Thyroid Function Test (TSH, T3, T4) and Associated Factors in Early Gestation Among Pregnant Women Attending ANC Clinic in Nekemte Referral Hospital Western Oromia, Ethiopia.

Introduction: Thyroid function testing in pregnancy is an area of concern for pregnant women, doctors and laboratories. Some women are known to have thyroid disease before pregnancy and require monitoring to ensure no harm comes to them or their baby. Thyroid disorders can have adverse reproductive and pregnancy implications. Thus, the aim of this study was to assess blood level of thyroid function test during early gestation and assess associated factors among pregnant women attending antenatal care clinic in Nekemte Referral Hospital. Methods: Facility-based Cross-sectional study design was deployed to collect information from 300 Pregnant women (<20 gestation weeks) attending antenatal clinic from March to May, 2017. Interviewer-administered structured questionnaire was used to obtain information and blood samples were collected from pregnant women for estimation of thyroid function test after getting written consent. Data was entered to Epi info version 3.5.3, cleaned and analyzed using SPSS version 24 and Multivariable analysis were used to examine the association between dependent and independent variables. Odds ratios (ORs) and their 95% condence intervals (CIs) were calculated. Signicance was declared at P-value 0.05. Result: A total of 300 pregnant women were interviewed and blood sample collected from them for thyroid function test with a response rate of 90.1%. Among the study subjects 14(4.7%) have Subclinical Hypothyroidism (SCH). Husband occupation, presence of Radio in the household, presence of television in their house and treating drinking water at household level were among factors associated with development of Hypothyroidism. Conclusion and Recommendation: In this study, Subclinical Hypothyroidism (SCH) among pregnant women in early gestation is signicant 14(4.7%) above the expected 2.5% of American thyroid association cut point. Husband occupation, Presence of media for information and treatment of drinking water were the associated factors. So, health workers should always update pregnant women on importance of consuming proper utilization of iodized salt. Health workers shall also teach pregnant women on personal and environmental hygiene.


Introduction
Thyroid function testing in pregnancy is an area of concern for pregnant women, doctors and laboratories [1,-3]. The changes in thyroid function tests with gestational age were elegantly shown in a study of 13,599 singleton pregnancies assessed at one week intervals from week 6 to term where serum TSH fell to a trough at week 10 followed by a progressive increase to term [4]. Some women are known to have thyroid disease before pregnancy and require monitoring to ensure no harm comes to them or their baby. Others may have unrecognised disease and there has been debate about the merits of screening, the potential harm caused by mild hypothyroidism, and how women should be tested [5,6]. Thyroid disease is the second most common endocrine disease to affect women of reproductive age. Thyroid disorders can have adverse reproductive and pregnancy implications. Although gestational hyperthyroidism is uncommon (0.2%), gestational hypothyroidism occurs in higher prevalence (2.5%) and can lead to neonatal and child neuro developmental de cits and maternal obstetric complications [7,8].
Maternal overt and subclinical thyroid disorders and dysfunction are associated with complications of pregnancy and both short-and long-term consequences for the mother and child. These risks seem to be increased in women with euthyroid autoimmune thyroid disease. Hypothyroidism during pregnancy is associated with gestational hypertension and low birth weight. Women who are on thyroid replacement therapy before pregnancy may require an increase in dosage during pregnancy [8,9]. It is commonly thought that total thyroxine (TT4) and total triiodothyronine (TT3) concentration increase in the setting of pregnancy-induced increases in serum TBG concentrations. Free T3 (FT3) and free T4 (FT4) levels are slightly lower in the second and third trimesters. Thyroid-stimulating hormone (TSH) levels are lownormal in the rst trimester, with normalization by the second trimester [10]. Accurate serum FT4 measurements are needed to differentiate overt hypothyroidism from subclinical hypothyroidism and for the diagnosis of euthyroid hypothyroxinaemia, the controversial diagnostic group that some claim is associated with adverse pregnancy outcomes [11].
