Iodine adequacy in reproductive age women living in the Western region of Saudi Arabia

Background Despite the signicance of iodine deciency in women of reproductive age due its associated serious maternal and foetal complications, surveys related to this vulnerable population in the Kingdom of Saudi Arabia (KSA) are lacking. This study, therefore, aimed to measure the frequency alongside the potential socioeconomic factors contributing towards iodine inadequacy in Saudi women of childbearing age from the Western province of KSA. Methods Urinary iodine concentrations IUIC) were measured in random spot samples collected from 1222 pregnant women and 400 age-matched non-pregnant/non-lactating women. The socioeconomic characteristics were obtained through a structured questionnaire. The classication of iodine suciency was based on the WHO criteria for UIC in pregnant (150–249 μg/L) and non-pregnant women (100–199 μg/L). the μg/L; IQR: WHO recommended other μg/L; IQR: 199) of the WHO Conclusions This study is the rst to show high prevalence of mild iodine deciency among reproductive age women, which could represent a serious public health This study also advocates to establish iodine intake in pregnant women to avoid the perilous maternal-foetal health consequences of iodine deciency.


Background
Iodine is essential for the synthesis of thyroid hormones and suboptimal intake of this nutrient at the periconceptual and/or during pregnancy has been linked with maternal goitre and hypothyroidism [1,2].
Iodine de ciency during pregnancy also signi cantly increase the risk of delayed neurodevelopment and poor cognitive functions in the offspring [3,4]. Although many countries have implemented the universal salt iodisation (USI) policy to ensure adequate access to iodine by the general public [5,6], numerous reports from different developed and developing countries have demonstrated high rates of iodine insu ciency among pregnant women [7][8][9][10][11]. Accordingly, many researchers have emphasised the need for alternative means (e.g. iodine supplements) to ensure adequate iodine intake in women of reproductive age [7][8][9][10][11] and the World Health Organisation (WHO) has recommended the increase of iodine intake from 150 to 250 μg/day during pregnancy [12].
About 90% of ingested iodine from diet and/or supplement is excreted by the kidney and, therefore, measuring urinary iodine concentration (UIC) is the recommended biochemical approach for assessing iodine status [13][14][15] However, compliance with the collection of 24-hour urine is low and spot urine analysis as well as estimating 24-hour urine iodine excretion  are alternative more convenient and accurate methods for measuring iodine intake [13][14][15]. Surveying school-age children (SAC) is also the currently accepted mean for evaluating iodine status within a population since they are easy to access, and it is believed they re ect the nutritional status of their families [16]. Yet again, the most recent report by the Iodine Global Network (IGN) in 2019 has indicated that 29 out of the 40 countries with data on iodine status among SAC and pregnant women have reported su ciency in the former group, whereas de ciency was common during pregnancy [5]. Hence, it has been proposed that surveying pregnant women should be conducted independent from SAC to precisely measure their iodine adequacy status [7][8][9][10][11].
In the Kingdom of Saudi Arabia (KSA), thyroid disorders are frequent among the general population at different ages [17][18][19][20]. Goitre was also found to be common in Saudi children especially in those living in high altitude areas of the kingdom [21,22]. Alissa et al. (2009) reported iodine de ciency in 85% and 83% of their healthy participants and hypothyroid patients from the Western region of KSA, respectively [23].
However, KSA is currently classi ed by the IGN (2019) as iodine su cient according to a national survey conducted among SAC in 2012 [5]. In contrast, numerous more recent studies on Saudi SAC have revealed that the majority were iodine de cient and goitre was prevalent among them [24,25]. Moreover, only 70% of the Saudi households consume iodised salt according to another recent national survey and the numbers are below the WHO references [26]. Concomitantly, we have previously shown that 26.8% and 4.8% of 500 rst trimesteric pregnant Saudi women from the Western region had hypothyroidism and isolated hypothyroxinaemia, respectively [27]. Nevertheless, currently there is no report on the status of iodine among Saudi women of childbearing age.
Hence, this study measured iodine adequacy in Saudi pregnant females at each trimester and the results were compared with non-pregnant non-lactating women of reproductive age. Additionally, the socioeconomic characteristics were collected to identify the factors that could contribute to iodine de ciency in this high-risk population. A better understanding about iodine status in this vulnerable group could enlighten the health authorities and policymakers regarding the magnitude of iodine de ciency during pregnancy and may possibly support the development of appropriate policies regarding screening and treatment of pregnancy associated iodine inadequacy in the kingdom.

