1 ng/dL, 0.010 μIU/mL and 7 IU/mL respectively. Intra-assay coefficients of variation for FT3, FT4, TSH and TPO Ab were 3.8%, 2.20%, 5.2% and 5.6%, respectively. Laboratory reference range for FT3, FT4 and TSH were 2.3-4.2 pg/mL, 0.89-1.76 ng/dL and 0.35-5.5 μIU/L, respectively. Normal range for TPO antibody was <35 IU/mL and value greater than or equal to 35 IU/mL indicate elevated TPO Ab. As per AACE guidelines pregnant women with TSH level ≤ 2.5 μIU/mL in first trimester and TSH level ≤ 3.0 μIU/mL in second and third trimester were classified as having normal thyroid status [7]. Urinary iodine levels were measured by method of Sandell and Kolthoff [8].

According to the aims and objectives of the study, the data was compiled and entered into MS Excel and analysed, using appropriate statistical tests in SPSS (statistical package for social sciences) version 20. For descriptive statistics, frequencies, percentages and median were calculated. To assess difference between categorical variables ‘Chi Square Test’ was used. Mann Whitney U test was used to compare the median of two separate sets of independent samples. For comparison of means of more than two samples-Kruskal Wallis H test was used. The p-values were two tailed and probability level of significant difference was set at <0.05.

381 consecutive pregnant women attending antenatal clinic (ANC) were screened for their thyroid status and 285(74.8%), 82(21.52%), 11(2.89%) and 3(0.79%) were diagnosed as euthyroid, SCH, overt hypothyroid and hyperthyroid respectively. The 285 euthyroid pregnant women were selected as study population and were studied for Median urinary iodine (MUI) excretion. The mean age of euthyroid pregnant women was 23.47 ± 3.2 year with mean BMI of 22.66 ± 3.96 kg/m². MUI level of euthyroid women was 119.2 μg/L (IQR=35.8-310.95) with 2.5^th percentile being 5.55 μg/L and 97.5^th percentile being 1058.68 μg/L. Out of 285 euthyroid women, 153(53.68%) euthyroid were having MUI level below 150 μg/L.

Among 285 euthyroid women, 89(31.23%) were from 1^st trimester, 98(34.39%) were from 2^nd and 3^rd trimester each. Mean age of women in 1^st, 2^nd and 3^rd trimester was 23.19 ± 2.88, 23.33 ± 3.29 and 23.88 ± 3.45 year respectively. Trimester specific MUI levels of euthyroid women were 193.25 μg/L, 111.8 μg/L and 97.65 μg/L in 1^st, 2^nd and 3^rd trimester respectively, showing a decreasing trend from 1^st to 3^rd trimester. 39(43.82%), 53(54.08%) and 61(62.24%) women of 1^st, 2^nd and 3^rd trimester were having MUI level below 150 μg/L.

When euthyroid women were divided as per Anti-TPO antibody status, 255(89.47%) and 30(10.53%) were found to be Anti-TPO antibody negative and positive respectively. The mean age of Anti-TPO antibody negative and positive women were 23.52 ± 3.2 and 23.1 ± 3.22 year respectively without being statistically significant (p=0.5). MUI levels of Anti-TPO antibody positive and negative euthyroid women were 153.8 μg/L and 118.4 μg/L respectively without being statistically significant (p=0.98). 138(54.12%) of Anti-TPO antibody negative and 15(50%) of Anti-TPO antibody positive were having MUI level below 150 μg/L. Table 1 summarises the frequency distribution of MUI level among euthyroid pregnant women according to trimester of pregnancy and TPO antibody status.

Table 1: Comparison of frequency distribution of MUI level among euthyroid pregnant women.

Evaluation of thyroid disease in pregnancy is important for gestational maternal health, obstetrical outcome and subsequent mental and physical development of the child. Prevalence of thyroid disorders is more in Asian countries compared to west [9]. Iodine is a micronutrient whose main role is in the synthesis of thyroid hormones and deficiency of which leads to a series of functional and developmental abnormalities grouped under the heading of “Iodine Deficiency Disorders (IDD)”. There are very few studies with conflicting results regarding the level of median urinary iodine level during different trimester of pregnancy. Some studies have shown similar values of urinary iodine in both pregnant and non-pregnant women while the others have shown increased excretion of urinary iodine in pregnant women [10-14]. On the contrary, few studies had shown that urinary iodine decreases during gestation [15,16].