Pregnant women with chronic autoimmune thyroiditis have a higher incidence of spontaneous miscarriage. Women with high TSH levels had a > 3-fold increase in risk of very preterm delivery, and in some analyses, gravidas who tested positive for antithyroglobulin antibody (TgAb) at entry to prenatal care also had a > 2-fold increased risk of very preterm delivery [8,9]. During the rst two trimesters of pregnancy the fetus is entirely dependent on the maternal thyroid hormone supply as the fetal thyroid does not develop until 13-15 weeks of gestation [12]. As the fetus progresses into the third trimester, it develops the ability to produce its own thyroid hormones but it is still dependent on maternal iodine for hormone synthesis [13]. Iodine de cient status in the mother is associated with impaired fetal development, both mental and physical. Epidemiological studies and case reports show that even a relatively minor degree of maternal hypothyroxenemia during the rst half of gestation is potentially dangerous for optimal fetal neurodevelopment [14].
Thyroid disorders are prevalent in women of child-bearing age and for this reason commonly present in pregnancy and the puerperium. Uncorrected thyroid dysfunction in pregnancy has adverse effects on foetal and maternal well-being. The deleterious effects of thyroid dysfunction also extend beyond pregnancy and delivery to affect neurointellectual development in the early life of the child [15].There is a greater prevalence of subclinical hypothyroidism in women with delivery before 32 weeks and there is even an association between thyroid autoimmunity and adverse obstetric outcome, which is independent of thyroid function [16]. Higher maternal TSH levels even within the normal reference range are associated with an increased risk of miscarriages, foetal and neonatal distress as well as preterm delivery [9,17].
It is essential therefore to have reliable accurate tests of thyroid function in pregnancy as maternal thyroid dysfunction may affect maternal health, foetal health and obstetric outcome. Gestational thyroid physiology affects thyroid tests and it is becoming clear that normative gestational related reference ranges for thyroid hormones are required for diagnosis. Therefore, the aim of the current study was to assess blood level of thyroid function test during early gestation (less than 20weeks) and assess associated factors among pregnant women attending Antenatal care clinic in Nekemte referral Hospital Western, Ethiopia.

Methods
The study has been carried out in Nekemte Referral Hospital which found in Nekemte Town. Nekemte Town is located in Oromia Regional State, in East Wollega Zone, Nekemte Woreda, at a Distance of 330 Km from Addis Ababa. Its astronomical location is 9º 04' North Latitude and 36º 30' East Longitude.
Administratively the town is divided into six sub administrative units; namely Cheleki, Burka Jato, Bakenisa Kese, Kaso, Bake Jema and Darge sub city. There is one referral governmental hospital and there are also two health centers 3 higher private clinics and 5 medium clinics in Nekemte town. The total population of the town as projected from 2007/2008 census is the town has a total population of 96,555 of which 47,282 (48.97%) are males & 49,273(51.03%) are females being distributed in its six sub cities.
Facility based Cross-sectional study design has been used to screen pregnant women for thyroid function test during early gestation of the pregnancy and blood samples were collected from pregnant women for estimation of thyroid hormones among pregnant women (< 20weeks) attending antenatal clinic from March to May, 2017.This comprises all pregnant women in Nekemte Referral Hospital catchment area. Study populations were all pregnant women's attending Antenatal clinic in Nekemte referral hospital.
A total of 300 pregnant women were enrolled for the study. Pregnant women who come before 20 weeks' gestation for antenatal care (ANC) during the study period were included in the study. Pregnant women with acute illness (active bleeding, acute febrile illness and diarrheal diseases), and revisits (follow ups) during study period was excluded. Literatures were reviewed to get information on the thyroid function test and associated factors among pregnant women.