Study design
This non-randomised cross-sectional study was conducted from March 2017 to May 2019 and the ethical approval (AMSEC 21-16-02-2017) was obtained from the Faculty of Applied Medical Sciences Ethics Committee in Umm Al-Qura University. The study population (n = 1662) consisted of 1222 apparently healthy pregnant Saudi women (18-44 years) at the different trimesters and who were recruited from the antenatal care unit in the Medical Centre of Umm Al-Qura University in Makkah city. Another 400 agematched non-pregnant non-lactating Saudi women and who had at least a single previous successful normal pregnancy were also recruited from the same centre during the routine vaccination of their children. All the participants had no current symptoms/signs or history of thyroid disorders, chronic diseases (e.g. hypertension, diabetes mellitus, etc.), gestational related medical disorders (e.g. preeclampsia, gestational diabetes, anaemia, etc.) or autoimmune diseases.
A fresh random urine sample (5 ml) was collected in a sterile urine container between 9:00 am and 1:00 pm from each woman and the samples were stored at -70 °C till transported to the Research Laboratories of the Faculty of Applied Medical Sciences in Umm Al-Qura University for processing. Additionally, the socioeconomic characteristics were obtained from all the participants through a structured questionnaire that included information related to age (years), pre-pregnancy and/or current weight (Kg) and height (cm) to calculate the body mass index (BMI), parity, type of salt intake, the use of daily iodine-containing supplements, family size, education level, employment status, total monthly income, smoking, residency and gestational age at the time of sample collection from the pregnant population. The question related to the use of iodised salt was categorised as previously reported into non-iodised, iodised and I don't know [28].

Urine Creatinine concentrations
Urine creatinine concentrations were measured on Cobas e411 (Roche Diagnostics International Ltd; Risch-Rotkreuz, Switzerland) according to the manufacturer's protocols. The predicted 24-hour urine creatinine excretion (24-hr Cr; g/day) was calculated by the previously published equation as follow [29]: Urine iodine concentrations 1

. Preparation of working solutions
All the chemicals were analytical grade and were purchased from Sigma-Aldrich Co. (MO, USA). The preparation of the required working solutions for measuring urine iodine was conducted as previously described [15]. Brie y, the acid ashing solution was freshly prepared by adding perchloric acid to nitric acid at the ratio of 4:1. The 0.125 molar arsenious solution was prepared according to the previously described method by Yaping et al. [30] that consisted of dissolving 4.8 g arsenic trioxide in 500 ml of 0.3 NaOH by vigorous stirring at 40 °C for 48 hours. Subsequently, 10 ml of 96% sulfuric acid were carefully added followed by 30 g of sodium chloride. The ceric ammonium solution was made by dissolving 14 g of tetra-ammonium cerium (IV) sulfate dihydrate in 300 mL deionised water with the subsequent addition of 52 mL of 96% sulfuric acid [15].

Iodine calibrators
The calibrators were prepared by using potassium iodate (KIO 3 ) that contains 59.3% iodine. The stock solution of 16000 nmol/l was prepared by dissolving 3.423 g of KIO 3 in 1000 ml of deionized water. The stock standard solution, which is stable for months, was then stored at 7 °C in the refrigerator in a brown bottle. Seven calibrators were freshly made as serial dilutions using deionized water to prepare the following standards: 1600; 800; 400; 200; 100; 50; 25 nmol/l which are equivalent to 203; 102; 51; 25; 13; 6; 3 μg/l of iodine. No zero-standard was added since the concentrations of iodine are logarithmically graded for the standard curve [15].