Present study found MUI level of 119.2 μg/L among euthyroid pregnant women with 53.68% having MUI <150 μg/L indicating iodine deficiency among them. Similar deficiency was found by Ategbo, et al. [17] in Rajasthan where 56% out of 360 pregnant women were having MUI level below 150 μg/L and Majumder, et al. [18] in Kolkata with 88(37%) out of 237 pregnant women were having iodine deficiency. Although Grewal, et al. [19] conducted a study in Delhi and found that only 3(2%) out of 150 pregnant women were having urinary iodine levels <150 μg/L with median value of 304 μg/L. Another study conducted by Ainy, et al. [20] from Delhi found MUI concentration of 178.2 μg/L, 182.8 μg/L, and 167.5 μg/L during the first, second, and third trimesters respectively. All levels fell within the range (150-250 μg/L) recommended by WHO/UNICEF/ICCIDD, 2007 for pregnant women. Urinary iodine levels of pregnant women were higher than those of non-pregnant women (143.8 μg/L). The higher MUI in pregnant as well as nonpregnant in above studies from Delhi could be due small sample size, selectivity of study population, and failure to exclude women consuming vitamins and mineral which are important sources of nonsalt iodine intake.

Various physiological changes associated with increased iodine requirement as pregnancy advances include increase in renal blood flow and GFR results in increased urinary iodine excretion, increased maternal thyroid hormone synthesis to maintain euthyroidism and transplacental transfer of iodine to the fetus for fetal thyroid hormone synthesis. There are very few studies across the world studying and reporting trimester specific median urinary excretion. In present study median urinary iodine levels of euthyroid pregnant women in 1^st, 2^nd and 3^rd trimester were found to be 193.2 μg/L, 111.8 μg/L and 97.65 μg/L respectively. Decrease was 42.13% from 1^st to 2^nd trimester and additional 7.32% from 2^nd to 3^rd trimester (p=0.69) which was not statistically significant. Brander, et al. [21] from Switzerland showed decrease in UIC from 267 μg/L, 206 μg/L to 172 μg/L from 1^st, 2^nd to 3^rd trimester respectively. Ainy et al. involving 298 Tehranian pregnant women found decreasing MUI from 1^st (193) to 2^nd (159) to 3^rd (141) trimester [22]. The increased thyroidal iodide clearance and shift to fetal-placental unit as pregnancy advances may explain the observation of progressive MUI decrease during gestation. However, other studies shows increased urinary iodine excretion as pregnancy advances. A study done in United Kingdom by Bath, et al. [23] found median Urinary iodine level (mcg/l) of 42, 52 and 69.4 in 1^st, 2^nd and 3^rd trimesters respectively. Chakraborty, et al. [24] found median urinary iodine level of 137.5, 135 and 160 μg/L in 1^st, 2^nd and 3^rd trimesters respectively, while study conducted by Grewal, et al. [19], involving 50 women from each trimester of pregnancy found median urinary iodine levels in 1^st, 2^nd and 3^rd trimester as 285, 318 and 304 μg/L, respectively. Various likely reasons cited for these differences in median urinary iodine excretion as pregnancy advances includes increased utilization of iodine to meet the high demands for thyroid hormone production during first trimester at least partly because of stimulation of thyroid gland by human chorionic gonadotropin reducing the proportion excreted into the urine and increase in dietary iodine intake, particularly from dairy products and non-dietary iodine intake inform of vitamin and minerals as pregnancy progresses which would had resulted in increased iodine excretion with advancing gestation. In the present study we have studies women who were not consuming vitamins and minerals containing iodine and found that median urinary iodine decreases as pregnancy advances.

No study in past have studied and compared the median urinary iodine level among TPO antibody positive and negative euthyroid pregnant women. In present study we found that although anti-TPO positive euthyroid women had higher median urinary iodine (153.8 μg/L) compared to TPO negative women (118.4 μg/L) although this difference was statistically insignificant (p=0.98). Studies from China and Japan had shown the higher incidence of thyroiditis among subjects taking more than adequate iodine intake [25,26]. Various mechanisms assumed for the development of iodine-induced autoimmune thyroiditis include increased the immunogenicity of thyroglobulin (Tg) in presence of high iodine intake, thereby precipitating an autoimmune process at both the T-and B-cell level, direct toxic effect of iodine on thyroid cells and lastly direct stimulation of immune and immunity-related cells by iodine.

To conclude with in post iodization era although Haryana is considered to be an iodine sufficient state with almost 100% of houses consuming iodised salt, in the present study, we found that 53.68% of pregnant women were having median urinary iodine levels in iodine deficient range as per WHO guidelines. Some of the short comings of present study include small sample size (although absolute no of participant was greater than other available studies), non-estimation of salt iodine content and no collection of information about salt storage. So there is need to estimate urinary iodine from a larger sample of pregnant women across different districts of not only Haryana but even other parts of country which are supposed to be iodine sufficient as Haryana to find out real current status of iodination in our population.