Simple random sampling procedure was applied to select pregnant women from those who attend ANC daily. Data was collected using pretested interviewer administered questionnaire, which contains sociodemographic characteristics, obstetric and medical history, simpli ed food frequency questionnaire (FFQ) developed by Hellen Keller International (9). This questionnaire was adopted and modi ed by former Ethiopian Health and Nutrition Research Institute EHNRI (10). Interview based data was collected by experienced and trained nurses working at different Hospital. The questionnaire was administered after obtaining written informed consent from the study participants and orientation had been given on the objective of the study and detail explaining the procedure of specimen collection. Finally, completeness of questionnaire was checked daily and nally, completed questionnaire was returned to the principal investigators.
Venous blood (5 ml) was collected using a plain serum separating tube from pregnant women during their rst visit to the hospital for thyroid hormone analysis. After 30 minutes, the remaining blood samples were centrifuged at 5000 rpm for 5 minutes and serum was separated and stored at 2-8 0 cfor 5days. The separated serum sample was transported by data collectors in batch of average of 30 samples with ice pack placed in sample transport box and sent to National Medium clinic (Nekemte) for thyroid hormone analysis (TSH, T3, T4,). The thyroid hormone analysis was done using ELISA (Enzyme Linked Immuno Sorbant Assay) by senior laboratory personnel who have experience on the test and done as per manufacture instruction set for free T3, T4 and TSH test analysis. The result of thyroid hormone (T3, T4 and TSH) recorded at National Medium clinic laboratory was collected and sent to Nekemte referral hospital ANC clinic to be lled on participants' questionnaire form laboratory part by data collectors. To assure the data quality, data collection tool was prepared after intensive reviewing of relevant literatures and similar studies. Pre-testing of the questionnaire was carried out on the 10% of sample size in other Hospital. Based on the result obtained necessary modi cation was done. The completeness of the data was checked by the data collector during data collection and also immediately after data collection by the supervisor and principal investigators. Reagent preparation and test procedure were done as per the manufacturer protocol. Blood sample collected below the standard and miss labeled were rejected.

Data Analysis
After data collection, each questionnaire was checked for completeness and consistencies. Data entries were done by using Epi info version3.5.3. The entered data was cleaned and analyzed by using SPSS version 24. Descriptive statistics such as frequency, percentage, mean and standard deviation were used to describe selected variables. Bivariate analysis using binary logistic regression were carried out one by one for each independent variable and p < 0.25 were considered as cut off point to label the signi cance of the variables to be included in multivariate logistic regression. Variables which were signi cant in the bivariate analysis were exported to multivariate logistic regression analysis. Multivariate logistic regression analysis has been carried out to identify the most important in uencing factors of thyroid function test in pregnant women by controlling the effects of possible confounding variables. Finally, the variable that have signi cant association were identi ed by computing odds ratio, with 95% con dence interval and P value < 0.05.

Ethical consideration
The proposal was submitted to Wollega University to obtain Ethical clearance from Institutional review board of the University. Then permission letter was obtained and submitted to east Wollega Zonal Health Bureaus and different concerned authorities to obtain their cooperation. A written permission was obtained from east Wollega zonal health bureau and using this formal letter, the Hospital was asked for their cooperation to participate in this study through o cial letters and nally selected study participants were communicated after getting permission from the Hospital medical director/managers. Written informed consent was obtained from each study participants. For this purpose, a consent form was attached to each questionnaire which explains about the purpose of the study, con dentiality, and the respondent's full right of voluntary participation. Different measures have been taken to assure the con dentiality of study subject's response such as writing their names or any identi cation in the questionnaire was omitted.

Results
A total of 300 pregnant women were interviewed and blood sample collected from them for thyroid function test. Almost all 296(98.7%) of respondents are currently in union and 219(73.0%) of them are protestant Christian follower. The mean age of the respondents was 24.2(± 3.9 SD) years, which ranges from18 to 38 years. From the total respondents, 118(39.3%) attended higher education, while 97(32.4%) had attended secondary level and 12(4.0%) of mothers were unable to read and write. Approximately 155(51.7% of mothers were house wife during the survey and in line with this 111(37.1%) of the spouse of the respondents were government employee. Majority of the family 265(88.6%) and 211(70.6%) had own radio and television respectively with median monthly household income of 3500 Ethiopian Birr (ETB) and varying from 200 to 40000 ETB as indicated in the table (Table 1).