Laboratory procedures for measuring urine iodine concentrations
The UIC in the collected samples were measured in 96-well plates as previously described and according to the principles of the Sandell-Kolthoff method [15]. Brie y, 250 μl of the ashing acid solution were added to each urine sample subsequent to the addition of glucose (20 μl) and sulfuric acid (1 ml), and the samples were then incubated in a dry bath incubator at 200 °C for 25 minutes as an initial step to eliminate any potential interfering substance through oxidation. The urine samples together with the iodine calibrators were then transferred to a fully automated ELISA machine to perform the catalytic step in 96-well clear polystyrene at-bottomed microplates (Thermo Fisher Scienti c). Each of the calibrators and samples (100 μl/well) was pipetted in duplicate and the arsenious acid solution (100 μl) followed by 50 μl of the yellow ceric (IV) ammonium sulfate solution were then added to each well. The plate was subsequently incubated for 20 minutes at 37 °C and the absorbance was then determined at the 405 nm wavelength and the readings were corrected by the 620 nm wavelength [15]. The 24-hr UIE was calculated according to the previously published equation by Perrine et al. [14] as follow: Spot urine [Iodine/Creatinine (μg/g)] × predicted 24-hour Cr (g/day).
The WHO reference intervals for the classi cation of iodine adequacy according to the amount of 24-hr UIE were applied as follow: de ciency (< 100 and < 150 ug/day), su ciency (100-199 and 150-249 ug/day) and above requirements (≥ 200 and ≥ 250 ug/day) for non-pregnant and pregnant women, respectively [13].

Statistical analysis
Statistical analysis was performed by SPSS software version 25 (New York, USA), and P < 0.05 was considered statistically signi cant. The Kolmogorov-Smirnov test for normality and Levene test for homogeneity were performed. Continuous data were expressed either as mean ± standard deviation (SD) or median with the interquartile range (IQR) depending on data normality, whereas ordinal and discontinuous data was shown as numbers and percentages. Cross-tabulation followed by Chi square (χ 2 ) test was used for frequency analysis. Based on data normality, the independent Student's t-test or Mann-Whitney U test were applied for comparing continuous data between two groups. One-way ANOVA or Kruskal-Wallis based on data normality followed by either Tukey's HSD or Games-Howell post-hoc tests based on variance equality were used to compare between more than two groups. Multinomial regression analysis was used to identify the socioeconomic predictors of iodine status.
Overall, there was no signi cant difference between the non-pregnant (n = 400) and pregnant populations (n = 1222) in relation to age (28.7 ± 5.7 vs. 29.1 ± 7.3 years; P = 0.3). However, the periconceptional BMI was signi cantly higher in the pregnant population (25.7 ± 4.7 Kg/m 2 ) compared with the current BMI of the non-pregnant women (24.03 ± 5.3 Kg/m 2 , P < 0.001). Furthermore, there were signi cant differences in the distributions of age groups, BMI classes, parity groups, monthly income categories and the percentage of women using iodine supplements between the non-pregnant and pregnant women ( Figure  1).
Among the 1222 (75.3%) pregnant population, 364 (22.4%) were in the rst, 352 (21.7%) in the second and 506 (31.2%) in the third trimesters of pregnancy. Moreover, there were signi cant differences between the non-pregnant women and the three trimesteric groups in age, BMI, family size, monthly income, parity groups, educational levels, frequency of active smokers and the use of daily iodine supplements (Table  1).
Spot and estimated daily iodine excretion Although spot urine iodine ( Figure 2A) and Cr ( Figure 2B) concentrations were signi cantly (P < 0.01) higher in the pregnant compared with the non-pregnant women, the spot I/Cr ratio was similar between both groups ( Figure 2C). Moreover, the estimated 24-hr urine Cr ( Figure 2D) and UIE ( Figure 2E) were signi cantly higher in the pregnant compared with the non-pregnant females. Notably, the median value of the 24-hr UIE in the non-pregnant women (101.64 μg/L; IQR: 73.72) was within the lower limit of the recommended WHO intervals for adequate iodine intake (100-199 μg/L). On the other hand, the 24-hr UIE in the pregnant population (112.99 μg/L; IQR: 104.56) was well below the recommended levels de ned by the WHO for iodine su ciency intake (150-249 μg/L) during pregnancy ( Figure 2E).
By further analysis, the spot UIC was only signi cantly higher in the 2 nd and 3 rd trimester groups ( Figure   3A), whereas all the trimesteric groups had marked elevations in the spot urine Cr levels ( Figure 3B), compared with the non-pregnant group. Nevertheless, the spot I/Cr ratio was comparable between the different groups ( Figure 3D). Although, the 24-hr UIE was markedly elevated in the 2 nd and 3 rd trimesteric groups compared with the non-pregnant group ( Figure 3E