During the study period majority 164(54.7%) of the respondents were in rst trimester of pregnancy and most 155(51.7%) of them were nulliparous and 152(50.7%) of them were gravid during data collection. For most of them 125(84.5%) the interval between last birth and current pregnancy is more than two years and with mean family size of three (3.1 ± 1.2). Among the respondents 25(8.3%) had history of abortion. Ever family planning use among study groups were 211(70.3%) and commonly used method was injectable 121(57.3%). Concerning menstrual regularity 268( 89.3%) of them had regular menstrual cycle ( Table 2). The mean weight of the respondents is 58.5Kg (± 10.7SD) with mean height and body mass index of 160.5 cm (± 8.8SD) and 22.6 Kg/m 2 (± 3.2SD) respectively (Table 3).
According to our observation based on TSH test, 240(80.0%) of the respondents have normal thyroid stimulating hormone test, 38(12.7%) of the respondents have below normal range TSH test and 22(7.3%) of them have high or above normal TSH test. Two subjects have TSH above 10 µIU/ml. In line with TSH test, the test of FT4 conducted and in 53(17.7%) of the cases the test was normal and majority 247(82.3%) of the subjects have high T4 test results. In addition to this 31(10.3%) of the study subjects have suppressed TSH level and increased FT4 levels at the earlier age of the pregnancy (Figs. 1 and 2). On other hand 236 (78.7%) of the study subjects have above normal T3 test result and 64(21.3%) of the remaining were fall in normal range of the test result. And also 210(70.0%) of the study subjects were observed with both T3 and T4 test raised above normal range.
Accordingly, Maternal OH can be de ned as TSH concentration > 2.5 µIU/ml and FT4 concentration below normal range. In maternal SCH, patient may not have symptoms but the concentration of TSH is above 2.5 µIU/ml with normal FT4 concentrations. Among the study subject 14(4.7%) have Subclinical Hypothyroidism (SCH) and one (0.3%) subject has overt hypothyroidism. Concerning hyperthyroidism, Subclinical hyperthyroidism is de ned as serum TSH concentration below the lower limit of reference range, with fT3 & fT4 concentration within normal range. Overt hyperthyroidism is de ned as serum TSH concentration below the lower limit of reference range, with increase in fT3 & fT4 concentration. Among the study subjects, six (2%) of them had subclinical hyperthyroidism and 3(1%) of the subjects were develop overt hyperthyroidism. Only one subject found with TSH level > 2.5 µIU/ml and FT4 less than normal range. Of the study groups, one (0.3%) of them has overt hypothyroidism. Mean TSH, FT4 and FT3 were found to be 2.0 ± 1.9 µIU/ml, 13.2 ± 2.9 pmol/dl and 2.3 ± 0.6 µg/dl respectively.
Socio-demographic characteristics of pregnant women were the rst set of factors assessed for their association with Sub Clinical Hypothyroidism. Results from bivariate analysis indicate that maternal education, paternal occupation, those who regularly treat water for drinking, family income and presence of Television and Radio in the house were the factors that associated with Hypothyroidism. Concerning education of the pregnant women, taking primary or less educated as the reference, pregnant women who attended secondary school were 2.7 times more likely to develop Hypothyroidism (OR = 2.7;95%CI: 0.5, 13.9). Concerning paternal occupation, taking others category as reference pregnant women whose husband occupation farmer is 10% less likely to develop hypothyroidism and pregnant women whose husband occupation belongs to government and private employee were 20% less likely to develop hypothyroidism.