Iodine status in the non-pregnant and pregnant women
According to the estimated 24-hr UIE, the overall prevalence was 59.4% (n = 963) for iodine insu ciency, 35.3% (n = 573) for iodine su ciency and 5.3% (n = 86) for above requirements in the 1622 study participants. Additionally, 49.7% (n = 199/400) of the non-pregnant population and 62.5% (n = 764/1222) of the pregnant women were classi ed as iodine insu cient. In contrast, the frequencies of taking iodine above-requirement were 8.5% (34/400) among the non-pregnant and 4.3% (52/1222) within the pregnant participants.
Additionally, the rates of iodine insu ciency increased signi cantly by 26% in the 1 st and 50% in the 3 rd trimesteric groups compared with the non-pregnant group (Table 2). However, the rates of insu ciency in the 2 nd trimester were comparable with those of the non-pregnant and 1 st trimester group and markedly lower than the 3 rd trimester group (Table 2). On the other hand, the numbers of women with su cient iodine decreased signi cantly in the 1 st and 2 nd trimester groups than those of the non-pregnant and 3 rd trimester groups. Coherently, the frequency of cases with iodine intake above requirements diminished markedly in the 1 st and 2 nd trimesters in comparison with the non-pregnant women ( Table 2).
Factors associated with insu cient or above requirement iodine intake The multinomial regression analysis showed that each of the trimesteric groups had signi cantly higher risk for developing iodine insu ciency compared with the non-pregnant women and the highest risk was detected in the 1 st trimester group (Table 3). Moreover, those women who reported the use of non-iodised salt showed two-fold increase in the risk of insu ciency compared with those using iodised salt. Opposingly, high BMI and the use of iodine supplements were associated with signi cantly lower risk for iodine insu ciency (Table 3). On the other hand, an increase in BMI was the only independent factor associated with a signi cant increase in the risk of taking iodine above the required levels. Alternatively, women in the 2 nd trimester and the use of non-iodised salt signi cantly decreased the risk of having excessive iodine intake (Table 3).

Discussion
The daily requirements of nutritional iodine increase immensely during pregnancy to supply the demands of the growing foetus as well as the increase in iodine clearance by the kidney [27]. Therefore, insu cient iodine intake in women, especially during pregnancy, represents a major worldwide health concern [31]. To the best of our knowledge, this study is the rst to measure iodine adequacy in a cohort of reproductive age Saudi women and the results were compared between the pregnant and non-pregnant participants. The data showed that the median of 24-hr UIE in the non-pregnant population was at the lowest recommended limit, whereas it was markedly inferior in the pregnant women than the minimal cutoff for iodine su ciency advocated by the WHO [13]. Although the majority of non-pregnant (73%; n = 292/400) and pregnant women (71.5%; n = 874/1222) reported the use of iodised salt, the rates of iodine de ciency were 49.7% (n = 199) and 62.5% (n = 764), respectively. Moreover, only 2.8% of the nonpregnant and 27.6% of the pregnant women were using iodine supplements.
The currently available reports regarding iodine nutritional state among the Saudi population are controversial and national surveys are rare. The general Saudi public is considered by the latest IGN report in 2019 as iodine su cient based on the ndings of a 2012 national survey of SAC [5,16]. Additionally, another study that included 311 SAC enrolled from a previously reported severe iodine de cient region in the Southwestern of KSA showed a signi cant enhancement in iodine status following the implementation of salt iodisation program [32]. Conversely, a more recent report from the same geographical region has revealed in 2015 severe iodine de ciency (median UIC 17 μg/L) in 3046 SAC among whom 24% had goitre [24]. Another study on 1887 SAC from Al-Taif city in Makkah province has also demonstrated a rate of 71% for nutritional iodine de ciency (median UIC 84 μg/L) and 7.4% of the study population were goitrous [25]. Coherently, a more recent national survey has shown that 70% of the Saudi households were using iodised salt, which does not meet the target of 90% set by the WHO [26]. Additionally, we have previously reported 26.8% hypothyroidism and 4.8% isolated hypothyroxinaemia in 500 pregnant Saudi women from the Western region [27]. The present study correlates with the prior reports that have underscored the magnitude of iodine de ciency in several regions of KSA, including Makkah province, as well as thyroid disorder in pregnant women [24][25][26][27].
Our results also suggest that the Saudi women of childbearing age, at least in the Western region, appear to suffer from mild to moderate iodine de ciency and the rates could increase signi cantly during pregnancy. Additionally, this study provides further support to the notion that the levels of UIC in SAC may not accurately re ect iodine status for pregnant women [7][8][9][10][11]. In consolidation, the IGN has revealed that 29 countries have reported iodine de ciency in pregnant women, whereas their SAC population was su cient [33]. Studies from the United States have also demonstrated a signi cant increase in the rates of iodine insu ciency among pregnant women despite the use of iodised salt [31]. Furthermore, a research group from Austria has similarly reported that 86.3% of 246 pregnant women were iodine insu cient even with the use of iodised salt and/or iodine supplements [7]. Coherently, the median of estimated 24-hr UIE in a Danish pregnant population was below the WHO limits with or without the use of daily supplements containing 175 μg/L of iodine [9]. More recently, another Israeli group has exposed that the median UIC was 61 μg/L in 1074 pregnant women and 85% of them were classi ed as iodine insu cient [10]. Likewise, a study from a Chinese province with iodine adequacy following the implementation of salt iodisation has shown that 50% of the enrolled 8159 pregnant women had iodine insu ciency [11]. Taken together, our study and the earlier reports advocate that the health authorities in each country should consider measuring iodine intake in women of reproductive age independently from SAC to precisely estimate the de ciency rates in this vulnerable group [7][8][9][10][11]. Furthermore, educational programs should be developed to increase the awareness of women of childbearing age, especially those who are pregnant or planning for pregnancy, about the signi cance of iodine for them as well as for their offspring wellbeing [34,35].
The status of nutritional iodine could be in uenced by numerous factors in reproductive age women [36][37][38]. Herein, the risk of developing iodine de ciency was highest during the rst trimester of pregnancy.