Household average family income has also showed association in binary analyses. Household with monthly income of less than one thousand birr were 2.5 times more likely to develop hypothyroidism 2.5(95%CI: 1.5, 65). The presence of television and radio in their home also showed difference in developing hypothyroidism during pregnancy. Pregnant women who have television in their house were less likely to develop hypothyroidism by crude odd ration of 0.7(95%CI: 0.6, 11.8). Pregnant women who have radio in their house were 40% less likely to develop hypothyroidism 0.4(95%CI: 0.1, 1.7). Those pregnant women who could not treat water in their house for drinking were 2.6 times more likely to develop hypothyroidism 2.6(95%CI: 1.5, 9.9). But, marital status, religion, ethnic group, education level, occupation of respondent, family size and age of respondent were not show an association with hypothyroidism.
After adjusting for the other variables, Husband occupation, those who regularly treat water for drinking, family income and presence of Television and Radio in house retain association in multivariate with Hypothyroidism. Concerning spouse occupation, taking others category as reference, pregnant women whose Husband occupation was government employee was 10% less likely to develop hypothyroidism (AOR = 0.1; 95%CI: 0.02, 0.47). Other variable which shown association with hypothyroidism was presence of radio in house, those pregnant women who has Radio in the household were 20% less likely to develop hypothyroidism (AOR = 0.2; 95%CI: 0.04, 0.9). Presence of television also shown association in multivariate, pregnant women who has television in their house were 30% less likely to develop hypothyroidism. Concerning household water treatment, those pregnant women who could not treat drinking water at household level were 2.4 times more likely to develop Hypothyroidism than those who treat water for drinking (AOR = 2.4; 95%CI:1.2, 8.8) (Table 4). Among pregnant women, 27(9.0%) had history of acute febrile illness before or during pregnancy 18(6.0%) of pregnant were treated for malaria and 290(96.7%) of respondents have been screened for HIV/AIDS.

Discussion
Thyroid hormone is important for the normal development of a baby during pregnancy. Some studies have shown that even a mildly underactive thyroid (subclinical hypothyroidism) in pregnant women between 11-13 weeks of pregnancy is a risk factor for miscarriage [18].
Other studies have shown that having high levels of thyroid antibodies in the blood, which are associated with one form of hypothyroidism, during pregnancy can also increase the risk of miscarriage [19]. So, this study tried to screen pregnant women for thyroid function test during early gestation and assess associated factors among pregnant women attending antenatal care clinic at Nekemte Referral Hospital.
According to this nding, based on TSH test, 240(80.0%) of the respondents have normal thyroid stimulating hormone test, 38(12.7%) of the respondents have below normal range TSH test and 22(7.3%) of them have high or above normal TSH test. Two subjects have TSH above 10µIU/ml. A total of 86.7% of pregnant woman (492/573) showed normal thyroid function tests. This is in line with study done in Turkey [20].
In line with TSH test, the test of FT4 conducted and in 53(17.7%) of the cases the test was normal and majority 247(82.3%) of the subjects have high T4 test results. In addition to this 31(10.3%) of the study subjects have suppressed TSH level and increased FT4 levels at the earlier age of the pregnancy. On other hand 236 (78.7%) of the study subjects have above normal T3 test result and 64(21.3%) of the remaining were fall in normal range of the test result. And also 210(70.0%) of the study subjects were observed with both T3 and T4 test raised above normal range. This is different from study done in Sudan [21]. The possible difference is may be because of nutritional status difference, geographical location multiple pregnancy, birth interval and previous bleeding history which is not the case in current study.
Accordingly, OH can be de ned as TSH concentration > 2.5 µIU/ml and FT4 concentration below normal range. In maternal Subclinical Hypothyroidism (SCH), patient may not have symptoms but, the concentration of TSH is above 2.5 µIU/ml with normal FT4 concentrations. This is in contrast with previous study in which the average ranges of TSH and T4 were (0.5-6.5 mIU/L) and T4 (10.5-20.4 Pmol/L) respectively and Subclinical and overt hypothyroidism was found in 56 (13.7%) of women [21]. This difference might be due to study site and study population difference.