Although the risk decreased as pregnancy progressed, women in the 2 nd and 3 rd trimesters showed > 3.5fold increase in the risk of iodine inadequacy. In harmony, a recent Irish study has equally unveiled that the median of UIC was signi cantly lowest during the rst trimester and despite an increase in the urine levels during the following trimesters, the UIC remained markedly below the WHO recommended levels [39]. An explanation for the observed higher risk of iodine de ciency during the rst trimester could also be related to inappropriate nutrition intake due to loss of appetite and/or increase vomiting [40]. Several studies conducted among the Saudi population have also indicated that most of pregnant women were malnourished and their consumption of essential nutrients were below the recommended daily requirements [41][42][43][44]. Saudi women from the Western region had also signi cantly lower intake of micronutrients during the rst trimester, thus their offspring had an increased risk of developing birth defects [45][46][47]. Our ndings agree with the earlier observations since they showed that only 27.6% of the enrolled pregnant population were using iodine supplements, which was associated with signi cantly lower risk of developing iodine de ciency during pregnancy. Accordingly, this study reinforces the many calls for improving awareness regarding the importance of iodine intake from dietary and/or supplement sources, especially at the early stages of pregnancy [39,[48][49][50].
The present results also revealed that the pregnant women consuming non-iodised salt had a 2-fold increase in the risk of developing inadequate iodine intake, whereas they showed > 50% decrease in the risk of taking iodine above their requirements during pregnancy. The WHO and UNICEF have adopted the USI policy since 1994 to ensure that the general public adequately consume su cient iodine [51].
Although the salt iodisation is implemented in KSA, the household consumption of iodised salt (70%) was found to be lower than the WHO recommendations of 90% usage by the general population to avoid iodine de ciency [26]. More recently, the WHO has also recommended salt reduction to 5 gram/day for adults, including pregnant and lactating women, to lessen the likelihood of developing hypertension and heart diseases [6]. Suggested plans to simultaneously maintain iodine adequacy with decreasing salt intake include fortifying salt with higher amounts of iodine [6]. Alternatively, Australia and New Zealand have adopted a different strategy by fortifying bread to ensure the delivery of adequate iodine and several studies have reported that the median UIC in adults, including pregnant women, met the WHO recommended intervals post-forti cation [52,53]. Therefore, the reported lower use of iodised salt in KSA as well as the advised reduction of salt intake accentuate the importance of developing other vehicle(s) for iodine to decrease the incidence of iodine de ciency disorders [52,53]. Furthermore, women of reproductive age (150 µg/day) and pregnant women (250 µg/day) could bene t from daily iodine supplements as a temporarily method till developing a solid and effective national salt/bread iodisation program [26,52,53].
The present study also showed a weak positive association between BMI and UIC, which correlates with previous reports from Bangladesh and Romania [54, 55]. A possible explanation could be that pregnant and non-pregnant women with high BMI were consuming higher foods rich in iodine than lean individuals. Additionally, pregnant women often change their dietary habits and eat more sh and milk, the richest sources of iodine, and the tendency for consuming these foods is higher in obese than lean women [56]. Opposingly, several other studies either have reported negative association between BMI and UIC [9,57] or have shown no correlation between body weight and iodine intake [8,39]. These discrepancies between the studies could be linked to differences in eating habits and dietary patterns between the different populations as well as between cities of each country [58, 59].
There are several limitations for your study. Although the number of participants is larger compared with several other reports on pregnant women [7][8][9], our study participants were enrolled only from a single site. Additionally, we did not measure the dietary intake and the thyroid function parameters to correlate them with the iodine status in the study population. However, this is a phase 1 study and we have plans to conduct further research with a special focus on pregnant women to measure the interactions between nutritional habits, thyroid functions and iodine intake.

Conclusions
This is the rst study to demonstrated nutritional iodine inadequacy in women of reproductive age from the Western region of Saudi Arabia despite the use of iodised salt and the prevalence of iodine de ciency increased signi cantly with pregnancy. Similar to other countries, this study highlighted the need to survey iodine intake in pregnant women independently from schoolchildren. Although the use of iodine supplements signi cantly decreased the risk of developing iodine de ciency among the targeted populations, less than 30% of pregnant women reported using supplements rich in iodine. Hence, our data advocates the necessity to develop/implement effective national programs in the different regions of KSA to overcome any potential complications arising from suboptimal iodine intake during pregnancy. Suggested actions include developing educational and awareness campaigns regarding the importance of iodine and to encourage adequate nutritional schemes based on diets rich in iodine (e.g. sh, milk, iodized salt intake and iodine forti ed foods). However, further studies are mandatory to assess the factual magnitude as well as the potential complications of iodine de ciency during pregnancy in the kingdom.

Competing of Interest
The authors declare no con ict of interest.     The distribution (%) of the socio-economic characteristics in the non-pregnant (n = 400) and the pregnant study populations. (* = P < 0.05 compared with the non-pregnant women by Chi-square test).