Among the study subjects, 14(4.7%) have Subclinical Hypothyroidism (SCH) and one (0.3%) subject has overt hypothyroidism. This is comparable with other study, in which the prevalence of thyroid dysfunction in pregnancy was 10.8% with hypothyroidism being 9.2%, out of which 8.5% were cases of subclinical hypothyroidism and 0.7% was cases of overt hypothyroidism [22]. But in contrast with previous study done elsewhere in which (10.5%) had subclinical hypothyroidism, 3.2% had overt hypothyroidism [21] and 0.5% had hypothyroidism, 8.9% had subclinical hypothyroidism, and 2.8% had hyperthyroidism [20] and the prevalence of hypothyroidism and hyperthyroidism was 12 and 1.25%, respectively [23]. This difference might be due to difference in study site and study population.
In the present study, among the study subjects, 6(2%) of them had subclinical hyperthyroidism and 3(1%) of the subjects were develop overt hyperthyroidism. Only one subject found with TSH level > 2.5 µIU/ml and FT4 less than normal range. Of the study groups one (0.3%) of them has overt hyperthyroidism. This is contrast with other study done elsewhere; in which 260 pregnant women (19.41%) had TSH values more than 3.0 mIU/L and of these, 216 had normal T 4 value, hence labeled as subclinical hypothyroidism and 44 had low T 4 , hence termed overt hypothyroidism. Three pregnant women had overt hyperthyroidism and 11 had subclinical hyperthyroidism. Nine women had low T 4 values-Isolated hypothyroidism [24] and also forty-three women (10.5%) had subclinical hypothyroidism, thirteen (3.2%) had overt hypothyroidism [21]. This difference might be due to study population and study area difference. Different factors have been assessed for association with hypothyroidism in multivariate logistic regression. Factor showed association with hypothyroidism during pregnancy were spouse occupation, pregnant women whose Husband occupation was government employee were 10% less likely to develop hypothyroidism this is probability related to consumption of food rich in iodine content. But there were no similar studies done elsewhere which indicated the signi cance of these factors with hypothyroidism and hyperthyroidism to compare with the present study.
Other variable which shown association with hypothyroidism was presence of radio in house, those pregnant women who has Radio in the household were 20% less likely to develop hypothyroidism. This is may be related to awareness of the family as radio can be source of information on consuming iodine rich food and utilization of iodized salt in household level. Presence of television also shown association in multivariate, pregnant women who has television in their house were 30% less likely to develop hypothyroidism. This is also related to information announced on television is more familiar and practical focus for pregnant women easily understand practice on iodine utilization. However, there were no similar studies done elsewhere which indicated the signi cance of these factors with hypothyroidism and hyperthyroidism to compare with the present study.
On other hand pregnant women who would not treat drinking water at household level were 2.4 times more likely to develop hypothyroidism than those who treat water for drinking. This is may be related to some infectious diseases that may have effect on iodine utilization and also some infectious diseases may cause thyroid diseases disorder. This was not the case in the previous study done showed signi cant association between overt hypothyroidism and history of infertility, history of miscarriage, stillbirth, preterm deliveries, BMI, maternal age and iodine de ciency [21,22]. But in the present study, marital status, religion, ethnic group, education level, occupation of respondent, family size and age of respondent were not show an association with hypothyroidism. But there were no similar studies done elsewhere to compare with.

Strength And Limitation Of The Study
The strength of this study is its inclusion of early age pregnancy and laboratory-based study was conducted. The limitation of this study was its cross sectional design effect and urine iodine test was not done.

Conclusion And Recommendation
According to this study 14(4.7%) have SCH and one (0.3%) subject has overt hypothyroidism and 6(2%) study subjects of them had subclinical hyperthyroidism and 3(1%) of the subjects were develop overt hyperthyroidism. Husband occupation, presence of radio in house, presence of television, pregnant women who would not treat drinking water at household level was associated with development of hypothyroidism. Health workers should always update pregnant women on importance of consuming proper utilization of iodized salt, on personal and environmental hygiene and on danger sign of pregnancy as it may occur due to hypothyroidism.